P97/VCP promotes the recycling of endocytic cargo
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Hela Cell
SUBMITTER: 
Alexander Buchberger
LAB HEAD: Alexander Buchberger
PROVIDER: PXD041169 | Pride | 2023-09-28
REPOSITORIES: Pride
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Project description:Through substitution mapping studies, we previously identified that a <330kb region from a rat strain with no renal pathology (the Lewis rat), which when introgressed onto the genetic background of a rat with renal disease (the Dahl Salt-sensitive (S) rat), caused an increase rather than the expected decrease in proteinuria. The purpose of this study was to prioritize a candidate gene and further delineate the mechanism underlying the observed increased in proteinuria. A higher level of proteinuria independent of dietary salt was observed in the congenic rat at a very young age (50-52 day old). The critical congenic segment was further mapped to <42.5kb containing a single candidate gene, rififylin. Rififylin was expressed 1.59 fold higher in the congenic strain compared with S. Overexpression of rififylin is known to delay recycling of endosomes. Renal transcriptome analysis indicated that Atp1a1 one of the most highly differentially expressed genes. Atp1a1 was 5.33 fold higher in the congenic strain compared with S. The protein product of Atp1a1, the alpha subunit of Na+K+ATPase, was also significantly higher in the endosomes of proximal tubules from the congenic strain compared with S. To determine whether the higher amounts of this protein in the endosomes is due to a delay in recycling of endosomes caused by the overexpression of rififylin in the congenic strain, recycling of exogenously labeled-transferrin by single cell cultures of proximal tubules was monitored by confocal microscopy. Recycling of transferrin was significantly delayed in the congenic strain compared with S. These results suggest that impaired endosomal recycling in the proximal tubules from the congenic strain caused by the overexpression of rififylin is a novel molecular mechanism linked to the observed increase in proteinuria of the congenic strain.
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Project description:Through substitution mapping studies, we previously identified that a <330kb region from a rat strain with no renal pathology (the Lewis rat), which when introgressed onto the genetic background of a rat with renal disease (the Dahl Salt-sensitive (S) rat), caused an increase rather than the expected decrease in proteinuria. The purpose of this study was to prioritize a candidate gene and further delineate the mechanism underlying the observed increased in proteinuria. A higher level of proteinuria independent of dietary salt was observed in the congenic rat at a very young age (50-52 day old). The critical congenic segment was further mapped to <42.5kb containing a single candidate gene, rififylin. Rififylin was expressed 1.59 fold higher in the congenic strain compared with S. Overexpression of rififylin is known to delay recycling of endosomes. Renal transcriptome analysis indicated that Atp1a1 one of the most highly differentially expressed genes. Atp1a1 was 5.33 fold higher in the congenic strain compared with S. The protein product of Atp1a1, the alpha subunit of Na+K+ATPase, was also significantly higher in the endosomes of proximal tubules from the congenic strain compared with S. To determine whether the higher amounts of this protein in the endosomes is due to a delay in recycling of endosomes caused by the overexpression of rififylin in the congenic strain, recycling of exogenously labeled-transferrin by single cell cultures of proximal tubules was monitored by confocal microscopy. Recycling of transferrin was significantly delayed in the congenic strain compared with S. These results suggest that impaired endosomal recycling in the proximal tubules from the congenic strain caused by the overexpression of rififylin is a novel molecular mechanism linked to the observed increase in proteinuria of the congenic strain. Three male S control and 3 male congenic S.LEW(10)x12x2x3x5 rats born on the same day were selected, weaned at 30 days of age, and caged with 1 congenic and 1 S rat per cage. They were raised on a low-salt (0.3%) diet (Harlan Teklad diet TD 7034; Harlan–Sprague-Dawley) and sacrificed at 53 days of age and total RNAs were isolated from the kidney. The isolated RNA from each animal was used for the cRNA preparation. cRNA was prepared and fragmented as suggested by Affymetrix technical manual, and simultaneously hybridized (15 µg adjusted cRNA for each chip) to Rat Expression Array 230 2.0 (3' IVT Expression Analysis). Statistical analyses of the microarray data were performed with BH adjustment using R statistical package (version 2.8.1).
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Project description:Membrane trafficking is defined as the vesicular transport of molecules throughout the cell. In intestinal enterocytes, defects in endocytic/recycling pathways impair their function and are linked to genetic diseases. How does trafficking regulate intestinal tissue homeostasis is poorly understood. Using the Drosophila intestine as an in vivo model system, we investigated enterocyte-specific functions for early endosomal trafficking in gut homeostasis. We focused on the small GTPase Rab21 that regulates specific early endosomal trafficking steps. Rab21-depleted guts showed severe intestinal morphology abnormalities, with deregulated homeostasis associated with a gain in mitotic cells and increased cell death. Increased in both apoptosis and Yki signaling were responsible for compensatory proliferation and tissue inflammation. Using a RNAi screen, we identified autophagy and specific membrane trafficking regulators phenocopying Rab21 loss. We further showed that Rab21-induced hyperplasia was rescued by inhibition of Egfr signaling, and we identified improperly trafficked cargoes in enterocytes depleted of Rab21. Our data shed light on an important role for the early endosomal protein Rab21, and early endosomal trafficking in enterocytes-mediated intestinal homeostasis.
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