The SBASE protein domain library, release 3.0: a collection of annotated protein sequence segments.

SBASE 3.0 is the third release of SBASE, a collection of annotated protein domain sequences. SBASE entries represent various structural, functional, ligand-binding and topogenic segments of proteins as defined by their publishing authors. SBASE can be used for establishing domain homologies using different database-search tools such as FASTA [Lipman and Pearson (1985) Science, 227, 1436-1441], and BLAST3 [Altschul and Lipman (1990) Proc. Natl. Acad. Sci. USA, 87, 5509-5513] which is especially useful in the case of loosely defined domain types for which efficient consensus patterns can not be established. The present release contains 41,749 entries provided with standardized names and cross-referenced to the major protein and nucleic acid databanks as well as to the PROSITE catalogue of protein sequence patterns. The entries are clustered into 2285 groups using the BLAST algorithm for computing similarity measures. SBASE 3.0 is freely available on request to the authors or by anonymous 'ftp' file transfer from < ftp.icgeb.trieste.it >. Individual records can be retrieved with the gopher server at < icgeb.trieste.it > and with a www-server at < http:@www.icgeb.trieste.it >. Automated searching of SBASE by BLAST can be carried out with the electronic mail server < sbase@icgeb.trieste.it >. Another mail server < domain@hubi.abc.hu > assigns SBASE domain homologies on the basis of SWISS-PROT searches. A comparison of pertinent search strategies is presented.     

The SBASE protein domain library, Release 4.0: a collection of annotated protein sequence segments.

SBASE 4.0 is the fourth release of SBASE, a collection of annotated protein domain sequences that represent various structural, functional, ligand binding and topogenic segments of proteins. SBASE was designed to facilitate the detection of functional homologies and can be searched with standard database search tools, such as FASTA and BLAST3. The present release contains 61 137 entries provided with standardized names and cross-referenced to all major protein, nucleic acid and sequence pattern collections. The entries are clustered into 13 155 groups in order to facilitate detection of distant similarities. SBASE 4.0 is freely available by anonymous ftp file transfer from ftp.icgeb.trieste.it. Individual records can be retrieved with the gopher server at icgeb.trieste.it and with a World Wide Web server at http://www.icgeb.trieste.it. Automated searching of SBASE with BLAST can be carried out with the electronic mail server sbase@icgeb.trieste.it, which now also provides a graphic representation of the homologies. A related mail server, domain@hubi.abc.hu, assigns SBASE domain homologies on the basis of SWISS-PROT searches.     

Cloning and nucleotide sequence of the structural gene encoding for human tryptophanyl-tRNA synthetase.

A structural gene encoding bovine (b) tryptophanyl-tRNA synthetase (WRS) has recently been cloned and sequenced [Garret et al., Biochemistry 30 (1991) 7809-7817]. Using part of this sequence as a hybridisation probe we have cloned and sequenced a structural gene encoding human polypeptide highly homologous with two mammalian proteins, bWRS [Garret et al., Biochemistry 30 (1991) 7809-7817; EMBL accession No. X52113] and rabbit peptide chain release factor [Lee et al., Proc. Natl. Acad. Sci. USA 87 (1990) 3508-3512]. Identification of the sequence encoding a human WRS is based on (i) the presence of 'HIGH' and 'KMSKS' structural motifs typical for class-I aminoacyl-tRNA synthetases [Eriani et al., Nature 347 (1990) 203-206]; (ii) coincidence of the number of SH groups per subunit estimated experimentally [Muench et al., Science 187 (1975) 1089-1091] and deduced from the cDNA sequence (six in both cases); (iii) close resemblance of two WRS polypeptides sequenced earlier [Muench et al., Science 187 (1975) 1089-1091] and the predicted structure in two different regions.     

The SBASE protein domain library, release 7.0: a collection of annotated protein sequence segments

SBASE 7.0 is the seventh release of the SBASE protein domain library sequences that contains 237 937 annotated structural, functional, ligand-binding and topogenic segments of proteins, cross-referenced to all major sequence databases and sequence pattern collections. The entries are clustered into over 1811 groups and are provided with two WWW-based search facilities for on-line use. SBASE 7.0 is freely available by anonymous 'ftp' file transfer from ftp.icgeb. trieste.it. Automated searching of SBASE with BLAST can be carried out with the WWW servers http://www.icgeb.trieste.it/sbase/and http://sbase.abc.hu/sbase/     

The SBASE protein domain library, release 8.0: a collection of annotated protein sequence segments

SBASE 8.0 is the eighth release of the SBASE library of protein domain sequences that contains 294 898 annotated structural, functional, ligand-binding and topogenic segments of proteins, cross-referenced to most major sequence databases and sequence pattern collections. The entries are clustered into over 2005 statistically validated domain groups (SBASE-A) and 595 non-validated groups (SBASE-B), provided with several WWW-based search and browsing facilities for online use. A domain-search facility was developed, based on non-parametric pattern recognition methods, including artificial neural networks. SBASE 8.0 is freely available by anonymous 'ftp' file transfer from ftp.icgeb.trieste.it. Automated searching of SBASE can be carried out with the WWW servers http://www.icgeb.trieste.it/sbase/ and http://sbase.abc. hu/sbase/.     

The SBASE protein domain library, release 5.0: a collection of annotated protein sequence segments.

SBASE 5.0 is the fifth release of SBASE, a collection of annotated protein domain sequences that represent various structural, functional, ligand-binding and topogenic segments of proteins. SBASE was designed to facilitate the detection of functional homologies and can be searched with standard database-search programs. The present release contains over 79863 entries provided with standardized names and is cross-referenced to all major sequence databases and sequence pattern collections. The information is assigned to individual domains rather than to entire protein sequences, thus SBASE contains substantially more cross-references and links than do the protein sequence databases. The entries are clustered into >16 000 groups in order to facilitate the detection of distant similarities. SBASE 5.0 is freely available by anonymous 'ftp' file transfer from <ftp.icgeb.trieste.it >. Automated searching of SBASE with BLAST can be carried out with the WWW-server <http://www.icgeb.trieste.it/sbase/ >. and with the electronic mail server <sbase@icgeb.trieste.it >which now also provides a graphic representation of the homologies. A related WWW-server <http://www.abc.hu/blast.html > and e-mail server <domain@hubi.abc.hu > predicts SBASE domain homologies on the basis of SWISS-PROT searches.     

ST7 is a novel low-density lipoprotein receptor-related protein (LRP) with a cytoplasmic tail that interacts with proteins related to signal transduction pathways

In 1997, McCormick and co-workers identified a novel putative tumor suppressor gene, designated ST7, encoding a unique protein with transmembrane receptor characteristics [Qing et al. (1999) Oncogene 18, 335-342]. Using degenerate primers corresponding to the highly conserved region of the ligand-binding domains of members of the low-density lipoprotein receptor (LDLR) superfamily, Ishii et al. [Genomics (1998) 51, 132-135] discovered a low-density lipoprotein receptor-related protein (LRP) that closely resembles ST7. Later, another LRP closely resembling ST7 and LRP3 was found (murine LRP9) [Sugiyama et al. (2000) Biochemistry 39, 15817-15825]. These results strongly suggested that ST7 was also a novel member of the low-density lipoprotein receptor superfamily. Proteins of this superfamily have been shown to function in endocytosis and/or signal transduction. To evaluate the relationship of ST7 to the LDLR superfamily proteins and to determine whether ST7 may function in endocytosis and/or signal transduction, we used proteomic tools to analyze the functional motifs present in the protein. Our results indicate that ST7 is a member of a subfamily of the LDLR superfamily and that its cytoplasmic domain contains several motifs implicated in endocytosis and signal transduction. Use of the yeast two-hybrid system to identify proteins that associate with ST7's cytoplasmic domain revealed that this domain interacts with three proteins involved in signal transduction and/or endocytosis, viz., receptor for activated protein C kinase 1 (RACK1), muscle integrin binding protein (MIBP), and SMAD anchor for receptor activation (SARA), suggesting that ST7, like other proteins in the LDLR superfamily, functions in these two pathways. Clearly, ST7 is an LRP, and therefore, it should now be referred to as LRP12.     

Amplified expression and large-scale purification of protein G'.

PCR was used to isolate the gene fragment coding for Protein G' (SpG'), a truncated bacterial cell surface protein from Streptococcus G148 which binds to the Fc region of IgG and expressed in E. coli [Goward et al. (1990) Biochem. J. 267: 171-177]. The PCR primer was designed to change the TTG initiation triplet to ATG and to incorporate it into an NdeI restriction site (CATATG), allowing the gene to be cloned in frame into an NdeI restriction site immediately downstream of a trp promoter. Expression of SpG' was estimated as about 30% total soluble cell protein which compares very favourably to the less than 1% total soluble cell protein obtained from the original system [Goward, et al. (1990) Biochem. J. 267: 171-177]. Homogeneous SpG' was recovered by a single anion-exchange chromatography step on Q-Sepharose FF in a process which avoided use of an affinity adsorbent. Even though SpG' consists of almost identical repetitive domains from amino acid sequence analysis, different proteolytic sensitivity of each domain was observed indicating their structural dissimilarity.     

Sequence of the gene (pheA) encoding phenol monooxygenase from Pseudomonas sp. EST1001: expression in Escherichia coli and Pseudomonas putida.

The plasmid pEST1412 contains the genes, pheA and pheB, encoding phenol monooxygenase (PMO) and catechol 1,2-dioxygenase (C12]), respectively. Thse were originally cloned from the plasmid DNA of Pseudomonas sp. EST1001 [Kivisaar et al., Plasmid 24 (1990) 25-36]. Although pheA and pheB are cotranscribed using the promoter sequences derived from Tn4652 and the level of expression of C120 activities from pEST1412 was equal both in Escherichia coli and in Pseudomonas putida, the level of PMO activity measured in the cell-free extracts of E. coli was lower than that in P. putida. The nucleotide sequence of the 2.0-kb PstI-HindIII fragment of pEST1412 carrying pheA was determined. A 1821-bp ORF was found in this DNA. The structural gene (tfdB) encoding 2,4-dichlorophenol hydroxylase from pJP4 has been sequenced [Perkins et al., J. Bacteriol. 172 (1990) 2351-2359]. Comparison of the deduced amino acid sequences of tfdB and pheA revealed highly conserved regions in the protein products of these genes.     

Extracting hydrophobic free energies from experimental data: relationship to protein folding and theoretical models

Solubility and vapor pressure measurements of hydrocarbons in water are generally thought to provide estimates of the strength of the hydrophobic effect in the range 20-30 cal/(mol.A2). Our reassessment of the solubility data on the basis of new developments in solution thermodynamics suggests that the hydrophobic surface free energy for hydrocarbon solutes is 46-47 cal/(mol.A2), although the actual value depends strongly on curvature effects [Nicholls et al. (1991) Proteins (in press); Sharp et al. (1991) Science 252, 106-109]. The arguments to support such a significant increase in the estimate of the hydrophobic effect stem partly from theoretical considerations and partly from the experimental results of De Young and Dill [(1990) J. Phys. Chem. 94, 801-809] on benzene partition between water and alkane solvents. Previous estimates of the hydrophobic effect derive from an analysis of solute partition data, which does not fully account for changes in volume entropy. We show here how the ideal gas equations, combined with experimental molar volumes, can account for such changes. Revised solubility scales for the 20 amino acids, based on cyclohexane to water and octanol to water transfer energies, are derived. The agreement between these scales, particularly the octanol scale, and mutant protein stability measurements from Kellis et al. [(1989) Biochemistry 28, 4914-4922] and Shortle et al. [(1990) Biochemistry 29, 8033-8041] is good. The increased strength of the hydrophobic interaction has implications for the energetics of protein folding, substrate binding, and nucleic acid base stacking and the interpretation of computer simulations.     

Expression of the 53 kD forked protein rescues F-actin bundle formation and mutant bristle phenotypes in Drosophila.

forked mutations affect bristle development in Drosophila pupae, resulting in short, thick, gnarled bristles in the adult. The forked proteins are components of 200-300-microm-long actin fiber bundles that are present transiently during pupal development [Petersen et al., 1994: Genetics 136:173-182]. These bundles are composed of segments of 3-10 microm long, and forked protein is localized along the actin fiber bundle segments and accumulates at the junctions connecting them longitudinally. In the forked mutants, f(36a) and f(hd), F-actin bundles are greatly reduced in number and size, and bundle segmentation is absent. The p-element, P[w(+), falter] contains a 5.3-kb fragment of the forked gene that encodes the 53-kD forked protein [Lankenau et al., 1996: Mol Cell Biol 16:3535-3544]. Expression of only the 53-kD forked protein is sufficient to rescue the actin bundle and bristle phenotypes of f(36a) and f(hd) mutant flies. The 5.3-kb forked sequence, although smaller than the 13-kb region previously shown to rescue forked mutants [Petersen et al., 1994: Genetics 136:173-182], does contain the core forked sequence that encodes actin binding and bundling domains in cultured mammalian cells [Grieshaber and Petersen, 1999: J Cell Sci 112:2203-2211]. These data show that the 53-kD forked protein is sufficient for normal bristle development and that the domains shown previously to be important for actin bundling in cell culture may be all that are required for normal actin bundle formation in developing Drosophila bristles.     

Sequence of a canine cDNA clone encoding a Ran/TC4 GTP-binding protein

We report the isolation and characterization of a canine cDNA encoding a 216-amino acid GTP-binding protein of the Ras superfamily. The protein is almost identical to the human TC4 [Drivas et al., Mol. Cell. Biol. 10 (1990) 1793–1798] and Ran [Bischoffand Ponstingi, Proc. Natl. Acad. Sci. USA 88 (1991) 10830–10834; Nature 354 (1991) 80–82] proteins, the latter of which has been found to be involved in cell cycle control. Furthermore, the protein is highly similar to the fission yeast spi1 gene product [Matsumoto and Beach, Cell 66 (1991) 347–360]. The high degree of evolutionary conservation in this protein suggests that it plays a vital role in the eukaryotic cell.     

Stable synthesis of viral protein 2 of infectious bursal disease virus in Saccharomyces cerevisiae.

Viral protein 2 (VP2) from infectious bursal disease virus and its precursor polyprotein (N-VP2-VP4-VP3-C), in the absence of their native N-terminal region (19 amino acids), required fusion of yeast presequences for their stable synthesis in Saccharomyces cerevisiae [Jagadish et al., Gene 95 (1990) 179-186]. Restoration of the missing 19 aa resulted in stable synthesis of VP2, indicating the significance of the N-terminal region in protein stability.     

The Methanocaldococcus jannaschii protein Mj0968 is not a P-type ATPase

The Methanocaldococcus jannaschii (formerly Methanococcus jannaschii) protein Mj0968 has been reported to represent a soluble P-type ATPase [Ogawa et al., FEBS Lett. 471 (2000) 99-102]. In this study, we report that the heterologously expressed Mj0968-His(10) protein exhibits high rates of phosphatase activity, whereas only very low ATPase activity was measured. Replacement of the aspartate residue in the DSAGT motif (D7A), which becomes phosphorylated during the reaction cycle of P-type ATPases, does not affect the V(max), but only the K(M) of the reaction. Labeling studies with [gamma-(32)P]ATP and [alpha-(32)P]ATP revealed that the previously reported labeling experiments [Ogawa et al., 2000] do not necessarily show phosphorylation of Mj0968, but rather point to ATP binding. Binding studies with trinitrophenyl adenosine nucleotides showed low apparent K(d) values for those molecules. These results provide evidence that the native function of Mj0968 seems to be that of a phosphatase, rather than that of an ATP-hydrolyzing enzyme.     

Phosphorylation of plant actin-depolymerising factor by calmodulin-like domain protein kinase.

The actin-depolymerising factor (ADF)/cofilin group of proteins are stimulus-responsive actin-severing proteins, members of which are regulated by reversible phosphorylation. The phosphorylation site on the maize ADF, ZmADF3, is Ser-6 but the kinase responsible is unknown [Smertenko et al., Plant J. 14 (1998) 187-193]. We have partially purified the ADF kinase(s) and found it to be calcium-regulated and inhibited by N-(6-aminohexyl)-[(3)H]5-chloro-1-naphthalenesulphonamide. Immunoblotting reveals that calmodulin-like domain protein kinase(s) (CDPK) are enriched in the purified preparation and addition of anti-CDPK to in vitro phosphorylation assays results in the inhibition of ADF phosphorylation. These data strongly suggest that plant ADF is phosphorylated by CDPK(s), a class of protein kinases unique to plants and protozoa.     

Structural basis for the processive protein degradation by tricorn protease

Cell survival critically depends on the efficient use of available resources. This includes both the clearance and the recycling of those protein components that have become futile or defective. Several proteins sequentially accomplish this complex task. The proteasome serves as an initial protein shredder and generates peptides of 7-12 amino acids in length. In general, these products are useless burden to the cell and need further processing. A few years ago, a proteolytic system was identified in the model organism Thermoplasma acidophilum which indeed performs this processing [Tamura et al., Science 274 (1996), 1385-1389]. The hexameric core protein of this modular system, referred to as tricorn protease, is a 720 kDa protease which is able to assemble further into a giant icosahedral capsid, as determined by electron microscopy. Recently, we determined the crystal structure of the tricorn core particle at 2.0 A resolution [Brandstetter et al., Nature 414 (2001), 466-469]. Here we describe the structural and mechanistic basis for tricorn's processive degradation mode, including a novel electrostatic substrate-to-product sink, and suggest how further components might interact with the tricorn protease to complete the cellular waste recycling process.     

Cloning and sequence determination of a cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase.

A partial cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase (ACMPK) was isolated from a lambda ZAP expression library by immunoselection using monospecific polyclonal antibodies to the enzyme. The sequence of both strands of the cDNA (CMKa) was determined. The deduced amino acid (aa) sequence contained all eleven consensus domains found in serine/threonine protein kinases [Hanks et al., Science 241 (1988) 42-52], as well as a putative calmodulin-binding domain. The cDNA contained an intron, lacked an in-frame start codon, and was not polyadenylated. A full-length copy of CMKa was subsequently isolated from a lambda gt10 library of A. nidulans cDNA using a restriction fragment of the first clone as a probe. It contained an in-frame start codon, an open reading frame (ORF) of 1242 bp and was polyadenylated. The ORF encoded a protein of 414 aa residues with an M(r) of 46,895 and an isoelectric point pI = 6.4. These values are in good agreement with that observed for the native enzyme [Bartelt et al., Proc. Natl. Acad. Sci. USA 85 (1988) 3279-3283]. When aligned to optimize homology, 29% of the predicted aa sequence of ACMPK is identical to that of the alpha-subunit of rat brain calmodulin-dependent protein kinase II. ACMPK shares 40 and 44% identity in aa sequence with YCMK1 and YCMK2, respectively, two Ca2+/calmodulin-dependent protein kinases recently cloned from Saccharomyces cerevisiae [Pausch et al., EMBO J. 10 (1991) 1511-1522]. Results of Southern analysis of restriction digests of genomic DNA indicate that ACMPK is encoded by a single-copy gene.     

Segments of bacteriophage lambda (orf 221) and phi 80 are homologous to genes coding for mammalian protein phosphatases

The amino acid sequences of mammalian protein phosphatase 1 and 2A were compared pairwise with every sequence in the National Biomedical Research Foundation protein sequence database using an exhaustive searching programme [Coulson et al., Comp. J. 30 (1987) 420-424]. The N-terminal half of the protein encoded by an open reading frame, orf 221, in bacteriophage lambda (nt 43,224-43,886 in the map of Daniels et al. [in Hendrix et al. (Eds.), Lambda II. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1983, pp. 519-676] shows 35% identity to either protein phosphatase 1 or 2A in this region. If conservative replacements are included the overall homology rises to 49%. A gene in phi 80 also shows 35% identity with the mammalian protein phosphatases. The results indicate that orf 221 of phage lambda and the homologous phi 80 gene may encode protein phosphatases. The possible roles of protein phosphorylation in the propagation of bacteriophage are discussed.     

Evidence for the extent of insertion of the active site loop of intact alpha 1 proteinase inhibitor in beta-sheet A.

The extent of insertion of beta-strand s4A into sheet A in intact serpin alpha 1-proteinase inhibitor (alpha 1PI has been probed by peptide annealing experiments [Schulze et al. (1990) Eur. J. Biochem. 194, 51-56]. Twelve synthetic peptides of systematically varied length corresponding in sequence to the unprimed (N-terminal) side of the active site loop were complexed with alpha 1PI. The complexes were then characterized by circular dichroism spectroscopy and tested for inhibitory activity. Four peptides formed complexes which retained inhibitory activity, one of which was nearly as effective as the native protein. Comparison with the three dimensional structures of cleaved alpha 1PI [L?bermann et al. (1984) J. Mol. Biol. 177, 531-556] and plakalbumin [Wright et al. (1990) J. Mol. Biol. 213, 513-528] supports a model in which alpha 1PI requires the insertion of a single residue, Thr345, into sheet A for activity.     

The epithelial Na(+) channel (ENaC) is related to the hypertonicity-induced Na(+) conductance in rat hepatocytes

The epithelial Na(+) channel (ENaC) is composed of the subunits alpha, beta, and gamma [Canessa et al., Nature 367 (1994) 463-467] and typically exhibits a high affinity to amiloride [Canessa et al., Nature 361 (1993) 467-470]. When expressed in Xenopus oocytes, conflicting results were reported concerning the osmo-sensitivity of the channel [Ji et al., Am. J. Physiol. 275 (1998) C1182-C1190; Hawayda and Subramanyam, J. Gen. Physiol. 112 (1998) 97-111; Rossier, J. Gen. Physiol. 112 (1998) 95-96]. Rat hepatocytes were the first system in which amiloride-sensitive sodium currents in response to hypertonic stress were reported [Wehner et al., J. Gen. Physiol. 105 (1995) 507-535; Wehner et al., Physiologist 40 (1997) A-4]. Moreover, all three ENaC subunits are expressed in these cells [B?hmer et al., Cell. Physiol. Biochem. 10 (2000) 187-194]. Here, we injected specific antisense oligonucleotides directed against alpha-rENaC into single rat hepatocytes in confluent primary culture and found an inhibition of hypertonicity-induced Na(+) currents by 70%. This is the first direct evidence for a role of the ENaC in cell volume regulation.