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J. Biol. Chem., Vol. 282, Issue 51, 36980-36986, December 21, 2007

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DNA Accelerates the Inhibition of Human Cathepsin V by Serpins^*

Poh Chee Ong¹, Sheena McGowan¹, Mary C. Pearce, James A. Irving², Wan-Ting Kan, Sergei A. Grigoryev^¶3, Boris Turk^||4, Gary A. Silverman^**, Klaudia Brix⁵, Stephen P. Bottomley, James C. Whisstock, An NHMRC Principle Research Fellow and a Monash University Senior Logan Fellow. Joint senior author⁶, and Robert N. Pike, Joint senior author⁷

From the Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia, the Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom, the ^¶Department of Biochemistry and Molecular Biology, Penn State University College of Medicine, Hershey, Pennsylvania 17033, the ^||Department of Biochemistry and Molecular Biology, J. Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia, the ^**Newborn Medicine Program, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and the School of Engineering and Science, Jacobs University Bremen, Campus Ring 6, D-28759 Bremen, Germany

A balance between proteolytic activity and protease inhibition is crucial to the appropriate function of many biological processes. There is mounting evidence for the presence of both papain-like cysteine proteases and serpins with a corresponding inhibitory activity in the nucleus. Well characterized examples of cofactors fine tuning serpin activity in the extracellular milieu are known, but such modulation has not been studied for protease-serpin interactions within the cell. Accordingly, we present an investigation into the effect of a DNA-rich environment on the interaction between model serpins (MENT and SCCA-1), cysteine proteases (human cathepsin V and human cathepsin L), and cystatin A. DNA was indeed found to accelerate the rate at which MENT inhibited cathepsin V, a human orthologue of mammalian cathepsin L, up to 50-fold, but unexpectedly this effect was primarily effected via the protease and secondarily by the recruitment of the DNA as a "template" onto which cathepsin V and MENT are bound. Notably, the protease-mediated effect was found to correspond both with an altered substrate turnover and a conformational change within the protease. Consistent with this, cystatin inhibition, which relies on occlusion of the active site rather than the substrate-like behavior of serpins, was unaltered by DNA. This represents the first example of modulation of serpin inhibition of cysteine proteases by a co-factor and reveals a mechanism for differential regulation of cathepsin proteolytic activity in a DNA-rich environment.

Received for publication, August 21, 2007 , and in revised form, October 5, 2007.

^* This work was supported in part by the National Health and Medical Research Council (NHMRC) of Australia and the Australian Research Council (ARC). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1.

¹ Both authors contributed equally to this work.

² An NHMRC C. J. Martin Postdoctoral Research Fellow.

³ Supported by National Institutes of Health Grant GM-59118.

⁴ Supported by grants from the Slovene Research Agency.

⁵ Supported by Deutsche Forschungsgemeinschaft Grant Br 1308/6-1, 6-2.

⁶ To whom correspondence may be addressed. Tel.: 61-3-99053747; Fax: 61-3-99054699; E-mail: james.whisstock{at}med.monash.edu.au. ⁷ To whom correspondence may be addressed. Tel.: 61-3-99053923; Fax: 61-3-99054699; E-mail: rob.pike{at}med.monash.edu.au.

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