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At least 307 records · Page 17Linked to original sources
PubMed2005

Evolutionary conservation of minor U12-type spliceosome between plants and humans.

Splicing of rare, U12-type or AT-AC introns is mediated by a distinct spliceosome that assembles from U11, U12, U4atac, U6atac, and U5 snRNPs. Although in human cells the protein composition of minor and major snRNPs is similar, differences, particularly in U11 and U12 snRNPs, have been recently described. We have identified an Arabidopsis U11 snRNP-specific 35K protein as an interacting partner of an RS-domain-containing cyclophilin. By using a transient expression system in Arabidopsis protoplasts, we show that the 35K protein incorporates into snRNP. Oligo affinity selection and glycerol gradient centrifugation revealed that the Arabidopsis 35K protein is present in monomeric U11 snRNP and in U11/U12-di snRNP. The interaction of the 35K protein with Arabidopsis SR proteins together with its strong sequence similarity to U1-70K suggests that its function in splicing of minor introns is analogous to that of U1-70K. Analysis of Arabidopsis and Oryza sativa genome sequences revealed that all U11/U12-di-snRNP-specific proteins are conserved in dicot and monocot plants. In addition, we have identified an Arabidopsis gene encoding the homolog of U4atac snRNA and a second Arabidopsis gene encoding U6atac snRNA. Secondary structure predictions indicate that the Arabidopsis U4atac is able to form dimeric complexes with both Arabidopsis U6atac snRNAs. As revealed by RNaseA/T1 protection assay, the U4atac snRNA gene is expressed as an ~160-nt RNA, whereas the second U6atac snRNA gene seems to be a pseudogene. Taken together, our data indicate that recognition and splicing of minor, AT-AC introns in plants is highly similar to that in humans.

Amino Acid Motifs↗
PubMed1999

Evidence for a novel cardiac-enriched retinoid X receptor partner.

Recent studies indicate that retinoid-mediated pathways play a pivotal role in cardiac morphogenesis and function. To identify proteins that serve as interacting partners of the retinoid X receptor alpha (RXRalpha) in heart, DNA-protein binding studies were performed with an RXR-responsive element (NRRE-1) derived from the medium chain acyl-CoA dehydrogenase gene promoter and nuclear protein extracts prepared from adult rat heart. NRRE-1 is a pleiotropic RXR-responsive element comprised of three potential recognition sites for class II members of the nuclear receptor superfamily. Gel mobility shift assays performed with an NRRE-1 probe in the absence or presence of bacterially overproduced RXRalpha and nuclear protein extracts prepared from adult rat heart, liver, or brain identified a cardiac-specific, RXR-dependent DNA-protein interaction. The NRRE-1-RXR.cardiac-enriched RXR-interacting protein (CERIP) complex exhibited a distinct mobility compared with NRRE-1-RXR.peroxisome proliferator-activated receptor, NRRE-1-RXR.retinoic acid receptor, or NRRE-1-RXR.thyroid receptor complexes. Mutational analysis demonstrated that two of the three potential binding half-sites of NRRE-1 (an everted repeat separated by an 8-base pair spacer) are required for the NRRE-1-RXR. CERIP interaction. Gel mobility shift assays demonstrated that CERIP interacted with RXRalpha and RXRgamma but not with RXRbeta, indicating a receptor subtypespecific binding preference and suggesting an RXR AB region-dependent interaction. The RXR.CERIP complex did not form on NRRE-1 when a mutant GST-RXRalpha fusion protein lacking the NH(2)-terminal AB region (but containing the receptor dimerization domain) of RXRalpha was added in place of the full-length RXRalpha, confirming a role for the AB region in the RXR. CERIP interaction. DNA-protein cross-linking studies demonstrated that CERIP is a DNA-binding protein of approximately 110 kDa. These results provide evidence for the existence of a cardiac-enriched DNA-binding protein that interacts with RXRalpha via the AB region and suggest a mechanism whereby cardiac retinoid signaling is controlled in an RXR subtype-specific manner.

Acyl-CoA Dehydrogenase↗
PubMed2004

In vivo increase of solubility of overexpressed Hha protein by tandem expression with interacting protein H-NS.

Gene cloning in appropriate vectors followed by protein overexpression in Escherichia coli is the common means for protein purification. This approach has many advantages but also some drawbacks; one of these is that many proteins fail to achieve a soluble conformation when overexpressed in E. coli. Hha protein belongs to a family of nucleoid-associated proteins functionally related to the H-NS family of proteins. Hha-like proteins and H-NS-like proteins are able to semidirectly bind to each other. We show in this work that overexpressed Hha or HisHha protein (a functional derivative of Hha containing a 6x His tag at the amino end) from a T7-polymerase promoter in BL21 DE3 E. coli strains results in the vast majority of the protein accumulated in insoluble aggregates (inclusion bodies). We also show that tandem overexpression of HisHha and H-NS increases the solubility of HisHha and prevents the formation of inclusion bodies. Single amino acid substitutions in the HisHha protein, which impair interaction with H-NS, render insoluble protein even when tandem-expressed with H-NS, tandem expression of an insoluble protein and an interacting partner is an experimental strategy which could be useful to increase the solubility of other overexpressed proteins in E. coli.

Bacterial Proteins↗
PubMed1997

Evolutionary conservation and predicted structure of the Drosophila extra sex combs repressor protein.

The Drosophila extra sex combs (esc) protein, a member of the Polycomb group (PcG), is a transcriptional repressor of homeotic genes. Genetic studies have shown that esc protein is required in early embryos at about the time that other PcG proteins become engaged in homeotic gene repression. The esc protein consists primarily of multiple copies of the WD repeat, a motif that has been implicated in protein-protein interaction. To further investigate the domain organization of esc protein, we have isolated and characterized esc homologs from divergent insect species. We report that esc protein is highly conserved in housefly (72% identical to Drosophila esc), butterfly (55% identical), and grasshopper (56% identical). We show that the butterfly homolog provides esc function in Drosophila, indicating that the sequence similarities reflect functional conservation. Homology modeling using the crystal structure of another WD repeat protein, the G-protein beta-subunit, predicts that esc protein adopts a beta-propeller structure. The sequence comparisons and modeling suggest that there are seven WD repeats in esc protein which together form a seven-bladed beta-propeller. We locate the conserved regions in esc protein with respect to this predicted structure. Site-directed mutagenesis of specific loops, predicted to extend from the propeller surface, identifies conserved parts of esc protein required for function in vivo. We suggest that these regions might mediate physical interaction with esc partner proteins.

Amino Acid Sequence↗
PubMed2026

Genome-wide identification of the carotenoid cleavage dioxygenase gene family in wheat and analysis of the TaDREB-7A-TaNCED9a regulatory module conferring drought tolerance.

Carotenoid cleavage dioxygenases (CCDs) play critical roles in plant growth, development, and abiotic stress responses, yet their genome-wide identification and drought response mechanisms remain unexplored in wheat. In this study, 34 TaCCD genes were identified in wheat, distributed across 15 chromosomes and phylogenetically classified into five subfamilies. Gene structure analysis indicated that members within each subfamily shared conserved motifs and similar intron-exon arrangements. Cis-regulatory element analysis suggested the potential roles of these genes in stress adaptation, developmental processes, and hormone signaling. Moreover, prediction of tertiary structures and protein-protein interactions revealed unique structural features and potential interacting partners of the TaCCD proteins. In addition, TaNCED9a, a member of the TaCCD family, showed the highest transcript level in wheat roots among all detected TaCCD genes and was significantly induced by drought stress. Subcellular localization assay indicated that TaNCED9a was located in chloroplasts. Downregulation of TaNCED9a expression led to reduced drought resistance in wheat, accompanied by an accumulation of reactive oxygen species and a decrease in endogenous abscisic acid levels. Using yeast one-hybrid, dual-luciferase, and tobacco transient co-expression assays, the upstream regulatory factor TaDREB-7A was identified, which can regulate the expression of TaNCED9a. Additionally, a KASP molecular marker was developed to identify the superior haplotype TaNCED9a-HapI, which exhibited a significantly higher germination rate compared to TaNCED9a-HapII under drought conditions, and was predominant in wheat. These results offer valuable insights into the TaCCD gene family's response mechanisms to drought stress in wheat, simultaneously identifying promising genetic resources for enhancing drought tolerance through molecular breeding.

CCD↗
PubMed2005

Advanced genetic strategies for recombinant protein expression in Escherichia coli.

Preparations enriched by a specific protein are rarely easily obtained from natural host cells. Hence, recombinant protein production is frequently the sole applicable procedure. The ribosomal machinery, located in the cytoplasm is an outstanding catalyst of recombinant protein biosynthesis. Escherichia coli facilitates protein expression by its relative simplicity, its inexpensive and fast high-density cultivation, the well-known genetics and the large number of compatible tools available for biotechnology. Especially the variety of available plasmids, recombinant fusion partners and mutant strains have advanced the possibilities with E. coli. Although often simple for soluble proteins, major obstacles are encountered in the expression of many heterologous proteins and proteins lacking relevant interaction partners in the E. coli cytoplasm. Here we review the current most important strategies for recombinant expression in E. coli. Issues addressed include expression systems in general, selection of host strain, mRNA stability, codon bias, inclusion body formation and prevention, fusion protein technology and site-specific proteolysis, compartment directed secretion and finally co-overexpression technology. The macromolecular background for a variety of obstacles and genetic state-of-the-art solutions are presented.

Cloning, Molecular↗
PubMed2006

14-3-3 proteins in membrane protein transport.

14-3-3 proteins affect the cell surface expression of several unrelated cargo membrane proteins, e.g., MHC II invariant chain, the two-pore potassium channels KCNK3 and KCNK9, and a number of different reporter proteins exposing Arg-based endoplasmic reticulum localization signals in mammalian and yeast cells. These multimeric membrane proteins have a common feature in that they all expose coatomer protein complex I (COPI)- and 14-3-3-binding motifs. 14-3-3 binding depends on phosphorylation of the membrane protein in some and on multimerization of the membrane protein in other cases. Evidence from mutant proteins that are unable to interact with either COPI or 14-3-3 and from yeast cells with an altered 14-3-3 content suggests that 14-3-3 proteins affect forward transport in the secretory pathway. Mechanistically, this could be explained by clamping, masking, or scaffolding. In the clamping mechanism, 14-3-3 binding alters the conformation of the signal-exposing tail of the membrane protein, whereas masking or scaffolding would abolish or allow the interaction of the membrane protein with other proteins or complexes. Interaction partners identified as putative 14-3-3 binding partners in affinity purification approaches constitute a pool of candidate proteins for downstream effectors, such as coat components, coat recruitment GTPases, Rab GTPases, GTPase-activating proteins (GAPs), guanine-nucleotide exchange factors (GEFs) and motor proteins.

14-3-3 Proteins↗
PubMed2004

Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1).

Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.

Animals↗
PubMed2004

A cDNA library functional screening strategy based on fluorescent protein complementation assays to identify novel components of signaling pathways.

Progress towards a deeper understanding of cellular biochemical networks demands the development of methods to both identify and validate component proteins of these networks. Here, we describe a cDNA library screening strategy that achieves these aims, based on a protein-fragment complementation assay (PCA) using green fluorescent protein (GFP) as a reporter. The strategy combines a simple cell-based cDNA-screening approach (interactions of a "bait" protein of interest with "prey" cDNA products) with specific functional assays that use the same system and provide initial validation of the cDNA products as being biologically relevant. We applied this strategy to identify novel interacting partners of the protein kinase PKB/Akt. This method provides very general means of identifying and validating genes involved in any cellular process and is particularly designed for identifying enzyme substrates or regulatory proteins for which the enzyme specificity can only be defined by their interactions with other proteins in cells in which the proteins are normally expressed.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2006

Evolutionary and physiological importance of hub proteins.

It has been claimed that proteins with more interaction partners (hubs) are both physiologically more important (i.e., less dispensable) and, owing to an assumed high density of binding sites, slow evolving. Not all analyses, however, support these results, probably because of biased and less-than reliable global protein interaction data. Here we provide the first examination of these issues using a comprehensive literature-curated dataset of well-substantiated protein interactions in Saccharomyces cerevisiae. Whereas use of less reliable yeast two-hybrid data alone can reject the possibility that local connectivity correlates with measures of dispensability, in higher quality datasets a relatively robust correlation is observed. In contrast, local connectivity does not correlate with the rate of protein evolution even in reliable datasets. This perhaps surprising lack of correlation with evolutionary rate appears in part to arise from the fact that hub proteins do not have a higher density of residues associated with binding. However, hub proteins do have at least one other set of unusual features, namely rapid turnover and regulation, as manifest in high mRNA decay rates and a large number of phosphorylation sites. This, we suggest, is an adaptation to minimize unwanted activation of pathways that might be mediated by adventitious binding to hubs, were they to actively persist longer than required at any given time point. We conclude that hub proteins are more important for cellular growth rate and under tight regulation but are not slow evolving.

Biological Evolution↗
PubMed2002

Target validation and drug discovery using genomic and protein-protein interaction technologies.

After the successful completion of the human genome project, mapping of the human proteome has become the next important challenge facing the biotech and pharmaceutical industries. Identification of the 'right' target(s) is now a critical part of the process because of the cost of drug discovery. Compounding this situation is the fact that the pharmaceutical industry faces a further challenge of being able to sustain current and historical growth rates. Hence, the discovery of new drug targets is important for developing new drug leads that can become preclinical drug candidates. Proteomics is the next phase of the effort whereby the human genome can be understood. However, mapping the human proteome presents a daunting challenge. Proteomics involves several essential components with the most significant being the discovery and description of all protein-protein interactions. Once this compendium is available, a secondary and equally important initiative will be to decipher proteins that are differentially expressed in any given disease condition. At this point, the critical focus will be to select the most relevant proteins, understand their partner interactions and then further winnow them to the point where they are relevant pharmaceutical target candidates. This paradigm can be compared to finding the relevant 'needle in the proteome haystack'. This review describes the use of genomic and protein-protein interaction technologies to identify and validate these 'needles' as the first step in the drug discovery process.

Animals↗
PubMed2003

A porphobilinogen deaminase (PBGD) Ran-binding protein interaction is implicated in nuclear trafficking of PBGD in differentiating glioma cells.

Porphobilinogen deaminase (PBGD) is a rate-limiting enzyme of the heme biosynthesis pathway, whose level is elevated in various human tumors. PBGD was observed in both nuclear and cytoplasmic fractions of C6 glioma cells by immunostaining. During mitosis, chromatids were intensely stained for PBGD in comparison to the interphase chromatin. Using the yeast two-hybrid system, we identified RanBPM, the nuclear Ran-binding protein, as an interacting partner of PBGD. During butyrate-induced differentiation of C6, both nuclear and cytoplasmic PBGD levels declined as did Ran protein and its nucleotide exchange factor RCC1. N,N'-hexamethylene bis-acetamide-dependent differentiation resulted in an increase of the cytoplasmic PBGD, whereas nuclear PBGD, Ran protein and RCC1 remained unchanged. mRNA levels of PBGD remained unchanged during stimulation with both butyrate and N,N'-hexamethylene bis-acetamide. The enzymatic activity of PBGD and protoporphyrin IX synthesis in C6 cells were dependent on the differentiation induction agent. We conclude that PBGD possibly has a nuclear role in addition to its cytosolic enzymatic activity required for heme synthesis, which is related to cell transformation and differentiation.

Active Transport, Cell Nucleus↗
PubMed1993

Protein-protein interaction studies using immobilized oligohistidine fusion proteins.

Using a human transcription factor, upstream stimulatory factor, as a model system, we developed a method for the rapid and efficient purification of proteins that interact with a cloned polypeptide expressed as a fusion with an oligohistidine domain. The complex between the oligohistidine fusion protein and its interacting partner was loaded onto a column of chelating resin charged with Ni2+. The bound complex could be eluted as a whole with high concentrations of imidazole. Alternatively, the secondary protein could be released separately using appropriate elution conditions. This procedure is both simple and efficient, and it presents distinct advantages over other affinity purification methods. In addition, this same method can be used to study protein-protein interactions.

Animals↗
PubMed2004

Modification of de novo DNA methyltransferase 3a (Dnmt3a) by SUMO-1 modulates its interaction with histone deacetylases (HDACs) and its capacity to repress transcription.

The de novo DNA methyltransferase Dnmt3a is one of three mammalian DNA methyltransferases that has been shown to play crucial roles in embryonic development, genomic imprinting and transcriptional silencing. Despite its importance, very little is known about how the enzymatic activity and transcriptional repression functions of Dnmt3a are regulated. Here we show that Dnmt3a interacts with multiple components of the sumoylation machinery, namely the E2 sumo conjugating enzyme Ubc9 and the E3 sumo ligases PIAS1 and PIASxalpha, all of which are involved in conjugating the small ubiquitin-like modifier polypeptide, SUMO-1, to its target proteins. Dnmt3a is modified by SUMO-1 in vivo and in vitro and the region of Dnmt3a responsible for interaction maps to the N-terminal regulatory domain. Functionally, sumoylation of Dnmt3a disrupts its ability to interact with histone deacetylases (HDAC1/2), but not with another interaction partner, Dnmt3b. Conditions that enhance the sumoylation of Dnmt3a in vivo abolish its capacity to repress transcription. These studies reveal a new level of regulation governing Dnmt3a whereby a post-translational modification can dramatically regulate its interaction with specific protein partners and alter its ability to repress transcription.

Animals↗
PubMed2003

Molecular basis for a dominant inactivation of RUNX1/AML1 by the leukemogenic inversion 16 chimera.

The Runt domain transcription factor, PEBP2/CBF, is a heterodimer composed of 2 subunits. The DNA-binding alpha subunit, or RUNX protein, interacts with a partner PEBP2beta/CBFbeta through the evolutionarily conserved Runt domain. Each of the genes encoding RUNX1 and PEBP2beta/CBFbeta is frequently involved in acute myeloid leukemia. The chimeric protein, CBFbeta(PEBP2beta)/SMMHC, is generated as a result of inversion of chromosome 16 in such a way to retain the heterodimerization domain of PEBP2beta at the amino-terminal side fused to the C-terminal coiled-coil region of smooth muscle myosin heavy chain (SMMHC). Here we show that, in the chimeric protein, the second heterodimerization domain is created by the fusion junction, enabling the chimeric protein to interact with RUNX1 at far greater affinity than PEBP2beta and inactivate the RUNX1/AML1 function. To explain why and how heterozygous CBFB/MYH11 can inactivate homozygous RUNX1 near to completion, we propose a new model for this chimeric protein that consists of a Y-shaped dimer with unpaired N-terminal halves followed by a coiled-coil for the C-terminal region.

Animals↗
PubMed2002

Identification of putative interaction partners for the Xenopus Polycomb-group protein Xeed.

The extra sex combs (esc) gene of Drosophila and its mammalian homologue embryonic ectoderm development (eed) play pivotal roles in establishing Polycomb-group (Pc-G) mediated transcriptional silencing of regulatory genes during early development. We have carried out a two-hybrid screen in yeast to identify maternally expressed proteins that interact directly with the product of the Xenopus eed homologue, Xeed. Xeed-interacting proteins that were recovered in this screen included a maternal Xenopus histone deacetylase (HDACm), the Xeed protein itself, and a Xenopus homologue of Enhancer of zeste (XEZ) - a second member of the Pc-G that is closely related by sequence similarity to histone methyltransferases. We have also identified a novel interaction between Xeed and a component of the Xenopus basal transcription machinery, TAF(II)32. We show for the first time that each of these proteins interacts with the Xeed polypeptide, both in the yeast two-hybrid assay and in vitro using purified recombinant proteins. XEZ, HDACm and TAF(II)32 mRNAs are all strongly co-expressed with Xeed mRNA in the fertilized egg, further suggesting that their encoded proteins could interact with Xeed during early embryonic development. Our observations support a multi-step model for the onset of transcriptional silencing in which Xeed binds to and inhibits the function of the transcription initiation complex and also recruits proteins that mediate the acquisition by associated chromatin of epigenetically heritable, post-translational modifications.

Amino Acid Sequence↗
PubMed1996

Destabilizing interactions between the partners of a bifunctional fusion protein.

Hybrid MalE-GVP is a bifunctional protein in vitro since it binds maltose as protein MalE of Escherichia coli and since it is dimeric and specifically binds single-stranded DNA as protein GVP of phage M13. The oxidation rate of a unique cysteine residue was used to compare the stabilities of GVP in its free and hybrid forms, under conditions where MalE was either folded or unfolded by a denaturing agent. The results showed that both the covalent link and tertiary non-covalent interactions between MalE and GVP destabilized GVP in MalE-GVP. To test whether GVP had identical structures in its free and hybrid forms, mutations were used as local conformational probes. The effects of these mutations on the capabilities of MalE-GVP to dimerize and to bind single-stranded DNA were assayed in vitro. They were compatible with the effects of the same mutations on the global activity of free GVP in vivo and with the effects that could be predicted from the known data on free GVP, in particular its crystal structure. Thus, one partner of a hybrid protein can be destabilized by the other partner while maintaining its structural and functional characteristics.

ATP-Binding Cassette Transporters↗
PubMed1995

Phosphorylation of elongation factor Tu prevents ternary complex formation.

The elongation factor Tu (EF-Tu) is a member of the GTP/GDP-binding proteins and interacts with various partners during the elongation cycle of protein biosynthesis thereby mediating the correct binding of amino-acylated transfer RNA (aa-tRNA) to the acceptor site (A-site) of the ribosome. After GTP hydrolysis EF-Tu is released in its GDP-bound state. In vivo, EF-Tu is post-translationally modified by phosphorylation. Here we report that the phosphorylation of EF-Tu by a ribosome associated kinase activity is drastically enhanced by EF-Ts. The antibiotic kirromycin, known to block EF-Tu function, inhibits the modification. This effect is specific, since kirromycin-resistant mutants do become phosphorylated in the presence of the antibiotic. On the other hand, phosphorylated wild-type EF-Tu does not bind kirromycin. Most interestingly, the phosphorylation of EF-Tu abolishes its ability to bind aa-tRNA. In the GTP conformation the site of modification is located at the interface between domains 1 and 3 and is involved in a strong interdomain hydrogen bond. Introduction of a charged phosphate group at this position will change the interaction between the domains, leading to an opening of the molecule reminiscent of the GDP conformation. A model for the function of EF-Tu phosphorylation in protein biosynthesis is presented.

Anti-Bacterial Agents↗