Mutant p97 exhibits species-specific changes of its ATPase activity and compromises the UBXD9-mediated monomerisation of p97 hexamers.
Mutant p97 exhibits species-specific changes of its ATPase activity and compromises the UBXD9-mediated monomerisation of p97 hexamers.
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突变体 p97 表现出其 ATP 酶活性的物种特异性变化，并损害 UBXD9 介导的 p97 六聚体单体化
DOI：
10.1016/j.ejcb.2016.03.004
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发表时间：
2016
影响因子：
6.6
通讯作者：
Eichinger L
中科院分区：
文献类型：
--
作者：
Rijal R;Arhzaouy K;Strucksberg K-H;Cross M;Hofmann A;Schröder R;Clemen CS;Eichinger L
p97（VCP）是一种同源六聚体三A ATP酶，其发挥过多的细胞过程。p97的杂合错义突变导致至少五种人类神经退行性疾病。然而，p97突变的具体分子后果迄今为止是广泛未知的。我们的人和网柄藻p97的硅结构模型显示，致病的人R93 C、R155 H和R155 C以及网柄藻R154 C、E219 K、R154 C/E219 K p97突变构成了表面暴露位置的变异。p97单体和六聚体的凝胶内ATP酶活性测量揭示了显着的突变和物种特异性差异。虽然所有的人p97突变都导致ATP酶活性增加，但在与人R155 C直向同源的网柄菌R154 C突变体中没有检测到任何变化。E219 K突变导致活性几乎完全丧失，这在R154 C/E219 K双突变体中部分恢复，表明p97结构域间通讯。通过免疫共沉淀实验，我们鉴定了一个含有UBX结构域的Dictyostelium蛋白作为一种新的p97相互作用伴侣。我们将所有含有UBX结构域的Dictyostelium蛋白进行了分类，并将其命名为UBXD 9。拉下试验和表面等离子体共振分析Dictyostelium UBXD 9或人类直系同源物TUG/ASPL/UBXD 9证明了与p97的直接相互作用以及结合亲和力的物种，突变和ATP依赖性差异。蔗糖密度梯度分析表明，人类和网柄藻UBXD 9蛋白都能非常有效地将野生型p97六聚体分解成单体，但在较小程度上将突变型p97六聚体分解成单体。我们的研究结果与p97点突变导致酶活性和分子相互作用差异的情况一致，长期导致迟发性和进行性多系统疾病。
p97 (VCP) is a homo-hexameric triple-A ATPase that exerts a plethora of cellular processes. Heterozygous missense mutations of p97 cause at least five human neurodegenerative disorders. However, the specific molecular consequences of p97 mutations are hitherto widely unknown. Ourin silicostructural models of human andDictyosteliump97 showed that the disease-causing human R93C, R155H, and R155C as well asDictyosteliumR154C, E219K, R154C/E219K p97 mutations constitute variations in surface-exposed locations. In-gel ATPase activity measurements of p97 monomers and hexamers revealed significant mutation- and species-specific differences. While all human p97 mutations led to an increase in ATPase activity, no changes could be detected for theDictyosteliumR154C mutant, which is orthologous to human R155C. The E219K mutation led to an almost complete loss of activity, which was partially recuperated in the R154C/E219K double-mutant indicating p97 inter-domain communication. By means of co-immunoprecipitation experiments we identified an UBX-domain containingDictyosteliumprotein as a novel p97 interaction partner. We categorized all UBX-domain containingDictyosteliumproteins and named the interaction partner UBXD9. Pull-down assays and surface plasmon resonance analyses ofDictyosteliumUBXD9 or the human orthologue TUG/ASPL/UBXD9 demonstrated direct interactions with p97 as well as species-, mutation- and ATP-dependent differences in the binding affinities. Sucrose density gradient assays revealed that both human andDictyosteliumUBXD9 proteins very efficiently disassembled wild-type, but to a lesser extent mutant p97 hexamers into monomers. Our results are consistent with a scenario in which p97 point mutations lead to differences in enzymatic activities and molecular interactions, which in the long-term result in a late-onset and progressive multisystem disease.
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影响因子：
3.7
作者：
Arhzaouy K;Strucksberg KH;Tung SM;Tangavelou K;Stumpf M;Faix J;Schröder R;Clemen CS;Eichinger L
通讯作者：
Eichinger L
人红细胞高分子量可溶性 Mg2-ATP 酶的生化和超微结构表征。
影响因子：
5.6
作者：
J. Harris
通讯作者：
J. Harris
用于天然产物分离的功能色谱技术。
影响因子：
3.2
作者：
Lau,EricC;Mason,DamianJ;Eichhorst,Nicole;Engelder,Pearce;Mesa,Celestina;KithsiriWijeratne,EM;Gunaherath,GMKamalB;Gunatilaka,AALeslie;LaClair,JamesJ;Chapman,Eli
通讯作者：
Chapman,Eli
影响因子：
3.3
作者：
Dalal, S;Rosser, MFN;Hanson, PI
通讯作者：
Hanson, PI
影响因子：
5.7
作者：
Davies, Jason M.;Brunger, Axel T.;Weis, William I.
通讯作者：
Weis, William I.