糖尿病肾病相关生物学标志物的研究进展

糖尿病肾病(diabetic nephropathy,DN)是引起糖尿病相关性死亡的主要原因之一。尿微量白蛋白是目前诊断DN最常用的临床指标,然而部分DN患者虽然已经有了晚期肾组织病理上的改变,但尿微量白蛋白仍然处于正常范围内。因此,寻找新的灵敏、特异的生物学指标对于DN的诊断及判断疾病进程有重要意义。近年来,肾损伤相关标志物、与DN发病机制相关的生物标志物等已经逐渐成为临床研究的热点,而蛋白质组学技术的发展也为寻找DN相关的生物标志物提供了新的指导方向。     

Oncogenomics: prospects for the future

Genomics has generated a wealth of data that is now being used to identify additional molecular alterations associated with cancer development. Mapping these alterations in the cancer genome is a critical first step in dissecting oncological pathways. There are two ways in which cancer research has changed in recent years. The first is the progressive elucidation of the genomic basis of cancer. This has been accomplished by the generation of detailed information using procedures such as global expression profiling. The second is a renewed emphasis on the role of epigenetic modifications in the etiology of cancer. Changes in DNA methylation and chromatin modification patterns are some of the epigenetic factors that cause gene deregulation in cancer. In this article, current and evolving genomic applications and the hypotheses underlying the modality for cancer therapy will be reviewed.     

Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells

Newly synthesized cellular proteins can be tagged with a variety of metabolic labels that distinguish them from preexisting proteins and allow them to be identified and tracked. Many such labels are incorporated into proteins via the endogenous cellular machinery and can be used in numerous cell types and organisms. Though broad applicability has advantages, we aimed to develop a strategy to restrict protein labeling to specified mammalian cells that express a transgene. Here we report that heterologous expression of a mutant methionyl-tRNA synthetase from Escherichia coli permits incorporation of azidonorleucine (Anl) into proteins made in mammalian (HEK293) cells. Anl is incorporated site-selectively at N-terminal positions (in competition with initiator methionines) and is not found at internal sites. Site selectivity is enabled by the fact that the bacterial synthetase aminoacylates mammalian initiator tRNA, but not elongator tRNA. N-terminally labeled proteins can be selectively conjugated to a variety of useful probes; here we demonstrate use of this system in enrichment and visualization of proteins made during various stages of the cell cycle. N-terminal incorporation of Anl may also be used to engineer modified proteins for therapeutic and other applications.     

Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling

Galectin-3 is a glycan-binding protein (GBP) that binds β-galactoside glycan structures to orchestrate a variety of important biological events, including the activation of hepatic stellate cells and regulation of immune responses. While the requisite glycan epitopes needed to bind galectin-3 have long been elucidated, the cellular glycoproteins that bear these glycan signatures remain unknown. Given the importance of the three-dimensional (3D) arrangement of glycans in dictating GBP interactions, strategies that allow the identification of GBP receptors in live cells, where the native glycan presentation and glycoprotein expression are preserved, have significant advantages over static and artificial systems. Here we describe the integration of a proximity labeling method and quantitative mass spectrometry to map the glycan and glycoprotein interactors for galectin-3 in live human hepatic stellate cells and peripheral blood mononuclear cells. Understanding the identity of the glycoproteins and defining the structures of the glycans will empower efforts to design and develop selective therapeutics to mitigate galectin-3–mediated biological events.

The noncovalent interactions between glycan-binding proteins (GBPs) and glycans dictate many important biological events. Among such GBPs is galectin-3, a 26-kDa β-galactoside GBP that plays key roles in many physiological and pathological events (1). In hepatic fibrosis, a disease that manifests as the excessive buildup of scar tissue, liver-resident macrophages secrete galectin-3 (2, 3), which then binds cell surface glycans on quiescent hepatic stellate cells (HSCs), activating them to transdifferentiate into a muscle-like phenotype. Galectin-3–null mice exhibit attenuated liver fibrosis even after induced injury, highlighting its critical role (3). Galectin-3 is also known to interact with cells of the innate immune system (4, 5) to regulate apoptosis (6) or control dendritic cell differentiation (7). In these cases, as well as in other cases in which galectin-3 is involved, the full complement of interacting glycoprotein receptors remains unknown.Despite significant advances in glycoscience, the study of GBP–glycan interactions and the identification of glycan-mediated counter-receptors remains a recurring challenge. Capturing these binding events often requires some form of artificial reconstitution to amplify individually weak interactions into high-avidity binding. Indeed, glycan microarrays with defined mixtures of homogenous glycans or recombinant GBPs have significantly propelled our understanding of glycan-mediated function (8). Conventional immunoprecipitation and lectin affinity techniques using cell lysates have similarly been used to reveal an initial catalog of 100 to 185 galectin-3–associated proteins (9–14). However, these manipulations alter the cell’s native and three-dimensional (3D) configuration and multivalent arrangement, both of which are critically important in the study of GBP–glycan interactions (15, 16).Another key issue involves the underlying glycoprotein ligand. Although many glycoproteins carry the glycan epitope for binding a GBP, only a limited set should be recognized as physiologically relevant receptors, owing to the physical constraints imposed by the living cell (17). While often overlooked, the glycoprotein carrying the glycan can impart specific biological functions to a GBP–glycan binding event (17). Recent work has put forth the concept of “professional glycoprotein ligands,” in which a specific set of glycoproteins (instead of a broadly defined glycome) can exhibit exquisite binding and functional roles (18). Thus, determining the identity of the underlying core protein that anchors the glycan can be greatly empowering. Not only can it provide an understanding of the 3D arrangement of the glycan (if the 3D structure of the core protein is known), but it can also provide additional insight into its expression levels in different cell types and tissues, further informing strategies for selective drug development.Thus, comprehensive approaches that permit the study of GBP–glycan interactions in live cells while simultaneously facilitating identification of the physiological glycoprotein receptors have great potential to impact glycoscience. We hypothesize that proximity labeling strategies (19) using an engineered ascorbate peroxidase, APEX2 (20), could be compatible for elucidating glycan-mediated GBP–glycoprotein interactions. In this approach (Fig. 1), APEX2 is fused to a protein of interest, followed by the treatment of cells with biotin-phenol and subsequently with hydrogen peroxide (H₂O₂). Under these conditions, APEX2 catalyzes the formation of highly reactive, short-lived (<1 ms), and proximally restricted (<20 nm) biotin-phenoxyl radicals that covalently tag nearby electron-rich residues. The biotinylated proteins can then be enriched and identified using quantitative mass spectrometry (MS)-based proteomics. Because the (glyco)proteins adjacent to the APEX2 fusion protein are preferentially biotinylated, the resulting MS data provide a readout of its immediate environment.Open in a separate windowFig. 1.Schematic illustration of the identification of galectin-3 (Gal-3) interacting proteins by in situ proximity labeling. Recombinant APEX2 and galectin-3 fusion proteins are applied to living cells where galectin-3 can freely diffuse to bind its cognate ligands. On addition of biotin phenol (yellow circle with “B”; 30 min) and hydrogen peroxide (H₂O₂; 1 min), APEX2 catalyzes the formation of highly-reactive biotin-phenoxyl radicals that react within a short range (<20 nm) of the galectin-3 complex within a short time frame (<1 ms). The biotin-tagged protein interactors can then be identified using MS-based proteomics.We reasoned that proximity labeling could offer significant advantages over other approaches to determining GBP–glycan interactions, including the opportunity to perform the labeling in live cells and the ability to tag weakly bound glycan-mediated interactors, as the covalent biotinylation reaction ensures that the enrichment step no longer relies on weak GBP–glycan interactions alone. When coupled with inhibitors, the proximity labeling strategy can also distinguish between glycan-mediated and non–glycan-mediated interactors. Integration of this approach with quantitative MS-based proteomics would also expedite the assignment of the interacting proteins and provide calculable measures to distinguish interactors from nonspecific binders.Here we report that the use of an APEX2 and galectin-3 fusion protein (PX-Gal3) provides a sensitive and comprehensive approach to mapping the proteome-wide glycan-mediated galectin-3 interactome in live human HSCs and peripheral blood mononuclear cells (PBMCs). We found that the exogenous incubation of cells with PX-Gal3 in HSCs leads to glycan-dependent interactions, whereas its cellular overexpression does not. We further validated the interactions between galectin-3 and candidate proteins in vitro and discovered that some proteins are direct glycan-mediated receptors. Using MS-based glycomics, we also examined the glycomes of HSC surfaces, PX-Gal3 tagged glycoproteins, and an individual glycoprotein receptor for galectin-3. Our results highlight the utility of the in situ proximity labeling approach in discovering physiologically relevant GBP interactors in living cells.     

早期矽肺损伤的蛋白质组学研究及 α 晶状体球蛋白 B 的表达与验证

目的：建立二氧化硅诱导早期肺损伤大鼠肺组织的二维凝胶电泳图谱,筛选在此过程中起关键作用的靶 蛋白分子并进行验证。方法：制作二氧化硅诱导大鼠早期肺损伤模型(实验组),通过HE染色观察肺部形态变化,用 双向凝胶电泳法建立二维凝胶图谱,用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分析鉴定差异表达蛋白 分子,用Western印迹验证差异表达蛋白分子在肺组织中的表达。结果：成功制作二氧化硅早期肺损伤模型,建立实 验组及对照组肺组织二维凝胶电泳图谱,二者蛋白表达及分布存在差异,发现差异点40个,选取重复性好,在凝胶 上位置和表达适中的共20个进行MALDI-TOF-MS鉴定及数据库搜索并筛选,鉴定出差异蛋白13种,包括α晶状体球蛋 白B、间隙蛋白I、肥大细胞蛋白酶等,进一步用Western印迹证实实验组α晶状体球蛋白B表达明显增加,与蛋白质组 学结果一致。结论：α晶状体球蛋白B在早期矽肺组织中表达明显升高,提示其可能在矽肺的发生发展中起关键作用。     

急性肝衰竭大鼠肝脏的蛋白质组学研究

目的 筛选急性药物性肝衰竭大鼠肝脏组织的差异表达蛋白质.方法 将24只SD大鼠分为两组 ,12只通过腹腔注射10 g/L的D-氨基半乳糖建立大鼠急性肝衰竭模型(实验组) ,12只腹腔注射生理盐水作为对照组.提取两组大鼠肝脏组织的蛋白质 ,定量后进行IEF和十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)双向凝胶电泳(2-DE)分离 ,通过软件找到差异蛋白点并使用基质辅助激光解吸电离飞行时间质谱法(MALDI-TOF-MS)进行鉴定.结果 成功鉴定出27个有效的差异蛋白质点 ,其中实验组较对照组上调15个 ,下调12个.结论 急性药物性肝衰竭模型大鼠肝脏酪蛋白激酶Iα(CKⅠα)、酪氨酸蛋白激酶(PTK)、增殖细胞核抗原(PCNA)等蛋白的表达较正常大鼠存在显著差异.     

Shenfu Injection(参附注射液) Suppresses Inflammation by Targeting Haptoglobin and Pentraxin 3 in Rats with Chronic Ischemic Heart Failure

Objective:To investigate the regulatory effects of Shenfu Injection(SFI,参附注射液) on hemodynamic parameters and serum proteins in rats with post-infarction chronic heart failure(CHF).Methods:Forty-five healthy Wistar rats were randomized into three groups:sham,heart failure(model) and SFI group.The CHF was induced by left coronary artery ligation.Seven days after the surgical operation,animals in the sham group and the model group received saline(6.2 mL/kg/d),while animals in the SFI group received SFI(6.2 mL/kg·d) intraperitoneally.Four weeks later,cardiac hemodynamic parameters were measured via the carotid route.The expression of serum proteins was analyzed by two-dimensional electrophoresis and matrixassisted laser desorption/ionization time-of-flight mass spectrometer(MALDI-TOF MS).Results:Recording of hemodynamic parameters showed that left ventricular systolic pressure(LVSP),maximum rate of left ventricular pressure(+dp/dt_(max)) rise,and maximum rate of left ventricular pressure(-dp/dt_(max)) decrease,while the left ventricular end diastolic pressure(LVEDP) rose in the model group compared to those in the sham group(P0.05).The results of the MALDI-TOF MS indicated that haptoglobin(HP),pentraxin 3(PTX3) and alpha-1-antitrypsin were up-regulated,while serum albumin and 40 S ribosomal protein were down-regulated in the model group(P0.05).Compared with the model group,LVSP,+dp/dt_(max)and-dp/dt_(max) were higher,while LVEDP was lower in the SFI group(P0.05).Expression levels of HP and PTX3 were lower than in the model group(P0.05).Conclusion:SFI could improve hemodynamic function and decrease inflammatory reactions in the pathophysiology of CHF.The serum proteins HP and PTX3 could be potential biomarkers for chronic ischemic heart failure,and they could also be the serum protein targets of SFI.