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.

Lysine-Targeted Inhibitors and Chemoproteomic Probes.

Covalent inhibitors are widely used in drug discovery and chemical biology. Although covalent inhibitors are frequently designed to react with noncatalytic cysteines, many ligand binding sites lack an accessible cysteine. Here, we review recent advances in the chemical biology of lysine-targeted covalent inhibitors and chemoproteomic probes. By analyzing crystal structures of proteins bound to common metabolites and enzyme cofactors, we identify a large set of mostly unexplored lysines that are potentially targetable with covalent inhibitors. In addition, we describe mass spectrometry-based approaches for determining proteome-wide lysine ligandability and lysine-reactive chemoproteomic probes for assessing drug-target engagement. Finally, we discuss the design of amine-reactive inhibitors that form reversible covalent bonds with their protein targets.

Comparative Flavivirus-Host Protein Interaction Mapping Reveals Mechanisms of Dengue and Zika Virus Pathogenesis.

Mosquito-borne flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), are a growing public health concern. Systems-level analysis of how flaviviruses hijack cellular processes through virus-host protein-protein interactions (PPIs) provides information about their replication and pathogenic mechanisms. We used affinity purification-mass spectrometry (AP-MS) to compare flavivirus-host interactions for two viruses (DENV and ZIKV) in two hosts (human and mosquito). Conserved virus-host PPIs revealed that the flavivirus NS5 protein suppresses interferon stimulated genes by inhibiting recruitment of the transcription complex PAF1C and that chemical modulation of SEC61 inhibits DENV and ZIKV replication in human and mosquito cells. Finally, we identified a ZIKV-specific interaction between NS4A and ANKLE2, a gene linked to hereditary microcephaly, and showed that ZIKV NS4A causes microcephaly in Drosophila in an ANKLE2-dependent manner. Thus, comparative flavivirus-host PPI mapping provides biological insights and, when coupled with in vivo models, can be used to unravel pathogenic mechanisms.

Decoding Mammalian Ribosome-mRNA States by Translational GTPase Complexes.

In eukaryotes, accurate protein synthesis relies on a family of translational GTPases that pair with specific decoding factors to decipher the mRNA code on ribosomes. We present structures of the mammalian ribosome engaged with decoding factor⋅GTPase complexes representing intermediates of translation elongation (aminoacyl-tRNA⋅eEF1A), termination (eRF1⋅eRF3), and ribosome rescue (Pelota⋅Hbs1l). Comparative analyses reveal that each decoding factor exploits the plasticity of the ribosomal decoding center to differentially remodel ribosomal proteins and rRNA. This leads to varying degrees of large-scale ribosome movements and implies distinct mechanisms for communicating information from the decoding center to each GTPase. Additional structural snapshots of the translation termination pathway reveal the conformational changes that choreograph the accommodation of decoding factors into the peptidyl transferase center. Our results provide a structural framework for how different states of the mammalian ribosome are selectively recognized by the appropriate decoding factor⋅GTPase complex to ensure translational fidelity.

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.

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.

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.

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.

Targeting Viral Proteostasis Limits Influenza Virus, HIV, and Dengue Virus Infection.

Viruses are obligate parasites and thus require the machinery of the host cell to replicate. Inhibition of host factors co-opted during active infection is a strategy hosts use to suppress viral replication and a potential pan-antiviral therapy. To define the cellular proteins and processes required for a virus during infection is thus crucial to understanding the mechanisms of virally induced disease. In this report, we generated fully infectious tagged influenza viruses and used infection-based proteomics to identify pivotal arms of cellular signaling required for influenza virus growth and infectivity. Using mathematical modeling and genetic and pharmacologic approaches, we revealed that modulation of Sec61-mediated cotranslational translocation selectively impaired glycoprotein proteostasis of influenza as well as HIV and dengue viruses and led to inhibition of viral growth and infectivity. Thus, by studying virus-human protein-protein interactions in the context of active replication, we have identified targetable host factors for broad-spectrum antiviral therapies.

Sequence-dependent sorting of recycling proteins by actin-stabilized endosomal microdomains.

The functional consequences of signaling receptor endocytosis are determined by the endosomal sorting of receptors between degradation and recycling pathways. How receptors recycle efficiently, in a sequence-dependent manner that is distinct from bulk membrane recycling, is not known. Here, in live cells, we visualize the sorting of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, from bulk recycling proteins and the degrading delta-opioid receptor. Our results reveal a remarkable diversity in recycling routes at the level of individual endosomes, and indicate that sequence-dependent recycling is an active process mediated by distinct endosomal subdomains distinct from those mediating bulk recycling. We identify a specialized subset of tubular microdomains on endosomes, stabilized by a highly localized but dynamic actin machinery, that mediate this sorting, and provide evidence that these actin-stabilized domains provide the physical basis for a two-step kinetic and affinity-based model for protein sorting into the sequence-dependent recycling pathway.

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.

Engineered Covalent Inactivation of TFIIH-Kinase Reveals an Elongation Checkpoint and Results in Widespread mRNA Stabilization.

During transcription initiation, the TFIIH-kinase Kin28/Cdk7 marks RNA polymerase II (Pol II) by phosphorylating the C-terminal domain (CTD) of its largest subunit. Here we describe a structure-guided chemical approach to covalently and specifically inactivate Kin28 kinase activity in vivo. This method of irreversible inactivation recapitulates both the lethal phenotype and the key molecular signatures that result from genetically disrupting Kin28 function in vivo. Inactivating Kin28 impacts promoter release to differing degrees and reveals a "checkpoint" during the transition to productive elongation. While promoter-proximal pausing is not observed in budding yeast, inhibition of Kin28 attenuates elongation-licensing signals, resulting in Pol II accumulation at the +2 nucleosome and reduced transition to productive elongation. Furthermore, upon inhibition, global stabilization of mRNA masks different degrees of reduction in nascent transcription. This study resolves long-standing controversies on the role of Kin28 in transcription and provides a rational approach to irreversibly inhibit other kinases in vivo.

Chromatin Kinases Act on Transcription Factors and Histone Tails in Regulation of Inducible Transcription.

The inflammatory response requires coordinated activation of both transcription factors and chromatin to induce transcription for defense against pathogens and environmental insults. We sought to elucidate the connections between inflammatory signaling pathways and chromatin through genomic footprinting of kinase activity and unbiased identification of prominent histone phosphorylation events. We identified H3 serine 28 phosphorylation (H3S28ph) as the principal stimulation-dependent histone modification and observed its enrichment at induced genes in mouse macrophages stimulated with bacterial lipopolysaccharide. Using pharmacological and genetic approaches, we identified mitogen- and stress-activated protein kinases (MSKs) as primary mediators of H3S28ph in macrophages. Cell-free transcription assays demonstrated that H3S28ph directly promotes p300/CBP-dependent transcription. Further, MSKs can activate both signal-responsive transcription factors and the chromatin template with additive effects on transcription. Specific inhibition of MSKs in macrophages selectively reduced transcription of stimulation-induced genes. Our results suggest that MSKs incorporate upstream signaling inputs and control multiple downstream regulators of inducible transcription.

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.

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.

Masters Athlete Screening Study (MASS): Insights Into the Psychological Impact of Cardiovascular Preparticipation Screening.

OBJECTIVE: To determine the psychological impact of a cardiovascular disease (CVD) diagnosis identified during preparticipation screening (PPS) of masters athletes. DESIGN: Cross-sectional study. SETTING: Masters athletes diagnosed with CVD through the Masters Athletes Screening Study. PARTICIPANTS: Sixty-seven athletes (89.6% male, mean age at diagnosis 60.1 ± 7.1 years, range 40-76) with diagnoses of coronary artery disease (CAD) (73.1%), high premature ventricular contraction burden (9.0%), mitral valve prolapse (7.5%), atrial fibrillation (AF) (3.0%), bicuspid aortic valve (3.0%), aortic dilatation (1.5%), coronary anomaly (1.5%), and rheumatic heart disease (1.5%). Three participants had multiple diagnoses. INTERVENTION: Online survey distributed to masters athletes identified with CVD. MAIN OUTCOME MEASURES: Assessment of psychological distress [Impact of Event Scale-Revised (IES-R)], perceptions of screening, and preferred support by CVD type. RESULTS: The median total IES-R and subscale scores were within the normal range {median [interquartile range (IQR)] total 2.0 [0-6.0]; intrusion 1.0 [0-3.0]; avoidance 0 [0-3.0]; hyperarousal 0 [0-1.0]}. Athletes with bicuspid aortic valve [20.5 (IQR, 4.0-37.0)], AF [7.0 (IQR, 0-14.0)], and severe CAD [5.5 (IQR, 1.0-12.0)] had the highest total IES-R scores. One individual with bicuspid aortic valve reported a significant stress reaction. Ten athletes (14.9%) had scores >12. Ninety-three percent of athletes were satisfied having undergone PPS. Preferred type of support varied by cardiovascular diagnosis. CONCLUSIONS: The majority of masters athletes diagnosed with CVD through PPS do not experience significant levels of psychological distress. Athletes diagnosed with more severe types of CVD should be monitored for psychological distress. Support should be provided through a multidisciplinary and individualized approach.

Platelet-Rich Plasma Applications for Achilles Tendon Repair: A Bridge between Biology and Surgery.

Achilles tendon ruptures are very common tendon ruptures and their incidence is increasing in modern society, resulting in work incapacity and months off sport, which generate a need for accelerated and successful therapeutic repair strategy. Platelet-rich plasma (PRP) is emerging as adjuvant human blood-derived constructs to assist Achilles tendon rupture treatment. However, myriad PRP preparation methods in conjunction with poor standardization in the modalities of their applications impinge on the consistent effectiveness of clinical and structural outcomes regarding their therapeutic efficacy. The purpose of this review is to provide some light on the application of PRP for Achilles tendon ruptures. PRP has many characteristics that make it an attractive treatment. Elements such as the inclusion of leukocytes and erythrocytes within PRP, the absence of activation and activation ex vivo or in vivo, the modality of application, and the adjustment of PRP pH can influence the biology of the applied product and result in misleading therapeutic conclusions. The weakest points in demonstrating their consistent effectiveness are primarily the result of myriad PRP preparation methods and the poor standardization of modalities for their application. Selecting the right biological scaffold and applying it correctly to restitutio ad integrum of ruptured Achilles tendons remains a daunting and complex task.

Senescent Phenotype Induced by p90RSK-NRF2 Signaling Sensitizes Monocytes and Macrophages to Oxidative Stress in HIV-Positive Individuals.

BACKGROUND: The incidence of cardiovascular disease is higher in HIV-positive (HIV+) patients than it is in the average population, and combination antiretroviral therapy (cART) is a recognized risk factor for cardiovascular disease. However, the molecular mechanisms that link cART and cardiovascular disease are currently unknown. Our study explores the role of the activation of p90RSK, a reactive oxygen species-sensitive kinase, in engendering senescent phenotype in macrophages and accelerating atherogenesis in patients undergoing cART. METHODS: Peripheral whole blood from cART-treated HIV+ individuals and nontreated HIV-negative individuals was treated with H2O2 (200 µmol/L) for 4 minutes, and p90RSK activity in CD14+ monocytes was measured. Plaque formation in the carotids was also analyzed in these individuals. Macrophage senescence was determined by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. The involvement of p90RSK-NRF2 signaling in cART-induced senescence was assessed by p90RSK-specific inhibitor (FMK-MEA) or dominant-negative p90RSK (DN-p90RSK) and NRF2 activator (NRF2A). Further, the severity of atherosclerosis was determined in myeloid cell-specific wild-type and DN-p90RSK transgenic mice. RESULTS: Monocytes from HIV+ patients exhibited higher levels of p90RSK activity and were also more sensitive to reactive oxygen species than monocytes from HIV-negative individuals. A multiple linear regression analysis involving cART, Reynolds cardiovascular risk score, and basal p90RSK activity revealed that cART and basal p90RSK activity were the 2 significant determinants of plaque formation. Many of the antiretroviral drugs individually activated p90RSK, which simultaneously triggered all components of the macrophage senescent phenotype. cART inhibited antioxidant response element reporter activity via ERK5 S496 phosphorylation. NRF2A reversed the H2O2-induced overactivation of p90RSK in cART-treated macrophages by countering the induction of senescent phenotype. Last, the data obtained from our gain- or loss-of-function mice conclusively showed the crucial role of p90RSK in inducing senescent phenotype in macrophages and atherogenesis. CONCLUSIONS: cART increased monocyte/macrophage sensitivity to reactive oxygen species- in HIV+ individuals by suppressing NRF2-ARE activity via p90RSK-mediated ERK5 S496 phosphorylation, which coordinately elicited senescent phenotypes and proinflammatory responses. As such, our report underscores the importance of p90RSK regulation in monocytes/macrophages as a viable biomarker and therapeutic target for preventing cardiovascular disease, especially in HIV+ patients treated with cART.

Ligand Conformational Bias Drives Enantioselective Modification of a Surface-Exposed Lysine on Hsp90.

Targeted covalent modification of surface-exposed lysines is challenging due to their low intrinsic reactivity and high prevalence throughout the proteome. Strategies for optimizing the rate of covalent bond formation by a reversibly bound inhibitor (kinact) typically involve increasing the reactivity of the electrophile, which increases the risk of off-target modification. Here, we employ an alternative approach for increasing kinact of a lysine-targeted covalent Hsp90 inhibitor, independent of the reversible binding affinity (Ki) or the intrinsic electrophilicity. Starting with a noncovalent ligand, we appended a chiral, conformationally constrained linker, which orients an arylsulfonyl fluoride to react rapidly and enantioselectively with Lys58 on the surface of Hsp90. Biochemical experiments and high-resolution crystal structures of covalent and noncovalent ligand/Hsp90 complexes provide mechanistic insights into the role of ligand conformation in the observed enantioselectivity. Finally, we demonstrate selective covalent targeting of cellular Hsp90, which results in a prolonged heat shock response despite concomitant degradation of the covalent ligand/Hsp90 complex. Our work highlights the potential of engineering ligand conformational constraints to dramatically accelerate covalent modification of a distal, poorly nucleophilic lysine on the surface of a protein target.

Discovery of Lysine-Targeted eIF4E Inhibitors through Covalent Docking.

Eukaryotic translation initiation factor 4E (eIF4E) binds the m7GTP cap structure at the 5'-end of mRNAs, stimulating the translation of proteins implicated in cancer cell growth and metastasis. eIF4E is a notoriously challenging target, and most of the reported inhibitors are negatively charged guanine analogues with negligible cell permeability. To overcome these challenges, we envisioned a covalent targeting strategy. As there are no cysteines near the eIF4E cap binding site, we developed a covalent docking approach focused on lysine. Taking advantage of a "make-on-demand" virtual library, we used covalent docking to identify arylsulfonyl fluorides that target a noncatalytic lysine (Lys162) in eIF4E. Guided by cocrystal structures, we elaborated arylsulfonyl fluoride 2 to 12, which to our knowledge is the first covalent eIF4E inhibitor with cellular activity. In addition to providing a new tool for acutely inactivating eIF4E in cells, our computational approach may offer a general strategy for developing selective lysine-targeted covalent ligands.