STRUCTURAL STUDIES OF SIR2 REACTION INTERMEDIATE COMPLEXES AND POLYUBIQUITIN: HI

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 Sirtuin家族是NAD+依赖的蛋白去乙酰基酶家族，调节多种生物途径，包括衰老、基因沉默、脂肪代谢、神经变性和HIV转录。NAD+的利用是一种很好的调节机制，但对Sir2酶提出了许多机制上的挑战。为了应对这些挑战，我们采取了基于结构的方法来剖析SiR_2反应机理，并解决了几个重要的SiR_2？反应中间络合物包括SiR_2-乙酰化肽-NAD+米氏络合物、SiR_2-乙酰化肽-过渡态类似物和SiR_2-O-烷基酰胺酸酯中间络合物。Sir2化学的另一个有趣的方面是了解小分子是如何激活这些酶的。我们已经生产了一种Sir2-乙酰化肽-NAD+-异烟酰胺的晶体，以了解异烟酰胺如何激活Sir2酶。 蛋白质的泛素修饰被用作许多细胞过程的信号。修饰可以是单个泛素或多泛素链，其由一个泛素的C末端与相邻泛素上的特定赖氨酸之间的异肽键定义。Lys48连接的多泛素链将其底物定位于26S蛋白酶体以进行降解。泛素降解途径是药物靶点研究的一个活跃领域。市场上有一种治疗多发性骨髓瘤的药物(VELCADE)。这种药物通过抑制蛋白酶体起作用。许多实验室正在寻找蛋白酶体上游途径中蛋白质的小分子抑制剂。我们有Lys48连接的聚泛素晶体结合到一个小分子抑制剂上。这种抑制物可防止多聚泛素标记的特定底物被降解。晶体结构将有助于了解这种抑制剂是如何发挥作用的，以及可能通过什么工程使其成为更好的抑制剂。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The sirtuin family of NAD+ dependent protein deacetylases regulates numerous biological pathways including aging, gene silencing, fat metabolism, neurodegeneration and HIV transcription. Utilization of NAD+ is a wonderful regulatory mechanism, but poses numerous mechanistic challenges for Sir2 enzymes. To address these challenges, we have taken a structure based approach to dissect the Sir2 reaction mechanism, and have solved the crystal structures of several important Sir2 ? reaction intermediate complexes including a Sir2-acetylated peptide-NAD+ Michaelis complex, a Sir2-acetylated peptide-transition state analogue complex, and a Sir2-O-alkylamidate intermediate complex. Another fascinating aspect of Sir2 chemistry is understanding how small molecules activate these enzymes. We have produced crystals of a Sir2-acetylated peptide-NAD+-isonicotinamide to understand how isonicotinamide activates Sir2 enzymes. Ubiquitin modification of proteins is used as a signal for many cellular processes. The modification can be a single ubiquitin or a polyubiquitin chain, which is defined by an isopeptide bond between the C-terminus of one ubiquitin and a specific lysine on a neighboring ubiquitin. Lys48-linked polyubiquitin chains target their substrates to the 26S proteasome for degradation. The ubiquitin degradation pathway is an active research field for drug targets. A drug (Velcade) is on the market which is used to treat multiple myeloma. This drug acts by inhibiting the proteasome. Small molecule inhibitors of the proteins in the pathway upstream of the proteasome are being sought by many labs. We have crystals of Lys48-linked polyubiquitin bound to a small molecule inhibitor. This inhibitor prevents specific substrates tagged by polyubiquitin from being degraded. The crystal structure will aid in understanding how this inhibitor acts, and possibly what can be engineered to make it a better inhibitor.