Lysosomal targetomics of ghr KO mice shows chaperone-mediated autophagy degrades nucleocytosolic acetyl-coA enzymes.

Mice deficient in GHR (growth hormone receptor; ghr KO) have a dramatic lifespan extension and elevated levels of hepatic chaperone-mediated autophagy (CMA). Using quantitative proteomics to identify protein changes in purified liver lysosomes and whole liver lysates, we provide evidence that elevated CMA in ghr KO mice downregulates proteins involved in ribosomal structure, translation initiation and elongation, and nucleocytosolic acetyl-coA production. Following up on these initial proteomics findings, we used a cell culture approach to show that CMA is necessary and sufficient to regulate the abundance of ACLY and ACSS2, the two enzymes that produce nucleocytosolic (but not mitochondrial) acetyl-coA. Inhibition of CMA in NIH3T3 cells has been shown to lead to aberrant accumulation of lipid droplets. We show that this lipid droplet phenotype is rescued by knocking down ACLY or ACSS2, suggesting that CMA regulates lipid droplet formation by controlling ACLY and ACSS2. This evidence leads to a model of how constitutive activation of CMA can shape specific metabolic pathways in long-lived endocrine mutant mice.Abbreviations: CMA: chaperone-mediated autophagy; DIA: data-independent acquisition; ghr KO: growth hormone receptor knockout; GO: gene ontology; I-WAT: inguinal white adipose tissue; KFERQ: a consensus sequence resembling Lys-Phe-Glu-Arg-Gln; LAMP2A: lysosomal-associated membrane protein 2A; LC3-I: non-lipidated MAP1LC3; LC3-II: lipidated MAP1LC3; PBS: phosphate-buffered saline; PI3K: phosphoinositide 3-kinase.

Cross-linked cytochrome P450 BM3 aggregates promoted by Ru(II)-diimine complexes bearing aldehyde groups.

Cross-linked enzyme aggregate (CLEA) methodology has been applied to immobilize cytochrome P450 BM3 variants (F87A and 21B3) with peroxygenase activity. Several Ru(II)-diimine complexes were found to be suitable cross-linking agents, surpassing the traditional glutaraldehyde and dextran aldehyde. They offer modular numbers of aldehyde functionalities and a more rigid framework than their organic counterparts. The F87A CLEAs display significant activity loss compared to the protein in solution. Meanwhile, for the 21B3 CLEAs, high activity recovery (up to 95%) is obtained. In order to minimize enzyme leaching from the CLEA, sodium cyanoborohydride was used to reduce the CLEAs imine bonds. The reduced CLEAs were active for several rounds of reactions leading to an overall increase in protein activity of 170% compared to the free protein in solution.