Functional and morphological recovery of dystrophic muscles in mice treated with deacetylase inhibitors.

Pharmacological interventions that increase myofiber size counter the functional decline of dystrophic muscles. We show that deacetylase inhibitors increase the size of myofibers in dystrophin-deficient (MDX) and alpha-sarcoglycan (alpha-SG)-deficient mice by inducing the expression of the myostatin antagonist follistatin in satellite cells. Deacetylase inhibitor treatment conferred on dystrophic muscles resistance to contraction-coupled degeneration and alleviated both morphological and functional consequences of the primary genetic defect. These results provide a rationale for using deacetylase inhibitors in the pharmacological therapy of muscular dystrophies.

Enhancement of daunomycin toxicity by the differentiation inducer hexamethylene bisacetamide in erythroleukemia cells.

Cytotoxic effects of daunomycin were investigated upon differentiation of Friend erythroleukemia cells induced with hexamethylene bisacetamide, a process during which a 20-fold increase in the hemoglobin content occurred. Daunomycin proved to be more toxic to differentiated Friend cells than to their undifferentiated counterparts. No changes in the daunomycin uptake rates of the two cell types were detectable. Externally added catalase and desferrioxamine mesylate protected against the additional cytotoxicity of daunomycin in differentiated cells, pointing to hydrogen peroxide and iron ions as mediators of the toxic effect. Daunomycin-dependent, cyanide-insensitive oxygen consumption of control and induced cells did not differ significantly, and the rate of formation of the daunomycin semiquinone radical electron paramagnetic resonance signal was similar in both cell types, indicating that the difference in toxicity was not due to increased drug activation by plasma membrane enzymes. Differentiated cells had a lowered catalase content; the cellular iron content was shown to increase by 2.8-fold upon cell differentiation, with hemoglobin-bound iron being about 50% of the total. Altogether, the results suggest increased intracellular hydrogen peroxide generation mediated by hemoglobin, combined with a decrease in catalase activity and an increase in accessible iron, as responsible for the higher sensitivity to daunomycin shown by differentiated Friend cells. This represents the first experimental system where the increase in anthracycline cytotoxicity upon cell differentiation can be attributed to redox activation and the formation of reactive oxygen species.

The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism.

The solution structure of the hepatitis C virus (BK strain) NS3 protein N-terminal domain (186 residues) has been solved by NMR spectroscopy. The protein is a serine protease with a chymotrypsin-type fold, and is involved in the maturation of the viral polyprotein. Despite the knowledge that its activity is enhanced by the action of a viral protein cofactor, NS4A, the mechanism of activation is not yet clear. The analysis of the folding in solution and the differences from the crystallographic structures allow the formulation of a model in which, in addition to the NS4A cofactor, the substrate plays an important role in the activation of the catalytic mechanism. A unique structural feature is the presence of a zinc-binding site exposed on the surface, subject to a slow conformational exchange process.

Structural characterization of the interactions of optimized product inhibitors with the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein by NMR and modelling studies.

The interactions of peptide inhibitors, obtained by the optimization of N-terminal cleavage products of natural substrates, with the protease of human hepatitis C virus (HCV) are characterized by NMR and modelling studies. The S-binding region of the enzyme and the bound conformation of the ligands are experimentally determined. The NMR data are then used as the experimental basis for modelling studies of the structure of the complex. The S-binding region involves the loop connecting strands E2 and F2, and appears shallow and solvent-exposed. The ligand binds in an extended conformation, forming an antiparallel beta-sheet with strand E2 of the protein, with the P1 carboxylate group in the oxyanion hole.

Conformational changes in the NS3 protease from hepatitis C virus strain Bk monitored by limited proteolysis and mass spectrometry.

Conformational changes occurring within the NS3 protease domain from the hepatitis C virus Bk strain (NS3(1-180)) under different physico-chemical conditions either in the absence or in the presence of its cofactor Pep4A were investigated by limited proteolysis experiments. Because the surface accessibility of the protein is affected by conformational changes, when comparative experiments were carried out on NS3(1-180) either at different glycerol concentrations or in the presence of Pep4A, differential peptide maps were obtained from which protein regions involved in the structural changes could be inferred. The surface topology of isolated NS3(1-180) in solution was essentially consistent with the crystal structure of the protein with the N-terminal segment showing a high conformational flexibility. At higher glycerol concentration, the protease assumed a more compact structure showing a decrease in the accessibility of the N-terminal segment that either was forced to interact with the protein or originate intermolecular interactions with neighboring molecules. Binding of the cofactor Pep4A caused the displacement of the N-terminal arm from the protein moiety, leading this segment to again adopt an open and flexible conformation, thus suggesting that the N-terminus of the protease contributes only marginally to the stability of the complex. The observed conformational changes might be directly correlated with the activation mechanism of the protease by either the cosolvent or the cofactor peptide because they lead to tighter packing of the substrate binding site.

Chaperonins dependent increase of Cu,Zn superoxide dismutase production in Escherichia coli.

Over-expression of the chaperonins GroEL and GroES significantly suppressed the temperature-dependent pattern of expression of Cu,Zn superoxide dismutases in Escherichia coli and increased the yield of active enzyme. The results obtained indicate that chaperonins prevent degradation of metal-deficient enzyme molecules. GroEL was shown to form a complex with unfolded Cu,Zn superoxide dismutase in vitro, confirming that GroEL can interact with beta-stranded proteins.

Hepatitis C virus protease inhibitors: current progress and future challenges.

Hepatitis C is a predominantly chronic viral infection, affecting 1-3% of the world population. The causative agent, the hepatitis C virus (HCV), has a positive strand-RNA genome that is utilized, in infected cells, as an mRNA to drive the synthesis of a large polyprotein precursor. This precursor subsequently undergoes proteolytic maturation to generate all of the functional, both structural and nonstructural proteins necessary for viral replication and assembly. The proteolytic activity that is responsible for the generation of the mature viral polymerase as well as for most of the cleavages occurring in the nonstructural region of the polyprotein is expressed by the virus itself and is contained in its nonstructural protein 3 (NS3). Here, the N-terminal 180 amino acids form a chymotrypsin-like serine protease domain. Full activation of this protease is achieved only after complexation with another viral protein, the cofactor protein NS4A. Together, NS3 and NS4A form the active, heterodimeric serine protease that presently is the target of medicinal chemistry efforts aiming at the development of inhibitors with potential antiviral activity. We here review the recent progress in our understanding of the structure and function of the enzyme and in the development of selective and potent NS3 protease inhibitors.

The hepatitis C virus NS3 proteinase: structure and function of a zinc-containing serine proteinase.

The hepatitis C virus (HCV) NS3 protein contains a serine proteinase domain implicated in the maturation of the viral polyprotein. NS3 forms a stable heterodimer with NS4A, a viral membrane protein that acts as an activator of the NS3 proteinase. The three-dimensional structure of the NS3 proteinase complexed with an NS4A-derived peptide has been determined. The NS3 proteinase adopts a chymotrypsin-like fold. A beta-strand contributed by NS4A is clamped between two beta-strands within the N terminus of NS3. Consistent with the requirement for extraordinarily long peptide substrates (P6-P4'), the structure of the NS3 proteinase reveals a very long, solvent-exposed substrate-binding site. The primary specificity pocket of the enzyme is shallow and closed at its bottom by Phe-154, explaining the preference of the NS3 proteinase for cysteine residues in the substrate P1 position. Another important feature of the NS3 proteinase is the presence of a tetrahedral zinc-binding site formed by residues Cys-97, Cys-99, Cys-145 and His-149. The zinc-binding site has a role in maintaining the structural stability and guiding the folding of the NS3 serine proteinase domain. Inhibition of the NS3 proteinase activity is regarded as a promising strategy to control the disease caused by HCV. Remarkably, the NS3 proteinase is susceptible to inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A/NS4B, NS4B/NS5A and NS5A/NS5B cleavage sites. The Ki values of the inhibitory products are lower than the K(m) values of the respective substrates and follow the order NS4A < NS5A < NS4B. Starting from the observation that the NS3 proteinase undergoes product inhibition, very potent, active site-directed inhibitors have been generated using a combinatorial peptide chemistry approach.

Oxidative stress induced by a di-Schiff base copper complex is both mediated and modulated by glutathione.

The reductive activation of N,N'-bis(2-pyridylmethylene)-1-4-butanediamine (N,N',N",N"')-Cu(II)-diperchlorate (CuPUPY), a di-Schiff base copper complex with antineoplastic properties, was investigated in vitro in the presence of glutathione, ascorbate, NADH or NADPH. Glutathione and ascorbate but not the pyridine dinucleotides were able to reduce the compound. The apparent second order rate constants of the reduction reaction (9.6 +/- 2.0 M-1 sec-1 for ascorbate and 94.7 +/- 1.9 M-1 sec-1 for glutathione) indicate that glutathione is more effective by about one order of magnitude in reducing CuPUPY than ascorbate. Reduction by glutathione triggered a CuPUPY-supported redox-cycle with oxygen yielding H2O2. Whereas reduction by ascorbate was reversible, CuPUPY reduced by glutathione reacted with excess reduced glutathione (GSH) in a ligand exchange reaction yielding a GSH-Cu(I) complex which was reoxidized by O2, forming a complex between copper(II) and oxidized glutathione. These results suggest a dual role for the reduced thiol tripeptide; promoting oxidative stress induced by CuPUPY at low concentrations and inhibiting it at high concentrations. This hypothesis was verified by showing that optimum glutathione/CuPUPY ratios are needed in order to obtain maximum oxidative damage to both DNA and albumin in vitro. Evidence was obtained for the occurrence of the same reaction pathway in human K562 erythroleukemia cells: CuPUPY was more toxic to cells in which glutathione synthesis was inhibited by buthionine sulfoximine. Moreover, ESR spectroscopy revealed alterations in the hyperfine structure of the Cu(II) spectrum, consistent with the occurrence of ligand-exchange reactions in K562 cells.

Cytotoxicity of a low molecular weight Cu2Zn2 superoxide dismutase active center analog in human erythroleukemia cells.

The cytotoxicity of SOD-mimics was studied in human K562 erythroleukemia cells. CuPUPY, a low molecular weight copper complex with properties typical of a Cu2Zn2 SOD active center analog was shown to display pronounced toxicity upon incubation with human K562 erythroleukemia cells, while the ligand, CuSO4 or CuEDTA did not affect vitality. Externally added catalase decreased the cytotoxic effects of CuPUPY by 50% indicating an involvement of hydrogen peroxide in toxicity. An increased oxygen uptake and glutathione oxidation by K562 cells in the presence of CuPUPY suggested that toxicity might be due to a copper-mediated redox-cycle. In fact addition of glutathione to a solution of CuPUPY resulted in glutathione oxidation, O2-consumption and H2O2-generation. CuPUPY proved to be less toxic to human lymphocytes than to K562 cells. This selectivity may be related to the low content of antioxidative enzymes in K562 cells.

Unraveling the hidden catalytic activity of vertebrate class IIa histone deacetylases.

Previous findings have suggested that class IIa histone deacetylases (HDACs) (HDAC4, -5, -7, and -9) are inactive on acetylated substrates, thus differing from class I and IIb enzymes. Here, we present evidence supporting this view and demonstrate that class IIa HDACs are very inefficient enzymes on standard substrates. We identified HDAC inhibitors unable to bind recombinant human HDAC4 while showing inhibition in a typical HDAC4 enzymatic assay, suggesting that the observed activity rather reflects the involvement of endogenous copurified class I HDACs. Moreover, an HDAC4 catalytic domain purified from bacteria was 1,000-fold less active than class I HDACs on standard substrates. A catalytic Tyr is conserved in all HDACs except for vertebrate class IIa enzymes where it is replaced by His. Given the high structural conservation of HDAC active sites, we predicted the class IIa His-Nepsilon2 to be too far away to functionally substitute the class I Tyr-OH in catalysis. Consistently, a Tyr-to-His mutation in class I HDACs severely reduced their activity. More importantly, a His-976-Tyr mutation in HDAC4 produced an enzyme with a catalytic efficiency 1,000-fold higher than WT, and this "gain of function phenotype" could be extended to HDAC5 and -7. We also identified trifluoroacetyl-lysine as a class IIa-specific substrate in vitro. Hence, vertebrate class IIa HDACs may have evolved to maintain low basal activities on acetyl-lysines and to efficiently process restricted sets of specific, still undiscovered natural substrates.

Copper-glutathione complexes under physiological conditions: structures in solution different from the solid state coordination.

The physiologically important copper complexes of oxidized glutathione have been examined by electron spin resonance (ESR) spectroscopy in aqueous solution at neutral pH. Low temperature measurements show that the Cu(II) binding site in oxidized glutathione has the same ligand arrangement as in copper complexes of S-methylglutathione, glutamine, glutamate and glycine. The site is composed of the amino nitrogens and the carboxyl oxygens of two gamma-glutamyl residues; there is no interaction with amide nitrogens, the sulphur bond or the glycyl carboxyl groups. At high metal to ligand ratios a binuclear species exists, in which each Cu(II) binds only to one gamma-glutamyl residue. The previously reported forbidden transition detected at g = 4 is due to non-specific aggregation and not to spin coupling of intramolecular sites. Liquid solution ESR spectra show the Cu(II)-glutathione complex has a lower mobility than the corresponding Cu(II)-S-methylglutathione species. From the degree of spectral anisotropy the complex with glutathione is calculated to exist as a dimer. These results demonstrate that the physiologically relevant complex between copper and oxidized glutathione in solution is completely different from the known solid state structure determined by crystallography.

Role of zinc-coordination and of the glutathione redox couple in the redox susceptibility of human transcription factor Sp1.

We show that thiol-groups confer redox-susceptibility to the zinc-finger transcription factor Sp1 and that this redox-susceptibility is prevented by DNA-binding and depends on zinc-coordination of the protein. Apo-Sp1 contained in metal depleted nuclear extracts of human K562 cells exhibited a markedly increased susceptibility towards oxidizing and alkylating agents, as compared to holo-Sp 1. Moreover, DNA binding of apo-Sp1, but not of the holo-protein, was dramatically decreased in the presence of GSH/GSSG ratios within the physiological range. We compared these results with the redox behaviour of two other transcription factors, OTF-1 and NF1, which was found to be different in several aspects from that of Sp1.

Mechanisms of hepatitis C virus NS3 proteinase inhibitors.

The NS3 serine proteinase is regarded as one of the preferred targets for the development of therapeutic agents against hepatitis C virus (HCV). Possible mechanisms of NS3 inhibitors include: (i) interference with the activation of the enzyme by its NS4A cofactor; (ii) binding to the structural zinc site; and (iii) binding to the active site. These mechanisms have been explored in detail by structural analysis of the enzyme. (i) The NS4A cofactor binds to the amino-terminal beta-barrel domain of the NS3 proteinase bringing about several conformational changes that result in enzyme activation. The interaction between NS3 and NS4A involves a very large surface area and therefore it is not a likely target for the development of inhibitors. (ii) The NS3 proteinase contains a structural zinc binding site. Spectroscopic studies have shown that changes in the conformation of this metal-binding site correlate with changes in the specific activity of the enzyme, and the NS3 proteinase is inhibited by compounds capable of extracting zinc from its native coordination sphere. (iii) Based on the observation that the NS3 proteinase undergoes inhibition by its cleavage products, potent, active site-directed inhibitors have been generated. Kinetic studies, site-directed mutagenesis, and molecular modelling have been used to characterize the interactions between the NS3 proteinase and its product inhibitors.

In vitro activity of hepatitis C virus protease NS3 purified from recombinant Baculovirus-infected Sf9 cells.

A recombinant Baculovirus expression system was used for the production of a 20-kDa protein encompassing the hepatitis C virus NS3 protease domain. The protein was purified to apparent homogeneity after detergent extraction of cell homogenates. It was shown to be a monomer in solution and to cleave the in vitro translated precursor proteins NS4A-NS4B and NS5A-NS5B, but not the NS4B-NS5A or the NS3-NS4A precursors. The enzyme also cleaved a 20-mer peptide corresponding to the NS4A-NS4B junction with kcat/Km = 174 m(-1) s(-1). Peptides harboring NS4A sequences comprising amino acids 21-54 (Pep4A21-54) and 21-34 (Pep4A21-34) were found to induce an up to 2.8-fold acceleration of cleavage. Kinetic analysis revealed that this acceleration was due to an increase in kcat whereas no significant effect on Km could be detected. Pep4A21-54 was also an absolute requirement for cleavage of in vitro translated NS4B-NS5A by the purified protease. From these data we conclude that: (i) the purified protease domain shows substrate specificity and cleavage requirements similar to those previously reported on the basis of transfection experiments, (ii) activation of the purified protease by the NS4A co-factor can be mimicked by synthetic peptide analogs, and (iii) a central hydrophobic region of NS4A with a minimum core of 14 amino acids is responsible for the interaction with NS3.

Copper-dependent metabolism of Cu,Zn-superoxide dismutase in human K562 cells. Lack of specific transcriptional activation and accumulation of a partially inactivated enzyme.

The regulation of Cu,Zn-superoxide dismutase by copper was investigated in human K562 cells. Copper ions caused a dose- and time-dependent increase, up to 3-fold, of the steady-state level of Cu,Zu-superoxide dismutase mRNA. A comparable increase was also observed for actin and ribosomal protein L32 mRNAs, but not for metallothionein mRNA which was augmented more than 50-fold and showed a different induction pattern. The copper-induced mRNAs were actively translated as judged from their enhanced loading on polysomes, the concomitantly increased cellular protein levels and an augmented incorporation of [3H]lysine into acid-precipitable material. Cu,Zn-superoxide dismutase protein followed this general trend, as demonstrated by dose- and time-dependent increases in immunoreactive and enzymically active protein. However, a specific accumulation of Cu,Zn-superoxide dismutase was noticed in cells grown in the presence of copper, that was not detectable for other proteins. Purification of the enzyme demonstrated that Cu,Zn-superoxide dismutase was present as a reconstitutable, copper-deficient protein with high specific activity (kcat./Cu = 0.89 x 10(9) M-1.s-1) in untreated K562 cells and as a fully metallated protein with low specific activity (kcat./Cu = 0.54 x 10(9) M-1.s-1) in copper-treated cells. Pulse-chase experiments using [3H]lysine indicated that turnover rates of Cu,Zn-superoxide dismutase in K562 cells were not affected by growth in copper-enriched medium, whereas turnover of total protein was significantly enhanced as a function of metal supplementation. From these results we conclude that: (i) unlike in yeast [Carrì, Galiazzo, Ciriolo and Rotilio (1991) FEBS Lett. 278, 263-266] Cu,Zn-superoxide dismutase is not specifically regulated by copper at the transcriptional level in human K562 cells, suggesting that this type of regulation has not been conserved during the evolution of higher eukaryotes; (ii) copper ions cause an inactivation of the enzyme in intact K562 cells; and (iii) the metabolic stability of Cu,Zn-superoxide dismutase results in its relative accumulation under conditions that lead to increased protein turnover.

The nonstructural proteins of the hepatitis C virus: structure and functions.

The hepatitis C virus is the major causative agent of nonA-nonB hepatitis worldwide. Although this virus cannot be cultivated in cell culture, several of its features have been elucidated in the past few years. The viral genome is a single-stranded, 9.5kb long RNA molecule of positive polarity. The viral genome is translated into a single polyprotein of about 3000 amino acids. The virally encoded polyprotein undergoes proteolytic processing by a combination of cellular and viral proteolytic enzymes in order to yield all the mature viral gene products. The gene order of HCV has been determined to be C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. The mature structural proteins, C, E1 and E2 have been shown to arise from the viral polyprotein via proteolytic processing by host signal peptidases. Conversely, generation of the mature nonstructural proteins relies on the activity of viral proteases. Thus, cleavage at the NS2/NS3 junction is accomplished by a metal-dependent autoprotease encoded within NS2 and the N-terminus of NS3. The remaining cleavages downstream from this site are effected by a serine protease contained within the N-terminal region of NS3. Besides the protease domain, NS3 also contains an RNA helicase domain at its C-terminus. NS3 forms a heterodimeric complex with NS4A. The latter is a membrane protein that has been shown to act as a cofactor of the protease. Whereas the NS5B protein has been shown to be the viral RNA-dependent RNA polymerase, no function has yet been attributed to NS4B and NS5A. The latter is a cytoplasmic phosphoprotein and appears to be involved in mediating the resistance of the hepatitis C virus to the action of interferon.