Huff Mary E, Page Lesley J, Balch William E, Kelly Jeffery W
Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute BCC265, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
J Mol Biol. 2003 Nov 14;334(1):119-27. doi: 10.1016/j.jmb.2003.09.029.
Mutation of aspartic acid 187 to asparagine (D187N) or tyrosine (D187Y) in domain 2 of the actin-modulating protein gelsolin causes the neurodegenerative disease familial amyloidosis of Finnish type (FAF). These mutations render plasma gelsolin susceptible to aberrant proteolysis by furin in the trans-Golgi network, the initial proteolytic event in the formation of 71 and 53 residue fragments that assemble into amyloid fibrils. Ca(2+) binding stabilizes wild-type domain 2 gelsolin against denaturation and proteolysis, but the FAF variants are unable to bind and be stabilized by Ca(2+). Though the chain of events initiating FAF has been elucidated recently, uncertainty remains about the mechanistic details that allow the FAF variants to be processed. To test the hypothesis that impaired Ca(2+) binding in the D187 variants, but not other factors specific to residue 187, increases susceptibility to aberrant proteolysis and subsequent amyloidogenesis, we designed the gelsolin variant E209Q to remove a different Ca(2+) ligand from the same Ca(2+) site that is affected in the FAF variants. Here, we show that E209Q domain 2 does not bind Ca(2+) and is not stabilized against denaturation or furin proteolysis, analogous to the behavior exhibited by the FAF variants. Transfection of full-length E209Q into COS cells results in secretion of both the full-length and furin-processed fragments, as observed with D187N and D187Y. Mutation of the furin consensus sequence in D187N and E209Q gelsolin prevents cleavage during secretion, indicating that inhibition of proprotein convertases (furin) represents a viable therapeutic approach for the treatment of FAF. Mutations that diminish domain 2 Ca(2+) binding allow furin access to an otherwise protected cleavage site, initiating the proteolytic cascade that leads to gelsolin amyloidogenesis and FAF.
肌动蛋白调节蛋白凝溶胶蛋白第2结构域中天冬氨酸187突变为天冬酰胺（D187N）或酪氨酸（D187Y）会导致神经退行性疾病芬兰型家族性淀粉样变性（FAF）。这些突变使血浆凝溶胶蛋白易受反式高尔基体网络中弗林蛋白酶异常蛋白水解的影响，这是形成组装成淀粉样原纤维的71和53个残基片段的初始蛋白水解事件。Ca(2+)结合可稳定野生型第2结构域凝溶胶蛋白，防止其变性和蛋白水解，但FAF变体无法结合Ca(2+)并被其稳定。尽管最近已经阐明了引发FAF的一系列事件，但关于允许FAF变体被加工的机制细节仍存在不确定性。为了检验以下假设：D187变体中Ca(2+)结合受损，而非187位残基的其他特定因素，会增加对异常蛋白水解和随后淀粉样蛋白生成的易感性，我们设计了凝溶胶蛋白变体E209Q，以从与FAF变体受影响的相同Ca(2+)位点去除不同的Ca(2+)配体。在此，我们表明E209Q第2结构域不结合Ca(2+)，并且在变性或弗林蛋白酶蛋白水解作用下不稳定，这与FAF变体的行为类似。将全长E209Q转染到COS细胞中会导致全长和弗林蛋白酶加工片段的分泌，这与D187N和D187Y的情况相同。D187N和E209Q凝溶胶蛋白中弗林蛋白酶共有序列的突变可防止分泌过程中的切割，这表明抑制前蛋白转化酶（弗林蛋白酶）是治疗FAF的一种可行治疗方法。减少第2结构域Ca(2+)结合的突变使弗林蛋白酶能够进入原本受保护的切割位点，启动导致凝溶胶蛋白淀粉样蛋白生成和FAF的蛋白水解级联反应。
