BACKGROUND Mutations in the gene encoding inverted formin-2 (INF2), a member of the formin family of actin regulatory proteins, are among the most common causes of autosomal dominant FSGS. INF2 is regulated by interaction between its N-terminal diaphanous inhibitory domain (DID) and its C-terminal diaphanous autoregulatory domain (DAD). INF2 also modulates activity of other formins, such as the mDIA subfamily, and promotes stable microtubule assembly. Why the disease-causing mutations are restricted to the N terminus and how they cause human disease has been unclear. METHODS We examined INF2 isoforms present in podocytes and evaluated INF2 cleavage as an explanation for immunoblot findings. We evaluated the expression of INF2 N- and C-terminal fragments in human kidney disease conditions. We also investigated the localization and functions of the DID-containing N-terminal fragment in podocytes and assessed whether the FSGS-associated R218Q mutation impairs INF2 cleavage or the function of the N-fragment. RESULTS The INF2-CAAX isoform is the predominant isoform in podocytes. INF2 is proteolytically cleaved, a process mediated by cathepsin proteases, liberating the N-terminal DID to function independently. Although the N-terminal region normally localizes to podocyte foot processes, it does not do so in the presence of FSGS-associated INF2 mutations. The C-terminal fragment localizes to the cell body irrespective of INF2 mutations. In podocytes, the N-fragment localizes to the plasma membrane, binds mDIA1, and promotes cell spreading in a cleavage-dependent way. The disease-associated R218Q mutation impairs these N-fragment functions but not INF2 cleavage. CONCLUSIONS INF2 is cleaved into an N-terminal DID-containing fragment and a C-terminal DAD-containing fragment. Cleavage allows the N-terminal fragment to function independently and helps explain the clustering of FSGS-associated mutations.

中文翻译：

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导致GSGS的FSGS突变损害足细胞中已分裂的INF2 N片段功能。

背景技术编码反式formin-2（INF2）（肌动蛋白调节蛋白的formin家族成员）的基因突变是常染色体显性FSGS的最常见原因。INF2通过其N端透明抑制域（DID）和C端透明自调节域（DAD）之间的相互作用进行调节。INF2还调节其他福尔马蛋白的活性，例如mDIA亚家族，并促进稳定的微管组装。为何致病突变为何仅限于N末端以及它们如何引起人类疾病，目前尚不清楚。方法我们检查了足细胞中存在的INF2亚型，并评估了INF2的裂解，以解释免疫印迹的发现。我们评估了人类肾脏疾病条件下INF2 N和C末端片段的表达。我们还调查了足细胞中含DID的N端片段的定位和功能，并评估了FSGS相关的R218Q突变是否损害INF2裂解或N片段的功能。结果INF2-CAAX亚型是足细胞中的主要亚型。INF2被蛋白水解切割，这是由组织蛋白酶蛋白酶介导的过程，释放N端DID以独立发挥作用。尽管N端区域通常位于足细胞足突，但在FSGS相关的INF2突变的情况下，N末端区域不存在。不论INF2突变如何，C末端片段都位于细胞体内。在足细胞中，N片段定位在质膜上，结合mDIA1，并以分裂依赖的方式促进细胞扩散。与疾病相关的R218Q突变会损害这些N片段功能，但不会破坏INF2的裂解。结论INF2被切割成一个包含N端DID的片段和一个包含C端DAD的片段。切割允许N末端片段独立发挥作用，并有助于解释与FSGS相关的突变的聚类。