STRUCTURAL STUDIES OF SIR2 REACTION INTERMEDIATE COMPLEXES AND POLYUBIQUITIN: HI

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 NAD+依赖性蛋白脱乙酰酶的sirtuin家族调节许多生物学途径，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Sir 2酶提出了许多机械挑战。 为了解决这些挑战，我们已经采取了基于结构的方法来剖析Sir 2反应机制，并解决了几个重要的Sir 2？反应中间体复合物，包括Sir 2-乙酰化肽-NAD + Michaelis复合物、Sir 2-乙酰化肽-过渡态类似物复合物和Sir 2-O-烷基酰胺化物中间体复合物。 Sir 2化学的另一个迷人的方面是了解小分子如何激活这些酶。 我们已经生产了Sir 2-乙酰化肽-NAD +-异烟酰胺的晶体，以了解异烟酰胺如何激活Sir 2酶。 蛋白质的泛素修饰被用作许多细胞过程的信号。所述修饰可以是单个泛素或多聚泛素链，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。Lys 48连接的多聚泛素链将其底物靶向26 S蛋白酶体进行降解。泛素降解途径是药物靶点的一个活跃研究领域。市场上有一种药物（万珂）用于治疗多发性骨髓瘤。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通路中蛋白质的小分子抑制剂。我们有Lys 48连接的多聚泛素晶体与小分子抑制剂结合。这种抑制剂防止由聚泛素标记的特异性底物被降解。晶体结构将有助于理解这种抑制剂如何起作用，以及可能如何设计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.