Ubiquitination of proteins involved in metabolism and immunomodulatory drug sensitivity in lymphocytes
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Proteins undergo post-translational modifications, such as ubiquitination and phosphorylation, which can alter their activity, localisation and stability, making a cell responsive to its internal and external environment. Ubiquitin ligases and kinases comprise large enzyme families which catalyse such reactions. The SCF-type E3 ubiquitin ligase sub-family utilise F-box proteins as the substrate targeting component. However, alongside promoting ubiquitination, the F-box protein Fbxo7 can also function as a scaffold and stabilise a subset of proteins, including Cdk6. Cdk6 is activated through binding the D-type cyclins, and historically, a key role has been as a cell cycle regulatory kinase that inactivates G1 checkpoint proteins. More recently, wider roles for Cdk6 have been identified, including as an inhibitor of glycolysis. Notably, Cdk6 has pro-survival activity in T acute lymphoblastic leukaemia (T-ALL) cells due to the phosphorylation and inhibition of glycolytic enzymes, including the rate-limiting gatekeeper, phosphofructokinase (PFKP). Our screens for Fbxo7-interacting partners identified a set of candidates that overlapped as cyclin D/Cdk6 substrates and included PFKP. Further study revealed that Fbxo7 promotes two post-translational modifications on PFKP, ubiquitination and phosphorylation, and specifically promotes Cdk6 activity. Analysis in T-ALL cells suggest that Fbxo7 inhibits the assembly of active PFKP complexes to ultimately inhibit glycolysis. This is confirmed in a murine model of reduced Fbxo7 expression, whose CD4+ T cells show higher levels of glycolytic flux, alongside various other metabolic defects including altered nucleotide biosynthesis and arginine metabolism. This places Fbxo7 as a negative regulator of glycolysis and unveils other diverse roles in metabolism, which may contribute to viability and activation defects observed in these Fbxo7-deficient murine T cells. Given that Fbxo7 negatively regulates glycolysis via PFKP, I also investigated how glucose regulates Fbxo7, as feedback loops in glucose signalling are commonplace in metabolic networks. I discovered Fbxo7 is a dose-dependent, glucose responsive protein in numerous cell types, which is both transcriptionally downregulated and targeted for autophagy in response to glucose starvation. Moreover, data suggest that Fbxo7 is responsive to other stresses, including oxidative stress, placing Fbxo7 as a nexus to link various cellular stress responses to metabolic reprogramming. In addition to PFKP, Fbxo7 has also been shown to recognise a protein called cereblon (CRBN), which is another E3 ubiquitin ligase. CRBN is of clinical relevance because its expression is required for the efficacy of immunomodulatory drugs (IMiDs) in multiple myeloma (MM), which primarily enable the recognition of neo-substrates by CRBN to elicit their anti-cancer effects. We sought to investigate a role for Fbxo7 in MM cells. We show that Fbxo7 promotes CRBN ubiquitination and propose that this targets CRBN for proteasomal degradation, which may have relevance for IMiD sensitivity. Together, these data identify two novel substrates for Fbxo7 ubiquitination and reveal a role for Fbxo7 in lymphocytes. We demonstrate that Fbxo7 expression is responsive to cellular stress and propose that Fbxo7 levels may fine-tune metabolism under different physiological and pathological conditions.