Biology subjects

Enrich, C.

Publications and source records attributed to Enrich, C..

3 recordsLinked to original sources

bioRxiv2022

Proteostatic regulation of caveolins avoids premature oligomerisation and preserves ER homeostasis

Caveolin-1 (CAV1) and CAV3 are membrane sculpting proteins driving formation of plasma membrane caveolae. Caveola formation is unique as it requires oligomerisation of newly synthesised caveolins through the biosynthetic-secretory pathway. Here, we combine structural, biochemical, and microscopy analyses to examine the early proteostasis of caveolin family members and mutants. We describe striking trafficking differences between newly synthesised caveolins, with CAV1 rapidly exported to the Golgi but CAV3 showing ER retention and targeting to lipid droplets. Only monomeric/low oligomeric caveolins are efficiently exported from the ER, with oligomers assembling beyond the cis-Golgi and disease-causing mutations leading to detrimental non-functional complexes. Caveolins in the ER are maintained at low levels by active proteasomal degradation, avoiding premature oligomerisation and ER stress. Increasing lipid availability, cholesterol for CAV1 and fatty acids for CAV3, enhances trafficking and reduces proteasomal degradation. In conclusion, we identify proteostatic mechanisms that modulate stability and trafficking of newly synthesised caveolins, protecting cells against ER stress but perturbed in caveolin-related disease. SummaryUnderstanding the unique proteostasis of caveolins has important implications for cell biology and physiopathology. Combining structural, microscopy, and biochemical analyses, we uncover new insights into the mechanisms that differentiate the early biosynthetic steps of caveolin family members, isoforms, and pathogenic mutants.

cell biology↗

bioRxiv2021

Direct reprogramming of human fibroblasts into insulin-producing cells by transcription factors

Direct lineage reprogramming of one somatic cell into another bypassing an intermediate pluripotent state has emerged as an alternative to embryonic or induced pluripotent stem cell differentiation to generate clinically relevant cell types. One cell type of clinical interest is the pancreatic {beta} cell that secretes insulin and whose loss and/or dysfunction leads to diabetes. Generation of functional {beta}-like cells from developmentally related somatic cell types (pancreas, liver, gut) has been achieved via enforced expression of defined sets of transcription factors. However, clinical applicability of these findings is challenging because the starting cell types are not easily obtainable. Skin fibroblasts are accessible and easily manipulated cells that could be a better option, but available studies indicate that their competence to give rise to {beta} cells through similar direct reprogramming approaches is limited. Here, using human skin fibroblasts and a protocol that ensures high and consistent expression of adenovirus-encoded reprogramming factors, we show that the transcription factor cocktail consisting of Pdx1, Ngn3, MafA, Pax4 and Nkx2-2 activates key {beta} cell genes and down-regulates the fibroblast transcriptional program. The converted cells produce insulin and exhibit intracellular calcium responses to glucose and/or membrane depolarization. Furthermore, they secrete insulin in response to glucose in vitro and after transplantation in vivo. These findings demonstrate that transcription factor-mediated direct reprogramming of human fibroblasts is a feasible strategy to generate insulin-producing cells.

developmental biology↗

bioRxiv2021

CRISPR screens for lipid regulators reveal a role for ER-bound SNX13 in lysosomal cholesterol export

We report here two genome-wide CRISPR screens carried out to identify genes that when knocked out, alter levels of lysosomal cholesterol or bis(monoacylglycero)phosphate. In addition, these screens were also carried out under conditions of NPC1 inhibition to identify modifiers of NPC1 function in lysosomal cholesterol export. The screens confirm tight co- regulation of cholesterol and bis(monoacylglycero)phosphate levels in cells, and reveal an unexpected role for the ER-localized, SNX13 protein as a negative regulator of lysosomal cholesterol export. In the absence of NPC1 function, SNX13 knockout decreases lysosomal cholesterol, and is accompanied by triacylglycerol-rich lipid droplet accumulation and increased lysosomal bis(monoacylglycero)phosphate. These experiments provide unexpected insight into the regulation of lysosomal lipids and modification of these processes by novel gene products. SUMMARYGenome-wide CRISPR screens carried out with and without NPC1 function identify shared pathways that coordinately control lysosomal cholesterol and bis(monoacylglycero)phosphate. ER-localized SNX13 protein plays an unexpected regulatory role in modifying NPC1 function to regulate cellular cholesterol localization.

cell biology↗