Molecular Regulation of Inducible Nitric Oxide Synthase

Molecular Regulation of Inducible Nitric Oxide Synthase

Bibliographic Details
Main Author: Wang, Tingting
Language:English
Published: The Ohio State University / OhioLINK 2012
Subjects:
Biochemistry
Biology
Biomedical Research
Cellular Biology
Molecular Biology
iNOS
CaM
CaMKII
Ca<sup>2+</sup>
sepsis
NO
Hsp90
SPSB2
induction
aggregation
degradation
ubiquitination
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=osu1353684453
id ndltd-OhioLink-oai-etd.ohiolink.edu-osu1353684453
record_format oai_dc
collection NDLTD
language English
sources NDLTD
topic Biochemistry
Biology
Biomedical Research
Cellular Biology
Molecular Biology
iNOS
CaM
CaMKII
Ca<sup>2+</sup>
sepsis
NO
Hsp90
SPSB2
induction
aggregation
degradation
ubiquitination
spellingShingle Biochemistry
Biology
Biomedical Research
Cellular Biology
Molecular Biology
iNOS
CaM
CaMKII
Ca<sup>2+</sup>
sepsis
NO
Hsp90
SPSB2
induction
aggregation
degradation
ubiquitination
Wang, Tingting
Molecular Regulation of Inducible Nitric Oxide Synthase
author Wang, Tingting
author_facet Wang, Tingting
author_sort Wang, Tingting
title Molecular Regulation of Inducible Nitric Oxide Synthase
title_short Molecular Regulation of Inducible Nitric Oxide Synthase
title_full Molecular Regulation of Inducible Nitric Oxide Synthase
title_fullStr Molecular Regulation of Inducible Nitric Oxide Synthase
title_full_unstemmed Molecular Regulation of Inducible Nitric Oxide Synthase
title_sort molecular regulation of inducible nitric oxide synthase
publisher The Ohio State University / OhioLINK
publishDate 2012
url http://rave.ohiolink.edu/etdc/view?acc_num=osu1353684453
work_keys_str_mv AT wangtingting molecularregulationofinduciblenitricoxidesynthase
_version_ 1719431076286300160
spelling ndltd-OhioLink-oai-etd.ohiolink.edu-osu13536844532021-08-03T06:06:46Z Molecular Regulation of Inducible Nitric Oxide Synthase Wang, Tingting Biochemistry Biology Biomedical Research Cellular Biology Molecular Biology iNOS CaM CaMKII Ca<sup>2+</sup> sepsis NO Hsp90 SPSB2 induction aggregation degradation ubiquitination <p>Nitric oxide (NO) generated by inducible NO synthase (iNOS) plays critical roles in inflammation and host defense. iNOS expression is induced by inflammatory stimuli and exhibits constant activity once expressed. Hence NO production from iNOS has been thought to be primarily controlled via enzyme expression. In this study, we identify an array of novel mechanisms showing that iNOS is regulated at multiple levels including gene expression, protein aggregation, and protein degradation. </p><p>Calmodulin (CaM) has always been thought as an iNOS cofactor facilitating electron transfer amid NO syntheses. We now show that CaM is essential for iNOS induction. CaM inhibition or knockdown prevented iNOS expressions in macrophages stimulated by inflammatory mediators. Further studies revealed that CaM acted through Ca<sup>2+</sup>/CaM-dependent kinase II (CaMKII), which functioned as a cardinal initiator in iNOS gene transactivation via both LPS-NF-¿¿¿¿¿B and IFN-γ-STAT1 pathways. Our study also found that iNOS gene transactivation required cytosolic Ca<sup>2+</sup> elevations. CaM or CaMKII inhibition was found to prevent iNOS induction in endotoxemic mice and improved survival rates. These studies extend the role of CaM from an enzyme cofactor to an essential modulator in iNOS gene transactivation. </p><p>Once expressed, biological iNOS output is determined by the levels of functional enzyme, which is influenced by protein stability. Our studies found that while initially existing as a soluble protein, iNOS progressively formed inactive protein aggregates. Blocking NO production prevented iNOS aggregation inside cells. iNOS aggresome formation could be recaptured by exposing cells to exogenous NO. The finding that NO per se induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.</p><p>After elucidating NO-mediated iNOS aggregation in physiological conditions, we then investigated iNOS protein stability under pathological circumstances upon heat shock protein 90 (Hsp90) or CaM inhibition. Hsp90 was previously reported to play an important role in iNOS function and gene expression. We now find that dissociation of Hsp90 from iNOS led to iNOS aggregation and these aggregates were cleared by ubiquitin-proteasome system. Further studies revealed that the SPRY domain-containing SOCS box protein 2 (SPSB2), but not the C-terminus of heat shock protein 70-interacting protein (CHIP), was essential for aggregated iNOS turnover in Hsp90-inhibited cells. Our studies found that CaM inhibition disrupted CaM-iNOS interaction, exposing the hydrophobic CaM-binding domain on iNOS which mediated iNOS aggregation. These studies identified novel roles of Hsp90 and CaM in modulating iNOS protein stability.</p><p> To gain a complete understanding of iNOS regulation, we investigated the ubiquitination site responsible for proteasomal degradation of iNOS. The N-terminal 1-100 amino acids were found to be essential for iNOS degradation. However, arginine replacement of all lysines within this region failed to prevent iNOS degradation. Further studies identified several non-lysine residues among the N-terminal region of iNOS crucial for its ubiquitination and proteasomal degradation. </p><p>In summary, our investigations change the notion that iNOS biology is largely dependent on the levels of its gene expression. The knowledge gained from this work provided a comprehensive understanding of iNOS regulation via three interrelated aspects including protein induction, stability and degradation.</p> 2012-12-18 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1353684453 http://rave.ohiolink.edu/etdc/view?acc_num=osu1353684453 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

Similar Items