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

Viral M45 and necroptosis-associated proteins form heteromeric amyloid assemblies

The murine cytomegalovirus protein M45 protects infected mouse cells from necroptotic death and, when heterologously expressed, can protect human cells from necroptosis induced by tumour necrosis factor receptor (TNFR) activation. Here, we show that the N-terminal 90 residues of the M45 protein, which contain a RIP homotypic interaction motif (RHIM), are sufficient to confer protection against TNFR-induced necroptosis. This N-terminal region of M45 drives rapid self-assembly into homo-oligomeric amyloid fibrils and interacts with the RHIMs of the human kinases RIPK1 and RIPK3, and the Z-DNA binding protein 1 (ZBP1), to form heteromeric amyloid fibrils in vitro. Mutation of the tetrad residues in the M45 RHIM attenuates homo- and hetero-amyloid assembly by M45, suggesting that the amyloidogenic nature of the M45 RHIM underlies its biological activity. The M45 RHIM preferentially interacts with RIPK3 and ZBP1 over RIPK1 and alters the properties of the host RHIM protein assemblies. Our results indicate that M45 mimics the interactions made by RIPK1 or ZBP1 with RIPK3, thereby forming heteromeric amyloid structures, which may explain its ability to inhibit necroptosis

Varicella zoster virus encodes a viral decoy RHIM to inhibit cell death

Herpesviruses are known to encode a number of inhibitors of host cell death, including RIP Homotypic Interaction Motif (RHIM)-containing proteins. Varicella zoster virus (VZV) is a member of the alphaherpesvirus subfamily and is responsible for causing chickenpox and shingles. We have identified a novel viral RHIM in the VZV capsid triplex protein, open reading frame (ORF) 20, that acts as a host cell death inhibitor. Like the human cellular RHIMs in RIPK1 and RIPK3 that stabilise the necrosome in TNF-induced necroptosis, and the viral RHIM in M45 from murine cytomegalovirus that inhibits cell death, the ORF20 RHIM is capable of forming fibrillar functional amyloid complexes. Notably, the ORF20 RHIM forms hybrid amyloid complexes with human ZBP1, a cytoplasmic sensor of viral nucleic acid. Although VZV can inhibit TNF-induced necroptosis, the ORF20 RHIM does not appear to be responsible for this inhibition. In contrast, the ZBP1 pathway is identified as important for VZV infection. Mutation of the ORF20 RHIM renders the virus incapable of efficient spread in ZBP1- expressing HT-29 cells, an effect which can be reversed by the inhibition of caspases. Therefore we conclude that the VZV ORF20 RHIM is important for preventing ZBP1-driven apoptosis during VZV infection, and propose that it mediates this effect by sequestering ZBP1 into decoy amyloid assemblies