Gang Hao

S-Nitrosylation Is Emerging as a Specific and Fundamental Posttranslational Protein Modification: Head-to-Head Comparison with O-Phosphorylation

Nitric oxide (NO) is a free-radical product of mammalian cell metabolism that plays diverse and important roles in the regulation of cell function. Biological actions of NO arise as a direct consequence of chemical reactions between NO or NO-derived species and protein targets. Reactions of NO with transition metals in target proteins have garnered the most attention to date as the principal mechanism of NO signaling; nonetheless, S-nitrosylation of protein Cys residues is rapidly moving to center stage in importance. In general, however, there has been a delay in adequate appreciation of the role of S-nitrosylation in biological signaling by NO. This lag is attributed to a poor understanding of the basis for selective targeting of NO to particular thiols, and methodological limitations in accurately quantifying this modification--recent breakthroughs in concepts and methods diminish these barriers. Here, we consider the wheres and whys of protein S-nitrosylation and its basis for specificity. Protein S-nitrosylation potentially represents a ubiquitous and fundamental mechanism for posttranslational control of protein activity on a par with that of O-phosphorylation. Nitric oxide (NO) is a mammalian cell product that has been implicated in the control of essentially all cellular functions. Key to NOs bioactivity is its chemical reactivity. Recent studies suggest that a biologically important reaction of NO is with SH-groups on cysteine residues of proteins--this modification has been termed S-nitrosylation. Until recently, our appreciation of the importance of S-nitrosylation as a mechanism for posttranslational regulation of protein activity has been hindered by an inadequate understanding of how S-nitrosylation may be targeted to specific protein thiols and the lack of a simple method for identification of proteins that are S-nitrosylated in vivo. This review highlights emerging concepts and a new technique that may help to overcome these obstacles. A head-to-head comparison suggests that S-nitrosylation, like O-phosphorylation, may similarly play a fundamental role in the post-translational control of protein activity and cellular function.

Urinary proteomic analysis of chronic allograft nephropathy.

The pathogenesis of progressive renal allograft injury, which is termed chronic allograft nephropathy (CAN), remains obscure and is currently defined by histology. Prospective protocol‐biopsy trials have demonstrated that clinical and standard laboratory tests are insufficiently sensitive indicators of the development and progression of CAN. The study aim was to determine if CAN could be characterized by urinary proteomic data and identify the proteins associated with disease. The urinary proteome of 75 renal transplant recipients and 20 healthy volunteers was analyzed using surface enhanced laser desorption and ionization MS. Patients could be classified into subgroups with normal histology and Banff CAN grades 2‐3 with a sensitivity of 86% and a specificity of 92% by applying the classification algorithm Adaboost to urinary proteomic data. Several urinary proteins associated with advanced CAN were identified including α1‐microglobulin, β2‐microglobulin, prealbumin, and endorepellin, the antiangiogenic C‐terminal fragment of perlecan. Increased urinary endorepellin was confirmed by ELISA and increased tissue expression of the endorepellin/perlecan ratio by immunofluoresence analysis of renal biopsies. In conclusion, analysis of urinary proteomic data has further characterized the more severe CAN grades and identified urinary endorepellin, as a potential biomarker of advanced CAN.

Heme catalyzes tyrosine 385 nitration and inactivation of prostaglandin H2 synthase-1 by peroxynitrite

The mechanism by which the inflammatory enzyme prostaglandin H2 synthase-1 (PGHS-1) deactivates remains undefined. This study aimed to determine the stabilizing parameters of PGHS-1 and identify factors leading to deactivation by nitric oxide species (NOx). Purified PGHS-1 was stabilized when solubilized in β-octylglucoside (rather than Tween-20 or CHAPS) and when reconstituted with hemin chloride (rather than hematin). Peroxynitrite (ONOO−) activated the peroxidase site of PGHS-1 independently of the cyclooxygenase site. After ONOO− exposure, holoPGHS-1 could not metabolize arachidonic acid and was structurally compromised, whereas apoPGHS-1 retained full activity once reconstituted with heme. After incubation of holoPGHS-1 with ONOO−, heme absorbance was diminished but to a lesser extent than the loss in enzymatic function, suggesting the contribution of more than one process to enzyme inactivation. Hydroperoxide scavengers improved enzyme activity, whereas hydroxyl radical scavengers provided no protection from the effects of ONOO−. Mass spectral analyses revealed that tyrosine 385 (Tyr 385) is a target for nitration by ONOO− only when heme is present. Multimer formation was also observed and required heme but could be attenuated by arachidonic acid substrate. We conclude that the heme plays a role in catalyzing Tyr 385 nitration by ONOO− and the demise of PGHS-1.

Neutral Loss of Isocyanic Acid in Peptide CID Spectra: A Novel Diagnostic Marker for Mass Spectrometric Identification of Protein Citrullination

Protein citrullination is emerging as an important signaling mechanism that modulates a variety of biological processes. This protein modification constitutes only a 1 Da mass shift, and can be readily confused with other common protein modifications that yield an identical mass shift. In an attempt to develop a robust methodology for detection of protein citrullination sites, we analyzed synthetic citrulline-containing peptides by electrospray ionization tandem mass spectrometry. Collision-induced dissociation (CID) spectra revealed abundant neutral loss of 43 Da from citrullinated peptide precursor ions, which was reconciled by elimination of the HNCO moiety (isocyanic acid) from the citrulline ureido group. The elimination occurs readily in multiple charge states of precursor ions and also in b and y ions. HNCO loss in CID spectra provides a novel diagnostic marker for citrullination, and its utility was demonstrated by the discovery of Arg197 as the specific site of citrullination on nucleophosmin upon peptidylarginine deiminase 4 treatment.