Christian Delamarche

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.

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.

Aquaglyceroporins, one channel for two molecules.

In the light of the recently published structure of GlpF and AQP1, we have analysed the nature of the residues which could be involved in the formation of the selectivity filter of aquaporins, glycerol facilitators and aquaglyceroporins. We demonstrate that the functional specificity for major intrinsic protein (MIP) channels can be explained on one side by analysing the polar environment of the residues that form the selective filter. On the other side, we show that the channel selectivity could be associated with the oligomeric state of the membrane protein. We conclude that a non-polar environment in the vicinity of the top of helix 5 could allow aquaglyceroporins and GlpF to exist as monomers within the hydrophobic environment of the membrane.

AMYPdb: A database dedicated to amyloid precursor proteins

BackgroundMisfolding and aggregation of proteins into ordered fibrillar structures is associated with a number of severe pathologies, including Alzheimers disease, prion diseases, and type II diabetes. The rapid accumulation of knowledge about the sequences and structures of these proteins allows using of in silico methods to investigate the molecular mechanisms of their abnormal conformational changes and assembly. However, such an approach requires the collection of accurate data, which are inconveniently dispersed among several generalist databases.ResultsWe therefore created a free online knowledge database (AMYPdb) dedicated to amyloid precursor proteins and we have performed large scale sequence analysis of the included data. Currently, AMYPdb integrates data on 31 families, including 1,705 proteins from nearly 600 organisms. It displays links to more than 2,300 bibliographic references and 1,200 3D-structures. A Wiki system is available to insert data into the database, providing a sharing and collaboration environment. We generated and analyzed 3,621 amino acid sequence patterns, reporting highly specific patterns for each amyloid family, along with patterns likely to be involved in protein misfolding and aggregation.ConclusionAMYPdb is a comprehensive online database aiming at the centralization of bioinformatic data regarding all amyloid proteins and their precursors. Our sequence pattern discovery and analysis approach unveiled protein regions of significant interest. AMYPdb is freely accessible [1].

MetAmyl: A METa-Predictor for AMYLoid Proteins

The aggregation of proteins or peptides in amyloid fibrils is associated with a number of clinical disorders, including Alzheimers, Huntingtons and prion diseases, medullary thyroid cancer, renal and cardiac amyloidosis. Despite extensive studies, the molecular mechanisms underlying the initiation of fibril formation remain largely unknown. Several lines of evidence revealed that short amino-acid segments (hot spots), located in amyloid precursor proteins act as seeds for fibril elongation. Therefore, hot spots are potential targets for diagnostic/therapeutic applications, and a current challenge in bioinformatics is the development of methods to accurately predict hot spots from protein sequences. In this paper, we combined existing methods into a meta-predictor for hot spots prediction, called MetAmyl for METapredictor for AMYLoid proteins. MetAmyl is based on a logistic regression model that aims at weighting predictions from a set of popular algorithms, statistically selected as being the most informative and complementary predictors. We evaluated the performances of MetAmyl through a large scale comparative study based on three independent datasets and thus demonstrated its ability to differentiate between amyloidogenic and non-amyloidogenic polypeptides. Compared to 9 other methods, MetAmyl provides significant improvement in prediction on studied datasets. We further show that MetAmyl is efficient to highlight the effect of point mutations involved in human amyloidosis, so we suggest this program should be a useful complementary tool for the diagnosis of these diseases.

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.

MIPDB : A relational database dedicated to MIP family proteins

Background information. The MIPs (major intrinsic proteins) constitute a large family of membrane proteins that facilitate the passive transport of water and small neutral solutes across cell membranes. Since water is the most abundant molecule in all living organisms, the discovery of selective water‐transporting channels called AQPs (aquaporins) has led to new knowledge on both the physiological and molecular mechanisms of membrane permeability. The MIPs are identified in Archaea, Bacteria and Eukaryota, and the rapid accumulation of new sequences in the database provides an opportunity for large‐scale analysis, to identify functional and/or structural signatures or to infer evolutionary relationships. To help perform such an analysis, we have developed MIPDB (database for MIP proteins), a relational database dedicated to members of the MIP family.

Pore selectivity analysis of an aquaglyceroporin by stopped-flow spectrophotometry on bacterial cell suspensions.

Background information. Transport of water and small neutral solutes across plasma membranes is facilitated by AQP (aquaporin) and aquaglyceroporin channels, which belong to the MIP (major intrinsic protein) family. So far, more than 800 MIP proteins have been identified on the basis of sequence homology, but only less than 10% of them have been functionally characterized. In most studies, the channel properties of MIP proteins have been determined by using Xenopus oocyte swelling assays or stopped‐flow spectrophotometry on proteoliposomes. As both methods sometimes present disadvantages, we developed an alternative method for analysing MIP function.

Characterization of the Pasteurella multocida skp and firA genes.

A 2.9-kb fragment of the Pasteurella multocida (Pm) genome encoding proteins p25 (25 kDa) and p28 (28 kDa) has previously been cloned and expressed in Escherichia coli (Ec). In the present paper, the nucleotide (nt) sequence of a 1.8-kb subfragment encoding the two proteins is described. The cloned fragment contains three open reading frames (ORFs). ORF1 is incomplete. ORF2 is homologous to the skp gene of Ec. ORF3 overlaps ORF2 and is highly homologous to the firA gene of Ec. The skp and firA genes are part of an operon governing the first steps of lipid A synthesis. Comparing the nt sequence with the N-terminal sequences of p25 and p28 revealed that the two proteins are encoded by ORF2 (skp). The preprotein p28 is converted into p25 by cleavage of a 23-amino-acid leader peptide. Though it serologically cross-reacts with porin H of Pm, p25 is not related to known bacterial porins.

A functional water channel protein in the pathogenic bacterium Brucella abortus

The gene for a new bacterial aquaporin, AqpX, was cloned from the pathogenic Gram-negative bacterium Brucella abortus. The gene was mapped on the large chromosome of B. abortus. It is flanked by one upstream and two downstream copies of the Brucella repeated sequence Bru-RS. Prediction from the nucleotide sequence indicated that the protein is a member of the MIP family, which comprises channels for water and/or solute transport. Expression in Xenopus oocytes and cryoelectron microscopy of Escherichia coli cells transformed with the aqpX gene confirmed that the protein is an efficient water channel. Glycerol uptake experiments in E. coli also showed that the protein is not able to transport glycerol.