The widely accepted oxidative stress theory of aging postulates that aging results from accumulation of oxidative damage. Surprisingly, data from the longest-living rodent known, naked molerats [MRs; mass 35 g; maximum lifespan (MLSP) > 28.3 years], when compared with mice (MLSP 3.5 years) exhibit higher levels of lipid peroxidation, protein carbonylation, and DNA oxidative damage even at a young age. We hypothesize that age-related changes in protein structural stability, oxidation, and degradation are abrogated over the lifespan of the MR. We performed a comprehensive study of oxidation states of protein cysteines [both reversible (sulfenic, disulfide) and indirectly irreversible (sulfinic/sulfonic acids)] in liver from young and old C57BL/6 mice (6 and 28 months) and MRs (2 and >24 years). Furthermore, we compared interspecific differences in urea-induced protein unfolding and ubiquitination and proteasomal activity. Compared with data from young mice, young MRs have 1.6 times as much free protein thiol groups and similar amounts of reversible oxidative damage to cysteine. In addition, they show less urea-induced protein unfolding, less protein ubiquitination, and higher proteasome activity. Mice show a significant age-related increase in cysteine oxidation and higher levels of ubiquitination. In contrast, none of these parameters were significantly altered over 2 decades in MRs. Clearly MRs have markedly attenuated age-related accrual of oxidation damage to thiol groups and age-associated up-regulation of homeostatic proteolytic activity. These pivotal mechanistic interspecies differences may contribute to the divergent aging profiles and strongly implicate maintenance of protein stability and integrity in successful aging.cysteine oxidation ͉ Heterocephalus glaber ͉ mechanisms of aging ͉ proteasome activity ͉ protein homeostasis

Ewing sarcoma is an aggressive paediatric cancer of the bone and soft tissue. It results from a chromosomal translocation, predominantly t(11;22)(q24:q12), that fuses the N-terminal transactivation domain of the constitutively expressed EWSR1 protein with the C-terminal DNA binding domain of the rarely expressed FLI1 protein. Ewing sarcoma is highly sensitive to genotoxic agents such as etoposide, but the underlying molecular basis of this sensitivity is unclear. Here we show that Ewing sarcoma cells display alterations in regulation of damage-induced transcription, accumulation of R-loops and increased replication stress. In addition, homologous recombination is impaired in Ewing sarcoma owing to an enriched interaction between BRCA1 and the elongating transcription machinery. Finally, we uncover a role for EWSR1 in the transcriptional response to damage, suppressing R-loops and promoting homologous recombination. Our findings improve the current understanding of EWSR1 function, elucidate the mechanistic basis of the sensitivity of Ewing sarcoma to chemotherapy (including PARP1 inhibitors) and highlight a class of BRCA-deficient-like tumours.

Replication stress" describes phenomena that alter DNA replication rates [1][2][3] . Multiple architectural challenges within the confined nuclear volume must be navigated during replication to prevent or repair replication stress. Cellular mechanisms potentiating changes in nuclear architecture that facilitate DNA replication remain unclear. Here we show that the ATR, IPMK and mTOR kinases regulate actin polymerisation in human cells to alter nuclear architecture and promote replication fork repair. We demonstrate that replication stress activates mTOR, in an ATR and IPMK-dependent manner, to induce polymerisation of nuclear filamentous actin (F-actin). mTOR and ATR then counteract replication stress-induced nuclear envelope deformation and increase nuclear volume through their regulation of actin dynamics.Additionally, we reveal that FANCD2 labelled replication forks colocalise with actin filaments in late S-phase. mTOR and ATR then regulate the mobility, speed and directionality of stalled replication foci within the three-dimensional nuclear architecture. Importantly, we find nuclear F-actin also acts as a substrate for the directed migration of stalled replication foci to the nuclear periphery. Suppressing mTOR and ATR-dependent actin forces prevents replication fork restart and promotes chromosome segregation errors in primary and cancer cell lines. Together, .

Altered structure, and hence function, of cellular macromolecules caused by oxidation can contribute to loss of physiological function with age. Here, we tested whether the lifespan of bats, which generally live far longer than predicted by their size, could be explained by reduced protein damage relative to short-lived mice. We show significantly lower protein oxidation (carbonylation) in Mexican free-tailed bats (Tadarida brasiliensis) relative to mice, and a trend for lower oxidation in samples from cave myotis bats (Myotis velifer) relative to mice. Both species of bat show in vivo and in vitro resistance to protein oxidation under conditions of acute oxidative stress. These bat species also show low levels of protein ubiquitination in total protein lysates along with reduced proteasome activity, suggesting diminished protein damage and removal in bats. Lastly, we show that bat-derived protein fractions are resistant to urea-induced protein unfolding relative to the level of unfolding detected in fractions from mice. Together, these data suggest that long lifespan in some bat species might be regulated by very efficient maintenance of protein homeostasis.