炎性小体中ASC斑点形成机理及调控机制北大核心CSCD

在一系列微生物感染及内外源性刺激物的作用下,细胞质中多种蛋白复合物组装成炎性小体,其主要功能是活化半胱天冬酶-1,引起一系列促炎细胞因子的分泌和半胱天冬酶-1依赖性的细胞死亡。凋亡相关斑点样蛋白(ASC)是炎性小体中连接胞浆内受体和半胱天冬酶-1的接头蛋白,在炎性小体活化中ASC聚集成大分子的二聚体,被称为ASC斑点(ASC-speck)。ASC斑点的形成对半胱天冬酶-1的活化至关重要,调控ASC斑点的形成是炎性小体相关疾病的治疗和预防的新途径。本文从ASC斑点形成的分子机理,以及磷酸化、泛素化和去泛素化、离子通道等方面,对近年来ASC斑点的调控机制相关的研究进展进行综合评述,总结了ASC斑点的形成机理及主要调控机制,最后结合作者相关研究成果和观点对该领域的研究前景进行了展望。     

PcG蛋白转录抑制复合物组份的功能及构成的时空特征

PcG蛋白主要以PRC1和PRC2两组复合物的形式存在,通过参与核小体组蛋白翻译后修饰等机制,发挥调控靶基因转录的功能. PRC1复合体中的RING1A/B具有使组蛋白H2AK119泛素化的活性;PRC2中的EZH2具有使组蛋白H3K27三甲基化的活性,形成PRC1锚定到核小体上的位点. PcG蛋白的表达特征具有发育阶段和细胞类型时空特异性. 长链非编码RNA等反式作用因子能募集PcG蛋白结合于靶基因,发挥靶向作用. 本文就PcG蛋白功能、构成的时空特异性、募集机制及其与疾病发生的关系研究进展做一综述.     

PcG蛋白转录抑制复合物组分的功能及构成的时空特征

PcG蛋白主要以PRC1和PRC2两组复合物的形式存在,通过参与核小体组蛋白翻译后修饰等机制,发挥调控靶基因转录的功能.PRC1复合体中的RING1A/B具有使组蛋白H2AK119泛素化的活性;PRC2中的EZH2具有使组蛋白H3K27三甲基化的活性,形成PRC1锚定到核小体上的位点.PcG蛋白的表达特征具有发育阶段和细胞类型时空特异性.长链非编码RNA等反式作用因子能募集PcG蛋白结合于靶基因,发挥靶向作用.本文就PcG蛋白功能、构成的时空特异性、募集机制及其与疾病发生的关系研究进展做一综述.     

基于位置权重矩阵的核小体识别及功能分析

为研究高通量的人类CD4+T细胞的核小体定位模式,使用迭代算法对核小体定位模式进行分类,并利用位置权重矩阵方法分别构建稳定核小体定位序列、动态核小体定位序列和连接区序列模型,通过十倍交叉验证评估模型性能,并与Segal方法与弯曲度方法进行比较,发现位置权重矩阵方法在敏感性、精度和准确性方面都具有一定优越性。同时采用滑窗法在全基因组选取候选序列进行核小体识别,挖掘核小体定位相关基因,并进行基因生物学进程功能富集分析,发现稳定与动态核小体、真实与潜在核小体对应的基因所参与调控的生物学过程各有不同,但也有一些生物学过程为不同类别核小体所共有,例如对细胞内大分子的调控功能。     

技术与方法体外组装含有组蛋白变体H2A.Z及H3.3的核小体

核小体是构成真核生物染色质的基本结构单位,组蛋白变体H2A.Z及H3.3对染色质结构及基因转录过程发挥着重要的调控作用。体内研究核小体及染色质结构受到诸多因素限制,体外重构含有H2A.Z及H3.3的核小体结构是研究与组蛋白变体相关基因表达调控的重要方法之一。实验表达纯化了6种组蛋白,在复性的过程中装配了含有H2A.Z和H3.3的组蛋白八聚体。基于DNA序列10bp周期性及序列模体设计了3条易于形成核小体的DNA序列,通过PCR大量扩增的方法,回收了标记Cy3荧光分子的目的DNA序列。采用盐透析法体外组装了含有H2A.Z和H3.3的核小体结构,利用荧光标记、EB染色及考马斯亮蓝染色检测了含有组蛋白变体的核小体形成效率及形成过程的吉布斯自由能变化。结果发现,设计的3条DNA序列可以有效地组装形成含有组蛋白电梯的核小体结构,而且随着组蛋白八聚体与DNA比例的增加,核小体的形成效率显著提高;采用Cy3荧光标记可以灵敏且定量地计算组装过程的吉布斯自由能。该方法的建立对研究组蛋白变体相关的结构生物学及转录调控等具有一定的意义。     

体外组装含有组蛋白变体H2A.Z及H3.3的核小体

核小体是构成真核生物染色质的基本结构单位,组蛋白变体H2A.Z及H3.3对染色质结构及基因转录过程发挥着重要的调控作用。体内研究核小体及染色质结构受到诸多因素限制,体外重构含有H2A.Z及H3.3的核小体结构是研究与组蛋白变体相关基因表达调控的重要方法之一。实验表达纯化了6种组蛋白,在复性的过程中装配了含有H2A.Z和H3.3的组蛋白八聚体。基于DNA序列10bp周期性及序列模体设计了3条易于形成核小体的DNA序列,通过PCR大量扩增的方法,回收了标记Cy3荧光分子的目的 DNA序列。采用盐透析法体外组装了含有H2A.Z和H3.3的核小体结构,利用荧光标记、EB染色及考马斯亮蓝染色检测了含有组蛋白变体的核小体形成效率及形成过程的吉布斯自由能变化。结果发现,设计的3条DNA序列可以有效地组装形成含有组蛋白电梯的核小体结构,而且随着组蛋白八聚体与DNA比例的增加,核小体的形成效率显著提高;采用Cy3荧光标记可以灵敏且定量地计算组装过程的吉布斯自由能。该方法的建立对研究组蛋白变体相关的结构生物学及转录调控等具有一定的意义。     

NLRP3炎症小体的负向调控机制

NLRP3炎症小体是由NOD样受体(NOD-like receptor, NLR) NLRP3、接头蛋白ASC和胱冬肽酶-1(Caspase-1)所形成的多聚蛋白复合体,能够感受来自病原微生物的病原相关分子模式(pathogen-associated molecular patterns, PAMPs)和胞内自身危险信号-危险相关分子模式(danger-associated molecular patterns, DAMPs),促进细胞因子IL-1β和IL-18的成熟和分泌、引起细胞焦亡,从而在多种生理、病理过程中发挥重要作用. NLRP3炎症小体是目前研究最深入的炎症小体,其表达水平和活化强度与多种疾病的发生、发展密切相关,如感染性疾病、痛风、Ⅱ型糖尿病、动脉粥样硬化、阿尔兹海默症及癌症等.因此,阐明NLRP3炎症小体活化的调控机制,对于揭示这些疾病发生、发展的机理,寻找免疫调节治疗的新途径具有重要意义.本文详细介绍了NLRP3炎症小体的负向调控机制.     

核质蛋白——重要的分子伴侣

分子伴侣一词最初是指一类在细胞核内介导蛋白和核酸相互作用的生物分子.核质蛋白是第一个发现和命名的核内分子伴侣,它在体内以五聚体的形式存在,参与核小体的装配、染色质的重建以及细胞凋亡中染色质的聚缩等重要生命活动.其序列中富含的酸性氨基酸区带是核质蛋白行使功能的重要结构域,这些酸性区带通过屏蔽组蛋白中的正电荷,使得组蛋白能逐步有序地结合到DNA上装配成核小体.核质蛋白晶体结构的测定又将研究推向分子机制的深入探讨和作用模型的建立. 本文主要对核质蛋白的结构和生物学性质、核质蛋白的相关功能以及活性调节的研究进展作一综述.     

NLRP3炎症小体活化、调控机制及相关疾病机制

炎症作为机体的一种自我保护机制有助于清除感染或者有害物质,但是炎症反应失调也会导致疾病发生.NLRP3炎症小体是由胞内模式识别受体NOD样受体家族成员NLRP3形成的一个胞内蛋白复合物,能够诱导IL-1β和IL-18等促炎因子的成熟和分泌,从而促进炎症反应的发生.NLRP3炎症小体可以被多种病原微生物和危险信号活化,并且参与多种人类重大疾病的发生过程,因而NLRP3炎症小体近年来受到了极大的关注.本文就NLRP3炎症小体的活化和调控机制、NLRP3炎症小体在疾病中的作用及靶向NLRP3炎症小体进行相关疾病干预的研究进展进行简要综述.     

酿酒酵母YPH499 Ⅲ号染色体上ARS形成核小体能力的比较

核小体定位是复制起始调控的重要因素,但是核小体定位是如何调控真核生物的复制起始,目前还不是很清楚。研究酵母Ⅲ号染色体上的不同活性复制起始序列形成核小体能力对探究真核生物DNA复制起始机制有着重要的生物学意义。酿酒酵母Ⅲ号染色体上的10个复制起始序列分为高活性和低活性两组复制起始序列,利用核小体体外组装技术,将回收纯化的高活性和低活性复制起始序列分别与组蛋白八聚体在体外进行梯度盐透析组装形成核小体,并进行Biotin标记检测,然后用Image J软件分析不同复制起始序列组装形成核小体能力的强弱。结果表明,用Image J软件对核小体组装能力强弱进行分析:ARS304ARS303ARS313ARS302ARS306ARS314,ARS305,ARS307,ARS309,ARS315。低活性复制起始序列较高活性复制起始序列更易形成核小体;复制起始位点偏好出现于核小体缺乏区。     

Interferon-Induced Antiviral Mx1 GTPase Is Associated with Components of the SUMO-1 System and Promyelocytic Leukemia Protein Nuclear Bodies

Mx proteins are interferon-induced large GTPases, some of which have antiviral activity against a variety of viruses. The murine Mx1 protein accumulates in the nucleus of interferon-treated cells and is active against members of the Orthomyxoviridae family, such as the influenza viruses and Thogoto virus. The mechanism by which Mx1 exerts its antiviral action is still unclear, but an involvement of undefined nuclear factors has been postulated. Using the yeast two-hybrid system, we identified cellular proteins that interact with Mx1 protein. The Mx1 interactors were mainly nuclear proteins. They included Sp100, Daxx, and Bloom's syndrome protein (BLM), all of which are known to localize to specific subnuclear domains called promyelocytic leukemia protein nuclear bodies (PML NBs). In addition, components of the SUMO-1 protein modification system were identified as Mx1-interacting proteins, namely the small ubiquitin-like modifier SUMO-1 and SAE2, which represents subunit 2 of the SUMO-1 activating enzyme. Analysis of the subcellular localization of Mx1 and some of these interacting proteins by confocal microscopy revealed a close spatial association of Mx1 with PML NBs. This suggests a role of PML NBs and SUMO-1 in the antiviral action of Mx1 and may allow us to discover novel functions of this large GTPase.     

Nuclear remodelling during viral infections

Because of their limited coding capacity, viruses are not able to encode all proteins that are required for their replication. Therefore, they depend on a wide variety of cellular functions and structures, such as the host cell nucleus. It has been shown that DNA, as well as RNA viruses, exploit the nucleus because it provides essential machinery for viral replication. On the other hand, the nucleus undergoes significant remodelling during viral usurpation or exploitation. Moreover, it is becoming increasingly clear that some subnuclear structures, such as promyelocytic leukaemia nuclear bodies, act as an antiviral defence mechanism, and several viruses antagonize this intracellular defence by modifying subnuclear structures. This article reviews the main alterations that take place in nucleus during viral infections.     

Resistance to Virus Infection Conferred by the Interferon-Induced Promyelocytic Leukemia Protein

The interferon (IFN)-induced promyelocytic leukemia (PML) protein is specifically associated with nuclear bodies (NBs) whose functions are yet unknown. Two of the NB-associated proteins, PML and Sp100, are induced by IFN. Here we show that overexpression of PML and not Sp100 induces resistance to infections by vesicular stomatitis virus (VSV) (a rhabdovirus) and influenza A virus (an orthomyxovirus) but not by encephalomyocarditis virus (a picornavirus). Inhibition of viral multiplication was dependent on both the level of PML expression and the multiplicity of infection and reached 100-fold. PML was shown to interfere with VSV mRNA and protein synthesis. Compared to the IFN mediator MxA protein, PML had less powerful antiviral activity. While nuclear body localization of PML did not seem to be required for the antiviral effect, deletion of the PML coiled-coil domain completely abolished it. Taken together, these results suggest that PML can contribute to the antiviral state induced in IFN-treated cells.     

Chromatin insulator bodies are nuclear structures that form in response to osmotic stress and cell death

Chromatin insulators assist in the formation of higher-order chromatin structures by mediating long-range contacts between distant genomic sites. It has been suggested that insulators accomplish this task by forming dense nuclear foci termed insulator bodies that result from the coalescence of multiple protein-bound insulators. However, these structures remain poorly understood, particularly the mechanisms triggering body formation and their role in nuclear function. In this paper, we show that insulator proteins undergo a dramatic and dynamic spatial reorganization into insulator bodies during osmostress and cell death in a high osmolarity glycerol–p38 mitogen-activated protein kinase–independent manner, leading to a large reduction in DNA-bound insulator proteins that rapidly repopulate chromatin as the bodies disassemble upon return to isotonicity. These bodies occupy distinct nuclear territories and contain a defined structural arrangement of insulator proteins. Our findings suggest insulator bodies are novel nuclear stress foci that can be used as a proxy to monitor the chromatin-bound state of insulator proteins and provide new insights into the effects of osmostress on nuclear and genome organization.     

Entrapment of viral capsids in nuclear PML cages is an intrinsic antiviral host defense against varicella-zoster virus

The herpesviruses, like most other DNA viruses, replicate in the host cell nucleus. Subnuclear domains known as promyelocytic leukemia protein nuclear bodies (PML-NBs), or ND10 bodies, have been implicated in restricting early herpesviral gene expression. These viruses have evolved countermeasures to disperse PML-NBs, as shown in cells infected in vitro, but information about the fate of PML-NBs and their functions in herpesvirus infected cells in vivo is limited. Varicella-zoster virus (VZV) is an alphaherpesvirus with tropism for skin, lymphocytes and sensory ganglia, where it establishes latency. Here, we identify large PML-NBs that sequester newly assembled nucleocapsids (NC) in neurons and satellite cells of human dorsal root ganglia (DRG) and skin cells infected with VZV in vivo. Quantitative immuno-electron microscopy revealed that these distinctive nuclear bodies consisted of PML fibers forming spherical cages that enclosed mature and immature VZV NCs. Of six PML isoforms, only PML IV promoted the sequestration of NCs. PML IV significantly inhibited viral infection and interacted with the ORF23 capsid surface protein, which was identified as a target for PML-mediated NC sequestration. The unique PML IV C-terminal domain was required for both capsid entrapment and antiviral activity. Similar large PML-NBs, termed clastosomes, sequester aberrant polyglutamine (polyQ) proteins, such as Huntingtin (Htt), in several neurodegenerative disorders. We found that PML IV cages co-sequester HttQ72 and ORF23 protein in VZV infected cells. Our data show that PML cages contribute to the intrinsic antiviral defense by sensing and entrapping VZV nucleocapsids, thereby preventing their nuclear egress and inhibiting formation of infectious virus particles. The efficient sequestration of virion capsids in PML cages appears to be the outcome of a basic cytoprotective function of this distinctive category of PML-NBs in sensing and safely containing nuclear aggregates of aberrant proteins.     

Nuclear choreography: interpretations from living cells

The advent of green fluorescent protein technology, its use in photobleaching experiments and the development of methods to rapidly acquire images and analyze complex datasets have opened the door to unraveling the mechanisms of nuclear functions in living cells. Studies over the past few years have characterized the movement of chromatin, nuclear proteins and nuclear bodies and, in some cases, correlated their dynamics with energy dependence, cell cycle progression, developmental changes, factor targeting and nuclear position. The mechanisms by which nuclear components move or are restrained have important implications for understanding not only the efficacy of nuclear functions but also the regulation of developmental programs and cellular growth.