Direct Regulation of Extracellular Proteostasis by the Unfolded Protein Response

 描述(申请人提供)：失稳的淀粉样蛋白的错误折叠和细胞外聚集与30多种蛋白质聚集性(即淀粉样蛋白)疾病的退行性表型有着千丝万缕的联系，这些疾病包括阿尔茨海默病、克雅氏病和系统性淀粉样变性。重要的药理学和遗传学证据证实，在这些疾病中，蛋白质聚集和有丝分裂后组织退化之间存在因果关系。细胞外蛋白质聚集在淀粉样蛋白疾病病理中的重要性刺激了大量的实验工作，重点是确定调节细胞外环境中蛋白质稳态(或蛋白质稳态)的生物和细胞途径。其中一个途径是未折叠蛋白反应(UPR)，这是一种应激反应信号通路，负责调节内质网(ER)胁迫下分泌途径中的蛋白平衡。我们实验室和其他实验室之前的结果表明，UPR通过减少哺乳动物细胞分泌淀粉样蛋白来间接影响不稳定的淀粉样蛋白的细胞外聚集，从而降低可用于浓度依赖聚集的细胞外蛋白质水平。在这里，我们假设UPR的激活也通过增加细胞外伴侣的表达和分泌来直接调节细胞外蛋白平衡，这些伴侣阻止了不稳定的、易于聚集的蛋白质的蛋白毒性聚集。我们已经确定内质网靶向的Hsp40辅助伴侣ERdj3是一种UPR调节的、分泌的伴侣蛋白，它促进细胞外蛋白稳定以响应内质网应激。我们发现ERdj3减弱了疾病相关的、易于聚集的分泌蛋白的聚集和蛋白毒性，包括A？40和毒性蛋白(TPrP)。此外，我们发现ERdj3与不稳定的、易于聚集的蛋白质在ER蛋白稳态途径被淹没的条件下共同分泌，提供了一种先发制人的机制来保护细胞外环境免受蛋白毒性细胞外蛋白聚集的影响。在这个应用中，我们使用生物物理、生化和细胞生物学的方法来扩展这些发现，以确定UPR依赖的ERDj3分泌保护细胞外环境免受蛋白毒性蛋白构象影响的分子机制。通过这些努力，我们将确定UPR调节的ERdj3分泌的特定方面，直接参与防止与淀粉样蛋白疾病病理相关的失稳分泌蛋白的蛋白毒性聚集。这些结果将证明，UPR信号的改变，如那些发生在正常衰老过程中或对淀粉样病相关基因突变的反应，可以促进参与淀粉样病发病机制的衰老依赖的细胞外蛋白聚集。此外，我们将确定UPR信号通路的成分，这些成分可以作为治疗靶点，促进细胞外蛋白稳定和防止细胞外蛋白聚集，从而揭示出一种新的策略，以减轻参与淀粉样蛋白疾病病理过程的分泌性蛋白的蛋白毒性。

 DESCRIPTION (provided by applicant): The misfolding and extracellular aggregation of destabilized, amyloidogenic proteins is inextricably linked to degenerative phenotypes in over 30 protein aggregation (i.e., amyloid) diseases including Alzheimer's disease, Creutzfeldt-Jakob disease and the systemic amyloidoses. Significant pharmacologic and genetic evidence confirms a causal relationship between protein aggregation and degeneration of post-mitotic tissues in these diseases. The importance of extracellular protein aggregation in amyloid disease pathology has stimulated significant experimental effort focused on defining the organismal and cellular pathways that regulate protein homeostasis (or proteostasis) in the extracellular environment. One such pathway is the Unfolded Protein Response (UPR) - the stress-responsive signaling pathways responsible for regulating proteostasis within the secretory pathway in response to endoplasmic reticulum (ER) stress. Previous results from our lab and others have shown that the UPR indirectly influences extracellular aggregation of destabilized, amyloidogenic proteins by reducing their secretion from mammalian cells, thus decreasing extracellular protein levels available for concentration-dependent aggregation. Here, we hypothesize that UPR activation also directly regulates extracellular proteostasis through the increased expression and secretion of extracellular chaperones that prevent the proteotoxic aggregation of destabilized, aggregation-prone proteins. We have identified the ER-targeted HSP40 co-chaperone ERdj3 as a UPR regulated, secreted chaperone that promotes extracellular proteostasis in response to ER stress. We show that ERdj3 attenuates the aggregation and proteotoxicity of disease-associated, aggregation-prone secreted proteins including Aß40 and toxic prion protein (TPrP). Additionally, we show that ERdj3 is co-secreted in a complex with destabilized, aggregation-prone proteins under conditions where ER proteostasis pathways are overwhelmed, providing a mechanism to preemptively protect the extracellular environment from proteotoxic extracellular protein aggregation. In this application, we expand on these findings using biophysical, biochemical and cell biological approaches to define the molecular mechanisms by which UPR-dependent ERdj3 secretion protects the extracellular environment against proteotoxic protein conformations. Through these efforts, we will identify specific aspects of UPR-regulated ERdj3 secretion directly involved in preventing the proteotoxic aggregation of destabilized secreted proteins associated with amyloid disease pathology. These results will demonstrate that altered UPR signaling, such as those that occur during normal aging or in response to amyloid-disease associated genetic mutations, can facilitate aging-dependent extracellular protein aggregation involved in amyloid disease pathogenesis. Furthermore, we will identify components of UPR signaling pathways that can be therapeutically targeted to promote extracellular proteostasis and prevent extracellular protein aggregation, revealing a new strategy to attenuate proteotoxicity of secreted proteins involved in the pathology of amyloid diseases.