Occurrence of protein structure elements in conserved sequence regions

BACKGROUND: Conserved protein sequence regions are extremely useful for identifying and studying functionally and structurally important regions. By means of an integrated analysis of large-scale protein structure and sequence data, structural features of conserved protein sequence regions were identified. RESULTS: Helices and turns were found to be underrepresented in conserved regions, while strands were found to be overrepresented. Similar numbers of loops were found in conserved and random regions. CONCLUSION: These results can be understood in light of the structural constraints on different secondary structure elements, and their role in protein structural stabilization and topology. Strands can tolerate fewer sequence changes and nonetheless keep their specific shape and function. They thus tend to be more conserved than helices, which can keep their shape and function with more changes. Loop behavior can be explained by the presence of both constrained and freely changing loops in proteins. Our detailed statistical analysis of diverse proteins links protein evolution to the biophysics of protein thermodynamic stability and folding. The basic structural features of conserved sequence regions are also important determinants of protein structure motifs and their function

Ady „Minden-Titkai”

Age-related expression of the NPPB gene in heart left ventricle show overexpression in young women. Women under and above 60, n = 54 and n = 22, respectively. Men under and above 60, n = 103 and n = 39, respectively. (PDF 92 kb

ZIPK: A Unique Case of Murine-Specific Divergence of a Conserved Vertebrate Gene

Zipper interacting protein kinase (ZIPK, also known as death-associated protein kinase 3 [DAPK3]) is a Ser/Thr kinase that functions in programmed cell death. Since its identification eight years ago, contradictory findings regarding its intracellular localization and molecular mode of action have been reported, which may be attributed to unpredicted differences among the human and rodent orthologs. By aligning the sequences of all available ZIPK orthologs, from fish to human, we discovered that rat and mouse sequences are more diverged from the human ortholog relative to other, more distant, vertebrates. To test experimentally the outcome of this sequence divergence, we compared rat ZIPK to human ZIPK in the same cellular settings. We found that while ectopically expressed human ZIPK localized to the cytoplasm and induced membrane blebbing, rat ZIPK localized exclusively within nuclei, mainly to promyelocytic leukemia oncogenic bodies, and induced significantly lower levels of membrane blebbing. Among the unique murine (rat and mouse) sequence features, we found that a highly conserved phosphorylation site, previously shown to have an effect on the cellular localization of human ZIPK, is absent in murines but not in earlier diverging organisms. Recreating this phosphorylation site in rat ZIPK led to a significant reduction in its promyelocytic leukemia oncogenic body localization, yet did not confer full cytoplasmic localization. Additionally, we found that while rat ZIPK interacts with PAR-4 (also known as PAWR) very efficiently, human ZIPK fails to do so. This interaction has clear functional implications, as coexpression of PAR-4 with rat ZIPK caused nuclear to cytoplasm translocation and induced strong membrane blebbing, thus providing the murine protein a possible adaptive mechanism to compensate for its sequence divergence. We have also cloned zebrafish ZIPK and found that, like the human and unlike the murine orthologs, it localizes to the cytoplasm, and fails to bind the highly conserved PAR-4 protein. This further supports the hypothesis that murine ZIPK underwent specific divergence from a conserved consensus. In conclusion, we present a case of species-specific divergence occurring in a specific branch of the evolutionary tree, accompanied by the acquisition of a unique protein–protein interaction that enables conservation of cellular function

One-Block CYRCA: an automated procedure for identifying multiple-block alignments from single block queries

One-Block CYRCA is an automated procedure for identifying multiple-block alignments from single block queries (). It is based on the LAMA and CYRCA block-to-block alignment methods. The procedure identifies whether the query blocks can form new multiple-block alignments (block sets) with blocks from a database or join pre-existing database block sets. Using pre-computed LAMA block alignments and CYRCA sets from the Blocks database reduces the computation time. LAMA and CYRCA are highly sensitive and selective methods that can augment many other sequence analysis approaches

Refining intra-protein contact prediction by graph analysis

<p>Abstract</p> <p>Background</p> <p>Accurate prediction of intra-protein residue contacts from sequence information will allow the prediction of protein structures. Basic predictions of such specific contacts can be further refined by jointly analyzing predicted contacts, and by adding information on the relative positions of contacts in the protein primary sequence.</p> <p>Results</p> <p>We introduce a method for graph analysis refinement of intra-protein contacts, termed GARP. Our previously presented intra-contact prediction method by means of pair-to-pair substitution matrix (P2PConPred) was used to test the GARP method. In our approach, the top contact predictions obtained by a basic prediction method were used as edges to create a weighted graph. The edges were scored by a mutual clustering coefficient that identifies highly connected graph regions, and by the density of edges between the sequence regions of the edge nodes. A test set of 57 proteins with known structures was used to determine contacts. GARP improves the accuracy of the P2PConPred basic prediction method in whole proteins from 12% to 18%.</p> <p>Conclusion</p> <p>Using a simple approach we increased the contact prediction accuracy of a basic method by 1.5 times. Our graph approach is simple to implement, can be used with various basic prediction methods, and can provide input for further downstream analyses.</p

Nuclear Import of Ho Endonuclease Utilizes Two Nuclear Localization Signals and Four Importins of the Ribosomal Import System *

Activity of Ho, the yeast mating switch endonuclease, is restricted to a narrow time window of the cell cycle. Ho is unstable and despite being a nuclear protein is exported to the cytoplasm for proteasomal degradation. We report here the molecular basis for the highly efficient nuclear import of Ho and the relation between its short half-life and passage through the nucleus. The Ho nuclear import machinery is functionally redundant, being based on two bipartite nuclear localization signals, recognized by four importins of the ribosomal import system. Ho degradation is regulated by the DNA damage response and Ho retained in the cytoplasm is stabilized, implying that Ho acquires its crucial degradation signals in the nucleus. Ho arose by domestication of a fungal VMA1 intein. A comparison of the primary sequences of Ho and fungal VMA1 inteins shows that the Ho nuclear localization signals are highly conserved in all Ho proteins, but are absent from VMA1 inteins. Thus adoption of a highly efficient import strategy occurred very early in the evolution of Ho. This may have been a crucial factor in establishment of homothallism in yeast, and a key event in the rise of the Saccharomyces sensu stricto. Ho endonuclease initiates a mating type switch in Saccharomyces cerevisiae and related yeasts by making a site-specific double strand break in a 24-bp cognate site in the mating type gene, MAT. Repair of the double strand break is by gene conversion using one of the silent cassettes of mating type information (HML␣ or HMRa) as a template. Repair occurs before replication of the MAT locus and each daughter cell has the new mating type with a regenerated Ho cognate site (1). Ho activity is tightly regulated: HO is transcribed briefly at the end of G 1 , its transcription is restricted to haploid mother cells, i.e. cells that have divided at least once (2), and the protein is rapidly degraded by the ubiquitin-26 S proteasome system (3). Cells in which Ho is retained in the nucleus beyond its normal time window of activity show perturbation of the cell cycle (4). Ho is marked for degradation by functions of the DNA damage response (DDR), 7 specifically the MEC1, RAD9, and CHK1 pathway (5). Despite being a nuclear protein, Ho must exit the nucleus to be degraded in the proteasomes. The DDR functions are important for Ho phosphorylation: phosphorylation of threonine 225 is crucial for Ho nuclear export and additional phosphorylations are required for recruitment of Ho for ubiquitylation. Ho is ubiquitylated by the SCF (Skp1-Cdc53-F-box protein) E3 ubiquitin ligase complex, to which it is recruited by the F-box protein Ufo1 (6). In mec1 mutants Ho is stabilized and accumulates in the nucleus; conversely trapping Ho in the nucleus by deletion of its nuclear exportin, Msn5, leads to stabilization of the protein (4). Ddi1 binds ubiquitylated Ho and is required for interaction of Ho with the proteasome; in its absence Ho is stabilized. The finding that Ho is not degraded within the nucleus, but in the cytoplasm, is further strengthened by the direct demonstration of accumulation of ubiquitylated Ho in the cytoplasm of ⌬ddi1 mutants (7). Ho nuclear import is very rapid and efficient. Ectopic expression of HO leads to rapid cleavage of MAT (8), and to a mating type switch at any phase of the cell cycle in both mother and daughter cells. This indicates that there is no impediment to its nuclear import (9). Macromolecules are conveyed through nuclear pore complexes in the nuclear envelope by soluble karyopherins. Karyopherins comprise two structurally related families, ␣-and ␤-karyopherins. These recognize specific nuclear localization sequence (NLS) peptide motifs in the cargo molecule: NLSs may comprise a short stretch of basic residues (classical/ cNLS), or two basic clusters 10 -12 residues apart (bipartite NLS) (10). Cargoes may be recognized by an adaptor protein, ␣-karyopherin/Srp1, which mediates their binding to the transport receptor, ␤-karyopherin/ Kap95 (11). Additionally, a family of about 14 ␤-karyopherins bind an array of cargoes directly and also makes contacts with the nucleoporin subunits of the nuclear pore complexes. Directionality of transport is determined by interaction with the GTPase Ran/yeast Gsp1. RanGTP is at a high concentration in the nucleus due to the asymmetric distribution of the Ran regulators. The nuclear guanine nucleotide exchange factor, RanGEF/yeast Prp20, converts RanGDP to RanGTP, whereas the GTPase activating protein, RanGAP/yeast Rna1, is localized in the cytoplasm and catalyzes the hydrolysis of RanGTP. Importin-cargo complexes assemble in the cytoplasm and after translocation into the nucleus they dissociate upon binding of RanGTP to the importin (12). To investigate how the efficient nuclear import that supports the unique biological function of Ho is achieved we located and analyzed its nuclea

Activity, specificity and structure of I-Bth0305I: a representative of a new homing endonuclease family

Novel family of putative homing endonuclease genes was recently discovered during analyses of metagenomic and genomic sequence data. One such protein is encoded within a group I intron that resides in the recA gene of the Bacillus thuringiensis 0305ϕ8–36 bacteriophage. Named I-Bth0305I, the endonuclease cleaves a DNA target in the uninterrupted recA gene at a position immediately adjacent to the intron insertion site. The enzyme displays a multidomain, homodimeric architecture and footprints a DNA region of ∼60 bp. Its highest specificity corresponds to a 14-bp pseudopalindromic sequence that is directly centered across the DNA cleavage site. Unlike many homing endonucleases, the specificity profile of the enzyme is evenly distributed across much of its target site, such that few single base pair substitutions cause a significant decrease in cleavage activity. A crystal structure of its C-terminal domain confirms a nuclease fold that is homologous to very short patch repair (Vsr) endonucleases. The domain architecture and DNA recognition profile displayed by I-Bth0305I, which is the prototype of a homing lineage that we term the ‘EDxHD’ family, are distinct from previously characterized homing endonucleases

USH3A transcripts encode clarin-1, a four-transmembrane-domain protein with a possible role in sensory synapses

[EN] Usher syndrome type 3 (USH3) is an autosomal recessive disorder characterised by the association of post-lingual progressive hearing loss, progressive visual loss due to retinitis pigmentosa and variable presence of vestibular dysfunction. Because the previously defined transcripts do not account for all USH3 cases, we performed further analysis and revealed the presence of additional exons embedded in longer human and mouse USH3A transcripts and three novel USH3A mutations. Expression of Ush3a transcripts was localised by whole mount in situ hybridisation to cochlear hair cells and spiral ganglion cells. The full length USH3A transcript encodes clarin-1, a four-transmembrane-domain protein, which defines a novel vertebrate-specific family of three paralogues. Limited sequence homology to stargazin, a cerebellar synapse four-transmembrane-domain protein, suggests a role for clarin-1 in hair cell and photoreceptor cell synapses, as well as a common pathophysiological pathway for different Usher syndromes.We are grateful to all patients and their family members who participated in this study. We would also like to thank Ronna Hertzano for the preparation of the mouse inner ear cDNA. This work was funded by an Infrastructure grant of the Israeli Ministry of Science Culture and Sports, the Crown Human Genome Center at The Weizmann Institute of Science, the Alfried Krupp Foundation and by the Finnish Eye and Tissue Bank Foundation, the Finnish Eye Foundation, the Maud Kuistila Memorial Foundation, the Oskar Oflund Foundation, Finnish State grant TYH9235, the European Commission (QLG2-CT-1999-00988) (KB Araham) and by the Foundation Fighting Blindness. JS Beckman holds the, Hermann Mayer professorial chair and D Lancet holds the Ralf and Lois Silver professorial chair.Adato, A.; Vreugde, S.; Joensuu, T.; Avidan, N.; Hamalainen, R.; Belenkiy, O.; Olender, T.... (2002). USH3A transcripts encode clarin-1, a four-transmembrane-domain protein with a possible role in sensory synapses. European Journal of Human Genetics. 10(6):339-350. https://doi.org/10.1038/sj.ejhg.520083133935010

New Modularity of DAP-Kinases: Alternative Splicing of the DRP-1 Gene Produces a ZIPk-Like Isoform

DRP-1 and ZIPk are two members of the Death Associated Protein Ser/Thr Kinase (DAP-kinase) family, which function in different settings of cell death including autophagy. DAP kinases are very similar in their catalytic domains but differ substantially in their extra-catalytic domains. This difference is crucial for the significantly different modes of regulation and function among DAP kinases. Here we report the identification of a novel alternatively spliced kinase isoform of the DRP-1 gene, termed DRP-1β. The alternative splicing event replaces the whole extra catalytic domain of DRP-1 with a single coding exon that is closely related to the sequence of the extra catalytic domain of ZIPk. As a consequence, DRP-1β lacks the calmodulin regulatory domain of DRP-1, and instead contains a leucine zipper-like motif similar to the protein binding region of ZIPk. Several functional assays proved that this new isoform retained the biochemical and cellular properties that are common to DRP-1 and ZIPk, including myosin light chain phosphorylation, and activation of membrane blebbing and autophagy. In addition, DRP-1β also acquired binding to the ATF4 transcription factor, a feature characteristic of ZIPk but not DRP-1. Thus, a splicing event of the DRP-1 produces a ZIPk like isoform. DRP-1β is highly conserved in evolution, present in all known vertebrate DRP-1 loci. We detected the corresponding mRNA and protein in embryonic mouse brains and in human embryonic stem cells thus confirming the in vivo utilization of this isoform. The discovery of module conservation within the DAPk family members illustrates a parsimonious way to increase the functional complexity within protein families. It also provides crucial data for modeling the expansion and evolution of DAP kinase proteins within vertebrates, suggesting that DRP-1 and ZIPk most likely evolved from their ancient ancestor gene DAPk by two gene duplication events that occurred close to the emergence of vertebrates