The role of chloride in supporting the degradative capacity of phagosomes and lysosomes

氯化物在支持吞噬体和溶酶体降解能力中的作用

• 批准号: RGPIN-2022-04485
• 负责人: Freeman, Spencer
• 金额: $ 2.7万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744131
• 关键词: role; chloride; supporting; degradative; capacity

Main Objective The health and metabolism of cells depends on their ability to breakdown large molecules in membrane-bound compartments (e.g. lysosomes) using enzymes. How the fluid environment of these compartments influences enzymatic activity is, however, poorly characterized. We propose that the enzymes resident to lysosomes depend on Cl- to function optimally, a fundamental and evolutionarily conserved process. The main objective of the proposed research is to therefore determine the molecular mechanism(s) underlying the transport of Cl- into the acidic compartments of cells. Summary of background and progress Cells use more than 30-70% of their energy to maintain chemical gradients across membranes. The gradients are exploited to drive the secondary transport of solutes/metabolites without an additional energy cost. A prime example occurs in the endocytic pathway where H+-pumping V-ATPases establish and maintain a H+ gradient and the acidic pH of lysosomes. The H+ gradient then facilitates an outward flux of organic solutes via H+-cotransporters and the inward movement of Cl- via a single exchanger, ClC7. Cl- influx had been proposed to facilitate acidification but may have additional, underappreciated roles in lysosome function. In our unpublished research, we find that ClC7 is only incorporated into the limiting membrane of mature endocytic organelles after they have reached their maximum acidic pH. Rather than regulating the activity of the V-ATPase, our results indicate that high luminal [Cl-] enables the optimal function of lysosome-resident hydrolases. In our unpublished findings, we show that eliminating ClC7 does not alter the normally acidic pH of lysosomes but instead prevents their degradation of endocytosed cargo. Taken together, our unpublished results suggest that the major function of luminal Cl- in acidic endosomal compartments is to facilitate their hydrolase activity. Interestingly, the loss of Cl- exchangers in yeast and mammalian cells leads to growth defects and lysosomal storage disorders respectively, suggesting that organellar enzymes may have evolved alongside with the anions of the luminal fluid. We hypothesize that a high Cl- concentration in the endocytic pathway is driven by the exchange activity of ClCs to support the activity of hydrolytic enzymes. Specific Objectives 1. Measure (phago)lysosomal [Cl-] and the contribution of ClC7 to establishment of luminal [Cl-]. 2. Determine the Cl- dependence of hydrolytic enzymes in vitro and the effect of altering Cl- in vivo. 3. Study the role of luminal Cl- on the membrane traffic that underlies compartment resolution. Impact. This work will help to explain phenotypes arising from ClC7 mutations that lead to lysosomal storage disorders and growth defects. More broadly, these studies stand to improve our understanding of the H+ gradient across organellar membranes as the provider of the force driving the secondary transport of critical solutes, including Cl-.

主要目的细胞的健康和代谢取决于它们使用酶分解膜结合区室（例如溶酶体）中的大分子的能力。然而，这些隔室的流体环境如何影响酶活性的特征很差。我们建议，酶居民的溶酶体依赖于Cl-发挥最佳作用，一个基本的和进化保守的过程。因此，拟议研究的主要目标是确定Cl-转运到细胞酸性区室的分子机制。 背景和进展概述细胞使用超过30-70%的能量来维持跨膜的化学梯度。利用梯度来驱动溶质/代谢物的二次转运，而无需额外的能量成本。一个主要的例子发生在内吞途径中，其中H+泵V-ATP酶建立并维持H+梯度和溶酶体的酸性pH。H+梯度，然后促进通过H+-cotransporters和Cl-通过一个单一的交换器，ClC 7的向内运动的有机溶质的向外通量。Cl-内流被认为促进了酸化，但可能在溶酶体功能中具有额外的未被充分认识的作用。 在我们未发表的研究中，我们发现，ClC 7只纳入成熟的内吞细胞器的限制膜后，他们已经达到其最大的酸性pH值。而不是调节V-ATP酶的活性，我们的研究结果表明，高流明[Cl-]使溶酶体驻留水解酶的最佳功能。在我们未发表的研究结果中，我们表明消除ClC 7不会改变溶酶体的正常酸性pH值，而是阻止其内吞货物的降解。两者合计，我们未发表的结果表明，管腔氯-酸性内体隔室的主要功能是促进其水解酶活性。有趣的是，酵母和哺乳动物细胞中Cl-交换剂的损失分别导致生长缺陷和溶酶体贮积症，这表明细胞器酶可能与腔液的阴离子一起进化。我们推测，高Cl-浓度的内吞途径是由CLC的交换活性，以支持水解酶的活性。 具体目标1。测量（噬菌体）溶酶体[Cl-]和ClC 7对建立腔[Cl-]的贡献。 2.测定体外水解酶的Cl-依赖性和改变体内Cl-的影响。 3.研究管腔氯离子对隔室分辨率下的膜运输的作用。冲击这项工作将有助于解释由ClC 7突变引起的表型，导致溶酶体胆积症和生长缺陷。更广泛地说，这些研究的立场，以提高我们的理解的H+梯度跨细胞器膜的供应商的驱动力的关键溶质，包括Cl-的二次运输。