The HIV capsid mimics karyopherin engagement of FG-nucleoporins

HIV can infect non-dividing cells because the viral capsid can overcome the selective barrier of the nuclear pore complex and deliver the genome directly into the nucleus1,2. Remarkably, the intact HIV capsid is more than 1,000 times larger than the size limit prescribed by the diffusion barrier of the nuclear pore3. This barrier in the central channel of the nuclear pore is composed of intrinsically disordered nucleoporin domains enriched in phenylalanine–glycine (FG) dipeptides. Through multivalent FG interactions, cellular karyopherins and their bound cargoes solubilize in this phase to drive nucleocytoplasmic transport4. By performing an in vitro dissection of the nuclear pore complex, we show that a pocket on the surface of the HIV capsid similarly interacts with FG motifs from multiple nucleoporins and that this interaction licences capsids to penetrate FG-nucleoporin condensates. This karyopherin mimicry model addresses a key conceptual challenge for the role of the HIV capsid in nuclear entry and offers an explanation as to how an exogenous entity much larger than any known cellular cargo may be able to non-destructively breach the nuclear envelope

Pleckstrin homology domain leucine-rich repeat protein phosphatases set the amplitude of receptor tyrosine kinase output

Growth factor receptor levels are aberrantly high in diverse cancers, driving the proliferation and survival of tumor cells. Understanding the molecular basis for this aberrant elevation has profound clinical implications. Here we show that the pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) suppresses receptor tyrosine kinase (RTK) signaling output by a previously unidentified epigenetic mechanism unrelated to its previously described function as the hydrophobic motif phosphatase for the protein kinase AKT, protein kinase C, and S6 kinase. Specifically, we show that nuclear-localized PHLPP suppresses histone phosphorylation and acetylation, in turn suppressing the transcription of diverse growth factor receptors, including the EGF receptor. These data uncover a much broader role for PHLPP in regulation of growth factor signaling beyond its direct inactivation of AKT: By suppressing RTK levels, PHLPP dampens the downstream signaling output of two major oncogenic pathways, the PI3 kinase/AKT and the Rat sarcoma (RAS)/ERK pathways. Our data are consistent with a model in which PHLPP modifies the histone code to control the transcription of RTKs

Structural basis of TIR-domain-assembly formation in MAL- and MyD88-dependent TLR4 signaling

Toll-like receptor (TLR) signaling is a key innate immunity response to pathogens. Recruitment of signaling adapters such as MAL (TIRAP) and MyD88 to the TLRs requires Toll/interleukin-1 receptor (TIR)-domain interactions, which remain structurally elusive. Here we show that MAL TIR domains spontaneously and reversibly form filaments in vitro. They also form cofilaments with TLR4 TIR domains and induce formation of MyD88 assemblies. A 7-Å-resolution cryo-EM structure reveals a stable MAL protofilament consisting of two parallel strands of TIR-domain subunits in a BB-loop-mediated head-to-tail arrangement. Interface residues that are important for the interaction are conserved among different TIR domains. Although large filaments of TLR4, MAL or MyD88 are unlikely to form during cellular signaling, structure-guided mutagenesis, combined with in vivo interaction assays, demonstrated that the MAL interactions defined within the filament represent a template for a conserved mode of TIR-domain interaction involved in both TLR and interleukin-1 receptor signaling

A cell-free approach to accelerate the study of protein-protein interactions in vitro

Protein-protein interactions are highly desirable targets in drug discovery, yet only a fraction of drugs act as binding inhibitors. Here, we review the different technologies used to find and validate protein-protein interactions. We then discuss how the novel combination of cell-free protein expression, AlphaScreen and single-molecule fluorescence spectroscopy can be used to rapidly map protein interaction networks, determine the architecture of protein complexes, and find new targets for drug discovery

Value of copeptin and the S-100b protein assay in ruling out the diagnosis of stroke-induced dizziness pattern in emergency departments

International audienceBackground: Dizziness is a frequent reason for visiting emergency departments (EDs). Differentiating stroke from other causes is challenging for physicians. The role of biomarkers has been poorly assessed. We evaluated whether copeptin and S100b protein (PS100b) assessment, alone or in combination, could rule out stroke in patients visiting EDs for dizziness.Methods: We included patients 18 years of age or older, visiting the adult ED of a French university hospital for a new episode of dizziness evolving for less than 72 h. All patients underwent standardized physical examination (HINT [Head Impulse test, Nystagmus, test of skew deviation] maneuvers), copeptin and S-100b protein (PS100) measurement and injected brain imaging. Stroke diagnosis involved diffusion-weighted magnetic resonance imaging or, if not available, neurological examination and contrast brain CT scan compatible with the diagnosis.Results: Of the 135 patients participating in the study, 13 (10%) had stroke. The sensitivity, specificity and positive and negative predictive values of copeptin/PS100 combination were 100% (95%CI, 77-100%), 48% (40-57%), 14% (11-27%) and 100% (94-100%), respectively. Values for copeptin alone were 77% (CI95% 0.50-0.91), 50% (CI95% 0.49-0.58), 14% (CI95% 0.08-0.24), 93% (CI95% 0.87-0.98), and for PS100 alone were 54% (CI95% 0.29-0.77), 97% (CI95% 0.92-0.99), 64% (CI95% 0.35-0.84), 95% (CI95% 0.90-0.98).Conclusions: Absence of copeptin and PS100 elevation seems to ruling out the diagnosis of stroke in patients visiting the ED for a new episode of dizziness. These results need to be confirmed in a large-scale study

Corrigendum to Single-molecule fluorescence reveals the oligomerisation and folding steps driving the prion-like behaviour of ASC [J. Mol. Biol. 430 (4) (February 16, 2018) 491–508]

The authors would like to include the following authors and their contributions and funding details: Author list: Yann Gambin, Nichole Giles, Ailís O'Carroll , Mark E. Polinkovsky, Wayne Johnston, Dominic J.B Hunter, Kirill Alexandrov, Kate Schroder, Emma Sierecki. Affiliations: EMBL Australia Node in Single Molecule Science, University of New South Wales, Kensington, NSW 2052, Australia. The Institute for Molecular Bioscience, University of Queensland, St Lucia, QLD 4072, Australia. IMB Centre for Inflammation and Disease Research, University of Queensland, St Lucia, QLD 4072, Australia. Author's contributions: N.G., Y.G. and E.S. carried out the cell-free experiments. D.H. contributed the cell-free reagent. N.G., A.O.C., M.E.P., Y.G. and E.S. performed single-molecule coincidence analysis and AlphaScreen experiments and analyzed data. W.J. contributed to the development of the cell-free expression system. K.A. contributed to the development of the cell-free expression system and to formulation of the research objective. K.S. contributed to design and conceptualization of the study, and expertise in inflammasome biology. E.S. and Y.G. designed the study. Y.G., E.S., K.S. and N.G. drafted the manuscript. All authors read and approved the final manuscript. Funding: The authors acknowledge the facilities and the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland. Y.G. and K.S. are supported by Australian Research Council Future Fellowships (FT110100478 to Y.G., FT130100361 to K.S.). The National Health and Medical Research Council supported the research (Program Grants 511005 and APP1037320 to K.A., and APP1100771 to Y.G. and E.S.). The authors would like to apologize for any inconvenience caused

Herpes simplex virus encoded ICP6 protein forms functional amyloid assemblies with necroptosis-associated host proteins

The viral protein ICP6, encoded by herpes simplex virus 1 (HSV-1), harbours a RIP-homotypic interaction motif (RHIM), that plays a role in viral inhibition of host cell death pathways. Other members of the Herpesviridae family also encode RHIM-containing proteins that interfere with host-cell death pathways, including the M45 protein from murine cytomegalovirus, and ORF20 protein from varicella zoster virus. We have used amyloid assembly assays, electron microscopy and single molecule fluorescence spectroscopy to show that the ICP6 RHIM is amyloidogenic and can interact with host RHIM-containing proteins to form heteromeric amyloid complexes, in a manner similar to that of M45 and ORF20 RHIMs. The core tetrad sequence of the ICP6 RHIM is important for both amyloid formation and interaction with host RHIM-containing proteins. Notably, we show that the amyloid forming capacity of the ICP6 RHIM is affected by the redox environment. We propose that the formation of an intramolecular disulfide bond within ICP6 triggers the formation of amyloid assemblies that are distinct from previously characterised viral amyloids M45 and ORF20. Formation of viral-host heteromeric amyloid assemblies may underlie a general mechanism of viral adaptation against host immune machineries