ABSTRACT
The exon-1 peptide of huntingtin has 51 Gln repeats and produces the symptoms of Huntington's disease in transgenic mice. Aggregation of the yeast Sup35 protein into prions has been attributed to its glutamine-rich and asparagine-rich domain. Here, we show that poly-L-asparagine forms polar zippers similar to those of poly-L-glutamine. In solution at acid pH, the glutamine-rich and asparagine-rich 18-residue Sup35 peptide, rendered soluble by the addition of two aspartates at the amino end and two lysines at the carboxyl end, gives a beta-sheet CD spectrum; it aggregates at neutral pH. A poly-alanine peptide D(2)A(10)K(2) gives an alpha-helical CD spectrum at all pHs and does not aggregate; a peptide with the sequence of the C-terminal helix of the alpha-chain of human hemoglobin, preceded by two aspartates and followed by two lysines, exhibits a random coil spectrum and does not aggregate either. Alignment of several beta-strands with the sequence of the 42-residue Alzheimer's amyloid beta-peptide shows that they can be linked together by a network of salt bridges. We also asked why single amino acid replacements can so destabilize the native structures of proteins that they unfold and form amyloids. The difference in free energy of a protein molecule between its native, fully ordered structure and an amorphous mixture of randomly coiled chains is only of the order of 10 kcal/mol. Theory shows that destabilization of the native structure by no more than 2 kcal/mol can increase the probability of nucleation of disordered aggregates from which amyloids could grow 130,000-fold.
Subject(s)
Alanine/chemistry , Amyloid beta-Peptides/chemistry , Asparagine/chemistry , Fungal Proteins/metabolism , Glutamine/chemistry , Peptide Fragments/chemistry , Prions , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Amino Acids/chemistry , Animals , Circular Dichroism , Exons , Hemoglobins/chemistry , Humans , Hydrogen-Ion Concentration , Light , Lysine/chemistry , Mice , Mice, Transgenic , Molecular Sequence Data , Peptide Termination Factors , Peptides/chemistry , Protein Binding , Protein Structure, Tertiary , Scattering, Radiation , Thermodynamics , Ultraviolet Rays , X-Ray DiffractionABSTRACT
The huntingtin interacting protein (HIP1) is enriched in membrane-containing cell fractions and has been implicated in vesicle trafficking. It is a multidomain protein containing an N-terminal ENTH domain, a central coiled-coil forming region and a C-terminal actin-binding domain. In the present study we have identified three HIP1 associated proteins, clathrin heavy chain and alpha-adaptin A and C. In vitro binding studies revealed that the central coiled-coil domain is required for the interaction of HIP1 with clathrin, whereas DPF-like motifs located upstream to this domain are important for the binding of HIP1 to the C-terminal 'appendage' domain of alpha-adaptin A and C. Expression of full length HIP1 in mammalian cells resulted in a punctate cytoplasmic immunostaining characteristic of clathrin-coated vesicles. In contrast, when a truncated HIP1 protein containing both the DPF-like motifs and the coiled-coil domain was overexpressed, large perinuclear vesicle-like structures containing HIP1, huntingtin, clathrin and endocytosed transferrin were observed, indicating that HIP1 is an endocytic protein, the structural integrity of which is crucial for maintenance of normal vesicle size in vivo.
Subject(s)
Carrier Proteins/physiology , Clathrin/chemistry , DNA-Binding Proteins , Endocytosis/physiology , Adaptor Protein Complex alpha Subunits , Animals , COS Cells , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Line , Clathrin/metabolism , Clathrin-Coated Vesicles/metabolism , Humans , Membrane Proteins/metabolism , Microscopy, Fluorescence , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , TransferrinABSTRACT
The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin-proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14-3-3, and alpha-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin-proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.
Subject(s)
Acetylcysteine/analogs & derivatives , Heat-Shock Proteins , Inclusion Bodies/metabolism , Mutation , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Peptide Fragments/metabolism , Proteins , 14-3-3 Proteins , Acetylcysteine/pharmacology , Carrier Proteins/metabolism , Cell Line , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Endoplasmic Reticulum Chaperone BiP , Exons , Humans , Huntingtin Protein , Huntington Disease/metabolism , Immunoblotting , Inclusion Bodies/ultrastructure , Membrane Proteins/metabolism , Microscopy, Fluorescence , Models, Biological , Molecular Chaperones/metabolism , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/metabolism , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Peptide Fragments/genetics , Poly(A)-Binding Proteins , Proteasome Endopeptidase Complex , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Synucleins , T-Cell Intracellular Antigen-1 , Transgenes , Tyrosine 3-Monooxygenase/metabolism , Vimentin/metabolism , alpha-SynucleinABSTRACT
Unwinding of double-stranded DNA into single-stranded intermediates required for various fundamental life processes is catalyzed by helicases, a family of mono-, di- or hexameric motor proteins fueled by nucleoside triphosphate hydrolysis. The three-dimensional crystal structure of the hexameric helicase RepA encoded by plasmid RSF1010 has been determined by X-ray diffraction at 2.4 A resolution. The hexamer shows an annular structure with 6-fold rotational symmetry and a approximately 17 A wide central hole, suggesting that single-stranded DNA may be threaded during unwinding. Homologs of all five conserved sequence motifs of the DnaB-like helicase family are found in RepA, and the topography of the monomer resembles RecA and the helicase domain of the bacteriophage T7 gp4 protein. In a modeled complex, ATP molecules are located at the subunit interfaces and clearly define adenine-binding and ATPase catalytic sites formed by amino acid residues located on adjacent monomers; most remarkable is the "arginine finger" Arg207 contributing to the active site in the adjacent monomer. This arrangement of active-site residues suggests cooperativity between monomers in ATP hydrolysis and helicase activity of RepA. The mechanism of DNA unwinding remains elusive, as RepA is 6-fold symmetric, contrasting the recently published asymmetric structure of the bacteriophage T7 gp4 helicase domain.
Subject(s)
DNA Helicases/chemistry , DNA-Binding Proteins , Gram-Negative Bacteria/enzymology , Gram-Negative Bacteria/genetics , Plasmids/genetics , Proteins/chemistry , Trans-Activators , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Motifs , Amino Acid Sequence , Arginine/chemistry , Binding Sites , Conserved Sequence , Crystallography, X-Ray , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Primase/chemistry , Dimerization , Models, Molecular , Molecular Sequence Data , Protein Structure, Quaternary , Protein Structure, Tertiary , Protein Subunits , Proteins/genetics , Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence AlignmentABSTRACT
The accumulation of insoluble protein aggregates in intra and perinuclear inclusions is a hallmark of Huntington's disease (HD) and related glutamine-repeat disorders. A central question is whether protein aggregation plays a direct role in the pathogenesis of these neurodegenerative diseases. Here we show by using a filter retardation assay that the mAb 1C2, which specifically recognizes the elongated polyglutamine (polyQ) stretch in huntingtin, and the chemical compounds Congo red, thioflavine S, chrysamine G, and Direct fast yellow inhibit HD exon 1 protein aggregation in a dose-dependent manner. On the other hand, potential inhibitors of amyloid-beta formation such as thioflavine T, gossypol, melatonin, and rifampicin had little or no inhibitory effect on huntingtin aggregation in vitro. The results obtained by the filtration assay were confirmed by electron microscopy, SDS/PAGE, and MS. Furthermore, cell culture studies revealed that the Congo red dye at micromolar concentrations reduced the extent of HD exon 1 aggregation in transiently transfected COS cells. Together, these findings contribute to a better understanding of the mechanism of huntingtin fibrillogenesis in vitro and provide the basis for the development of new huntingtin aggregation inhibitors that may be effective in treating HD.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Huntington Disease/therapy , Nerve Tissue Proteins/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Peptides/antagonists & inhibitors , Animals , Benzoates/pharmacology , Benzothiazoles , Biphenyl Compounds/pharmacology , COS Cells , Congo Red/pharmacology , Gossypol/pharmacology , Humans , Huntingtin Protein , Melatonin/pharmacology , Rifampin/pharmacology , Thiazoles/pharmacologySubject(s)
Amyloid/isolation & purification , Filtration , Nerve Tissue Proteins/isolation & purification , Nuclear Proteins/isolation & purification , Peptides/isolation & purification , Amino Acid Sequence , Amyloid/chemistry , Escherichia coli , Glutathione Transferase/chemistry , Glutathione Transferase/genetics , Humans , Huntingtin Protein , Immunoblotting/methods , Membranes, Artificial , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Peptides/chemistry , Protein Conformation , Recombinant Fusion Proteins/chemistrySubject(s)
Peptides/analysis , Animals , Cell Nucleus/metabolism , Huntington Disease/pathology , Immunohistochemistry/methods , Inclusion Bodies/metabolism , Macromolecular Substances , Mice , Mice, Transgenic , Microscopy, Electron/methods , Muscle, Skeletal/metabolism , Neurons/metabolism , Peptides/chemistryABSTRACT
We have shown previously by electron microscopy that the purified glutathione S-transferase (GST)-Huntington's disease (HD) exon 1 fusion protein with 51 glutamine residues (GST-HD51) is an oligomer, and that site-specific proteolytic cleavage of this fusion protein results in the formation of insoluble more highly ordered protein aggregates with a fibrillar or ribbon-like morphology (E. Scherzinger et al. (1997) Cell 90, 549-558). Here we report that a truncated GST HD exon 1 fusion protein with 51 glutamine residues, which lacks the proline-rich region C-terminal to the polyglutamine (polyQ) tract (GST-HD51 delta P) self-aggregates into high-molecular-mass protein aggregates without prior proteolytic cleavage. Electron micrographs of these protein aggregates revealed thread-like fibrils with a uniform diameter of ca. 25 nm. In contrast, proteolytic cleavage of GST-HD51 delta P resulted in the formation of numerous clusters of high-molecular-mass fibrils with a different, ribbon-like morphology. These structures were reminiscent of prion rods and beta-amyloid fibrils in Alzheimer's disease. In agreement with our previous results with full-length GST-HD exon 1, the truncated fusion proteins GST-HD20 delta P and GST-HD30 delta P did not show any tendency to form more highly ordered structures, either with or without protease treatment.
Subject(s)
Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Trinucleotide Repeat Expansion/genetics , Cloning, Molecular , Escherichia coli , Exons , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Humans , Huntingtin Protein , Huntington Disease/genetics , Microscopy, Electron , Nerve Tissue Proteins/ultrastructure , Nuclear Proteins/ultrastructure , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/ultrastructure , Sequence DeletionABSTRACT
Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine [poly(Q)] repeat expansion in the first exon of the huntingtin protein. Previously, we showed that N-terminal huntingtin peptides with poly(Q) tracts in the pathological range (51-122 glutamines), but not with poly(Q) tracts in the normal range (20 and 30 glutamines), form high molecular weight protein aggregates with a fibrillar or ribbon-like morphology, reminiscent of scrapie prion rods and beta-amyloid fibrils in Alzheimer's disease. Here we report that the formation of amyloid-like huntingtin aggregates in vitro not only depends on poly(Q) repeat length but also critically depends on protein concentration and time. Furthermore, the in vitro aggregation of huntingtin can be seeded by preformed fibrils. Together, these results suggest that amyloid fibrillogenesis in Huntington's disease, like in Alzheimer's disease, is a nucleation-dependent polymerization.
Subject(s)
Huntington Disease/pathology , Nerve Tissue Proteins/biosynthesis , Nuclear Proteins/biosynthesis , Peptide Fragments/metabolism , Peptides/metabolism , Amino Acid Sequence , Amyloid/metabolism , Amyloid/ultrastructure , Animals , COS Cells , Cloning, Molecular , Escherichia coli , Humans , Huntingtin Protein , Huntington Disease/metabolism , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nuclear Proteins/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , TransfectionABSTRACT
The mechanism by which aggregated polygins cause the selective neurodegeneration in Huntington's disease (HD) is unknown. Here, we show that the SH3GL3 protein, which is preferentially expressed in brain and testis, selectively interacts with the HD exon 1 protein (HDex1p) containing a glutamine repeat in the pathological range and promotes the formation of insoluble polyglutamine-containing aggregates in vivo. The C-terminal SH3 domain in SH3GL3 and the proline-rich region in HDex1p are essential for the interaction. Coimmunoprecipitations and immunofluorescence studies revealed that SH3GL3 and HDex1p colocalize in transfected COS cells. Additionally, an anti-SH3GL3 antibody was also able to coimmunoprecipitate the full-length huntingtin from an HD human brain extract. The characteristics of the interaction between SH3GL3 and huntingtin and the colocalization of the two proteins suggest that SH3GL3 could be involved in the selective neuronal cell death in HD.
Subject(s)
Carrier Proteins/genetics , Carrier Proteins/metabolism , Glutamine/genetics , Huntington Disease/genetics , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , src Homology Domains/genetics , Adaptor Proteins, Signal Transducing , Animals , Brain Chemistry , COS Cells , Carrier Proteins/isolation & purification , Exons/genetics , Gene Expression , Glutamine/metabolism , Humans , Huntingtin Protein , Nerve Tissue Proteins/isolation & purification , Nerve Tissue Proteins/metabolism , Nuclear Proteins/isolation & purification , Nuclear Proteins/metabolism , Peptide Fragments/genetics , Peptide Fragments/metabolism , Precipitin Tests , Proline , RNA, Messenger/analysis , Rabbits , Repetitive Sequences, Nucleic Acid , Subcellular Fractions/chemistry , Yeasts/geneticsABSTRACT
We have developed a technique to establish catalogues of protein products of arrayed cDNA clones identified by DNA hybridisation or sequencing. A human fetal brain cDNA library was directionally cloned in a bacterial vector that allows IPTG-inducible expression of His6-tagged fusion proteins. Using robot technology, the library was arrayed in microtitre plates and gridded onto high-density in situ filters. A monoclonal antibody recognising the N-terminal RGSH6sequence of expressed proteins (RGS.His antibody, Qiagen) detected 20% of the library as putative expression clones. Two example genes, GAPDH and HSP90alpha, were identified on high-density filters using DNA probes and antibodies against their proteins.
Subject(s)
Antibodies, Monoclonal , DNA, Complementary/genetics , Gene Library , Proteins/genetics , Proteins/immunology , Brain Chemistry , Cloning, Molecular , Fetus , Gene Expression , Humans , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunologyABSTRACT
It has been reported that the ataxin-3 protein containing a polyglutamine sequence in the pathological range (61-84Q) is localized within the nucleus of neuronal cells, whereas ataxin-3 with a normal repeat length (12-37Q) is predominantly a cytoplasmic protein. In this study, the subcellular localization of the full-length ataxin-3 protein with a glutamine sequence in the normal range (Q3KQ22) was analysed in two mammalian cell lines. Using two affinity-purified polyclonal antibodies raised against the N- or C-terminal portion of ataxin-3, the protein was detected predominantly, but not exclusively, in the nucleus of COS-7 as well as neuroblastoma cells by immunofluorescence and confocal laser scanning microscopy (CLSM). The distribution of the protein in these cellular compartments was confirmed by biochemical subcellular fractionations. Furthermore, CLSM revealed that the ataxin-3 protein present in the nucleus of neuroblastoma cells is associated with the inner nuclear matrix. Our results taken together with the finding of a nuclear localization signal in ataxin-3 indicate that the ataxin-3 protein per se translocates to the nucleus and that an expanded glutamine repeat is not essential for this transport.
Subject(s)
Cell Nucleus/metabolism , Nerve Tissue Proteins/metabolism , Nuclear Matrix/metabolism , Animals , Antibodies/immunology , Ataxin-3 , Biological Transport , COS Cells , Cell Fractionation , Escherichia coli , Glutamine/metabolism , Humans , Microscopy, Confocal , Microscopy, Fluorescence , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/immunology , Nuclear Localization Signals , Nuclear Proteins , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Repressor Proteins , Transfection , Tumor Cells, CulturedABSTRACT
An initial stage of fibrillogenesis in solutions of glutathione S-transferase-huntingtin (GST-HD) fusion proteins has been studied by using dynamic light scattering. Two GST-HD systems with poly-L-glutamine (polyGln) extensions of different lengths (20 and 51 residues) have been examined. For both systems, kinetics of z-average translation diffusion coefficients (Dapp) and their angular dependence have been obtained. Our data reveal that aggregation does occur in both GST-HD51 and GST-HD20 solutions, but that it is much more pronounced in the former. Thus, our approach provides a powerful tool for the quantitative assay of GST-HD fibrillogenesis in vitro.
Subject(s)
Glutathione Transferase/chemistry , Huntington Disease/metabolism , Nerve Tissue Proteins/chemistry , Nuclear Proteins/chemistry , Protein Conformation , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Humans , Huntingtin Protein , Light , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Spectrum AnalysisABSTRACT
The RepA protein of the mobilizable broad host range plasmid RSF1010 has a key function in its replication. RepA is one of the smallest known helicases. The protein forms a homohexamer of 29,896-Da subunits. A variety of methods were used to analyze the quaternary structure of RepA. Gel filtration and cross-linking experiments demonstrated the hexameric structure, which was confirmed by electron microscopy and image reconstruction. These results agree with recent data obtained from RepA crystals diffracting at 3.5-A resolution (Röleke, D., Hoier, H., Bartsch, C., Umbach, P., Scherzinger, E., Lurz, R., and Saenger, W. (1997) Acta Crystallogr. Sec. D 53, 213-216). The RepA helicase has 5' --> 3' polarity. As do most true replicative helicases, RepA prefers a tailed substrate with an unpaired 3'-tail mimicking a replication fork. Optimal unwinding activity was achieved at the remarkably low pH of 5.5. In the presence of Mg2+ (Mn2+) ions, the RepA activity is fueled by ATP, dATP, GTP, and dGTP and less efficiently by CTP and dCTP. UTP and dTTP are poor effectors. Nonhydrolyzable ATP analogues, ADP, and pyrophosphate inhibit the helicase activity, whereas inorganic phosphate does not. The presence of Escherichia coli single-stranded DNA-binding protein stimulates unwinding at physiological pH 2-3-fold, whereas the RSF1010 replicon-specific primase, RepB' protein, has no effect, either in the presence or in the absence of single-stranded DNA-binding protein.
Subject(s)
DNA Helicases/genetics , DNA Replication , Plasmids , Proteins/genetics , Trans-Activators , Adenosine Triphosphate/analogs & derivatives , Chromatography, Gel , DNA Helicases/antagonists & inhibitors , DNA Helicases/chemistry , DNA Helicases/metabolism , DNA Helicases/ultrastructure , DNA-Binding Proteins/metabolism , Diphosphates/pharmacology , Enzyme Inhibitors/pharmacology , Microscopy, Electron , Molecular Weight , Proteins/metabolism , Substrate SpecificityABSTRACT
Huntington's disease (HD) is one of an increasing number of human neurodegenerative disorders caused by a CAG/polyglutamine-repeat expansion. The mutation occurs in a gene of unknown function that is expressed in a wide range of tissues. The molecular mechanism responsible for the delayed onset, selective pattern of neuropathology, and cell death observed in HD has not been described. We have observed that mice transgenic for exon 1 of the human HD gene carrying (CAG)115 to (CAG)156 repeat expansions develop pronounced neuronal intranuclear inclusions, containing the proteins huntingtin and ubiquitin, prior to developing a neurological phenotype. The appearance in transgenic mice of these inclusions, followed by characteristic morphological change within neuronal nuclei, is strikingly similar to nuclear abnormalities observed in biopsy material from HD patients.
Subject(s)
Brain/pathology , Cell Nucleus/ultrastructure , Huntington Disease/genetics , Huntington Disease/pathology , Inclusion Bodies/ultrastructure , Nerve Tissue Proteins/genetics , Neurons/ultrastructure , Nuclear Proteins/genetics , Animals , Cell Nucleus/pathology , Disease Progression , Exons , Homozygote , Humans , Huntingtin Protein , Huntington Disease/physiopathology , Immunohistochemistry , Inclusion Bodies/pathology , Mice , Mice, Transgenic , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/biosynthesis , Neurons/pathology , Nuclear Proteins/analysis , Nuclear Proteins/biosynthesis , Repetitive Sequences, Nucleic Acid , Ubiquitins/analysis , Weight LossABSTRACT
The mechanism by which an elongated polyglutamine sequence causes neurodegeneration in Huntington's disease (HD) is unknown. In this study, we show that the proteolytic cleavage of a GST-huntingtin fusion protein leads to the formation of insoluble high molecular weight protein aggregates only when the polyglutamine expansion is in the pathogenic range. Electron micrographs of these aggregates revealed a fibrillar or ribbon-like morphology, reminiscent of scrapie prions and beta-amyloid fibrils in Alzheimer's disease. Subcellular fractionation and ultrastructural techniques showed the in vivo presence of these structures in the brains of mice transgenic for the HD mutation. Our in vitro model will aid in an eventual understanding of the molecular pathology of HD and the development of preventative strategies.
Subject(s)
Huntington Disease/genetics , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Amino Acid Sequence , Amyloid , Animals , Base Sequence , Brain/pathology , Cell Nucleus/pathology , DNA Primers , Exons , Glutathione Transferase , Humans , Huntingtin Protein , Huntington Disease/pathology , Kidney/pathology , Macromolecular Substances , Mice , Mice, Transgenic , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nuclear Proteins/chemistry , Polymerase Chain Reaction , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistryABSTRACT
Helicases are ATP-driven enzymes essential for DNA unwinding. The broad host range plasmid RSFI010 harbours a gene (repA) encoding for one of the smallest known oligomeric helicases, RepA, a homo-hexamer with 30 kDa subunits. Electron micrographs indicate that the overall shape of RepA resembles a hexagon with globular monomers at the corners, diameter 140 A, and a central channel. Below pH 6, the molecules aggregate into tubular structures. The enzyme has been purified and crystallized using the hanging-drop vapour-diffusion method with polyethyleneglycol monomethylether as precipitating agent. The crystals exhibit the monoclinic space group P2(1) with unit-cell parameters a = 105.8, b = 180.3, c = 115.4 A, beta = 95.2 degrees, and diffract to 3.5 A resolution using rotating-anode Cu Kalpha radiation. Assuming two 180 kDa molecules per asymmetric unit, the volume per unit weight is V(m) = 3.06 A Da(-1), equivalent to a solvent content of 60%. A self-rotation search indicates that the sixfold axis of the hexamer is parallel to the ac plane and inclined at about 2 degrees to the c axis. The two hexamers are oriented head-to-head with point-group symmetry D(6).
ABSTRACT
We report the discovery of the huntingtin interacting protein I (HIP-I) which binds specifically to the N-terminus of human huntingtin, both in the two-hybrid screen and in in vitro binding experiments. For the interaction in vivo, a protein region downstream of the polyglutamine stretch in huntingtin is essential. The HIP1 cDNA isolated by the two-hybrid screen encodes a 55 kDa fragment of a novel protein. Using an affinity-purified polyclonal antibody raised against recombinant HIP-I, a protein of 116 kDa was detected in brain extracts by Western blot analysis. The predicted amino acid sequence of the HIP-I fragment exhibits significant similarity to cytoskeleton proteins, suggesting that HIP-I and huntingtin play a functional role in the cell filament networks. The HIP1 gene is ubiquitously expressed in different brain regions at low level. HIP-I is enriched in human brain but can also be detected in other human tissues as well as in mouse brain. HIP-I and huntingtin behave almost identically during subcellular fractionation and both proteins are enriched in the membrane containing fractions.
Subject(s)
DNA-Binding Proteins , Huntington Disease/genetics , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/isolation & purification , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Amino Acid Sequence , Blotting, Northern , Blotting, Western , Brain Chemistry , Cloning, Molecular , Cytoskeleton/chemistry , Electrophoresis, Polyacrylamide Gel , Gene Expression , Humans , Huntingtin Protein , Huntington Disease/metabolism , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Peptides/chemistry , Recombinant Fusion Proteins/metabolism , Repetitive Sequences, Nucleic Acid , Saccharomyces cerevisiae/genetics , Sequence AnalysisABSTRACT
A site-specific and strand-specific nick, introduced into the RSF1010 plasmid origin of transfer (oriT), initiates unidirectional DNA transfer during bacterial conjugation. We have previously reproduced this nicking at the duplex oriT in vitro using purified preparations of the three known RSF1010-mobilization proteins: MobA (78-kDa form of RSF1010 primase), MobB and MobC [Scherzinger, E., Lurz, R., Otto, S. & Dobrinski, B. (1992) Nucleic Acids Res. 20, 41-48]. In this study we report the purification of MobA to apparent homogeneity and demonstrate that this 78-kDa protein by itself is capable of creating the oriT-specific nick if the DNA is present in the single-stranded form. By studying the cleavage of sets of oligodeoxyribonucleotides varying successively by single nucleotides at the 5' or 3' end, the minimal substrate for cleavage has been defined. The results identify the MobA recognition sequence within the 11-residue oligonucleotide AAGTGCGC-CCT which is cleaved at the 3' side of the G at position 7. During the cleavage reaction, MobA becomes covalently linked to the 5'-phosphate end of each broken DNA molecule and retains its activity for the rejoining reaction. It can transfer the attached DNA to an incoming acceptor strand provided that the DNA molecule contains at its 3' end at least the seven nucleotides upstream of the nick site. The covalent MobA-DNA linkage has been determined by two-dimensional thin-layer electrophoresis to be a tyrosyl phosphate. Extensive digestion of the 32P-labeled MobA-oligonucleotide complex with lysine carboxypeptidase yielded a single DNA-bound peptide which was purified and sequenced. The resulting peptide sequence consists of amino acid residues at positions 22-30 in the MobA sequence and identifies Tyr24 as the residue linked to DNA in the covalent complex.