Svein-Ole Mikalsen

Connexins, gap junctional intercellular communication and kinases

Summry— A number of kinases and signal transduction pathways are known to affect gap junctional intercellular communication and/or phosphorylation of connexins. Most of the information is available for protein kinase A, protein kinase C, mitogen‐activated protein kinase, and the tyrosine kinase Src. Much less is known for protein kinase G, Ca2+‐calmodulin dependent protein kinase, and casein kinase. However, the present lack of knowledge is not necessarily synonymous with lack of importance in the regulation of intercellular communication and phosphorylation of connexins. Kinases and the phosphorylation of connexins may be involved in the regulation of gap junctional intercellular communication at all levels ranging from the expression of connexin genes to the degradation of the gap junction channels. The exact role of the phosphorylation depends both on the kinase and the connexin involved, as well as the cellular context.

Computational methods for mass spectrometry proteomics

Computational methods for mass spectrometry proteomics , Computational methods for mass spectrometry proteomics , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

Evolutionary selection pressure and family relationships among connexin genes.

Abstract We suggest an extension of connexin orthology relationships across the major vertebrate lineages. We first show that the conserved domains of mammalian connexins (encoding the N-terminus, four transmembrane domains and two extracellular loops) are subjected to a considerably more strict selection pressure than the full-length sequences or the variable domains (the intracellular loop and C-terminal tail). Therefore, the conserved domains are more useful for the study of family relationships over larger evolutionary distances. The conserved domains of connexins were collected from chicken, Xenopus tropicalis, zebrafish, pufferfish, green spotted pufferfish, Ciona intestinalis and Halocynthia pyriformis (two tunicates). A total of 305 connexin sequences were included in this analysis. Phylogenetic trees were constructed, from which the orthologies and the presumed evolutionary relationships between the sequences were deduced. The tunicate connexins studied had the closest, but still distant, relationships to vertebrate connexin36, 39.2, 43.4, 45 and 47. The main structure in the connexin family known from mammals pre-dates the divergence of bony fishes, but some additional losses and gains of connexin sequences have occurred in the evolutionary lineages of subsequent vertebrates. Thus, the connexin gene family probably originated in the early evolution of chordates, and underwent major restructuring with regard to gene and subfamily structures (including the number of genes in each subfamily) during early vertebrate evolution.

The connexin gene family in mammals

Abstract Unannotated mammalian genome databases (dog, cow, opossum) were searched for candidate connexin genes, using sequences from annotated genomes (man, mouse). All 18 ‘multi-species’connexin genes, i.e., orthologs of connexin26, 29/31.3 (duplicated in opossum), 30, 30.2/31.9, 30.3, 31, 31.1, 32, 36, 37, 39/40.1, 40, 43, 45, 44/46, 47, 50, and 57/62, were found in dog, cow and opossum. Connexin25 and 58 have been considered specific for man, but evident orthologs of connexin25 were found in dog, cow and opossum, and orthologs of connexin58 were found in dog and cow. Moreover, a connexin43-like sequence (approx. 80% identical to connexin43) was found in man, chimpanzee, dog and cow. In the three former species, the sequences were located on the X chromosome. In man, chimpanzee and cow, there were stop codons in all reading frames; these sequences are therefore judged as pseudogenes, called here Cx43pX. In the dog, the sequence contained an open reading frame for a protein of 35.7 kDa (connexin35.7). We suggest that these sequences are orthologs of connexin33, previously considered as a rodent-specific connexin gene. Thus, connexin25, 33 and 58 are not species-specific genes. However, the opossum may possess a candidate, connexin39.2, without obvious orthologs in other mammals. Furthermore, pseudogenes of primate connexin31.3 and opossum connexin35 (one of the two orthologs of primate connexin31.3) were detected. These results suggest that the structure of the mammalian connexin gene family should be revised, especially with regard to the so-called ‘species-specific’connexins.

Connexin43 is overexpressed in ApcMin/+-mice adenomas and colocalises with COX-2 in myofibroblasts

The expression of gap junction proteins, connexins, in the intestine and their role in tumorigenesis are poorly characterised. Truncating mutations in the tumour suppressor gene adenomatous polyposis coli (APC) are early and important events, both in inheritable (familial adenomatous polyposis, FAP) and spontaneous forms of intestinal cancer. Multiple intestinal neoplasia (Min) mice, a FAP model with inherited heterozygous mutation in Apc, spontaneously develop numerous intestinal adenomas. We recently reported reduced expression of connexin32 in Paneth cells of Min‐mice. We further examine the expression of connexin43 (Cx43) and other connexins as a function of heterozygous and homozygous Apc mutation in normal intestinal tissues and adenomas of Min‐mice. Qualitative analysis of connexin mRNA in intestine revealed a similar expression pattern in Min‐ and wild‐type (wt) mice. Connexin26 and connexin40 proteins were found in equal amounts in Min and wt epithelia of large and small intestine, respectively. Interestingly, the connexin43 level was increased in the stroma of Min‐mice adenomas, in close proximity to epithelial cells with nuclear β‐catenin staining. Cx43 and COX‐2 were located to the same areas of the adenomas, and immunostaining exhibited coexpression in the myofibroblasts. Prostaglandin E2 induces Cx43 expression and COX‐2 is the rate‐limiting enzyme in the prostaglandin synthesis. However, the COX‐2‐specific inhibitor, celecoxib, did not reduce Cx43 expression. Although both Cx43 and COX‐2 are target genes for β‐catenin, they were overexpressed in stromal cells but not in epithelial tumour cells. We hypothesise that gap junctions may be of importance in the transfer of signals between epithelium and stroma.

MassSorter: a tool for administrating and analyzing data from mass spectrometry experiments on proteins with known amino acid sequences

BackgroundProteomics is the study of the proteome, and is critical to the understanding of cellular processes. Two central and related tasks of proteomics are protein identification and protein characterization. Many small laboratories are interested in the characterization of a small number of proteins, e.g., how posttranslational modifications change under different conditions.ResultsWe have developed a software tool called MassSorter for administrating and analyzing data from peptide mass fingerprinting experiments on proteins with known amino acid sequences. It is meant for small scale mass spectrometry laboratories that are interested in posttranslational modifications of known proteins. Several experiments can be compared simultaneously, and the matched and unmatched peak values are clearly indicated. The hits can be sorted according to m/z values (default) or according to the sequence of the protein. Filters defined by the user can mark autolytic protease peaks and other contaminating peaks (keratins, proteins co-migrating with the protein of interest, etc.). Unmatched peaks can be further analyzed for unexpected modifications by searches against a local version of the UniMod database. They can also be analyzed for unexpected cleavages, a highly useful feature for proteins that undergo maturation by proteolytic cleavage, creating new N- or C-terminals. Additional tools exist for visualization of the results, like sequence coverage, accuracy plots, different types of statistics, 3D models, etc. The program and a tutorial are freely available for academic users at http://www.bioinfo.no/software/massSorter.ConclusionMassSorter has a number of useful features that can promote the analysis and administration of MS-data.

Protease-dependent fractional mass and peptide properties.

Mass spectrometric analyses of peptides mainly rely on cleavage of proteins with proteases that have a defined specificity. The specificities of the proteases imply that there is not a random distribution of amino acids in the peptides. The physico–chemical effects of this distribution have been partly analyzed for tryptic peptides, but to a lesser degree for other proteases. Using all human proteins in Swiss-Prot, the relationships between peptide fractional mass, pI and hydrophobicity were investigated. The distribution of the fractional masses and the average regression lines for the fractional masses were similar, but not identical, for the peptides generated by the proteases trypsin, chymotrypsin and gluC, with the steepest regression line for gluC. The fractional mass regression lines for individual proteins showed up to ±100 ppm in mass difference from the average regression line and the peptides generated showed protease-dependent properties. We here show that the fractional mass and some other properties of the peptides are dependent on the protease used for generating the peptides. With the increasing accuracy of mass spectrometry instruments, it is possible to exploit the information embedded in the fractional mass of unknown peaks in peptide mass fingerprint spectra.

Blind search for post-translational modifications and amino acid substitutions using peptide mass fingerprints from two proteases

BackgroundMass spectrometric analysis of peptides is an essential part of protein identification and characterization, the latter meaning the identification of modifications and amino acid substitutions. There are two main approaches for characterization: (i) using a predefined set of possible modifications and substitutions or (ii) performing a blind search. The first option is straightforward, but can not detect modifications or substitutions outside the predefined set. A blind search does not have this limitation, and therefore has the potential of detecting both known and unknown modifications and substitutions. Combining the peptide mass fingerprints from two proteases result in overlapping sequence coverage of the protein, thereby offering alternative views of the protein and a novel way of indicating post-translational modifications and amino acid substitutions.ResultsWe have developed an algorithm and a software tool, MassShiftFinder, that performs a blind search using peptide mass fingerprints from two proteases with different cleavage specificities. The algorithm is based on equal mass shifts for overlapping peptides from the two proteases used, and can indicate both post-translational modifications and amino acid substitutions. In most cases it is possible to suggest a restricted area within the overlapping peptides where the mass shift can occur. The program is available at http://www.bioinfo.no/software/massShiftFinder.ConclusionWithout any prior assumptions on their presence the described algorithm is able to indicate post-translational modifications or amino acid substitutions in MALDI-TOF experiments on identified proteins, and can thereby direct the involved peptides to subsequent TOF-TOF analysis. The algorithm is designed for detailed and low-throughput characterization of single proteins.

MassSorter: peptide mass fingerprinting data analysis.

MassSorter is a software tool that sorts, systemizes, and analyzes data from peptide mass fingerprinting (PMF) experiments on proteins with known amino acid sequences. Several experiments can be simultaneously analyzed for sequence coverage and posttranslational modifications occurring during sample handling, induced chemical modifications, and unexpected cleavages. Experimental m/z values are compared with m/z values from an in silico digestion, taking modifications into account. Filters can be defined by users for marking autolytic protease peaks and other contaminating peaks. MassSorter functions as a database of all the detected peptides. It includes tools for visualization of the results, such as sequence coverage, accuracy plots, statistics, and 3D models.

Supplementary material to the paper “Evolutionary selection pressure and family relationships among connexin genes”

We suggest an extension of connexin orthology relationships across the major vertebrate lineages. We first show that the conserved domains of mammalian connexins (encoding the N-terminus, four transmembrane domains and two extracellular loops) are subjected to a considerably more strict selection pressure than the full-length sequences or the variable domains (the intracellular loop and C-terminal tail). Therefore, the conserved domains are more useful for the study of family relationships over larger evolutionary distances. The conserved domains of connexins were collected from chicken, Xenopus tropicalis, zebrafish, pufferfish, green spotted pufferfish, Ciona intestinalis and Halocynthia pyriformis (two tunicates). A total of 305 connexin sequences were included in this analysis. Phylogenetic trees were constructed, from which the orthologies and the presumed evolutionary relationships between the sequences were deduced. The tunicate connexins studied had the closest, but still distant, relationships to vertebrate connexin 36, 39.2, 43.4, 45 and 47. The main structure in the connexin family known from mammals pre-dates the divergence of bony fishes, but some additional losses and gains of connexin sequences have occurred in the evolutionary lineages of subsequent vertebrates. Thus, the connexin gene family probably originated in the early evolution of chordates, and underwent major restructuring with regard to gene and subfamily structures (including the number of genes in each subfamily) during early vertebrate evolution.