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PubMed1985

Location of C-protein, H-protein and X-protein in rabbit skeletal muscle fibre types.

The locations of C-protein, H-protein and X-protein in rabbit psoas, plantaris and soleus muscles have been investigated with fluorescently tagged specific antibodies. Two systems have been examined: isolated myofibrils allowed the locations of these proteins within the sarcomere to be determined, while cryosections allowed a comparison of the amounts of these proteins between different types of fibre in the three muscles. Using antibody-labelled cryosections, we find that the amounts of each of these proteins depends closely on the fibre type. In all the muscles studied, C-protein is present in the largest amounts in fast white and fast intermediate fibres and is absent from slow red fibres, while X-protein is absent from fast white fibres and is present in the largest amounts in fast and slow red fibres. In psoas muscle, H-protein is present in the largest amounts in fast white fibres and is absent in fast and slow red fibres. In plantaris muscle, however, H-protein is absent from fast white fibres but occurs in some slow red fibres. All psoas myofibrils label with anti-C and anti-H and a minority label with anti-X. In each case the pattern of labelling is a zone in each half of the A-band. Measured across the middle of the A-band, the zones for H-protein are much closer together than for C-protein; the centre-to-centre spacings are 0.35 micron for anti-H and 0.64 micron for anti-C. The fluorescent zones for X-protein are slightly but significantly closer (0.52 micron) than those for C-protein. All soleus myofibrils label with anti-X but the centre-to-centre spacing was greater (0.67 micron). With plantaris myofibrils, where labelling occurs with anti-C or anti-H, the spacings resemble those in psoas myofibrils, but with anti-X the spacing resembles that in soleus myofibrils. The spacing of the fluorescent zones in an A-band, whether produced by anti-C, anti-X or anti-H does not vary with sarcomere length. We conclude that X-protein and H-protein, like C-protein, are thick filament components. With both fibres and myofibrils, there is no simple relationship between the amount of X-protein and the amount of C-protein. Many fast intermediate fibres in psoas and plantaris muscle label as strongly with anti-C as do fast white fibres but also label as strongly with anti-X as do fast and slow red fibres.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

The Bead blot: a method for identifying ligand-protein and protein-protein interactions using combinatorial libraries of peptide ligands.

Small molecules that bind proteins can be used as ligands for protein purification and for investigating protein-protein and protein-drug interactions. Unfortunately, many methods used to identify new ligands to desired proteins suffer from common shortcomings, including the requirement that the target protein be purified and/or the requirement that the ligands be selected under conditions different from those under which it will be used. We have developed a new method called the Bead blot that can (i) select ligands to unpurified proteins, including trace proteins, present in complex materials (e.g., unfractionated plasma); (ii) select ligands to multiple proteins under a variety of conditions in a single experiment; and (iii) be used with libraries of different types of ligands. In the Bead blot, a library of ligands, synthesized on chromatography resin beads, is incubated with a starting material containing a target protein for which a ligand is sought. The proteins in the material bind to their complementary ligands according to specific affinity interactions. Then the protein-loaded beads are immobilized in a porous matrix, and the proteins are directionally eluted from the beads and captured on a membrane superimposed on the beads. The location of the target protein on the membrane is determined, and because the position of the protein(s) on the membrane reflects the position of the bead(s) in the matrix, the bead that originally bound the protein is identified, with subsequent elucidation of the ligand sequence. Ligands to several targets can be identified in one experiment. Here we demonstrate the broad utility of this method by the selection of ligands that purify plasma protein complexes or that remove pathogens from whole blood with very high affinity constants. We also select ligands to a protein based on competitive elution.

Amino Acid Sequence↗
PubMed1994

Maternal protein reserves and their influence on lactational performance in rats. 4. Tissue protein synthesis and turnover associated with mobilization of maternal protein.

The present study was undertaken to investigate the changes in muscle protein turnover involved in the rapid mobilization of protein in rats subjected to severe protein restriction during lactation. Estimates of mammary gland and liver protein synthesis were also made during lactation. Multiparous female Sprague-Dawley rats, caged individually following mating, were offered a high-protein diet (H; 215 g crude protein (N x 6.25; CP)/kg dry matter (DM)) ad lib. until parturition. Following parturition, half the females continued to receive diet H, whilst the remainder were offered a diet low in protein (L; 90 g CP/kg DM) ad lib. On days 2, 4, 8 and 12 of lactation, groups of females were used in the estimation of tissue protein synthesis (flooding dose of [3H]phenylalanine) immediately after a milk sample had been obtained. Rates of muscle protein synthesis were unchanged during lactation in group H. The feeding of diet L during lactation reduced the muscle protein synthesis on day 12 to rates that were lower than group H and also the rate on diet L on day 2 (P < 0.01). However, this fall in muscle protein synthesis was not rapid and muscle fractional synthesis rate (FSR) was different from group H only from day 8 (P < 0.05). Estimated rates of mammary protein synthesis appeared to be generally unchanged by dietary treatment or stage of lactation. Liver FSR was also unchanged by dietary protein supply or stage of lactation. The effect of dietary protein restriction on liver size and protein content during lactation influenced liver absolute synthesis rate (ASR), and on days 8 and 12 of lactation liver ASR was lower in group L than in group H (P < 0.001). The loss of muscle protein in rats fed on diet L during lactation (133 mg) occurred mainly between days 2 and 8 of lactation and was primarily associated with a dramatic increase in degradation (13.0% per d), with the decline in synthesis having a much smaller role. A decline in muscle protein degradation during the latter half of lactation was part of the mechanism that prevented excessive muscle protein catabolism. It is thought that the estimation of mammary protein synthesis in the present study was impaired by the milk sampling procedure previously used.

Animals↗
PubMed2003

Prediction of protein function using protein-protein interaction data.

Assigning functions to novel proteins is one of the most important problems in the postgenomic era. Several approaches have been applied to this problem, including the analysis of gene expression patterns, phylogenetic profiles, protein fusions, and protein-protein interactions. In this paper, we develop a novel approach that employs the theory of Markov random fields to infer a protein's functions using protein-protein interaction data and the functional annotations of protein's interaction partners. For each function of interest and protein, we predict the probability that the protein has such function using Bayesian approaches. Unlike other available approaches for protein annotation in which a protein has or does not have a function of interest, we give a probability for having the function. This probability indicates how confident we are about the prediction. We employ our method to predict protein functions based on "biochemical function," "subcellular location," and "cellular role" for yeast proteins defined in the Yeast Proteome Database (YPD, www.incyte.com), using the protein-protein interaction data from the Munich Information Center for Protein Sequences (MIPS, mips.gsf.de). We show that our approach outperforms other available methods for function prediction based on protein interaction data. The supplementary data is available at www-hto.usc.edu/~msms/ProteinFunction.

Bayes Theorem↗
PubMed1996

The WRPW motif of the hairy-related basic helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and protein-protein interaction domain.

Hairy-related proteins include the Drosophila Hairy and Enhancer of Split proteins and mammalian Hes proteins. These proteins are basic helix-loop-helix (bHLH) transcriptional repressors that control cell fate decisions such as neurogenesis or myogenesis in both Drosophila melanogaster and mammals. Hairy-related proteins are site-specific DNA-binding proteins defined by the presence of both a repressor-specific bHLH DNA binding domain and a carboxyl-terminal WRPW (Trp-Arg-Pro-Trp) motif. These proteins act as repressors by binding to DNA sites in target gene promoters and not by interfering with activator proteins, indicating that these proteins are active repressors which should therefore have specific repression domains. Here we show the WRPW motif to be a functional transcriptional repression domain sufficient to confer active repression to Hairy-related proteins or a heterologous DNA-binding protein, Ga14. This motif was previously shown to be necessary for interactions with Groucho, a genetically defined corepressor for Drosophila Hairy-related proteins. Here we show that the WRPW motif is sufficient to recruit Groucho or the TLE mammalian homologs to target gene promoters. We also show that Groucho and TLE proteins actively repress transcription when directly bound to a target gene promoter and identify a novel, highly conserved transcriptional repression domain in these proteins. These results directly demonstrate that Groucho family proteins are active transcriptional corepressors for Hairy-related proteins and are recruited by the 4-amino acid protein-protein interaction domain, WRPW.

Amino Acid Sequence↗
PubMed2005

Simulation studies of protein-induced bilayer deformations, and lipid-induced protein tilting, on a mesoscopic model for lipid bilayers with embedded proteins.

Biological membranes are complex and highly cooperative structures. To relate biomembrane structure to their biological function it is often necessary to consider simpler systems. Lipid bilayers composed of one or two lipid species, and with embedded proteins, provide a model system for biological membranes. Here we present a mesoscopic model for lipid bilayers with embedded proteins, which we have studied with the help of the dissipative particle dynamics simulation technique. Because hydrophobic matching is believed to be one of the main physical mechanisms regulating lipid-protein interactions in membranes, we considered proteins of different hydrophobic length (as well as different sizes). We studied the cooperative behavior of the lipid-protein system at mesoscopic time- and lengthscales. In particular, we correlated in a systematic way the protein-induced bilayer perturbation, and the lipid-induced protein tilt, with the hydrophobic mismatch (positive and negative) between the protein hydrophobic length and the pure lipid bilayer hydrophobic thickness. The protein-induced bilayer perturbation was quantified in terms of a coherence length, xi(P), of the lipid bilayer hydrophobic thickness profile around the protein. The dependence on temperature of xi(P), and the protein tilt-angle, were studied above the main-transition temperature of the pure system, i.e., in the fluid phase. We found that xi(P) depends on mismatch, i.e., the higher the mismatch is, the longer xi(P) becomes, at least for positive values of mismatch; a dependence on the protein size appears as well. In the case of large model proteins experiencing extreme mismatch conditions, in the region next to the so-called lipid annulus, there appears an undershooting (or overshooting) region where the bilayer hydrophobic thickness is locally lower (or higher) than in the unperturbed bilayer, depending on whether the protein hydrophobic length is longer (or shorter) than the pure lipid bilayer hydrophobic thickness. Proteins may tilt when embedded in a too-thin bilayer. Our simulation data suggest that, when the embedded protein has a small size, the main mechanism to compensate for a large hydrophobic mismatch is the tilt, whereas large proteins react to negative mismatch by causing an increase of the hydrophobic thickness of the nearby bilayer. Furthermore, for the case of small, peptidelike proteins, we found the same type of functional dependence of the protein tilt-angle on mismatch, as was recently detected by fluorescence spectroscopy measurements.

Computer Simulation↗
PubMed1989

Phosphorylation of P400 protein by cyclic AMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase II.

Purified P400 protein was phosphorylated by both purified Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase). Because P400 protein was suggested to function as an integral membrane protein, we investigated the phosphorylation of P400 protein using crude mitochondrial and microsomal fractions (P2/P3 fraction). Incubation of the P2/P3 fraction from mouse cerebellum with cyclic AMP or the catalytic subunit of A-kinase stimulated the phosphorylation of P400 protein. The phosphorylation of P400 protein was not observed in the P2/P3 fraction from mouse forebrain. Cyclic AMP and A-kinase enhanced the phosphorylation of several proteins, including P400 protein, suggesting that P400 protein is one of the best substrates for A-kinase in the P2/P3 fraction. Although endogenous and exogenous CaM kinase II stimulated the phosphorylation of some proteins in the P2/P3 fraction, the phosphorylation of P400 protein was weak. Immunoprecipitation with the monoclonal antibody to P400 protein confirmed that the P400 protein itself was definitely phosphorylated by the catalytic subunit of A-kinase and CaM kinase II. A-kinase phosphorylated only the seryl residue in P400 protein. Immunoblot analysis of the cells in primary culture of mouse cerebellum confirmed the expression of P400 protein, which migrated at the same position on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as that in the P2/P3 fraction. Incubation of the cultured cerebellar cells with [32P]orthophosphate resulted in the labeling of P400 protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Protein S and C4b-binding protein: components involved in the regulation of the protein C anticoagulant system.

The protein C anticoagulant system provides important control of the blood coagulation cascade. The key protein is protein C, a vitamin K-dependent zymogen which is activated to a serine protease by the thrombin-thrombomodulin complex on endothelial cells. Activated protein C functions by degrading the phospholipid-bound coagulation factors Va and VIIIa. Protein S is a cofactor in these reactions. It is a vitamin K-dependent protein with multiple domains. From the N-terminal it contains a vitamin K-dependent domain, a thrombin-sensitive region, four EGF) epidermal growth factor (EGF)-like domains and a C-terminal region homologous to the androgen binding proteins. Three different types of post-translationally modified amino acid residues are found in protein S, 11 gamma-carboxy glutamic acid residues in the vitamin K-dependent domain, a beta-hydroxylated aspartic acid in the first EGF-like domain and a beta-hydroxylated asparagine in each of the other three EGF-like domains. The EGF-like domains contain very high affinity calcium binding sites, and calcium plays a structural and stabilising role. The importance of the anticoagulant properties of protein S is illustrated by the high incidence of thrombo-embolic events in individuals with heterozygous deficiency. Anticoagulation may not be the sole function of protein S, since both in vivo and in vitro, it forms a high affinity non-covalent complex with one of the regulatory proteins in the complement system, the C4b-binding protein (C4BP). The complexed form of protein S has no APC cofactor function. C4BP is a high molecular weight multimeric protein with a unique octopus-like structure. It is composed of seven identical alpha-chains and one beta-chain. The alpha- and beta-chains are linked by disulphide bridges. The cDNA cloning of the beta-chain showed the alpha- and beta-chains to be homologous and of common evolutionary origin. Both subunits are composed of multiple 60 amino acid long repeats (short complement or consensus repeats, SCR) and their genes are located in close proximity on chromosome 1, band 1q32. Available experimental data suggest the beta-chain to contain the single protein S binding site on C4BP, whereas each of the alpha-chains contains a binding site for the complement protein, C4b. As C4BP lacking the beta-chain is unable to bind protein S, the beta-chain is required for protein S binding, but not for the assembly of the alpha-chains during biosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2005

Oligomeric protein structure networks: insights into protein-protein interactions.

BACKGROUND: Protein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues) with special emphasis to protein interfaces. RESULTS: A variety of interactions such as hydrogen bond, salt bridges, aromatic and hydrophobic interactions, which occur at the interfaces are identified in a consolidated manner as amino acid clusters at the interface, from this study. Moreover, the characterization of the highly connected hub-forming residues at the interfaces and their comparison with the hubs from the non-interface regions and the non-hubs in the interface regions show that there is a predominance of charged interactions at the interfaces. Further, strong and weak interfaces are identified on the basis of the interaction strength between amino acid residues and the sizes of the interface clusters, which also show that many protein interfaces are stronger than their monomeric protein cores. The interface strengths evaluated based on the interface clusters and hubs also correlate well with experimentally determined dissociation constants for known complexes. Finally, the interface hubs identified using the present method correlate very well with experimentally determined hotspots in the interfaces of protein complexes obtained from the Alanine Scanning Energetics database (ASEdb). A few predictions of interface hot spots have also been made based on the results obtained from this analysis, which await experimental verification. CONCLUSION: The construction and analysis of oligomeric protein structure networks and their comparison with monomeric protein structure networks provide insights into protein association. Further, the interface hubs identified using the present method can be effective targets for interface de-stabilizing mutations. We believe this analysis will significantly enhance our knowledge of the principles behind protein association and also aid in protein design.

Animals↗
PubMed2002

Detecting protein-protein interactions using Renilla luciferase fusion proteins.

We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.

Amino Acid Sequence↗
PubMed2003

Somatomedin-1 binding protein-3: insulin-like growth factor-1 binding protein-3, insulin-like growth factor-1 carrier protein.

Somatomedin-1 binding protein-3 [insulin-like growth factor-1 binding protein-3, SomatoKine] is a recombinant complex of insulin-like growth factor-1 (rhIGF-1) and binding protein-3 (IGFBP-3), which is the major circulating somatomedin (insulin-like growth factor) binding protein; binding protein-3 regulates the delivery of somatomedin-1 to target tissues. Somatomedin-1 binding protein-3 has potential as replacement therapy for somatomedin-1 which may become depleted in indications such as major surgery, organ damage/failure and traumatic injury, resulting in catabolism. It also has potential for the treatment of osteoporosis; diseases associated with protein wasting including chronic renal failure, cachexia and severe trauma; and to attenuate cardiac dysfunction in a variety of disease states, including after severe burn trauma. Combined therapy with somatomedin-1 and somatomedin-1 binding protein-3 would prolong the duration of action of somatomedin-1 and would reduce or eliminate some of the undesirable effects associated with somatomedin-1 monotherapy. Somatomedin-1 is usually linked to binding protein-3 in the normal state of the body, and particular proteases clip them apart in response to stresses and release somatomedin-1 as needed. Therefore, somatomedin-1 binding protein-3 is a self-dosing system and SomatoKine would augment the natural supply of these linked compounds. Somatomedin-1 binding protein-3 was developed by Celtrix using its proprietary recombinant protein production technology. Subsequently, Celtrix was acquired by Insmed Pharmaceuticals on June 1 2000. Insmed and Avecia, UK, have signed an agreement for the manufacturing of SomatoKine and its components, IGF-1 and binding protein-3. CGMP clinical production of SomatoKine and its components will be done in Avecia's Advanced Biologics Centre, Billingham, UK, which manufactures recombinant-based medicines and vaccines with a capacity of up to 1000 litres. In 2003, manufacturing of SomatoKine is planned to move to Avecia's larger facility with a capacity of 10 000 litres. Somatomedin-1 binding protein-3 was originally licenced to Welfide for Japan. On October 1 2001, Welfide Corporation merged with Mitsubishi-Tokyo Pharmaceuticals to form Mitsubishi Pharma Corporation. The new company is a subsidiary of Mitsubishi Chemical. In April 2003 Insmed initiated a named patient programme in Europe, that will make available somatomedin-1 binding protein-3 for the treatment of growth hormone insensitivity syndrome (GHIS)--Laron syndrome. The treatment of patients was initiated in Scandinavia, with authorisation pending in several other European countries. Somatomedin-1 binding protein-3 will be made available to those GHIS patients who, in the opinion of their doctor, may benefit from IGF-1 therapy. At precommercial scale quantities, the drug will be available on a limited basis. Safety data generated from the named patient programme will be used to support marketing applications in 2004. A phase II dose-ranging study in children with GHIS was completed at Saint Bartholomew's and the Royal London School of Medicine, London, UK. A single dose of somatomedin-1 binding protein-3 delivered the same amount of IGF-1 as two daily injections of unbound IGF-1. There were no adverse events reported. GHIS is a genetic condition in which patients do not produce adequate quantities of IGF because of a failure to respond to the growth hormone signal. This results in a slower growth rate and short stature. Insmed has acquired an exclusive licence to Pharmacia's regulatory filings concerning yeast-derived IGF-1. These filings were used by Pharmacia to receive marketing approvals in several European countries and also in the investigational New Drug Application with the US FDA. This licence will facilitate the development of SomatoKine for the treatment of children with GHIS. In January 2003, Insmed announced positive results from a double-blind, placebo-controlled, dose-ranging study of SomatoKine in adolescent patients with type 1 diabetes mellitus redolescent patients with type 1 diabetes mellitus receiving insulin therapy. The study was conducted at the University of Cambridge, Cambridge, UK, under the supervision of Professor D. Dunger. It has also been granted orphan drug status for the treatment of GHIS--Laron syndrome in the US and in Europe. Celtrix has been granted 11 US patents for its recombinant protein production technology, which it used for developing somatomedin-1 binding protein-3. Subsequently, Celtrix was acquired by Insmed Pharmaceuticals on June 1 2000. Following the acquisition, Insmed announced that it intends to maintain the US rights to Celtrix's products portfolio. These US patents will expire between 2010 through 2017. Insmed is holding a US patent (expires in 2019) for the use of SomatoKine in the treatment of both type 1 and type 2 diabetes mellitus.

Child↗
PubMed1995

Identification of candidate residues for interaction of protein S with C4b binding protein and activated protein C.

Protein S is a plasma factor essential for prevention of thrombosis, partly due to its activity as a cofactor for the plasma anticoagulant protease-activated protein C. To expand knowledge about structure-function relationships in homologous protein S molecules, studies of protein S from different species have been performed. Protein S anti-coagulant activity in human, monkey, bovine, and porcine plasma has been inactivated by purified human C4b binding protein (C4BP) with dose-dependence, suggesting that each protein S can bind human C4BP and that only the free form of each is anti-coagulantly active. Purified porcine protein S has a 10-fold higher Kd for human C4BP than has human protein S. Protein S residues 420-434 provide an essential binding site for the negative regulator C4BP. cDNA sequences show that protein S residues 420-434 are highly conserved in all four species with the notable exception of Lys-429-Ile in porcine protein S. Differences between porcine and human protein S, e.g. Lys-429-Ile, Lys-43-Ala, Ser-197-Leu, Ser 199-Phe, Glu-463-Gly, Lys-571-Glu, Asn-602-Ile, Gln-607-Pro, may contribute to the decreased affinity of porcine protein S for human C4BP. Moreover, the species specificity of cofactor activities of various species of protein S is determined for human versus bovine-activated protein C, and these results, combined with sequence comparisons, agree with previous evidence that the thrombin-sensitive region and the first epidermal growth factor domain of protein S, i.e. residues 47-116, are responsible for recognition of activated protein C.

Amino Acid Sequence↗
PubMed2003

Apparent dependence of protein evolutionary rate on number of interactions is linked to biases in protein-protein interactions data sets.

BACKGROUND: Several studies have suggested that proteins that interact with more partners evolve more slowly. The strength and validity of this association has been called into question. Here we investigate how biases in high-throughput protein-protein interaction studies could lead to a spurious correlation. RESULTS: We examined the correlation between evolutionary rate and the number of protein-protein interactions for sets of interactions determined by seven different high-throughput methods in Saccharomyces cerevisiae. Some methods have been shown to be biased towards counting more interactions for abundant proteins, a fact that could be important since abundant proteins are known to evolve more slowly. We show that the apparent tendency for interactive proteins to evolve more slowly varies directly with the bias towards counting more interactions for abundant proteins. Interactions studies with no bias show no correlation between evolutionary rate and the number of interactions, and the one study biased towards counting fewer interactions for abundant proteins actually suggests that interactive proteins evolve more rapidly. In all cases, controlling for protein abundance significantly decreases the observed correlation between interactions and evolutionary rate. Finally, we disprove the hypothesis that small data set size accounts for the failure of some interactions studies to show a correlation between evolutionary rate and the number of interactions. CONCLUSIONS: The only correlation supported by a careful analysis of the data is between evolutionary rate and protein abundance. The reported correlation between evolutionary rate and protein-protein interactions cannot be separated from the biases of some protein-protein interactions studies to count more interactions for abundant proteins.

Bacterial Proteins↗
PubMed2001

A novel approach for the identification of protein-protein interaction with integral membrane proteins.

Protein-protein interaction plays a major role in all biological processes. The currently available genetic methods such as the two-hybrid system and the protein recruitment system are relatively limited in their ability to identify interactions with integral membrane proteins. Here we describe the development of a reverse Ras recruitment system (reverse RRS), in which the bait used encodes a membrane protein. The bait is expressed in its natural environment, the membrane, whereas the protein partner (the prey) is fused to a cytoplasmic Ras mutant. Protein-protein interaction between the proteins encoded by the prey and the bait results in Ras membrane translocation and activation of a viability pathway in yeast. We devised the expression of the bait and prey proteins under the control of dual distinct inducible promoters, thus enabling a rapid selection of transformants in which growth is attributed solely to specific protein-protein interaction. The reverse RRS approach greatly extends the usefulness of the protein recruitment systems and the use of integral membrane proteins as baits. The system serves as an attractive approach to explore novel protein-protein interactions with high specificity and selectivity, where other methods fail.

Animals↗
PubMed2002

Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions.

We have previously described a strategy for detecting protein protein interactions based on protein interaction assisted folding of rationally designed fragments of enzymes. We call this strategy the protein fragment complementation assay (PCA). Here we describe PCAs based on the enzyme TEM-1 beta-lactamase (EC: 3.5.2.6), which include simple colorimetric in vitro assays using the cephalosporin nitrocefin and assays in intact cells using the fluorescent substrate CCF2/AM (ref. 6). Constitutive protein protein interactions of the GCN4 leucine zippers and of apoptotic proteins Bcl2 and Bad, and the homodimerization of Smad3, were tested in an in vitro assay using cell lysates. With the same in vitro assay, we also demonstrate interactions of protein kinase PKB with substrate Bad. The in vitro assay is facile and amenable to high-throughput modes of screening with signal-to-background ratios in the range of 10:1 to 250:1, which is superior to other PCAs developed to date. Furthermore, we show that the in vitro assay can be used for quantitative analysis of a small molecule induced protein interaction, the rapamycin-induced interaction of FKBP and yeast FRB (the FKBP-rapamycin binding domain of TOR (target of rapamycin)). The assay reproduces the known dissociation constant and number of sites for this interaction. The combination of in vitro colorimetric and in vivo fluorescence assays of beta-lactamase in mammalian cells suggests a wide variety of sensitive and high-throughput large-scale applications, including in vitro protein array analysis of protein protein or enzyme protein interactions and in vivo applications such as clonal selection for cells expressing interacting protein partners.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2006

Detection and analysis of protein-protein interactions in organellar and prokaryotic proteomes by native gel electrophoresis: (Membrane) protein complexes and supercomplexes.

It is an essential and challenging task to unravel protein-protein interactions in their actual in vivo context. Native gel systems provide a separation platform allowing the analysis of protein complexes on a rather proteome-wide scale in a single experiment. This review focus on blue-native (BN)-PAGE as the most versatile and successful gel-based approach to separate soluble and membrane protein complexes of intricate protein mixtures derived from all biological sources. BN-PAGE is a charge-shift method with a running pH of 7.5 relying on the gentle binding of anionic CBB dye to all membrane and many soluble protein complexes, leading to separation of protein species essentially according to their size and superior resolution than other fractionation techniques can offer. The closely related colorless-native (CN)-PAGE, whose applicability is restricted to protein species with intrinsic negative net charge, proved to provide an especially mild separation capable of preserving weak protein-protein interactions better than BN-PAGE. The essential conditions determining the success of detecting protein-protein interactions are the sample preparations, e.g. the efficiency/mildness of the detergent solubilization of membrane protein complexes. A broad overview about the achievements of BN- and CN-PAGE studies to elucidate protein-protein interactions in organelles and prokaryotes is presented, e.g. the mitochondrial protein import machinery and oxidative phosphorylation supercomplexes. In many cases, solubilization with digitonin was demonstrated to facilitate an efficient and particularly gentle extraction of membrane protein complexes prone to dissociation by treatment with other detergents. In general, analyses of protein interactomes should be carried out by both BN- and CN-PAGE.

Electrophoresis, Polyacrylamide Gel↗
PubMed1995

snRNP Sm proteins share two evolutionarily conserved sequence motifs which are involved in Sm protein-protein interactions.

The spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5 share eight proteins B', B, D1, D2, D3, E, F and G which form the structural core of the snRNPs. This class of common proteins plays an essential role in the biogenesis of the snRNPs. In addition, these proteins represent the major targets for the so-called anti-Sm auto-antibodies which are diagnostic for systemic lupus erythematosus (SLE). We have characterized the proteins F and G from HeLa cells by cDNA cloning, and, thus, all human Sm protein sequences are now available for comparison. Similar to the D, B/B' and E proteins, the F and G proteins do not possess any of the known RNA binding motifs, suggesting that other types of RNA-protein interactions occur in the snRNP core. Strikingly, the eight human Sm proteins possess mutual homology in two regions, 32 and 14 amino acids long, that we term Sm motifs 1 and 2. The Sm motifs are evolutionarily highly conserved in all of the putative homologues of the human Sm proteins identified in the data base. These results suggest that the Sm proteins may have arisen from a single common ancestor. Several hypothetical proteins, mainly of plant origin, that clearly contain the conserved Sm motifs but exhibit only comparatively low overall homology to one of the human Sm proteins, were identified in the data base. This suggests that the Sm motifs may also be shared by non-spliceosomal proteins. Further, we provide experimental evidence that the Sm motifs are involved, at least in part, in Sm protein-protein interactions. Specifically, we show by co-immunoprecipitation analyses of in vitro translated B' and D3 that the Sm motifs are essential for complex formation between B' and D3. Our finding that the Sm proteins share conserved sequence motifs may help to explain the frequent occurrence in patient sera of anti-Sm antibodies that cross-react with multiple Sm proteins and may ultimately further our understanding of how the snRNPs act as auto-antigens and immunogens in SLE.

Amino Acid Sequence↗
PubMed1997

The interaction between the endothelial cell protein C receptor and protein C is dictated by the gamma-carboxyglutamic acid domain of protein C.

The endothelial cell protein C receptor (EPCR) binds to both protein C and activated protein C (APC) with similar affinity. Removal of the Gla domain of protein C results in the loss of most of the binding affinity. This observation is compatible with at least two models: 1) the Gla domain of protein C interacts with phospholipid on cell surfaces to stabilize interaction with EPCR or 2) the Gla domain of protein C makes specific protein-protein interactions with EPCR. The latter model predicts that chimeric proteins containing the protein C Gla domain should interact with EPCR. To test this, we constructed a prothrombin chimera in which the Gla domain and aromatic stack of prothrombin were replaced with the corresponding region of protein C. The 125I-labeled chimera (Kd = 176 nM) and 125I-APC (Kd = 65 nM) both bound specifically to 293 cells stably transfected with EPCR, but both bound poorly to sham-transfected cells. The chimera also blocked APC binding to EPCR-transfected cells in a dose-dependent fashion (Ki approximately 139 nM) similarly to protein C (Ki approximately 75 nM). Chimera binding to EPCR-transfected cells was blocked by soluble EPCR, demonstrating direct protein-protein interaction between the chimera and EPCR. Consistent with this conclusion, the isolated Gla domain of protein C blocked APC binding to EPCR-transfected cells (IC50 = 2 microM). No inhibition was observed with the isolated prothrombin Gla domain. A protein C chimera with the prothrombin Gla domain and aromatic stack failed to bind to EPCR detectably. These data suggest that the Gla domain of protein C is responsible for much of the binding energy and specificity of the protein C-EPCR interaction.

1-Carboxyglutamic Acid↗