Proteomics analysis of IBS-D with spleen and kidney yang deficiency

Objective:To investigate the molecular mechanism underlying the development of diarrhea-predominant irritable bowel syndrome (IBS-D) with spleen and kidney yang deficiency (SKYD) using a proteomics approach.Methods:Male Sprague-Dawley rats (n =22) were divided into IBS-D (n =12) and normal control (n =10) groups.SKYD was then modeled in IBS-D rats by a combination of acetic acid enema,bondage,rectal dilation,tail stimulation,and Senna gavage.Colon tissue samples were subsequently collected and examined by Q Exactive mass spectrometry to identify differentially expressed proteins between the two groups.Results:The occurrence of SKYD/IBS-D was associated with ribosomal protein S23 (Rps23),protein phosphatase 2 catalytic subunit alpha (Pp2a),and growth factor receptor-bound protein 2 (Grb2),which are involved in the ribosome,neurotrophin signaling,and Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathways.Conclusion:These data suggest that SKYD/IBS-D pathophysiology likely involves inflammation,cell growth,apoptosis,stress granule formation,immune activation,loss of epithelial cell integrity,and visceral hypersensitivity.

RUVBL2, a novel AS160-binding protein, regulates insulinstimulated GLUT4 translocation

In fat and muscle cells, insulin-stimulated glucose uptake is mainly mediated by glucose transporter 4 （GLUT4）, which translocates from intracellular compartments to the cell surface in response to insulin stimulation. AS160 is one of the substrates of Akt and plays important roles in insulin-regulated GLUT4 translocation. In this study, RuvB- like protein 2 （RUVBL2） is identified as a new AS160-binding protein using mammalian tandem affinity purification （TAP） combined with mass spectrometry. In 3T3-L1 adipocytes, RUVBL2 is highly expressed and is mainly distrib- uted in the cytosol. Depletion of RUVBL2 in adipocytes inhibits insulin-stimulated GLUT4 translocation and glucose uptake through reducing insulin-stimulated ASI60 phosphorylation. However, introduction of human RUVBL2 can reverse this inhibitory effect. These data suggest that RUVBL2 plays an important role in insulin-stimulated GLUT4 translocation through its interaction with AS160.

Antidiabetic Effects of Gegen Qinlian Decoction via the Gut Microbiota Are Attributable to Its Key Ingredient Berberine

Gegen Qinlian Decoction(GQD),a traditional Chinese medicine(TCM)formula,has long been used for the treatment of common metabolic diseases,including type 2 diabetes mellitus.However,the main limitation of its wider application is ingredient complexity of this formula.Thus,it is critically important to identify the major active ingredients of GQD and to illustrate mechanisms underlying its action.Here,we compared the effects of GQD and berberine,a hypothetical key active pharmaceutical ingredient of GQD,on a diabetic rat model by comprehensive analyses of gut microbiota,short-chain fatty acids,proinflammatory cytokines,and ileum transcriptomics.Our results show that berberine and GQD had similar effects on lowering blood glucose levels,modulating gut microbiota,inducing ileal gene expression,as well as relieving systemic and local inflammation.As expected,both berberine and GQD treatment significantly altered the overall gut microbiota structure and enriched many butyrate-producing bacteria,including Faecalibacterium and Roseburia,thereby attenuating intestinal inflammation and lowering glucose.Levels of short-chain fatty acids in rat feces were also significantly elevated after treatment with berberine or GQD.Moreover,concentration of serum proinflammatory cytokines and expression of immune-related genes,including Nfkb1,Stat1,and Ifnrg1,in pancreatic islets were significantly reduced after treatment.Our study demonstrates that the main effects of GQD can be attributed to berberine via modulating gut microbiota.The strategy employed would facilitate further standardization and widespread application of TCM in many diseases.

Quantitative Proteomics Using Isobaric Labeling:A Practical Guide

In the past decade,relative proteomic quantification using isobaric labeling technology has developed into a key tool for comparing the expression of proteins in biological samples.Although its multiplexing capacity and flexibility make this a valuable technology for addressing various biological questions,its quantitative accuracy and precision still pose significant challenges to the reliability of its quantification results.Here,we give a detailed overview of the different kinds of isobaric mass tags and the advantages and disadvantages of the isobaric labeling method.We also discuss which precautions should be taken at each step of the isobaric labeling workflow,to obtain reliable quantification results in large-scale quantitative proteomics experiments.In the last section,we discuss the broad applications of the isobaric labeling technology in biological and clinical studies,with an emphasis on thermal proteome profiling and proteogenomics.

Oleic Acid and Eicosapentaenoic Acid Reverse Palmitic Acid-induced Insulin Resistance in Human HepG2 Cells via the Reactive Oxygen Species/JUN Pathway

Oleic acid(OA),a monounsaturated fatty acid(MUFA),has previously been shown to reverse saturated fatty acid palmitic acid(PA)-induced hepatic insulin resistance(IR).However,its underlying molecular mechanism is unclear.In addition,previous studies have shown that eicosapentaenoic acid(EPA),aω-3 polyunsaturated fatty acid(PUFA),reverses PA-induced muscle IR,but whether EPA plays the same role in hepatic IR and its possible mechanism involved need to be further clarified.Here,we confirmed that EPA reversed PA-induced IR in HepG2 cells and compared the proteomic changes in HepG2 cells after treatment with different free fatty acids(FFAs).A total of 234 proteins were determined to be differentially expressed after PA+OA treatment.Their functions were mainly related to responses to stress and endogenous stimuli,lipid metabolic process,and protein binding.For PA+EPA treatment,the PA-induced expression changes of 1326 proteins could be reversed by EPA,415 of which were mitochondrial proteins,with most of the functional proteins involved in oxidative phosphorylation(OXPHOS)and tricarboxylic acid(TCA)cycle.Mechanistic studies revealed that the protein encoded by JUN and reactive oxygen species(ROS)play a role in OA-and EPA-reversed PA-induced IR,respectively.EPA and OA alleviated PA-induced abnormal adenosine triphosphate(ATP)production,ROS generation,and calcium(Ca^(2+))content.Importantly,H_(2)O_(2)-activated production of ROS increased the protein expression of JUN,further resulting in IR in HepG2 cells.Taken together,we demonstrate that ROS/JUN is a common response pathway employed by HepG2 cells toward FFA-regulated IR.

Mitochondria in the pathogenesis of diabetes:a proteomic view

Diabetes mellitus is a complex metabolic disorder characterized by chronic hyperglycemia due to absolute or relative lack of insulin.Though great efforts have been made to investigate the pathogenesis of diabetes,the underlying mechanism behind the development of diabetes and its complications remains unexplored.Cumulative evidence has linked mitochondrial modification to the pathogenesis of diabetes and its complications and they are also observed in various tissues affected by diabetes.Proteomics is an attractive tool for the study of diabetes since it allows researchers to compare normal and diabetic samples by identifying and quantifying the differentially expressed proteins in tissues,cells or organelles.Great progress has already been made in mitochondrial proteomics to elucidate the role of mitochondria in the pathogenesis of diabetes and its complications.Further studies on the changes of mitochondrial protein specifically post-translational modifications during the diabetic state using proteomic tools,would provide more information to better understand diabetes.

Protomer Roles in Chloroplast Chaperonin Assembly and Function

The individual roles of three chloroplast CPN60 protomers （CPN60α, CPN60β1, and CPN60β2） and whether and how they are assembled into functional chaperonin complexes are investigated in Chlamydomonas reinhardtii. Protein complexes containing all three potential subunits were identified in Chlamydomonas, and their co-expression in Escherichia coil yielded a homogeneous population of oligomers containing all three subunits （CPN60α1β11β2）, with a molecular weight consistent with a tetradecameric structure. While homo-oligomers of CPN60β could form, they were dramatically reduced when CPN60α was present and homo-oligomers of CPN60β2 were readily changed into hetero-oligomers in the presence of ATP and other protomers. ATP hydrolysis caused CPN60 oligomers to disassemble and drove the purified protomers to reconstitute oligomers in vitro, suggesting that the dynamic nature of CPN60 oligomers is dependent on ATP. Only hetero-oligomeric CPN60α1β1β2, containing CPN60α, CPN60β1, and CPN60β2 subunits in a 5：6：3 ratio, cooperated functionally with GroES. The combination of CPN60α and CPN60β subunits, but not the individual subunits alone, complemented GroEL function in E. coil with subunit recognition specificity. Down-regulation of the CPN60α subunit in Chlamydomonas resulted in a slow growth defect and an inability to grow autotrophically, indicating the essential role of CPN60α in vivo.

A subpopulation of CD146^(+) macrophages enhances antitumor immunity by activating the NLRP3 inflammasome

As one of the main tumor-infiltrating immune cell types, tumor-associated macrophages (TAMs) determine the efficacy of immunotherapy. However, limited knowledge about their phenotypically and functionally heterogeneous nature restricts their application in tumor immunotherapy. In this study, we identified a subpopulation of CD146+ TAMs that exerted antitumor activity in both human samples and animal models. CD146 expression in TAMs was negatively controlled by STAT3 signaling. Reducing this population of TAMs promoted tumor development by facilitating myeloid-derived suppressor cell recruitment via activation of JNK signaling. Interestingly, CD146 was involved in the NLRP3 inflammasome-mediated activation of macrophages in the tumor microenvironment, partially by inhibiting transmembrane protein 176B (TMEM176B), an immunoregulatory cation channel. Treatment with a TMEM176B inhibitor enhanced the antitumor activity of CD146+ TAMs. These data reveal a crucial antitumor role of CD146+ TAMs and highlight the promising immunotherapeutic approach of inhibiting CD146 and TMEM176B.

DSSylation, a novel protein modification targets proteins induced by oxidative stress, and facilitates their degradation in cells

Timely removal of oxidatively damaged proteins is crit- ical for cells exposed to oxidative stresses; however, cellular mechanism for clearing oxidized proteins is not clear. Our study reveals a novel type of protein modifi- cation that may play a role in targeting oxidized proteins and remove them. In this process, DSS1 （deleted in split hand/split foot 1）, an evolutionally conserved small protein, is conjugated to proteins induced by oxidative stresses in vitro and in vivo, implying oxidized proteins are DSS1 clients. A subsequent ubiquitination targeting DSSl-protein adducts has been observed, suggesting the client proteins are degraded through the ubiquitin- proteasome pathway. The DSS1 attachment to its clients is evidenced to be an enzymatic process modulated by an unidentified ATPase. We name this novel protein modification as DSSylation, in which DSS1 plays as amodifier, whose attachment may render target proteins a signature leading to their subsequent ubiquitination, thereby recruits proteasome to degrade them.

Quantitative proteomics analysis of parthenogenetically induced pluripotent stem cells

Parthenogenetic embryonic stem(pES)cells isolated from parthenogenetic activation of oocytes and embryos,also called parthenogenetically induced pluripotent stem cells,exhibit pluripotency evidenced by both in vitro and in vivo differentiation potential.Differential proteomic analysis was performed using differential in-gel electrophoresis and isotope-coded affinity tag-based quantitative proteomics to investigate the molecular mechanisms underlying the developmental pluripotency of pES cells and to compare the protein expression of pES cells generated from either the in vivo-matured ovulated(IVO)oocytes or from the in vitro-matured(IVM)oocytes with that of fertilized embryonic stem(fES)cells derived from fertilized embryos.A total of 76 proteins were upregulated and 16 proteins were downregulated in the IVM pES cells,whereas 91 proteins were upregulated and 9 were downregulated in the IVO pES cells based on a minimal 1.5-fold change as the cutoff value.No distinct pathways were found in the differentially expressed proteins except for those involved in metabolism and physiological processes.Notably,no differences were found in the protein expression of imprinted genes between the pES and fES cells,suggesting that genomic imprinting can be corrected in the pES cells at least at the early passages.The germline competent IVM pES cells may be applicable for germ cell renewal in aging ovaries if oocytes are retrieved at a younger age.