Nuclear proteome analysis suggests spatial and temporal compartmentalization of metabolic enzymes during Trypanosoma cruzi cell cycle

Trypanosoma cruzi , the etiological agent of Chagas Disease (CD), represents a significant public health concern and serves as a valuable model for investigating the cell cycle in early-diverging eukaryotes. Its unique cellular features,including the absence of chromosome condensation and the coordination of nuclear division with specialized organelles, provide insights into non-canonical regulatory mechanisms, and underscore the pivotal role of the nucleus in maintaining genomic integrity and regulating fundamental processes such as DNA replication and gene expression. To investigate nuclear dynamics throughout the cell cycle, we synchronized T. cruzi epimastigotes at the G1/S transition using hydroxyurea (HU), followed their progression through S and G2/M phases, and quantitatively analyzed isolated nuclear proteins by LC-MS/MS. We identified 2,937 nuclear-enriched proteins, revealing distinct, phase-specific expression patterns. The G1/S phase was marked by increased levels of metabolic enzymes including those related to energy and nucleotide/nucleoside metabolisms. The S phase showed elevated abundance of canonical and variant histones, consistent with chromatin remodeling and DNA replication. The G2/M phase was enriched in proteins involved in protein synthesis and microtubule dynamics, essential for mitosis. Notably, metabolic enzymes represented approximately 6% of the proteins identified in the purified nuclear fraction, consistent with previously published proteomic analyses of T. cruzi nuclear-enriched fractions. Hexokinase, citrate synthase, and pyruvate kinase activities were detected in extracts from this fraction, while immunofluorescence supported the association of hexokinase and citrate synthase with the nuclear region. These results suggest that metabolic enzymes and/or their metabolites may modulate nuclear processes in T. cruzi , potentially influencing the epigenome and gene expression regulation, as proposed in other eukaryotic models. Altogether, our findings reveal dynamic remodeling of the nuclear proteome during the T. cruzi cell cycle and point to a previously underappreciated role for metabolic enzymes likely regulating nuclear functions in a cell cycle-dependent manner.