Macrolactamization of Glycosylated Peptide Thioesters by the Thioesterase Domain of Tyrocidine Synthetase

SummaryThe 35 kDa thioesterase (TE) domain excised from the megadalton tyrocidine synthetase (Tyc Syn) retains autonomous capacity to macrocyclize peptidyl thioesters to D-Phe1-L-Leu10-macrolactams. Since a number of nonribosomal peptides undergo O-glycosylation events during tailoring to gain biological activity, the Tyc Syn TE domain was evaluated for cyclization capacity with glycosylated peptidyl-S-NAC substrates. First, Tyr7 was replaced with Tyr(β-D-Gal) and Tyr(β-D-Glc) as well as with Ser-containing β-linked D-Gal, D-Glc, D-GlcNAc, and D-GlcNH2, and these new analogs were shown to be cyclized with comparable kcat/Km catalytic efficiency. Similarly, Gal- or tetra-O-acetyl-Gal-Ser could also be substituted at residues 5, 6, and 8 in the linear decapeptidyl-S-NAC sequences and cyclized without substantial loss in catalytic efficiency by Tyc Syn TE. The cyclic glycopeptides retained antibiotic activity as membrane perturbants in MIC assays, opening the possibility for library construction of cyclic glycopeptides by enzymatic macrocyclization

A Versatile Approach for Siteâ Specific Lysine Acylation in Proteins

Using amber suppression in coordination with a mutant pyrrolysylâ tRNA synthetaseâ tRNAPyl pair, azidonorleucine is genetically encoded in E. coli. Its genetic incorporation followed by traceless Staudinger ligation with a phosphinothioester allows the convenient synthesis of a protein with a siteâ specifically installed lysine acylation. By simply changing the phosphinothioester identity, any lysine acylation type could be introduced. Using this approach, we demonstrated that both lysine acetylation and lysine succinylation can be installed selectively in ubiquitin and synthesized histone H3 with succinylation at its K4 position (H3K4su). Using an H3K4suâ H4 tetramer as a substrate, we further confirmed that Sirt5 is an active histone desuccinylase. Lysine succinylation is a recently identified postâ translational modification. The reported technique makes it possible to explicate regulatory functions of this modification in proteins.Azidonorleucine, an azideâ containing amino acid, is genetically encoded and incorporated into model proteins. This incorporation followed by traceless Staudinger ligation potentiates the synthesis of proteins with a myriad of siteâ specific lysine acylations.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137311/1/anie201611415-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137311/2/anie201611415_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137311/3/anie201611415.pd

Sirt6 regulates TNFα secretion via hydrolysis of long chain fatty acyl lysine

The Sir2 family of enzymes or sirtuins are known as nicotinamide adenine dinucleotide (NAD)-dependent deacetylases1 and have been implicated in the regulation of transcription, genome stability, metabolism, and lifespan2, 3. However, four of the seven mammalian sirtuins have very weak deacetylase activity in vitro. Here we show that human Sirt6 efficiently removes long chain fatty acyl groups, such as myristoyl, from lysine residues. The crystal structure of Sirt6 reveals a large hydrophobic pocket that can accommodate long chain fatty acyl groups. We demonstrate further that Sirt6 promotes the secretion of tumor necrosis factor α (TNFα) by removing the fatty acyl modification on K19 and K20 of TNFα. Protein lysine fatty acylation has been known to occur in mammalian cells, but the function and regulatory mechanisms of this modification were unknown. Our data suggest that protein lysine fatty acylation is a novel mechanism that regulates protein secretion. The discovery of Sirt6 as an enzyme that controls protein lysine fatty acylation provides new opportunities to investigate the physiological function of the previously ignored protein posttranslational modification

Metabolic Characterization of a Sirt5 deficient mouse model

Sirt5, localized in the mitochondria, is a member of sirtuin family of NAD(+)-dependent deacetylases. Sirt5 was shown to deacetylate and activate carbamoyl phosphate synthase 1. Most recently, Sirt5 was reported to be the predominant protein desuccinylase and demalonylase in the mitochondria because the ablation of Sirt5 enhanced the global succinylation and malonylation of mitochondrial proteins, including many metabolic enzymes. In order to determine the physiological role of Sirt5 in metabolic homeostasis, we generated a germline Sirt5 deficient (Sirt5(-/-)) mouse model and performed a thorough metabolic characterization of this mouse line. Although a global protein hypersuccinylation and elevated serum ammonia during fasting were observed in our Sirt5(-/-) mouse model, Sirt5 deficiency did not lead to any overt metabolic abnormalities under either chow or high fat diet conditions. These observations suggest that Sirt5 is likely to be dispensable for the metabolic homeostasis under the basal conditions

Reversible lysine fatty acylation of an anchoring protein mediates adipocyte adrenergic signaling.

N-myristoylation on glycine is an irreversible modification that has long been recognized to govern protein localization and function. In contrast, the biological roles of lysine myristoylation remain ill-defined. We demonstrate that the cytoplasmic scaffolding protein, gravin-α/A kinase–anchoring protein 12, is myristoylated on two lysine residues embedded in its carboxyl-terminal protein kinase A (PKA) binding domain. Histone deacetylase 11 (HDAC11) docks to an adjacent region of gravin-α and demyristoylates these sites. In brown and white adipocytes, lysine myristoylation of gravin-α is required for signaling via β(2)- and β(3)-adrenergic receptors (β-ARs), which are G protein–coupled receptors (GPCRs). Lysine myristoylation of gravin-α drives β-ARs to lipid raft membrane microdomains, which results in PKA activation and downstream signaling that culminates in protective thermogenic gene expression. These findings define reversible lysine myristoylation as a mechanism for controlling GPCR signaling and highlight the potential of inhibiting HDAC11 to manipulate adipocyte phenotypes for therapeutic purposes

Construção de um protótipo de Data Warehouse como suporte ao sistema de informação numa instituição de ensino superior

Uma das dificuldades que se verifica na extracção de informação numa organização é a falta de integração dos dados existentes dispersos em diversos formatos: ficheiros de processadores de texto, folhas de cálculo, bases de dados, entre outras fontes. A partir deste problema, este trabalho propõe a estruturação de um modelo de Data Warehouse com o objectivo de organizar, armazenar e integrar as informações provenientes de outros formatos e sistemas, numa única base de dados para uma futura utilização no suporte à tomada de decisão. Existem, neste momento, na comunidade de Data Warehousing duas principais abordagens, uma preconizada por William H. Inmon, mais centrada nos dados, e outra por Ralph Kimball, mais centrada no projecto. Assim, com a metodologia proposta foi desenvolvido um caso de estudo com a finalidade de verificar e avaliar a aplicabilidade da metodologia no Instituto Politécnico de Tomar; ABSTRACT: One difficulty that exists in the extraction of information in organizations is the lack of integration of existing data scattered in various formats: word processing files, spreadsheets, databases, among other sources. From this problem, this paper proposes to structure a model of Data Warehouse in order to organize, store and integrate information from other systems and formats in a single database for future use in supporting decision making. There are at present in the community of Data Warehousing two main approaches, one advocated by William H. Inmon, more data-centric, and one by Ralph Kimball, more focused on the project. So with the proposed methodology was developed a case study in order to verify and evaluate the applicability of the methodology at the Polytechnic Institute of Tomar

Trapoxin A Analogue as a Selective Nanomolar Inhibitor of HDAC11

Histone deacetylases (HDACs) are enzymes that regulate many important biological pathways. There is a need for the development of isoform-selective HDAC inhibitors for further biological applications. Here, we report the development of trapoxin A analogues as potent and selective inhibitors of HDAC11, an enzyme that can efficiently remove long-chain fatty acyl groups from proteins. In particular, we show that one of the trapoxin A analogues, TD034, has nanomolar potency in enzymatic assays. We show that in cells, TD034 is active at low micromolar concentrations and inhibits the defatty acylation of SHMT2, a known HDAC11 substrate. The high potency and selectivity of TD034 would permit further development of HDAC11 inhibitors for biological and therapeutic applications

The Acyl-CoA Specificity of Human Lysine Acetyltransferase KAT2A

Protein post-translational modification sserve to regulate a broad range of cellular functions including signal transduction, transcription, and metabolism. Protein lysine residue sundergo many post-translational acylations and are regulated by a range of enzymes, such as histone acetyl transferases (HATs)and histone deacetylases (HDACs).KAT2A,well characterized as a lysine acetyltransferase for both histone and nonhistone substrates, has been reported to tolerate addition alacyl-CoA substrates, such as succinyl-CoA, and shows nonacetyl transferase activity in specific biologicalcontexts.In thiswork, we investigatethe acyl-CoA substrate preference of KAT2A and attempt to determine whether and to what extent addition alacyl-CoA substrates may be utilized by KAT2A in a cellula rcontext. We show that while KAT2A can bind and utilize malonyl-CoA, its activity with succinyl-CoA or glutaryl-CoA is very weak, and acetylation is still the most efficient activity for KAT2A in vitro and in cells.ISSN:0006-2960ISSN:1520-499