酵母菌中琥珀酸去氫酶D的直系同源物：臨床和演化意義

Abstract

人類SDH由四個次單元SDHA、SDHB、SDHC、SDHD組成，是TCA循環和電子傳遞鏈進行的樞紐酵素，其中SDHD會和SDHC一起鑲嵌在粒線體內層膜上負責將電子傳遞給complex III。副神經節瘤 (PGL)為罕見的腫瘤疾病，30%的PGL病人具有SDH遺傳性基因突變，其中近半數為SDHD異常，臨床症狀主要為好發於頭頸部的良性瘤，但目前腫瘤形成的機制仍未知，利用酵母菌具有和人類基因高度同源的優勢，我們以酵母菌做為動物模式探討PGL的致病機制。酵母菌的SDH同樣由四個次單元組成，分別為Sdh1、Sdh2、Sdh3和Sdh4，比對hSDHD和酵母菌的蛋白序列後發現一個功能未知的蛋白，Shh4/Ylr164W，相較於Sdh4有較高的序列相似度，在實驗室先前的研究中發現，單獨拿掉shh4對酵母菌的生長影響不大，但在sdh4缺失的酵母菌中拿掉shh4時明顯降低酵母菌的生長速度，此外在進入生長停滯期時，同時拿掉sdh4和shh4比單獨拿掉sdh4有更顯著的粒線體膜電位下降，因此推測Shh4在酵母菌中扮演Sdh4備份蛋白的角色，演化上人類的SDHD較有可能從SHH4演變而來，SDH4則因較無抵抗環境壓力的能力而逐漸被淘汰。為了確認此推論，我們比較不同壓力下蛋白質的表現時，發現Shh4在特定壓力下表現量會比Sdh4高；拿掉Sdh4時Shh4表現量明顯上升；以及Sdh3的穩定度在同時拿掉Sdh4和Shh4時較單獨拿掉Sdh4不穩定，由核基因的突變率也觀察到相同趨勢，以及在人類細胞中表現臨床點突變基因時發現蛋白質不穩定的表現相似於在SDH4缺失的酵母菌中Shh4表現點突變的型態，因此進一步推測基因缺失造成細胞內活性氧化物上升，使得基因益加不穩定是導致腫瘤形成的原因。

Gene duplication and divergence are common in evolution. The mitochondrial succinate dehydrogenase (SDH) is an essential complex of the electron transport chain and of the tricarboxylic acid cycle. Mutation in human succinate dehydrogenase subunit SDHD frequently leads to cancer formation. In addition to the originally discovered and assigned yeast SDHD subunit Sdh4, another conserved homolog Shh4 has recently been noticed in the budding yeast. Interestingly, Shh4 is upregulated under several stresses and can substitute for Sdh4 in sdh4∆ cells. Mitochondria membrane potential and stability of SDH complex are further compromised and ROS production and mutation frequency are enhanced in sdh4∆ shh4∆ double mutants than sdh4∆ cells. Missense mutations found in the human SDHD gene in cancer patients were created in Sdh4 and Shh4. Surprisingly, growth defect merely appeared in the chimera of human deficient SDHD in Shh4 and this defect is due to the instability of the protein. Altogether, the sequence comparison and functional analysis favor a hypothesis that the succinate dehydrogenase in humans is evolved from yeast Shh4. Besides, SDH mutation leads to the disassembly of SDHC-SDHD complex, mitochondria damage, ROS production, genome instability and tumor formation.