普利昂胜肽序列差異對其纖維結構及交叉播種屏障的影響

Abstract

普利昂疾病(Prion disease)是一種可由錯誤摺疊的蛋白質傳染的致死性神經退化性疾病，並可在好幾種哺乳類動物之間跨種傳染，其致病機制源於正常細胞膜上的普利昂蛋白PrPC (cellular prion protein)轉變為錯誤摺疊的PrPSc (scrapie prion protein)後，堆疊形成富含β-sheet的類澱粉纖維並在腦部累積，最後造成腦細胞死亡並在腦中留下很多海綿狀的空洞。 在不同物種間的跨種傳染存在著因序列差異所導致的物種感染屏障(sequence-dependent transmission barrier)，例如牛會感染貓和人，卻不會感染狗和豬。人的普利昂蛋白的第129號胺基酸有多型性，可以是methionine (M) 或是valine (V)，根據截至目前為止之流行病學報告，因狂牛症而感染新型庫賈氏症(new variant Creutzfeldt-Jakob disease，v-CJD)的患者中只有一名是129M/V異型接合子, 其他新型庫賈氏症患者全都是129M同型接合子。 本研究著重探討胺基酸序列與類澱粉纖維結構及引晶效率的關係，由於普利昂蛋白的危險等級為3，因此我們僅研究沒有生物體感染性，但對於普利昂纖維形成很重要的序列108-144，分別合成牛、貓、及人類(129M與129V)等物種之普利昂胜肽去形成類澱粉纖維。研究結果顯示四種普利昂胜肽纖維展現了具差異性的Thioflavin T螢光強度與親和性，依此特性進一步將其分成三個組別；交叉引晶(cross-seeding)方法表明人類129V普利昂胜肽(huPrP(108-144)129V)有較高的引晶障礙(seeding barrier)並只有人類129M普利昂胜肽(huPrP(108-144)129M)有能力感染huPrP(108-144)129V。另一方面，我們亦利用冷凍電子顯微鏡去進行所形成的牛普利昂胜肽(bPrP(108-144))類澱粉纖維的結構分析。

Prion disease is a fatal neurodegenerative disease that can be transmitted between multiple mammalian species through misfolded proteins. The pathogenic mechanism involves the conversion of normal protein PrPC (cellular prion protein) into pathological isoform PrPSc (scrapie prion protein), which undergoes self-propagating misfolding and accumulates as β-sheet-rich amyloid-like fibrils in neural tissues. This accumulation ultimately leads to neuronal death and sponge-like cavities in the brain. Interspecies transmission is caused by sequence-dependent infection barriers, such as the ability of cattle to infect cats and humans but not dogs and pigs. Human prion proteins exhibit polymorphism at the 129th amino acid position, either methionine (M) or valine (V). To date, epidemiological data indicate that among those infected with variant Creutzfeldt-Jakob disease (v-CJD) from mad cow disease, all patients were 129M homozygotes except for one 129M/V heterozygote. This research focuses on investigating the relationship between amino acid sequences, amyloid-like fibril structures, and seeding efficiency. The study concentrates on the non-infectious sequence 108-144, which is crucial for fibril formation. Synthesized prion peptides from bovine, cat, and human (129M and 129V) were used for analysis. The study results identified varying levels of Thioflavin T fluorescence intensity and binding profile across four different prion peptide fibers, leading to their classification into three groups. Cross-seeding studies revealed that huPrP(108-144)129V exhibits a higher seeding barrier, with only the huPrP(108-144)129M capable of inducing fibril formation in huPrP(108-144)129V. Additionally, cryogenic electron microscopy (cryo-EM) was employed to resolve the fibril structure of bPrP(108-144).