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PubMed2004

Quantitative functional analysis of protein complexes on surfaces.

A major challenge in cell and molecular physiology research is to understand the mechanisms of biological processes in terms of the interactions, activities and regulation of the underlying proteins. Functional and mechanistic analyses of the large number of proteins that participate in the regulation of cellular processes will require new approaches and techniques for high throughput and multiplexed functional analyses of protein interactions, protein conformational dynamics and protein activity. In this review we focus on the development and application of proteomics and associated technologies for quantitative functional analysis of proteins and their complexes that include: (1) the application of surface plasmon resonance (SPR) imaging for multiplexed, label-free analyses of protein interactions, binding constants for biomolecular interactions and protein activities; and (2) high content analysis of protein motions within functional multiprotein complexes.

Animals↗

PubMed2002

Analysis of tyrosine phosphorylation sites in signaling molecules by a phosphotyrosine-specific immonium ion scanning method.

Signal transduction pathways involve cascades of events, such as formation of second messengers and protein complexes that alter the activities of proteins. This can ultimately lead to changes in gene expression in response to the stimuli. Reversible phosphorylation of proteins is an important mechanism for activating or inhibiting enzymes and for the assembly of multiprotein complexes. Here, we describe a mass spectrometry-based phosphotyrosine-specific immonium ion scanning (PSI scanning) method for selective detection of tyrosine-phosphorylated peptides. Once the tyrosine-phosphorylated peptides are identified, they can be directly sequenced in the same experiment to localize the phosphorylation site. We provide protocols for isolation and preparation of samples for analysis, and detailed instructions for operation of a quadrupole time-of-flight (TOF) mass spectrometer for this method. Because of its simplicity and specificity, PSI scanning is likely to become an important tool in proteomic studies of pathways involving tyrosine phosphorylation.

Alkylation↗

PubMed2006

FGF-2 protects small cell lung cancer cells from apoptosis through a complex involving PKCepsilon, B-Raf and S6K2.

Patients with small cell lung cancer (SCLC) die because of chemoresistance. Fibroblast growth factor-2 (FGF-2) increases the expression of antiapoptotic proteins, XIAP and Bcl-X(L), and triggers chemoresistance in SCLC cells. Here we show that these effects are mediated through the formation of a specific multiprotein complex comprising B-Raf, PKCepsilon and S6K2. S6K1, Raf-1 and other PKC isoforms do not form similar complexes. RNAi-mediated downregulation of B-Raf, PKCepsilon or S6K2 abolishes FGF-2-mediated survival. In contrast, overexpression of PKCepsilon increases XIAP and Bcl-X(L) levels and chemoresistance in SCLC cells. In a tetracycline-inducible system, increased S6K2 kinase activity triggers upregulation of XIAP, Bcl-X(L) and prosurvival effects. However, increased S6K1 kinase activity has no such effect. Thus, S6K2 but not S6K1 mediates prosurvival/chemoresistance signalling.

Antineoplastic Agents↗

PubMed2001

All phox homology (PX) domains from Saccharomyces cerevisiae specifically recognize phosphatidylinositol 3-phosphate.

Phox homology (PX) domains are named for a 130-amino acid region of homology shared with part of two components of the phagocyte NADPH oxidase (phox) complex. They are found in proteins involved in vesicular trafficking, protein sorting, and lipid modification. It was recently reported that certain PX domains specifically recognize phosphatidylinositol 3-phosphate (PtdIns-3-P) and drive recruitment of their host proteins to the cytoplasmic leaflet of endosomal and/or vacuolar membranes where this phosphoinositide is enriched. We have analyzed phosphoinositide binding by all 15 PX domains encoded by the Saccharomyces cerevisiae genome. All yeast PX domains specifically recognize PtdIns-3-P in protein-lipid overlay experiments, with just one exception (a significant sequence outlier). In surface plasmon resonance studies, four of the yeast PX domains bind PtdIns-3-P with high (micromolar range) affinity. Although the remaining PX domains specifically recognize PtdIns-3-P, they bind this lipid with only low affinity. Interestingly, many proteins with "low affinity" PX domains are known to form large multimeric complexes, which may increase the overall avidity for membranes. Our results establish that PtdIns-3-P, and not other phosphoinositides, is the target of all PX domains in S. cerevisiae and suggest a role for PX domains in assembly of multiprotein complexes at specific membrane surfaces.

Amino Acid Sequence↗

PubMed1989

Asymmetric DNA bending induced by the yeast multifunctional factor TUF.

TUF is a yeast regulatory factor that binds to conserved DNA sequence elements involved in gene activation or silencing as well as in telomere function. Using gel electrophoresis analyses, we show here that TUF induces DNA bending at a site located upstream of the recognition sequence (rpg box). Several point mutations in the rpg box reduced TUF binding strength without affecting the extent of bending. Selective proteolysis of TUF.DNA complexes further suggested the existence of two separate protein domains involved in DNA bending and specific DNA recognition. DNA bending may be an important feature of multifunctional factors that could help them to recruit other proteins for the formation of multiprotein complexes.

Cloning, Molecular↗

PubMed2006

Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex assembled on DNA and its implications for DNA DSB repair.

DNA-PKcs is a large (approximately 470 kDa) kinase that plays an essential role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). DNA-PKcs is recruited to DSBs by the Ku70/Ku80 heterodimer, with which it forms the core of a multiprotein complex that promotes synapsis of the broken DNA ends. We have purified the human DNA-PKcs/Ku70/Ku80 holoenzyme assembled on a DNA molecule. Its three-dimensional (3D) structure at approximately 25 Angstroms resolution was determined by single-particle electron microscopy. Binding of Ku and DNA elicits conformational changes in the FAT and FATC domains of DNA-PKcs. Dimeric particles are observed in which two DNA-PKcs/Ku70/Ku80 holoenzymes interact through the N-terminal HEAT repeats. The proximity of the dimer contacts to the likely positions of the DNA ends suggests that these represent synaptic complexes that maintain broken DNA ends in proximity and provide a platform for access of the various enzymes required for end processing and ligation.

Antigens, Nuclear↗

PubMed2003

Aph-1, Pen-2, and Nicastrin with Presenilin generate an active gamma-Secretase complex.

Gamma-Secretase cleaves the Amyloid Precursor Protein (APP) in its transmembrane domain, releasing the amyloid peptide Abeta. Abeta is the main constituent of the amyloid plaques in the brains of patients suffering from Alzheimer's disease. Several other type I integral membrane proteins are also cleaved by this protease. Recent work indicates that gamma-Secretase is a multiprotein complex consisting of Presenilin, Nicastrin, Aph-1, and Pen-2 and that all four proteins are necessary for full proteolytic activity. Since several paralogs and alternatively spliced variants of at least Presenilin and Aph-1 have been identified as well, it is anticipated that gamma-Secretase is not a homogeneous activity. Gamma-Secretase is an interesting but complex drug target that challenges classical thinking about proteolytic processing and intracellular signaling.

Amyloid Precursor Protein Secretases↗

PubMed2006

Unravelling the dynamics of RNA degradation by ribonuclease II and its RNA-bound complex.

RNA degradation is a determining factor in the control of gene expression. The maturation, turnover and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a major exoribonuclease that intervenes in all of these fundamental processes; it can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex. RNase II-like enzymes are found in all three kingdoms of life, but there are no structural data for any of the proteins of this family. Here we report the X-ray crystallographic structures of both the ligand-free (at 2.44 A resolution) and RNA-bound (at 2.74 A resolution) forms of Escherichia coli RNase II. In contrast to sequence predictions, the structures show that RNase II is organized into four domains: two cold-shock domains, one RNB catalytic domain, which has an unprecedented alphabeta-fold, and one S1 domain. The enzyme establishes contacts with RNA in two distinct regions, the 'anchor' and the 'catalytic' regions, which act synergistically to provide catalysis. The active site is buried within the RNB catalytic domain, in a pocket formed by four conserved sequence motifs. The structure shows that the catalytic pocket is only accessible to single-stranded RNA, and explains the specificity for RNA versus DNA cleavage. It also explains the dynamic mechanism of RNA degradation by providing the structural basis for RNA translocation and enzyme processivity. We propose a reaction mechanism for exonucleolytic RNA degradation involving key conserved residues. Our three-dimensional model corroborates all existing biochemical data for RNase II, and elucidates the general basis for RNA degradation. Moreover, it reveals important structural features that can be extrapolated to other members of this family.

Catalysis↗

PubMed2000

The WD-repeat protein pfs2p bridges two essential factors within the yeast pre-mRNA 3'-end-processing complex.

In the yeast Saccharomyces cerevisiae, pre-mRNA 3'-end processing requires six factors: cleavage factor IA (CF IA), cleavage factor IB (CF IB), cleavage factor II (CF II), polyadenylation factor I (PF I), poly(A) polymerase (Pap1p) and poly(A)-binding protein I (Pab1p). We report the characterization of Pfs2p, a WD-repeat protein previously identified in a multiprotein complex carrying PF I-Pap1p activity. The 3'-end-processing defects of pfs2 mutant strains and the results of immunodepletion and immunoinactivation experiments indicate an essential function for Pfs2p in cleavage and polyadenylation. With a one-step affinity purification method that exploits protein A-tagged Pfs2p, we showed that this protein is part of a CF II-PF I complex. Pull-down experiments with GST fusion proteins revealed direct interactions of Pfs2p with subunits of CF II-PF I and CF IA. These results show that Pfs2p plays an essential role in 3'-end formation by bridging different processing factors and thereby promoting the assembly of the processing complex.

Amino Acid Sequence↗

PubMed2004

Molecular architecture of bacteriophage T4.

In studying bacteriophage T4--one of the basic models of molecular biology for several decades--there has come a Renaissance, and this virus is now actively used as object of structural biology. The structures of six proteins of the phage particle have recently been determined at atomic resolution by X-ray crystallography. Three-dimensional reconstruction of the infection device--one of the most complex multiprotein components--has been developed on the basis of cryo-electron microscopy images. The further study of bacteriophage T4 structure will allow a better understanding of the regulation of protein folding, assembly of biological structures, and also mechanisms of functioning of the complex biological molecular machines.

Animals↗

PubMed1997

Geldanamycin, a heat shock protein 90-binding benzoquinone ansamycin, inhibits steroid-dependent translocation of the glucocorticoid receptor from the cytoplasm to the nucleus.

When they are translated, steroid receptors are assembled into a multiprotein complex containing hsp90, p23, an immunophilin, and often some hsp70. Some of the receptors, such as that for progesterone, have nuclear localization signals that are functional in the absence of hormone, and they move into the nucleus where they exist in the same multiprotein heterocomplex with hsp90. Other receptors, such as the glucocorticoid receptor, are localized predominantly in the cytoplasm in the absence of hormone and move into the nucleus in a hormone-dependent fashion. We have previously proposed that hsp90 and the immunophilin play a role in receptor trafficking [Pratt, W. B. (1993) J. Biol. Chem. 268, 21455-21458]. In this work, we show that treatment of L cells with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, impedes dexamethasone-dependent trafficking of the glucocorticoid receptor from the cytoplasm to the nucleus. Because geldanamycin treatment of hormone-free cells causes a rapid loss of steroid binding activity, receptors were prebound with dexamethasone by incubating cells with hormone at 0 degrees C prior to shifting the temperature to 37 degrees C for 20 min to permit receptor transformation and translocation in the presence or absence of geldanamycin. Geldanamycin does not cause steroid to dissociate from prebound receptors, and it does not inhibit hormone-mediated receptor transformation assayed by conversion to the DNA-binding state. However, as reported previously for the progesterone receptor, geldanamycin blocks assembly of the glucocorticoid receptor-hsp90 heterocomplex at an intermediate state of assembly where the receptor is bound to hsp70 and p60, both of which are required components in the assembly mechanism. Our observations support the proposal that dynamic association of receptors with hsp90 is required for receptor translocation from the cytoplasm to the nucleus.

Animals↗

PubMed2001

CARD11 and CARD14 are novel caspase recruitment domain (CARD)/membrane-associated guanylate kinase (MAGUK) family members that interact with BCL10 and activate NF-kappa B.

The caspase recruitment domain (CARD) is a protein-binding module that mediates the assembly of CARD-containing proteins into apoptosis and NF-kappaB signaling complexes. We report here that CARD protein 11 (CARD11) and CARD protein 14 (CARD14) are novel CARD-containing proteins that belong to the membrane-associated guanylate kinase (MAGUK) family, a class of proteins that functions as molecular scaffolds for the assembly of multiprotein complexes at specialized regions of the plasma membrane. CARD11 and CARD14 have homologous structures consisting of an N-terminal CARD domain, a central coiled-coil domain, and a C-terminal tripartite domain comprised of a PDZ domain, an Src homology 3 domain, and a GUK domain with homology to guanylate kinase. The CARD domains of both CARD11 and CARD14 associate specifically with the CARD domain of BCL10, a signaling protein that activates NF-kappaB through the IkappaB kinase complex in response to upstream stimuli. When expressed in cells, CARD11 and CARD14 activate NF-kappaB and induce the phosphorylation of BCL10. These findings suggest that CARD11 and CARD14 are novel MAGUK family members that function as upstream activators of BCL10 and NF-kappaB signaling.

Adaptor Proteins, Signal Transducing↗

PubMed1995

DnaX complex of Escherichia coli DNA polymerase III holoenzyme. Physical characterization of the DnaX subunits and complexes.

A physical characterization of the tau and gamma subunits of the Escherichia coli DNA polymerase III holoenzyme and their complexes with the delta, delta', chi, and psi subunits is presented. The native molecular mass of the tau and gamma subunits was determined to be 255,000 and 189,000 Da, respectively, by sedimentation equilibrium analytical ultracentrifugation. Both values indicate a tetrameric quaternary structure. The tau and gamma complexes were reconstituted and purified using two different methods. Both complexes assembled readily and were reconstituted at subunit concentrations approaching physiological levels. The stoichiometries of the tau and gamma complexes, as determined by quantitative densitometry of SDS-polyacrylamide gels, were found to be tau 4 delta 1 delta' 1 chi 1 psi 1 and gamma 4 delta 1 delta' 1 chi 1 psi 1. BIAcore analysis demonstrated that the formation of large multiprotein complexes of holoenzyme subunits depends on the presence of the tau subunit; gamma could not substitute. We present a model for a gamma-less form of DNA polymerase III holoenzyme that has asymmetrical structural features that may be responsible for the functional asymmetry observed in holoenzyme. The stoichiometry of the reconstituted DNA polymerase III* component of holoenzyme in this model is (alpha epsilon theta)2DnaX4 delta 1 delta' 1 chi 1 psi 1.

Coenzymes↗

PubMed2003

DDB complexities.

A group of recent publications contribute new insights concerning the role of the DNA damage-binding protein complex (DDB) in DNA repair. Mutations in the 48kDa DDB2 subunit are now found in all confirmed cases of xeroderma pigmentosum complementation group E. Several studies have reported a connection between the 127kDa DDB1 subunit and proteins involved in ubiquitin-mediated proteolysis. One such multiprotein complex containing DDB1 and DDB2 is closely related to a complex containing DDB1 and the Cockayne syndrome group A (CSA) protein. There is accumulating evidence for several levels of cellular regulation of DDB, including translocation to the nucleus, proteolytic degradation of DDB2 protein, and transcriptional induction of DDB2 mRNA. Although the mechanism is not yet known, it appears that DDB assists in nucleotide excision repair in chromatin.

DNA Damage↗

PubMed2005

Action of designer cellulosomes on homogeneous versus complex substrates: controlled incorporation of three distinct enzymes into a defined trifunctional scaffoldin.

In recent work, we reported the self-assembly of a comprehensive set of defined "bifunctional" chimeric cellulosomes. Each complex contained the following: (i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. The observed enhanced synergy on recalcitrant substrates by the bifunctional designer cellulosomes was ascribed to two major factors: substrate targeting and proximity of the two catalytic components. In the present work, the capacity of the previously described chimeric cellulosomes was amplified by developing a third divergent cohesin-dockerin device. The resultant trifunctional designer cellulosomes were assayed on homogeneous and complex substrates (microcrystalline cellulose and straw, respectively) and found to be considerably more active than the corresponding free enzyme or bifunctional systems. The results indicate that the synergy between two prominent cellulosomal enzymes (from the family-48 and -9 glycoside hydrolases) plays a crucial role during the degradation of cellulose by cellulosomes and that one dominant family-48 processive endoglucanase per complex is sufficient to achieve optimal levels of synergistic activity. Furthermore cooperation within a cellulosome chimera between cellulases and a hemicellulase from different microorganisms was achieved, leading to a trifunctional complex with enhanced activity on a complex substrate.

Bacterial Proteins↗

PubMed2000

Isolation and characterization of a Shigella flexneri invasin complex subunit vaccine.

The invasiveness and virulence of Shigella spp. are largely due to the expression of plasmid-encoded virulence factors, among which are the invasion plasmid antigens (Ipa proteins). After infection, the host immune response is directed primarily against lipopolysaccharide (LPS) and the virulence proteins (IpaB, IpaC, and IpaD). Recent observations have indicated that the Ipa proteins (IpaB, IpaC, and possibly IpaD) form a multiprotein complex capable of inducing the phagocytic event which internalizes the bacterium. We have isolated a complex of invasins and LPS from water-extractable antigens of virulent shigellae by ion-exchange chromatography. Western blot analysis of the complex indicates that all of the major virulence antigens of Shigella, including IpaB, IpaC, and IpaD, and LPS are components of this macromolecular complex. Mice or guinea pigs immunized intranasally with purified invasin complex (invaplex), without any additional adjuvant, mounted a significant immunoglobulin G (IgG) and IgA antibody response against the Shigella virulence antigens and LPS. The virulence-specific response was very similar to that previously noted in primates infected with shigellae. Guinea pigs (keratoconjunctivitis model) or mice (lethal lung model) immunized intranasally on days 0, 14, and 28 and challenged 3 weeks later with virulent shigellae were protected from disease (P<0.01 for both animal models).

Adhesins, Bacterial↗

PubMed2004

UV laser cross-linking: a real-time assay to study dynamic protein/DNA interactions during chromatin remodeling.

We describe the use of laser ultraviolet (UV) cross-linking to study the interaction of transcription factors with in vitro assembled chromatinized DNA templates in real time. Because the laser source delivers a high density of photons in a single ns pulse, the cross-linking reaction is completed in less than 1 microseconds, allowing the investigator to freeze rapid dynamic changes in protein-DNA interactions. Using this approach, we have sampled the dynamic equilibrium of the glucocorticoid receptor (GR) and the chromatin remodeling complex (SWI/SNF) during adenosine triphosphate (ATP)-dependent chromatin remodeling on a chromatinized mouse mammary tumor virus promoter in vitro. UV laser cross-linking shows that the GR and SWI/SNF complex undergoes a periodic binding and displacement event during the process of chromatin remodeling. The assay provides unique information regarding the equilibrium of protein-DNA interactions in real time and can be easily adapted to study the dynamic events in the assembly and disassembly of other multiprotein complexes on chromatin or DNA templates.

Animals↗

PubMed2006

Replitase: complete machinery for DNA synthesis.

Replication of nuclear DNA in eukaryotes presents a tremendous challenge, not only due to the size and complexity of the genome, but also because of the time constraint imposed by a limited duration of S phase during which the entire genome has to be duplicated accurately and only once per cell division cycle. A challenge of this magnitude can only be met by the close coupling of DNA precursor synthesis to replication. Prokaryotic systems provide evidence for multienzyme and multiprotein complexes involved in DNA precursor synthesis and DNA replication. In addition, fractionation of nuclear proteins from proliferating mammalian cells shows co-sedimentation of enzymes involved in DNA replication with those required for synthesis of deoxynucleoside triphosphates (dNTPs). Such complexes can be isolated only from cells that are in S phase, but not from cells in G(0)/G(1) phases of cell cycle. The kinetics of deoxynucleotide metabolism supporting DNA replication in intact and permeabilized cells reveals close coupling and allosteric interaction between the enzymes of dNTP synthesis and DNA replication. These interactions contribute to channeling and compartmentation of deoxynucleotides in the microvicinity of DNA replication. A multienzyme and multiprotein megacomplex with these unique properties is called "replitase." In this article, we summarize some of the relevant evidence to date that supports the concept of replitase in mammalian cells, which originated from the observations in Dr. Pardee's laboratory. In addition, we show that androgen receptor (AR), which plays a critical role in proliferation and viability of prostate cancer cells, is associated with replitase, and that identification of constituents of replitase in androgen-dependent versus androgen-independent prostate cancer cells may provide insights into androgen-regulated events that control proliferation of prostate cancer cells and potentially offer an effective strategy for the treatment of prostate cancer.

Animals↗