Background and Aims In patients with acute liver failure (ALF) who suffer from massive hepatocyte loss, liver progenitor cells (LPCs) take over key hepatocyte functions, which ultimately determines... Show moreBackground and Aims In patients with acute liver failure (ALF) who suffer from massive hepatocyte loss, liver progenitor cells (LPCs) take over key hepatocyte functions, which ultimately determines survival. This study investigated how the expression of hepatocyte nuclear factor 4 alpha (HNF4 alpha), its regulators, and targets in LPCs determines clinical outcome of patients with ALF. Approach and Results Clinicopathological associations were scrutinized in 19 patients with ALF (9 recovered and 10 receiving liver transplantation). Regulatory mechanisms between follistatin, activin, HNF4 alpha, and coagulation factor expression in LPC were investigated in vitro and in metronidazole-treated zebrafish. A prospective clinical study followed up 186 patients with cirrhosis for 80 months to observe the relevance of follistatin levels in prevalence and mortality of acute-on-chronic liver failure. Recovered patients with ALF robustly express HNF4 alpha in either LPCs or remaining hepatocytes. As in hepatocytes, HNF4 alpha controls the expression of coagulation factors by binding to their promoters in LPC. HNF4 alpha expression in LPCs requires the forkhead box protein H1-Sma and Mad homolog 2/3/4 transcription factor complex, which is promoted by the TGF-beta superfamily member activin. Activin signaling in LPCs is negatively regulated by follistatin, a hepatocyte-derived hormone controlled by insulin and glucagon. In contrast to patients requiring liver transplantation, recovered patients demonstrate a normal activin/follistatin ratio, robust abundance of the activin effectors phosphorylated Sma and Mad homolog 2 and HNF4 alpha in LPCs, leading to significantly improved coagulation function. A follow-up study indicated that serum follistatin levels could predict the incidence and mortality of acute-on-chronic liver failure. Conclusions These results highlight a crucial role of the follistatin-controlled activin-HNF4 alpha-coagulation axis in determining the clinical outcome of massive hepatocyte loss-induced ALF. The effects of insulin and glucagon on follistatin suggest a key role of the systemic metabolic state in ALF. Show less
Mukherjee, R.; Bhattacharya, A.; Bojkova, D.; Mehdipour, A.R.; Shin, D.; Khan, K.S.; ... ; Dikic, I. 2021
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 ... Show moreApart 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-kappa B 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. Show less
Kim, R.Q.; Misra, M.; Gonzalez, A.; Tomaskovic, I.; Shin, D.; Schindelin, H.; ... ; Noort, G.J.V. van 2020
Legionnaires' disease is caused by infection with the intracellularly replicating Gram-negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host... Show moreLegionnaires' disease is caused by infection with the intracellularly replicating Gram-negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host proteins by generating a phosphoribosyl linkage between substrate proteins and ubiquitin by making use of an ADPribosylated ubiquitin (Ub(ADPr)) intermediate. The family of SidE effector enzymes that catalyze this reaction is counteracted by Legionella hydrolases, which are called Dups. This unusual ubiquitination process is important for Legionella proliferation and understanding these processes on a molecular level might prove invaluable in finding new treatments. Herein, a modular approach is used for the synthesis of triazole-linked Ub(ADPr), and analogues thereof, and their affinity towards the hydrolase DupA is determined and hydrolysis rates are compared to natively linked Ub(ADPr). The inhibitory effects of modified Ub on the canonical eukaryotic E1-enzyme Uba1 are investigated and rationalized in the context of a high-resolution crystal structure reported herein. Finally, it is shown that synthetic Ub(ADPr) analogues can be used to effectively pull-down overexpressed DupA from cell lysate. Show less
Legionella pneumophila causes a severe pneumonia known as Legionnaires' disease. During the infection, Legionella injects more than 300 effector proteins into host cells. Among them are enzymes... Show moreLegionella pneumophila causes a severe pneumonia known as Legionnaires' disease. During the infection, Legionella injects more than 300 effector proteins into host cells. Among them are enzymes involved in altering the host-ubiquitination system. Here, we identified two LegionellaOTU (ovarian tumor)-like deubiquitinases (LOT-DUBs; LotB [Lpg1621/Ceg23] and LotC [Lpg2529]). The crystal structure of the LotC catalytic core (LotC(14-310)) was determined at 2.4 angstrom. Unlike the classical OTU-family, the LOT-family shows an extended helical lobe between the Cysloop and the variable loop, which defines them as a unique class of OTU-DUBs. LotB has an additional ubiquitin-binding site (S1'), which enables the specific cleavage of Lys63-linked polyubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of LotB and LotC identified different categories of host-interacting proteins and substrates. Together, our results provide new structural insights into bacterial OTU-DUBs and indicate distinct roles in host-pathogen interactions. Show less
Kim, R.Q.; Misra, M.; Gonzalez, A.; Tomašković, I.; Shin, D.; Schindelin, H.; ... ; Heden- van Noort, G.J. van der 2020
Legionnaires’ disease is caused by infection with the intracellularly replicating Gram‐negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host... Show moreLegionnaires’ disease is caused by infection with the intracellularly replicating Gram‐negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host proteins by generating a phosphoribosyl linkage between substrate proteins and ubiquitin by making use of an ADPribosylated ubiquitin (UbADPr) intermediate. The family of SidE effector enzymes that catalyze this reaction is counteracted by Legionella hydrolases, which are called Dups. This unusual ubiquitination process is important for Legionella proliferation and understanding these processes on a molecular level might prove invaluable in finding new treatments. Herein, a modular approach is used for the synthesis of triazole‐linked UbADPr, and analogues thereof, and their affinity towards the hydrolase DupA is determined and hydrolysis rates are compared to natively linked UbADPr. The inhibitory effects of modified Ub on the canonical eukaryotic E1‐enzyme Uba1 are investigated and rationalized in the context of a high‐resolution crystal structure reported herein. Finally, it is shown that synthetic UbADPr analogues can be used to effectively pull‐down overexpressed DupA from cell lysate. Show less
Biochemical, structural and functional studies on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) papain-like protease PLpro reveal that it regulates host antiviral responses by... Show moreBiochemical, structural and functional studies on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) papain-like protease PLpro reveal that it regulates host antiviral responses by preferentially cleaving the ubiquitin-like interferon-stimulated gene 15 protein (ISG15) and identify this protease as a potential therapeutic target for coronavirus disease 2019 (COVID-19).The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread(1,2). PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses(3-5). Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-kappa B pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity. Show less
