Molecular mechanisms involved in the activation and regulation of the alpha 1-adrenergic receptor subtypes.

The adrenergic receptors (ARs) belong to the superfamily of membrane-bound G protein coupled receptors (GPCRs). Our investigation has focused on the structure-function relationship of the alpha 1b-AR subtype used as the model system for other GPCRs. Site-directed mutagenesis studies have elucidated the structural domains of the alpha 1b-AR involved in ligand binding, G protein coupling or desensitization. In addition, a combined approach using site-directed mutagenesis and molecular dynamics analysis of the alpha 1b-AR has provided information about the potential mechanisms underlying the activation process of the receptor, i.e. its transition from the 'inactive' to the 'active' conformation.

Whole genome sequencing in patients with retinitis pigmentosa reveals pathogenic DNA structural changes and NEK2 as a new disease gene.

We performed whole genome sequencing in 16 unrelated patients with autosomal recessive retinitis pigmentosa (ARRP), a disease characterized by progressive retinal degeneration and caused by mutations in over 50 genes, in search of pathogenic DNA variants. Eight patients were from North America, whereas eight were Japanese, a population for which ARRP seems to have different genetic drivers. Using a specific workflow, we assessed both the coding and noncoding regions of the human genome, including the evaluation of highly polymorphic SNPs, structural and copy number variations, as well as 69 control genomes sequenced by the same procedures. We detected homozygous or compound heterozygous mutations in 7 genes associated with ARRP (USH2A, RDH12, CNGB1, EYS, PDE6B, DFNB31, and CERKL) in eight patients, three Japanese and five Americans. Fourteen of the 16 mutant alleles identified were previously unknown. Among these, there was a 2.3-kb deletion in USH2A and an inverted duplication of ∼446 kb in EYS, which would have likely escaped conventional screening techniques or exome sequencing. Moreover, in another Japanese patient, we identified a homozygous frameshift (p.L206fs), absent in more than 2,500 chromosomes from ethnically matched controls, in the ciliary gene NEK2, encoding a serine/threonine-protein kinase. Inactivation of this gene in zebrafish induced retinal photoreceptor defects that were rescued by human NEK2 mRNA. In addition to identifying a previously undescribed ARRP gene, our study highlights the importance of rare structural DNA variations in Mendelian diseases and advocates the need for screening approaches that transcend the analysis of the coding sequences of the human genome.

Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation.

Site-directed mutagenesis and molecular dynamics simulations of the alpha 1B-adrenergic receptor (AR) were combined to explore the potential molecular changes correlated with the transition from R (inactive state) to R (active state). Using molecular dynamics analysis we compared the structural/dynamic features of constitutively active mutants with those of the wild type and of an inactive alpha 1B-AR to build a theoretical model which defines the essential features of R and R. The results of site-directed mutagenesis were in striking agreement with the predictions of the model supporting the following hypothesis. (i) The equilibrium between R and R depends on the equilibrium between the deprotonated and protonated forms, respectively, of D142 of the DRY motif. In fact, replacement of D142 with alanine confers high constitutive activity to the alpha 1B-AR. (ii) The shift of R143 of the DRY sequence out of a conserved 'polar pocket' formed by N63, D91, N344 and Y348 is a feature common to all the active structures, suggesting that the role of R143 is fundamental for mediating receptor activation. Disruption of these intramolecular interactions by replacing N63 with alanine constitutively activates the alpha 1B-AR. Our findings might provide interesting generalities about the activation process of G protein-coupled receptors.

Structural variation-associated expression changes are paralleled by chromatin architecture modifications.

Copy number variants (CNVs) influence the expression of genes that map not only within the rearrangement, but also to its flanks. To assess the possible mechanism(s) underlying this "neighboring effect", we compared intrachromosomal interactions and histone modifications in cell lines of patients affected by genomic disorders and control individuals. Using chromosome conformation capture (4C-seq), we observed that a set of genes flanking the Williams-Beuren Syndrome critical region (WBSCR) were often looping together. The newly identified interacting genes include AUTS2, mutations of which are associated with autism and intellectual disabilities. Deletion of the WBSCR disrupts the expression of this group of flanking genes, as well as long-range interactions between them and the rearranged interval. We also pinpointed concomitant changes in histone modifications between samples. We conclude that large genomic rearrangements can lead to chromatin conformation changes that extend far away from the structural variant, thereby possibly modulating expression globally and modifying the phenotype. GEO SERIES ACCESSION NUMBER: GSE33784, GSE33867.

The complete amino acid sequence for brain beta spectrin (beta fodrin): relationship to globin sequences.

The amino acid sequence of mouse brain beta spectrin (beta fodrin), deduced from the nucleotide sequence of complementary DNA clones, reveals that this non-erythroid beta spectrin comprises 2363 residues, with a molecular weight of 274,449 Da. Brain beta spectrin contains three structural domains and we suggest the position of several functional domains including f-actin, synapsin I, ankyrin and spectrin self association sites. Analysis of deduced amino acid sequences indicated striking homology and similar structural characteristics of brain beta spectrin repeats beta 11 and beta 12 to globins. In vitro analysis has demonstrated that heme is capable of specific attachment to brain spectrin, suggesting possible new functions in electron transfer, oxygen binding, nitric oxide binding or heme scavenging.

Licorice-induced hypertension and common variants of genes regulating renal sodium reabsorption.

AIM: To study if gene alterations affecting renal sodium reabsorption associate with susceptibility to licorice-induced hypertension.METHODS: Finnish subjects (n = 30) with a previously documented incident of licorice-induced hypertension were recruited for the study using a newspaper announcement. Their previous clinical and family histories as well as serum electrolyte levels were examined. DNA samples from all individuals were screened for variants of the genes encoding 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) and alpha-, beta-, and gamma-subunits of the epithelial sodium channel (ENaC).RESULTS: Upon licorice predisposition, the patients had a mean blood pressure of 201/118 mmHg. Circulating potassium, renin, and aldosterone levels were low. No significant DNA variations were identified in the 11betaHSD2 gene. Four subjects were heterozygous for beta- and gammaENaC variants previously shown to be associated with hypertension. Furthermore, a novel G insertion (2004-2005insG) in the SCNN1A gene encoding the alphaENaC was identified in two subjects. The frequency of these ENaC variants was significantly higher in subjects with licorice-induced hypertension (6/30 i.e. 20%) than in blood donors (11/301 i.e. 3.7%, P = 0.002).CONCLUSIONS: Defects of the 11betaHSD2 gene do not constitute a likely cause for licorice-induced hypertension. Variants of the ENaC subunits may render some individuals sensitive to licorice-induced metabolic alterations and hypertension.

Experimental evidences for universality of acoustic emission avalanche distributions during structural transitions

Acoustic emission avalanche distributions are studied in different alloy systems that exhibit a phase transition from a bcc to a close-packed structure. After a small number of thermal cycles through the transition, the distributions become critically stable (exhibit power-law behavior) and can be characterized by an exponent alpha. The values of alpha can be classified into universality classes, which depend exclusively on the symmetry of the resulting close-packed structure.

A natural structural variant of the mouse TCR beta-chain displays intrinsic receptor function and antigen specificity.

The Cbeta0 alternate cassette exon is located between the Jbeta1 and Cbeta1 genes in the mouse TCR beta-locus. In T cells with a VDJbeta1 rearrangement, the Cbeta0 exon may be included in TCRbeta transcripts (herein called TCRbeta-Cbeta0 transcripts), potentially inserting an additional 24 aa between the V and C domains of the TCR beta-chain. These TCRbeta splice isoforms may be differentially regulated after Ag activation, because we detected TCRbeta-Cbeta0 transcripts in a high proportion (>60%) of immature and mature T cells having VDJbeta1 rearrangements but found a substantially reduced frequency (<35%) of TCRbeta-Cbeta0 expression among CD8 T cells selected by Ag in vivo. To study the potential activity of the TCRbeta-Cbeta0 splice variant, we cloned full-length TCR cDNAs by single-cell RT-PCR into retroviral expression vectors. We found that the TCRbeta-Cbeta0 splice isoform can function during an early stage of T cell development normally dependent on TCR beta-chain expression. We also demonstrate that T hybridoma-derived cells expressing a TCRbeta-Cbeta0 isoform together with the clonally associated TCR alpha-chain recognize the same cognate peptide-MHC ligand as the corresponding normal alphabetaTCR. This maintenance of receptor function and specificity upon insertion of the Cbeta0 peptide cassette signifies a remarkable adaptability for the TCR beta-chain, and our findings open the possibility that this splice isoform may function in vivo.

Two-step chromatographic purification of human plasma alpha(1)-acid glycoprotein: its application to the purification of rare phenotype samples of the protein and their study by chromatography on immobilized metal chelate affinity adsorbent.

Alpha1-Acid glycoprotein (AAG) or orosomucoid was purified to homogeneity from human plasma by a separate two-step method using chromatography on immobilized Cibacron Blue F3G-A to cross-linked agarose and chromatography on hydroxyapatite. The conditions for the pre-purification of AAG by chromatography on immobilized Cibacron Blue F3G-A were first optimized using different buffer systems with different pH values. The overall yield of the combined techniques was 80% and ca. 12 mg of AAG were purified from an initial total amount of ca. 15 mg in a ca. 40 ml sample of human plasma. This method was applied to the purification of AAG samples corresponding to the three main phenotypes of the protein (FI*S/A, F1/A and S/A), from individual human plasma previously phenotyped for AAG. A study by isoelectric focusing with carrier ampholytes showed that the microheterogeneity of the purified F1*S/A, F1/A and S/A AAG samples was similar to that of AAG in the corresponding plasma, thus suggesting that no apparent desialylation of the glycoprotein occurred during the purification steps. This method was also applied to the purification of AAG samples corresponding to rare phenotypes of the protein (F1/A*AD, S/A*X0 and F1/A*C1) and the interactions of these variants with immobilized copper(II) ions were then studied at pH 7, by chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the different variants encoded by the first of the two genes coding for AAG in humans (i.e. the F1 and S variants) interacted non-specifically with the immobilized ligand, whereas those encoded by the second gene of AAG (i.e. the A, AD, X0 and C1 variants) strongly bound to immobilized Cu(II) ions. These results suggested that chromatography on an immobilized affinity Cu(II) adsorbent could be helpful to distinguish between the respective products of the two highly polymorphic genes which code for human AAG.

Subgroup-specific structural variation across 1,000 medulloblastoma genomes.

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-β signalling in Group 3, and NF-κB signalling in Group 4, suggest future avenues for rational, targeted therapy.

Enantioselective transformation of alpha-hexachlorocyclohexane by the dehydrochlorinases LinA1 and LinA2 from the soil bacterium Sphingomonas paucimobilis B90A.

Sphingomonas paucimobilis B90A contains two variants, LinA1 and LinA2, of a dehydrochlorinase that catalyzes the first and second steps in the metabolism of hexachlorocyclohexanes (R. Kumari, S. Subudhi, M. Suar, G. Dhingra, V. Raina, C. Dogra, S. Lal, J. R. van der Meer, C. Holliger, and R. Lal, Appl. Environ. Microbiol. 68:6021-6028, 2002). On the amino acid level, LinA1 and LinA2 were 88% identical to each other, and LinA2 was 100% identical to LinA of S. paucimobilis UT26. Incubation of chiral alpha-hexachlorocyclohexane (alpha-HCH) with Escherichia coli BL21 expressing functional LinA1 and LinA2 S-glutathione transferase fusion proteins showed that LinA1 preferentially converted the (+) enantiomer, whereas LinA2 preferred the (-) enantiomer. Concurrent formation and subsequent dissipation of beta-pentachlorocyclohexene enantiomers was also observed in these experiments, indicating that there was enantioselective formation and/or dissipation of these enantiomers. LinA1 preferentially formed (3S,4S,5R,6R)-1,3,4,5,6-pentachlorocyclohexene, and LinA2 preferentially formed (3R,4R,5S,6S)-1,3,4,5,6-pentachlorocyclohexene. Because enantioselectivity was not observed in incubations with whole cells of S. paucimobilis B90A, we concluded that LinA1 and LinA2 are equally active in this organism. The enantioselective transformation of chiral alpha-HCH by LinA1 and LinA2 provides the first evidence of the molecular basis for the changed enantiomer composition of alpha-HCH in many natural environments. Enantioselective degradation may be one of the key processes determining enantiomer composition, especially when strains that contain only one of the linA genes, such as S. paucimobilis UT26, prevail.

The C-terminal domain of Plasmodium falciparum merozoite surface protein 3 self-assembles into alpha-helical coiled coil tetramer.

Proteins located on the surface of the pathogenic malaria parasite Plasmodium falciparum are objects of intensive studies due to their important role in the invasion of human cells and the accessibility to host antibodies thus making these proteins attractive vaccine candidates. One of these proteins, merozoite surface protein 3 (MSP3) represents a leading component among vaccine candidates; however, little is known about its structure and function. Our biophysical studies suggest that the 40 residue C-terminal domain of MSP3 protein self-assembles into a four-stranded alpha-helical coiled coil structure where alpha-helices are packed "side-by-side". A bioinformatics analysis provides an extended list of known and putative proteins from different species of Plasmodium which have such MSP3-like C-terminal domains. This finding allowed us to extend some conclusions of our studies to a larger group of the malaria surface proteins. Possible structural and functional roles of these highly conserved oligomerization domains in the intact merozoite surface proteins are discussed.

Plasmid electrotransfer of eye ciliary muscle: principles and therapeutic efficacy using hTNF-alpha soluble receptor in uveitis.

Due to its small size and particular isolating barriers, the eye is an ideal target for local therapy. Recombinant protein ocular delivery requires invasive and painful repeated injections. Alternatively, a transfected tissue might be used as a local producer of transgene-encoded therapeutic protein. We have developed a nondamaging electrically mediated plasmid delivery technique (electrotransfer) targeted to the ciliary muscle, which is used as a reservoir tissue for the long-lasting expression and secretion of therapeutic proteins. High and long-lasting reporter gene expression was observed, which was restricted to the ciliary muscle. Chimeric TNF-alpha soluble receptor (hTNFR-Is) electrotransfer led to elevated protein secretion in aqueous humor and to drastic inhibition of clinical and histological inflammation scores in rats with endotoxin-induced uveitis. No hTNFR-Is was detected in the serum, demonstrating the local delivery of proteins using this method. Plasmid electrotransfer to the ciliary muscle, as performed in this study, did not induce any ocular pathology or structural damage. Local and sustained therapeutic protein production through ciliary muscle electrotransfer is a promising alternative to repeated intraocular protein administration for a large number of inflammatory, degenerative, or angiogenic diseases.

Coexpression of the T-cell receptor constant alpha domain triggers tumor reactivity of single-chain TCR-transduced human T cells.

Transfer of tumor antigen-specific T-cell receptors (TCRs) into human T cells aims at redirecting their cytotoxicity toward tumors. Efficacy and safety may be affected by pairing of natural and introduced TCRalpha/beta chains potentially leading to autoimmunity. We hypothesized that a novel single-chain (sc)TCR framework relying on the coexpression of the TCRalpha constant alpha (Calpha) domain would prevent undesired pairing while preserving structural and functional similarity to a fully assembled double-chain (dc)TCR/CD3 complex. We confirmed this hypothesis for a murine p53-specific scTCR. Substantial effector function was observed only in the presence of a murine Calpha domain preceded by a TCRalpha signal peptide for shuttling to the cell membrane. The generalization to a human gp100-specific TCR required the murinization of both C domains. Structural and functional T-cell avidities of an accessory disulfide-linked scTCR gp100/Calpha were higher than those of a dcTCR. Antigen-dependent phosphorylation of the proximal effector zeta-chain-associated protein kinase 70 at tyrosine 319 was not impaired, reflecting its molecular integrity in signaling. In melanoma-engrafted nonobese diabetic/severe combined immunodeficient mice, adoptive transfer of scTCR gp100/Calpha transduced T cells conferred superior delay in tumor growth among primary and long-term secondary tumor challenges. We conclude that the novel scTCR constitutes a reliable means to immunotherapeutically target hematologic malignancies.

Structural investigations into the interaction of hemoglobin and part structures with bacterial endotoxins.

An understanding of details of the interaction mechanisms of bacterial endotoxins (lipopolysaccharide, LPS) with the oxygen transport protein hemoglobin is still lacking, despite its high biological relevance. Here, a biophysical investigation into the endotoxin:hemoglobin interaction is presented which comprises the use of various rough mutant LPS as well as free lipid A; in addition to the complete hemoglobin molecule from fetal sheep extract, also the partial structure alpha-chain and the heme-free sample are studied. The investigations comprise the determination of the gel-to-liquid crystalline phase behaviour of the acyl chains of LPS, the ultrastructure (type of aggregate structure and morphology) of the endotoxins, and the incorporation of the hemoglobins into artificial immune cell membranes and into LPS. Our data suggest a model for the interaction between Hb and LPS in which hemoglobins do not react strongly with the hydrophilic or with the hydrophobic moiety of LPS, but with the complete endotoxin aggregate. Hb is able to incorporate into LPS with the longitudinal direction parallel to the lipid A double-layer. Although this does not lead to a strong disturbance of the LPS acyl chain packing, the change of the curvature leads to a slightly conical molecular shape with a change of the three-dimensional arrangement from unilamellar into cubic LPS aggregates. Our previous results show that cubic LPS structures exhibit strong endotoxic activity. The property of Hb on the physical state of LPS described here may explain the observation of an increase in LPS-mediating endotoxicity due to the action of Hb.