蛋白激酶Ca相互作用蛋白的结构与功能

蛋白激酶Cα相互作用蛋白(proteininteractingwithCαkinase,PICK1)是蛋白激酶Cα(proteinkinaseCα,PKCα)的靶蛋白之一,也是在PKCα和突触后膜受体蛋白间起重要作用的衔接蛋白。PICK1分别由PDZ结构域、BAR结构域以及卷曲螺旋区和酸性氨基酸区组成。PICK1中的PDZ结构域和受体蛋白、转运蛋白、衔接蛋白的相互作用报道较多,BAR结构域则与支架蛋白、质膜等相互作用。PICK1在突触可塑性、神经递质传递、外周神经感觉、细胞生长和黏连等方面发挥重要作用。本文对PICK1的结构和功能进行综述。     

PICK1蛋白C端酸性区域对其功能的调节

蛋白激酶C相互作用蛋白1(protein interacting with Ckinase1,PICK1)是调节AMPA(alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)受体在细胞膜上的数量与分布,引起LTP与LTD现象的重要蛋白.本文利用基因克隆、荧光光谱以及免疫分析等方法,分析了PICK1蛋白C末端酸性区对BAR结构域与膜脂结合能力以及PICK1分子内BAR(Bin/amphiphysin/RVS)结构域与PDZ结构域相互作用的影响,研究了钙离子结合C末端酸性区后对上述相互作用的调节.结果显示,C末端酸性区的存在使BAR结构域与膜脂的结合能力减弱大约10倍,但PICK1分子内的BAR与PDZ结构域的相互作用与不含C末端的酸性区相比增强了大约4倍.另一方面,C末端酸性区的存在,伴随钙离子浓度的提高,有助于增强BAR与膜脂的结合,却削弱了PDZ和BAR结构域的作用.当钙离子浓度增加到500μmol/L时,BARC的脂质结合能力以及和PDZ的亲和力与不含酸性区相当.     

PICK1的结构与功能研究进展

PICK1蛋白是一个从线虫到人都高度保守膜周蛋白,在多种组织中表达,尤以脑和睾丸的表达最高.在细胞内,PICK1定位于核周区和诸如神经突触的特化细胞结构中.PICK1蛋白含一个PDZ结构域和一个BAR结构域,PDZ结构域能和许多膜蛋白结合.而BAR结构域能与脂质分子(主要为磷酸肌醇)相结合,通过这种机制PICK1可调节相关蛋白的亚细胞定位和膜表达.由于各蛋白与PICK1相互作用的PDZ结合基序不同,可利用与特定蛋白结合基序相同的PDZ结合多肤竞争性地结合PDZ结构域,特异性地阻断该蛋白的作用,从而特异性地增强或减弱PICK1在某组织中的作用,为PICK1的临床应用提供了药理基础.     

PICK1：一个功能多样的药靶蛋白

蛋白质是生命功能的执行者.生命体中某些关键蛋白的功能异常往往是导致疾病发生的根本原因.这些疾病相关蛋白极有可能成为药物靶点,为新药研发和疾病治疗提供重要线索. PICK1蛋白（protein interacting with Cα kinase 1）结合能力广泛、功能多样以及在多种重要疾病（如：癌症、精神分裂症、疼痛、帕金森综合症等）的发生发展过程中发挥潜在的作用,使其成为一个可能的药靶蛋白. PICK1与绝大多数配体蛋白的相互作用是通过其PDZ结构域与配体C末端区域的结合介导的,使PICK1的PDZ结构域成为一个潜在的药物靶点.因此,可以利用生物小分子物质特异性地结合PICK1的PDZ结构域,干扰或阻断PICK1与配体蛋白的天然相互作用,最终达到治疗相关疾病的目的.     

PICKI的结构与功能研究进展

PICKI蛋白是一个从线虫到人都高度保守膜周蛋白，在多种组织中表达，尤以脑和睾丸的表达最高。在细胞内，PICKI定位于核周区和诸如神经突触的特化细胞结构中。PICKI蛋白含一个PDZ结构域和一个BAR结构域，PDZ结构域能和许多膜蛋白结合，而BAR结构域能与脂质分子（主要为磷酸肌醇）相结合，通过这种机制PICKI可调节相关蛋白的亚细胞定位和膜表达。由于各蛋白与PICKI相互作用的PDZ结合基序不同，可利用与特定蛋白结合基序相同的PDZ结合多肽竞争性地结合PDZ结构域，特异性地阻断该蛋白的作用，从而特异性地增强或减弱PICKI在某组织中的作用，为PICKI的临床应用提供了药理基础。     

PICK1中N端酸性区域对其PDZ结构域脂质结合能力的调节

蛋白激酶Cα相互作用蛋白1(protein interacting with Cα kinase 1, PICK1)是衔接膜上受体和蛋白激酶Cα的重要蛋白.利用荧光光谱结合定点突变技术 、蛋白与脂质覆盖法等方法,分析了PICK1蛋白N末端区域几个酸性氨基酸残基对PDZ 结构域与膜脂结合的影响,以及钙离子结合N末端酸性区域对PDZ脂结合能力的调节. 结果显示, 带有上游酸性区域的PDZ结构域（NPDZ）的脂质结合能力仅相当PDZ结构 域的15%,相比单独的PDZ结构域与脂质的解离常数Kd(PDZ)为1.58×103 μg·L-1, NPDZ与脂质解离常数Kd(NPDZ)为3.3×104μg·L-1,其中在N末端酸性残基中D8与 D12两个天冬氨酸是影响脂质结合能力减弱的关键残基,若将二者分别突变为丙氨酸 后,NPDZ与脂质的解离常数分别为：Kd (D8/A)=4.42×103μg·L-1;Kd (D12/A) =1.73×103μg·L-1接近于PDZ结构域与脂质结合能力;钙离子会增强NPDZ脂结合能力,当钙离子浓度达到30 μmol/L时,NPDZ的脂结合能力提高2.3倍,但只相当于PDZ的50% 的结合能力.     

多功能的胞内衔接蛋白syntenin

Syntenin蛋白是在原核生物及真核生物中广泛存在的一类胞内衔接蛋白（adaptor proteins）. Syntenin由N端结构域（N-terminal domain,NTD）、两个串联的PDZ结构域(postsynaptic density protein, disc large and zonula occludens, PDZ)和C端结构域（C-terminal domain,CTD）组成,在生物进化过程中相对保守. Syntenin蛋白的PDZ结构域可与不同膜受体C端的PDZ结合基序（PDZ-binding motif,PBM）特异性结合, PDZ结构域结合受体的多样性导致了syntenin功能的多样性. 本文综述了syntenin蛋白的发现与分布及其结构特征,对syntenin在肿瘤转移、细胞质膜蛋白组装、参与动物免疫等领域的研究成果进行了较为详细的综述,同时介绍了syntenin在参与动物胚胎发育调控、血管生成和轴突生长等方面的研究进展.     

受体、突触和记忆

大脑中神经元突触间的信号传递是由许多神经递质受体介导的。在过去，Richard L．Huganir实验室一直致力于神经递质受体功能调节的分子机制。而最近，该实验室又聚焦到大脑中一种最主要的兴奋性受体的研究——谷氨酸受体。谷氨酸受体主要可以分为两大类：AMPA受体和NMDA受体。AMPA受体主要介导了快速的兴奋性突触传递；而NMDA受体则在神经可塑性和发育中起到重要作用。实验发现，AMPA受体和NMDA受体都可以被一系列的蛋白激酶磷酸化，而磷酸化的水平则直接影响了这些受体的功能特性，包括通道电导和受体膜定位等。AMPA受体磷酸化的水平同时还在学习和记忆的细胞模型中发生改变，如长时程增强（LTP）和长时程抑制（LTD）。此外，AMPA受体中GluR1亚单位的磷酸化对于各种形式的可塑性以及空间记忆的维持有重要的作用。实验室主要研究突触部位谷氨酸受体在亚细胞水平的定位和聚集的分子机制。最近，一系列可以直接或间接与AMPA和NMDA受体相互作用的蛋白质得以发现，其中包括一个新发现的蛋白家族GRIPs（glutamate receptor interacting proteins）。GRIPs可以直接和AMPA受体的GluR2／3亚单位的C端结合。GRIPs包含7个PDZ结构域，可以介导蛋白与蛋白直接的相互连接，从而把各个AMPA受体交互连接在一起并与其他蛋白相连。另外，GluR2亚单位的c端还可以和兴奋性突触中的蛋白激酶C结合蛋白（PICK1）的PDZ结构域相互作用。另外，GluR2亚单位的C端也可以与一种参与膜融合的蛋白NSF相互作用。这些与AMPA受体相互作用的蛋白质对于受体在膜上的运输以及定位有至关重要的作用。同时，受体与PICK1和GRIP的结合对于小脑运动学习中的LTD有重要作用。总体上说，该实验室发现了一系列可以调节神经递质受体功能的分子机制，这些工作提示受体功能的调节可能是?     

PSD—95对NMDA受体信号转导的整合作用

PSD－95是新近在谷氨酸能突触的突触后致密物（PSD）中发现的一种特殊蛋白质,含有3个N末端的PDZ结构域,一个SH3结构域和一个C末端的GK结构域。PSD－95通过不同结构域与其它蛋白相互作用,不仅能够串集NMDA受体及其信号通路中的相关蛋白分子,组成受体－信号分子－调节分子－靶分子复合物,还可通过突触前后粘附分子的相互作用,参与突触连接的形成和维持,在介导和整合NMDA受体信号转导中具有关键性作用。     

钙/钙调蛋白依赖性丝氨酸蛋白激酶的结构和功能

钙/钙调蛋白依赖性丝氨酸蛋白激酶(calcium/calmodulin-dependent serine protein kinase, CASK)属于膜相关鸟苷酸激酶（membrane associated guanylate kinase, MAGUK）家族.CASK具有多个不同蛋白质结合结构域，在细胞膜的特定区域，与其他蛋白质形成多种蛋白质复合体，参与组成细胞骨架.它通过衔接细胞外信号蛋白和细胞内骨架蛋白，协助功能蛋白质的转运和定位，以及细胞内的信号传递.此外CASK还可以进入细胞核影响基因转录调控，以及作用在神经突触膜上参与神经递质的释放.     

快速构建蛋白质结构域克隆库的方法

对蛋白质组学的研究有许多不同的切入方法 .从研究的生物学意义和可行性考虑 ,提出从蛋白结构域入手进行蛋白质组学研究 .SH2 (Srchomology 2 )结构域是细胞信号转导中重要的元件之一 ,人SH2结构域共有约 12 0种 ,对其进行研究将深刻揭示细胞信号转导的规律 .为了得到人所有的SH2结构域序列及克隆 ,首先在公共数据库里检索出了人所有的SH2结构域序列 ,利用国际上现有的共享资源IMAGE(IntegratedMolecularAnalysisofGenomesandTheirExpression)克隆为PCR模板 ,解决了从cDNA文库中难以克隆低丰度结构域的问题 .利用有方向性的TOPO克隆技术提高克隆效率 ,从而快速高效地构建了包括 6 0个SH2结构域的克隆库 .克隆库可以方便地转换到GATEWAY系统具有各种用途的载体上 ,为SH2结构域的蛋白质组学研究奠定了坚实的基础     

Caveolin-1 activates Rab5 and enhances endocytosis through direct interaction

Caveolin-1, a constitutive protein of the caveolae, is implicated in processes of vesicular transport during caveolae-mediated endocytosis. However, the molecular mechanisms of caveolae-mediated endocytosis are not yet clearly defined. Here, we show the physiological role of the Rab5-caveolin-1 interaction during caveolae-mediated endocytosis. Rab5 was found in caveolae-enriched fractions and Rab5 directly bound to caveolin-1. Furthermore, binding sites of Rab5 to caveolin-1 were identified in the scaffold (SD), transmembrane (TM), and C-terminus (CC) domains, and the Rab5 binding domain of caveolin-1 was required for CTXB uptake. Subsequently, we performed a GST-R5BD pull-down assay to determine whether the Rab5 binding domain of caveolin-1 is involved in Rab5 activity or not. The results showed that overexpression of the Rab5 binding domain of caveolin-1 increase the amount of Rab5-GTP in Cos-1 cells. These findings imply that caveolin-1 controls the Rab5 activity during the caveolae-mediated endocytosis.     

The relationship between domain duplication and recombination

Protein domains represent the basic evolutionary units that form proteins. Domain duplication and shuffling by recombination are probably the most important forces driving protein evolution and hence the complexity of the proteome. While the duplication of whole genes as well as domain-encoding exons increases the abundance of domains in the proteome, domain shuffling increases versatility, i.e. the number of distinct contexts in which a domain can occur. Here, we describe a comprehensive, genome-wide analysis of the relationship between these two processes. We observe a strong and robust correlation between domain versatility and abundance: domains that occur more often also have many different combination partners. This supports the view that domain recombination occurs in a random way. However, we do not observe all the different combinations that are expected from a simple random recombination scenario, and this is due to frequent duplication of specific domain combinations. When we simulate the evolution of the protein repertoire considering stochastic recombination of domains followed by extensive duplication of the combinations, we approximate the observed data well. Our analyses are consistent with a stochastic process that governs domain recombination and thus protein divergence with respect to domains within a polypeptide chain. At the same time, they support a scenario in which domain combinations are formed only once during the evolution of the protein repertoire, and are then duplicated to various extents. The extent of duplication of different combinations varies widely and, in nature, will depend on selection for the domain combination based on its function. Some of the pair-wise domain combinations that are highly duplicated also recur frequently with other partner domains, and thus represent evolutionary units larger than single protein domains, which we term "supra-domains".     

植物TIR-NB-LRR类型抗病基因各结构域的研究进展

植物抗病反应是一个多基因调控的复杂过程,在这个过程中R基因发挥了非常重要的作用。根据其氨基酸基序组成以及跨膜结构域的不同,R基因可以分为多种类型,其中NBS-LRR类型是植物基因组中最大的基因家族之一。TIR-NB-LRR类型的抗病基因又是NB-LRR类型中的一大类,也是目前抗病基因研究的热点。该文总结了TIR-NB-LRR类型抗病基因各个结构域的功能和相关的研究进展。相关研究表明,TIR结构域主要通过自身或异源的二聚体化介导抗性信号的转导,但也有部分研究表明,该结构域可能参与病原菌的特异性识别。NBS结构域常被认为具有"分子开关"的功能,它可以通过结合ADP或ATP来调节植物抗病蛋白的构象变化,从而调节下游抗病信号的传导。LRR结构域在植物与病原菌互作的过程中可以通过与病原菌的无毒蛋白直接或间接互作来特异识别病原菌。也有研究发现,LRR结构域具有调节信号传导的功能。这些信息将为研究植物抗病机理提供理论依据,也为将来通过基因编辑技术对作物进行定向抗病育种提供思路。     

GalaxyDomDock: An Ab Initio Domain–domain Docking Web Server for Multi-domain Protein Structure Prediction

A significant proportion of proteins comprise multiple domains. Domain–domain docking is a tool that predicts multi-domain protein structures when individual domain structures can be accurately predicted but when domain orientations cannot be predicted accurately. GalaxyDomDock predicts an ensemble of domain orientations from given domain structures by docking. Such information would also be beneficial in elucidating the functions of proteins that have multiple states with different domain orientations. GalaxyDomDock is an ab initio domain–domain docking method based on GalaxyTongDock, a previously developed protein–protein docking method. Infeasible domain orientations for the given linker are effectively screened out from the docked conformations by a geometric filter, using the Dijkstra algorithm. In addition, domain linker conformations are predicted by adopting a loop sampling method FALC. The proposed GalaxyDomDock outperformed existing ab initio domain–domain docking methods, such as AIDA and Rosetta, in performance tests on the Rosetta benchmark set of two-domain proteins. GalaxyDomDock also performed better than or comparable to AIDA on the AIDA benchmark set of two-domain proteins and two-domain proteins containing discontinuous domains, including the benchmark set in which each domain of the set was modeled by the recent version of AlphaFold. The GalaxyDomDock web server is freely available as a part of GalaxyWEB at http://galaxy.seoklab.org/domdock.     

Delineation of modular proteins: domain boundary prediction from sequence information

The delineation of domain boundaries of a given sequence in the absence of known 3D structures or detectable sequence homology to known domains benefits many areas in protein science, such as protein engineering, protein 3D structure determination and protein structure prediction. With the exponential growth of newly determined sequences, our ability to predict domain boundaries rapidly and accurately from sequence information alone is both essential and critical from the viewpoint of gene function annotation. Anyone attempting to predict domain boundaries for a single protein sequence is invariably confronted with a plethora of databases that contain boundary information available from the internet and a variety of methods for domain boundary prediction. How are these derived and how well do they work? What definition of 'domain' do they use? We will first clarify the different definitions of protein domains, and then describe the available public databases with domain boundary information. Finally, we will review existing domain boundary prediction methods and discuss their strengths and weaknesses.     

Regulators of G protein signaling: a bestiary of modular protein binding domains

Members of the newly discovered regulator of G protein signaling (RGS) families of proteins have a common RGS domain. This RGS domain is necessary for conferring upon RGS proteins the capacity to regulate negatively a variety of Galpha protein subunits. However, RGS proteins are more than simply negative regulators of signaling. RGS proteins can function as effector antagonists, and recent evidence suggests that RGS proteins can have positive effects on signaling as well. Many RGS proteins possess additional C- and N-terminal modular protein-binding domains and motifs. The presence of these additional modules within the RGS proteins provides for multiple novel regulatory interactions performed by these molecules. These regions are involved in conferring regulatory selectivity to specific Galpha-coupled signaling pathways, enhancing the efficacy of the RGS domain, and the translocation or targeting of RGS proteins to intracellular membranes. In other instances, these domains are involved in cross-talk between different Galpha-coupled signaling pathways and, in some cases, likely serve to integrate small GTPases with these G protein signaling pathways. This review discusses these C- and N-terminal domains and their roles in the biology of the brain-enriched RGS proteins. Methods that can be used to investigate the function of these domains are also discussed.     

Biophysical characterization of the domain association between cytosolic A and B domains of the mannitol transporter enzymes IIMtl in the presence and absence of a connecting linker

The mannitol transporter enzyme II^Mtl of the bacterial phosphotransferase system is a multi‐domain protein that catalyzes mannitol uptake and phosphorylation. Here we investigated the domain association between cytosolic A and B domains of enzyme II^Mtl, which are natively connected in Escherichia coli, but separated in Thermoanaerobacter tengcongensis. NMR backbone assignment and residual dipolar couplings indicated that backbone folds were well conserved between the homologous domains. The equilibrium binding of separately expressed domains, however, exhibited ～28‐fold higher affinity compared to the natively linked ones. Phosphorylation of the active site loop significantly contributed to the binding by reducing conformational dynamics at the binding interface, and a few key mutations at the interface were critical to further stabilize the complex by hydrogen bonding and hydrophobic interactions. The affinity increase implicated that domain associations in cell could be maintained at an optimal level regardless of the linker.     

泛素连接酶E3

蛋白质的泛素化修饰具有高度的特异性,它参与调节细胞内许多的生理活动。蛋白质的泛素化修饰涉及一系列的酶参与反应,包括泛素激活酶E1、结合酶E2以及连接酶E3。而其中泛素连接酶E3对靶蛋白的特异性识别起关键作用。泛素连接酶E3主要由HECT结构域家族、RING结构域家族和U-box结构域家族组成。现对泛素连接酶E3的分类、结构及其对靶蛋白的识别机制等进行综述。     

The dimeric form of the unphosphorylated response regulator BaeR

Bacterial response regulators (RRs) can regulate the expression of genes that confer antibiotic resistance; they contain a receiver and an effector domain and their ability to bind DNA is based on the dimerization state. This is triggered by phosphorylation of the receiver domain by a kinase. However, even in the absence of phosphorylation RRs can exist in equilibrium between monomers and dimers with phosphorylation shifting the equilibrium toward the dimer form. We have determined the crystal structure of the unphosphorylated dimeric BaeR from Escherichia coli. The dimer interface is formed by a domain swap at the receiver domain. In comparison with the unphosphorylated dimeric PhoP from Mycobacterium tuberculosis, BaeR displays an asymmetry of the effector domains.