Nur77 在缺氧/ 复氧诱导的心肌细胞凋亡中的作用及其机制的研究

目的:观察Nur77通过线粒体转位对缺氧/复氧(H/R)诱导的心肌细胞凋亡的影响。方法:原代培养l-2天SD大鼠心肌细胞,建立H/R模型。随机分为正常对照组、H/R组、Nur77组,采用免疫荧光检测横纹肌肌动蛋白(α-actin)鉴定心肌细胞;采用TUNEL染色法及Caspase-3酶活性检测心肌细胞凋亡情况;采用Western blot检测细胞核及线粒体Nur77蛋白表达、线粒体及胞浆Omi/HtrA2蛋白表达。结果:H/R组细胞核中Nur77蛋白表达明显低于正常对照组;而在线粒体中则相反。Nur77组线粒体中的Omi/HtrA2蛋白表达明显低于正常对照组;而在胞浆中则相反。结论:在心肌细胞H/R损伤时,Nur77线粒体转位促使Omi/HtrA2蛋白从线粒体释放入胞浆,从而导致心肌细胞凋亡。     

弗林蛋白酶——一种重要的前体蛋白内切蛋白酶

在真核生物细胞中,许多具有生物活性的多肽和蛋白是在其分泌过程中由前体蛋白经内切蛋白酶切割后激活形成的。弗林蛋白酶（Furin)就是这个内切蛋白酶家族重要成员之一,它可以识别剪切多种蛋白质,如生长因子、血清蛋白、基因金属蛋白酶、受体、病毒囊膜蛋白和细菌外毒 素等。近年来Furin得到了迅速而广泛的研究,本简介了它的表达与加工运输、生物学功能、与病毒侵染的关系,以及它的抑制剂。     

Cdk5-mediated Acn/Acinus phosphorylation regulates basal autophagy independently of metabolic stress

In neurons, autophagy counteracts consequences of aging. It is therefore of interest how basal rates of macroautophagy/autophagy can be controlled independently of metabolic stress. We recently investigated the regulation of basal, starvation-independent autophagy by Acn/Acinus, a multifunctional nuclear protein with proposed roles in apoptosis, alternative RNA splicing, and basal autophagy. We found that Acn is stabilized by phosphorylation of the conserved serine 437. The phosphomimetic Acn^S437D mutation causes no overt developmental phenotypes, but significantly elevates levels of basal autophagy and extends life spans. An RNAi screen identified Cdk5 as a kinase targeting S437, a role confirmed by gain- and loss-of-function mutants of Cdk5 or its obligatory cofactor Cdk5r1/p35. Flies lacking Cdk5 function display reduced basal autophagy and a shortened life span. Both of these phenotypes are suppressed by the phosphomimetic Acn^S437D mutation, indicating that phosphorylating serine 437 of Acn, and thereby maintaining basal levels of autophagy, is critical for Cdk5's function in maintaining neuronal health.     

Invariant chain processing is independent of cathepsin variation between primary human B cells/dendritic cells and B-lymphoblastoid cells

As part of the endocytic antigen processing pathway, proteolytic cleavage of the invariant chain (Ii) is important for the generation of class II-associated invariant chain peptide (CLIP). CLIP remains associated with the major histocompatibility complex (MHC) class II molecule to prevent premature loading of antigenic peptides. Cysteine proteases, such as Cathepsin S (CatS), CatL, or CatV, play a pivotal role in the final stage of Ii degradation depending on the cell type studied. Less is known regarding the early stages of Ii processing. We therefore explored whether the serine protease CatG is involved in the initial step of Ii degradation in primary antigen presenting cells (APC), since the cathepsin distribution differs between primary APC and cell lines. While primary human B cells and dendritic cells (DC) do harbor CatG, this protease is absent in B-lymphoblastoid cells (BLC) or monocyte-derived DC generated in vitro. In addition, other proteases, such as CatC, CatL, and the asparagine endoprotease (AEP), are active in BLC and monocyte-derived DC. Here we demonstrate that CatG progressively degraded Ii in vitro resulting in several intermediates. However, pharmacological inhibition of CatG in primary B cells and DC did not alter Ii processing, indicating that CatG is dispensable in Ii degradation. Interestingly, stalling of cysteine proteases by inhibition in BLC vs. primary B cells and DC did not result in any differences in the generation of distinct Ii intermediates between the cells tested, suggesting that Ii processing is independent of the cathepsin variation within professional human APC.     

Rapid characterization of the binding property of HtrA2/Omi PDZ domain by validation screening of PDZ ligand library

HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mammalian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial homeostasis. PDZ domain is one of the most important protein-protein interaction modules and is involved in a variety of important cellular functions, such as signal transduction, degradation of proteins, and formation of cytoskeleton. Recently, it was reported that the PDZ domain of HtrA2/Omi might regulate proteolytic activity through its interactions with ligand proteins. In this study, we rapidly characterized the binding properties of HtrA2/Omi PDZ domain by validation screening of the PDZ ligand library with yeast two-hybrid approach. Then, we predicted its novel ligand proteins in human proteome and reconfirmed them in the yeast two-hybrid system. Finally, we analyzed the smallest networks bordered by the shortest path length between the protein pairs of novel interactions to evaluate the confidence of the identified interactions. The results revealed some novel binding properties of HtrA2/Omi PDZ domain. Besides the reported Class II PDZ motif, it also binds to Class I and Class III motifs, and exhibits restricted variability at P⁻³, which means that the P⁻³ residue is selected according to the composition of the last three residues. Seven novel ligand proteins of HtrA2/Omi PDZ domain were discovered, providing significant clues for further clarifying the roles of HtrA2/Omi. Moreover, this study proves the high efficiency and practicability of the newly developed validation screening of candidate ligand library method for binding property characterization of peptide-binding domains.     

Precursors of the Cocoa-Specific Aroma Components are Derived from the Vicilin-Class (7S) Globulin of the Cocoa Seeds by Proteolytic Processing

Essential precursors of the cocoa-specific aroma components are formed during fermentation of cocoa seeds in the tropics. During the past decades, indications have accumulated that these aroma precursors are derived from seed proteins by acid-induced proteolysis. This has been recently corroborated by in vitro studies on the formation of the aroma-related components. It has been shown that the essential proteolysis products are derived from the vicilin-class (7S) globulins of the cocoa seeds by the cooperative action of an endogenous aspartic endoprotease and a carboxypeptidase.     

Rapid characterization of the binding property of HtrA2/Omi PDZ domain by validation screening of PDZ ligand library

HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mammalian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial homeostasis. PDZ domain is one of the most important protein-protein interaction modules and is involved in a variety of important cellular functions, such as signal transduction, degradation of proteins,and formation of cytoskeleton. Recently, it was reported that the PDZ domain of HtrA2/Omi might regulate proteolytic activity through its interactions with ligand proteins. In this study, we rapidly characterized the binding properties of HtrA2/Omi PDZ domain by validation screening of the PDZ ligand library with yeast two-hybrid approach. Then, we predicted its novel ligand proteins in human proteome and reconfirmed them in the yeast two-hybrid system. Finally, we analyzed the smallest networks bordered by the shortest path length between the protein pairs of novel interactions to evaluate the confidence of the identified interactions. The results revealed some novel binding properties of HtrA2/Omi PDZ domain. Besides the reported Class Ⅱ PDZ motif, it also binds to Class Ⅰ and Class Ⅲ motifs, and exhibits restricted variability at P-3, which means that the P-3 residue is selected according to the composition of the last three residues. Seven novel ligand proteins of HtrA2/Omi PDZ domain were discovered, providing significant clues for further clarifying the roles of HtrA2/Omi.Moreover, this study proves the high efficiency and practicability of the newly developed validation screening of candidate ligand library method for binding property characterization of peptide-binding domains.     

线粒体促凋亡因子Omi/HtrA2在肝癌组织中表达的研究

目的探讨丝氨酸蛋白酶Omi/HtrA2在肝癌组织、癌旁组织与正常肝组织中的表达及意义。方法应用免疫组化SABC法检测43例肝癌、30例癌旁组织及10例正常肝组织中Omi/HtrA2的表达。结果29例(67·44%)肝癌中Omi/HtrA2蛋白表达阳性,30例癌旁组织和10正常肝组织没有或只有少量很弱的表达。肝癌高分化组中Omi/HtrA2蛋白的表达明显高于中、低分化组(P<0·01)。另外,Omi/HtrA2表达与肿瘤大小及临床分期相关,但Omi/HtrA2表达与肝硬化、有无癌栓、HBsAg和AFP无关。结论肝细胞癌可能需要Omi/HtrA2的表达来促进凋亡,Omi/HtrA2的表达对肝癌的发展有重要作用。     

Mutations that are synthetically lethal with a<Emphasis Type="Italic"> gas1Δ</Emphasis> allele cause defects in the cell wall of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The GAS1 -related genes of fungi encode GPI-anchored proteins with -1,3-glucanosyltransferase activity. Loss of this activity results in defects in the assembly of the cell wall. We isolated mutants that show a synthetic defect when combined with a gas1 allele in Saccharomyces cerevisiae, and identified nine wild-type genes that rescue this defect. The indispensability of BIG1 and KRE6 for the viability of gas1 cells confirmed the important role of -1,6-glucan in cells that are defective in the processing of -1,3-glucan. The identification of the Wsc1p hypo-osmotic stress sensor and components of the PKC signal transduction pathway in our screen also confirmed that the cell wall integrity response attenuates the otherwise lethal gas1 defect. Unexpectedly, we found that the KEX2 gene is also required for the viability of the gas1 mutant. Kex2p is a Golgi/endosome-membrane-anchored protease that processes secretory preproteins. A cell wall defect was also found in the kex2 mutant, which was suppressible by multiple copies of the MKC7 or YAP3 gene, both of which encode other GPI-anchored proteases. Therefore, normal cell wall assembly requires proteolytic processing of secretory preproteins. Furthermore, the genes CSG2 and IPT1 were found to be required for normal growth of gas1 cells in the presence of 1 M sorbitol. This finding suggests that complex sphingolipids play a role in the hyper-osmotic response.Communicated by C. P. Hollenberg     

验证性筛选PDZ结构域结合配体文库快速研究HtrA2/Omi PDZ结构域的结合特性

HtrA2/Omi是一种线粒体丝氨酸蛋白酶, 在哺乳动物细胞中具有双重功能, 即诱导细胞凋亡和参与维持线粒体活性的动态平衡. PDZ结构域是最重要的蛋白质相互作用结构域之一, 参与多种生物学过程, 如细胞信号转导、蛋白质降解、细胞骨架组织等. 最近研究发现, HtrA2/Omi蛋白的PDZ结构域与配体的相互作用, 可以调节HtrA2/Omi蛋白自身的水解酶活性.以HtrA2/Omi PDZ结构域为研究对象, 用酵母双杂交系统验证性筛选PDZ结构域结合配体文库, 快速研究该结构域的结合特性, 并在人类全蛋白质组范围内预测并发现该结构域新的相互作用蛋白, 最后分析这些新的相互作用所能够形成的最小相互作用网络来评估其可信度. 研究结果揭示了HtrA2/Omi PDZ结构域新的结合特性, 即: 不仅能够结合已报道的II类PDZ配体而且还可以结合I类和III类PDZ配体, 并且配体-3位氨基酸具有一定范围内的可变性. 而且, 发现了7个新的HtrA2/Omi PDZ结构域相互作用蛋白, 为进一步阐明HtrA2/Omi蛋白的生物学功能提供了重要线索. 同时证明了验证性筛选目的结构域结合配体文库, 这一结构域结合特性研究新策略的实用性和高效性.