Human protein aging: modification and crosslinking through dehydroalanine and dehydrobutyrine intermediates

Nonenzymatic post‐translational modification (PTM) of proteins is a fundamental molecular process of aging. The combination of various modifications and their accumulation with age not only affects function, but leads to crosslinking and protein aggregation. In this study, aged human lens proteins were examined using HPLC–tandem mass spectrometry and a blind PTM search strategy. Multiple thioether modifications of Ser and Thr residues by glutathione (GSH) and its metabolites were unambiguously identified. Thirty‐four of 36 sites identified on 15 proteins were found on known phosphorylation sites, supporting a mechanism involving dehydroalanine (DHA) and dehydrobutyrine (DHB) formation through β‐elimination of phosphoric acid from phosphoserine and phosphothreonine with subsequent nucleophilic attack by GSH. In vitro incubations of phosphopeptides demonstrated that this process can occur spontaneously under physiological conditions. Evidence that this mechanism can also lead to protein–protein crosslinks within cells is provided where five crosslinked peptides were detected in a human cataractous lens. Nondisulfide crosslinks were identified for the first time in lens tissue between βB2‐ & βB2‐, βA4‐ & βA3‐, γS‐ & βB1‐, and βA4‐ & βA4‐crystallins and provide detailed structural information on in vivo crystallin complexes. These data suggest that phosphoserine and phosphothreonine residues represent susceptible sites for spontaneous breakdown in long‐lived proteins and that DHA‐ and DHB‐mediated protein crosslinking may be the source of the long‐sought after nondisulfide protein aggregates believed to scatter light in cataractous lenses. Furthermore, this mechanism may be a common aging process that occurs in long‐lived proteins of other tissues leading to protein aggregation diseases.     

Deleted in Breast Cancer 1 regulates cellular senescence during obesity

Chronic obesity leads to inflammation, tissue dysfunction, and cellular senescence. It was proposed that cellular senescence during obesity and aging drives inflammation and dysfunction. Consistent with this, clearance of senescent cells increases healthspan in progeroid mice. Here, we show that the protein Deleted in Breast Cancer‐1 (DBC1) regulates cellular senescence during obesity. Deletion of DBC1 protects preadipocytes against cellular senescence and senescence‐driven inflammation. Furthermore, we show protection against cellular senescence in DBC1 KO mice during obesity. Finally, we found that DBC1 participates in the onset of cellular senescence in response to cell damage by mechanism that involves binding and inhibition of HDAC3. We propose that by regulating HDAC3 activity during cellular damage, DBC1 participates in the fate decision that leads to the establishment of cellular senescence and consequently to inflammation and tissue dysfunction during obesity.     

Weekly administration of rapamycin improves survival and biomarkers in obese male mice on high‐fat diet

Recent discoveries have revealed the key role of mTOR (target of rapamycin) in aging. Furthermore, rapamycin extends lifespan in mice, especially in female mice. Here, we treated obese male mice on high‐fat diet with rapamycin given intermittently: either weekly (once a week) or alternating bi‐weekly (three injections every other week). While only marginally reducing obesity, intermittent administration of rapamycin significantly extended lifespan. Significance was achieved for weekly treated group and for the three rapamycin‐received groups combined. In weekly treatment group, 100% mice were alive by the age of 2 years, whereas 60% of mice died in untreated group by this age. The effect of weekly treatment on survival was highly significant and cannot be fully explained by partial reduction in obesity. Alternating bi‐weekly treatments seem to be less effective than weekly treatment, although effects of additional factors (see 3 ) may not be excluded. After one year of treatment, all survived mice were sacrificed 8 days after the last administration of rapamycin to avoid its direct interference with parameters examined. Fasting levels of cardiac and hepatic p‐S6, a marker of mTORC1 activity, were lower in weekly treatment group compared with control mice. In contrast, levels of p‐Akt (S473), glucose, triglycerides and insulin were unchanged, whereas leptin and IGF‐1 tended to be lower. Thus, weekly treatment with rapamycin may slow down aging in obese male mice on high‐fat diet.     

Gene profiling reveals association between altered Wnt signaling and loss of T‐cell potential with age in human hematopoietic stem cells

Functional decline of the hematopoietic system occurs during aging and contributes to clinical consequences, including reduced competence of adaptive immunity and increased incidence of myeloid diseases. This has been linked to aging of the hematopoietic stem cell (HSC) compartment and has implications for clinical hematopoietic cell transplantation as prolonged periods of T‐cell deficiency follow transplantation of adult mobilized peripheral blood (PB), the primary transplant source. Here, we examined the gene expression profiles of young and aged HSCs from human cord blood and adult mobilized PB, respectively, and found that Wnt signaling genes are differentially expressed between young and aged human HSCs, with less activation of Wnt signaling in aged HSCs. Utilizing the OP9‐DL1 in vitro co‐culture system to promote T‐cell development under stable Notch signaling conditions, we found that Wnt signaling activity is important for T‐lineage differentiation. Examination of Wnt signaling components and target gene activation in young and aged human HSCs during T‐lineage differentiation revealed an association between reduced Wnt signal transduction, increasing age, and impaired or delayed T‐cell differentiation. This defect in Wnt signal activation of aged HSCs appeared to occur in the early T‐progenitor cell subset derived during in vitro T‐lineage differentiation. Our results reveal that reduced Wnt signaling activity may play a role in the age‐related intrinsic defects of aged HSCs and early hematopoietic progenitors and suggest that manipulation of this pathway could contribute to the end goal of improving T‐cell generation and immune reconstitution following clinical transplantation.     

Fibroblasts derived from long‐lived insulin receptor substrate 1 null mice are not resistant to multiple forms of stress

Reduced signalling through the insulin/insulin-like growth factor-1 signalling (IIS) pathway is a highly conserved lifespan determinant in model organisms. The precise mechanism underlying the effects of the IIS on lifespan and health is currently unclear, although cellular stress resistance may be important. We have previously demonstrated that mice globally lacking insulin receptor substrate 1 (Irs1^−/−) are long-lived and enjoy a greater period of their life free from age-related pathology compared with wild-type (WT) controls. In this study, we show that primary dermal fibroblasts and primary myoblasts derived from Irs1^−/− mice are no more resistant to a range of oxidant and nonoxidant chemical stressors than cells derived from WT mice.     

Shc depletion stimulates brown fat activity in vivo and in vitro

Adipose tissue is an important metabolic organ that integrates a wide array of homeostatic processes and is crucial for whole‐body insulin sensitivity and energy metabolism. Brown adipose tissue (BAT) is a key thermogenic tissue with a well‐established role in energy expenditure. BAT dissipates energy and protects against both hypothermia and obesity. Thus, BAT stimulation therapy is a rational strategy for the looming pandemic of obesity, whose consequences and comorbidities have a huge impact on the aged. Shc‐deficient mice (ShcKO) were previously shown to be lean, insulin sensitive, and resistant to high‐fat diet and obesity. We investigated the contribution of BAT to this phenotype. Insulin‐dependent BAT glucose uptake was higher in ShcKO mice. Primary ShcKO BAT cells exhibited increased mitochondrial respiration; increased expression of several mitochondrial and lipid‐oxidative enzymes was observed in ShcKO BAT. Levels of brown fat‐specific markers of differentiation, UCP1, PRDM16, ELOVL3, and Cox8b, were higher in ShcKO BAT. In vitro, Shc knockdown in BAT cell line increased insulin sensitivity and metabolic activity. In vivo, pharmacological stimulation of ShcKO BAT resulted in higher energy expenditure. Conversely, pharmacological inhibition of BAT abolished the improved metabolic parameters, that is the increased insulin sensitivity and glucose tolerance of ShcKO mice. Similarly, in vitro Shc knockdown in BAT cell lines increased their expression of UCP1 and metabolic activity. These data suggest increased BAT activity significantly contributes to the improved metabolic phenotype of ShcKO mice.