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Biomedical subjects

A Lupas

Publications and source records attributed to A Lupas.

At least 19 recordsLinked to original sources
PubMed2001

Phylogenetic analyses do not support horizontal gene transfers from bacteria to vertebrates.

Horizontal gene transfer (HGT) has long been recognized as a principal force in the evolution of genomes. Genome sequences of Archaea and Bacteria have revealed the existence of genes whose similarity to loci in distantly related organisms is explained most parsimoniously by HGT events. In most multicellular organisms, such genetic fixation can occur only in the germ line. Therefore, it is notable that the publication of the human genome reports 113 incidents of direct HGT between bacteria and vertebrates, without any apparent occurrence in evolutionary intermediates, that is, non-vertebrate eukaryotes. Phylogenetic analysis arguably provides the most objective approach for determining the occurrence and directionality of HGT. Here we report a phylogenetic analysis of 28 proposed HGT genes, whose presence in the human genome had been confirmed by polymerase chain reaction (PCR). The results indicate that most putative HGT genes are present in more anciently derived eukaryotes (many such sequences available in non-vertebrate EST databases) and can be explained in terms of descent through common ancestry. They are, therefore, unlikely to be examples of direct HGT from bacteria to vertebrates.

Animals↗
PubMed2001

Systematic identification of selective essential genes in Helicobacter pylori by genome prioritization and allelic replacement mutagenesis.

A comparative genomic approach was used to identify Helicobacter pylori 26695 open reading frames (ORFs) which are conserved in H. pylori J99 but highly diverged in other eubacteria. A survey of selected pathways of central intermediary metabolism was also carried out, and genes with a potentially selective role in H. pylori were identified. Forty-five ORFs identified in these two analyses were screened using a rapid vector-free allelic replacement mutagenesis technique, and 33 were shown to be essential in vitro. Notably, 13 ORFs gave essentiality results which are unexpected in view of their known or proposed functions, and phylogenetic analysis was used to investigate the annotation of 7 such ORFs which are highly diverged. We propose that the products of a number of these H. pylori-specific essential genes may be suitable targets for novel anti-H. pylori therapies.

Alleles↗
PubMed2000

Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins.

The non-fimbrial adhesins, YadA of enteropathogenic Yersinia species, and UspA1 and UspA2 of Moraxella catarrhalis, are established pathogenicity factors. In electron micrographs, both surface proteins appear as distinct 'lollipop'-shaped structures forming a novel type of surface projection on the outer membranes. These structures, amino acid sequence analysis of these molecules and yadA gene manipulation suggest a tripartite organization: an N-terminal oval head domain is followed by a putative coiled-coil rod and terminated by a C-terminal membrane anchor domain. In YadA, the head domain is involved in autoagglutination and binding to host cells and collagen. Analysis of the coiled-coil segment of YadA revealed unusual pentadecad repeats with a periodicity of 3.75, which differs significantly from the 3.5 periodicity found in the Moraxella UspAs and other canonical coiled coils. These findings predict that the surface projections are formed by oligomers containing right- (Yersinia) or left-handed (Moraxella) coiled coils. Strikingly, sequence comparison revealed that related proteins are found in many proteobacteria, both human pathogenic and environmental species, suggesting a common role in adaptation to specific ecological niches.

Adhesins, Bacterial↗
PubMed2000

Genetic characterization of gram-positive homologs of the XerCD site-specific recombinases.

Homologs of the XerCD enzymes, which in Escherichia coli have been shown to be responsible for resolving chromosomal multimers prior to chromosome segregation, were identified in the genomes of Staphylococcus aureus and Streptococcus pneumoniae. Phylogenetic and conservation pattern analysis suggests that the S. aureus gene products are orthologs of XerC and D. A S. aureus xerC null mutant displayed in vitro characteristics consistent with the segregation defect reported for E. coli xer mutants, and was found to be attenuated in a murine infection model. Strikingly, the S. aureus xerD gene appears to be absolutely required for viability, and may therefore be the first example of an essential gene of the lambda integrase family. In contrast, phylogenetic and conservation pattern analysis show that the S. pneumoniae gene products are more closely related to phage integrases than to XerCD. S. pneumoniae xer1, 2 and 3 null mutants were each found to be attenuated in a murine infection model, suggesting that they may control processes which affect virulence.

Alleles↗
PubMed1999

The solution structure of VAT-N reveals a 'missing link' in the evolution of complex enzymes from a simple betaalphabetabeta element.

BACKGROUND: The VAT protein of the archaebacterium Thermoplasma acidophilum, like all other members of the Cdc48/p97 family of AAA ATPases, has two ATPase domains and a 185-residue amino-terminal substrate-recognition domain, VAT-N. VAT shows activity in protein folding and unfolding and thus shares the common function of these ATPases in disassembly and/or degradation of protein complexes. RESULTS: Using nuclear magnetic resonance (NMR) spectroscopy, we found that VAT-N is composed of two equally sized subdomains. The amino-terminal subdomain VAT-Nn (comprising residues Met1-Thr92) forms a double-psi beta-barrel whose pseudo-twofold symmetry is mirrored by an internal sequence repeat of 42 residues. The carboxy-terminal subdomain VAT-Nc (comprising residues Glu93-Gly185) forms a novel six-stranded beta-clam fold. Together, VAT-Nn and VAT-Nc form a kidney-shaped structure, in close agreement with results from electron microscopy. Sequence and structure analyses showed that VAT-Nn is related to numerous proteins including prokaryotic transcription factors, metabolic enzymes, the protease cofactors UFD1 and PrlF, and aspartic proteinases. These proteins map out an evolutionary path from simple homodimeric transcription factors containing a single copy of the VAT-Nn repeat to complex enzymes containing four copies. CONCLUSIONS: Our results suggest that VAT-N is a precursor of the aspartic proteinases that has acquired peptide-binding activity while remaining proteolytically incompetent. We propose that the binding site of the protein is similar to that of aspartic proteinases, in that it lies between the psi-loops of the amino-terminal beta-barrel and that it coincides with a crescent-shaped band of positive charge extending across the upper face of the molecule.

Adenosine Triphosphatases↗
PubMed1999

Novel molecular architecture of the multimeric archaeal PEP-synthase homologue (MAPS) from Staphylothermus marinus.

The phosphoenolpyruvate (PEP)-synthases belong to the family of structurally and functionally related PEP-utilizing enzymes. The only archaeal member of this family characterized thus far is the Multimeric Archaeal PEP-Synthase homologue from Staphylothermus marinus (MAPS). This protein complex differs from the bacterial and eukaryotic representatives characterized to date in its homomultimeric, as opposed to dimeric or tetrameric, structure. We have probed the molecular architecture of MAPS using limited proteolytic digestion in conjunction with electron microscopic, biochemical, and biophysical techniques. The 2.2 MDa particle was found to be organized in a concentric fashion. The 93.7 kDa monomers possess a pronounced tripartite domain structure and are arranged such that the N-terminal domains form an outer shell, the intermediate domains form an inner shell, and the C-terminal domains form a core structure responsible for the assembly into a multimeric complex. The core domain was shown to be capable of assembling into the native multimer by recombinant expression in Escherichia coli. Deletion mutants as well as a synthetic peptide were investigated for their state of oligomerization using native polyacrylamide gel electrophoresis, molecular sieve chromatography, analytical ultracentrifugation, circular dichroism (CD) spectroscopy, and chemical cross-linking. Our data confirmed the existence of a short C-terminal, alpha-helical oligomerization motif that had been suggested by multiple sequence alignments and secondary structure predictions. We propose that this motif bundles the monomers into six groups of four. An additional formation of 12 dimers between globular domains from different bundles leads to the multimeric assembly. According to our model, each of the six bundles of globular domains is positioned at the corners of an imaginary octahedron, and the helical C-terminal segments are oriented towards the centre of the particle. The edges of the octahedron represent the dimeric contacts. Phylogenetic analysis suggests that the ancient predecessor of this family of enzymes contained the C-terminal oligomerization motif as a feature that was preserved in some hyperthermophiles.

Amino Acid Sequence↗
PubMed1998

Characterization of ARC, a divergent member of the AAA ATPase family from Rhodococcus erythropolis.

A gene encoding a AAA ATPase was discovered in the 5' region of the second operon of 20 S proteasome subunits in the nocardioform actinomycete Rhodococcus erythropolis NI86/21. The gene was cloned and expressed in Escherichia coli. The protein, ARC (AAA ATPase forming Ring-shaped Complexes), is a divergent member of the AAA family. The deduced product of the arc gene is 591 residues long (66 kDa). The purified protein possesses a low, N-ethylmaleimide-sensitive ATPase activity and forms rings of six subunits, arranged symmetrically around a central opening or cavity. Two-dimensional crystals grown on lipid monolayers yielded images of the ATPase molecules in "end-on" orientation at 1.9 nm resolution.

Amino Acid Sequence↗
PubMed1998

The regulated outer membrane protein Omp21 from Comamonas acidovorans is identified as a member of a new family of eight-stranded beta-sheet proteins by its sequence and properties.

Omp21, a minor outer membrane protein of the soil bacterium Comamonas acidovorans, was purified from a spontaneous mutant lacking a surface layer and long-chain lipopolysaccharide. Omp21 synthesis is enhanced by oxygen depletion, and the protein has a variable electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to its heat-modifiable behavior. The structural gene omp21 encodes a precursor of 204 amino acids with a putative signal peptide of 21 amino acids. Mature Omp21 is a typical outer membrane protein with a high content of beta structure as determined by infrared spectroscopy. Sequence comparisons show that it belongs to a new outer membrane protein family, characterized by eight amphipathic beta strands, which includes virulence proteins, such as the neisserial opacity proteins, Salmonella typhimurium Rck, and Yersinia enterocolitica Ail, as well as the major outer membrane proteins OmpA from Escherichia coli and OprF from Pseudomonas aeruginosa.

Amino Acid Sequence↗
PubMed1997

The dodo gene family encodes a novel protein involved in signal transduction and protein folding.

Recent studies in yeast, Drosophila and humans have revealed the existence of a highly conserved gene encoding a novel protein, Dodo, comprised of four modules: a WW domain, involved in protein-protein interactions, a peptidyl-prolyl cis-trans isomerase (PPIase) domain belonging to a recently described third family of PPIases involved in protein folding and unfolding, a nuclear localization motif and finally, a long, surface-exposed alpha-helix that is likely to be involved in binding to a cell cycle serine/threonine kinase. The genetic, molecular, biochemical and structural data are reviewed in the context of the potential biological properties of this new protein family.

Amino Acid Sequence↗
PubMed1997

The thermosome: alternating alpha and beta-subunits within the chaperonin of the archaeon Thermoplasma acidophilum.

The thermosome of the archaeon Thermoplasma acidophilum is composed of two subunits, alpha and beta, which are arranged in two stacked, eight-membered rings. Electron cryo-microscopy in conjunction with image analysis revealed a 4-fold symmetry in the heterooligomeric alpha + beta thermosome isolated from Thermoplasma, but not in the homooligomeric alpha-only thermosome expressed in Escherichia coli. This indicates that alpha and beta-subunits alternate within the rings of the Thermoplasma thermosome rather than forming two different homooligomeric rings. In addition, a small subpopulation of 9-fold symmetric complexes was found among the recombinant alpha-only thermosomes, and a central mass most likely representing bound substrate molecules was observed in about half of the native and recombinant thermosome particles.

Adenosine Triphosphatases↗
PubMed1997

Cloning, sequencing and expression of VAT, a CDC48/p97 ATPase homologue from the archaeon Thermoplasma acidophilum.

A member of the AAA family of Mg2(+)-ATPases from the archaeon Thermoplasma acidophilum has been cloned and expressed in Escherichia coli. The protein, VCP-like ATPase of Thermoplasma acidophilum (VAT), is a homologue of SAV from Sulfolobus acidocaldarius and CdcH of Halobacterium salinarium, and belongs to the CDC48/VCP/p97 subfamily. The deduced product of the vat gene is 745 residues long (Mr 83,000), which has an optimal Mg2(+)-ATPase activity at 70 degrees C. Electron microscopy shows the purified protein to form single and double homo-hexameric rings. Although the symmetry is different, the appearance of the complexes formed of two rings resembles the 20S proteasome and Hsp60/GroEL.

Adenosine Triphosphatases↗
PubMed1997

The proteasome.

The proteasome is a macromolecular assembly that is designed to confine proteolytic activity to an inner cavity. Access to the central proteolytic nanocompartment is restricted to unfolded proteins, which necessitates a functional coupling of the 20S proteasome to a substrate-recognition and unfolding machinery. Significant progress has been made during the past two years in elucidating the structural principles and the enzymatic mechanism of the 20S proteasome.

Cysteine Endopeptidases↗
PubMed1997

Predicting coiled-coil regions in proteins.

The past several years have seen significant advances in our ability to recognize coiled coils from protein sequences and model their structures. New methods include a detection program based on pairwise residue correlations, a program that distinguishes two-stranded from three-stranded coiled coils and a routine for modelling the coordinates of the core residues in coiled coils. Several widely noted predictions, among them those for heterotrimeric G proteins and for cartilage oligomeric matrix protein, have been confirmed by crystal structures, and several new predictions have been made, including a model for the still hypothetical right-handed coiled coil.

Computer Simulation↗
PubMed1997

Self-compartmentalizing proteases.

Among the hundreds of proteases characterized so far, most of which are monomeric or dimeric, there is a small group that form compartments through self-association and that segregate their proteolytic active sites to the interior of these compartments. Although few in number, they represent the main agents of intracellular protein breakdown. They belong to different hydrolase families but have converged towards the same barrel-shaped architecture. Frequently, they are coupled to chaperone-like ATPases of similar quaternary structure that regulate the access to the proteolytic compartments and appear to have been recruited from the same branch of P-loop NTPases.

Amino Acid Sequence↗
PubMed1997

Eubacterial proteasomes.

Proteasomes are large, multisubunit proteases with highly conserved structures. The 26S proteasome of eukaryotes is an ATP-dependent enzyme of about 2 MDa, which acts as the central protease of the ubiquitin-dependent pathway of protein degradation. The core of the 26S complex is formed by the 20S proteasome, an ATP-independent, barrel-shaped protease of about 700 kDa, which has also been detected in archaebacteria and, more recently, in eubacteria. Currently, the distribution of 20S proteasomes in eubacteria appears limited to the actinomycetes, while most other eubacteria contain a related complex of simpler structure.

Amino Acid Sequence↗
PubMed1996

Classification of tyrosine kinases from Dictyostelium discoideum with two distinct, complete or incomplete catalytic domains.

Two new kinases of Dictyostelium discoideum were identified by screening of a (lambda)gt11 expression library with a phosphotyrosine specific antibody. Amino-acid sequences derived from cDNA and genomic clones indicate that DPYK3 is a protein of 150 kDa and DPYK4, a protein of 75 kDa. The C-terminal fragments of each protein were produced in Escherichia coli and shown to be autocatalytically phosphorylated at tyrosine residues. A common feature of these kinases is the presence of two different sequence stretches in tandem that are related to kinase catalytic domains. The sequence relationships of DPYK3 and 4 to other protein kinases, and the positions of their catalytic domain sequences within the phylogenetic tree of protein kinases were analysed. Domains I of both kinases and domain II of DPYK3 constitute, together with the catalytic domains of two previously described tyrosine kinases of D. discoideum, a branch of their own, separate from the tyrosine kinase domains in sensu strictu. Domain II in DPYK4 is found on a different branch close to serine/threonine kinases.

Amino Acid Sequence↗
PubMed1996

Multivalent antibody fragments with high functional affinity for a tumor-associated carbohydrate antigen.

We report in this work a human-derived self-assembling polypeptide based on the tetramerization domain of the human transcription factor p53, which can be fused to single-chain Fv Ab (scFv) fragments via a long and flexible hinge sequence of human origin, allowing exploitation of the functional affinity increase of binding to a ligand or cell surface with multimeric binding sites. We have demonstrated the use of this polypeptide by applying it to the construction of a tetrameric scFv against the tumor-associated carbohydrate Ag Lewis Y (Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3] GlcNAc beta 1-->3R). For comparison purposes, the corresponding scFv and dimeric mini-antibody, comprising the scFv fused via a flexible murine hinge to an artificial dimerization domain, were also created. The recombinant mini-antibody proteins were expressed in functional form in Escherichia coli and showed the expected m.w. of a dimer and tetramer, respectively. Analysis of Lewis Y-binding behavior by surface plasmon resonance revealed specific but very weak binding of the scFv fragment. In contrast, both dimeric and tetrameric scFv fusion proteins exhibited an enormous gain in functional affinity that was greatest in the case of the tetrameric mini-antibody.

Amino Acid Sequence↗