Mass Spectrometric Metabolic Profiling of the Multiple Myeloma Cell Line JJN-3 under APIM-peptide Stress

Abstract

The proliferating cell nuclear antigen (PCNA) is recognized as a hub protein, central to maintaining cellular homeostasis through its numerous protein interaction partners. Under conditions of genotoxic stress, there is a switch in PCNA-affinity to proteins involved in cellular stress responses, interacting through the AlkB homologue 2 PCNA-interacting motif (APIM). Targeting these interactions through synthetic APIM-containing peptides induces apoptosis in a cancer-cell-specific matter, and potentiates the effect of DNA-damaging agents in these cells. As APIM-peptides are seemingly affecting signaling transduction pathways regulating primary metabolism, this project was designed to investigate whether any corresponding effects were evident on the metabolome, lipidome and fluxome levels. Specifically, the response of the multiple myeloma cell line JJN-3 to APIM-peptide exposure was characterized, applying targeted and non-targeted mass-spectrometric profiling approaches. Four different stress-response studies were conducted, monitoring the effect of APIM-peptide exposure on the membrane lipid composition, primary carbon metabolism, metabolic carbon fluxes and the global metabolite pool within a 4- or 24-hour time frame. Furthermore, the glucose consumption was assessed employing enzymatic assays on growth medium samples. No changes were evident with respect to membrane-lipid composition. However, all cell cultures exposed to APIM-peptides, either alone or in combination with cisplatin, were significantly differing from untreated cultures with respect to the intracellular concentration of several primary carbon metabolites. Among these were intermediates of glycolysis, the pentose phosphate pathway (PPP) and the tricarboxylic acid- (TCA) cycle, as well as several amino acids. Further, these cultures were characterized by a lowered energy charge (EC), and low levels of GTP, CTP and UTP relative to untreated controls. These effects were evident for all APIM-treated cultures, regardless of cisplatin co-treatment. Interestingly, the 13C-label enrichment patterns were similar in stressed and control cells, indicating that intracellular metabolic flux distributions were not affected, even though the APIM-peptide treatment increased glucose consumption. Altogether, this clearly demonstrates an effect of APIM-peptide exposure in the system under study. Further, these observations indicate a role of APIM-mediated PCNA-interactions in the regulation of primary carbon metabolism that is yet to be unraveled, clearly justifying and motivating further research on this topic.