Biology subjects

Ishiwata-Endo, H.

Publications and source records attributed to Ishiwata-Endo, H..

4 recordsLinked to original sources

bioRxiv2023

A PARP inhibitor, rucaparib, improves cardiac dysfunction in ADP-ribose-acceptor hydrolase 3 (Arh3) deficiency

AimsPatients with ADP-ribose-acceptor hydrolase 3 (ARH3) deficiency exhibit stress-induced childhood-onset neurodegeneration with ataxia and seizures (CONDSIAS). ARH3 degrades protein-linked poly(ADP- ribose) (PAR) synthesized by poly(ADP-ribose)polymerase (PARP)-1 during oxidative stress, leading to cleavage of the ADP-ribose linked to protein. ARH3 deficiency leads to excess accumulation of PAR, resulting in PAR-dependent cell death or parthanatos. Approximately one-third of patients with homozygous mutant ARH3 die from cardiac arrest, which has been described as neurogenic, suggesting that ARH3 may play an important role in maintaining myocardial function. To address this question, cardiac function was monitored in Arh3-knockout (KO) and - heterozygous (HT) mice. Methods and resultsArh3-KO male mice displayed cardiac hypertrophy by histopathology and decreased cardiac contractility assessed by MRI. In addition, both genders of Arh3-KO and -HT mice showed decreased cardiac contractility by dobutamine stress test assessed by echocardiography. A direct role of ARH3 on myocardial function was seen with a Langendorff-perfused isolated heart model. Arh3-KO male mouse hearts showed decreased post-ischemic rate pressure products, increased size of ischemia-reperfusion (IR) infarcts, and elevated PAR levels. Consistently, in vivo IR injury showed enhanced infarct size in Arh3-KO mice in both genders. In addition, Arh3-HT male mice showed increased size of in vivo IR infarcts. Treatment with an FDA-approved PARP inhibitor, rucaparib, improved cardiac contractility during dobutamine-induced stress and exhibited reduced size of in vivo IR infarcts. To understand better the role of ARH3, CRISPR-Cas9 was used to generate different Arh3 genotypes of myoblasts and myotubes. Incubation with H2O2 decreased viability of Arh3-KO and -HT myoblasts and myotubes, resulting in PAR-dependent cell death that was reduced by PARP inhibitors or by transfection with the Arh3 gene. ConclusionARH3 regulates PAR homeostasis in myocardium to preserve function and protect against oxidative stress; PARP inhibitors reduce the myocardial dysfunction seen with Arh3 mutations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/527369v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@a76514org.highwire.dtl.DTLVardef@1dd3184org.highwire.dtl.DTLVardef@ddfd26org.highwire.dtl.DTLVardef@16e0cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

ADP-ribose-acceptor hydrolase 2 (Arh2) deficiency results in cardiac dysfunction, tumorigenesis, inflammation, and decreased survival

ADP-ribosylation is a reversible reaction with ADP-ribosyltransferases catalyzing the forward reaction and ADP-ribose-acceptor hydrolases (ARHs) hydrolyzing the ADP-ribose acceptor bond. ARH2 is a member of the 39-kDa ARH family (ARH1-3), which is expressed in heart and skeletal muscle. ARH2 failed to exhibit any in vitro enzymatic activity. To determine its possible in vivo activities, Arh2-knockout (KO) and - heterozygous (Het) mice were generated using CRISPR-Cas9. Arh2-KO mice exhibited decreased cardiac contractility by MRI, echocardiography and dobutamine stress with cardiomegaly and abnormal motor function. Arh2-Het mice showed results similar to those seen in Arh2-KO mice except for cardiomegaly. Arh2-KO and -Het mice and mouse embryonic fibroblasts (MEFs) developed spontaneous tumors and subcutaneous tumors in nude mice. We identified 13 mutations in Arh2-Het MEFs and heterozygous tumors, corresponding to human ARH2 mutations in cancers obtained from COSMIC. Of interest, the L116R mutation in Arh2 gene plays a critical role in aggressive tumorigenesis in nude mice, corresponding to human ARH2 mutations in stomach adenocarcinoma. Both genders of Arh2-KO and -Het mice showed increased unexpectedly deaths and decreased survival rate during a 24-month observation, caused by tumor, inflammation, non-inflammation (e.g., cardiomegaly, dental dysplasia), and congenital diseases. Thus, Arh2 plays a pivotal role in cardiac function, tumorigenesis, inflammation, and overall survival.

cancer biology↗

bioRxiv2023

Macrodomain Mac1 of SARS-CoV-2 Nonstructural Protein 3 Hydrolyzes Diverse ADP-ribosylated Substrates

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that resulted in more than 6-million deaths worldwide. The virus encodes several non-structural proteins (Nsps) that contain elements capable of disrupting cellular processes. Among these Nsp proteins, Nsp3 contains macrodomains, e.g., Mac1, Mac2, Mac3, with potential effects on host cells. Mac1 has been shown to increase SARS-CoV-2 virulence and disrupt ADP-ribosylation pathways in mammalian cells. ADP-ribosylation results from the transfer of the ADP-ribose moiety of NAD+ to various acceptors, e.g., proteins, DNA, RNA, contributing on a cells biological processes. ADP-ribosylation is the mechanism of action of bacterial toxins, e.g., Pseudomonas toxins, diphtheria toxin that disrupt protein biosynthetic and signaling pathways. On the other hand, some viral macrodomains cleavage ADP-ribose-acceptor bond, generating free ADP-ribose. By this reaction, the macrodomain-containing proteins interfere ADP-ribose homeostasis in host cells. Here, we examined potential hydrolytic activities of SARS-CoV-2 Mac1, 2, and 3 on substrates containing ADP-ribose. Mac1 cleaved -NAD+, but not {beta}-NAD+, consistent with stereospecificity at the C-1" bond. In contrast to ARH1 and ARH3, Mac1 did not require Mg2+ for optimal activity. Mac1 also hydrolyzed O-acetyl-ADP-ribose and ADP-ribose-1"-phosphat, but not Mac2 and Mac3. However, Mac1 did not cleave -ADP-ribose-(arginine) and ADP-ribose-(serine)-histone H3 peptide, suggesting that Mac1 hydrolyzes ADP-ribose attached to O- and N-linked functional groups, with specificity at the catalytic site in the ADP-ribose moiety. We conclude that SARS-CoV-2 Mac1 may exert anti-viral activity by reversing host-mediated ADP-ribosylation. New insights on Nsp3 activities may shed light on potential SARS-CoV-2 therapeutic targets. IMPORTANCESARS-CoV-2, the virus responsible for COVID-19, encodes 3 macrodomain-containing proteins, e.g., Mac1, Mac2, Mac3, within non-structural proteins 3 (Nsp3). Mac1 was shown previously to hydrolyze ADP-ribose-phosphate. Inactivation of Mac1 reduced viral proliferation. Here we report that Mac1, but not Mac2 and Mac3, has multiple activities, i.e., Mac1 hydrolyzed. -NAD+ and O-acetyl-ADP-ribose. However, Mac1 did not hydrolyze {beta}-NAD+, ADP-ribose-serine on a histone 3 peptide (aa1-21), and ADP-ribose-arginine, exhibiting substrate selectivity. These data suggest that Mac1 may have multi-function as a -NAD+ consumer for viral replication and a disruptor of host-mediated ADP-ribosylation pathways. Understanding Mac1s mechanisms of action is important to provide possible therapeutic targets for COVID-19.

biochemistry↗

bioRxiv2023

Mono-ADP-ribosyltransferase 1 (Artc1)-deficiency decreases tumorigenesis, increases inflammation, decreases cardiac contractility, and reduces survival

Arginine-specific mono-ADP-ribosylation is a reversible post-translational modification; arginine-specific, cholera toxin-like mono-ADP-ribosyltransferases (ARTCs) transfer ADP-ribose from NAD+ to arginine, followed by cleavage of ADP-ribose-(arginine)protein bond by ADP-ribosylarginine hydrolase 1 (ARH1), generating unmodified (arginine)protein. ARTC1 has been shown to enhance tumorigenicity as does Arh1 deficiency. In this study, Artc1-KO and Artc1/Arh1-double-KO mice showed decreased spontaneous tumorigenesis and increased age-dependent, multi-organ inflammation with upregulation of pro-inflammatory cytokine TNF-. In a xenograft model using tumorigenic Arh1-KO mouse embryonic fibroblasts (MEFs), tumorigenicity was decreased in Artc1-KO and heterozygous recipient mice, with tumor infiltration by CD8+ T cells and macrophages, leading to necroptosis, suggesting that ARTC1 promotes the tumor microenvironment. Furthermore, Artc1/Arh1-double-KO MEFs showed decreased tumorigenesis in nude mice, showing that tumor cells as well as tumor microenvironment require ARTC1. By echocardiography and MRI, Artc1-KO and heterozygous mice showed male-specific, reduced myocardial contractility. Furthermore, Artc1-KO male hearts exhibited enhanced susceptibility to myocardial ischemia-reperfusion-induced injury with increased receptor-interacting protein kinase 3 (RIP3) protein levels compared to WT mice, suggesting that ARTC1 suppresses necroptosis. Overall survival rate of Artc1-KO was less than their Artc1-WT counterparts, primarily due to enhanced immune response and inflammation. Thus, anti-ARTC1 agents may reduce tumorigenesis but may increase multi-organ inflammation and decrease cardiac contractility. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/527366v1_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@1cded54org.highwire.dtl.DTLVardef@9851ceorg.highwire.dtl.DTLVardef@22cd8aorg.highwire.dtl.DTLVardef@e42e7a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗