lysosomes, Uncategorized

Lysosome and chloride

This is going to be a very quick tour of a lysosome publication that challenges the dogma that pH alone controlss lysosome function.

Lyosomes operate at the acidic pH generated by the V-ATPase. The resulting transmembrane H+ gradient also energizes the secondary transport such as the 2Cl/H+ exchanger ClC-7. It is necessary for the resolution of phagolysosomes formed by macrophages. Deletion of ClC-7 had a negligible effect on phagosomal acidification. Instead, luminal Cl was found to be required for activation of a wide range of phagosomal hydrolases including proteases, nucleases, and glycosidases. [1] These authors claim that Cl anions operate “allosterically.” The bait in panel this publication was antibody coated sheep red blood cells. (RBC)

The RAW 264.7 macrophage cell line was compared with the same cell line in which the CLCN7 genes were knocked out with CRISPR. The macrophage were fed sheep RBC coated with antibody. No difference in pH was seen. In a chloride containing medium, the sRBC volume decreased with time. When cloride was replaced with nitrate, there was no decrease in sRBC volume.

Some representative hydrolades in lysosomes are more active at higher Clconcentrations. As a graduate student we performed all of our experiments in potassium propionate. Propionate is a short chain fatty acid whose carboxyl group was thought to more closely resemble the side chains of acid amino acids like glutamate and aspartate.

The concept of hard and soft anions and cations comes with how they are hydrated with water. K+ is the predominant intracellular cation. Like Na+ it is well hydrated. Cl- and carboxyl groups are hydrated differently than Cl. Kosmotrophes increase the stability of water and promotes stability of most proteins. Chaotrophes decrese the structure of water and may solubilize proteins that have large hydrophobic surfaces that are normally hidden from water due to the way the protein folds. Chaotrophes can unfold proteins.

This image is borrowed from a post on glyphosate. The point is that cloride in the lysome has the potential to induce changes in the hydrolases.

  • (NH(2)-aminobutyric-homophenylalanine)(2)-rhodamine demonstrated the best reactivity and selectivity profile in the FACS assay using the B721 human B-lymphoblastoid cell line. The resulting FACS assay was validated through correlation of the IC(50) values with a competitive radiolabeling assay against a series of small molecule inhibitors of cathepsin C. [2]
  • fluorescence-based lysozyme activity assay, Enzchek (Molecular Probes Inc, Eugene, OR) [3]
  • A probe for DNA strand breaks…[4]

This is a very quick summary of why lysosome pH is probably not the best way to high throughput screen small molecules for treating LSD. It makes complete sense that chloride is the larger driving force of lysosome activity. This post is not intended to recommend the best assays. The point is that the fluorescent high throughput assays for hydrolase activity are out there.

  • The kinase PIKFYVE generates the signaling lipid PI(3,5)P2 on the surface of endosomes and lysosomes
  • The phosphatase FIG4degrades it with the scaffold protein VAC14
  • Cells deficient in FIG4 or VAC14 have enlarged lysosomes bounded by membranes containing LAMP1 and LAMP2, and elevated levels of the autophagosome markers LC3-II and p62
  • Loss of FIG4 leads to decreased levels of PI(3,5)P2 in cells leading to osmotic swelling.
  • The swelling is attributed to the failure of PI(3,5)P2 to inhibit CIC7
  • Mutations of FIG4 and VAC14 have been associated with: Yunis-Varón Syndrome, Charcot-Marie-Tooth disease type 4J, and familial epilepsy with polymicrogyria, as well as hypopigmentation issues.
  • PI(3,5)P2 inhibits the ClC-7 lysosomes and late endosome 2 Cl exchanger.
  • The cryo-EM structure of human ClC-7 reveals a positively charged phosphoinositide binding pocket
  • Deficiency of FIG4 or VAC14 leads to reduction of PI(3,5)P2 that is predicted to result in overly active CIC7.
  • According to UniProt, Fig4 has serine-protein phosphatase activity acting on PIKfyve to stimulate its lipid kinase activity. Fig4 is required for maximal PI(3,5)P2 production by PKfyve.
  • This cannot be more confusing! PIKfyve is a lipid and protein kinase. It autophosphorylates itself thus also turinng itself off. [6] Fig4 is a lipid and protein phosphatase. It removes the phosphate group from PI(3,5)P2 and the phosphate off switch of PIKfyve.

These authors examined gene interaction between FIG4, VAC14, CLCN6 and CLCN7 by pairwise combination of mutant genes in cultured cells and mutant mice. They suggested that CIC7 inhibitors could compensate for FIG4 and VAC14 mutants related disorders.

Part of Cao 2023 Fig10 was made less confusing by importing information that Fig4 also activates PIKfyve from UniProt.

References

  1. Wu JZ, Zeziulia M, Kwon W, Jentsch TJ, Grinstein S, Freeman SA. ClC-7 drives intraphagosomal chloride accumulation to support hydrolase activity and phagosome resolution. J Cell Biol. 2023 Jun 5;222(6):e202208155. PMC free article
  2. Li J, Petrassi HM, Tumanut C, Masick BT, Trussell C, Harris JL. Substrate optimization for monitoring cathepsin C activity in live cells. Bioorg Med Chem. 2009 Feb 1;17(3):1064-70.
  3. Enzchek
  4. Minchew CL, Didenko VV. Dual Detection of Nucleolytic and Proteolytic Markers of Lysosomal Cell Death: DNase II-Type Breaks and Cathepsin D. Methods Mol Biol. 2017;1554:229-236.
  5. Cao X, Lenk GM, Mikusevic V, Mindell JA, Meisler MH. The chloride antiporter CLCN7 is a modifier of lysosome dysfunction in FIG4 and VAC14 mutants. PLoS Genet. 2023 Jun 26;19(6):e1010800. PMC free article
  6. Lees JA, Li P, Kumar N, Weisman LS, Reinisch KM. Insights into Lysosomal PI(3,5)P2 Homeostasis from a Structural-Biochemical Analysis of the PIKfyve Lipid Kinase Complex. Mol Cell. 2020 Nov 19;80(4):736-743.e4. PMC free article

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