RESUMO
Upon infection of host cells the Legionella pneumophila bacterium releases a multitude of effector enzymes into the host's cytoplasm that manipulate cellular host pathways, including the host-ubiquitination pathways. The effectors belonging to the SidE-family are involved in non-canonical phosphoribosyl serine ubiquitination (PR-ubiquitination) of host substrate proteins. This results in the recruitment of ER-remodeling proteins and the formation of a Legionella-containing vacuole which is crucial in the onset of legionnaires disease. PR-ubiquitination is a dynamic process reversed by other Legionella effectors called Dups. During PR-Ubiquitin phosphodiester hydrolysis Dups form a covalent intermediate with the phosphoribosyl ubiquitylated protein using its active site His67 residue. We envisioned that covalent probes to target Legionella effectors could be of value to study these effectors and contribute to deciphering the complex biology of Legionella infection. Hence we effectively installed a photo-activatable pyridinium warhead on the 5'-OH of triazole-linked ribosylated ubiquitin allowing crosslinking of the probe to the catalytic histidine residues in Legionella SidE or Dup enzymes. In vitro tests on recombinantly expressed DupA and SdeAPDE revealed that the probe was able to capture the enzymes covalently upon photo-activation.
RESUMO
PEX5, the peroxisomal protein shuttling receptor, binds newly synthesized proteins in the cytosol and transports them to the organelle. During its stay at the peroxisomal protein translocon, PEX5 is monoubiquitinated at its cysteine 11 residue, a mandatory modification for its subsequent ATP-dependent extraction back into the cytosol. The reason why a cysteine and not a lysine residue is the ubiquitin acceptor is unknown. Using an established rat liver-based cell-free in vitro system, we found that, in contrast to wild-type PEX5, a PEX5 protein possessing a lysine at position 11 is polyubiquitinated at the peroxisomal membrane, a modification that negatively interferes with the extraction process. Wild-type PEX5 cannot retain a polyubiquitin chain because ubiquitination at cysteine 11 is a reversible reaction, with the E2-mediated deubiquitination step presenting faster kinetics than PEX5 polyubiquitination. We propose that the reversible nonconventional ubiquitination of PEX5 ensures that neither the peroxisomal protein translocon becomes obstructed with polyubiquitinated PEX5 nor is PEX5 targeted for proteasomal degradation.
Assuntos
Cisteína , Lisina , Animais , Ratos , Proteínas de Transporte/metabolismo , Cisteína/metabolismo , Lisina/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/química , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Transporte Proteico , UbiquitinaçãoRESUMO
The coronavirus papain-like protease (PLpro) is crucial for viral replicase polyprotein processing. Additionally, PLpro can subvert host defense mechanisms by its deubiquitinating (DUB) and deISGylating activities. To elucidate the role of these activities during SARS-CoV-2 infection, we introduced mutations that disrupt binding of PLpro to ubiquitin or ISG15. We identified several mutations that strongly reduced DUB activity of PLpro, without affecting viral polyprotein processing. In contrast, mutations that abrogated deISGylating activity also hampered viral polyprotein processing and when introduced into the virus these mutants were not viable. SARS-CoV-2 mutants exhibiting reduced DUB activity elicited a stronger interferon response in human lung cells. In a mouse model of severe disease, disruption of PLpro DUB activity did not affect lethality, virus replication, or innate immune responses in the lungs. This suggests that the DUB activity of SARS-CoV-2 PLpro is dispensable for virus replication and does not affect innate immune responses in vivo. Interestingly, the DUB mutant of SARS-CoV replicated to slightly lower titers in mice and elicited a diminished immune response early in infection, although lethality was unaffected. We previously showed that a MERS-CoV mutant deficient in DUB and deISGylating activity was strongly attenuated in mice. Here, we demonstrate that the role of PLpro DUB activity during infection can vary considerably between highly pathogenic coronaviruses. Therefore, careful considerations should be taken when developing pan-coronavirus antiviral strategies targeting PLpro.
Assuntos
COVID-19 , Proteases Semelhantes à Papaína de Coronavírus , Humanos , Animais , Camundongos , Proteases Semelhantes à Papaína de Coronavírus/genética , SARS-CoV-2/metabolismo , Imunidade Inata , Papaína/genética , Papaína/metabolismo , Peptídeo Hidrolases/metabolismo , Replicação Viral , PoliproteínasRESUMO
Ubiquitin (Ub) chain formation by homologous to E6AP C-terminus (HECT)-family E3 ligases regulates vast biology, yet the structural mechanisms remain unknown. We used chemistry and cryo-electron microscopy (cryo-EM) to visualize stable mimics of the intermediates along K48-linked Ub chain formation by the human E3, UBR5. The structural data reveal a ≈ 620 kDa UBR5 dimer as the functional unit, comprising a scaffold with flexibly tethered Ub-associated (UBA) domains, and elaborately arranged HECT domains. Chains are forged by a UBA domain capturing an acceptor Ub, with its K48 lured into the active site by numerous interactions between the acceptor Ub, manifold UBR5 elements and the donor Ub. The cryo-EM reconstructions allow defining conserved HECT domain conformations catalyzing Ub transfer from E2 to E3 and from E3. Our data show how a full-length E3, ubiquitins to be adjoined, E2 and intermediary products guide a feed-forward HECT domain conformational cycle establishing a highly efficient, broadly targeting, K48-linked Ub chain forging machine.
Assuntos
Ubiquitina-Proteína Ligases , Ubiquitina , Humanos , Ubiquitina/química , Microscopia Crioeletrônica , Ubiquitina-Proteína Ligases/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinas/metabolismo , UbiquitinaçãoRESUMO
Although ubiquitin is found only in eukaryotes, several pathogenic bacteria and viruses possess proteins that hinder the host ubiquitin system. Legionella, a gram-negative intracellular bacterium, possesses an ovarian tumor (OTU) family of deubiquitinases (Lot DUBs). Herein, we describe the molecular characteristics of Lot DUBs. We elucidated the structure of the LotA OTU1 domain and revealed that entire Lot DUBs possess a characteristic extended helical lobe that is not found in other OTU-DUBs. The structural topology of an extended helical lobe is the same throughout the Lot family, and it provides an S1' ubiquitin-binding site. Moreover, the catalytic triads of Lot DUBs resemble those of the A20-type OTU-DUBs. Furthermore, we revealed a unique mechanism by which LotA OTU domains cooperate together to distinguish the length of the chain and preferentially cleave longer K48-linked polyubiquitin chains. The LotA OTU1 domain itself cleaves K6-linked ubiquitin chains, whereas it is also essential for assisting the cleavage of longer K48-linked polyubiquitin chains by the OTU2 domain. Thus, this study provides novel insights into the structure and mechanism of action of Lot DUBs.
Assuntos
Legionella , Neoplasias Ovarianas , Feminino , Humanos , Ubiquitina/metabolismo , Poliubiquitina/química , Poliubiquitina/metabolismo , Legionella/metabolismo , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Neoplasias Ovarianas/genéticaRESUMO
Deubiquitinating enzymes are key regulators in the ubiquitin system and an emerging class of drug targets. These proteases disassemble polyubiquitin chains and many deubiquitinases show selectivity for specific polyubiquitin linkages. However, most biochemical insights originate from studies of single diubiquitin linkages in isolation, whereas in cells all linkages coexist. To better mimick this diubiquitin substrate competition, we develop a multiplexed mass spectrometry-based deubiquitinase assay that can probe all ubiquitin linkage types simultaneously to quantify deubiquitinase activity in the presence of all potential diubiquitin substrates. For this, all eight native diubiquitins are generated and each linkage type is designed with a distinct molecular weight by incorporating neutron-encoded amino acids. Overall, 22 deubiquitinases are profiled, providing a three-dimensional overview of deubiquitinase linkage selectivity over time and enzyme concentration.
Assuntos
Enzimas Desubiquitinantes , Poliubiquitina , Ubiquitinação , Poliubiquitina/metabolismo , Enzimas Desubiquitinantes/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMO
Autophagy is a conserved cellular process involved in the degradation of intercellular materials. During this process, double-membrane vesicles called autophagosomes engulf cytoplasmic components ready for degradation. A key component in the formation of autophagosomes are the autophagy-related (Atg) proteins, including microtubule-associated protein light chain 3A (LC3A) and 3B (LC3B). After the C-terminus of LC3 is conjugated to a phospholipid, it promotes the elongation of the phagosome and provides a docking station for the delivery of proteins ready for degradation. Since dysregulation of the autophagy pathway has been associated with a variety of human diseases, components of this process have been considered as potential therapeutic targets. However, the mechanistic details of LC3-specific ligases and deconjugation enzymes are far from unraveled and chemical tools for activity profiling could aid in affording more insights into this process. Herein, we describe a native chemical ligation approach for the synthesis of two LC3 activity-based probes (ABPs). Initial studies show that the probes covalently interact with the cysteine protease ATG4B, showcasing the potential of these probes to unravel mechanistic and structural details.
RESUMO
Hybrid chains are a combination of ubiquitin (Ub) and Ub-like (UbL) proteins, expanding on the finely tuned Ub code. To decipher this intricate code, understanding of its assembly, architecture, as well as specific interactors of these Ub/UbL hybrid chains are important, warranting the development of suitable reagents. Here, we describe the chemical methodology to access linkage specific non-hydrolyzable Ub-NEDD8-based chains endowed with an affinity handle in all possible combinations of K48 hybrid chain dimers between Ub and NEDD8.
Assuntos
Polímeros , Ubiquitina , Ligação GenéticaRESUMO
Solid-phase peptide synthesis (SPPS) enables the synthesis of chemically modified peptides and proteins. Chemically synthesized ubiquitin(-like) proteins containing a fluorescent tag or reactive warhead have proven to be important tools in elucidating biological processes. Here, we describe the first fully synthetic method for the linear synthesis of two LC3 ubiquitin-like proteins using disaggregating building blocks and heated synthesis. Both LC3A and LC3B were synthesized and equipped with a fluorescent rhodamine tag, followed by folding of the proteins and liquid chromatography-mass spectrometry and SDS-PAGE analysis to prove that the quality of the synthetic material is comparable to expressed material.
Assuntos
Corantes , Temperatura Alta , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , Cromatografia Líquida , UbiquitinasRESUMO
The AAA ATPases PEX1â¢PEX6 extract PEX5, the peroxisomal protein shuttling receptor, from the peroxisomal membrane so that a new protein transport cycle can start. Extraction requires ubiquitination of PEX5 at residue 11 and involves a threading mechanism, but how exactly this occurs is unclear. We used a cell-free in vitro system and a variety of engineered PEX5 and ubiquitin molecules to challenge the extraction machinery. We show that PEX5 modified with a single ubiquitin is a substrate for extraction and extend previous findings proposing that neither the N- nor the C-terminus of PEX5 are required for extraction. Chimeric PEX5 molecules possessing a branched polypeptide structure at their C-terminal domains can still be extracted from the peroxisomal membrane thus suggesting that the extraction machinery can thread more than one polypeptide chain simultaneously. Importantly, we found that the PEX5-linked monoubiquitin is unfolded at a pre-extraction stage and, accordingly, an intra-molecularly cross-linked ubiquitin blocked extraction when conjugated to residue 11 of PEX5. Collectively, our data suggest that the PEX5-linked monoubiquitin is the extraction initiator and that the complete ubiquitin-PEX5 conjugate is threaded by PEX1â¢PEX6.
Assuntos
Proteínas de Membrana , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos , Ubiquitina , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Membrana/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Peroxissomos/metabolismo , Transporte Proteico , Ubiquitina/metabolismo , Ubiquitinação , Humanos , Sistema Livre de CélulasRESUMO
Development of (semi-)synthetic methods to prepare ubiquitin (Ub)-based reagents has proven to be helpful in the classification of deubiquitinating proteases (DUBs). To study DUB selectivity for one or more of the eight possible poly-Ub chains, fluorogenic assay reagents have been reported relying on the appearance of a fluorescent signal upon DUB-mediated cleavage of the reagent. In this protocol, we describe the use of such an assay to profile the selectivity of TRABID, a member of the OTU family of DUBs.
Assuntos
Corantes Fluorescentes , Ubiquitinas , Ubiquitinas/metabolismo , Ubiquitina/metabolismo , Endopeptidases/metabolismo , Peptídeo Hidrolases/metabolismo , UbiquitinaçãoRESUMO
Small ubiquitin-like modifiers (SUMOs) are conjugated to protein substrates in cells to regulate their function. The attachment of SUMO family members SUMO1-3 to substrate proteins is reversed by specific isopeptidases called SENPs (sentrin-specific protease). Whereas SENPs are SUMO-isoform or linkage type specific, comprehensive analysis is missing. Furthermore, the underlying mechanism of SENP linkage specificity remains unclear. We present a high-throughput synthesis of 83â isopeptide-linked SUMO-based fluorescence polarization reagents to study enzyme preferences. The assay reagents were synthesized via a native chemical ligation-desulfurization protocol between 11-mer peptides containing a γ-thiolysine and a SUMO3 thioester. Subsequently, five recombinantly expressed SENPs were screened using these assay reagents to reveal their deconjugation activity and substrate preferences. In general, we observed that SENP1 is the most active and nonselective SENP while SENP6 and SENP7 show the least activity. Furthermore, SENPs differentially process peptides derived from SUMO1-3, who form a minimalistic representation of diSUMO chains. To validate our findings, five distinct isopeptide-linked diSUMO chains were chemically synthesized and proteolysis was monitored using a gel-based read-out.
Assuntos
Corantes Fluorescentes , Ensaios de Triagem em Larga Escala , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Endopeptidases/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Proteólise , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/síntese química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/químicaRESUMO
Ubiquitination is a dynamic post-translational modification that regulates virtually all cellular processes by modulating function, localization, interactions and turnover of thousands of substrates. Canonical ubiquitination involves the enzymatic cascade of E1, E2 and E3 enzymes that conjugate ubiquitin to lysine residues giving rise to monomeric ubiquitination and polymeric ubiquitination. Emerging research has established expansion of the ubiquitin code by non-canonical ubiquitination of N-termini and cysteine, serine and threonine residues. Generic methods for identifying ubiquitin substrates using mass spectrometry based proteomics often overlook non-canonical ubiquitinated substrates, suggesting that numerous undiscovered substrates of this modification exist. Moreover, there is a knowledge gap between in vitro studies and comprehensive understanding of the functional consequence of non-canonical ubiquitination in vivo. Here, we discuss the current knowledge about non-lysine ubiquitination, strategies to map the ubiquitinome and their applicability for studying non-canonical ubiquitination substrates and sites. Furthermore, we elucidate the available chemical biology toolbox and elaborate on missing links required to further unravel this less explored subsection of the ubiquitin system.
RESUMO
We describe the development and optimization of a methodology to prepare peptides and proteins modified on the arginine residue with an adenosine-di-phosphate-ribosyl (ADPr) group. Our method comprises reacting an ornithine containing polypeptide on-resin with an α-linked anomeric isothiourea N-riboside, ensuing installment of a phosphomonoester at the 5'-hydroxyl of the ribosyl moiety followed by the conversion into the adenosine diphosphate. We use this method to obtain four regioisomers of ADP-ribosylated ubiquitin (UbADPr), each modified with an ADP-ribosyl residue on a different arginine position within the ubiquitin (Ub) protein (Arg42, Arg54, Arg72, and Arg74) as the first reported examples of fully synthetic arginine-linked ADPr-modified proteins. We show the chemically prepared Arg-linked UbADPr to be accepted and processed by Legionella enzymes and compare the entire suite of four Arg-linked UbADPr regioisomers in a variety of biochemical experiments, allowing us to profile the activity and selectivity of Legionella pneumophila ligase and hydrolase enzymes.
Assuntos
Adenosina Difosfato Ribose , Arginina , Adenosina Difosfato Ribose/química , Arginina/metabolismo , ADP-Ribosilação , Ubiquitina/química , Proteínas Ubiquitinadas/metabolismo , Peptídeos/químicaRESUMO
Chemical protein synthesis has proven to be a powerful tool to access homogenously modified proteins. The chemical synthesis of nanobodies (Nb) would create possibilities to design tailored Nbs with a range of chemical modifications such as tags, linkers, reporter groups, and subsequently, Nb-drug conjugates. Herein, we describe the total chemical synthesis of a 123 amino-acid Nb against GFP. A native chemical ligation- desulfurization strategy was successfully applied for the synthesis of this GFP Nb, modified with a propargyl (PA) moiety for on-demand functionalization. Biophysical characterization indicated that the synthetic GFP Nb-PA was correctly folded after internal disulfide bond formation. The synthetic Nb-PA was functionalized with a biotin or a sulfo-cyanine5 dye by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), resulting in two distinct probes used for functional inâ vitro validation in pull-down and confocal microscopy settings.
Assuntos
Azidas , Anticorpos de Domínio Único , Alcinos/química , Azidas/química , Biotina , Química Click , Cobre/química , Dissulfetos , Proteínas/químicaRESUMO
The ubiquitin-proteasome system is an essential regulator of many cellular processes including controlling protein homeostasis. The degradation of proteins by the multi-subunit proteasome complex is tightly regulated through a series of checkpoints, amongst which are a set of deubiquitinating proteases (DUBs). The proteasome-associated DUBs, UCH-L5 (Ubiquitin carboxyl-terminal hydrolase isozyme L5) and USP14 (Ubiquitin-specific protease 14), and the integral-DUB in the proteasome, Rpn11, is known to regulate proteasomal degradation by deubiquitination of distinct substrates. Although selective inhibitors for USP14 and Rpn11 have been recently developed, there are no known inhibitors that selectively bind to UCH-L5. The X-ray structure of the Ubiquitin (Ub) bound to UCH-L5 shows a ß-sheet hairpin in Ub that contains a crucial hydrophobic patch involved in the interaction with UCH-L5. Herein, we designed and developed both a Ub sequence-based linear- and cyclic- ß-sheet hairpin peptide that was found to preferably inhibit UCH-L5. We show that these peptides have low micromolar IC50 values and the cyclic peptide competes with the activity-based UbVME (Ubiquitin-Vinyl-Methyl-Ester) probe for UCH-L5, binding in a concentration-dependent manner. We further establish the selectivity profile of the cyclic peptide for UCH-L5 compared to other members of the UCH-DUB family and other cysteine DUBs in cell lysate. Furthermore, the cyclic peptide infiltrated cells resulting in the accumulation of polyUb chains, and was found to be non-toxic at the concentrations used here. Taken together, our data suggest that the cyclic peptide permeates the cell membrane, inhibits UCH-L5 by possibly blocking its deubiquitinating function, and contributes to the accumulation of polyubiquitinated substrates. The implications of inhibiting UCH-L5 in the context of the 26S proteasome render it an attractive candidate for further development as a potential selective inhibitor for therapeutic purposes.
RESUMO
BACKGROUND: Cross-presentation of exogenous antigens in HLA-class I molecules by professional antigen presenting cells (APCs) is crucial for CD8+ T cell function. Recent murine studies show that several non-professional APCs, including cancer-associated fibroblasts (CAFs) also possess this capacity. Whether human CAFs are able to cross-present exogenous antigen, which molecular pathways are involved in this process and how this ultimately affects tumor-specific CD8+ T cell function is unknown. METHODS: In this study, we investigated the ability of human colorectal cancer (CRC)-derived CAFs to cross-present neoantigen-derived synthetic long peptides (SLPs), corresponding to tumor-derived mutant peptides, and how this affects tumor-specific T-cell function. Processing of the SLP was studied by targeting components of the cross-presentation machinery through CRISPR/Cas9 and siRNA-mediated genetic ablation to identify the key molecules involved in fibroblast-mediated cross-presentation. Multispectral flow cytometry and killing assays were performed to study the effect of fibroblast cross-presentation on T cell function. RESULTS: Here, we show that human CRC-derived CAFs display an enhanced capacity to cross-present neoantigen-derived SLPs when compared with normal colonic fibroblasts. Cross-presentation of antigens by fibroblasts involved the lysosomal protease cathepsin S. Cathepsin S expression by CAFs was detected in situ in human CRC tissue, was upregulated in ex vivo cultured CRC-derived CAFs and showed increased expression in normal fibroblasts after exposure to CRC-conditioned medium. Cognate interaction between CD8+ T cells and cross-presenting CAFs suppressed T cell function, reflected by decreased cytotoxicity, reduced activation (CD137) and increased exhaustion (TIM3, LAG3 and CD39) marker expression. CONCLUSION: These data indicate that CAFs may directly suppress tumor-specific T cell function in an antigen-dependent fashion in human CRC.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias Colorretais , Animais , Fibroblastos Associados a Câncer/metabolismo , Catepsinas , Neoplasias Colorretais/genética , Apresentação Cruzada , Humanos , Lisossomos/metabolismo , Camundongos , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Regulação para CimaRESUMO
Protein ubiquitination is a key post-translational modification in regulating many fundamental cellular processes and dysregulation of these processes can give rise to a vast array of diseases. Unravelling the molecular mechanisms of ubiquitination hence is an important area in current ubiquitin research with as aim to understand this enigmatic process. The complexity of ubiquitin (Ub) signaling arises from the large variety of Ub conjugates, where Ub is attached to other Ub proteins, Ub-like proteins, and protein substrates. The chemical preparation of such Ub conjugates in high homogeneity and in adequate amounts contributes greatly to the deciphering of Ub signaling. The strength of these chemically synthesized conjugates lies in the chemo-selectivity in which they can be created that are sometimes difficult to obtain using biochemical methodology. In this review, we will discuss the progress in the chemical protein synthesis of state-of-the-art Ub and Ub-like chemical probes, their unique concepts and related discoveries in the ubiquitin field.
Assuntos
Processamento de Proteína Pós-Traducional , Ubiquitina , Ubiquitina/metabolismo , Ubiquitinação , Proteínas/metabolismo , Transdução de SinaisRESUMO
Tuberculosis is a global health problem caused by infection with the Mycobacterium tuberculosis (Mtb) bacteria. Although antibiotic treatment has dramatically reduced the impact of tuberculosis on the population, the existence and spreading of drug resistant strains urgently demands the development of new drugs that target Mtb in a different manner than currently used antibiotics. The prokaryotic ubiquitin-like protein (Pup) proteasome system is an attractive target for new drug development as it is unique to Mtb and related bacterial genera. Using a Pup-based fluorogenic substrate, we screened for inhibitors of Dop, the Mtb depupylating protease, and identified I-OMe-Tyrphostin AG538 (1) and Tyrphostin AG538 (2). The hits were validated and determined to be fast-reversible, non-ATP competitive inhibitors. We synthesized >25 analogs of 1 and 2 and show that several of the synthesized compounds also inhibit the depupylation actions of Dop on native substrate, FabD-Pup. Importantly, the pupylation activity of PafA, the sole Pup ligase in Mtb, was also inhibited by some of these compounds.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Desenvolvimento de Medicamentos , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tirfostinas/farmacologia , Ubiquitinas/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/enzimologia , Relação Estrutura-Atividade , Tirfostinas/síntese química , Tirfostinas/química , Ubiquitinas/metabolismoRESUMO
Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.