Document Detail


Calpain and proteasomal regulation of antiretroviral zinc finger protein OTK18 in human macrophages: visualization in live cells by intramolecular FRET.
MedLine Citation:
PMID:  19034669     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
As part of the innate immune defense against HIV infection, OTK18, a zinc finger protein, is upregulated in human macrophages and reduces viral replication through suppression of viral long-terminal repeat promoter activity. Although we know that the processing products of OTK18 accumulate in the cytoplasm of brain perivascular macrophages in advanced HIV encephalitis cases, the molecular mechanisms behind its post-translational processing are still poorly understood. To characterize OTK18 processing, we assessed a panel of protease inhibitors to identify the candidates involved in the OTK18 processing using human monocyte-derived macrophages (MDM) overexpressing OTK18 by recombinant adenoviral gene transfer. Viral infection of MDM strongly increased the processing of OTK18 into its N-terminal fragment. Treatment of OTK18-expressing MDM with calpain and proteasome inhibitors significantly accumulated either full-length or processed OTK18 fragments in time- and dose-dependent manners. A series of OTK18 truncation mutants and synthetic peptides were tested to locate the calpain cleavage sites after arginine 359. Finally, we developed an enhanced cyan and yellow fluorescent protein (ECFP and EYFP)-based intramolecular fluorescent resonance energy transfer (intramolecular FRET) system to monitor the OTK18 endoproteolysis in human microglia cell line. Inhibition of proteasome activity significantly increased the intramolecular FRET signal in the nucleus. These data suggest that calpain and proteasome are involved in OTK18 endoproteolysis and degradation. Additionally, intramolecular FRET has proven to be a useful tool for monitoring the processing in live cells.
Authors:
Lindsey B Martinez; Shannon M Walsh; Michael T Jacobsen; Shinji Sato; Jayme Wiederin; Pawel Ciborowski; Tsuneya Ikezu
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2008-11-26
Journal Detail:
Title:  Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology     Volume:  4     ISSN:  1557-1904     ISO Abbreviation:  J Neuroimmune Pharmacol     Publication Date:  2009 Mar 
Date Detail:
Created Date:  2009-02-17     Completed Date:  2009-05-11     Revised Date:  2014-09-19    
Medline Journal Info:
Nlm Unique ID:  101256586     Medline TA:  J Neuroimmune Pharmacol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  116-28     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Adenoviridae / genetics
Blotting, Western
Calpain / genetics,  physiology*
Cell Line
Cell Nucleus / metabolism
DNA-Binding Proteins / biosynthesis*,  genetics*
Fluorescence Resonance Energy Transfer
Gene Deletion
Green Fluorescent Proteins
Humans
Kruppel-Like Transcription Factors / genetics
Macrophages / drug effects,  metabolism*,  ultrastructure
Mutation / genetics
Proteasome Endopeptidase Complex / genetics,  physiology*
Reverse Transcriptase Polymerase Chain Reaction
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Tissue Culture Techniques
Transcription Factors / biosynthesis*,  genetics*
Transfection
Zinc Fingers / genetics
Grant Support
ID/Acronym/Agency:
P20 RR015635/RR/NCRR NIH HHS; P20 RR015635-086861/RR/NCRR NIH HHS; P20RR15635/RR/NCRR NIH HHS; R01 MH072539/MH/NIMH NIH HHS; R01 MH072539/MH/NIMH NIH HHS; R01 MH072539-01A1/MH/NIMH NIH HHS; R01 MH072539-02/MH/NIMH NIH HHS; R01 MH072539-03/MH/NIMH NIH HHS; R01 MH072539-04/MH/NIMH NIH HHS
Chemical
Reg. No./Substance:
0/Cyan Fluorescent Protein; 0/DNA-Binding Proteins; 0/Kruppel-Like Transcription Factors; 0/Transcription Factors; 0/ZNF175 protein, human; 147336-22-9/Green Fluorescent Proteins; EC 3.4.22.-/Calpain; EC 3.4.25.1/Proteasome Endopeptidase Complex
Comments/Corrections

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