Summary Calorie restriction (CR) extends lifespan in diverse species. Mitochondria play a key role in CR adaptation, however, the molecular details remain elusive. We developed and applied a quantitative mass spectrometry method to probe the liver mitochondrial acetyl-proteome during CR vs. control diet in mice that were wild-type or lacked the protein deacetylase SIRT3. Quantification of 3,285 acetylation sites −2,193 from mitochondrial proteins rendered a comprehensive atlas of the acetyl-proteome and enabled global site-specific, relative acetyl occupancy measurements between all four experimental conditions. Bioinformatic and biochemical analyses provided additional support for the effects of specific acetylation on mitochondrial protein function. Our results (1) reveal widespread reprogramming of mitochondrial protein acetylation in response to CR and SIRT3, (2) identify three biochemically distinct classes of acetylation sites, and (3) provide evidence that SIRT3 is a prominent regulator in CR adaptation by coordinately deacetylating proteins involved in diverse pathways of metabolism and mitochondrial maintenance.

Anti-silencing function 1 (Asf1) is a highly conserved chaperone of histones H3/H4 that assembles or disassembles chromatin during transcription, replication, and repair. The structure of the globular domain of Asf1 bound to H3/H4 determined by X-ray crystallography to a resolution of 1.7 Angstroms shows how Asf1 binds the H3/H4 heterodimer, enveloping the C terminus of histone H3 and physically blocking formation of the H3/H4 heterotetramer. Unexpectedly, the C terminus of histone H4 that forms a mini-beta sheet with histone H2A in the nucleosome undergoes a major conformational change upon binding to Asf1 and adds a beta strand to the Asf1 beta sheet sandwich. Interactions with both H3 and H4 were required for Asf1 histone chaperone function in vivo and in vitro. The Asf1-H3/H4 structure suggests a "strand-capture" mechanism whereby the H4 tail acts as a lever to facilitate chromatin disassembly/assembly that may be used ubiquitously by histone chaperones.

Summary The mechanisms whereby chromatin structure and cell cycle progression are restored after DNA repair are largely unknown. We show that chromatin is reassembled following double-strand break (DSB) repair and that this requires the histone chaperone Asf1. Absence of Asf1 causes persistent activation of the DNA damage checkpoint after DSB repair as a consequence of defective checkpoint recovery, leading to cell death. The contribution of Asf1 towards chromatin assembly after DSB repair is due to its role in promoting acetylation of free histone H3 on lysine 56 (K56) by the histone acetyl transferase Rtt109, because mimicking acetylation of K56 bypasses the requirement for Asf1 for chromatin reassembly and checkpoint recovery after repair, while mutations that prevent K56 acetylation block chromatin reassembly after repair. These results indicate that restoration of the chromatin following DSB repair is driven by acetylated H3 K56 and that this is a signal for the completion of repair.

Summary Changes to the chromatin structure accompany aging, but the molecular mechanisms underlying aging and the accompanying changes to the chromatin are unclear. Here we report a mechanism whereby altering chromatin structure regulates lifespan. We show that normal aging is accompanied by a profound loss of histone proteins from the genome. Indeed, yeast lacking the histone chaperone Asf1 or acetylation of histone H3 on lysine 56 are short lived and this appears to be at least partly due to their having decreased histone levels. Conversely, increasing the histone supply by inactivation of the Hir (histone information regulator) complex or overexpression of histones dramatically extends lifespan, via a pathway that is distinct from previously known pathways of lifespan extension. This study indicates that maintenance of the fundamental chromatin structure is critical for slowing down the aging process and reveals that increasing the histone supply extends lifespan.

Summary Maximal exercise-associated oxidative capacity is strongly correlated with health and longevity in humans. Rats selectively bred for high running capacity (HCR) have improved metabolic health and are longer-lived than their low capacity counterparts (LCR). Using metabolomic and proteomic profiling, we show that HCR efficiently oxidize fatty acids (FA) and branched-chain amino acid (BCAA), sparing glycogen and reducing accumulation of short- and medium-chain acylcarnitines. HCR mitochondria have reduced acetylation of mitochondrial proteins within oxidative pathways at rest, and there is rapid protein deacetylation with exercise, which is greater in HCR than LCR. Fluxomic analysis of valine degradation with exercise demonstrates a functional role of differential protein acetylation in HCR and LCR. Our data suggest efficient FA and BCAA utilization contribute to high intrinsic exercise capacity and the health and longevity benefits associated with enhanced fitness.