Specific Protein Families Research Articles

SKEMPI: a Structural Kinetic and Energetic database of Mutant Protein Interactions and its use in empirical models

Empirical models for the prediction of how changes in sequence alter protein-protein binding kinetics and thermodynamics can garner insights into many aspects of molecular biology. However, such models require empirical training data and proper validation before they can be widely applied. Previous databases contained few stabilizing mutations and no discussion of their inherent biases or how this impacts model construction or validation. We present SKEMPI, a database of 3047 binding free energy changes upon mutation assembled from the scientific literature, for protein-protein heterodimeric complexes with experimentally determined structures. This represents over four times more data than previously collected. Changes in 713 association and dissociation rates and 127 enthalpies and entropies were also recorded. The existence of biases towards specific mutations, residues, interfaces, proteins and protein families is discussed in the context of how the data can be used to construct predictive models. Finally, a cross-validation scheme is presented which is capable of estimating the efficacy of derived models on future data in which these biases are not present. The database is available online at http://life.bsc.es/pid/mutation_database/.

Phorbol esters enhance 1α,25-dihydroxyvitamin D3-regulated 25-hydroxyvitamin d-24-hydroxylase (CYP24A1) gene expression through ERK-mediated phosphorylation of specific protein 3 (Sp3) in Caco-2 cells

Phorbol 12-myristate 13-acetate (PMA) increased 1,25(OH)2D3-induced human 25 hydroxyvitamin d-24 hydroxylase (hCYP24A1) gene expression and vitamin D receptor (VDR) binding to the hCYP24A1 promoter. It did not alter transient receptor potential cation channel, subfamily V, member 6 (TRPV6) expression, VDR binding to the TRPV6 promoter, or VDR binding to a crude chromatin preparation. PMA activated Extracellular signal-Regulated Kinases (ERK) 1/2 and p38 mitogen activated protein kinases (MAPK) and inhibiting these kinases reduced 1,25(OH)2D3-induced and PMA-enhanced hCYP24A1 promoter activity. Mithramycin A inhibits Specific Protein (Sp) family member binding to DNA and reduced 1,25(OH)2D3-induced and PMA-enhanced hCYP24A1 promoter activity. Sp1 or Sp3 siRNA knockdown reduced 1,25(OH)2D3-regulated hCYP24A1 promoter activity but only Sp3 siRNA reduced PMA-enhanced hCYP24A1 promoter activity. PMA increased MAPK-dependent Sp3 phosphorylation, Sp3–VDR interactions, and Sp3 binding to the hCYP24A1 promoter. These data suggest that MAPK signaling contributes to 1,25(OH)2D3-induced and PMA-enhanced CYP24A1 gene transcription by modulating Sp3 function.

ESTs library from embryonic stages reveals tubulin and reflectin diversity in Sepia officinalis (Mollusca — Cephalopoda)

New molecular resources regarding the so-called “non-standard models” in biology extend the present knowledge and are essential for molecular evolution and diversity studies (especially during the development) and evolutionary inferences about these zoological groups, or more practically for their fruitful management. Sepia officinalis, an economically important cephalopod species, is emerging as a new lophotrochozoan developmental model. We developed a large set of expressed sequence tags (ESTs) from embryonic stages of S. officinalis, yielding 19,780 non-redundant sequences (NRS). Around 75% of these sequences have no homologs in existing available databases. This set is the first developmental ESTs library in cephalopods. By exploring these NRS for tubulin, a generic protein family, and reflectin, a cephalopod specific protein family, we point out for both families a striking molecular diversity in S. officinalis.

Mining Genomes of Marine Cyanobacteria for Elements of Zinc Homeostasis

Zinc is a recognized essential element for the majority of organisms, and is indispensable for the correct function of hundreds of enzymes and thousands of regulatory proteins. In aquatic photoautotrophs including cyanobacteria, zinc is thought to be required for carbonic anhydrase and alkaline phosphatase, although there is evidence that at least some carbonic anhydrases can be cambialistic, i.e., are able to acquire in vivo and function with different metal cofactors such as Co2+ and Cd2+. Given the global importance of marine phytoplankton, zinc availability in the oceans is likely to have an impact on both carbon and phosphorus cycles. Zinc concentrations in seawater vary over several orders of magnitude, and in the open oceans adopt a nutrient-like profile. Most studies on zinc handling by cyanobacteria have focused on freshwater strains and zinc toxicity; much less information is available on marine strains and zinc limitation. Several systems for zinc homeostasis have been characterized in the freshwater species Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803, but little is known about zinc requirements or zinc handling by marine species. Comparative metallo-genomics has begun to explore not only the putative zinc proteome, but also specific protein families predicted to have an involvement in zinc homeostasis, including sensors for excess and limitation (SmtB and its homologs as well as Zur), uptake systems (ZnuABC), putative intracellular zinc chaperones (COG0523) and metallothioneins (BmtA), and efflux pumps (ZiaA and its homologs).

Inhibition and Termination of Physiological Responses by GTPase Activating Proteins

Physiological processes are strictly organized in space and time. However, in cell physiology research, more attention is given to the question of space rather than to time. To function as a signal, environmental changes must be restricted in time; they need not only be initiated but also terminated. In this review, we concentrate on the role of one specific protein family involved in biological signal termination. GTPase activating proteins (GAPs) accelerate the endogenously low GTP hydrolysis rate of monomeric guanine nucleotide-binding proteins (GNBPs), limiting thereby their prevalence in the active, GTP-bound form. We discuss cases where defective or excessive GAP activity of specific proteins causes significant alteration in the function of the nervous, endocrine, and hemopoietic systems, or contributes to development of infections and tumors. Biochemical and genetic data as well as observations from human pathology support the notion that GAPs represent vital elements in the spatiotemporal fine tuning of physiological processes.

Molecular Mining of Alleles in Water Buffalo Bubalus bubalis and Characterization of the TSPY1 and COL6A1 Genes

BackgroundMinisatellites are an integral part of eukaryotic genomes and show variation in the complexity of their organization. Besides their presence in non-coding regions, a small fraction of them are part of the transcriptome, possibly participating in gene regulation, expression and silencing. We studied the minisatellite (TGG)n tagged transcriptome in the water buffalo Bubalus bubalis across various tissues and the spermatozoa, and characterized the genes TSPY1 and COL6A1 discovered in the process.ResultsMinisatellite associated sequence amplification (MASA) conducted using cDNA and oligonucleotide primer (TGG)5 uncovered 38 different mRNA transcripts from somatic tissues and gonads and 15 from spermatozoa. These mRNA transcripts corresponded to several known and novel genes. The majority of the transcripts showed the highest level of expression either in the testes or spermatozoa with exception of a few showing higher expression levels in the lungs and liver. Transcript SR1, which is expressed in all the somatic tissues and gonads, was found to be similar to the Bos taurus collagen type VI alpha 1 gene (COL6A1). Similarly, SR29, a testis-specific transcript, was found to be similar to the Bos taurus testis-specific Y-encoded protein-1 representing cancer/testis antigen 78 (CT78). Subsequently, full length coding sequences (cds) of these two transcripts were obtained. Quantitative PCR (q-PCR) revealed 182-202 copies of theTSPY1 gene in water buffalo, which localized to the Y chromosome.ConclusionsThe MASA approach enabled us to identify several genes, including two of clinical significance, without screening an entire cDNA library. Genes identified with TGG repeats are not part of a specific family of proteins and instead are distributed randomly throughout the genome. Genes showing elevated expression in the testes and spermatozoa may prove to be potential candidates for in-depth characterization. Furthermore, their possible involvement in fertility or lack thereof would augment animal biotechnology.

Expression profiling analyses of porcine MuRF1 gene and its association with muscle production traits

Muscle specific RING finger protein-1 (MuRF1) is a member of the muscle specific RING finger protein family, and it is specifically expressed in cardiac and skeletal muscle tissues and is involved in myocyte differentiation, development and morphogenesis. In this study the complete open reading frame (ORF) of the porcine MuRF1 gene consisting of 354 amino acids was obtained and it shared 93% and 90% identity with those of the human and mice, respectively. Using the INRA radiation hybrid panel (IMpRH) technique, the MuRF1 gene was assigned to SSC6q21-26, closely linked to microsatellite markers SW1823 and SW709. The tissue distribution patterns revealed that MuRF1 mRNA was exclusively expressed in cardiac and skeletal muscle tissues. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) results displayed that MuRF1 mRNA was up-regulated in Landrace pigs during the prenatal skeletal muscle development stages. A synonymous T/C single nucleotide polymorphism (SNP) was identified in MuRF1 exon 3 and then a Hin6I PCR - RFLP was developed for SNP genotyping in two pig populations. Association of the genotypes with growth and carcass traits showed that different genotypes of MuRF1 were genetically significantly associated with average daily gain from birth to 90 kg and loin muscle area in one experimental population. The study suggested that the porcine MuRF1 gene might affect muscle growth and development, and could be a potential candidate gene for muscle production traits in the pig. Keywords: Molecular cloning, expression profile, mapping, association analysis

An EST-based analysis identifies new genes and reveals distinctive gene expression features of Coffea arabica and Coffea canephora

BackgroundCoffee is one of the world's most important crops; it is consumed worldwide and plays a significant role in the economy of producing countries. Coffea arabica and C. canephora are responsible for 70 and 30% of commercial production, respectively. C. arabica is an allotetraploid from a recent hybridization of the diploid species, C. canephora and C. eugenioides. C. arabica has lower genetic diversity and results in a higher quality beverage than C. canephora. Research initiatives have been launched to produce genomic and transcriptomic data about Coffea spp. as a strategy to improve breeding efficiency.ResultsAssembling the expressed sequence tags (ESTs) of C. arabica and C. canephora produced by the Brazilian Coffee Genome Project and the Nestlé-Cornell Consortium revealed 32,007 clusters of C. arabica and 16,665 clusters of C. canephora. We detected different GC3 profiles between these species that are related to their genome structure and mating system. BLAST analysis revealed similarities between coffee and grape (Vitis vinifera) genes. Using KA/KS analysis, we identified coffee genes under purifying and positive selection. Protein domain and gene ontology analyses suggested differences between Coffea spp. data, mainly in relation to complex sugar synthases and nucleotide binding proteins. OrthoMCL was used to identify specific and prevalent coffee protein families when compared to five other plant species. Among the interesting families annotated are new cystatins, glycine-rich proteins and RALF-like peptides. Hierarchical clustering was used to independently group C. arabica and C. canephora expression clusters according to expression data extracted from EST libraries, resulting in the identification of differentially expressed genes. Based on these results, we emphasize gene annotation and discuss plant defenses, abiotic stress and cup quality-related functional categories.ConclusionWe present the first comprehensive genome-wide transcript profile study of C. arabica and C. canephora, which can be freely assessed by the scientific community at http://www.lge.ibi.unicamp.br/coffea. Our data reveal the presence of species-specific/prevalent genes in coffee that may help to explain particular characteristics of these two crops. The identification of differentially expressed transcripts offers a starting point for the correlation between gene expression profiles and Coffea spp. developmental traits, providing valuable insights for coffee breeding and biotechnology, especially concerning sugar metabolism and stress tolerance.

Phosphate import in plants: focus on the PHT1 transporters

The main source of phosphorus for plants is inorganic phosphate (Pi), which is characterized by its poor availability and low mobility. Uptake of this element from the soil relies heavily upon the PHT1 transporters, a specific family of plant plasma membrane proteins that were identified by homology with the yeast PHO84 Pi transporter. Since the discovery of PHT1 transporters in 1996, various studies have revealed that their function is controlled by a highly complex network of regulation. This review will summarize the current state of research on plant PHT1 multigenic families, including physiological, biochemical, molecular, cellular, and genetics studies.

Porcine MuRF2 and MuRF3: molecular cloning, expression and association analysis with muscle production traits

Muscle specific RING finger protein2 (MuRF2) and Muscle specific RING finger protein3(MuRF3) are two important members of the muscle specific RING finger protein family, which are especially expressed in cardiac and skeletal muscle tissues and play critical roles during the myocyte differentiation, development and morphogenesis. In this study, the molecular characteristics of porcine MuRF2 and MuRF3 gene were reported, and furthermore two variants of MuRF2 were identified. The tissue distribution pattern analyses revealed that MuRF2-b and MuRF3 mRNA was exclusively expressed in striated muscle tissues while MuRF2-a had a low-level expression in liver tissue. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) results displayed MuRF2 mRNA expression levels were significantly varied at three stages of fetal skeletal muscle in Landrace pigs, and the expression of MuRF2-a was lower than that of MuRF2-b in all stages. An essencial region of -396 to -22 for transcription was identified at the 5'UTR of porcine MuRF2 gene, while no active regulatory fragment found in the 5'UTR of mouse MuRF2. A single nucleotide polymorphism (SNP), c.915G>A was identified in MuRF2 exon 5. A HinfI PCR-RFLP was developed for SNP genotyping in two different pig populations. Association of the genotypes with growth and carcass traits showed that different genotypes of MuRF2 were significantly (P<0.05) associated with average daily gain on test, carcass weight and carcass length. The study suggested that the porcine MuRF2 and MuRF3 genes are involved in the muscle growth and development, and can be considered as potential candidate genes affecting muscle production traits in the pig.

Identification of three CCp genes in Babesia divergens: Novel markers for sexual stages parasites

Babesia divergens, a tick-borne protozoan parasite of red blood cells, is the main agent of bovine and human babesiosis in Europe. Very few data are available concerning its life cycle and sexual reproduction inside the tick vector, Ixodes ricinus. The aim of this study was to define some markers of the B.divergens sexual stage. An in silico post-genomic approach was used to analyze genomic, transcriptomic and proteomic data and to select specific sexual stage proteins of the related apicomplexan genus Plasmodium. Three proteins, based on sequence identity between the available genomes of Plasmodium and Babesia spp., were chosen, as members of a highly conserved and apicomplexan sexual stages specific protein family (CCp) potentially involved in adhesive functions. Degenerate primers were used to amplify and clone three B.divergens orthologs (bdccp1, bdccp2, and bdccp3) corresponding to newly identified genes in this parasite. The opportunities offered by such markers to study parasite development in its vector are discussed.

Expansion of the Protein Repertoire in Newly Explored Environments: Human Gut Microbiome Specific Protein Families

The microbes that inhabit particular environments must be able to perform molecular functions that provide them with a competitive advantage to thrive in those environments. As most molecular functions are performed by proteins and are conserved between related proteins, we can expect that organisms successful in a given environmental niche would contain protein families that are specific for functions that are important in that environment. For instance, the human gut is rich in polysaccharides from the diet or secreted by the host, and is dominated by Bacteroides, whose genomes contain highly expanded repertoire of protein families involved in carbohydrate metabolism. To identify other protein families that are specific to this environment, we investigated the distribution of protein families in the currently available human gut genomic and metagenomic data. Using an automated procedure, we identified a group of protein families strongly overrepresented in the human gut. These not only include many families described previously but also, interestingly, a large group of previously unrecognized protein families, which suggests that we still have much to discover about this environment. The identification and analysis of these families could provide us with new information about an environment critical to our health and well being.

Between proteins and phenotypes: annotation and interpretation of mutations

Between proteins and phenotypes: annotation and interpretation of mutations

Autophagy: molecular machinery, regulation, and implications for renal pathophysiology.

Autophagy is a cellular process of "self-eating." During autophagy, a portion of cytoplasm is enveloped in double membrane-bound structures called autophagosomes, which undergo maturation and fusion with lysosomes for degradation. At the core of the molecular machinery of autophagy is a specific family of genes or proteins called Atg. Originally identified in yeast, Atg orthologs are now being discovered in mammalian cells and have been shown to play critical roles in autophagy. Traditionally, autophagy is recognized as a cellular response to nutrient deprivation or starvation whereby cells digest cytoplasmic organelles and macromolecules to recycle nutrients for self-support. However, studies during the last few years have indicated that autophagy is a general cellular response to stress. Interestingly, depending on experimental conditions, especially stress levels, autophagy can directly induce cell death or act as a mechanism of cell survival. In this review, we discuss the molecular machinery, regulation, and function of autophagy. In addition, we analyze the recent findings of autophagy in renal systems and its possible role in renal pathophysiology.

Barley seed proteomics from spots to structures

Barley is a major cereal crop grown mainly for feed and malting. Two-dimensional gel electrophoresis has been used to analyse barley proteins for over 20 years and more recently, mass spectrometry was applied. In the absence of a genome sequence, barley gene and EST sequences combined with information from rice and other cereals facilitate identification of barley proteins. Several hundred barley seed proteins are identified and lower abundance proteins including membrane proteins are now being analysed. In the present review we focus on variation in protein profiles of seed tissues during grain filling, maturation, germination and radicle elongation. Cultivar comparisons and genetic mapping of polymorphic protein spots in doubled haploid populations provide a means to link the genome to the proteome and identify proteins that can influence grain quality. Many proteins appear in multiple forms on 2D-gels. Specific protein families, including peroxidases and alpha-amylases have been subjected to in-depth analysis resulting in characterisation of different isozymes, post-translational modifications and processing. A functional proteomics study focusing on the seed thioredoxin system has led to identification of thioredoxin target proteins, quantitative analysis of reduction of individual target disulphides and structural studies to gain insight into determinants for target protein recognition by thioredoxin.

UPF201 Archaeal Specific Family Members Reveal Structural Similarity to RNA-Binding Proteins but Low Likelihood for RNA-Binding Function

We have determined X-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (UPF0201; Pfam classification: DUF54) to advance our understanding of the genetic repertoire of archaea. Despite low pairwise amino acid sequence identities (10–40%) and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. Their common polypeptide chain fold, encompassing a five-stranded antiparallel β-sheet and five α-helices, proved to be quite unexpectedly similar to that of the RRM-type RNA-binding domain of the ribosomal L5 protein, which is responsible for binding the 5S- rRNA. Structure-based sequence alignments enabled construction of a phylogenetic tree relating UPF0201 family members to L5 ribosomal proteins and other structurally similar RNA binding proteins, thereby expanding our understanding of the evolutionary purview of the RRM superfamily. Analyses of the surfaces of these newly determined UPF0201 structures suggest that they probably do not function as RNA binding proteins, and that this domain specific family of proteins has acquired a novel function in archaebacteria, which awaits experimental elucidation.

Seeing small molecules in action with bioorthogonal chemistry

Chemical probes that target specific protein families offer powerful tools to accelerate drug discovery. Small molecules modified with uniquely reactive functional groups and detection tags provide novel tools to characterize complex proteomes functionally and also to help determine the specificity of small molecule inhibitors toward various enzyme/protein classes. This review highlights the application of bioorthogonal chemistries in combination with chemical probes, which together are offering unprecedented opportunities to dissect the functions of enzyme/protein families in vivo and enabling more precise target identification of small molecules. Advances in chemical probes and bioorthogonal reactions are poised to reveal new therapeutic targets and to facilitate the discovery and characterization of small molecules aimed at disease.

Role of Phosphatase of Regenerating Liver-3 in the Metastasis of Melanoma Cells

In plants, the actin cytoskeleton plays a major role in organelle movement, cargo transport, maintaining cell polarity and controlling the morphogenesis of endomembrane systems. All of these events require a direct connection between membrane structures and the cytoskeleton. Our knowledge in this field has been greatly advanced by a few recent discoveries including the identification of the plant specific NETWORKED family of proteins, which can mediate such linkages. Other proteins that are known to regulate actin nucleation and polymerization are also likely to be involved, but many key questions still remain unanswered. In this paper, we will focus on recent research on the interfaces between the actin cytoskeleton and membranes of the endoplasmic reticulum, the vacuole and autophagosomes.

Development of Resorufin Fluorescence Assays for monitoring the ATP‐ase Activity of Human Chaperone Hsp‐70

In response to environmental stress, there is an increase in the expression of heat shock proteins. Heat shock proteins are molecular chaperones that maintain cell homeostasis through various cellular mechanisms. Heat shock protein 70 is a specific family of heat shock proteins that uses an ATP‐driven mechanism to bind its client proteins. The overexpression of Hsp70 has been shown to prevent apoptosis and lead to tumorigenesis, but the inhibition of Hsp70 has been linked to an increase in apoptosis and tumor cell death. These anti‐apoptotic functions of Hsp‐70 prompted many groups of researchers toward development of sensitive assays for monitoring the ATP‐ase activity of this chaperone which can further be used for discovery of new potential competitive inhibitors of its enzymatic activity. This paper presents the development of resorufin fluorescence based assays for monitoring the ATP‐ase activity of Hsp‐70 by measuring the inorganic phosphate (Pi) or the adenosine diphosphate (ADP) production during the multi turn‐over enzymatic reactions. Enzyme‐coupled reactions were developed to convert the Pi or ADP in hydrogen peroxide which in turn was used to oxidize the reagent amplex red into resorufin which emits fluorescence at 590 nm upon excitation at 530 nm. The non‐hydrolysable ATP analog AMP‐PNP showed inhibition of human Hsp‐70 ATP‐ase activity in the low mM range.

ETV6 mutations and loss in AML-M0

ETV6 (ETS translocation-variant gene 6, located on chromosome 12p), also known as TEL, encodes a transcription repressor belonging to the E26 transforming specific (ETS) family of DNA-binding proteins. ETV6 is known as a proto-oncogene involved in translocation with over 40 partners.1 In acute myeloid leukemia (AML) only a few rare translocations result in transforming fusion proteins,1 indicating that the oncogenic role of ETV6 does not play a major part in AML. However, abnormalities of the short arm of chromosome 12 (12p) are found in about 5% of AML and myelodysplastic syndromes. Most abnormalities consist of total or partial loss of 12p usually affecting ETV6 and CDKN1B, implicating these genes as tumor-suppressor genes.1, 2, 3, 4 Recently, heterozygous mutations of ETV6, resulting in loss of repressor activity, were found in AML, adding to the view that ETV6 might have tumor-suppressor characteristics.5