Peptide Modification Diminishes HLA Class II-restricted CD4+ T Cell Recognition of Prostate Cancer Cells.

Prostate cancer poses an ongoing problem in the western world accounting for significant morbidity and mortality in the male population. Current therapy options are effective in treating most prostate cancer patients, but a significant number of patients progress beyond a manageable disease. For these patients, immunotherapy has emerged as a real option in the treatment of the late-stage metastatic disease. Unfortunately, even the most successful immunotherapy strategies have only led to a four-month increase in survival. One issue responsible for the shortcomings in cancer immunotherapy is the inability to stimulate helper CD4+ T cells via the HLA class II pathway to generate a potent antitumor response. Obstacles to proper HLA class II stimulation in prostate cancer vaccine design include the lack of detectable class II proteins in prostate tumors and the absence of defined class II specific prostate tumor antigens. Here, for the first time, we show that the insertion of a lysosomal thiol reductase (GILT) into prostate cancer cells directly enhances HLA class II antigen processing and results in increased CD4+ T cell activation by prostate cancer cells. We also show that GILT insertion does not alter the expression of prostate-specific membrane antigen (PSMA), an important target in prostate cancer vaccine strategies. Our study suggests that GILT expression enhances the presentation of the immunodominant PSMA459 epitope via the HLA class II pathway. Biochemical analysis showed that the PSMA459 peptide was cysteinylated under a normal physiologic concentration of cystine, and this cysteinylated form of PSMA459 inhibited T cell activation. Taken together, these results suggest that GILT has the potential to increase HLA class II Ag presentation and CD4+ T cell recognition of prostate cancer cells, and GILT-expressing prostate cancer cells could be used in designing cell therapy and/or vaccines against prostate cancer.

GILT Expression in Human Melanoma Cells Enhances Generation of Antigenic Peptides for HLA Class II-Mediated Immune Recognition.

Melanoma is an aggressive skin cancer that has become increasingly prevalent in western populations. Current treatments such as surgery, chemotherapy, and high-dose radiation have had limited success, often failing to treat late stage, metastatic melanoma. Alternative strategies such as immunotherapies have been successful in treating a small percentage of patients with metastatic disease, although these treatments to date have not been proven to enhance overall survival. Several melanoma antigens (Ags) proposed as targets for immunotherapeutics include tyrosinase, NY-ESO-1, gp-100, and Mart-1, all of which contain both human leukocyte antigen (HLA) class I and class II-restricted epitopes necessary for immune recognition. We have previously shown that an enzyme, gamma-IFN-inducible lysosomal thiol-reductase (GILT), is abundantly expressed in professional Ag presenting cells (APCs), but absent or expressed at greatly reduced levels in many human melanomas. In the current study, we report that increased GILT expression generates a greater pool of antigenic peptides in melanoma cells for enhanced CD4+ T cell recognition. Our results suggest that the induction of GILT in human melanoma cells could aid in the development of a novel whole-cell vaccine for the enhancement of immune recognition of metastatic melanoma.

Zonal regulation of collagen-type proteins and posttranslational modifications in prostatic benign and cancer tissues by imaging mass spectrometry.

BACKGROUND: The emergence of reactive stroma is a hallmark of prostate cancer (PCa) progression and a potential source for prognostic and diagnostic markers of PCa. Collagen is a main component of reactive stroma and changes systematically and quantitatively to reflect the course of PCa, yet has remained undefined due to a lack of tools that can define collagen protein structure. Here we use a novel collagen-targeting proteomics approach to investigate zonal regulation of collagen-type proteins in PCa prostatectomies. METHODS: Prostatectomies from nine patients were divided into zones containing 0%, 5%, 20%, 70% to 80% glandular tissue and 0%, 5%, 25%, 70% by mass of PCa tumor following the McNeal model. Tissue sections from zones were graded by a pathologist for Gleason score, percent tumor present, percent prostatic intraepithelial neoplasia and/or inflammation (INF). High-resolution accurate mass collagen targeting proteomics was done on a select subset of tissue sections from patient-matched tumor or nontumor zones. Imaging mass spectrometry was used to investigate collagen-type regulation corresponding to pathologist-defined regions. RESULTS: Complex collagen proteomes were detected from all zones. COL17A and COL27A increased in zones of INF compared with zones with tumor present. COL3A1, COL4A5, and COL8A2 consistently increased in zones with tumor content, independent of tumor size. Collagen hydroxylation of proline (HYP) was altered in tumor zones compared with zones with INF and no tumor. COL3A1 and COL5A1 showed significant changes in HYP peptide ratios within tumor compared with zones of INF (2.59 ± 0.29, P value: .015; 3.75 ± 0.96 P value .036, respectively). By imaging mass spectrometry COL3A1 showed defined localization and regulation to tumor pathology. COL1A1 and COL1A2 showed gradient regulation corresponding to PCa pathology across zones. Pathologist-defined tumor regions showed significant increases in COL1A1 HYP modifications compared with COL1A2 HYP modifications. Certain COL1A1 and COL1A2 peptides could discriminate between pathologist-defined tumor and inflammatory regions. CONCLUSIONS: Site-specific posttranslational regulation of collagen structure by proline hydroxylation may be involved in reactive stroma associated with PCa progression. Translational and posttranslational regulation of collagen protein structure has potential for new markers to understand PCa progression and outcomes.

Voltage-Dependent Anion Channels Influence Cytotoxicity of ME-344, a Therapeutic Isoflavone.

ME-344 is a second-generation cytotoxic isoflavone with anticancer activity promulgated through interference with mitochondrial functions. Using a click chemistry version of the drug together with affinity-enriched mass spectrometry, voltage-dependent anion channels (VDACs) 1 and 2 were identified as drug targets. To determine the importance of VDAC1 or 2 to cytotoxicity, we used lung cancer cells that were either sensitive (H460) or intrinsically resistant (H596) to the drug. In H460 cells, depletion of VDAC1 and VDAC2 by small interfering RNA impacted ME-344 effects by diminishing generation of reactive oxygen species (ROS), preventing mitochondrial membrane potential dissipation, and moderating ME-344-induced cytotoxicity and mitochondrial-mediated apoptosis. Mechanistically, VDAC1 and VDAC2 knockdown prevented ME-344-induced apoptosis by inhibiting Bax mitochondrial translocation and cytochrome c release as well as apoptosis in these H460 cells. We conclude that VDAC1 and 2, as mediators of the response to oxidative stress, have roles in modulating ROS generation, Bax translocation, and cytochrome c release during mitochondrial-mediated apoptosis caused by ME-344. SIGNIFICANCE STATEMENT: Dissecting preclinical drug mechanisms are of significance in development of a drug toward eventual Food and Drug Administration approval.

Exploring G protein-coupled receptor signaling networks using SILAC-based phosphoproteomics.

The type 1 parathyroid hormone receptor (PTH1R) is a key regulator of calcium homeostasis and bone turnover. Here, we employed SILAC-based quantitative mass spectrometry and bioinformatic pathways analysis to examine global changes in protein phosphorylation following short-term stimulation of endogenously expressed PTH1R in osteoblastic cells in vitro. Following 5min exposure to the conventional agonist, PTH(1-34), we detected significant changes in the phosphorylation of 224 distinct proteins. Kinase substrate motif enrichment demonstrated that consensus motifs for PKA and CAMK2 were the most heavily upregulated within the phosphoproteome, while consensus motifs for mitogen-activated protein kinases were strongly downregulated. Signaling pathways analysis identified ERK1/2 and AKT as important nodal kinases in the downstream network and revealed strong regulation of small GTPases involved in cytoskeletal rearrangement, cell motility, and focal adhesion complex signaling. Our data illustrate the utility of quantitative mass spectrometry in measuring dynamic changes in protein phosphorylation following GPCR activation.

Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion.

Alternative mRNA splicing is a mechanism to regulate protein isoform expression and is regulated by alternative splicing factors. The alternative splicing factor 45 (SPF45) is overexpressed in cancer, although few biological effects of SPF45 are known, and few splicing targets have been identified. We previously showed that Extracellular Regulated Kinase 2 (ERK2) phosphorylation of SPF45 regulates cell proliferation and adhesion to fibronectin. In this work, we show that Cdc2-like kinase 1 (Clk1) phosphorylates SPF45 on eight serine residues. Clk1 expression enhanced, whereas Clk1 inhibition reduced, SPF45-induced exon 6 exclusion from Fas mRNA. Mutational analysis of the Clk1 phosphorylation sites on SPF45 showed both positive and negative regulation of splicing, with a net effect of inhibiting SPF45-induced exon 6 exclusion, correlating with reduced Fas mRNA binding. However, Clk1 enhanced SPF45 protein expression, but not mRNA expression, whereas inhibition of Clk1 increased SPF45 degradation through a proteasome-dependent pathway. Overexpression of SPF45 or a phospho-mimetic mutant, but not a phospho-inhibitory mutant, stimulated ovarian cancer cell migration and invasion, correlating with increased fibronectin expression, ERK activation and enhanced splicing and phosphorylation of full-length cortactin. Our results demonstrate for the first time that SPF45 overexpression enhances cell migration and invasion, dependent on biochemical regulation by Clk1.

Disruption of HLA class II antigen presentation in Burkitt lymphoma: implication of a 47,000 MW acid labile protein in CD4+ T-cell recognition.

While Burkitt lymphoma (BL) has a well-known defect in HLA class I-mediated antigen presentation, the exact role of BL-associated HLA class II in generating a poor CD4(+) T-cell response remains unresolved. Here, we found that BL cells are deficient in their ability to optimally stimulate CD4(+) T cells via the HLA class II pathway. This defect in CD4(+) T-cell recognition was not associated with low levels of co-stimulatory molecules on BL cells, as addition of external co-stimulation failed to elicit CD4(+) T-cell activation by BL. Further, the defect was not caused by faulty antigen/class II interaction, because antigenic peptides bound with measurable affinity to BL-associated class II molecules. Interestingly, functional class II-peptide complexes were formed at acidic pH 5·5, which restored immune recognition. Acidic buffer (pH 5·5) eluate from BL cells contained molecules that impaired class II-mediated antigen presentation and CD4(+) T-cell recognition. Biochemical analysis showed that these molecules were greater than 30,000 molecular weight in size, and proteinaceous in nature. In addition, BL was found to have decreased expression of a 47,000 molecular weight enolase-like molecule that enhances class II-mediated antigen presentation in B cells, macrophages and dendritic cells, but not in BL cells. These findings demonstrate that BL likely has multiple defects in HLA class II-mediated antigen presentation and immune recognition, which may be exploited for future immunotherapies.

PIM1 phosphorylates ABI2 to enhance actin dynamics and promote tumor invasion.

Distinguishing key factors that drive the switch from indolent to invasive disease will make a significant impact on guiding the treatment of prostate cancer (PCa) patients. Here, we identify a novel signaling pathway linking hypoxia and PIM1 kinase to the actin cytoskeleton and cell motility. An unbiased proteomic screen identified Abl-interactor 2 (ABI2), an integral member of the wave regulatory complex (WRC), as a PIM1 substrate. Phosphorylation of ABI2 at Ser183 by PIM1 increased ABI2 protein levels and enhanced WRC formation, resulting in increased protrusive activity and cell motility. Cell protrusion induced by hypoxia and/or PIM1 was dependent on ABI2. In vivo smooth muscle invasion assays showed that overexpression of PIM1 significantly increased the depth of tumor cell invasion, and treatment with PIM inhibitors significantly reduced intramuscular PCa invasion. This research uncovers a HIF-1-independent signaling axis that is critical for hypoxia-induced invasion and establishes a novel role for PIM1 as a key regulator of the actin cytoskeleton.

AKAP signaling in reinstated cocaine seeking revealed by iTRAQ proteomic analysis.

To identify candidate proteins in the nucleus accumbens (NAc) as potential pharmacotherapeutic targets for treating cocaine addition, an 8-plex iTRAQ (isobaric tag for relative and absolute quantitation) proteomic screen was performed using NAc tissue obtained from rats trained to self-administer cocaine followed by extinction training. Compared with yoked-saline controls, 42 proteins in a postsynaptic density (PSD)-enriched subfraction of the NAc from cocaine-trained animals were identified as significantly changed. Among proteins of interest whose levels were identified as increased was AKAP79/150, the rat ortholog of human AKAP5, a PSD scaffolding protein that localizes signaling molecules to the synapse. Functional downregulation of AKAP79/150 by microinjecting a cell-permeable synthetic AKAP (A-kinase anchor protein) peptide into the NAc to disrupt AKAP-dependent signaling revealed that inhibition of AKAP signaling impaired the reinstatement of cocaine seeking. Reinstatement of cocaine seeking is thought to require upregulated surface expression of AMPA glutamate receptors, and the inhibitory AKAP peptide reduced the PSD content of protein kinase A (PKA) as well as surface expression of GluR1 in NAc. However, reduced surface expression was not associated with changes in PKA phosphorylation of GluR1. This series of experiments demonstrates that proteomic analysis provides a useful tool for identifying proteins that can regulate cocaine relapse and that AKAP proteins may contribute to relapse vulnerability by promoting increased surface expression of AMPA receptors in the NAc.

Site-specific microtubule-associated protein 4 dephosphorylation causes microtubule network densification in pressure overload cardiac hypertrophy.

In severe pressure overload-induced cardiac hypertrophy, a dense, stabilized microtubule network forms that interferes with cardiocyte contraction and microtubule-based transport. This is associated with persistent transcriptional up-regulation of cardiac alpha- and beta-tubulin and microtubule-stabilizing microtubule-associated protein 4 (MAP4). There is also extensive microtubule decoration by MAP4, suggesting greater MAP4 affinity for microtubules. Because the major determinant of this affinity is site-specific MAP4 dephosphorylation, we characterized this in hypertrophied myocardium and then assessed the functional significance of each dephosphorylation site found by mimicking it in normal cardiocytes. We first isolated MAP4 from normal and pressure overload-hypertrophied feline myocardium; volume-overloaded myocardium, which has an equal degree and duration of hypertrophy but normal functional and cytoskeletal properties, served as a control for any nonspecific growth-related effects. After cloning cDNA-encoding feline MAP4 and obtaining its deduced amino acid sequence, we characterized by mass spectrometry any site-specific MAP4 dephosphorylation. Solely in pressure overload-hypertrophied myocardium, we identified striking MAP4 dephosphorylation at Ser-472 in the MAP4 N-terminal projection domain and at Ser-924 and Ser-1056 in the assembly-promoting region of the C-terminal microtubule-binding domain. Site-directed mutagenesis of MAP4 cDNA was then used to switch each serine to non-phosphorylatable alanine. Wild-type and mutated cDNAs were used to construct adenoviruses; microtubule network density, stability, and MAP4 decoration were assessed in normal cardiocytes following an equivalent level of MAP4 expression. The Ser-924 --> Ala MAP4 mutant produced a microtubule phenotype indistinguishable from that seen in pressure overload hypertrophy, such that Ser-924 MAP4 dephosphorylation during pressure overload hypertrophy may be central to this cytoskeletal abnormality.

HLA class II defects in Burkitt lymphoma: bryostatin-1-induced 17 kDa protein restores CD4+ T-cell recognition.

While the defects in HLA class I-mediated Ag presentation by Burkitt lymphoma (BL) have been well documented, CD4+ T-cells are also poorly stimulated by HLA class II Ag presentation, and the reasons underlying this defect(s) have not yet been fully resolved. Here, we show that BL cells are deficient in their ability to optimally stimulate CD4+ T cells via the HLA class II pathway. The observed defect was not associated with low levels of BL-expressed costimulatory molecules, as addition of external co-stimulation failed to result in BL-mediated CD4+ T-cell activation. We further demonstrate that BL cells express the components of the class II pathway, and the defect was not caused by faulty Ag/class II interaction, because antigenic peptides bound with measurable affinity to BL-associated class II molecules. Treatment of BL with broystatin-1, a potent modulator of protein kinase C, led to significant improvement of functional class II Ag presentation in BL. The restoration of immune recognition appeared to be linked with an increased expression of a 17 kDa peptidylprolyl-like protein. These results demonstrate the presence of a specific defect in HLA class II-mediated Ag presentation in BL and reveal that treatment with bryostatin-1 could lead to enhanced immunogenicity.

Defining the Tumor Microenvironment by Integration of Immunohistochemistry and Extracellular Matrix Targeted Imaging Mass Spectrometry.

Breast stroma plays a significant role in breast cancer risk and progression yet remains poorly understood. In breast stroma, collagen is the most abundantly expressed protein and its increased deposition and alignment contributes to progression and poor prognosis. Collagen post-translation modifications such as hydroxylated-proline (HYP) control deposition and stromal organization. The clinical relevance of collagen HYP site modifications in cancer processes remains undefined due to technical issues accessing collagen from formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a targeted approach for investigating collagen and other extracellular matrix proteins from FFPE tissue. Here, we hypothesized that immunohistochemistry staining for fibroblastic markers would not interfere with targeted detection of collagen stroma peptides and could reveal peptide regulation influenced by specific cell types. Our initial work demonstrated that stromal peptide peak intensities when using MALD-IMS following IHC staining (αSMA, FAP, P4HA3 and PTEN) were comparable to serial sections of nonstained tissue. Analysis of histology-directed IMS using PTEN on breast tissues and TMAs revealed heterogeneous PTEN staining patterns and suggestive roles in stromal protein regulation. This study sets the foundation for investigations of target cell types and their unique contribution to collagen regulation within extracellular matrix niches.

Olfactory cleft mucus proteome in chronic rhinosinusitis: a case-control pilot study.

BACKGROUND: Mechanisms of smell loss in chronic rhinosinusitis (CRS) are still unclear and likely multifactorial. Little attention has been given to olfactory cleft (OC) mucus proteins involved in odorant binding and metabolizing enzymes and their potential role in smell loss. METHODS: Mucus from the OC was sampled from patients with CRS (n = 20) and controls (n = 10). Liquid chromatography and mass spectrometry were performed, followed by data processing so that protein groups could be identified, quantified, and compared. Hierarchical clustering and bioinformatic analysis were performed on significantly different proteins to explore for enrichment in known biologic pathways. RESULTS: A total of 2514 proteins were found in OC mucus from all 30 subjects. Significant differences in protein abundance were found between CRS and controls, including both CRSsNP (n = 351 proteins; log2 fold change range: -3.88 to 6.71) and CRSwNP (n = 298 proteins; log2 fold change range: -4.00 to -6.13). Significant differences were found between patients with normosmia and those with dysosmia (n = 183; log2 fold change range: -3.62 to -2.16) and across groups of interest for a number of odorant binding proteins and metabolizing enzymes. CONCLUSION: OC mucous in CRS displays a rich and abundant array of proteins, many of which have been implicated in odorant transport and metabolization in animal studies. Significant differences in the olfactory mucus proteome were seen between CRS subtypes and controls, as well as between those with normal and abnormal olfaction. Further study should confirm these findings and explore the role individual proteins play in odorant transport and metabolization. ©2020 ARSAAOA, LLC.

Collagen fiber regulation in human pediatric aortic valve development and disease.

Congenital aortic valve stenosis (CAVS) affects up to 10% of the world population without medical therapies to treat the disease. New molecular targets are continually being sought that can halt CAVS progression. Collagen deregulation is a hallmark of CAVS yet remains mostly undefined. Here, histological studies were paired with high resolution accurate mass (HRAM) collagen-targeting proteomics to investigate collagen fiber production with collagen regulation associated with human AV development and pediatric end-stage CAVS (pCAVS). Histological studies identified collagen fiber realignment and unique regions of high-density collagen in pCAVS. Proteomic analysis reported specific collagen peptides are modified by hydroxylated prolines (HYP), a post-translational modification critical to stabilizing the collagen triple helix. Quantitative data analysis reported significant regulation of collagen HYP sites across patient categories. Non-collagen type ECM proteins identified (26 of the 44 total proteins) have direct interactions in collagen synthesis, regulation, or modification. Network analysis identified BAMBI (BMP and Activin Membrane Bound Inhibitor) as a potential upstream regulator of the collagen interactome. This is the first study to detail the collagen types and HYP modifications associated with human AV development and pCAVS. We anticipate that this study will inform new therapeutic avenues that inhibit valvular degradation in pCAVS and engineered options for valve replacement.

Extracellular matrix alterations in low-grade lung adenocarcinoma compared with normal lung tissue by imaging mass spectrometry.

Lung adenocarcinoma (LUAD) is the second most common cancer, affecting both men and women. Fibrosis is a hallmark of LUAD occurring throughout progression with excess production of extracellular matrix (ECM) components that lead to metastatic cell processes. Understanding the ECM cues that drive LUAD progression has been limited due to a lack of tools that can access and report on ECM components within the complex tumor microenvironment. Here, we test whether low-grade LUAD can be distinguished from normal lung tissue using a novel ECM imaging mass spectrometry (ECM IMS) approach. ECM IMS analysis of a tissue microarray with 20 low-grade LUAD tissues and 20 normal lung samples from 10 patients revealed 25 peptides that could discriminate between normal and low-grade LUAD using area under the receiver-operating curve (AUC) ≥0.7, P value ≤.001. Principal component analysis demonstrated that 62.4% of the variance could be explained by sample origin from normal or low-grade tumor tissue. Additional work performed on a wedge resection with moderately differentiated LUAD demonstrated that the ECM IMS analytical approach could distinguish LUAD spectral features from spectral features of normal adjacent lung tissue. Conventional liquid chromatography with tandem mass spectrometry (LC-MS/MS) proteomics demonstrated that specific sites of hydroxylation of proline (HYP) were a main collagen post translational modification that was readily detected in LUAD. A distinct peptide from collagen 3A1 modified by HYP was increased 3.5 fold in low-grade LUAD compared with normal lung tissue (AUC 0.914, P value <.001). This suggests that regulation of collagen proline hydroxylation could be an important process during early LUAD fibrotic deposition. ECM IMS is a useful tool that may be used to define fibrotic deposition in low-grade LUAD.

Isoflavone ME-344 Disrupts Redox Homeostasis and Mitochondrial Function by Targeting Heme Oxygenase 1.

ME-344 is a second-generation isoflavone with unusual cytotoxic properties that is in clinical testing in cancer. To identify targets that contribute to its anticancer activity and therapeutic index, we used lung cancer cell lines that are naturally sensitive or resistant to ME-344. Drug-induced apoptosis was linked with enhanced levels of reactive oxygen species and this initiated a nuclear erythroid factor 2-like 2 signaling response, downstream of which, heme oxygenase 1 (HO-1) was also found to be time-dependently inhibited by ME-344. ME-344 specifically bound to, and altered, HO-1 structure and increased HO-1 translocation from the rough endoplasmic reticulum to mitochondria, but only in drug-sensitive cells. These effects did not occur in either drug-resistant or primary lung fibroblasts with lower HO-1 basal levels. HO-1 was confirmed as a drug target by using surface plasmon resonance technology and through interaction with a clickable ME-344 compound (M2F) and subsequent proteomic analyses, showing direct binding of ME-344 with HO-1. Proteomic analysis showed that clusters of mitochondrial proteins, including voltage-dependent anion-selective channels, were also impacted by ME-344. Human lung cancer biopsies expressed higher levels of Nrf2 and HO-1 compared with normal tissues. Overall, our data show that ME-344 inhibits HO-1 and impacts its mitochondrial translocation. Other mitochondrial proteins are also affected, resulting in interference in tumor cell redox homeostasis and mitochondrial function. These factors contribute to a beneficial therapeutic index and support continued clinical development of ME-344. SIGNIFICANCE: A novel cytotoxic isoflavone is shown to inhibit heme oxygenase, a desirable yet elusive target that disrupts redox homeostasis causing cell death.

Case of desmoplastic infantile ganglioglioma secreting ceruloplasmin.

Desmoplastic infantile ganglioglioma is a rare World Health Organization (WHO) grade I tumor commonly arising in early infancy and usually presenting with both solid and cystic components. We report a case of a large midline-enhancing desmoplastic infantile ganglioglioma in which newly formed cysts in communication with lateral ventricles contained highly proteinaceous fluid. Proteomic analysis of the fluid showed three proteins not normally found in cerebrospinal fluid. Immunohistochemical analysis of the tumor sample showed that the desmoplastic infantile ganglioglioma produced a high concentration of ceruloplasmin, which probably accounts for most of the 30- to 40-fold increase in protein compared with normal cerebrospinal fluid. To our knowledge, this is the first report of ceruloplasmin secretion by a brain tumor, and ongoing studies on the mechanism might yield novel approaches to reducing cyst production and protein content in an otherwise stable solid tumor.

Identification of phosphorylation sites on the E3 ubiquitin ligase UBR5/EDD.

UBR5 (ubiquitin protein ligase E3 component n-recognin 5)/EDD (E3 ligase identified by differential display) is an E3 ubiquitin ligase that is a potential biomarker for poor prognosis for recurrent, platinum-resistant ovarian cancer. UBR5 has a role in the DNA damage response and many such proteins are regulated by phosphorylation. UBR5 is a 309 kDa nuclear phosphoprotein that we previously identified as a substrate of the MAP kinase ERK2. With its 477 potential phosphorylation sites, little is known about UBR5 phosphorylation and how it may regulate protein function. Currently, thirty-four sites of phosphorylation on UBR5 have been reported in the literature, mostly identified by large scale proteomics studies of tissues or of cells after various treatments; however, no studies have specifically targeted the identification of UBR5 phosphorylation sites. In this study, we used Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to obtain a total sequence coverage of 64.3% from combining tryptic and GluC digests on UBR5 isolated from transfected COS-1 cells. We identified 24 sites of phosphorylation, 18 of which are novel sites. This data enhances our knowledge of UBR5 phosphorylation and provides a framework for the study of how phosphorylation affects UBR5 function.

Proteomic analysis of CTGF-activated lung fibroblasts: identification of IQGAP1 as a key player in lung fibroblast migration.

Connective tissue growth factor (CTGF, CCN2) is overexpressed in lung fibroblasts isolated from patients with interstitial lung disease (ILD) and systemic sclerosis (SSc, scleroderma) and is considered to be a molecular marker of fibrosis. To understand the significance of elevated CTGF, we investigated the changes in lung fibroblast proteome in response to CTGF overexpression. Using 2-dimensional gel electrophoresis followed by in-gel proteolytic digestion and mass spectrometric analysis, we identified 13 proteins affected by CTGF. Several of the CTGF-induced proteins, such as pro-alpha (I) collagen and cytoskeletal proteins vinculin, moesin, and ezrin, are known to be elevated in pulmonary fibrosis, whereas 9 of 13 proteins have not been studied in pulmonary fibrosis and are, therefore, novel CTGF-responsive molecules that may have important roles in ILD. Our study demonstrates that 1 of the novel CTGF-induced proteins, IQ motif containing GTPase activating protein (IQGAP) 1, is elevated in lung fibroblasts isolated from scleroderma patients with ILD. IQGAP1 is a scaffold protein that plays a pivotal role in regulating migration of endothelial and epithelial cells. Scleroderma lung fibroblasts and normal lung fibroblasts treated with CTGF demonstrated increased rate of migration in a wound healing assay. Depletion of IQGAP1 expression by small interfering RNA inhibited CTGF-induced migration and MAPK ERK1/2 phosphorylation in lung fibroblasts. MAPK inhibitor U0126 decreased CTGF-induced cell migration and did not interfere with CTGF-induced IQGAP1 expression, suggesting that MAPK pathway is downstream of IQGAP1. These findings further implicate the importance of CTGF in lung tissue repair and fibrosis and propose that CTGF-induced migration of lung fibroblasts to the damaged tissue is mediated via IQGAP1 and MAPK signaling pathways.

Succination of protein thiols during adipocyte maturation: a biomarker of mitochondrial stress.

Although obesity is a risk factor for development of type 2 diabetes and chemical modification of proteins by advanced glycoxidation and lipoxidation end products is implicated in the development of diabetic complications, little is known about the chemical modification of proteins in adipocytes or adipose tissue. In this study we show that S-(2-succinyl)cysteine (2SC), the product of chemical modification of proteins by the Krebs cycle intermediate, fumarate, is significantly increased during maturation of 3T3-L1 fibroblasts to adipocytes. Fumarate concentration increased > or =5-fold during adipogenesis in medium containing 30 mm glucose, producing a > or =10-fold increase in 2SC-proteins in adipocytes compared with undifferentiated fibroblasts grown in the same high glucose medium. The elevated glucose concentration in the medium during adipocyte maturation correlated with the increase in 2SC, whereas the concentration of the advanced glycoxidation and lipoxidation end products, N(epsilon)-(carboxymethyl)lysine and N(epsilon)-(carboxyethyl)lysine, was unchanged under these conditions. Adipocyte proteins were separated by one- and two-dimensional electrophoresis and approximately 60 2SC-proteins were detected using an anti-2SC polyclonal antibody. Several of the prominent and well resolved proteins were identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry. These include cytoskeletal proteins, enzymes, heat shock and chaperone proteins, regulatory proteins, and a fatty acid-binding protein. We propose that the increase in fumarate and 2SC is the result of mitochondrial stress in the adipocyte during adipogenesis and that 2SC may be a useful biomarker of mitochondrial stress in obesity, insulin resistance, and diabetes.