Moulun Luo

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• Corenblum, M. J., McRobbie-Johnson, A., Carruth, E., Bernard, K., Luo, M., Mandarino, L. J., Peterson, S., Sans-Fuentes, M. A., Billheimer, D., Maley, T., Eggers, E. D., & Madhavan, L. (2023). Parallel neurodegenerative phenotypes in sporadic Parkinson's disease fibroblasts and midbrain dopamine neurons. Progress in neurobiology, 229, 102501.
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  Understanding the mechanisms causing Parkinson's disease (PD) is vital to the development of much needed early diagnostics and therapeutics for this debilitating condition. Here, we report cellular and molecular alterations in skin fibroblasts of late-onset sporadic PD subjects, that were recapitulated in matched induced pluripotent stem cell (iPSC)-derived midbrain dopamine (DA) neurons, reprogrammed from the same fibroblasts. Specific changes in growth, morphology, reactive oxygen species levels, mitochondrial function, and autophagy, were seen in both the PD fibroblasts and DA neurons, as compared to their respective controls. Additionally, significant alterations in alpha synuclein expression and electrical activity were also noted in the PD DA neurons. Interestingly, although the fibroblast and neuronal phenotypes were similar to each other, they differed in their nature and scale. Furthermore, statistical analysis revealed potential novel associations between various clinical measures of the PD subjects and the different fibroblast and neuronal data. In essence, these findings encapsulate spontaneous, in-tandem, disease-related phenotypes in both sporadic PD fibroblasts and iPSC-based DA neurons, from the same patient, and generates an innovative model to investigate PD mechanisms with a view towards rational disease stratification and precision treatments.
• Luo, M., & Santulli, G. (2023). Editorial: The link between obesity, type 2 diabetes, and mitochondria. Frontiers in endocrinology, 14, 1229935.
• Barakati, N., Zapata Bustos, R., Coletta, D. K., Langlais, P. R., Kohler, L. N., Luo, M., Funk, J. L., Willis, W. T., & Mandarino, L. J. (2022). Fuel Selection in Skeletal Muscle Exercising at Low Intensity; Reliance on Carbohydrate in Very Sedentary Individuals. Metabolic syndrome and related disorders.
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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 ( 
• Finlayson, J., Barakati, N., Langlais, P. R., Funk, J., 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.
• Luo, M., Ma, W., Zapata-Bustos, R., Willis, W. T., & Mandarino, L. J. (2021). Deletion of Von Willebrand A Domain Containing Protein (VWA8) raises activity of mitochondrial electron transport chain complexes in hepatocytes. Biochemistry and biophysics reports, 26, 100928.
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  VWA8 (Von Willebrand A Domain Containing Protein 8) is a AAA+ ATPase that is localized to the mitochondrial matrix and is widely expressed in highly energetic tissues. Originally found to be higher in abundance in livers of mice fed a high fat diet, deletion of the VWA8 gene in differentiated mouse AML12 hepatocytes unexpectedly produced a phenotype of higher mitochondrial and nonmitochondrial oxidative metabolism, higher ROS (reactive oxygen species) production mainly from NADPH oxidases, and increased HNF4a expression. The purposes of this study were first, to determine whether higher mitochondrial oxidative capacity in VWA8 null hepatocytes is the product of higher capacity in all aspects of the electron transport chain and oxidative phosphorylation, and second, the density of cristae in mitochondria and mitochondrial content was measured to determine if higher mitochondrial oxidative capacity is accompanied by greater cristae area and mitochondrial abundance. Electron transport chain complexes I, II, III, and IV activities all were higher in hepatocytes in which the VWA8 gene had been deleted using CRISPR/Cas9. A comparison of abundance of proteins in electron transport chain complexes I, III and ATP synthase previously determined using an unbiased proteomics approach in hepatocytes in which VWA8 had been deleted showed agreement with the activity assays. Mitochondrial cristae, the site where electron transport chain complexes are located, were quantified using electron microscopy and stereology. Cristae density, per mitochondrial area, was almost two-fold higher in the VWA8 null cells (P 
• Zapata-Bustos, R., Finlayson, J., Langlais, P. R., Coletta, D. K., Luo, M., Grandjean, D., De Filippis, E. A., & Mandarino, L. (2021). Altered Transcription Factor Expression Responses to Exercise in Insulin Resistance. Frontiers in physiology, 12, 649461.
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  Insulin resistant muscle is resistant to gene expression changes induced by acute exercise. This study was undertaken to identify transcription factors that differentially respond to exercise in insulin resistance. Candidate transcription factors were identified from analysis of 5'-untranslated regions (5'-UTRs) of exercise responsive genes and from analysis of the 5'-UTRs of genes coding for proteins that differ in abundance in insulin resistance.
• 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. (2020). Single Mutation in the NFU1 Gene Metabolically Reprograms Pulmonary Artery Smooth Muscle Cells. Arteriosclerosis, thrombosis, and vascular biology, ATVBAHA120314655.
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  NFU1 is a mitochondrial iron-sulfur scaffold protein, involved in iron-sulfur assembly and transfer to complex II and LAS (lipoic acid synthase). Patients with the point mutation NFU1 and CRISPR/CAS9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-generated rats develop mitochondrial dysfunction leading to pulmonary arterial hypertension. However, the mechanistic understanding of pulmonary vascular proliferation due to a single mutation in NFU1 remains unresolved. Approach and Results: Quantitative proteomics of isolated mitochondria showed the entire phenotypic transformation of NFU1 rats with a disturbed mitochondrial proteomic landscape, involving significant changes in the expression of 208 mitochondrial proteins. The NFU1 mutation deranged the expression pattern of electron transport proteins, resulting in a significant decrease in mitochondrial respiration. Reduced reliance on mitochondrial respiration amplified glycolysis in pulmonary artery smooth muscle cell (PASMC) and activated GPD (glycerol-3-phosphate dehydrogenase), linking glycolysis to oxidative phosphorylation and lipid metabolism. Decreased PDH (pyruvate dehydrogenase) activity due to the lipoic acid shortage is compensated by increased fatty acid metabolism and oxidation. PASMC became dependent on extracellular fatty acid sources due to upregulated transporters such as CD36 and CPT (carnitine palmitoyltransferase)-1. Finally, the NFU1 mutation produced a dysregulated antioxidant system in the mitochondria, leading to increased reactive oxygen species levels. PASMC from NFU1 rats showed apoptosis resistance, increased anaplerosis, and attained a highly proliferative phenotype. Attenuation of mitochondrial reactive oxygen species by mitochondrial-targeted antioxidant significantly decreased PASMC proliferation.
• Luo, M., Ma, W., Sand, Z., Finlayson, J., Wang, T., Brinton, R. D., Willis, W. T., & Mandarino, L. J. (2020). Von Willebrand factor A domain-containing protein 8 (VWA8) localizes to the matrix side of the inner mitochondrial membrane. Biochemical and biophysical research communications, 521(1):158-163(158-163), 5. doi:10.1016/j.bbrc.2019.10.095
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  VWA8 is a poorly characterized mitochondrial AAA + ATPase protein. The specific submitochondrial localization of VWA8 remains unclear. The purpose of this study was to determine the specific submitochondrial compartment within which VWA8 resides in order to provide more insight into the function of this protein. Bioinformatics analysis showed that VWA8 has a 34 amino acid N-terminal Matrix-Targeting Signal (MTS) that is similar to those in proteins known to localize to the mitochondrial matrix. Experiments in C2C12 mouse myoblasts using confocal microscopy showed that deletion of the VWA8 MTS (vMTS) resulted in cytosolic, rather than mitochondrial, localization of VWA8. Biochemical analysis using differential sub-fractionation of mitochondria isolated from rat liver showed that VWA8 localizes to the matrix side of inner mitochondrial membrane, similar to the inner mitochondrial membrane protein Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETFDH). The results of these experiments show that the vMTS is essential for localization to the mitochondrial matrix and that once there, VWA8 localizes to the matrix side of inner mitochondrial membrane.
• 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.
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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., Barker, N. K., Langlais, P., Srivastava, A., Luo, M., Cooke, L. S., Weterings, E., & 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, 9(1), 18347.
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  We investigated biomarker CEACAM6, a highly abundant cell surface adhesion receptor that modulates the extracellular matrix (ECM) in pancreatic ductal adenocarcinoma (PDA). The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) RNA-Seq data from PDA patients were analyzed for CEACAM6 expression and evaluated for overall survival, association, enrichment and correlations. A CRISPR/Cas9 Knockout (KO) of CEACAM6 in PDA cell line for quantitative proteomics, mitochondrial bioenergetics and tumor growth in mice were conducted. We found CEACAM6 is over-expressed in primary and metastatic basal and classical PDA subtypes. Highest levels are in classical activated stroma subtype. CEACAM6 over-expression is universally a poor prognostic marker in KRAS mutant and wild type PDA. High CEACAM6 expression is associated with low cytolytic T-cell activity in both basal and classical PDA subtypes and correlates with low levels of T- markers. In HPAF-II cells knockout of CEACAM6 alters ECM-cell adhesion, catabolism, immune environment, transmembrane transport and autophagy. CEACAM6 loss increases mitochondrial basal and maximal respiratory capacity. HPAF-II CEACAM6-/- cells are growth suppressed by >65% vs. wild type in mice bearing tumors. CEACAM6, a key regulator affects several hallmarks of PDA including the fibrotic reaction, immune regulation, energy metabolism and is a novel therapeutic target in PDA.
• Kruse, R., Krantz, J., Barker, N., Coletta, R., Rafikov, R., Luo, M., Hoejlund, K., Mandarino, L. J., & Langlais, P. R. (2017). The CLASP2 Protein Interaction Network in Adipocytes Links CLIP2 to AGAP3, CLASP2 to G2L1, MARK2, and SOGA1, and Identifies SOGA1 as a Microtubule-Associated Protein. Molecular & cellular proteomics : MCP.
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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.
• Luo, M., Mengos, A. E., Ma, W., Finlayson, J., Bustos, R. Z., Xiao Zhu, Y., Shi, C. X., Stubblefield, T. M., Willis, W. T., & Mandarino, L. J. (2017). Characterization of the novel protein KIAA0564 (Von Willebrand Domain-containing Protein 8). Biochemical and biophysical research communications, 487(3), 545-551.
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  The VWA8 gene was first identified by the Kazusa cDNA project and named KIAA0564. Based on the observation, by similarity, that the protein encoded by KIAA0564 contains a Von Willebrand Factor 8 domain, KIAA0564 was named Von Willebrand Domain-containing Protein 8 (VWA8). The function of VWA8 protein is almost unknown. The purpose of this study was to characterize the tissue distribution, cellular location, and function of VWA8. In mice VWA8 protein was mostly distributed in liver, kidney, heart, pancreas and skeletal muscle, and is present as a long isoform and a shorter splice variant (VWA8a and VWA8b). VWA8 protein and mRNA were elevated in mouse liver in response to high fat feeding. Sequence analysis suggests that VWA8 has a mitochondrial targeting sequence and domains responsible for ATPase activity. VWA8 protein was targeted exclusively to mitochondria in mouse AML12 liver cells, and this was prevented by deletion of the targeting sequence. Moreover, the VWA8 short isoform overexpressed in insect cells using a baculovirus construct had in vitro ATPase activity. Deletion of the Walker A motif or Walker B motif in VWA8 mostly blocked ATPase activity, suggesting Walker A motif or Walker B motif are essential to the ATPase activity of VWA8. Finally, homology modeling suggested that VWA8 may have a structure most confidently similar to dynein motor proteins.
• Shi, C. X., Kortüm, K. M., Zhu, Y. X., Bruins, L. A., Jedlowski, P., Votruba, P. G., Luo, M., Stewart, R. A., Ahmann, J., Braggio, E., & Stewart, A. K. (2017). CRISPR Genome-Wide Screening Identifies Dependence on the Proteasome Subunit PSMC6 for Bortezomib Sensitivity in Multiple Myeloma. Molecular cancer therapeutics, 16(12), 2862-2870.
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  Bortezomib is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. Consequently, we employed CRISPR targeting 19,052 human genes to identify unbiased targets that contribute to bortezomib resistance. Specifically, we engineered an RPMI8226 multiple myeloma cell line to express Cas9 infected by lentiviral vector CRISPR library and cultured derived cells in doses of bortezomib lethal to parental cells. Sequencing was performed on surviving cells to identify inactivated genes responsible for drug resistance. From two independent whole-genome screens, we selected 31 candidate genes and constructed a second CRISPR sgRNA library, specifically targeting each of these 31 genes with four sgRNAs. After secondary screening for bortezomib resistance, the top 20 "resistance" genes were selected for individual validation. Of these 20 targets, the proteasome regulatory subunit PSMC6 was the only gene validated to reproducibly confer bortezomib resistance. We confirmed that inhibition of chymotrypsin-like proteasome activity by bortezomib was significantly reduced in cells lacking PSMC6. We individually investigated other members of the PSMC group (PSMC1 to 5) and found that deficiency in each of those subunits also imparts bortezomib resistance. We found 36 mutations in 19S proteasome subunits out of 895 patients in the IA10 release of the CoMMpass study (https://themmrf.org). Our findings demonstrate that the PSMC6 subunit is the most prominent target required for bortezomib sensitivity in multiple myeloma cells and should be examined in drug-refractory populations. .
• Zhu, Y. X., Shi, C. X., Bruins, L. A., Jedlowski, P., Wang, X., Kortüm, K. M., Luo, M., Ahmann, J. M., Braggio, E., & Stewart, A. K. (2017). Loss of Promotes Cell Survival in Myeloma. Cancer research, 77(16), 4317-4327.
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  is one of the most recurrently mutated genes in multiple myeloma; however its role in disease pathogenesis has not been determined. Here we demonstrate that wild-type (WT) overexpression induces substantial cytotoxicity in multiple myeloma cells. In contrast, mutations found in multiple myeloma patients abrogate this cytotoxicity, indicating a survival advantage conferred by the mutant phenotype. WT FAM46C overexpression downregulated , and and upregulated immunoglobulin (Ig) light chain and / Furthermore, pathway analysis suggests that enforced expression activated the unfolded protein response pathway and induced mitochondrial dysfunction. CRISPR-mediated depletion of endogenous enhanced multiple myeloma cell growth, decreased Ig light chain and HSPA5/BIP expression, activated ERK and antiapoptotic signaling, and conferred relative resistance to dexamethasone and lenalidomide treatments. Genes altered in -depleted cells were enriched for signaling pathways regulating estrogen, glucocorticoid, B-cell receptor signaling, and ATM signaling. Together these results implicate FAM46C in myeloma cell growth and survival and identify mutation as a contributor to myeloma pathogenesis and disease progression via perturbation in plasma cell differentiation and endoplasmic reticulum homeostasis. .
• Luo, M., Mengos, A. E., Mandarino, L. J., & Sekulic, A. (2016). Association of liprin β-1 with kank proteins in melanoma. Experimental dermatology, 25(4), 321-3.
• Xu, Q., Hou, Y. X., Langlais, P., Erickson, P., Zhu, J., Shi, C. X., Luo, M., Zhu, Y., Xu, Y., Mandarino, L. J., Stewart, K., & Chang, X. B. (2016). Expression of the cereblon binding protein argonaute 2 plays an important role for multiple myeloma cell growth and survival. BMC cancer, 16, 297.
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  Immunomodulatory drugs (IMiDs), such as lenalidomide, are therapeutically active compounds that bind and modulate the E3 ubiquitin ligase substrate recruiter cereblon, thereby affect steady-state levels of cereblon and cereblon binding partners, such as ikaros and aiolos, and induce many cellular responses, including cytotoxicity to multiple myeloma (MM) cells. Nevertheless, it takes many days for MM cells to die after IMiD induced depletion of ikaros and aiolos and thus we searched for other cereblon binding partners that participate in IMiD cytotoxicity.
• Langlais, P., Langlais, P., Zhu, Y., Zhu, Y., Zhu, Y., Zhu, Y., Zhu, J., Zhu, J., Xu, Q., Xu, Q., Stewart, K., Stewart, K., Shi, C., Shi, C., Mandarino, L. J., Mandarino, L. J., Luo, M., Luo, M., Langlais, P. R., , Langlais, P. R., et al. (2015). Abstract 3625: Expression of CRBN binding protein AGO2 plays important roles for myeloma cell growth. Cancer Research, 75, 3625-3625. doi:10.1158/1538-7445.am2015-3625
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  Immunomodulatory drugs (IMiDs) are therapeutically active compounds that bind to the E3 ubiquitin ligase substrate recruiter cereblon (CRBN) and induce many cellular responses, including cytotoxicity. We had found that the expression of CRBN in multiple myeloma (MM) cells provided the basis for their responses to IMiDs. In this report, we have identified argonaute 2 (AGO2) as a new member of CRBN-downstream binding protein and found that: 1) the steady-state levels of CRBN in IMiD-sensitive cells are significantly higher than in IMiD-resistant cells; 2) the steady-state levels of AGO2 in IMiD-sensitive cells are significantly less compared to IMiD-resistant cells, suggesting that CRBN plays a role in regulating AGO2. AGO2 is a component of microRNA (miRNA)-induced silencing complexes and plays an important role in miRNA maturation and function. The matured miRNAs are considered as key regulators of almost all cellular pathways. Treatment of IMiD-sensitive MM cells with lenalidomide, an IMiD, significantly increased CRBN, decreased both AGO2 and miRNAs and inhibited cell growth. In contrast, although treatment of IMiD-resistant MM cells with lenalidomide moderately increased CRBN and decreased both AGO2 and miRNAs, this treatment did not significantly inhibit cell growth. However, silencing of AGO2 with small hairpin RNA significantly decreased both AGO2 and miRNAs and inhibited cell growth regardless of whether the cells are sensitive or resistant to IMiDs. Therefore, AGO2 could be considered as a new drug target for overcoming IMiDs resistance in MM cells. Citation Format: Qinqin Xu, Yuexian Hou, Paul Langlais, Patrick Erickson, James Zhu, Changxin Shi, Moulun Luo, Yuanxiao Zhu, Lawrence Mandarino, Keith Stewart, Xiu-Bao Chang. Expression of CRBN binding protein AGO2 plays important roles for myeloma cell growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3625. doi:10.1158/1538-7445.AM2015-3625
• Zhu, Y. X., Votruba, P. G., Stewart, R. A., Stewart, A. K., Shi, C., Luo, M., Kortum, K. M., Jedlowski, P., Bruins, L. A., Braggio, E., & Ahmann, J. M. (2015). Crispr Sgrnas Genome-Wide Screen Identifies the Proteasome Regulatory Subunit PSMC6 As a Bortezomib Resistance Gene in Human Multiple Myeloma Cells. Blood, 126(23), 450-450. doi:10.1182/blood.v126.23.450.450
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  Background: The proteasome inhibitor Bortezomib (BTZ) is an efficient treatment option for both, newly diagnosed and relapsed/refractory multiple myeloma (MM). Despite the effectiveness, most patients eventually acquire drug resistance for reasons not fully understood Materials and methods: To better understand BTZ resistance mechanism we used a CRISPR library (GeCKO V2) targeting 19052 human genes, trying to identify genes responsible for BTZ resistance. CRISPR sgRNAs targeting the ERN1-XBP1 pathway were used as positive controls. We first infected RPMI 8226 myeloma cell line expressing Cas9 with the CRISPR library packaged into lenti-vectors and selected for resistance to BTZ. Surviving cells were subjected to next generation sequencing. Based on the initial screen results we constructed a second CRISPR sgRNA library including 31 genes, each gene targeted with four sgRNAs. After the second round screening and subsequent sequencing, we selected the top 20 genes for individual validation. Results: Proteasome regulatory gene PSMC6 was identified as the only reproducible gene conferring BTZ resistance. Interestingly, the same gene was independently found by a second group using a CRISPR approach (Sheffer M et al. ASH Abstract 273, 2014). Resistance was reproducible using a PSMC6 knockout by three individual CRISPR sgRNAs targeting exonic regions and one pair of SgRNA targeting intron region flanking exon for the deletion of exon one. PSMC6 knockout was verified by PCR and Sanger sequencing. Sensitivity to BTZ was rescued by over expression of PSMC6 cDNA in RPMI 8226 cells harboring a deletion of PSMC6 exon 1. MM cells lacking PSMC6 also developed resistance against Carfilzomib. Resistance was reproduced on a second MM cell line, KMS11. We did not see any significant difference of toxicity in PSMC6 deleted cells for other chemicals tested (tunicamycin, staurosporine, dexamethasone and melphalan). We demonstrated that the sensitivity of chymotrypsin-like activity of proteasome against BTZ was significantly reduced in cells lacking PSMC6. Consequently, MM cells without the PSMC6 gene were relatively resistant to apoptosis induced by BTZ, which was verified by Western blot for caspase 8 degradation and luminescent assay for caspase 3/7 activities. Clinically we could not correlate the PSMC6 expression level with the outcome of BTZ treatment in BTZ naive patients using publically available gene expression data. We initially used CRISPR sgRNAs targeting ER stress pathway (ERN1 and XBP1) as a positive control. However we could not derive any resistant cells from the experiment. We also could not identify any sgRNAs targeting the ERN1-XBP1 pathway from our whole exome screen and next generation sequencing. Since this contradicts published data, we decided to knockout ERN1 and XBP1 genes individually. Clones of cells with successful knockout of ERN1 in three cell lines (RPMI 8226, KMS11 and JJN3) and XBP1 in two cell lines (RPMI 8226 and KMS11) were tested for response to BTZ and Carfilzomib , however we did not find any drug response difference between the knockout and parent cells. We also found that the ERN1-XBP1 knockout cells did not show difference in response to ERN1 specific inhibitors (4u8C and STF-038010) and ER stress inducers (tunicamycin and thapsigargin) compared to parental cells. Conclusions: Human multiple myeloma cells lacking the PSMC6 gene develop significant resistance to apoptosis induced by BTZ. We have however not found a correlation of PSMC6 expression levels with outcome to BTZ treatment in BTZ naive patients. We are therefore currently investigating the PSMC6 mutation rate in relapsed MM patients after proteasome treatment. In contrast to previous reports showing that progenitor MM cells lacing XBP-1 or ERN-1 invoked BTZ resistance, we were not able to demonstrate a change in sensitivity after full CRISPR knock out of either ERN1 or XBP1. It has long been believed that ERN1-XBP1 pathway plays an important role for MM treatment, leading to the development of ERN1 specific inhibitors. However, we demonstrated that the toxicity of two ERN1-specific inhibitors appears independent of the ERN1-XBP1 pathway. We also demonstrated that the toxicity of two important ER stress inducers, tunicamycin and thapsigargin, is independent of the ERN1-XBP1 pathway. Disclosures Stewart:Celgene: Consultancy; Oncospire Inc.: Equity Ownership; BMS: Membership on an entity9s Board of Directors or advisory committees; Novartis: Consultancy.
• Zhu, Y. X., Braggio, E., Shi, C. X., Kortuem, K. M., Bruins, L. A., Schmidt, J. E., Chang, X. B., 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), 536-45.
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  Cereblon (CRBN) mediates immunomodulatory drug (IMiD) action in multiple myeloma (MM). Using 2 different methodologies, we identified 244 CRBN binding proteins and established relevance to MM biology by changes in their abundance after exposure to lenalidomide. Proteins most reproducibly binding CRBN (>fourfold vs controls) included DDB1, CUL4A, IKZF1, KPNA2, LTF, PFKL, PRKAR2A, RANGAP1, and SHMT2. After lenalidomide treatment, the abundance of 46 CRBN binding proteins decreased. We focused attention on 2 of these-IKZF1 and IKZF3. IZKF expression is similar across all MM stages or subtypes; however, IKZF1 is substantially lower in 3 of 5 IMiD-resistant MM cell lines. The cell line (FR4) with the lowest IKZF1 levels also harbors a damaging mutation and a translocation that upregulates IRF4, an IKZF target. Clinical relevance of CRBN-binding proteins was demonstrated in 44 refractory MM patients treated with pomalidomide and dexamethasone therapy in whom low IKZF1 gene expression predicted lack of response (0/11 responses in the lowest expression quartile). CRBN, IKZF1, and KPNA2 levels also correlate with significant differences in overall survival. Our study identifies CRBN-binding proteins and demonstrates that in addition to CRBN, IKZF1, and KPNA2, expression can predict survival outcomes.
• 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), 3342-50.
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  Protein phosphatase 1 (PP1) is one of the major phosphatases responsible for protein dephosphorylation in eukaryotes. So far, only few specific phosphorylation sites of PP1 regulatory subunit 12A (PPP1R12A) have been shown to regulate the PP1 activity. The effect of insulin on PPP1R12A phosphorylation is largely unknown. Utilizing a mass spectrometry based phosphorylation identification and quantification approach, we identified 21 PPP1R12A phosphorylation sites (7 novel sites, including Ser20, Thr22, Thr453, Ser478, Thr671, Ser678, and Ser680) and quantified 16 of them under basal and insulin stimulated conditions in hamster ovary cells overexpressing the insulin receptor (CHO/IR), an insulin sensitive cell model. Insulin stimulated the phosphorylation of PPP1R12A significantly at Ser477, Ser478, Ser507, Ser668, and Ser695, while simultaneously suppressing the phosphorylation of PPP1R12A at Ser509 (more than 2-fold increase or decrease compared to basal). Our data demonstrate that PPP1R12A undergoes insulin stimulated/suppressed phosphorylation, suggesting that PPP1R12A phosphorylation may play a role in insulin signal transduction. The novel PPP1R12A phosphorylation sites as well as the new insulin-responsive phosphorylation sites of PPP1R12A in CHO/IR cells provide targets for investigation of the regulation of PPP1R12A and the PPP1R12A-PP1cδ complex in insulin action and other signaling pathways in other cell models, animal models, and humans.
• Luo, M., Mengos, A. E., Stubblefield, T. M., & Mandarino, L. J. (2012). High Fat Diet-Induced Changes in Hepatic Protein Abundance in Mice. Journal of proteomics & bioinformatics, 5(3), 60-66.
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  Nonalcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance, type 2 diabetes, and dyslipidemia. The purpose of this study was to identify novel proteins and pathways that contribute to the pathogenesis and complications of NAFLD. C57BL/6J male mice were fed a 60% (HFD) or 10% (LFD) high or low fat diet. HFD induced obesity, hepatic steatosis and insulin resistance (euglycemic clamps, glucose infusion rate: LFD 50.5 ± 6.4 vs. HFD 14.2 ± 9.5 μg/ (g·min); = 12). Liver proteins were analyzed by mass spectrometry-based proteomics analysis. Numerous hepatic proteins were altered in abundance after 60% HFD feeding. Nine down-regulated and nine up-regulated proteins were selected from this list for detailed analysis based on the criteria of 1.5-fold difference, consistency across replicates, and having at least 2 spectra assigned. Proteins that decreased in abundance were acyl-coA desaturase-I (SCD-1), acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), pyruvate kinase isozymes R/L (PKLR), NADP-dependent malic enzyme (ME-1), ATP-citrate synthase (ACL), ketohexokinase (KHK), long-chain-fatty acid-CoA ligase-5 (ACSL-5) and carbamoyl-phosphate synthase-I (CPS-1). Those that increased were KIAA0564, apolipoprotein A-I (apoA-1), ornithine aminotransferase (OAT), multidrug resistance protein 2 (MRP-2), liver carboxylesterase-I (CES-1), aminopeptidase N (APN), fatty aldehyde dehydrogenase (FALDH), major urinary protein 2 (MUP-2) and KIAA0664. KIAA0564 and KIAA0664 proteins are uncharacterized and are novel proteins associated with NAFLD. The decreased abundance of normally highly abundant proteins like FAS and CPS-1 was confirmed by Coomassie Blue staining after bands were identified by MS/MS, and immunoblot analysis confirmed the increased abundance of KIAA0664 after 60% HFD feeding. In conclusion, this study shows NAFLD is characterized by changes in abundance of proteins related to cell injury, inflammation, and lipid metabolism. Two novel and uncharacterized proteins, KIAA0564 and KIAA0664, may provide insight into the pathogenesis of NAFLD induced by lipid oversupply.
• Zhang, X., Højlund, K., Luo, M., Meyer, C., Geetha, T., & Yi, Z. (2012). Novel tyrosine phosphorylation sites in rat skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MS. Journal of proteomics, 75(13), 4017-26.
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  Tyrosine phosphorylation plays a fundamental role in many cellular processes including differentiation, growth and insulin signaling. In insulin resistant muscle, aberrant tyrosine phosphorylation of several proteins has been detected. However, due to the low abundance of tyrosine phosphorylation (
• 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), e26171.
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  We sought to evaluate the reproducibility of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to measure the stable-isotope enrichment of in vivo-labeled muscle ATP synthase β subunit (β-F1-ATPase), a protein most directly involved in ATP production, and whose abundance is reduced under a variety of circumstances. Muscle was obtained from a rat infused with stable-isotope-labeled leucine. The muscle was homogenized, β-F1-ATPase immunoprecipitated, and the protein was resolved using 1D-SDS PAGE. Following trypsin digestion of the isolated protein, the resultant peptide mixtures were subjected to analysis by HPLC-ESI-MS/MS, which resulted in the detection of multiple β-F1-ATPase peptides. There were three β-F1-ATPase unique peptides with a leucine residue in the amino acid sequence, and which were detected with high intensity relative to other peptides and assigned with >95% probability to β-F1-ATPase. These peptides were specifically targeted for fragmentation to access their stable-isotope enrichment based on MS/MS peak areas calculated from extracted ion chromatographs for selected labeled and unlabeled fragment ions. Results showed best linearity (R(2) = 0.99) in the detection of MS/MS peak areas for both labeled and unlabeled fragment ions, over a wide range of amounts of injected protein, specifically for the β-F1-ATPase(134-143) peptide. Measured stable-isotope enrichment was highly reproducible for the β-F1-ATPase(134-143) peptide (CV = 2.9%). Further, using mixtures of synthetic labeled and unlabeled peptides we determined that there is an excellent linear relationship (R(2) = 0.99) between measured and predicted enrichment for percent enrichments ranging between 0.009% and 8.185% for the β-F1-ATPase(134-143) peptide. The described approach provides a reliable approach to measure the stable-isotope enrichment of in-vivo-labeled muscle β-F1-ATPase based on the determination of the enrichment of the β-F1-ATPase(134-143) peptide.
• 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(3), 457-66.
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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. HPLC-ESI-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.
• Langlais, P., Yi, Z., Finlayson, J., Luo, M., Mapes, R., De Filippis, E., Meyer, C., Plummer, E., Tongchinsub, P., Mattern, M., & Mandarino, L. J. (2011). Global IRS-1 phosphorylation analysis in insulin resistance. Diabetologia, 54(11), 2878-89.
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  IRS-1 serine phosphorylation is often elevated in insulin resistance models, but confirmation in vivo in humans is lacking. We therefore analysed IRS-1 phosphorylation in human muscle in vivo.
• 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.
• 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.
• Yi, Z., Luo, M., Mandarino, L. J., Reyna, S. M., Carroll, C. A., & Weintraub, S. T. (2006). Quantification of phosphorylation of insulin receptor substrate-1 by HPLC-ESI-MS/MS. Journal of the American Society for Mass Spectrometry, 17(4), 562-7.
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  Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) regulates the function and subsequent insulin signaling of this protein. Human IRS-1 has 1242 amino acid residues, including 182 serines and 60 threonines. The size, complexity, and relatively low abundance of this protein in biological samples make it difficult to map and quantify phosphorylation sites by conventional means. A mass spectrometry peak area based quantification approach has been developed and applied to assess the relative abundance of IRS-1 phosphorylation in the absence or presence of stimuli. In this method, the peak area for a phosphopeptide of interest is normalized against the average of peak areas for six selected representative IRS-1 peptides that serve as endogenous internal standards. Relative quantification of each phosphopeptide is then obtained by comparing the normalized peak area ratios for untreated and treated samples. Two non-IRS-1 peptides were added to each digest for use as HPLC retention time markers and additional standards as well as references to the relative quantity of IRS-1 in different samples. This approach does not require isotopic or chemical labeling and can be applied to various cell lines and tissues. Using this method, we assessed the relative changes in the quantities of two tryptic phosphopeptides isolated from human IRS-1 expressed in L6 cells incubated in the absence or presence of insulin or tumor necrosis factor-alpha. Substantial increases of phosphorylation were observed for Thr(446) upon stimulation. In contrast, no obvious change in the level of phosphorylation was observed for Ser(1078). This mass spectrometry based strategy provides a powerful means to quantify changes in the relative phosphorylation of peptides in response to various stimuli in a complex, low-abundance protein.
• 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.
• Yi, Z., Luo, M., Carroll, C. A., Weintraub, S. T., & Mandarino, L. J. (2005). Identification of phosphorylation sites in insulin receptor substrate-1 by hypothesis-driven high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Analytical chemistry, 77(17), 5693-9.
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  Serine phosphorylation of insulin receptor substrate-1 (IRS-1) can regulate tyrosine phosphorylation of IRS-1 and subsequent insulin signaling. The 182 serine and 60 threonine residues in IRS-1 make position-by-position analysis of potential phosphorylation sites by mutagenesis difficult. Tandem mass spectrometry provides a more efficient way to identify phosphorylated residues in IRS-1. Toward this end, we overexpressed glutathione S-transferase-IRS-1 fusion proteins in E. coli and treated them in vitro with various kinases followed by identification of phosphorylation sites using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Nine phosphorylation sites were detected in the tryptic digests of middle and C-terminal regions of IRS-1 treated with protein kinase A or extracellular signal-regulated kinase 2. Of these sites, five have not previously been detected by any method and provide novel candidates for identification in cells or in vivo.
• Guo, Y., Luo, M., & Lin, Z. (2002). Establishment of immune insulin resistance model in the rats by i.v. injection of BCG. Yao Xue Xue Bao, 37(5), 321-325.
• Luo, M., Guo, Y., & Lin, Z. (1999). Euglycemic Clamp Technique in Rats. Yao Xue Xue Bao, 34(255-259), 5.