分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: Werner syndrome (WS) is a premature aging disorder caused by WRN protein deficiency. Here, we report on the generation of a human WS model in human embryonic stem cells (ESCs). Differentiation of WRN-null ESCs to mesenchymal stem cells (MSCs) recapitulates features of premature cellular aging, a global loss of H3K9me3, and changes in heterochromatin architecture. We show that WRN associates with heterochromatin proteins SUV39H1 and HP1 alpha and nuclear lamina-heterochromatin anchoring protein LAP2 beta. Targeted knock-in of catalytically inactive SUV39H1 in wild-type MSCs recapitulates accelerated cellular senescence, resembling WRN-deficient MSCs. Moreover, decrease in WRN and heterochromatin marks are detected in MSCs from older individuals. Our observations uncover a role for WRN in maintaining heterochromatin stability and highlight heterochromatin disorganization as a potential determinant of human aging.
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-12
摘要: The lipid droplet (LD) is a cellular organelle that stores neutral lipids in cells and has been linked with metabolic disorders. Caenorhabditis elegans has many characteristics which make it an excellent animal model for studying LDs. However, unlike in mammalian cells, no LD structure-like/resident proteins have been identified in C. elegans, which has limited the utility of this model for the study of lipid storage and metabolism. Herein based on three lines of evidence, we identified that MDT-28 and DHS-3 previously identified in C. elegans LD proteome were two LD structure-like/resident proteins. First, MDT-28 and DHS-3 were found to be the two most abundant LD proteins in the worm. Second, the proteins were specifically localized to LDs and we identified the domains responsible for this targeting in both proteins. Third and most importantly, the depletion of MDT-28 induced LD clustering while DHS-3 deletion reduced triacylglycerol content (TAG). We further characterized the proteins finding that MDT-28 was ubiquitously expressed in the intestine, muscle, hypodermis, and embryos, whereas DHS-3 was expressed mainly in intestinal cells. Together, these two LD structure-like/resident proteins provide a basis for future mechanistic studies into the dynamics and functions of LDs in C. elegans. (C) 2015 Elsevier B.V. All rights reserved.
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-12
摘要: The individual roles of three chloroplast CPN60 protomers (CPN60 alpha, CPN60 beta 1, and CPN60 beta 2) 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 coli yielded a homogeneous population of oligomers containing all three subunits (CPN60 alpha beta 1 beta 2), with a molecular weight consistent with a tetradecameric structure. While homo-oligomers of CPN60 beta could form, they were dramatically reduced when CPN60 alpha was present and homo-oligomers of CPN60 beta 2 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 CPN60 alpha beta 1 beta 2, containing CPN60 alpha, CPN60 beta 1, and CPN60 beta 2 subunits in a 5: 6: 3 ratio, cooperated functionally with GroES. The combination of CPN60 alpha and CPN60 beta subunits, but not the individual subunits alone, complemented GroEL function in E. coli with subunit recognition specificity. Down-regulation of the CPN60 alpha subunit in Chlamydomonas resulted in a slow growth defect and an inability to grow autotrophically, indicating the essential role of CPN60 alpha in vivo.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: SILAC is based on direct addition of selected stable isotope amino acids into the cell culture medium, allowing superior quantitative analysis of the cellular proteome compared to other labeling methods. The great advantages of SILAC lie in its straight-forward implementation, quantitative accuracy, and reproducibility over chemical labeling or label-free quantification strategies, favoring its adoption for proteomic research. SILAC has been widely applied to characterize the proteomic changes between different biological samples, to investigate dynamic changes of protein PTMs, to distinguish specific interacting proteins in interaction proteomic analysis, and to analyze protein turnover in the proteome-wide scale. The present review summarizes the principles of SILAC technology, its applications in biological research, and the present state of this technology.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: Protein lysine malonylation, a newly identified protein post-translational modification (PTM), has been proved to be evolutionarily conserved and is present in both eukaryotic and prokaryotic cells. However, its potential roles associated with human diseases remain largely unknown. In the present study, we observed an elevated lysine malonylation in a screening of seven lysine acylations in liver tissues of db/db mice, which is a typical model of type 2 diabetes. We also detected an elevated lysine malonylation in ob/ob mice, which is another model of type 2 diabetes. We then performed affinity enrichment coupled with proteomic analysis on liver tissues of both wild-type (wt) and db/db mice and identified a total of 573 malonylated lysine sites from 268 proteins. There were more malonylated lysine sites and proteins in db/db than in wt mice. Five proteins with elevated malonylation were verified by immunoprecipitation coupled with Western blot analysis. Bioinformatic analysis of the proteomic results revealed the enrichment of malonylated proteins in metabolic pathways, especially those involved in glucose and fatty acid metabolism. In addition, the biological role of lysine malonylation was validated in an enzyme of the glycolysis pathway. Together, our findings support a potential role of protein lysine malonylation in type 2 diabetes with possible implications for its therapy in the future.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: Phosphoinositides, the phosphorylated derivatives of phosphatidylinositol (PtdIns), are key regulators of many fundamental biological processes, including cell growth, proliferation, and motility. Here, we present a novel method for rapid, sensitive, and simultaneous profiling of phosphatidylinositol trisphosphate (PtdInsP(3)), phosphatidylinositol bisphosphate (PtdInsP(2)), and phosphatidylinositol phosphate (PtdInsP) of different fatty acid compositions. This method is based on a technique called charged diacylglycerol fragment ion-specific multiple precursor ion scanning (DAG(+)-specific MPIS), coupled with prior phosphate methylation. Using DAG(+)-specific MPIS, we were able to identify 32 PtdIns, 28 PtdInsP, 30 PtdInsP(2), and 3 PtdInsP(3) molecular species from bovine brain extracts or prostatic cancer cell lines in an efficient and time-saving manner. Our analysis revealed a large range of fatty acyl compositions in phosphoinositides not obtained previously from mammalian samples. We also developed a method that involves isotopic labeling of endogenous phosphoinositides with deuterated diazomethane (CD2N2) for quantitation of phosphoinositides. CD2N2 was generated in situ through acid-catalyzed H/D exchange and methanolysis of trimethylsilyl diazomethane (TMS-diazomethane). Phosphoinositides, extracted from a PC3 prostatic cancer cell line, were labeled either with CH2N2 or CD2N2 and mixed in known proportions for DAG(+)-specific MPIS-based mass spectrometry (MS) analysis. The results indicate that isotopic labeling is capable of providing accurate quantitation of PtdInsP(3), PtdInsP(2), and PtdInsP with adequate linearity as well as high reproducibility with an average coefficient variation of 18.9%. More importantly, this new methods excluded the need for multiple phosphoinositide internal standards. DAG(+)-specific MPIS and isotopic labeling based MS analysis of phosphoinositides offers unique advantages over existing approaches and presents a powerful tool for research of phosphoinositide metabolism.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: One of the major challenges in prostate cancer therapy remains the development of effective treatments for castration-resistant prostate cancer (CRPC), as the underlying mechanisms for its progression remain elusive. Previous studies showed that androgen receptor (AR) is crucially involved in regulation of metabolism in prostate cancer (PCa) cells throughout the transition from early stage, androgen-sensitive PCa to androgen-independent CRPC. AR achieves such metabolic rewiring directively either via its transcriptional activity or via interactions with AMP-activated protein kinase (AMPK). However, due to the heterogeneous expression and activity status of AR in PCa cells, it remains a challenge to investigate the links between AR status and metabolic alterations. To this end, we compared the proteomes of three pairs of androgen-sensitive (AS) and androgen-independent (AI) PCa cell lines, namely, PC3-AR(+)/PC3, 22Rv1/Du145, and LNCaP/C42B, using an iTRAQ labeling approach. Our results revealed that most of the differentially expressed proteins between each pair function in metabolism, indicating a metabolic shift between AS and AT cells, as further validated by multiple reaction monitoring (MRM)-based quantification of nucleotides and relative comparison of fatty acids between these cell lines. Furthermore, increased adenylate kinase isoenzyme 1 (AK1) in AS relative to AT cells may result in activation of AMPK, representing a major regulatory factor involved in the observed metabolic shift in PCa cells.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-11
摘要: Lipid droplets (LDs) are ubiquitous organelles that serve as a neutral lipid reservoir and a hub for lipid metabolism. Manipulating LD formation, evolution, and mobilization in oleaginous species may lead to the production of fatty acid-derived biofuels and chemicals. However, key factors regulating LD dynamics remain poorly characterized. Here we purified the LDs and identified LD-associated proteins from cells of the lipid-producing yeast Rhodosporidium toruloides cultured under nutrient-rich, nitrogen-limited, and phosphorus-limited conditions. The LD proteome consisted of 226 proteins, many of which are involved in lipid metabolism and LD formation and evolution. Further analysis of our previous comparative transcriptome and proteome data sets indicated that the transcription level of 85 genes and protein abundance of 77 proteins changed under nutrient-limited conditions. Such changes were highly relevant to lipid accumulation and partially confirmed by reverse transcription-quantitative PCR. We demonstrated that the major LD structure protein Ldp1 is an LD marker protein being upregulated in lipid-rich cells. When overexpressed in Saccharomyces cerevisiae, Ldp1 localized on the LD surface and facilitated giant LD formation, suggesting that Ldp1 plays an important role in controlling LD dynamics. Our results significantly advance the understanding of the molecular basis of lipid overproduction and storage in oleaginous yeasts and will be valuable for the development of superior lipid producers.
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-11
摘要: Brown adipose tissue (BAT) maintains animal body temperature by non-shivering thermogenesis, which is through uncoupling protein 1 (UCP1) that uncouples oxidative phosphorylation and utilizes beta-oxidation of fatty acids released from triacylglycerol (TAG) in lipid droplets (LDs). Increasing BAT activity and "browning" other tissues such as white adipose tissue (WAT) can enhance the expenditure of excess stored energy, and in turn reduce prevalence of metabolic diseases. Although many studies have characterized the biology of BAT and brown adipocytes, BAT LDs especially their activation induced by cold exposure remain to be explored. We have isolated LDs from mouse interscapular BAT and characterized the full proteome using mass spectrometry. Both morphological and biochemical experiments showed that the LDs could tightly associate with mitochondria. Under cold treatment mouse BAT started expressing LD structure protein PLIN-2/ADRP and increased expression of PLIN1 Both hormone sensitive lipase (HSL) and adipose TAG lipase (ATGL) were increased in LDs. In addition, isolated BAT LDs showed increased levels of the mitochondrial protein UCP1, and prolonged cold exposure could stimulate BAT mitochondrial cristae biogenesis. These changes were in agreement with the data from transcriptional analysis. Our results provide the BAT LD proteome for the first time and show that BAT LDs facilitate heat production by coupling increasing TAG hydrolysis through recruitment of ATGL and HSL to the organelle and expression of another LD resident protein PLIN2/ADRP, as well as by tightly associating with activated mitochondria. These findings will benefit the study of BAT activation and the interaction between LDs and mitochondria. (C) 2015 Elsevier B.V. All rights reserved.
分类: 生物学 >> 生物物理学 >> 肿瘤学 提交时间: 2016-05-05
摘要: Wilms' tumors are one of the most common malignant, solid intra-abdominal tumors observed in children. Although potential tumor markers have been found, inflammatory cytokines interfere with the process of specific protein identification. The present study was undertaken to identify post-traumatic stress-related factors of Wilms' tumors and to verify the accuracy of early-stage tumor-specific serum protein markers. Serum samples were screened for differentially-expressed proteins using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). Potential markers were isolated and purified using solid-phase extraction (SPE) and SDS-PAGE. Following enzymatic digestion of the protein samples, the peptide fragments were detected with high performance liquid chromatography-mass spectrometry. The obtained peptide mass fingerprint was searched in the Swiss-Prot protein sequence database via the Mascot search engine. Differentially-expressed proteins were verified using western blot analysis. Differentially-expressed proteins with a mass/charge of 5,816 were screened out using SELDI-TOF-MS, and significant differences between the tumor and control groups, and the trauma and control groups were observed. Target proteins were isolated and purified using SPE and SDS-PAGE. Thioredoxin 1 (Trx1) was found to be differentially expressed. In the serum of children with Wilms' tumors, there was an increase in the level of the post-traumatic stress-related inflammatory factor, Trx1, as compared with the normal control group. Thus, the results of this study indicate that Trx1 presents a potential post-traumatic stress-related factor of Wilms' tumors.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-05
摘要: Excessive retention of neutral lipids in cardiac lipid droplets (LDs) is a common observation in cardiomyopathy. Thus, the systematic investigation of the cardiac LD proteome will help to dissect the underlying mechanisms linking cardiac steatosis and myocardial dysfunction. Here, after isolation of LDs from normal and dysfunctional Sprague-Dawley rat hearts, we identified 752 heart-associated LD proteins using iTRAQ quantitative proteomic method, including 451 proteins previously unreported on LDs. The most noteworthy finding was the identification of the membrane resealing protein, dysferlin. An analysis of dysferlin truncation mutants indicated that its C2 domain was responsible for its LD localization. Quantitative proteomic results further determined that 27 proteins were increased and 16 proteins were decreased in LDs from post pressure overload-induced dysfunctional hearts, compared with normal hearts. Notably, adipose triacylglycerol lipase (ATGL) was dramatically decreased and dysferlin was substantially increased on dysfunctional cardiac LDs. This study for the first time reveals the dataset of the heart LD proteome in healthy tissue and the variation of it under cardiac dysfunction. These findings highlight an association between the altered LD protein localization of dysferlin and ATGL and myocardial dysfunction.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-05
摘要: Accumulated studies demonstrate that saturated fatty acids (FAs) such as palmitic acid (PA) inhibit insulin signaling in skeletal muscle cells and monounsaturated fatty acids such as oleic acid (OA) reverse the effect of PA on insulin signaling. The detailed molecular mechanism of these opposite effects remains elusive. Here we provide a comparative proteomic study of skeletal myoblast cell line C2C12 that were untreated or treated with PA, and PA plus OA. A total of 3437 proteins were quantified using SILAC in this study and 29 proteins fall into the pattern that OA reverses PA effect. Expression of some these proteins were verified using qRT-PCR and Western blot. The most significant change was cyclooxygenase-2 (Cox-2). In addition to whole cell comparative proteomic study, we also compared lipid droplet (LD)-associated proteins and identified that Cox-2 was one of three major altered proteins under the FA treatment. This finding was then confirmed using immunofluorescence. Finally, Cox-2 selective inhibitor, celecoxib protected cells from PA-reduced insulin signaling Akt phosphorylation. Together, these results not only provide a dataset of protein expression change in FA treatment but also suggest that Cox-2 and lipid droplets (LDs) are potential players in PA- and OA-mediated cellular processes.