Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors

Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mm, 1 mm CHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4') was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations

Engineered Toxins “Zymoxins” Are Activated by the HCV NS3 Protease by Removal of an Inhibitory Protein Domain

The synthesis of inactive enzyme precursors, also known as “zymogens,” serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated “zymogenized” chimeric toxins (which we denote “zymoxins”). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the “zymoxin” approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected

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

Nitric oxide modulates chromatin folding in human endothelial cells via protein phosphatase 2A activation and class II histone deacetylases nuclear shuttling

Nitric oxide ( NO) modulates important endothelial cell (EC) functions and gene expression by a molecular mechanism which is still poorly characterized. Here we show that in human umbilical vein ECs (HUVECs) NO inhibited serum-induced histone acetylation and enhanced histone deacetylase ( HDAC) activity. By immunofluorescence and Western blot analyses it was found that NO induced class II HDAC4 and 5 nuclear shuttling and that class II HDACs selective inhibitor MC1568 rescued serum-dependent histone acetylation above control level in NO-treated HUVECs. In contrast, class I HDACs inhibitor MS27-275 had no effect, indicating a specific role for class II HDACs in NO-dependent histone deacetylation. In addition, it was found that NO ability to induce HDAC4 and HDAC5 nuclear shuttling involved the activation of the protein phosphatase 2A (PP2A). In fact, HDAC4 nuclear translocation was impaired in ECs expressing small-t antigen and exposed to NO. Finally, in cells engineered to express a HDAC4-Flag fusion protein, NO induced the formation of a macromolecular complex including HDAC4, HDAC3, HDAC5, and an active PP2A. The present results show that NO-dependent PP2A activation plays a key role in class II HDACs nuclear translocation

Benzodeazaoxaflavins as sirtuin inhibitors with antiproliferative properties in cancer stem cells

Inhibition of sirtuins has recently been proposed as a promising anticancer strategy. Some of the new benzodeazaoxaflavins (2a, 2b, and 2d) here reported as SIRT1/2 inhibitors were endowed with pro-apoptotic properties in human U937 leukemia cells and, most importantly, together with the prototype MC2141 (1) displayed antiproliferative effects in cancer stem cells from patients with colorectal carcinoma and glioblastoma multiforme, known to be highly tumorigenic, resistant to conventional cancer chemotherapy, and responsible, at least in part, for cancer relapse or recurrence. © 2012 American Chemical Society

Structural consequences of the familial amyotrophic lateral sclerosis SOD1 mutant His46Arg

The His46Arg (H46R) mutant of human copper-zinc superoxide dismutase (SOD1) is associated with an unusual, slowly progressing form of familial amyotrophic lateral sclerosis (FALS). Here we describe in detail the crystal structures of pathogenic H46R SOD1 in the Zn-loaded (Zn-H46R) and metal-free (apo-H46R) forms. The Zn-H46R structure demonstrates a novel zinc coordination that involves only three of the usual four liganding residues, His 63, His 80, and Asp 83 together with a water molecule. In addition, the Asp 124 “secondary bridge” between the copper- and zinc-binding sites is disrupted, and the “electrostatic loop” and “zinc loop” elements are largely disordered. The apo-H46R structure exhibits partial disorder in the electrostatic and zinc loop elements in three of the four dimers in the asymmetric unit, while the fourth has ordered loops due to crystal packing interactions. In both structures, nonnative SOD1–SOD1 interactions lead to the formation of higher-order filamentous arrays. The disordered loop elements may increase the likelihood of protein aggregation in vivo, either with other H46R molecules or with other critical cellular components. Importantly, the binding of zinc is not sufficient to prevent the formation of nonnative interactions between pathogenic H46R molecules. The increased tendency to aggregate, even in the presence of Zn, arising from the loss of the secondary bridge is consistent with the observation of an increased abundance of hyaline inclusions in spinal motor neurons and supporting cells in H46R SOD1 transgenic rats

Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF

Toll-like receptors (TLRs) bind pathogen-specific ligands early in infection, initiating signaling pathways that lead to expression of multiple protective cellular genes. Many viruses have evolved strategies that block the effector mechanisms induced through these signaling pathways, but viral interference with critical proximal receptor interactions has not been described. We show here that the NS3/4A serine protease of hepatitis C virus (HCV), a virus notorious for its ability to establish persistent intrahepatic infection, causes specific proteolysis of Toll-IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF or TICAM-1), an adaptor protein linking TLR3 to kinases responsible for activating IFN regulatory factor 3 (IRF-3) and NF-κB, transcription factors controlling a multiplicity of antiviral defenses. NS3/4A-mediated cleavage of TRIF reduces its abundance and inhibits polyI:C-activated signaling through the TLR3 pathway before its bifurcation to IRF-3 and NF-κB. This uniquely broad mechanism of immune evasion potentially limits expression of multiple host defense genes, thereby promoting persistent infections with this medically important virus