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PubMed2005

Purification and characterization of the Caenorhabditis elegans HCF protein and domains of human HCF.

The human cellular factor (HCF) is a multidomain protein that is implicated in processes of cell cycle progression, and it is recruited into a multicomponent assembly that triggers the expression of the herpes simplex virus genome. The amino-terminal domain of HCF has been proposed to form a "kelch" type beta-propeller fold, and the carboxy-terminal domain contains a repeat of a fibronectin-like motif. We describe the expression, purification, and characterization of the domains from the human HCF and of the full-length HCF from Caenorhabditis elegans. The purified recombinant C. elegans HCF can substitute for the human HCF in efficiently forming a multiprotein complex on a herpes simplex virus promoter element. As noted in earlier studies, a segment of human HCF encompassing the human kelch domain forms a stable complex on a viral promoter element. The purified fibronectin domain can also be recruited into this complex, but not into the stable complex formed with the minimal kelch domain. These results suggest that the fibronectin domain can interact with HCF in the transcriptional activating complex and that the association requires a region outside the putative beta-propeller.

Animals↗

PubMed1996

Binding of immunophilins to the 90 kDa heat shock protein (hsp90) via a tetratricopeptide repeat domain is a conserved protein interaction in plants.

In animal cell lysates, multiprotein complexes containing hsp90, hsp70, p60, p23, and several immunophilins can assemble steroid receptors and oncogenic protein kinases, such as v-Src and v-Raf, into heterocomplexes that contain hsp90 and either immunophilins or, in the case of protein kinases, p50. The complexes with hsp90 are required for the proper functioning of these signal transduction systems. Wheat germ lysate contains a similar protein folding activity that forms functional steroid receptor complexes with hsp90, but not all the components of this system have been identified. The plant chaperone system has conserved interactions with animal chaperones in that wheat hsp70 functions in the rabbit reticulocyte lysate heterocomplex assembly system and human p23 functions in the wheat germ lysate. Here, we ask if wheat germ lysate also contains immunophilins of the FK506-binding class (FKBPs) that bind to the hsp90 component of the chaperone complex via tetratricopeptide repeat (TPR) domains. To demonstrate the plant heterocomplex, we add purified mammalian p23, preadsorbed with the JJ3 antibody to protein A-Sepharose, to wheat germ lysate and allow ATP-dependent formation of an animal p23. plant hsp90 complex. The complex is then washed and incubated with the radiolabeled immunosuppressant drug [3H]FK506, which binds in a specific manner to a coimmunoadsorbed plant FKBP. Binding of the plant FKBP to plant hsp90 is prevented by adding to wheat germ lysate a purified fragment containing the TPR domains of human cyclophilin-40. Geldanamycin, a benzoquinone ansamycin that binds to animal hsp90s and prevents their chaperone activity, binds in a temperature-dependent manner to wheat hsp90 to block formation of the p23.hsp90.FKBP heterocomplex. These data show that immunophilin binding to hsp90 via TPR domains is conserved in the plant kingdom as well as in the animal kingdom and that geldanamycin will be an important tool for the study of hsp90-mediated protein chaperoning in plant cells.

Adenosine Triphosphate↗

PubMed1997

Purification and characterization of the DNA polymerase alpha associated exonuclease: the RTH1 gene product.

We report here the purification and mechanistic characterization of a 5'-3' exonuclease associated with DNA polymerase alpha from the yeast Saccharomyces cerevisiae. Earlier, we identified a 5' --> 3' exonuclease activity that copurified with yeast DNA polymerase alpha-primase in a multiprotein complex [Biswas, E. E., et al. (1993) Biochemistry, 32, 3020-3027]. Peptide sequence analysis of the purified 47 kDa exonuclease was carried out, and the peptide sequence was found to be identical to the S. cerevisiae gene YKL510 encoded polypeptide, which is also known as yeast RAD2 homolog 1 or RTH1 nuclease. The native exonuclease also had strong flap endonuclease activity similar to that observed with RTH1 nuclease and homologous yeast (RAD2) and mammalian enzymes. During our studies, we have discovered certain unique features of the mechanism of action of the native RTH1 nuclease. Studies presented here indicated that the exonuclease had specific pause sites during its 5'-3' exonuclease nucleotide excision. These pause sites were easily detected with long (approximately 50 bp) oligonucleotide substrates during exonucleolytic excision by the formation of a discontinuous ladder of excision product. We have further analyzed the mechanism of generation of the pause sites, as they could occur through a number of different pathways. Alignment of the pause sites with the nucleotide sequence of the oligonucleotide substrate indicated that the pause sites were dependent on the nucleotide sequence. Our analysis revealed that RTH1 nuclease pauses predominantly at G:C rich sequences. With poly(dA):oligo(dT)50 as substrate, the exonucleolytic products formed a continuous ladder with no evidence of pausing. The G:C rich DNA sequences are thermodynamically more stable than the A:T rich sequences, which may be in part responsible for pausing of the RTH1 5' --> 3' exonuclease at these sites.

Amino Acid Sequence↗

PubMed1998

Destabilized PCNA trimers suppress defective Rfc1 proteins in vivo and in vitro.

Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are two essential DNA polymerase accessory proteins that are required for numerous aspects of DNA metabolism including DNA replication, DNA repair, and telomere metabolism. PCNA is a homotrimeric ring-shaped sliding DNA clamp that can facilitate DNA replication by tethering DNA polymerase delta or DNA polymerase epsilon to the DNA template. RFC is the 5-subunit multiprotein complex that loads PCNA onto DNA at primer-template junctions in an ATP-dependent reaction. All five of the RFC subunits share a set of related sequences (RFC boxes) that include nucleotide-binding consensus sequences. We report here that a mutation in the gene encoding the large subunit of yeast RFC gives rise to DNA metabolism defects that can be observed in vivo and in vitro. The rfc1-1 substitution (D513N) lies within the widely conserved RFC box VIII consensus sequence and results in phenotypes including DNA replication defects, increased sensitivity to DNA damaging agents, and elongated telomeres. Mutant Rfc1-1 complexes exhibit in vitro DNA replication defects that are sensitive to ATP concentrations, and these defects can be suppressed by mutant PCNA proteins which contain substitutions that destabilize the homotrimeric sliding DNA clamp.

Adenosine Triphosphate↗

PubMed2006

Thermodynamic basis for promiscuity and selectivity in protein-protein interactions: PDZ domains, a case study.

Like other protein-protein interaction domains, PDZ domains are involved in many key cellular processes. These processes often require that specific multiprotein complexes be assembled, a task that PDZ domains accomplish by binding to specific peptide motifs in target proteins. However, a growing number of experimental studies show that PDZ domains (like other protein-protein interaction domains) can engage in a variety of interactions and bind distinct peptide motifs. Such promiscuity in ligand recognition raises intriguing questions about the molecular and thermodynamic mechanisms that can sustain it. To identify possible sources of promiscuity and selectivity underlying PDZ domain interactions, we performed molecular dynamics simulations of 20 to 25 ns on a set of 12 different PDZ domain complexes (for the proteins PSD-95, Syntenin, Erbin, GRIP, NHERF, Inad, Dishevelled, and Shank). The electrostatic, nonpolar, and configurational entropy binding contributions were evaluated using the MM/PBSA method combined with a quasi-harmonic analysis. The results revealed that PDZ domain interactions are characterized by overwhelmingly favorable nonpolar contributions and almost negligible electrostatic components, a mix that may readily sustain promiscuity. In addition, despite the structural similarity in fold and in recognition modes, the entropic and other dynamical aspects of binding were remarkably variable not only across PDZ domains but also for the same PDZ domain bound to distinct ligands. This variability suggests that entropic and dynamical components can play a role in determining selectivity either of PDZ domain interactions with peptide ligands or of PDZ domain complexes with downstream effectors.

Computer Simulation↗

PubMed2006

Coupling artificial actin cortices to biofunctionalized lipid monolayers.

We report the assembly of protein supramolecular structures at an air-water interface and coupling of artificial actin cortices to such structures. The coupling strategies adopted include electrostatic binding of actin to monolayers doped with lipids, exposing positively charged poly(ethylene glycol) headgroups; binding of biotinylated actin to lipids carrying biotin headgroups through avidin; binding of actin to membranes through biotinylated hisactophilin (a cellular actin-membrane coupler) using an avidin-biotin linkage; and coupling of actin to membranes carrying chelating lipids through a 15-nm-diameter protein capsid (bacterial lumazine synthase or LuSy) exhibiting histidine tags (which bind both to actin and to the chelating lipid). The distribution of the proteins in a direction normal to the interface was measured by neutron reflectivity under different conditions of pH and ionic strength. In the case of the first three binding methods, the thickness of the actin film was found to correspond to a single actin filament. Multilayers of actin could be formed only by using the multifunctional LuSy couplers that exhibit 60 hexahistidine tags and can thus act as actin cross-linkers. The LuSy-mediated binding can be reversibly switched by pH variations.

Actins↗

PubMed1997

Proteasome (prosome) associated endonuclease activity.

The 20S proteasome (prosome) is a highly organized multiprotein complex with approximate molecular weight of about 700 kDa. Whilst the role of the proteasome in the processing and turnover of cellular proteins is becoming clearer, its relationship with RNA remains still obscure. Here we focus on the nature and function of proteasome associated endonuclease activity. Thus the involvement of a proteasome alpha-type subunit in RNA-degradation, the catalytic requirements, the interaction of proteasomes with their RNA-substrate and the identification of a well defined cleavage site in the 3'UTR of short-lived cellular mRNAs will be described in detail. All data indicate that proteasomes associated endonuclease activity could be involved in post-transcriptional gene control at the level of translation.

Animals↗

PubMed2001

The structural maintenance of chromosomes (SMC) family of proteins in mammals.

Structural maintenance of chromosomes (SMC) family proteins play critical roles in chromosome structural changes. SMC proteins are known to be involved in two major chromosome structural organization events required for mitotic segregation of chromosomes: mitotic chromosome condensation and sister chromatid cohesion. In eukaryotes, two separate sets of SMC heterodimers form the cores of two distinct multiprotein complexes termed 'condensin' and 'cohesin', each specialized for condensation or cohesion, respectively. It is clear that both condensin and cohesin are conserved in mammals, including humans. The mammalian complexes demonstrate dynamic changes in intracellular distribution in a cell cycle-dependent manner. At any point in the cell cycle, the intracellular localization of the majority of mammalian cohesin and condensin appears to be complementary. Cohesin is associated with chromatin in interphase, while condensin is largely cytoplasmic. Similarly, in mitosis, cohesin is mostly excluded from chromosomes while condensin is distinctly bound to them. Cell cycle-dependent targeting of the two complexes appears to play a major role in regulating their cell cycle-specific activities, and how this redistribution is controlled is an area of active research. Finally, there is evidence that SMC proteins may be involved in DNA recombination and repair. This review focuses on what we have learned about SMC family proteins in humans and other mammalian species in comparison to those in lower eukaryotes. The authors present their own views with regard to some of the major outstanding questions surrounding the nature and functions of the SMC family of proteins.

Animals↗

PubMed2001

Clues to calcineurin function in mammalian fast-twitch muscle.

It is believed that brief, high amplitude Ca2+ transients, as found in fast-twitch muscles, are not sufficient to activate the calcineurin (Cn)-dependent signaling pathway involved in regulation of slow myosin and slow sarcoplasmic reticulum Ca2+-ATPase genes (Olson and Williams, Cell 101: 689-692, 2000). The results reported here try to fill the gap between this molecular knowledge, and the still fragmentary pieces of information on a possible different role of calcineurin in the same type of muscles. In the present work calcineurin was determined immunocytochemically by labeling fast- and slow-twitch fibers of representative rabbit muscles with anti-CnB antibodies, and was assessed by western blotting of isolated subcellular fractions. Calcineurin was found to be largely soluble and to be constitutively overexpressed in fast muscle as CnAalpha and CnAbeta isoforms, the latter appearing to be predominant. Particulate calcineurin was not only associated with myofibrils but also with membranes of various origins. Fluorescence microscopy showed that calcineurin was distributed in the same pattern with respect to sarcomeres in both types of fibers, and formed punctate dots spanning the I-Z-I region, rather than being exclusively located at the Z-line, a disposition described for cardiomyocytes (Frey et al., Proc Natl Acad Sci USA 97: 14,632-14,637, 2000). From knowledge that, in mammalian skeletal muscle fibers, junctional triads are located at the A-I band boundary, we explored the distribution of calcineurin between triadic components, after having verified that it was present in very low amounts in dystrophin-enriched sarcolemmal membranes. Our results demonstrate that a small but significant proportion of calcineurin coenriched with transverse tubules (TT), and copurified with the DHPR and with DHPR-associated PKA-AKAP15/18, thus suggesting that it is assembled as a multiprotein complex in the junctional membrane domain of TT. The membrane specificity of this association is further corroborated by the negative evidence for the presence of calcineurin in SR terminal cisternae. Calcineurin was separated from the DHPR and isolated as a AKAP15/18 subcomplex, including beta2 adrenergic receptor, in addition to PKA and calcineurin, following equilibrium centrifugation of detergent extracts on a linear sucrose gradient. We show that the alpha1 subunit skeletal isoform of the DHPR, is a substrate for calcineurin dephosphorylation, after previous phosphorylation by endogenous PKA.

Animals↗

PubMed2002

Localization of human SMC1 protein at kinetochores.

Proper cohesion of sister chromatids is prerequisite for correct segregation of chromosomes during cell division. The cohesin multiprotein complex, conserved in eukaryotes, is required for sister chromatid cohesion. Human cohesion is composed of a stable heterodimer of the structural maintenance of chromosomes (SMC) family proteins, hSMC1 and hSMC3, and non-SMC components, hRAD21 and SA1 (or SA2). In yeast, cohesion associates with chromosomes from late G1 to metaphase and is required for the establishment and maintenance of both chromosome arm and centromeric cohesion. However, in human cells, the majority of cohesion dissociates from chromosomes before mitosis. Although it was recently shown that a small amount of hRAD21 localizes to the centromeres during metaphase, the presence of other cohesion components at the centromere has not been demonstrated in human cells. Here we report the mitosis-specific localization of hSMC1 to the kinetochores. hSMC1 is targeted to the kinetochore region during prophase concomitant with kinetochore assembly and remains through anaphase. Importantly, hSMC1 is targeted only to the active centromere on dicentric chromosomes. These results suggest that hSMC1 is an integral component of the functional kinetochore structure during mitosis.

Antibody Specificity↗

PubMed2002

The relationship between alveolar epithelial proliferation and airway obstruction after ozone exposure.

BACKGROUND: Proliferating cell nuclear antigen (PCNA) is a component of multiprotein complexes that are expressed during cell proliferation. Ozone induces cell necrosis and a consequent increase in cell proliferation. METHODS: To investigate the effects of acute ozone inhalation on cell proliferation and airway obstruction in BALB/C mice, we examined enhanced pause (Penh) as an index of airway obstruction and PCNA expression by immunohistochemical staining. RESULTS: Compared with controls that received filtered air, the ozone-exposed groups had increased PCNA expression in the alveolar epithelial cells. In rank order, the highest PCNA index was found following 2.0 p.p.m. ozone exposure. In the 2.0 p.p.m. ozone group, there was a PCNA index of 16.83 +/- 0.57% (mean +/- SEM; P< 0.01), compared with 4.25 +/- 0.5% at 0.12 p.p.m., 6.83 +/- 0.60 at 0.5 p.p.m and 12.16 +/- 0.48% at 1 p.p.m. Following ozone exposure, Penh was increased in a dose-dependent manner. There was a significant correlation between the PCNA index in alveoli and Penh (r = 0.63, P< 0.01). CONCLUSIONS: These data suggest that ozone can induce alveolar epithelial cell proliferation in a dose-dependent manner, and that alveolar epithelial cell proliferation is correlated with airway obstruction.

Administration, Inhalation↗

PubMed2003

The TCR ADAPts to integrin-mediated cell adhesion.

Adapter proteins regulate leukocyte signal transduction through recruitment of effector molecules to multiprotein complexes. Recent studies in Adhesion and Degranulation promoting Adapter Protein (ADAP)-deficient mice have established that the cytoplasmic phosphoprotein ADAP is required for optimal, mature T-cell proliferation. Furthermore, ADAP plays a key role in T-cell antigen receptor (TCR)-mediated 'inside out' signaling leading to integrin activation and to enhanced cellular adhesion to integrin ligands. ADAP associates physically with molecules known to play roles in the regulation of TCR-stimulated actin polymerization. These associations support the hypothesis that ADAP functions in actin cytoskeletal reorganization leading to cellular adhesion and activation.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1.

Cohesion between sister chromatids is established during DNA replication and depends on a multiprotein complex called cohesin. Attachment of sister kinetochores to the mitotic spindle during mitosis generates forces that would immediately split sister chromatids were it not opposed by cohesion. Cohesion is essential for the alignment of chromosomes in metaphase but must be abolished for sister separation to start during anaphase. In the budding yeast Saccharomyces cerevisiae, loss of sister-chromatid cohesion depends on a separating protein (separin) called Esp1 and is accompanied by dissociation from the chromosomes of the cohesion subunit Scc1. Here we show that Esp1 causes the dissociation of Scc1 from chromosomes by stimulating its cleavage by proteolysis. A mutant Scc1 is described that is resistant to Esp1-dependent cleavage and which blocks both sister-chromatid separation and the dissociation of Scc1 from chromosomes. The evolutionary conservation of separins indicates that the proteolytic cleavage of cohesion proteins might be a general mechanism for triggering anaphase.

Anaphase↗

PubMed1998

Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer.

Malignant rhabdoid tumours (MRTs) are extremely aggressive cancers of early childhood. They can occur in various locations, mainly the kidney, brain and soft tissues. Cytogenetic and molecular analyses have shown that the deletion of region 11.2 of the long arm of chromosome 22 (22q11.2) is a recurrent genetic characteristic of MRTs, indicating that this locus may encode a tumour suppressor gene. Here we map the most frequently deleted part of chromosome 22q11.2 from a panel of 13 MRT cell lines. We observed six homozygous deletions that delineate the smallest region of overlap between the cell lines. This region is found in the hSNF5/INI1 gene, which encodes a member of the chromatin-remodelling SWI/SNF multiprotein complexes. We analysed the sequence of hSNF5/INI1 and found frameshift or nonsense mutations of this gene in six other cell lines. These truncating mutations of one allele were associated with the loss of the other allele. Identical alterations were observed in corresponding primary tumour DNAs but not in matched constitutional DNAs, indicating that they had been acquired somatically. The observation of bi-allelic alterations of hSNF5/INI1 in MRTs suggests that loss-of-function mutations of hSNF5/INI1 contribute to oncogenesis.

Child↗

PubMed1990

Sequencing and expression of complementary DNA for the general transcription factor BTF3.

The initiation of transcription of eukaryotic genes involves the ordered assembly of a multiprotein complex on proximal promoter elements such as the TATA box. In addition to RNA polymerase II (otherwise RNA pol II, RNA polymerase B), four general transcription factors are required for initiation of transcription: BTF1 (also referred to as TFIID) which has recently been cloned from yeast, BTF2, BTF3 and STF. The first step in assembly of the initiation complex is the stable binding of BTF1 to the TATA box, which is facilitated by STF. Neither BTF2 nor BTF3 bind directly to the promoter proximal elements, but BTF3 can form a stable complex with RNA pol II. We recently purified BTF3, which is a protein of relative molecular mass 27,000, but further studies have been hampered by its low abundance in cells. On the basis of sequences from peptides of BTF3, we have now cloned two complementary DNAs, one for a protein (BTF3a) with all the characteristics of purified BTF3, and one for a shorter protein (BTF3b) lacking the first 44 residues of BTF3a and which is transcriptionally inactive, despite its ability to bind RNA pol II.

Amino Acid Sequence↗

PubMed2000

Nuclear translocation and transcription regulation by the membrane-associated guanylate kinase CASK/LIN-2.

Membrane-associated guanylate kinases (MAGUKs) contain multiple protein-binding domains that allow them to assemble specific multiprotein complexes in particular regions of the cell. CASK/LIN-2, a MAGUK required for EGF receptor localization and signalling in Caenorhabditis elegans, contains a calmodulin-dependent protein kinase-like domain followed by PDZ, SH3 and guanylate kinase-like domains. In adult rat brain, CASK is concentrated at neuronal synapses and binds to the cell-surface proteins neurexin and syndecan and the cytoplasmic proteins Mint/LIN-10 and Veli/LIN-7. Here we report that, through its guanylate kinase domain, CASK interacts with Tbr-1, a T-box transcription factor that is involved in forebrain development. CASK enters the nucleus and binds to a specific DNA sequence (the T-element) in a complex with Tbr-1. CASK acts as a coactivator of Tbr-1 to induce transcription of T-element containing genes, including reelin, a gene that is essential for cerebrocortical development. Our findings show that a MAGUK which is usually associated with cell junctions has a transcription regulation function.

Animals↗

PubMed2000

The Gcn5 bromodomain co-ordinates nucleosome remodelling.

The access of transcription factors to eukaryotic promoters often requires modification of their chromatin structure, which is accomplished by the action of two general classes of multiprotein complexes. One class contains histone acetyltransferases (HATs), such as Gcn5 in the SAGA complex, which acetylate nucleosomal histones. The second class contains ATPases, such as Swi2 in the Swi/Snf complex, which provide the energy for nucleosome remodelling. In several promoters these two complexes cooperate but their functional linkage is unknown. A protein module that is present in all nuclear HATs, the bromodomain, could provide such a link. The recently reported in vitro binding of a HAT bromodomain with acetylated lysines within H3 and H4 amino-terminal peptides indicates that this interaction may constitute a targeting step for events that follow histone acetylation. Here we use a suitable promoter to show that bromodomain residues essential for acetyl-lysine binding are not required in vivo for Gcn5-mediated histone acetylation but are fundamental for the subsequent Swi2-dependent nucleosome remodelling and consequent transcriptional activation. We show that the Gcn5 bromodomain stabilizes the Swi/Snf complex on this promoter.

Acetylation↗

PubMed2000

Global histone acetylation and deacetylation in yeast.

Histone acetyltransferases and deacetylases can be targeted to promoters to activate or repress genes. For example, the histone acetyltransferase GCN5 is part of a yeast multiprotein complex that is recruited by the DNA-binding activator protein GCN4 (refs 1-3). The histone deacetylase RPD3 complex is recruited to DNA by the repressor UME6 (refs 4, 5); similar mechanisms exist in other eukaryotes. However, deletion of RPD3 also increases expression of the PHO5 gene that is repressed by nucleosomes, and regulated by GCN5 (ref. 10) but not by UME6. We have determined whether acetylation and deacetylation are promoter specific at PHO5, by using antibodies against acetylated lysine residues and chromatin immunoprecipitation to examine the acetylation state of a 4.25-kilobase region surrounding the PHO5 gene. Here we show that this region is acetylated extensively by ESA1 and GCN5 and deacetylated by HDA1 and RPD3, and that widespread histone modification affects three separate chromosomal regions examined, which total 22kb. Our data indicate that targeted modification occurs in a background of global acetylation and deacetylation that not only reduces basal transcription, but also allows a rapid return to the initial state of acetylation when targeting is removed.

Acetylation↗