Isolation and characterization of the nuclear serpin MENT

Sergei Grigoryev, Sheena McGowan

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

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.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages29-47
Number of pages19
DOIs
StatePublished - 2011

Publication series

NameMethods in Enzymology
Volume501
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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    Grigoryev, S., & McGowan, S. (2011). Isolation and characterization of the nuclear serpin MENT. In Methods in Enzymology (pp. 29-47). (Methods in Enzymology; Vol. 501). Academic Press Inc.. https://doi.org/10.1016/B978-0-12-385950-1.00003-1