Endocytic Trafficking and Human Diseases

内吞贩运与人类疾病

• 批准号: 7594352
• 负责人: rosa puertollano
• 金额: $ 153.73万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594352
• 关键词: Achlorhydria; Address; Adenoviruses; Affect; Aftercare; Alanine; Am 580; Ashkenazim; Bacteria; Biogenesis; Biological Assay; C-terminal; Cell Maturation; Cell membrane; Cell physiology; Cells; Chimera organism; Chimeric Proteins; Chromosomes; Cornea; Cyclic AMP-Dependent Protein Kinases; Defect; Destinations; Disease; Electron Microscopy; Epitopes; Event; Family; Fibroblasts; Forskolin; Future; Ganglioside Sialidase Deficiency Disease; Gangliosides; Genes; Glutathione S-Transferase; Glycosaminoglycans; H 89; Hair Cells; Hearing; Hela Cells; Human Pathology; Impairment; In Vitro; Incubated; Ion Channel; Length; Light; Lysosomes; Maps; Mediating; Melanosomes; Molecular; Mus; Muscle Tonus; Muscle hypotonia; Mutagenesis; Mutate; Mutation; N-terminal; Names; Open Reading Frames; Oryctolagus cuniculus; Pathway interactions; Patients; Phenotype; Phospholipids; Phosphorylation; Phosphorylation Site; Phosphotransferases; Pigmentation physiologic function; Plasmids; Play; Post-Translational Protein Processing; Protein Dephosphorylation; Protein Kinase; Protein Kinase Inhibitors; Protein-Serine-Threonine Kinases; Proteins; Psyche structure; Range; Reagent; Recycling; Regulation; Reporting; Retinal Degeneration; Role; Screening procedure; Sequence Alignment; Serine; Sorting - Cell Movement; Staurosporine; Strabismus; Structure; Suggestion; Tail; Testing; Threonine; Vacuole; Visual; Yeasts; base; cell type; follower of religion Jewish; genetic linkage analysis; human disease; in vivo; kinase inhibitor; knock-down; member; palmitoylation; polyclonal antibody; protein kinase inhibitor; receptor; research study; small hairpin RNA; trafficking; yeast two hybrid system

Mucolipidosis type IV (MLIV) is an autosomal recessive disease characterized by mental and psychomotor retardation, diminished muscle tone or hypotonia, achlorhydria, and visual problems including corneal clouding, retinal degeneration, sensitivity to light, and strabismus. Analysis of fibroblasts from MLIV patients by electron microscopy revealed the presence of enlarged vacuolar structures that accumulate mucopolysaccharides, phospholipids, and gangliosides. These enlarged vacuoles are present not only in fibroblasts but in many different cell types, suggesting a general impairment of the lysosomal function in MLIV patients. Linkage analysis carried out in 13 Ashkenazi Jewish families mapped the MLIV locus to chromosome 19p13.3-p13.210 and allowed the identification of mucolipin-1 (MCOLN1) as the gene responsible for the disease. MCOLN1 is an ion channel with homology to the transient receptor potential (TRP) channel superfamily. The selectivity of the MCOLN1 channel remains controversial, as different studies have suggested that it is permeable to Ca2+, Ca2+, K+ and Na+, and H+. The lysosomal defects observed in MLIV patients have led to the suggestion that MCOLN1 might play a role in the biogenesis of lysosomes. Recently, several groups have suggested additional roles for MCOLN1 in different cellular processes including regulation of lysosomal acidification and lysosomal secretion. To understand better the molecular mechanisms that regulate MCOLN1 activity, we investigated whether the N- and C-terminal tails of MCOLN1 are modified by phosphorylation. Soluble proteins representing the N-terminal tail (GST-MCOLN1-NTail; residues 1-66) and the C-terminal tail (GST-MCOLN1-CTail; residues 518-580) were synthesized in bacteria as GST fusion proteins. Following purification, these fusion proteins were incubated with HeLa cell lysates and subjected to in vitro kinase assays. Our results showed that GST-MCOLN1-CTail was efficiently phosphorylated in vitro by protein kinase activities present in HeLa cells lysates. Moreover, the phosphorylation of the GST-MCOLN1-CTail chimera was almost completely abolished after treatment with staurosporine, a broad range serine/threonine kinase inhibitor, suggesting that a serine/threonine kinase present in HeLa cell lysates phosphorylates the C-terminal tail of MCOLN1. Mutagenesis analysis allowed us to identify Ser557 as the major in vitro phosphorylation site in the MCOLN1 C-tail; Ser559 is also phosphorylated but to a much lesser extent. To determine the type of serine/threonine protein kinase responsible for the phosphorylation of MCOLN1, we examined the effects of various serine/threonine protein kinase inhibitors. Using our in vitro kinase assay, we found that addition of H89 to HeLa cell lysates effectively inhibited phosphorylation. Because H89 inhibition of MCOLN1 phosphorylation is indicative of protein kinase A (PKA) activity, we sought to determine whether MCOLN1 is phosphorylated after treatment of cells with forskolin (FSK), a PKA activator. As expected, phosphorylation of MCOLN1 C-tail and MCOLN1 full-length was significantly increased after FSK treatment both in vitro and in vivo. To test the effect of MCOLN1 phosphorylation on its activity, we analyzed changes in amplitudes of MCOLN1-dependent currents associated with inhibition or activation of PKA. Interestingly, cell treatment with H89 induced an increase in amplitudes of MCOLN1-dependent currents, while treatment with FSK inhibited the currents. Based on these results we conclude that inhibition of PKA and MCOLN1 dephosphorylation increases MCOLN1 channel activity while MCOLN1 phosphorylation inhibits it. The stimulatory effect of H89 on MCOLN1 function was not observed when Ser557 and Ser559 were mutated to alanine, indicating that these two residues are essential for PKA-mediated negative regulation of MCOLN1. This study constitutes the first report of regulation of a member of the mucolipin family by phosphorylation. Sequence alignment revealed that MCOLN1 shows high homology with two other proteins named, mucolipin-2 (MCOLN2) and mucolipin-3 (MCOLN3), and the three proteins have been grouped into the mucolipin family. MCOLN3 might also play a role in different human pathologies, as mutations in this gene are responsible for the varitint-waddler mouse phenotype that is characterized by defects in pigmentation and hearing loss22. MCOLN3 is located in hair cells, and it could be implicated in hair cell maturation and melanosome trafficking. In addition we have recently described that MCOLN2 MCOLN2 traffics through the Arf6-associated pathway and regulates recycling of CD59 to the plasma membrane. Thus, our results have provided important information indicating that mucolipins are important regulators of specific intracellular trafficking events. Future experiments include the search for cellular proteins that interact with the members of the mucolipin family. We are using a combination of yeast two-hybrid screening and pull down experiments that hopefully will result in the identification of effectors that regulate MCOLNs function. In addition we have generated a battery of reagents (including rabbit polyclonal antibodies against endogenous MCOLNs, adenovirus expressing shRNA sequences that can specifically knock down each of the MCOLNs, and plasmids encoding the ORF of the three MCOLNs fused to different epitope tags, as well as chimeras carrying mutations that affect MCOLN traffic and activity) that will allow us to continue the study of the role of MCOLNs on intracellular trafficking.

IV型粘脂沉积症（MLIV）是一种常染色体隐性遗传疾病，其特征为智力和精神发育迟缓、肌张力降低或张力减退、无氯血症和视力问题，包括角膜混浊、视网膜变性、对光敏感和斜视。通过电子显微镜对MLIV患者的成纤维细胞进行分析，发现存在积累粘多糖、磷脂和神经节苷脂的增大的空泡结构。这些扩大的空泡不仅存在于成纤维细胞中，而且存在于许多不同的细胞类型中，表明MLIV患者的溶酶体功能普遍受损。在13个德系犹太人家庭中进行的连锁分析将MLIV基因定位于染色体19p13.3-p13.210，并将粘脂蛋白-1（MCOLN 1）鉴定为引起该疾病的基因。MCOLN 1是与瞬时受体电位（TRP）通道超家族具有同源性的离子通道。MCOLN 1通道的选择性仍然存在争议，因为不同的研究表明它对Ca 2+、Ca 2+、K+和Na+以及H+是可渗透的。在MLIV患者中观察到的溶酶体缺陷表明MCOLN 1可能在溶酶体的生物发生中发挥作用。最近，几个研究小组提出了MCOLN 1在不同细胞过程中的额外作用，包括调节溶酶体酸化和溶酶体分泌。 为了更好地理解调节MCOLN 1活性的分子机制，我们研究了MCOLN 1的N-和C-末端尾部是否被磷酸化修饰。在细菌中合成代表N-末端尾（GST-MCOLN 1-NTail;残基1-66）和C-末端尾（GST-MCOLN 1-CTail;残基518-580）的可溶性蛋白作为GST融合蛋白。纯化后，将这些融合蛋白与HeLa细胞裂解物一起孵育，并进行体外激酶测定。我们的结果表明，GST-MCOLN 1-CTail在体外被HeLa细胞裂解物中存在的蛋白激酶活性有效磷酸化。此外，GST-MCOLN 1-CTail嵌合体的磷酸化在用星形孢菌素（一种广谱丝氨酸/苏氨酸激酶抑制剂）处理后几乎完全消除，这表明HeLa细胞裂解物中存在的丝氨酸/苏氨酸激酶使MCOLN 1的C末端尾磷酸化。突变分析使我们能够将Ser 557确定为MCOLN 1 C尾中的主要体外磷酸化位点; Ser 559也被磷酸化，但程度要小得多。为了确定负责MCOLN 1磷酸化的丝氨酸/苏氨酸蛋白激酶的类型，我们研究了各种丝氨酸/苏氨酸蛋白激酶抑制剂的作用。使用我们的体外激酶测定，我们发现，除了H89的HeLa细胞裂解物有效地抑制磷酸化。由于H89对MCOLN 1磷酸化的抑制是蛋白激酶A（PKA）活性的指示，我们试图确定用毛喉素（FSK）（PKA激活剂）处理细胞后MCOLN 1是否磷酸化。正如预期的那样，体外和体内FSK处理后，MCOLN 1 C尾和MCOLN 1全长的磷酸化显着增加。为了测试MCOLN 1磷酸化对其活性的影响，我们分析了与PKA抑制或激活相关的MCOLN 1依赖性电流振幅的变化。有趣的是，用H89处理细胞诱导MCOLN 1依赖性电流的幅度增加，而用FSK处理抑制电流。基于这些结果，我们得出结论，PKA和MCOLN 1去磷酸化的抑制增加MCOLN 1通道活性，而MCOLN 1磷酸化抑制它。当Ser 557和Ser 559突变为丙氨酸时，没有观察到H89对MCOLN 1功能的刺激作用，表明这两个残基是PKA介导的MCOLN 1负调控所必需的。这项研究构成了第一个报告的调节的一个成员的粘脂蛋白家族的磷酸化。 序列比对显示MCOLN 1与另外两种蛋白质MCOLN 2和MCOLN 3具有很高的同源性，这三种蛋白质被归类为MCOLN家族。MCOLN 3也可能在不同的人类病理学中发挥作用，因为该基因的突变导致varitint-waddler小鼠表型，其特征在于色素沉着和听力损失的缺陷22。MCOLN 3位于毛细胞中，可能与毛细胞成熟和黑素体运输有关。此外，我们最近描述了MCOLN 2 MCOLN 2通过Arf 6相关途径运输，并调节CD 59向质膜的再循环。因此，我们的研究结果提供了重要的信息，表明粘脂是重要的调节剂的特定的细胞内运输事件。 未来的实验包括寻找与粘脂家族成员相互作用的细胞蛋白。我们正在使用酵母双杂交筛选和下拉实验的组合，希望将导致在调节MCOLN功能的效应器的识别。此外我们还生产了一系列试剂（包括针对内源性MCOLN的兔多克隆抗体、表达可特异性敲低每种MCOLN的shRNA序列的腺病毒、以及编码融合至不同表位标签的三种MCOLN的ORF的质粒，以及携带影响MCOLN运输和活性的突变的嵌合体）这将使我们能够继续研究MCOLN在细胞内运输中的作用。