TY - JOUR
T1 - Calpain and proteasomal regulation of antiretroviral zinc finger protein OTK18 in human macrophages
T2 - Visualization in live cells by intramolecular FRET
AU - Martinez, Lindsey B.
AU - Walsh, Shannon M.
AU - Jacobsen, Michael T.
AU - Sato, Shinji
AU - Wiederin, Jayme
AU - Ciborowski, Pawel
AU - Ikezu, Tsuneya
N1 - Funding Information:
Acknowledgments We would like to thank M. Tardieu for human microglia cell line, Y. Tot for supporting the quantification of SE-FRET images, J. Buescher for assisting with tissue culture and purification of DNA plasmids, and Meg Marquardt for manuscript editing. This work is supported in part by NIH Grants R01 MH072539 (T.I.) and NCRR P20RR15635 (T.I.).
PY - 2009/3
Y1 - 2009/3
N2 - 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.
AB - 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.
KW - Endoproteolysis
KW - HIV-1
KW - Live imaging
KW - Macrophage
KW - Protease
KW - Transcription factor
UR - http://www.scopus.com/inward/record.url?scp=60549108880&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=60549108880&partnerID=8YFLogxK
U2 - 10.1007/s11481-008-9140-4
DO - 10.1007/s11481-008-9140-4
M3 - Article
C2 - 19034669
AN - SCOPUS:60549108880
SN - 1557-1890
VL - 4
SP - 116
EP - 128
JO - Journal of Neuroimmune Pharmacology
JF - Journal of Neuroimmune Pharmacology
IS - 1
ER -