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At least 19 recordsLinked to original sources
PubMed2003

Organismal complexity, protein complexity, and gene duplicability.

Although the evolutionary significance of gene duplication has long been recognized, it remains unclear what determines gene duplicability. We find protein complexity to be an important determinant because the proportion of unduplicated genes (P) increases with the number of subunits in a protein. However, P is high (>or=65%) for both monomers and multimers in yeast, but <or=30% in human except for subunits of large multimers, implying that organismal complexity is a stronger determinant of gene duplicability than protein complexity. The same conclusion is reached from a comparison of family sizes in yeast and human.

Dimerization↗
PubMed2001

Recruitment of coat-protein-complex proteins on to phagosomal membranes is regulated by a brefeldin A-sensitive ADP-ribosylation factor.

Particle internalization in macrophages is followed by a complex maturation process. We have previously observed that proteins bound to phagocytosed particles are sorted from phagosomes into a heterogeneous population of vesicles that fuse with endosomes. However, the mechanism and the protein machinery involved in the formation of these phagosome-derived vesicles are largely unknown. It has been shown that vesicles coated with coat protein complex type I (COPI) have a role in both secretion and endocytosis. To address the possibility that COPI proteins might participate in the formation of phagosome-derived vesicles we studied the recruitment of beta-COP to highly purified phagosomes. The binding of beta-COP to phagosomal membranes was regulated by nucleotides and inhibited by brefeldin A (BFA). An ADP-ribosylation factor 1 (ARF1) mutant defective in GTP hydrolysis supported the binding of beta-COP to phagosomes independently of added nucleotide. AlF(4) and Gbetagamma subunits, agents known to modulate heterotrimeric G-protein activity, were tested in the beta-COP binding assay. AlF(4) increased beta-COP association, whereas binding was inhibited by the addition of Gbetagamma subunits. Our results suggest that COP proteins are recruited to phagosomal membranes by a mechanism that involves heterotrimeric GTP-binding proteins and a BFA-sensitive ARF. In addition, our findings indicate that COPI proteins are involved in the recycling of components from phagosomes to the cell surface.

ADP-Ribosylation Factors↗
PubMed1986

Fluorescence quenching studies of apolipoprotein A-I in solution and in lipid-protein complexes: protein dynamics.

Fluorescence lifetime and intensity quenching studies of human plasma apolipoprotein A-I (apo A-I) in aqueous solution and in recombinant lipoprotein complexes with dimyristoylphosphatidylcholine (DMPC) indicate differences in conformational dynamics. In aqueous solution, the bimolecular quenching constants (k*) for lipid-free apo A-I fluorescence quenching by oxygen and acrylamide are 2.4 X 10(9) and 0.38 X 10(9) M-1 s-1, respectively. These values are independent of the oligomeric form of the protein. There is no correlation between the relatively small k* for apo A-I, which reflects rapid, low-amplitude protein fluctuations, and the labile conformational changes of apo A-I folding reactions, like denaturation, which occur on a slower time scale. In recombinant DMPC/apo A-I complexes (100:1 molar ratio) the protein increases in amphiphilic alpha-helical structure as it blankets the lipid matrix. The apparent k* for oxygen quenching of apo A-I fluorescence in the complex is large and increases in a temperature-dependent manner. We have introduced a two-compartment model, which discriminates the source of quencher molecules as aqueous or lipid, to describe oxygen quenching of DMPC/apo A-I fluorescence. The magnitude and temperature dependence of the apparent k* predominantly reflect the partitioning of oxygen between the two phases rather than being a probe of the lipid physical state. Calculations of the helical hydrophobic moment in apo A-I indicate that tryptophan residues 8 and 72 occur at the lipid-protein interface of amphiphilic alpha-helices, whereas the other two tryptophan residues (50, 108) lie on the nonpolar faces of amphiphilic helices.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2002

Deciphering protein complexes and protein interaction networks by tandem affinity purification and mass spectrometry: analytical perspective.

We employed a combination of tandem affinity purification and mass spectrometry for deciphering protein complexes and the protein interaction network in budding yeast. 53 genes were epitope-tagged, and their interaction partners were isolated by two-step immunoaffinity chromatography from whole cell lysates. 38 baits pulled down a total of 220 interaction partners, which are members of 19 functionally distinct protein complexes. We identified four proteins shared between complexes of different functionality thus charting segments of a protein interaction network. Concordance with the results of genome-wide two-hybrid screening was poor (14% of identified interactors overlapped) suggesting that the two approaches may provide complementary views on physical interactions within the proteome.

Chromatography, Affinity↗
PubMed2006

Impaired gene and protein expression of exocytotic soluble N-ethylmaleimide attachment protein receptor complex proteins in pancreatic islets of type 2 diabetic patients.

Exocytosis of insulin is dependent on the soluble N-ethylmaleimide attachment protein receptor (SNARE) complex proteins in the B-cells. We assessed insulin release as well as gene and protein expression of SNARE complex protein in isolated pancreatic islets of type 2 diabetic patients (n = 4) and nondiabetic control subjects (n = 4). In islets from the diabetic patients, insulin responses to 8.3 and 16.7 mmol/l glucose were markedly reduced compared with control islets (4.7 +/- 0.3 and 8.4 +/- 1.8 vs. 17.5 +/- 0.1 and 24.3 +/- 1.2 microU . islet(-1) . h(-1), respectively; P < 0.001). Western blot analysis revealed decreased amounts of islet SNARE complex and SNARE-modulating proteins in diabetes: syntaxin-1A (21 +/- 5% of control levels), SNAP-25 (12 +/- 4%), VAMP-2 (7 +/- 4%), nSec1 (Munc 18; 34 +/- 13%), Munc 13-1 (27 +/- 4%), and synaptophysin (64 +/- 7%). Microarray gene chip analysis, confirmed by quantitative PCR, showed that gene expression was decreased in diabetes islets: syntaxin-1A (27 +/- 2% of control levels), SNAP-25 (31 +/- 7%), VAMP-2 (18 +/- 3%), nSec1 (27 +/- 5%), synaptotagmin V (24 +/- 2%), and synaptophysin (12 +/- 2%). In conclusion, these data support the view that decreased islet RNA and protein expression of SNARE and SNARE-modulating proteins plays a role in impaired insulin secretion in type 2 diabetic patients. It remains unclear, however, to which extent this defect is primary or secondary to, e.g., glucotoxicity.

Aged↗
PubMed2006

An agent-based system to discover protein-protein interactions, identify protein complexes and proteins with multiple peptide mass fingerprints.

Proteins "work together" by actually binding to form multicomponent complexes that carry out specific functions. Proteomic analyses based on the mass spectrum are now key methods to determine the components in protein complexes. The protein-protein interaction or functional association may be known to exist among the extracted protein spots while analyzing the proteins on the 2D gel. In this study, we develop an agent-based system, namely AgentMultiProtIdent, which integrated two protein identification tools and a variety of databases storing relations among proteins and used to discover protein-protein interactions and protein functional associations, and identify protein complexes and proteins with multiple peptide mass fingerprints as input. The system takes Multiple Peptide Mass Fingerprints (PMFs) as a whole in the protein complex or protein identification. With the relations among proteins, it may greatly improve the accuracy of identification of protein complexes. Also, possible relationship of the multiple peptide mass fingerprints, such as ontology relation, can be discovered by our system, especially in the identification of protein complexes. The agent-based system is now available on the Web at http://dbms104.csie.ncu.edu.tw/ approximately protein/NEW2/.

Animals↗
PubMed2001

Identification of individual proteins in complex protein mixtures by high-resolution, high-mass-accuracy MALDI TOF-mass spectrometry analysis of in-solution thermal denaturation/enzymatic digestion.

Identification of individual proteins in complex protein mixtures by high-resolution (HR), high-mass-accuracy matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) is demonstrated for synthetic protein mixtures. Instead of chemical denaturation, thermal denaturation followed by in-solution trypsin digestion is used to achieve uniform digestion of the constituents of the protein mixture. Protein identification is carried out using protein database searches with search scoring systems, which seems more effective than conventional peptide mass mapping without using a scoring system. Identification of individual proteins by MALDI HR-TOF-MS peptide mass mapping dramatically reduces data acquisition/analysis time and does not require special equipment for sample preparation/transfer prior to mass spectral analysis.

Amino Acid Sequence↗
PubMed1979

Immunoassay of the adenosine deaminase complexing proteins of human tissues and body fluids.

A sensitive immunoassay for the adenosine deaminase binding protein (complexing protein) of human kidney has been developed. Impetus for the development of the assay was provided by the observations that (a) antibody to complexing protein does not react with the catalytically active adenosine deaminase monomer, and (b) binding of antibody to complexing protein does not affect the binding or catalytic activity of the enzyme monomer. Preformed immune precipitate prepared from rabbit anti-kidney complexing protein serum and goat anti-rabbit gamma-globulin serum is used to selectively insolubilize complexing protein. Quantitation is accomplished by measuring the intrinsic adenosine deaminating activity or adenosine deaminase binding capacity of the protein held in the immune precipitate. As little as 1 ng of kidney complexing protein can be accurately quantitated with the assay. The assay was used to demonstrate that complexing proteins from liver, lung, spleen, fibroblasts, plasma, and urine react with antibody to kidney complexing protein. The shared capacity to bind adenosine deaminase coupled with their antigenic similarity suggests that the complexing proteins of a number of human tissues and body fluids may be products of the same gene.

Adenosine Deaminase↗
PubMed2001

Duality in bromodomain-containing protein complexes.

Proteins that contain a motif called a bromodomain are implicated in both transcriptional activation and repression. The bromodomain of p/CAF, the only solution structure of a bromodomain that has been solved to date, reveals that the motif binds N-acetyl-lysine groups, presumably to anchor enzymatic functions to histones and by extension to chromatin. The enzymatic activities can either be encoded within the same polypeptide as the bromodomain motif, or associated with a multiprotein complex. Thus, a wide variety of chromatin-directed functions, including but not limited to phosphorylation, acetylation, methylation, transcriptional co-activation or recruitment, characterize the complexes that contain bromodomain motifs. Their versatility and ubiquity ensures diverse, rapid and flexible transcriptional responses.

Acetyltransferases↗
PubMed2003

Fanconi anemia protein complex: mapping protein interactions in the yeast 2- and 3-hybrid systems.

Fanconi anemia (FA) is an autosomal recessive syndrome characterized by progressive bone marrow failure and cancer predisposition. Eight FA complementation groups have been identified. The FANCA, FANCC, FANCE, FANCF, and FANCG proteins form a nuclear complex required for the monoubiquination of the FANCD2 protein. To investigate the architecture of the FA protein complex, the yeast 2-hybrid system was used to map contact points of the FANCA/FANCG, FANCC/FANCE, and FANCF/FANCG interactions. FANCG was shown to interact with both the amino-terminus of FANCA and the carboxyl-terminal region of FANCF. A FANCG mutant truncated at the carboxyl-terminus retained the ability to interact with FANCA. The interaction between FANCG and FANCF was ablated by a Leu71Pro mutant of FANCG. A central region of FANCE was sufficient for FANCC binding. A Leu554Pro mutant of FANCC failed to interact with FANCE. To further examine complex assembly, the yeast 3-hybrid system was used to investigate the ability of FANCG to act as a molecular bridge in mediating interaction between other FA proteins. FANCG was able to mediate interaction between FANCA and FANCF, as well as between monomers of FANCA. Direct interaction between FANCE and FANCD2 was also demonstrated in the yeast 2-hybrid system. This interaction involving an amino-terminal region of FANCD2 may provide a link between the FA protein complex and its downstream targets.

Binding Sites↗
PubMed2003

Coagulation, inflammation, and apoptosis: different roles for protein S and the protein S-C4b binding protein complex.

Protein S (PS) has an established role as an important cofactor to activated protein C (APC) in the degradation of coagulation cofactors Va and VIIIa. This anticoagulant role is evident from the consequences of its deficiency, when there is an increased risk of venous thromboembolism. In human plasma, PS circulates approximately 40% as free PS (FPS) and 60% in complex with C4b-binding protein (C4BP). Formation of this complex results in loss of PS cofactor function, and C4BP can then modulate the anticoagulant activity of APC. It had long been predicted that the complex could act as a bridge between coagulation and inflammation due to the involvement of C4BP in regulating complement activation. This prediction was recently supported by the demonstration of binding of the PS-C4BP complex to apoptotic cells. This review aims to summarize recent findings on the structure and functions of PS, the basis and importance of its deficiency, its interaction with C4BP, and the possible physiologic and pathologic importance of the PS-C4BP interaction.

Apoptosis↗
PubMed1996

Dissection of functional domains of the human DNA replication protein complex replication protein A.

Replication protein A (RPA) is a mammalian single-stranded DNA binding factor essential for DNA replication, repair, and recombination. It is composed of three subunits of 70, 34, and 13 kDa (Rpa1, Rpa2, and Rpa3, respectively). Deletion mapping of the Rpa2 subunit identified the domain required for interaction with Rpa1 and Rpa3 which does not include the N-terminal domain that is phosphorylated during S phase. Deletion mapping of Rpa1 defined three domains. The C-terminal third of the Rpa1 polypeptide binds Rpa2 which itself forms a bridge between Rpa1 and Rpa3. The N-terminal third of Rpa1 bound single-stranded DNA under low stringency conditions only (0.1 M NaCl), while a central domain binds to single-stranded DNA under both low and high stringency conditions (0.5 M NaCl). Binding to p53 requires the N-terminal third of Rpa1 with some contribution from the C-terminal third. The evolutionarily conserved putative zinc finger near the C terminus of Rpa1 was not required for binding to single-stranded DNA, Rpa2, or p53. However, all three subdomains of Rpa1 and the zinc finger were essential for supporting DNA replication in vitro. These experiments are a first step toward defining peptide components responsible for the many functions of the RPA protein complex.

Animals↗
PubMed2005

Loss of synaptonemal complex protein-1, a synaptonemal complex protein, contributes to the initiation of follicular assembly in the developing rat ovary.

In the rat ovary, germ and somatic cells become organized into primordial follicles 48-72 h after birth. Although several genes have been implicated in the control of early follicular growth, less is known about the factors involved in the formation of primordial follicles. Using the method of differential display of mRNAs, we found several genes differentially expressed at the time of follicular assembly. One of them encodes synaptonemal complex protein-1 (SCP1), a core component of the protein complex that maintains recombining chromosomes together during prophase I of the first meiotic division in germ cells. This association, evident during the pachytene stage, ends when chromosomal desynapsis begins in the diplotene stage at the end of prophase I. Oocytes become arrested in the diplotene/dictate stage before becoming enclosed into primordial follicles, suggesting that oocytes must complete meiotic prophase I before becoming competent to direct follicle assembly. We now show that attainment of the diplotene stage results in follicular formation. In developing rat ovaries, SCP1 mRNA expression is confined to oocytes and decreases precipitously within 24 h after birth, preceding the organization of primordial follicles. The premature loss of SCP1, achieved via treatment with an antisense oligodeoxynucleotide targeting SCP1 mRNA, resulted in more oocytes reaching the diplotene stage, as evidenced by a decrease in the number of oocytes containing germ cell nuclear antigen-1 (a nuclear protein whose expression ceases in diplotene) and an increase in the number of oocytes expressing MSY2 (a cytoplasmic Y box protein expressed in oocytes that have become arrested in diplotene). SCP1-deficient ovaries exhibited an increased number of newly formed follicles, suggesting that completion of meiotic prophase I endows oocytes with the ability to orchestrate follicular assembly.

Animals↗
PubMed1997

Novel membrane protein complexes for protein glycosylation in the yeast Golgi apparatus.

Three type II membrane proteins Anp1, Van1 and Mnn9 of Saccharomyces cerevisiae share significant sequence homology. Their precise biochemical activity has long been unknown though the mutant phenotype indicates their participation in protein glycosylation in the Golgi apparatus. To shed light on their molecular characteristics, interactions of these proteins were studied by immunoprecipitation after solubilizing the membrane by nonionic detergent. Our results indicated that there are at least two submembrane complexes containing these proteins: one contains Van1 and Mnn9 proteins and the other contains Anp1 and Mnn9 proteins. In addition, Hoc1 protein which has significant homology to Och1 protein colocalized with Anp1 and Mnn9 proteins. These complexes with similar but partially different constituents may represent essential parts of glycosylation machinery in the yeast Golgi compartments.

Alleles↗
PubMed1977

Immunological identification of complex proteins resolved by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis.

Immunological identification of an antigen resolved from a protein complex by sodium dodecyl sulfate polyacrylamide gel electrophoresis has been attained. The identification is based on the formation of immunoprecipitin lines after the antigen diffuses laterally from acrylamide gel transverse slices into a surrounding agarose gel. This technique was designed for study of contractile and regulatory protein complexes of non-muscle cells where the scarcity of tissue precludes easy purification or high yield of muscle-like proteins. It complements double-gel immunodiffusion or immunoelectrophoresis and its use may be extended to other protein complexes.

Actinin↗
PubMed1979

Studies on the binding of the ribosomal protein complex L7/12-L10 and protein L11 to the 5'-one third of 23S RNA: a functional centre of the 50S subunit.

The RNA binding sites of the protein complex of L7/12 dimers and L10, and of protein L11, occur within the 5'-one third of 23S RNA. Binding of the L7/12-L10 protein complex to the 23S RNA is stimulated by protein L11 and vice-versa. This is the second example to be established of mutual stimulation of RNA binding by two ribosomal proteins or protein complexes, and suggests that this may be an important principle governing ribosomal protein-RNA assembly. When the L7/12-L10 complex is bound to the RNA, L10 becomes strongly resistant to trypsin. Since the L7/12 dimer does not bind specifically to the 23S RNA, this suggests that L10 constitutes a major RNA binding site of the protein complex. Only one of the L7/12 dimers is bound strongly in the (L7/12-L10)-23S RNA complex; the other can dissociate with no concurrent loss of L10.

Escherichia coli↗