Zongming Fu

Milk Fat Globule Protein Epidermal Growth Factor-8. A Pivotal Relay Element Within the Angiotensin II and Monocyte Chemoattractant Protein-1 Signaling Cascade Mediating Vascular Smooth Muscle Cells Invasion

Advancing age induces aortic wall thickening that results from the concerted effects of numerous signaling proteins, many of which have yet to be identified. To search for novel proteins associated with aortic wall thickening, we have performed a comprehensive quantitative proteomic study to analyze aortic proteins from young (8 months) and old (30 months) rats and identified 50 proteins that significantly change in abundance with aging. One novel protein, the milk fat globule protein epidermal growth factor 8 (MFG-E8), increases 2.3-fold in abundance in old aorta. Transcription and translation analysis demonstrated that aortic MFG-E8 mRNA and protein levels increase with aging in several mammalian species including humans. Dual immunolabeling shows that MFG-E8 colocalizes with both angiotensin II and monocyte chemoattractant protein (MCP)-1 within vascular smooth muscle cells (VSMCs) of the thickened aged aortic wall. Exposure of early passage VSMCs from young aorta to angiotensin II markedly increases MFG-E8 and enhances invasive capacity to levels observed in VSMCs from old rats. Treatment of VSMCs with MFG-E8 increases MCP-1 expression and VSMCs invasion that are inhibited by the MCP-1 receptor blocker vCCI. Silencing MFG-E8 RNA substantially reduces MFG-E8 expression and VSMCs invasion capacity. The data indicate that arterial MFG-E8 significantly increases with aging and is a pivotal relay element within the angiotensin II/MCP-1/VSMC invasion signaling cascade. Thus, targeting of MFG-E8 within this signaling axis pathway is a potential novel therapy for the prevention and treatment of the age-associated vascular diseases such as atherosclerosis.

MFG-E8 activates proliferation of vascular smooth muscle cells via integrin signaling

An accumulation of milk fat globule EGF‐8 protein (MFG‐E8) occurs within the context of arterial wall inflammatory remodeling during aging, hypertension, diabetes mellitus, or atherosclerosis. MFG‐E8 induces VSMC invasion, but whether it affects VSMC proliferation, a salient feature of arterial inflammation, is unknown. Here, we show that in the rat arterial wall in vivo, PCNA and Ki67, markers of cell cycle activation, increase with age between 8 and 30 months. In fresh and early passage VSMC isolated from old aortae, an increase in CDK4 and PCNA, an increase in the acceleration of cell cycle S and G2 phases, decrease in the G1/G0 phase, and an increase in PDGF and its receptors confer elevated proliferative capacity, compared to young VSMC. Increased coexpression and physical interaction of MFG‐E8 and integrin αvβ5 occur with aging in both the rat aortic wall in vivo and in VSMC in vitro. In young VSMC in vitro, MFG‐E8 added exogenously, or overexpressed endogenously, triggers phosphorylation of ERK1/2, augmented levels of PCNA and CDK4, increased BrdU incorporation, and promotes proliferation, via αvβ5 integrins. MFG‐E8 silencing, or its receptor inhibition, or the blockade of ERK1/2 phosphorylation in these cells reduces PCNA and CDK4 levels and decelerates the cell cycle S phase, conferring a reduction in proliferative capacity. Collectively, these results indicate that MFG‐E8 in a dose‐dependent manner coordinates the expression of cell cycle molecules and facilitates VSMC proliferation via integrin/ERK1/2 signaling. Thus, an increase in MFG‐E8 signaling is a mechanism of the age‐associated increase in aortic VSMC proliferation.

Vinculin network-mediated cytoskeletal remodeling regulates contractile function in the aging heart.

Cardiac vinculin overexpression is a conserved aging response that is associated with enhanced myocardial performance and extended organismal life span. Sending in vinculin reinforcements A common charge for graceful aging is to stay “young at heart.” With age, the heart undergoes necessary remodeling to keep it functioning—or young—even though the heart experiences relatively little regeneration in the human lifetime. The mechanisms of remodeling in mammals remain unclear but, if known, could help develop new therapies to treat heart failure, a leading killer in the developed world. Kaushik et al. therefore performed a proteomic analysis in old and young monkeys and rats, and identified one protein at the heart of it all: vinculin. Vinculin is conserved across species, being present at cell-matrix and cell-cell adhesions and also anchoring the cardiomyocyte membrane to its actin cytoskeleton. Thus, Kaushik et al. hypothesized that vinculin accumulates with age to regulate cytoskeletal stiffening and heart cell contractility. This mechanism was confirmed in rats and in different strains of Drosophila, supporting the notion that particular aspects of heart remodeling are beneficial and prolong life span, rather than being maladaptive. By using several models and producing a large proteomic network centered on vinculin and other cytoskeletal proteins, the authors have put forth a valuable resource for better understanding cardiac aging and for selecting therapeutic targets to prevent heart failure and also keep the heart young and beating as we age. The human heart is capable of functioning for decades despite minimal cell turnover or regeneration, suggesting that molecular alterations help sustain heart function with age. However, identification of compensatory remodeling events in the aging heart remains elusive. We present the cardiac proteomes of young and old rhesus monkeys and rats, from which we show that certain age-associated remodeling events within the cardiomyocyte cytoskeleton are highly conserved and beneficial rather than deleterious. Targeted transcriptomic analysis in Drosophila confirmed conservation and implicated vinculin as a unique molecular regulator of cardiac function during aging. Cardiac-restricted vinculin overexpression reinforced the cortical cytoskeleton and enhanced myofilament organization, leading to improved contractility and hemodynamic stress tolerance in healthy and myosin-deficient fly hearts. Moreover, cardiac-specific vinculin overexpression increased median life span by more than 150% in flies. A broad array of potential therapeutic targets and regulators of age-associated modifications, specifically for vinculin, are presented. These findings suggest that the heart has molecular mechanisms to sustain performance and promote longevity, which may be assisted by therapeutic intervention to ameliorate the decline of function in aging patient hearts.

Identification and characterization of citrulline-modified brain proteins by combining HCD and CID fragmentation

Citrullination is a protein PTM of arginine residues catalyzed by peptidylarginine deiminase. Protein citrullination has been detected in the CNS and associated with a number of neurological diseases. However, identifying citrullinated proteins from complex mixtures and pinpointing citrullinated residues have been limited. Using RP LC and high‐resolution MS, this study determined in vitro citrullination sites of glial fibrillary acid protein (GFAP), neurogranin (NRGN/RC3), and myelin basic protein (MBP) and in vivo sites in brain protein extract. Human GFAP has five endogenous citrullination sites, R30, R36, R270, R406, and R416, and MBP has 14 in vivo citrullination sites. Human NRGN/RC3 was found citrullinated at residue R68. The sequence of citrullinated peptides and citrullination sites were confirmed from peptides identified in trypsin, Lys‐C, and Glu‐C digests. The relative ratio of citrullination was estimated by simultaneous identification of citrullinated and unmodified peptides from Alzheimers and control brain samples. The site occupancy of citrullination at the residue R68 of NRGN ranged from 1.6 to 9.5%. Compared to CID, higher‐energy collisional dissociation (HCD) mainly produced protein backbone fragmentation for citrullinated peptides. CID‐triggered HCD fragmentation is an optimal approach for the identification of citrullinated peptides in complex protein digests.

Improved protein extraction and protein identification from archival formalin-fixed paraffin-embedded human aortas

Evaluate combination of heat and elevated pressure to enhance protein extraction and quality of formalin‐fixed (FF), and FF paraffin‐embedded (FFPE) aorta for proteomics.

S100A11 Mediates Hypoxia-induced Mitogenic Factor (HIMF)-induced Smooth Muscle Cell Migration, Vesicular Exocytosis, and Nuclear Activation

Hypoxia-induced mitogenic factor (HIMF) is a newly discovered protein that is up-regulated in murine models of pulmonary arterial hypertension and asthma. Our previous study shows that HIMF is a potent mitogenic, angiogenic, and vasoconstrictive chemokine associated with pulmonary arterial hypertension. Two-dimensional gel electrophoresis was used to investigate downstream molecules in HIMF-induced cell signaling, demonstrating that S100A11, an EF-hand calcium-binding protein, was exclusively altered and was decreased (2.7 ± 0.2-fold, p < 0.05) in pulmonary artery smooth muscle cells (SMCs) treated with HIMF for 5 min compared with untreated cells (n = 4). Immunofluorescence showed that in control cells S100A11 is a cytosolic protein, which then aggregates and translocates both to the plasma membrane with subsequent exocytosis and to the nucleus upon HIMF stimulation. Annexin A2, a known S100A11 binding partner, also colocalized with S100A11 during HIMF-induced membrane trafficking. To investigate the intracellular function of S100A11, siRNA was used to knock down S100A11 expression in SMCs. The S100A11 knockdown significantly reduced HIMF-induced SMC migration but did not affect the SMC mitogenic action of HIMF. Our data show that S100A11 mediates HIMF-induced smooth muscle cell migration, vesicular exocytosis, and nuclear activation.

Multiple and Selective Reaction Monitoring Using Triple Quadrupole Mass Spectrometer: Preclinical Large Cohort Analysis.

Multiple reaction monitoring (MRM), sometimes referred to as selective reaction monitoring (SRM), is a mass spectrometry method that can target selective peptides for the detection and quantitation of a protein. Compared to traditional ELISA, MRM assays have a number of advantages including ease in multiplexing several proteins in the same assay and independence from the necessity for high-quality, expensive, and at times unreliable antibodies. Furthermore, MRM assays can be developed to quantify multiple proteoforms of a single protein allowing the quantification of allelic expression of a particular sequence polymorphism, protein isoform, as well as determining site occupancy of posttranslational modification(s). In this chapter, we describe our workflow for target peptide selection, assay optimization, and acquisition multiplexing. Our workflow is presented using the example of constrained MRM assays developed for the serum protein ApoL1 in its various proteoforms to highlight the specific technical considerations necessary for the difficult task of quantifying peptide targets based on highly specific amino acid sequences by MRM.

Can proteomics yield insight into aging aorta

The aging aorta exhibits structural and physiological changes that are reflected in the proteome of its component cells types. The advance in proteomic technologies has made it possible to analyze the quantity of proteins associated with the natural history of aortic aging. These alterations reflect the molecular and cellular mechanisms of aging and could provide an opportunity to predict vascular health. This paper focuses on whether discoveries stemming from the application of proteomic approaches of the intact aging aorta or vascular smooth muscle cells can provide useful insights. Although there have been limited studies to date, a number of interesting proteins have been identified that are closely associated with aging in the rat aorta. Such proteins, including milk fat globule‐EGF factor 8, matrix metalloproteinase type‐2, and vitronectin, could be used as indicators of vascular health, or even explored as therapeutic targets for aging‐related vascular diseases.

Protein S-Nitrosylation Controls Glycogen Synthase Kinase 3β Function Independent of Its Phosphorylation State

Rationale: GSK-3&bgr; (glycogen synthase kinase 3&bgr;) is a multifunctional and constitutively active kinase known to regulate a myriad of cellular processes. The primary mechanism to regulate its function is through phosphorylation-dependent inhibition at serine-9 residue. Emerging evidence indicates that there may be alternative mechanisms that control GSK-3&bgr; for certain functions. Objectives: Here, we sought to understand the role of protein S-nitrosylation (SNO) on the function of GSK-3&bgr;. SNO-dependent modulation of the localization of GSK-3&bgr; and its ability to phosphorylate downstream targets was investigated in vitro, and the network of proteins differentially impacted by phospho- or SNO-dependent GSK-3&bgr; regulation and in vivo SNO modification of key signaling kinases during the development of heart failure was also studied. Methods and Results: We found that GSK-3&bgr; undergoes site-specific SNO both in vitro, in HEK293 cells, H9C2 myoblasts, and primary neonatal rat ventricular myocytes, as well as in vivo, in hearts from an animal model of heart failure and sudden cardiac death. S-nitrosylation of GSK-3&bgr; significantly inhibits its kinase activity independent of the canonical phospho-inhibition pathway. S-nitrosylation of GSK-3&bgr; promotes its nuclear translocation and access to novel downstream phosphosubstrates which are enriched for a novel amino acid consensus sequence motif. Quantitative phosphoproteomics pathway analysis reveals that nuclear GSK-3&bgr; plays a central role in cell cycle control, RNA splicing, and DNA damage response. Conclusions: The results indicate that SNO has a differential effect on the location and activity of GSK-3&bgr; in the cytoplasm versus the nucleus. SNO modification of GSK-3&bgr; occurs in vivo and could contribute to the pathobiology of heart failure and sudden cardiac death.

Molecular Profile of Priapism Associated with Low Nitric Oxide Bioavailability

Priapism is a disorder in which prolonged penile erection persists uncontrollably, potentially leading to tissue damage. Priapism commonly afflicts patient populations with severely low nitric oxide (NO) bioavailability. Because NO is a primary mediator of erection, the molecular mechanisms involved in priapism pathophysiology associated with low NO bioavailability are not well-understood. The objective of this study was to identify dysregulated molecular targets and signaling pathways in penile tissue of a mouse model of low NO bioavailability that have potential relevance to priapism. Neuronal plus endothelial NO synthase double knockout mice (NOS1/3-/-) were used as a model of low NO bioavailability. Priapic-like activity was demonstrated in the NOS1/3-/- mice relative to wild-type (WT) mice by the measurement of prolonged erections following cessation of electrical stimulation of the cavernous nerve. Penile tissue was processed and analyzed by reverse-phase liquid chromatography tandem mass spectrometry. As a result, 1279 total proteins were identified and quantified by spectral counting, 46 of which were down-regulated and 110 of which were up-regulated in NOS1/3-/- versus WT (P < 0.05). Ingenuity Pathway Analysis of differentially expressed proteins revealed increased protein kinase A and G-protein coupled receptor signaling in NOS1/3-/- penises, which represent potential mechanisms contributing to priapism for secondary to low NO bioavailability.