CHEMICAL SYNTHESIS OF SKP1 DERIVED GLYCOPEPTIDES FOR BIOCHEMICAL STUDIES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 真核生物已经进化出许多机制来感知氧气的可获得性并相应地调节细胞功能。动物体内的一个主要途径是转录因子亚单位缺氧诱导因子-1a(HIFA)1的氧依赖的Pro-4-羟基化，这使其成为E3VHLUb连接酶的靶标，该连接酶传递26S-蛋白酶体随后降解的信号。在霉菌属中，动物HIFA型脯氨酰4-羟基酶的同源基因P4H1也参与了O2的感受。P4H1的遗传破坏增加了顶峰所需的O2-，这是一个多细胞的鼻涕虫转化为子实体的发育过程。相反，P4H1的过表达降低了对O2-的需求。这些和其他结果提出了一个模型，在该模型中，环境O2是一个线索，影响活动的鼻涕在迁移到土壤表面后分化为无柄的子实体的决定，促进孢子扩散到潜在更有利的环境。 网柄网柄菌缺乏明显的HIFA同源基因。DictyostelialP4H1的一个已知靶标是Skp1，它是E3SCFUb连接酶中的一个接头。SCF(Skp1/cullin 1/F-box Protein)和VHL(von-Hippel Lindau)-型Ub-连接酶在进化上是相关的，有趣的是后者的一个亚基VHL蛋白识别一个羟基脯氨酸靶标，而在网柄网柄菌中，它是前者的一个亚基Skp1，即4-羟化的Pro。羟基化后，HyPro143被5个胞质糖基转移酶活性依次修饰，导致糖帽延伸到5个糖。先前的一项研究表明，在pgtA中中断的突变体对O2-的需求是正常的，这是添加第二糖和后续糖所必需的。这表明，除了第一种糖GlcNAc外，糖基化途径的其余部分可能发挥了另一种作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Eukaryotic organisms have evolved numerous mechanisms to sense O2 availability and regulate cellular function accordingly. A major pathway in animals is the O2-dependent prolyl 4-hydroxylation of the transcriptional factor subunit hypoxia inducible factor-1a (HIFa)1, which renders it a target of the E3VHLUb-ligase which signals subsequent degradation by the 26S-proteasome. In the mycetozoan Dictyostelium, the ortholog of animal HIFa-type prolyl 4-hydroxylases, P4H1, is also involved in O2 sensing. Genetic disruption of P4H1 increases the O2-requirement for culmination, the developmental process in which the multicellular slug converts to a fruiting body. In contrast, overexpression of P4H1 reduces the O2-requirement. These and other results suggest a model in which environmental O2 is a cue that influences the decision of the motile slug to differentiate, after migration to the soil surface, into a sessile fruiting body that promotes spore dispersal to potentially more favorable environments. Dictyostelium lacks an obvious ortholog of HIFa. A known target of Dictyostelium P4H1 is Skp1, an adaptor in E3SCFUb-ligases. SCF (Skp1/Cullin 1/F-box protein)- and VHL (von-Hippel Lindau)-type Ub-ligases are evolutionarily related and it is interesting that a subunit of the latter, VHL protein, recognizes a hydroxyproline target whereas in Dictyostelium, it is a subunit of the former type, Skp1, that is prolyl 4-hydroxylated. Subsequent to hydroxylation, HyPro143 is sequentially modified by 5 cytoplasmic glycosyltransferase activities, resulting in a glycan cap extending to up to 5 sugars. A previous study showed that the O2-requirement for a mutant disrupted in pgtA, required for addition of the second and subsequent sugars, was normal. This suggested that, except possibly for the first sugar, GlcNAc, the remainder of the glycosylation pathway fulfilled an alternative role.