Role of cellular metabolism in keratin variant-mediated liver disease progression

描述（由申请人提供）：本提案的主要目标是测试代谢缺陷可以揭示消化上皮细胞中细胞骨架的突变相关致病作用的可能性。角蛋白是最大的中间丝蛋白亚类，形成高度稳定的细胞骨架结构，提供机械支撑并增强细胞在压力下的弹性。角蛋白和其他IF蛋白的细胞保护功能已在动物模型中得到广泛证实，并反映在与IF基因突变相关的大量组织特异性人类疾病中。编码消化道上皮中主要IF蛋白的角蛋白8（K8）的基因中的杂合突变是急性和慢性肝病的易感因素。已知的K8变体的致病作用，表现出结构丝的变化，仅表现在细胞损伤，但这是知之甚少的生物学基础。该提案的目的是确定K8变异体肝脏发病机制背后的特定分子事件，特别关注K8聚集对能量代谢变化的反应。我们的计算机模拟分析的初步数据显示，已知的K8变体聚集在最有可能形成原纤维结构的蛋白质区域内，这通常被认为是细胞毒性的。此外，我们发现K8溶解度、寡聚化和聚集受位点特异性K8乙酰化调节，以响应葡萄糖和烟酰胺腺嘌呤二核苷酸（NAD）可用性的变化以及细胞质NAD依赖性脱乙酰酶沉默调节蛋白-2（SIRT 2）的活性。这些发现提出了代谢变化可能引发K8突变的病理表现的可能性，并使我们假设突变诱导的消化上皮角蛋白聚集是细胞代谢控制下的毒性事件。提出的具体目的旨在：（i）评价细胞代谢对肝病相关角蛋白突变体聚集特性的影响;和（ii）评估细胞代谢对肝病相关角蛋白突变体毒性的影响。在完成拟议的研究后，我们希望知道代谢变化是否可以打破突变K8细丝解体和有毒聚集体形成之间的平衡。这些信息，反过来，将作为未来研究的基础，旨在针对缺陷的角蛋白，例如，使用药理学伴侣或动力学稳定剂的基本角蛋白丝亚基作为潜在的新方法来治疗角蛋白相关疾病。

DESCRIPTION (provided by applicant): The main goal of this proposal is to test the possibility that metabolic deficiencies can unmask mutation-related pathogenic roles of the cytoskeleton in digestive epithelia. Keratins, which represent the largest sub-class of intermediate filament (IF) proteins, form highly stable cytoskeletal structures that provide mechanical support and reinforce cellular resilience under stress. The cell-protective functions of keratins and other IF proteins have been extensively demonstrated in animal models, and are reflected in the vast number of tissue-specific human diseases linked to IF gene mutations. Heterozygous mutations in the gene that encodes keratin 8 (K8), the major IF protein in digestive epithelia, are susceptibility factors for acute and chronic liver diseases. The pathogenic effects of the known K8 variants, which exhibit structural filament changes, are only manifested upon cell injury, but the biological basis for this is poorly understood. The objective of this proposal is to identify specific molecular events behind the liver pathogenesis of K8 variants, with a particular focus on K8 aggregation in response to changes in energy metabolism. Our preliminary data from in silico analysis reveal that the known K8 variants cluster within regions of the protein that are most likely to form protofibrillar structures, which are generally considered to be cytotoxic. Furthermore, we have found that K8 solubility, oligomerization, and aggregation are regulated by site-specific K8 acetylation in response to changes in glucose and nicotinamide adenine dinucleotide (NAD) availability, as well as activity of the cytoplasmic NAD- dependent deacetylase sirtuin-2 (SIRT2). These findings raise the possibility that metabolic changes may trigger the pathologic manifestation of K8 mutations and lead us to hypothesize that mutation-induced keratin aggregation in digestive epithelia is a toxic event under the control of cellular metabolism. The proposed specific aims are designed to: (i) evaluate the influence of cellular metabolism on the aggregation properties of liver disease - associated keratin mutants; and, (ii) assess the effects of cellular metabolism on the toxicity of liver disease - associated keratin mutants. Upon completion of the proposed studies we expect to know if metabolic changes can tip the balance between mutant K8 filament disorganization and formation of toxic aggregates. This information, in turn, will serve as the basis for future studies aimed at targeting defective keratins by, for example, the use of pharmacological chaperones or kinetic stabilizers of the basic keratin filament subunits as potential novel approaches to treat keratin-related disorders.