Long-lived States in an intrinsically disordered protein domain

Long-lived states (LLS) are relaxation-favoured eigenstates of J-coupled magnetic nuclei. LLS were measured, along with classical 1H and 15 N relaxation rate constants, in aminoacids of the N-terminal Unique domain of the c-Src kinase (USrc), which is disordered in vitro under physiological conditions. The relaxation rates of LLS are a probe for motions and interactions in biomolecules. LLS of the aliphatic protons of glycines, with lifetimes ca. four times longer than their spin-lattice relaxation times, are reported for the first time in an intrinsically disordered protein domain (IDP). LLS relaxation experiments were integrated with 2D spectroscopy methods, further adapting them for studies on proteins.

Molecular cloning of a novel human I-mfa domain-containing protein that differently regulates human T-cell leukemia virus type I and HIV-1 expression.

Regulation of viral genome expression is the result of complex cooperation between viral proteins and host cell factors. We report here the characterization of a novel cellular factor sharing homology with the specific cysteine-rich C-terminal domain of the basic helix-loop-helix repressor protein I-mfa. The synthesis of this new factor, called HIC for Human I-mfa domain-Containing protein, is controlled at the translational level by two different codons, an ATG and an upstream non-ATG translational initiator, allowing the production of two protein isoforms, p32 and p40, respectively. We show that the HIC protein isoforms present different subcellular localizations, p32 being mainly distributed throughout the cytoplasm, whereas p40 is targeted to the nucleolus. Moreover, in trying to understand the function of HIC, we have found that both isoforms stimulate in T-cells the expression of a luciferase reporter gene driven by the human T-cell leukemia virus type I-long terminal repeat in the presence of the viral transactivator Tax. We demonstrate by mutagenesis that the I-mfa-like domain of HIC is involved in this regulation. Finally, we also show that HIC is able to down-regulate the luciferase expression from the human immunodeficiency virus type 1-long terminal repeat induced by the viral transactivator Tat. From these results, we propose that HIC and I-mfa represent two members of a new family of proteins regulating gene expression and characterized by a particular cysteine-rich C-terminal domain.

Characterization of HbpR binding by site-directed mutagenesis of its DNA-binding site and by deletion of the effector domain.

In the presence of 2-hydroxybiphenyl, the enhancer binding protein, HbpR, activates the sigma54-dependent P(hbpC) promoter and controls the initial steps of 2-hydroxybiphenyl degradation in Pseudomonas azelaica. In the activation process, an oligomeric HbpR complex of unknown subunit composition binds to an operator region containing two imperfect palindromic sequences. Here, the HbpR-DNA binding interactions were investigated by site-directed mutagenesis of the operator region and by DNA-binding assays using purified HbpR. Mutations that disrupted the twofold symmetry in the palindromes did not affect the binding affinity of HbpR, but various mutations along a 60 bp region, and also outside the direct palindromic sequences, decreased the binding affinity. Footprints of HbpR on mutant operator fragments showed that a partial loss of binding contacts occurs, suggesting that the binding of one HbpR 'protomer' in the oligomeric complex is impaired whilst leaving the other contacts intact. An HbpR variant, devoid of its N-terminal sensing A-domain, was unable to activate transcription from the hbpC promoter while maintaining protection of the operator DNA in footprints. Wild-type HbpR was unable to activate transcription from the hbpC promoter when delta A-HbpR was expressed in the same cell, suggesting the formation of (repressing) hetero-oligomers. This model implies that HbpR can self-associate on its operator DNA without effector recognition or ATP binding. Furthermore, our findings suggest that the N-terminal sensing domain of HbpR is needed to activate the central ATPase domain rather than to repress a constitutively active C domain, as is the case for the related regulatory protein XylR.

Structure-function analysis of the human TFIIB-related factor II protein reveals an essential role for the C-terminal domain in RNA polymerase III transcription.

The transcription factors TFIIB, Brf1, and Brf2 share related N-terminal zinc ribbon and core domains. TFIIB bridges RNA polymerase II (Pol II) with the promoter-bound preinitiation complex, whereas Brf1 and Brf2 are involved, as part of activities also containing TBP and Bdp1 and referred to here as Brf1-TFIIIB and Brf2-TFIIIB, in the recruitment of Pol III. Brf1-TFIIIB recruits Pol III to type 1 and 2 promoters and Brf2-TFIIIB to type 3 promoters such as the human U6 promoter. Brf1 and Brf2 both have a C-terminal extension absent in TFIIB, but their C-terminal extensions are unrelated. In yeast Brf1, the C-terminal extension interacts with the TBP/TATA box complex and contributes to the recruitment of Bdp1. Here we have tested truncated Brf2, as well as Brf2/TFIIB chimeric proteins for U6 transcription and for assembly of U6 preinitiation complexes. Our results characterize functions of various human Brf2 domains and reveal that the C-terminal domain is required for efficient association of the protein with U6 promoter-bound TBP and SNAP(c), a type 3 promoter-specific transcription factor, and for efficient recruitment of Bdp1. This in turn suggests that the C-terminal extensions in Brf1 and Brf2 are crucial to specific recruitment of Pol III over Pol II.

Evolution of the ferric reductase domain (FRD) superfamily: modularity, functional diversification, and signature motifs.

A heme-containing transmembrane ferric reductase domain (FRD) is found in bacterial and eukaryotic protein families, including ferric reductases (FRE), and NADPH oxidases (NOX). The aim of this study was to understand the phylogeny of the FRD superfamily. Bacteria contain FRD proteins consisting only of the ferric reductase domain, such as YedZ and short bFRE proteins. Full length FRE and NOX enzymes are mostly found in eukaryotic cells and all possess a dehydrogenase domain, allowing them to catalyze electron transfer from cytosolic NADPH to extracellular metal ions (FRE) or oxygen (NOX). Metazoa possess YedZ-related STEAP proteins, possibly derived from bacteria through horizontal gene transfer. Phylogenetic analyses suggests that FRE enzymes appeared early in evolution, followed by a transition towards EF-hand containing NOX enzymes (NOX5- and DUOX-like). An ancestral gene of the NOX(1-4) family probably lost the EF-hands and new regulatory mechanisms of increasing complexity evolved in this clade. Two signature motifs were identified: NOX enzymes are distinguished from FRE enzymes through a four amino acid motif spanning from transmembrane domain 3 (TM3) to TM4, and YedZ/STEAP proteins are identified by the replacement of the first canonical heme-spanning histidine by a highly conserved arginine. The FRD superfamily most likely originated in bacteria.

The DNA-binding domain (DBD) is essential for NR2E3 dimerization and interaction with Crx

Purpose:NR2E3 (PNR) is an orphan nuclear receptor essential for proper photoreceptor determination and differentiation. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS) and, more recently, with autosomal dominant retinitis pigmentosa (adRP). NR2E3 acts in concert with the transcription factors Crx and Nrl to repress cone-specific genes and activate rod-specific genes. NR2E3 and Crx have been shown to physically interact by their DNA-binding domain (DBD), which may also be implicated in the dimerization process of the nuclear receptor. However, neither NR2E3 homodimerization nor NR2E3/Crx complex formation has been investigated in detail. Methods:In this present work, we analyzed the dimerization of the NR2E3 protein and its interaction with Crx by bioluminescence resonance energy transfer (BRET2) which utilizes Renilla luciferase (hRluc) protein and its substrate DeepBlueC as an energy donor and a mutant green fluorescent protein (GFP2) as the acceptor. We investigated, on whole intact cells, the role of NR2E3 DBD-mutations in dimerization and association with Crx. Results:We clearly showed that NR2E3 formed homodimers in HEK-293T cells. Moreover, all causative NR2E3 mutations present in the DBD of the protein showed an alteration in dimerization, except for the R76Q and the R104W mutants. Interestingly, the adRP-linked G56R mutant was the only DBD-NR2E3 mutant that showed a correct interaction with Crx. Finally, we observed a decrease in rhodospin gene transactivation for all DBD-NR2E3 mutants tested and no potentiation for the adRP-linked G56R mutant. In addition, the p.G56R mutant enhanced the transrepression of M-opsin promoter, while all other DBD-NR2E3 mutants did not repress M-opsin transactivation. Conclusions:A defect, either in the dimer formation or in the interaction of NR2E3 with Crx, leads to abnormal transcriptional activity on rhodopsin and M-opsin promoter and to an atypical retinal development; while the titration of Crx by p.G56R-NR2E3 leads to low levels of rhodopsin and M-opsin expression and may be responsible for the strong adRP phenotype.

Prevalence and dimensions of sexual orientation in Swiss adolescents: a cross-sectional survey of 16 to 20-year-old students.

AIM: Sexual orientation plays an important part in building identity during adolescence. The aim of this study was to describe patterns of sexual orientation, including sexual attraction, fantasies, affiliations and behaviour. METHODS: The study was based on the analysis of data from computerized self-administered questionnaires of a Swiss national survey on the sexual life of 16 to 20-year-old adolescents (n = 2,075 girls and 2,208 boys.). RESULTS: Overall, 95.0% of girls and 96.2% of boys described themselves as predominantly heterosexual; 1.4% of girls and 1.7% of boys as predominantly homosexual or bisexual; and 2.8% of teenagers (girls: 3.6%; boys: 2.1%) were "unsure" of their sexual orientation. The reported prevalence of homosexual attraction (girls: 2.0%; boys: 2.9%) exceeded homosexual fantasies (girls: 0.4%; boys: 0.5%) and affiliations (girls: 0.3%; boys: 0.5%). Among the 4205 respondents, 31 girls (1.5% of girls) and 56 boys (2.5% of boys) reported sexual behaviour (experience or penetrative intercourse) with a person of the same sex. Among 1.5% of girls and 2.5% of boys who reported sexual behaviour with a person of the same sex, 65% of boys and 80% of girls nevertheless considered themselves as heterosexual. CONCLUSION: For a comprehensive understanding of sexual orientation in adolescence a differentiated look at dimensions of sexual orientation is indispensable. This applies to clinical settings, public health and research.

Multicentric carpotarsal osteolysis is caused by mutations clustering in the amino-terminal transcriptional activation domain of MAFB.

Multicentric carpotarsal osteolysis (MCTO) is a rare skeletal dysplasia characterized by aggressive osteolysis, particularly affecting the carpal and tarsal bones, and is frequently associated with progressive renal failure. Using exome capture and next-generation sequencing in five unrelated simplex cases of MCTO, we identified previously unreported missense mutations clustering within a 51 base pair region of the single exon of MAFB, validated by Sanger sequencing. A further six unrelated simplex cases with MCTO were also heterozygous for previously unreported mutations within this same region, as were affected members of two families with autosomal-dominant MCTO. MAFB encodes a transcription factor that negatively regulates RANKL-induced osteoclastogenesis and is essential for normal renal development. Identification of this gene paves the way for development of novel therapeutic approaches for this crippling disease and provides insight into normal bone and kidney development.

The N-terminal domain of Plasmodium falciparum circumsporozoite protein represents a target of protective immunity.

The N-terminal domain of the circumsporozoite protein (CSP) has been largely neglected in the search for a malaria vaccine in spite of being a target of inhibitory antibodies and protective T cell responses in mice. Thus, in order to develop this region as a vaccine candidate to be eventually associated with other candidates and, in particular, with the very advanced C-terminal counterpart, synthetic constructs representing N- and C-terminal regions of Plasmodium falciparum and Plasmodium berghei CSP were administered as single or combined formulations in mice. We show that the antisera generated against the combinations inhibit sporozoite invasion of hepatocytes in vitro better than antisera against single peptides. Furthermore, two different P. falciparum CSP N-terminal constructs (PfCS22-110 and PfCS65-110) were recognized by serum samples from people living in malaria-endemic regions. Importantly, recognition of the short N-terminal peptide (PfCS65-110) by sera from children living in a malaria-endemic region was associated with protection from disease. Taken together, these results underline the potential of using such fragments as malaria vaccine candidates.

Processing of metacaspase into a cytoplasmic catalytic domain mediating cell death in Leishmania major.

Metacaspases are cysteine peptidases that could play a role similar to caspases in the cell death programme of plants, fungi and protozoa. The human protozoan parasite Leishmania major expresses a single metacaspase (LmjMCA) harbouring a central domain with the catalytic dyad histidine and cysteine as found in caspases. In this study, we investigated the processing sites important for the maturation of LmjMCA catalytic domain, the cellular localization of LmjMCA polypeptides, and the functional role of the catalytic domain in the cell death pathway of Leishmania parasites. Although LmjMCA polypeptide precursor form harbours a functional mitochondrial localization signal (MLS), we determined that LmjMCA polypeptides are mainly localized in the cytoplasm. In stress conditions, LmjMCA precursor forms were extensively processed into soluble forms containing the catalytic domain. This domain was sufficient to enhance sensitivity of parasites to hydrogen peroxide by impairing the mitochondrion. These data provide experimental evidences of the importance of LmjMCA processing into an active catalytic domain and of its role in disrupting mitochondria, which could be relevant in the design of new drugs to fight leishmaniasis and likely other protozoan parasitic diseases.

Hepatitis C virus RNA replication requires a conserved structural motif within the transmembrane domain of the NS5B RNA-dependent RNA polymerase.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), the viral RNA-dependent RNA polymerase (RdRp), is a tail-anchored protein with a highly conserved C-terminal transmembrane domain (TMD) that is required for the assembly of a functional replication complex. Here, we report that the TMD of the HCV RdRp can be functionally replaced by a newly identified analogous membrane anchor of the GB virus B (GBV-B) NS5B RdRp. Replicons with a chimeric RdRp consisting of the HCV catalytic domain and the GBV-B membrane anchor replicated with reduced efficiency. Compensatory amino acid changes at defined positions within the TMD improved the replication efficiency of these chimeras. These observations highlight a conserved structural motif within the TMD of the HCV NS5B RdRp that is required for RNA replication.