Mechanisms Targeting the Unfolded Protein Response in Asthma.

Lung cells are constantly exposed to various internal and external stressors that disrupt protein homeostasis. To cope with these stimuli, cells evoke a highly conserved adaptive mechanism called the unfolded protein response (UPR). UPR stressors can impose greater protein secretory demands on the endoplasmic reticulum (ER), resulting in the development, differentiation, and survival of these cell types to meet these increasing functional needs. Dysregulation of the UPR leads to the development of the disease. The UPR and ER stress are involved in several human conditions, such as chronic inflammation, neurodegeneration, metabolic syndrome, and cancer. Furthermore, potent and specific compounds that target the UPR pathway are under development as future therapies. The focus of this review is to thoroughly describe the effects of both internal and external stressors on the ER in asthma. Furthermore, we discuss how the UPR signaling pathway is activated in the lungs to overcome cellular damage. We also present an overview of the pathogenic mechanisms, with a brief focus on potential strategies for pharmacological interventions.

The Measurement of Whole-Body Glucose Homeostasis in Mice.

The resident immune cells (e.g., macrophages) present in major metabolic tissues, such as adipose and liver tissues, play crucial roles in respective tissue homeostasis through cross talk with metabolic tissues, and consequently in metabolic homeostasis at the systemic level, and their dysregulation contributes to metabolic dysregulation at large, as well as many associated diseases. Moreover, the cross talk between different resident immune cells and metabolic tissues is not limited to an intra-organ level but also includes interorgan cross talk, as they work in a coordinated manner throughout the body, such as in adipose tissue, skeletal muscle, and liver. Thus, it is important to determine the impact of altered immune functions on metabolic homeostasis and vice versa, to enhance our knowledge of immunometabolic biology. Glucose and insulin tolerance tests are simple methods that enable the measurement and analysis of the overall glucose homeostasis at the systemic level. Here we describe the process of performing metabolic tests for glucose homeostasis in mice, as mouse models are often used for defining the mechanistic underpinnings of physiology and pathophysiology related to immunometabolism, and in preclinical studies.

The roles of apoptosis, autophagy and unfolded protein response in arbovirus, influenza virus, and HIV infections.

Virus infection induces different cellular responses in infected cells. These include cellular stress responses like autophagy and unfolded protein response (UPR). Both autophagy and UPR are connected to programed cell death I (apoptosis) in chronic stress conditions to regulate cellular homeostasis via Bcl2 family proteins, CHOP and Beclin-1. In this review article we first briefly discuss arboviruses, influenza virus, and HIV and then describe the concepts of apoptosis, autophagy, and UPR. Finally, we focus upon how apoptosis, autophagy, and UPR are involved in the regulation of cellular responses to arboviruses, influenza virus and HIV infections. Abbreviation: AIDS: Acquired Immunodeficiency Syndrome; ATF6: Activating Transcription Factor 6; ATG6: Autophagy-specific Gene 6; BAG3: BCL Associated Athanogene 3; Bak: BCL-2-Anatagonist/Killer1; Bax; BCL-2: Associated X protein; Bcl-2: B cell Lymphoma 2x; BiP: Chaperon immunoglobulin heavy chain binding Protein; CARD: Caspase Recruitment Domain; cART: combination Antiretroviral Therapy; CCR5: C-C Chemokine Receptor type 5; CD4: Cluster of Differentiation 4; CHOP: C/EBP homologous protein; CXCR4: C-X-C Chemokine Receptor Type 4; Cyto c: Cytochrome C; DCs: Dendritic Cells; EDEM1: ER-degradation enhancing-a-mannosidase-like protein 1; ENV: Envelope; ER: Endoplasmic Reticulum; FasR: Fas Receptor;G2: Gap 2; G2/M: Gap2/Mitosis; GFAP: Glial Fibrillary Acidic Protein; GP120: Glycoprotein120; GP41: Glycoprotein41; HAND: HIV Associated Neurodegenerative Disease; HEK: Human Embryonic Kidney; HeLa: Human Cervical Epithelial Carcinoma; HIV: Human Immunodeficiency Virus; IPS-1: IFN-ß promoter stimulator 1; IRE-1: Inositol Requiring Enzyme 1; IRGM: Immunity Related GTPase Family M protein; LAMP2A: Lysosome Associated Membrane Protein 2A; LC3: Microtubule Associated Light Chain 3; MDA5: Melanoma Differentiation Associated gene 5; MEF: Mouse Embryonic Fibroblast; MMP: Mitochondrial Membrane Permeabilization; Nef: Negative Regulatory Factor; OASIS: Old Astrocyte Specifically Induced Substrate; PAMP: Pathogen-Associated Molecular Pattern; PERK: Pancreatic Endoplasmic Reticulum Kinase; PRR: Pattern Recognition Receptor; Puma: P53 Upregulated Modulator of Apoptosis; RIG-I: Retinoic acid-Inducible Gene-I; Tat: Transactivator Protein of HIV; TLR: Toll-like receptor; ULK1: Unc51 Like Autophagy Activating Kinase 1; UPR: Unfolded Protein Response; Vpr: Viral Protein Regulatory; XBP1: X-Box Binding Protein 1.

Myocardial Cell Signaling During the Transition to Heart Failure: Cellular Signaling and Therapeutic Approaches.

Cardiovascular disease leading to heart failure (HF) remains a leading cause of morbidity and mortality worldwide. Improved pharmacological and interventional coronary procedures have led to improved outcomes following acute myocardial infarction. This success has translated into an unforeseen increased incidence in HF. This review summarizes the signaling pathways implicated in the transition to HF following cardiac injury. In addition, we provide an update on cell death signaling and discuss recent advances in cardiac fibrosis as an independent event leading to HF. Finally, we discuss cell-based therapies and their possible use to avert the deteriorating nature of HF. © 2019 American Physiological Society. Compr Physiol 9:75-125, 2019.

Glioblastoma and chemoresistance to alkylating agents: Involvement of apoptosis, autophagy, and unfolded protein response.

Despite advances in neurosurgical techniques and radio-/chemotherapy, the treatment of brain tumors remains a challenge. This is particularly true for the most frequent and fatal adult brain tumor, glioblastoma (GB). Upon diagnosis, the average survival time of GB patients remains only approximately 15months. The alkylating drug temozolomide (TMZ) is routinely used in brain tumor patients and induces apoptosis, autophagy and unfolded protein response (UPR). Here, we review these cellular mechanisms and their contributions to TMZ chemoresistance in brain tumors, with a particular emphasis on TMZ chemoresistance in glioma stem cells and GB.

Mevalonate Cascade and its Regulation in Cholesterol Metabolism in Different Tissues in Health and Disease.

The cholesterol biosynthesis pathway, also referred to as the mevalonate (MVA) pathway, is responsible for the biosynthesis of two key isoprenoids: farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Post-translational modification of small GTPases by FPP and GGPP has captured much attention due to their potential contribution to cancer, cardiovascular and neurodegenerative diseases. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) catalyzes the conversion of HMG-CoA to MVA, and is the rate-limiting step in the biosynthesis of cholesterol. Statins are HMGCR inhibitors that are used extensively in the treatment of hypercholesterolemia. Inhibitors of the MVA pathway exhibit anti-tumor effects and may reduce cancer incidence and cancer-related mortality in humans. In this review, we will focus on the mevalonate cascade and its regulation in cholesterol metabolism as well as polymorphisms of the MVA cascade in cancer development, infectious and cardiovascular disease (CVD).

Prohibitin in Adipose and Immune Functions.

Prohibitin (PHB) was discovered in a quest to find genes with antiproliferative functions. However, the attribute of PHB that is responsible for its antiproliferative function remains elusive. Meanwhile, recent studies have established PHB as a pleiotropic protein with roles in metabolism, immunity, and senescence. PHB has cell compartment-specific functions, acting as a scaffolding protein in mitochondria, an adaptor molecule in membrane signaling, and a transcriptional coregulator in the nucleus. However, it remains unclear whether different functions and locations of PHB are interrelated or independent from each other, or if PHB works in a tissue-specific manner. Here, we discuss new findings on the role of PHB in adipose-immune interaction and an unexpected role in sex differences in adipose and immune functions.

Prohibitin: an unexpected role in sex dimorphic functions.

Sex differences are known to exist in adipose and immune functions in the body, and sex steroid hormones are known to be involved in sexually dimorphic biological and pathological processes related to adipose-immune interaction. However, our knowledge of proteins that mediate such differences is poor. Two novel obese mice models, Mito-Ob and m-Mito-Ob, that have been reported recently have revealed an unexpected role of a pleiotropic protein, prohibitin (PHB), in sex differences in adipose and immune functions. This discovery points towards a role of pleiotropic proteins and their potential interplay with sex steroid hormones in mediating sexually dimorphic adipose-immune interaction.

Prohibitin-induced, obesity-associated insulin resistance and accompanying low-grade inflammation causes NASH and HCC.

Obesity increases the risk for nonalcoholic steatohepatitis (NASH) and hepatocarcinogenesis. However, the underlying mechanisms involved in the disease process remain unclear. Recently, we have developed a transgenic obese mouse model (Mito-Ob) by prohibitin mediated mitochondrial remodeling in adipocytes. The Mito-Ob mice develop obesity in a sex-neutral manner, but obesity-associated adipose inflammation and metabolic dysregulation in a male sex-specific manner. Here we report that with aging, the male Mito-Ob mice spontaneously develop obesity-linked NASH and hepatocellular carcinoma (HCC). In contrast, the female Mito-Ob mice maintained normal glucose and insulin levels and did not develop NASH and HCC. The anti-inflammatory peptide ghrelin was significantly upregulated in the female mice and down regulated in the male mice compared with respective control mice. In addition, a reduction in the markers of mitochondrial content and function was found in the liver of male Mito-Ob mice with NASH/HCC development. We found that ERK1/2 signaling was significantly upregulated whereas STAT3 signaling was significantly down regulated in the tumors from Mito-Ob mice. These data provide a proof-of-concept that the metabolic and inflammatory status of the adipose tissue and their interplay at the systemic and hepatic level play a central role in the pathogenesis of obesity-linked NASH and HCC.

Apoptosis, autophagy and unfolded protein response pathways in Arbovirus replication and pathogenesis.

Arboviruses are pathogens that widely affect the health of people in different communities around the world. Recently, a few successful approaches toward production of effective vaccines against some of these pathogens have been developed, but treatment and prevention of the resulting diseases remain a major health and research concern. The arbovirus infection and replication processes are complex, and many factors are involved in their regulation. Apoptosis, autophagy and the unfolded protein response (UPR) are three mechanisms that are involved in pathogenesis of many viruses. In this review, we focus on the importance of these pathways in the arbovirus replication and infection processes. We provide a brief introduction on how apoptosis, autophagy and the UPR are initiated and regulated, and then discuss the involvement of these pathways in regulation of arbovirus pathogenesis.

Obesity-related abnormalities couple environmental triggers with genetic susceptibility in adult-onset T1D.

The incidence of adult-onset T1D in low-risk non-HLA type has increased several folds, whereas the contemporaneous incidence in high-risk HLA-type remains stable. Various factors behind this selective increase in T1D in young adults remain unclear. Obesity and its associated abnormalities appear to be an important determinant; however, the underlying mechanism involved is not understood. Recently, we have developed two novel transgenic obese mice models, Mito-Ob and m-Mito-Ob, by expressing a pleiotropic protein prohibitin (PHB) and a phospho mutant form of PHB (Y114F-PHB or m-PHB) from the aP2 gene promoter, respectively. Both mice models develop obesity in a sex-neutral manner, independent of diet; but obesity associated chronic low-grade inflammation and insulin resistance in a male sex-specific manner. Interestingly, on a high fat diet (HFD) only male m-Mito-Ob mice displayed marked mononuclear cell infiltration in pancreas and developed insulitis that mimic adult-onset T1D. Male Mito-Ob mice that share the metabolic phenotype of male m-Mito-Ob mice, and female m-Mito-Ob that harbor m-PHB similar to male m-Mito-Ob mice, did not develop insulitis. Thus, insulitis development in male m-Mito-Ob in response to HFD requires both, obesity-related abnormalities and m-PHB. Collectively, this data provides a proof-of-concept that obesity-associated abnormalities couple environmental triggers with genetic susceptibility in adult-onset T1D and reveals PHB as a potential susceptibility gene for T1D.

Assessment of posttranslational modification of mitochondrial proteins.

Mitochondria play vital roles in the maintenance of cellular homeostasis. They are a storehouse of cellular energy and antioxidative enzymes. Because of its immense role and function in the development of an organism, this organelle is required for the survival. Defects in mitochondrial proteins lead to complex mitochondrial disorders and heterogeneous diseases such as cancer, type 2 diabetes, and cardiovascular and neurodegenerative diseases. It is widely known in the literature that some of the mitochondrial proteins are regulated by posttranslational modifications. Hence, designing methods to assess these modifications in mitochondria will be an important way to study the regulatory roles of mitochondrial proteins in greater detail. In this chapter, we outlined procedures to isolate mitochondria from cells and separate the mitochondrial proteins by two-dimensional gel electrophoresis and identify the different posttranslational modifications in them by using antibodies specific to each posttranslational modification.

Prohibitin overexpression in adipocytes induces mitochondrial biogenesis, leads to obesity development, and affects glucose homeostasis in a sex-specific manner.

Adipocytes are the primary cells in the body that store excess energy as triglycerides. To perform this specialized function, adipocytes rely on their mitochondria; however, the role of adipocyte mitochondria in the regulation of adipose tissue homeostasis and its impact on metabolic regulation is not understood. We developed a transgenic mouse model, Mito-Ob, overexpressing prohibitin (PHB) in adipocytes. Mito-Ob mice developed obesity due to upregulation of mitochondrial biogenesis in adipocytes. Of note, Mito-Ob female mice developed more visceral fat than male mice. However, female mice exhibited no change in glucose homeostasis and had normal insulin and high adiponectin levels, whereas male mice had impaired glucose homeostasis, compromised brown adipose tissue structure, and high insulin and low adiponectin levels. Mechanistically, we found that PHB overexpression enhances the cross talk between the mitochondria and the nucleus and facilitates mitochondrial biogenesis. The data suggest a critical role of PHB and adipocyte mitochondria in adipose tissue homeostasis and reveal sex differences in the effect of PHB-induced adipocyte mitochondrial remodeling on whole-body metabolism. Targeting adipocyte mitochondria may provide new therapeutic opportunities for the treatment of obesity, a major risk factor for type 2 diabetes.

Targeting the mevalonate cascade as a new therapeutic approach in heart disease, cancer and pulmonary disease.

The cholesterol biosynthesis pathway, also known as the mevalonate (MVA) pathway, is an essential cellular pathway that is involved in diverse cell functions. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) is the rate-limiting step in cholesterol biosynthesis and catalyzes the conversion of HMG-CoA to MVA. Given its role in cholesterol and isoprenoid biosynthesis, the regulation of HMGCR has been intensely investigated. Because all cells require a steady supply of MVA, both the sterol (i.e. cholesterol) and non-sterol (i.e. isoprenoid) products of MVA metabolism exert coordinated feedback regulation on HMGCR through different mechanisms. The proper functioning of HMGCR as the proximal enzyme in the MVA pathway is essential under both normal physiologic conditions and in many diseases given its role in cell cycle pathways and cell proliferation, cholesterol biosynthesis and metabolism, cell cytoskeletal dynamics and stability, cell membrane structure and fluidity, mitochondrial function, proliferation, and cell fate. The blockbuster statin drugs ('statins') directly bind to and inhibit HMGCR, and their use for the past thirty years has revolutionized the treatment of hypercholesterolemia and cardiovascular diseases, in particular coronary heart disease. Initially thought to exert their effects through cholesterol reduction, recent evidence indicates that statins also have pleiotropic immunomodulatory properties independent of cholesterol lowering. In this review we will focus on the therapeutic applications and mechanisms involved in the MVA cascade including Rho GTPase and Rho kinase (ROCK) signaling, statin inhibition of HMGCR, geranylgeranyltransferase (GGTase) inhibition, and farnesyltransferase (FTase) inhibition in cardiovascular disease, pulmonary diseases (e.g. asthma and chronic obstructive pulmonary disease (COPD)), and cancer.

Autophagy and apoptosis dysfunction in neurodegenerative disorders.

Autophagy and apoptosis are basic physiologic processes contributing to the maintenance of cellular homeostasis. Autophagy encompasses pathways that target long-lived cytosolic proteins and damaged organelles. It involves a sequential set of events including double membrane formation, elongation, vesicle maturation and finally delivery of the targeted materials to the lysosome. Apoptotic cell death is best described through its morphology. It is characterized by cell rounding, membrane blebbing, cytoskeletal collapse, cytoplasmic condensation, and fragmentation, nuclear pyknosis, chromatin condensation/fragmentation, and formation of membrane-enveloped apoptotic bodies, that are rapidly phagocytosed by macrophages or neighboring cells. Neurodegenerative disorders are becoming increasingly prevalent, especially in the Western societies, with larger percentage of members living to an older age. They have to be seen not only as a health problem, but since they are care-intensive, they also carry a significant economic burden. Deregulation of autophagy plays a pivotal role in the etiology and/or progress of many of these diseases. Herein, we briefly review the latest findings that indicate the involvement of autophagy in neurodegenerative diseases. We provide a brief introduction to autophagy and apoptosis pathways focusing on the role of mitochondria and lysosomes. We then briefly highlight pathophysiology of common neurodegenerative disorders like Alzheimer's diseases, Parkinson's disease, Huntington's disease and Amyotrophic lateral sclerosis. Then, we describe functions of autophagy and apoptosis in brain homeostasis, especially in the context of the aforementioned disorders. Finally, we discuss different ways that autophagy and apoptosis modulation may be employed for therapeutic intervention during the maintenance of neurodegenerative disorders.

Phosphorylation of transglutaminase 2 (TG2) at serine-216 has a role in TG2 mediated activation of nuclear factor-kappa B and in the downregulation of PTEN.

BACKGROUND: Transglutaminase 2 (TG2) and its phosphorylation have been consistently found to be upregulated in a number of cancer cell types. At the molecular level, TG2 has been associated with the activation of nuclear factor-kappa B (NF-κB), protein kinase B (PKB/Akt) and in the downregulation of phosphatase and tensin homologue deleted on chromosome 10 (PTEN). However, the underlying mechanism involved is not known. We have reported that protein kinase A (PKA) induced phosphorylation of TG2 at serine-216 (Ser(216)) regulates TG2 function and facilitates protein-protein interaction. However, the role of TG2 phosphorylation in the modulation of NF-κB, Akt and PTEN is not explored. METHODS: In this study we have investigated the effect of TG2 phosphorylation on NF-κB, Akt and PTEN using embryonic fibroblasts derived from TG2 null mice (MEF(tg2-/-)) overexpressing native TG2 or mutant-TG2 (m-TG2) lacking Ser(216) phosphorylation site with and without dibutyryl cyclic-AMP (db-cAMP) stimulation. Functional consequences on cell cycle and cell motility were determined by fluorescence activated cell sorting (FACS) analysis and cell migration assay respectively. RESULTS: PKA activation in TG2 overexpressing MEF(tg2-/-) cells resulted in an increased activation of NF-κB and Akt phosphorylation in comparison to empty vector transfected control cells as determined by the reporter-gene assay and immunoblot analysis respectively. These effects were not observed in MEF(tg2-/-) cells overexpressing m-TG2. Similarly, a significant downregulation of PTEN at both, the mRNA and protein levels were found in cells overexpressing TG2 in comparison to empty vector control and m-TG2 transfected cells. Furthermore, Akt activation correlated with the simultaneous activation of NF-κB and a decrease in PTEN suggesting that the facilitatory effect of TG2 on Akt activation occurs in a PTEN-dependent manner. Similar results were found with MCF-7 and T-47D breast cancer cells overexpressing TG2 and m-TG2 further supporting the role of TG2 phosphorylation in NF-κB activation and in the downregulation of PTEN. CONCLUSIONS: Collectively, these data suggest that phosphorylation of TG2 at Ser(216) plays a role in TG2 mediated activation of NF-κB, Akt and in the downregulation of PTEN. Blocking TG2 phosphorylation may provide a novel strategy to attenuate NF-κB activation and downregulation of PTEN in TG2 overexpressing cancers.

Functional characterization of naturally occurring transglutaminase 2 mutants implicated in early-onset type 2 diabetes.

Transglutaminase 2 (TG2) is an enzyme with diverse biological functions. TG2 catalyzes transamidation reactions, has intrinsic kinase activity, and acts as a G-protein in intracellular signaling. TG2 (Tgm2)-null mice are glucose intolerant and have impaired glucose-stimulated insulin secretion (GSIS). Furthermore, three naturally occurring missense mutations in the human TGM2 gene, corresponding to amino acid substitutions of Met330Arg, Ile331Asn, and Asn333Ser in the TG2 protein, have been reported and found to be associated with early-onset type 2 diabetes. However, their effect on TG2 function is not fully understood. To determine this, we have reproduced naturally occurring mutations in TG2 using site-directed mutagenesis. Overexpression of Myc-TG2 mutants in INS-1E cells resulted in a reduction of GSIS in comparison with cells overexpressing wild-type Myc-TG2 (WT-TG2). The maximum reduction was found in cells overexpressing Ile331Asn-TG2 (32%) followed by Met330Arg-TG2 (20%), and the least in Asn333Ser-TG2 (7%). Enzymatic analysis revealed that TG2 mutants have impaired transamidation and kinase activities in comparison with WT-TG2. GTP-binding assays showed that TG2 mutants also have altered GTP-binding ability, which is found to be modulated in response to glucose stimulation. Collectively, these data suggest that naturally occurring mutations in TG2 affect transamidation, kinase, and GTP-binding functions of TG2. While reduced insulin secretion, as a result of naturally occurring mutations in TG2, is due to the impairment of more than one biological function of TG2, it is the transamidation function that appears to be impaired during the first phase, whereas the GTP-binding function affects the second phase of insulin secretion.

Overexpression of phospho mutant forms of transglutaminase 2 downregulates epidermal growth factor receptor.

Simultaneous upregulation of transglutaminase 2 (TG2) and epidermal growth factor receptor (EGFR) have been reported in a number of systems. Moreover, TG2 has been identified as a downstream target gene for EGF/EGFR. However, it is not known whether the relationship between EGFR and TG2 is only one-way or collaborative. Using embryonic fibroblasts derived from TG2 null mice (MEF(tg2-/-)), co-overexpressing native TG2 and EGFR, here we report that TG2 differentially regulates EGFR protein in the presence and absence of EGF. In the absence of EGF, TG2 facilitates EGFR downregulation whereas in the presence of EGF, TG2 has opposite effect on EGFR and facilitates Akt phosphorylation. TG2 mediated ligand-independent downregulation of EGFR was not observed in MEF(tg2-/-) cells overexpressing Ser212Ala phospho mutant form of TG2 suggesting a role of TG2 phosphorylation in this process. However, similar to native TG2, Ser212Ala-TG2 mutant was also able to attenuate ligand-dependent down regulation of EGFR in MEF(tg2-/-) cells. Interestingly, overexpression of Ser216Ala-TG2 mutant led to downregulation of EGFR in MEF(tg2-/-) cells irrespective of the ligand. These results were further confirmed in breast cancer cells expressing high levels of EGFR. Collectively, data presented here suggests that the relationship between EGFR and TG2 is collaborative and phosphorylation of TG2 play a key role in it. Phospho mutant forms of TG2 reported in this study may be utilized as a part of a novel strategy to downregulate EGFR in cancers with enhanced EGFR signaling.

O-GlcNAc modification: why so intimately associated with phosphorylation?

Post-translational modification of proteins at serine and threonine side chains by ß-N-acetylglucosamine (O-GlcNAc) mediated by the enzyme ß-N-acetylglucosamine transferase has been emerging as a fundamental regulatory mechanism encompassing a wide range of proteins involved in cell division, metabolism, transcription and cell signaling. Furthermore, an extensive interplay between O-GlcNAc modification and serine/threonine phosphorylation in a variety of proteins has been reported to exist. However, our understanding of the regulatory mechanisms involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins is still elusive. Recent success in the mapping of O-GlcNAc modification sites in proteins as a result of technological advancement in mass spectrometry have revealed two important clues which may be inherently connected to the regulation of O-GlcNAc modification and its interplay with phosphorylation in proteins. First, almost all O-GlcNAc modified proteins are known phospho proteins. Second, the prevalence of tyrosine phosphorylation among O-GlcNAc modified proteins is exceptionally higher (~68%) than its normal occurrence (~2%) alone. We hypothesize that phosphorylation may be a requisite for O-GlcNAc modification and tyrosine phosphorylation plays a role in the interplay between O-GlcNAc modification and serine/threonine phosphorylation in proteins. In other words, the interplay between O-GlcNAc modification and phosphorylation is not limited to serine/threonine phosphorylation but also includes tyrosine phosphorylation. Our hypothesis provides an opportunity to understand the underlying mechanism involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins. Furthermore, implication of our hypothesis extends to tyrosine kinase signaling.

Altered O-GlcNAc modification and phosphorylation of mitochondrial proteins in myoblast cells exposed to high glucose.

Hyperglycemia induced increased posttranslational modification of proteins by O-linked-ß-N-acetyl glucosamine (O-GlcNAcylation) and mitochondrial dysfunction has been independently implicated in the development of insulin resistance. It is not known whether repertoire of O-GlcNAcylated proteins includes mitochondrial proteins and their altered O-GlcNAcylation impinges on their phosphorylation mediated normal functioning thus contribute to mitochondrial dysfunction and insulin resistance. We have explored the O-GlcNAcylation of mitochondrial proteins from myoblast cells under basal (4mM) and high glucose (30mM) conditions using a combination of proteomic approaches. Furthermore, we have assessed the accompanied changes in the phosphorylation of mitochondrial proteins. We report that a number of mitochondrial proteins are O-GlcNAcylated under basal condition which is altered under high glucose condition. In addition, we report that exposure to high glucose not only changes the O-GlcNAcylation of mitochondrial proteins but also changes their phosphorylation profiles. The dynamic and complex interplay between O-GlcNAcylation and phosphorylation of mitochondrial proteins was further validated by immunoblot analysis of HSP60, prohibitin, and voltage-dependent anion channel 1 as candidate proteins. O-GlcNAcylation of mitochondrial proteins may play a role in normal functioning of mitochondria. High glucose induced changes in O-GlcNAcylation and phosphorylation of mitochondrial proteins may be associated with mitochondrial dysfunction and insulin resistance.