Clathrin-independent entry of baculovirus triggers uptake of E. coli in non-phagocytic human cells

Peer reviewe

Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors

Target or substrate Rab GTPases are identified for 17 proteins with DENN domains

CtIP tetramer assembly is required for DNA-end resection and repair.

Mammalian CtIP protein has major roles in DNA double-strand break (DSB) repair. Although it is well established that CtIP promotes DNA-end resection in preparation for homology-dependent DSB repair, the molecular basis for this function has remained unknown. Here we show by biophysical and X-ray crystallographic analyses that the N-terminal domain of human CtIP exists as a stable homotetramer. Tetramerization results from interlocking interactions between the N-terminal extensions of CtIP's coiled-coil region, which lead to a 'dimer-of-dimers' architecture. Through interrogation of the CtIP structure, we identify a point mutation that abolishes tetramerization of the N-terminal domain while preserving dimerization in vitro. Notably, we establish that this mutation abrogates CtIP oligomer assembly in cells, thus leading to strong defects in DNA-end resection and gene conversion. These findings indicate that the CtIP tetramer architecture described here is essential for effective DSB repair by homologous recombination.We thank M. Kilkenny for help with the collection of X-ray diffraction data, A. Sharff and P. Keller for help with X-ray data processing and J.D. Maman for assistance with SEC-MALS. This work was supported by a Wellcome Trust Senior Research Fellowship award in basic biomedical sciences (L.P.), an Isaac Newton Trust research grant (L.P. and O.R.D.) and a Cambridge Overseas Trust PhD studentship (M.D.S.). Research in the laboratory of S.P.J. is funded by Cancer Research UK (CRUK; programme grant C6/A11224), the European Research Council and the European Community Seventh Framework Programme (grant agreement no. HEALTH-F2-2010-259893 (DDResponse)). Core funding is provided by Cancer Research UK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, supplemented by CRUK. J.V.F. is funded by Cancer Research UK programme grant C6/A11224 and the Ataxia Telangiectasia Society. R.B. and J.C. are funded by Cancer Research UK programme grant C6/A11224. Y.G. and M.D. are funded by the European Research Council grant DDREAM.This is the accepted manuscript of a paper published in Nature Structural & Molecular Biology, 22, 150–157 (2015) doi: 10.1038/nsmb.293

Characterization of a Novel Interaction between Bcl-2 Members Diva and Harakiri

Interactions within proteins of the Bcl-2 family are key in the regulation of apoptosis. The death-inducing members control apoptotic mechanisms partly by antagonizing the prosurvival proteins through heterodimer formation. Structural and biophysical studies on these complexes are providing important clues to understand their function. To help improve our knowledge on protein-protein interactions within the Bcl-2 family we have studied the binding between two of its members: mouse Diva and human Harakiri. Diva has been shown to perform both prosurvival and killing activity. In contrast, Harakiri induces cell death by interacting with antiapoptotic Bcl-2 members. Here we show using ELISA and NMR that Diva and Harakiri can interact in vitro. Combining the NMR data with the previously reported three-dimensional structure of Diva we find that Harakiri binds to a specific region in Diva. This interacting surface is equivalent to the known binding area of prosurvival Bcl-2 members from the reported structures of the complexes, suggesting that Diva could function at the structural level similarly to the antiapoptotic proteins of the Bcl-2 family. We illustrate this result by building a structural model of the heterodimer using molecular docking and the NMR data as restraints. Moreover, combining circular dichroism and NMR we also show that Harakiri is largely unstructured with residual (13%) α-helical conformation. This result agrees with intrinsic disorder previously observed in other Bcl-2 members. In addition, Harakiri constructs of different length were studied to identify the region critical for the interaction. Differential affinity for Diva of these constructs suggests that the amino acid sequence flanking the interacting region could play an important role in binding

Immunoprecipitation of steroidogenic enzyme autoantigens with autoimmune polyglandular syndrome type I (APS I) sera; further evidence for independent humoral immunity to P450c17 and P450c21

Three steroidogenic P450 cytochromes, steroid 17α-hydroxylase (P450c17), steroid 21-hydroxylase (P450c21) and side-chain cleavage enzyme (P450scc), have been described as autoantigens in APS I. In this study we report an immunoprecipitation assay for the detection of autoantibodies to these three enzymes using in vitro35S-labelled antigens. Overall, 33 out of 46 (72%) patients with APS I had autoantibodies to at least one of the three proteins and each protein was recognized by patient sera with equal frequency. A higher rate of autoantibody positivity was observed in APS I patients with Addison's disease compared with patients without Addison's disease (85% versus 39%). All 11 patients with ovarian failure had anti-P450c17 or anti-P450scc antibodies. The immunoprecipitation results with P450c17, P450c21 and P450scc correlated well with the results obtained by immunoblotting assays. In addition, the steroidogenic enzymes 11β-hydroxylase (P450c11β), aromatase (P450arom), 3β-hydroxysteroid dehydrogenase (3βHSD) and adrenodoxin were studied by immunoprecipitation assay, but no reaction was found either with 46 APS I or with 26 healthy control sera. To study the suggested immunological cross-reactivity between P450c17 and P450c21 enzymes, nine APS I patient sera were preabsorbed with bacterially expressed P450c17 or P450c21 and subsequently used in immunoprecipitation assay. The absorption experiments clearly indicated that the preincubation inhibited only the reactivity of corresponding antigen, suggesting independent autoantibody response to the two enzymes. Our results suggest that the immune response to some but not to all steroidogenic enzymes is a specific feature of APS I that may be pathogenically significant