Yonekura, H. | bioRxiv Science

bioRxiv2025

HUWE1 stimulates mTORC1 activity by enhancing Rheb interaction with mTORC1 and supports de novo pyrimidine synthesis

mTORC1 is a central regulator of cell growth that is directly activated by the small GTPase Rheb on the lysosomal membrane in response to growth factors. We recently reported that polyubiquitinated Rheb facilitates its interaction with mTORC1, leading to mTORC1 activation. However, the molecular mechanisms underlying the ubiquitination of Rheb and its interaction with mTORC1 for activation are not fully understood. In this study, we demonstrate that HUWE1, an E3 ubiquitin ligase, preferentially interacts with ubiquitinated Rheb and is essential for the interaction between Rheb and mTOR, as well as for mTORC1 activation. Additionally, HUWE1 is necessary for Rheb to interact with CAD, a crucial enzyme involved in de novo pyrimidine biosynthesis, and for its activation through the mTORC1-S6K1 pathway. Knocking down HUWE1 in cultured cells or in the liver tissues of mice inhibits mTORC1 activity without affecting the phosphorylation of Akt or TSC2, nor does it affect the lysosomal localization of TSC2 or mTORC1. Furthermore, HUWE1 specifically enhances the expression of CAD without influencing other enzymes involved in de novo pyrimidine synthesis and maintains UMP levels in the hepatocytes of liver tissues. These findings indicate that HUWE1 serves as a key organizer of the Rheb ubiquitin complex, amplifying mTORC1 activity and playing a vital role in stimulating de novo pyrimidine synthesis by enhancing CAD expression and activity. HighlightsO_LIHUWE1 and CAD preferentially interact with ubiquitinated Rheb. C_LIO_LIHUWE1 further ubiquitinates Rheb and supports Rheb interaction with mTORC1 and CAD. C_LIO_LIHUWE1 stimulates mTORC1 activity through its E3 ligase activity. C_LIO_LIHUWE1 also supports pyrimidine synthesis by enhancing CAD expression. C_LI

biochemistry↗

bioRxiv2019

Nuclear Expression and DNA Binding Capacity of Receptor for Advanced Glycation End Products in Renal Tissue

The AGER gene encodes for a number of RAGE isoforms, with the membrane bound signal transduction and \"decoy\" circulating soluble RAGE being the best characterised. Here we demonstrate a novel nuclear isoform of RAGE in mice and human kidney cortex which by cell and size fractionation we determined to be approximately 37kda. This nuclear RAGE isoform is functional and binds to DNA sequences within the upstream 5 promoter region of its own gene, AGER. This binding was shown to be abrogated by mutating the DNA consensus binding sequences during electromobility shift assay (EMSA) and was independent of NF-{square}B or AP-1 binding. Cotransfection of expression constructs encoding various RAGE isoforms along with AGER gene promoter reporter-plasmids identified that the most likely source of the nuclear isoform of RAGE was a cleavage product of the nt-RAGE isoform. In obese mice with impaired kidney function, there was increased binding of nuclear RAGE within the A. Region of ager gene promoter with corresponding increases in membrane bound RAGE in renal cells. These findings were reproduced in vitro using proximal tubule cells. Hence, we postulate that RAGE expression is in part, self-regulated by the binding of a nuclear RAGE isoform to the promoter of the AGER gene (encoding RAGE) in the kidney. We also suggest that this RAGE self-regulation is altered under pathological conditions and this may have implications for chronic kidney disease.

molecular biology↗