Rachita Dash

Gut Microbes. 2024 Jan-Dec;16(1):2438465. doi: 10.1080/19490976.2024.2438465. Epub 2024 Dec 11.

ABSTRACT

Gram-positive Enterococcus faecium exhibited higher susceptibility (>4-fold) to polymyxin B (PMB), the canonical antimicrobial peptide against Gram-negative bacteria, under anaerobic condition than aerobic condition. Anaerobically grown E. faecium exhibited high vulnerability to PMB, leading to alteration of cell surface and morphology, as observed based on their high dansyl-PMB affinity (>2.9-fold), a proportion (>8.5-fold) of propidium iodide-stained cells, and observation of scanning electron microscopy results. Interestingly, our transcriptomic and chemical analyses revealed that enterocin B, produced anaerobically, imposes a burden on the cellular envelope when cells are exposed to PMB. This scenario was also supported by PMB susceptibility tests and killing curves, which showed that ΔentB knockout mutant cells were more resistant to PMB (32 µg/mL) compared to wild-type cells (4 µg/mL) under anaerobic condition. Fluorescent D-amino acid and BOCILLIN™-fluorescent profiling of transpeptidase activities in ΔentB mutant cells under anaerobic condition revealed similar levels of activity to those observed in WT cells under aerobic condition. The high level of secreted bacteriocins in WT under anaerobic condition likely led to significant membrane depolarization and loosening of the peptidoglycan layer, making the cells more permeable to PMB. Overall, our findings suggest that anaerobically produced bacteriocins, in conjunction with PMB, contribute to the killing of E. faecium by destabilizing its cell envelope.

PMID:39663231 | PMC:PMC11651277 | DOI:10.1080/19490976.2024.2438465

bioRxiv [Preprint]. 2024 Nov 28:2024.11.28.625793. doi: 10.1101/2024.11.28.625793.

ABSTRACT

Class I MHC molecules present peptides derived from intracellular antigens on the cell surface for immune surveillance, and specific targeting of these peptide-MHC (pMHC) complexes could have considerable utility for treating diseases. Such targeting is challenging as it requires readout of the few outward facing peptide antigen residues and the avoidance of extensive contacts with the MHC carrier which is present on almost all cells. Here we describe the use of deep learning-based protein design tools to denovo design small proteins that arc above the peptide binding groove of pMHC complexes and make extensive contacts with the peptide. We identify specific binders for ten target pMHCs which when displayed on yeast bind the on-target pMHC tetramer but not closely related peptides. For five targets, incorporation of designs into chimeric antigen receptors leads to T-cell activation by the cognate pMHC complexes well above the background from complexes with peptides derived from proteome. Our approach can generate high specificity binders starting from either experimental or predicted structures of the target pMHC complexes, and should be widely useful for both protein and cell based pMHC targeting.

PMID:39651227 | PMC:PMC11623666 | DOI:10.1101/2024.11.28.625793

Chembiochem. 2025 Jan 2;26(1):e202400922. doi: 10.1002/cbic.202400922. Epub 2024 Dec 5.

ABSTRACT

Dietary fiber (DF)-based interventions are crucial in establishing a health-promoting gut microbiota. However, directly investigating DFs' in vivo interactions with intestinal bacteria remains challenging due to the lack of suitable tools. Here, we develop an in vivo metabolic labeling-based strategy, which enables not only imaging and identifying the bacteria that bind with specific DF in the intestines, but also quantifying DF's impact on their metabolic status. Four DFs, including galactan, rhamnogalacturonan and two inulins, are fluorescently derivatized and used for in vivo labeling to visually record DFs' interactions with gut bacteria. The subsequent cell-sorting, 16S rDNA sequencing, and fluorescence in situ hybridization identify the taxa that bind each DF. We then select a DF-binding species newly identified herein and verify its DF-catabolizing capability in vitro. Furthermore, we find that the indigenous metabolic status of Gram-positive bacteria, whether inulin-binders or not, is significantly enhanced by the inulin supplement. This trend is not observed in Gram-negative microbiota, even for the inulin-binders, demonstrating the ability of our methods in differentiating the primary, secondary DF-degraders from cross-feeders, a question that is difficult to answer by using other methods. Our strategy provides a novel chemical biology tool for deciphering the complex DF-bacteria interactions in the gut.

PMID:39538366 | DOI:10.1002/cbic.202400922

bioRxiv [Preprint]. 2024 Nov 18:2024.11.18.622547. doi: 10.1101/2024.11.18.622547.

ABSTRACT

The development of macrocyclic binders to therapeutic proteins typically relies on large-scale screening methods that are resource-intensive and provide little control over binding mode. Despite considerable progress in physics-based methods for peptide design and deep-learning methods for protein design, there are currently no robust approaches for de novo design of protein-binding macrocycles. Here, we introduce RFpeptides, a denoising diffusion-based pipeline for designing macrocyclic peptide binders against protein targets of interest. We test 20 or fewer designed macrocycles against each of four diverse proteins and obtain medium to high-affinity binders against all selected targets. Designs against MCL1 and MDM2 demonstrate K_D between 1-10 μM, and the best anti-GABARAP macrocycle binds with a K_D of 6 nM and a sub-nanomolar IC₅₀ in vitro. For one of the targets, RbtA, we obtain a high-affinity binder with K_D < 10 nM despite starting from the target sequence alone due to the lack of an experimentally determined target structure. X-ray structures determined for macrocycle-bound MCL1, GABARAP, and RbtA complexes match very closely with the computational design models, with three out of the four structures demonstrating Ca RMSD of less than 1.5 Å to the design models. In contrast to library screening approaches for which determining binding mode can be a major bottleneck, the binding modes of RFpeptides-generated macrocycles are known by design, which should greatly facilitate downstream optimization. RFpeptides thus provides a powerful framework for rapid and custom design of macrocyclic peptides for diagnostic and therapeutic applications.

PMID:39605685 | PMC:PMC11601608 | DOI:10.1101/2024.11.18.622547

Nat Commun. 2024 Nov 22;15(1):10130. doi: 10.1038/s41467-024-54481-x.

ABSTRACT

D-amino acids, found in excess in a minority of organisms and crucial for marine invertebrates, contrast with the more common L-amino acids in most life forms. The local prebiotic origin of D-amino acid enantiomeric excess in natural systems remains an unsolved conundrum. Herein, we demonstrate the formation of enantiomeric excess (ee) D-amino acids through photocatalytic reductive amination of α-keto acids on natural pyrite. Various amino acids with ee values in the range of 14.5-42.4%, are formed. The wavy arrangement of atoms on the surface of pyrite is speculated to lead to the preferential formation of D-amino acids. This work reveals the intrinsic asymmetric photocatalytic activity of pyrite, which could expand understandings on mechanism of asymmetric catalysis and chirality of inorganic crystals. Furthermore, it provides a plausible pathway for the prebiotic formation of D-amino acids, adding further evidence to the origin of D-amino acids enantiomeric excess in natural systems.

PMID:39578467 | PMC:PMC11584652 | DOI:10.1038/s41467-024-54481-x

J Biol Chem. 2024 Nov 13;300(12):107991. doi: 10.1016/j.jbc.2024.107991. Online ahead of print.

ABSTRACT

Understanding how natural and engineered peptides enter cells would facilitate the elucidation of biochemical mechanisms underlying cell biology and is pivotal for developing effective intracellular targeting strategies. In this study, we demonstrate that our peptide stapling technique, fluorine-thiol displacement reaction (FTDR), can produce flexibly constrained peptides with significantly improved cellular uptake, particularly into the nucleus. This platform confers enhanced flexibility, which is further amplified by the inclusion of a D-amino acid, while maintaining environment-dependent α helicity, resulting in highly permeable peptides without the need for additional cell-penetrating motifs. Targeting the estrogen receptor α (ERα)-coactivator interaction prevalent in estrogen receptor-positive (ER+) breast cancers, we showcased that FTDR-stapled peptides, notably SRC2-LD, achieved superior internalization, including cytoplasmic and enriched nuclear uptake, compared to peptides stapled by ring-closing metathesis. These FTDR-stapled peptides use different mechanisms of cellular uptake, including energy-dependent transport such as actin-mediated endocytosis and macropinocytosis. As a result, FTDR peptides exhibit enhanced antiproliferative effects despite their slightly decreased target affinity. Our findings challenge existing perceptions of cell permeability, emphasizing the possibly incomplete understanding of the structural determinants vital for cellular uptake of peptide-like macromolecules. Notably, while α helicity and lipophilicity are positive indicators, they alone are insufficient to determine high-cell permeability, as evidenced by our less helical, more flexible, and less lipophilic FTDR-stapled peptides.

PMID:39547512 | DOI:10.1016/j.jbc.2024.107991

Mass Spectrom Rev. 2024 Nov 18. doi: 10.1002/mas.21916. Online ahead of print.

ABSTRACT

One of the great triumphs of mass spectrometry-based peptide and protein characterization is the characterization of their modifications as most modifications have a characteristic mass shift. What happens when the modification does not change the mass of the peptide? Here, the characterization of several peptide and proteins modifications that do not involve a mass shift are highlighted. Protein and peptide synthesis on ribosomes involves L-amino acids; however, posttranslational modifications (PTMs) can convert these L-amino acids into their D-isomers. As another example, nonenzymatic PTM of aspartate leads to the formation of three different isomers, with isoaspartate being the most prevalent. Both modifications do not alter the mass of the peptide and yet can have profound impact on the physicochemical characteristics of the peptide. Several MS and ion mobility techniques are highlighted, as are other methods such as chromatography, enzymatic enrichment, and labeling. The challenges inherent to these analytical methods and prospective developments in bioinformatics and computational strategies are discussed for these zero-dalton PTMs.

PMID:39558451 | DOI:10.1002/mas.21916

Chembiochem. 2024 Nov 13:e202400922. doi: 10.1002/cbic.202400922. Online ahead of print.

ABSTRACT

Dietary fiber (DF)-based interventions are crucial in establishing a health-promoting gut microbiota. However, directly investigating DFs' in vivo interactions with intestinal bacteria remains challenging due to the lack of suitable tools. Here, we develop an in vivo metabolic labeling-based strategy, which enables not only imaging and identifying the bacteria that bind with specific DF in the intestines, but also quantifying DF's impact on their metabolic status. Four DFs, including galactan, rhamnogalacturonan and two inulins, are fluorescently derivatized and used for in vivo labeling to visually record DFs' interactions with gut bacteria. The subsequent cell-sorting, 16S rDNA sequencing, and fluorescence in situ hybridization identify the taxa that bind each DF. We then select a DF-binding species newly identified herein and verify its DF-catabolizing capability in vitro. Furthermore, we find that the indigenous metabolic status of Gram-positive bacteria, whether inulin-binders or not, is significantly enhanced by the inulin supplement. This trend is not observed in Gram-negative microbiota, even for the inulin-binders, demonstrating the ability of our methods in differentiating the primary, secondary DF-degraders from cross-feeders, a question that is difficult to answer by using other methods. Our strategy provides a novel chemical biology tool for deciphering the complex DF-bacteria interactions in the gut.

PMID:39538366 | DOI:10.1002/cbic.202400922

Nat Commun. 2024 Nov 11;15(1):9746. doi: 10.1038/s41467-024-53554-1.

ABSTRACT

Peptidylarginine deiminase IV (PADI4, PAD4) deregulation promotes the development of autoimmunity, cancer, atherosclerosis and age-related tissue fibrosis. PADI4 additionally mediates immune responses and cellular reprogramming, although the full extent of its physiological roles is unexplored. Despite detailed molecular knowledge of PADI4 activation in vitro, we lack understanding of its regulation within cells, largely due to a lack of appropriate systems and tools. Here, we develop and apply a set of potent and selective PADI4 modulators. Using the mRNA-display-based RaPID system, we screen >10¹² cyclic peptides for high-affinity, conformation-selective binders. We report PADI4_3, a cell-active inhibitor specific for the active conformation of PADI4; PADI4_7, an inert binder, which we functionalise for the isolation and study of cellular PADI4; and PADI4_11, a cell-active PADI4 activator. Structural studies with PADI4_11 reveal an allosteric binding mode that may reflect the mechanism that promotes cellular PADI4 activation. This work contributes to our understanding of PADI4 regulation and provides a toolkit for the study and modulation of PADI4 across (patho)physiological contexts.

PMID:39528459 | PMC:PMC11555231 | DOI:10.1038/s41467-024-53554-1

ACS Appl Mater Interfaces. 2024 Nov 20;16(46):63195-63206. doi: 10.1021/acsami.4c12609. Epub 2024 Nov 6.

ABSTRACT

Surface modification of materials with proteins has various biological applications, and hence the methodology for surface modification needs to accommodate a wide range of proteins that differ in structure, size, and function. Presented here is a methodology that uses the Affinity Bioorthogonal Chemistry (ABC) tag, 3-(2-pyridyl)-6-methyltetrazine (PyTz), for the site-selective modification and purification of proteins and subsequent attachment of the protein to trans-cyclooctene (TCO)-functionalized hydrogel microfibers. This method of surface modification is shown to maintain the functionality of the protein after conjugation with proteins of varying size and functionalities, namely, HaloTag, NanoLuc luciferase (NanoLuc), and fibronectin type III domains 9-10 (FNIII 9-10). The method also supports surface modification with multiple proteins, which is shown by the simultaneous conjugation of HaloTag and NanoLuc on the microfiber surface. The ability to control the relative concentrations of multiple proteins presented on the surface is shown with the use of HaloTag and superfolder GFP (sfGFP). This application of the ABC-tagging methodology expands on existing surface modification methods and provides flexibility in the site-selective protein conjugation methods used along with the rapid kinetics of tetrazine ligation.

PMID:39503333 | PMC:PMC11824234 | DOI:10.1021/acsami.4c12609