Mutagenesis of a stacking contact in the MS2 coat protein-RNA complex.

The thermodynamic contribution of a stacking interaction between Tyr85 in MS2 coat protein and a single-stranded pyrimidine in its RNA binding site has been examined. Mutation of Tyr85 to Phe, His, Cys, Ser and Ala decreased the RNA affinity by 1-3 kcal/mol under standard binding conditions. Since the Phe, His and Cys 85 proteins formed UV photocrosslinks with iodouracil-containing RNA at the same rate as the wild-type protein, the mutant proteins interact with RNA in a similar manner. The pH dependence of KD for the Phe and His proteins differs substantially from the wild-type protein, suggesting that the titration of position 85 contributes substantially to the binding properties. Experiments with specifically substituted phosphorothioate RNAs confirm a hydrogen bond between the hydroxyl group of tyrosine and a phosphate predicted by the crystal structure.     

The active essential CFNS3d protein complex

The NS2B-NS3 protease complex is essential for the replication of dengue virus, which is the etiologic agent of dengue and hemorrhagic fevers, diseases that are a burden for the tropical and subtropical areas of the world. The active form of the NS3 protease linked to the 40 residues of the NS2B cofactor shows highly flexible and disordered region(s) that are responsible for its high propensity to aggregate at the concentrations necessary for NMR spectroscopy studies or for crystallization. Limited proteolysis of this active form of the protease enabled us to obtain a folded and new essential form of the NS2B-NS3 protease complex. We found that the region from residues D50 to E80 of NS2B interacts directly and strongly with the NS3 protease domain. The proteolytic activity of the noncovalently binding complex was determined by a rapid and continuous fluorescence resonance energy transfer activity assay using a depsipeptide substrate. The new protein-cofactor complex obtained, encompassing the NS2B fragment (D50-E80) and the NS3 protease, shows proteolytic activity. The (1)H-(15)N-heteronuclear single quantum coherence spectrum of the isotopically enriched protein complex shows good cross-peak dispersion; this is indicative of a stable folded state. Our results significantly complement the X-ray structure of the NS2B-NS3pro complex published recently. Moreover, these results open the way to performing direct structural and interaction studies in solution on a new active NS2B-NS3pro complex with libraries of substrates and inhibitors in order to identify new drugs that prevent viral polyprotein processing.     

Identification of a minimal segment of complexin II essential for preferential distribution in axons

Complexin II (CPLX2) is a soluble pre-synaptic protein believed to regulate neurotransmitter release from pre-synaptic terminals. CPLX2 is localized in pre-synaptic terminals in mature brain, but the mechanism of selective localization remains unclear. Here we identified an essential segment of CPLX2 for preferential axonal distribution. Myc-tagged CPLX2 was expressed in cultured rat hippocampal neurons and its distribution between axons and dendrites was compared by immunocytochemistry and image analysis. Fluorescence signals were detected in both axons and dendrites; however, their respective distribution varied significantly. Despite the fact that signal intensity decreased almost linearly from the base to the tip of the dendrite, a substantial level was sustained along the axon, even at a position near the tip. Image analyses using a series of mutants indicated that the deletion of 19 amino acid residues, G71-P89, within the 'central core' for binding to soluble N-ethylmaleimide sensitive factor attachment protein receptor proteins resulted in the loss of preferential axonal distribution. The enhanced green fluorescent protein derivative fused with the G71-P89 fragment exhibited a similar localization to that of wild type CPLX2, indicating that the G71-P89 region of CPLX2 is essential and sufficient for preferential axonal distribution.     

Mhc diversity in two passerine birds: no evidence for a minimal essential Mhc

Humans express an array of Mhc genes, while the chicken has an Mhc that is relatively small and compact with fewer expressed genes. Here we ask whether the "minimal essential Mhc" of the chicken is representative for birds. We investigated the RFLP genotypes in 55 great reed warblers Acrocephalus arundinaceus and 10 willow warblers Phylloscopus trochilus to obtain an overview of the number of class II B genes. There were 13-17 bands per individual in the great reed warblers and 25-30 in the willow warblers, and every individual had a unique RFLP genotype. The high number of RFLP bands indicates that both species have a large number of class II B genes although some may be pseudogenes. Seven different class II B sequences were detected in a great reed warbler cDNA library. There was considerable sequence divergence between the cDNA sequences in exon 2 (peptide-binding region, PBR), whereas they were very similar in exon 3. The cDNA sequences were easily alignable to a classical chicken class II B sequence, and balancing selection was acting in the PBR. One of the cDNA sequences had two deletions and is likely nonfunctional. Finally, the polymorphic class I and class II B RFLP fragments seemed to be linked in the five studied great reed warbler families. These and previous results suggest that birds of the order Passeriformes in general have more Mhc class I and II B genes than birds of the order Galliformes. This difference could be caused by their phylogenetic past, and/or by variance in the selection pressure for maintaining a high number of Mhc genes.     

The HIV-1 gp41 N-terminal heptad repeat plays an essential role in membrane fusion

For many different enveloped viruses the crystal structure of the fusion protein core has been established. A striking conservation in the tertiary and quaternary arrangement of these core structures is repeatedly revealed among members of diverse families. It has been proposed that the primary role of the core involves structural rearrangements which facilitate apposition between viral and target cell membranes. Forming the internal trimeric coiled coil of the core, the N-terminal heptad repeat (NHR) of HIV-1 gp41 was suggested to have additional roles, due to its ability to bind biological membranes. The NHR is adjacent to the N-terminal hydrophobic fusion peptide (FP), which alone can fuse biological membranes. To investigate the role of the NHR in membrane fusion, we synthesized and functionally characterized HIV-1 gp41 peptides corresponding to the FP and NHR alone, as well as continuous peptides made of both FP and NHR (wild type and mutant). We show here that a consecutive, 70-residue peptide consisting of both the FP and NHR (gp41/1-70) has dramatic fusogenic properties. The effect of including the complete NHR, as compared to shorter 23-, 33-, or 52-residue N-terminal peptides, is illustrated by a leap in lipid mixing of phosphatidylcholine (PC) large unilamellar vesicles (LUV) and clearly delineates the synergistic role of the NHR in the fusion event. Furthermore, a mutation in the NHR that renders the virus noninfectious is reflected by a significant reduction in in vitro lipid mixing induced by the mutant, gp41/1-70 (I62D). Additional spectroscopic studies, characterizing membrane binding and apposition induced by the peptides, help to clarify the role of the NHR in membrane fusion.     

The SUN41 and SUN42 genes are essential for cell separation in Candida albicans

Completion of the yeast cell cycle involves extensive remodelling of the cell wall upon separation of mother and daughter cells. We have studied two members of the ascomycete-specific SUN gene family in Candida albicans. Inactivation of SUN41 yields defects in cell separation and hyphal elongation while inactivation of SUN42 results in minor phenotypic alterations. Simultaneous inactivation of SUN41 and SUN42 is synthetically lethal due to lysis of mother cells after septation. Electronic microscopy reveals cell wall defects mainly localized in the region surrounding the septa. This phenotype is osmoremediable and the conditional double mutants show increased sensitivity to cell wall or cell membrane perturbing agents. The essential function shared by Sun41p and Sun42p is conserved among yeasts because UTH1, a Saccharomyces cerevisiae SUN gene, suppresses the lethality of SUN41 and SUN42 conditional mutants. Investigation of functional genomic data obtained in S. cerevisiae reveals links between members of the SUN gene family and the RAM pathway regulating cell wall-degrading enzymes specifically involved during cell separation. Thus, the main function of ascomycetous Sun proteins appears linked to cell wall remodelling, with a probable role in counter-balancing cell wall degradation to avoid cell lysis upon cell separation.     

The nascent polypeptide-associated complex is essential for autophagic flux

The ribosome-associated nascent polypeptide-associated complex (NAC) is involved in multiple cotranslational processes, including protein transport into the ER and mitochondria, and also acts as a chaperone to assist protein folding. Here we demonstrated that NAC is also essential for autophagic degradation of a variety of protein aggregates in C. elegans. Loss of function of NAC impairs lysosome function, resulting in accumulation of autophagic substrates in enlarged autolysosomes. Knockdown of mammalian NAC also causes accumulation of nondegradative autolysosomes. Our study revealed that NAC plays an evolutionarily conserved role in the autophagy pathway and thus in maintaining protein homeostasis under physiological conditions.     

The nascent polypeptide-associated complex is essential for autophagic flux

The ribosome-associated nascent polypeptide-associated complex (NAC) is involved in multiple cotranslational processes, including protein transport into the ER and mitochondria, and also acts as a chaperone to assist protein folding. Here we demonstrated that NAC is also essential for autophagic degradation of a variety of protein aggregates in C. elegans. Loss of function of NAC impairs lysosome function, resulting in accumulation of autophagic substrates in enlarged autolysosomes. Knockdown of mammalian NAC also causes accumulation of nondegradative autolysosomes. Our study revealed that NAC plays an evolutionarily conserved role in the autophagy pathway and thus in maintaining protein homeostasis under physiological conditions.     

The C9orf72-SMCR8-WDR41 complex is a GAP for small GTPases

ABSTRACT

Massive expansions of the hexanucleotide in C9orf72 are the primary genetic origins of familial amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). Current studies have found that this repeat sequence participates in the disease process by producing neurotoxic substances and reducing the level of C9orf72 protein; however, the progress in the functional study of C9orf72 is slow. Recently, a stable complex, consisting of C9orf72, SMCR8, and WDR41, has been implicated in regulating membrane trafficking and macroautophagy. We reported the cryo-electron microscopy (cryo-EM) structure of the C9orf72-SMCR8-WDR41 complex (CSW complex), unveiling that the CSW complex is a dimer of heterotrimers. Intriguingly, in the heterotrimer of the C9orf72-SMCR8-WDR41, C9orf72 interacts with SMCR8 in a manner similar to the FLCN-FNIP2 complex. Nevertheless, WDR41 is connected to the DENN domain of SMCR8 through its N-terminal β-strand and C-terminal helix but does not directly interact with C9orf72. Notably, the C9orf72-SMCR8 complex was demonstrated to act as a GAP for RAB8A and RAB11A in vitro.