Lysine acetylation is a conserved protein post-translational modification that links acetyl-coenzyme A metabolism and cellular signalling. Recent advances in the identification and quantification of lysine acetylation by mass spectrometry have increased our understanding of lysine acetylation, implicating it in many biological processes through the regulation of protein interactions, activity and localization. In addition, proteins are frequently modified by other types of acylations, such as formylation, butyrylation, propionylation, succinylation, malonylation, myristoylation, glutarylation and crotonylation. The intricate link between lysine acylation and cellular metabolism has been clarified by the occurrence of several such metabolite-sensitive acylations and their selective removal by sirtuin deacylases. These emerging findings point to new functions for different lysine acylations and deacylating enzymes and also highlight the mechanisms by which acetylation regulates various cellular processes.

Macroautophagy is a process that leads to the bulk degradation of subcellular constituents by producing autophagosomes/autolysosomes. It is believed that Atg5 (ref. 4) and Atg7 (ref. 5) are essential genes for mammalian macroautophagy. Here we show, however, that mouse cells lacking Atg5 or Atg7 can still form autophagosomes/autolysosomes and perform autophagy-mediated protein degradation when subjected to certain stressors. Although lipidation of the microtubule-associated protein light chain 3 (LC3, also known as Map1lc3a) to form LC3-II is generally considered to be a good indicator of macroautophagy, it did not occur during the Atg5/Atg7-independent alternative process of macroautophagy. We also found that this alternative process of macroautophagy was regulated by several autophagic proteins, including Unc-51-like kinase 1 (Ulk1) and beclin 1. Unlike conventional macroautophagy, autophagosomes seemed to be generated in a Rab9-dependent manner by the fusion of isolation membranes with vesicles derived from the trans-Golgi and late endosomes. In vivo, Atg5-independent alternative macroautophagy was detected in several embryonic tissues. It also had a function in clearing mitochondria during erythroid maturation. These results indicate that mammalian macroautophagy can occur through at least two different pathways: an Atg5/Atg7-dependent conventional pathway and an Atg5/Atg7-independent alternative pathway.

Summary Reversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Here, we use a label-free quantitative proteomic approach to characterize the lysine succinylome in liver mitochondria and its regulation by the desuccinylase SIRT5. A total of 1190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including β-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5−/− animals. Loss of SIRT5 leads to accumulation of medium- and long-chain acylcarnitines and decreased β-hydroxybutyrate production in vivo. In addition, we demonstrate that SIRT5 regulates succinylation of the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) both in vivo and in vitro. Finally, mutation of hypersuccinylated residues K83 and K310 on HMGCS2 to glutamic acid strongly inhibits enzymatic activity. Taken together, these findings establish SIRT5 as a global regulator of lysine succinylation in mitochondria and present a mechanism for inhibition of ketogenesis through HMGCS2.

SUMMARY Protein acylation links energetic substrate flux with cellular adaptive responses. SIRT5 is a NAD+-dependent lysine deacylase and removes both succinyl and malonyl groups. Using affinity enrichment and label free quantitative proteomics, we characterized the SIRT5-regulated lysine malonylome in wild-type (WT) and Sirt5−/− mice. 1,137 malonyllysine sites were identified across 430 proteins, with 183 sites (from 120 proteins) significantly increased in Sirt5−/− animals. Pathway analysis identified glycolysis as the top SIRT5-regulated pathway. Importantly, glycolytic flux was diminished in primary hepatocytes from Sirt5−/− compared to WT mice. Substitution of malonylated lysine residue 184 in glyceraldehyde 3-phosphate dehydrogenase with glutamic acid, a malonyllysine mimic, suppressed its enzymatic activity. Comparison with our previous reports on acylation reveals that malonylation targets a different set of proteins than acetylation and succinylation. These data demonstrate that SIRT5 is a global regulator of lysine malonylation and provide a mechanism for regulation of energetic flux through glycolysis.

Macroautophagy is a highly conserved intracellular process responsible for the degradation of subcellular constituents. Macroautophagy was recently suggested to be involved in the removal of mitochondria from reticulocytes during the final stage of erythrocyte differentiation. Although Atg5 and Atg7 are indispensable for macroautophagy, their role in mitochondrial clearance remains controversial. We recently discovered that mammalian cells use conventional Atg5/Atg7-dependent macroautophagy as well as an alternative Unc-51-like kinase 1 (Ulk1)-dependent Atg5/Atg7-independent macroautophagy process. We hypothesized that the latter may be involved in mitochondrial clearance from reticulocytes during erythrocyte differentiation. Here we report that fetal definitive reticulocytes from Ulk1-deficient and Ulk1/Atg5 double-deficient mice retain their mitochondria, whereas the mitochondria are engulfed and digested within autophagic structures in wild-type and Atg5-deficient mice. Mitochondrial retention by Ulk1-deficient reticulocytes is far less marked in primitive and adult definitive reticulocytes. These data indicate that Ulk1-dependent Atg5-independent macroautophagy is the dominant process of mitochondrial clearance from fetal definitive reticulocytes.

Programmed cell death is a crucial process in the normal development and physiology of metazoans, and it can be divided into several categories that include type I death (apoptosis) and type II death (autophagic cell death). The Bcl-2 family proteins are well-characterized regulators of apoptosis, among which multidomain pro-apoptotic members (such as Bax and Bak) function as a mitochondrial gateway at which various apoptotic signals converge. Although embryonic fibroblasts from Bax/Bak doubleknockout (DKO) mice are resistant to apoptosis, we have previously reported that these cells still die by autophagy in response to various death stimuli. In this study, we found that jun N-terminal kinase (JNK) was activated in etoposide-and staurosporine-treated, but not serumstarved, Bax/Bak DKO cells, and that autophagic cell death was suppressed by the addition of a JNK inhibitor and by a dominant-negative mutant of JNK. Studies with sek1À/À cells revealed that disruption of JNK prevented the induction of autophagic cell death. Coactivation of JNK and autophagy induced autophagic cell death. Activation of JNK occurred downstream of the induction of autophagy, and was dependent on the autophagic process. These results indicate that JNK activation is crucial for the autophagic death of Bax/ Bak DKO cells.

Macroautophagy/autophagy is considered as an evolutionarily conserved cellular catabolic process. In this study, we aimed to elucidate the role of autophagy in vascular smooth muscle cells (SMCs) on atherosclerosis. SMCs cultured from mice with SMC-specific deletion of the essential autophagy gene atg7 (Atg7cKO) showed reduced serum-induced cell growth, increased cell death, and decreased cell proliferation rate. Furthermore, 7-ketocholestrerol enhanced apoptosis and the expression of CCL2 (chemokine [C-C motif] ligand 2) with the activation of TRP53, the mouse ortholog of human and rat TP53, in SMCs from Atg7cKO mice. In addition, Atg7cKO mice crossed with Apoe (apolipoprotein E)-deficient mice (apoeKO; Atg7cKO:apoeKO) showed reduced medial cellularity and increased TUNEL-positive cells in the descending aorta at 10 weeks of age. Intriguingly, Atg7cKO: apoeKO mice fed a Western diet containing 1.25% cholesterol for 14 weeks showed a reduced survival rate. Autopsy of the mice demonstrated the presence of aortic rupture. Analysis of the descending aorta in Atg7cKO:apoeKO mice showed increased plaque area, increased TUNEL-positive area, decreased SMC-positive area, accumulation of macrophages in the media, and adventitia and perivascular tissue, increased CCL2 expression in SMCs in the vascular wall, medial disruption, and aneurysm formation. In conclusion, our data suggest that defective autophagy in SMCs enhances atherosclerotic changes with outward arterial remodeling.

Programmed cell death, which is required for the development and homeostasis of metazoans, includes mechanisms such as apoptosis, autophagic cell death, and necrotic (or type III) death. Members of the Bcl2 family regulate apoptosis, among which Bax and Bak act as a mitochondrial gateway. Although embryonic fibroblasts from Bax/Bak double-knockout (DKO) mice are resistant to apoptosis, we previously demonstrated that these cells die through an autophagy-dependent mechanism in response to various types of cellular stressors. To determine the physiological role of autophagy-dependent cell death, we generated Atg5/Bax/Bak triple-knockout (TKO) mice, in which autophagy is greatly suppressed compared with DKO mice. Embryonic fibroblasts and thymocytes from TKO mice underwent autophagy much less frequently, and their viability was much higher than DKO cells in the presence of certain cellular stressors, providing genetic evidence that DKO cells undergo Atg5-dependent death. Compared with wild-type embryos, the loss of interdigital webs was significantly delayed in DKO embryos and was even further delayed in TKO embryos. Brain malformation is a distinct feature observed in DKO embryos on the 129 genetic background, but not in those on a B6 background, whereas such malformations appeared in TKO embryos even on a B6 background. Taken together, our data suggest that Atg5-dependent cell death contributes to the embryonic development of DKO mice, implying that autophagy compensates for the deficiency in apoptosis.