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Local anesthetics (LAs) inhibit endoplasmic reticulum-associated protein degradation, however the mechanisms remain elusive. Here, we show that the clinically used LAs pilsicainide and lidocaine bind directly to the 20S proteasome and inhibit its activity. Molecular dynamic calculation indicated that these LAs were bound to the β5 subunit of the 20S proteasome, and not to the other active subunits, β1 and β2. Consistently, pilsicainide inhibited only chymotrypsin-like activity, whereas it did not inhibit the caspase-like and trypsin-like activities. In addition, we confirmed that the aromatic ring of these LAs was critical for inhibiting the proteasome. These LAs stabilized p53 and suppressed proliferation of p53-positive but not of p53-negative cancer cells.

alpha-Synuclein is one of the causative proteins of the neurodegenerative disorder, Parkinson's disease. Deposits of alpha-synuclein called Lewy bodies are a hallmark of this disorder, which is implicated in its progression. In order to understand the mechanism of amyloid fibril formation of alpha-synuclein in more detail, in this study we have isolated a specific, ~20 residue peptide region of the alpha-synuclein fibril core, using a combination of Edman degradation and mass-spectroscopy analyses of protease-resistant samples. Starting from this core peptide sequence, we then synthesized a series of peptides that undergo aggregation and fibril formation under similar conditions. Interestingly, in a derivative peptide where a crucial phenylalanine residue was changed to a glycine, the ability to initiate spontaneous fibril formation was abolished, while the ability to extend from preexisting fibril seeds was conserved. This fibril extension occurred irrespective of the source of the initial fibril seed, as demonstrated in experiments using fibril seeds of insulin, lysozyme, and GroES. This interesting ability suggests that this peptide might form the basis for a possible diagnostic tool useful in detecting the presence of various fibrillogenic factors.

金沢大学がん研究所がん分子細胞制御, The assembly of RAD51 recombinase on DNA substrates at sites of breakage is essential for their repair by homologous recombination repair (HRR). The signaling pathway that triggers RAD51 assembly at damage sites to form subnuclear foci is unclear. Here, we provide evidence that c-ABL, a tyrosine kinase activated by DNA damage which phosphorylates RAD51 on Tyr-315, works at a previously unrecognized, proximal step to initiate RAD51 assembly. We first show that c-ABL associates with chromatin after DNA damage in a manner dependent on its kinase activity. Using RAD51 mutants that are unable to oligomerize to form a nucleoprotein filament, we separate RAD51 assembly on DNA to form foci into two steps: stable chromatin association followed by oligomerization. We show that phosphorylation on Tyr-315 by c-ABL is required for chromatin association of oligomerization-defective RAD51 mutants, but is insufficient to restore oligomerization. Our findings suggest a new model for the regulation of early steps of HRR. © 2009 Elsevier Inc. All rights reserved.

Experimental probing of a protein-folding energy land-scape can be challenging, and energy landscapes comprising multiple intermediates have not yet been defined. Here, we quasi-statically unfolded single molecules of staphylococcal nuclease by constant-rate mecahnical stretching with a feedback positioning system. Multiple discrete transition states were detected as force peaks, and only some of the multiple transition states emerged stochastically in each trial. This finding was confirmed by molecular dynamics simulations, and agreed with another result of the simulations which showed that individual trajectories took highly heterogeneous pathways. The presence of Ca2+ did not changethe location of the transition states, but changed the frequency of the emergence. Transition states emerged frequency of the emergence. Transition states emerged more frequently in stabilized domains. The simulations also confirmed this feature, and showed that the stabilized domains had rugged energy surfaces. The mean energy required per residue to disrupt secondary structures was a few times the thermal energy (1-3 kBT), which agreed with the stochastic feature. Thus, single-molecule quasi-static measurement has achieved notable success in detecting stochastic features of a huge number of possible conformations of a protein.

The DMC1 protein, a meiosis-specific DNA recombinase, catalyzes strand exchange between homologous chromosomes. In rice, two Dmc1 genes, Dmc1A and Dmc1B, have been reported. Although the Oryza sativa DMC1A protein has been partially characterized, however the biochemical properties of the DMC1B protein have not been defined. In the present study, we expressed the Oryza sativa DMC1A and DMC1B proteins in bacteria and purified them. The purified DMC1A and DMC1B proteins formed helical filaments along single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and promoted robust strand exchange between ssDNA and dsDNA over five thousand base pairs in the presence of RPA, as a co-factor. The DMC1A and DMC1B proteins also promoted strand exchange in the absence of RPA with long DNA substrates containing several thousand base pairs. In contrast, the human DMC1 protein strictly required RPA to promote strand exchange with these long DNA substrates. The strand-exchange activity of the Oryza sativa DMC1A protein was much higher than that of the DMC1B protein. Consistently, the DNA-binding activity of the DMC1A protein was higher than that of the DMC1B protein. These biochemical differences between the DMC1A and DMC1B proteins may provide important insight into their functional differences during meiosis in rice.

The Rad51 protein, which catalyzes homologous-pairing and strand-exchange reactions, is an essential enzyme for homologous recombinational repair (HRR) and meiotic homologous recombination in eukaryotes. In humans, the conventional Rad51 (HsRad51) protein has a Lys residue at position 313; however, the HsRad51-Q313 protein, in which the Lys313 residue is replaced by Gln, was reported as an isoform, probably corresponding to a polymorphic variant. In this study, we purified the HsRad51-K313 and HsRad51-Q313 isoforms and analyzed their biochemical activities in vitro. Compared to the conventional HsRad51-K313 protein, the HsRad51-Q313 protein exhibited significantly enhanced strand-exchange activity under conditions with Ca2+, although the difference was not observed without Ca2+. A double-stranded DNA (dsDNA) unwinding assay revealed that the HsRad51-Q313 protein clearly showed enhanced DNA unwinding activity, probably due to its enhanced filament-formation ability. Mutational analyses of the HsRad51-Lys313 residue revealed that positively charged residues (Lys and Arg), but not negatively charged, polar and hydrophobic residues (Glu, Gln and Met, respectively), at position 313 reduced the strand-exchange and DNA unwinding abilities of the HsRad51 protein. These results suggest that the electrostatic environment around position 313 is important for the regulation of the HsRad51 recombinase activity.

In order to understand how inter-subunit association stabilizes oligomeric proteins, a single polypeptide chain variant of heptameric co-chaperonin GroES (tandem GroES) was constructed from Escherichia coli heptameric GroES by linking consecutively the C-terminal of one subunit to the N-terminal of the adjacent subunit with a small linker peptide. The tandem GroES (ESC7) showed properties similar to wild-type GroES in structural aspects and co-chaperonin activity. In unfolding and refolding equilibrium experiments using guanidine hydrochloride (Gdn-HCl) as a denaturant at a low protein concentration (50 microg ml(-1)), ESC7 showed a two-state transition with a greater resistance toward Gdn-HCl denaturation (Cm=1.95 M) compared to wild-type GroES (Cm=1.1 M). ESC7 was found to be about 10 kcal mol(-1) more stable than the wild-type GroES heptamer at 50 microg ml(-1). Kinetic unfolding and refolding experiments of ESC7 revealed that the increased stability was mainly attributed to a slower unfolding rate. Also a transient intermediate was detected in the refolding reaction. Interestingly, at the physiological GroES concentration (>1 mg ml(-1)), the free energy of unfolding for GroES heptamer exceeded that for ESC7. These results showed that at low protein concentrations (

Na+チャネル遮断薬ピルジカイニド (Pil) がKv1.5チャネル蛋白を含む半減期の短い蛋白の増加作用を有することを見出したので, その機序をリドカイン (Lido) と比較して報告する.PilとLidoはプロテアソームで分解されるp53やIKK2蛋白を用量依存性に安定化し, このp53蛋白への作用は変異Mdmによりユビキチン・プロテアソーム系が遮断されると生じない, PilとLidoは用量依存性に20Sプロテアソーム活性を抑制するが, l-verapamilやE-4031にはこの作用は認められなかった.さらにPilはLidoに比較してより低濃度 (臨床使用濃度) でp53蛋白を安定化した.同様にPilとLidoはプロテアソームで分解されるKv1.5チャネル蛋白を安定化して細胞膜での/Kurを増加し, この作用は発現された培養COS7細胞Kv1.5チャネルのみならずラツト心房筋Kv1.5チャネルでも認められた, またKv1.5チャネル蛋白の安定化作用はPilがLidoより強い.その分子機序をin silicoドッキング試験から検討し, PilとLidoはともに20sプロテアソームを構成するβ5サブユニットの基質結合部位に作用して, その活性を抑制すると推測でき, PilがLidoに比較して結合性が高いことも再現できた, これらの結果からNa+チヤネル遮断薬Pilが臨床使用濃度でプロテアソーム活性を抑制し, Kv1.5チャネル蛋白を含む半減期の短い蛋白を増加させる作用を発揮することが明らかにされた.

Chaperonin 10 (cpn10) is a well-conserved subgroup of the molecular chaperone family. GroES, the cpn10 from Escherichia coli, is composed of seven 10kDa subunits, which form a dome-like oligomeric ring structure. From our previous studies, it was found that GroES unfolded completely through a three-state unfolding mechanism involving a partly folded monomer and that this reaction was reversible. In order to study whether these unfolding-refolding characteristics were conserved in other cpn10 proteins, we have examined the structural stabilities of cpn10s from rat mitochondria (RatES) and from hyperthermophilic eubacteria Thermotoga maritima (TmaES), and compared the values to those of GroES. From size-exclusion chromatography experiments in the presence of various concentrations of Gdn-HCl at 25 degrees C, both cpn10s showed unfolding-refolding characteristics similar to those of GroES, i.e. two-stage unfolding reactions that include formation of a partially folded monomer. Although the partially folded monomer of TmaES was considerably more stable compared to GroES and RatES, it was found that the overall stabilities of all three cpn10s were achieved significantly by inter-subunit interactions. We studied this contribution of inter-subunit interactions to overall stability in the GroES heptamer by introducing a mutation that perturbed subunit association, specifically the interaction between the two anti-parallel beta-strands at the N and C termini of this protein. From analyses of the mutants' stabilities, it was revealed that the anti-parallel beta-strands at the subunit interface are crucial for subunit association and stabilization of the heptameric GroES protein.

To overcome the ensemble-averaging barrier, single-molecule experiments have been performed, but energy landscapes comprising multiple intermediates have not yet been defined. We performed mechanical unfolding of staphylococcal nuclease using intermoleculer force microscopy, modified AFM with high resolution and feedback control of the positioning. The force dropped vertically just after its peak, and multiple transition states were detected as force peaks. The multiple and stochastic intermediates found in the present study provide new important information on protein folding.

The RAD51 protein is a central player in homologous recombinational repair. The RAD51B protein is one of five RAD51 paralogs that function in the homologous recombinational repair pathway in higher eukaryotes. In the present study, we found that the human EVL (Ena/Vasp-like) protein, which is suggested to be involved in actin-remodeling processes, unexpectedly binds to the RAD51 and RAD51B proteins and stimulates the RAD51-mediated homologous pairing and strand exchange. The EVL knockdown cells impaired RAD51 assembly onto damaged DNA after ionizing radiation or mitomycin C treatment. The EVL protein alone promotes single-stranded DNA annealing, and the recombination activities of the EVL protein are further enhanced by the RAD51B protein. The expression of the EVL protein is not ubiquitous, but it is significantly expressed in breast cancer-derived MCF7 cells. These results suggest that the EVL protein is a novel recombination factor that may be required for repairing specific DNA lesions, and that may cause tumor malignancy by its inappropriate expression.

It is difficult to determine the structural stability of the individual subunits or protomers of many proteins in the cell that exist in an oligomeric or complexed state. In this study, we used single-molecule force spectroscopy on seven subunits of covalently linked cochaperonin GroES (ESC7) to evaluate the structural stability of the subunit. A modified form of ESC7 was immobilized on a mica surface. The force-extension profile obtained from the mechanical unfolding of this ESC7 showed a distinctive sawtooth pattern that is typical for multimodular proteins. When analyzed according to the worm-like chain model, the contour lengths calculated from the peaks in the profile suggested that linked-GroES subunits unfold in distinct steps after the oligomeric ring structure of ESC7 is disrupted. The evidence that structured subunits of ESC7 withstand external force to some extent even after the perturbation of the oligomeric ring structure suggests that a stable monomeric intermediate is an important component of the equilibrium unfolding reaction of GroES.

The subunit structure of chaperonin GroEL is divided into three domains; the apical domain, the intermediate domain, and the equatorial domain. Each domain has a specific role in the chaperonin mechanism. The ‘hinge 2’ site of GroEL contains three glycine residues, Gly192, Gly374, and Gly375, connecting the apical domain and the intermediate domain. In this study, to understand the importance of the hinge 2 amino acid residues in chaperonin function, we substituted each of these three glycine residues to tryptophan. The GroEL mutants G374W and G375W were functionally similar to wild-type GroEL. However, GroEL G192W showed a significant decrease in the ability to assist the refolding of stringent substrate proteins. Interestingly, from biochemical assays and characterization using surface plasmon resonance analysis, we found that GroEL G192W was capable of binding GroES even in the absence of ATP to form a very stable GroEL–GroES complex, which could not be dissociated even upon addition of ATP. Electron micrographs showed that GroEL G192W intrinsically formed an asymmetric double ring structure with one ring locked in the ‘open’ conformation, and it is postulated that GroES binds to this open ring in the absence of ATP. Trans-binding of both substrate protein and GroES was observed for this binary complex, but simultaneous binding of both substrate and GroES (a mechanism that ensures substrate encapsulation) was impaired. We postulate that alteration of Gly192 severely compromises an essential movement that allows efficient encapsulation of unfolded protein intermediates.

The Rad51 protein, which catalyzes homologous-pairing and strand-exchange reactions, is an essential enzyme for homologous recombinational repair (HRR) and meiotic homologous recombination in eukaryotes. In humans, the conventional Rad51 (HsRad51) protein has a Lys residue at position 313; however, the HsRad51-Q313 protein, in which the Lys313 residue is replaced by Gln, was reported as an isoform, probably corresponding to a polymorphic variant. In this study, we purified the HsRad51-K313 and HsRad51-Q313 isoforms and analyzed their biochemical activities in vitro. Compared to the conventional HsRad51-K313 protein, the HsRad51-Q313 protein exhibited significantly enhanced strand-exchange activity under conditions with Ca(2+), although the difference was not observed without Ca(2+). A double-stranded DNA (dsDNA) unwinding assay revealed that the HsRad51-Q313 protein clearly showed enhanced DNA unwinding activity, probably due to its enhanced filament-formation ability. Mutational analyses of the HsRad51-Lys313 residue revealed that positively charged residues (Lys and Arg), but not negatively charged, polar and hydrophobic residues (Glu, Gln and Met, respectively), at position 313 reduced the strand-exchange and DNA unwinding abilities of the HsRad51 protein. These results suggest that the electrostatic environment around position 313 is important for the regulation of the HsRad51 recombinase activity.

International audience; The M200V polymorphism of the human DMC1 protein, which is an essential, meiosis-specific DNA recombinase, was found in an infertile patient, raising the question of whether this homozygous human DMC1-M200V polymorphism may cause infertility by affecting the function of the human DMC1 protein. In the present study, we determined the crystal structure of the human DMC1-M200V variant in the octameric-ring form. Biochemical analyses revealed that the human DMC1-M200V variant had reduced stability, and was moderately defective in catalyzing in vitro recombination reactions. The corresponding M194V mutation introduced in the Schizosaccharomyces pombe dmc1 gene caused a significant decrease in the meiotic homologous recombination frequency. Together, these structural, biochemical and genetic results provide extensive evidence that the human DMC1-M200V mutation impairs its function, supporting the previous interpretation that this single-nucleotide polymorphism is a source of human infertility.

A noncollinear optical parametric amplifier is presented that generates transform-limited sub-10-fs pulses that are tunable in both the visible and the near infrared (NIR).?The pulse-front-matched pump geometry realizes tilt-free signal amplification, and pulses as short as 6.1 fs can be obtained from 550 to 700 nm. The large angular dispersion of the idler specific to the group-velocity-matching interaction is effectively eliminated by a grating–telescope compensator, and 9-fs NIR pulses are also successfully obtained from 900 to 1300 nm. This is believed to be the first tunable sub-10-fs light source.

Heat shock protein 10 (hsp10) is a member of the molecular chaperones and works with hsp60 in mediating various protein folding reactions. GroES is a representative protein of hsp10 from Escherichia coli. Recently, we found that GroES formed a typical amyloid fibril from a guanidine hydrochloride (Gdn-HCl) unfolded state at neutral pH. Here, we report that other hsp10 homologues, such as human hsp10 (Hhsp10), rat mitochondrial hsp10 (Rhsp10), Gp31 from T4 phage, and hsp10 from the hyperthermophilic bacteria Thermotoga maritima, also form amyloid fibrils from an unfolded state. Interestingly, whereas GroES formed fibrils from either the Gdn-HCl unfolded state (at neutral pH) or the acidic unfolded state (at pH 2.0-3.0), Hhsp10, Rhsp10, and Gp31 formed fibrils from only the acidic unfolded state. Core peptide regions of these protein fibrils were determined by proteolysis treatment followed by a combination of Edman degradation and mass spectroscopy analyses of the protease-resistant peptides. The core peptides of GroES fibrils were identical for fibrils formed from the Gdn-HCl unfolded state and those formed from the acidic unfolded state. However, a peptide with a different sequence was isolated from fibrils of Hhsp10 and Rhsp10. With the use of synthesized peptides of the determined core regions, it was also confirmed that the identified regions were capable of fibril formation. These findings suggested that GroES homologues formed typical amyloid fibrils under acidic unfolding conditions but that the fibril core structures were different, perhaps owing to differences in local amino acid sequences.

The human Rad51 protein (HsRad51) catalyzes homologous pairing and strand exchange between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) during recombinational repair of double-stranded DNA breaks. An HsRad51 mutation that results in the substitution of Gln for Arg150 (R150Q) was found in bilateral breast cancer patients; however, the consequences of this R150Q mutation have not been elucidated. To determine how this HsRad51(R150Q) mutation affects HsRad51 function, in the present study, we purified the HsRad51(R150Q) mutant. The purified HsRad51(R150Q) was completely proficient in the ATP-hydrolyzing activity. A gel filtration analysis revealed that HsRad51(R150Q) also retained the polymer formation ability. In contrast, the ssDNA- and dsDNA-binding abilities of HsRad51(R150Q) were clearly reduced, as compared to those of HsRad51. These differences in the DNA-binding properties between HsRad51(R150Q) and HsRad51 may be important to account for the tumorigenesis in breast cancer patients with the HsRad51(R150Q) mutation.

The human Rad51 protein, a eukaryotic ortholog of the bacterial RecA protein, is a key enzyme that functions in homologous recombination and recombinational repair of double strand breaks. The Rad51 protein contains two flexible loops, L1 and L2, which are proposed to be sites for DNA binding, based on a structural comparison with RecA. In the present study, we performed mutational and fluorescent spectroscopic analyses on the L1 and L2 loops to examine their role in DNA binding. Gel retardation and DNA-dependent ATP hydrolysis measurements revealed that the substitution of the tyrosine residue at position 232 (Tyr232) within the L1 loop with alanine, a short side chain amino acid, significantly decreased the DNA-binding ability of human Rad51, without affecting the protein folding or the salt-induced, DNA-independent ATP hydrolysis. Even the conservative replacement with tryptophan affected the DNA binding, indicating that Tyr232 is involved in DNA binding. The importance of the L1 loop was confirmed by the fluorescence change of a tryptophan residue, replacing the Asp231, Ser233, or Gly236 residue, upon DNA binding. The alanine replacement of phenylalanine at position 279 (Phe279) within the L2 loop did not affect the DNA-binding ability of human Rad51, unlike the Phe203 mutation of the RecA L2 loop. The Phe279 side chain may not be directly involved in the interaction with DNA. However, the fluorescence intensity of the tryptophan replacing the Rad51-Phe279 residue was strongly reduced upon DNA binding, indicating that the L2 loop is also close to the DNA-binding site.