Role of endolysosomal channels in calcium homeostasis and trafficking

粘脂（或TRPML）构成与瞬时受体电位超家族具有同源性的内体阳离子通道家族。在哺乳动物中，粘脂家族包括三个成员，粘脂-1、粘脂-2和粘脂-3（MCOLN 1 -3）。MCOLN 1是该家族中最具特征的成员，因为这种蛋白质的突变与称为IV型粘脂沉积症（MLIV）的人类疾病相关。我们和其他人已经表明，MCOLN 1在细胞中的主要作用是介导晚期内体和溶酶体的钙外流，从而促进细胞器融合和调节内体运输。 MCOLN 3中的功能获得性突变导致小鼠中的varitint-waddler（Va）表型，其特征在于听力损失、前庭功能障碍和毛色稀释。Va表型由MCOLN 3的孔区域中的点状突变（A419 P）引起，所述点状突变将通道锁定在开放构象中，导致钙大量进入细胞内并通过凋亡诱导细胞死亡。野生型MCOLN 3的过表达导致内体途径的严重改变，包括内体的增大和聚集、延迟的EGF受体降解和受损的自噬体成熟，从而表明MCOLN 3在内体功能的调节中起重要作用。为了更好地理解MCOLN 3的生理作用，我们通过表达通道死亡显性负突变体（458 DD/KK）或通过敲低内源性MCOLN 3来抑制MCOLN 3功能，并测量几个内体参数，包括管腔钙、pH和内体融合。我们发现MCOLN 3活性的损伤导致了内腔钙在内体的显著积累。这种积累导致内体酸化的严重缺陷以及增加的内体融合。我们的研究结果揭示了MCOLN 3在调节内体途径的钙稳态中的突出作用，并证实了腔钙对于适当酸化和膜运输的重要性。 MCOLN 2的细胞功能远未被表征。为了解决MCOLN 2在生理相关细胞类型中的功能，我们首先分析了MCOLN 2在不同小鼠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MCOLN 2在转录水平上受到严格调控。虽然MCOLN 2表达在静息巨噬细胞中可以忽略不计，但其mRNA和蛋白水平在体外和体内响应TLR活化而显著增加。相反，MCOLN 1和MCOLN 3水平在TLR激活后没有变化。免疫荧光分析表明，内源性MCOLN 2主要定位于回收内体在培养和原代细胞，与MCOLN 1和MCOLN 3，其分布到晚期和早期内体途径，分别。为了更好地理解MCOLN 2的体内功能，我们产生了MCOLN 2敲除小鼠。我们发现，在MCOLN 2基因敲除小鼠中，几种趋化因子，特别是CCL 2的产生严重减少。此外，MCOLN 2基因敲除小鼠表现出外周巨噬细胞对腹腔内（IP）注射LPS和活细菌的应答受损，表明免疫应答中存在潜在缺陷。总的来说，我们的研究揭示了MCOLN家族成员的调节和分布的有趣差异，并确定了MCOLN 2在先天免疫应答中的新作用。 内溶酶体途径，特别是MCOLN 1和MCOLN 3，在调节自噬体降解速率方面也发挥重要作用。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，我们已经表征了两种新型抗癌化合物，它们通过破坏自噬通量和降低mTORC 1活性在胰腺导管腺癌细胞中显示出抗增殖活性。

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.