Proteome Analysis Database: online application of InterPro and CluSTr for the functional classification of proteins in whole genomes

The SWISS-PROT group at EBI has developed the Proteome Analysis Database utilising existing resources and providing comparative analysis of the predicted protein coding sequences of the complete genomes of bacteria, archaea and eukaryotes (http://www.ebi.ac.uk/proteome/). The two main projects used, InterPro and CluSTr, give a new perspective on families, domains and sites and cover 31–67% (InterPro statistics) of the proteins from each of the complete genomes. CluSTr covers the three complete eukaryotic genomes and the incomplete human genome data. The Proteome Analysis Database is accompanied by a program that has been designed to carry out InterPro proteome comparisons for any one proteome against any other one or more of the proteomes in the database.

Functional identification and reconstitution of an odorant receptor in single olfactory neurons

The olfactory system is remarkable in its capacity to discriminate a wide range of odorants through a series of transduction events initiated in olfactory receptor neurons. Each olfactory neuron is expected to express only a single odorant receptor gene that belongs to the G protein coupled receptor family. The ligand–receptor interaction, however, has not been clearly characterized. This study demonstrates the functional identification of olfactory receptor(s) for specific odorant(s) from single olfactory neurons by a combination of Ca2+-imaging and reverse transcription–coupled PCR analysis. First, a candidate odorant receptor was cloned from a single tissue-printed olfactory neuron that displayed odorant-induced Ca2+ increase. Next, recombinant adenovirus-mediated expression of the isolated receptor gene was established in the olfactory epithelium by using green fluorescent protein as a marker. The infected neurons elicited external Ca2+ entry when exposed to the odorant that originally was used to identify the receptor gene. Experiments performed to determine ligand specificity revealed that the odorant receptor recognized specific structural motifs within odorant molecules. The odorant receptor-mediated signal transduction appears to be reconstituted by this two-step approach: the receptor screening for given odorant(s) from single neurons and the functional expression of the receptor via recombinant adenovirus. The present approach should enable us to examine not only ligand specificity of an odorant receptor but also receptor specificity and diversity for a particular odorant of interest.

Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma

Inactivation of the genes involved in DNA mismatch repair is associated with microsatellite instability (MSI) in colorectal cancer. We report that hypermethylation of the 5′ CpG island of hMLH1 is found in the majority of sporadic primary colorectal cancers with MSI, and that this methylation was often, but not invariably, associated with loss of hMLH1 protein expression. Such methylation also occurred, but was less common, in MSI− tumors, as well as in MSI+ tumors with known mutations of a mismatch repair gene (MMR). No hypermethylation of hMSH2 was found. Hypermethylation of colorectal cancer cell lines with MSI also was frequently observed, and in such cases, reversal of the methylation with 5-aza-2′-deoxycytidine not only resulted in reexpression of hMLH1 protein, but also in restoration of the MMR capacity in MMR-deficient cell lines. Our results suggest that microsatellite instability in sporadic colorectal cancer often results from epigenetic inactivation of hMLH1 in association with DNA methylation.

Molecular cloning and functional analysis of SUT-1, a sulfate transporter from human high endothelial venules

High endothelial venules (HEV) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. One of the major characteristics of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. Here, we show that HEVEC express two functional classes of sulfate transporters defined by their differential sensitivity to the anion-exchanger inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), and we report the molecular characterization of a DIDS-resistant sulfate transporter from human HEVEC, designated SUT-1. SUT-1 belongs to the family of Na+-coupled anion transporters and exhibits 40–50% amino acid identity with the rat renal Na+/sulfate cotransporter, NaSi-1, as well as with the human and rat Na+/dicarboxylate cotransporters, NaDC-1/SDCT1 and NaDC-3/SDCT2. Functional expression studies in cRNA-injected Xenopus laevis oocytes showed that SUT-1 mediates high levels of Na+-dependent sulfate transport, which is resistant to DIDS inhibition. The SUT-1 gene mapped to human chromosome 7q33. Northern blotting analysis revealed that SUT-1 exhibits a highly restricted tissue distribution, with abundant expression in placenta. Reverse transcription–PCR analysis indicated that SUT-1 and the diastrophic dysplasia sulfate transporter (DTD), one of the two known human DIDS-sensitive sulfate transporters, are coexpressed in HEVEC. SUT-1 and DTD could correspond, respectively, to the DIDS-resistant and DIDS-sensitive components of sulfate uptake in HEVEC. Together, these results demonstrate that SUT-1 is a distinct human Na+-coupled sulfate transporter, likely to play a major role in sulfate incorporation in HEV.

A natural classification of the basic helix–loop–helix class of transcription factors

A natural (evolutionary) classification is provided for 242 basic helix–loop–helix (bHLH) motif-containing proteins. Phylogenetic analyses of amino acid sequences describe the patterns of evolutionary change within the motif and delimit evolutionary lineages. These evolutionary lineages represent well known functional groups of proteins and can be further arranged into five groups based on binding to DNA at the hexanucleotide E-box, the amino acid patterns in other components of the motif, and the presence/absence of a leucine zipper. The hypothesized ancestral amino acid sequence for the bHLH transcription factor family is given together with the ancestral sequences of the subgroups. It is suggested that bHLH proteins containing a leucine zipper are not a natural, monophyletic group.

Functional antioxidant responsive elements

Exposure of human and rodent cells to a wide variety of chemoprotective compounds confers resistance against a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of protective enzymes like glutathione S-transferases (GST). Antioxidant responsive elements (AREs) mediate the transcriptional induction of a battery of genes which comprise much of this chemoprotective response system. Past studies identified a necessary ARE “core” sequence of RTGACnnnGC, but this sequence alone is insufficient to mediate induction. In this study, the additional sequences necessary to define a sufficient, functional ARE are identified through systematic mutational analysis of the murine GST Ya ARE. Introduction of the newly identified necessary nucleotides into the regions flanking a nonresponsive, ARE-like, GST-Mu promoter sequence produced an inducible element. A screen of the GenBank database with the newly identified ARE consensus identified 16 genes which contained the functional ARE consensus sequence in their promoters. Included within this group was an ARE sequence from the murine ferritin-L promoter that mediated induction when tested. In an electrophoretic mobility-shift assay, the ferritin-L ARE was bound by ARE–binding protein 1, a protein previously identified as the likely mediator of the chemoprotective response. A three-level ARE classification system is presented to account for the distinct induction strengths observed in our mutagenesis studies. A model of the ARE as a composite regulatory site, where multiple transcription factors interact, is presented to account for the complex characteristics of ARE-mediated chemoprotective gene expression.

Leptin can induce proliferation, differentiation, and functional activation of hemopoietic cells

Many cytokines exert their biological effect through members of the hemopoietin receptor family. Using degenerate oligonucleotides to the common WSXWS motif, we have cloned from human hemopoietic cell cDNA libraries various forms of the receptor that was recently shown to bind the obesity hormone, leptin. mRNAs encoding long and short forms of the human leptin receptor were found to be coexpressed in a range of human and murine hemopoietic organs, and a subset of cells from these tissues bound leptin at the cell surface. Ectopic expression in murine Ba/F3 and M1 cell lines revealed that the long, but not the short, form of the leptin receptor can signal proliferation and differentiation, respectively. In cultures of murine or human marrow cells, human leptin exhibited no capacity to stimulate cell survival or proliferation, but it enhanced cytokine production and phagocytosis of Leishmania parasites by murine peritoneal macrophages. Our data provide evidence that, in addition to its role in fat regulation, leptin may also be able to regulate aspects of hemopoiesis and macrophage function.

Functional integrity of mitochondrial genomes in human platelets and autopsied brain tissues from elderly patients with Alzheimer’s disease

To determine whether pathogenic mutations in mtDNA are involved in phenotypic expression of Alzheimer’s disease (AD), the transfer of mtDNA from elderly patients with AD into mtDNA-less (ρ0) HeLa cells was carried out by fusion of platelets or synaptosomal fractions of autopsied brain tissues with ρ0 HeLa cells. The results showed that mtDNA in postmortem brain tissue survives for a long time without degradation and could be rescued in ρ0 HeLa cells. Next, the cybrid clones repopulated with exogenously imported mtDNA from patients with AD were used for examination of respiratory enzyme activity and transfer of mtDNA with the pathogenic mutations that induce mitochondrial dysfunction. The presence of the mutated mtDNA was restricted to brain tissues and their cybrid clones that formed with synaptosomes as mtDNA donors, whereas no cybrid clones that isolated with platelets as mtDNA donors had detectable mutated mtDNA. However, biochemical analyses showed that all cybrid clones with mtDNA imported from platelets or brain tissues of patients with AD restored mitochondrial respiration activity to almost the same levels as those of cybrid clones with mtDNA from age-matched normal controls, suggesting functional integrity of mtDNA in both platelets and brain tissues of elderly patients with AD. These observations warrant the reassessment of the conventional concept that the accumulation of pathogenic mutations in mtDNA throughout the aging process is responsible for the decrease of mitochondrial respiration capacity with age and with the development of age-associated neurodegenerative diseases.

Phloem sap proteins from Cucurbita maxima and Ricinus communis have the capacity to traffic cell to cell through plasmodesmata

In angiosperms, the functional enucleate sieve tube system of the phloem appears to be maintained by the surrounding companion cells. In this study, we tested the hypothesis that polypeptides present within the phloem sap traffic cell to cell from the companion cells, where they are synthesized, into the sieve tube via plasmodesmata. Coinjection of fluorescently labeled dextrans along with size-fractionated Cucurbita maxima phloem proteins, ranging in size from 10 to 200 kDa, as well as injection of individual fluorescently labeled phloem proteins, provided unambiguous evidence that these proteins have the capacity to interact with mesophyll plasmodesmata in cucurbit cotyledons to induce an increase in size exclusion limit and traffic cell to cell. Plasmodesmal size exclusion limit increased to greater than 20 kDa, but less than 40 kDa, irrespective of the size of the injected protein, indicating that partial protein unfolding may be a requirement for transport. A threshold concentration in the 20–100 nM range was required for cell-to-cell transport indicating that phloem proteins have a high affinity for the mesophyll plasmodesmal binding site(s). Parallel experiments with glutaredoxin and cystatin, phloem sap proteins from Ricinus communis, established that these proteins can also traffic through cucurbit mesophyll plasmodesmata. These results are discussed in terms of the requirements for regulated protein trafficking between companion cells and the sieve tube system. As the threshold value for plasmodesmal transport of phloem sap proteins falls within the same range as many plant hormones, the possibility is discussed that some of these proteins may act as long-distance signaling molecules.

A rapid classification protocol for the CATH Domain Database to support structural genomics

In order to support the structural genomic initiatives, both by rapidly classifying newly determined structures and by suggesting suitable targets for structure determination, we have recently developed several new protocols for classifying structures in the CATH domain database (http://www.biochem.ucl.ac.uk/bsm/cath). These aim to increase the speed of classification of new structures using fast algorithms for structure comparison (GRATH) and to improve the sensitivity in recognising distant structural relatives by incorporating sequence information from relatives in the genomes (DomainFinder). In order to ensure the integrity of the database given the expected increase in data, the CATH Protein Family Database (CATH-PFDB), which currently includes 25 320 structural domains and a further 160 000 sequence relatives has now been installed in a relational ORACLE database. This was essential for developing more rigorous validation procedures and for allowing efficient querying of the database, particularly for genome analysis. The associated Dictionary of Homologous Superfamilies [Bray,J.E., Todd,A.E., Pearl,F.M.G., Thornton,J.M. and Orengo,C.A. (2000) Protein Eng., 13, 153–165], which provides multiple structural alignments and functional information to assist in assigning new relatives, has also been expanded recently and now includes information for 903 homo­logous superfamilies. In order to improve coverage of known structures, preliminary classification levels are now provided for new structures at interim stages in the classification protocol. Since a large proportion of new structures can be rapidly classified using profile-based sequence analysis [e.g. PSI-BLAST: Altschul,S.F., Madden,T.L., Schaffer,A.A., Zhang,J., Zhang,Z., Miller,W. and Lipman,D.J. (1997) Nucleic Acids Res., 25, 3389–3402], this provides preliminary classification for easily recognisable homologues, which in the latest release of CATH (version 1.7) represented nearly three-quarters of the non-identical structures.

A fully automatic evolutionary classification of protein folds: Dali Domain Dictionary version 3

The Dali Domain Dictionary (http://www.ebi.ac.uk/dali/domain) is a numerical taxonomy of all known structures in the Protein Data Bank (PDB). The taxonomy is derived fully automatically from measurements of structural, functional and sequence similarities. Here, we report the extension of the classification to match the traditional four hierarchical levels corresponding to: (i) supersecondary structural motifs (attractors in fold space), (ii) the topology of globular domains (fold types), (iii) remote homologues (functional families) and (iv) homologues with sequence identity above 25% (sequence families). The computational definitions of attractors and functional families are new. In September 2000, the Dali classification contained 10 531 PDB entries comprising 17 101 chains, which were partitioned into five attractor regions, 1375 fold types, 2582 functional families and 3724 domain sequence families. Sequence families were further associated with 99 582 unique homologous sequences in the HSSP database, which increases the number of effectively known structures several-fold. The resulting database contains the description of protein domain architecture, the definition of structural neighbours around each known structure, the definition of structurally conserved cores and a comprehensive library of explicit multiple alignments of distantly related protein families.

The COG database: new developments in phylogenetic classification of proteins from complete genomes

The database of Clusters of Orthologous Groups of proteins (COGs), which represents an attempt on a phylogenetic classification of the proteins encoded in complete genomes, currently consists of 2791 COGs including 45 350 proteins from 30 genomes of bacteria, archaea and the yeast Saccharomyces cerevisiae (http://www.ncbi.nlm.nih.gov/COG). In addition, a supplement to the COGs is available, in which proteins encoded in the genomes of two multicellular eukaryotes, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, and shared with bacteria and/or archaea were included. The new features added to the COG database include information pages with structural and functional details on each COG and literature references, improvements of the COGNITOR program that is used to fit new proteins into the COGs, and classification of genomes and COGs constructed by using principal component analysis.

iProClass: an integrated, comprehensive and annotated protein classification database

The iProClass database is an integrated resource that provides comprehensive family relationships and structural and functional features of proteins, with rich links to various databases. It is extended from ProClass, a protein family database that integrates PIR superfamilies and PROSITE motifs. The iProClass currently consists of more than 200 000 non-redundant PIR and SWISS-PROT proteins organized with more than 28 000 superfamilies, 2600 domains, 1300 motifs, 280 post-translational modification sites and links to more than 30 databases of protein families, structures, functions, genes, genomes, literature and taxonomy. Protein and family summary reports provide rich annotations, including membership information with length, taxonomy and keyword statistics, full family relationships, comprehensive enzyme and PDB cross-references and graphical feature display. The database facilitates classification-driven annotation for protein sequence databases and complete genomes, and supports structural and functional genomic research. The iProClass is implemented in Oracle 8i object-relational system and available for sequence search and report retrieval at http://pir.georgetow n.edu/iproclass/.

TIGRFAMs: a protein family resource for the functional identification of proteins

TIGRFAMs is a collection of protein families featuring curated multiple sequence alignments, hidden Markov models and associated information designed to support the automated functional identification of proteins by sequence homology. We introduce the term ‘equivalog’ to describe members of a set of homologous proteins that are conserved with respect to function since their last common ancestor. Related proteins are grouped into equivalog families where possible, and otherwise into protein families with other hierarchically defined homology types. TIGRFAMs currently contains over 800 protein families, available for searching or downloading at www.tigr.org/TIGRFAMs. Classification by equivalog family, where achievable, complements classification by orthology, superfamily, domain or motif. It provides the information best suited for automatic assignment of specific functions to proteins from large-scale genome sequencing projects.

Biomechanical activation of vascular endothelium as a determinant of its functional phenotype

One of the striking features of vascular endothelium, the single-cell-thick lining of the cardiovascular system, is its phenotypic plasticity. Various pathophysiologic factors, such as cytokines, growth factors, hormones, and metabolic products, can modulate its functional phenotype in health and disease. In addition to these humoral stimuli, endothelial cells respond to their biomechanical environment, although the functional implications of this biomechanical paradigm of activation have not been fully explored. Here we describe a high-throughput genomic analysis of modulation of gene expression observed in cultured human endothelial cells exposed to two well defined biomechanical stimuli—a steady laminar shear stress and a turbulent shear stress of equivalent spatial and temporal average intensity. Comparison of the transcriptional activity of 11,397 unique genes revealed distinctive patterns of up- and down-regulation associated with each type of stimulus. Cluster analyses of transcriptional profiling data were coupled with other molecular and cell biological techniques to examine whether these global patterns of biomechanical activation are translated into distinct functional phenotypes. Confocal immunofluorescence microscopy of structural and contractile proteins revealed the formation of a complex apical cytoskeleton in response to laminar shear stress. Cell cycle analysis documented different effects of laminar and turbulent shear stresses on cell proliferation. Thus, endothelial cells have the capacity to discriminate among specific biomechanical forces and to translate these input stimuli into distinctive phenotypes. The demonstration that hemodynamically derived stimuli can be strong modulators of endothelial gene expression has important implications for our understanding of the mechanisms of vascular homeostasis and atherogenesis.