Global signal peptide profiling reveals principles of selective Sec61 inhibition.

Cotransins target the Sec61 translocon and inhibit the biogenesis of an undefined subset of secretory and membrane proteins. Remarkably, cotransin inhibition depends on the unique signal peptide (SP) of each Sec61 client, which is required for cotranslational translocation into the endoplasmic reticulum. It remains unknown how an SP's amino acid sequence and biophysical properties confer sensitivity to structurally distinct cotransins. Here we describe a fluorescence-based, pooled-cell screening platform to interrogate nearly all human SPs in parallel. We profiled two cotransins with distinct effects on cancer cells and discovered a small subset of SPs, including the oncoprotein human epidermal growth factor receptor 3 (HER3), with increased sensitivity to the more selective cotransin, KZR-9873. By comparing divergent mouse and human orthologs, we unveiled a position-dependent effect of arginine on SP sensitivity. Our multiplexed profiling platform reveals how cotransins can exploit subtle sequence differences to achieve SP discrimination.

Functional knee brace use for 21 h leads to a longer duration to achieve peak vertical ground reaction forces and the removal of the brace after 17.5 h results in faster loading of the knee joint.

PURPOSE: To investigate the landing strategies used after discontinuing and continuing the use of a functional knee brace (FKB) while performing a drop jump. METHODS: Following published methodology and power analysis, 23 uninjured male athletes, mean age of 19.4 ± 3.0 years, performed seven tests, during three test conditions (nonbraced, braced and removed brace or continued brace use), over 6 days of 12 testing sessions (S) for a total of 38.5 h. Each subject was provided with a custom-fitted FKB. This study focuses on the single leg drop jump kinetics during S12 when subjects were randomly selected to remove the FKB after 17.5 h or continued use of FKB. The time to peak vertical ground reaction forces (PVGRF) and PVGRF were recorded on landing in eight trials. RESULTS: After brace removal, a significantly shorter mean time to PVGRF was recorded (9.4 ± 22.9 msec (3.9%), p = 0.005, 95% confidence interval (95% CI): -168.1, 36.1), while continued brace use required a nonsignificant (n.s.) longer mean duration to achieve PVGRF (19.4 ± 53.6 msec (8.9%), n.s., 95% CI: -49.7, 73.4). No significant mean PVGRF difference was found in brace removal (25.3 ± 65.8 N) and continued brace use (25.1 ± 23.0 N). CONCLUSION: Removal of FKB after 17.5 h of use led to a significantly shorter time to achieve PVGRF, while continued brace use for 21 h required a longer duration to achieve PVGRF, suggesting faster and slower knee joint loading, respectively. Understanding the concerns associated with the use of FKB and the kinetics of the knee joint will assist clinicians in counselling athletes about the risks and benefits of using an FKB. LEVEL OF EVIDENCE: Level II.

The eRF1 degrader SRI-41315 acts as a molecular glue at the ribosomal decoding center.

Translation termination is an essential cellular process, which is also of therapeutic interest for diseases that manifest from premature stop codons. In eukaryotes, translation termination requires eRF1, which recognizes stop codons, catalyzes the release of nascent proteins from ribosomes and facilitates ribosome recycling. The small molecule SRI-41315 triggers eRF1 degradation and enhances translational readthrough of premature stop codons. However, the mechanism of action of SRI-41315 on eRF1 and translation is not known. Here we report cryo-EM structures showing that SRI-41315 acts as a metal-dependent molecular glue between the N domain of eRF1 responsible for stop codon recognition and the ribosomal subunit interface near the decoding center. Retention of eRF1 on ribosomes by SRI-41315 leads to ribosome collisions, eRF1 ubiquitylation and a higher frequency of translation termination at near-cognate stop codons. Our findings reveal a new mechanism of release factor inhibition and additional implications for pharmacologically targeting eRF1.

Predicting the Risk of Injuries Through Assessments of Asymmetric Lower Limb Functional Performance: A Prospective Study of 415 Youth Taekwondo Athletes.

Background: The impact of interlimb asymmetries on sport injuries is unclear because of inconsistent findings, and there is a lack of research on youth athletes and the sport of taekwondo. Purpose: To examine the effects of functional interlimb asymmetries on noncontact lower limb injuries in youth athletes. Study Design: Cohort study; Level of evidence, 2. Methods: A total of 415 taekwondo athletes (318 boys and 97 girls) aged 6 to 17 years underwent baseline testing to determine interlimb asymmetries through the single-leg countermovement jump (CMJ), hop, and triple hop tests as well as the Star Excursion Balance Test. The athletes were then evaluated for 12 months to observe the occurrence of noncontact lower limb injuries. Results: During the study, 98 athletes (70 boys and 28 girls) sustained at least 1 noncontact lower limb injury. Athletes with higher interlimb asymmetries in single-leg CMJ height showed a significantly increased risk of noncontact lower limb injuries (boys: odds ratio [OR], 1.053 [95% CI, 1.027-1.080], P < .001; girls: OR, 1.070 [95% CI, 1.016-1.128], P = .011). Asymmetry in single-leg CMJ height of ≥15.28% was found to be the cutoff point for predicting noncontact lower limb injuries in boys (OR, 4.652 [95% CI, 2.577-8.398]; P < .001). Conclusion: This study highlights the utility of interlimb asymmetries in unilateral jump performance as a tool for assessing the risk of noncontact lower limb injuries in youth taekwondo athletes of both sexes. A proper evaluation of interlimb asymmetries may improve prevention strategies for youth athletes.

Direct mapping of ligandable tyrosines and lysines in cells with chiral sulfonyl fluoride probes.

Advances in chemoproteomic technology have revealed covalent interactions between small molecules and protein nucleophiles, primarily cysteine, on a proteome-wide scale. Most chemoproteomic screening approaches are indirect, relying on competition between electrophilic fragments and a minimalist electrophilic probe with inherently limited proteome coverage. Here we develop a chemoproteomic platform for direct electrophile-site identification based on enantiomeric pairs of clickable arylsulfonyl fluoride probes. Using stereoselective site modification as a proxy for ligandability in intact cells, we identify 634 tyrosines and lysines within functionally diverse protein sites, liganded by structurally diverse probes. Among multiple validated sites, we discover a chiral probe that modifies Y228 in the MYC binding site of the epigenetic regulator WDR5, as revealed by a high-resolution crystal structure. A distinct chiral probe stimulates tumour cell phagocytosis by covalently modifying Y387 in the recently discovered immuno-oncology target APMAP. Our work provides a deep resource of ligandable tyrosines and lysines for the development of covalent chemical probes.

Large library docking for novel SARS-CoV-2 main protease non-covalent and covalent inhibitors.

Antiviral therapeutics to treat SARS-CoV-2 are needed to diminish the morbidity of the ongoing COVID-19 pandemic. A well-precedented drug target is the main viral protease (MPro ), which is targeted by an approved drug and by several investigational drugs. Emerging viral resistance has made new inhibitor chemotypes more pressing. Adopting a structure-based approach, we docked 1.2 billion non-covalent lead-like molecules and a new library of 6.5 million electrophiles against the enzyme structure. From these, 29 non-covalent and 11 covalent inhibitors were identified in 37 series, the most potent having an IC50 of 29 and 20 µM, respectively. Several series were optimized, resulting in low micromolar inhibitors. Subsequent crystallography confirmed the docking predicted binding modes and may template further optimization. While the new chemotypes may aid further optimization of MPro inhibitors for SARS-CoV-2, the modest success rate also reveals weaknesses in our approach for challenging targets like MPro versus other targets where it has been more successful, and versus other structure-based techniques against MPro itself.

Signal peptide mimicry primes Sec61 for client-selective inhibition.

Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been hampered by excessive toxicity. Here we describe KZR-8445, a cyclic depsipeptide that targets the Sec61 translocon and selectively disrupts secretory and membrane protein biogenesis in a signal peptide-dependent manner. KZR-8445 potently inhibits the secretion of pro-inflammatory cytokines in primary immune cells and is highly efficacious in a mouse model of rheumatoid arthritis. A cryogenic electron microscopy structure reveals that KZR-8445 occupies the fully opened Se61 lateral gate and blocks access to the lumenal plug domain. KZR-8445 binding stabilizes the lateral gate helices in a manner that traps select signal peptides in the Sec61 channel and prevents their movement into the lipid bilayer. Our results establish a framework for the structure-guided discovery of novel therapeutics that selectively modulate Sec61-mediated protein biogenesis.

mRNA decoding in human is kinetically and structurally distinct from bacteria.

In all species, ribosomes synthesize proteins by faithfully decoding messenger RNA (mRNA) nucleotide sequences using aminoacyl-tRNA substrates. Current knowledge of the decoding mechanism derives principally from studies on bacterial systems1. Although key features are conserved across evolution2, eukaryotes achieve higher-fidelity mRNA decoding than bacteria3. In human, changes in decoding fidelity are linked to ageing and disease and represent a potential point of therapeutic intervention in both viral and cancer treatment4-6. Here we combine single-molecule imaging and cryogenic electron microscopy methods to examine the molecular basis of human ribosome fidelity to reveal that the decoding mechanism is both kinetically and structurally distinct from that of bacteria. Although decoding is globally analogous in both species, the reaction coordinate of aminoacyl-tRNA movement is altered on the human ribosome and the process is an order of magnitude slower. These distinctions arise from eukaryote-specific structural elements in the human ribosome and in the elongation factor eukaryotic elongation factor 1A (eEF1A) that together coordinate faithful tRNA incorporation at each mRNA codon. The distinct nature and timing of conformational changes within the ribosome and eEF1A rationalize how increased decoding fidelity is achieved and potentially regulated in eukaryotic species.

Masters athlete screening study (MASS): incidence of cardiovascular disease and major adverse cardiac events and efficacy of screening over five years.

BACKGROUND: The efficacy of cardiovascular screening in Masters athletes (MAs) (≥35 y), and whether screening decreases their risk of major adverse cardiac events (MACEs) is unknown. PURPOSE: To evaluate the effectiveness of yearly cardiovascular screening, and the incidence of cardiovascular disease (CVD) and MACE over five years. METHODS AND RESULTS: MAs (≥35 y) without previous history of CVD underwent yearly cardiovascular screening. Participants with an abnormal screen underwent further evaluations. In the initial year, 798 MAs (62.7% male, 55 ± 10 y) were screened; 11.4% (n = 91) were diagnosed with CVD. Coronary artery disease (CAD) was the most common diagnosis (n = 64; 53%). During follow-up, there were an additional 89 CVD diagnoses with an incidence rate of 3.58/100, 4.14/100, 3.74/100, 1.19/100, for years one to four, respectively. The most common diagnoses during follow-up were arrhythmias (n = 33; 37%). Increasing age (OR = 1.047, 95% confidence interval (CI): 1.003-1.094; P = 0.0379), Framingham Risk Score (FRS) (OR = 1.092, 95% CI: 1.031-1.158; P = 0.003), and LDL cholesterol (OR = 1.709, 95% CI: 1.223-2.401; P = 0.002) were predictive of CAD, whereas moderate intensity activity (min/wk) (OR = 0.997, 95% CI: 0.996-0.999; P = 0.002) was protective. Ten MACE (2.8/1000 athlete-years) occurred. All of these MAs were male, and 90% had ≥10% FRS. All underwent further evaluations with only two identified to have obstructive CAD. CONCLUSION: MACE occurred despite yearly screening. All MAs who had an event had an abnormal screen; however, cardiac functional tests failed to detect underlying CAD in most cases. It may be appropriate to offer computed coronary tomography angiography in MAs with ≥10% FRS to overcome the limitations of functional testing, and to assist with lifestyle and treatment modifications.

The efficacy of heart screening in Masters athletes (MAs) (≥35 y) is not well understood. This study of 798 MAs reported 10 major adverse cardiac event (MACE) over 5 years (2.8/1000 athlete-years), despite undergoing yearly screening. The MAs who had a MACE occurred only in males whom had an abnormal screen with 90% having an intermediate or higher cardiovascular risk. All of these MAs underwent further testing, however, stress tests (i.e. echocardiogram, electrocardiogram, nuclear) failed to detect underlying heart disease in most cases. Therefore, it may be appropriate to offer computed coronary tomography angiography in MAs with intermediate or higher cardiovascular risk to overcome the limitations of functional testing in this population, and to assist with lifestyle and treatment modifications.

An E3 ligase network engages GCN1 to promote the degradation of translation factors on stalled ribosomes.

Ribosomes frequently stall during mRNA translation, resulting in the context-dependent activation of quality control pathways to maintain proteostasis. However, surveillance mechanisms that specifically respond to stalled ribosomes with an occluded A site have not been identified. We discovered that the elongation factor-1α (eEF1A) inhibitor, ternatin-4, triggers the ubiquitination and degradation of eEF1A on stalled ribosomes. Using a chemical genetic approach, we unveiled a signaling network comprising two E3 ligases, RNF14 and RNF25, which are required for eEF1A degradation. Quantitative proteomics revealed the RNF14 and RNF25-dependent ubiquitination of eEF1A and a discrete set of ribosomal proteins. The ribosome collision sensor GCN1 plays an essential role by engaging RNF14, which directly ubiquitinates eEF1A. The site-specific, RNF25-dependent ubiquitination of the ribosomal protein RPS27A/eS31 provides a second essential signaling input. Our findings illuminate a ubiquitin signaling network that monitors the ribosomal A site and promotes the degradation of stalled translation factors, including eEF1A and the termination factor eRF1.

IFITM proteins assist cellular uptake of diverse linked chemotypes.

The search for cell-permeable drugs has conventionally focused on low-molecular weight (MW), nonpolar, rigid chemical structures. However, emerging therapeutic strategies break traditional drug design rules by employing flexibly linked chemical entities composed of more than one ligand. Using complementary genome-scale chemical-genetic approaches we identified an endogenous chemical uptake pathway involving interferon-induced transmembrane proteins (IFITMs) that modulates the cell permeability of a prototypical biopic inhibitor of MTOR (RapaLink-1, MW: 1784 g/mol). We devised additional linked inhibitors targeting BCR-ABL1 (DasatiLink-1, MW: 1518 g/mol) and EIF4A1 (BisRoc-1, MW: 1466 g/mol), uptake of which was facilitated by IFITMs. We also found that IFITMs moderately assisted some proteolysis-targeting chimeras and examined the physicochemical requirements for involvement of this uptake pathway.

Didemnin B and ternatin-4 differentially inhibit conformational changes in eEF1A required for aminoacyl-tRNA accommodation into mammalian ribosomes.

Rapid and accurate mRNA translation requires efficient codon-dependent delivery of the correct aminoacyl-tRNA (aa-tRNA) to the ribosomal A site. In mammals, this fidelity-determining reaction is facilitated by the GTPase elongation factor-1 alpha (eEF1A), which escorts aa-tRNA as an eEF1A(GTP)-aa-tRNA ternary complex into the ribosome. The structurally unrelated cyclic peptides didemnin B and ternatin-4 bind to the eEF1A(GTP)-aa-tRNA ternary complex and inhibit translation but have different effects on protein synthesis in vitro and in vivo. Here, we employ single-molecule fluorescence imaging and cryogenic electron microscopy to determine how these natural products inhibit translational elongation on mammalian ribosomes. By binding to a common site on eEF1A, didemnin B and ternatin-4 trap eEF1A in an intermediate state of aa-tRNA selection, preventing eEF1A release and aa-tRNA accommodation on the ribosome. We also show that didemnin B and ternatin-4 exhibit distinct effects on the dynamics of aa-tRNA selection that inform on observed disparities in their inhibition efficacies and physiological impacts. These integrated findings underscore the value of dynamics measurements in assessing the mechanism of small-molecule inhibition and highlight potential of single-molecule methods to reveal how distinct natural products differentially impact the human translation mechanism.

Synthesis and single-molecule imaging reveal stereospecific enhancement of binding kinetics by the antitumour eEF1A antagonist SR-A3.

Ternatin-family cyclic peptides inhibit protein synthesis by targeting the eukaryotic elongation factor-1α. A potentially related cytotoxic natural product ('A3') was isolated from Aspergillus, but only 4 of its 11 stereocentres could be assigned. Here, we synthesized SR-A3 and SS-A3-two out of 128 possible A3 epimers-and discovered that synthetic SR-A3 is indistinguishable from naturally derived A3. Relative to SS-A3, SR-A3 exhibits an enhanced residence time and rebinding kinetics, as revealed by single-molecule fluorescence imaging of elongation reactions catalysed by eukaryotic elongation factor-1α in vitro. An increased residence time-stereospecifically conferred by the unique ß-hydroxyl in SR-A3-was also observed in cells. Consistent with its prolonged duration of action, thrice-weekly dosing with SR-A3 led to a reduced tumour burden and increased survival in an aggressive Myc-driven mouse lymphoma model. Our results demonstrate the potential of SR-A3 as a cancer therapeutic and exemplify an evolutionary mechanism for enhancing cyclic peptide binding kinetics via stereospecific side-chain hydroxylation.

Reversible lysine-targeted probes reveal residence time-based kinase selectivity.

The expansion of the target landscape of covalent inhibitors requires the engagement of nucleophiles beyond cysteine. Although the conserved catalytic lysine in protein kinases is an attractive candidate for a covalent approach, selectivity remains an obvious challenge. Moreover, few covalent inhibitors have been shown to engage the kinase catalytic lysine in animals. We hypothesized that reversible, lysine-targeted inhibitors could provide sustained kinase engagement in vivo, with selectivity driven in part by differences in residence time. By strategically linking benzaldehydes to a promiscuous kinase binding scaffold, we developed chemoproteomic probes that reversibly and covalently engage >200 protein kinases in cells and mice. Probe-kinase residence time was dramatically enhanced by a hydroxyl group ortho to the aldehyde. Remarkably, only a few kinases, including Aurora A, showed sustained, quasi-irreversible occupancy in vivo, the structural basis for which was revealed by X-ray crystallography. We anticipate broad application of salicylaldehyde-based probes to proteins that lack a druggable cysteine.

Inhibition of the Sec61 translocon overcomes cytokine-induced glucocorticoid resistance in T-cell acute lymphoblastic leukaemia.

Glucocorticoid (GC) resistance is a poor prognostic factor in T-cell acute lymphoblastic leukaemia (T-ALL). Interleukin-7 (IL-7) mediates GC resistance via GC-induced upregulation of IL-7 receptor (IL-7R) expression, leading to increased pro-survival signalling. IL-7R reaches the cell surface via the secretory pathway, so we hypothesized that inhibiting the translocation of IL-7R into the secretory pathway would overcome GC resistance. Sec61 is an endoplasmic reticulum (ER) channel that is required for insertion of polypeptides into the ER. Here, we demonstrate that KZR-445, a novel inhibitor of Sec61, potently attenuates the dexamethasone (DEX)-induced increase in cell surface IL-7R and overcomes IL-7-induced DEX resistance.

Association between Inter-Limb Asymmetries in Lower-Limb Functional Performance and Sport Injury: A Systematic Review of Prospective Cohort Studies.

BACKGROUND: Inter-limb asymmetry in lower-limb functional performance has been associated with increased risk of sport injury; however, findings are not always consistent. PURPOSE: To conduct a systematic review on whether inter-limb asymmetry in lower-limb functional performance can predict sport injury. METHODS: Four electronic databases (MEDLINE, EMBASE, Web of Science, and SportDiscus) were systematically searched for prospective cohort studies reporting the association between inter-limb asymmetry in lower-limb functional performance and sport injury. RESULTS: A total of 28 prospective cohort studies were included in the analyses. Collectively, the findings were highly inconsistent, and a clear statement on the association between each asymmetry and sport injury was difficult. CONCLUSIONS: Highly inconsistent findings make it difficult to create clear recommendations on the relationship between the inter-limb asymmetry in lower-limb functional performance (power, muscle flexibility, and dynamic balance) and sport injury. The influence of potential factors (selection of tests/parameters, participant characteristics, definition of injury, and ways of calculating asymmetry) should be considered when using previous findings.

Tailored physical activity behavior change interventions: challenges and opportunities.

A physically active lifestyle provides innumerable benefits; yet, few individuals are physically active enough to reap those benefits. Tailored physical activity interventions may address low rates of physical activity by offering individualized strategies that consider a person's characteristics, needs, preferences, and/or context, rather than the traditional one-size-fits-all approach. However, the tailoring methodology is in its nascency, and an understanding of how best to develop such interventions is needed. In this commentary, we identify future directions to enhance the impact of tailored interventions designed to increase physical activity participation. A multi-country collaborative was established to review the literature and discuss an agenda for future research. Two overarching research opportunities are suggested for improving the development of tailored, behavioral physical activity interventions: (a) optimize the engagement of diverse knowledge users in intervention co-design and (b) examine ethical considerations that may impact the use of technology to support tailored physical activity delivery. Specifically, there is a need for better reporting and evaluation of knowledge user involvement alongside targeting diversity in the inclusion of knowledge users. Furthermore, while technology boasts many opportunities to increase the scale and precision of interventions, examinations of how it impacts recipients' experiences of and participation in tailored interventions are needed to ensure the benefits of technology use outweigh the risks. A better understanding of these research areas will help ensure that the diverse needs of individuals are met, technology is appropriately used to support tailoring, and ultimately it improves the effectiveness of tailored physical activity interventions.

Being physically active has many social, emotional, and health benefits, but very few individuals are active enough to see those benefits. Using interventions that are tailored, in other words, individualized to a person's characteristics, needs, preferences, and/or situation, may help improve physical activity participation rates. However, a better understanding of how to do tailoring is needed. Our collaboration reviewed the literature and convened to suggest two key opportunities to better understand how tailored approaches to physical activity can be done: (a) improve engagement of those who the research is intended for and (b) understand the ethical impacts and patient/provider experience of using technology to support tailoring.

Identifying the Cellular Target of Cordyheptapeptide A and Synthetic Derivatives.

Cordyheptapeptide A is a lipophilic cyclic peptide from the prized Cordyceps fungal genus that shows potent cytotoxicity in multiple cancer cell lines. To better understand the bioactivity and physicochemical properties of cordyheptapeptide A with the ultimate goal of identifying its cellular target, we developed a solid-phase synthesis of this multiply N-methylated cyclic heptapeptide which enabled rapid access to both side chain- and backbone-modified derivatives. Removal of one of the backbone amide N-methyl (N-Me) groups maintained bioactivity, while membrane permeability was also preserved due to the formation of a new intramolecular hydrogen bond in a low dielectric solvent. Based on its cytotoxicity profile in the NCI-60 cell line panel, as well as its phenotype in a microscopy-based cytological assay, we hypothesized that cordyheptapeptide was acting on cells as a protein synthesis inhibitor. Further studies revealed the molecular target of cordyheptapeptide A to be the eukaryotic translation elongation factor 1A (eEF1A), a target shared by other lipophilic cyclic peptide natural products. This work offers a strategy to study and improve cyclic peptide natural products while highlighting the ability of these lipophilic compounds to effectively inhibit intracellular disease targets.

Risk Factors for Non-Contact Lower-Limb Injury: A Retrospective Survey in Pediatric-Age Athletes.

BACKGROUND: Risk factors for non-contact lower-limb injury in pediatric-age athletes and the effects of lateral dominance in sport (laterally vs. non-laterally dominant sports) on injury have not been investigated. PURPOSE: To identify risk factors for non-contact lower-limb injury in pediatric-age athletes. METHODS: Parents and/or legal guardians of 2269 athletes aged between 6-17 years were recruited. Each participant completed an online questionnaire that contained 10 questions about the athlete's training and non-contact lower-limb injury in the preceding 12 months. RESULTS: The multivariate logistic regression model determined that lateral dominance in sport (adjusted OR (laterally vs. non-laterally dominant sports), 1.38; 95% CI, 1.10-1.75; p = 0.006), leg preference (adjusted OR (right vs. left-leg preference), 0.71; 95% CI, 0.53-0.95; p = 0.023), increased age (adjusted OR, 1.21; 95% CI, 1.16-1.26; p = 0.000), training intensity (adjusted OR, 1.77; 95% CI, 1.43-2.19; p = 0.000), and training frequency (adjusted OR, 1.36; 95% CI, 1.25-1.48; p = 0.000) were significantly associated with non-contact lower-limb injury in pediatric-age athletes. Length of training (p = 0.396) and sex (p = 0.310) were not associated with a non-contact lower-limb injury. CONCLUSIONS: Specializing in laterally dominant sports, left-leg preference, increase in age, training intensity, and training frequency indicated an increased risk of non-contact lower-limb injury in pediatric-age athletes. Future research should take into account exposure time and previous injury.

Corrigendum: p90RSK-MAGI1 Module Controls Endothelial Permeability by Post-translational Modifications of MAGI1 and Hippo Pathway.

[This corrects the article DOI: 10.3389/fcvm.2020.542485.].