scholarly journals Role of helical structure and dynamics in oligoadenylate synthetase 1 (OAS1) mismatch tolerance and activation by short dsRNAs

2022 ◽  
Vol 119 (3) ◽  
pp. e2107111119
Author(s):  
Samantha L. Schwartz ◽  
Debayan Dey ◽  
Julia Tanquary ◽  
Camden R. Bair ◽  
Anice C. Lowen ◽  
...  
Keyword(s):  
Critical Role ◽  
Helical Structure ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Difference Spectra ◽  
Independent Manner ◽  
Double Stranded Rna ◽  
Dynamics Simulations ◽  
Thermal Difference

The 2’-5’-oligoadenylate synthetases (OAS) are innate immune sensors of cytosolic double-stranded RNA (dsRNA) that play a critical role in limiting viral infection. How these proteins are able to avoid aberrant activation by cellular RNAs is not fully understood, but adenosine-to-inosine (A-to-I) editing has been proposed to limit accumulation of endogenous RNAs that might otherwise cause stimulation of the OAS/RNase L pathway. Here, we aim to uncover whether and how such sequence modifications can restrict the ability of short, defined dsRNAs to activate the single-domain form of OAS, OAS1. Unexpectedly, we find that all tested inosine-containing dsRNAs have an increased capacity to activate OAS1, whether in a destabilizing (I•U) or standard Watson–Crick-like base pairing (I–C) context. Additional variants with strongly destabilizing A•C mismatches or stabilizing G–C pairs also exhibit increased capacity to activate OAS1, eliminating helical stability as a factor in the relative ability of the dsRNAs to activate OAS1. Using thermal difference spectra and molecular dynamics simulations, we identify both increased helical dynamics and specific local changes in helical structure as important factors in the capacity of short dsRNAs to activate OAS1. These helical features may facilitate more ready adoption of the distorted OAS1-bound conformation or stabilize important structures to predispose the dsRNA for optimal binding and activation of OAS1. These studies thus reveal the molecular basis for the greater capacity of some short dsRNAs to activate OAS1 in a sequence-independent manner.

scholarly journals NLRP7 Promotes Choriocarcinoma Growth and Progression through the Establishment of an Immunosuppressive Microenvironment

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2999
Author(s):  
Deborah Reynaud ◽  
Roland Abi Nahed ◽  
Nicolas Lemaitre ◽  
Pierre-Adrien Bolze ◽  
Wael Traboulsi ◽  
...  
Keyword(s):  
Immune Response ◽  
Tumor Cells ◽  
Major Gene ◽  
Critical Role ◽  
Orthotopic Model ◽  
Independent Manner ◽  
Maternal Immune Response ◽  
The Impact

The inflammatory gene NLRP7 is the major gene responsible for recurrent complete hydatidiform moles (CHM), an abnormal pregnancy that can develop into gestational choriocarcinoma (CC). However, the role of NLRP7 in the development and immune tolerance of CC has not been investigated. Three approaches were employed to define the role of NLRP7 in CC development: (i) a clinical study that analyzed human placenta and sera collected from women with normal pregnancies, CHM or CC; (ii) an in vitro study that investigated the impact of NLRP7 knockdown on tumor growth and organization; and (iii) an in vivo study that used two CC mouse models, including an orthotopic model. NLRP7 and circulating inflammatory cytokines were upregulated in tumor cells and in CHM and CC. In tumor cells, NLRP7 functions in an inflammasome-independent manner and promoted their proliferation and 3D organization. Gravid mice placentas injected with CC cells invalidated for NLRP7, exhibited higher maternal immune response, developed smaller tumors, and displayed less metastases. Our data characterized the critical role of NLRP7 in CC and provided evidence of its contribution to the development of an immunosuppressive maternal microenvironment that not only downregulates the maternal immune response but also fosters the growth and progression of CC.

scholarly journals SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial-derived cells and cardiomyocytes

2021 ◽  
Vol 118 (16) ◽  
pp. e2022643118
Author(s):  
Yize Li ◽  
David M. Renner ◽  
Courtney E. Comar ◽  
Jillian N. Whelan ◽  
Hanako M. Reyes ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Innate Immune ◽  
Alveolar Type ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Protein Kinase R ◽  
Double Stranded Rna ◽  
Induced Pluripotent ◽  
Host Virus Interactions

Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase–ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection; induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung; and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, whereas PKR activation is evident in iAT2 and iCM. In SARS-CoV-2–infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however, activation of OAS-RNase L and PKR is observed. This is in contrast to Middle East respiratory syndrome (MERS)-CoV, which effectively inhibits IFN signaling and OAS-RNase L and PKR pathways, but is similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host–virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.

Phosphorylation of Ser136 is critical for potent bone sialoprotein-mediated nucleation of hydroxyapatite crystals

2010 ◽  
Vol 428 (3) ◽  
pp. 385-395 ◽  
Author(s):  
Gurpreet S. Baht ◽  
Jason O'Young ◽  
Antonia Borovina ◽  
Hong Chen ◽  
Coralee E. Tye ◽  
...  
Keyword(s):  
Critical Role ◽  
Bone Sialoprotein ◽  
Site Directed Mutagenesis ◽  
Crystal Face ◽  
Mineralized Tissues ◽  
Dynamics Simulations ◽  
A Site ◽  
Functional Analyses ◽  
Kinase Ck2

Acidic phosphoproteins of mineralized tissues such as bone and dentin are believed to play important roles in HA (hydroxyapatite) nucleation and growth. BSP (bone sialoprotein) is the most potent known nucleator of HA, an activity that is thought to be dependent on phosphorylation of the protein. The present study identifies the role phosphate groups play in mineral formation. Recombinant BSP and peptides corresponding to residues 1–100 and 133–205 of the rat sequence were phosphorylated with CK2 (protein kinase CK2). Phosphorylation increased the nucleating activity of BSP and BSP-(133–205), but not BSP-(1–100). MS analysis revealed that the major site phosphorylated within BSP-(133–205) was Ser136, a site adjacent to the series of contiguous glutamate residues previously implicated in HA nucleation. The critical role of phosphorylated Ser136 in HA nucleation was confirmed by site-directed mutagenesis and functional analyses. Furthermore, peptides corresponding to the 133–148 sequence of rat BSP were synthesized with or without a phosphate group on Ser136. As expected, the phosphopeptide was a more potent nucleator. The mechanism of nucleation was investigated using molecular-dynamics simulations analysing BSP-(133–148) interacting with the {100} crystal face of HA. Both phosphorylated and non-phosphorylated sequences adsorbed to HA in extended conformations with alternating residues in contact with and facing away from the crystal face. However, this alternating-residue pattern was more pronounced when Ser136 was phosphorylated. These studies demonstrate a critical role for Ser136 phosphorylation in BSP-mediated HA nucleation and identify a unique mode of interaction between the nucleating site of the protein and the {100} face of HA.

scholarly journals Reverse Genetics Reveals a Role of Rotavirus VP3 Phosphodiesterase Activity in Inhibiting RNase L Signaling and Contributing to Intestinal Viral Replication In Vivo

2020 ◽  
Vol 94 (9) ◽  
Author(s):  
Yanhua Song ◽  
Ningguo Feng ◽  
Liliana Sanchez-Tacuba ◽  
Linda L. Yasukawa ◽  
Lili Ren ◽  
...  
Keyword(s):  
Small Intestine ◽  
Viral Replication ◽  
Reverse Genetics ◽  
Innate Immune ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Rotavirus Vaccines

ABSTRACT Our understanding of how rotavirus (RV) subverts host innate immune signaling has greatly increased over the past decade. However, the relative contribution of each virus-encoded innate immune antagonist has not been fully studied in the context of RV infection in vivo. Here, we present both in vitro and in vivo evidence that the host interferon (IFN)-inducible 2′-5′-oligoadenylate synthetase (OAS) and RNase L pathway effectively suppresses the replication of heterologous RV strains. VP3 from homologous RVs relies on its 2′-5′-phosphodiesterase (PDE) domain to counteract RNase L-mediated antiviral signaling. Using an RV reverse-genetics system, we show that compared to the parental strain, VP3 PDE mutant RVs replicated at low levels in the small intestine and were shed less in the feces of wild-type mice, and such defects were rescued in Rnasel−/− suckling mice. Collectively, these findings highlight an important role of VP3 in promoting viral replication and pathogenesis in vivo in addition to its well-characterized function as the viral RNA-capping enzyme. IMPORTANCE Rotaviruses are significant human pathogens that result in diarrhea, dehydration, and deaths in many children around the world. Rotavirus vaccines have suboptimal efficacy in low- to middle-income countries, where the burden of the diseases is the most severe. With the ultimate goal of improving current vaccines, we aim to better understand how rotavirus interacts with the host innate immune system in the small intestine. Here, we demonstrate that interferon-activated RNase L signaling blocks rotavirus replication in a strain-specific manner. In addition, virus-encoded VP3 antagonizes RNase L activity both in vitro and in vivo. These studies highlight an ever-evolving arms race between antiviral factors and viral pathogens and provide a new means of targeted attenuation for next-generation rotavirus vaccine design.

scholarly journals Double-stranded RNA-binding protein E3 controls translation of viral intermediate RNA, marking an essential step in the life cycle of modified vaccinia virus Ankara

2006 ◽  
Vol 87 (5) ◽  
pp. 1145-1155 ◽  
Author(s):  
Holger Ludwig ◽  
Yasemin Suezer ◽  
Zoe Waibler ◽  
Ulrich Kalinke ◽  
Barbara S. Schnierle ◽  
...  
Keyword(s):  
Life Cycle ◽  
Vaccinia Virus ◽  
Human Cells ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Double Stranded Rna ◽  
Modified Vaccinia Virus Ankara ◽  
Late Transcription

Infection of human cells with modified vaccinia virus Ankara (MVA) activates the typical cascade-like pattern of viral early-, intermediate- and late-gene expression. In contrast, infection of human HeLa cells with MVA deleted of the E3L gene (MVA-ΔE3L) results in high-level synthesis of intermediate RNA, but lacks viral late transcription. The viral E3 protein is thought to bind double-stranded RNA (dsRNA) and to act as an inhibitor of dsRNA-activated 2′-5′-oligoadenylate synthetase (2′-5′OA synthetase)/RNase L and protein kinase (PKR). Here, it is demonstrated that viral intermediate RNA can form RNase A/T1-resistant dsRNA, suggestive of activating both the 2′-5′OA synthetase/RNase L pathway and PKR in various human cell lines. Western blot analysis revealed that failure of late transcription in the absence of E3L function resulted from the deficiency to produce essential viral intermediate proteins, as demonstrated for vaccinia late transcription factor 2 (VLTF 2). Substantial host cell-specific differences were found in the level of activation of either RNase L or PKR. However, both rRNA degradation and phosphorylation of eukaryotic translation initiation factor-2α (eIF2α) inhibited the synthesis of VLTF 2 in human cells. Moreover, intermediate VLTF 2 and late-protein production were restored in MVA-ΔE3L-infected mouse embryonic fibroblasts from Pkr 0/0 mice. Thus, both host-response pathways may be involved, but activity of PKR is sufficient to block the MVA molecular life cycle. These data imply that an essential function of vaccinia virus E3L is to secure translation of intermediate RNA and, thereby, expression of other viral genes.

scholarly journals Structural basis of the heterodimerization of the MST and RASSF SARAH domains in the Hippo signalling pathway

2014 ◽  
Vol 70 (7) ◽  
pp. 1944-1953 ◽  
Author(s):  
Eunha Hwang ◽  
Hae-Kap Cheong ◽  
Ameeq Ul Mushtaq ◽  
Hye-Yeon Kim ◽  
Kwon Joo Yeo ◽  
...  
Keyword(s):  
Structural Information ◽  
Critical Role ◽  
Three Dimensional ◽  
Helical Structure ◽  
Structural Features ◽  
Signalling Pathway ◽  
Structural Basis ◽  
X Ray Crystallography ◽  
Hippo Signalling

Despite recent progress in research on the Hippo signalling pathway, the structural information available in this area is extremely limited. Intriguingly, the homodimeric and heterodimeric interactions of mammalian sterile 20-like (MST) kinases through the so-called `SARAH' (SAV/RASSF/HPO) domains play a critical role in cellular homeostasis, dictating the fate of the cell regarding cell proliferation or apoptosis. To understand the mechanism of the heterodimerization of SARAH domains, the three-dimensional structures of an MST1–RASSF5 SARAH heterodimer and an MST2 SARAH homodimer were determined by X-ray crystallography and were analysed together with that previously determined for the MST1 SARAH homodimer. While the structure of the MST2 homodimer resembled that of the MST1 homodimer, the MST1–RASSF5 heterodimer showed distinct structural features. Firstly, the six N-terminal residues (Asp432–Lys437), which correspond to the short N-terminal 310-helix h1 kinked from the h2 helix in the MST1 homodimer, were disordered. Furthermore, the MST1 SARAH domain in the MST1–RASSF5 complex showed a longer helical structure (Ser438–Lys480) than that in the MST1 homodimer (Val441–Lys480). Moreover, extensive polar and nonpolar contacts in the MST1–RASSF5 SARAH domain were identified which strengthen the interactions in the heterodimer in comparison to the interactions in the homodimer. Denaturation experiments performed using urea also indicated that the MST–RASSF heterodimers are substantially more stable than the MST homodimers. These findings provide structural insights into the role of the MST1–RASSF5 SARAH domain in apoptosis signalling.

scholarly journals RRM2B-Mediated Regulation of Mitochondrial Activity and Inflammation under Oxidative Stress

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Er-Chieh Cho ◽  
Mei-Ling Kuo ◽  
Jia-hui Cheng ◽  
Yu-Chi Cheng ◽  
Yi-Chen Hsieh ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Signaling Pathway ◽  
Ribonucleotide Reductase ◽  
Inflammatory Diseases ◽  
Critical Role ◽  
Mitochondrial Activity ◽  
Independent Manner ◽  
Mitochondrial Homeostasis

RRM2B is a critical ribonucleotide reductase (RR) subunit that exists as p53-inducible and p53-dependent molecule. The p53-independent regulation of RRM2B has been recently studied, and FOXO3 was identified as a novel regulator of RRM2B. However, the p53-independent regulation of RRM2B, particularly under oxidative stress, remains largely unknown. In this study, we investigated the role of RRM2B underoxidative stress-induced DNA damage and further examined the regulation of mitochondrial and inflammatory genes by RRM2B. Our study is the first to report the critical role of RRM2B in mitochondrial homeostasis and the inflammation signaling pathway in a p53-independent manner. Furthermore, our study provides novel insights into the role of the RR in inflammatory diseases.

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