COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite ...previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.
Thyrotoxicosis has multiple etiologies, manifestations, and potential therapies. Appropriate treatment requires an accurate diagnosis and is influenced by coexisting medical conditions and patient ...preference. This document describes evidence-based clinical guidelines for the management of thyrotoxicosis that would be useful to generalist and subspecialty physicians and others providing care for patients with this condition.
The American Thyroid Association (ATA) previously cosponsored guidelines for the management of thyrotoxicosis that were published in 2011. Considerable new literature has been published since then, and the ATA felt updated evidence-based guidelines were needed. The association assembled a task force of expert clinicians who authored this report. They examined relevant literature using a systematic PubMed search supplemented with additional published materials. An evidence-based medicine approach that incorporated the knowledge and experience of the panel was used to update the 2011 text and recommendations. The strength of the recommendations and the quality of evidence supporting them were rated according to the approach recommended by the Grading of Recommendations, Assessment, Development, and Evaluation Group.
Clinical topics addressed include the initial evaluation and management of thyrotoxicosis; management of Graves' hyperthyroidism using radioactive iodine, antithyroid drugs, or surgery; management of toxic multinodular goiter or toxic adenoma using radioactive iodine or surgery; Graves' disease in children, adolescents, or pregnant patients; subclinical hyperthyroidism; hyperthyroidism in patients with Graves' orbitopathy; and management of other miscellaneous causes of thyrotoxicosis. New paradigms since publication of the 2011 guidelines are presented for the evaluation of the etiology of thyrotoxicosis, the management of Graves' hyperthyroidism with antithyroid drugs, the management of pregnant hyperthyroid patients, and the preparation of patients for thyroid surgery. The sections on less common causes of thyrotoxicosis have been expanded.
One hundred twenty-four evidence-based recommendations were developed to aid in the care of patients with thyrotoxicosis and to share what the task force believes is current, rational, and optimal medical practice.
Protein ubiquitination regulates many cellular processes via attachment of structurally and functionally distinct ubiquitin (Ub) chains. Several atypical chain types have remained poorly ...characterized because the enzymes mediating their assembly and receptors with specific binding properties have been elusive. We found that the human HECT E3 ligases UBE3C and AREL1 assemble K48/K29- and K11/K33-linked Ub chains, respectively, and can be used in combination with DUBs to generate K29- and K33-linked chains for biochemical and structural analyses. Solution studies indicate that both chains adopt open and dynamic conformations. We further show that the N-terminal Npl4-like zinc finger (NZF1) domain of the K29/K33-specific deubiquitinase TRABID specifically binds K29/K33-linked diUb, and a crystal structure of this complex explains TRABID specificity and suggests a model for chain binding by TRABID. Our work uncovers linkage-specific components in the Ub system for atypical K29- and K33-linked Ub chains, providing tools to further understand these unstudied posttranslational modifications.
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•The HECT E3 ligases UBE3C and AREL1 assemble K29- and K33-linked polyubiquitin, respectively•K29- and K33-linked chains adopt open conformations in solution•The N-terminal NZF1 domain of TRABID specifically recognizes K29/K33-diubiquitin•A structure of a K33 filament bound to NZF1 domains explains TRABID specificity
Michel et al. reveal that UBE3C and AREL1 assemble K29- and K33-linked polyubiquitin, respectively, on substrates and as unanchored chains. They further identify a K29/K33-specific ubiquitin binding domain in TRABID and structurally characterize how TRABID recognizes K29/K33 filaments.
Unidentified infrared emission bands are ubiquitous in many astronomical sources. These bands are widely, if not unanimously, attributed to collective emissions from polycyclic aromatic hydrocarbon ...(PAH) molecules, yet no single species of this class has been identified in space. Using spectral matched filtering of radio data from the Green Bank Telescope, we detected two nitrile-group-functionalized PAHs, 1- and 2-cyanonaphthalene, in the interstellar medium. Both bicyclic ring molecules were observed in the TMC-1 molecular cloud. In this paper, we discuss potential in situ gas-phase PAH formation pathways from smaller organic precursor molecules.
Interrogating biological systems is often limited by access to biological probes. The emergence of “click chemistry” has revolutionized bioconjugate chemistry by providing facile reaction conditions ...amenable to both biologic molecules and small molecule probes such as fluorophores, toxins, or therapeutics. One particularly popular version is the copper-catalyzed azide–alkyne cycloaddition (AAC) reaction, which has spawned new alternatives such as the strain-promoted azide–alkyne cycloaddition reaction, among others. This focused review highlights practical approaches to AAC reactions for the synthesis of peptide or protein bioconjugates and contrasts current challenges and limitations in light of recent advances in the field. The conical success of antibody drug conjugates has expanded the toolbox of linkers and payloads to facilitate practical applications of bioconjugation to create novel therapeutics and biologic probes. The AAC reaction in particular is poised to enable a large set of functionalized molecules as a combinatorial approach to high-throughput bioconjugate generation, screening, and honing of lead compounds.
The protein kinase PINK1 was recently shown to phosphorylate ubiquitin (Ub) on Ser65, and phosphoUb activates the E3 ligase Parkin allosterically. Here, we show that PINK1 can phosphorylate every Ub ...in Ub chains. Moreover, Ser65 phosphorylation alters Ub structure, generating two conformations in solution. A crystal structure of the major conformation resembles Ub but has altered surface properties. NMR reveals a second phosphoUb conformation in which β5‐strand slippage retracts the C‐terminal tail by two residues into the Ub core. We further show that phosphoUb has no effect on E1‐mediated E2 charging but can affect discharging of E2 enzymes to form polyUb chains. Notably, UBE2R1‐ (CDC34), UBE2N/UBE2V1‐ (UBC13/UEV1A), TRAF6‐ and HOIP‐mediated chain assembly is inhibited by phosphoUb. While Lys63‐linked poly‐phosphoUb is recognized by the TAB2 NZF Ub binding domain (UBD), 10 out of 12 deubiquitinases (DUBs), including USP8, USP15 and USP30, are impaired in hydrolyzing phosphoUb chains. Hence, Ub phosphorylation has repercussions for ubiquitination and deubiquitination cascades beyond Parkin activation and may provide an independent layer of regulation in the Ub system.
Synopsis
Ubiquitin phosphorylation by PINK1 kinase is involved in Parkin activation. Structural and biochemical work now shows that it also affects ubiquitin conformation, chain assembly and deconjugation, thus potentially affecting ubiquitin signaling more broadly.
PINK1 phosphorylates Ser65 both in free ubiquitin and in polyubiquitin chains.
Parkin is activated by phospho‐ubiquitin only in the presence of unphosphorylated ubiquitin.
Ser65 phospho‐ubiquitin interconverts between a major, canonical ubiquitin conformation and a previously unknown minor conformation.
Due to beta‐strand slippage, the ubiquitin C‐terminal tail is retracted in the minor conformation.
Ser65 phospho‐ubiquitin is charged onto E2 enzymes, but discharging with and without E3 can be inhibited.
Ser65 phospho‐ubiquitin‐incorporating polyubiquitin chains are less well hydrolyzed by all classes of DUB enzymes.
PINK1‐mediated ubiquitin phosphorylation is not only involved in Parkin activation, but has the potential to affect ubiquitin conjugation and signaling at various levels.
Direct recognition of invading pathogens by innate immune cells is a critical driver of the inflammatory response. However, cells of the innate immune system can also sense their local ...microenvironment and respond to physiological fluctuations in temperature, pH, oxygen and nutrient availability, which are altered during inflammation. Although cells of the immune system experience force and pressure throughout their life cycle, little is known about how these mechanical processes regulate the immune response. Here we show that cyclical hydrostatic pressure, similar to that experienced by immune cells in the lung, initiates an inflammatory response via the mechanically activated ion channel PIEZO1. Mice lacking PIEZO1 in innate immune cells showed ablated pulmonary inflammation in the context of bacterial infection or fibrotic autoinflammation. Our results reveal an environmental sensory axis that stimulates innate immune cells to mount an inflammatory response, and demonstrate a physiological role for PIEZO1 and mechanosensation in immunity.
Several ubiquitin chain types have remained unstudied, mainly because tools and techniques to detect these posttranslational modifications are scarce. Linkage-specific antibodies have shaped our ...understanding of the roles and dynamics of polyubiquitin signals but are available for only five out of eight linkage types. We here characterize K6- and K33-linkage-specific “affimer” reagents as high-affinity ubiquitin interactors. Crystal structures of affimers bound to their cognate chain types reveal mechanisms of specificity and a K11 cross-reactivity in the K33 affimer. Structure-guided improvements yield superior affinity reagents suitable for western blotting, confocal fluorescence microscopy and pull-down applications. This allowed us to identify RNF144A and RNF144B as E3 ligases that assemble K6-, K11-, and K48-linked polyubiquitin in vitro. A protocol to enrich K6-ubiquitinated proteins from cells identifies HUWE1 as a main E3 ligase for this chain type, and we show that mitofusin-2 is modified with K6-linked polyubiquitin in a HUWE1-dependent manner.
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•Respective linkage-specific affimers recognize K6- or K33-/K11-linked chains•Structures of affimer:diUb complexes reveal mechanisms of linkage specificity•Improved affimers can be used in western blotting, confocal microscopy, and pull-downs•Pull-downs with K6-specific affimers reveal HUWE1 to be a major K6 ligase in cells
Michel et al. describe linkage-specific tools for the study of atypical K6 and K33/K11 ubiquitin linkages. Affimers have applications in western blotting, confocal microscopy, and pull-downs. Using a K6-specific affimer, they reveal that HUWE1 is a major source of cellular K6 chains and decorates Mfn2 with K6 chains.
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