Molecular pathogenesis of spondylocheirodysplastic Ehlers-Danlos syndrome caused by mutant ZIP13 proteins.

The zinc transporter protein ZIP13 plays critical roles in bone, tooth, and connective tissue development, and its dysfunction is responsible for the spondylocheirodysplastic form of Ehlers-Danlos syndrome (SCD-EDS, OMIM 612350). Here, we report the molecular pathogenic mechanism of SCD-EDS caused by two different mutant ZIP13 proteins found in human patients: ZIP13(G64D), in which Gly at amino acid position 64 is replaced by Asp, and ZIP13(ΔFLA), which contains a deletion of Phe-Leu-Ala. We demonstrated that both the ZIP13(G64D) and ZIP13(ΔFLA) protein levels are decreased by degradation via the valosin-containing protein (VCP)-linked ubiquitin proteasome pathway. The inhibition of degradation pathways rescued the protein expression levels, resulting in improved intracellular Zn homeostasis. Our findings uncover the pathogenic mechanisms elicited by mutant ZIP13 proteins. Further elucidation of these degradation processes may lead to novel therapeutic targets for SCD-EDS.

Ligands for pheromone-sensing neurons are not conformationally activated odorant binding proteins.

Pheromones form an essential chemical language of intraspecific communication in many animals. How olfactory systems recognize pheromonal signals with both sensitivity and specificity is not well understood. An important in vivo paradigm for this process is the detection mechanism of the sex pheromone (Z)-11-octadecenyl acetate (cis-vaccenyl acetate [cVA]) in Drosophila melanogaster. cVA-evoked neuronal activation requires a secreted odorant binding protein, LUSH, the CD36-related transmembrane protein SNMP, and the odorant receptor OR67d. Crystallographic analysis has revealed that cVA-bound LUSH is conformationally distinct from apo (unliganded) LUSH. Recombinantly expressed mutant versions of LUSH predicted to enhance or diminish these structural changes produce corresponding alterations in spontaneous and/or cVA-evoked activity when infused into olfactory sensilla, leading to a model in which the ligand for pheromone receptors is not free cVA, but LUSH that is "conformationally activated" upon cVA binding. Here we present evidence that contradicts this model. First, we demonstrate that the same LUSH mutants expressed transgenically affect neither basal nor pheromone-evoked activity. Second, we compare the structures of apo LUSH, cVA/LUSH, and complexes of LUSH with non-pheromonal ligands and find no conformational property of cVA/LUSH that can explain its proposed unique activated state. Finally, we show that high concentrations of cVA can induce neuronal activity in the absence of LUSH, but not SNMP or OR67d. Our findings are not consistent with the model that the cVA/LUSH complex acts as the pheromone ligand, and suggest that pheromone molecules alone directly activate neuronal receptors.

Physical interaction between bacterial heat shock protein (Hsp) 90 and Hsp70 chaperones mediates their cooperative action to refold denatured proteins.

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.

Long synthetic peptides as biologically active proteins: the example of the chemokines.

Chemokines constitute an expanding protein family of over 40 members which exhibit a wide variety of biological activities and are involved in many normal physiological processes, such as cellular migration, differentiation and activation, but also in pathological situations, such as inflammation and metastasis. Over the last few years, we have developed methods to manufacture long synthetic peptides of up to 130 residues, and to achieve the formation of native-like cysteine pairings. This ability prompted us to undertake the total chemical synthesis of chemokines. So far, we have successfully produced over 30 chemokine species, which exhibit biological activities similar to, or greater than, those reported by others. Chemical synthesis offers a clear advantage over recombinant technologies for the introduction of fluorochromes and haptens at molecularly defined positions. In addition, approval of chemically synthesized products for use in humans is straightforward compared with material produced by biological methods.

Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination.

Inherited mutations in human PALB2 are associated with a predisposition to breast and pancreatic cancers. PALB2's tumor-suppressing effect is thought to be based on its ability to facilitate BRCA2's function in homologous recombination. However, the biochemical properties of PALB2 are unknown. Here we show that human PALB2 binds DNA, preferentially D-loop structures, and directly interacts with the RAD51 recombinase to stimulate strand invasion, a vital step of homologous recombination. This stimulation occurs through reinforcing biochemical mechanisms, as PALB2 alleviates inhibition by RPA and stabilizes the RAD51 filament. Moreover, PALB2 can function synergistically with a BRCA2 chimera (termed piccolo, or piBRCA2) to further promote strand invasion. Finally, we show that PALB2-deficient cells are sensitive to PARP inhibitors. Our studies provide the first biochemical insights into PALB2's function with piBRCA2 as a mediator of homologous recombination in DNA double-strand break repair.

Reconstitution of the strand invasion step of double-strand break repair using human Rad51 Rad52 and RPA proteins.

The human Rad51 recombinase is essential for the repair of double-strand breaks in DNA that occur in somatic cells after exposure to ionising irradiation, or in germ line cells undergoing meiotic recombination. The initiation of double-strand break repair is thought to involve resection of the double-strand break to produce 3'-ended single-stranded (ss) tails that invade homologous duplex DNA. Here, we have used purified proteins to set up a defined in vitro system for the initial strand invasion step of double-strand break repair. We show that (i) hRad51 binds to the ssDNA of tailed duplex DNA molecules, and (ii) hRad51 catalyses the invasion of tailed duplex DNA into homologous covalently closed DNA. Invasion is stimulated by the single-strand DNA binding protein RPA, and by the hRad52 protein. Strikingly, hRad51 forms terminal nucleoprotein filaments on either 3' or 5'-ssDNA tails and promotes strand invasion without regard for the polarity of the tail. Taken together, these results show that hRad51 is recruited to regions of ssDNA occurring at resected double-strand breaks, and that hRad51 shows no intrinsic polarity preference at the strand invasion step that initiates double-strand break repair.

Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses.

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.

Proteins influencing foam formation in wine and beer: the role of yeast

This review focuses on the role of proteins in the production and maintenance of foam in both sparkling wines and beer. The quality of the foam in beer but especially in sparkling wines depends, among other factors, on the presence of mannoproteins released from the yeast cell walls during autolysis. These proteins are hydrophobic, highly glycosylated, and their molecular masses range from 10 to 200 kDa characteristics that allow mannoproteins to surround and thus stabilize the gas bubbles of the foam. Both the production and stabilization of foam also depend on other proteins. In wine, these include grape-derived proteins such as vacuolar invertase; in beer, barley-derived proteins, such as LTP1, protein Z, and hordein-derived polypeptides, are even more important in this respect than mannoproteins

The Munc13 proteins differentially regulate readily releasable pool dynamics and calcium-dependent recovery at a central synapse.

The Munc13 gene family encodes molecules located at the synaptic active zone that regulate the reliability of synapses to encode information over a wide range of frequencies in response to action potentials. In the CNS, proteins of the Munc13 family are critical in regulating neurotransmitter release and synaptic plasticity. Although Munc13-1 is essential for synaptic transmission, it is paradoxical that Munc13-2 and Munc13-3 are functionally dispensable at some synapses, although their loss in other synapses leads to increases in frequency-dependent facilitation. We addressed this issue at the calyx of Held synapse, a giant glutamatergic synapse that we found to express all these Munc13 isoforms. We studied their roles in the regulation of synaptic transmission and their impact on the reliability of information transfer. Through detailed electrophysiological analyses of Munc13-2, Munc13-3, and Munc13-2-3 knock-out and wild-type mice, we report that the combined loss of Munc13-2 and Munc13-3 led to an increase in the rate of calcium-dependent recovery and a change in kinetics of release of the readily releasable pool. Furthermore, viral-mediated overexpression of a dominant-negative form of Munc13-1 at the calyx demonstrated that these effects are Munc13-1 dependent. Quantitative immunohistochemistry using Munc13-fluorescent protein knock-in mice revealed that Munc13-1 is the most highly expressed Munc13 isoform at the calyx and the only one highly colocalized with Bassoon at the active zone. Based on these data, we conclude that Munc13-2 and Munc13-3 isoforms limit the ability of Munc13-1 to regulate calcium-dependent replenishment of readily releasable pool and slow pool to fast pool conversion in central synapses.

In conditions of limited chromophore supply rods entrap 11-cis-retinal leading to loss of cone function and cell death.

RPE65 is a retinoid isomerase required for the production of 11-cis-retinal, the chromophore of both cone and rod visual pigments. We recently established an R91W knock-in mouse strain as homologous animal model for patients afflicted by this mutation in RPE65. These mice have impaired vision and can only synthesize minute amounts of 11-cis-retinal. Here, we investigated the consequences of this chromophore insufficiency on cone function and pathophysiology. We found that the R91W mutation caused cone opsin mislocalization and progressive geographic cone atrophy. Remnant visual function was mostly mediated by rods. Ablation of rod opsin corrected the localization of cone opsin and improved cone retinal function. Thus, our analyses indicate that under conditions of limited chromophore supply rods and cones compete for 11-cis-retinal that derives from regeneration pathway(s) which are reliant on RPE65. Due to their higher number and the instability of cone opsin, rods are privileged under this condition while cones suffer chromophore deficiency and degenerate. These findings reinforce the notion that in patients any effective gene therapy with RPE65 needs to target the cone-rich macula directly to locally restore the cones' chromophore supply outside the reach of rods.

A novel TMPRSS3 missense mutation in a DFNB8/10 family prevents proteolytic activation of the protein.

Pathogenic mutations in TMPRSS3, which encodes a transmembrane serine protease, cause non-syndromic deafness DFNB8/10. Missense mutations map in the low density-lipoprotein receptor A (LDLRA), scavenger-receptor cysteine-rich (SRCR), and protease domains of the protein, indicating that all domains are important for its function. TMPRSS3 undergoes proteolytic cleavage and activates the ENaC sodium channel in a Xenopus oocyte model system. To assess the importance of this gene in non-syndromic childhood or congenital deafness in Turkey, we screened for mutations affected members of 25 unrelated Turkish families. The three families with the highest LOD score for linkage to chromosome 21q22.3 were shown to harbor P404L, R216L, or Q398X mutations, suggesting that mutations in TMPRSS3 are a considerable contributor to non-syndromic deafness in the Turkish population. The mutant TMPRSS3 harboring the novel R216L missense mutation within the predicted cleavage site of the protein fails to undergo proteolytic cleavage and is unable to activate ENaC, thus providing evidence that pre-cleavage of TMPRSS3 is mandatory for normal function.

Contribution à la connaissance du métabolisme secondaire des Thymelaeaceae et investigation phytochimique de l'une d'elles: "Gnidia involucrata" Steud. ex A. Rich

Dans le but de mieux connaître le métabolisme secondaire de la famille des Thymelaeaceae et de découvrir de nouveaux composés naturels à intérêt thérapeutique, 30 extraits provenant de 8 espèces africaines ont été soumis à un criblage chimique et biologique. Les cibles biologiques suivantes ont servi à l?évaluation de l?activité des extraits étudiés : la moisissure phytopathogène Cladosporium cucumerinum, la levure commensale Candida albicans, la bactérie opportuniste Bacillus subtilis, la larve du moustique vecteur de la fièvre jaune Aedes aegypti et l?hôte intermédiaire mollusque de la schistosomiase urinaire Biomphalaria glabrata. Les propriétés antiradicalaires et inhibitrices de l?acétylcholinestérase de ces extraits ont également été dépistées. Des analyses sur CCM avec révélation chimique, ainsi que des expériences LC/DAD-UV, ont permis demettre en évidence la présence de tanins, de flavonoïdes et de xanthones dans les extraits polaires. Sur la base des résultats de ces analyses préliminaires, l?investigation phytochimique des extraits méthanoliques des racines et des parties aériennes de Gnidia involucrata a été entreprise. Cette démarche a permis l?isolement de 8 composés naturels et leur caractérisation complète au moyen de méthodes spectroscopiques (UV, MS, CD, 1H- et 13C-NMR). Les activités de ces produits purs ont été évaluées et il est apparu qu?ils possédaient presque tous des propriétés antiradicalaires intéressantes, supérieures à celles du BHT, un antioxydant de synthèse (E 321) utilisé dans l?industrie alimentaire. Deux benzophénones simples, respectivement O- et C-glucosylées, ont été isolées des parties aériennes de G. involucrata au côté de la mangiférine, une C-glycosylxanthone ubiquitaire. Ces découvertes sont remarquables à plusieurs titres : (1) les benzophénones simples (nonprénylées) sont très rares dans la nature ; (2) c?est la première fois qu?une Oglycosylbenzophénone a été décrite ; (3) aucune xanthone n?avait été mise en évidence auparavant dans la famille et (4) les benzophénones semblent ne pas être que des produits intermédiaires dans la biosynthèse des xanthones. Trois 3,8??-biflavanones du type GB ont été isolées des racines et des parties aériennes de la même plante, dont deux stéréoisomères se trouvant en mélange. Une analyse LC/CD a permis d?attribuer les configurations absolues des quatre carbones asymétriques de chaque molécule. Cette classe de métabolites secondaires est réputée pour ses propriétés analgésiques et sa présence chez les Thymelaeaceae est prometteuse. Des techniques couplées de pointe ont été utilisées dans ce travail et ont montré leur apport inestimable dans le domaine de la recherche phytochimique. Une analyse LC/ MSn a ainsi permis de mettre en évidence on-line trois C-glycosylflavones ? l?isoorientine, l?isovitexine et la vitexine ? dans les extraits méthanoliques bruts de G. involucrata. De plus, les parties aériennes de cette même plante ont servi de matériel pour le développement d?une nouvelle méthode d?analyse d?extraits bruts : la LC/1H-NMR time-slice. Cette approche consiste à « découper » le temps d?analyse par des interruptions régulières du flux LC, durant lesquelles les données NMR nécessaires sont acquises. Le problème de la faible sensibilité relative de la LC/NMR a été partiellement résolu par ce biais et a permis d?envisager l?utilisation de la NMR au sein de systèmes de couplages multiples en série avec d?autres méthodes spectrales (UV, MS, IR, CD,?).<br/><br/>With the aim of acquiring a better knowledge of the secondary metabolism of the family Thymelaeaceae and of the discovering of new natural therapeutics, 30 extracts from 8 African plant species were submitted to chemical and biological screening. The following biological targets were used to estimate the activity of the extracts under study: the phytopathogenic fungus Cladosporium cucumerinum, the commensal yeast Candida albicans, the opportunistic bacteria Bacillus subtilis, larvae of the yellow fever-transmitting mosquito Aedes aegypti and the intermediate snail host of urinary schistosomiasis Biomphalaria glabrata. The antiradical and acetylcholinesterase-inhibiting properties of these extracts were also investigated. TLC analyses followed by chemical detection, together with LC/DAD-UV experiments, showed the presence of tannins, flavonoids and xanthones in the polar extracts. On the basis of these results, a phytochemical investigation of the methanol extracts of the roots and the aerial parts of Gnidia involucrata was undertaken. This procedure led to the isolation of 8 natural products, which were then characterised by spectroscopic means (UV, MS, CD, 1H- and 13C-NMR). The activities of the pure compounds were then further evaluated: almost all of them exhibited very interesting antiradical properties, superior to those of BHT, a synthetic antioxidant (E 321) used in the food industry. Two simple benzophenones, one O- and one C-glycosylated, were isolated from the aerial parts of G. involucrata, together with mangiferin, a ubiquitous C-glycosylxanthone. These findings are of multiple importance: (1) simple (non-prenylated) benzophenones are very rare in nature; (2) it is the first time that an O-glycosylbenzophenone has been described; (3) no xanthones have been previously reported in the family and (4) benzophenones do not seem to be exclusive intermediates in the biosynthesis of xanthones. Three 3,8??-biflavanones of the GB type were isolated from the roots and the aerial parts of the same plant, among them two stereoisomers in mixture. A LC/CD analysis allowed the assignment of the absolute configurations of all four stereocenters in both molecules. This class of secondary metabolite is well known for its analgesic properties and its presence in the Thymelaeaceae is very promising. Advanced hyphenated techniques were used in this work and showed their inestimable contribution to the field of phytochemical research. A LC/MSn analysis, for example, allowed the on-line characterisation of three C-glycosylflavones ? isoorientin, isovitexin and vitexin ? in the crude methanol extracts of G. involucrata. Furthermore, the aerial parts of this plant were used as material for the development of a new analytical method for crude plant extracts: time-slice LC/1H-NMR. This approach consisted in "slicing" the analytical procedure by interrupting the LC flow at given intervals, during which the necessary NMR data were acquired. The relative lack of sensitivity of LC/NMR was partially surmounted by this means, allowing one to envisage the use of NMR in a multiple hyphenated system, together with other spectroscopic methods (UV, MS, IR, CD,?)

A Study on Fractionation and Ultrafiltration of Proteins with Characterized Modified and Unmodified Membranes

Membrane filtration has become increasingly attractive in the processing of both foodand biotechnological products. However, the poor selectivity of the membranes and fouling are the critical factors limiting the development of UF systems for the specific fractionation of protein mixtures. This thesis gives an overview on fractionation of proteins from model protein solutions or from biological solutions. An attempt was made to improve the selectivity of the available membranes by modifying the membranes and by exploiting the different electrostatic interactions between the proteins and the membrane pore surfaces. Fractionation and UF behavior of proteins in the model solutions and in the corresponding biological solutions were compared. Characterization of the membranes and protein adsorptionto the membrane were investigated with combined flux and streaming potential studies. It has been shown that fouling of the membranes can be reduced using "self-rejecting" membranes at pH values where electrostatic repulsion is achieved between the membrane and the proteins in solution. This effect is best shown in UF of dilute single protein solutions at low ionic strengths and low pressures. Fractionation of model proteins in single, binary, and ternary solutionshas been carried out. The results have been compared to the results obtained from fractination of biological solutions. It was generally observed that fractination of proteins from biological solutions are more difficult to carry out owingto the presence of non studied protein components with different properties. Itcan be generally concluded that it is easier to enrich the smaller protein in the permeate but it is also possible to enrich the larger protein in the permeateat pH values close to the isoelectric point of the protein. It should be possible to find an optimal flux and modification to effectively improve the fractination of proteins even with very similar molar masses.

Critical flux and fouling in ultrafiltration of proteins

In this thesis different parameters influencing critical flux in protein ultrafiltration and membrane foul-ing were studied. Short reviews of proteins, cross-flow ultrafiltration, flux decline and criticalflux and the basic theory of Partial Least Square analysis (PLS) are given at the beginning. The experiments were mainly performed using dilute solutions of globular proteins, commercial polymeric membranes and laboratory scale apparatuses. Fouling was studied by flux, streaming potential and FTIR-ATR measurements. Critical flux was evaluated by different kinds of stepwise procedures and by both con-stant pressure and constant flux methods. The critical flux was affected by transmembrane pressure, flow velocity, protein concentration, mem-brane hydrophobicity and protein and membrane charges. Generally, the lowest critical fluxes were obtained at the isoelectric points of the protein and the highest in the presence of electrostatic repulsion between the membrane surface and the protein molecules. In the laminar flow regime the critical flux increased with flow velocity, but not any more above this region. An increase in concentration de-creased the critical flux. Hydrophobic membranes showed fouling in all charge conditionsand, furthermore, especially at the beginning of the experiment even at very low transmembrane pressures. Fouling of these membranes was thought to be due to protein adsorption by hydrophobic interactions. The hydrophilic membranes used suffered more from reversible fouling and concentration polarisation than from irreversible foul-ing. They became fouled at higher transmembrane pressures becauseof pore blocking. In this thesis some new aspects on critical flux are presented that are important for ultrafiltration and fractionation of proteins.

ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins

Background: Annotations of completely sequenced genomes reveal that nearly half of the genes identified are of unknown function, and that some belong to uncharacterized gene families. To help resolve such issues, information can be obtained from the comparative analysis of homologous genes in model organisms. Results: While characterizing genes from the retinitis pigmentosa locus RP26 at 2q31-q33, we have identified a new gene, ORMDL1, that belongs to a novel gene family comprising three genes in humans (ORMDL1, ORMDL2 and ORMDL3), and homologs in yeast, microsporidia, plants, Drosophila, urochordates and vertebrates. The human genes are expressed ubiquitously in adult and fetal tissues. The Drosophila ORMDL homolog is also expressed throughout embryonic and larval stages, particularly in ectodermally derived tissues. The ORMDL genes encode transmembrane proteins anchored in the endoplasmic reticulum (ER). Double knockout of the two Saccharomyces cerevisiae homologs leads to decreased growth rate and greater sensitivity to tunicamycin and dithiothreitol. Yeast mutants can be rescued by human ORMDL homologs. Conclusions: From protein sequence comparisons we have defined a novel gene family, not previously recognized because of the absence of a characterized functional signature. The sequence conservation of this family from yeast to vertebrates, the maintenance of duplicate copies in different lineages, the ubiquitous pattern of expression in human and Drosophila, the partial functional redundancy of the yeast homologs and phenotypic rescue by the human homologs, strongly support functional conservation. Subcellular localization and the response of yeast mutants to specific agents point to the involvement of ORMDL in protein folding in the ER.