Perk eIF2a Kinase Integrates Proinsulin Quality Control and Insulin Secretion

 描述(由申请人提供)：永久性新生儿糖尿病和2型细胞失代偿的单基因形式突出了内质网(ER)中胰岛素原处理的重要性和脆弱性。胰岛素原折叠、内质网相关蛋白降解(ERAD)介导的质量和数量控制以及胰岛素原从内质网到高尔基体的运输等过程曾经被认为是管家的功能，但这些过程都受到代谢状态和对胰岛素的需求的高度调控。我们已经确定PERK eIF2a激酶是内质网胰岛素原加工的关键调节因子，它的功能丧失会导致Wolcott Rallison综合征的永久性新生儿糖尿病。我们假设了一个新的且有争议的模型，即PERK通过控制关键的ER伴侣和包括GRP78和ERp72在内的折叠蛋白的表达来调节胰岛素原的加工，这些折叠蛋白反过来控制胰岛素原是通过ERAD途径降解还是被运输到高尔基体进行蛋白质分解加工和包装成分泌颗粒。在这项提案中，我们将通过操纵PERK-eIF2a通路和关键ER伴侣的表达以及评估胰岛素原的命运和处理状态来进一步测试这一模型。我们还将确定PERK-eIF2a如何调节胰岛素原的处理，以响应血糖水平的波动，以提供适当的胰岛素产生水平。基于eIF2a去磷酸化抑制剂guanabenz可以逆转Perk缺乏的？细胞中出现的严重的胰岛素原聚集的初步结果，我们建议测试其预防或逆转与Wolcott Rallison综合征和显性-阴性作用胰岛素突变(Midy)相关的严重永久性新生儿糖尿病的能力。这一策略也可能适用于与2型糖尿病失代偿相关的？细胞功能障碍的治疗。最终目的是确定依赖于PERK的内质网伴侣调节的分子机制，这对于理解PERK在调节？细胞功能中起核心作用的调节环境是至关重要的。

 DESCRIPTION (provided by applicant): Monogenic forms of permanent neonatal diabetes and type 2 ß-cell decompensation have highlighted the importance and vulnerability of proinsulin processing in the endoplasmic reticulum (ER). Once thought to be housekeeping functions, the processes of proinsulin folding, ER associated protein degradation (ERAD)- mediated quality and quantity controand trafficking of proinsulin from the ER to the Golgi are all highly regulated as a function of the metabolic state and demand for insulin. We have identified the PERK eIF2a kinase, whose loss of function results in permanent neonatal diabetes in the Wolcott Rallison syndrome, as a key regulator of proinsulin processing in the ER. We postulated a novel and controversial model that PERK regulates proinsulin processing by controlling the expression of key ER chaperone and folding proteins including GRP78 and ERp72, which, in turn, control whether proinsulin is degraded by the ERAD pathway or trafficked forward to the Golgi for proteolytic processing and packaging into secretory granules. In this proposal we will further test this model by manipulating the PERK-eIF2a pathway and the expression of key ER chaperones and assessing the fate and processing state of proinsulin. We will also determine how PERK- eIF2a modulates proinsulin processing in response to fluctuating glucose levels in order to provide the appropriate level of insulin production. Based on preliminary results that Guanabenz, an eIF2a dephosphorylation inhibitor, can reverse the severe proinsulin aggregation seen in PERK deficient ß-cells, we proposed to test its ability to prevent or reverse the severe permanent neonatal diabetes associated with the Wolcott Rallison Syndrome and dominant-negative acting insulin mutants (MIDY). This strategy may also be applicable to treatment of ß-cell dysfunctions associated with type 2 diabetes decompensation. The final Aim is designed to identify the molecular mechanisms underlying PERK-dependent regulation of ER chaperones and is essential for understanding the regulatory context in which PERK plays a central role in regulating ß-cell functions.