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OBJECTIVE: In the fibrotic kidneys, the extent of a formed deleterious microenvironment is determined by cellular mechanical forces. This process requires metabolism for energy. However, how cellular mechanics and metabolism are connected remains unclear. METHODS: A multi-disciplinary approach was employed: the fibrotic kidney disease models were induced by renal ischemia-reperfusion injury and unilateral ureteral obstruction in Calponin 2 (CNN2) knockdown mice. Proteomics, bioinformatics, and in vivo and in vitro molecular experimental pathology studies were performed. RESULT: Our proteomics revealed that actin filament binding and cell metabolism are the two most dysregulated events in the fibrotic kidneys. As a prominent actin stabilizer, CNN2 was predominantly expressed in fibroblasts and pericytes. In CKD patients, CNN2 levels was markedly induced in blood. In mice, CNN2 knockdown preserves kidney function and alleviates fibrosis. Global proteomics profiled that CNN2 knockdown enhanced the activities of the key rate-limiting enzymes and regulators of fatty acid oxidation (FAO) in the diseased kidneys. Inhibiting carnitine palmitoyltransferase 1α in the FAO pathway resulted in lipid accumulation and extracellular matrix deposition in the fibrotic kidneys, which were restored after CNN2 knockdown. Bioinformatics and chromatin immunoprecipitation showed that CNN2 interactor, estrogen receptor 2 (ESR2), binds peroxisome proliferator-activated receptor-α (PPARα) to transcriptionally regulate FAO downstream target genes expression amid kidney fibrosis. In vitro, ESR2 knockdown repressed the mRNA levels of PPARα and the key genes in the FAO pathway. Conversely, activation of PPARα reduced CNN2-induced matrix inductions. CONCLUSIONS: Our results suggest that balancing cell mechanics and metabolism is crucial to develop therapeutic strategies to halt kidney fibrosis.

Influenza-related cardiac complications have been increasingly defined in the clinical setting. Recent reports support a direct link between laboratory-confirmed influenza infection and adverse cardiac events. Here, we define candidate molecular mediators underlying influenza virus-induced cardiac pathogenesis after the resolution of pulmonary infection, as well as the potential role of necroptosis in this process using mice deficient in its effector molecule MLKL. Global proteome and phosphoproteome analyses using high resolution accurate mass-based LC-MS/MS and label-free quantitation was used. Both the global proteome and the phosphoproteome profiles were significantly altered in IAV-infected mouse hearts, with significantly altered expression of mitochondrial and muscle contraction protein levels and phosphorylation states. Intriguingly, necroptosis deficient mice showed partial protection from IAV-mediated proteomic changes. We confirmed changes in energy state and mitochondrial dysfunction in vitro and define molecular candidates for therapeutics targeting necroptosis and oxidative stress in cardiac tissue after influenza infection.

Shotgun metaproteomics has potential to reveal the functional landscape of microbial communities, but lacks appropriate methods for complex samples with unknown compositions. In the absence of prior taxonomic information, tandem mass spectra would be searched against large pan-microbial databases, which requires heavy computational workload and reduces sensitivity. We present ProteoStorm, an efficient database search framework for large-scale metaproteomics studies, which identifies high-confidence peptide-spectrum matches (PSMs) while achieving a two to three orders-of-magnitude speedup over popular tools. A reanalysis of a urinary tract infection (UTI) dataset of 110 individuals revealed a complex pattern of polymicrobial expression, including sub-types of urinary tract infections, cases of bacterial vaginosis, and evidence of no underlying disease. Importantly, compared to the initial UTI study that restricted the search database to a manually-curated list of 20 genera, ProteoStorm identified additional genera that were previously unreported, including a case of infection with the rare pathogen Propionimicrobium.

Over 100 mutations in Cu/Zn-superoxide dismutase (SOD1) result in familial amyotrophic lateral sclerosis. Dimer dissociation is the first step in SOD1 aggregation, and studies suggest nearly every amino acid residue in SOD1 is dynamically connected to the dimer interface. Post-translational modifications of SOD1 residues might be expected to have similar effects to mutations, but few modifications have been identified. Here we show, using SOD1 isolated from human erythrocytes, that human SOD1 is phosphorylated at threonine 2 and glutathionylated at cysteine 111. A second SOD1 phosphorylation was observed and mapped to either Thr-58 or Ser-59. Cysteine 111 glutathionylation promotes SOD1 monomer formation, a necessary initiating step in SOD1 aggregation, by causing a 2-fold increase in the Kd. This change in the dimer stability is expected to result in a 67% increase in monomer concentration, 315 nm rather than 212 nm at physiological SOD1 concentrations. Because protein glutathionylation is associated with redox regulation, our finding that glutathionylation promotes SOD1 monomer formation supports a model in which increased oxidative stress promotes SOD1 aggregation.

By using an unambiguous in vivo deuterated-leucine labeling quantitative proteomic approach, at close to the physiologically relevant level, we systematically profiled multiple proteins interacting with 14-3-3epsilon, the isoform with least characterized protein interactions in 14-3-3 family in mammalian cells. Among the 19 proteins interacting with 14-3-3epsilon identified, 6 of them including SKb1Hs, p54nrb, serine/threonine kinase 38, MEP50, 14-3-3theta and cofilin 2 were the previously unknown interacting partners with 14-3-3epsilon. The newly identified interactor cofilin 2 was also validated in co-transfection and co-immunoprecipitation. In contrast, with the same stringent criteria only three known partners were identified by conventional tandem affinity purification (TAP) approach. Therefore the 'in-spectra' quantitative marker of deuterated-leucine assisted to precisely identify those genuine interacting partners with minimum requirement of validation using other molecular approaches.

Corrigendum to ‘‘An optimized magnetite microparticle-based phosphopeptide enrichment strategy for identifying multiple phosphorylation sites in an immunoprecipitated protein’’ [Anal. Biochem. 408 (2011) 19–31] Yi Huang , Qihui Shi , Chia-Kuang Tsung , Harsha P. Gunawardena , Ling Xie , Yanbao Yu , Hongjun Liang , Pengyuan Yang , Galen D. Stucky c,⇑⇑, Xian Chen a,b,⇑ Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, People’s Republic of China Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA