Regulation of one carbon metabolism and epigenetics by SIRT5

NAD+依赖性沉默调节蛋白家族脱酰基酶调节细胞和生物体内平衡的各个方面。 其中，SIRT 5仍然是一个有点神秘的蛋白质。SIRT 5主要是线粒体sirtuin 具有非典型催化性质，从赖氨酸上除去琥珀酰，丙二酰和戊二酰基团， 其底物蛋白质。尽管SIRT 5是靶向这些翻译后蛋白的主要细胞活性， 修饰（PTM），细胞和整个动物中的SIRT 5缺陷仅引起非常轻微的表型， 特别是在基础条件下。因此，SIRT 5及其靶点PTM的生理功能仍然存在 有点神秘。使用基于shRNA和CRISPR/Cas9的方法，我们发现sirtuin SIRT 5是 对于特定癌症类型的生存至关重要，包括黑色素瘤和尤因肉瘤（EWS）。整个 转录组分析表明，SIRT 5影响生存和应激相关基因的表达 包括黑色素瘤谱系基因MITF和FOXO 3A转录因子。通过代谢产物 通过分析，我们已经鉴定了由SIRT 5耗尽所赋予的一碳代谢（1CM）中的缺陷。1CM 由一系列复杂的生化反应组成，这些反应是产生通用甲基供体S- 腺苷甲硫氨酸（SAM），组蛋白和DNA甲基化的共同底物。在黑素瘤和EWS细胞中 当SIRT 5耗尽时，H3 K4 me 3水平降低，这是通过用SIRT 5小分子处理再现的表型。 分子降解剂。值得注意的是，SAM重组完全拯救了 黑色素瘤中与SIRT 5缺失相关的致死性。长期目标是阐明SIRT 5的作用 维持细胞内环境稳定。本提案的目的是阐明SIRT 5在以下方面的作用： 调控1CM和核基因表达。中心假设是SIRT 5调节 1CM酶促进SAM生成、H3 K4三甲基化和适当的基因表达。的理由 1CM是细胞SAM和其他关键细胞代谢物的来源，因此， 1CM调节的新机制具有重要的生物学意义。这项工作将在 三个具体目标。首先，SIRT 5在调节1CM中的作用将从机制上阐明， 使用代谢物追踪，生物化学和其他补充方法，最初侧重于 MTHFD 1 L酶作为候选SIRT 5靶标。第二，H3 K4 me 3在介导细胞凋亡中的作用。 SIRT 5对基因表达的作用将使用定向ChIP和全局ChIP-seq进行阐明。第三，作用 SIRT 5在调节H3 K4 me 3水平和响应于1C应激的存活中的特征将在正常情况下进行。 细胞和小鼠。该应用程序是创新的，因为目前没有文献将SIRT 5与1CM联系起来，并且大多数 文献中描述的SIRT 5缺失的表型是非常适度的。这项工作意义重大， 由于SIRT 5在调节1CM中的功能的阐明将确定1CM生物学的新方面，以及 可能会带来新的治疗机会。

NAD+-dependent sirtuin-family deacylases regulate diverse aspects of cellular and organismal homeostasis. Among these, SIRT5 has remained a somewhat enigmatic protein. SIRT5 is a primarily mitochondrial sirtuin that possesses atypical catalytic properties, removing succinyl, malonyl, and glutaryl groups from lysines on its substrate proteins. Although SIRT5 is the dominant cellular activity that targets these post-translational modifications (PTMs), SIRT5 deficiency in cells and whole animals provokes only very mild phenotypes, particularly under basal conditions. Thus, physiologic functions of SIRT5 and its target PTMs have remained somewhat mysterious. Using shRNA and CRISPR/Cas9-based approaches, we find that the sirtuin SIRT5 is critical for survival of specific cancer types, including melanoma and Ewing sarcoma (EWS). Whole transcriptome profiling demonstrates that SIRT5 affects expression of genes integral to survival and stress responses, including the melanoma lineage gene MITF and the FOXO3A transcription factor. Via metabolite profiling, we have identified defects in one carbon metabolism (1CM) conferred by SIRT5 depletion. 1CM consists of a complex set of biochemical reactions required for generation of the universal methyl donor S- adenosyl methionine (SAM), the co-substrate for histone and DNA methylation. In melanoma and EWS cells depleted for SIRT5, H3K4me3 levels are reduced, a phenotype recapitulated by treatment with a SIRT5 small molecule degrader that we recently developed. Remarkably, SAM reconstitution completely rescues the lethality associated with SIRT5 depletion in melanoma. The long-term goal is to elucidate the roles of SIRT5 in maintaining cellular homeostasis. The objective of this proposal is to elucidate roles for SIRT5 in regulating 1CM and nuclear gene expression. The central hypothesis is that SIRT5 regulates activities of 1CM enzymes to promote SAM generation, H3K4 trimethylation and proper gene expression. The rationale is that 1CM is the source of cellular SAM and other key cellular metabolites, and hence characterization of new mechanisms of 1CM regulation is of fundamental biological importance. The work will take place in the context of three Specific Aims. First, roles of SIRT5 in regulating 1CM will be elucidated mechanistically, using metabolite tracing, biochemistry, and other complementary approaches, focusing initially on the MTHFD1L enzyme as a candidate SIRT5 target. Second, the role of H3K4me3 in mediating the effects of SIRT5 on gene expression will be elucidated, using directed ChIP and global ChIP-seq. Third, the role of SIRT5 in regulating H3K4me3 levels and survival in response to 1C stress will be characterized in normal cells and mice. The application is innovative, in that no literature currently links SIRT5 to 1CM, and most phenotypes of SIRT5 depletion described in the literature are remarkably modest. The work is significant, as elucidation of SIRT5 functions in regulating 1CM will identify new aspects of 1CM biology, as well as potentially illuminating new therapeutic opportunities.