Role of endolysosomal channels in calcium homeostasis and trafficking

内溶酶体通道在钙稳态和运输中的作用

• 批准号: 9572295
• 负责人: Rosa Puertollano-Moro
• 金额: $ 45.26万
• 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/9572295
• 关键词: Address; Adenocarcinoma Cell; Apoptosis; Autophagocytosis; Autophagosome; Bacteria; CCL2 gene; Calcium; Cations; Cell Death; Cell physiology; Cells; Collaborations; Color; Defect; Dominant-Negative Mutation; Endosomes; Epidermal Growth Factor Receptor; Event; Family; Family member; Functional disorder; Ganglioside Sialidase Deficiency Disease; Genetic Transcription; Homeostasis; Immune response; Immunofluorescence Immunologic; Impairment; In Vitro; Injection of therapeutic agent; Innate Immune Response; Ions; Kidney; Knockout Mice; Laboratories; Ligands; Lymphoid; Lysosomes; Mammals; Measures; Mediating; Membrane; Messenger RNA; Molecular Conformation; Mus; Mutation; Organ; Organelles; Pancreatic Ductal Adenocarcinoma; Pathway interactions; Peripheral; Peritoneal; Phenotype; Physiological; Physiological Processes; Play; Production; Proteins; Recruitment Activity; Recycling; Regulation; Rest; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Transduction; System; TRP channel; Tissues; cell type; chemokine; gain of function mutation; hearing impairment; human disease; in vivo; interest; knock-down; late endosome; macrophage; member; novel; overexpression; receptor; response; trafficking; tumor

Mucolipins (or TRPMLs) constitute a family of endosomal cation channels with homology to the transient receptor potential superfamily. In mammals, the mucolipin family includes three members, mucolipin-1, -2, and -3 (MCOLN1-3). MCOLN1 is the best-characterized member of the family due to the fact that mutations in this protein are associated with a human disease known as mucolipidosis type IV (MLIV). We and others have shown that the primary role of MCOLN1 in cells is to mediate calcium efflux from late endosomes and lysosomes, thus promoting organelle fusion and regulating endosomal trafficking. Gain-of-function mutation in MCOLN3 causes the varitint-waddler (Va) phenotype in mice, which is characterized by hearing loss, vestibular dysfunction, and coat color dilution. The Va phenotype results from a punctual mutation (A419P) in the pore region of MCOLN3 that locks the channel in an open conformation causing massive entry of calcium inside cells and inducing cell death by apoptosis. Overexpression of wild-type MCOLN3 produces severe alterations of the endosomal pathway, including enlargement and clustering of endosomes, delayed EGF receptor degradation, and impaired autophagosome maturation, thus suggesting that MCOLN3 plays an important role in the regulation of endosomal function. To understand better the physiological role of MCOLN3, we inhibited MCOLN3 function by expression of a channel-dead dominant negative mutant (458DD/KK) or by knockdown of endogenous MCOLN3 and measure several endosomal parameters including luminal calcium, pH, and endosomal fusion. We found impairment of MCOLN3 activity caused a significant accumulation of luminal calcium at endosomes. This accumulation led to severe defects in endosomal acidification as well as to increased endosomal fusion. Our findings reveal a prominent role for MCOLN3 in regulating calcium homeostasis at the endosomal pathway and confirm the importance of luminal calcium for proper acidification and membrane trafficking. The cellular function of MCOLN2 is far less characterized. To address MCOLN2 function in a physiologically relevant cell type, we first analyzed MCOLN2 expression in different mouse tissues and organs and found that it was predominantly expressed in lymphoid organs and kidney. Quantitative RT-PCR revealed tight regulation of MCOLN2 at the transcriptional level. While MCOLN2 expression was negligible in resting macrophages, its mRNA and protein levels dramatically increased in response to TLR activation both in vitro and in vivo. Conversely, MCOLN1 and MCOLN3 levels did not change upon TLR activation. Immunofluorescence analysis demonstrated that endogenous MCOLN2 primarily localized to recycling endosomes both in culture and primary cells, in contrast with MCOLN1 and MCOLN3, which distribute to the late and early endosomal pathway, respectively. To better understand the in vivo function of MCOLN2, we generated a MCOLN2-knockout mouse. We found that the production of several chemokines, in particular CCL2, was severely reduced in MCOLN2-knockout mice. Furthermore, MCOLN2-knockout mice displayed impaired recruitment of peripheral macrophages in response to intra peritoneal (IP) injections of LPS and live bacteria, suggesting a potential defect in the immune response. Overall, our study reveals interesting differences in the regulation and distribution of the members of the MCOLN family and identifies a novel role for MCOLN2 in the innate immune response. The endolysosomal pathway, and in particular MCOLN1 and MCOLN3, play also an important role regulating the rate of autophagosome degradation. Recent evidence suggests that the elimination of damaged cellular components mediated by autophagy contributes to the progression of certain tumors. In collaboration with the laboratory of Dr. Liqiang Chen, we have characterized two novel anticancer compounds that showed antiproliferative activity in pancreatic ductal adenocarcinoma cells by disrupting autophagy flux and reducing mTORC1 activity.

粘膜脂(或TRPML)构成了一个与瞬时受体电位超家族同源的内体阳离子通道家族。在哺乳动物中，粘蛋白家族包括三个成员，即粘蛋白-1、-2和-3(MCOLN1-3)。MCOLN1是该家族中特征最好的成员，因为这种蛋白的突变与一种称为IV型粘脂沉积症(MLIV)的人类疾病有关。我们和其他人已经证明，MCOLN1在细胞中的主要作用是介导晚期内小体和溶酶体的钙外流，从而促进细胞器融合和调节内小体运输。 MCOLN3的功能获得突变导致小鼠的Va表型，其特征是听力损失、前庭功能障碍和毛色变淡。Va表型是由MCOLN3孔区的一个点突变(A419P)引起的，该突变将通道锁定在开放的构象中，导致钙大量进入细胞内，并通过凋亡诱导细胞死亡。野生型MCOLN3的过表达导致内体途径的严重改变，包括内小体的增大和聚集，EGF受体降解延迟，自噬小体成熟受阻，因此MCOLN3在内体功能的调节中起重要作用。为了更好地了解MCOLN3的生理作用，我们通过表达通道死亡的显性负性突变体(458DD/KK)或通过敲除内源MCOLN3来抑制MCOLN3的功能，并测量了几个内体参数，包括管腔钙、pH和内体融合。我们发现，MCOLN3活性的受损导致了腔内钙在内吞体内的显著积累。这种堆积导致内体酸化的严重缺陷以及内体融合的增加。我们的发现揭示了MCOLN3在调节内体途径的钙稳态方面的显著作用，并证实了腔钙对适当的酸化和膜转运的重要性。 对MCOLN2的细胞功能的研究还远远不够。为了解决MCOLN2在生理相关细胞类型中的功能，我们首先分析了MCOLN2在小鼠不同组织和器官中的表达，发现它主要在淋巴器官和肾脏中表达。定量RT-PCR显示MCOLN2在转录水平上有严格的调控。在静息的巨噬细胞中，MCOLN2的表达微乎其微，但其在体外和体内的TLR激活后，其mRNA和蛋白水平显著增加。相反，MCOLN1和MCOLN3水平在TLR激活时没有变化。免疫荧光分析表明，在培养细胞和原代细胞中，内源性MCOLN2主要定位于循环内体，而MCOLN1和MCOLN3分别分布于晚期和早期的内体途径。为了更好地了解MCOLN2在体内的功能，我们产生了MCOLN2基因敲除小鼠。我们发现，在MCOLN2基因敲除的小鼠中，几种趋化因子的产生严重减少，特别是CCL2。此外，MCOLN2基因敲除小鼠对腹膜内注射内毒素和活细菌的反应表现出外周巨噬细胞募集功能受损，这表明免疫反应存在潜在缺陷。总体而言，我们的研究揭示了MCOLN家族成员在调节和分布方面的有趣差异，并确定了MCOLN2在先天性免疫反应中的一个新角色。 内溶酶体途径，特别是MCOLN1和MCOLN3，也在调节自噬体内降解速率方面发挥着重要作用。最近的证据表明，自噬介导的受损细胞成分的消除有助于某些肿瘤的进展。与陈立强博士的实验室合作，我们鉴定了两种新的抗癌化合物，它们通过破坏自噬通量和降低mTORC1活性在胰腺导管腺癌细胞中显示出抗增殖活性。