Alterations in Lipid Profile of the Aging Kidney Identified by MALDI Imaging Mass Spectrometry.

During aging, the kidney undergoes functional and physiological changes that are closely affiliated with chronic kidney disease (CKD). There is increasing evidence supporting the role of lipid or lipid-derived mediators in the pathogenesis of CKD and other aging-related diseases. To understand the role of lipids in various metabolic processes during kidney aging, we conducted matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) analysis in kidneys harvested from young (2 months old, n = 3) and old mice (24 months old, n = 3). MALDI-IMS analysis showed an increase in ceramide level and a decrease in sphingomyelin (SM) and phosphatidylcholine (PC) levels in kidneys of old mice. The increased expression of cPLA2 and SMPD1 protein in aged kidney was confirmed by immunohistochemistry and Western blot analysis. Our MALDI-IMS data showed the altered distribution of lipids in aged kidney as indicative of aging-related functional changes of the kidney. Combined analysis of MALDI-IMS and IHC confirmed lipidomic changes and expression levels of responsible enzymes as well as morphological changes.

Docosahexaenoic acid-mediated protein aggregates may reduce proteasome activity and delay myotube degradation during muscle atrophy in vitro.

Proteasomes are the primary degradation machinery for oxidatively damaged proteins that compose a class of misfolded protein substrates. Cellular levels of reactive oxygen species increase with age and this cellular propensity is particularly harmful when combined with the age-associated development of various human disorders including cancer, neurodegenerative disease and muscle atrophy. Proteasome activity is reportedly downregulated in these disease conditions. Herein, we report that docosahexaenoic acid (DHA), a major dietary omega-3 polyunsaturated fatty acid, mediates intermolecular protein cross-linkages through oxidation, and the resulting protein aggregates potently reduce proteasomal activity both in vitro and in cultured cells. Cellular models overexpressing aggregation-prone proteins such as tau showed significantly elevated levels of tau aggregates and total ubiquitin conjugates in the presence of DHA, thereby reflecting suppressed proteasome activity. Strong synergetic cytotoxicity was observed when the cells overexpressing tau were simultaneously treated with DHA. Antioxidant N-acetyl cysteine significantly desensitized the cells to DHA-induced oxidative stress. DHA significantly delayed the proteasomal degradation of muscle proteins in a cellular atrophy model. Thus, the results of our study identified DHA as a potent inducer of cellular protein aggregates that inhibit proteasome activity and potentially delay systemic muscle protein degradation in certain pathologic conditions.

The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins.

The N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-end rule pathway. Using para-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-end rule pathway), we identified that blocking the Arg/N-end rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, ATE1-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of the ER heat shock protein HSPA5 that initially targets cargo proteins, along with SQSTM1, to the autophagosome. At the late stage of autophagy, however, proteasomal degradation of arginylated HSPA5 might function as a critical checkpoint for the proper progression of autophagic flux in the cells. Consistently, the inhibition of the Arg/N-end rule pathway with PCA significantly elevated levels of MAPT and huntingtin aggregates, accompanied by increased numbers of LC3 and SQSTM1 puncta. Cells treated with the Arg/N-end rule inhibitor became more sensitized to proteotoxic stress-induced cytotoxicity. SILAC-based quantitative proteomics also revealed that PCA significantly alters various biological pathways, including cellular responses to stress, nutrient, and DNA damage, which are also closely involved in modulation of autophagic responses. Thus, our results indicate that the Arg/N-end rule pathway may function to actively protect cells from detrimental effects of cellular stresses, including proteotoxic protein accumulation, by positively regulating autophagic flux.