Genetics Of Familial Mediterranean Fever/ Related Condit

家族性地中海热/相关病症的遗传学

• 批准号: 6967700
• 负责人: Daniel L Kastner
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6967700
• 关键词: X ray crystallography; amyloidosis; apoptosis; cell line; cysteine endopeptidases; cytoskeleton; family genetics; gene mutation; gene targeting; genetic disorder; genetically modified animals; human subject; hyperthermia; inflammation; interleukin 1; laboratory mouse; linkage mapping; microarray technology; molecular cloning; patient oriented research; polymerase chain reaction; protein localization; protein protein interaction; protein structure function; transcription factor; transfection

Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by self-limited attacks of fever with serosal, synovial, or cutaneous inflammation, sometimes complicated by systemic amyloidosis. In 1992 our laboratory mapped the FMF locus to chromosome 16p13.3, and in 1997 we isolated the underlying gene, MEFV, and demonstrated that it is highly expressed in granulocytes. During the 6 years leading up to the present reporting period, we have focused on several areas, including FMF population genetics, the regulation of FMF gene expression in leukocyte subpopulations, the biochemistry and cell biology of pyrin (the FMF protein), and the development of animal models of FMF. During the year directly antecedent to the present reporting period, the laboratory focused on two major projects. The major findings of the first project, a study of a mouse strain expressing a truncated form of pyrin, indicated that pyrin plays a major role in the regulation of caspase-1 activation and IL-1beta processing, and that pyrin regulates macrophage apoptosis through a caspase-8-dependent, IL-1beta-independent pathway. The second project, an analysis of proteins that interact with pyrin, established that the cytoskeletal protein PSTPIP1 binds pyrin, and that PSTPIP1 mutations associated with the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne (PAPA) lead to markedly increased pyrin-binding and IL-1beta activation. Results of the Last Year A major focus of the last year has been the study of the direct interaction of pyrin with caspase-1, and its potential implications in normal biology and in FMF. In a series of experiments that had begun during the FY03 reporting period, we demonstrated that antibodies to caspase-1 coimmunoprecipitate endogenous pyrin in THP-1 human monocytic cells. Conversely, we found that polyclonal antipyrin antibodies specifically precipitate caspase-1, but not caspases 2-10, from THP-1 lysates. To rule out any possible role of the ASC protein (apoptosis-associated specklike protein with a caspase recruitment domain), which can bind both pyrin and caspase-1, GST-pulldown experiments were also performed in PT67 cells, which do not express ASC even by sensitive RT-PCR. Further GST-pulldowns using deletion mutants demonstrated that the C-terminal B30.2 domain of pyrin and the catalytic p20 and p10 domains of caspase-1 are the major interaction domains. To study the biochemical consequences of this interaction, radiolabeled pyrin was incubated with purified caspase-1. In these assays we observed a time- and dose-dependent cleavage of pyrin from its 95 kDa native form to an approximately 55 kDa cleavage fragment. The cleavage of pyrin was blocked by a specific inhibitor of caspase-1 in a dose-dependent fashion. The actual cleavage site was determined by Edman sequencing, and was found to be located between Asp330 and Ser331 in pyrin. Site-directed mutagenesis of Asp330 completely abolished the caspase-1-mediated cleavage of pyrin. We next examined the subcellular localization of GFP-tagged pyrin and its N-terminal (aa 1-330) and C-terminal (aa 331-781) cleavage fragments. Whereas full-length pyrin and the C-terminal fragment were restricted to the cytoplasm, the N-terminal fragment was found in both the nucleus and the cytoplasm. Moreover, in luciferase reporter assays, the N-terminal pyrin cleavage fragment had dramatic NF-kappaB activating effects, relative to the modest stimulation seen with full-length pyrin. The NF-kappaB stimulatory activity of both full-length pyrin and the N-terminal fragment was dependent on the presence of ASC, and inactivating mutations in the PYRIN domains of either pyrin or ASC abrogated NF-kappa B activation. Finally, we examined the effect of FMF-associated mutations in pyrin?s C-terminal B30.2 domain on caspase-1 interaction, on caspase-1-mediated cleavage, and on NF-kappaB activation. Disease-associated B30.2 mutants associated less well than wild type with caspase-1, but were cleaved more efficiently. When FMF-associated pyrin mutants were cotransfected with caspase-1 in a luciferase reporter assay, there as an approximately 10-fold increase in NF-kappaB activation, relative to wild type protein. Moreover, absolute and relative quantities of cleaved pyrin were substantially increased in leukocytes from FMF patients, compared with healthy controls. Conclusions and Significance These data establish a novel inflammatory pathway in which the cleavage of pyrin by caspase-1 induces NF-kappaB activation. The heightened susceptibility of FMF-associated pyrin mutants to cleavage may help account for the autoinflammatory phenotype of FMF, and suggests a possible mechanism whereby carriers of even a single MEFV mutation sometimes exhibit clinical or biochemical evidence of episodic inflammation. Such heterozygote effects may also confer increased resistance to bacterial pathogens, possibly accounting for the high FMF carrier frequencies in certain populations. The biochemical mechanism by which N-terminal pyrin induces NF-kappaB activation is unknown, but our data renew interest in pyrin as a possible transcription factor. Our findings also suggest that the N-terminal fragment of pyrin is a potentially attractive target for anti-inflammatory drug discovery. During the next year, our objectives will be: (1) to elucidate the biochemical mechanism by which N-terminal pyrin activates NF-kappaB; (2) to extend studies in animal models, particularly a recently-developed line of pyrin-null mice; (3) to undertake structural studies of pyrin and related proteins; (4) to study gene expression profiles of patients with FMF during and between attacks; and (5) to undertake clinical trials of IL-1 inhibition in FMF and related disorders.

家族性地中海热（FMF）是一种隐性遗传疾病，其特征是伴有浆膜，滑膜或皮肤炎症的自限性发作，有时会因全身性淀粉样变性而复杂。 1992年，我们的实验室将FMF基因座映射到16p13.3染色体，并在1997年分离了基础基因MEFV，并证明它在粒细胞中高度表达。在当前报告期之前的6年中，我们专注于多个领域，包括FMF种群遗传学，白细胞亚群中FMF基因表达的调节，吡啶（FMF蛋白）的生物化学和细胞生物学以及FMF动物模型的发展。在本报告期间直接前期的一年中，实验室着重于两个主要项目。第一个项目的主要发现是对表达pyRIN截断形式的小鼠菌株的研究，表明吡啶在调节caspase-1激活和IL-1BETA处理中起主要作用，并且pyrin调节通过caspase-8依赖性的IL-1Beta-1beta-littedent依赖的caspase-8依赖的巨噬细胞凋亡。第二个项目是对与吡啶相互作用的蛋白质的分析，确定细胞骨架蛋白PSTPIP1结合了吡啶，并且与肩肾上腺腺癌和痤疮（PAPA）的Pyogenic关节炎综合征相关的PSTPIP1突变导致pyoderma蛋白（PAPA）可显着增加pyrin-lind-binting and Il-binding和iil-1beta Activation。 去年的结果 去年的主要重点是研究吡啶与caspase-1的直接相互作用及其在正常生物学和FMF中的潜在影响。在03财年的报告期开始的一系列实验中，我们证明了caspase-1的抗体在THP-1人类单核细胞中的内源性吡啶。相反，我们发现来自THP-1裂解物的多克隆抗蛋白抗体特异性沉淀caspase-1，而不是caspase-1，而不是caspase 2-10。为了排除ASC蛋白（与caspase募集结构域的凋亡相关的斑点样蛋白）的任何可能作用，它可以在PT67细胞中结合吡啶和caspase-1，GST-Pulldown实验，即使通过敏感RT-PCR表达ASC也不会表达ASC。使用缺失突变体的进一步的GST-Pulldowns表明，caspase-1的pyrin的C端B30.2结构域以及催化P20和P10域是主要的相互作用域。 为了研究这种相互作用的生化后果，将放射性标记的吡啶与纯化的caspase-1孵育。在这些测定中，我们观察到pyrin从其95 kDa天然形式到约55 kDa裂解片段的时间和剂量依赖性裂解。吡啶的裂解被caspase-1的特定抑制剂以剂量依赖性的方式阻塞。实际的切割位点是通过Edman测序确定的，发现位于Pyrin中的ASP330和Ser331之间。 ASP330的位置定向诱变完全消除了caspase-1介导的pyrin裂解。 接下来，我们检查了GFP标记的吡啶及其N末端（AA 1-330）和C末端（AA 331-781）的裂解片段的亚细胞定位。全长吡啶和C末端片段仅限于细胞质，但在核和细胞质中都发现了N末端片段。此外，在萤光素酶报告基因测定中，N末端吡啶裂解片段具有戏剧性的NF-kappab激活效果，相对于全长吡啶的适度刺激。全长吡啶和N末端片段的NF-kappab刺激活性取决于ASC的存在，以及在吡啶或ASC废除的NF-kappa B激活的吡啶结构域中的灭活突变。 最后，我们研究了FMF相关突变在吡啶的C末端B30.2域对caspase-1相互作用，caspase-1介导的裂解以及NF-kappab激活的影响。与caspase-1相比，与疾病相关的B30.2突变体不如野生型良好，但更有效地裂解。当在荧光素酶报告基准测定中与caspase-1共转染FMF相关的吡啶突变体时，相对于野生型蛋白，NF-kappab激活的增加约10倍。此外，与健康对照组相比，FMF患者的白细胞中切割的吡啶的绝对和相对量显着增加。 结论和意义 这些数据建立了一种新颖的炎症途径，其中caspase-1 pyrin裂解会诱导NF-kappab激活。 FMF相关的吡啶突变体对裂解的敏感性提高可能有助于解释FMF的自炎表型，并提出了一种可能的机制，即使单个MEFV突变的载体有时也会表现出临床或生物化学证据。这种杂合作用也可能赋予对细菌病原体的耐药性，这可能考虑了某些人群中的高FMF载体频率。 N末端吡啶诱导NF-kappab激活的生化机制尚不清楚，但我们对吡啶的数据更新为可能的转录因子。我们的发现还表明，吡啶的N末端片段是抗炎药物发现的潜在吸引人的靶标。 在第二年，我们的目标将是：（1）阐明N端吡啶激活NF-kappab的生化机制； （2）扩展在动物模型中的研究，特别是最近开发的pyrin-null小鼠系； （3）进行吡啶和相关蛋白的结构研究； （4）研究FMF患者在发作期间和两次攻击之间的基因表达谱； （5）在FMF和相关疾病中对IL-1抑制进行临床试验。