Timothy D. Cummins

M-Type Phospholipase A2 Receptor as Target Antigen in Idiopathic Membranous Nephropathy

BACKGROUND Idiopathic membranous nephropathy, a common form of the nephrotic syndrome, is an antibody-mediated autoimmune glomerular disease. Serologic diagnosis has been elusive because the target antigen is unknown. METHODS We performed Western blotting of protein extracts from normal human glomeruli with serum samples from patients with idiopathic or secondary membranous nephropathy or other proteinuric or autoimmune diseases and from normal controls. We used mass spectrometry to analyze the reactive protein bands and confirmed the identity and location of the target antigen with a monospecific antibody. RESULTS Serum samples from 26 of 37 patients (70%) with idiopathic but not secondary membranous nephropathy specifically identified a 185-kD glycoprotein in nonreduced glomerular extract. Mass spectrometry of the reactive protein band detected the M-type phospholipase A(2) receptor (PLA(2)R). Reactive serum specimens recognized recombinant PLA(2)R and bound the same 185-kD glomerular protein as did the monospecific anti-PLA(2)R antibody. Anti-PLA(2)R autoantibodies in serum samples from patients with membranous nephropathy were mainly IgG4, the predominant immunoglobulin subclass in glomerular deposits. PLA(2)R was expressed in podocytes in normal human glomeruli and colocalized with IgG4 in immune deposits in glomeruli of patients with membranous nephropathy. IgG eluted from such deposits in patients with idiopathic membranous nephropathy, but not in those with lupus membranous or IgA nephropathy, recognized PLA(2)R. CONCLUSIONS A majority of patients with idiopathic membranous nephropathy have antibodies against a conformation-dependent epitope in PLA(2)R. PLA(2)R is present in normal podocytes and in immune deposits in patients with idiopathic membranous nephropathy, indicating that PLA(2)R is a major antigen in this disease.

Proteomic and functional characterisation of platelet microparticle size classes

UNLABELLED Activated platelets release large lipid-protein complexes termed microparticles. These platelet microparticles (PMP) are composed of vesicular fragments of the plasma membrane and alpha-granules. PMP facilitate coagulation, promote platelet and leukocyte adhesion to the subendothelial matrix, support angiogenesis and stimulate vascular smooth muscle proliferation. OBJECTIVES PMP were separated into 4 size classes to facilitate identification of active protein and lipid components. PMP were obtained from activated human platelets and separated into 4 size classes by gel filtration chromatography. Proteins were identified using 2-dimensional, liquid chromatography tandem mass spectrometry. Functional effects on platelets were determined using the PFA-100 and on endothelial cells by measuring transendothelial cell electrical resistance. PMP size classes differed significantly in their contents of plasma membrane receptors and adhesion molecules, chemokines, growth factors and protease inhibitors. The two smallest size classes (3 and 4) inhibited collagen/adenosine-diphosphate-mediated platelet thrombus formation, while fractions 2 and 4 stimulated barrier formation by endothelial cells. Heat denaturation blocked the effect of fraction 4 on endothelial cell function, but not fraction 2 implying that the active component in fraction 4 is a protein and in fraction 2 is a heat-stable protein or lipid but not sphingosine-1-phosphate. Proteomic and functional analysis of PMP size fractions has shown that PMP can be separated into different size classes that differ in protein components, protein/lipid ratio, and functional effects on platelets and endothelial cells. This analysis will facilitate identification of active components in the PMP and clarify their involvement in diseases such as atherosclerosis and cancer.

Microfiltration isolation of human urinary exosomes for characterization by MS

Purpose: The purpose of this study was to address the hypothesis that small vesicular urinary particles known as exosomes could be selectively microfiltered using low protein‐binding size exclusion filters, thereby simplifying their use in clinical biomarker discovery studies.

Overexpression of Endothelial Nitric Oxide Synthase Prevents Diet-Induced Obesity and Regulates Adipocyte Phenotype

Rationale: Endothelial dysfunction is a characteristic feature of diabetes and obesity in animal models and humans. Deficits in nitric oxide production by endothelial nitric oxide synthase (eNOS) are associated with insulin resistance, which is exacerbated by high-fat diet. Nevertheless, the metabolic effects of increasing eNOS levels have not been studied. Objective: The current study was designed to test whether overexpression of eNOS would prevent diet-induced obesity and insulin resistance. Methods and Results: In db/db mice and in high-fat diet-fed wild-type C57BL/6J mice, the abundance of eNOS protein in adipose tissue was decreased without significant changes in eNOS levels in skeletal muscle or aorta. Mice overexpressing eNOS (eNOS transgenic mice) were resistant to diet-induced obesity and hyperinsulinemia, although systemic glucose intolerance remained largely unaffected. In comparison with wild-type mice, high-fat diet-fed eNOS transgenic mice displayed a higher metabolic rate and attenuated hypertrophy of white adipocytes. Overexpression of eNOS did not affect food consumption or diet-induced changes in plasma cholesterol or leptin levels, yet plasma triglycerides and fatty acids were decreased. Metabolomic analysis of adipose tissue indicated that eNOS overexpression primarily affected amino acid and lipid metabolism; subpathway analysis suggested changes in fatty acid oxidation. In agreement with these findings, adipose tissue from eNOS transgenic mice showed higher levels of PPAR-&agr; and PPAR-&ggr; gene expression, elevated abundance of mitochondrial proteins, and a higher rate of oxygen consumption. Conclusions: These findings demonstrate that increased eNOS activity prevents the obesogenic effects of high-fat diet without affecting systemic insulin resistance, in part, by stimulating metabolic activity in adipose tissue.

PDGF-mediated autophagy regulates vascular smooth muscle cell phenotype and resistance to oxidative stress

Vascular injury and chronic arterial diseases result in exposure of VSMCs (vascular smooth muscle cells) to increased concentrations of growth factors. The mechanisms by which growth factors trigger VSMC phenotype transitions remain unclear. Because cellular reprogramming initiated by growth factors requires not only the induction of genes involved in cell proliferation, but also the removal of contractile proteins, we hypothesized that autophagy is an essential modulator of VSMC phenotype. Treatment of VSMCs with PDGF (platelet-derived growth factor)-BB resulted in decreased expression of the contractile phenotype markers calponin and α-smooth muscle actin and up-regulation of the synthetic phenotype markers osteopontin and vimentin. Autophagy, as assessed by LC3 (microtubule-associated protein light chain 3 α; also known as MAP1LC3A)-II abundance, LC3 puncta formation and electron microscopy, was activated by PDGF exposure. Inhibition of autophagy with 3-methyladenine, spautin-1 or bafilomycin stabilized the contractile phenotype. In particular, spautin-1 stabilized α-smooth muscle cell actin and calponin in PDGF-treated cells and prevented actin filament disorganization, diminished production of extracellular matrix, and abrogated VSMC hyperproliferation and migration. Treatment of cells with PDGF prevented protein damage and cell death caused by exposure to the lipid peroxidation product 4-hydroxynonenal. The results of the present study demonstrate a distinct form of autophagy induced by PDGF that is essential for attaining the synthetic phenotype and for survival under the conditions of high oxidative stress found to occur in vascular lesions.

Metabolomic Analysis of Pressure-overloaded and Infarcted Mouse Hearts

Background—Cardiac hypertrophy and heart failure are associated with metabolic dysregulation and a state of chronic energy deficiency. Although several disparate changes in individual metabolic pathways have been described, there has been no global assessment of metabolomic changes in hypertrophic and failing hearts in vivo. Hence, we investigated the impact of pressure overload and infarction on myocardial metabolism. Methods and Results—Male C57BL/6J mice were subjected to transverse aortic constriction or permanent coronary occlusion (myocardial infarction [MI]). A combination of LC/MS/MS and GC/MS techniques was used to measure 288 metabolites in these hearts. Both transverse aortic constriction and MI were associated with profound changes in myocardial metabolism affecting up to 40% of all metabolites measured. Prominent changes in branched-chain amino acids were observed after 1 week of transverse aortic constriction and 5 days after MI. Changes in branched-chain amino acids after MI were associated with myocardial insulin resistance. Longer duration of transverse aortic constriction and MI led to a decrease in purines, acylcarnitines, fatty acids, and several lysolipid and sphingolipid species but a marked increase in pyrimidines as well as ascorbate, heme, and other indices of oxidative stress. Cardiac remodeling and contractile dysfunction in hypertrophied hearts were associated with large increases in myocardial, but not plasma, levels of the polyamines putrescine and spermidine as well as the collagen breakdown product prolylhydroxyproline. Conclusions—These findings reveal extensive metabolic remodeling common to both hypertrophic and failing hearts that are indicative of extracellular matrix remodeling, insulin resistance and perturbations in amino acid, and lipid and nucleotide metabolism.

Metabolic remodeling of white adipose tissue in obesity

Adipose tissue metabolism is a critical regulator of adiposity and whole body energy expenditure; however, metabolic changes that occur in white adipose tissue (WAT) with obesity remain unclear. The purpose of this study was to understand the metabolic and bioenergetic changes occurring in WAT with obesity. Wild-type (C57BL/6J) mice fed a high-fat diet (HFD) showed significant increases in whole body adiposity, had significantly lower V̇(O₂), V̇(CO₂), and respiratory exchange ratios, and demonstrated worsened glucose and insulin tolerance compared with low-fat-fed mice. Metabolomic analysis of WAT showed marked changes in lipid, amino acid, carbohydrate, nucleotide, and energy metabolism. Tissue levels of succinate and malate were elevated, and metabolites that could enter the Krebs cycle via anaplerosis were mostly diminished in high-fat-fed mice, suggesting altered mitochondrial metabolism. Despite no change in basal oxygen consumption or mitochondrial DNA abundance, citrate synthase activity was decreased by more than 50%, and responses to FCCP were increased in WAT from mice fed a high-fat diet. Moreover, Pgc1a was downregulated and Cox7a1 upregulated after 6 wk of HFD. After 12 wk of high-fat diet, the abundance of several proteins in the mitochondrial respiratory chain or matrix was diminished. These changes were accompanied by increased Parkin and Pink1, decreased p62 and LC3-I, and ultrastructural changes suggestive of autophagy and mitochondrial remodeling. These studies demonstrate coordinated restructuring of metabolism and autophagy that could contribute to the hypertrophy and whitening of adipose tissue in obesity.

Comparison of Proteins Expressed on Secretory Vesicle Membranes and Plasma Membranes of Human Neutrophils

Secretory vesicles are neutrophil intracellular storage granules formed by endocytosis. Understanding the functional consequences of secretory vesicle exocytosis requires knowledge of their membrane proteins. The current study was designed to use proteomic technologies to develop a more complete catalog of secretory vesicle membrane proteins and to compare the proteomes of secretory vesicle and plasma membranes. A total of 1118 proteins were identified, 573 (51%) were present only in plasma membrane-enriched fractions, 418 (37%) only in secretory vesicle-enriched membrane fractions, and 127 (11%) in both fractions. Gene Ontology categorized 373 of these proteins as integral membrane proteins. Proteins typically associated with other intracellular organelles, including nuclei, mitochondria, and ribosomes, were identified in both membrane fractions. Ingenuity Pathway Knowledge Base analysis determined that the majority of canonical and functional pathways were significantly associated with proteins from both plasma membrane-enriched and secretory vesicle-enriched fractions. There were, however, some canonical signaling pathways that involved proteins only from plasma membranes or secretory vesicles. In conclusion, a number of proteins were identified that may elucidate mechanisms and functional consequences of secretory vesicle exocytosis. The small number of common proteins suggests that the hypothesis that secretory vesicles are formed from plasma membranes by endocytosis requires more critical evaluation.

Yeast SREBP Cleavage Activation Requires the Golgi Dsc E3 Ligase Complex

Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1-4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2-conjugating enzyme Ubc4, the Dsc1 RING E3 ligase, and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation.

Proteomic analysis of renal calculi indicates an important role for inflammatory processes in calcium stone formation

Even though renal stones/calculi occur in approximately 10% of individuals, they are an enormous economic burden to the entire US health system. While the relative metabolic composition of renal calculi is generally known, there is no clear understanding of the genetics of renal stone formation, nor are there clear prognostic indicators of renal stone formation. The application of proteomics to the analysis of renal calculi axiomatically holds that insight into renal stone pathobiology can be gained by a more comprehensive understanding of renal calculus protein composition. We analyzed isolated renal stone matrix proteins with mass spectrometric and immunohistochemical methods identifying 158 proteins with high confidence, including 28 common proteins. The abundant proteins included those identified previously in stones and proteins identified here for the first time, such as myeloid lineage-specific, integral membrane and lipid regulatory proteins. Pathway analyses of all proteins identified suggested that a significant fraction of the most abundant matrix proteins participate in inflammatory processes. These proteomic results support the hypothesis that stone formation induces a cellular inflammatory response and the protein components of this response contribute to the abundant stone matrix proteome.