Document Detail


Isolation and Characterization of the Nuclear Serpin MENT.
MedLine Citation:
PMID:  22078529     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
A balance between proteolytic activity and protease inhibition is required to maintain the appropriate function of biological systems in which proteases play a role. The Myeloid and Erythroid Nuclear Termination protein, MENT, is a nonhistone heterochromatin-associated serpin that is an effective inhibitor of the papain-like cysteine proteases. Our laboratories have extensively investigated the dual functions of this protein, namely, chromatin condensation and protease inhibition. Unlike other serpins to date, MENT contains a unique insertion between the C- and D-helices known as the "M-loop." This loop contains two critical functional motifs that allow the nuclear function of MENT, namely, nuclear localization and DNA binding. However, the nuclear function of MENT is not restricted to the activities of the M-loop alone. In vitro, MENT brings about the dramatic remodeling of chromatin into higher-order structures by forming protein bridges via its reactive center loop. Further, we have determined that in a protease-mediated effect, DNA can act as a cofactor to accelerate the rate at which MENT can inhibit its target proteases. In this chapter, we discuss the isolation of MENT from native chicken blood as well as recombinant protein produced in Escherichia coli. Various techniques including in vitro functional assays and biophysical characterization are explained that can be used to elucidate the ability of the protein to interact with DNA and other deoxynucleoprotein complexes. In situ chromatin precipitation using natively purified MENT is also detailed.
Authors:
Sergei Grigoryev; Sheena McGowan
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Methods in enzymology     Volume:  501     ISSN:  1557-7988     ISO Abbreviation:  Meth. Enzymol.     Publication Date:  2011  
Date Detail:
Created Date:  2011-11-14     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0212271     Medline TA:  Methods Enzymol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  29-47     Citation Subset:  IM    
Copyright Information:
Copyright © 2011 Elsevier Inc. All rights reserved.
Affiliation:
Department of Biochemistry & Molecular Biology, Penn State University College of Medicine, Milton S. Hershey Medical Center, University Drive, Hershey, Pennsylvania, USA.
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