Sumo-dependent substrate targeting of the SUMO protease Ulp1

<p>Abstract</p> <p>Background</p> <p>In the yeast <it>Saccharomyces cerevisiae</it>, the essential small ubiquitin-like modifier (SUMO) protease Ulp1 is responsible for both removing SUMO/Smt3 from specific target proteins and for processing precursor SUMO into its conjugation-competent form. Ulp1 localizes predominantly to nuclear pore complexes but has also been shown to deconjugate sumoylated septins at the bud-neck of dividing cells. How Ulp1 is directed to bud-neck localized septins and other cytoplasmic deconjugation targets is not well understood.</p> <p>Results</p> <p>Using a structure/function approach, we set out to elucidate features of Ulp1 that are required for substrate targeting. To aid our studies, we took advantage of a catalytically inactive mutant of Ulp1 that is greatly enriched at the septin ring of dividing yeast cells. We found that the localization of Ulp1 to the septins requires both SUMO and specific structural features of Ulp1's catalytic domain. Our analysis identified a 218-amino acid, substrate-trapping mutant of the catalytic domain of Ulp1, Ulp1(3)^(C580S), that is necessary and sufficient for septin localization. We also used the targeting and SUMO-binding properties of Ulp1(3)^(C580S)to purify Smt3-modified proteins from cell extracts.</p> <p>Conclusions</p> <p>Our study provides novel insights into how the Ulp1 SUMO protease is actively targeted to its substrates <it>in vivo </it>and <it>in vitro</it>. Furthermore, we found that a substrate-trapping Ulp1(3)^(C580S)interacts robustly with human SUMO1, SUMO2 and SUMO2 chains, making it a potentially useful tool for the analysis and purification of SUMO-modified proteins.</p

Relief of the Dma1-mediated checkpoint requires Dma1 autoubiquitination and dynamic localization

© 2018 Jones, Chen, et al. Chromosome segregation and cell division are coupled to prevent aneuploidy and cell death. In the fission yeast Schizosaccharomyces pombe, the septation initiation network (SIN) promotes cytokinesis, but upon mitotic checkpoint activation, the SIN is actively inhibited to prevent cytokinesis from occurring before chromosomes have safely segregated. SIN inhibition during the mitotic checkpoint is mediated by the E3 ubiquitin ligase Dma1. Dma1 binds to the CK1-phosphorylated SIN scaffold protein Sid4 at the spindle pole body (SPB), and ubiquitinates it. Sid4 ubiquitination antagonizes the SPB localization of the Polo-like kinase Plo1, the major SIN activator, so that SIN signaling is delayed. How this checkpoint is silenced once spindle defects are resolved has not been clear. Here we establish that Dma1 transiently leaves SPBs during anaphase B due to extensive autoubiquitination. The SIN is required for Dma1 to return to SPBs later in anaphase. Blocking Dma1 removal from SPBs by permanently tethering it to Sid4 prevents SIN activation and cytokinesis. Therefore, controlling Dma1’s SPB dynamics in anaphase is an essential step in S. pombe cell division and the silencing of the Dma1-dependent mitotic checkpoint

Rescuing AAV gene transfer from neutralizing antibodies with an IgG-degrading enzyme

Preexisting humoral immunity to recombinant adeno-associated virus (AAV) vectors restricts the treatable patient population and efficacy of human gene therapies. Approaches to clear neutralizing antibodies (NAbs), such as plasmapheresis and immunosuppression, are either ineffective or cause undesirable side effects. Here, we describe a clinically relevant strategy to rapidly and transiently degrade NAbs before AAV administration using an IgG-degrading enzyme (IdeZ). We demonstrate that recombinant IdeZ efficiently cleaved IgG in dog, monkey, and human antisera. Prophylactically administered IdeZ cleaved circulating human IgG in mice and prevented AAV neutralization in vivo. In macaques, a single intravenous dose of IdeZ rescued AAV transduction by transiently reversing seropositivity. Importantly, IdeZ efficiently cleaved NAbs and rescued AAV transduction in mice passively immunized with individual human donor sera representing a diverse population. Our antibody clearance approach presents a potentially new paradigm for expanding the prospective patient cohort and improving efficacy of AAV gene therapy

Ring finger protein 121 is a potent regulator of adeno-associated viral genome transcription.

Adeno-associated viruses (AAV) are Dependoparvoviruses that have shown promise as recombinant vectors for gene therapy. While infectious pathways of AAV are well studied, gaps remain in our understanding of host factors affecting vector genome expression. Here, we map the role of ring finger protein 121 (RNF121), an E3 ubiquitin ligase, as a key regulator of AAV genome transcription. CRISPR-mediated knockout of RNF121 (RNF121 KO) in different cells markedly decreased AAV transduction regardless of capsid serotype or vector dose. Recombinant AAV transduction is partially rescued by overexpressing RNF121, but not by co-infection with helper Adenovirus. Major steps in the AAV infectious pathway including cell surface binding, cellular uptake, nuclear entry, capsid uncoating and second strand synthesis are unaffected. While gene expression from transfected plasmids or AAV genomes is unaffected, mRNA synthesis from AAV capsid-associated genomes is markedly decreased in RNF121 KO cells. These observations were attributed to transcriptional arrest as corroborated by RNAPol-ChIP and mRNA half-life measurements. Although AAV capsid proteins do not appear to be direct substrates of RNF121, the catalytic domain of the E3 ligase appears essential. Inhibition of ubiquitin-proteasome pathways revealed that blocking Valosin Containing Protein (VCP/p97), which targets substrates to the proteasome, can selectively and completely restore AAV-mediated transgene expression in RNF121 KO cells. Expanding on this finding, transcriptomic and proteomic analysis revealed that the catalytic subunit of DNA PK (DNAPK-Cs), a known activator of VCP, is upregulated in RNF121 KO cells and that the DNA damage machinery is enriched at sites of stalled AAV genome transcription. We postulate that a network of RNF121, VCP and DNA damage response elements function together to regulate transcriptional silencing and/or activation of AAV vector genomes

Capsid-mediated control of adeno-associated viral transcription determines host range

Summary: Adeno-associated virus (AAV) is a member of the genus Dependoparvovirus, which infects a wide range of vertebrate species. Here, we observe that, unlike most primate AAV isolates, avian AAV is transcriptionally silenced in human cells. By swapping the VP1 N terminus from primate AAVs (e.g., AAV8) onto non-mammalian isolates (e.g., avian AAV), we identify a minimal component of the AAV capsid that controls viral transcription and unlocks robust transduction in both human cells and mouse tissue. This effect is accompanied by increased AAV genome chromatin accessibility and altered histone methylation. Proximity ligation analysis reveals that host factors are selectively recruited by the VP1 N terminus of AAV8 but not avian AAV. Notably, these include AAV essential factors implicated in the nuclear factor κB pathway, chromatin condensation, and histone methylation. We postulate that the AAV capsid has evolved mechanisms to recruit host factors to its genome, allowing transcriptional activation in a species-specific manner

The Chesapeake Bay Program Modeling System: Overview and Recommendations For Future Development

The Chesapeake Bay is the largest, most productive, and most biologically diverse estuary in the continental United States providing crucial habitat and natural resources for culturally and economically important species. Pressures from human population growth and associated development and agricultural intensification have led to excessive nutrient and sediment inputs entering the Bay, negatively affecting the health of the Bay ecosystem and the economic services it provides. The Chesapeake Bay Program (CBP) is a unique program formally created in 1983 as a multi-stakeholder partnership to guide and foster restoration of the Chesapeake Bay and its watershed. Since its inception, the CBP Partnership has been developing, updating, and applying a complex linked modeling system of watershed, airshed, and estuary models as a planning tool to inform strategic management decisions and Bay restoration efforts. This paper provides a description of the 2017 CBP Modeling System and the higher trophic level models developed by the NOAA Chesapeake Bay Office, along with specific recommendations that emerged from a 2018 workshop designed to inform future model development. Recommendations highlight the need for simulation of watershed inputs, conditions, processes, and practices at higher resolution to provide improved information to guide local nutrient and sediment management plans. More explicit and extensive modeling of connectivity between watershed landforms and estuary sub-areas, estuarine hydrodynamics, watershed and estuarine water quality, the estuarine-watershed socioecological system, and living resources will be important to broaden and improve characterization of responses to targeted nutrient and sediment load reductions. Finally, the value and importance of maintaining effective collaborations among jurisdictional managers, scientists, modelers, support staff, and stakeholder communities is emphasized. An open collaborative and transparent process has been a key element of successes to date and is vitally important as the CBP Partnership moves forward with modeling system improvements that help stakeholders evolve new knowledge, improve management strategies, and better communicate outcomes