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PubMed2002

The spectrin repeat: a structural platform for cytoskeletal protein assemblies.

Spectrin repeats are three-helix bundle structures which occur in a large number of diverse proteins, either as single copies or in tandem arrangements of multiple repeats. They can serve structural purposes, by coordination of cytoskeletal interactions with high spatial precision, as well as a 'switchboard' for interactions with multiple proteins with a more regulatory role. We describe the structure of the alpha-actinin spectrin repeats as a prototypical example, their assembly in a defined antiparallel dimer, and the interactions of spectrin repeats with multiple other proteins. The alpha-actinin rod domain shares several features common to other spectrin repeats. (1) The rod domain forms a rigid connection between two actin-binding domains positioned at the two ends of the alpha-actinin dimer. The exact distance and rigidity are important, for example, for organizing the muscle Z-line and maintaining its architecture during muscle contraction. (2) The spectrin repeats of alpha-actinin have evolved to make tight antiparallel homodimer contacts. (3) The spectrin repeats are important interaction sites for multiple structural and signalling proteins. The interactions of spectrin repeats are, however, diverse and defy any simple classification of their preferred interaction sites, which is possible for other domains (e.g. src-homology domains 3 or 2). Nevertheless, the binding properties of the repeats perform important roles in the biology of the proteins where they are found, and lead to the assembly of complex, multiprotein structures involved both in cytoskeletal architecture as well as in forming large signal transduction complexes.

Actinin↗

PubMed2005

Functional complementation of Trypanosoma brucei RNA in vitro editing with recombinant RNA ligase.

The approximately 20S RNA ligase-containing complex (L-complex) in trypanosomatid mitochondria interacts by means of RNA linkers with at least two other multiprotein complexes to mediate the editing of mitochondrial cryptogene transcripts. The L-complex contains approximately 16 proteins, including the two RNA-editing ligases (RELs), REL1 and REL2. Leishmania tarentolae REL1 and REL2 and Trypanosoma brucei REL1 were expressed as enzymatically active tandem affinity purification-tagged proteins in a Baculovirus system. When these proteins were added to mitochondrial lysates from T. brucei procyclic cells that were depleted of the cognate endogenous ligase by RNA interference down-regulation of expression, the added proteins were integrated into the L-complex, and, in the case of REL1, there was a complementation of in vitro-precleaved U-insertion and U-deletion editing activities of the 20S L-complex. Integration of the recombinant proteins did not occur or occurred at a very low level with noncognate ligase-depleted L-complex or with wild-type L-complex. A C-terminal region of the T. brucei recombinant REL1 downstream of the catalytic domain was identified as being involved in integration into the L-complex. The ability to perform functional complementation in vitro provides a powerful tool for molecular dissection of the editing reaction.

Animals↗

PubMed2005

Functional interaction of reverse gyrase with single-strand binding protein of the archaeon Sulfolobus.

Reverse gyrase is a unique hyperthermophile-specific DNA topoisomerase that induces positive supercoiling. It is a modular enzyme composed of a topoisomerase IA and a helicase domain, which cooperate in the ATP-dependent positive supercoiling reaction. Although its physiological function has not been determined, it can be hypothesized that, like the topoisomerase-helicase complexes found in every organism, reverse gyrase might participate in different DNA transactions mediated by multiprotein complexes. Here, we show that reverse gyrase activity is stimulated by the single-strand binding protein (SSB) from the archaeon Sulfolobus solfataricus. Using a combination of in vitro assays we analysed each step of the complex reverse gyrase reaction. SSB stimulates all the steps of the reaction: binding to DNA, DNA cleavage, strand passage and ligation. By co-immunoprecipitation of cell extracts we show that reverse gyrase and SSB assemble a complex in the presence of DNA, but do not make stable protein-protein interactions. In addition, SSB stimulates reverse gyrase positive supercoiling activity on DNA templates associated with the chromatin protein Sul7d. Furthermore, SSB enhances binding and cleavage of UV-irradiated substrates by reverse gyrase. The results shown here suggest that these functional interactions may have biological relevance and that the interplay of different DNA binding proteins might modulate reverse gyrase activity in DNA metabolic pathways.

Archaeal Proteins↗

PubMed2006

Pericellular proteases in angiogenesis and vasculogenesis.

Pericellular proteases play an important role in angiogenesis and vasculogenesis. They comprise (membrane-type) matrix metalloproteinases [(MT-)MMPs], serine proteases, cysteine cathepsins, and membrane-bound aminopeptidases. Specific inhibitors regulate them. Major roles in initiating angiogenesis have been attributed to MT1-matrix metalloproteinase (MMP), MMP-2, and MMP-9. Whereas MT-MMPs are membrane-bound by nature, MMP-2 and MMP-9 can localize to the membrane by binding to alphavbeta3-integrin and CD44, respectively. Proteases switch on neovascularization by activation, liberation, and modification of angiogenic growth factors and degradation of the endothelial and interstitial matrix. They also modify the properties of angiogenic growth factors and cytokines. Neovascularization requires cell migration, which depends on the assembly of protease-protein complexes at the migrating cell front. MT1-MMP and urokinase (u-PA) form multiprotein complexes in the lamellipodia and focal adhesions of migrating cells, facilitating proteolysis and sufficient support for endothelial cell migration and survival. Excessive proteolysis causes loss of endothelial cell-matrix interaction and impairs angiogenesis. MMP-9 and cathepsin L stimulate the recruitment and action of blood- or bone-marrow-derived accessory cells that enhance angiogenesis. Proteases also generate fragments of extracellular matrix and hemostasis factors that have anti-angiogenic properties. Understanding the complexity of protease activities in angiogenesis contributes to recognizing new targets for stimulation or inhibition of neovascularization in disease.

ADAM Proteins↗

PubMed2001

Functional synergy between Rab5 effector Rabaptin-5 and exchange factor Rabex-5 when physically associated in a complex.

Rab GTPases are central elements of the vesicular transport machinery. An emerging view is that downstream effectors of these GTPases are multiprotein complexes that include nucleotide exchange factors to ensure coupling between GTPase activation and effector function. We have previously shown that Rab5, which regulates various steps of transport along the early endocytic pathway, is activated by a complex consisting of Rabex-5, a Rab5 nucleotide exchange factor, and the effector Rabaptin-5. We postulated that the physical association of these two proteins is necessary for their activity in Rab5-dependent endocytic membrane transport. To evaluate the functional implications of such complex formation, we have reconstituted it with the use of recombinant proteins and characterized its properties. First, we show that Rabaptin-5 increases the exchange activity of Rabex-5 on Rab5. Second, Rab5-dependent recruitment of Rabaptin-5 to early endosomes is completely dependent on its physical association with Rabex-5. Third, complex formation between Rabaptin-5 and Rabex-5 is essential for early endosome homotypic fusion. These results reveal a functional synergy between Rabaptin-5 and Rabex-5 in the complex and have implications for the function of analogous complexes for Rab and Rho GTPases.

Animals↗

PubMed2001

SRYand architectural gene regulation: the kinetic stability of a bent protein-DNA complex can regulate its transcriptional potency.

Protein-directed DNA bending is proposed to regulate assembly of higher-order DNA-multiprotein complexes (enhanceosomes and repressosomes). Because transcriptional initiation is a nonequilibrium process, gene expression may be modulated by the lifetime of such complexes. The human testis-determining factor SRY contains a specific DNA-bending motif, the high-mobility group (HMG) box, and is thus proposed to function as an architectural factor. Here, we test the hypothesis that the kinetic stability of a bent HMG box-DNA complex can in itself modulate transcriptional potency. Our studies employ a cotransfection assay in a mammalian gonadal cell line as a model for SRY-dependent transcriptional activation. Whereas sex-reversal mutations impair SRY-dependent gene expression, an activating substitution is identified that enhances SRY's potency by 4-fold. The substitution (I13F in the HMG box; fortuitously occurring in chimpanzees) affects the motif's cantilever side chain, which inserts between base pairs to disrupt base pairing. An aromatic F13 cantilever prolongs the lifetime of the DNA complex to an extent similar to its enhanced function. By contrast, equilibrium properties (specific DNA affinity, specificity, and bending; thermodynamic stability and cellular expression) are essentially unchanged. This correlation between potency and lifetime suggests a mechanism of kinetic control. We propose that a locked DNA bend enables multiple additional rounds of transcriptional initiation per promoter. This model predicts the occurrence of a novel class of clinical variants: bent but unlocked HMG box-DNA complexes with native affinity and decreased lifetime. Aromatic DNA-intercalating agents exhibit analogous kinetic control of transcriptional elongation whereby chemotherapeutic potencies correlate with drug-DNA dissociation rates.

Amino Acid Sequence↗

PubMed2001

Biotinylation of protein complexes may lead to aggregation as well as to loss of subunits as revealed by Blue Native PAGE.

Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a high-resolution method for studying native protein complexes. Here, the migration behaviour of the B cell antigen receptor (BCR) in BN-PAGE with and without prior biotinylation of the cell surface, from which the complexes were purified, are compared. Non-biotinylated complexes appear as a single band, whereas biotinylated complexes display several bands, indicating that biotinylation leads to aggregation of complexes as well as to loss of subunits. Thus, BN-PAGE has limitations in studying multiprotein complexes. These results are used to gain insight into the stoichiometry of the complex.

Biotin↗

PubMed2001

A gamma-tubulin antibody against a plant peptide sequence localises to cell division-specific microtubule arrays and organelles in plants.

Gamma tubulin (gamma-tubulin) is involved in microtubule initiation in the eukaryotes. In animal cells it is localised to centrosomes and to other, non-centrosomal sites of microtubule initiation. In addition, cytoplasmic complexes containing gamma-tubulin (gamma-TuRCs; gamma-somes) have been described, which are multiprotein complexes involved in microtubule initiation. Most localisations of gamma-tubulin in plants have previously been achieved using an antibody directed towards a conserved peptide sequence found in animal cells, showing co-localisation with all microtubule arrays throughout the cell cycle. Because different antibodies may give various patterns of subcellular localisation, in the present study we raised a polyclonal antibody ('Hayley') to the plant peptide sequence EDFATQGGDRKDVFFY (bold letters indicate plant-specific amino acids) to further investigate the subcellular distribution in plants. Immunoblotting using wheat root tip protein extracts revealed a 58 kDagamma-tubulin-like peptide as has been described before. Immunofluorescence microscopy of wheat root-tip cells, however, revealed localisation of gamma-tubulin to a subset of mitotic microtubule arrays and the cytokinetic phragmoplast, but not to interphase cortical arrays or the preprophase band of microtubules. This lack of labelling may be caused by a restriction of antibody access during interphase, but more likely by a cell division-specific conformational change in the gamma-tubulin molecule. Our antibody also gave an organelle-like labelling, not described before, which may represent storage forms or precursors of gamma-tubulin, perhaps related to plastid-based microtubule initiation in hepatics and hornworts.

Amino Acid Sequence↗

PubMed1994

Interaction mating reveals binary and ternary connections between Drosophila cell cycle regulators.

We characterized interactions between Drosophila melanogaster cell cycle regulatory proteins by a yeast interaction-mating technique. The results were displayed as two-dimensional matrices that revealed individual binary interactions between proteins. Each protein (Cdi, cyclin-dependent kinase interactor) interacted with a distinct spectrum of cyclin-dependent kinases (Cdk) from Drosophila and other organisms. Some Cdis interacted with other Cdis, indicating that these proteins may form trimeric complexes that include Cdks. Similar analysis of interaction matrices may be generally useful in detecting other multiprotein complexes and in establishing connectivity between individual complex members. Moreover, such analysis may also help assign function to newly identified proteins, identify domains involved in protein-protein interactions, and aid the dissection of genetic regulatory networks.

Animals↗

PubMed2000

A human condensin complex containing hCAP-C-hCAP-E and CNAP1, a homolog of Xenopus XCAP-D2, colocalizes with phosphorylated histone H3 during the early stage of mitotic chromosome condensation.

Structural maintenance of chromosomes (SMC) family proteins play critical roles in structural changes of chromosomes. Previously, we identified two human SMC family proteins, hCAP-C and hCAP-E, which form a heterodimeric complex (hCAP-C-hCAP-E) in the cell. Based on the sequence conservation and mitotic chromosome localization, hCAP-C-hCAP-E was determined to be the human ortholog of the Xenopus SMC complex, XCAP-C-XCAP-E. XCAP-C-XCAP-E is a component of the multiprotein complex termed condensin, required for mitotic chromosome condensation in vitro. However, presence of such a complex has not been demonstrated in mammalian cells. Coimmunoprecipitation of the endogenous hCAP-C-hCAP-E complex from HeLa extracts identified a 155-kDa protein interacting with hCAP-C-hCAP-E, termed condensation-related SMC-associated protein 1 (CNAP1). CNAP1 associates with mitotic chromosomes and is homologous to Xenopus condensin component XCAP-D2, indicating the presence of a condensin complex in human cells. Chromosome association of human condensin is mitosis specific, and the majority of condensin dissociates from chromosomes and is sequestered in the cytoplasm throughout interphase. However, a subpopulation of the complex was found to remain on chromosomes as foci in the interphase nucleus. During late G(2)/early prophase, the larger nuclear condensin foci colocalize with phosphorylated histone H3 clusters on partially condensed regions of chromosomes. These results suggest that mitosis-specific function of human condensin may be regulated by cell cycle-specific subcellular localization of the complex, and the nuclear condensin that associates with interphase chromosomes is involved in the reinitiation of mitotic chromosome condensation in conjunction with phosphorylation of histone H3.

Amino Acid Sequence↗

PubMed2003

Activation functions 1 and 2 of nuclear receptors: molecular strategies for transcriptional activation.

Nuclear receptors (NRs) comprise a family of ligand inducible transcription factors. To achieve transcriptional activation of target genes, DNA-bound NRs directly recruit general transcription factors (GTFs) to the preinitiation complex or bind intermediary factors, so-called coactivators. These coactivators often constitute subunits of larger multiprotein complexes that act at several functional levels, such as chromatin remodeling, enzymatic modification of histone tails, or modulation of the preinitiation complex via interactions with RNA polymerase II and GTFs. The binding of NR to coactivators is often mediated through one of its activation domains. Many NRs have at least two activation domains, the ligand-independent activation function (AF)-1, which resides in the N-terminal domain, and the ligand-dependent AF-2, which is localized in the C-terminal domain. In this review, we summarize and discuss current knowledge regarding the molecular mechanisms of AF-1- and AF-2-mediated gene activation, focusing on AF-1 and AF-2 conformation and coactivator binding.

Animals↗

PubMed2000

The LIM-only protein DRAL/FHL2 binds to the cytoplasmic domain of several alpha and beta integrin chains and is recruited to adhesion complexes.

LIM proteins contain one or more double zinc finger structures (LIM domains) mediating specific contacts between proteins that participate in the formation of multiprotein complexes. We report that the LIM-only protein DRAL/FHL2, with four and a half LIM domains, can associate with alpha(3A), alpha(3B), alpha(7A), and several beta integrin subunits as shown in yeast two-hybrid assays as well as after overexpression in human cells. The amino acid sequence immediately following the conserved membrane-proximal region in the integrin alpha subunits or the C-terminal region with the conserved NXXY motif of the integrin beta subunits are critical for binding DRAL/FHL2. Furthermore, the DRAL/FHL2 associates with itself and with other molecules that bind to the cytoplasmic domain of integrin alpha subunits. Deletion analysis of DRAL/FHL2 revealed that particular LIM domains or LIM domain combinations bind the different proteins. These results, together with the fact that full-length DRAL/FHL2 is found in cell adhesion complexes, suggest that it is an adaptor/docking protein involved in integrin signaling pathways.

3T3 Cells↗

PubMed1994

A novel chaperone complex for steroid receptors involving heat shock proteins, immunophilins, and p23.

In the absence of hormone, the avian progesterone receptor exists in a large multiprotein complex that is inactive but able to bind and respond to progestins. This inactive complex can be reconstituted in vitro by incubation of receptor monomer in rabbit reticulocyte lysate in the presence of ATP and magnesium. This results in receptor binding to the two heat shock proteins, hsp90 and hsp70, the FK506 binding-proteins, FKBP54 and FKBP52, the cyclosporin A-binding protein, cyclophilin-40, and the recently characterized protein p23. Immune isolation of p23 from rabbit reticulocyte lysate in the absence of receptor reveals an ATP-dependent complex containing the major proteins associated with steroid receptors. Depletion of p23 from lysate prevents the assembly of progesterone receptor complexes, and the addition of purified p23 restores this activity, indicating that the p23 protein complex is an essential precursor to the formation of progesterone receptor complexes.

Amino Acid Isomerases↗

PubMed2003

Synapse signalling complexes and networks: machines underlying cognition.

All thoughts and actions are encoded in patterns of neuronal electrical activity. Circuits of nerve cells connected by synapses are dedicated to processing information in these patterns. Information is not only transmitted across the synapse but also monitored by postsynaptic molecular machines. These machines are macromolecular complexes of approximately 100 proteins organised into a network of protein interactions. The network can be mathematically described as a scale-free network. Components of the complexes are necessary for decoding the neural code and converting electrical information into biochemical changes. The network properties of these complexes may explain many of the features of neuronal plasticity and cognitive function in rodents. Importantly, these multiprotein complexes and their network properties shed new light on the basis of human cognitive diseases including schizophrenia, autism, Huntington's disease and mental retardation. Supplementary material for this article can be found on the BioEssays website http://www.interscience.wiley.com/jpages/0265-9247/suppmat/index.html.

Animals↗

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↗

PubMed1999

The Arp2/3 complex: a multifunctional actin organizer.

The actin-related proteins (Arps) constitute a recently characterized family of proteins, many of which function as members of multiprotein complexes. The discovery that two family members, Arp2 and Arp3, act as multifunctional organizers of actin filaments in all eukaryotes has generated much excitement. Over the past two years, newly discovered properties of the Arp2/3 complex have suggested a central role in the control of actin polymerization. First, it promotes actin assembly on the surface of the motile intracellular pathogen Listeria monocytogenes. Second, it can nucleate and cross-link actin filaments in vitro. Third, it localizes with dynamic actin-rich spots of mammalian cells suggesting a role in protrusion; it is found in cortical actin patches in the budding and fission yeasts where it may control patch movement and cortical actin function. Clearly, the complex has a central role in actin cytoskeletal function and will be the subject of much research in the coming years.

Actin-Related Protein 2↗

PubMed2002

Programming off and on states in chromatin: mechanisms of Polycomb and trithorax group complexes.

Polycomb and trithorax group proteins are evolutionarily conserved chromatin components that maintain stable states of gene expression. Recent studies have identified and characterized several multiprotein complexes containing these transcriptional regulators. Advances in understanding molecular activities of these complexes in vitro, and functional domains present in their subunits, suggest that they control transcription through multistep mechanisms that involve nucleosome modification, chromatin remodeling, and interaction with general transcription factors.

Animals↗

PubMed2003

Catalysis and electron transfer in protein crystals: the binary and ternary complexes of methylamine dehydrogenase with electron acceptors.

Polarized absorption microspectrophotometry has been used to detect catalysis and intermolecular electron transfer in single crystals of two multiprotein complexes: (1) the binary complex between Paracoccus denitrificans methylamine dehydrogenase, which contains tryptophan-tryptophylquinone (TTQ) as a cofactor, and its redox partner, the blue copper protein amicyanin; (2) the ternary complex between the same two proteins and cytochrome c-551i. Continuous wave electron paramagnetic resonance has been used to compare the state of copper in polycrystalline powders of the two systems. While catalysis and intermolecular electron transfer from reduced TTQ to copper are too fast to be accessible to our measurements, heme reduction occurs over a period of several minutes. The observed rate constant is about four orders of magnitude lower than in solution. The analysis of the temperature dependence of this apparent constant provides values for the parameters H(AB), related to electronic coupling between the two centers, and lambda, the reorganizational energy, that are compatible with electron transfer being the rate-determining step. From these parameters and the known distance between copper and heme, it is possible to calculate the parameter beta, which depends on the nature of the intervening medium, obtaining a value typical of electron transfer across a protein matrix. These findings suggest that the ternary complex in solution might achieve a higher efficiency than the rigid crystal structure thanks to an as yet unidentified role of protein dynamics.

Catalysis↗