Fluorogenic Trp(redBODIPY) cyclopeptide targeting keratin 1 for imaging of aggressive carcinomas.

Keratin 1 (KRT1) is overexpressed in squamous carcinomas and associated with aggressive pathologies in breast cancer. Herein we report the design and preparation of the first Trp-based red fluorogenic amino acid, which is synthetically accessible in a few steps and displays excellent photophysical properties, and its application in a minimally-disruptive labelling strategy to prepare a new fluorogenic cyclopeptide for imaging of KRT1+ cells in whole intact tumour tissues.

Investigating Ebola virus pathogenicity using molecular dynamics.

BACKGROUND: Ebolaviruses have been known to cause deadly disease in humans for 40 years and have recently been demonstrated in West Africa to be able to cause large outbreaks. Four Ebolavirus species cause severe disease associated with high mortality in humans. Reston viruses are the only Ebolaviruses that do not cause disease in humans. Conserved amino acid changes in the Reston virus protein VP24 compared to VP24 of other Ebolaviruses have been suggested to alter VP24 binding to host cell karyopherins resulting in impaired inhibition of interferon signalling, which may explain the difference in human pathogenicity. Here we used protein structural analysis and molecular dynamics to further elucidate the interaction between VP24 and KPNA5. RESULTS: As a control experiment, we compared the interaction of wild-type and R137A-mutant (known to affect KPNA5 binding) Ebola virus VP24 with KPNA5. Results confirmed that the R137A mutation weakens direct VP24-KPNA5 binding and enables water molecules to penetrate at the interface. Similarly, Reston virus VP24 displayed a weaker interaction with KPNA5 than Ebola virus VP24, which is likely to reduce the ability of Reston virus VP24 to prevent host cell interferon signalling. CONCLUSION: Our results provide novel molecular detail on the interaction of Reston virus VP24 and Ebola virus VP24 with human KPNA5. The results indicate a weaker interaction of Reston virus VP24 with KPNA5 than Ebola virus VP24, which is probably associated with a decreased ability to interfere with the host cell interferon response. Hence, our study provides further evidence that VP24 is a key player in determining Ebolavirus pathogenicity.

Changes associated with Ebola virus adaptation to novel species.

MOTIVATION: Ebola viruses are not pathogenic but can be adapted to replicate and cause disease in rodents. Here, we used a structural bioinformatics approach to analyze the mutations associated with Ebola virus adaptation to rodents to elucidate the determinants of host-specific Ebola virus pathogenicity. RESULTS: We identified 33 different mutations associated with Ebola virus adaptation to rodents in the proteins GP, NP, L, VP24 and VP35. Only VP24, GP and NP were consistently found mutated in rodent-adapted Ebola virus strains. Fewer than five mutations in these genes seem to be required for the adaptation of Ebola viruses to a new species. The role of mutations in GP and NP is not clear. However, three VP24 mutations located in the protein interface with karyopherin α5 may enable VP24 to inhibit karyopherins and subsequently the host interferon response. Three further VP24 mutations change hydrogen bonding or cause conformational changes. Hence, there is evidence that few mutations including crucial mutations in VP24 enable Ebola virus adaptation to new hosts. Since Reston virus, the only non-human pathogenic Ebolavirus species circulates in pigs in Asia, this raises concerns that few mutations may result in novel human pathogenic Ebolaviruses. CONTACT: m.n.wass@kent.ac.uk , m.michaelis@kent.ac.uk or j.s.rossman@kent.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Conserved differences in protein sequence determine the human pathogenicity of Ebolaviruses.

Reston viruses are the only Ebolaviruses that are not pathogenic in humans. We analyzed 196 Ebolavirus genomes and identified specificity determining positions (SDPs) in all nine Ebolavirus proteins that distinguish Reston viruses from the four human pathogenic Ebolaviruses. A subset of these SDPs will explain the differences in human pathogenicity between Reston and the other four ebolavirus species. Structural analysis was performed to identify those SDPs that are likely to have a functional effect. This analysis revealed novel functional insights in particular for Ebolavirus proteins VP40 and VP24. The VP40 SDP P85T interferes with VP40 function by altering octamer formation. The VP40 SDP Q245P affects the structure and hydrophobic core of the protein and consequently protein function. Three VP24 SDPs (T131S, M136L, Q139R) are likely to impair VP24 binding to human karyopherin alpha5 (KPNA5) and therefore inhibition of interferon signaling. Since VP24 is critical for Ebolavirus adaptation to novel hosts, and only a few SDPs distinguish Reston virus VP24 from VP24 of other Ebolaviruses, human pathogenic Reston viruses may emerge. This is of concern since Reston viruses circulate in domestic pigs and can infect humans, possibly via airborne transmission.

VarMod: modelling the functional effects of non-synonymous variants.

Unravelling the genotype-phenotype relationship in humans remains a challenging task in genomics studies. Recent advances in sequencing technologies mean there are now thousands of sequenced human genomes, revealing millions of single nucleotide variants (SNVs). For non-synonymous SNVs present in proteins the difficulties of the problem lie in first identifying those nsSNVs that result in a functional change in the protein among the many non-functional variants and in turn linking this functional change to phenotype. Here we present VarMod (Variant Modeller) a method that utilises both protein sequence and structural features to predict nsSNVs that alter protein function. VarMod develops recent observations that functional nsSNVs are enriched at protein-protein interfaces and protein-ligand binding sites and uses these characteristics to make predictions. In benchmarking on a set of nearly 3000 nsSNVs VarMod performance is comparable to an existing state of the art method. The VarMod web server provides extensive resources to investigate the sequence and structural features associated with the predictions including visualisation of protein models and complexes via an interactive JSmol molecular viewer. VarMod is available for use at http://www.wasslab.org/varmod.

Heme oxygenase-2/adiponectin protein-protein interaction in metabolic syndrome.

Insulin resistance with adipose tissue dysfunction and dysregulation in the production and secretion of adipokines is one of the hallmarks of metabolic syndrome. We have previously reported that increased levels of the heme oxygenase (HO) system, HO-1/HO-2 results in increased levels of adiponectin. Despite documentation of the existence of the anti-inflammatory axis HO-adiponectin, a possible protein-protein interaction between HO and adiponectin has not been examined. Here, we investigated the existence of protein interactions between HO-2 and adiponectin in the maintenance of adipocyte function during metabolic syndrome by integrating phenotypic and in silico studies. Compared to WT animals, HO-2 null mice displayed an increase in both visceral and subcutaneous fat content and reduced circulating adiponectin levels. The decrease in adiponectin was reversed by upregulation of HO-1. HO-2 depletion was associated with increased adipogenesis in cultured mesenchymal stem cells (MSCs) and decreased adiponectin levels in the culture media. In addition, HO-1 siRNA decreased adiponectin release. HO-2 was found to bind to the monomeric form of adiponectin, according to poses and calculated energies. HO-2-adiponectin interactions were validated by the two-hybrid system assay. In conclusion, protein-protein interactions between HO-2 and adiponectin highlight the role of HO-2 as a molecular chaperone for adiponectin assembly, while HO-1 increases adiponectin levels. Thus, crosstalk between HO-2 and HO-1 could be manipulated in a therapeutic approach to ameliorate the deleterious effects of obesity and the metabolic syndrome.

Evaluation of imidazole-based compounds as heme oxygenase-1 inhibitors.

Imidazole-based compounds previously synthesized in our laboratory were selected and reconsidered as inhibitors of heme oxygenase-1 obtained from the microsomal fractions of rat spleens. Most of tested compounds were good inhibitors with IC(50) values in the low micromolar range. Compounds were also assayed on membrane-free full-length recombinant human heme oxygenase-1; all tested compounds were unable to interact with human heme oxygenase-1 at 100 µm concentrations with the exception of compounds 11 and 13 that inhibited the enzyme of 54% and 20%, respectively. The binding of the most active compound 11 with heme or heme-conjugated human heme oxygenase-1 was also examined by spectral analyses. When heme was not conjugated to human heme oxygenase-1, compound 11 caused changes in the heme spectrum only at concentration 50-fold (100 µm) higher than that required to inhibit rat heme oxygenase-1; when heme was conjugated to human heme oxygenase-1, compound 11 was able to form a heme-compound 11 complex also at low micromolar concentrations. To obtain information on the binding mode of the tested compounds with enzyme, docking studies and pharmacophore analysis were performed. Template docking results were in agreement with experimental inhibition data and with a structure-based pharmacophoric model. These data may be exploitable to design new OH-1 inhibitors.