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標題: | 探討神經退化性疾病中的蛋白質恆定及摺疊機制 Analysis of proteostasis and protein folding mechanisms in neurodegenerative diseases |
作者: | Huan-Yun Chen 陳奐勻 |
指導教授: | 鄧述諄(Shu-Chun Teng) |
關鍵字: | 巴金森氏病,伴侶蛋白, BAG5,co-chaperone,p53,a-synuclein,Parkinson's disease,STUB1,CHIP,DNAJB11,SDF2,BiP, |
出版年 : | 2020 |
學位: | 博士 |
摘要: | 巴金森氏病(Parkinson’s disease)是一種常見的神經退化性疾病,主要病理特徵為異常折疊之突變的α-synuclein 本身形成集聚(self-aggregation)於腦黑質 (substantia nigra)並形成胞質內路易氏體(lewy body)。目前已知老化、基因遺傳及後天環境因素均會影響a-synuclein異常折疊集聚進而促進巴金森氏病發生。上述因子可能會造成a-synuclein異常折疊集聚,細胞中則有伴侶蛋白(chaperone)協助細胞內分子組裝和協助蛋白質摺疊的蛋白質。儘管迄今已有許多證據顯示:伴侶蛋白(chaperone)在維持細胞內蛋白質恆定(proteostasis)扮演重要角色,但伴侶蛋白的作用如何造成巴金森氏病致病機轉,目前仍不甚清楚。本研究中我們在不同環境壓力下分析了多個熱休克蛋白70(HSP70)及熱休克蛋白90(HSP90)相關因子的表現;結果發現在經過癌妥滅靜脈注射液劑(etoposide)及過氧化氫(H2O2)處理後的人類神經瘤母細胞(SH-SY5Y),共伴侶蛋白(co-chaperone) BAG5表現量會大幅提升。此外,外界壓力誘導表現的腫瘤抑制蛋白p53會結合至BAG5啟動子(promoter)以促進BAG5大量表現。在細胞實驗及人體臨床試驗中進一步發現,經壓力適量誘導產生的BAG5會與異常折疊集聚的a-synuclein及熱休克蛋白70結合進而促進熱休克蛋白70針對異常堆疊的蛋白質重新進行折疊;然而若過量表現的BAG5則會使熱休克蛋白70重新折疊蛋白質的能力下降。由此結果顯示: BAG5表現與否會影響a-synucleing是否異常折疊集聚。綜合上述結果發現,外在壓力會影響腫瘤抑制蛋白p53、共伴侶蛋白(co-chaperone) BAG5及熱休克蛋白70三者間的作用,此發現亦提供巴金森氏病致病機轉及治療上新的契機。
STUB1編碼CHIP蛋白,同時具有E3泛素連接酶及共伴侶蛋白(co-chaperone)的角色,參與到多個蛋白異常折疊集聚的降解。在遺傳性小腦性運動失調疾病(autosomal recessive cerebellar ataxia, ARCA)中已發現STUB1在多個位點有基因突變發生。本研究透過台灣本土的一個遺傳性小腦性運動失調疾病的家庭成員基因圖譜定序分析發現:STUB1在這些發病個體上均具有一個新的STUB1位點的基因突變 (c.832del, p.Glu278fs)。在人類神經瘤母細胞(SH-SY5Y)及神經母細胞瘤細胞(BE2M17)大量表現STUB1(c.832del, p.Glu278fs)後發現:相較於具有正常功能的STUB1,此STUB1點突變無法有效清除細胞中-synuclein異常折疊集聚。此外在先前的研究已顯示:此STUB1點突變破壞的蛋白質結構域(protein domain)為E2泛素接合酵素(ubiquitin-conjugating enzyme, E2)結合的區域;因此後續的研究除了會探討此STUB1點突變是否會影響蛋白異常折疊集聚的降解,同時也會針對E2泛素接合酵素結合能力進行分析。 細胞可以透過重新調整(remodeling)內質網(endoplasmic reticulum)上蛋白質品質管制的訊息路徑(quality control pathways)進而回應外在的壓力刺激。內質網上有許多伴侶蛋白(chaperone)可協助新合成的蛋白質進行折疊。DNAJB11就是在內質網上特定的共伴侶蛋白(co-chaperone),透過DNAJB11可協助自然態蛋白質(native protein)進入內質網、並將未折疊的蛋白質攜帶給熱休克蛋白70的其中一員,BiP (immunoglobulin heavy chain binding protein in pre-B cells)進行折疊,最後在協助其離開內質網。根據先前的研究,人胚胎腎細胞293 (HEK293T)利用輻射(irradiation)處理以模仿DNA損傷(DNA damage)環境,結果發現其中一個共伴侶蛋白(co-chaperone), DNAJB11其上的T188位點為可能被ATM/ATR kinase 磷酸化的位點。透過蛋白質功能性分析發現:DNAJB11其上的T188位點可能影響另一個共伴侶蛋白(co-chaperone) SDF2的穩定度及DNAJB11與SDF2 complex形成,進而影響未折疊的蛋白質攜帶至Bip進行重新摺疊的能力。由此現象推測:DNAJB11 T188的磷酸化可能會影響Bip進行蛋白質摺疊的能力,進而導致不正常的蛋白質堆疊。 Parkinson’s disease (PD) is a common neurodegenerative disorder with the pathological hallmark of a-synuclein aggregation. Dysregulation of a-synuclein homeostasis caused by aging, genetic, and environmental factors underlies the pathogenesis of PD. While chaperones are essential for proteostasis, whether modulation of cochaperones may participate in PD formation has not been fully characterized. Here, I first assessed the expression of several HSP70- and HSP90-related factors under various stresses and found that BAG5 expression is distinctively elevated in etoposide- or H2O2-treated SH-SY5Y cells. Stress-induced p53 binds to the BAG5 promoter directly to stimulate BAG5. Induced BAG5 binds a-synuclein and HSP70 in both cell cultures and brain lysates from PD patients. Overexpressed BAG5 may result in the loss of its ability to promote HSP70. Importantly, a-synuclein aggregation in SH-SY5Y cells requires BAG5. BAG5 expression is also detected in transgenic SNCA mutant mice and in PD patients. Together, my data reveal stress-induced p53-BAG5-HSP70 regulation that provides a potential therapeutic angle for PD. STUB1, encoding the protein CHIP (C-terminus of HSC70-interacting protein), is a dual-function protein as a co-chaperone and an E3 ligase. Mutations in the gene STUB1 have recently been suggested as a cause of recessive ataxia. Here I aim to investigate the effect of the heterozygous missense STUB1 (c.832del, p.Glu278fs) mutation found in Taiwanese Spinocerebellar ataxia (SCA) patients. With a combination of homozygosity mapping and exome sequencing, we identified a mutation of STUB1 in a family with ataxia and cognitive impairment, a heterozygous missense variant (c.832del, p.Glu278fs) that segregated in two affected siblings and a consanguineous parent. The p.Glu278fs missense impairs CHIP’s ability to ubiquitinate a-synuclein aggregation in both SH-SY5Y and BE2-M17 cells. Previous studies have shown that this domain is critical for the interaction between CHIP and the E2 ligase. I am determining how STUB1 mutations cause disturb the E2-E3 interaction and the E3 ligase function. Finally, cells response to stresses through remodeling of ER quality control pathways. ER is an essential sub‐cellular compartment of the folding of newly synthesized proteins, which is assisted by ER-resident chaperone proteins. DNAJB11, an ER‐targeted HSP40 co‐chaperone, is required for assisting nascent proteins entering ER lumen, delivering them to the BiP chaperone and facilitating their exit from the ER. A previous large-scale phosphoproteomic analysis in response to DNA damage has identified DNAJB11 threonine 188 is a possible phosphorylation site of ATM and ATR kinases. DNAJB11 forms a complex with SDF2 to facilitate the unfolded protein transfer to BiP. However, a DNAJB11 T188A mutant inhibits the unfolded a-synuclein A53T client binding and thus may prevent the delivery of misfolded cargo from the DNAJB11-SDF2 complex to BiP. These findings suggest that DNAJB11 T188 phosphorylation may act as a key regulatory stop in the BiP chaperone cycle to prevent the aggregation of aggregrated proteins. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8091 |
DOI: | 10.6342/NTU202004318 |
全文授權: | 同意授權(全球公開) |
電子全文公開日期: | 2025-11-05 |
顯示於系所單位: | 微生物學科所 |
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