Efficient Biochemical Method for Characterizing and Classifying Related Amyloidogenic Peptides.

Amyloidosis is a group of proteinopathies characterized by the systemic or organ-specific deposition of proteins in the form of amyloid fibers. Nearly 40 proteins play a role in these pathologies, and the structures of the associated fibers are beginning to be determined by Cryo-EM. However, the molecular events underlying the process, such as fiber nucleation and elongation, are poorly understood, which impairs developing efficient therapies.

Hsp90 directly interacts, in vitro, with amyloid structures and modulates their assembly and disassembly.

Background: The 90kDa heat shock protein (Hsp90) participates in regulating the homeostasis of cellular proteins and was considered one of the key chaperones involved in the control and regulation of amyloid deposits. Hsp90 interacts with the amyloid protein tau through tau aggregation-prone regions, including the VQIVYK hexapeptide motif. This hexapeptide, which self-aggregates, forming amyloid fibrils, is widely used to model amyloid formation mechanisms.

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 Alzheimer's, Huntington's 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.

AMYPdb: a database dedicated to amyloid precursor proteins.

Background: Misfolding and aggregation of proteins into ordered fibrillar structures is associated with a number of severe pathologies, including Alzheimer's 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.

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.

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.

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.

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).

Oligomerization of water and solute channels of the major intrinsic protein (MIP) family.

Water and small solute fluxes through cell membranes are ensured in many tissues by selective pores that belong to the major intrinsic protein family (MIP). This family includes the water channels or aquaporins (AQP) and the neutral solute facilitators such as the glycerol facilitator (GlpF). We have compared the characteristics of representatives of each subfamily.

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.

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.

Color and graphic display (CGD): programs for multiple sequence alignment analysis in spreadsheet software.

Interpretation of multiple sequence alignments is of major interest for the prediction of functional and structural domains in proteins or for the organization of related sequences in families and subfamilies. However, a necessity for the bench scientist is the use of outstanding programs in a friendly computing environment. This paper describes Color and Graphic Display (CGD), a set of modules that runs as part of the Microsoft Excel spreadsheet to color and analyze multiple sequence alignments.

Oligomerization state of MIP proteins expressed in Xenopus oocytes as revealed by freeze-fracture electron-microscopy analysis.

The MIP (major intrinsic protein) family is a widespread family of membrane proteins exhibiting two major types of channel properties: aquaporins and solute facilitators. In the present study, freeze-fracture electron microscopy was used to investigate the oligomerization state of two MIP proteins heterologously expressed in the plasma membrane of Xenopus laevis oocytes: AQPcic, an aquaporin from the insect Cicadella viridis, and GlpF, a glycerol facilitator from Escherichia coli. Swelling assays performed on oocytes 48 and 72 h following cRNA microinjections showed that these proteins were functionally expressed.

A symbolic-numeric approach to find patterns in genomes. Application to the translation initiation sites of E. coli.

DNA sequence data provided by genome sequencing programs open new research prospects. In this respect, computational investigations are of major importance to discover new 'functional/structural patterns' and to improve biological process knowledge. For example, even though the principal steps of translation initiation in prokaryotes are known, it is difficult to point out the exact pattern of the mRNA that is recognized by the ribosome.

Visualization of AqpZ-mediated water permeability in Escherichia coli by cryoelectron microscopy.

Transport of water across the plasma membrane is a fundamental process occurring in all living organisms. In bacteria, osmotic movement of water across the cytoplasmic membrane is needed to maintain cellular turgor; however, the molecular mechanisms of this process are poorly defined. Involvement of aquaporin water channels in bacterial water permeability was suggested by the recent discovery of the aquaporin gene, aqpZ, in Escherichia coli.

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.

Oligomerization state of water channels and glycerol facilitators. Involvement of loop E.

The major intrinsic protein (MIP) family includes water channels aquaporins (AQPs) and facilitators for small solutes such as glycerol (GlpFs). Velocity sedimentation on sucrose gradients demonstrates that heterologous AQPcic expressed in yeast or Xenopus oocytes behaves as an homotetramer when extracted by n-octyl beta-D-glucopyranoside (OG) and as a monomer when extracted by SDS. We performed an analysis of GlpF solubilized from membranes of Escherichia coli or of mRNA-injected Xenopus oocytes.

Prediction of functional residues in water channels and related proteins.

In this paper, we present an updated classification of the ubiquitous MIP (Major Intrinsic Protein) family proteins, including 153 fully or partially sequenced members available in public databases. Presently, about 30 of these proteins have been functionally characterized, exhibiting essentially two distinct types of channel properties: (1) specific water transport by the aquaporins, and (2) small neutral solutes transport, such as glycerol by the glycerol facilitators. Sequence alignments were used to predict amino acids and motifs discriminant in channel specificity.

Molecular cloning and characterization of an insect aquaporin functional comparison with aquaporin 1.

We previously described the structural organization of P25, a member of the major-intrinsic-protein family found in the digestive tract of homopteran sap-sucking insects [Beuron, F., Le Cahérec, F., Guillam, M.

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.

Structural analysis of a MIP family protein from the digestive tract of Cicadella viridis.

Homopteran insects, and especially Cicadella viridis, display in their digestive tract a specialized epithelial differentiation, the filter chamber (FC) acting as a water-shunting complex. The main intrinsic membrane protein of the FC is a 25,000-Da polypeptide (P25). In this paper we demonstrate that this P25 polypeptide is a member of the MIP family of membrane channel proteins, and that P25 forms homotetramers in the native membranes.

Cloning and expression of two Pasteurella multocida genes in Escherichia coli.

A library of cloned Pasteurella multocida (toxigenic strain 9222, serotype D2) genomic sequences was constructed in Escherichia coli by incorporating TaqI digestion fragments into the plasmid vector pUC19. Immunological screening with antibodies directed against porin H, the major protein of the P multocida outer membrane, allowed the identification of a recombinant plasmid containing a 2.9-kbp DNA insert.

Protein IIIa of Rhizobium leguminosarum is probably a porin.

The cloning, sequencing and expression of the gene encoding the 36-kilodalton (kDa) outer membrane protein of Rhizobium leguminosarum has been recently described in the literature (De Maagd RA et al (1992) J Bacteriol 174, 214-221). We present evidence that this protein is a porin from a sub-type covalently bound to the peptidoglycan.

Aging and Alzheimer's disease: protease inhibitors in cerebrospinal fluid.

Recent advances suggested that proteases and their inhibitors could be implicated in the genesis and/or maturation of insoluble deposits associated with Alzheimer's disease (AD). This study was designed to measure the level of alpha 1-antichymotrypsin (ACT) and alpha 1-antitrypsin (AT) in cerebrospinal fluid (CSF) of patients with AD and nondemented humans at various ages. Our analysis failed to demonstrate a significant relationship between inhibitor content and disease.