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Results for “Multiprotein Complexes”

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PubMed1992

Control of steroid receptor function and cytoplasmic-nuclear transport by heat shock proteins.

As targeted proteins that move within the cell, the steroid receptors have become very useful probes for understanding the linked phenomena of protein folding and transport. From the study of steroid receptor-associated proteins it has become clear over the past two years that these receptors are bound to a multiprotein complex containing at least two heat shock proteins, hsp90 and hsp56. Attachment of receptors to this complex in a cell-free system appears to require the protein unfolding/folding activity of a third heat shock protein, hsp70. Like the oncogenic tyrosine kinase pp60src, steroid receptors bind to this complex of chaperone proteins at the time of their translation. Binding of the receptor to the hsp90 component of the system occurs through the hormone binding domain and is under strict hormonal control. The hormone binding domain of the receptor acts as a transferable regulatory unit that confers both tight hormonal control and hsp90 binding onto chimaeric proteins. The model of folding and transport being developed for steroid receptors leads to some general suggestions regarding the folding and transport of targeted proteins in the cell.

Animals↗

PubMed1992

Cyclins A and B1 in the human cell cycle.

Cyclins are a family of proteins involved in the regulation of the eukaryotic cell cycle. The first cyclins to be isolated were the A- and B-type cyclins and we have been studying their behaviour in human somatic cells. The levels of both cyclin A and B1 are regulated transcriptionally as well as post-translationally; both are rapidly and specifically degraded in mitosis. Cyclin A synthesis commences at the start of S phase and the protein is predominantly nuclear, whereas cyclin B1 appears during S phase and is primarily cytoplasmic. Cyclin B1 moves into the nucleus just at the start of mitosis and associates with condensed chromosomes and the mitotic spindle. Both cyclin A and cyclin B1 bind to and activate a protein serine/threonine kinase subunit; cyclin A associates with p33cdk2 and with p34cdc2, whereas cyclin B1 seems to bind exclusively to p34cdc2. Cyclin A-associated kinase activity appears much earlier in the cell cycle than that of cyclin B1, which appears only at the G2 to M transition. Therefore cyclin A may play a role in the events of S phase as well as G2 and M phases. Cyclin A forms a cell cycle-dependent complex with p33cdk2 and the transcription factor E2F, although the function of this complex is not yet clear. We conclude that cyclins A and B1 may differentially regulate the cell cycle in several ways. They form complexes with distinct protein kinases and these complexes are active at different times in the cell cycle; they form distinct multiprotein complexes, such as with the transcription factor E2F; and they are localized to different parts of the cell where different substrates will be available to them.

Amino Acid Sequence↗

PubMed1991

Studies on the activator 1 protein complex, an accessory factor for proliferating cell nuclear antigen-dependent DNA polymerase delta.

Activator 1 (A1) is a multiprotein complex which is essential for proliferating cell nuclear antigen (PCNA)-dependent DNA polymerase delta (pol delta) activity and efficient in vitro DNA synthesis in the SV40 dipolymerase replication system. In this report, we describe the isolation of A1 from HeLa cytosolic extracts. A1 stimulated pol delta activity in singly primed phi X174 DNA or (dA)4500.oligo(dT)12-18 in reactions containing PCNA, single-stranded DNA binding protein (SSB), and ATP. Using this assay, A1 has been extensively purified. Purified preparations contained five discrete subunits of 145, 40, 38, 37, and 36.5 kDa. ATP hydrolysis to ADP and Pi is essential for A1-dependent pol delta activity, and we have shown that A1 contains an intrinsic ATPase which is stimulated by DNA. The DNA-dependent hydrolysis of ATP can be stimulated by PCNA and further activated by PCNA plus the human single-stranded DNA binding protein. These stimulatory effects were observed with (dA)4500.oligo(dT)12-18, but were not detected with each poly-deoxynucleotide alone. Furthermore, A1 formed a complex with (dA)4500.oligo(dT)12-18 which could be measured by nitrocellulose binding. No complex with (dA)4500 or oligo(dT)12-18 alone was detected by this procedure. Data are also presented which indicate that A1, in conjunction with PCNA, functions as a primer-recognition factor for pol delta, increasing its ability to utilize low levels of primer ends, but it does not increase the size of the DNA products. A1 also markedly reduced the amount of PCNA required for pol delta activity on a multiply primed DNA suggesting that PCNA interacts with A1 at the primer end. These multiple effects of A1 closely resemble the properties of the multisubunit protein RF-C described by Tsurimoto and Stillman (Tsurimoto, T., and Stillman, B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1023-1027).

Adenosine Triphosphatases↗

PubMed1991

Immunological characterization of chromatin assembly factor I, a human cell factor required for chromatin assembly during DNA replication in vitro.

Chromatin assembly factor I (CAF-I) is a multisubunit protein complex purified from the nuclei of human cells and required for chromatin assembly during DNA replication in vitro. Purified CAF-I promotes chromatin assembly in a reaction that is dependent upon, and coupled with, DNA replication and is therefore likely to reflect events that occur during S phase in vivo. In order to investigate the regulation and mechanism of CAF-I and the replication-dependent chromatin assembly process, we have used the purified protein to raise monoclonal antibodies. In this report we describe the characterization of a panel of monoclonal antibodies which recognize different subunits of the CAF-I complex. We use immunoprecipitation analysis to show that CAF-I exists as a multiprotein complex in vivo and that some of the polypeptides are phosphorylated. In addition, immunocytochemistry demonstrates that CAF-I is localized to the nucleus of human cells. Finally, monoclonal antibodies directed against the individual subunits of CAF-I immunodeplete chromatin assembly activity from nuclear extracts, confirming that CAF-I is a multisubunit protein required for chromatin assembly in vitro.

Antibodies, Monoclonal↗

PubMed1990

High-molecular-weight forms of aminoacyl-tRNA synthetases and tRNA modification enzymes in Escherichia coli.

The presence of high-molecular-weight complexes of aminoacyl-tRNA synthetases in Escherichia coli has been reported (C. L. Harris, J. Bacteriol. 169:2718-2723, 1987). In the current study, Bio-Gel A-5M gel chromatography of 105,000 x g supernatant preparations from E. coli Q13 indicated high molecular weights for both tRNA methylase (300,000) and tRNA sulfurtransferase (450,000). These tRNA modification enzymes did not appear to exist in the same multienzymic complex. On the other hand, 4-thiouridine sulfurtransferase eluted with aminoacyl-tRNA synthetase activity on Bio-Gel A-5M, and both of these activities were cosedimented after further centrifugation of cell supernatants at 160,000 x g for 18 h. Despite this evidence for association of the sulfurtransferase with the synthetase complex, isoleucyl-tRNA synthetase and tRNA sulfurtransferase were totally resolved from each other by DEAE-Sephacel chromatography. Subsequent gel chromatography showed little change in their elution positions on agarose. Hence, either nonspecific aggregation occurred here, or the modification enzymes studied are not members of the aminoacyl-tRNA synthetase complex in E. coli. These findings do suggest that some bacterial tRNA modification enzymes are present in multiprotein complexes of high molecular weight.

Amino Acyl-tRNA Synthetases↗

PubMed1990

Kinetics of photosynthetic electron transfer in artificial vesicles reconstituted with purified complexes from Rhodobacter capsulatus. II. Direct electron transfer between the reaction center and the bc1 complex and role of cytochrome c2.

1. The cyclic photosynthetic chain of Rhodobacter capsulatus has been reconstituted incorporating into phospholipid liposomes containing ubiquinone-10 two multiprotein complexes: the reaction center and the ubiquinol-cytochrome-c2 reductase (or bc1 complex). 2. In the presence of cytochrome c2 added externally, at concentrations in the range 10-10(4) nM, a flash-induced cyclic electron transfer can be observed. In the presence of antimycin, an inhibitor of the quinone-reducing site of the bc1 complex, the reduction of cytochrome b561 is a consequence of the donation of electrons to the photo-oxidized reaction center. At low ionic strength (10 mM KCl) and at concentrations of cytochrome c2 lower than 1 microM, the rate of this reaction is limited by the concentration of cytochrome c2. At higher concentrations the reduction rate of cytochrome b561 is controlled by the concentration of quinol in the membrane, and, therefore, is increased when the ubiquinone pool is progressively reduced. At saturating concentrations of cytochrome c2 and optimal redox poise, the half-time for cytochrome b561 reduction is about 3 ms. 3. At high ionic stength (200 mM KCl), tenfold higher concentrations of cytochrome c2 are required for promoting equivalent rates of cytochrome-b561 reduction. If the absolute values of these rates are compared with those of the cytochrome-c2-reaction-center electron transfer, it can be concluded that the reaction of oxidized cytochrome c2 with the bc1 complex is rate-limiting and involves electrstatic interactions. 4. A significant rate of intercomplex electron transfer can be observed also in the absence of cytochrome c2; in this case the electron donor to the recation center is the cytochrome c1 of the oxidoreductase complex. The oxidation of cytochrome c1 triggers a normal electron transfer within the bc1 complex. The intercomplex reaction follows second-order kinetics and is slowed at high ionic strength, suggesting a collisional interaction facilitated by electrostatic attraction. From the second-order rate constant of this process, a minimal bidimensional diffusion coefficient for the complexes in the membrane equal to 3 X 10(-11) cm2 s-1 can be evaluated.

Bacterial Proteins↗

PubMed1992

Binding of the human E2F transcription factor to the retinoblastoma protein but not to cyclin A is abolished in HPV-16-immortalized cells.

The adenovirus E1A, SV40 large T and papillomavirus E7 proteins immortalize primary cells by virtue of their ability to bind the retinoblastoma gene product (pRB) and other cellular proteins, including cyclin A and the prRB-related protein, p107. It has been demonstrated that these viral oncogene products will prevent the inhibition of positive growth regulators by pRB, one of them being the E2F transcription factor. Here we show that the interactions of pRB and cyclin A with E2F are present also in normal keratinocytes and in primary human fibroblasts. In human keratinocytes immortalized by human papillomavirus 16 (HPV-16), expressing high levels of HPV-16 E7 protein, complexes between E2F and pRB are disrupted. In this cell line, as well as in HeLa cells which express HPV-18 E7, complexes containing E2F and cyclin A are maintained, indicating that this interaction is not sensitive to the viral oncoprotein and that cyclin A can associate with E2F independently of pRB. In vitro binding experiments suggest that the E7 gene product is able to preferentially abolish the interaction of pRB with E2F, leaving the cyclin A complexes intact. Our findings suggest that E7-dependent immortalization of human cells is associated with modifications of E2F multiprotein complexes.

Base Sequence↗

PubMed1979

Enzymology of ubiquinone-utilizing electron transfer complexes in nonionic detergent.

The enzymology of isolated succinate: ubiquinone reductase and ubiquinone: cytochrome c reductase in nonionic detergents (alkyl polyoxyethylene derivatives) was studied. In the membrane the two multiprotein complexes and their hydrophobic substrates ubiquinone and dihydroubiquinone, are embedded in a common lipid bilayer. In detergent solutions the complexes are each inserted into micelles. Detergent micelles also serve as a solvent for the complexes hydrophobic substrates. As a consequence the isolated complexes are in a discontinuous phase with respect to their hydrophobic substrates and with respect to each other. Three types of assays were used. Firstly, single enzyme assays in which the hydrophobic substrates had to transfer from free micelles to the complex-bound micelles in order for enzyme reactions to occur. Secondly, assays in which the enzymic reactions were coupled to auxiliary nonenzymic reactions which rapidly converted the hydrophobic products back into substrates within the complex-bound micelle. Dichloroindophenol was used for the oxidation of dihydroubiquinone and dihydroduroquinone for the reduction of ubiquinone. Thirdly, assays in which the succinate: ubiquinone reductase reaction was coupled with the ubiquinone: cytochrome c reductase reaction. With the first type of assay, the kinetics of the substrate transfer reaction was dependent upon the type of detergent. In detergents with small polyoxyethylene head groups the transfer reactions were rate-limiting, and in detergents with large polyoxyethylene head groups the transfer reactions were fast and the enzymic reactions were rate-limiting...

Animals↗

PubMed1990

The transcription complex of the Xenopus somatic 5 S RNA gene. A functional analysis of protein-DNA interactions outside of the internal control region.

We have defined protein-DNA interactions associated with the transcription complex of a Xenopus somatic 5 S RNA gene under efficient in vitro transcription conditions. Transcription factor IIIA, the internal control region of the 5 S RNA gene and specific DNA sequences 5' and 3' of the internal control region are all involved in the formation of a multiprotein complex. Specific protein-DNA interactions outside of the 5 S RNA gene itself, revealed by DNase I footprinting, have no apparent role in the transcription process. Sequences within the 5 S RNA gene, 5' of the internal control region, are not essential for transcription by RNA polymerase III in vitro, but do contribute to its efficiency.

Animals↗

PubMed2026

Targeting the Disease Response With NlpD and LytM for Effective Nonantibiotic Treatment of Urinary Tract Infections.

BACKGROUND: Finding new ways of treating bacterial infections is essential. The NlpD protein, which inhibits RNA polymerase II (Pol II), has shown therapeutic efficacy against urinary tract infection. This study investigated the mechanism of Pol II inhibition and protection by NlpD and its LytM peptide. METHODS: Recombinant NlpD and LytM were screened for interactions with constituents of the Pol II complex, using AlphaFold predictions and protein interaction technology. Treatment effects were quantified in infected tissues and regulated host response pathways identified by genome-wide transcriptomics analysis in models of acute pyelonephritis and acute cystitis in Irf3-/- and Asc-/- mice, respectively. RESULTS: LytM was shown to interact with constituents of the Pol II multiprotein complex, inhibiting the CDK12 kinase from phosphorylating the Pol II subunit RPB1 and disrupting Pol II complex formation by interfering with the interaction between PAF1C and RPB1. The protection by LytM against acute pyelonephritis was accompanied by a reduction in gene expression in infected kidneys from >1900 significantly regulated genes (fold change >6) in the placebo group to about 150 in LytM-treated mice. The inhibition of gene expression in infected kidneys particularly targeted the excessive innate immune response. A similar effect was observed in acute cystitis. Bacterial clearance was accelerated in both model by LytM treatment, with effects against antibiotic-sensitive and resistant Escherichia coli strains. CONCLUSIONS: The results suggest that inhibiting the disease response of the host, using NlpD or LytM, may offer an efficient alternative to antibiotics in these models.

Animals↗

PubMed1991

Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.

A discontinuous electrophoretic system for the isolation of membrane proteins from acrylamide gels has been developed using equipment for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Coomassie dyes were introduced to induce a charge shift on the proteins and aminocaproic acid served to improve solubilization of membrane proteins. Solubilized mitochondria or extracts of heart muscle tissue, lymphoblasts, yeast, and bacteria were applied to the gels. From cells containing mitochondria, all the multiprotein complexes of the oxidative phosphorylation system were separated within one gel. The complexes were resolved into the individual polypeptides by second-dimension Tricine-SDS-PAGE or extracted without SDS for functional studies. The recovery of all respiratory chain complexes was almost quantitative. The percentage recovery of functional activity depended on the respective protein complex studied and was zero for some complexes, but almost quantitative for others. The system is especially useful for small scale purposes, e.g., separation of radioactively labeled membrane proteins, N-terminal protein sequencing, preparation of proteins for immunization, and diagnostic studies of inborn neuromuscular diseases.

Aminocaproates↗

PubMed1990

Mitochondrial rhodanese: membrane-bound and complexed activity.

We have proposed that phosphorylated and dephosphorylated forms of the mitochondrial sulfurtransferase, rhodanese, function as converter enzymes that interact with membrane-bound iron-sulfur centers of the electron transport chain to modulate the rate of mitochondrial respiration (Ogata, K., Dai, X., and Volini, M. (1989) J. Biol. Chem. 204, 2718-2725). In the present studies, we have explored some structural aspects of the mitochondrial rhodanese system. By sequential extraction of lysed mitochondria with phosphate buffer and phosphate buffer containing 20 mM cholate, we have shown that 30% of the rhodanese activity of bovine liver is membrane-bound. Resolution of cholate extracts on Sephadex G-100 indicates that part of the bound rhodanese is complexed with other mitochondrial proteins. Tests with the complex show that it forms iron-sulfur centers when incubated with the rhodanese sulfur-donor substrate thiosulfate, iron ions, and a reducing agent. Experiments on the rhodanese activity of rat liver mitochondria give similar results. Taken together, the findings indicate that liver rhodanese is in part bound to the mitochondrial membrane as a component of a multiprotein complex that forms iron-sulfur centers. The findings are consistent with the role we propose for rhodanese in the modulation of mitochondrial respiratory activity.

Animals↗

PubMed1992

Transfection of CD14 into 70Z/3 cells dramatically enhances the sensitivity to complexes of lipopolysaccharide (LPS) and LPS binding protein.

Bacterial endotoxin (lipopolysaccharide [LPS]) causes fatal shock in humans and experimental animals. The shock is mediated by cytokines released by direct LPS stimulation of cells of monocytic origin (monocyte/macrophage [MO]). Recent studies have supported the concept that the plasma protein, LPS binding protein (LBP), plays an important role in controlling MO responses to LPS. Specifically, evidence has been presented to suggest that CD14, a membrane protein present in MO, serves as a receptor for complexes of LPS and the plasma protein LPS binding protein (LBP). In this function CD14 mediates attachment of LPS-bearing particles opsonized with LBP and appears to play an important role in regulating cytokine production induced by complexes of LPS and LBP. The CD14-, murine pre-B cell line 70Z/3 responds to LPS by synthesis of kappa light chains and consequent expression of surface IgM. To better understand the role of CD14 in controlling cellular responses to LPS, we investigated the effect of transfection of CD14 into 70Z/3 cells on LPS responsiveness. We report here that transfection of human or rabbit CD14 cDNA into 70Z/3 cells results in membrane expression of a glycosyl-phosphatidylinositol-anchored CD14. When LPS is complexed with LBP, CD14-bearing 70Z/3 cells bind more LPS than do the parental or 70Z/3 cells transfected with vector only. Remarkably, the expression of CD14 lowers the amount of LPS required to stimulate surface IgM expression by up to 10,000-fold when LPS dose-response curves in the CD14-, parental and CD14-bearing, transfected 70Z/3 cells are compared. In contrast, the response of CD14-bearing 70Z/3 cells and the parental 70Z/3 cell line (CD14-) to interferon gamma is indistinguishable. LPS stimulation of the parental and CD14-bearing 70Z/3 cells results in activation of NF-kB. These data provide evidence to support the concept that the LPS receptor in cells that constitutively express CD14 may be a multiprotein complex containing CD14 and membrane protein(s) common to a diverse group of LPS-responsive cells.

Acute-Phase Proteins↗

PubMed1992

DNA replication and the cell cycle.

The replication of DNA in the eukaryotic cell cycle is one of the most highly regulated events in cell growth and division. Biochemical studies on the replication of the genome of the small DNA virus simian virus 40 (SV40) have resulted in the identification of a number of DNA replication proteins from human cells. One of these, Replication Protein A (RPA), was phosphorylated in a cell cycle-dependent manner, beginning at the onset of DNA replication. RPA was phosphorylated in vitro by the cell cycle-regulated cdc2 protein kinase. This kinase also stimulated the unwinding of the SV40 origin of DNA replication during initiation of DNA replication in vitro, suggesting a mechanism by which cdc2 kinase may regulate DNA replication. Functional homologues of the DNA replication factors have been identified in extracts from the yeast Saccharomyces cerevisiae, enabling a genetic characterization of the role of these proteins in the replication of cellular DNA. A cellular origin binding protein had not been characterized. To identify proteins that function like T antigen at cellular origins of DNA replication, we examined the structure of a yeast origin of DNA replication in detail. This origin consists of four separate functional elements, one of which is essential. A multiprotein complex that binds to the essential element has been identified and purified. This protein complex binds to all known cellular origins from S. cerevisiae and may function as an origin recognition complex.

Animals↗

PubMed2026

Characterizing the Activity of Inflammasome-Related Genes and Their Association With Oncological Outcomes in Prostate Cancer.

BACKGROUND: Inflammation plays a critical role in cancer cell proliferation; however, the specific role of inflammasomes, multiprotein complexes that regulate inflammation-associated signaling pathways, in prostate cancer (PCa) remains insufficiently explored. This study aims to characterize the expression of inflammasome-related genes in PCa and evaluate their association with clinical outcomes. METHODS: De-identified transcriptome data from the Decipher GRID RP, a cohort of 52,266 radical prostatectomy (RP) samples tested (2016-2024) with the Decipher prostate genomic classifier (Veracyte, San Diego, CA), were retrieved from the GRID registry (NCT02609269). Expression analysis of 34 genes involved in inflammatory pathways was conducted to associate their expression with clinical and genomic variables. Outcomes analyses were conducted on a retrospective cohort of 855 patients treated with RP (META855). RESULTS: Analysis of inflammasome gene expression in the GRID RP cohort revealed that most genes exhibit low baseline expression, whereas HSP90AB1, APP, TXN, and TXNIP demonstrate strong expression signals. Additionally, higher expression of most genes was associated with Gleason Grade Group 4-5 and very high Decipher scores. On survival analysis of the META855 cohort, higher expressions of AIM2 and HSP90AB1 were significantly associated with worse metastasis-free survival. Conversely, both high and low expression levels of NLRP3 were associated with better metastasis-free survival outcomes following RP compared to average expression. On multivariable Cox regression analysis, higher expressions of AIM2 (HR 1.75) and HSP90AB1 (HR 1.60) were significantly associated with shorter time to metastasis following RP. CONCLUSIONS: There is molecular heterogeneity within pro-inflammatory genes among patients with PCa. Our findings showed there is a potential association between the expression levels of certain inflammasomes, such as AIM2, HSP90AB1, and NLRP3, and oncological outcomes following RP.

Aged↗

PubMed1992

Estrogen receptor-induced bending of the Xenopus vitellogenin A2 gene hormone response element.

DNA bending is increasingly proposed as an essential step for the establishment of the multiprotein complexes required for transcription initiation. Polyamines and metallic cations, known to promote DNA-bending, enhance the binding of purified estrogen receptor (ER) to the estrogen response element (ERE) of the Xenopus vitellogenin A2 gene. Using both circular permutation electrophoretic mobility and cyclization assays, we provide evidence that ER bends the DNA at the estrogen response element. The same bending occurs as a result of estrogen receptor protein binding independently of its conformational changes induced by hormone or anti-hormone. We suggest a role of the observed DNA bending in estrogen-regulated transcription.

Animals↗

PubMed1991

The regulation of adenylyl cyclase by receptor-operated G proteins.

The receptor regulated adenylyl cyclase system is a multiprotein complex which is a member of the family of the receptor-effector systems whose signal is transduced by heterotrimeric GTP-binding proteins. The system consists of stimulatory and inhibitory receptors (Rs and Ri), stimulatory and inhibitory G proteins (Gs and Gi) and the adenylyl cyclase enzyme (C). While quite specific in situ, receptors (stimulatory or inhibitory) from one source can activate the appropriate G protein from other cell types or species which in turn can act on C from other sources. Studies with chimeric proteins have shown that the various specificities (stimulatory or inhibitory) can be mapped to defined domains in both receptors and G proteins. The mechanism by which the heterotrimeric G proteins couple to the stimulatory and inhibitory signals is discussed in detail. Specifically, the data supporting collision coupling vs the shuttle mechanism is reviewed, as well as the role of beta gamma subunits in both the stimulatory and inhibitory signals.

Adenylyl Cyclases↗

PubMed1992

Blotting and band-shifting: techniques for studying protein-protein interactions.

The type II cAMP-dependent protein kinase (PKA) is localized in certain cellular compartments through association with specific A-kinase anchoring proteins (AKAPs). A variety of blotting and electrophoresis techniques have been developed to study the protein-protein interactions that occur between the regulatory (R) subunit of PKA and AKAPs. These methods have also been used for a variety of purposes such as detecting calmodulin-binding proteins, comparing wild-type- and mutant-form binding affinities and estimating the molecular weight of multiprotein complexes.

Animals↗