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1.
Sci Rep ; 8(1): 10438, 2018 Jul 11.
Article in English | MEDLINE | ID: mdl-29992979

ABSTRACT

HIV-1 protease (PR) is a homodimeric enzyme that is autocatalytically cleaved from the Gag-Pol precursor. Known PR inhibitors bind the mature enzyme several orders of magnitude more strongly than the PR precursor. Inhibition of PR at the precursor level, however, may stop the process at its rate-limiting step before the proteolytic cascade is initiated. Due to its structural heterogeneity, limited solubility and autoprocessing, the PR precursor is difficult to access by classical methods, and limited knowledge regarding precursor inhibition is available. Here, we describe a cell-based assay addressing precursor inhibition. We used a reporter molecule containing the transframe (TFP) and p6* peptides, PR, and N-terminal fragment of reverse transcriptase flanked by the fluorescent proteins mCherry and EGFP on its N- and C- termini, respectively. The level of FRET between EGFP and mCherry indicates the amount of unprocessed reporter, allowing specific monitoring of precursor inhibition. The inhibition can be quantified by flow cytometry. Additionally, two microscopy techniques confirmed that the reporter remains unprocessed within individual cells upon inhibition. We tested darunavir, atazanavir and nelfinavir and their combinations against wild-type PR. Shedding light on an inhibitor's ability to act on non-mature forms of PR may aid novel strategies for next-generation drug design.


Subject(s)
Anti-HIV Agents/pharmacology , HIV Protease Inhibitors/pharmacology , HIV-1/enzymology , Protein Precursors/antagonists & inhibitors , Atazanavir Sulfate/pharmacology , Cell Line , Darunavir/pharmacology , Flow Cytometry , Fluorescence Resonance Energy Transfer , Fluorescent Dyes , Humans , Nelfinavir/pharmacology , Proteolysis
2.
Sci Rep ; 6: 30443, 2016 07 27.
Article in English | MEDLINE | ID: mdl-27461074

ABSTRACT

Although Ddi1-like proteins are conserved among eukaryotes, their biological functions remain poorly characterized. Yeast Ddi1 has been implicated in cell cycle regulation, DNA-damage response, and exocytosis. By virtue of its ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, it has been proposed to serve as a proteasomal shuttle factor. All Ddi1-like family members also contain a highly conserved retroviral protease-like (RVP) domain with unknown substrate specificity. While the structure and biological function of yeast Ddi1 have been investigated, no such analysis is available for the human homologs. To address this, we solved the 3D structures of the human Ddi2 UBL and RVP domains and identified a new helical domain that extends on either side of the RVP dimer. While Ddi1-like proteins from all vertebrates lack a UBA domain, we identify a novel ubiquitin-interacting motif (UIM) located at the C-terminus of the protein. The UIM showed a weak yet specific affinity towards ubiquitin, as did the Ddi2 UBL domain. However, the full-length Ddi2 protein is unable to bind to di-ubiquitin chains. While proteomic analysis revealed no activity, implying that the protease requires other factors for activation, our structural characterization of all domains of human Ddi2 sets the stage for further characterization.


Subject(s)
Aspartic Acid Proteases/chemistry , Aspartic Acid Proteases/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Structural Homology, Protein , Amino Acid Motifs , Amino Acid Sequence , Conserved Sequence , Crystallography, X-Ray , Evolution, Molecular , HEK293 Cells , Humans , Models, Molecular , Polyubiquitin/metabolism , Protein Binding , Protein Domains , Protein Interaction Mapping , Protein Multimerization , Proteolysis , Scattering, Small Angle , Sequence Analysis, Protein , Solutions
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