The origin recognition complex protein family

The proteins of the origin recognition complex are found throughout all eukaryotes and have roles beyond that of DNA replication

Assessing the Evolution of Gene Expression Using Microarray Data

Classical studies of the evolution of gene function have predominantly focused on mutations within protein coding regions. With the advent of microarrays, however, it has become possible to evaluate the transcriptional activity of a gene as an additional characteristic of function. Recent studies have revealed an equally important role for gene regulation in the retention and evolution of duplicate genes. Here we review approaches to assessing the evolution of gene expression using microarray data, and discuss potential influences on expression divergence. Currently, there are no established standards on how best to identify and quantify instances of expression divergence. There have also been few efforts to date that incorporate suspected influences into mathematical models of expression divergence. Such developments will be crucial to a comprehensive understanding of the role gene duplications and expression evolution play in the emergence of complex traits and functional diversity. An integrative approach to gene family evolution, including both orthologous and paralogous genes, has the potential to bring strong predictive power both to the functional annotation of extant proteins and to the inference of functional characteristics of ancestral gene family members

A quantitative model of the initiation of DNA replication in Saccharomyces cerevisiae predicts the effects of system perturbations.

BackgroundEukaryotic cell proliferation involves DNA replication, a tightly regulated process mediated by a multitude of protein factors. In budding yeast, the initiation of replication is facilitated by the heterohexameric origin recognition complex (ORC). ORC binds to specific origins of replication and then serves as a scaffold for the recruitment of other factors such as Cdt1, Cdc6, the Mcm2-7 complex, Cdc45 and the Dbf4-Cdc7 kinase complex. While many of the mechanisms controlling these associations are well documented, mathematical models are needed to explore the network's dynamic behaviour. We have developed an ordinary differential equation-based model of the protein-protein interaction network describing replication initiation.ResultsThe model was validated against quantified levels of protein factors over a range of cell cycle timepoints. Using chromatin extracts from synchronized Saccharomyces cerevisiae cell cultures, we were able to monitor the in vivo fluctuations of several of the aforementioned proteins, with additional data obtained from the literature. The model behaviour conforms to perturbation trials previously reported in the literature, and accurately predicts the results of our own knockdown experiments. Furthermore, we successfully incorporated our replication initiation model into an established model of the entire yeast cell cycle, thus providing a comprehensive description of these processes.ConclusionsThis study establishes a robust model of the processes driving DNA replication initiation. The model was validated against observed cell concentrations of the driving factors, and characterizes the interactions between factors implicated in eukaryotic DNA replication. Finally, this model can serve as a guide in efforts to generate a comprehensive model of the mammalian cell cycle in order to explore cancer-related phenotypes

Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d

<p>Abstract</p> <p>Background</p> <p>Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB) for coplanar aromatic motifs similar to those found in known glycan-binding proteins.</p> <p>Results</p> <p>The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO) enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192) in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry.</p> <p>Conclusions</p> <p>Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, <it>Phytophthora</it>. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.</p

The evolution of the class A scavenger receptors

<p>Abstract</p> <p>Background</p> <p>The class A scavenger receptors are a subclass of a diverse family of proteins defined based on their ability to bind modified lipoproteins. The 5 members of this family are strikingly variable in their protein structure and function, raising the question as to whether it is appropriate to group them as a family based on their ligand binding abilities.</p> <p>Results</p> <p>To investigate these relationships, we defined the domain architecture of each of the 5 members followed by collecting and annotating class A scavenger receptor mRNA and amino acid sequences from publicly available databases. Phylogenetic analyses, sequence alignments, and permutation tests revealed a common evolutionary ancestry of these proteins, indicating that they form a protein family. We postulate that 4 distinct gene duplication events and subsequent domain fusions, internal repeats, and deletions are responsible for the diverse protein structures and functions of this family. Despite variation in domain structure, there are highly conserved regions across all 5 members, indicating the possibility that these regions may represent key conserved functional motifs.</p> <p>Conclusions</p> <p>We have shown with significant evidence that the 5 members of the class A scavenger receptors form a protein family. We have indicated that these receptors have a common origin which may provide insight into future functional work with these proteins.</p

Proteomic analysis of the response of the plant growth-promoting bacterium Pseudomonas putida UW4 to nickel stress

<p>Abstract</p> <p>Background</p> <p>Plant growth-promoting bacteria can alleviate the inhibitory effects of various heavy metals on plant growth, via decreasing levels of stress-induced ethylene. However, little has been done to detect any mechanisms specific for heavy metal resistance of this kind of bacteria. Here, we investigate the response of the wild-type plant growth-promoting bacterium <it>Pseudomonas putida </it>UW4 to nickel stress using proteomic approaches. The mutant strain <it>P. putida </it>UW4/AcdS^-, lacking a functional 1-aminocyclopropane-1-carboxylic acid deaminase gene, was also assessed for its response to nickel stress.</p> <p>Results</p> <p>Two dimensional difference in-gel electrophoresis (DIGE) was used to detect significantly up- or down- regulated proteins (<it>p </it>< 0.05, | ratio | > 1.5) in <it>P. putida </it>in response to the presence of 2 mM Ni. Out of a total number of 1,702 proteins detected on the analytical gels for <it>P. putida </it>UW4, the expression levels of 82 (4.82%) proteins increased significantly while the expression of 81 (4.76%) proteins decreased significantly. Of 1,575 proteins detected on the analytical gels for <it>P. putida </it>UW4/AcdS^-, the expression levels of 74 (4.70%) proteins increased and 51 (3.24%) proteins decreased significantly. Thirty-five proteins whose expression was altered were successfully identified by mass spectrometry and sequence comparisons with related species. Nineteen of the identified proteins were detected as differentially expressed in both wild-type and mutant expression profiles.</p> <p>Conclusion</p> <p>Functional assessment of proteins with significantly altered expression levels revealed several mechanisms thought to be involved in bacterial heavy metal detoxification, including general stress adaptation, anti-oxidative stress and heavy metal efflux proteins. This information may contribute to the development of plant growth-promoting bacteria mediated phytoremediation processes.</p

Insights into the evolutionary origins of clostridial neurotoxins from analysis of the Clostridium botulinum strain A neurotoxin gene cluster

<p>Abstract</p> <p>Background</p> <p>Clostridial neurotoxins (CNTs) are the most deadly toxins known and causal agents of botulism and tetanus neuroparalytic diseases. Despite considerable progress in understanding CNT structure and function, the evolutionary origins of CNTs remain a mystery as they are unique to <it>Clostridium </it>and possess a sequence and structural architecture distinct from other protein families. Uncovering the origins of CNTs would be a significant contribution to our understanding of how pathogens evolve and generate novel toxin families.</p> <p>Results</p> <p>The <it>C. botulinum </it>strain A genome was examined for potential homologues of CNTs. A key link was identified between the neurotoxin and the flagellin gene (CBO0798) located immediately upstream of the BoNT/A neurotoxin gene cluster. This flagellin sequence displayed the strongest sequence similarity to the neurotoxin and NTNH homologue out of all proteins encoded within <it>C. botulinum </it>strain A. The CBO0798 gene contains a unique hypervariable region, which in closely related flagellins encodes a collagenase-like domain. Remarkably, these collagenase-containing flagellins were found to possess the characteristic HEXXH zinc-protease motif responsible for the neurotoxin's endopeptidase activity. Additional links to collagenase-related sequences and functions were detected by further analysis of CNTs and surrounding genes, including sequence similarities to collagen-adhesion domains and collagenases. Furthermore, the neurotoxin's HCRn domain was found to exhibit both structural and sequence similarity to eukaryotic collagen jelly-roll domains.</p> <p>Conclusion</p> <p>Multiple lines of evidence suggest that the neurotoxin and adjacent genes evolved from an ancestral collagenase-like gene cluster, linking CNTs to another major family of clostridial proteolytic toxins. Duplication, reshuffling and assembly of neighboring genes within the BoNT/A neurotoxin gene cluster may have lead to the neurotoxin's unique architecture. This work provides new insights into the evolution of <it>C. botulinum </it>neurotoxins and the evolutionary mechanisms underlying the origins of virulent genes.</p

Differential chromatin proteomics of the MMS-induced DNA damage response in yeast

<p>Abstract</p> <p>Background</p> <p>Protein enrichment by sub-cellular fractionation was combined with differential-in-gel-electrophoresis (DIGE) to address the detection of the low abundance chromatin proteins in the budding yeast proteome. Comparisons of whole-cell extracts and chromatin fractions were used to provide a measure of the degree of chromatin association for individual proteins, which could be compared across sample treatments. The method was applied to analyze the effect of the DNA damaging agent methyl methanesulfonate (MMS) on levels of chromatin-associated proteins.</p> <p>Results</p> <p>Up-regulation of several previously characterized DNA damage checkpoint-regulated proteins, such as Rnr4, Rpa1 and Rpa2, was observed. In addition, several novel DNA damage responsive proteins were identified and assessed for genotoxic sensitivity using either DAmP (decreased abundance by mRNA perturbation) or knockout strains, including Acf2, Arp3, Bmh1, Hsp31, Lsp1, Pst2, Rnr4, Rpa1, Rpa2, Ste4, Ycp4 and Yrb1. A strain in which the expression of the Ran-GTPase binding protein Yrb1 was reduced was found to be hypersensitive to genotoxic stress.</p> <p>Conclusion</p> <p>The described method was effective at unveiling chromatin-associated proteins that are less likely to be detected in the absence of fractionation. Several novel proteins with altered chromatin abundance were identified including Yrb1, pointing to a role for this nuclear import associated protein in DNA damage response.</p