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: 8558019
• 负责人: rosa puertollano
• 金额: $ 51.15万
• 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/8558019
• 关键词: Animal Model; Apoptosis; Autophagosome; Binding; Calcium; Cations; Cell Death; Cell physiology; Cells; Color; Defect; Development; Dominant-Negative Mutation; Endosomes; Epidermal Growth Factor Receptor; Event; Exons; Family; Family member; Functional disorder; GPI Membrane Anchors; Ganglioside Sialidase Deficiency Disease; Hela Cells; Homeostasis; Impairment; Introns; Ions; Knockout Mice; Ligands; Lysosomes; Mammals; Measures; Mediating; Membrane; Membrane Protein Traffic; Molecular Conformation; Mus; Mutation; Organelles; Pathway interactions; Phenotype; Physiological; Physiological Processes; Play; Proteins; RNA Splicing; Recycling; Regulation; Role; Signal Transduction; Site; System; TRP channel; Tissues; Tubular formation; gain of function mutation; hearing impairment; human disease; in vivo; interest; late endosome; member; overexpression; protein expression; receptor; recombinase; trafficking

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 proposed 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. We have previously described that, in HeLa cells, MCOLN2 distributes at the tubular recycling endosomes of the Arf6-regulated pathway and regulates recycling of specific glycosylphosphatidylinositol-anchored proteins (GPIs). To further characterize the physiological role of MCOLN2, we have initiated the development of in vivo animal models. In particular, we have generated MCOLN2 knockout mice by inserting a trapping cassette SA-βgeo-pA (splice acceptor-beta-geo-polyA) flanked by Flp-recombinase target FRT sites within an intron upstream of a critical exon. We are currently phenotyping the MCOLN2 KO mice and assessing the tissue expression of this protein.

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。我们和其他人提出，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。我们之前已经描述过，在HeLa细胞中，MCOLN2分布在Arf6调控途径的管状循环内小体，并调控特定糖基磷脂酰肌醇锚定蛋白(GGIs)的循环。为了进一步研究MCOLN2的生理作用，我们启动了体内动物模型的开发。特别是，我们通过在关键外显子上游的内含子上游插入一个剪接受体-β-Geo-Pola(拼接受体-β-Geo-Polya)两侧的FLP-重组酶靶FRT位点，获得了MCOLN2基因敲除小鼠。我们目前正在对MCOLN2KO小鼠进行表型鉴定，并评估该蛋白的组织表达。