PCNA regulates primary metabolism by scaffolding metabolic enzymes

Abstract

The essential roles of proliferating cell nuclear antigen (PCNA) as a scaffold protein in DNA replication and repair are well established, while its cytosolic roles are less explored. Two metabolic enzymes, alpha-enolase (ENO1) and 6-phosphogluconate dehydrogenase (6PGD), both contain PCNA interacting motifs. Mutation of the PCNA interacting motif APIM in ENO1 (F423A) impaired its binding to PCNA and resulted in reduced cellular levels of ENO1 protein, reduced growth rate, reduced glucose consumption, and reduced activation of AKT. Metabolome and signalome analysis reveal large consequences of impairing the direct interaction between PCNA and ENO1. Metabolites above ENO1 in glycolysis accumulated while lower glycolytic and TCA cycle metabolite pools decreased in the APIM-mutated cells; however, their overall energetic status were similar to parental cells. Treating haematological cancer cells or activated primary monocytes with a PCNA targeting peptide drug containing APIM (ATX-101) also lead to a metabolic shift characterized by reduced glycolytic rate. In addition, we show that ATX-101 treatments reduced the ENO1 - PCNA interaction, the ENO1, GAPDH and 6PGD protein levels, as well as the 6PGD activity. Here we report for the first time that PCNA acts as a scaffold for metabolic enzymes, and thereby act as a direct regulator of primary metabolism.

Activation of multiple stress responses in Staphylococcus aureus substantially lowers the minimal inhibitory concentration when combining two novel antibiotic drug candidates

“…Selecting for the signalome reduces the complexity of the results, making it easier to identify key proteins and signaling pathways, and allows for the detection of dynamic changes caused by post-translation modifications that alter protein activity. The MIB assay has successfully been used to study the signalome in both eukaryotic and prokaryotic cells by pulling down activated signaling proteins, such as kinases, ATP/GTP-binding proteins, and proteins in complex with the latter two ( Petrovic et al, 2017 ; Søgaard et al, 2019 ; Sumabe et al, 2021 ; Røst et al, 2023 ). Increased protein pulldown upon treatment suggests increased activation while reduced pulldown indicates inhibition, destabilization, or reduced activation of the protein.…”

“…PCNA, the eukaryotic DNA-sliding clamp, has recently been shown to also have scaffolding functions outside replication and DNA repair, e.g., in cell signaling, redox balance, and metabolism ( Olaisen et al, 2015 ; Ohayon et al, 2019 ; Røst et al, 2023 ). As DNA sliding clamps are highly conserved, BTP-001’s mode of action (MoA) may also involve inhibiting scaffolding roles of the β-clamp outside the replisome.…”

“…In this study, we investigated the individual and combined MoAs of JK-274 and BTP-001 on S. aureus using signalomics supplemented with metabolomics. For signalomics, we used the multiplexed kinase inhibitory bead (MIB) assay which detects the level of activated signaling proteins, many of which are involved in stress responses ( Duncan et al, 2012 ; Petrovic et al, 2017 ; Røst et al, 2023 ). The MIB assay was optimized for bacterial cell extracts by testing several in-house compounds designed to mimic kinase inhibitors.…”