17 research outputs found

    Characterisation of peroxisomes in the fission Yeast Schizosaccharomyces pombe and slime mold Dictyostelium discoideum

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    Peroxisome is a compartment that is found in most eukaryotic organisms' cells. It has several crucial roles, such as fatty acid beta (╬▓) oxidation and hydrogen peroxide (H2O2) detoxification. It contains many essential enzymes, including oxidase and catalase, and has several metabolic and non-metabolic pathways, depending on the environment and the organisms within its cells. This study investigates the role of peroxisomes in two organisms, S. pombe and D. discoideum. Although S. pombe is a well-studied yeast, there is only one study of this yeast that has focused on peroxisomes. This study offers a few crucial observations, including that S. pombe contains peroxisomes, that GFP containing a well-characterized PTS1 (SKL) is efficiently imported, and that peroxisome numbers increase in cells grown on a fatty acid as the sole carbon source, suggesting a role for peroxisomes in fatty acid degradation. The starting point in my research was initially a bioinformatics screen. This screening recognized the enzymes imported into peroxisomes based on the presence of a potential peroxisomal targeting signal. A few proteins were found. However, the low number of proteins with a classical PTS might be the result of different targeting signals that are not recognized by our bioinformatics parameters. Indeed, in other organisms, there are proteins without PTS1 that still use Pex5 for import. The first example is S. cerevisiae Acyl-CoA oxidase. In a global yeast two-hybrid screen, S. pombe Pex5 was found to bind S. pombe Str3 and Lys3. Consequently, we think that there is conserved targeting of a peroxisomal protein lacking a PTS1 and PTS2 imported into the peroxisome by Pex5. One of these is the Str3 case. Interestingly, proteins involved in peroxisomal fatty acid ╬▓ -oxidation are absent from the S. pombe genome, casting doubt on the conclusions from the previous study and explaining the low number of potential peroxisomal enzymes. In D. discoideum, this study investigates the dynamic regulation of peroxisome numbers in response to growth conditions and identifies peroxisomal import and contents through a proximity labeling approach (BioID). Overall, this study sheds light on the roles and regulation of peroxisomes in these two organisms

    The Viral Macrodomain Counters Host Antiviral ADP-Ribosylation

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    This work is licensed under a Creative Commons Attribution 4.0 International License.Macrodomains, enzymes that remove ADP-ribose from proteins, are encoded by several families of RNA viruses and have recently been shown to counter innate immune responses to virus infection. ADP-ribose is covalently attached to target proteins by poly-ADP-ribose polymerases (PARPs), using nicotinamide adenine dinucleotide (NAD+) as a substrate. This modification can have a wide variety of effects on proteins including alteration of enzyme activity, proteinÔÇôprotein interactions, and protein stability. Several PARPs are induced by interferon (IFN) and are known to have antiviral properties, implicating ADP-ribosylation in the host defense response and suggesting that viral macrodomains may counter this response. Recent studies have demonstrated that viral macrodomains do counter the innate immune response by interfering with PARP-mediated antiviral defenses, stress granule formation, and pro-inflammatory cytokine production. Here, we will describe the known functions of the viral macrodomains and review recent literature demonstrating their roles in countering PARP-mediated antiviral responses

    Coronavirus infection and PARP expression dysregulate the NAD metabolome: An actionable component of innate immunity

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    Poly(ADP-ribose) polymerase (PARP) superfamily members covalently link either a single ADP-ribose (ADPR) or a chain of ADPR units to proteins using NAD as the source of ADPR. Although the well-known poly(ADP-ribosylating) (PARylating) PARPs primarily function in the DNA damage response, many noncanonical mono(ADP-ribosylating) (MARylating) PARPs are associated with cellular antiviral responses. We recently demonstrated robust up-regulation of several PARPs following infection with murine hepatitis virus (MHV), a model coronavirus. Here we show that SARS-CoV-2 infection strikingly up-regulates MARylating PARPs and induces the expression of genes encoding enzymes for salvage NAD synthesis from nicotinamide (NAM) and nicotinamide riboside (NR), while down-regulating other NAD biosynthetic pathways. We show that overexpression of PARP10 is sufficient to depress cellular NAD and that the activities of the transcriptionally induced enzymes PARP7, PARP10, PARP12 and PARP14 are limited by cellular NAD and can be enhanced by pharmacological activation of NAD synthesis. We further demonstrate that infection with MHV induces a severe attack on host cell NAD+ and NADP+. Finally, we show that NAMPT activation, NAM, and NR dramatically decrease the replication of an MHV that is sensitive to PARP activity. These data suggest that the antiviral activities of noncanonical PARP isozyme activities are limited by the availability of NAD and that nutritional and pharmacological interventions to enhance NAD levels may boost innate immunity to coronaviruses

    SARS-CoV-2 Mac1 is an essential virulence factor

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    Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in this set of proteins is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions (ΔMac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV ΔMac1 BACs, SARS-CoV-2 ΔMac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 ΔMac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, ΔMac1 was quickly cleared causing minimal pathology without any morbidity. ΔMac1 SARS-CoV-2 induced increased levels of interferon (IFN) and interferon-stimulated gene (ISG) expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. ΔMac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target.National Institutes of Health (NIH) grant P20GM103648 (RC) National Institutes of Health (NIH) grant 2P01AI060699 (LE) National Institutes of Health (NIH) grant P20GM113117 (ARF) National Institutes of Health (NIH) grant K22AI134993 (ARF) National Institutes of Health (NIH) grant R35GM138029 (ARF) National Science Foundation (NSF) grant 2135167 (RLU) University of Kansas General Research Fund (GRF) and Start-up funds (ARF) NIH Graduate Training at the Biology-Chemistry Interface grant T32GM132061 (CMK) University of Kansas College of Liberal Arts and Sciences Graduate Research Fellowship (CMK) Government of Spain (PID2019-107001RB-I00 AEI/FEDER, UE) LE European Commission (H2020-SC1-2019, ISOLDA Project nº 848166-2) LEN

    Functions of hepatitis C virus glycoprotein E2 variable regions

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    HCV chronically infects ~3% of the global human population, including 200,000 Australians, causing recurring, progressively worsening liver disease, and cirrhosis and hepatocellular carcinoma. Preventative vaccines are not available. However, the advent of direct acting antivirals for the treatment of HCV results in viral clearance in at least 90% of treated individuals. Hepatitis C virus encodes two structural glycoproteins; E1 and E2. Glycoproteins E1 and E2 associate covalently and non-covalently to form heterodimers that mediate HCV binding to cell surface receptors. The E2 ectodomain region spanning residues 384-661 constitutes the receptor binding domain (RBD; E2661) that includes three highly variable regions: hypervariable region 1 (HVR1), HVR2 and the intergenotypic variable region (igVR). The HVR1 is known to elicit type-specific neutralizing antibodies (NAb). The HVR2 is located downstream of HVR1 within a region flanked by two cysteine residues (Cys-459 and Cys-486). HVR2 is not a direct target of the antibody response and the reason for variation within its sequence is unknown. Similar to HVR2, the igVR is flanked by two cysteine residues (Cys-569 and Cys-581). The igVR is relatively conserved within subtypes but the sequence and length of this region varies widely between genotypes. Patients infected with HCV develop NAb during their infection. The natural targets for NAb are the envelope glycoproteins E1 and E2 especially within the E2 RBD. The majority of NAb are directed to the binding sites within E2 for the major cell surface receptor CD81. In this study, the role of HVRs in modulating the exposure of both neutralizing and non-neutralizing epitopes within E2 has been examined. Examination of sequence evolution within HCV infected patients revealed that in addition to HVR1, both HVR2 and igVR are under selective pressure. In the case of the igVR, this contrasts to earlier reports suggesting that the igVR does not vary within genotypes. Mutations in the igVR were shown to directly modulate the exposure of non-neutralizing antibody epitopes in the patient that cleared their infection. In addition, the CD81 binding site was more occluded in E2 RBDs isolated during the chronic phase of the disease and was partly attributable to mutation in HVR2. In another approach, intragenotypic HVR2 and igVR replacement attenuate genotype 2 (G.2) HCVcc virus, whereas intergenotypic HVR2 and igVR chimeras between G.2a and G.1a results in non-infectious HCVcc. Passaging attenuated virus restored replication due to acquisition of adaptive mutations in the E2 transmembrane domain. With the aid of neutralizing antibodies, we also show that neutralizing epitope I encompassing 411-428 is more accessible when replacing HVR1 of G.1a to that of G.1b, but becomes occluded by replacing igVR. In contrast, neutralizing epitope III covering residues 512-529 is occluded by HVR1 and becomes inaccessible by exchanging igVR of G.1a to that of G.1b. The results show that HCV variable regions play a role in modulating the exposure of NAb epitopes and access to the CD81 binding site on the E2 RBD

    Ein Rechtsstreit in Gie├čen vor 500 Jahren

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    The E2 glycoprotein of Hepatitis C virus (HCV) is a major target of the neutralizing antibody (NAb) response with the majority of epitopes located within its receptor binding domain (RBD; 384-661). Within E2 are three variable regions located at the N-terminus (HVR1; 384-411), and internally at 460-480 (HVR2) and 570-580 [intergenotypic variable region (igVR)], all of which lie outside a conserved core domain that contains the CD81 binding site, essential for attachment of virions to host cells and a major target of NAbs. In this study, we examined the evolution of the E1 and E2 region in two patients infected with genotype 3a virus. Whereas one patient was able to clear the acute infection, the other developed a chronic infection. Mutations accumulated at multiple positions within the N-terminal HVR1 as well as within the igVR in both patients over time, whereas mutations in HVR2 were observed only in the chronically infected patient. Mutations within or adjacent to the CD81 contact site were observed in both patients but were less frequent and more conservative in the patient that cleared his/her infection. The evolution of CD81 binding function and antigenicity was examined with longitudinal E2 RBD sequences. The ability of the RBD to bind CD81 was completely lost by week 108 in the patient that developed chronic HCV. In the second patient, the ability of the week 36 RBD, just prior to viral clearance, to bind CD81 was reduced ~50% relative to RBD sequences obtained earlier. The binding of a NAb specific to a conserved epitope located within E2 residues 411-428 was significantly reduced by week 108 despite complete conservation of its epitope suggesting that E2 antigenicity is allosterically modulated. The exposure of non-neutralizing antibody epitopes was similarly explored and we observed that the epitope of 3 out of 4 non-NAbs were significantly more exposed in the RBDs representing the late timepoints in the chronic patient. By contrast, the exposure of non-neutralizing epitopes was reduced in the patient that cleared his/her infection and could in part be attributed to sequence changes in the igVR. These studies reveal that during HCV infection, the exposure of the CD81 binding site on E2 becomes increasingly occluded, and the antigenicity of the E2 RBD towards both neutralizing and non-neutralizing antibodies is modulated via allosteric mechanisms

    Binding of neutralizing MAb24 to E2 RBD glycoproteins.

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    <p>E2 RBD glycoproteins were normalized and applied to the ELISA plates coated with GNA-Lectin. Bound E2 RBD glycoproteins from (A) patient A and (B) patient B were detected with serially titrated neutralizing anti E2 MAb24 and secondary anti-mouse HRP antibodies. (C+D). The optical density at the third point of the curves is shown for patient A (C) and patient B in (D) as means ┬▒ SD from two independent experiments performed in duplicate.</p

    Ability of recombinant E2 RBD proteins to bind CD81.

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    <p>Normalized amounts of E2 RBD glycoproteins were applied to ELISA plates coated with the recombinant form of the CD81 large extracellular loop, MBP-LEL<sup>113ÔÇô201</sup>. The E2 RBD glycoproteins from patient A (A) and patient B (B) were used in ELISA to detect binding to MBP-LEL<sup>113ÔÇô201</sup>. Bound glycoproteins were detected with anti His antibody. Optical density was measured at 450 nm with background subtraction at 620 nm. The optical density at the second point of the curves is shown for patient A (C) and patient B (D) as means ┬▒ SEM from four independent experiments. <i>P</i> < 0.05 values were calculated by paired <i>t</i> test using GraphPad Prism 6 software.</p

    Binding of non-neutralizing MAbs to E2 RBD glycoproteins.

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    <p>E2 RBD glycoproteins were normalized and applied to the ELISA plates coated with GNA-Lectin. Bound E2 RBD glycoproteins were detected with anti E2 MAbs 6, 13, 22 and 25 and secondary anti-mouse HRP. The optical density at the second point of the curves is shown for the patient A and patient B as means ┬▒ SEM from three independent experiments. <i>P</i> < 0.05 values were calculated by paired <i>t</i> test using GraphPad Prism 6 software.</p
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