Nedd4-2-Mediated Ubiquitination Facilitates Processing of Surfactant Protein-C
Nedd4-2-Mediated Ubiquitination Facilitates Processing of Surfactant Protein-C
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DOI：
10.1165/rcmb.2009-0058oc
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发表时间：
2010-02-01
影响因子：
6.4
通讯作者：
Weaver, Timothy E.
中科院分区：
文献类型：
--
作者：
Conkright, Juliana J.;Apsley, Karen S.;Weaver, Timothy E.
我们以前提出了一个模型的表面活性蛋白（SP）-C的生物合成，其中内化的前蛋白从限制膜的多泡体的内部囊泡代表的加工和分泌的SP-C的一个关键步骤。为了验证这一假设，进行了SP-C的N-末端前肽的丙氨酸诱变。将编码突变体前蛋白的腺病毒感染到从Sftpc（-/-）小鼠分离的11型细胞中，并在感染后24小时分析培养基中分泌的SP-C。（SPPDYS 17）-P-12的突变完全阻断了SP-C的分泌。PPDY（PY基序）先前已显示结合神经前体细胞表达的发育下调的（Nedd）4样E3泛素连接酶的WW结构域。纯化的重组谷胱甘肽S-转移酶-SP-C前肽（残基1-35）强烈结合重组Nedd 4 -2，Nedd 4弱结合;（SPPDYS 17）-P-12突变消除了SP-C与Nedd 4 -2的结合。固定化的重组Nedd 4 - 2 WW结构域从小鼠11型细胞裂解物中捕获SP-C前蛋白;在反向下拉中，11型细胞中的内源性SP-C被重组Nedd 4 -2捕获。为了确定Nedd 4 -2和SP-C的相互作用是否导致泛素化，从瞬时转染的人胚肾293细胞中免疫沉淀SP-C前蛋白，并用泛素抗体通过SDS-PAGE/Western印迹分析。两种泛素化形式的SP-C被检测到，泛素化被阻断突变的K6，但不是K34，在SP-C前肽。K6的突变还抑制人胚肾293细胞中SP-C前蛋白加工为成熟肽。Nedd 4 -2介导的泛素化调节SP-C的腔内重新定位，导致SP-C的加工并最终分泌。
We previously proposed a model of surfactant protein (SP)-C biosynthesis in which internalization of the proprotein from the limiting membrane of the multivesicular body to internal vesicles represents a key step in the processing and secretion of SP-C. To test this hypothesis, alanine mutagenesis of the N-terminal propeptide of SP-C was performed. Adenoviruses encoding mutant proproteins were infected into type 11 cells isolated from Sftpc(-/-) mice, and media analyzed for secreted SP-C 24 hours after infection. Mutation of (SPPDYS17)-P-12 completely blocked secretion of SP-C. PPDY (PY motif) has previously been shown to bind WW domains of neural precursor cell-expressed developmentally down-regulated (Nedd) 4-like E3 ubiquitin ligases. Purified recombinant glutathione S-transferase-SP-C propeptide (residues 1-35) bound recombinant Nedd4-2 strongly, and Nedd4 weakly; the (SPPDYS17)-P-12 mutation abrogated binding of SP-C to Nedd4-2. Immobilized recombinant Nedd4-2WW domain captured SP-C proprotein from mouse type 11 cell lysates; in the reverse pulldown, endogenous SP-C in type 11 cells was captured by recombinant Nedd4-2. To determine if the interaction of Nedd4-2 and SP-C resulted in ubiquitination, the SP-C proprotein was immunoprecipitated from transiently transfected human embryonic kidney 293 cells, and analyzed by SDS-PAGE/Western blotting with ubiquitin antibody. Two ubiquitinated forms of SP-C were detected; ubiquitination was blocked by mutation of K6, but not K34, in the SP-C propeptide. Mutation of K6 also inhibited processing of SP-C proprotein to the mature peptide in human embryonic kidney 293 cells. Nedd4-2-mediated ubiquitination regulates lumenal relocation of SP-C, leading to processing and, ultimately, secretion of SP-C.