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: 8746647
• 负责人: rosa puertollano
• 金额: $ 48.33万
• 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/8746647
• 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，因为该蛋白质中的突变与称为IV型粘脂沉积症（MLIV）的人类疾病相关。我们和其他人提出，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在调节内体途径钙稳态方面的突出作用，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。我们以前已经描述过，在HeLa细胞中，MCOLN 2分布在Arf 6调节途径的管状再循环内体，并调节特定糖基磷脂酰肌醇锚定蛋白（GPIs）的再循环。为了进一步表征MCOLN 2的生理作用，我们已经启动了体内动物模型的开发。特别地，我们通过在关键外显子上游的内含子内插入侧翼为Flp-重组酶靶FRT位点的捕获盒SA-geo-pA（剪接受体-β-geo-polyA）来产生MC 0 LN 2敲除小鼠。我们目前正在对MCOLN 2 KO小鼠进行表型分析，并评估该蛋白的组织表达。