Wrenches in the works: drug discovery targeting the SCF ubiquitin ligase and APC/C complexes

Recently, the ubiquitin proteasome system (UPS) has matured as a drug discovery arena, largely on the strength of the proven clinical activity of the proteasome inhibitor Velcade in multiple myeloma. Ubiquitin ligases tag cellular proteins, such as oncogenes and tumor suppressors, with ubiquitin. Once tagged, these proteins are degraded by the proteasome. The specificity of this degradation system for particular substrates lies with the E3 component of the ubiquitin ligase system (ubiquitin is transferred from an E1 enzyme to an E2 enzyme and finally, thanks to an E3 enzyme, directly to a specific substrate). The clinical effectiveness of Velcade (as it theoretically should inhibit the output of all ubiquitin ligases active in the cell simultaneously) suggests that modulating specific ubiquitin ligases could result in an even better therapeutic ratio. At present, the only ubiquitin ligase leads that have been reported inhibit the degradation of p53 by Mdm2, but these have not yet been developed into clinical therapeutics. In this review, we discuss the biological rationale, assays, genomics, proteomics and three-dimensional structures pertaining to key targets within the UPS (SCFSkp2 and APC/C) in order to assess their drug development potential

Resistance of Subtype C HIV-1 Strains to Anti-V3 Loop Antibodies

HIV-1's subtype C V3 loop consensus sequence exhibits increased resistance to anti-V3 antibody-mediated neutralization as compared to the subtype B consensus sequence. The dynamic 3D structure of the consensus C V3 loop crown, visualized by ab initio folding, suggested that the resistance derives from structural rigidity and non-β-strand secondary protein structure in the N-terminal strand of the β-hairpin of the V3 loop crown, which is where most known anti-V3 loop antibodies bind. The observation of either rigidity or non-β-strand structure in this region correlated with observed resistance to antibody-mediated neutralization in a series of chimeric pseudovirus (psV) mutants. The results suggest the presence of an epitope-independent, neutralization-relevant structural difference in the antibody-targeted region of the V3 loop crown between subtype C and subtype B, a difference that we hypothesize may contribute to the divergent pattern of global spread between these subtypes. As antibodies to a variable loop were recently identified as an inverse correlate of risk for HIV infection, the structure-function relationships discussed in this study may have relevance to HIV vaccine research

Young and Early Career Investigators: Report from a Global HIV Vaccine Enterprise Working Group

The scientific challenges facing HIV-1 vaccine development are unprecedented in the history of vaccinology. As a result, investigators, funders, and other stakeholders generally agree that “game-changing” ideas are required. While innovation can certainly arise from investigators at all career stages, young and early-career investigators, defined as those under 40 years of age or within 10 years of their final degree or clinical training, are especially key contributors of novel and transformative ideas. Young and early-career investigators bring energy, enthusiasm, and fresh perspectives that are unbiased by prevailing dogma and that are essential to scientific progress

Indirect Detection of an Epitope-Specific Response to HIV-1 gp120 Immunization in Human Subjects

A specific response of human serum neutralizing antibodies (nAb) to a conformational epitope as a result of vaccination of human subjects with the surface envelope glycoprotein (gp120) of HIV-1 has not previously been documented. Here, we used computational analysis to assess the epitope-specific responses of human subjects, which were immunized with recombinant gp120 immunogens in the VAX003 and VAX004 clinical trials. Our computational methodology—a variation of sieve analysis—compares the occurrence of specific nAb targeted conformational 3D epitopes on viruses from infected individuals who received vaccination to the occurrence of matched epitopes in the viruses infecting placebo subjects. We specifically studied seven crystallographically defined nAb targeted conformational epitopes in the V3 loop, an immunogenic region of gp120. Of the six epitopes present in the immunogens and targeted by known monoclonal neutralizing antibodies, only the one targeted by the anti-V3 nAb 2219 exhibited a significant reduction in occurrence in vaccinated subjects compared to the placebo group. This difference occurred only in the VAX003 Thailand cohort. No difference was seen between vaccinated and placebo groups for the occurrence of an epitope that was not present in the immunogen. Thus, it can be theorized that a specific 2219-like human neutralizing antibody immune response to AIDSVAX immunization occurred in the VAX003 cohort, and that this response protected subjects from a narrow subset of HIV-1 viruses circulating in Thailand in the 1990s and bearing the conformational epitope targeted by the neutralizing antibody 2219

Ubiquitylation of the ER-Shaping Protein Lunapark via the CRL3KLHL12 Ubiquitin Ligase Complex

Summary: Cullin-RING ligases (CRLs) control key cellular processes by promoting ubiquitylation of a multitude of soluble cytosolic and nuclear proteins. Subsets of CRL complexes are recruited and activated locally at cellular membranes; however, few CRL functions and substrates at these distinct cellular compartments are known. Here, we use a proteomic screen to identify proteins that are ubiquitylated at cellular membranes and found that Lunapark, an endoplasmic reticulum (ER)-shaping protein localized to ER three-way junctions, is ubiquitylated by the CRL3KLHL12 ubiquitin ligase. We demonstrate that Lunapark interacts with mechanistic target of rapamycin complex-1 (mTORC1), a central cellular regulator that coordinates growth and metabolism with environmental conditions. We show that mTORC1 binds Lunapark specifically at three-way junctions, and lysosomes, where mTORC1 is activated, make contact with three-way junctions where Lunapark resides. Inhibition of Lunapark ubiquitylation results in neurodevelopmental defects indicating that KLHL12-dependent ubiquitylation of Lunapark is required for normal growth and development

CL-705G: a novel chemical Kir6.2-specific KATP channel opener

Background: KATP channels have diverse roles, including regulation of insulin secretion and blood flow, and protection against biological stress responses and are excellent therapeutic targets. Different subclasses of KATP channels exist in various tissue types due to the unique assemblies of specific pore-forming (Kir6.x) and accessory (SURx) subunits. The majority of pharmacological openers and blockers act by binding to SURx and are poorly selective against the various KATP channel subclasses.Methods and Results: We used 3D models of the Kir6.2/SUR homotetramers based on existing cryo-EM structures of channels in both the open and closed states to identify a potential agonist binding pocket in a functionally critical area of the channel. Computational docking screens of this pocket with the Chembridge Core chemical library of 492,000 drug-like compounds yielded 15 top-ranked “hits”, which were tested for activity against KATP channels using patch clamping and thallium (Tl+) flux assays with a Kir6.2/SUR2A HEK-293 stable cell line. Several of the compounds increased Tl+ fluxes. One of them (CL-705G) opened Kir6.2/SUR2A channels with a similar potency as pinacidil (EC50 of 9 µM and 11 μM, respectively). Remarkably, compound CL-705G had no or minimal effects on other Kir channels, including Kir6.1/SUR2B, Kir2.1, or Kir3.1/Kir3.4 channels, or Na+ currents of TE671 medulloblastoma cells. CL-705G activated Kir6.2Δ36 in the presence of SUR2A, but not when expressed by itself. CL-705G activated Kir6.2/SUR2A channels even after PIP2 depletion. The compound has cardioprotective effects in a cellular model of pharmacological preconditioning. It also partially rescued activity of the gating-defective Kir6.2-R301C mutant that is associated with congenital hyperinsulinism.Conclusion: CL-705G is a new Kir6.2 opener with little cross-reactivity with other channels tested, including the structurally similar Kir6.1. This, to our knowledge, is the first Kir-specific channel opener

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Recovering Protein-Protein and Domain-Domain Interactions from Aggregation of IP-MS Proteomics of Coregulator Complexes

Coregulator proteins (CoRegs) are part of multi-protein complexes that transiently assemble with transcription factors and chromatin modifiers to regulate gene expression. In this study we analyzed data from 3,290 immuno-precipitations (IP) followed by mass spectrometry (MS) applied to human cell lines aimed at identifying CoRegs complexes. Using the semi-quantitative spectral counts, we scored binary protein-protein and domain-domain associations with several equations. Unlike previous applications, our methods scored prey-prey protein-protein interactions regardless of the baits used. We also predicted domain-domain interactions underlying predicted protein-protein interactions. The quality of predicted protein-protein and domain-domain interactions was evaluated using known binary interactions from the literature, whereas one protein-protein interaction, between STRN and CTTNBP2NL, was validated experimentally; and one domain-domain interaction, between the HEAT domain of PPP2R1A and the Pkinase domain of STK25, was validated using molecular docking simulations. The scoring schemes presented here recovered known, and predicted many new, complexes, protein-protein, and domain-domain interactions. The networks that resulted from the predictions are provided as a web-based interactive application at http://maayanlab.net/HT-IP-MS-2-PPI-DDI/

Control of chromosome stability by the \u3b2-TrCP\u2013REST\u2013Mad2 axis

REST/NRSF (repressor-element-1-silencing transcription factor/ neuron-restrictive silencing factor) negatively regulates the tran- scription of genes containing RE1 sites1,2. REST is expressed in non-neuronal cells and stem/progenitor neuronal cells, in which it inhibits the expression of neuron-specific genes. Overexpression of REST is frequently found in human medulloblastomas and neuroblastomas3\u20137, in which it is thought to maintain the stem character of tumour cells. Neural stem cells forced to express REST and c-Myc fail to differentiate and give rise to tumours in the mouse cerebellum3. Expression of a splice variant of REST that lacks the carboxy terminus has been associated with neuronal tumours and small-cell lung carcinomas8\u201310, and a frameshift mutant (REST-FS), which is also truncated at the C terminus, has oncogenic properties11. Here we show, by using an unbiased screen, that REST is an interactor of the F-box protein b-TrCP. REST is degraded by means of the ubiquitin ligase SCFb-TrCP dur- ing the G2 phase of the cell cycle to allow transcriptional derepres- sion of Mad2, an essential component of the spindle assembly checkpoint. The expression in cultured cells of a stable REST mutant, which is unable to bind b-TrCP, inhibited Mad2 expres- sion and resulted in a phenotype analogous to that observed in Mad21/2 cells. In particular, we observed defects that were con- sistent with faulty activation of the spindle checkpoint, such as shortened mitosis, premature sister-chromatid separation, chro- mosome bridges and mis-segregation in anaphase, tetraploidy, and faster mitotic slippage in the presence of a spindle inhibitor. An indistinguishable phenotype was observed by expressing the oncogenic REST-FS mutant11, which does not bind b-TrCP. Thus, SCFb-TrCP-dependent degradation of REST during G2 permits the optimal activation of the spindle checkpoint, and consequently it is required for the fidelity of mitosis. The high levels of REST or its truncated variants found in certain human tumours may contri- bute to cellular transformation by promoting genomic instability

Geographic patterns of tree dispersal modes in Amazonia and their ecological correlates

Unidad de excelencia María de Maeztu CEX2019-000940-MAim: To investigate the geographic patterns and ecological correlates in the geographic distribution of the most common tree dispersal modes in Amazonia (endozoochory, synzoochory, anemochory and hydrochory). We examined if the proportional abundance of these dispersal modes could be explained by the availability of dispersal agents (disperser-availability hypothesis) and/or the availability of resources for constructing zoochorous fruits (resource-availability hypothesis). Time period: Tree-inventory plots established between 1934 and 2019. Major taxa studied: Trees with a diameter at breast height (DBH) ≥ 9.55 cm. Location: Amazonia, here defined as the lowland rain forests of the Amazon River basin and the Guiana Shield. Methods: We assigned dispersal modes to a total of 5433 species and morphospecies within 1877 tree-inventory plots across terra-firme, seasonally flooded, and permanently flooded forests. We investigated geographic patterns in the proportional abundance of dispersal modes. We performed an abundance-weighted mean pairwise distance (MPD) test and fit generalized linear models (GLMs) to explain the geographic distribution of dispersal modes. Results: Anemochory was significantly, positively associated with mean annual wind speed, and hydrochory was significantly higher in flooded forests. Dispersal modes did not consistently show significant associations with the availability of resources for constructing zoochorous fruits. A lower dissimilarity in dispersal modes, resulting from a higher dominance of endozoochory, occurred in terra-firme forests (excluding podzols) compared to flooded forests. Main conclusions: The disperser-availability hypothesis was well supported for abiotic dispersal modes (anemochory and hydrochory). The availability of resources for constructing zoochorous fruits seems an unlikely explanation for the distribution of dispersal modes in Amazonia. The association between frugivores and the proportional abundance of zoochory requires further research, as tree recruitment not only depends on dispersal vectors but also on conditions that favour or limit seedling recruitment across forest types