Characterization and molecular basis of the oligomeric structure of HIV-1 Nef protein

International audienceThe Nef protein of human immunodeficiency virus type I~HIV-1! is an important determinant for the onset of AIDS disease. The self-association properties of HIV-1 Nef are analyzed by chemical cross-linking, dynamic light scattering, equilibrium analytical ultracentrifugation, and NMR spectroscopy. The experimental data show that the HIV-1 Nef core domain forms stable homo-dimers and trimers in solution, but not higher oligomers. These Nef homomers are not covalently linked by disulfide bridges, and the equilibrium between these forms is dependent on the Nef concentration. We further provide the molecular basis for the Nef core dimers and trimers obtained by analysis of crystallographic models. Oligomerization of biological polypeptides is a common tool used to trigger events in cellular signaling and endocytosis, both of which are targeted by Nef. The quaternary structure of Nef may be of physiological importance and may help to connect its cellular targets or to increase affinity of the viral molecule for its ligands. The herein described models for Nef dimers and trimers will allow further mutational studies to elucidate their role in vivo. These results provide novel insight into the structural and functional relationships of this important viral protein. Moreover, the oligomer interface may represent a novel target for the design of antiviral agents

ROP18 Is a Rhoptry Kinase Controlling the Intracellular Proliferation of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite for which the discharge of apical organelles named rhoptries is a key event in host cell invasion. Among rhoptry proteins, ROP2, which is the prototype of a large protein family, is translocated in the parasitophorous vacuole membrane during invasion. The ROP2 family members are related to protein-kinases, but only some of them are predicted to be catalytically active, and none of the latter has been characterized so far. We show here that ROP18, a member of the ROP2 family, is located in the rhoptries and re-localises at the parasitophorous vacuole membrane during invasion. We demonstrate that a recombinant ROP18 catalytic domain (amino acids 243–539) possesses a protein-kinase activity and phosphorylate parasitic substrates, especially a 70-kDa protein of tachyzoites. Furthermore, we show that overexpression of ROP18 in transgenic parasites causes a dramatic increase in intra-vacuolar parasite multiplication rate, which is correlated with kinase activity. Therefore, we demonstrate, to our knowledge for the first time, that rhoptries can discharge active protein-kinases upon host cell invasion, which can exert a long-lasting effect on intracellular parasite development and virulence

MLLT1 YEATS domain mutations in clinically distinctive Favourable Histology Wilms tumours.

Wilms tumour is an embryonal tumour of childhood that closely resembles the developing kidney. Genomic changes responsible for the development of the majority of Wilms tumours remain largely unknown. Here we identify recurrent mutations within Wilms tumours that involve the highly conserved YEATS domain of MLLT1 (ENL), a gene known to be involved in transcriptional elongation during early development. The mutant MLLT1 protein shows altered binding to acetylated histone tails. Moreover, MLLT1-mutant tumours show an increase in MYC gene expression and HOX dysregulation. Patients with MLLT1-mutant tumours present at a younger age and have a high prevalence of precursor intralobar nephrogenic rests. These data support a model whereby activating MLLT1 mutations early in renal development result in the development of Wilms tumour

QAUST: Protein Function Prediction Using Structure Similarity, Protein Interaction, and Functional Motifs

The number of available protein sequences in public databases is increasing exponentially. However, a significant percentage of these sequences lack functional annotation, which is essential for the understanding of how biological systems operate. Here, we propose a novel method, Quantitative Annotation of Unknown STructure (QAUST), to infer protein functions, specifically Gene Ontology (GO) terms and Enzyme Commission (EC) numbers. QAUST uses three sources of information: structure information encoded by global and local structure similarity search, biological network information inferred by protein–protein interaction data, and sequence information extracted from functionally discriminative sequence motifs. These three pieces of information are combined by consensus averaging to make the final prediction. Our approach has been tested on 500 protein targets from the Critical Assessment of Functional Annotation (CAFA) benchmark set. The results show that our method provides accurate functional annotation and outperforms other prediction methods based on sequence similarity search or threading. We further demonstrate that a previously unknown function of human tripartite motif-containing 22 (TRIM22) protein predicted by QAUST can be experimentally validated

Interleukin-26 activates macrophages and facilitates killing of Mycobacterium tuberculosis

Tuberculosis-causing Mycobacterium tuberculosis (Mtb) is transmitted via airborne droplets followed by a primary infection of macrophages and dendritic cells. During the activation of host defence mechanisms also neutrophils and T helper 1 (TH1) and TH17 cells are recruited to the site of infection. The TH17 cell-derived interleukin (IL)-17 in turn induces the cathelicidin LL37 which shows direct antimycobacterial effects. Here, we investigated the role of IL-26, a TH1- and TH17-associated cytokine that exhibits antimicrobial activity. We found that both IL-26 mRNA and protein are strongly increased in tuberculous lymph nodes. Furthermore, IL-26 is able to directly kill Mtb and decrease the infection rate in macrophages. Binding of IL-26 to lipoarabinomannan might be one important mechanism in extracellular killing of Mtb. Macrophages and dendritic cells respond to IL-26 with secretion of tumor necrosis factor (TNF)-α and chemokines such as CCL20, CXCL2 and CXCL8. In dendritic cells but not in macrophages cytokine induction by IL-26 is partly mediated via Toll like receptor (TLR) 2. Taken together, IL-26 strengthens the defense against Mtb in two ways: firstly, directly due to its antimycobacterial properties and secondly indirectly by activating innate immune mechanisms

Regulation of the PI3K pathway through a p85α monomer–homodimer equilibrium

© Cheung et al. The canonical action of the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K) is to associate with the p110α catalytic subunit to allow stimuli-dependent activation of the PI3K pathway. We elucidate a p110α-independent role of homodimerized p85α in the positive regulation of PTEN stability and activity. p110α-free p85α homodimerizes via two intermolecular interactions (SH3:proline-rich region and BH:BH) to selectively bind unphosphorylated activated PTEN. As a consequence, homodimeric but not monomeric p85α suppresses the PI3K pathway by protecting PTEN from E3 ligase WWP2-mediated proteasomal degradation. Further, the p85α homodimer enhances the lipid phosphatase activity and membrane association of PTEN. Strikingly, we identified cancer patient-derived oncogenic p85α mutations that target the homodimerization or PTEN interaction surface. Collectively, our data suggest the equilibrium of p85α monomerdimers regulates the PI3K pathway and disrupting this equilibrium could lead to disease development.Link_to_subscribed_fulltex

The ancestral retinoic acid receptor was a low-affinity sensor triggering neuronal differentiation.

Retinoic acid (RA) is an important intercellular signaling molecule in vertebrate development, with a well-established role in the regulation of hox genes during hindbrain patterning and in neurogenesis. However, the evolutionary origin of the RA signaling pathway remains elusive. To elucidate the evolution of the RA signaling system, we characterized RA metabolism and signaling in the marine annelid Platynereis dumerilii, a powerful model for evolution, development, and neurobiology. Binding assays and crystal structure analyses show that the annelid retinoic acid receptor (RAR) binds RA and activates transcription just as vertebrate RARs, yet with a different ligand-binding pocket and lower binding affinity, suggesting a permissive rather than instructive role of RA signaling. RAR knockdown and RA treatment of swimming annelid larvae further reveal that the RA signal is locally received in the medial neuroectoderm, where it controls neurogenesis and axon outgrowth, whereas the spatial colinear hox gene expression in the neuroectoderm remains unaffected. These findings suggest that one early role of the new RAR in bilaterian evolution was to control the spatially restricted onset of motor and interneuron differentiation in the developing ventral nerve cord and to indicate that the regulation of hox-controlled anterior-posterior patterning arose only at the base of the chordates, concomitant with a high-affinity RAR needed for the interpretation of a complex RA gradient

Pre-mRNA splicing repression triggers abiotic stress signaling in plants

[EN] Alternative splicing (AS) of precursor RNAs enhances transcriptome plasticity and proteome diversity in response to diverse growth and stress cues. Recent work has shown that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various inhibitors of AS. Here, we show that the macrolide pladienolide B (PB) inhibits constitutive splicing and AS in plants. Also, our RNA sequencing (RNA-seq) data revealed that PB mimics abiotic stress signals including salt, drought and abscisic acid (ABA). PB activates the abiotic stress-and ABA-responsive reporters RD29A::LUC and MAPKKK18::uidA in Arabidopsis thaliana and mimics the effects of ABA on stomatal aperture. Genome-wide analysis of AS by RNA-seq revealed that PB perturbs the splicing machinery and leads to a striking increase in intron retention and a reduction in other forms of AS. Interestingly, PB treatment activates the ABA signaling pathway by inhibiting the splicing of clade A PP2C phosphatases while still maintaining to some extent the splicing of ABA-activated SnRK2 kinases. Taken together, our data establish PB as an inhibitor and modulator of splicing and a mimic of abiotic stress signals in plants. Thus, PB reveals the molecular underpinnings of the interplay between stress responses, ABA signaling and post-transcriptional regulation in plants.We wish to thank members of the Laboratory for Genome Engineering at King Abdullah University of Science and Technology for helpful discussions and comments on the manuscript. We wish to thank Moussa Benhamed for helpful discussions and suggestions and for providing key materials. We wish to thank Sean Cutler for providing Arabidopsis seeds of MAKPKKK18-uidA. This study was supported by King Abdullah University of Science and Technology. Work in PR's laboratory was funded by grant BIO2014-52537-R from MINECO. Work in PD's laboratory is funded by grant PTDC/BIA-PLA/1084/2014 from FCT. The authors declare no conflicts of interest.Ling, Y.; Alshareef, S.; Butt, H.; Lozano Juste, J.; Li, L.; Galal, AA.; Moustafa, A.... (2017). Pre-mRNA splicing repression triggers abiotic stress signaling in plants. The Plant Journal. 89(2):291-309. https://doi.org/10.1111/tpj.13383S29130989

Clinical, genetic, and functional characterization of the glycine receptor β-subunit A455P variant in a family affected by hyperekplexia syndrome

Hyperekplexia is a rare neurological disorder characterized by exaggerated startle response affecting newborns with the hallmark characteristics of hypertonia, apnea, and noise or touch-induced non-epileptic seizures. The genetic causes of the disease can vary and several associated genes and mutations have been reported to affect glycine receptors (GlyRs); however, the mechanistic links between GlyRs and hyperekplexia are not yet understood. Here, we describe a patient with hyperekplexia from a consanguineous family. Extensive genetic screening using exome sequencing coupled with autozygome analysis and iterative filtering supplemented by in silico prediction identified that the patient carries the homozygous missense mutation A455P in GLRB, which encodes the GlyR β-subunit. To unravel the physiological and molecular effects of A455P on GlyRs, we used electrophysiology in a heterologous system as well as immunocytochemistry, confocal microscopy, and cellular biochemistry. We found a reduction in glycine-evoked currents in N2A cells expressing the mutation compared to wild type cells. Western blot analysis also revealed a reduced amount of GlyR β protein both in cell lysates and isolated membrane fractions. In line with the above observations, co-immunoprecipitation assays suggested that the GlyR α1-subunit retained co-assembly with βA455P to form membrane-bound heteromeric receptors. Finally, structural modelling showed that the A455P mutation affected the interaction between the GlyR β-subunit transmembrane domain 4 and the other helices of the subunit. Taken together, our study identifies and validates a novel loss-of-function mutation in GlyRs whose pathogenicity is likely to cause hyperekplexia in affected individuals

The Genome Sequence of the Wild Tomato Solanum pimpinellifolium Provides Insights Into Salinity Tolerance

Solanum pimpinellifolium, a wild relative of cultivated tomato, offers a wealth of breeding potential for desirable traits such as tolerance to abiotic and biotic stresses. Here, we report the genome assembly and annotation of S. pimpinellifolium ‘LA0480.’ Moreover, we present phenotypic data from one field experiment that demonstrate a greater salinity tolerance for fruit- and yield-related traits in S. pimpinellifolium compared with cultivated tomato. The ‘LA0480’ genome assembly size (811 Mb) and the number of annotated genes (25,970) are within the range observed for other sequenced tomato species. We developed and utilized the Dragon Eukaryotic Analyses Platform (DEAP) to functionally annotate the ‘LA0480’ protein-coding genes. Additionally, we used DEAP to compare protein function between S. pimpinellifolium and cultivated tomato. Our data suggest enrichment in genes involved in biotic and abiotic stress responses. To understand the genomic basis for these differences in S. pimpinellifolium and S. lycopersicum, we analyzed 15 genes that have previously been shown to mediate salinity tolerance in plants. We show that S. pimpinellifolium has a higher copy number of the inositol-3-phosphate synthase and phosphatase genes, which are both key enzymes in the production of inositol and its derivatives. Moreover, our analysis indicates that changes occurring in the inositol phosphate pathway may contribute to the observed higher salinity tolerance in ‘LA0480.’ Altogether, our work provides essential resources to understand and unlock the genetic and breeding potential of S. pimpinellifolium, and to discover the genomic basis underlying its environmental robustness