Understanding how ciliary gene mutations affect the processing and activity of Gli2 and Gli3 transcription factors

摘要 初级纤毛是一个孤立的微管为基础的细胞器，突出于细胞表面， 存在于大多数脊椎动物细胞中。它在肾脏和视网膜等器官中充当感觉细胞器， 也在转导细胞外信号如Hedgehog（Hh）中起作用，Hh是一种分泌的信号分子， 是胚胎发育和细胞增殖和分化所必需的。纤毛结构缺陷， 功能与各种各样的发育异常有关，统称为“纤毛病”。 脊椎动物中的Hh信号发生在初级纤毛中，并且主要由Gli 2和Gli 3锌指介导。 含有转录因子。Gli 2主要是一种激活剂，而Gli 3主要是一种阻遏物，尽管它也 表现出弱的激活剂功能。与它们的功能一致，大多数全长Gli 3（Gli 3FL）是蛋白水解的， 在没有Hh信号传导的情况下，加工产生C末端截短的抑制子，而只有少量的抑制子 处理Gli 2FL的级分。Gli 2/Gli 3加工是由六个蛋白质中的前四个的磷酸化诱导的。 通过蛋白激酶A（PKA）和然后通过糖原合成酶激酶3在其C-末端的丝氨酸/苏氨酸残基 （GSK 3）和酪蛋白激酶1（CK 1）。然后，多磷酸化的Gli 2/Gli 3被泛素化，并部分地 被蛋白酶体降解Hh信号通过抑制PKA介导的Gli 2/Gli 3加工抑制Gli 2/Gli 3加工 Gli 2FL/Gli 3FL通过抑制前四个PKA位点的磷酸化而激活， C-末端有第5和第6个PKA位点。 纤毛结构和功能的缺陷主要影响Hh信号传导。睫状体的一个几乎普遍的特征是 基因突变体在分子水平上是Gli 2/Gli 3加工减少。有趣的是， 睫状体基因突变体中的Gli 2/Gli 3活性根据突变的睫状体基因而从无变化到升高。这 表明纤毛蛋白调节Hh信号传导的机制是多样和复杂的。而 纤毛突变对Hh信号传导的影响是公认的，这些突变的分子机制 其影响至今尚不清楚。该提案的目标是阐明分子机制， 纤毛基因突变体中Gli 2/Gli 3的加工、稳定性和活性发生改变。

Abstract The primary cilium is a solitary microtubule-based organelle that protrudes from the cell surface and is found on most vertebrate cells. It serves as a sensory organelle in organs such as the kidney and retina and also functions in transducing extracellular signals such as Hedgehog (Hh), a secreted signaling molecule that is essential for embryo development and cell proliferation and differentiation. Defects in cilia structure and function are associated with a diverse array of developmental abnormalities, collectively termed “ciliopathies”. Hh signaling in vertebrates occurs in primary cilia and is primarily mediated by Gli2 and Gli3 zinc finger- containing transcription factors. Gli2 is primarily an activator, whereas Gli3 is mostly a repressor, though it also exhibits a weak activator function. Consistent with their functions, most full-length Gli3 (Gli3FL) is proteolytically processed to generate a C-terminally truncated repressor in the absence of Hh signaling, while only a small fraction of Gli2FL is processed. Gli2/Gli3 processing is induced by phosphorylation of the first four of the six serine/threonine residues at their C-termini by protein kinase A (PKA) and then by glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). The multi-phosphorylated Gli2/Gli3 are then ubiquitinated and partially degraded by the proteasome. Hh signaling inhibits Gli2/Gli3 processing by suppressing PKA-mediated phosphorylation of the first four PKA sites and also activates Gli2FL/Gli3FL by inhibiting the phosphorylation of the fifth and sixth PKA sites in their C-termini. Defects in cilia structure and function mostly affect Hh signaling. One near universal hallmark of ciliary gene mutants at the molecular level is the reduced Gli2/Gli3 processing. Interestingly, however, the levels of Gli2/Gli3 activity in ciliary gene mutants vary from none to elevated depending on mutated ciliary genes. This suggests that the mechanism by which ciliary proteins regulate Hh signaling is diverse and complex. While the effect of ciliary mutations on Hh signaling is well established, the molecular mechanisms underlying these effects are thus far unknown. The goal of this proposal is to elucidate the molecular mechanisms by which Gli2/Gli3 processing, stability, and activity are altered in ciliary gene mutants.