Stability and activity of human immunodeficiency virus protease: comparison of the natural dimer with a homologous, single-chain tethered dimer.

Cheng, Yih-Shyun E.; Yin, Fay H.; Foundling, S.; Blomstrom, Dale C.; Kettner, Charles A.

doi:10.1073/pnas.87.24.9660

Cited by 117 publications

(105 citation statements)

References 22 publications

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“…The synthetic fluorescent cationic peptide substrate 2-aminobenzoyl-Ala-Thr-His-Gln-Val-Tyr-Phe(NO 2 )-Val-Arg-Lys-Ala (28) was incubated with truncated or full-length HERV-K10 at 25°C in an assay buffer containing 50 mM MES, pH 5.0, 1 M NaCl, 20% glycerol, 1 mM EDTA. The synthesis of the substrate has been described elsewhere (28). The enzymatic reaction was terminated with 0.2 M ammonium hydroxide.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Inhibition of Human Endogenous Retrovirus-K10 Protease in Cell-free and Cell-based Assays

Kuhelj¹,

Rizzo²,

Chang

et al. 2001

Journal of Biological Chemistry

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A full-length and C-terminally truncated version of human endogenous retrovirus (HERV)-K10 protease were expressed in Escherichia coli and purified to homogeneity. Both versions of the protease efficiently processed HERV-K10 Gag polyprotein substrate. HERV-K10 Gag was also cleaved by human immunodeficiency virus, type 1 (HIV-1) protease, although at different sites. To identify compounds that could inhibit protein processing dependent on the HERV-K10 protease, a series of cyclic ureas that had previously been shown to inhibit HIV-1 protease was tested. Several symmetric bisamides acted as very potent inhibitors of both the truncated and full-length form of HERV-K10 protease, in subnanomolar or nanomolar range, respectively. One of the cyclic ureas, SD146, can inhibit the processing of in vitro translated HERV-K10 Gag polyprotein substrate by HERV-K10 protease. In addition, in virus-like particles isolated from the teratocarcinoma cell line NCCIT, there is significant accumulation of Gag and Gag-Pol precursors upon treatment with SD146, suggesting the compound efficiently blocks HERV-K Gag processing in cells. This is the first report of an inhibitor able to block cell-associated processing of Gag polypeptides of an endogenous retrovirus.

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Section: Methodsmentioning

confidence: 99%

“…Expression and Purification of HIV-1 Protease-HIV-1 protease was expressed in E. coli and then renatured from inclusion bodies as described previously (28).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Human Endogenous Retrovirus-K10 Protease in Cell-free and Cell-based Assays

Kuhelj¹,

Rizzo²,

Chang

et al. 2001

Journal of Biological Chemistry

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“…It also impairs immune function and the release of cytokines and growth factors, hindering the progression of inflammation and scarring 24 . In addition, hypothermia reduces the recruitment and function of neutrophils and the production of natural killer cells 25 , thereby decreasing antibody-mediated cell damage. In this study, we found that perioperative hypothermia reduced the activity of antioxidant enzymes in tissues from colonic anastomoses in rats.…”

Section: Level Of Lipid Peroxidation (Tbars)mentioning

confidence: 99%

Effects of perioperative hypothermia and reactive oxygen species in the healing of colonic anastomosis in rats

Oliveira

et al. 2014

Acta Cir. Bras.

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PURPOSE:We evaluated the hypothesis that induced perioperative hypothermia (32 ± 1ºC) affects the redox balance in the tissue of colonic anastomosis in rats by modifying biochemical enzymatic and non-enzymatic markers related to oxidative stress. METHODS:Forty-eight male Wistar rats were randomly divided into eight experimental groups of six animals each and underwent laparotomy, sigmoid section and immediate anastomosis. Four groups were operated under normothermia (36 ± 1ºC), and the other four under hypothermia (32 ± 1ºC). The animals were reoperated on days 3, 7 and 14 postoperatively, and two groups underwent SHAM at 3 days. From the scar tissue samples, the activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) was evaluated, and the levels of non-enzymatic markers of oxidative stress, such as reduced glutathione (GSH) and lipid peroxidation, were measured by the thiobarbituric acid reactive substances (TBARS) assay. The means were compared between groups corresponding to each day of sampling and euthanasia. RESULTS:The hypothermic groups showed a significant reduction on the activity of SOD on day 7 postoperatively, on the activity of CAT on days 7 and 14 postoperatively and on the levels of GSH on day 7 postoperatively. The level of lipid peroxidation was increased in the hypothermia group on day 7 postoperatively and decreased on day 14 compared with the normothermic groups. CONCLUSION:Perioperative hypothermia reduced the activity of the antioxidant enzymes catalase and superoxide dismutase, glutathione levels and increased lipid peroxidation in the scar tissue of colonic anastomoses in rats.

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“…The gene construct coded for two copies of the HIV monomer linked by the nucleotide sequence that codes for Gly-Gly-Ser-Ser-Gly. 48, 51 The nucleotide sequence that coded for each monomer was unique; the use of two different sequences allowed for site-directed mutagenesis to be targeted to a specific monomer. Cysteines at positions 67, 95, 167, and 195 were mutated to Leu, Met, Leu, and Met, respectively.…”

Section: Mutagenesis Protein Purification and Crystallizationmentioning

confidence: 99%

“…Therefore, all experimental data was collected using single chain HIV-1 protease in which the two monomers were tethered with a five amino acid flexible linker (GGSSG). [48][49][50][51] Kinetic experiments for the WT and Pr3 protease were carried out and resulting kinetic parameters for substrate cleavage were compared. Because of minimal peptide solubility, limited data was obtained for substrates with K M s >50 lM.…”

Section: Experimental Determination Of Binding and Kinetic Parametersmentioning

confidence: 99%

Structural, kinetic, and thermodynamic studies of specificity designed HIV‐1 protease

et al. 2012

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HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.

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Stability and activity of human immunodeficiency virus protease: comparison of the natural dimer with a homologous, single-chain tethered dimer.

Cited by 117 publications

References 22 publications

Inhibition of Human Endogenous Retrovirus-K10 Protease in Cell-free and Cell-based Assays

Inhibition of Human Endogenous Retrovirus-K10 Protease in Cell-free and Cell-based Assays

Effects of perioperative hypothermia and reactive oxygen species in the healing of colonic anastomosis in rats

Structural, kinetic, and thermodynamic studies of specificity designed HIV‐1 protease

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