Synthesis, Enzymatic Peptide Incorporation, and Applications of Diazirine-Containing Isoprenoid Diphosphate Analogues.

Prenylated proteins contain C or C isoprenoids linked to cysteine residues positioned near their C-termini. Here we describe the preparation of isoprenoid diphosphate analogues incorporating diazirine groups that can be used to probe interactions between prenylated proteins and other proteins that interact with them. Studies using synthetic peptides and whole proteins demonstrate that these diazirine analogues are efficient substrates for prenyltransferases.

Translation of peptidoglycan metabolites into immunotherapeutics.

The discovery of defined peptidoglycan metabolites that activate host immunity and their specific receptors has revealed fundamental insights into host-microbe recognition and afforded new opportunities for therapeutic development against infection and cancer. In this review, we summarise the discovery of two key peptidoglycan metabolites, γ-d-glutamyl--diaminopimelic acid (iE-DAP) and muramyl dipeptide and their respective receptors, Nod1 and Nod2, and review progress towards translating these findings into therapeutic agents. Notably, synthetic derivatives of peptidoglycan metabolites have already yielded approved drugs for chemotherapy-induced leukopenia and paediatric osteosarcoma; however, the broad effects of peptidoglycan metabolites on host immunity suggest additional translational opportunities for new therapeutics towards other cancers, microbial infections and inflammatory diseases.

Chemical Reporters for Exploring Microbiology and Microbiota Mechanisms.

The advances made in bioorthogonal chemistry and the development of chemical reporters have afforded new strategies to explore the targets and functions of specific metabolites in biology. These metabolite chemical reporters have been applied to diverse classes of bacteria including Gram-negative, Gram-positive, mycobacteria, and more complex microbiota communities. Herein we summarize the development and application of metabolite chemical reporters to study fundamental pathways in bacteria as well as microbiota mechanisms in health and disease.

secreted antigen A generates muropeptides to enhance host immunity and limit bacterial pathogenesis.

We discovered that (), a ubiquitous commensal bacterium, and its secreted peptidoglycan hydrolase (SagA) were sufficient to enhance intestinal barrier function and pathogen tolerance, but the precise biochemical mechanism was unknown. Here we show has unique peptidoglycan composition and remodeling activity through SagA, which generates smaller muropeptides that more effectively activates nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in mammalian cells. Our structural and biochemical studies show that SagA is a NlpC/p60-endopeptidase that preferentially hydrolyzes crosslinked Lys-type peptidoglycan fragments.

Peptidoglycan Metabolite Photoaffinity Reporters Reveal Direct Binding to Intracellular Pattern Recognition Receptors and Arf GTPases.

The peptidoglycan fragments γ-d-glutamyl- meso-diaminopimelic acid (iE-DAP) and muramyl-dipeptide (MDP) are microbial-specific metabolites that activate intracellular pattern recognition receptors and stimulate immune signaling pathways. While extensive structure-activity studies have demonstrated that these bacterial cell wall metabolites trigger NOD1- and NOD2-dependent signaling, their direct binding to these innate immune receptors or other proteins in mammalian cells has not been established. To characterize these fundamental microbial metabolite-host interactions, we synthesized a series of peptidoglycan metabolite photoaffinity reporters and evaluated their cross-linking to NOD1 and NOD2 in mammalian cells.

A secreted bacterial peptidoglycan hydrolase enhances tolerance to enteric pathogens.

The intestinal microbiome modulates host susceptibility to enteric pathogens, but the specific protective factors and mechanisms of individual bacterial species are not fully characterized. We show that secreted antigen A (SagA) from Enterococcus faecium is sufficient to protect Caenorhabditis elegans against Salmonella pathogenesis by promoting pathogen tolerance. The NlpC/p60 peptidoglycan hydrolase activity of SagA is required and generates muramyl-peptide fragments that are sufficient to protect C.

Analogs of farnesyl diphosphate alter CaaX substrate specificity and reactions rates of protein farnesyltransferase.

Attempts to identify the prenyl-proteome of cells or changes in prenylation following drug treatment have used 'clickable' alkyne-modified analogs of the lipid substrates farnesyl- and geranylgeranyl-diphosphate (FPP and GGPP). We characterized the reactivity of four alkyne-containing analogs of FPP with purified protein farnesyltransferase and a small library of dansylated peptides using an in vitro continuous spectrofluorimetric assay. These analogs alter prenylation specificity and reactivity suggesting that in vivo results obtained using these FPP analogs should be interpreted cautiously.

Synthetic isoprenoid analogues for the study of prenylated proteins: Fluorescent imaging and proteomic applications.

Protein prenylation is a posttranslational modification catalyzed by prenyltransferases involving the attachment of farnesyl or geranylgeranyl groups to residues near the C-termini of proteins. This irreversible covalent modification is important for membrane localization and proper signal transduction. Here, the use of isoprenoid analogues for studying prenylated proteins is reviewed.

Error-prone translesion synthesis past DNA-peptide cross-links conjugated to the major groove of DNA via C5 of thymidine.

DNA-protein cross-links (DPCs) are exceptionally bulky, structurally diverse DNA adducts formed in cells upon exposure to endogenous and exogenous bis-electrophiles, reactive oxygen species, and ionizing radiation. If not repaired, DPCs can induce toxicity and mutations. It has been proposed that the protein component of a DPC is proteolytically degraded, giving rise to smaller DNA-peptide conjugates, which can be subject to nucleotide excision repair and replication bypass.

Engineering protein farnesyltransferase for enzymatic protein labeling applications.

Creating covalent protein conjugates is an active area of research due to the wide range of uses for protein conjugates spanning everything from biological studies to protein therapeutics. Protein Farnesyltransferase (PFTase) has been used for the creation of site-specific protein conjugates, and a number of PFTase substrates have been developed to facilitate that work. PFTase is an effective catalyst for protein modification because it transfers Farnesyl diphosphate (FPP) analogues to protein substrates on a cysteine four residues from the C-terminus.

Synthesis of site-specific DNA-protein conjugates and their effects on DNA replication.

DNA-protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome upon exposure to common antitumor drugs, environmental/occupational toxins, ionizing radiation, and endogenous free-radical-generating systems. As a result of their considerable size and their pronounced effects on DNA-protein interactions, DPCs can interfere with DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological consequences of these ubiquitous lesions are not fully understood due to the difficulty of generating DNA substrates containing structurally defined, site-specific DPCs.

Rapid analysis of protein farnesyltransferase substrate specificity using peptide libraries and isoprenoid diphosphate analogues.

Protein farnesytransferase (PFTase) catalyzes the farnesylation of proteins with a carboxy-terminal tetrapeptide sequence denoted as a Ca1a2X box. To explore the specificity of this enzyme, an important therapeutic target, solid-phase peptide synthesis in concert with a peptide inversion strategy was used to prepare two libraries, each containing 380 peptides. The libraries were screened using an alkyne-containing isoprenoid analogue followed by click chemistry with biotin azide and subsequent visualization with streptavidin-AP.

Diazirine-containing photoactivatable isoprenoid: synthesis and application in studies with isoprenylcysteine carboxyl methyltransferase.

Photoaffinity labeling is a useful technique employed to identify protein-ligand and protein-protein noncovalent interactions. Photolabeling experiments have been particularly informative for probing membrane-bound proteins where structural information is difficult to obtain. The most widely used classes of photoactive functionalities include aryl azides, diazocarbonyls, diazirines, and benzophenones.

Synthesis and screening of peptide libraries with free C-termini.

Peptide libraries are useful tools to investigate the relationship between structure and function of proteins. The creation of peptide libraries with free C-termini presents unique synthetic challenges. In this review, methods for creating peptide libraries using either solid-phase peptide synthesis or phage display are described.

Butyrophilin 3A1 plays an essential role in prenyl pyrophosphate stimulation of human Vγ2Vδ2 T cells.

Most human γδ T cells express Vγ2Vδ2 TCRs and play important roles in microbial and tumor immunity. Vγ2Vδ2 T cells are stimulated by self- and foreign prenyl pyrophosphate intermediates in isoprenoid synthesis. However, little is known about the molecular basis for this stimulation.

Bayesian approach of nearfield acoustic reconstruction with particle filters.

This paper demonstrates that inverse source reconstruction can be performed using a methodology of particle filters that relies primarily on the Bayesian approach of parameter estimation. In particular, the proposed approach is applied in the context of nearfield acoustic holography based on the equivalent source method (ESM). A state-space model is formulated in light of the ESM.

Photoactive analogs of farnesyl diphosphate and related isoprenoids: design and applications in studies of medicinally important isoprenoid-utilizing enzymes.

Farnesyl diphosphate (FPP) is an important metabolic intermediate in the biosynthesis of a variety of molecules including sesquiterpenes and the side chains of a number of cofactors. FPP is also the source of isoprenoid side chains found attached to proteins. Enzymes that employ FPP as a substrate are of interest because they are involved in the semisynthesis of drugs as well as targets for drug design.

Solid-phase synthesis of C-terminal peptide libraries for studying the specificity of enzymatic protein prenylation.

Prenylation is an essential post-translational modification in all eukaryotes. Here we describe the synthesis of a 340-member library of peptides containing free C-termini on cellulose membranes. The resulting library was then used to probe the specificity of protein farnesyltransferase from S.

p-Cresyl sulphate and indoxyl sulphate predict progression of chronic kidney disease.

Background: Indoxyl sulphate (IS) and p-cresyl sulphate (PCS) are uraemic toxins that have similar protein binding, dialytic clearance and proinflammatory features. However, only a few prospective studies have evaluated possible associations between these two retained solutes and renal disease progression in chronic kidney disease (CKD) patients.

Methods: This prospective observational study evaluated independent associations between serum total IS and PCS with renal progression in a selected cohort of patients having different stages of CKD.

Synthesis and evaluation of a new fluorescent transglycosylase substrate: lipid II-based molecule possessing a dansyl-C20 polyprenyl moiety.

The preparation of a novel fluorescent lipid II-based substrate for transglycosylases (TGases) is described. This substrate has characteristic structural features including a shorter lipid chain, a fluorophore tag at the end of the lipid chain rather than on the peptide chain, and no labeling with a radioactive atom. This fluorescent substrate is readily utilized in TGase activity assays to characterize TGases and also to evaluate the activities of TGase inhibitors.

Solid-phase organic synthesis of polyisoprenoid alcohols with traceless sulfone linker.

Solid-phase organic synthesis of polyprenols with a traceless sulfone linker is described. The polymer-bound benezenesulfinate is first linked with the "tail" building blocks of isoprenyl chlorides via S-alkylation. With use of dimsyl anion as an appropriate base, the polymer-bound alpha-sulfonyl carbanion is generated and coupled with other "body" building blocks in an efficient manner.