p62在蛋白質堆疊物的降解作用之調控機制

Abstract

神經退化性疾病的共同特徵，是在神經內或周圍有不正常的蛋白質堆疊，許多研究指出，這些異常堆疊的蛋白質，是導致神經退化的主因。目前已知選擇性的細胞自噬作用(selective autophagy)，可以特定清除這些堆疊物，做為一種保護神經的機制。有證據顯示，在哺乳動物細胞中，這樣的選擇性是經由p62/sequestosome1做為受體:它可以跟自噬體上的LC3結合，且被觀察到會共同沉積含特定致病蛋白的堆疊物中，例如亨丁頓舞蹈症(Huntington’s disease, HD)的突變型Huntingtin (Htt)，以及阿茲海默氏症(Alzheimer’s disease, AD)的Tau。 本論文的第一部分，延續了之前在酵母菌模式生物的發現，找出在LC3上負責與p62結合的胺基酸位點，顯示從酵母菌到哺乳動物都利用相同的分子機制，將受體連結到自噬體中。而我們的實驗也證實，當LC3與p62無法形成連結時，突變Htt型蛋白堆疊便無法經由選擇性細胞自噬作用所清除，進一步支持p62可作為細胞自噬作用降解特定蛋白質堆疊的受體。 p62在神經退化性疾病的重要性，促使我們去研究它基因表現上的調控，與其蛋白質功能的相關性。本論文的第二部份，陳述我們發現presenilin-1 (PS1)可經由活化Akt/AP-1訊息傳導途徑，來促進p62基因的轉錄表現。我們也測試了幾個在家族遺傳性AD病人中特有的PS1基因突變型，發現當細胞表現這些突變的PS1時，卻無法維持其p62基因的正常表現，導致細胞沒有足夠的p62來幫助清除Tau蛋白質。此研究找到了PS1調控p62表現進而影響Tau降解的新功能，也更加確認p62在神經退化性疾病中，作為清除蛋白質堆疊的重要分子。 本論文的研究成果，證實LC3-p62的結合，在選擇性細胞自噬作用中擔任舉足輕重的角色，負責將誘發神經退化性疾病的蛋白質堆疊送至自噬體中。另一方面，也找到調控p62表現的新途徑，幫助我們更進一步了解AD及其他神經退化性疾病的致病機轉。

The accumulation of certain misfolded protein aggregates in the brain is a common feature in various neurodegenerative diseases, and is accepted as a major causative factor of neurodegeneration. Aggrephagy, the process by which protein aggregates are selectively degraded through macroautophagy, plays an essential role in protecting neurons from aggregate-induced neurotoxicity. Recent findings have identified p62/sequestosome1 as a cargo receptor that interacts with the autophagosomal membrane associated protein LC3, and recruits ubiquitin-positive protein aggregates into autophagosomes. The finding that p62 is co-localized with inclusion bodies in the brains of patients with Huntington’s disease (HD) and Alzheimer’s disease (AD) suggests a critical role for p62 in neurodegeneration. Previous findings have identified residues in a yeast LC3 homologue, Atg8, that are essential for interaction of Atg8 with the cargo receptor Atg19 in selective autophagic processes. In the first part of my thesis, I describe our attempts to determine whether such interaction is evolutionally conserved from yeast to mammals. By using an amino acid replacement approach, we determined that three residues in LC3 corresponding to those in Atg8 were essential for p62 binding. Furthermore, while disruption of the LC3-p62 complex formation did not alter overall autophagic activity, it was sufficient to impede the autophagy-mediated clearance of aggregation–prone mutant Huntingtin (Htt), the cytotoxic protein which induces the pathological phenotypes of HD. The protective role of p62 in the clearance of aggregation-prone proteins prompted us to investigate how p62 expression is regulated under pathological conditions. In the second part of my thesis, I describe our discovery that p62 expression is transcriptionally regulated by presenilin 1 (PS1), a protein which is mutated in the majority of patients with early-onset familial Alzheimer’s disease (FAD). The PI3K/Akt/AP-1 pathway was found to be required for PS1-mediated regulation of p62 expression. Moreover, down-regulation of p62 by either PS1 deficiency or over-expression of FAD-linked PS1 mutants compromised clearance of aggregation-prone Tau, which forms intracellular neurofibrillary tangles in the AD brain; these findings thus confirm the essential role of p62 in the clearance of neurotoxic protein aggregates. Together, our studies emphasize the importance of the LC3-p62 interaction in selective autophagy, and the requirement of p62 for the removal of neurodegeneration-associated protein aggregates. Furthermore, the identification of PS1-dependent transcriptional regulation of p62 expression uncovers a novel PS1/p62-mediated molecular mechanism underlying the pathogenesis of AD and related neurodegenerative diseases.