Browsing by Subject "Peptide Hydrolases"
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Item Discovering GCNA: A Novel Regulator of Germline Genomic Stability(2018-10-15) Bhargava, Varsha; Mendell, Joshua T.; Buszczak, Michael; Olson, Eric N.; Tu, BenjaminGerm cells transfer genetic information across generations. Any change in germ line DNA is inherited by succeeding generations. Therefore, germ cell DNA must be protected from both internal and external assault. An advantage of sexual reproduction stems from the ability to generate variation by exchange of chromosomal segments during meiosis. During meiosis, hundreds of double-stranded DNA breaks are initiated at once, which if generated in most other cell types would introduce chromosomal aberrations. Germ cells, however, execute the formation of these breaks while preventing their deleterious effects from becoming pervasive throughout the genome. The mechanisms underlying the robustness of germ cells in the face of DNA damage, however, are poorly understood. We initiated an in vivo CRISPR-Cas9 knockout screen for genes highly enriched in the Drosophila female germ line. From this screen, we identified Germ Cell Nuclear Acidic Peptidase (GCNA) as a conserved regulator of genome stability across multiple species. Loss of GCNA results in replication stress, chromosomal instability, and an accumulation of DNA-protein crosslinks (DPCs). Disruption of GCNA leads to an accumulation of nuclear Top2 and Top2 DPCs. This work shows GCNA protects germ cells from damage and provides novel insights into the conserved networks that promote genome integrity across generations.Item GCNA: Guardian of the Genome(2020-05-01T05:00:00.000Z) Goldstein, Courtney DaVee; Abrams, John M.; Buszczak, Michael; Brekken, Rolf A.; Olson, Eric N.The propagation of species depends on the ability of germ cells to protect their genome in the face of numerous exogenous and endogenous threats. While germ cells employ a number of know repair pathways, specialized mechanisms that ensure high-fidelity replication, chromosome segregation, and repair of germ cell genomes remain incompletely understood. Here, we identify Germ cell nuclear acidic peptidase (GCNA) as a conserved regulator of genome stability in flies, worms, zebrafish and human germ cell tumors. GCNA contains an acidic intrinsically disordered region (IDR) and a protease-like SprT domain. In addition to chromosomal instability and replication stress, Gcna mutants accumulate DNA-protein crosslinks (DPCs). GCNA acts in parallel with a second SprT domain protein Spartan. Structural analysis reveals that while the SprT domain is needed to limit meiotic and replicative damage, much of GCNA's function maps to its IDR. This work shows GCNA protects germ cells from various sources of damage, providing novel insights into conserved mechanisms that promote genome integrity across generations.Item Hepatitis C Virus NS3/4A Protease and the Intracellular Antiviral Response: Mapping Complex Virus-Host Interactions(2009-06-17) Johnson, Cynthia L.; Gale, Michael, Jr.Virus infection triggers an innate immune response characterized by host cell production of interferon (IFN). Intermediates of viral replication, including dsRNA, initiate a signaling cascade that is amplified within the cell and alerts neighboring cells of viral invaders. Recognition of dsRNA intermediates occurs through retinoic acid inducible gene-I (RIG-I). RIG-I elicits an antiviral state by binding to the IFN-beta Promoter Stimulator-1 (IPS-1) adaptor protein, activating the latent downstream transcription factors IRF-3 and NF-kappaB. These transcription factors bind to the promoter region of effector genes including IFN-beta, producing an antiviral amplification loop within and around the infected cell. This response is critical for immunity to infection. Hepatitis C virus (HCV) is a serious global health problem with 170 million people chronically infected. HCV persistence is linked to viral regulation of innate host defenses by the nonstructural 3/4A protein complex (NS3/4A) cleavage of IPS-1. NS3 structural composition includes an amino-terminal serine protease and a carboxy-terminal RNA helicase. A structure-function analysis of NS3/4A truncation and deletion mutations was conducted. Mutants lacking the helicase domain retained the ability to control RIG-I signaling, but this regulation was abrogated by truncation of the protease domain. Furthermore, treatment of HCV-infected cells with a NS3/4A protease inhibitor prevented IPS-1 proteolysis, restored RIG-I signaling, and decreased viral protein levels. These results indicate that the NS3/4A protease domain alone can target IPS-1 on the mitochondrial membrane. Current dogma holds that NS3/4A is located on the endoplasmic reticulum, thus the mechanism of NS3/4A targeting IPS-1, a mitochondrial membrane protein, remains unexplained. We have shown that NS3/4A distributes on mitochondria independently of the previously identified NS4A membrane localization motif, in a manner dependent on the first twenty amino acids of the NS3 protease domain. The functional domains of IPS-1 that direct the immune response have not been elucidated. We conducted a structure-function study of IPS-1 that revealed distinct processes of IRF-3 and NF-kappaB activation. Mutational analyses further identified areas of IPS-1 critical for mitochondrial localization, dimerization, and uncoupling IRF-3 and NF-kappaB signaling. These findings improve our understanding of IPS-1 function in innate immunity to virus infection.