Rachita Dash

Chemistry. 2023 Mar 7;29(14):e202203923. doi: 10.1002/chem.202203923. Epub 2023 Feb 6.

ABSTRACT

Macrocyclisation provides a means of stabilising the conformation of peptides, often resulting in improved stability, selectivity, affinity, and cell permeability. In this work, a new approach to peptide macrocyclisation is reported, using a cyanobenzothiazole-containing amino acid that can be incorporated into peptides by both in vitro translation and solid phase peptide synthesis, meaning it should be applicable to peptide discovery by mRNA display. This cyclisation proceeds rapidly, with minimal by-products, is selective over other amino acids including non N-terminal cysteines, and is compatible with further peptide elaboration exploiting such an additional cysteine in bicyclisation and derivatisation reactions. Molecular dynamics simulations show that the new cyclisation group is likely to influence the peptide conformation as compared to previous thioether-based approaches, through rigidity and intramolecular aromatic interactions, illustrating their complementarity.

PMID:36529683 | DOI:10.1002/chem.202203923

J Bacteriol. 2023 Jan 26;205(1):e0042422. doi: 10.1128/jb.00424-22. Epub 2022 Dec 21.

ABSTRACT

The peptidoglycan of mycobacteria has two types of direct cross-links, classical 4-3 cross-links that occur between diaminopimelate (DAP) and alanine residues, and nonclassical 3-3 cross-links that occur between DAP residues on adjacent peptides. The 3-3 cross-links are synthesized by the concerted action of d,d-carboxypeptidases and l,d-transpeptidases (Ldts). Mycobacterial genomes encode several Ldt proteins that can be classified into six classes based upon sequence identity. As a group, the Ldt enzymes are resistant to most β-lactam antibiotics but are susceptible to carbapenem antibiotics, with the exception of LdtC, a class 5 enzyme. In previous work, we showed that loss of LdtC has the greatest effect on the carbapenem susceptibility phenotype of Mycobacterium smegmatis (also known as Mycolicibacterium smegmatis) compared to other ldt deletion mutants. In this work, we show that a M. smegmatis mutant lacking the five ldt genes other than ldtC has a wild-type phenotype with the exception of increased susceptibility to rifampin. In contrast, a mutant lacking all six ldt genes has pleiotropic cell envelope defects, is temperature sensitive, and has increased susceptibility to a variety of antibiotics. These results indicate that LdtC is capable of functioning as the sole l,d-transpeptidase in M. smegmatis and suggest that it may represent a carbapenem-resistant pathway for peptidoglycan biosynthesis. IMPORTANCE Mycobacteria have several enzymes to catalyze nonclassical 3-3 linkages in the cell wall peptidoglycan. Understanding the biology of these cross-links is important for the development of antibiotic therapies to target peptidoglycan biosynthesis. Our work provides evidence that LdtC can function as the sole enzyme for 3-3 cross-link formation in M. smegmatis and suggests that LdtC may be part of a carbapenem-resistant l,d-transpeptidase pathway.

PMID:36541811 | PMC:PMC9879121 | DOI:10.1128/jb.00424-22

J Bacteriol. 2023 Jan 26;205(1):e0038222. doi: 10.1128/jb.00382-22. Epub 2022 Dec 12.

ABSTRACT

Peptidoglycan (PG) is a unique and essential component of the bacterial cell envelope. It is made up of several linear glycan polymers cross-linked through covalently attached stem peptides making it a fortified mesh-like sacculus around the bacterial cytosolic membrane. In most bacteria, including Escherichia coli, the stem peptide is made up of l-alanine (l-Ala¹), d-glutamate (d-Glu²), meso-diaminopimelic acid (mDAP³), d-alanine (d-Ala⁴), and d-Ala⁵ with cross-links occurring either between d-ala⁴ and mDAP³ or between two mDAP³ residues. Of these, the cross-links of the 4-3 (d-Ala⁴-mDAP³) type are the most predominant and are formed by penicillin-binding D,D-transpeptidases, whereas the formation of less frequent 3-3 linkages (mDAP³-mDAP³) is catalyzed by L,D-transpeptidases. In this study, we found that the frequency of the 3-3 cross-linkages increased upon cold shock in exponentially growing E. coli and that the increase was mediated by an L,D-transpeptidase, LdtD. We found that a cold-inducible RNA helicase DeaD enhanced the cellular LdtD level by facilitating its translation resulting in an increased abundance of 3-3 cross-linkages during cold shock. However, DeaD was also required for optimal expression of LdtD during growth at ambient temperature. Overall, our study finds that E. coli undergoes PG remodeling during cold shock by altering the frequency of 3-3 cross-linkages, implying a role for these modifications in conferring fitness and survival advantage to bacteria growing in diverse environmental conditions. IMPORTANCE Most bacteria are surrounded by a protective exoskeleton called peptidoglycan (PG), an extensively cross-linked mesh-like macromolecule. In bacteria, such as Escherichia coli, the cross-links in the PG are of two types: a major fraction is of 4-3 type whereas a minor fraction is of 3-3 type. Here, we showed that E. coli exposed to cold shock had elevated levels of 3-3 cross-links due to the upregulation of an enzyme, LdtD, that catalyzed their formation. We showed that a cold-inducible RNA helicase DeaD enhanced the cellular LdtD level by facilitating its translation, resulting in increased 3-3 cross-links during cold shock. Our results suggest that PG remodeling contributes to the survival and fitness of bacteria growing in conditions of cold stress.

PMID:36507682 | PMC:PMC9879098 | DOI:10.1128/jb.00382-22

Biochem Biophys Res Commun. 2023 Jan 22;641:186-191. doi: 10.1016/j.bbrc.2022.12.037. Epub 2022 Dec 13.

ABSTRACT

Activation of N-methyl-_d-aspartate receptors (NMDARs) requires binding of a co-agonist in addition to _l-glutamate. _d-serine binds to the co-agonist site on GluN1 subunits of NMDARs and modulates glutamatergic neurotransmission. While loss of GluN1 subunits in mice results in neonatal death due to respiratory failure, animals that lack a _d-serine synthetic enzyme, serine racemase (SR), show grossly normal growth. However, SR-independent origins of _d-serine in the brain remain unclarified. In the present study, we investigated the origin of brain _d-serine in mice. Loss of SR significantly reduced _d-serine in the cerebral cortex, but a portion of _d-serine remained in both neonates and adults. Although _d-serine was also produced by intestinal bacteria, germ-free experiments did not influence _d-serine levels in the cerebral cortex. In addition, treatment of SR-knockout mice with antibiotics showed a significant reduction of intestinal _d-serine, but no reduction in the brain. On the other hand, restriction of dietary intake reduced systemic circulation of _d-serine and resulted in a slight decrease of _d-serine in the cerebral cortex, but did not account for brain _d-serine found in the SR-knockout mice. Therefore, our findings show that endogenous _d-serine of non-SR origin exists in the brain. Such previously unrecognized, SR-independent, endogenous _d-serine may contribute baseline activity of NMDARs, especially in developing brain, which has minimal SR expression.

PMID:36535077 | DOI:10.1016/j.bbrc.2022.12.037

Chembiochem. 2022 Dec 5. doi: 10.1002/cbic.202200590. Online ahead of print.

NO ABSTRACT

PMID:36471561 | DOI:10.1002/cbic.202200590

Int J Pept Res Ther. 2023;29(1):7. doi: 10.1007/s10989-022-10478-y. Epub 2022 Dec 1.

NO ABSTRACT

PMID:36471676 | PMC:PMC9713128 | DOI:10.1007/s10989-022-10478-y

J Vis Exp. 2022 Nov 30;(189). doi: 10.3791/64256.

ABSTRACT

Here, with the aim of developing a novel anti-cancer treatment, seven dipeptides were designed that contained methylated tryptophan and/or methylated arginine and were produced using Fmoc solid-phase peptide synthesis. Overexpression of the Src tyrosine kinase enzyme has been implicated in the development of different cancers. Dipeptides containing unnatural amino acids such as methylated arginine (RCH3), dimethylated arginine (R(CH3)2), and/or methylated tryptophan (WCH3) residues have earlier been shown to inhibit Src kinase. In this study, three such dipeptides, W-RCH3, WCH3-RCH3, and W-R(CH3)2, were tested using acellular assays and were found to have IC50 values (the concentration at which 50% inhibition occurs) of 510 nM, 916 nM, and 1 µM, respectively. These values were comparable to those obtained for cyclic penta- to nano-W-R peptides ([W-R]5-[W-R]9) synthesized in previous studies. However, the unmethylated versions of the dipeptides did not show any inhibitory activity against Src kinase. All of these dipeptides (50 µM) did not show any cytotoxicity after incubation up to 72 h with three different cancer cell lines, including leukemia (CCRF-CEM), breast adenocarcinoma (MDA-MB-231), and ovarian adenocarcinoma (SK-OV-3) cell lines, indicating the limited permeability of the peptides through the cell membrane. Therefore, further study is needed to improve the permeability of these peptides for anticancer applications, such as by using a peptide carrier or additional peptide functionalization. In summary, this study provides a protocol to synthesize and test peptides that inhibit Src kinase activity, and thus possess promising anticancer ability, as demonstrated using acellular and cellular assays.

PMID:36533825 | DOI:10.3791/64256

Biochim Biophys Acta Mol Cell Res. 2022 Nov 29;1870(2):119405. doi: 10.1016/j.bbamcr.2022.119405. Online ahead of print.

NO ABSTRACT

PMID:36455781 | DOI:10.1016/j.bbamcr.2022.119405

J Enzyme Inhib Med Chem. 2023 Dec;38(1):387-397. doi: 10.1080/14756366.2022.2149745.

NO ABSTRACT

PMID:36446617 | PMC:PMC9718554 | DOI:10.1080/14756366.2022.2149745

Langmuir. 2022 Dec 6. doi: 10.1021/acs.langmuir.2c02520. Online ahead of print.

NO ABSTRACT

PMID:36472987 | DOI:10.1021/acs.langmuir.2c02520

J Med Chem. 2022 Nov 28. doi: 10.1021/acs.jmedchem.2c01469. Online ahead of print.

NO ABSTRACT

PMID:36442155 | DOI:10.1021/acs.jmedchem.2c01469

Microorganisms. 2022 Oct 5;10(10):1969. doi: 10.3390/microorganisms10101969.

ABSTRACT

The human gut is host to almost 3000 microbial species, of which 90% are bacteria. Quorum sensing (QS) molecules generated by intestinal bacteria are important in establishing species- and strain-level structures within the gut microbiome but are also used to communicate with the host. Although we do not know which QS molecules have the most direct interaction with intestinal and sensory neurons, it is clear they affect our physiological and mental health. Signals produced by bacteria are diverse and include autoinducers (AIs), homoserine lactones (HSLs), quinolines, peptides, toxins and proteases. These signaling molecules activate specific receptors in the bacterial cell wall and trigger sensors in the cytoplasm that regulate gene expressions. A better understanding of the gene structures encoding the production of QS molecules is of importance when selecting strains with neurogenerative and other probiotic properties. Furthermore, QS molecules may be used as biomarkers in the diagnosis of inflammable bowel disease (IBD), irritable bowel syndrome (IBS) and colorectal cancer (CRC). In the future, it should be possible to use QS biomarkers to diagnose neurological and psychiatric diseases such as anxiety and depression, major depressive disorder (MDD), schizophrenia, bipolar disorder, autism and obsessive-compulsive disorder (OCD).

PMID:36296244 | PMC:PMC9611604 | DOI:10.3390/microorganisms10101969

Protein Sci. 2022 Oct 18:e4478. doi: 10.1002/pro.4478. Online ahead of print.

ABSTRACT

The cell biology and biochemistry of peptide exchange on MHC-I proteins are of great interest in the study of immunodominance, which requires iterative optimization of peptide affinity, and cross-presentation of pathogen and tumor antigens, in which endogenous peptides are exchanged for exogenous ones. Even though several methods exist to catalyze peptide exchange on recombinant MHC-I proteins, the cellular conditions and mechanisms allowing for peptide exchange in vivo remain unclear. Here, we demonstrate that low pH, as present in endosomes, indeed triggers peptide exchange, and we dissect the individual steps of the exchange reaction. We find that low pH stabilizes the peptide-empty forms of MHC-I that occur as intermediates of the exchange reaction, and that is synergizes with dipeptides and with disulfide-mediated stabilization of MHC-I. This article is protected by copyright. All rights reserved.

PMID:36258668 | DOI:10.1002/pro.4478

Nat Commun. 2022 Oct 18;13(1):6174. doi: 10.1038/s41467-022-33808-6.

ABSTRACT

Developing an effective binder for a specific ubiquitin (Ub) chain is a promising approach for modulating various biological processes with potential applications in drug discovery. Here, we combine the Random Non-standard Peptides Integrated Discovery (RaPID) method and chemical protein synthesis to screen an extended library of macrocyclic peptides against synthetic Lys63-linked Di-Ub to discover a specific binder for this Ub chain. Furthermore, next-generation binders are generated by chemical modifications. We show that our potent cyclic peptide is cell-permeable, and inhibits DNA damage repair, leading to apoptotic cell death. Concordantly, a pulldown experiment with the biotinylated analog of our lead cyclic peptide supports our findings. Collectively, we establish a powerful strategy for selective inhibition of protein-protein interactions associated with Lys63-linked Di-Ub using cyclic peptides. This study offers an advancement in modulating central Ub pathways and provides opportunities in drug discovery areas associated with Ub signaling.

PMID:36257952 | PMC:PMC9579194 | DOI:10.1038/s41467-022-33808-6