Cross-linking mass spectrometry discovers, evaluates, and corroborates structures and protein-protein interactions in the human cell.

Significant recent advances in structural biology, particularly in the field of cryoelectron microscopy, have dramatically expanded our ability to create structural models of proteins and protein complexes. However, many proteins remain refractory to these approaches because of their low abundance, low stability, or-in the case of complexes-simply not having yet been analyzed. Here, we demonstrate the power of using cross-linking mass spectrometry (XL-MS) for the high-throughput experimental assessment of the structures of proteins and protein complexes. This included those produced by high-resolution but in vitro experimental data, as well as in silico predictions based on amino acid sequence alone. We present the largest XL-MS dataset to date, describing 28,910 unique residue pairs captured across 4,084 unique human proteins and 2,110 unique protein-protein interactions. We show that models of proteins and their complexes predicted by AlphaFold2, and inspired and corroborated by the XL-MS data, offer opportunities to deeply mine the structural proteome and interactome and reveal mechanisms underlying protein structure and function.

A broad-spectrum macrocyclic peptide inhibitor of the SARS-CoV-2 spike protein.

The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used messenger RNA (mRNA) display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor-binding domain, N-terminal domain, and S2 region, distal to the angiotensin-converting enzyme 2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target.

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 O2-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. ADO, in contrast, is ubiquitously expressed, suggesting it can regulate the stability of additional Nt-cys proteins in an O2-dependent manner. However, the role of individual chemical groups, active site metal, amino acid composition, and globular structure on protein substrate association remains elusive. To help identify new targets and examine the underlying biochemistry of the system, we conducted a series of biophysical experiments to investigate the binding requirements of established ADO substrates RGS5 and interleukin-32. We demonstrate, using surface plasmon response and enzyme assays, that a free, unmodified Nt-thiol and Nt-amine are vital for substrate engagement through active site metal coordination, with residues next to Nt-cys moderately impacting association and catalytic efficiency. Additionally, we show, through 1H-15N heteronuclear single quantum coherence nuclear magnetic resonance titrations, that the globular portion of RGS5 has limited impact on ADO association, with interactions restricted to the N-terminus. This work establishes key features involved in ADO substrate binding, which will help identify new protein targets and, subsequently, elucidate its role in hypoxic adaptation.

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. To begin to address this knowledge gap in an unbiased manner, we carried out mRNA display screens against a BD-the N-terminal BD of BRD3-using peptide libraries that contained either one or two acetyllysine residues. We discovered peptides with very strong consensus sequences and with affinities that are significantly higher than typical BD-peptide interactions. X-ray crystal structures also revealed modes of binding that have not been seen with natural ligands. Intriguingly, however, our sequences are not found in the human proteome, perhaps suggesting that strong binders to BDs might have been selected against during evolution.

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 (Mpro) 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 Mpro to enhance its cell permeability and in vitro antiviral activity. By identifying mutation-tolerant amino acid residues within the peptide sequence, we describe the development of a second-generation Mpro inhibitor bearing five cyclohexylalanine residues. This cyclic peptide analogue exhibited significantly improved cell permeability and antiviral activity compared to the parent peptide. This approach highlights the importance of optimizing cyclic peptide hits for activity against intracellular targets such as the SARS-CoV-2 Mpro.

Changes in Potency and Subtype Selectivity of Bivalent NaV Toxins are Knot-Specific.

Disulfide-rich peptide toxins have long been studied for their ability to inhibit voltage-gated sodium channel subtype NaV1.7, a validated target for the treatment of pain. In this study, we sought to combine the pore blocking activity of conotoxins with the gating modifier activity of spider toxins to design new bivalent inhibitors of NaV1.7 with improved potency and selectivity. To do this, we created an array of heterodimeric toxins designed to target human NaV1.7 by ligating a conotoxin to a spider toxin and assessed the potency and selectivity of the resulting bivalent toxins. A series of spider-derived gating modifier toxins (GpTx-1, ProTx-II, gHwTx-IV, JzTx-V, CcoTx-1, and Pn3a) and two pore-blocker µ-conotoxins, SxIIIC and KIIIA, were used for this study. We employed either enzymatic ligation with sortase A for C- to N-terminal ligation or click chemistry for N- to N-terminal ligation. The bivalent peptide resulting from ligation of ProTx-II and SxIIIC (Pro[LPATG6]Sx) was shown to be the best combination as native ProTx-II potency at hNaV1.7 was conserved following ligation. At hNaV1.4, a synergistic effect between the pore blocker and gating modifier toxin moieties was observed, resulting in altered sodium channel subtype selectivity compared to the parent peptides. Further studies including mutant bivalent peptides and mutant hNaV1.7 channels suggested that gating modifier toxins have a greater contribution to the potency of the bivalent peptides than pore blockers. This study delineated potential benefits and drawbacks of designing pharmacological hybrid peptides targeting hNaV1.7.

Cyclic peptides can engage a single binding pocket through highly divergent modes.

Cyclic peptide library screening technologies show immense promise for identifying drug leads and chemical probes for challenging targets. However, the structural and functional diversity encoded within such libraries is largely undefined. We have systematically profiled the affinity, selectivity, and structural features of library-derived cyclic peptides selected to recognize three closely related targets: the acetyllysine-binding bromodomain proteins BRD2, -3, and -4. We report affinities as low as 100 pM and specificities of up to 106-fold. Crystal structures of 13 peptide-bromodomain complexes reveal remarkable diversity in both structure and binding mode, including both α-helical and ß-sheet structures as well as bivalent binding modes. The peptides can also exhibit a high degree of structural preorganization. Our data demonstrate the enormous potential within these libraries to provide diverse binding modes against a single target, which underpins their capacity to yield highly potent and selective ligands.

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.

Evaluating Use Cases for Human Challenge Trials in Accelerating SARS-CoV-2 Vaccine Development.

Human challenge trials (HCTs) have been proposed as a means to accelerate SARS-CoV-2 vaccine development. We identify and discuss 3 potential use cases of HCTs in the current pandemic: evaluating efficacy, converging on correlates of protection, and improving understanding of pathogenesis and the human immune response. We outline the limitations of HCTs and find that HCTs are likely to be most useful for vaccine candidates currently in preclinical stages of development. We conclude that, while currently limited in their application, there are scenarios in which HCTs would be extremely beneficial. Therefore, the option of conducting HCTs to accelerate SARS-CoV-2 vaccine development should be preserved. As HCTs require many months of preparation, we recommend an immediate effort to (1) establish guidelines for HCTs for COVID-19; (2) take the first steps toward HCTs, including preparing challenge virus and making preliminary logistical arrangements; and (3) commit to periodically re-evaluating the utility of HCTs.

Sustainability of an Enhanced Recovery After Surgery initiative for elective colorectal resections in a community hospital.

Background: In March 2016, an Enhanced Recovery After Surgery (ERAS) initiative was implemented for all elective colorectal resections at an urban hospital in St. John's, Newfoundland and Labrador, Canada. An ERAS coordinator supervised and enforced guideline compliance for 6 months. The aim of this study was to evaluate the sustainability of the ERAS program after supervision of guideline compliance was eliminated. Methods: Patient outcomes and guideline compliance were compared between surgeries performed under standard practice (April 2014 to March 2015) and those performed during and after the implementation of the ERAS initiative (March 2016 to August 2016 was the implementation phase and September 2016 to February 2017 was the sustainability phase). Results: Hospital length of stay decreased from 7.26 days at baseline to 5.44 days during the implementation phase of the ERAS program (p < 0.001). There was no significant difference between length of stay at baseline and during the 6-month sustainability phase of the ERAS program (7.10 d). There were no significant differences in rates of readmission or mortality during and after implementation. Rate of ileus decreased significantly from 13.8% during the implementation phase to 4.6% during the sustainability phase (p = 0.036). Total guideline compliance increased from 52.2% at baseline to 80.7% during the implementation phase (p < 0.001), and decreased to 74.7% during the sustainability phase (p < 0.001). Adherence to postoperative guidelines regressed: 79.2% in the implementation phase and 68.6% in the sustainability phase (p < 0.001). Conclusion: La durée des séjours à l'hôpital a diminué après l'adoption du programme de RAAC, lorsque le coordonnateur du programme était présent. Les méthodes de maintien des lignes directrices après leur adoption seront cruciales au succès de programmes similaires à l'avenir.

Contexte: En mars 2016, une initiative de récupération améliorée après la chirurgie (RAAC) a été mise en place pour toutes les résections colorectales électives effectuées dans un hôpital urbain de St. John's, à Terre-Neuve-et-Labrador, au Canada. Un coordonnateur du projet de RAAC a supervisé l'application des directives pendant 6 mois. Cette étude visait à évaluer la viabilité du programme une fois que l'application des directives n'était plus surveillée. Méthodes: Nous avons comparé les issues pour les patients et le respect des directives pour les chirurgies réalisées selon les pratiques habituelles (avril 2014 à mars 2015) et pour celles réalisées pendant et après l'adoption du programme de RAAC (mars 2016 à août 2016 ­ mise en oeuvre ­ et septembre 2016 à février 2017 ­ évaluation de la viabilité). Résultats: La durée du séjour à l'hôpital est passée de 7,26 jours à 5,44 jours pendant la phase de mise en oeuvre du programme (p < 0,001). Il n'y avait pas de différence significative entre la durée du séjour au début du programme et pendant les 6 mois de la phase d'évaluation de la viabilité (7,10 jours). Les taux de réadmission et de mortalité avant et après la mise en place du programme n'ont pas changé de manière significative. Le taux d'iléus a connu une baisse significative, passant de 13,8 % pendant la phase de mise en oeuvre à 4,6 % pendant l'évaluation de la viabilité (p = 0,036). Le respect des directives est passé de 52,2 % au début de la mise en oeuvre à 80,7 % pendant cette même phase (p < 0,001), pour ensuite descendre à 74,7 % pendant la phase suivante (p < 0,001). Le respect de lignes directrices postopératoires a régressé : il était de 79,2 % pendant la phase de mise en oeuvre et de 68,6 % pendant la phase d'évaluation de la viabilité (p < 0,001). Conclusion: La durée des séjours à l'hôpital a augmenté après l'adoption du programme de RAAC, lorsque le coordonnateur du programme était présent. Les méthodes de maintien des lignes directrices après leur adoption seront cruciales au succès de programmes similaires à l'avenir.

Solid-state (13)C NMR and synchrotron SAXS/WAXS studies of uniaxially-oriented polyethylene.

We report solid-state (13)C NMR and synchrotron wide-and small-angle X-ray scattering experiments (WAXS, SAXS) on metallocene linear low density polyethylene films (e.g., Exceed™ 1018 mLLDPE; nominally 1MI, 0.918 density ethylene-hexene metallocene copolymer) as a function of uniaxial draw ratio, λ. Combined, these experiments provide an unambiguous, quantitative molecular view of the orientation of both the crystalline and amorphous phases in the samples as a function of draw. Together with previously reported differential scanning calorimetry (DSC), gas transport measurements, transmission electron microscopy (TEM), optical birefringence, small angle X-ray scattering (SAXS) as well as other characterization techniques, this study of the state of orientation in both phases provides insight concerning the development of unusually high barrier properties of the most oriented samples (λ=10). In this work, static (non-spinning) solid-state NMR measurements indicate that in the drawn Exceed(TM) films both the crystalline and amorphous regions are highly oriented. In particular, chemical shift data show the amorphous phase is comprised increasingly of so-called "taut tie chains" (or tie chains under any state of tautness) in the mLLDPE with increasing draw ratio - the resonance lines associated with the amorphous phase shift to where the crystalline peaks are observed. In the sample with highest total draw (λ=10), virtually all of the chains in the non-crystalline region have responded and aligned in the machine (draw) direction. Both monoclinic and orthorhombic crystalline peaks are observed in high-resolution, solid-state magic-angle spinning (MAS) NMR measurements of the oriented PE films. The orientation is comparable to that obtained for ultra-high molecular weight HDPE fibers described as "ultra-oriented" in the literature. Furthermore, the presence of a monoclinic peak in cold-drawn samples suggests that there is an appreciable internal stress associated with the LLDPE. The results are confirmed and independently quantified by Herman's Orientation Function values derived from the WAXS measurements. The degree of orientation approaches theoretically perfect alignment of chains along the draw direction. We deduce from this observation that a high fraction of the non-crystalline chains are either tie chains that directly connect adjacent lamellae or are interlocking loops from adjacent lamellae. In either case, the chains are load-bearing and are consistent with the idea of "taut tie chains". We note that transmission electron micrographs recorded for the ultra-oriented Exceed showed the lamellae are often appreciably thinner and shorter than they are for cast or blown Exceed 1018. Combined with higher crystallinity, the thinner lamellae statistically favor more tie chains. Finally, the remarkably large decrease in permeability of the λ=10 film is primarily attributed to the high degree of orientation (and loss of entropy) of the amorphous phase.

Providers with Limited Experience Perform Better in Advanced Life Support with Assistance Using an Interactive Device with an Automated External Defibrillator Linked to a Ventilator.

BACKGROUND: Medical teams with limited experience in performing advanced life support (ALS) or with a low frequency of cardiopulmonary resuscitation (CPR) while on duty, often have difficulty complying with CPR guidelines. OBJECTIVE: This study evaluated whether the quality of CPR of trained medical students, who served as an example of teams with limited experience in ALS, could be improved with device assistance. The primary outcome was the hands-off time (i.e., the percentage of the entire CPR time without chest compressions). The secondary outcome was seven time intervals, which should be as short as possible, and the quality of ventilations and chest compressions on the mannequin. METHODS: We compared standard CPR equipment to an interactive device with visual and acoustic instructions for ALS workflow measures to guide briefly trained medical students through the ALS algorithm in a full-scale mannequin simulation study with a randomized crossover study design. The study equipment consisted of an automatic external defibrillator and ventilator that were electronically linked and communicating as a single system. Included were regular medical students in the third to sixth years of medical school of one class who provided written informed consent for voluntary participation and for the analysis of their CPR performance data. No exclusion criteria were applied. For statistical measures of evaluation we used an analysis of variance for crossover trials accounting for treatment effect, sequence effect, and carry-over effect, with adjustment for prior practical experience of the participants. RESULTS: Forty-two medical students participated in 21 CPR sessions, each using the standard and study equipment. Regarding the primary end point, the study equipment reduced the hands-off time from 40.1% (95% confidence interval [CI] 36.9-43.4%) to 35.6% (95% CI 32.4-38.9%, p = 0.031) compared with the standard equipment. Within the prespecified secondary end points, study equipment reduced the time interval until the first rescuer changeover from 273 s (95% CI 244-302 s) to 223 s (95% CI 194-253 s, p = 0.001) and increased the percentage of ventilations with a correct tidal volume of 400-600 mL from 34.3% (95% CI 19.0-49.6%) to 60.9% (95% CI 45.6-76.2%, p = 0.018). CONCLUSIONS: The assist device increased the rescuers' CPR quality. CPR providers with limited experience or a limited frequency of CPR performance (i.e., rural Emergency Medical Services crew) may potentially benefit from this assist device.

Optimising the use of the prostate- specific antigen blood test in asymptomatic men for early prostate cancer detection in primary care: report from a UK clinical consensus.

BACKGROUND: Screening is not recommended for prostate cancer in the UK. Asymptomatic men aged ≥50 years can request a prostate-specific antigen (PSA) test following counselling on potential harms and benefits. There are areas of clinical uncertainty among GPs, resulting in the content and quality of counselling varying. AIM: To produce a consensus that can influence guidelines for UK primary care on the optimal use of the PSA test in asymptomatic men for early prostate cancer detection. DESIGN AND SETTING: Prostate Cancer UK facilitated a RAND/UCLA consensus. METHOD: Statements covering five topics were developed with a subgroup of experts. A panel of 15 experts in prostate cancer scored (round one) statements on a scale of one (strongly disagree) to nine (strongly agree). Panellists met to discuss statements before rescoring (round two). A lived experience panel of seven men scored a subset of statements with outcomes fed into the main panel. RESULTS: Of the initial 94 statements reviewed by the expert panel, a final 48/85 (56%) achieved consensus. In the absence of screening, there was consensus on proactive approaches to initiate discussions about the PSA test with men who were at higher-than-average risk. CONCLUSION: Improvements in the prostate cancer diagnostic pathway may have reduced some of the harms associated with PSA testing; however, several areas of uncertainty remain in relation to screening, including optimal PSA thresholds for referral and intervals for retesting. There is consensus on proactive approaches to testing in higher-than-average risk groups. This should prompt a review of current guidelines.

Discovery and characterization of cyclic peptides selective for the C-terminal bromodomains of BET family proteins.

DNA-encoded cyclic peptide libraries can yield high-potency, high-specificity ligands against target proteins. We used such a library to seek ligands that could distinguish between paralogous bromodomains from the closely related bromodomain and extra-terminal domain family of epigenetic regulators. Several peptides isolated from a screen against the C-terminal bromodomain of BRD2, together with new peptides discovered in previous screens against the corresponding domain from BRD3 and BRD4, bound their targets with nanomolar and sub-nanomolar affinities. X-ray crystal structures of several of these bromodomain-peptide complexes reveal diverse structures and binding modes, which nevertheless display several conserved features. Some peptides demonstrate significant paralog-level specificity, although the physicochemical explanations for this specificity are often not clear. Our data demonstrate the power of cyclic peptides to discriminate between very similar proteins with high potency and hint that differences in conformational dynamics might modulate the affinity of these domains for particular ligands.

Evaluation of Peptide Ligation Strategies for the Synthesis of the Bivalent Acid-Sensing Ion Channel Inhibitor Hi1a.

Hi1a is a naturally occurring bivalent spider-venom peptide that is being investigated as a promising molecule for limiting ischemic damage in strokes, myocardial infarction, and organ transplantation. However, the challenges associated with the synthesis and production of the peptide in large quantities have slowed the progress in this area; hence, access to synthetic Hi1a is an essential milestone for the development of Hi1a as a pharmacological tool and potential therapeutic.

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.

Pore structure controls stability and molecular flux in engineered protein cages.

Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein cages have featured in the construction of nanoreactors and synthetic organelles, relatively little is known about the underlying molecular parameters that govern stability and flux through their pores. In this work, we systematically designed 24 variants of the Thermotoga maritima encapsulin cage, featuring pores of different sizes and charges. Twelve pore variants were successfully assembled and purified, including eight designs with exceptional thermal stability. While negatively charged mutations were better tolerated, we were able to form stable assemblies covering a full range of pore sizes and charges, as observed in seven new cryo-EM structures at 2.5- to 3.6-Å resolution. Molecular dynamics simulations and stopped-flow experiments revealed the importance of considering both pore size and charge, together with flexibility and rate-determining steps, when designing protein cages for controlling molecular flux.

A dataset of non-pharmaceutical interventions on SARS-CoV-2 in Europe.

During the second half of 2020, many European governments responded to the resurging transmission of SARS-CoV-2 with wide-ranging non-pharmaceutical interventions (NPIs). These efforts were often highly targeted at the regional level and included fine-grained NPIs. This paper describes a new dataset designed for the accurate recording of NPIs in Europe's second wave to allow precise modelling of NPI effectiveness. The dataset includes interventions from 114 regions in 7 European countries during the period from the 1st August 2020 to the 9th January 2021. The paper includes NPI definitions tailored to the second wave following an exploratory data collection. Each entry has been extensively validated by semi-independent double entry, comparison with existing datasets, and, when necessary, discussion with local epidemiologists. The dataset has considerable potential for use in disentangling the effectiveness of NPIs and comparing the impact of interventions across different phases of the pandemic.

Site-selective photocatalytic functionalization of peptides and proteins at selenocysteine.

The importance of modified peptides and proteins for applications in drug discovery, and for illuminating biological processes at the molecular level, is fueling a demand for efficient methods that facilitate the precise modification of these biomolecules. Herein, we describe the development of a photocatalytic method for the rapid and efficient dimerization and site-specific functionalization of peptide and protein diselenides. This methodology, dubbed the photocatalytic diselenide contraction, involves irradiation at 450 nm in the presence of an iridium photocatalyst and a phosphine and results in rapid and clean conversion of diselenides to reductively stable selenoethers. A mechanism for this photocatalytic transformation is proposed, which is supported by photoluminescence spectroscopy and density functional theory calculations. The utility of the photocatalytic diselenide contraction transformation is highlighted through the dimerization of selenopeptides, and by the generation of two families of protein conjugates via the site-selective modification of calmodulin containing the 21st amino acid selenocysteine, and the C-terminal modification of a ubiquitin diselenide.