RESUMEN
Stress granules (SGs) and processing-bodies (PBs, P-bodies) are ubiquitous and widely studied ribonucleoprotein (RNP) granules involved in cellular stress response, viral infection, and the tumor microenvironment. While proteomic and transcriptomic investigations of SGs and PBs have provided insights into molecular composition, chemical tools to probe and modulate RNP granules remain lacking. Herein, we combine an immunofluorescence (IF)-based phenotypic screen with chemoproteomics to identify sulfonyl-triazoles (SuTEx) capable of preventing or inducing SG and PB formation through liganding of tyrosine (Tyr) and lysine (Lys) sites in stressed cells. Liganded sites were enriched for RNA-binding and protein-protein interaction (PPI) domains, including several sites found in RNP granule-forming proteins. Among these, we functionally validate G3BP1 Y40, located in the NTF2 dimerization domain, as a ligandable site that can disrupt arsenite-induced SG formation in cells. In summary, we present a chemical strategy for the systematic discovery of condensate-modulating covalent small molecules.
Asunto(s)
Gránulos Citoplasmáticos , ADN Helicasas , ADN Helicasas/química , ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Gránulos Citoplasmáticos/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteómica , ARN Helicasas/químicaRESUMEN
Lipids in complex, protein-enriched films at air/liquid interfaces reduce surface tension. In the absence of this benefit, the light refracting and immunoprotective tear film on eyes would collapse. Premature collapse, coupled with chronic inflammation compromising visual acuity, is a hallmark of dry eye disease affecting 7 to 10% of individuals worldwide. Although collapse seems independent of mutation (unlike newborn lung alveoli), selective proteome and possible lipidome changes have been noted. These include elevated tissue transglutaminase and consequent inactivation through C-terminal cross-linking of the tear mitogen lacritin, leading to significant loss of lacritin monomer. Lacritin monomer restores homeostasis via autophagy and mitochondrial fusion and promotes basal tearing. Here, we discover that lacritin monomer C-terminal processing, inclusive of cysteine, serine, and metalloproteinase activity, generates cationic amphipathic α-helical proteoforms. Such proteoforms (using synthetic peptide surrogates) act like alveolar surfactant proteins to rapidly bind and stabilize the tear lipid layer. Immunodepletion of C- but not N-terminal proteoforms nor intact lacritin, from normal human tears promotes loss of stability akin to human dry eye tears. Stability of these and dry eye tears is rescuable with C- but not N-terminal proteoforms. Repeated topical application in rabbits reveals a proteoform turnover time of 7 to 33 h with gradual loss from human tear lipid that retains bioactivity without further processing. Thus, the processed C-terminus of lacritin that is deficient or absent in dry eye tears appears to play a key role in preventing tear film collapse and as a natural slow release mechanism that restores epithelial homeostasis.
Asunto(s)
Síndromes de Ojo Seco/fisiopatología , Proteínas del Ojo/metabolismo , Glicoproteínas/fisiología , Isoformas de Proteínas/fisiología , Lágrimas/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Glándulas Tarsales/fisiología , ConejosRESUMEN
Covalent probes serve as valuable tools for global investigation of protein function and ligand binding capacity. Despite efforts to expand coverage of residues available for chemical proteomics (e.g., cysteine and lysine), a large fraction of the proteome remains inaccessible with current activity-based probes. Here, we introduce sulfur-triazole exchange (SuTEx) chemistry as a tunable platform for developing covalent probes with broad applications for chemical proteomics. We show modifications to the triazole leaving group can furnish sulfonyl probes with ~5-fold enhanced chemoselectivity for tyrosines over other nucleophilic amino acids to investigate more than 10,000 tyrosine sites in lysates and live cells. We discover that tyrosines with enhanced nucleophilicity are enriched in enzymatic, protein-protein interaction and nucleotide recognition domains. We apply SuTEx as a chemical phosphoproteomics strategy to monitor activation of phosphotyrosine sites. Collectively, we describe SuTEx as a biocompatible chemistry for chemical biology investigations of the human proteome.
Asunto(s)
Sondas Moleculares/química , Proteómica/métodos , Azufre/química , Triazoles/química , Tirosina/análisis , Tirosina/química , Células A549 , Sitios de Unión , Flúor/química , Gutatión-S-Transferasa pi/genética , Gutatión-S-Transferasa pi/metabolismo , Células HEK293 , Humanos , Sondas Moleculares/síntesis química , Fosforilación , Fosfotirosina/química , Fosfotirosina/metabolismo , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Ácidos Sulfínicos/química , Tirosina/metabolismoRESUMEN
Sulfonyl-triazoles have emerged as a new reactive group for covalent modification of tyrosine sites on proteins through sulfur-triazole exchange (SuTEx) chemistry. The extent to which this sulfur electrophile can be tuned for developing ligands with cellular activity remains largely underexplored. Here, we performed fragment-based ligand discovery in live cells to identify SuTEx compounds capable of liganding tyrosine sites on diverse protein targets. We verified our quantitative chemical proteomic findings by demonstrating concentration-dependent activity of SuTEx ligands, but not inactive counterparts, against recombinant protein targets directly in live cells. Our structure-activity relationship studies identified the SuTEx ligand HHS-0701 as a cell-active inhibitor capable of blocking prostaglandin reductase 2 (PTGR2) biochemical activity.
Asunto(s)
15-Oxoprostaglandina 13-Reductasa/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Azufre/farmacología , Triazoles/farmacología , 15-Oxoprostaglandina 13-Reductasa/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Células HEK293 , Humanos , Ligandos , Estructura Molecular , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Azufre/química , Compuestos de Azufre , Triazoles/químicaRESUMEN
Tuning reactivity of sulfur electrophiles is key for advancing click chemistry and chemical probe discovery. To date, activation of the sulfur electrophile for protein modification has been ascribed principally to stabilization of a fluoride leaving group (LG) in covalent reactions of sulfonyl fluorides and arylfluorosulfates. We recently introduced sulfur-triazole exchange (SuTEx) chemistry to demonstrate the triazole as an effective LG for activating nucleophilic substitution reactions on tyrosine sites of proteins. Here, we probed tunability of SuTEx for fragment-based ligand discovery by modifying the adduct group (AG) and LG with functional groups of differing electron-donating and -withdrawing properties. We discovered the sulfur electrophile is highly sensitive to the position of modification (AG versus LG), which enabled both coarse and fine adjustments in solution and proteome activity. We applied these reactivity principles to identify a large fraction of tyrosine sites (â¼30%) on proteins (â¼44%) that can be liganded across >1500 probe-modified sites quantified by chemical proteomics. Our proteomic studies identified noncatalytic tyrosine and phosphotyrosine sites that can be liganded by SuTEx fragments with site specificity in lysates and live cells to disrupt protein function. Collectively, we describe SuTEx as a versatile covalent chemistry with broad applications for chemical proteomics and protein ligand discovery.
Asunto(s)
Proteínas/química , Azufre/química , Triazoles/química , Tirosina/química , Células HEK293 , Humanos , Ligandos , Estructura Molecular , Proteómica , Relación Estructura-ActividadRESUMEN
Diacylglycerol kinases (DGKs) are metabolic kinases involved in regulating cellular levels of diacylglycerol and phosphatidic lipid messengers. The development of selective inhibitors for individual DGKs would benefit from discovery of protein pockets available for inhibitor binding in cellular environments. Here we utilized a sulfonyl-triazole probe (TH211) bearing a DGK fragment ligand for covalent binding to tyrosine and lysine sites on DGKs in cells that map to predicted small molecule binding pockets in AlphaFold structures. We apply this chemoproteomics-AlphaFold approach to evaluate probe binding of DGK chimera proteins engineered to exchange regulatory C1 domains between DGK subtypes (DGKα and DGKζ). Specifically, we discovered loss of TH211 binding to a predicted pocket in the catalytic domain when C1 domains on DGKα were exchanged that correlated with impaired biochemical activity as measured by a DAG phosphorylation assay. Collectively, we provide a family-wide assessment of accessible sites for covalent targeting that combined with AlphaFold revealed predicted small molecule binding pockets for guiding future inhibitor development of the DGK superfamily.
RESUMEN
Proteomic methods for RNA interactome capture (RIC) rely principally on crosslinking native or labeled cellular RNA to enrich and investigate RNA-binding protein (RBP) composition and function in cells. The ability to measure RBP activity at individual binding sites by RIC, however, has been more challenging due to the heterogenous nature of peptide adducts derived from the RNA-protein crosslinked site. Here, we present an orthogonal strategy that utilizes clickable electrophilic purines to directly quantify protein-RNA interactions on proteins through photoaffinity competition with 4-thiouridine (4SU)-labeled RNA in cells. Our photo-activatable-competition and chemoproteomic enrichment (PACCE) method facilitated detection of >5500 cysteine sites across ~3000 proteins displaying RNA-sensitive alterations in probe binding. Importantly, PACCE enabled functional profiling of canonical RNA-binding domains as well as discovery of moonlighting RNA binding activity in the human proteome. Collectively, we present a chemoproteomic platform for global quantification of protein-RNA binding activity in living cells.
Asunto(s)
Proteómica , ARN , Humanos , ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Sitios de Unión , Péptidos/metabolismoRESUMEN
Background: Patients with rheumatoid arthritis (RA) experience joint swelling and cartilage destruction resulting in chronic pain, functional disability, and compromised joint function. Current RA treatments, including glucocorticoid receptor agonists, produce adverse side effects and lack prolonged treatment efficacy. Cannabinoids (i.e., cannabis-like signaling molecules) exert anti-inflammatory and analgesic effects with limited side effects compared to traditional immunosuppressants, making them excellent targets for the development of new arthritic therapeutics. Monoacylglycerol lipase (MAGL) inhibition reduces inflammation in mouse models of acute inflammation, through cannabinoid receptor dependent and independent pathways. The current study investigated the efficacy of inhibiting synthetic and catabolic enzymes that regulate the endocannabinoid 2-arachidonoylglycerol (2-AG) in blocking paw inflammation, pain-related behaviors, and functional loss caused by collagen-induced arthritis (CIA). Methods: Male DB1A mice subjected to CIA were administered the glucocorticoid agonist dexamethasone (DEX), MAGL inhibitor JZL184 (8 or 40 mg/kg, s.c.), alone or in combination, or diacylglycerol lipase ß (DAGLß) inhibitor KT109 (40 mg/kg, s.c.). CIA-induced deficits were assayed by arthritic clinical scoring, paw thickness measurements, and behavioral tests of pain and paw function. Results: DEX or dual administration with JZL184 reduced paw thickness and clinical scores, and JZL184 dose-dependently attenuated grip strength and balance beam deficits caused by CIA. Traditional measures of pain-induced behaviors (hyperalgesia and allodynia) were inconsistent. The antiarthritic effects of JZL184 (40 mg/kg) were largely blocked by coadministration of the CB2 antagonist SR144528, and the DAGLß inhibitor KT109 had no effect on CIA, indicating that these effects likely occurred through CB2 activation. Conclusions: MAGL inhibition reduced paw inflammation and pain-depressed behavioral signs of arthritis, likely through an endocannabinoid mechanism requiring CB2. These data support the development of MAGL as a target for therapeutic treatment of inflammatory arthritis.
Asunto(s)
Ácidos Araquidónicos/fisiología , Artritis Experimental/tratamiento farmacológico , Benzodioxoles/farmacología , Endocannabinoides/fisiología , Glicéridos/fisiología , Inflamación/tratamiento farmacológico , Monoacilglicerol Lipasas/antagonistas & inhibidores , Piperidinas/farmacología , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/enzimología , Dexametasona/farmacología , Edema/tratamiento farmacológico , Pie , Hiperalgesia/tratamiento farmacológico , Inflamación/inducido químicamente , Masculino , Ratones , Ratones Endogámicos DBARESUMEN
The asymmetric synthesis of all four of the known natural phlegmarines and one synthetic derivative has been accomplished in 19-22 steps from 4-methoxy-3-(triisopropylsilyl)pyridine. Chiral N-acylpyridinium salt chemistry was used twice to set the stereocenters at the C-9 and C-2' positions of the phlegmarine skeleton. Key reactions include the use of a mixed Grignard reagent for the second N-acylpyridinium salt addition, zinc/acetic acid reduction of a complex dihydropyridone, and a von Braun cyanogen bromide N-demethylation of a late intermediate. These syntheses confirmed the absolute stereochemistry of all of the known phlegmarines.
Asunto(s)
Alcaloides/síntesis química , Alcaloides/química , Indicadores y Reactivos/química , Espectroscopía de Resonancia Magnética , Conformación Molecular , Estructura Molecular , EstereoisomerismoRESUMEN
Series of aminopyridinecarboxamide-based inhibitors were synthesized and tested against human recombinant IKK-2 and in IL-1beta stimulated synovial fibroblasts. The 2-amino-5-chloropyridine-4-carboxamides were identified as the most potent inhibitors with improved cellular activity.
Asunto(s)
Aminopirina/química , Aminopirina/farmacología , Antiinflamatorios/química , Antiinflamatorios/farmacología , Quinasa I-kappa B/antagonistas & inhibidores , Interleucina-1beta/antagonistas & inhibidores , Amidas/química , Amidas/farmacología , Secuencia de Aminoácidos , Artritis Reumatoide/tratamiento farmacológico , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Humanos , Quinasa I-kappa B/metabolismo , Proteínas I-kappa B/química , Interleucina-1beta/metabolismo , Cápsula Articular/citología , Datos de Secuencia Molecular , Relación Estructura-ActividadRESUMEN
A series of 21 novel 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarboxamides were synthesized and evaluated for the inhibition of IKK-2. In spite of their often modest activity on the enzyme, six selected analogs showed significant inhibition of the production of inflammatory cytokine IL-8 in IL-1beta stimulated rheumatoid arthritis-derived synovial fibroblasts, demonstrating their potential usefulness as NF-kappaB regulators.