An mRNA-display derived cyclic peptide scaffold reveals the substrate binding interactions of an N-terminal cysteine oxidase.

N-terminal cysteine oxidases (NCOs) act as enzymatic oxygen (O) sensors, coordinating cellular changes to hypoxia in animals and plants. They regulate the O-dependent stability of proteins bearing an N-terminal cysteine residue through the N-degron pathway. Despite their important role in hypoxic adaptation, which renders them potential therapeutic and agrichemical targets, structural information on NCO substrate binding remains elusive.

FUT10 and FUT11 are protein O-fucosyltransferases that modify protein EMI domains.

O-Fucosylation plays crucial roles in various essential biological events. Alongside the well-established O-fucosylation of epidermal growth factor-like repeats by protein O-fucosyltransferase 1 (POFUT1) and thrombospondin type 1 repeats by POFUT2, we recently identified a type of O-fucosylation on the elastin microfibril interface (EMI) domain of Multimerin-1 (MMRN1). Here, using AlphaFold2 screens, co-immunoprecipitation, enzymatic assays combined with mass spectrometric analysis and CRISPR-Cas9 knockouts, we demonstrate that FUT10 and FUT11, originally annotated in UniProt as α1,3-fucosyltransferases, are actually POFUTs responsible for modifying EMI domains; thus, we renamed them as POFUT3 and POFUT4, respectively.

Discovery of Selective Cyclic d-Sulfopeptide Ligands of the Chemokine CCL22 via Mirror-Image mRNA Display with Genetic Reprogramming.

Chemokines are small proteins involved in recruiting leukocytes to sites of inflammation via interactions with specific cell surface receptors. CCL22 is a chemokine known to play a critical role in inflammatory diseases such as atopic dermatitis and asthma; inhibition of this chemokine therefore represents an attractive therapeutic strategy. Herein, we describe the discovery of cyclic d-sulfopeptide inhibitors of CCL22 identified through mirror-image mRNA display with genetic reprogramming.

Intranasal Self-Adjuvanted Lipopeptide Vaccines Elicit High Antibody Titers and Strong Cellular Responses against SARS-CoV-2.

Despite concerted efforts to tackle the COVID-19 pandemic, the persistent transmission of SARS-CoV-2 demands continued research into novel vaccination strategies to combat the virus. In light of this, intranasally administered peptide vaccines, particularly those conjugated to an immune adjuvant to afford so-called "self-adjuvanted vaccines", remain underexplored. Here, we describe the synthesis and immunological evaluation of self-adjuvanting peptide vaccines derived from epitopes of the spike glycoprotein of SARS-CoV-2 covalently fused to the potent adjuvant, PamCys, that targets toll-like receptor 2 (TLR2).

The enzymatic oxygen sensor cysteamine dioxygenase binds its protein substrates through their N-termini.

The non-heme iron-dependent dioxygenase 2-aminoethanethiol (aka cysteamine) dioxygenase (ADO) has recently been identified as an enzymatic oxygen sensor that coordinates cellular changes to hypoxia by regulating the stability of proteins bearing an N-terminal cysteine (Nt-cys) through the N-degron pathway. It catalyzes O-dependent Nt-cys sulfinylation, which promotes proteasomal degradation of the target. Only a few ADO substrates have been verified, including regulators of G-protein signaling (RGS) 4 and 5, and the proinflammatory cytokine interleukin-32, all of which exhibit cell and/or tissue specific expression patterns.

Expressed Protein Ligation in Flow.

The development of a flow chemistry platform for the generation of modified protein targets via expressed protein ligation (EPL) is described. The flow EPL platform enables efficient ligation reactions with high recoveries of target protein products and superior reaction rates compared to corresponding batch processes. The utility of the flow EPL technology was first demonstrated through the semisynthesis of the tick-derived chemokine-binding protein ACA-01 containing two tyrosine sulfate modifications.

Exploiting Hydrophobic Amino Acid Scanning to Develop Cyclic Peptide Inhibitors of the SARS-CoV-2 Main Protease with Antiviral Activity.

The development of novel antivirals is crucial not only for managing current COVID-19 infections but for addressing potential future zoonotic outbreaks. SARS-CoV-2 main protease (M) is vital for viral replication and viability and therefore serves as an attractive target for antiviral intervention. Herein, we report the optimization of a cyclic peptide inhibitor that emerged from an mRNA display selection against the SARS-CoV-2 M to enhance its cell permeability and in vitro antiviral activity.

Discovery of High Affinity Cyclic Peptide Ligands for Human ACE2 with SARS-CoV-2 Entry Inhibitory Activity.

The development of effective antiviral compounds is essential for mitigating the effects of the COVID-19 pandemic. Entry of SARS-CoV-2 virions into host cells is mediated by the interaction between the viral spike (S) protein and membrane-bound angiotensin-converting enzyme 2 (ACE2) on the surface of epithelial cells. Inhibition of this viral protein-host protein interaction is an attractive avenue for the development of antiviral molecules with numerous spike-binding molecules generated to date.

mRNA display reveals a class of high-affinity bromodomain-binding motifs that are not found in the human proteome.

Bromodomains (BDs) regulate gene expression by recognizing protein motifs containing acetyllysine. Although originally characterized as histone-binding proteins, it has since become clear that these domains interact with other acetylated proteins, perhaps most prominently transcription factors. The likely transient nature and low stoichiometry of such modifications, however, has made it challenging to fully define the interactome of any given BD.

Electrochemical Modification of Polypeptides at Selenocysteine.

Mild strategies for the selective modification of peptides and proteins are in demand for applications in therapeutic peptide and protein discovery, and in the study of fundamental biomolecular processes. Herein, we describe the development of an electrochemical selenoetherification (e-SE) platform for the efficient site-selective functionalization of polypeptides. This methodology utilizes the unique reactivity of the 21st amino acid, selenocysteine, to effect formation of valuable bioconjugates through stable selenoether linkages under mild electrochemical conditions.

Exploiting Chemical Protein Synthesis to Study the Role of Tyrosine Sulfation on Anticoagulants from Hematophagous Organisms.

Tyrosine sulfation is a post-translational modification (PTM) that modulates function by mediating key protein-protein interactions. One of the early proteins shown to possess this PTM was hirudin, produced in the salivary glands of the medicinal leech , whereby tyrosine sulfation led to a ∼10-fold improvement in α-thrombin inhibitory activity. Outside of this pioneering discovery, the involvement of tyrosine sulfation in modulating the activity of salivary proteins from other hematophagous organisms was unknown.

Synthesis and applications of mirror-image proteins.

The homochirality of biomolecules in nature, such as DNA, RNA, peptides and proteins, has played a critical role in establishing and sustaining life on Earth. This chiral bias has also given synthetic chemists the opportunity to generate molecules with inverted chirality, unlocking valuable new properties and applications. Advances in the field of chemical protein synthesis have underpinned the generation of numerous 'mirror-image' proteins (those comprised entirely of D-amino acids instead of canonical L-amino acids), which cannot be accessed using recombinant expression technologies.

Mucosal TLR2-activating protein-based vaccination induces potent pulmonary immunity and protection against SARS-CoV-2 in mice.

Current vaccines against SARS-CoV-2 substantially reduce mortality, but protection against infection is less effective. Enhancing immunity in the respiratory tract, via mucosal vaccination, may provide protection against infection and minimise viral spread. Here, we report testing of a subunit vaccine in mice, consisting of SARS-CoV-2 Spike protein with a TLR2-stimulating adjuvant (PamCys), delivered to mice parenterally or mucosally.

Synthetic vaccines targeting Mincle through conjugation of trehalose dibehenate.

The covalent fusion of immunostimulatory adjuvants to immunogenic antigens is a promising strategy for the development of effective synthetic vaccines for infectious diseases. Herein, we describe the conjugation of a mycobacterial peptide antigen from the 6 kDa early secretory antigenic target (ESAT6) to a suitably functionalised trehalose dibehenate (TDB), a potent glycolipid adjuvant targeting macrophage inducible C-type lectin (Mincle).

Expressed Protein Selenoester Ligation.

Herein, we describe the development and application of a novel expressed protein selenoester ligation (EPSL) methodology for the one-pot semi-synthesis of modified proteins. EPSL harnesses the rapid kinetics of ligation reactions between modified synthetic selenopeptides and protein aryl selenoesters (generated from expressed intein fusion precursors) followed by in situ chemoselective deselenization to afford target proteins at concentrations that preclude the use of traditional ligation methods. The utility of the EPSL technology is showcased through the efficient semi-synthesis of ubiquitinated polypeptides, lipidated analogues of the membrane-associated GTPase YPT6, and site-specifically phosphorylated variants of the oligomeric chaperone protein Hsp27 at high dilution.

Tyrosine-O-sulfation is a widespread affinity enhancer among thrombin interactors.

Tyrosine-O-sulfation is a common post-translational modification (PTM) of proteins following the cellular secretory pathway. First described in human fibrinogen, tyrosine-O-sulfation has long been associated with the modulation of protein-protein interactions in several physiological processes. A number of relevant interactions for hemostasis are largely dictated by this PTM, many of which involving the serine proteinase thrombin (FIIa), a central player in the blood-clotting cascade.

Synthetic protein conjugate vaccines provide protection against in mice.

The global incidence of tuberculosis remains unacceptably high, with new preventative strategies needed to reduce the burden of disease. We describe here a method for the generation of synthetic self-adjuvanted protein vaccines and demonstrate application in vaccination against Two vaccine constructs were designed, consisting of full-length ESAT6 protein fused to the TLR2-targeting adjuvants PamCys-SK or PamCys-SK These were produced by chemical synthesis using a peptide ligation strategy. The synthetic self-adjuvanting vaccines generated powerful local CD4 T cell responses against ESAT6 and provided significant protection in the lungs from virulent aerosol challenge when administered to the pulmonary mucosa of mice.

Revealing the functional roles of tyrosine sulfation using synthetic sulfopeptides and sulfoproteins.

The decoration of proteins with post-translational modifications (PTMs) serves as a mechanism to expand the functional repertoire of the proteome. Tyrosine sulfation is a PTM that has been shown to be a key regulator of extracellular protein-protein interactions in a select number of examples. However, the challenges associated with identifying and characterising the functional consequences of tyrosine sulfation have hindered our ability to understand the full scope of its role in the wider proteome when compared with that of other PTMs, for example, phosphorylation and glycosylation.