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PubMed2003

A computational study of Shewanella oneidensis MR-1: structural prediction and functional inference of hypothetical proteins.

The genomes of many organisms have been sequenced in the last 5 years. Typically about 30% of predicted genes from a newly sequenced genome cannot be given functional assignments using sequence comparison methods. In these situations three-dimensional structural predictions combined with a suite of computational tools can suggest possible functions for these hypothetical proteins. Suggesting functions may allow better interpretation of experimental data (e.g., microarray data and mass spectroscopy data) and help experimentalists design new experiments. In this paper, we focus on three hypothetical proteins of Shewanella oneidensis MR-1 that are potentially related to iron transport/metabolism based on microarray experiments. The threading program PROSPECT was used for protein structural predictions and functional annotation, in conjunction with literature search and other computational tools. Computational tools were used to perform transmembrane domain predictions, coiled coil predictions, signal peptide predictions, sub-cellular localization predictions, motif prediction, and operon structure evaluations. Combined computational results from all tools were used to predict roles for the hypothetical proteins. This method, which uses a suite of computational tools that are freely available to academic users, can be used to annotate hypothetical proteins in general.

ATP-Binding Cassette Transporters↗

PubMed2001

Cloning and identification of MYPT3: a prenylatable myosin targetting subunit of protein phosphatase 1.

To identify novel protein phosphatase 1 (PP1)-interacting proteins, a yeast two-hybrid 3T3-L1 adipocyte cDNA library was screened with the catalytic subunit of PP1 as bait. In the present work, the isolation, identification and initial biochemical characterization of a novel PP1-interacting protein, MYPT3, which is homologous with the myosin phosphatase targetting subunit (MYPT) family, is described. MYPT3 aligns >99% with a region of mouse genomic DNA clone RP23-156P23 and localizes to chromosome 15, between markers at 44.1-46.5 cM, as demonstrated by radiation hybrid mapping. The gene consists of ten exons that encode for a 524-amino acid sequence with a predicted molecular mass of 57529 Da. The N-terminal region of MYPT3 consists of a consensus PP1-binding site and multiple ankyrin repeats. MYPT3 is distinguished from related approximately 110-130 kDa MYPT subunits by its molecular mass of 58 kDa, and a unique C-terminal region that contains several potential signalling motifs and a CaaX prenylation site. We have shown that affinity-purified glutathione S-transferase (GST)-MYPT3 is prenylated by purified recombinant farnesyltransferase in vitro. Endogenous PP1 from 3T3-L1 lysates specifically interacts with MYPT3. Additionally, purified PP1 activity was inhibited by GST-MYPT3 toward phosphorylase a, myosin light chain and myosin substrate in vitro. Overall, our findings identify a novel prenylatable subunit of PP1 that defines a new subfamily of MYPT.

3T3 Cells↗

PubMed2003

Plastid ultrastructure defines the protein import pathway in dinoflagellates.

Eukaryotic cells contain a variety of different compartments that are distinguished by their own particular function and characteristic set of proteins. Protein targeting mechanisms to organelles have an additional layer of complexity in algae, where plastids may be surrounded by three or four membranes instead of two as in higher plants. The mechanism of protein import into dinoflagellates plastids, however, has not been previously described despite the importance of plastid targeting in a group of algae responsible for roughly half the ocean's net primary production. Here, we show how nuclear-encoded proteins enter the triple membrane-bound plastids of the dinoflagellate Gonyaulax. These proteins all contain an N-terminal leader sequence with two distinct hydrophobic regions flanking a region rich in hydroxylated amino acids (S/T). We demonstrate that plastid proteins transit through the Golgi in vivo, that the first hydrophobic region in the leader acts as a typical signal peptide in vitro, and that the S/T-rich region acts as a typical plastid transit sequence in transgenic plants. We also show that the second hydrophobic region acts as a stop transfer sequence so that plastid proteins in Golgi-derived vesicles are integral membrane proteins with a predominant cytoplasmic component. The dinoflagellate mechanism is thus different from that used by the phylogenetically related apicomplexans, and instead, is similar to that of the phylogenetically distant Euglena, whose plastids are also bound by three membranes. We conclude that the protein import mechanism is dictated by plastid ultrastructure rather than by the evolutionary history of the cell.

5' Untranslated Regions↗

PubMed2004

Protection from cytosolic prion protein toxicity by modulation of protein translocation.

Failure to promptly dispose of undesirable proteins is associated with numerous diseases. In the case of cellular prion protein (PrP), inhibition of the proteasome pathway can generate a highly aggregation-prone, cytotoxic form of PrP implicated in neurodegeneration. However, the predominant mechanisms that result in delivery of PrP, ordinarily targeted to the secretory pathway, to cytosolic proteasomes have been unclear. By accurately measuring the in vivo fidelity of protein translocation into the endoplasmic reticulum (ER), we reveal a slight inefficiency in PrP signal sequence function that generates proteasomally degraded cytosolic PrP. Attenuating this source of cytosolic PrP completely eliminates the dependence on proteasomes for PrP degradation. This allows cells to tolerate both higher expression levels and decreased proteasomal capacity without succumbing to the adverse consequences of misfolded PrP. Thus, the generation of potentially toxic cytosolic PrP is controlled primarily during its initial translocation into the ER. These results suggest that a substantial proportion of the cell's constitutive proteasomal burden may consist of proteins that, like PrP, fail to cotranslationally enter the secretory pathway with high fidelity.

Animals↗

PubMed2004

Ribosome-inactivating activity and cDNA cloning of antiviral protein isoforms of Chenopodium album.

We have characterized a novel type I ribosome-inactivating protein (CAP30) from the leaves of Chenopodium album. Purified native CAP30 depurinated the ribosomes of Chenopodium, tomato, and tobacco leaves in vitro. To further characterize this protein, cDNA clones were isolated from a leaf cDNA library using a DNA probe derived from the N-terminal amino acid sequence. Two full-length cDNA clones, CAP30A and CAP30B, were isolated. The two clones were highly homologous (91.4% identity over 280 amino acids) at the deduced amino acid level. Both contain a putative signal peptide of 25 amino acid and a conserved domain commonly found in ribosome-inactivating proteins. This suggests that CAP30 is a single-chain ribosome-inactivating protein. Expression of CAP30 mRNA peaked twice, at 12 and 72 h, after tobacco mosaic virus (TMV) infection or wounding. Transformed Escherichia coli cells expressing pre- or mature CAP had greatly reduced growth rates. These results suggest that CAP30 functions as a broad-spectrum defense-related protein with both antiviral and anti-microbial activity.

Amino Acid Sequence↗

PubMed2005

Identification of the preprotein binding domain of SecA.

SecA, the preprotein translocase ATPase, has a helicase DEAD motor. To catalyze protein translocation, SecA possesses two additional flexible domains absent from other helicases. Here we demonstrate that one of these "specificity domains" is a preprotein binding domain (PBD). PBD is essential for viability and protein translocation. PBD mutations do not abrogate the basal enzymatic properties of SecA (nucleotide binding and hydrolysis), nor do they prevent SecA binding to the SecYEG protein conducting channel. However, SecA PBD mutants fail to load preproteins onto SecYEG, and their translocation ATPase activity does not become stimulated by preproteins. Bulb and Stem, the two sterically proximal PBD substructures, are physically separable and have distinct roles. Stem binds signal peptides, whereas the Bulb binds mature preprotein regions as short as 25 amino acids. Binding of signal or mature region peptides or full-length preproteins causes distinct conformational changes to PBD and to the DEAD motor. We propose that (a) PBD is a preprotein receptor and a physical bridge connecting bound preproteins to the DEAD motor, and (b) preproteins control the ATPase cycle via PBD.

Adenosine Triphosphatases↗

PubMed2004

Cell surface expression of the prion protein in yeast does not alter copper utilization phenotypes.

Prion diseases are fatal neurodegenerative disorders that result from conversion of a normal, cell surface glycoprotein (PrP(C)) into a conformationally altered isoform (PrP(Sc)) that is thought to be infectious. Although a great deal is known about the role of PrP(Sc) in the disease process, the physiological function of PrP(C) has remained enigmatic. In this report, we have used the yeast Saccharomyces cerevisiae to test one hypothesized function of PrP(C), as a receptor for the uptake or efflux of copper ions. We first modified the PrP signal peptide by replacing its hydrophobic core with the signal sequence from the yeast protein dipeptidyl aminopeptidase B, so that the resulting protein was targeted cotranslationally to the secretory pathway when synthesized in yeast. PrP molecules with the modified signal peptide were efficiently glycosylated, glycolipid-anchored, and localized to the plasma membrane. We then tested whether PrP expression altered the growth deficiency phenotypes of yeast strains harboring deletions in genes that encode key components of copper utilization pathways, including transporters, chaperones, pumps, reductases, and cuproenzymes. We found that PrP did not rescue any of these mutant phenotypes, arguing against a direct role for the protein in copper utilization. Our results provide further clarification of the physiological function of PrP(C), and lay the groundwork for using PrP-expressing yeast to study other aspects of prion biology.

Animals↗

PubMed1999

TAP binds to the constitutive transport element (CTE) through a novel RNA-binding motif that is sufficient to promote CTE-dependent RNA export from the nucleus.

The constitutive transport element (CTE) of the simian type D retroviruses overcomes nuclear retention and allows nuclear export of unspliced viral RNAs by recruiting TAP, a host factor which is thought to be required for export of cellular mRNAs. In this report, we show that the first 372 amino acid residues of TAP, comprising a stretch of leucine-rich repeats, are both necessary and sufficient for binding to the CTE RNA and promoting its export to the cytoplasm. Moreover, like the full-length protein, this domain migrates to the cytoplasm upon nuclear co-injection with the CTE RNA. Together, these results indicate that the CTE-binding domain includes the signals for nuclear export. We also describe a derivative of TAP that bears a triple amino acid substitution within the CTE-binding domain and substantially reduces the export of mRNAs from the nucleus. This provides further evidence for a role for TAP in this process. Thus, the CTE-binding domain of TAP defines a novel RNA-binding motif which has dual functions, both recognizing the CTE RNA and interacting with other components of the nuclear transport machinery.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.

We describe and validate a new membrane protein topology prediction method, TMHMM, based on a hidden Markov model. We present a detailed analysis of TMHMM's performance, and show that it correctly predicts 97-98 % of the transmembrane helices. Additionally, TMHMM can discriminate between soluble and membrane proteins with both specificity and sensitivity better than 99 %, although the accuracy drops when signal peptides are present. This high degree of accuracy allowed us to predict reliably integral membrane proteins in a large collection of genomes. Based on these predictions, we estimate that 20-30 % of all genes in most genomes encode membrane proteins, which is in agreement with previous estimates. We further discovered that proteins with N(in)-C(in) topologies are strongly preferred in all examined organisms, except Caenorhabditis elegans, where the large number of 7TM receptors increases the counts for N(out)-C(in) topologies. We discuss the possible relevance of this finding for our understanding of membrane protein assembly mechanisms. A TMHMM prediction service is available at http://www.cbs.dtu.dk/services/TMHMM/.

Animals↗

PubMed1997

A nodule-specific gene family from Alnus glutinosa encodes glycine- and histidine-rich proteins expressed in the early stages of actinorhizal nodule development.

Two cDNAs representing different members (agNt84 and ag164) of a gene family encoding glycine- and histidine-rich proteins have been isolated from cDNA libraries from Alnus glutinosa root nodules. Expression of the corresponding genes could only be detected in nodules. With in situ hybridization, the expression in nodules was found to occur in young, infected cells of the prefixation zone (zone 2). The encoded proteins contain putative signal peptides for targeting to the endomembrane system, sharing sequence similarity with signal peptides from plant glycine-rich proteins, among them nodulin 24, a nodule-specific protein from soybean. This similarity suggests that, analogous to nodulin-24, proteins encoded by agNt84/ag164 may be located at the interface between the host plant membrane and the matrix surrounding the endosymbiont. The 3' untranslated regions of the cDNAs contain unusual poly(AT)n stretches that may play a role in the regulation of RNA stability. The protein encoded by agNt84 cDNA was expressed in Escherichia coli as a fusion with maltose-binding protein, and was shown to have the ability to bind to a nickel-chelating resin, indicating that it may function as a metal-binding protein.

Actinomycetales↗

PubMed2005

A hyperthermophilic laccase from Thermus thermophilus HB27.

A copper-inducible laccase activity was detected in Thermus thermophilus HB27. The enzyme was partially purified and separated by SDS-PAGE. After staining, a gel slice containing a approximately 53-kDa protein was excised and treated with trypsin, and the in-gel digests were analyzed by mass spectrometry. By mass fingerprinting, the peptides were found to share identity with the TTC1370 protein of the thermophile, which was tentatively annotated as a laccase in the whole genome analysis, albeit experimental evidence was lacking. The assigned mass nearest to the N-terminal sequence was that from Gln23 to Lys31. By signal peptide prediction, TTC1370 protein was assumed to be a secretory protein starting from Gln23. The DNA encoding the mature protein was then cloned and expressed in Escherichia coli. The recombinant enzyme, expressed as an apoprotein, was dialyzed against copper-containing buffer to yield a holoprotein. The holoprotein was purified to homogeneity, which displayed a blue color typical of laccases and oxidized canonical laccase substrates such as guaiacol and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate). The enzyme was most notable for its striking thermophilicity; the optimal reaction temperature was approximately 92 degrees C and the half-life of thermal inactivation at 80 degrees C was >14 h, ranking it as the most thermophilic laccase reported thus far.

Amino Acid Sequence↗

PubMed2003

Purification and properties of the formate dehydrogenase and characterization of the fdhA gene of Sulfurospirillum multivorans.

The soluble periplasmic subunit of the formate dehydrogenase FdhA of the tetrachloroethene-reducing anaerobe Sulfurospirillum multivorans was purified to apparent homogeneity and the gene ( fdhA) was identified and sequenced. The purified enzyme catalyzed the oxidation of formate with oxidized methyl viologen as electron acceptor at a specific activity of 1683 nkat/mg protein. The apparent molecular mass of the native enzyme was determined by gel filtration to be about 100 kDa, which was confirmed by the fdhA nucleotide sequence. fdhA encodes for a pre-protein that differs from the truncated mature protein by an N-terminal 35-amino-acid signal peptide containing a twin arginine motif. The amino acid sequence of FdhA revealed high sequence similarities to the larger subunits of the formate dehydrogenases of Campylobacter jejuni, Wolinella succinogenes, Escherichia coli (FdhN, FdhH, FdhO), and Methanobacterium formicicum. According to the nucleotide sequence, FdhA harbors one Fe(4)/S(4) cluster and a selenocysteine residue as well as conserved amino acids thought to be involved in the binding of a molybdopterin guanidine dinucleotide cofactor.

Amino Acid Sequence↗

PubMed1996

Adhesive protein cDNA sequence of the mussel Mytilus coruscus and its evolutionary implications.

cDNA encoding the adhesive protein of the mussel Mytilus coruscus (Mcfp1) was isolated. The coding region encoded 848 amino acids (a.a.) comprising the 20-a.a. signal peptide, the 21-a.a. nonrepetitive linker, and the 805-a.a. repetitive domain. Although the first 204 nucleotides and the 3'-untranslated region of Mcfp1 cDNA were homologous to corresponding parts of M. galloprovincialis adhesive protein (Mgfp1) cDNA, the other parts diverged. The representative repeat motif of the repetitive domain, YKPK(I/P)(S/T)YPP(T/S), was similar but slightly different from the repeat motif of Mgfp1. The codon usage patterns for the same amino acids were different in different positions of the decapeptide motif. Almost identical nucleotide sequences encoding the two to 13 repeats appeared several times in the repetitive region, which suggests that the adhesive protein genes of mussels have evolved through the duplication of these repeat units.

Amino Acid Sequence↗

PubMed2003

Disruption of a receptor-mediated mechanism for intracellular sorting of proinsulin in familial hyperproinsulinemia.

In familial hyperproinsulinemia, specific mutations in the proinsulin gene are linked with a profound increase in circulating plasma proinsulin levels. However, the molecular and cellular basis for this disease remains uncharacterized. Here we investigated how these mutations may disrupt the sorting signal required to target proinsulin to the secretory granules of the regulated secretory pathway, resulting in the unregulated release of proinsulin. Using a combination of molecular modeling and site-directed mutagenesis, we have identified structural molecular motifs in proinsulin that are necessary for correct sorting into secretory granules of endocrine cells. We show that membrane carboxypeptidase E (CPE), previously identified as a prohormone-sorting receptor, is essential for proinsulin sorting. This was demonstrated through short interfering RNA-mediated depletion of CPE and transfection with a dominant negative mutant of CPE in a beta-cell line. Mutant proinsulins found in familial hyperproinsulinemia failed to bind to CPE and were not sorted efficiently. These findings provide evidence that the elevation of plasma proinsulin levels found in patients with familial hyperproinsulinemia is caused by the disruption of CPE-mediated sorting of mutant proinsulins to the regulated secretory pathway.

Carboxypeptidase H↗

PubMed1990

Identification and sequencing of the spheroidin gene of Choristoneura biennis entomopoxvirus.

Entomopoxviruses are a class of insect viruses whose virions are embedded in cytoplasmic occlusion bodies. The major component of these protective complexes is a protein called spheroidin. An open reading frame encoding the spheroidin gene of Choristoneura biennis entomopoxvirus has been identified and sequenced in our laboratory. This protein coding region is 1023 nucleotides long and specifies a polypeptide of 38,500 Da. Spheroidin was purified by SDS polyacrylamide gel electrophoresis, electroeluted, and its amino terminus sequence was determined on a gas phase sequencer. We observed that the first 20 N-terminal amino acids were absent in the mature processed form of the spheroidin molecule. Examination of these 20 residues revealed their hydrophobic nature and close resemblance to the consensus signal peptide sequence which is commonly found on membrane proteins. The DNA sequence of the spheroidin gene predicted a processed polypeptide with a molecular weight of 36 kDa. However, spheroidin was observed to aggregate in complexes composed of 50-kDa monomers. Intermolecular disulfide bonds were shown to play major roles in the formation and structure of these viral occlusion bodies. The difference in molecular weight between the predicted protein and its counterpart in infected cells is likely due to post-translational modifications. Indeed, two potential asparagine-linked glycosylation sites are present on the spheroidin molecule. The 5' flanking regions of the spheroidin gene and the vaccinia major core protein precursor gene P4b were shown to share substantial homology.

Amino Acid Sequence↗

PubMed2005

Complex protein targeting to dinoflagellate plastids.

Protein trafficking pathways to plastids are directed by N-terminal targeting peptides. In plants this consists of a relatively simple transit peptide, while in organisms with secondary plastids (which reside within the endomembrane system) a signal peptide is appended to the transit peptide. Despite amino acid compositional differences between organisms, often due to nucleotide biases, the features of plastid targeting sequences are generally consistent within species. Dinoflagellate algae deviate from this trend. We have conducted an expressed sequence tag (EST) survey of the peridinin-plastid containing dinoflagellate Heterocapsa triquetra to identify and characterize numerous targeting presequences of plastid proteins encoded in the nucleus. Consistent with targeting systems present in other secondary plastid-containing organisms, these all possess a canonical signal peptide at their N termini, however two major classes of transit peptides occur. Both classes possess a common N-terminal portion of the transit peptide, but one class of transit peptides contains a hydrophobic domain that has been reported to act as a stop-transfer membrane anchor, temporarily arresting protein insertion into the endoplasmic reticulum. A second class of transit peptide lacks this feature. These two classes are represented approximately equally, and for any given protein the class is conserved across all dinoflagellate taxa surveyed to date. This dichotomy suggests that two mechanisms, perhaps even trafficking routes, may direct proteins to dinoflagellate plastids. A four-residue phenylalanine-based motif is also a consistent feature of H. triquetra transit peptides, which is an ancient feature predating red algae and galucophytes that was lost in green plastids.

Amino Acid Sequence↗

PubMed2004

A co-translational model to explain the in vivo import of proteins into HeLa cell mitochondria.

The dual signal approach, i.e. a mitochondrial signal at the N-terminus and an ER (endoplasmic reticulum) or a peroxisomal signal at the C-terminus of EGFP (enhanced green fluorescent protein), was employed in transfected HeLa cells to test for a co-translational import model. The signal peptide from OTC (ornithine transcarbamylase) or arginase II was fused to the N-terminus of EGFP, and an ER or peroxisomal signal was fused to its C-terminus. The rationale was that if the free preprotein remained in the cytosol, it could be distributed between the two organelles by using a post-translational pathway. The resulting fusion proteins were imported exclusively into mitochondria, suggesting that co-translational import occurred. Native preALDH (precursor of rat liver mitochondrial aldehyde dehydrogenase), preOTC and rhodanese, each with the addition of a C-terminal ER or peroxisomal signal, were also translocated only to the mitochondria, again showing that a co-translational import pathway exists for these native proteins. Import of preALDH(sp)-DHFR, a fusion protein consisting of the leader sequence (signal peptide) of preALDH fused to DHFR (dihydrofolate reductase), was studied in the presence of methotrexate, a substrate analogue for DHFR. It was found that 70% of the preALDH(sp)-DHFR was imported into mitochondria in the presence of methotrexate, implying that 70% of the protein utilized the co-translational import pathway and 30% used the post-translational import pathway. Thus it appears that co-translational import is a major pathway for mitochondrial protein import. A model is proposed to explain how competition between binding factors could influence whether or not a cytosolic carrier protein, such as DHFR, uses the co- or post-translational import pathway.

Aldehyde Dehydrogenase↗

PubMed1999

Bradykinin-potentiating peptides and C-type natriuretic peptides from snake venom.

Cloning of cDNAs encoding bradykinin-potentiating peptides (BPPs)-C-type natriuretic peptide (CNP) precursor or its homologue was performed for cDNA libraries of Bothrops jararaca (South American snake), Trimeresurus flavoviridis, Trimeresurus gramineus and Agkistrodon halys blomhoffi (Asian snakes), all belonging to Crotalinae subfamily. Each cDNA library was constructed from the venom glands of a single snake to preclude ambiguity by intraspecies variation in venom components. Thirteen positive clones derived from B. jararaca were divided into two types depending on restriction sites. Differences in the nucleotide sequence arise at three locations and two of them accompanied amino acid conversions. Despite the differences, both types of cDNA clones encode the BPP-CNP precursor of 256 amino acid residues. Sequence analysis demonstrated that cDNA clones from three Asian snakes encode homologues of the BPP-CNP precursor from B. jararaca. In a precursor polypeptide, a signal sequence (approximately 25 aa) at the N-terminus is followed by sequences of BPP or the analogue (5-13 aa) with flanking spacer sequences (indefinite number of aa), an intervening linker sequence (approximately 144 aa) with unidentified function, and a CNP sequence (22 aa) with a preceding processing signal sequence (10 aa). cDNA clones from A. halys blomhoffi encode two distinct peptides in place of BPP, and T. flavoviridis and T. gramineus were shown to have considerably different sequences in the BPP domain from those known as BPP sequences. The present results provide evidence for a wide distribution of the orthologous gene expressing a series of bioactive peptides among Crotalinae subfamily.

3' Untranslated Regions↗