Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity.

Sirtuins (SIRTs) are critical enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, mitochondrial SIRT4 and SIRT5 have little to no deacetylase activity, and a robust catalytic activity for SIRT4 has been elusive. Here, we establish SIRT4 as a cellular lipoamidase that regulates the pyruvate dehydrogenase complex (PDH). Importantly, SIRT4 catalytic efficiency for lipoyl- and biotinyl-lysine modifications is superior to its deacetylation activity. PDH, which converts pyruvate to acetyl-CoA, has been known to be primarily regulated by phosphorylation of its E1 component. We determine that SIRT4 enzymatically hydrolyzes the lipoamide cofactors from the E2 component dihydrolipoyllysine acetyltransferase (DLAT), diminishing PDH activity. We demonstrate SIRT4-mediated regulation of DLAT lipoyl levels and PDH activity in cells and in vivo, in mouse liver. Furthermore, metabolic flux switching via glutamine stimulation induces SIRT4 lipoamidase activity to inhibit PDH, highlighting SIRT4 as a guardian of cellular metabolism.

Intricate Roles of Mammalian Sirtuins in Defense against Viral Pathogens.

For a number of years, sirtuin enzymes have been appreciated as effective "sensors" of the cellular environment to rapidly transmit information to diverse cellular pathways. Much effort was placed into exploring their roles in human cancers and aging. However, a growing body of literature brings these enzymes to the spotlight in the field of virology. Here, we discuss sirtuin functions in the context of viral infection, which provide regulatory points for therapeutic intervention against pathogens.

Rosa26-GFP direct repeat (RaDR-GFP) mice reveal tissue- and age-dependence of homologous recombination in mammals in vivo.

Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals.

Probing phosphorylation-dependent protein interactions within functional domains of histone deacetylase 5 (HDAC5).

Class IIa histone deacetylases (HDACs) are critical transcriptional regulators, shuttling between nuclear and cytoplasmic cellular compartments. Within the nucleus, these HDACs repress transcription as components of multiprotein complexes, such as the nuclear corepressor and beclin-6 corepressor (BCoR) complexes. Cytoplasmic relocalization relieves this transcriptional repressive function. Class IIa HDAC shuttling is controlled, in part, by phosphorylations flanking the nuclear localization signal (NLS). Furthermore, we have reported that phosphorylation within the NLS by the kinase Aurora B modulates the localization and function of the class IIa HDAC5 during mitosis. While we identified numerous additional HDAC5 phosphorylations, their regulatory functions remain unknown. Here, we studied phosphorylation sites within functional HDAC5 domains, including the deacetylation domain (DAC, Ser755), nuclear export signal (NES, Ser1108), and an acidic domain (AD, Ser611). We have generated phosphomutant cell lines to investigate how absence of phosphorylation at these sites impacts HDAC5 localization, enzymatic activity, and protein interactions. Combining molecular biology and quantitative MS, we have defined the interactions and HDAC5-containing complexes mediated by site-specific phosphorylation and quantified selected changes using parallel reaction monitoring. These results expand the current understanding of HDAC regulation, and the functions of this critical family of proteins within human cells.

Myelin-associated glycoprotein protects neurons from excitotoxicity.

In addition to supporting rapid nerve conduction, myelination nurtures and stabilizes axons and protects them from acute toxic insults. One myelin molecule that protects and sustains axons is myelin-associated glycoprotein (MAG). MAG is expressed on the innermost wrap of myelin, apposed to the axon surface, where it interacts with axonal receptors that reside in lateral membrane domains including gangliosides, the glycosylphosphatidylinositol-anchored Nogo receptors, and ß1-integrin. We report here that MAG protection extends beyond the axon to the neurons from which those axons emanate, protecting them from excitotoxicity. Compared to wild type mice, Mag-null mice displayed markedly increased seizure activity in response to intraperitoneal injection of kainic acid, an excitotoxic glutamate receptor agonist. Mag-null mice also had larger lesion volumes in response to intrastriatal injection of the excitotoxin NMDA. Prior injection of a soluble form of MAG partially protected Mag-null mice from NMDA-induced lesions. Hippocampal neurons plated on proteins extracted from wild-type rat or mouse myelin were resistant to kainic acid-induced excitotoxicity, whereas neurons plated on proteins from Mag-null myelin were not. Protection was reversed by anti-MAG antibody and replicated by addition of soluble MAG. MAG-mediated protection from excitotoxicity was dependent on Nogo receptors and ß1-integrin. We conclude that MAG engages membrane-domain resident neuronal receptors to protect neurons from excitotoxicity, and that soluble MAG mitigates excitotoxic damage in vivo.

Protein lipoylation: an evolutionarily conserved metabolic regulator of health and disease.

Lipoylation is a rare, but highly conserved lysine posttranslational modification. To date, it is known to occur on only four multimeric metabolic enzymes in mammals, yet these proteins are staples in the core metabolic landscape. The dysregulation of these mitochondrial proteins is linked to a range of human metabolic disorders. Perhaps most striking is that lipoylation itself, the proteins that add or remove the modification, as well as the proteins it decorates are all evolutionarily conserved from bacteria to humans, highlighting the importance of this essential cofactor. Here, we discuss the biological significance of protein lipoylation, the importance of understanding its regulation in health and disease states, and the advances in mass spectrometry-based proteomic technologies that can aid these studies.

Sirtuin Lipoamidase Activity Is Conserved in Bacteria as a Regulator of Metabolic Enzyme Complexes.

Lipoic acid is an essential metabolic cofactor added as a posttranslational modification on several multimeric enzyme complexes. These protein complexes, evolutionarily conserved from bacteria to humans, are core regulators of cellular metabolism. While the multistep enzymatic process of adding lipoyl modifications has been well characterized in Escherichia coli, the enzyme required for the removal of these lipoyl moieties (i.e., a lipoamidase or delipoylase) has not yet been identified. Here, we describe our discovery of sirtuins as lipoamidases in bacteria and establish their conserved substrates. Specifically, by using a series of knockout, overexpression, biochemical, in vitro, proteomic, and functional assays, we determined the substrates of sirtuin CobB in E. coli as components of the pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KDH), and glycine cleavage (GCV) complexes. In vitro assays provided direct evidence for this specific CobB activity and its NAD+ dependence, a signature of all sirtuins. By designing a targeted quantitative mass spectrometry method, we further measured sirtuin-dependent, site-specific lipoylation on these substrates. The biological significance of CobB-modulated lipoylation was next established by its inhibition of both PDH and KDH activities. By restricting the carbon sources available to E. coli, we demonstrated that CobB regulates PDH and KDH under several growth conditions. Additionally, we found that SrtN, the sirtuin homolog in Gram-positive Bacillus subtilis, can also act as a lipoamidase. By demonstrating the evolutionary conservation of lipoamidase activity across sirtuin homologs, along with the conservation of common substrates, this work emphasizes the significance of protein lipoylation in regulating central metabolic processes.IMPORTANCE Here, we demonstrate that sirtuin lipoamidase activity exists in both Gram-positive and Gram-negative bacteria and establishing its conservation from bacteria to humans. Specifically, we discovered that CobB and SrtN act as lipoamidases in E. coli and B. subtilis, respectively. Intriguingly, not only is this sirtuin enzymatic activity conserved, but also the lipoylated substrates and functions are conserved, as bacterial sirtuins negatively regulate the lipoylation levels and activities of PDH and KDH. Considering that PDH and KDH regulate two carbon entry points into the tricarboxylic acid cycle, our finding highlights lipoylation as a conserved molecular toggle that regulates central metabolic pathways. Indeed, our findings from tests in which we limited nutrient availability support this. Furthermore, this study illustrates how the integration of technologies from different disciplines provides avenues to uncover enzymatic activities at the core of cellular metabolism regulation.

Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA repair inhibitors.

A key modality of non-surgical cancer management is DNA damaging therapy that causes DNA double-strand breaks that are preferentially toxic to rapidly dividing cancer cells. Double-strand break repair capacity is recognized as an important mechanism in drug resistance and is therefore a potential target for adjuvant chemotherapy. Additionally, spontaneous and environmentally induced DSBs are known to promote cancer, making DSB evaluation important as a tool in epidemiology, clinical evaluation and in the development of novel pharmaceuticals. Currently available assays to detect double-strand breaks are limited in throughput and specificity and offer minimal information concerning the kinetics of repair. Here, we present the CometChip, a 96-well platform that enables assessment of double-strand break levels and repair capacity of multiple cell types and conditions in parallel and integrates with standard high-throughput screening and analysis technologies. We demonstrate the ability to detect multiple genetic deficiencies in double-strand break repair and evaluate a set of clinically relevant chemical inhibitors of one of the major double-strand break repair pathways, non-homologous end-joining. While other high-throughput repair assays measure residual damage or indirect markers of damage, the CometChip detects physical double-strand breaks, providing direct measurement of damage induction and repair capacity, which may be useful in developing and implementing treatment strategies with reduced side effects.