SUMO chain formation is required for response to replication arrest in S. pombe

SUMO is a ubiquitin-like protein that is post-translationally attached to one or more lysine residues on target proteins. Despite having only 18% sequence identity with ubiquitin, SUMO contains the conserved betabetaalphabetabetaalphabeta fold present in ubiquitin. However, SUMO differs from ubiquitin in having an extended N-terminus. In S. pombe the N-terminus of SUMO/Pmt3 is significantly longer than those of SUMO in S. cerevisiae, human and Drosophila. Here we investigate the role of this N-terminal region. We have used two dimensional gel electrophoresis to demonstrate that S. pombe SUMO/Pmt3 is phosphorylated, and that this occurs on serine residues at the extreme N-terminus of the protein. Mutation of these residues (in pmt3-1) results in a dramatic reduction in both the levels of high Mr SUMO-containing species and of total SUMO/Pmt3, indicating that phosphorylation of SUMO/Pmt3 is required for its stability. Despite the significant reduction in high Mr SUMO-containing species, pmt3-1 cells do not display an aberrant cell morphology or sensitivity to genotoxins or stress. Additionally, we demonstrate that two lysine residues in the N-terminus of S. pombe SUMO/Pmt3 (K14 and K30) can act as acceptor sites for SUMO chain formation in vitro. Inability to form SUMO chains results in aberrant cell and nuclear morphologies, including stretched and fragmented chromatin. SUMO chain mutants are sensitive to the DNA synthesis inhibitor, hydroxyurea (HU), but not to other genotoxins, such as UV, MMS or CPT. This implies a role for SUMO chains in the response to replication arrest in S. pomb

Enhanced In Vitro Refolding of Fibroblast Growth Factor 15 with the Assistance of SUMO Fusion Partner

Fibroblast growth factor 15 (Fgf15) is the mouse orthologue of human FGF19. Fgf15 is highly expressed in the ileum and functions as an endocrine signal to regulate liver function, including bile acid synthesis, hepatocyte proliferation and insulin sensitivity. In order to fully understand the function of Fgf15, methods are needed to produce pure Fgf15 protein in the prokaryotic system. However, when expressed in Escherichia coli (E. coli), the recombinant Fgf15 protein was insoluble and found only in inclusion bodies. In the current study, we report a method to produce recombinant Fgf15 protein in E. coli through the use of small ubiquitin-related modifier (SUMO) fusion tag. Even though the SUMO has been shown to strongly improve protein solubility and expression levels, our studies suggest that the SUMO does not improve Fgf15 protein solubility. Instead, proper refolding of Fgf15 protein was achieved when Fgf15 was expressed as a partner protein of the fusion tag SUMO, followed by in vitro dialysis refolding. After refolding, the N-terminal SUMO tag was cleaved from the recombinant Fgf15 fusion protein by ScUlp1 (Ubiquitin-Like Protein-Specific Protease 1 from S. cerevisiae). With or without the SUMO tag, the refolded Fgf15 protein was biologically active, as revealed by its ability to reduce hepatic Cyp7a1 mRNA levels in mice. In addition, recombinant Fgf15 protein suppressed Cyp7a1 mRNA levels in a dose-dependent manner. In summary, we have developed a successful method to express functional Fgf15 protein in prokaryotic cells

Severe childhood malaria syndromes defined by plasma proteome profiles

BACKGROUND Cerebral malaria (CM) and severe malarial anemia (SMA) are the most serious life-threatening clinical syndromes of Plasmodium falciparum infection in childhood. Therefore it is important to understand the pathology underlying the development of CM and SMA, as opposed to uncomplicated malaria (UM). Different host responses to infection are likely to be reflected in plasma proteome-patterns that associate with clinical status and therefore provide indicators of the pathogenesis of these syndromes. METHODS AND FINDINGS Plasma and comprehensive clinical data for discovery and validation cohorts were obtained as part of a prospective case-control study of severe childhood malaria at the main tertiary hospital of the city of Ibadan, an urban and densely populated holoendemic malaria area in Nigeria. A total of 946 children participated in this study. Plasma was subjected to high-throughput proteomic profiling. Statistical pattern-recognition methods were used to find proteome-patterns that defined disease groups. Plasma proteome-patterns accurately distinguished children with CM and with SMA from those with UM, and from healthy or severely ill malaria-negative children. CONCLUSIONS We report that an accurate definition of the major childhood malaria syndromes can be achieved using plasma proteome-patterns. Our proteomic data can be exploited to understand the pathogenesis of the different childhood severe malaria syndromes

Structural insight into SUMO chain recognition and manipulation by the ubiquitin ligase RNF4

The small ubiquitin-like modifier (SUMO) can form polymeric chains that are important signals in cellular processes such as meiosis, genome maintenance and stress response. The SUMO-targeted ubiquitin ligase RNF4 engages with SUMO chains on linked substrates and catalyses their ubiquitination, which targets substrates for proteasomal degradation. Here we use a segmental labelling approach combined with solution nuclear magnetic resonance (NMR) spectroscopy and biochemical characterization to reveal how RNF4 manipulates the conformation of the SUMO chain, thereby facilitating optimal delivery of the distal SUMO domain for ubiquitin transfer

Unexpected Roles of a Tether Harboring a Tyrosine Gatekeeper Residue in Modular Nitrite Reductase Catalysis

© 2019 American Chemical Society. It is generally assumed that tethering enhances rates of electron harvesting and delivery to active sites in multidomain enzymes by proximity and sampling mechanisms. Here, we explore this idea in a tethered 3-domain, trimeric copper-containing nitrite reductase. By reverse engineering, we find that tethering does not enhance the rate of electron delivery from its pendant cytochrome c to the catalytic copper-containing core. Using a linker that harbors a gatekeeper tyrosine in a nitrite access channel, the tethered haem domain enables catalysis by other mechanisms. Tethering communicates the redox state of the haem to the distant T2Cu center that helps initiate substrate binding for catalysis. It also tunes copper reduction potentials, suppresses reductive enzyme inactivation, enhances enzyme affinity for substrate, and promotes intercopper electron transfer. Tethering has multiple unanticipated beneficial roles, the combination of which fine-tunes function beyond simplistic mechanisms expected from proximity and restrictive sampling models

DNA topoisomerases participate in fragility of the oncogene RET

Fragile site breakage was previously shown to result in rearrangement of the RET oncogene, resembling the rearrangements found in thyroid cancer. Common fragile sites are specific regions of the genome with a high susceptibility to DNA breakage under conditions that partially inhibit DNA replication, and often coincide with genes deleted, amplified, or rearranged in cancer. While a substantial amount of work has been performed investigating DNA repair and cell cycle checkpoint proteins vital for maintaining stability at fragile sites, little is known about the initial events leading to DNA breakage at these sites. The purpose of this study was to investigate these initial events through the detection of aphidicolin (APH)-induced DNA breakage within the RET oncogene, in which 144 APHinduced DNA breakpoints were mapped on the nucleotide level in human thyroid cells within intron 11 of RET, the breakpoint cluster region found in patients. These breakpoints were located at or near DNA topoisomerase I and/or II predicted cleavage sites, as well as at DNA secondary structural features recognized and preferentially cleaved by DNA topoisomerases I and II. Co-treatment of thyroid cells with APH and the topoisomerase catalytic inhibitors, betulinic acid and merbarone, significantly decreased APH-induced fragile site breakage within RET intron 11 and within the common fragile site FRA3B. These data demonstrate that DNA topoisomerases I and II are involved in initiating APH-induced common fragile site breakage at RET, and may engage the recognition of DNA secondary structures formed during perturbed DNA replication

Analysis of Global Sumoylation Changes Occurring during Keratinocyte Differentiation

Sumoylation is a highly dynamic process that plays a role in a multitude of processes ranging from cell cycle progression to mRNA processing and cancer. A previous study from our lab demonstrated that SUMO plays an important role in keratinocyte differentiation. Here we present a new method of tracking the sumoylation state of proteins by creating a stably transfected HaCaT keratinocyte cell line expressing an inducible SNAP-SUMO3 protein. The SNAP-tag allows covalent fluorescent labeling that is denaturation resistant. When combined with two-dimensional gel electrophoresis, the SNAP-tag technology provides direct visualization of sumoylated targets and can be used to follow temporal changes in the global cohort of sumoylated proteins during dynamic processes such as differentiation. HaCaT keratinocyte cells expressing SNAP-SUMO3 displayed normal morphological and biochemical features that are consistent with typical keratinocyte differentiation. SNAP-SUMO3 also localized normally in these cells with a predominantly nuclear signal and some minor cytoplasmic staining, consistent with previous reports for untagged SUMO2/3. During keratinocyte differentiation the total number of proteins modified by SNAP-SUMO3 was highest in basal cells, decreased abruptly after induction of differentiation, and slowly rebounded beginning between 48 and 72 hours as differentiation progressed. However, within this overall trend the pattern of change for individual sumoylated proteins was highly variable with both increases and decreases in amount over time. From these results we conclude that sumoylation of proteins during keratinocyte differentiation is a complex process which likely reflects and contributes to the biochemical changes that drive differentiation

An affordable, quality-assured community-based system for high-resolution entomological surveillance of vector mosquitoes that reflects human malaria infection risk patterns.

ABSTRACT: BACKGROUND: More sensitive and scalable entomological surveillance tools are required to monitor low levels of transmission that are increasingly common across the tropics, particularly where vector control has been successful. A large-scale larviciding programme in urban Dar es Salaam, Tanzania is supported by a community-based (CB) system for trapping adult mosquito densities to monitor programme performance. Methodology An intensive and extensive CB system for routine, longitudinal, programmatic surveillance of malaria vectors and other mosquitoes using the Ifakara Tent Trap (ITT-C) was developed in Urban Dar es Salaam, Tanzania, and validated by comparison with quality assurance (QA) surveys using either ITT-C or human landing catches (HLC), as well as a cross-sectional survey of malaria parasite prevalence in the same housing compounds. RESULTS: Community-based ITT-C had much lower sensitivity per person-night of sampling than HLC (Relative Rate (RR) [95% Confidence Interval (CI)] = 0.079 [0.051, 0.121], P < 0.001 for Anopheles gambiae s.l. and 0.153 [0.137, 0.171], P < 0.001 for Culicines) but only moderately differed from QA surveys with the same trap (0.536 [0.406,0.617], P = 0.001 and 0.747 [0.677,0.824], P < 0.001, for An. gambiae or Culex respectively). Despite the poor sensitivity of the ITT per night of sampling, when CB-ITT was compared with QA-HLC, it proved at least comparably sensitive in absolute terms (171 versus 169 primary vectors caught) and cost-effective (153US$versus 187US$ per An. gambiae caught) because it allowed more spatially extensive and temporally intensive sampling (4284 versus 335 trap nights distributed over 615 versus 240 locations with a mean number of samples per year of 143 versus 141). Despite the very low vectors densities (Annual estimate of about 170 An gambiae s.l bites per person per year), CB-ITT was the only entomological predictor of parasite infection risk (Odds Ratio [95% CI] = 4.43[3.027,7. 454] per An. gambiae or Anopheles funestus caught per night, P =0.0373). Discussion and conclusion CB trapping approaches could be improved with more sensitive traps, but already offer a practical, safe and affordable system for routine programmatic mosquito surveillance and clusters could be distributed across entire countries by adapting the sample submission and quality assurance procedures accordingly

SUMO Pathway Dependent Recruitment of Cellular Repressors to Herpes Simplex Virus Type 1 Genomes

Components of promyelocytic leukaemia (PML) nuclear bodies (ND10) are recruited to sites associated with herpes simplex virus type 1 (HSV-1) genomes soon after they enter the nucleus. This cellular response is linked to intrinsic antiviral resistance and is counteracted by viral regulatory protein ICP0. We report that the SUMO interaction motifs of PML, Sp100 and hDaxx are required for recruitment of these repressive proteins to HSV-1 induced foci, which also contain SUMO conjugates and PIAS2β, a SUMO E3 ligase. SUMO modification of PML and elements of its tripartite motif (TRIM) are also required for recruitment in cells lacking endogenous PML. Mutants of PML isoform I and hDaxx that are not recruited to virus induced foci are unable to reproduce the repression of ICP0 null mutant HSV-1 infection mediated by their wild type counterparts. We conclude that recruitment of ND10 components to sites associated with HSV-1 genomes reflects a cellular defence against invading pathogen DNA that is regulated through the SUMO modification pathway

Deciphering Museums, Politics and Impact

This paper makes a contribution towards deciphering the relationship between museums, politics and impact. I suggest that this is akin to that between three languages in the early nineteenth century: Greek, Demotic and Hieroglyphs. I argue that museums should be taken much more seriously by the discipline of politics and international relations. This paper begins with an analysis of the REF 2014 Impact Case Studies submitted under the Politics and International Studies Unit of Assessment. Thereafter, it looks at how museums have been examined in the field of politics and international relations. Finally, it outlines some of the benefits and opportunities of scholars in the field engaging with museums in terms of their research, as potential collaborators, and as partners for knowledge transfer and impactful activities – within and outwith the strictures of the UK Research Excellence Framework (REF)