Rationale:: Cardiac progenitor cells are an attractive cell type for tissue regeneration, but their mechanism for myocardial remodeling is still unclear. Objective:: This investigation determines how chronological age influences the phenotypic characteristics and the secretome of human cardiac progenitor cells (CPCs), and their potential to recover injured myocardium. Methods and Results:: Adult (aCPCs) and neonatal (nCPCs) cells were derived from patients aged >40 years or <1 month, respectively, and their functional potential was determined in a rodent myocardial infarction model. A more robust in vitro proliferative capacity of nCPCs, compared with aCPCs, correlated with significantly greater myocardial recovery mediated by nCPCs in vivo. Strikingly, a single injection of nCPC-derived total conditioned media was significantly more effective than nCPCs, aCPC-derived TCM, or nCPC-derived exosomes in recovering cardiac function, stimulating neovascularization, and promoting myocardial remodeling. High-resolution accurate mass spectrometry with reverse phase liquid chromatography fractionation and mass spectrometry was used to identify proteins in the secretome of aCPCs and nCPCs, and the literature-based networking software identified specific pathways affected by the secretome of CPCs in the setting of myocardial infarction. Examining the TCM, we quantified changes in the expression pattern of 804 proteins in nCPC-derived TCM and 513 proteins in aCPC-derived TCM. The literature-based proteomic network analysis identified that 46 and 6 canonical signaling pathways were significantly targeted by nCPC-derived TCM and aCPC-derived TCM, respectively. One leading candidate pathway is heat-shock factor-1, potentially affecting 8 identified pathways for nCPC-derived TCM but none for aCPC-derived TCM. To validate this prediction, we demonstrated that the modulation of heat-shock factor-1 by knockdown in nCPCs or overexpression in aCPCs significantly altered the quality of their secretome. Conclusions:: A deep proteomic analysis revealed both detailed and global mechanisms underlying the chronological age-based differences in the ability of CPCs to promote myocardial recovery via the components of their secretome.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme best known for its role in glycolysis. However, extra-glycolytic functions of GAPDH have been described, including regulation of protein expression via RNA binding. GAPDH binds to numerous adenine-uridine rich elements (AREs) from various mRNA 3′-untranslated regions in vitro and in vivo despite its lack of a canonical RNA binding motif. How GAPDH binds to these AREs is still unknown. Here we discovered that GAPDH binds with high affinity to the core ARE from tumor necrosis factor-α mRNA via a two-step binding mechanism. We demonstrate that a mutation at the GAPDH dimer interface impairs formation of the second RNA-GAPDH complex and leads to changes in the RNA structure. We investigated the effect of this interfacial mutation on GAPDH oligomerization by crystallography, small-angle x-ray scattering, nano-electrospray ionization native mass spectrometry, and hydrogen-deuterium exchange mass spectrometry. We show that the mutation does not significantly affect GAPDH tetramerization as previously proposed. Instead, the mutation promotes short-range and long-range dynamic changes in regions located at the dimer and tetramer interface and in the NAD⁺ binding site. These dynamic changes are localized along the P axis of the GAPDH tetramer, suggesting that this region is important for RNA binding. Based on our results, we propose a model for sequential GAPDH binding to RNA via residues located at the dimer and tetramer interfaces.The glycolytic enzyme GAPDH is a non-canonical RNA-binding protein. Results A dimer interface mutation impairs the two-step binding of GAPDH to AU-rich elements from TNF-α mRNA and leads to RNA structural changes. Conclusion Dimer and tetramer interface residues are important for GAPDH-RNA binding. Significance We propose a novel model for GAPDH binding to AU-rich RNA via the oligomeric interfaces.

Dithiolethiones are five-membered sulfur-containing cyclic scaffolds that exhibit antioxidative, anti-inflammatory, antithrombic and chemotherapeutic activities. Dithiolethiones display the chemopreventive and cytoprotective effects by activating the antioxidant response element and mounting the transcription of cytoprotective phase II enzymatic machinery. In addition, several classes of dithiolethiones efficiently modulate the activities of proteins that play crucial roles in normal and cancer cells, including glutathione S-transferase, cyclooxygenases and master regulator NF-κB. The present paper summarizes synthetic aspects, pharmacological potentials and biological attributes of dithiolethiones and its derivatives. Additionally, this review concludes with a discussion on how the current state-of-the-art technologies may help in defining a structure–activity relationship of dithiolethiones, thereby facilitating the design and synthesis of potent drug candidates. Graphical abstract