Paul R Langlais

Interests

Teaching

Insulin action, post-translational modification, signal transduction, cytoskeletal dynamics, glucose uptake, mass spectrometry, quantitative proteomics

Research

The role of insulin is to lower blood glucose levels by stimulating glucose uptake into muscle and adipose tissue. Resistance to insulin, a phenomenon directly involved in the pathogenesis of type 2 diabetes, remains to be understood. Basic research has yet to fully discover how insulin action is elicited. Research in the laboratory of Paul R. Langlais, Ph.D., focuses on the identification and characterization of proteins involved in insulin signal transduction and also tests whether the dysfunction of these proteins is involved in the pathogenesis of insulin resistance and type 2 diabetes.Dr. Langlais specializes in the use of mass spectrometry to perform proteomics, a technique that allows for large-scale quantitative analysis of protein abundances between different treatments. This approach led him to the discovery that CLIP-associating protein 2 (CLASP2) is responsive to insulin stimulation, and his now-published findings support the involvement of CLASP2 in insulin-stimulated glucose uptake. A series of research articles were published by the Langlais Lab characterizing insulin action in the context of regulating microtubule-associated proteins, in addition to identifying new proteins previously unknown to function in this system.Dr. Langlais leads the University of Arizona Quantitative Proteomics Laboratory, a collaborative environment aimed at offering investigators the infrastructure to perform large-scale quantitative proteomics studies.

Courses

Scholarly Contributions

Chapters

• Langlais, P. R., & Mandarino, L. J. (2019). Proteomic Profiling of Human Skeletal Muscle in Health and Disease. In Cell Omics approaches to understanding muscle biology.

Journals/Publications

• Victor, R. A., Lipinski, A., Langlais, P. R., & Schwartz, J. C. (2026). Identifying Subcellular Structure Components in Escherichia Coli by Crosslinking and SEC-MS. Proteomics, e70105.
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  Cells are comprised of a broad spectrum of structures that compartmentalize biochemical and signaling mechanisms. These structures can be comprised of many biomolecules, but especially lipids, proteins, and nucleic acids. Techniques are limited to quantify or discover new subcellular structures. We explored whether a proteomics approach using chemical crosslinking followed by size-exclusion chromatography and mass spectrometry (SEC-MS) of whole cell lysates can address this challenge. Formaldehyde crosslinking was used to preserve the weak molecular interactions responsible for many protein and nucleic acid assemblies. In this study, we perform the first formaldehyde crosslinking-assisted SEC-MS in a bacterial system. We demonstrate that when expressed ectopically in E. coli, large structures of a known assembly protein, FUS, can be detected through SEC-MS. We then show that E. coli proteins are enriched in particles of large or medium size due to formaldehyde crosslinking, which is the first analysis by formaldehyde and SEC-MS for a bacterial system. Last, analysis identified previously characterized E. coli protein assemblies and condensates, as well as potentially novel associations of prokaryote metabolism with large subcellular bodies. We propose this unbiased method can be used to stimulate or supplement targeted methods for discovery of new cellular bodies in a wide range of cell types.
• Cecil, J. H., Padilla, C. M., Lipinski, A. A., Langlais, P., Luo, X., & Capaldi, A. P. (2025). The molecular logic of Gtr1/2- and Pib2-dependent TORC1 regulation in budding yeast. eLife, 13.
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  The Target of Rapamycin kinase Complex 1 (TORC1) regulates cell growth and metabolism in eukaryotes. Previous studies have shown that, in , nitrogen and amino acid signals activate TORC1 via the highly conserved small GTPases, Gtr1/2, and the phosphatidylinositol 3-phosphate binding protein, Pib2. However, it was unclear if/how Gtr1/2 and Pib2 cooperate to control TORC1. Here, we report that this dual regulator system pushes TORC1 into at least three distinct signaling states: (i) a Gtr1/2 on, Pib2 on, rapid growth state in nutrient replete conditions; (ii) a Gtr1/2 inhibited, Pib2 on, adaptive/slow growth state in poor-quality growth medium; and (iii) a Gtr1/2 off, Pib2 off, quiescent state in starvation conditions. We suggest that other signaling pathways work in a similar way to drive a multilevel response via a single kinase, but the behavior has been overlooked since most studies follow signaling to a single reporter protein.
• Diezel, C. C., Majuta, L. A., Bahramnejad, E., Karlage, K. L., Partin, J. L., Barbour, S. M., Peterson, I. L., Flowers, M., von Hippel, S. T., Haveman, R., Villarroel, E., Villarroel, I., Langlais, P. R., Largent-Milnes, T. M., Vanderah, T. W., & Riegel, A. C. (2025). Molecular Dynamics in the Ventral Tegmental Area during Chronic Pain-Induced Negative Affect. bioRxiv : the preprint server for biology.
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  Chronic pain frequently coexists with negative affect, with about 60% of patients suffering from both. This dual condition complicates treatment and exacerbates both disorders, highlighting the urgent need for innovative therapeutic strategies. Chronic pain negative affect (CPNA) involves complex neurobiological changes, including increased hyperexcitability of the ventral tegmental area (VTA), a critical region involved in reward, mood, and pain processing. To elucidate CPNA's underlying mechanisms, we employed a multidisciplinary approach using immunohistochemistry, lipidomic analysis, and proteomic screening to investigate VTA molecular alterations in mice subjected to partial sciatic nerve ligation (pSNL) at one and four weeks post-injury. Our results revealed a significant, sex-dependent increase in Kv7.2 channel expression in dopamine neurons, alongside a notable reduction in endocannabinoid 2-arachidonoylglycerol (2-AG) levels, which plays a vital role in mood regulation. This neurochemical shift associated with an increase in negative affect-like behaviors, as determined by the forced swim test. Furthermore, pharmacological intervention utilizing either exogenous 2-AG or retigabine, a Kv7 channel opener, effectively alleviated pain-related negative affect symptoms. Proteomic profiling further uncovered alterations within the CaMKK2 pathway, involving crucial proteins such as PLCγ2, AMPKγ2, and AMPKβ1, and CaMK1α, with changes in abundance and phosphorylation activity that could be reversed with the next-generation CaMK1α antagonist CS640. This research provides the first comprehensive analyses of VTA adaptations linked to CPNA, yielding significant insights into molecular changes impacting VTA neuronal integrity and signaling throughout CPNA progression.
• Lopez-Pier, M. A., Marino, V. A., Vazquez-Loreto, A. C., Skaria, R. S., Cannon, D. K., Hoyer-Kimura, C. H., Solomon, A. E., Lipovka, Y., Doubleday, K., Pier, M., Chu, M., Mayfield, R., Behunin, S. M., Hu, T., Langlais, P. R., McKinsey, T. A., & Konhilas, J. P. (2025). Myocardial transcriptomic and proteomic landscapes across the menopausal continuum in a murine model of chemically induced accelerated ovarian failure. Physiological Genomics, 57(Issue 7). doi:10.1152/physiolgenomics.00133.2024
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  Risk of cardiovascular disease (CVD) in women increases with the menopausal transition. Using a chemical model (4-vinylcyclohexene diepoxide; VCD) of accelerated ovarian failure, we previously demonstrated that menopausal females are more susceptible to CVD compared with peri-or premenopausal females like humans. Yet, the cellular and molecular mechanisms underlying this shift in CVD susceptibility across the pre-to peri-to menopause continuum remain understudied. In this work using the VCD mouse model, we phenotyped cellular and molecular signatures from hearts at each hormonally distinct stage that included transcriptomic, proteomic, and cell biological analyses. The transcriptional profile of premenopausal hearts clustered separately from perimenopausal and menopausal hearts, which clustered more similarly. Proteomics also revealed hormonal clustering; perimenopausal hearts grouped more closely with premenopausal than menopausal hearts. Both proteomes and transcriptomes showed similar trends in genes associated with atherothrombosis, contractility, and impaired nuclear signaling between pre-, peri-, and menopausal murine hearts. Further analysis of posttranslational modifications (PTMs) showed hormone-dependent shifts in the phosphoproteome and acetylome. To further interro-gate these findings, we triggered pathological remodeling using angiotensin II (Ang II). Phosphorylation of AMP-activated protein kinase (AMPK) signaling and histone deacetylase (HDAC) activity were found to be dependent on hormonal status and Ang II stimulation. Finally, knockdown of anti-inflammatory regulatory T cells (Treg) exacerbated Ang II-dependent fibrosis implicating HDAC-mediated epi-genetic suppression of Treg activity. Taken together, we demonstrated unique cellular and molecular profiles underlying the cardiac phenotype of pre-, peri-, and menopausal mice supporting the necessity to study CVD in females across the hormonal transition. NEW & NOTEWORTHY Cycling and perimenopausal females are protected from cardiovascular disease (CVD) whereas menopausal females are more susceptible to CVD and other pathological sequalae. The cellular and molecular mechanisms underlying loss of CVD protection across the pre-to peri-to menopause transition remain understudied. Using the murine 4-vinylcyclohexene diepoxide (VCD) model of menopause we highlight cellular and molecular signatures from hearts at each hormonally distinct stage that included transcriptomic, proteomic, and cell biological analyses.
• Padilla, C. M., Lim, J., Lipinski, A. A., Langlais, P. R., & Capaldi, A. P. (2025). Multilayered regulation of TORC1 signaling by Ait1, Gcn2, and SEAC/GATOR during nitrogen limitation and starvation. Nature communications, 17(1), 220.
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  The Target of Rapamycin kinase Complex I (TORC1) is a central hub in the cell growth and metabolic control network of eukaryotes. How its upstream regulators cooperate to tune signaling across environmental conditions remains unclear. Here, we combine phosphoproteomics, TORC1 activity assays, and targeted genetic perturbations to dissect TORC1 regulation in Saccharomyces cerevisiae during transitions from a high-quality nitrogen source (glutamine) to a low-quality nitrogen source (proline), and on to complete nitrogen starvation. In proline medium, Ait1 and Gcn2 attenuate TORC1 activity, establishing a partially inhibited "Low Nitrogen Adaptive" state marked by extensive metabolic reprogramming without growth arrest. In contrast, during nitrogen starvation, SEAC, Ait1, and Gcn2 cooperate to drive TORC1 into a fully inhibited state, triggering widespread dephosphorylation of its downstream targets and entry into quiescence. Our results define a multilayered regulatory circuit that governs graded TORC1 control-a design likely conserved across eukaryotes.
• Straub, L. G., Funcke, J. B., Joffin, N., Joung, C., Al-Ghadban, S., Zhao, S., Zhu, Q., Kruglikov, I. L., Zhu, Y., Langlais, P. R., Gordillo, R., Herbst, K. L., & Scherer, P. E. (2025). Defining lipedema's molecular hallmarks by multi-omics approach for disease prediction in women. Metabolism: Clinical and Experimental, 168(Issue). doi:10.1016/j.metabol.2025.156191
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  Lipedema is a chronic disease in females characterized by pathologic subcutaneous adipose tissue expansion and hitherto remains without druggable targets. In this observational study, we investigated the molecular hallmarks of lipedema using an unbiased multi-omics approach. We found adipokine dysregulation in lipedema patients participating in a cross-sectional clinical study (ClinicalTrial.gov, NCT02838277), pointing towards the adipocyte as a key player. Analyses of newly generated transcriptomic (SRA, PRJNA940039) and proteomic (ProteomeXchange, PXD058489) datasets of early- and late-stage lipedema samples revealed a local downregulation of factors involved in inflammation. Concomitantly, factors involved in cellular respiration, oxidative phosphorylation, as well as in mitochondrial organization were upregulated. Measuring a cytokine and chemokine panel in the serum of non-menopausal women, we observed little systemic changes in inflammatory markers, but a trend towards increased VEGF. Metabolomic and lipidomic analyses highlighted altered circulating glutamic acid, glutathione, and sphingolipid levels, suggesting a broader dysregulation of metabolic and inflammatory processes. We subsequently benchmarked a set of models to accurately predict lipedema using serum factor measurements (sLPM). Our study of the molecular signature of lipedema thus provides not only potential targets for therapeutic intervention, but also candidate markers of disease development and progression.
• Wright, E. B., Larsen, E. G., Padilla-Rodriguez, M., Langlais, P. R., & Bhattacharya, M. R. (2025). TMEM184B modulates endolysosomal acidification via the vesicular proton pump. Journal of Cell Science, 138(Issue 15). doi:10.1242/jcs.263908
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  Disruption of endolysosomal acidification causes toxic protein accumulation and neuronal dysfunction linked to neurodevelopmental and neurodegenerative disorders. However, the molecular mechanisms regulating neuronal endolysosomal pH remain unclear. Transmembrane protein 184B (TMEM184B) is a conserved seven-pass transmembrane protein that is essential for synaptic function, and its sequence disruption is associated with neurodevelopmental disorders. Here, we identify TMEM184B as a key regulator of endolysosomal acidification. TMEM184B localizes to early and late endosomes, and proteomic analysis confirms that TMEM184B interacts with endosomal proteins, including the vacuolar ATPase (V-ATPase), a multi-subunit proton pump crucial for lumenal acidification. Tmem184b-mutant mouse cortical neurons have reduced endolysosomal acidification compared to wild-type neurons. We find reductions in V-ATPase complex assembly in Tmem184b-mutant mouse brains, suggesting that TMEM184B facilitates endosomal flux by promoting V-ATPase activity. These findings establish TMEM184B as a regulator of neuronal endolysosomal acidification and provide mechanistic insight into its role in TMEM184B-associated nervous system disorders.
• van der Pijl, R., Gohlke, J., Strom, J., Peters, E., Shen, S., Conijn, S., Hourani, Z., Lange, S., Chen, J., Langlais, P., Labeit, S., Granzier, H., & Ottenheijm, C. (2025). The titin N2A-MARP signalosome constrains muscle longitudinal hypertrophy in response to stretch. bioRxiv : the preprint server for biology.
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  Titin-based mechanosensing is a key driver of trophic signaling in muscle, yet the downstream pathways linking titin sensing to muscle remodeling remain poorly understood. To investigate these signaling mechanisms, we utilized unilateral diaphragm denervation (UDD), an in vivo model that induces titin-stiffness-dependent hypertrophy via mechanical stretch. Using UDD in rats and mice, we characterized the longitudinal hypertrophic response and distinguished stretch-induced signaling from denervation effects by performing global transcriptomic and proteomic analyses following UDD and bilateral diaphragm denervation (BDD) in rats. Our findings identified upregulation of titin-associated muscle ankyrin repeat proteins (MARPs). Subsequent phosphorylation enrichment mass spectrometry in mouse diaphragm highlighted the involvement of the N2A-element. UDD in MARP knockout (KO) mice resulted in enhanced longitudinal hypertrophy, with Western blot analysis revealing activation of the mTOR pathway. Furthermore, pharmacological inhibition of mTORC1 with rapamycin suppressed longitudinal hypertrophy, demonstrating that mTOR signaling regulates titin-mediated hypertrophic growth in a MARP-dependent manner. These findings establish MARPs as key modulators of titin-based mechanotransduction and highlight mTORC1 as a central regulator of longitudinal muscle hypertrophy.
• Flores, P., Ahmed, T., Podgorski, J., Ortiz, H., Langlais, P., Mythreye, K., & Lee, N. (2024). Phosphoproteomic profiling identifies DNMT1 as a key substrate of beta IV spectrin-dependent ERK/MAPK signaling in suppressing angiogenesis. Biochemical and Biophysical Research Communications, 711. doi:10.1016/j.bbrc.2024.149916
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  βIV-spectrin is a membrane-associated cytoskeletal protein that maintains the structural stability of cell membranes and integral proteins such as ion channels and transporters. Its biological functions are best characterized in the brain and heart, although recently we discovered a fundamental new role in the vascular system. Using cellular and genetic mouse models, we reported that βIV-spectrin acts as a critical regulator of developmental and tumor-associated angiogenesis. βIV-spectrin was shown to selectively express in proliferating endothelial cells (EC) and suppress VEGF/VEGFR2 signaling by enhancing receptor internalization and degradation. Here we examined how these events impact the downstream kinase signaling cascades and target substrates. Based on quantitative phosphoproteomics, we found that βIV-spectrin significantly affects the phosphorylation of epigenetic regulatory enzymes in the nucleus, among which DNA methyltransferase 1 (DNMT1) was determined as a top substrate. Biochemical and immunofluorescence results showed that βIV-spectrin inhibits DNMT1 function by activating ERK/MAPK, which in turn phosphorylates DNMT1 at S717 to impede its nuclear localization. Given that DNMT1 controls the DNA methylation patterns genome-wide, and is crucial for vascular development, our findings suggest that epigenetic regulation is a key mechanism by which βIV-spectrin suppresses angiogenesis.
• Levine, A., Liktor-Busa, E., Balasubramanian, S., Palomino, S., Burtman, A., Couture, S., Lipinski, A., Langlais, P., & Largent-Milnes, T. (2024). Depletion of Endothelial-Derived 2-AG Reduces Blood-Endothelial Barrier Integrity via Alteration of VE-Cadherin and the Phospho-Proteome. International Journal of Molecular Sciences, 25(1). doi:10.3390/ijms25010531
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  Mounting evidence supports the role of the endocannabinoid system in neurophysiology, including blood–brain barrier (BBB) function. Recent work has demonstrated that activation of endocannabinoid receptors can mitigate insults to the BBB during neurological disorders like traumatic brain injury, cortical spreading depression, and stroke. As alterations to the BBB are associated with worsening clinical outcomes in these conditions, studies herein sought to examine the impact of endocannabinoid depletion on BBB integrity. Barrier integrity was investigated in vitro via bEnd.3 cell monolayers to assess endocannabinoid synthesis, barrier function, calcium influx, junctional protein expression, and proteome-wide changes. Inhibition of 2-AG synthesis using DAGLα inhibition and siRNA inhibition of DAGLα led to loss of barrier integrity via altered expression of VE-cadherin, which could be partially rescued by exogenous application of 2-AG. Moreover, the deleterious effects of DAGLα inhibition on BBB integrity showed both calcium and PKC (protein kinase C)-dependency. These data indicate that disruption of 2-AG homeostasis in brain endothelial cells, in the absence of insult, is sufficient to disrupt BBB integrity thus supporting the role of the endocannabinoid system in neurovascular disorders.
• Li, N. C., Iannuzo, N., Christenson, S. A., Langlais, P. R., Kraft, M., Ledford, J. G., & Li, X. (2024). Investigation of lactotransferrin messenger RNA expression levels as an anti-type 2 asthma biomarker. The Journal of allergy and clinical immunology, 154(3), 609-618.
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  Lactotransferrin (LTF) has an immunomodulatory function, and its expression levels are associated with asthma susceptibility.
• Miller, K. L., Liu, X., McSwain, M. G., Jauregui, E. J., Langlais, P. R., & Craig, Z. R. (2024). Quantitative label-free proteomic analysis of mouse ovarian antral follicles following oral exposure to a human-relevant mixture of three phthalates. Toxicological sciences : an official journal of the Society of Toxicology, 201(2), 226-239.
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  Dibutyl phthalate (DBP), di-2-ethylhexyl phthalate (DEHP), and benzyl butyl phthalate (BBP) are used in personal and medical care products. In the ovary, antral follicles are essential for steroidogenesis and ovulation. DBP, BBP, and DEHP are known to inhibit mouse antral follicle growth and ovulation in vitro, and associate with decreased antral follicle counts in women. Given that the in vivo effects of a three-phthalate mixture on antral follicles are unknown, we evaluated the effects of a human-relevant mixture of DBP, BBP, and DEHP on ovarian follicles through proteome profiling analysis. Adult CD-1 female mice were fed corn oil (vehicle), or two dose levels of a phthalate mixture based on estimated exposures in general (32 µg/kg/d; PHT 32) and occupationally exposed (500 µg/kg/d; PHT 500) populations for 10 d. Antral follicles (>250 µm) were isolated and subjected to proteome profiling via label-free tandem mass spectrometry. A total of 5,417 antral follicle proteins were detected, of which 194 were differentially abundant between vehicle and PHT 32, and 136 between vehicle and PHT 500. Bioinformatic analysis revealed significantly different responses between the two phthalate doses. Protein abundance differences in the PHT 32 exposure mapped to cytoplasm, mitochondria, and lipid metabolism; whereas those in the PHT 500 exposure mapped to cytoplasm, nucleus, and phosphorylation. When both doses altered proteins mapped to common processes, the associated predicted transcription factors were different. These findings provide novel mechanistic insight into phthalate-associated, ovary-driven reproductive outcomes in women.
• Ortiz, H. R., Cruz Flores, P., Podgorski, J., Ramonett, A., Ahmed, T., Hempel, N., Charest, P. G., Ellis, N. A., Langlais, P. R., Montfort, W. R., Mythreye, K., Kumar, S., & Lee, N. Y. (2024). Extracellular signals induce dynamic ER remodeling through αTAT1-dependent microtubule acetylation. Neoplasia (New York, N.Y.), 53, 101003.
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  Dynamic changes in the endoplasmic reticulum (ER) morphology are central to maintaining cellular homeostasis. Microtubules (MT) facilitate the continuous remodeling of the ER network into sheets and tubules by coordinating with many ER-shaping protein complexes, although how this process is controlled by extracellular signals remains unknown. Here we report that TAK1, a kinase responsive to various growth factors and cytokines including TGF-β and TNF-α, triggers ER tubulation by activating αTAT1, an MT-acetylating enzyme that enhances ER-sliding. We show that this TAK1/αTAT1-dependent ER remodeling promotes cell survival by actively downregulating BOK, an ER membrane-associated proapoptotic effector. While BOK is normally protected from degradation when complexed with IP3R, it is rapidly degraded upon their dissociation during the ER sheets-to-tubules conversion. These findings demonstrate a distinct mechanism of ligand-induced ER remodeling and suggest that the TAK1/αTAT1 pathway may be a key target in ER stress and dysfunction.
• Plank, M., Carmiol, N., Mitri, B., Lipinski, A. A., Langlais, P. R., & Capaldi, A. P. (2024). Systems level analysis of time and stimuli specific signaling through PKA. Molecular biology of the cell, 35(4), ar60.
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  It is well known that eukaryotic cells create gradients of cAMP across space and time to regulate the cAMP dependent protein kinase (PKA) and, in turn, growth and metabolism. However, it is unclear how PKA responds to different concentrations of cAMP. Here, to address this question, we examine PKA signaling in in different conditions, timepoints, and concentrations of the chemical inhibitor 1-NM-PP1, using phosphoproteomics. These experiments show that there are numerous proteins that are only phosphorylated when cAMP and PKA activity are at/near their maximum level, while other proteins are phosphorylated even when cAMP levels and PKA activity are low. The data also show that PKA drives cells into distinct growth states by acting on proteins with different thresholds for phosphorylation in different conditions. Analysis of the sequences surrounding the 118 PKA-dependent phosphosites suggests that the phosphorylation thresholds are set, at least in part, by the affinity of PKA for each site.
• van den Berg, M., Shi, Z., Claassen, W. J., Hooijman, P., Lewis, C. T., Andersen, J. L., van der Pijl, R. J., Bogaards, S. J., Conijn, S., Peters, E. L., Begthel, L. P., Uijterwijk, B., Lindqvist, J., Langlais, P. R., Girbes, A. R., Stapel, S., Granzier, H., Campbell, K. S., Ma, W., , Irving, T., et al. (2024). Super-relaxed myosins contribute to respiratory muscle hibernation in mechanically ventilated patients. Science Translational Medicine, 16(Issue 758). doi:10.1126/scitranslmed.adg3894
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  Patients receiving mechanical ventilation in the intensive care unit (ICU) frequently develop contractile weakness of the diaphragm. Consequently, they may experience difficulty weaning from mechanical ventilation, which increases mortality and poses a high economic burden. Because of a lack of knowledge regarding the molecular changes in the diaphragm, no treatment is currently available to improve diaphragm contractility. We compared diaphragm biopsies from ventilated ICU patients (N = 54) to those of non-ICU patients undergoing thoracic surgery (N = 27). By integrating data from myofiber force measurements, x-ray diffraction experiments, and biochemical assays with clinical data, we found that in myofibers isolated from the diaphragm of ventilated ICU patients, myosin is trapped in an energy-sparing, super-relaxed state, which impairs the binding of myosin to actin during diaphragm contraction. Studies on quadriceps biopsies of ICU patients and on the diaphragm of previously healthy mechanically ventilated rats suggested that the super-relaxed myosins are specific to the diaphragm and not a result of critical illness. Exposing slow- and fast-twitch myofibers isolated from the diaphragm biopsies to small-molecule compounds activating troponin restored contractile force in vitro. These findings support the continued development of drugs that target sarcomere proteins to increase the calcium sensitivity of myofibers for the treatment of ICU-acquired diaphragm weakness.
• van den Berg, M., Shi, Z., Claassen, W. J., Hooijman, P., Lewis, C. T., Andersen, J. L., van der Pijl, R. J., Bogaards, S. J., Conijn, S., Peters, E. L., Begthel, L. P., Uijterwijk, B., Lindqvist, J., Langlais, P. R., Girbes, A. R., Stapel, S., Granzier, H., Campbell, K. S., Ma, W., , Irving, T., et al. (2024). Super-relaxed myosins contribute to respiratory muscle hibernation in mechanically ventilated patients. Science translational medicine, 16(758), eadg3894.
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  Patients receiving mechanical ventilation in the intensive care unit (ICU) frequently develop contractile weakness of the diaphragm. Consequently, they may experience difficulty weaning from mechanical ventilation, which increases mortality and poses a high economic burden. Because of a lack of knowledge regarding the molecular changes in the diaphragm, no treatment is currently available to improve diaphragm contractility. We compared diaphragm biopsies from ventilated ICU patients ( = 54) to those of non-ICU patients undergoing thoracic surgery ( = 27). By integrating data from myofiber force measurements, x-ray diffraction experiments, and biochemical assays with clinical data, we found that in myofibers isolated from the diaphragm of ventilated ICU patients, myosin is trapped in an energy-sparing, super-relaxed state, which impairs the binding of myosin to actin during diaphragm contraction. Studies on quadriceps biopsies of ICU patients and on the diaphragm of previously healthy mechanically ventilated rats suggested that the super-relaxed myosins are specific to the diaphragm and not a result of critical illness. Exposing slow- and fast-twitch myofibers isolated from the diaphragm biopsies to small-molecule compounds activating troponin restored contractile force in vitro. These findings support the continued development of drugs that target sarcomere proteins to increase the calcium sensitivity of myofibers for the treatment of ICU-acquired diaphragm weakness.
• Ahmed, T., Ramonett, A., Kwak, E. A., Kumar, S., Flores, P. C., Ortiz, H. R., Langlais, P. R., Hund, T. J., Mythreye, K., & Lee, N. Y. (2023). Endothelial tip/stalk cell selection requires BMP9-induced βIV-spectrin expression during sprouting angiogenesis. Molecular Biology of the Cell, 34(Issue 7). doi:10.1091/mbc.e23-02-0064
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  βIV-Spectrin is a membrane cytoskeletal protein with specialized roles in the nervous system and heart. Recent evidence also indicates a fundamental role for βIV-spectrin in angiogenesis as its endothelial-specific gene deletion in mice enhances embryonic lethality due to hypervascularization and hemorrhagic defects. During early vascular sprouting, βIVspectrin is believed to inhibit tip cell sprouting in favor of the stalk cell phenotype by mediating VEGFR2 internalization and degradation. Despite these essential roles, mechanisms governing βIV-spectrin expression remain unknown. Here we identify bone morphogenetic protein 9 (BMP9) as a major inducer of βIV-spectrin gene expression in the vascular system. We show that BMP9 signals through the ALK1/Smad1 pathway to induce βIV-spectrin expression, which then recruits CaMKII to the cell membrane to induce phosphorylation-dependent VEGFR2 turnover. Although BMP9 signaling promotes stalk cell behavior through activation of hallmark stalk cell genes ID-1/3 and Hes-1 and Notch signaling cross-talk, we find that βIV-spectrin acts upstream of these pathways as loss of βIV-spectrin in neonate mice leads to retinal hypervascularization due to excessive VEGFR2 levels, increased tip cell populations, and strong Notch inhibition irrespective of BMP9 treatment. These findings demonstrate βIV-spectrin as a BMP9 gene target critical for tip/stalk cell selection during nascent vessel sprouting.
• Ahmed, T., Ramonett, A., Kwak, E., Cruz-Flores, P., Ortiz, H. R., Langlais, P. R., Hund, T. J., Mythreye, K., & Lee, N. Y. (2023). Endothelial tip/stalk cell selection requires BMP9-induced β_IV-spectrin expression during sprouting angiogenesis. Molecular Biology of the Cell.
• Barakati, N., Bustos, R. Z., Coletta, D. K., Langlais, P. R., Kohler, L. N., Luo, M., Funk, J. L., Willis, W. T., & Mandarino, L. J. (2023). Fuel Selection in Skeletal Muscle Exercising at Low Intensity; Reliance on Carbohydrate in Very Sedentary Individuals. Metabolic syndrome and related disorders, 21(1), 16-24.
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  Resting skeletal muscle in insulin resistance prefers to oxidize carbohydrate rather than lipid, exhibiting metabolic inflexibility. Although this is established in resting muscle, complexities involved in directly measuring fuel oxidation using indirect calorimetry across a muscle bed have limited studies of this phenomenon in working skeletal muscle. During mild exercise and at rest, whole-body indirect calorimetry imperfectly estimates muscle fuel oxidation. We provide evidence that a method termed "ΔRER" can reasonably estimate fuel oxidation in skeletal muscle activated by exercise. Completely sedentary volunteers ( = 20, age 31 ± 2 years, V̇O 24.4 ± 1.5 mL O per min/kg) underwent glucose clamps to determine insulin sensitivity and graded exercise consisting of three periods of mild steady-state cycle ergometry (15, 30, 45 watts, or 10%, 20%, and 30% of maximum power) with measurements of whole-body gas exchange. ΔRER, the RER in working muscle, was calculated as (V̇CO2 -V̇CO)/(V̇O - V̇O), from which the fraction of fuel accounted for by lipid was estimated. Lactate levels were low and stable during steady-state exercise. Muscle biopsies were used to estimate mitochondrial content. The rise of V̇O at onset of exercise followed a monoexponential function, with a time constant of 51 ± 7 sec, typical of skeletal muscle; the average O cost of work was about 12 mL O/watt/min, representing a mechanical efficiency of about 24%. At work rates of 30 or 45 watts, active muscle relied predominantly on carbohydrate, independent of insulin sensitivity within this group of very sedentary volunteers. The fraction of muscle fuel oxidation from fat was predicted by power output ( 
• Grijalva, A., Benson, D. R., Fink, J., Zhang, X., Coletta, D., Langlais, P., Pandey, A. C., Mangiola, M., Koevary, J., Lancaster, J., & Goldman, S. (2023). Abstract 17096: Immune Modulation to Treat Ischemic Heart Failure. Circulation, 148(Suppl_1). doi:10.1161/circ.148.suppl_1.17096
• Iannuzo, N., Dy, A. B., Guerra, S., Langlais, P. R., & Ledford, J. (2023). The Impact of CC16 on Pulmonary Epithelial-Driven Host Responses during Mycoplasma Pneumoniae Infection in Mouse Tracheal Epithelial Cells. Cells.
• Jensen, C., Clements, A. N., Liou, H., Ball, L. E., Bethard, J. R., Langlais, P. R., Toth, R. K., Chauhan, S. S., Casillas, A. L., Daulat, S. R., Kraft, A., Cress, A. E., Miranti, C., Mouneimne, G., Rogers, G. C., & Warfel, N. A. (2023). PIM kinases regulate actin dynamics and tumor cell invasion in hypoxia. Journal of Cell Biology.
• Kwak, E., Ahmed, T., Cruz-Flores, P., Ortiz, H. R., Langlais, P. R., & Lee, N. Y. (2023). Beta IV spectrin inhibits the metastatic growth of melanoma by suppressing VEGFR2-driven tumor angiogenesis. Cancer Med.
• Ledford, J., Langlais, P. R., Cusanovich, D. A., Li, X., Guerra, S., Johnson, M. D., Li, N., Welfley, H., & Iannuzo, N. (2023). CC16 drives VLA-2-dependent SPLUNC1 expression. Frontiers in Immunology.
• Ou, J., Lewandowski, E., Hu, Y., Lipinski, A., Aljasser, A., Colon-Ascanio, M., Morgan, R., Jacobs, L., Zhang, X., Bikowitz, M., Langlais, P. R., Tan, H., Wang, J., Chen, Y., & Choy, J. S. (2023). A yeast-based system to study SARS-CoV-2 Mpro structure and to identify nirmatrelvir resistant mutaations. PLoS Pathogens.
• Parker, S. S., Ly, K. T., Grant, A. D., Wang, A., Parker, J. D., Roman, M. R., Padi, M., Wolgemuth, C. W., Langlais, P. R., & Mouneimne, G. (2023). EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in developing neurons. Journal of Cell Biology.
• Sangam, s., Sun, X., Schwantes-An, T. H., Yegambaram, M., Lu, Q., Zagorski, J., Morrisroe, S. A., Shi, Y., Cook, T., Fisher, A., Frump, A. L., Coleman, A., Sun, Y., Liang, S., Crawford, H., Lutz, K. A., Muan, A. D., Pauciulo, M. W., Karnes, J. H., , Chaudhary, K. R., et al. (2023). SOX17 Deficiency Mediates Pulmonary Hypertension: At the Crossroads of Sex, Metabolism, and Genetics. Am J Respir Crit Care Med.
• Skaria, R. S., Lopez-Pier, M. A., Kathuria, B. S., Leber, C. J., Langlais, P. R., Aras, S. G., Khalpey, Z. I., Hitscherich, P. G., Chnari, E., Long, M., Churko, J., Runyan, R. B., & Konhilas, J. P. (2023). Epicardial Placement of Human Placental Membrane Protects from Heart Injury in a Swine Model of Myocardial Infarction. Physiol Rep.
• Zapata Bustos, R., Coletta, D. K., Galons, J., Davidson, L., Langlais, P. R., Funk, J. L., Willis, W. T., & Mandarino, L. J. (2023). Nonequilibrium thermodynamics and mitochondrial protein content predict insulin sensitivity and fuel selection during exercise in human skeletal muscle. Front Physiol.
• Ahmed, T., Cruz-Flores, P., Pan, C. C., Ortiz, H. R., Lee, Y. S., Langlais, P. R., Mythreye, K., & Lee, N. Y. (2022). EPDR1 is a noncanonical effector of insulin-mediated angiogenesis regulated by an endothelial-specific TGF-β receptor complex.. Journal of Biological Chemistry.
• Cornejo, N. R., Amofah, B., Lipinksi, A. A., Langlais, P. R., Ghosh, I., & Jewett, J. C. (2022). Direct intracellular delivery of benzene diazonium ions as observed by increased tyrosine phosphorylation. Biochemistry.
• Keresztes, A., Olson, K., Nguyen, P., Lopez-Pier, M. A., Hecksel, R., Barker, N. K., Liu, Z., Hruby, V., Konhilas, J. P., Langlais, P. R., & Streicher, J. M. (2022). Antagonism of the Mu-Delta Opioid Receptor Heterodimer Enhances Opioid Anti-Nociception by Activating Src and CaMKII Signaling. Pain.
• Kwak, E. A., Pan, C. C., Ramonett, A., Kumar, S., Cruz-Flores, P., Ahmed, T., Ortiz, H. R., Lochhead, J. L., Ellis, N., Mouneimne, G., Lee, Y. S., Vanderah, T. W., Milnes, T. M., Mohler, P. J., Hund, T. J., Langlais, P. R., Mythreye, K., & Lee, N. Y. (2022). BIV-spectrin as a stalk cell-intrinsic regulator of VEGF signaling. Nature Communications.
• Ramonett, A., Kwak, E. A., Ahmed, T., Flores, P. C., Ortiz, H. R., Lee, Y. S., Vanderah, T. W., Milnes, T. M., Kashatus, D. F., Langlais, P. R., Mythreye, K., & Lee, N. Y. (2022). Regulation of mitochondrial fission by GIPC-mediated Drp1 retrograde transport. Molecular biology of the cell, 33(1), ar4.
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  Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission, a large cytoplasmic GTPase recruited to the mitochondrial surface via transmembrane adaptors to initiate scission. While Brownian motion likely accounts for the local interactions between Drp1 and the mitochondrial adaptors, how this essential enzyme is targeted from more distal regions like the cell periphery remains unknown. Based on proteomic interactome screening and cell-based studies, we report that GAIP/RGS19-interacting protein (GIPC) mediates the actin-based retrograde transport of Drp1 toward the perinuclear mitochondria to enhance fission. Drp1 interacts with GIPC through its atypical C-terminal PDZ-binding motif. Loss of this interaction abrogates Drp1 retrograde transport resulting in cytoplasmic mislocalization and reduced fission despite retaining normal intrinsic GTPase activity. Functionally, we demonstrate that GIPC potentiates the Drp1-driven proliferative and migratory capacity in cancer cells. Together, these findings establish a direct molecular link between altered GIPC expression and Drp1 function in cancer progression and metabolic disorders.
• Vizcarra, V. S., Barber, K. R., Franca-Solomon, G., Majuta, L., Smith, A., Langlais, P. R., Milnes, T. M., Vanderah, T. W., & Riegel, A. C. (2022). Targeting 5-HT2A receptors and Kv7 channels in PFC to attenuate chronic neuropathic pain in rats using a spared nerve injury model. . Neuroscience Letters.
• Wahl, J. R., Vivek, A., Palomino, S. M., Almuslim, M., Cottier, K. E., Langlais, P. R., Streicher, J. M., Vanderah, T. W., & Milnes, T. M. (2022). Extracellular Alterations in pH and K+ Modify the Murine Brain Endothelial Cell Total and Phospho-Proteome. Biology of sex differences.
• Batty, S. R., & Langlais, P. R. (2021). Microtubules in insulin action: what's on the tube?. Trends in endocrinology and metabolism: TEM, 32(10), 776-789.
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  Microtubules (MT) have a role in the intracellular response to insulin stimulation and subsequent glucose transport by glucose transporter 4 (GLUT4), which resides in specialized storage vesicles that travel through the cell. Before GLUT4 is inserted into the plasma membrane for glucose transport, it undergoes complex trafficking through the cell via the integration of cytoskeletal networks. In this review, we highlight the importance of MT elements in insulin action in adipocytes through a summary of MT depolymerization studies, MT-based GLUT4 movement, molecular motor proteins involved in GLUT4 trafficking, as well as MT-related phenomena in response to insulin and links between insulin action and MT-associated proteins.
• Blawn, K. T., Kellohen, K. L., Galloway, E. A., Verkhovsky, V. G., Wahl, J., Vivek, A., Barker, N. K., Cottier, K. E., Vallecillo, T. G., Langlais, P. R., Liktor-Busa, E., Vanderah, T. W., & Milnes, T. M. (2021). Sex hormones regulate NHE1 functional expression and brain endothelial proteome to control paracellular integrity of the blood endothelial barrier. Biology of Sex Differences.
• Casillas, A. L., Chauhan, S. S., Toth, R. K., Sainz, A. G., Clements, A. N., Jensen, C. C., Langlais, P. R., Miranti, C. K., Cress, A. E., & Warfel, N. A. (2021). Direct phosphorylation and stabilization of HIF-1a by PIM1 kinase drives angiogenesis in solid tumors. Oncogene.
• Dy, A. B., Langlais, P. R., Barker, N. K., Addison, K. J., Tanyaratsrisakul, S., Boitano, S. A., Christenson, S. A., Kraft, M., Meyers, D., Bleecker, E., Li, X., & Ledford, J. (2021). Myeloid-associated differentiation marker is a novel SP-A-associated transmembrane protein whose expression on airway epithelial cells correlates with asthma severity. Scientific reports, 11(1), 23392.
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  Surfactant protein A (SP-A) is well-known for its protective role in pulmonary immunity. Previous studies from our group have shown that SP-A mediates eosinophil activities, including degranulation and apoptosis. In order to identify potential binding partners on eosinophils for SP-A, eosinophil lysates were subjected to SP-A pull-down and tandem mass spectrometry (MS/MS) analysis. We identified one membrane-bound protein, myeloid-associated differentiation marker (MYADM), as a candidate SP-A binding partner. Blocking MYADM on mouse and human eosinophils ex vivo prevented SP-A from inducing apoptosis; blocking MYADM in vivo led to increased persistence of eosinophilia and airway hyper-responsiveness in an ovalbumin (OVA) allergy model and increased airways resistance and mucus production in a house dust mite (HDM) asthma model. Examination of a subset of participants in the Severe Asthma Research Program (SARP) cohort revealed a significant association between epithelial expression of MYADM in asthma patients and parameters of airway inflammation, including: peripheral blood eosinophilia, exhaled nitric oxide (FeNO) and the number of exacerbations in the past 12 months. Taken together, our studies provide the first evidence of MYADM as a novel SP-A-associated protein that is necessary for SP-A to induce eosinophil apoptosis and we bring to light the potential importance of this previously unrecognized transmembrane protein in patients with asthma.
• Finlayson, J., Barakati, N., Langlais, P. R., Funk, J. L., Zapata Bustos, R., Coletta, D. K., Luo, M., Willis, W. T., & Mandarino, L. J. (2021). Site-specific acetylation of adenine nucleotide translocase 1 at lysine 23 in human muscle. Analytical biochemistry, 630, 114319.
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  Evidence suggests acetylation of human adenine nucleotide translocase 1 (ANT1) at lysine 23 (Lys23) reduces binding of ADP. Lys23 contributes to the positive charge that facilitates this interaction. This study was undertaken to characterize ANT1 abundance and acetylation by a novel method using small amounts of human skeletal muscle biopsies. Lysates of whole muscle or mitochondria from the same tissue were prepared from needle biopsies of vastus lateralis muscle of healthy volunteers. Lysed proteins were resolved on gels, the section containing ANT1 (surrounding 30 Kd) was excised, digested with trypsin, spiked with labeled unacetylated and acetylated synthetic standard peptides and analyzed by mass spectrometry. Natural logarithm transformation of data linearized ion intensities over a 10-fold range of peptide mass. Coefficients of variation ranged from 7 to 30% for ANT1 abundance and Lys23 acetylation. In three volunteers, ANT1 content was 8.36 ± 0.33 nmol/g wet weight muscle and 0.64 ± 0.05 nmol/mg mitochondria, so mitochondrial content was 13.3 ± 2.4 mg mitochondria per gram muscle. Acetylation of Lys23 averaged 14.3 ± 4.2% and 4.87 ± 1.84% in whole muscle and mitochondria, respectively. This assay makes it possible to assess effects of acetylation on the function of ANT1 in human muscle.
• He, S., Ryu, J., Liu, J., Luo, H., Lv, Y., Langlais, P. R., Wen, J., Dong, F., Sun, Z., Xia, W., Lynch, J. L., Duggirala, R., Nicolson, B., Zhang, M., Shi, Y., Zhang, F., Liu, F., Bai, J., & Dong, L. Q. (2021). LRG1 is an adipokine that mediates obesity-induced hepatosteatosis and insulin resistance. J Clin Invest..
• James, J., Zemskova, M., Eccles, C. A., Varghese, M. V., Niihori, M., Barker, N. K., Luo, M., Mandarino, L. J., Langlais, P. R., Rafikova, O., & Rafikov, R. (2021). Single Mutation in the NFU1 Gene Metabolically Reprograms Pulmonary Artery Smooth Muscle Cells. Arteriosclerosis, Thrombosis, and Vascular Biology.
• Levine, A., Liktor-Busa, E., Lipinski, A. A., Couture, S., Balasubramanian, S., Aicher, S. A., Langlais, P. R., Vanderah, T. W., & Milnes, T. M. (2021). Sex differences in the expression of the endocannabinoid system within V1M cortex and PAG of Sprague Dawley rats. Biology of sex differences, 12(1), 60.
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  Several chronic pain disorders, such as migraine and fibromyalgia, have an increased prevalence in the female population. The underlying mechanisms of this sex-biased prevalence have yet to be thoroughly documented, but could be related to endogenous differences in neuromodulators in pain networks, including the endocannabinoid system. The cellular endocannabinoid system comprises the endogenous lipid signals 2-AG (2-arachidonoylglycerol) and AEA (anandamide); the enzymes that synthesize and degrade them; and the cannabinoid receptors. The relative prevalence of different components of the endocannabinoid system in specific brain regions may alter responses to endogenous and exogenous ligands.
• Polverino, F., Wu, T. D., Rojas-Quintero, J., Wang, X., Mayo, J., Tomchaney, M., Tram, J., Packard, S., Zhang, D., Cleveland, K., Cordoba-Lanus, E., Owen, C. A., Fawzy, A., Kinney, G. L., Hersh, C. P., Hansel, N. N., Doubleday, K., Sauler, M., Tesfaigzi, Y., , Ledford, J., et al. (2020). Metformin: Experimental and Clinical Evidence for a Potential Role in Emphysema Treatment. American Journal of Respiratory and Critical Care Medicine.
• Uhlorn, J. A., Husband, N. A., Romero-Aleshire, M. J., Moffett, C., Lindsay, M. L., Langlais, P. R., & Brooks, H. L. (2021). CD4+ T Cell-Specific Proteomic Pathways Identified in Progression of Hypertension Across Postmenopausal Transition. Journal of the American Heart Association.
• Zapata Bustos, R., Finlayson, J., Langlais, P. R., Coletta, D. K., Luo, M., Grandjean, D., De Filippis, E. A., & Mandarino, L. J. (2021). Altered Transcription Factor Expression Responses to Exercise in Insulin Resistance. Frontiers in Physiology.
• Zhang, H., Lipinski, A. L., Smith, A. F., Moutal, A., Khanna, R., Langlais, P. R., Milnes, T. M., & Vanderah, T. W. (2020). The effects of chronic morphine on the endogenous cannabinoid system in the ventral tegmental area. Frontiers In Pharmacology.
• van der Pijl, R. J., van den Berg, M., van de Locht, M., Shen, S., Bogaards, S. J., Conijn, S., Langlais, P. R., Chen, J., Hooijman, P. E., Labeit, S., Heunks, L. M., Granzier, H. L., & Ottenheijm, C. A. (2021). MARP1 (Muscle ankyrin repeat protein 1) locks titin to the sarcomeric thin filament and is a newly discovered passive force regulator. J Gen Physiol.
• Burt, J. M., Taylor, S. Z., Jacobsen, N. L., Pontifex, T. K., & Langlais, P. R. (2020). Serine 319 phosphorylation is necessary and sufficient to induce a Cx37 conformation that leads to arrested cell cycling.. J Cell Sci., 133. doi:doi:10.1242/jcs.240721
• Duron, D. I., Lei, W., Barker, N. K., Stine, C., Mishra, S., Blagg, B. S., Langlais, P. R., & Streicher, J. M. (2020). Inhibition of spinal cord Hsp90 enhances morphine anti-nociception by activating an ERK/RSK pathway.. Science Signaling.
• Harris, S., Langlais, P. R., Granger, K., Napierski, N., Touma, K., Moran, H., & Strom, J. (2020). A novel “cut and paste” method for in situ replacement of cMyBP-C reveals a new role for cMyBP-C in the regulation of contractile oscillations. Circulation Research.
• James, J., Varghese, M. V., Vasilyev, M., Langlais, P. R., Tofovic, S. P., Rafikova, O., & Rafikov, R. (2020). Complex III Inhibition-Induced Pulmonary Hypertension Affects the Mitochondrial Proteomic Landscape. International Journal of Molecular Sciences.
• Moinpour, M., Barker, N. K., Guzman, L. E., Jewett, J. C., Langlais, P. R., & Schwartz, J. C. (2020). Discriminating changes in protein structure using tyrosine conjugation. Protein Science.
• Pendleton, A. L., Antolic, A. T., Kelly, A. C., Davis, M. A., Camacho, L. E., Doubleday, K., Anderson, M. J., Langlais, P. R., Lynch, R. M., & Limesand, S. W. (2020). Lower oxygen consumption and Complex I activity in mitochondria isolated from skeletal muscle of fetal sheep with intrauterine growth restriction. American Journal of Physioly - Endocrinology & Metabolism.
• Jacobsen, N. L., Pontifex, T. K., Langlais, P. R., & Burt, J. M. (2019). Phosphorylation-dependent intra-domain interaction of the Cx37 carboxyl-terminus controls cell survival. Cancers, 11(Issue 2). doi:10.3390/cancers11020188
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  Differential phosphorylation of the carboxyl-terminus of connexin 37 (Cx37-CT) regulates phenotypic switching between cell growth phenotypes (cell death, cell cycle arrest, proliferation). The specific phosphorylation events in the Cx37-CT that are necessary for these growth regulatory effects are currently unknown. Through the combined use of deletion and site specific (de)phospho-mimetic Cx37-CT mutants, our data suggest a phosphorylation-dependent interaction between the mid-tail (aa 273–317) and end-tail (aa 318–333) portions of the Cx37-CT that regulates cell survival. As detected by mass spectrometry, Cx37 was phosphorylated at serines 275, 321, and 328; phosphomimetic mutations of these sites resulted in cell death when expressed in rat insulinoma cells. Alanine substitution at S328, but not at S275 or S321, also triggered cell death. Cx37-S275D uniquely induced the death of only low density, non-contact forming cells, but neither hemichannel open probability nor channel conductance distinguished death-inducing mutants. As channel function is necessary for cell death, together the data suggest that the phosphorylation state of the Cx37-CT controls an intra-domain interaction within the CT that modifies channel function and induces cell death.
• Jacobsen, N. L., Pontifex, T. K., Langlais, P. R., & Burt, J. M. (2019). Phosphorylation-dependent intra-domain interaction of the Cx37 carboxyl-terminus controls cell survival. Cancers.
• Krantz, J., Parker, S. S., Barker, N. K., Deer, C. G., Mouneimne, G., & Langlais, P. R. (2019). Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes.. Molecular & cellular proteomics : MCP.
• Luo, M., Willis, W. T., Coletta, D. K., Langlais, P. R., Mengos, A., Ma, W., Finlayson, J., Wagner, G. R., Baier, L. J., Nair, A., & Mandarino, L. J. (2019). Deletion of the Mitochondrial Protein VWA8 Induces Oxidative Stress and an HNF4α Compensatory Response in Hepatocytes.. Biochemistry.
• Luo, M., Willis, W. T., Coletta, D. K., Langlais, P. R., Mengos, A., Ma, W., Finlayson, J., Wagner, G. R., Shi, C. X., & Mandarino, L. J. (2019). Deletion of the Mitochondrial Protein VWA8 Induces Oxidative Stress and an HNF4α Compensatory Response in Hepatocytes. Biochemistry, 58(Issue 49). doi:10.1021/acs.biochem.9b00863
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  von Willebrand A domain-containing protein 8 (VWA8) is a poorly characterized, mitochondrial matrix-targeted protein with an AAA ATPase domain and ATPase activity that increases in livers of mice fed a high-fat diet. This study was undertaken to use CRISPR/Cas9 to delete VWA8 in cultured mouse hepatocytes and gain insight into its function. Unbiased omics techniques and bioinformatics were used to guide subsequent assays, including the assessment of oxidative stress and the determination of bioenergetic capacity. Metabolomics analysis showed VWA8 null cells had higher levels of oxidative stress and protein degradation; assays of hydrogen peroxide production revealed higher levels of production of reactive oxygen species (ROS). Proteomics and transcriptomics analyses showed VWA8 null cells had higher levels of expression of mitochondrial proteins (electron transport-chain Complex I, ATP synthase), peroxisomal proteins, and lipid transport proteins. The pattern of higher protein abundance in the VWA8 null cells could be explained by a higher level of hepatocyte nuclear factor 4 α (HNF4α) expression. Bioenergetic assays showed higher rates of carbohydrate oxidation and mitochondrial and nonmitochondrial lipid oxidation in intact and permeabilized cells. Inhibitor assays localized sites of ROS production to peroxisomes and NOX1/4. The rescue of VWA8 protein restored the wild-type phenotype, and treatment with antioxidants decreased the level of HNF4α expression. Thus, loss of VWA8 produces a mitochondrial defect that may be sensed by NOX4, leading to an increase in the level of ROS that results in a higher level of HNF4α. The compensatory HNF4α response results in a higher oxidative capacity and an even higher level of ROS production. We hypothesize that VWA8 is an AAA ATPase protein that plays a role in mitochondrial protein quality.
• Pandey, R., Zhou, M., Islam, S., Chen, B., Langlais, P. R., Srivastava, A., Cooke, L. S., Weterings, E., Von Hoff, D., & Mahadevan, D. (2019). Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) in Pancreatic Ductal Adenocarcinoma (PDA): An integrative analysis of a novel therapeutic target. Scientific Reports.
• Rafikov, R., McBride, M. L., Zemskova, M., Kurdyukov, S., McClain, N., Niihori, M., Langlais, P. R., & Rafikova, O. (2019). INOSITOL MONOPHOSPHATASE 1 (IMPA1) AS A NOVEL INTERACTING PARTNER OF RAGE IN PULMONARY HYPERTENSION.. American Journal of Physiology - Lung Cellular and Molecular Physiology.
• Tran, L., Langlais, P. R., Hoffman, N., Roust, L., & Katsanos, C. (2019). Mitochondrial ATP synthase β-subunit production rate and ATP synthase specific activity are reduced in skeletal muscle of humans with obesity.. Experimental Physiology.
• Husband, N. A., Moffett, C., Romero‐Aleshire, M. J., Uhlorn, J. A., Uhrlaub, J., Barker, N., Nunez, F., Nikolich‐Zugich, J., Langlais, P., & Brooks, H. L. (2018). Differential Responses in the Splenic CD4⁺ T Cell Proteome Following Ang II‐Induced Hypertension in VCD‐Treated Menopausal Mice. The FASEB Journal, 32(S1). doi:10.1096/fasebj.2018.32.1_supplement.716.18
• Kras, K. A., Langlais, P. R., Hoffman, N., Roust, L. R., Benjamin, T. R., De Filippis, E. A., Dinu, V., & Katsanos, C. S. (2018). Obesity modifies the stoichiometry of mitochondrial proteins in a way that is distinct to the subcellular localization of the mitochondria in skeletal muscle.. Metabolism.
• Rafikova, O., Williams, E. E., McBride, M. L., Zemskova, M., Srivastava, A., Nair, V., Desai, A., Langlais, P. R., Zemskov, E., Simon, M., Mandarino, L. J., & Rafikov, R. (2018). Hemolysis-induced lung vascular leakage contributes to the development of pulmonary hypertension. American Journal of Respiratory Cell and Molecular Biology.
• Suntravat, M., Langlais, P. R., Sanchez, E. E., & Nielsen, V. G. (2018). CatroxMP-II: a heme-modulated fibrinogenolytic metalloproteinase isolated from Crotalus atrox venom.. Biometals.
• Willis, W. T., Miranda-Grandjean, D., Hudgens, J., Willis, E. A., Finlayson, J., De Filippis, E. A., Zapata Bustos, R., Langlais, P. R., Mielke, C., & Mandarino, L. J. (2018). Dominant and sensitive control of oxidative flux by the ATP-ADP carrier in human skeletal muscle mitochondria: Effect of lysine acetylation.. Archives of Biochemistry and Biophysics.
• Xie, X., Sinha, S., Yi, Z., Langlais, P. R., Madan, M., Bowen, B. P., Willis, W., & Meyer, C. (2018). Role of adipocyte mitochondria in inflammation, lipemia and insulin sensitivity in humans: Effects of pioglitazone treatment. International Journal of Obesity, 42(Issue 2). doi:10.1038/ijo.2017.192
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  Background/Objectives:To gain further insight into the role of adipocyte mitochondria in systemic lipid metabolism, inflammation and insulin sensitivity in humans and to provide a better understanding of the mechanisms of action of the peroxisome proliferator-activated receptor gamma agonist pioglitazone.Subjects/Methods:Mitochondrial DNA (mtDNA) copy number, mitochondrial distribution, mitochondrial and overall cellular protein abundances as well as intrinsic mitochondrial function of subcutaneous adipocytes were assessed by real-time quantitative PCR, MitoTracker staining, global proteomics analyses and NADH cytochrome c reductase activity in insulin-sensitive, normal-glucose-tolerant (NGT) individuals and age, gender, adiposity-matched insulin-resistant individuals with abnormal glucose tolerant (AGT) before and after 3 months of pioglitazone treatment.Results:mtDNA copy number/adipocyte and mtDNA copy number/adipocyte volume were ∼55% and ∼4-fold lower in AGT than in NGT, respectively, and correlated positively with the M-value of euglycemic clamps and high-density lipoprotein, and negatively with fasting plasma triglyceride, tumor necrosis factor-α and interleukin-6 levels in the entire cohort. mtDNA copy number/adipocyte volume also correlated positively with plasma adiponectin. Pioglitazone, which improved insulin sensitivity, plasma lipids and inflammation, increased the mitochondrial copy number, and led to a redistribution of mitochondria from a punctate to a more reticular pattern as observed in NGT. This was accompanied by disproportionately increased abundances of mitochondrial proteins, including those involved in fat oxidation and triglyceride synthesis. Pioglitazone also increased the abundance of collagen VI and decreased the abundance of cytoskeletal proteins. NADH cytochrome c reductase activity of isolated adipocyte mitochondria was similar in AGT and NGT and unaltered by pioglitazone.Conclusions:Adipocyte mitochondria are deficient in insulin-resistant individuals and correlate with systemic lipid metabolism, inflammation and insulin sensitivity. Pioglitazone induces mitochondrial biogenesis and reorganization as well as the synthesis of mitochondrial proteins including those critical for lipid metabolism. It also alters extracellular matrix and cytoskeletal proteins. The intrinsic function of adipocyte mitochondria appears unaffected in insulin resistance and by pioglitazone.
• Kruse, R., Krantz, J., Barker, N., Coletta, R., Rafikov, R., Luo, M., Hoejlund, K., Mandarino, L. J., & Langlais, P. R. (2017). Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein. Molecular & cellular proteomics : MCP, 16(10), 1718-1735. doi:10.1074/mcp.RA117.000011
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  CLASP2 is a microtubule-associated protein that undergoes insulin-stimulated phosphorylation and co-localization with reorganized actin and GLUT4 at the plasma membrane. To gain insight to the role of CLASP2 in this system, we developed and successfully executed a streamlined interactome approach and built a CLASP2 protein network in 3T3-L1 adipocytes. Using two different commercially available antibodies for CLASP2 and an antibody for epitope-tagged, overexpressed CLASP2, we performed multiple affinity purification coupled with mass spectrometry (AP-MS) experiments in combination with label-free quantitative proteomics and analyzed the data with the bioinformatics tool Significance Analysis of Interactome (SAINT). We discovered that CLASP2 co-immunoprecipitates (co-IPs) the novel protein SOGA1, the microtubule-associated protein kinase MARK2, and the microtubule/actin-regulating protein G2L1. The GTPase-activating proteins AGAP1 and AGAP3 were also enriched in the CLASP2 interactome, although subsequent AGAP3 and CLIP2 interactome analysis suggests a preference of AGAP3 for CLIP2. Follow-up MARK2 interactome analysis confirmed reciprocal co-IP of CLASP2 and also revealed MARK2 can co-IP SOGA1, glycogen synthase, and glycogenin. Investigating the SOGA1 interactome confirmed SOGA1 can reciprocal co-IP both CLASP2 and MARK2 as well as glycogen synthase and glycogenin. SOGA1 was confirmed to colocalize with CLASP2 and also with tubulin, which identifies SOGA1 as a new microtubule-associated protein. These results introduce the metabolic function of these proposed novel protein networks and their relationship with microtubules as new fields of cytoskeleton-associated protein biology.
• Xie, X., Sinha, S., Yi, Z., Langlais, P. R., Madan, M., Bowen, B. P., Willis, W., & Meyer, C. (2017). Role of adipocyte mitochondria in inflammation, lipemia and insulin sensitivity in humans: effects of pioglitazone treatment. International journal of obesity (2005).
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  To gain further insight into the role of adipocyte mitochondria in systemic lipid metabolism, inflammation and insulin sensitivity in humans and to provide a better understanding of the mechanisms of action of the peroxisome proliferator-activated receptor gamma agonist pioglitazone.
• Campbell, L. E., Langlais, P. R., Day, S. E., Coletta, R. L., Benjamin, T. R., De Filippis, E. A., Madura, J. A., Mandarino, L. J., Roust, L. R., & Coletta, D. K. (2016). Identification of novel changes in human skeletal muscle proteome after Roux-en-Y gastric bypass surgery. Diabetes, 65(Issue 9). doi:10.2337/db16-0004
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  The mechanisms of metabolic improvements after Rouxen- Y gastric bypass (RYGB) surgery are not entirely clear. Therefore, the aim of our study was to investigate the role of obesity and RYGB on the human skeletal muscle proteome. Basal muscle biopsies were obtained from seven obese (BMI >40 kg/m2) female subjects (45.1 ± 3.6 years) pre- and 3 months post-RYGB, and euglycemic- hyperinsulinemic clamps were used to assess insulin sensitivity. Four age-matched (48.5 ± 4.7 years) lean (BMI
• Campbell, L. E., Langlais, P. R., Day, S. E., Coletta, R. L., Benjamin, T. R., De, F., Madura, J., Mandarino, L. J., Roust, L. R., & Coletta, D. K. (2016). Identification of Novel Changes in Human Skeletal Muscle Proteome After Roux-en-Y Gastric Bypass Surgery. DIABETES, 65(9), 2724-2731.
• Kras, K. A., Willis, W. T., Barker, N., Czyzyk, T., Langlais, P. R., & Katsanos, C. S. (2016). Subsarcolemmal mitochondria isolated with the proteolytic enzyme nagarse exhibit greater protein specific activities and functional coupling. Biochemistry and biophysics reports, 6, 101-107.
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  Skeletal muscle mitochondria are arranged as a reticulum. Insight into the functional characteristics of such structure is achieved by viewing the network as consisting of "subsarcolemmal" (SS) and "intermyofibrillar" (IMF) regions. During the decades, most, but not all, published studies have reported higher (sometimes over 2-fold) enzyme and enzyme-pathway protein-specific activities in IMF compared to SS mitochondria. We tested the hypothesis that non-mitochondrial protein contamination might account for much of the apparently lower specific activities of isolated SS mitochondria. Mouse gastrocnemii (n = 6) were suspended in isolation medium, minced, and homogenized according to procedures typically used to isolate SS mitochondria. However, the supernatant fraction, collected after the first slow-speed (800×g) centrifugation, was divided equally: one sample was exposed to nagarse (MITO+), while the other was not (MITO-). Nagarse treatment reduced total protein yield by 25%, while it increased protein-specific respiration rates (nmol O2 min(-1) mg(-1)), by 38% under "resting" (state 4) and by 84% under maximal (state 3) conditions. Nagarse therefore increased the respiratory control ratio (state 3/state 4) by 30%. In addition, the ADP/O ratio was increased by 9% and the activity of citrate synthase (U/mg) was 49% higher. Mass spectrometry analysis indicated that the MITO+ preparation contained less contamination from non-mitochondrial proteins. We conclude that nagarse treatment of SS mitochondria removes not only non-mitochondrial proteins but also the protein of damaged mitochondria, improves indices of functional integrity, and the resulting protein-specific activities.
• Xie, X., Yi, Z., Sinha, S., Madan, M., Bowen, B. P., Langlais, P., Ma, D., Mandarino, L., & Meyer, C. (2016). Proteomics analyses of subcutaneous adipocytes reveal novel abnormalities in human insulin resistance. Obesity (Silver Spring, Md.), 24(7), 1506-14.
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  To provide a more global view of adipocyte changes in human insulin resistance by proteomics analyses.
• Xu, Q., Hou, Y., Langlais, P., Erickson, P., Zhu, J., Shi, C., Luo, M., Zhu, Y., Xu, Y. e., Mandarino, L. J., Stewart, K., & Chang, X. (2016). Expression of the cereblon binding protein argonaute 2 plays an important role for multiple myeloma cell growth and survival. BMC CANCER, 16.
• McLean, C. S., Mielke, C., Cordova, J. M., Langlais, P. R., Bowen, B., Miranda, D., Coletta, D. K., & Mandarino, L. J. (2015). Gene and MicroRNA Expression Responses to Exercise; Relationship with Insulin Sensitivity. PLOS ONE, 10(5).
• Aqel, B., Langlais, P., Vargas, H. E., Carey, E. J., Leonard, M., & Mandarino, L. J. (2014). Prospective Assessment of the Abundance of Drug Metabolizing Enzymes in the P450 Pathway in Patients with Non Alcoholic Fatty Liver Disease (NAFLD). HEPATOLOGY, 60, 759A-759A.
• Mielke, C., Lefort, N., McLean, C. G., Cordova, J. M., Langlais, P. R., Bordner, A. J., Te, J. A., Ozkan, S. B., Willis, W. T., & Mandarino, L. J. (2014). Adenine Nucleotide Translocase Is Acetylated in Vivo in Human Muscle: Modeling Predicts a Decreased ADP Affinity and Altered Control of Oxidative Phosphorylation. BIOCHEMISTRY, 53(23), 3817-3829.
• Mielke, C., Lefort, N., McLean, C. G., Cordova, J. M., Langlais, P. R., Bordner, A. J., Te, J. A., Ozkan, S. B., Willis, W. T., & Mandarino, L. J. (2014). Adenine nucleotide translocase is acetylated in vivo in human muscle: Modeling predicts a decreased ADP affinity and altered control of oxidative phosphorylation. Biochemistry, 53(Issue 23). doi:10.1021/bi401651e
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  Proteomics techniques have revealed that lysine acetylation is abundant in mitochondrial proteins. This study was undertaken (1) to determine the relationship between mitochondrial protein acetylation and insulin sensitivity in human skeletal muscle, identifying key acetylated proteins, and (2) to use molecular modeling techniques to understand the functional consequences of acetylation of adenine nucleotide translocase 1 (ANT1), which we found to be abundantly acetylated. Eight lean and eight obese nondiabetic subjects had euglycemic clamps and muscle biopsies for isolation of mitochondrial proteins and proteomics analysis. A number of acetylated mitochondrial proteins were identified in muscle biopsies. Overall, acetylation of mitochondrial proteins was correlated with insulin action (r = 0.60; P < 0.05). Of the acetylated proteins, ANT1, which catalyzes ADP-ATP exchange across the inner mitochondrial membrane, was acetylated at lysines 10, 23, and 92. The extent of acetylation of lysine 23 decreased following exercise, depending on insulin sensitivity. Molecular dynamics modeling and ensemble docking simulations predicted the ADP binding site of ANT1 to be a pocket of positively charged residues, including lysine 23. Calculated ADP-ANT1 binding affinities were physiologically relevant and predicted substantial reductions in affinity upon acetylation of lysine 23. Insertion of these derived binding affinities as parameters into a complete mathematical description of ANT1 kinetics predicted marked reductions in adenine nucleotide flux resulting from acetylation of lysine 23. Therefore, acetylation of ANT1 could have dramatic physiological effects on ADP-ATP exchange. Dysregulation of acetylation of mitochondrial proteins such as ANT1 therefore could be related to changes in mitochondrial function that are associated with insulin resistance. © 2014 American Chemical Society.
• Xie, X., Langlais, P., Zhang, X., Heckmann, B. L., Saarinen, A. M., Mandarino, L. J., & Liu, J. (2014). Identification of a novel phosphorylation site in adipose triglyceride lipase as a regulator of lipid droplet localization. AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM, 306(12), E1449-E1459.
• Zhu, Y. X., Braggio, E., Shi, C., Kortuem, K. M., Bruins, L. A., Schmidt, J. E., Chang, X., Langlais, P., Luo, M., Jedlowski, P., LaPlant, B., Laumann, K., Fonseca, R., Bergsagel, P. L., Mikhael, J., Lacy, M., Champion, M. D., & Stewart, A. K. (2014). Identification of cereblon-binding proteins and relationship with response and survival after IMiDs in multiple myeloma. BLOOD, 124(4), 536-545.
• Chao, A., Zhang, X., Ma, D., Langlais, P., Luo, M., Mandarino, L. J., Zingsheim, M., Pham, K., Dillon, J., & Yi, Z. (2012). Site-specific phosphorylation of protein phosphatase 1 regulatory subunit 12A stimulated or suppressed by insulin. JOURNAL OF PROTEOMICS, 75(11), 3342-3350.
• Geetha, T., Langlais, P., Caruso, M., & Yi, Z. (2012). Protein phosphatase 1 regulatory subunit 12A and catalytic subunit delta, new members in the phosphatidylinositide 3 kinase insulin-signaling pathway. JOURNAL OF ENDOCRINOLOGY, 214(3), 437-443.
• Langlais, P., Dillon, J. L., Mengos, A., Baluch, D. P., Ardebili, R., Miranda, D. N., Xie, X., Heckmann, B. L., Liu, J., & Mandarino, L. J. (2012). Identification of a Role for CLASP2 in Insulin Action. JOURNAL OF BIOLOGICAL CHEMISTRY, 287(46), 39245-39253.
• Liu, M., Zhou, L., Wei, L. i., Villarreal, R., Yang, X., Hu, D., Riojas, R. A., Holmes, B. M., Langlais, P. R., Lee, H., & Dong, L. Q. (2012). Phosphorylation of Adaptor Protein Containing Pleckstrin Homology Domain, Phosphotyrosine Binding Domain, and Leucine Zipper Motif 1 (APPL1) at Ser(430) Mediates Endoplasmic Reticulum (ER) Stress-induced Insulin Resistance in Hepatocytes. JOURNAL OF BIOLOGICAL CHEMISTRY, 287(31), 26087-26093.
• Liu, M., Zhou, L., Wei, L., Villarreal, R., Yang, X., Hu, D., Riojas, R. A., Holmes, B. M., Langlais, P. R., Lee, H., & Dong, L. Q. (2012). Phosphorylation of adaptor protein containing pleckstrin homology domain, phosphotyrosinebinding domain, and leucine zipper motif 1 (APPL1) at Ser 430 mediates endoplasmic reticulum (ER) stress-induced insulin resistance in hepatocytes. Journal of Biological Chemistry, 287(Issue 31). doi:10.1074/jbc.m112.372292
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  APPL1 is an adaptor protein that plays a critical role in regulating adiponectin and insulin signaling. However, how APPL1 is regulated under normal and pathological conditions remains largely unknown. In this study, we show that APPL1 undergoes phosphorylation at Ser430 and that this phosphorylation is enhanced in the liver of obese mice displaying insulin resistance. In cultured mouse hepatocytes, APPL1 phosphorylation at Ser 430 is stimulated by phorbol 12-myristate 13-acetate, an activator of classicPKCisoforms, and by the endoplasmic reticulum (ER) stress inducer, thapsigargin. Overexpression of wild-type but not dominant negative PKCα increases APPL1 phosphorylation at Ser430 in mouse hepatocytes. In addition, suppressing PKCα expression by shRNA in hepatocytes reduces ER stressinduced APPL1 phosphorylation at Ser430 as well as the inhibitory effect of ER stress on insulin-stimulated Akt phosphorylation. Consistent with a negative regulatory role of APPL1 phosphorylation at Ser 430 in insulin signaling, overexpression of APPL1S430D but not APPL1S430A impairs the potentiating effect of APPL1 on insulin-stimulated Akt phosphorylation at Thr308. Taken together, our results identify APPL1 as a novel target in ER stress-induced insulin resistance and PKCα as the kinase mediating ER stress-induced phosphorylation of APPL1 at Ser430. © 2012 by The American Society for Biochemistry and Molecular Biology Inc.
• Pham, K., Langlais, P., Zhang, X., Chao, A., Zingsheim, M., & Yi, Z. (2012). Insulin-stimulated phosphorylation of protein phosphatase 1 regulatory subunit 12B revealed by HPLC-ESI-MS/MS. PROTEOME SCIENCE, 10.
• Everman, S., Yi, Z., Langlais, P., Mandarino, L. J., Luo, M., Roberts, C., & Katsanos, C. S. (2011). Reproducibility of an HPLC-ESI-MS/MS Method for the Measurement of Stable-Isotope Enrichment of in Vivo-Labeled Muscle ATP Synthase Beta Subunit. PLOS ONE, 6(10).
• Geetha, T., Langlais, P., Luo, M., Mapes, R., Lefort, N., Chen, S. C., Mandarino, L. J., & Yi, Z. (2011). Label-free proteomic identification of endogenous, insulin-stimulated interaction partners of insulin receptor substrate-1. Journal of the American Society for Mass Spectrometry, 22(Issue 3). doi:10.1007/s13361-010-0051-2
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  Protein-protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein-protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein-protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLCESI- MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction. © The Author(s) 2011.
• Geetha, T., Langlais, P., Luo, M., Mapes, R., Lefort, N., Chen, S., Mandarino, L. J., & Yi, Z. (2011). Label-Free Proteomic Identification of Endogenous, Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY, 22(3), 457-466.
• Langlais, P., Yi, Z., & Mandarino, L. J. (2011). The Identification of Raptor as a Substrate for p44/42 MAPK. ENDOCRINOLOGY, 152(4), 1264-1273.
• Langlais, P., Mandarino, L. J., & Yi, Z. (2010). Label-free Relative Quantification of Co-eluting Isobaric Phosphopeptides of Insulin Receptor Substrate-1 by HPLC-ESI-MS/MS. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY, 21(9), 1490-1499.
• Sharoff, C. G., Langlais, P., Yi, Z., & Mandarino, L. J. (2010). Regulation of novel sites on AS160 by insulin and AICAR in human skeletal muscle. The FASEB Journal, 24(S1). doi:10.1096/fasebj.24.1_supplement.783.4
• Hojlund, K., Bowen, B. P., Hwang, H., Flynn, C. R., Madireddy, L., Geetha, T., Langlais, P., Meyer, C., Mandarino, L. J., & Yi, Z. (2009). In vivo Phosphoproteome of Human Skeletal Muscle Revealed by Phosphopeptide Enrichment and HPLC-ESI-MS/MS. JOURNAL OF PROTEOME RESEARCH, 8(11), 4954-4965.
• Højlund, K., Bowen, B. P., Hwang, H., Flynn, C. R., Madireddy, L., Geetha, T., Langlais, P., Meyer, C., Mandarino, L. J., & Yi, Z. (2009). In vivo phosphoproteome of human skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MS. Journal of Proteome Research, 8(Issue 11). doi:10.1021/pr9007267
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  Protein phosphorylation plays an essential role in signal transduction pathways that regulate substrate and energy metabolism, contractile function, and muscle mass in human skeletal muscle. Abnormal phosphorylation of signaling enzymes has been identified in insulin-resistant muscle using phospho-epitope-specific antibodies, but its role in other skeletal muscle disorders remains largely unknown. This may be in part due to insufficient knowledge of relevant targets. Here, we therefore present the first large-scale in vivo phosphoproteomic study of human skeletal muscle from 3 lean, healthy volunteers. Trypsin digestion of 3-5mg human skeletal muscle protein was followed by phosphopeptide enrichment using SCX and TiO2. The resulting phosphopeptides were analyzed by HPLC-ESI-MS/ MS. Using this unbiased approach, we identified 306 distinct in vivo phosphorylation sites in 127 proteins, including 240 phosphoserines, 53 phosphothreonines, and 13 phosphotyrosines in at least 2 out of 3 subjects. In addition, 61 ambiguous phosphorylation sites were identified in at least 2 out of 3 subjects. The majority of phosphoproteins detected are involved in sarcomeric function, excitation-contraction coupling (the Ca2+-cycle), glycolysis, and glycogen metabolism. Of particular interest, we identified multiple novel phosphorylation sites on several sarcomeric Z-disk proteins known to be involved in signaling and muscle disorders. These results provide numerous new targets for the investigation of human skeletal muscle phosphoproteins in health and disease and demonstrate feasibility of phosphoproteomics research of human skeletal muscle in vivo. © 2009 American Chemical Society.
• Hojlund, K., Yi, Z., Hwang, H., Bowen, B., Lefort, N., Flynn, C. R., Langlais, P., Weintraub, S. T., & Mandarino, L. J. (2008). Characterization of the human skeletal muscle proteome by one-dimensional gel electrophoresis and HPLC-ESI-MS/MS. MOLECULAR & CELLULAR PROTEOMICS, 7(2), 257-267.
• Hojlund, K., Yi, Z., Lefort, N., Langlais, P., Bowen, B., Levin, K., Beck-Nielsen, H., & Mandarino, L. J. (2008). Identification of multiple phosphorylation sites on ATP synthase beta subunit in human muscle. Increased phosphorylation at Thr213 and Tyr361 in obesity and type 2 diabetes. DIABETES, 57, A19-A19.
• Luo, M., Langlais, P., Yi, Z., Lefort, N., De Filippis, E. A., Hwang, H., Christ-Roberts, C. Y., & Mandarino, L. J. (2007). Phosphorylation of human insulin receptor substrate-1 at Serine 629 plays a positive role in insulin signaling. Endocrinology, 148(10), 4895-905.
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  The function of insulin receptor substrate-1 (IRS-1) is regulated by both tyrosine and serine/threonine phosphorylation. Phosphorylation of some serine/threonine residues in IRS-1 dampens insulin signaling, whereas phosphorylation of other serine/threonine residues enhances insulin signaling. Phosphorylation of human IRS-1 at Ser(629) was increased by insulin in Chinese hamster ovary cells expressing the insulin receptor (1.26 +/- 0.09-fold; P < 0.05) and L6 cells (1.35 +/- 0.29-fold; P < 0.05) expressing human IRS-1. Sequence analysis surrounding Ser(629) revealed conformity to the consensus phosphorylation sequence recognized by Akt. Phosphorylation of IRS-1 at Ser(629) in cells was decreased upon treatment with either an Akt inhibitor or by coexpression with kinase dead Akt, whereas Ser(629) phosphorylation was increased by coexpression with constitutively active Akt. In addition, Ser(629) of IRS-1 is directly phosphorylated by Akt in vitro. In cells, preventing phosphorylation of Ser(629) by a Ser(629)Ala mutation resulted in increased phosphorylation of Ser(636), a known negative regulator of IRS-1, without affecting phosphorylation of Tyr(632) or Ser(616). Cells expressing the Ser(629)Ala mutation, along with increased Ser(636) phosphorylation, had decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol 3'-kinase with IRS-1 and decreased phosphorylation of Akt at Ser(473). Finally, in vitro phosphorylation of a Ser(629)-containing IRS-1 fragment with Akt reduces the subsequent ability of ERK to phosphorylate Ser(636/639). These results suggest that a feed-forward mechanism may exist whereby insulin activation of Akt leads to phosphorylation of IRS-1 at Ser(629), resulting in decreased phosphorylation of IRS-1 at Ser(636) and enhanced downstream signaling. Understanding the complex phosphorylation patterns of IRS-1 is crucial to elucidating the factors contributing to insulin resistance and, ultimately, the pathogenesis of type 2 diabetes.
• Mapes, R., Yi, Z., Langlais, P., Riojas, R., Liu, F., Dong, L. Q., & Mandarino, L. J. (2007). APPL1, an adiponectin receptor binding partner, is phosphorylated at ser(401) in human skeletal muscle in vivo. DIABETES, 56, A508-A508.
• Yi, Z., Flynn, C. R., Langlais, P., Mandarino, L. J., & Hojlund, K. (2007). Comprehensive characterization of the human skeletal muscle proteome by LC-MS/MS. DIABETES, 56, A397-A397.
• Yi, Z., Langlais, P., De Filippis, E. A., Luo, M., Flynn, C. R., Schroeder, S., Weintraub, S. T., Mapes, R., & Mandarino, L. J. (2007). Global assessment of regulation of phosphorylation of insulin receptor substrate-1 by insulin in vivo in human muscle. Diabetes, 56(6), 1508-16.
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  Research has focused on insulin receptor substrate (IRS)-1 as a locus for insulin resistance. Tyrosine phosphorylation of IRS-1 initiates insulin signaling, whereas serine/threonine phosphorylation alters the ability of IRS-1 to transduce the insulin signal. Of 1,242 amino acids in IRS-1, 242 are serine/threonine. Serine/threonine phosphorylation of IRS-1 is affected by many factors, including insulin. The purpose of this study was to perform global assessment of phosphorylation of serine/threonine residues in IRS-1 in vivo in humans.
• Mao, X., Kikani, C. K., Riojas, R. A., Langlais, P., Wang, L., Ramos, F. J., Fang, Q., Christ-Roberts, C. Y., Hong, J. Y., Kim, R. Y., Liu, F., & Dong, L. Q. (2006). APPL1 binds to adiponectin receptors and mediates adiponectin signalling and function. Nature Cell Biology, 8(Issue 5). doi:10.1038/ncb1404
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  Adiponectin, also known as Acrp30, is an adipose tissue-derived hormone with anti-atherogenic, anti-diabetic and insulin sensitizing properties. Two seven-transmembrane domain-containing proteins, AdipoR1 and AdipoR2, have recently been identified as adiponectin receptors, yet signalling events downstream of these receptors remain poorly defined. By using the cytoplasmic domain of AdipoR1 as bait, we screened a yeast two-hybrid cDNA library derived from human fetal brain. This screening led to the identification of a phosphotyrosine binding domain and a pleckstrin homology domain-containing adaptor protein, APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding (PTB) domain and leucine zipper motif). APPL1 interacts with adiponectin receptors in mammalian cells and the interaction is stimulated by adiponectin. Overexpression of APPL1 increases, and suppression of APPL1 level reduces, adiponectin signalling and adiponectin-mediated downstream events (such as lipid oxidation, glucose uptake and the membrane translocation of glucose transport 4 (GLUT4)). Adiponectin stimulates the interaction between APPL1 and Rab5 (a small GTPase) interaction, leading to increased GLUT4 membrane translocation. APPL1 also acts as a critical regulator of the crosstalk between adiponectin signalling and insulin signalling pathways. These results demonstrate a key function for APPL1 in adiponectin signalling and provide a molecular mechanism for the insulin sensitizing function of adiponectin. © 2006, Nature Publishing Group Ltd. All rights reserved.
• Mao, X., Kikani, C. K., Riojas, R. A., Langlais, P., Wang, L., Ramos, F. J., Fang, Q., Christ-Roberts, C. Y., Hong, J. Y., Kim, R., Liu, F., & Dong, L. Q. (2006). APPL1 binds to adiponectin receptors and mediates adiponectin signalling and function. Nature cell biology, 8(5), 516-23.
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  Adiponectin, also known as Acrp30, is an adipose tissue-derived hormone with anti-atherogenic, anti-diabetic and insulin sensitizing properties. Two seven-transmembrane domain-containing proteins, AdipoR1 and AdipoR2, have recently been identified as adiponectin receptors, yet signalling events downstream of these receptors remain poorly defined. By using the cytoplasmic domain of AdipoR1 as bait, we screened a yeast two-hybrid cDNA library derived from human fetal brain. This screening led to the identification of a phosphotyrosine binding domain and a pleckstrin homology domain-containing adaptor protein, APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding (PTB) domain and leucine zipper motif). APPL1 interacts with adiponectin receptors in mammalian cells and the interaction is stimulated by adiponectin. Overexpression of APPL1 increases, and suppression of APPL1 level reduces, adiponectin signalling and adiponectin-mediated downstream events (such as lipid oxidation, glucose uptake and the membrane translocation of glucose transport 4 (GLUT4)). Adiponectin stimulates the interaction between APPL1 and Rab5 (a small GTPase) interaction, leading to increased GLUT4 membrane translocation. APPL1 also acts as a critical regulator of the crosstalk between adiponectin signalling and insulin signalling pathways. These results demonstrate a key function for APPL1 in adiponectin signalling and provide a molecular mechanism for the insulin sensitizing function of adiponectin.
• Ramos, F. J., Langlais, P. R., Hu, D., Dong, L. Q., & Liu, F. (2006). Grb10 mediates insulin-stimulated degradation of the insulin receptor: a mechanism of negative regulation. American journal of physiology. Endocrinology and metabolism, 290(6), E1262-6.
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  Growth factor receptor-bound protein 10 (Grb10) is an adapter protein that interacts with a number of tyrosine-phosphorylated growth factor receptors, including the insulin receptor (IR). To investigate the role of Grb10 in insulin signaling, we generated cell lines in which the expression levels of Grb10 are either overexpressed by stable transfection or suppressed by RNA interference. We found that suppressing endogenous Grb10 expression led to increased IR protein levels, whereas overexpression of Grb10 led to reduced IR protein levels. Altering Grb10 expression levels had no effect on the mRNA levels of IR, suggesting that the modulation occurs at the protein level. Reduced IR levels were also observed in cells with prolonged insulin treatment, and this reduction was inhibited in Grb10-deficient cells. The insulin-induced IR reduction was greatly reversed by MG-132, a proteasomal inhibitor, but not by chloroquine, a lysosomal inhibitor. IR underwent insulin-stimulated ubiquitination in cells, and this ubiquitination was inhibited in the Grb10-suppressed cell line. Together, our results suggest that, in addition to inhibiting IR kinase activity by directly binding to the IR, Grb10 also negatively regulates insulin signaling by mediating insulin-stimulated degradation of the receptor.
• Riojas, R. A., Kikani, C. K., Wang, C., Mao, X., Zhou, L., Langlais, P. R., Hu, D., Roberts, J. L., Dong, L. Q., & Liu, F. (2006). Fine tuning PDK1 activity by phosphorylation at Ser163. The Journal of biological chemistry, 281(31), 21588-93.
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  3-Phosphoinositide-dependent protein kinase-1 (PDK1) mediates phosphorylation and activation of members of the AGC protein kinase family and plays an essential role in insulin signaling and action. However, whether and how PDK1 activity is regulated in cells remains largely uncharacterized. In the present study, we show that PDK1 undergoes insulin-stimulated and phosphatidylinositol 3-kinase-dependent phosphorylation at Ser244 in the activation loop and at a novel site: Ser163 in the hinge region between the two lobes of the kinase domain. Sequence alignment studies revealed that the residue corresponding to Ser163 of PDK1 in all other AGC kinases is glutamate, suggesting that a negative charge at this site may be important for PDK1 function. Replacing Ser163 with a negatively charged residue, glutamate, led to a 2-fold increase in PDK1 activity. Molecular modeling studies suggested that phosphorylated Ser163 may form additional hydrogen bonds with Tyr149 and Gln223. In support of this, mutation of Tyr149 to Ala is sufficient to reduce PDK1 activity. Taken together, our results suggest that PDK1 phosphorylation of Ser163 may provide a mechanism to fine-tune PDK1 activity and function in cells.
• Yi, Z., Luo, M., Reyna, S. M., Weintraub, S. T., Langlais, P., & Mandarino, L. J. (2006). Quantification of serine phosphorylation of IRS-1 by HPLC-ESI-MS/MS. DIABETES, 55, A305-A305.
• Langlais, P., Wang, C., Dong, L. Q., Carroll, C. A., Weintraub, S. T., & Liu, F. (2005). Phosphorylation of Grb10 by mitogen-activated protein kinase: identification of Ser150 and Ser476 of human Grb10zeta as major phosphorylation sites. Biochemistry, 44(24), 8890-7.
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  Grb10 is a Src-homology 2 (SH2) and Pleckstrin-homology (PH) domain-containing protein that binds to several autophosphorylated receptor tyrosine kinases including the insulin receptor (IR). Our previous studies showed that Grb10 underwent insulin-stimulated serine phosphorylation, yet the kinase(s) responsible for phosphorylation and the sites of the phosphorylation remain unknown. In this report, we show that Grb10 is a direct substrate of the p42/44 mitogen-activated protein kinase (MAPK). In addition, we found that inhibition of the MAPK signaling pathway reduced Grb10 phosphorylation in cells. Using site-directed mutagenesis, phosphopeptide mapping, and capillary HPLC-electrospray-tandem mass spectrometry analysis, we identified Ser(150), Ser(418), and Ser(476) of human Grb10zeta as MAPK-mediated in vitro phosphorylation sites. In vivo labeling and two-dimensional phosphopeptide mapping studies revealed that Ser(150) and Ser(476) of human Grb10zeta are phosphorylated in intact cells. Replacing Ser(150) and Ser(476) with alanines reduced the inhibitory effect of human Grb10zeta on insulin-stimulated IRS1 tyrosine phosphorylation. Taken together, our findings suggest that phosphorylation of the adaptor protein may provide a feedback inhibitory mechanism by which Grb10 regulates insulin signaling.
• Luo, M., Reyna, S., Wang, L., Yi, Z., Carroll, C., Dong, L. Q., Langlais, P., Weintraub, S. T., & Mandarino, L. J. (2005). Identification of insulin receptor substrate 1 serine/threonine phosphorylation sites using mass spectrometry analysis: regulatory role of serine 1223. Endocrinology, 146(10), 4410-6.
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  Insulin receptor substrate 1 (IRS-1), an intracellular substrate of the insulin receptor tyrosine kinase, also is heavily phosphorylated on serine and threonine residues, and several serine phosphorylation sites alter the function of IRS-1. Because of the large number of serine/threonine residues, position-by-position analysis of these potential phosphorylation sites by mutagenesis is difficult. To circumvent this, we have employed matrix-assisted laser desorption/ionization time-of-flight and HPLC-electrospray ionization tandem mass spectrometry techniques to scan for serine and threonine residues that are phosphorylated in full-length human IRS-1 ectopically expressed in cells using an adenoviral vector. This approach revealed 12 phosphorylation sites on serine or threonine residues, 10 of which were novel sites. Seven of these sites were in proline-directed motifs, whereas five were in arginine-directed sites. Sequence inspection suggested that phosphorylation of Ser1223 might alter the interaction of IRS-1 with the protein tyrosine phosphatase Src homology domain 2 (SH2)-containing phosphatase-2 (SHP-2). Mutation of Ser1223 to alanine to prevent phosphorylation resulted in increased association of SHP-2 with IRS-1, decreased insulin-stimulated tyrosine phosphorylation of IRS-1 in CHO/IR cells, and decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol-3-kinase with IRS-1. This mutation had no effect on association of IRS-1 with the insulin receptor. Sequence analysis showed the Ser1223 region to be widely conserved evolutionarily. These data suggest that phosphorylation of Ser1223 dampens association of IRS-1 with SHP-2, thereby increasing net insulin-stimulated tyrosine phosphorylation.
• Langlais, P., Dong, L. Q., Ramos, F. J., Hu, D., Li, Y., Quon, M. J., & Liu, F. (2004). Negative regulation of insulin-stimulated mitogen-activated protein kinase signaling by Grb10. Molecular endocrinology (Baltimore, Md.), 18(2), 350-8.
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  Grb10 is a Pleckstrin homology and Src homology 2 (SH2) domain-containing protein that binds to the tyrosine-phosphorylated insulin receptor in response to insulin stimulation. Loss of Grb10 function in mice results in fetal and placental overgrowth; however, the molecular mechanism remains unknown. In the present study, we show that overexpression of Grb10 in Chinese hamster ovary cells expressing the insulin receptor or in 3T3-L1 adipocytes reduced insulin-stimulated phosphorylation of MAPK. Overexpression of Grb10 in rat primary adipocytes also inhibited insulin-stimulated phosphorylation of the MAPK downstream substrate Elk1. To determine the mechanism by which Grb10 inhibited insulin-stimulated MAPK signaling, we examined whether Grb10 affects the phosphorylation of MAPK upstream signaling components. We found that overexpression of Grb10 inhibited the insulin-stimulated phosphorylation of Shc, a positive regulator of the MAPK signaling pathway. The inhibitory effect was diminished when the SH2 domain of Grb10 was deleted. The negative role of Grb10 in insulin signaling was established by suppression of endogenous Grb10 by RNA interference in HeLa cells overexpressing the insulin receptor, which enhanced insulin-stimulated phosphorylation of MAPK, Shc, and Akt. Taken together, our findings suggest that Grb10 functions as a negative regulator in the insulin-stimulated MAPK signaling pathway. In addition, the inhibitory effect of Grb10 on the MAPK pathway is most likely due to a direct block of insulin-stimulated Shc tyrosine phosphorylation.
• Li, Y., Langlais, P., Gamper, N., Liu, F., & Shapiro, M. S. (2004). Dual phosphorylations underlie modulation of unitary KCNQ K(+) channels by Src tyrosine kinase. The Journal of biological chemistry, 279(44), 45399-407.
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  Src tyrosine kinase suppresses KCNQ (M-type) K(+) channels in a subunit-specific manner representing a mode of modulation distinct from that involving G protein-coupled receptors. We probed the molecular and biophysical mechanisms of this modulation using mutagenesis, biochemistry, and both whole-cell and single channel modes of patch clamp recording. Immunoprecipitation assays showed that Src associates with KCNQ2-5 subunits but phosphorylates only KCNQ3-5. Using KCNQ3 as a background, we found that mutation of a tyrosine in the amino terminus (Tyr-67) or one in the carboxyl terminus (Tyr-349) abolished Src-dependent modulation of heterologously expressed KCNQ2/3 heteromultimers. The tyrosine phosphorylation was much weaker for either the KCNQ3-Y67F or KCNQ3-Y349F mutants and wholly absent in the KCNQ3-Y67F/Y349F double mutant. Biotinylation assays showed that Src activity does not alter the membrane abundance of channels in the plasma membrane. In recordings from cell-attached patches containing a single KCNQ2/3 channel, we found that Src inhibits the open probability of the channels. Kinetic analysis was consistent with the channels having two discrete open times and three closed times. Src activity reduced the durations of the longest open time and lengthened the longest closed time of the channels. The implications for the mechanisms of channel regulation by the dual phosphorylations on both channel termini are discussed.
• McClung, J. P., Roneker, C. A., Mu, W., Lisk, D. J., Langlais, P., Liu, F., & Lei, X. G. (2004). Development of insulin resistance and obesity in mice overexpressing cellular glutathione peroxidase. Proceedings of the National Academy of Sciences of the United States of America, 101(24), 8852-7.
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  Insulin resistance, a hallmark of type 2 diabetes, is associated with oxidative stress. However, the role of reactive oxygen species or specific antioxidant enzymes in its development has not been tested under physiological conditions. The objective of our study was to investigate the impact of overexpression of glutathione peroxidase 1 (GPX1), an intracellular selenoprotein that reduces hydrogen peroxide (H(2)O(2)) in vivo, on glucose metabolism and insulin function. The GPX1-overexpressing (OE) and WT male mice (n = 80) were fed a selenium-adequate diet (0.4 mg/kg) from 8 to 24 weeks of age. Compared with the WT, the OE mice developed (P < 0.05) hyperglycemia (117 vs. 149 mg/dl), hyperinsulinemia (419 vs. 1,350 pg/ml), and elevated plasma leptin (5 vs. 16 ng/ml) at 24 weeks of age. Meanwhile, these mice were heavier (37 vs. 27 g, P < 0.001) and fatter (37% vs. 17% fat, P < 0.01) than the WT mice. At 30-60 min after an insulin challenge, the OE mice had 25% less (P < 0.05) of a decrease in blood glucose than the WT mice. Their insulin resistance was associated with a 30-70% reduction (P < 0.05) in the insulin-stimulated phosphorylations of insulin receptor (beta-subunit) in liver and Akt (Ser(473) and Thr(308)) in liver and soleus muscle. Here we report the development of insulin resistance in mammals with elevated expression of an antioxidant enzyme and suggest that increased GPX1 activity may interfere with insulin function by overquenching intracellular reactive oxygen species required for insulin sensitizing.
• McClung, J. P., Roneker, C. A., Mu, W., Lisk, D. J., Langlais, P., Liu, F., & Lei, X. G. (2004). Development of insulin resistance and obesity in mice overexpressing cellular glutathione peroxidase. Proceedings of the National Academy of Sciences of the United States of America, 101(Issue 24). doi:10.1073/pnas.0308096101
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  Insulin resistance, a hallmark of type 2 diabetes, is associated with oxidative stress. However, the role of reactive oxygen species or specific antioxidant enzymes in its development has not been tested under physiological conditions. The objective of our study was to investigate the impact of overexpression of glutathione peroxidase 1 (GPX1), an intracellular selenoprotein that reduces hydrogen peroxide (H2O2) in vivo, on glucose metabolism and insulin function. The GPX1-overexpressing (OE) and WT male mice (n = 80) were fed a selenium-adequate diet (0.4 mg/kg) from 8 to 24 weeks of age. Compared with the WT, the OE mice developed (P < 0.05) hyperglycemia (117 vs. 149 mg/dl), hyperinsulinemia (419 vs. 1,350 pg/ml), and elevated plasma leptin (5 vs. 16 ng/ml) at 24 weeks of age. Meanwhile, these mice were heavier (37 vs. 27 g, P < 0.001) and fatter (37% vs. 17% fat, P < 0.01) than the WT mice. At 30-60 min after an insulin challenge, the OE mice had 25% less (P < 0.05) of a decrease in blood glucose than the WT mice. Their insulin resistance was associated with a 30-70% reduction (P < 0.05) in the insulin-stimulated phosphorylations of insulin receptor (β-subunit) in liver and Akt (Ser473 and Thr308) in liver and soleus muscle. Here we report the development of insulin resistance in mammals with elevated expression of an antioxidant enzyme and suggest that increased GPX1 activity may interfere with insulin function by overquenching intracellular reactive oxygen species required for insulin sensitizing.
• Wick, K. R., Werner, E. D., Langlais, P., Ramos, F. J., Dong, L. Q., Shoelson, S. E., & Liu, F. (2003). Grb10 inhibits insulin-stimulated insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase/Akt signaling pathway by disrupting the association of IRS-1/IRS-2 with the insulin receptor. The Journal of biological chemistry, 278(10), 8460-7.
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  Grb10 has been proposed to inhibit or activate insulin signaling, depending on cellular context. We have investigated the mechanism by which full-length hGrb10gamma inhibits signaling through the insulin receptor substrate (IRS) proteins. Overexpression of hGrb10gamma in CHO/IR cells and in differentiated adipocytes significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. Inhibition occurred rapidly and was sustained for 60 min during insulin stimulation. In agreement with inhibited signaling through the IRS/PI 3-kinase pathway, we found hGrb10gamma to both delay and reduce phosphorylation of Akt at Thr(308) and Ser(473) in response to insulin stimulation. Decreased phosphorylation of IRS-1/2 may arise from impaired catalytic activity of the receptor, since hGrb10gamma directly associates with the IR kinase regulatory loop. However, yeast tri-hybrid studies indicated that full-length Grb10 blocks association between IRS proteins and IR, and that this requires the SH2 domain of Grb10. In cells, hGrb10gamma inhibited insulin-stimulated IRS-1 tyrosine phosphorylation in a dose-dependent manner, but did not affect IR catalytic activity toward Tyr(972) in the juxtamembrane region and Tyr(1158/1162/1163) in the regulatory domain. We conclude that binding of hGrb10gamma to IR decreases signaling through the IRS/PI 3-kinase/AKT pathway by physically blocking IRS access to IR.
• Wick, M. J., Dong, L. Q., Hu, D., Langlais, P., & Liu, F. (2001). Insulin receptor-mediated p62dok tyrosine phosphorylation at residues 362 and 398 plays distinct roles for binding GTPase-activating protein and Nck and is essential for inhibiting insulin-stimulated activation of Ras and Akt. The Journal of biological chemistry, 276(46), 42843-50.
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  A GTPase-activating protein (GAP)-associated 60-kDa protein has been found to undergo rapid tyrosine phosphorylation in response to insulin stimulation. However, whether this protein is a direct in vivo substrate for the insulin receptor (IR) tyrosine kinase and whether the tyrosine phosphorylation plays a role in insulin signaling remain to be established. Here we show that the insulin-stimulated tyrosine phosphorylation of the GAP-associated protein, now identified as p62(dok), is inhibited by Grb10, an adaptor protein that binds directly to the kinase domain of the IR, both in vitro and in cells. Replacing Tyr(362) and Tyr(398) with phenylalanine greatly decreased the IR-catalyzed p62(dok) tyrosine phosphorylation in vitro, suggesting that these two residues are the major IR-mediated phosphorylation sites. However, mutations at Tyr(362) and Tyr(398) only partially blocked insulin-stimulated p62(dok) tyrosine phosphorylation in cells, indicating that p62(dok) is also a target for other cellular tyrosine kinase(s) in addition to the IR. Replacing Tyr(362) with phenylalanine abolished the interaction between p62(dok) and Nck. Mutations at Tyr(362/398) of p62(dok) disrupted the interaction between p62(dok) and GAP and decreased the inhibitory effect of p62(dok) on the insulin-stimulated activation of Ras and Akt, but not mitogen-activated protein kinase. Furthermore, the inhibitory effect of p62(dok) on Akt phosphorylation could be blocked by coexpression of a constitutively active Ras. Taken together, our findings indicate that p62(dok) is a direct substrate for the IR tyrosine kinase and that phosphorylation at Tyr(362) and Tyr(398) plays an essential role for p62(dok) to interact with its effectors and negatively regulate the insulin signaling pathway.
• Langlais, P., Dong, L. Q., Hu, D., & Liu, F. (2000). Identification of Grb10 as a direct substrate for members of the Src tyrosine kinase family. Oncogene, 19(25), 2895-903.
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  Treatment of cells with insulin and protein tyrosine phosphatase inhibitors such as vanadate and pervanadate resulted in the tyrosine phosphorylation of Grb10, a Src homology 2 (SH2) and pleckstrin homology domain-containing adaptor protein which binds to a number of receptor tyrosine kinases including the insulin receptor (IR). Although Grb10 binds directly to the kinase domain of the IR, our data show that Grb10 is not a direct substrate for the IR tyrosine kinase. Consistent with this finding, Grb10 tyrosine phosphorylation in cells was inhibited by herbimycin A, a relatively specific inhibitor for members of the Src tyrosine kinase family, and by the expression of dominant negative Src or Fyn. In addition, Grb10 tyrosine phosphorylation was stimulated by expression of constitutively active Src or Fyn in cells and by incubation with purified Src or Fyn in vitro. The insulin stimulated or Src/Fyn-mediated tyrosine phosphorylation in vivo was significantly reduced when Grb10 tyrosine 67 was changed to glycine. This mutant form of Grb10 bound with higher affinity to the IR in cells than that of the wild-type protein, suggesting that tyrosine phosphorylation of Grb10 may normally negatively regulate its binding to the IR. Our data show that Grb10 is a new substrate for members of the Src tyrosine kinase family and that the tyrosine phosphorylation of the protein may play a potential role in cell signaling processes mediated by these kinases. Oncogene (2000).
• Dong, L. Q., Zhang, R. B., Langlais, P., He, H., Clark, M., Zhu, L., & Liu, F. (1999). Primary structure, tissue distribution, and expression of mouse phosphoinositide-dependent protein kinase-1, a protein kinase that phosphorylates and activates protein kinase Czeta. The Journal of biological chemistry, 274(12), 8117-22.
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  Phosphoinositide-dependent protein kinase-1 (PDK1) is a recently identified serine/threonine kinase that phosphorylates and activates Akt and p70(S6K), two downstream kinases of phosphatidylinositol 3-kinase. To further study the potential role of PDK1, we have screened a mouse liver cDNA library and identified a cDNA encoding the enzyme. The predicted mouse PDK1 (mPDK1) protein contained 559 amino acids and a COOH-terminal pleckstrin homology domain. A 7-kilobase mPDK1 mRNA was broadly expressed in mouse tissues and in embryonic cells. In the testis, a high level expression of a tissue-specific 2-kilobase transcript was also detected. Anti-mPDK1 antibody recognized multiple proteins in mouse tissues with molecular masses ranging from 60 to 180 kDa. mPDK1 phosphorylated the conserved threonine residue (Thr402) in the activation loop of protein kinase C-zeta and activated the enzyme in vitro and in cells. Our findings suggest that there may be different isoforms of mPDK1 and that the protein is an upstream kinase that activates divergent pathways downstream of phosphatidylinositol 3-kinase.

Presentations

• Tabsh, K. K., Pendleton, A. L., Barker, N. K., Langlais, P. R., Limesand, S. W., & Hill, M. G. (2020, March/Spring). Confirmation of Inflammatory Biomarkers from Maternal Plasma in Women with Intrahepatic Cholestasis of Pregnancy. Society for Reproductive Investigation 67th Annual Scientific Meeting.
• Barker, N., Krantz, J., Parker, S., Mouneimne, G., & Langlais, P. R. (2018, June/Summer). Characterization of the G2L1 Interactome Leads to the Discovery that CLIP2, G2L1, and EB1 Undergo Insulin-Stimulated Phosphorylation. American Diabetes Association. Orlando, Florida.
• Kras, K., Langlais, P. R., Willis, W. T., Mandarino, L., De Filippis, E., Roust, L., & Katsanos, C. (2016, June). Differential enrichment of Sub-sarcolemmal mitochondria with individual proteins between lean and obese, insulin-resistant subjects. American Diabetes Association Annual Meeting.

Poster Presentations

• Lefkowitz, E., Benson, D., Zozobrado, L., Ref, J., Bravo, F., Lancaster, J., Koevary, J., Langlais, P. R., Hicks, M., & Goldman, S. (2025).

  Biologic Therapy Promotes Muscle Regeneration in-vitro following Volumetric Muscle Loss Injury by RHO GTPase and CSPG2 Modulation 

  . 2026 Western Medical Research Conference.
• Benson, D., Singh, S., Grijalva, A., Fink, J., Davis-Gorman, G., Zhang, X., Deeb, M., O’Donnell, D., Tulino, A., Zozobrado, L., Coletta, D. K., Langlais, P. R., Mangiola, M., Pandey, A., Zile, M., Bradshaw, A., Arunachalam, P., Fain, M., Nikolich, J., , Koevary, J., et al. (2025).

  Evaluation of Predictive Signatures in Clinical and Pre-Clinical Models of Chronic Ischemic Heart Failure for the Development and Translation of Diagnostics, Prognostics, and Therapeutics for Tissue Repair

  . 2025 Sarver Heart Center Research Symposium.
• Benson, D., Singh, S., Grijalva, A., Fink, J., Davis-Gorman, G., Zhang, X., Deeb, M., O’Donnell, D., Tulino, A., Zozobrado, L., Coletta, D. K., Langlais, P. R., Mangiola, M., Pandey, A., Zile, M., Bradshaw, A., Arunachalam, P., Fain, M., Nikolich, J., , Koevary, J., et al. (2025).

  Evaluation of Predictive Signatures in Clinical and Pre-Clinical Models of Chronic Ischemic Heart Failure for the Development and Translation of Diagnostics, Prognostics, and Therapeutics for Tissue Repair

  . American Heart Association Basic Cardiovascular Sciences Scientific Sessions 2025.
• Berthiaume Fox, K., Li, J., Santiago Raj, P. V., Langlais, P. R., & Ledford, J. (2025).

  CC16 Regulates Airway Epithelial Metabolism and Mitochondrial Function Due to Bronchoconstrictive Stress in Asthma

  . 2025 Biomedical Engineering Society Annual Meeting.
• Chen, S., Diesel, C., Partin, J., Karlage, K. L., Milnes, T. M., Langlais, P. R., Vanderah, T. W., & Riegel, A. C. (2025).

  Inhibition of CaMKIα In Mice With Chronic Neuropathic Pain Reduces Anxiety-related Behaviors Without Altering Depression or Cognitive Behaviors

  . 2025 UA Research Day.
• Crudo, E. M., Sullivan, A. M., Briggs, C. C., George, A. E., Lu, E. J., Coletta, D. K., & Langlais, P. R. (2025).

  Investigating the Role of RHG21 in Insulin-Stimulated GLUT4 Translocation

  . 2025 Sharing Opportunities and Achievements in Research in DOM (SOAR in DOM) Forum.
• Crudo, E. M., Sullivan, A. M., Briggs, C. C., George, A. E., Lu, E. J., Coletta, D. K., & Langlais, P. R. (2025).

  Investigating the Role of RHG21 in Insulin-Stimulated GLUT4 Translocation

  . 2025 University of Arizona Physiology Students Symposium.
• Diesel, C., Bahramnejad, E., Majuta, L., Karlage, K. L., Haveman, R., Milnes, T. M., Langlais, P. R., Vanderah, T. W., & Riegel, A. C. (2025).

  Proteomic Analysis Reveals Acute and Chronic Neuropathic Pain Differentially Affect Protein Abundance in the Ventral Tegmental Area 

  . 2025 UA Research Day.
• Giphart, A., Lu, E. J., Montoya, J. A., McGraw, M. B., Barbour, S., Lee, N. Y., & Langlais, P. R. (2025).

  Investigating the Potential Influence of ATAT1 and TAK1 on Insulin Resistance

  . 2025 University of Arizona Undergraduate Biology Research Symposium.
• Hauck, G., Vasquez, C., Lynn, M., Klass, M., Langlais, P. R., Hamilton, S., & Tardiff, J. C. (2025).

  Calmodulin Kinase II is a Mutation-Specific Driver of Disease in Hypertrophic Cardiomyopathy

  . American Heart Association Basic Cardiovascular Sciences Scientific Sessions 2025.
• Lasher, J., Lu, E. J., Lipinksi, A. A., & Langlais, P. R. (2025).

  Use of Artificial Intelligence in Quantitative Proteomics Data Analysis 

  . 2025 Sharing Opportunities and Achievements in Research in DOM (SOAR in DOM) Forum.
• Lasher, J., Lu, E. J., Lipinksi, A. A., & Langlais, P. R. (2025).

  Use of Artificial Intelligence in Quantitative Proteomics Data Analysis 

  . 2025 University of Arizona Franke Honors College Pinnacle Symposium.
• Lasher, J., Lu, E. J., Lipinksi, A. A., & Langlais, P. R. (2025).

  Use of Artificial Intelligence in Quantitative Proteomics Data Analysis 

  . 2025 University of Arizona Physiology Students Symposium.
• Mendoza-Siqueros, F., Roa, M. C., McGraw, M. B., Lu, E. J., Montoya, J. A., Giphart, A., Barbour, S., Coletta, D. K., Lee, N. Y., & Langlais, P. R. (2025).

  Insulin Promotes Microtubule Stabilization and Trafficking in Adipocytes: A New Perspective on Insulin Resistance

  . University of Arizona Undergraduate Research Opportunities Consortium Symposium.
• Mendoza-Siqueros, F., Roa, M. C., McGraw, M. B., Lu, E. J., Montoya, J. A., Giphart, A., Barbour, S., Coletta, D. K., Lee, N. Y., & Langlais, P. R. (2025).

  The Link Between Inflammation and Insulin Resistance: How TAK1 and Microtubule Dynamics Affect Glucose Transport

  . 2025 American Society for Microbiology's (ASM) Conference in Support of Multidisciplinary Science and Workforce Development.
• Zhao, W., Chrisenberry, D., Varela, M., Luna-Ramirez, R., Langlais, P. R., Brown, L., & Limesand, S. W. (2025).

  Exogenous oxygen and glucose supplementation remodels the mitochondrial proteome in skeletal muscle of FGR fetal sheep

  . 2025 Perinatal Biology Symposium.
• Barbour, S., McGraw, M. B., Lu, E. J., Giphart, A., Montoya, J. A., & Langlais, P. R. (2024, August). Understanding TAK1’s Possible Role in Insulin-Stimulated Glucose Uptake. University of Arizona Undergraduate Research Opportunities Consortium Symposium.
• Diesel, C., Bahramnejad, E., Majuta, L., Partin, J., Haskell, M., Karlage, K., Hellinger, R., Liktor-Busa, E., Milnes, T. M., Langlais, P. R., Vanderah, T. W., & Riegel, A. C. (2024, October). Investigation of the Neurobiological Underpinnings of Chronic Pain-Induced Depression. 17th Arizona Physiological Society Annual Meeting.
• Diesel, C., Bahramnejad, E., Majuta, L., Umbertus, D., Karlage, K., Young, S., Liktor-Busa, E., Milnes, T. M., Langlais, P. R., Vanderah, T. W., & Riegel, A. C. (2024, April). Endocannabinoid Signaling and Activation of Kv7 ion Channels Regulates Pain-Related Depression-like Behaviors in Mice. University of Arizona College of Medicine Research Day Symposium.
• Gackle, M. E., Lu, E., Goodmanson, M. J., Zelms, K. R., Rojas, N. G., McGraw, M. B., Nash, J. C., Lasher, J. D., Amaro, A., Moberly, A. P., & Langlais, P. R. (2024, January). Assessing Candidate Proteins Identified by Quantitative Proteomics for a Role in Insulin-Stimulated Glucose Uptake. Undergraduate Biology Research Program.
• Gackle, M. E., Lu, E., Goodmanson, M. J., Zelms, K. R., Rojas, N. G., McGraw, M. B., Nash, J. C., Lasher, J. D., Amaro, A., Moberly, A. P., Cruz-Flores, P., Lee, N. Y., & Langlais, P. R. (2024, April). Assessing Candidate Proteins Identified by Quantitative Proteomics for a Role in Insulin-Stimulated Glucose Uptake. University of Arizona College of Medicine Research Day Symposium.
• Lu, E. J., Montoya, J. A., McGraw, M. B., Barbour, S., Giphart, A., Coletta, D. K., Lee, N. Y., & Langlais, P. R. (2024, October). A Microtubule-Acetylation Specific Inhibitor Induces Insulin Resistance. 17th Arizona Physiological Society Annual Meeting.
• McGraw, M. B., Lu, E. J., Montoya, J. A., Giphart, A., Barbour, S., Peper, T. J., Coletta, D. K., Lee, N. Y., & Langlais, P. R. (2024, November). Inhibition of Microtubule Stabilization Causes Insulin Resistance . American College of Physicians Arizona Chapter Annual Scientific Meeting.
• Montoya, J. A., Barbour, S., McGraw, M. B., Giphart, A., Lu, E. J., & Langlais, P. R. (2024, August). Investigating a Potential Role for TAK1 in Insulin Action . University of Arizona FRONTERA Undergraduate Research Program.
• Odeneye, R. E., Lipinski, A. A., Coletta, D. K., & Langlais, P. R. (2024, January). Proteomic Profiling of Differentiated Myotubes Identifies Protein Candidates Potentially Involved in Insulin-Stimulated Glucose Uptake. University of Arizona Physiology Undergraduate Biology Research Symposium.
• Willis, W., Willis, B., Langlais, P. R., & Mandarino, L. J. (2024, August). Interpretation of 31P-MRS Assessment of (delta)GATP:JATP Elasticity in Human Skeletal Muscle Requires Estimation of the Oxidative Fuel Mixture. 22nd European Bioenergetics Conference 2024. Innsbruck, Austria.
• Zelms, K. R., Gackle, M. E., Goodmanson, M. J., Lu, E. J., Rojas, N. G., McGraw, M. B., Nash, J. C., Lasher, J. D., Amaro, A., Moberly, A. P., & Langlais, P. R. (2024, April). Assessing Candidate Proteins Identified by Quantitative Proteomics For a Role in Insulin-Stimulated Glucose Uptake. UA W.A. Franke Honors College Pinnacle Annual Poster Session.
• Grijalva, A., Benson, D., Fink, J., Zhang, X., Coletta, D. K., Langlais, P. R., Pandey, A., Mangiola, A., Koevary, J., Lancaster, J., & Goldman, S. (2023, November). Immune Modulation to Treat Ischemic Heart Failure. American Heart Association Annual Meeting.
• McGraw, M. B., Nash, J. C., Rojas, N. G., Amaro, A., Gackle, M. E., Goodmanson, M. J., Zelms, K. R., Moberly, A. P., & Langlais, P. R. (2023, April). New Components of the Insulin Signaling Pathway: Investigating the Role of Novel Proteins in GLUT4 Translocation. UA W.A. Franke Honors College Pinnacle Annual Poster Session.
• Nash, J. C., McGraw, M. B., Rojas, N. G., Amaro, A., Gackle, M. E., Goodmanson, M. J., Zelms, K. R., Moberly, A. P., & Langlais, P. R. (2023, January). Increased Adipocyte Differentiation Potentially Leading to Increased Expression of Proteins Involved in Glucose Transporter 4 Translocation. Undergraduate Biology Research Program.
• Odeneye, R. E., Lipinski, A. A., Coletta, D. K., & Langlais, P. R. (2023, August). Proteomic Profiling of Differentiated Myotubes Identifies Protein Candidates Potentially Involved in Insulin-Stimulated Glucose Uptake. Undergraduate Biology Research Program.
• Rojas, N. G., McGraw, M. B., Nash, J. C., Goodmanson, M. J., Amaro, A., Zelms, K. R., Gackle, M. E., Moberly, A. P., & Langlais, P. R. (2023, April). Proteomic Profiling of Mature White Adipocytes Identifies Protein Candidates Potentially Involved in Insulin-Stimulated Glucose Uptake. UA W.A. Franke Honors College Pinnacle Annual Poster Session.
• Hoyer-Kimura, C., Konhilas, J. P., Langlais, P. R., Pier, M., Salcedo, V., & Fricks, J. (2022, October). Estrogen Status in Menopause and Sex Differences  within the Gut Promote Functionally Distinct Changes. Arizona Physiological Society.
• Iannuzo, N., Langlais, P. R., Guerra, S., & Ledford, J. (2022, June). CC16 DEFICIENCY IMPACTS PULMONARY EPITHELIAL-DRIVEN RESPONSES DURING MYCOPLASMA PNEUMONIAE INFECTION.. Aspen Lung Conference.
• Miller, K., Liu, X., McSwain, M., Jaurgui, E., Langlais, P. R., & Craig, Z. R. (2022, June). Effects of a Phthalate Mixture on Hormone and Ovarian Antral Follicle Protein Abundance  . Gordon Research Conference - Environmental Endocrine Disruptors.
• Moberly, A. P., Krantz, J., Roman, M., Batty, S. R., Surber, E., Lee, N. Y., & Langlais, P. R. (2022, January). The Role of G2L1 in Microtubule and Actin-based Insulin-Stimulated Delivery of Glucose Transporter 4 to the Plasma Membrane. Undergraduate Biology Research Program.
• Victor, R. A., Lipinski, A. A., Langlais, P. R., & Ledford, J. (2022, June). Identifying novel phase separation proteins in E. coli and human cells using SEC-MS. RNA Society Meeting.
• Akram, A., Iannuzo, N., Langlais, P. R., & Ledford, J. (2021, July). CC16 Deficiency in Context of Early-life Infections and Epithelial-driven Responses. KEYS Conference.
• Batty, S. R., Roman, M., McCrary, D., Moberly, A. P., Lipinski, A. A., Barker, N. K., Krantz, J., & Langlais, P. R. (2021, January). Identification of a Role for G2L1 in Insulin Action. Undergraduate Biology Research Program.
• Moberly, A. P., McCrary, D., Lipinski, A. A., Roman, M., Batty, S. R., Kwak, E., Lee, N. Y., & Langlais, P. R. (2021, January). Isolation of Microtubules and Microtubule Associated Proteins through Subcellular Fractionation. Undergraduate Biology Research Program.
• Vizcarra, V. S., Barber, K. R., Franca-Solomon, G., Smith-Flint, A., Majuta, L., Langlais, P. R., Milnes, T. M., Vanderah, T. W., & Riegel, A. C. (2021, November). Targeted manipulation of PFC 5HT2A receptors and KV7 channels attenuates chronic neuropathic pain in rats.. Society for Neuroscience.
• Gabriel, K., Duron, D., Barker, N. K., Langlais, P. R., & Streicher, J. M. (2020, April/Spring). Does DUSP15 promote activation of ERK MAPK signaling after Hsp90 inhibition in the spinal cord to promote opioid anti-nociception?. Experimental Biology.
• Hurtado, K., Keresztes, A., Barker, N. K., Langlais, P. R., & Streicher, J. M. (2020, April/Spring). Does the Mu-Delta Opioid Receptor Heterodimer Repress Akt Kinase to Reduce Opioid Anti-Nociception?. Experimental Biology.
• James, J., Varghese, M. V., Niihori, M., Zemskova, M., Langlais, P. R., Rafikova, O., & Rafikov, R. (2020, May/Summer). The NFU1 G206C mutation metabolically reprograms pulmonary artery smooth muscle cells, promotes proliferation and apoptosis resistance. American Thoracic Society.
• Kraft, M., Francisco, D., Childress, B., Kimura, H., & Langlais, P. R. (2020, May/Summer). Significant Differences in Airway Epithelial Cell Phosphorylated Proteins are Present in Unstimulated Asthma Compared to Cells from Non-asthmatic Participants. American Thoracic Society.
• Pendleton, A. L., Davis, M. A., Kelly, A. C., Camacho, L. E., Anderson, M. J., Langlais, P. R., & Limesand, S. W. (2020, March/Spring). Decreased Mitochondrial Complex I Activity in Skeletal Muscle of Growth Restricted Ovine Fetuses with Placental Insufficiency. Society for Reproductive Investigation 67th Annual Scientific Meeting.
• Sieffert, M. M., Keresztes, A., Barker, N. K., Langlais, P. R., & Streicher, J. M. (2020, April/Spring). Investigation of the Signaling Mechanisms of the Mu-Delta Opioid Receptor Heterodimer. Experimental Biology.
• Zapata Bustos, R., Langlais, P. R., Coletta, D. K., De Filippis, E. A., Grandjean, D. N., & Mandarino, L. J. (2020, June/Summer). Lower Response of Connective Tissue Growth Factor (CTGF) to Exercise Characterizes Insulin Resistant Muscle. American Diabetes Association 80th scientific sessions. Chicago, IL: American Diabetes Association.
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  Rocio Zapata-Bustos, Paul Langlais, Dawn Coletta, Elena A. De Filippis, Danielle Grandjean, Lawrence J. MandarinoLower Response of Connective Tissue Growth Factor (CTGF) to Exercise Characterizes Insulin Resistant Muscle
• Barker, N. K., Krantz, J. L., & Langlais, P. R. (2019, June/Summer). Characterization of Insulin-Regulated CLASP2 Phosphorylation. American Diabetes Association.
• Dy, A. B., Barker, N. K., Langlais, P. R., & Ledford, J. (2019, Sept/Fall). Identification and functional testing of a novel receptor for SP-A on eosinophils.. 2019 European Respiratory Society Congress.
• Dy, A. B., Langlais, P. R., & Ledford, J. (2019, August/Summer). Identification and functional testing of a novel receptor for SP-A on eosinophils.. Gordon Conference – Lung Development, Injury and Repair.
• James, J., Zemskova, M., Eccles, C. A., Varghese, M. V., Niihori, M., Barker, N. K., Langlais, P. R., Rafikova, O., & Rafikov, R. (2019, Novermber/Fall). A single mutation in NFU1 metabolically reprograms pulmonary artery smooth muscle cells and drives proliferation with apoptosis resistance. The Society for Redox Biology and Medicine's 26th Annual Conference.
• Keresztes, A., Olson, K., Hguyen, P., Barker, N. K., Liu, Z., Langlais, P. R., Hruby, V., & Streicher, J. M. (2019, July/Summer). The Mu-Delta Opioid Receptor Heterodimer Acts as a Negative Feedback Brake to Reduce Opioid Anti-Nociception by Repression of CaMKII and Src Signaling. International Narcotics Research Conference - 50TH ANNIVERSARY MEETING.
• Keresztes, A., Olson, K., Hguyen, P., Barker, N. K., Liu, Z., Langlais, P. R., Hruby, V., & Streicher, J. M. (2019, May/Summer). Mu-Delta Opioid Receptor Heterodimer Antagonists: Novel Ligands that Enhance Opioid Analgesia while Reducing Opioid Withdrawal. 2019 NIH Pain Consortium Symposium - Pain Across the Lifespan.
• Langlais, P. R., Parker, S. S., Krantz, J., Kwak, E., Barker, N. K., Deer, C. G., Lee, N. Y., & Mouneimne, G. (2019, June/Summer). What’s on the Tube? Microtubule-Associated Proteins, Microtubule Stabilization, and Insulin Action.. Federation of American Societies For Experimental Biology - The Regulation of Glucose Metabolism Conference.
• Pandey, R., Zhou, M., Islam, S., Chen, B., Langlais, P. R., Srivastava, A., Cooke, L. S., Weterings, E., Von Hoff, D., & Mahadevan, D. (2019, March/Spring). Targeting carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) reshapes the tumor-stroma in pancreatic ductal adenocarcinoma (PDA). American Association for Cancer Research Annual Meeting 2019.
• Tabsh, K. K., Barker, N. K., Langlais, P. R., & Hill, M. G. (2019, March/Spring). Proteomic profile of maternal plasma in women with intrahepatic cholestasis of pregnancy.. Society for Reproductive Investigation 66th Annual Scientific Meeting.
• Uhlorn, J. A., Husband, N. A., Romero-Aleshire, M. J., Moffett, C. K., Kelly, A. C., Langlais, P. R., Nikolich-Zugich, J., & Brooks, H. L. (2019, September/Fall). Transcriptomic and Proteomic Analysis of CD4+ T Cells to Identify Sex Differences in Angiotensin II Signaling Pathways.. American Heart Association – Hypertension 2019 Scientific Sessions.
• Haider, N., Langlais, P. R., Moly, P. K., Barker, N. K., & Zhou, W. (2018, May). Resistin induced Differential Global Protein Expression in Human Macrophage. Vascular Discovery 2018 Scientific Sessions.
• Husband, N. A., Moffett, C., Romero-Aleshire, M. J., Uhlorn, J. A., Uhrlaub, J., Nunez, F., Nikolich-Zugich, J., Langlais, P. R., & Brooks, H. L. (2018, April). Angiotensin II-Induced Hypertension in VCD-Treated Menopausal Female Mice Elicits Significant Changes to the Splenic CD4+ Cell Proteome. Experimental Biology.
• Lopez-Pier, M., Cannon, D., Langlais, P. R., & Konhilas, J. P. (2018, October/Fall). AMP activated Protein Kinase and Estrogen-depednent mechanisms underlying increased susceptibility to CVD during Menopause. University of Arizona Biomedical Engineering Student Poster Session.
• Rafikova, O., Mandarino, L. J., Zemskov, E., Langlais, P. R., Desai, A., Srivastava, A., & Rafikov, R. (2018, May). Free heme-mediated endothelial barrier dysfunction contributes to the development of pulmonary hypertension. American Thoracic Society.
  More info

  Our data indicate that PH patients have increased levels of free Hb in plasma that correlate with disease severity and progression. There is also a significant accumulation of free Hb and depletion of haptoglobin in the sugen/hypoxia rat model. In rats, perivascular edema was observed during first two weeks of PH concomitant with increased infiltration of inflammatory cells. In the cell culture model of HLMVECs, we found that not hemoglobin but free heme-induced endothelial permeability via activation of the p38/HSP27 signaling pathway. Indeed, the rat model also exhibited an increased activation of p38/HSP27 during the initial phase of PH. Surprisingly, despite the increased levels of hemolysis and heme-mediated signaling; there was no heme oxygenase-1 activation. This can be explained by observed destabilization of HIF1a during the first two weeks of PAH regardless of hypoxic conditions. We found that heme-mediated effects on endothelium, at least in part, depend on Heme Carrier Protein 1 (HCP-1) and pharmacological inhibition of HCP-1 by sulfasalazine reduced barrier disruptive potential of the heme. Sulfasalazine administration to sugen/hypoxia rats results in attenuation of PH by a reduction in vascular remodeling in the lungs as well as decreasing right heart hypertrophy.
• Rafikova, O., Rafikov, R., Langlais, P. R., & McBride, M. L. (2018, May). Receptor for Advanced Glycation Endproducts (RAGE) regulates metabolic reprogramming induced over-proliferation in pulmonary hypertension. American Thoracic Society.
  More info

  Our data indicate that PH patients have increased levels of free Hb in plasma that correlate with disease severity and progression. There is also a significant accumulation of free Hb and depletion of haptoglobin in the sugen/hypoxia rat model. In rats, perivascular edema was observed during first two weeks of PH concomitant with increased infiltration of inflammatory cells. In the cell culture model of HLMVECs, we found that not hemoglobin but free heme-induced endothelial permeability via activation of the p38/HSP27 signaling pathway. Indeed, the rat model also exhibited an increased activation of p38/HSP27 during the initial phase of PH. Surprisingly, despite the increased levels of hemolysis and heme-mediated signaling; there was no heme oxygenase-1 activation. This can be explained by observed destabilization of HIF1a during the first two weeks of PAH regardless of hypoxic conditions. We found that heme-mediated effects on endothelium, at least in part, depend on Heme Carrier Protein 1 (HCP-1) and pharmacological inhibition of HCP-1 by sulfasalazine reduced barrier disruptive potential of the heme. Sulfasalazine administration to sugen/hypoxia rats results in attenuation of PH by a reduction in vascular remodeling in the lungs as well as decreasing right heart hypertrophy.
• Satyal, M. K., Bowser, S., McMillan, R. P., Davy, B. M., Langlais, P. R., Helm, R. F., Davy, K. P., & Hulver, M. W. (2018, November/Fall). Short-Term High-Fat Diet Alters Human Skeletal Muscle ERK 1/2 Phosphorylation. The Obesity Society @ Obesity Week.
• Satyal, M. K., Krantz, J., Parker, S., & Langlais, P. R. (2018, September/Fall). Human skeletal muscle phosphoproteome response to high-fat feeding.. American Physiological Societey - Maryland.
• Vasilyev, M., Langlais, P. R., Rafikova, O., & Rafikov, R. (2018, Novermber/Fall). Inhibition of respiratory chain Complex III irreversibly changes mitochondria proteomic landscape. The Society for Redox Biology and Medicine's 25th Annual Conference.
• Barker, N., Krantz, J., & Langlais, P. R. (2017, November). Targeted Quantitative Phosphoproteomics Discovers a Link between the Cytoskeleton-Associated Proteins SOGA1, G2L1, AGAP3, EB1, CLIP2, & MARK2 and the Insulin Signaling Pathway. UA College of Medicine Junior Investigators Symposium.
• Katsanos, C. S., Kras, K. A., Langlais, P. R., Willis, W. T., De Filippis, E. A., & Roust, L. R. (2017, June). Effects of Obesity on Skeletal Muscle Biokgical Pathways Associated with Subsarcolemal Versus Intermyofibrillar Mitochondria Revealed by Proteomic Analysis. American Diabetes Association Annual Meeting.
• Langlais, P. R., & Mandarino, L. J. (2017, November). UA Department of Medicine Quantitative Proteomics Lab. UA College of Medicine Junior Investigators Symposium.
• Langlais, P. R., & Mandarino, L. J. (2017, October). UA Department of Medicine Quantitative Proteomics Lab. UA College of Medicine Innovation Symposium.
• McBride, M. L., Williams, E. R., Langlais, P. R., Mandarino, L. J., Rafikov, R., & Rafikova, O. (2017, June). Inositol Monophosphate 1 (IMPA1) And Rage Interaction: The Role Of Novel Proliferative Pathway In Pulmonary Hypertension. American Heart Association.
• Rafikov, R., Srivastava, A., Desai, A., Langlais, P. R., Zemskov, E., Mandarino, L. J., & Rafikova, O. (2017, June). Hemolysis-mediated vascular permeability in lungs contributes to the development of pulmonary hypertension. American Heart Association.
  More info

  Our data indicate that PH patients have increased levels of free Hb in plasma that correlate with disease severity and progression. There is also a significant accumulation of free Hb and depletion of haptoglobin in the sugen/hypoxia rat model. In rats, perivascular edema was observed during first two weeks of PH concomitant with increased infiltration of inflammatory cells. In the cell culture model of HLMVECs, we found that not hemoglobin but free heme-induced endothelial permeability via activation of the p38/HSP27 signaling pathway. Indeed, the rat model also exhibited an increased activation of p38/HSP27 during the initial phase of PH. Surprisingly, despite the increased levels of hemolysis and heme-mediated signaling; there was no heme oxygenase-1 activation. This can be explained by observed destabilization of HIF1a during the first two weeks of PAH regardless of hypoxic conditions. We found that heme-mediated effects on endothelium, at least in part, depend on Heme Carrier Protein 1 (HCP-1) and pharmacological inhibition of HCP-1 by sulfasalazine reduced barrier disruptive potential of the heme. Sulfasalazine administration to sugen/hypoxia rats results in attenuation of PH by a reduction in vascular remodeling in the lungs as well as decreasing right heart hypertrophy.
• Zapata Bustos, R., Finlayson, J., Langlais, P. R., De Filippis, E. A., & Mandarino, L. J. (2017, November). Global acetylome analysis of muscle reveals differentially lysine acetylated sites in obesity.. Junior Investigator Poster Forum. Tucson, Arizona.: College of Medicine, University of Arizona.
  More info

  Zapata-Bustos R, Finlayson J, Langlais P, De Filippis EA, Mandarino LJ. Global acetylome analysis of muscle reveals differentially lysine acetylated sites in obesity. Junior Investigator Poster Forum, November 17, 2017. Tucson, Arizona.
• Sangam, S., Gupta, A., Yilmaz, S., Yuan, J. X., Langlais, P. R., & Desai, A. A. (2018, May). Endothelial Targets of Sox17 in Pulmonary Arterial Hypertension: A Quantitative Proteomic Approach. American Thoracic Society.

Other Teaching Materials

• Langlais, P. R. (2018. 7 Lessons to Keep in Mind If You Are Considering a Career as an Academic in Discovery-based Research. University of Texas Health Science Center at San Antonio.