Stéphane Deschamps

Switch from an Aquaporin to a Glycerol Channel by Two Amino Acids Substitution

The MIP (major intrinsic protein) proteins constitute a channel family of currently 150 members that have been identified in cell membranes of organisms ranging from bacteria to man. Among these proteins, two functionally distinct subgroups are characterized: aquaporins that allow specific water transfer and glycerol channels that are involved in glycerol and small neutral solutes transport. Since the flow of small molecules across cell membranes is vital for every living organism, the study of such proteins is of particular interest. For instance, aquaporins located in kidney cell membranes are responsible for reabsorption of 150 liters of water/day in adult human. To understand the molecular mechanisms of solute transport specificity, we analyzed mutant aquaporins in which highly conserved residues have been substituted by amino acids located at the same positions in glycerol channels. Here, we show that substitution of a tyrosine and a tryptophan by a proline and a leucine, respectively, in the sixth transmembrane helix of an aquaporin leads to a switch in the selectivity of the channel, from water to glycerol.

The developing female genital tract: from genetics to epigenetics

The mammalian female reproductive tract develops from the Mullerian ducts which differentiate, in a cranial to caudal direction, into oviducts, uterine horns, cervix and the anterior vagina. The developmental processes taking place during this organogenesis are notably under the control of steroid hormones, such as members of the Wnt and Hox families, which regulate key developmental genes. At later stages, steroid hormones also participate in the development of the female genital tract. Chemical compounds homologous to steroids can thus act as agonists or antagonists in fetuses exposed to them. These so-called endocrine disruptors are nowadays found in increasing amounts in the environment and may therefore have a particular impact on such developing organs. Epidemiological studies have revealed that endocrine disruptors have had drastic effects on female health and fertility during the last decades. Furthermore, these adverse effects might be transmitted to subsequent generations through epigenetic modifications. Given the potential hazard of inherited epigenetic marks altering reproduction and/or human health, such molecular mechanisms must be urgently investigated. This review aims to summarize the cellular and molecular mechanisms involved in female genital tract development, to highlight key genes involved in this process and to present epigenetic mechanisms triggered by endocrine disruptors and their consequences in regard to female reproductive tract development.

Functional characterization of a microbial aquaglyceroporin

The major intrinsic proteins (MIPs) constitute a widespread membrane channel family essential for osmotic cell equilibrium. The MIPs can be classified into three functional subgroups: aquaporins, glycerol facilitators and aquaglyceroporins. Bacterial MIP genes have been identified in archaea as well as in Gram-positive and Gram-negative eubacteria. However, with the exception of Escherichia coli, most bacterial MIPs have been analysed by sequence homology. Since no MIP has yet been functionally characterized in Gram-positive bacteria, we have studied one of these members from Lactococcus lactis. This MIP is shown to be permeable to glycerol, like E. coli GlpF, and to water, like E. coli AqpZ. This is the first characterization of a microbial MIP that has a mixed function. This result provides important insights to reconstruct the evolutionary history of the MIP family and to elucidate the molecular pathway of water and other solutes in these channels.

Expression of the sarcoplasmic reticulum Ca2+-ATPase in yeast

We describe here an easy system for the production of mg amounts of the rabbit Ca2+‐ATPase SERCA 1a in the yeast S. cerevisiae. The protein is present in several membranes, including the plasma membrane of the yeast, in a native conformation. It can be purified by immunoprecipitation and can be phosphorylated from ATP in a Ca2+‐dependent manner. Using a temperature‐sensitive secretion mutant strain, the fully active protein can also be obtained in secretory vesicles.

A Yeast Recombinant Aquaporin Mutant That Is Not Expressed or Mistargeted in Xenopus Oocyte Can Be Functionally Analyzed in Reconstituted Proteoliposomes

We have recently identified AQPcic (foraquaporin cicadella), an insect aquaporin found in the digestive tract of homopteran insects and involved in the elimination of water ingested in excess with the dietary sap (Le Cahérec, F., Deschamps, S., Delamarche, C., Pellerin, I., Bonnec, G., Guillam, M. T., Gouranton, J., Thomas, D., and Hubert, J. F. (1996) Eur. J. Biochem.241, 707–715). Like many other aquaporins, AQPcic is inhibited by mercury reagents. In this study, we have demonstrated that residue Cys82 is essential for mercury inhibition. Another mutant version of AQPcic (AQP-C134S), expression of which in Xenopus laevis failed to produce an active molecule, was successfully expressed in Saccharomyces cerevisiae. Using stopped-flow analysis of reconstituted proteoliposomes, we demonstrated that the biological activity and Hg sensitivity of yeast-expressed wild type and mutant type AQPcic was readily assessed. Therefore, we propose that the yeast system is a valid alternative to Xenopus oocytes for studying particular mutants of aquaporin.

Role of C-terminal Domain and Transmembrane Helices 5 and 6 in Function and Quaternary Structure of Major Intrinsic Proteins ANALYSIS OF AQUAPORIN/GLYCEROL FACILITATOR CHIMERIC PROTEINS

We previously observed that aquaporins and glycerol facilitators exhibit different oligomeric states when studied by sedimentation on density gradients following nondenaturing detergent solubilization. To determine the domains of major intrinsic protein (MIP) family proteins involved in oligomerization, we constructed protein chimeras corresponding to the aquaporin AQPcic substituted in the loop E (including the proximal part of transmembrane domain (TM) 5) and/or the C-terminal part (including the distal part of TM 6) by the equivalent domain of the glycerol channel aquaglyceroporin (GlpF) (chimeras called AGA, AAG, and AGG). The analogous chimeras of GlpF were also constructed (chimeras GAG, GGA, and GAA). cRNA corresponding to all constructs were injected into Xenopus oocytes. AQPcic, GlpF, AAG, AGG, and GAG were targeted to plasma membranes. Water or glycerol membrane permeability measurements demonstrated that only the AAG chimera exhibited a channel function corresponding to water transport. Analysis of all proteins expressed either in oocytes or in yeast by velocity sedimentation on sucrose gradients following solubilization by 2% n-octyl glucoside indicated that only AQPcic and AAG exist in tetrameric forms. GlpF, GAG, and GAA sediment in a monomeric form, whereas GGA and AGG were found mono/dimeric. These data bring new evidence that, within the MIP family, aquaporins and GlpFs behave differently toward nondenaturing detergents. We demonstrate that the C-terminal part of AQPcic, including the distal half of TM 6, can be substituted by the equivalent domain of GlpF (AAG chimera) without modifying the transport specificity. Our results also suggest that interactions of TM 5 of one monomer with TM 1 of the adjacent monomer are crucial for aquaporin tetramer stability.

Purification of two thermostable components of messenger ribonucleoprotein particles (mRNPs) from Xenopus laevis oocytes, belonging to a novel class of RNA‐binding proteins

We have purified and partially sequenced two proteins from Xenopus laevis previtellogenic oocytes, belonging to messenger ribonucleoprotein particles (mRNPs). The purification procedure rests on the thermostability of these proteins, which remain soluble after heating the cell extracts at 80°C. The thermostable proteins can be identified with two of the most abundant components (mRNP3 and mRNP4) of the mRNPs, described by Darnbrough and Ford (1981) [Eur. J. Biochem. 118, 415–424]. mRNP3 and mRNP4 are homologous to each other, but to no other protein of known sequence. The abundance and semi‐periodic distribution of proline residues in mRNF3 and mRNF4 sequences suggest that these RNA‐binding proteins adopt an unusual type of conformation

Identification of a new type of PBX1 partner that contains zinc finger motifs and inhibits the binding of HOXA9-PBX1 to DNA.

PBX1 belongs to the TALE-class of homeodomain protein and has a wide functional diversity during development. Indeed, PBX1 is required for haematopoiesis as well as for multiple developmental processes such as skeletal patterning and organogenesis. It has furthermore been shown that PBX1 functions as a HOX cofactor during development. More recent data suggest that PBX1 may act even more broadly by modulating the activity of non-homeodomain transcription factors. To better understand molecular mechanisms triggered by PBX1 during female genital tract development, we searched for additional PBX1 partners that might be involved in this process. Using a two hybrid screen, we identified a new PBX1 interacting protein containing several zinc finger motifs that we called ZFPIP for Zinc Finger PBX1 Interacting Protein. We demonstrated that ZFPIP is expressed in embryonic female genital tract but also in other PBX1 expression domains such as the developing head and the limb buds. We further showed that ZFPIP is able to bind physically and in vivo to PBX1 and moreover, that it prevents the binding of HOXA9/PBX complexes to their consensus DNA site. We suggest that ZFPIP is a new type of PBX1 partner that could participate in PBX1 function during several developmental pathways.

Involvement of ITIH5, a candidate gene for congenital uterovaginal aplasia (Mayer-Rokitansky-Küster-Hauser syndrome), in female genital tract development.

The ITI (inter-trypsine inhibitor) gene family includes five genes (ITIH1 to ITIH5) that encode proteins involved in the dynamics of the extracellular matrix (ECM). ITIH5 was found inactivated by partial deletion in a case of congenital uterovaginal aplasia, a human rare disease also called Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. The aim of the present study was to analyze the expression of ITIH5 in the uterus in adult life and during embryogenesis in order to establish the involvement of this gene in both normal and pathological conditions of uterus development. This was achieved in mice by reverse transcription-quantitative PCR, whole-mount hybridization, and Western blot analysis. Itih5 expression was much stronger in female genital tract primordia (Müllerian ducts) and derivatives than elsewhere in the body. This gene was strongly expressed during pregnancy and development of the female genital tract, indicating that the encoded protein probably had an important function in the uterus during these periods. Two different specific isoforms of the protein were detected in Müllerian derivatives during embryogenesis and in adults. Although ITIH genes are expected to be predominantly expressed in the liver, ITIH5 is mainly expressed in the uterus during development and adult life. This tends to indicate an additional and specific role of this gene in the female reproductive tract, and furthermore reinforces ITIH5 as a putative candidate gene for MRKH syndrome.

mRNP3 and mRNP4 are phosphorylatable by casein kinase II in Xenopus oocytes, but phosphorylation does not modify RNA‐binding affinity

mRNP3 and mRNP4 (also called FRGY2) are two mRNA‐binding proteins which are major constituents of the maternal RNA storage particles of Xenopus laevis oocytes. The phosphorylation of mRNP3–4 has been implicated in the regulation of mRNA masking. In this study, we have investigated their phosphorylation by casein kinase II and its consequence on their affinity for RNA. Comparison of the phosphopeptide map of mRNP3–4 phosphorylated in vivo with that obtained after phosphorylation in vitro by purified Xenopus laevis casein kinase II strongly suggests that casein kinase II is responsible for the in vivo phosphorylation of mRNP3–4 in oocytes. The phosphorylation occurs on a serine residue in a central domain of the proteins. The affinity of mRNP3–4 for RNA substrates remained unchanged after the treatment with casein kinase II or calf intestine phosphatase in vitro. This suggests that phosphorylation of these proteins does not regulate their interaction with RNA but rather controls their interactions with other proteins.