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Modified oligonucleotides containing 5-methylcytidine and/or 2-aminoadenosine form tighter hybrids with DNA and are, therefore, more efficient primers for DNA sequencing as compared to their natural counterparts. Strings of contiguous modified pentanucleotides can be used for DNA sequencing by primer walking.
The "J" domains of eukaryotic DnaJ-like proteins specify interaction with various Hsp70s. The conserved tripeptide, HPD, present in all J domains has been shown to be important for the interaction between yeast and bacterial DnaJ/Hsp70 protein pairs. We have characterized mutations in the HPD motif of the synaptic vesicle protein cysteine-string protein (Csp). Mutation of the histidine (H43Q) or aspartic acid (D45A) residues of this motif reduced the ability of Csp to stimulate the ATPase activity of mammalian Hsc70. The H43Q and D45A mutant proteins were not able to stimulate the ATPase activity of Hsc70 to any significant extent. The mutant proteins were characterized by competition assays, tryptic digestion analysis, and direct binding analysis from which it was seen that these proteins were defective in binding to Hsc70. Thus, the HPD motif of Csp is required for binding to Hsc70. We also analyzed the interaction between Csp and a model substrate protein, denatured firefly luciferase. Both Csp1 and the C-terminally truncated isoform Csp2 were able to prevent aggregation of heat-denatured luciferase, and they also cooperated with Hsc70 to prevent aggregation. In addition, complexes of Csp1 or Csp2 with Hsc70 and luciferase were isolated, confirming that these proteins interact and that Csps can bind directly to denatured proteins. Csp1 and Csp2 isoforms must differ in some aspect other than interaction with Hsc70 and substrate protein. These results show that both Csp1 and Csp2 can bind a partially unfolded protein and act as chaperones. This suggests that Csps may have a general chaperone function in regulated exocytosis.
Cysteine string proteins (Csps) are J-domain chaperone proteins anchored at the surface of synaptic vesicles. Csps are involved in neurotransmitter release and may modulate presynaptic calcium channel activity, although the molecular mechanisms are unknown. Interactions between Csps, proteins of the synaptic core (SNARE) complex, and P/Q-type calcium channels were therefore explored. Co-immunoprecipitation suggested that Csps occur in complexes containing synaptobrevin (VAMP), but not syntaxin 1, SNAP-25, nor P/Q-type calcium channels labeled with 125I-omega-conotoxin MVIIC. However binding experiments with 35S-labeled Csp1 demonstrated an interaction (apparent KD = 700 nM at pH 7.4 and 4 degreesC) with a fusion protein containing a segment of the cytoplasmic loop linking homologous domains II-III of the alpha1A calcium channel subunit (BI isoform, residues 780-969). Binding was specific as it was displaced by unlabeled Csp1, and no interactions were detected with fusion proteins containing other calcium channel domains, VAMP, or syntaxin 1A. A Csp binding site on the P/Q-type calcium channel is thus located within the 200 residue synaptic protein interaction site that can also bind syntaxin I, SNAP-25, and synaptotagmin I. Csp may act as a molecular chaperone to direct assembly or disassembly of exocytotic complexes at the calcium channel.
Cyclic AMP-dependent protein kinase (PKA) enhances regulated exocytosis in neurons and most other secretory cells. To explore the molecular basis of this effect, known exocytotic proteins were screened for PKA substrates. Both cysteine string protein (CSP) and soluble NSF attachment protein-alpha (alpha-SNAP) were phosphorylated by PKA in vitro, but immunoprecipitation of cellular alpha-SNAP failed to detect (32)P incorporation. In contrast, endogenous CSP was phosphorylated in synaptosomes, PC12 cells, and chromaffin cells. In-gel kinase assays confirmed PKA to be a cellular CSP kinase, with phosphorylation occurring on Ser(10). PKA phosphorylation of CSP reduced its binding to syntaxin by 10-fold but had little effect on its interaction with HSC70 or G-protein subunits. Furthermore, an in vivo role for Ser(10) phosphorylation at a late stage of exocytosis is suggested by analysis of chromaffin cells transfected with wild type or non-phosphorylatable mutant CSP. We propose that PKA phosphorylation of CSP could modulate the exocytotic machinery, by selectively altering its availability for protein-protein interactions.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel whose phosphorylation regulates both channel gating and its trafficking at the plasma membrane. Cysteine string proteins (Csps) are J-domain-containing, membrane-associated proteins that have been functionally implicated in regulated exocytosis. Therefore, we evaluated the possibility that Csp is involved in regulated CFTR trafficking. We found Csp expressed in mammalian epithelial cell lines, several of which express CFTR. In Calu-3 airway cells, immunofluorescence colocalized Csp with calnexin in the endoplasmic reticulum and with CFTR at the apical membrane domain. CFTR coprecipitated with Csp from Calu-3 cell lysates. Csp associated with both core-glycosylated immature and fully glycosylated mature CFTRs (bands B and C); however, in relation to the endogenous levels of the B and C bands expressed in Calu-3 cells, the Csp interaction with band B predominated. In vitro protein binding assays detected physical interactions of both mammalian Csp isoforms with the CFTR R-domain and the N terminus, having submicromolar affinities. In Xenopus oocytes expressing CFTR, Csp overexpression decreased the chloride current and membrane capacitance increases evoked by cAMP stimulation and decreased the levels of CFTR protein detected by immunoblot. In mammalian cells, the steady-state expression of CFTR band C was eliminated, and pulse-chase studies showed that Csp coexpression blocked the conversion of immature to mature CFTR and stabilized band B. These results demonstrate a primary role for Csp in CFTR protein maturation. The physical interaction of this Hsc70-binding protein with immature CFTR, its localization in the endoplasmic reticulum, and the decrease in production of mature CFTR observed during Csp overexpression reflect a role for Csp in CFTR biogenesis. The documented role of Csp in regulated exocytosis, its interaction with mature CFTR, and its coexpression with CFTR at the apical membrane domain of epithelial cells may reflect also a role for Csp in regulated CFTR trafficking at the plasma membrane.
Cysteine string protein (CSP), a 34-kDa molecular chaperone, is expressed on synaptic vesicles in neurons and on secretory vesicles in endocrine, neuroendocrine, and exocrine cells. CSP can be found in a complex with two other chaperones, the heat shock cognate protein Hsc70, and small glutamine-rich tetratricopeptide repeat domain protein (SGT). CSP function is vital in synaptic transmission; however, the precise nature of its role remains controversial. We have previously reported interactions of CSP with both heterotrimeric GTP-binding proteins (G proteins) and N-type calcium channels. These associations give rise to a tonic G protein inhibition of the channels. Here we have examined the effects of huntingtin fragments (exon 1) with (huntingtin(exon1/exp)) and without (huntingtin(exon1/nonexp)) expanded polyglutamine (polyQ) tracts on the CSP chaperone system. In vitro huntingtin(exon1/exp) sequestered CSP and blocked the association of CSP with G proteins. In contrast, huntingtin(exon1/nonexp) did not interact with CSP and did not alter the CSP/G protein association. Similarly, co-expression of huntingtin(exon1/exp) with CSP and N-type calcium channels eliminated CSP's tonic G protein inhibition of the channels, while coexpression of huntingtin(exon1/nonexp) did not alter the robust inhibition promoted by CSP. These results indicate that CSP's modulation of G protein inhibition of calcium channel activity is blocked in the presence of a huntingtin fragment with expanded polyglutamine tracts.
In many species, binding of sperm to the egg initiates cortical granule exocytosis, an event that contributes to a sustained block of polyspermy. Interestingly, cortical granule exocytosis can be elicited in immature Xenopus oocytes by the protein kinase C activator, phorbol-12-myristate-13-acetate. In this study, we investigated the role of cysteine string protein (csp) in phorbol-12-myristate-13-acetate-evoked cortical granule exocytosis. Prior work indicated that csp is associated with cortical granules of Xenopus oocytes. In oocytes exhibiting >20-fold overexpression of full-length Xenopus csp, cortical granule exocytosis was reduced by approximately 80%. However, csp overexpression did not affect constitutive exocytosis. Subcellular fractionation and confocal fluorescence microscopy revealed that little or none of the overexpressed csp was associated with cortical granules. This accumulation of csp at sites other than cortical granules suggested that mislocalized csp might sequester a protein that is important for regulated exocytosis. Because the NH2-terminal region of csp includes a J-domain, which interacts with constitutively expressed 70-kDa heat shock proteins (Hsc 70), we evaluated the effect of overexpressing the J-domain of csp. Although the native J-domain of csp inhibited cortical granule exocytosis, point mutations that interfere with J-domain binding to Hsc 70 eliminated this inhibition. These data indicate that csp interaction with Hsc 70 molecular chaperones is vital for regulated secretion in Xenopus oocytes.
Protein kinase B/Akt has been implicated in the insulin-dependent exocytosis of GLUT4-containing vesicles, and, more recently, insulin secretion. To determine if Akt also regulates insulin-independent exocytosis, we used adrenal chromaffin cells, a popular neuronal model. Akt1 was the predominant isoform expressed in chromaffin cells, although lower levels of Akt2 and Akt3 were also found. Secretory stimuli in both intact and permeabilized cells induced Akt phosphorylation on serine 473, and the time course of Ca2+-induced Akt phosphorylation was similar to that of exocytosis in permeabilized cells. To determine if Akt modulated exocytosis, we transfected chromaffin cells with Akt constructs and monitored catecholamine release by amperometry. Wild-type Akt had no effect on the overall number of exocytotic events, but slowed the kinetics of catecholamine release from individual vesicles, resulting in an increased quantal size. This effect was due to phosphorylation by Akt, because it was not seen in cells transfected with kinase-dead mutant Akt. As overexpression of cysteine string protein (CSP) results in a similar alteration in release kinetics and quantal size, we determined if CSP was an Akt substrate. In vitro 32P-phosphorylation studies revealed that Akt phosphorylates CSP on serine 10. Using phospho-Ser10-specific antisera, we found that both transfected and endogenous cellular CSP is phosphorylated by Akt on this residue. Taken together, these findings reveal a novel role for Akt phosphorylation in regulating the late stages of exocytosis and suggest that this is achieved via the phosphorylation of CSP on serine 10.
Sumoylation regulates the activities of several members of the ETS transcription factor family. To provide a molecular framework for understanding this regulation, we have characterized the conjugation of Ets-1 with SUMO-1. Ets-1 is modified in vivo predominantly at a consensus sumoylation motif containing Lys-15. This lysine is located within the unstructured N-terminal segment of Ets-1 preceding its PNT domain. Using NMR spectroscopy, we demonstrate that the Ets-1 sumoylation motif associates with the substrate binding site on the SUMO-conjugating enzyme UBC9 (K(d) approximately 400 microm) and that the PNT domain is not involved in this interaction. Ets-1 with Lys-15 mutated to an arginine still binds UBC9 with an affinity similar to the wild type protein, but is no longer sumoylated. NMR chemical shift and relaxation measurements reveal that the covalent attachment of mature SUMO-1, via its flexible C-terminal Gly-97, to Lys-15 of Ets-1 does not perturb the structure or dynamic properties of either protein. Therefore sumoylated Ets-1 behaves as "beads-on-a-string" with the two proteins tethered by flexible polypeptide segments containing the isopeptide linkage. Accordingly, SUMO-1 may mediate interactions of Ets-1 with signaling or transcriptional regulatory macromolecules by acting as a structurally independent docking module, rather than through the induction of a conformational change in either protein upon their covalent linkage. We also hypothesize that the flexibility of the linking polypeptide sequence may be a general feature contributing to the recognition of SUMO-modified proteins by their downstream effectors.
We examined the role of the cysteine string protein (Csp) in cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis in relation to another J-domain protein, Hdj-2, a recognized CFTR cochaperone. Increased expression of Csp produced a dose-dependent reduction in mature (band C) CFTR and an increase in immature (band B) CFTR. Exogenous expression of Hdj-2 also increased CFTR band B, but unlike Csp, Hdj-2 increased band C as well. The Csp-induced block of CFTR maturation required Hsp70, because a J-domain mutant (H43Q) that interferes with the ability of Csp to stimulate Hsp70 ATPase activity relieved the Csp-induced block of CFTR maturation. Nevertheless, Csp H43Q still increased immature CFTR. Csp-induced band B CFTR was found adjacent to the nucleus, co-localizing with calnexin, and it remained detergent-soluble. These data indicate that Csp did not block CFTR maturation by promoting the aggregation or degradation of immature CFTR. Csp knockdown by RNA interference produced a 5-fold increase in mature CFTR and augmented cAMP-stimulated CFTR currents. Thus, the production of mature CFTR is inversely related to the expression level of Csp. Both Csp and Hdj-2 associated with the CFTR R-domain in vitro, and Hdj-2 binding was displaced by Csp, suggesting common interaction sites. Combined expression of Csp and Hdj-2 mimicked the effect of Csp alone, a block of CFTR maturation. But together, Csp and Hdj-2 produced additive increases in CFTR band B, and this did not depend on their interactions with Hsp70, consistent with direct chaperone actions of these proteins. Like Hdj-2, Csp reduced the aggregation of NBD1 in vitro in the absence of Hsp70. Our data suggest that both Csp and Hdj-2 facilitate the biosynthesis of immature CFTR, acting as direct CFTR chaperones, but in addition, Csp is positioned later in the CFTR biogenesis cascade where it regulates the production of mature CFTR by limiting its exit from the endoplasmic reticulum.
The molecular systematics of vertebrates has been based entirely on alignments of primary structures of macromolecules; however, higher order features of DNA sequences not used in traditional studies also contain valuable phylogenetic information. Recent molecular data sets conflict over the phylogenetic placement of flightless birds (ratites - paleognaths), but placement of this clade critically influences interpretation of character change in birds. To help resolve this issue, we applied a new bioinformatics approach to the largest molecular data set currently available. We distilled nearly one megabase (1 million base pairs) of heterogeneous avian genomic DNA from 20 birds and an alligator into genomic signatures, defined as the complete set of frequencies of short sequence motifs (strings), thereby providing a way to directly compare higher order features of nonhomologous DNA sequences. Phylogenetic analysis and principal component analysis of the signatures strongly support the traditional hypothesis of basal ratites and monophyly of the nonratite birds (neognaths) and imply that ratite genomes are linguistically primitive within birds, despite their base compositional similarity to neognath genomes. Our analyses show further that the phylogenetic signal of genomic signatures are strongest among deep splits within vertebrates. Despite clear problems with phylogenetic analysis of genomic signatures, our study raises intriguing issues about the biological and genomic differences that fundamentally differentiate paleognaths and neognaths.
A method is presented for determining within strict bounds the probability of matching a regular expression with a match start point in a given section of a random data string. The method in general requires time and space exponential in the number of optional characters in the regular expression, but in practice was used to determine bounds for probabilities of matching all the ProSite patterns without difficulty.
The problem of reconstructing of a symbol string given the data about precedence of fixed length substrings arises in the method of nucleic acid sequencing by nested strand hybridization. We reformulate the problem in the graph-theoretical terms, describe the structure of the set of solutions, and present polynomial time algorithms that check existence and uniqueness of a solution or finiteness of the solution set, and then construct the solutions.
We present a general technique for proving NP-hardness (under randomized polynomial time reductions) of string folding problems over a finite alphabet. All previous such intractability results have required an unbounded alphabet size. These problems correspond to the protein folding problem in variants of the hydrophobic-hydrophilic (or HP) model with a fixed number of monomer types. Our proof also establishes the MAX SNP-hardness of these problems (again under randomized polynomial time reductions). This means that obtaining even an approximate solution to the protein folding problem, to within some fixed constant factor, is NP-hard. Our technique involves replacing the symbols of an unbounded alphabet by code-words over a fixed alphabet, and has two novel aspects. The first is the essential use of the approximation hardness of the source problem in the reduction, even for the proof of NP-hardness. The second is the concept of spatial codes, a variant of classical error-correcting codes in which different codewords are required to have large "distance" from one another even when they are arbitrarily embedded in three-dimensional space.
Cysteine string protein (Csp) is essential for neurotransmitter release in Drosophila. It has been suggested that Csp functions by regulating the activity of presynaptic Ca2+ channels, thus controlling exocytosis. We have examined the effect of overexpressing Csp1 in PC12 cells, a neuroendocrine cell line. PC12 cell clones overexpressing Csp1 did not show any changes in morphology, granule number or distribution, or in the levels of other key exocytotic proteins. This overexpression did not affect intracellular Ca2+ signals after depolarization, suggesting that Csp1 has no gross effect on Ca2+ channel activity in PC12 cells. In contrast, we show that Csp1 overexpression enhances the extent of exocytosis from permeabilized cells in response to Ca2+ or GTPgammaS in the absence of Ca2+. Because secretion from permeabilized cells is not influenced by Ca2+ channel activity, this represents the first demonstration that Csp has a direct role in regulated exocytosis.
MOTIVATION: A large number of new DNA sequences with virtually unknown functions are generated as the Human Genome Project progresses. Therefore, it is essential to develop computer algorithms that can predict the functionality of DNA segments according to their primary sequences, including algorithms that can predict promoters. Although several promoter-predicting algorithms are available, they have high false-positive detections and the rate of promoter detection needs to be improved further. RESULTS: In this research, PromFD, a computer program to recognize vertebrate RNA polymerase II promoters, has been developed. Both vertebrate promoters and non-promoter sequences are used in the analysis. The promoters are obtained from the Eukaryotic Promoter Database. Promoters are divided into a training set and a test set. Non-promoter sequences are obtained from the GenBank sequence databank, and are also divided into a training set and a test set. The first step is to search out, among all possible permutations, patterns of strings 5-10 bp long, that are significantly over-represented in the promoter set. The program also searches IMD (Information Matrix Database) matrices that have a significantly higher presence in the promoter set. The results of the searches are stored in the PromFD database, and the program PromFD scores input DNA sequences according to their content of the database entries. PromFD predicts promoters-their locations and the location of potential TATA boxes, if found. The program can detect 71% of promoters in the training set with a false-positive rate of under 1 in every 13,000 bp, and 47% of promoters in the test set with a false-positive rate of under 1 in every 9800 bp. PromFD uses a new approach and its false-positive identification rate is better compared with other available promoter recognition algorithms. The source code for PromFD is in the 'c+2' language.
MOTIVATION: Classification of proteins sequences into functional and structural families based on sequence homology is a central problem in computational biology. Discriminative supervised machine learning approaches provide good performance, but simplicity and computational efficiency of training and prediction are also important concerns. RESULTS: We introduce a class of string kernels, called mismatch kernels, for use with support vector machines (SVMs) in a discriminative approach to the problem of protein classification and remote homology detection. These kernels measure sequence similarity based on shared occurrences of fixed-length patterns in the data, allowing for mutations between patterns. Thus, the kernels provide a biologically well-motivated way to compare protein sequences without relying on family-based generative models such as hidden Markov models. We compute the kernels efficiently using a mismatch tree data structure, allowing us to calculate the contributions of all patterns occurring in the data in one pass while traversing the tree. When used with an SVM, the kernels enable fast prediction on test sequences. We report experiments on two benchmark SCOP datasets, where we show that the mismatch kernel used with an SVM classifier performs competitively with state-of-the-art methods for homology detection, particularly when very few training examples are available. Examination of the highest-weighted patterns learned by the SVM classifier recovers biologically important motifs in protein families and superfamilies.
MOTIVATION: Remote homology detection between protein sequences is a central problem in computational biology. Discriminative methods involving support vector machines (SVMs) are currently the most effective methods for the problem of superfamily recognition in the Structural Classification Of Proteins (SCOP) database. The performance of SVMs depends critically on the kernel function used to quantify the similarity between sequences. RESULTS: We propose new kernels for strings adapted to biological sequences, which we call local alignment kernels. These kernels measure the similarity between two sequences by summing up scores obtained from local alignments with gaps of the sequences. When tested in combination with SVM on their ability to recognize SCOP superfamilies on a benchmark dataset, the new kernels outperform state-of-the-art methods for remote homology detection. AVAILABILITY: Software and data available upon request.