EspF of Enterohemorrhagic Escherichia coli Enhances Apoptosis via Endoplasmic Reticulum Stress in Intestinal Epithelial Cells: An Isobaric Tags for Relative and Absolute Quantitation-Based Comparative Proteomic Analysis.

There have been large foodborne outbreaks related to Enterohemorrhagic Escherichia coli (EHEC) around the world. Among its virulence proteins, the EspF encoded by locus of enterocyte effacement is one of the most known functional effector proteins. In this research, we infected the HT-29 cells with the EHEC wild type strain and EspF-deficient EHEC strain. Via the emerging technique isobaric tags for relative and absolute quantitation (iTRAQ), we explored the pathogenic characteristics of EspF within host cells. Our data showed that the differences regarding cellular responses mainly contained immune regulation, protein synthesis, signal transduction, cellular assembly and organization, endoplasmic reticulum (ER) stress, and apoptosis. Notably, compared with the EspF-deficient strain, the protein processing in the ER and ribosome were upregulated during wild type (WT) infection. Our findings proved that the EspF of Enterohemorrhagic Escherichia coli induced ER stress in intestinal epithelial cells; the ER stress-dependent apoptosis pathway was also activated within the host cells. This study provides insight into the virulence mechanism of protein EspF, which will deepen our general understanding of A/E pathogens and their interaction with host proteins.

A coronary artery disease-associated tRNAThr mutation altered mitochondrial function, apoptosis and angiogenesis.

The tissue specificity of mitochondrial tRNA mutations remains largely elusive. In this study, we demonstrated the deleterious effects of tRNAThr 15927G>A mutation that contributed to pathogenesis of coronary artery disease. The m.15927G>A mutation abolished the highly conserved base-pairing (28C-42G) of anticodon stem of tRNAThr. Using molecular dynamics simulations, we showed that the m.15927G>A mutation caused unstable tRNAThr structure, supported by decreased melting temperature and slower electrophoretic mobility of mutated tRNA. Using cybrids constructed by transferring mitochondria from a Chinese family carrying the m.15927G>A mutation and a control into mitochondrial DNA (mtDNA)-less human umbilical vein endothelial cells, we demonstrated that the m.15927G>A mutation caused significantly decreased efficiency in aminoacylation and steady-state levels of tRNAThr. The aberrant tRNAThr metabolism yielded variable decreases in mtDNA-encoded polypeptides, respiratory deficiency, diminished membrane potential and increased the production of reactive oxygen species. The m.15927G>A mutation promoted the apoptosis, evidenced by elevated release of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 3, 7, 9 and PARP. Moreover, the lower wound healing cells and perturbed tube formation were observed in mutant cybrids, indicating altered angiogenesis. Our findings provide new insights into the pathophysiology of coronary artery disease, which is manifested by tRNAThr mutation-induced alterations.