The SARS‐CoV‐2 coronavirus encodes an essential papain‐like protease domain as part of its non‐structural protein (nsp)‐3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ...ubiquitin‐like ISG15 protein modifications as well as, with lower activity, Lys48‐linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin‐binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non‐covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self‐processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS‐CoV‐2 infection model.
Synopsis
Crystal structures explain the specificity of SARS‐CoV‐2 papain‐like protease, PLpro, for ISG15 and Lys48‐linked diubiquitin, and specific inhibition of PLpro by small molecules, shows strong antiviral effects.
SARS‐CoV-2 PLpro preferentially cleaves ISG15 and also targets longer Lys48‐linked ubiquitin chains.
Preference for ISG15 is provided by the S1 ubiquitin binding site in PLpro, while Lys48‐polyubiquitin specificity is provided by the S2 ubiquitin binding site.
In a high‐throughput screen, FDA approved drugs and late stage clinical compounds are unable to inhibit PLpro in vitro.
Known SARS PLpro inhibitors also target SARS2 PLpro and show anti‐viral efficacy.
Crystal structure and biochemical analysis explains the specificity of SARS‐CoV‐2 PLpro for ISG15 and longer Lys48‐linked ubiquitin chains leading to the identification of inhibitors that show promising antiviral activity in a SARS‐CoV‐2 infection model.
The E3 ubiquitin ligase Parkin is a key effector of the removal of damaged mitochondria by mitophagy. Parkin determines cell fate in response to mitochondrial damage, with its loss promoting early ...onset Parkinson's disease and potentially also cancer progression. Controlling a cell's apoptotic response is essential to co‐ordinate the removal of damaged mitochondria. We report that following mitochondrial damage‐induced mitophagy, Parkin directly ubiquitinates the apoptotic effector protein BAK at a conserved lysine in its hydrophobic groove, a region that is crucial for BAK activation by BH3‐only proteins and its homo‐dimerisation during apoptosis. Ubiquitination inhibited BAK activity by impairing its activation and the formation of lethal BAK oligomers. Parkin also suppresses BAX‐mediated apoptosis, but in the absence of BAX ubiquitination suggesting an indirect mechanism. In addition, we find that BAK‐dependent mitochondrial outer membrane permeabilisation during apoptosis promotes PINK1‐dependent Parkin activation. Hence, we propose that Parkin directly inhibits BAK to suppress errant apoptosis, thereby allowing the effective clearance of damaged mitochondria, but also promotes clearance of apoptotic mitochondria to limit their potential pro‐inflammatory effect.
Synopsis
The ubiquitin ligase Parkin plays a protective role in neurodegenerative disease by removing damaged mitochondria and preventing apoptosis, and by limiting the functions of pro‐apoptotic effector proteins BAK and BAX. Defective control of apoptosis may contribute to the pathogenesis of early onset Parkinson's Disease caused by Parkin mutations.
Parkin activity is induced by mitochondrial damage during apoptosis.
Parkin mono‐ and di‐ubiquitinates BAK at a conserved lysine in its hydrophobic groove.
Ubiquitination reduces BAK oligomerisation and apoptotic activity on mitochondria.
Parkin prevents BAX mitochondrial localisation and apoptotic activity independent of ubiquitination.
Certain Parkinson's Disease‐associated Parkin mutants cannot ubiquitinate BAK and restrain it on mitochondria.
Autophagic clearance of damaged mitochondria and suppression of apoptotic effector proteins are coordinated functions of the Parkin ubiquitin ligase that are lost upon Parkinson's Disease‐associated mutations.
Parkin and mitophagy in cancer Bernardini, J P; Lazarou, M; Dewson, G
Oncogene,
03/2017, Letnik:
36, Številka:
10
Journal Article
Recenzirano
Mitophagy, the selective engulfment and clearance of mitochondria, is essential for the homeostasis of a healthy network of functioning mitochondria and prevents excessive production of cytotoxic ...reactive oxygen species from damaged mitochondria. The mitochondrially targeted PTEN-induced kinase-1 (PINK1) and the E3 ubiquitin ligase Parkin are well-established synergistic mediators of the mitophagy of dysfunctional mitochondria. This pathway relies on the ubiquitination of a number of mitochondrial outer membrane substrates and subsequent docking of autophagy receptor proteins to selectively clear mitochondria. There are also alternate Parkin-independent mitophagy pathways mediated by BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 and Nip-3 like protein X as well as other effectors. There is increasing evidence that ablation of mitophagy accelerates a number of pathologies. Familial Parkinsonism is associated with loss-of-function mutations in PINK1 and Parkin. A growing number of studies have observed a correlation between impaired Parkin activity and enhanced cancer development, leading to the emerging concept that Parkin activity, or mitophagy in general, is a tumour suppression mechanism. This review examines the molecular mechanisms of mitophagy and highlights the potential links between Parkin and the hallmarks of cancer that may influence tumour development and progression.
An increasing number of genetic diseases are linked to deregulation of E3 ubiquitin ligases. Loss-of-function mutations in the RING-between-RING (RBR) family E3 ligase RNF216 (TRIAD3) cause ...Gordon-Holmes syndrome (GHS) and related neurodegenerative diseases. Functionally, RNF216 assembles K63-linked ubiquitin chains and has been implicated in regulation of innate immunity signaling pathways and synaptic plasticity. Here, we report crystal structures of key RNF216 reaction states including RNF216 in complex with ubiquitin and its reaction product, K63 di-ubiquitin. Our data provide a molecular explanation for chain-type specificity and reveal the molecular basis for disruption of RNF216 function by pathogenic GHS mutations. Furthermore, we demonstrate how RNF216 activity and chain-type specificity are regulated by phosphorylation and that RNF216 is allosterically activated by K63-linked di-ubiquitin. These molecular insights expand our understanding of RNF216 function and its role in disease and further define the mechanistic diversity of the RBR E3 ligase family.
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•Structures of the E3 ligase RNF216 explain K63 ubiquitin chain-type specificity•RNF216 catalytic activity and chain-type specificity are enhanced by phosphorylation•RNF216 is allosterically activated by K63-linked di-ubiquitin•Human disease-causing mutations map to ubiquitin-interacting sites
Cotton et al. report crystal structures of the RBR E3 ubiquitin ligase RNF216, explaining its specificity for generating K63-linked ubiquitin chains and the molecular effect of Gordon-Holmes syndrome mutations. Phosphorylation of RNF216 enhances ligase activity and ubiquitin chain-type specificity. Furthermore, RNF216 is allosterically activated by K63 di-ubiquitin.
Wide variation in mortality rates among critically ill patients with coronavirus disease 2019 (COVID-19) has been reported. This study evaluated whether healthcare-associated infections (HAI) are a ...risk factor for death among patients with severe COVID-19 in the intensive care unit (ICU).
This retrospective cohort study included patients with severe COVID-19 hospitalized in the ICU of four hospitals in the city of Curitiba, Brazil. Patients with COVID-19 who died during ICU hospitalization were compared with those who were discharged. A second analysis compared patients who developed HAI in the ICU with those who did not. Multiple logistic regression models were used to control for confounders.
In total, 400 patients were included, and 123 (31%) patients developed HAI. The most common HAI was lower respiratory tract infection (67%). Independent risk factors for death were: age odds ratio (OR) 1.75, 95% confidence interval (CI) 1.43–2.15; P<0.0001; clinical severity score (OR 2.21, 95% CI 1.70–2.87; P<0.0001); renal replacement therapy (OR 12.8, 95% CI 5.78–28.6; P<0.0001); and HAI (OR 5.9, 95% CI 3.31–10.5; P<0.0001). A longer interval between symptom onset and hospital admission was protective against death (OR 0.93, 95% CI 0.88–0.98; P=0.017). The only independent factors associated with HAI were high C-reactive protein and low PaO2/FiO2 ratio.
No factors that could point to a high-risk group for HAI acquisition were identified. However, age, dialysis and HAI increased the risk of death in ICU patients with severe COVID-19; of these, HAI is the only preventable risk factor.
This paper presents a model developed in the Netherlands for the estimation of damage caused by floods. The model attempts to fill the gap in the international literature about integrated flood ...damage modelling and develop an integrated framework for the assessment of both direct hazard-induced damages and indirect economic damages such as the interruption of production flows outside the flood affected area, as well as loss of life due to flooding. The scale of damage assessment varies from a specified flood-prone area in a river basin or a coastal region to the country's entire economy. The integrative character of the presented model is featured by the combination of information on land use and economic data, and data on flood characteristics and stage-damage functions, where the geographical dimension is supported by modern GIS to obtain a damage estimate for various damage categories. The usefulness of the model is demonstrated in a case study estimating expected flood damage in the largest flood-prone area in the Netherlands.
ABSTRACT The events recorded by ARGO-YBJ in more than five years of data collection have been analyzed to determine the diffuse gamma-ray emission in the Galactic plane at Galactic longitudes 25° < l ...< 100° and Galactic latitudes . The energy range covered by this analysis, from ∼350 GeV to ∼2 TeV, allows the connection of the region explored by Fermi with the multi-TeV measurements carried out by Milagro. Our analysis has been focused on two selected regions of the Galactic plane, i.e., 40° < l < 100° and 65° < l < 85° (the Cygnus region), where Milagro observed an excess with respect to the predictions of current models. Great care has been taken in order to mask the most intense gamma-ray sources, including the TeV counterpart of the Cygnus cocoon recently identified by ARGO-YBJ, and to remove residual contributions. The ARGO-YBJ results do not show any excess at sub-TeV energies corresponding to the excess found by Milagro, and are consistent with the predictions of the Fermi model for the diffuse Galactic emission. From the measured energy distribution we derive spectral indices and the differential flux at 1 TeV of the diffuse gamma-ray emission in the sky regions investigated.
The extended TeV gamma-ray source ARGO J2031+4157 (or MGRO J2031+41) is positionally consistent with the Cygnus Cocoon discovered by Fermi-LAT at GeV energies in the Cygnus superbubble. Reanalyzing ...the ARGO-YBJ data collected from 2007 November to 2013 January, the angular extension and energy spectrum of ARGO J2031+4157 are evaluated. After subtracting the contribution of the overlapping TeV sources, the ARGO-YBJ excess map is fitted with a two-dimensional Gaussian function in a square region of 10degrees x 10degrees, finding a source extension sigma sub(ext)= 1degrees.8 + or - 0degrees.5. The observed differential energy spectrum is dN/dE = (2.5 + or - 0.4) x 10 super(-11) (E/1 TeV) super(-2.6+ or -0.3) photons cm super(-2) s super(-1) TeV super(-1), in the energy range 0.2-10 TeV. The angular extension is consistent with that of the Cygnus Cocoon as measured by Fermi-LAT and the spectrum also shows a good connection with the one measured in the 1-100 GeV energy range. These features suggest to identify ARGO J2031+4157 as the counterpart of the Cygnus Cocoon at TeV energies. The Cygnus Cocoon, located in the star-forming region of Cygnus X, is interpreted as a cocoon of freshly accelerated cosmic rays related to the Cygnus superbubble. The spectral similarity with supernova remnants (SNRs) indicates that the particle acceleration inside a superbubble is similar to that in an SNR. The spectral measurements from 1 GeV to 10 TeV allows for the first time to determine the possible spectrum slope of the underlying particle distribution. A hadronic model is adopted to explain the spectral energy distribution.
ABSTRACT This paper reports on the measurement of the large-scale anisotropy in the distribution of cosmic-ray arrival directions using the data collected by the air shower detector ARGO-YBJ from ...2008 January to 2009 December, during the minimum of solar activity between cycles 23 and 24. In this period, more than 2 × 1011 showers were recorded with energies between ∼1 and 30 TeV. The observed two-dimensional distribution of cosmic rays is characterized by two wide regions of excess and deficit, respectively, both of relative intensity ∼10−3 with respect to a uniform flux, superimposed on smaller size structures. The harmonic analysis shows that the large-scale cosmic-ray relative intensity as a function of R.A. can be described by the first and second terms of a Fouries series. The high event statistics allow the study of the energy dependence of the anistropy, showing that the amplitude increases with energy, with a maximum intensity at ∼10 TeV, and then decreases while the phase slowly shifts toward lower values of R.A. with increasing energy. The ARGO-YBJ data provide accurate observations over more than a decade of energy around this feature of the anisotropy spectrum.
Inhibitors of apoptosis (IAPs) proteins are critical regulators of innate immune signaling pathways and therefore have potential as drug targets. X-linked IAP (XIAP) and cellular IAP1 and IAP2 (cIAP1 ...and cIAP2) are E3 ligases that have been shown to be required for signaling downstream of NOD2, an intracellular receptor for bacterial peptidoglycan. We used genetic and biochemical approaches to compare the responses of IAP-deficient mice and cells to NOD2 stimulation. In all cell types tested, XIAP is the only IAP required for signaling immediately downstream of NOD2, while cIAP1 and cIAP2 are dispensable for NOD2-induced nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) activation. However, mice lacking cIAP1 or TNFR1 have a blunted cytokine response to NOD2 stimulation. We conclude that cIAPs regulate NOD2-dependent autocrine TNF signaling in vivo and highlight the importance of physiological context in the interplay of innate immune signaling pathways.
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•XIAP is the only IAP required for NF-κB and MAPK activation downstream of NOD2•NOD2-driven cytokine production relies on a TNF-dependent amplification loop•Dendritic-like cells are the first responders to MDP stimulation in vivo
Stafford et al. show that XIAP is the only IAP required for the initial RIPK2/NOD2-dependent response to MDP, while autocrine TNF is required to amplify cytokine production in a cIAP1-dependent manner.
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