Mark E Mccomb

Boston University | BU · Department of Biochemistry

Aims S-glutathionylation is a reversible oxidative modification of protein cysteines that plays a critical role in redox signaling. Glutaredoxin-1 (Glrx), a glutathione-specific thioltransferase, removes protein S-glutathionylation. Glrx, though a cytosolic protein, can activate a nuclear protein Sirtuin-1 (SirT1) by removing its S-glutathionylatio...

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear...

Osteocytes, the bone cells embedded in the mineralized matrix, control bone modeling, and remodeling through direct contact with adjacent cells and via paracrine and endocrine factors that affect cells in the bone marrow microenvironment or distant organs. Osteocytes express numerous G protein-coupled receptors (GPCRs) and thus mice lacking the sti...

Glutaredoxin-1 (Glrx) is a small cytosolic enzyme that removes S-glutathionylation, glutathione adducts of protein cysteine residues, thus modulating redox signaling and gene transcription. Although Glrx up-regulation prevented endothelial cell (EC) migration and global Glrx transgenic mice had impaired ischemic vascularization, the effects of cell...

The cellular isoform of the prion protein (PrPC) serves as precursor to the infectious isoform (PrPSc), and as a cell-surface receptor, which binds misfolded protein oligomers as well as physiological ligands such as Cu2+ ions. PrPC consists of two domains: a flexible N-terminal domain and a structured C-terminal domain. Both the physiological and...

Sirtuin-1 (SirT1) catalyzes NAD⁺-dependent protein lysine deacetylation and is a critical regulator of energy and lipid metabolism, mitochondrial biogenesis, apoptosis, and senescence. Activation of SirT1 mitigates metabolic perturbations associated with diabetes and obesity. Pharmacologic molecules, cellular redox, and nutritional states can regul...

Parkinson's disease (PD) is a neurological disorder characterized by the progressive loss of functional dopaminergic neurons in the nigrostriatal pathway in the brain. While current treatments provide only symptomatic relief, gene therapy has the potential to slow or halt the degeneration of nigrostriatal dopamine neurons in PD patients. Adeno-asso...

Ki-1/57 is a nuclear and cytoplasmic regulatory protein first identified in malignant cells from Hodgkin’s lymphoma. It is involved in gene expression regulation on both transcriptional and mRNA metabolism levels. Ki-1/57 belongs to the family of intrinsically unstructured proteins, undergoes phosphorylation by PKC and methylation by PRMT1. Previou...

Glycomics and glycoproteomics analyses by mass spectrometry require efficient front-end separation methods to enable deep characterization of heterogeneous glycoform populations. Chromatography methods are generally limited in their ability to resolve glycoforms using mobile phases that are compatible with online liquid chromatography-mass spectrom...

Deregulated cellular metabolism is a hallmark of tumors. Cancer cells increase glucose and glutamine flux to provide energy needs and macromolecular synthesis demands. Several studies have been focused on the importance of glycolysis and pentose phosphate pathway. However, a neglected but very important branch of glucose metabolism is the hexosamin...

One mode of γ-globin gene silencing involves a GATA-1/FOG-1/Mi2β repressor complex that binds to the -566 GATA site relative to the Aγ-globin gene CAP site. However, the mechanism as to how this repressor complex is assembled at -566 GATA site is unknown. In this study, we demonstrate that the O-GlcNAc processing enzymes, O-GlcNAc transferase (OGT)...

O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification on serine and threonine residues of many proteins, plays crucial regulatory roles in diverse biological events. As a nutrient sensor, O-GlcNAc modification (O-GlcNAcylation) on nuclear and cytoplasmic proteins underlies the pathology of diabetic complications, including ca...

Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential c...

IPA predicted multiple protein networks associated with oxidative changes caused by catalase overexpression. The 11 ‘node’ proteins are highlighted in grey. Legend to network analysis: enzyme (diamond), transmembrane receptor (vertical oval), transcriptional regulator (horizontal oval), phosphatase (triangle), transporter (trapezoid), kinase (trian...

Cardiac mitochondrial maximal and uncoupled oxygen consumption were similar in both groups. (A) Maximal (State III) and uncoupled (oligomycin 2μM) (State IV) complex I substrate-driven oxygen consumption rate; (B) Maximal (State III) and uncoupled (oligomycin 2 μM) (State IV) complex II substrate-driven oxygen consumption rate. Data represents mean...

Multiplexed quantitation of reversible cysteine oxidation in Cat Tg and WT mice. (A) TMT-switch labeling strategy. To determine the total available (free and reducible) cysteines, half of each left ventricle was completely reduced and labeled with iodoTMT; to label only reversibly oxidized cysteines, all free cysteines were blocked by IAM during ly...

Validation of TMT-tagged peptides. (A) Peptide frequency histogram of the coefficient of variation (CV) for changes in total available cysteine thiols (●), reversibly oxidized cysteine thiols (□) and their occupancy (∆). (B) Distribution of TMT-tagged peptides. A total of 2264 peptides with modifications were detected by LC-MS/MS analysis, of which...

Complete list of proteins with a change in thiol oxidation in Cat Tg vs. WT. Accession number, gene ID, sites of modification and peptide sequences were retrieved from the Uniprot knowledgebase. Fold changes in Cat Tg vs. WT, were calculated from ratio of reporter ions for changes in total available cysteine as (m/z 129)/(m/z 127), reversibly oxidi...

Proteins with a change in thiol occupancy by >2-fold in Cat Tg vs. WT mice. Accession number, gene ID, sites of modification and peptide sequences were retrieved from the Uniprot knowledgebase. Fold changes in Cat Tg vs. WT, were calculated from ratio of reporter ions for changes in total available cysteine as (m/z 129)/(m/z 127), reversibly oxidiz...

Proteins involved in five canonical pathways predicted in IPA, with a change in thiol occupancy by >1.3-fold in Cat Tg vs. WT. Accession number, gene ID, sites of modification and peptide sequences were retrieved from the Uniprot knowledgebase. Fold changes in Cat Tg vs. WT, were calculated from ratio of reporter ions for changes in total available...

The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic ag...

Hemoglobinopathies are the most common inherited disorders in humans and are thus the target of screening programs worldwide. Over the past decade, mass spectrometry (MS) has gained a more important role as a clinical means to diagnose variants, and a number of approaches have been proposed for characterization. Here we investigate the use of matri...

Pulmonary arterial hypertension (PAH) is a disease characterized by increased pulmonary vascular resistance and remodeling. Increase in the population of vascular smooth muscle cells is among the key events contributing to the remodeling. Endothelin-1 (ET-1), a potent vasoconstrictor, is linked to the etiology and progression of PAH. Here we analyz...

Human NEK7 is a regulator of cell division and plays an important role in growth and survival of mammalian cells. Human NEK6 and NEK7 are closely related, consisting of a conserved C-terminal catalytic domain and a nonconserved and disordered N-terminal regulatory domain, crucial to mediate the interactions with their respective proteins. Here, in...

Using a novel cysteine thiol labeling strategy coupled with mass spectrometric analysis, we identified and quantified the changes in global reversible cysteine oxidation of proteins in the left ventricle of hearts from mice with metabolic syndrome-associated diastolic dysfunction. This phenotype was induced by feeding a high-fat, high-sucrose, type...

The identification of protein post‐translational modifications (PTMs) is an increasingly important component of proteomics and biomarker discovery, but very few tools exist for performing fast and easy characterization of global PTM changes and differential comparison of PTMs across groups of data obtained from liquid chromatography–tandem mass spe...

Pulmonary hypertension (PH), an independent risk factor for mortality in sickle cell disease (SCD), has epidemiologic links to systemic complications such as leg ulcers, priapism and renal disease suggestive of a diffuse vasculopathy. The pathogenesis of SCD related vasculopathy is unclear but altered redox biology is thought to be an important dis...

Phenotype/genotype correlation can provide important insight into the pathophysiology of hemoglobin disorders and molecular mechanisms regulating globin gene expression. We present 2 unusual patients with β-globin gene cluster deletions of 44 and 589 kb. In the first case, the findings suggest that competition for LCR interaction among β-like globi...

Protein phosphorylation is a dynamic post-translational modification. Mass spectrometry-based quantitation was performed to determine the phosphoproteome profile of epithelial cells in response to injury, nucleotide, or epidermal growth factor. Phosphotyrosine enrichment used immunoprecipitation and immobilized metal affinity chromatography. Nucleo...

One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. Accurate comparison of glycoforms and quantification of glycosites is an essential step in this direction. Mass spec...

The evolutionarily conserved aryl hydrocarbon receptor (AhR) has been studied for its role in environmental chemical-induced toxicity. However, recent studies have demonstrated that the AhR may regulate the hematopoietic and immune systems during development in a cell-specific manner. These results, together with the absence of an in vitro model sy...

During mitosis, proteins undergo a variety of post-translational modifications (PTM) in order to alter function and localization. Our objective is to determine the interplay between O-GlcNAcylation and phosphorylation on proteins during mitotic progression. O-GlcNAc is the attachment of a single N-acetylglucosamine moiety to serine and threonine re...

Unfavorable metabolic conditions (metabolic disorders) associated with obesity, diabetes, and hyperlipidemia are major causes for cardiovascular disease (CVD). One major environmental cause of this may be attributed to poor diet, aka the American diet. The early detection and monitoring of the adverse effects of metabolic disease on the heart and v...

Global changes in reversible cysteine oxidation of cardiac proteins from mice fed a high fat, high sucrose (HFHS) diet for 8 months were quantified using novel 6-plex Iodo-based isobaric labels in a switch assay and high resolution mass spectrometry. 400 reversibly oxidized proteins were identified, 56 with cysteines exhibiting more than 1.5-fold c...

Unfavorable metabolic conditions (metabolic disorders) associated with obesity and diabetes are major causes for cardiovascular disease. Early detection of the adverse effects of metabolic disease remain elusive. We believe that nonspecific changes which occur in plasma proteins, indicators of inflammation and oxidants, may act as evidence of syste...

There is an increasing interest in the identification and characterization of protein posttranslational modifications (PTMs). The vast amount of information obtained within a typical differential proteomics makes the measure of PTMs challenging. We have begun to explore counting methods for differential analysis of PTM changes in proteomes. We have...

Post-translational modifications are essential in regulating the function, stability, and localization of proteins. One such modification is the O-GlcNAc modification. O-GlcNAc is the attachment of a single N-acetylglucosamine moiety to serine and threonine residues on nuclear and cytoplasmic proteins. O-GlcNAc is dynamically cycling on Ser/Thr res...

Unfavorable metabolic conditions (metabolic disorders) associated with obesity and diabetes are major causes for cardiovascular disease. One major cause may be attributed to poor diet, aka the American diet model. Early detection of the adverse effects of metabolic disease remain elusive. We believe that nonspecific changes which occur in plasma pr...

Significance: Oxidative post-translational modifications (OPTMs) have been demonstrated as contributing to cardiovascular physiology and pathophysiology. These modifications have been identified using antibodies as well as advanced proteomic methods, and the functional importance of each is beginning to be understood using transgenic and gene dele...

Post-translational modifications (PTMs) are essential in regulating the function, stability, and localization of proteins. One such modification is the O-GlcNAc. O-GlcNAc is the attachment of a single N-acetylglucosamine moiety to serine and threonine residues on nuclear and cytoplasmic proteins. The modification is dynamically processed on protein...

The biological functions of glycoconjugate glycans arise in the context of structural heterogeneity resulting from non-template driven biosynthetic reactions. Such heterogeneity is particularly apparent for the glycosaminoglycan (GAG) classes, of which heparan sulfate (HS) is of particular interest for its properties in binding to many classes of g...

Here we demonstrate a new paradigm in redox signaling, whereby oxidants resulting from metabolic stress directly alter protein palmitoylation by oxidizing reactive cysteine thiolates. In mice fed a high-fat, high-sucrose diet and in cultured endothelial cells (ECs) treated with high palmitate and high glucose (HPHG), there was decreased HRas palmit...

Type II citrate synthases (CSs), found in gram-negative bacteria such as Escherichia coli, are subject to strong and specific allosteric inhibition by NADH. Crystallographic studies have shown that the NADH binding sites are located at macromolecular contacts, so that tight binding requires the intact hexameric structure. Although other adenylates,...

Transthyretin (TTR) amyloidosis and hemoglobinopathies are the archetypes of molecular diseases where point mutation characterization is diagnostically critical. We have developed a Top-down analytical platform for variant and/or modified protein sequencing and are examining the feasibility of using this platform for the analysis of hemoglobin/TTR...

Non-porous polyurethane (PU) membranes and porous PU thin films are used as sample supports for MALDI-TOFMS. Mass spectra obtained are compared with those acquired using metal targets and the crushed matrix method. The compounds characterized are wheat proteins which consist of moderately water-soluble gliadins, and of water-insoluble low molecular...

The enzyme citrate synthase from E. coli is a protein with a molecular weight (Mr) of 47 885 Da (wild type). This enzyme has been studied extensively, and its amino acid sequence has been characterized. This model protein has been used in this work for development and validation of methods involving capillary electrophoresis (CE) and electrospray i...

RP-15

Sirtuin-1 (SIRT1) is an NAD(+)-dependent protein deacetylase that is sensitive to oxidative signals. Our purpose was to determine whether SIRT1 activity is sensitive to the low molecular weight nitrosothiol, S-nitrosoglutathione (GSNO), which can transduce oxidative signals into physiological responses. SIRT1 formed mixed disulfides with GSNO-Sepha...

The selective capture of target peptides poses a great challenge to modern chemists and biologists, especially when enriching them from proteome samples possessing extremes in concentration dynamic range and sequence diversity. While approaches based on traditional techniques such as biotin-avidin pairing offer versatile tools to design strategies...

In order that biological meaning may be derived and testable hypotheses may be built from proteomics experiments, assignments of proteins identified by mass spectrometry or other techniques must be supplemented with additional notation, such as information on known protein functions, protein-protein interactions, or biological pathway associations....

The extreme pathological diversity of non-Hodgkin's lymphomas has made their accurate histological assessment difficult. New diagnostics and treatment modalities are urgently needed for these lymphomas, particularly in drug development for cancer-specific targets. Previously, we showed that a subset of B cell lymphoma, diffuse large B cell lymphoma...