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Originally published In Press as doi:10.1074/jbc.M706991200 on October 8, 2007

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*Formula

Poh Chee Ong{ddagger}1, Sheena McGowan{ddagger}1, Mary C. Pearce{ddagger}, James A. Irving{ddagger}§2, Wan-Ting Kan{ddagger}, Sergei A. Grigoryev3, Boris Turk||4, Gary A. Silverman**, Klaudia Brix{ddagger}{ddagger}5, Stephen P. Bottomley{ddagger}, James C. Whisstock, An NHMRC Principle Research Fellow and a Monash University Senior Logan Fellow. Joint senior author{ddagger}6, and Robert N. Pike, Joint senior author{ddagger}7

From the {ddagger}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 {ddagger}{ddagger}School of Engineering and Science, Jacobs University Bremen, Campus Ring 6, D-28759 Bremen, Germany

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The papain-like cysteine protease, cathepsin L, has long been known as a degradative enzyme of endocytic organelles of mammalian cells, participating in the important role of protein turnover and antigen processing in these compartments (1, 2). Cathepsin L is also associated with an endosomal processing step during invasion of the cell by the Ebola virus (3), the SARS coronavirus (4), and the murine hepatitis coronavirus (5). Gene duplication in primates has given rise to two isoforms of this protein, termed cathepsin L and cathepsin V (6); an abnormal skin phenotype exhibited by mice lacking the cathepsin L gene was rescued by a transgenic strain bearing human cathepsin V but not human cathepsin L (7), suggesting specific and distinct roles for these isoenzymes.

Recently, an internally translated form of murine cathepsin L with a shortened preproregion was shown to traffic to the nucleus (8). Nuclear murine cathepsin L was able to cleave the transcription factor CDP/Cux and thereby influence progression through the cell cycle (8). These studies and others (9–12), have begun to uncover a hitherto unsuspected role for cathepsins outside endocytic compartments. Further, the observation that these cathepsins are unstable at neutral pH and hence that their proteolytic activity should be confined to acidic organelles of the cell has been shown to be an artifact of buffer choice in in vitro experiments (11–14). It is clear that in conditions mimicking physiological pH and ionic strength, these enzymes retain activity for longer than previously thought (13–16). Accordingly, human cathepsin L is also able to play an important role in tumor metastasis in the extracellular milieu, where overexpression of this protein leads to misdirected trafficking to the cell surface via the secretory pathway (for a review, see Ref. 17).

The means by which a protease is regulated is integral to its biological role. The presence of cathepsins in the nucleus almost certainly necessitates a corresponding inhibitor of nuclear cathepsin activity. We have shown that MENT (myeloid and erythrocyte nuclear termination stage-specific protein), a serpin predominantly localized to the nucleus of avian cells, is capable of inhibiting human cathepsins L and V in vitro (15). MENT was originally identified as a chromatin remodeling protein in the nucleus of terminally differentiated avian granulocytes and erythrocytes (18). Accordingly, it also has the ability to bind DNA (19) and can effectively condense chromatin both in vivo and in vitro (15, 18–20). Furthermore, the human cysteine protease-inhibiting serpin, SCCA-1 (squamous cell carcinoma antigen-1), has also been shown to localize to the nucleus of certain cell types (21).

The serpin inhibitory mechanism relies on conformational change, and several serpins, most notably the plasma serpins, antithrombin and heparin cofactor II (22), become more efficient protease inhibitors in the presence of a bound cofactor. Further, certain proteases also bind cofactors to mediate interactions with substrates and serpin-like inhibitors (22, 23). Thus, given the observation that both papain-like cysteine proteases and serpins can occur in the nucleus, we have investigated whether the interaction between serpins and the papain-like cysteine proteases are influenced by DNA. Our data reveal that, in the presence of DNA, cathepsin V is inhibited 10–50-fold more rapidly by serpins due to both a change in the enzyme and templating effects of longer DNA molecules with DNA-binding serpins. We show that this effect is most likely mediated via a structural change in the protease. Further, for serpins that interact with DNA, we show that a templating effect is also apparent. Together, our data suggest that nuclear cathepsins may be preferentially primed by DNA for inhibition by nuclear serpins.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Inhibitors, Enzymes, Substrates, DNA, and Buffers—The MENT protein was expressed and purified as described previously (19). MENT concentration was determined using absorbance at 280 nm with reference to the predicted extinction coefficient (0.805/mg ml-1). SCCA-1 expressed in Escherichia coli was a kind gift from Dr. Ruby Law (Department of Biochemistry and Molecular Biology, Monash University). Stefin A (cystatin A) was expressed in an E. coli system as described previously (24). Human cathepsins V and L were expressed in Pichia pastoris (system kindly donated by Dr. Dieter Bromme) and purified as described previously (25). All cathepsins were preactivated by incubation in cathepsin buffer (0.1 M acetate, 1 mM EDTA, 0.1% (w/v) Brij-35, 0.02% (w/v) sodium azide, 10 mM cysteine, pH 5.5) for at least 20 min at room temperature before use. The active enzyme concentration was determined with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane (E-64) (Sigma) titration. The DNA probes (Gene Works Pty. Ltd.) used in the enzymatic reactions and in electrophoretic mobility shift assays are described in supplemental Table 1.

Determination of the Association Rate Constant (ka) for Serpin-Cysteine Protease Interaction in the Presence and Absence of Cofactors—The discontinuous method of determining ka values between serpins and papain-like cysteine protease was undertaken in the pH 5.5 cathepsin buffer at 30 °C using the well mode in a Fluorostar Galaxy plate reader (BMG Labtechnologies) with excitation/emission wavelengths of 370/460 nm. Residual activity of cathepsin V in the presence of ds65mer DNA had to be measured within 20 s, since the inhibition by MENT in the presence of DNA was essentially complete within 1 min. For any given assay, the enzyme concentration was held constant at 0.1 nM, whereas the serpin concentration was at least 5-fold higher, with the concentration varying between 0.5 and 60 nM (maximum of 5 nM serpin in the presence of DNA). The DNA concentrations for the discontinuous assays were usually 10 nM, at least 2-fold in excess over the serpin concentration.

Following the addition of serpin to the protease in the presence or absence of cofactor, the residual protease activity at various time points was determined in a final volume of 200 µl of 40 µM N-benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin substrate (Z-Phe-Arg-NHMec)8 (Sigma) in cathepsin buffer. Substrate turnover was monitored at 1-s intervals over a short period of time. The natural logarithm of the initial velocity was then plotted against the elapsed time since serpin addition, and the resulting relationship was quantitated using linear least squares regression in PRISM (GraphPad Software). The gradient of this line represents the observed rate of inhibition for a given concentration of serpin (-kobs). The association rate constant (ka (M-1 s-1)) was then calculated from the slope of a plot of -kobs values against inhibitor concentration.

Determination of the Stoichiometry of Inhibition (SI) in the Absence and Presence of Cofactors—For each assay, 50 µl of 20 nM activated cathepsin was mixed with a constant concentration of the cofactors that were used in the discontinuous kinetic assays for 5 min at 37 °C, followed by the addition of increasing concentrations of serpin. The mixture was then incubated at 37 °C for 20–45 min. The reactions were diluted 10-fold with cathepsin buffer, and 100 µl of 40 µM Z-Phe-Arg-NHMec substrate was added to each incubation mixture. The rate of increase in fluorescence, which is proportional to the amount of residual protease activity, was immediately monitored, and this value was plotted against the molar ratio of serpin to cathepsin using PRISM (GraphPad Software). The stoichiometry of inhibition was determined as the x intercept value calculated from linear regression analysis as previously described (26).

Determination of Association Rate Constants for Cystatin A Inhibition of Cathepsin V—The ka value for cystatin A inhibition of cathepsin V was determined by continuously monitoring the rate of substrate cleavage by cathepsin V over a period of time in the presence of varying concentrations of cystatin A. Cathepsin V was preactivated at 37 °C for at least 15 min prior to performing the assay. In each assay, cathepsin V (0.05 nM), DNA (2 µM), and substrate (Z-Phe-Arg-NHMec) were kept constant. Cystatin concentrations were varied from 0 to 2 nM. Cystatin A was incubated with the substrate for 5 min at 30 °C before adding cathepsin V to the reaction mixture. Upon the addition of cathepsin V, the rate of substrate cleavage by cathepsin V was monitored continuously for 30 min. The data were collected and fitted using the equation described in Ref. 15. The ka value was determined by taking into account substrate competition.

Determination of Kinetic Constants for Cleavage of Z-Phe-Arg-AMC Substrate—The final concentrations of cathepsin V and ds65mer DNA were 0.1 and 10 nM, respectively, whereas final substrate concentrations ranged from 5 to 200 µM in cathepsin V buffer. Initial velocities were determined from the linear portion of the release of fluorescence, detected with excitation/emission wavelengths of 370/460 nm at 37 °C. Each experiment was performed in triplicate. Nonlinear regression of velocity against substrate concentration was performed with Graph Prism 3 according to the equation, v = Vmax[S]/Km + [S].


Figure 1
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  		FIGURE 1.
Determination of association rate constants for cathepsin V/MENT reactions in the presence and absence of DNA. The initial velocity of substrate turnover by cathepsin V (0.1 nM) was determined in the presence of various concentrations of MENT at different time points, in the absence (A) and presence of ds65-mer DNA (B). The natural logarithm of the initial velocity (ln slope) was plotted against the time point (s) and fitted using linear least squares regression. The resulting negative linear slope reflects the observed rate of inhibition for each individual serpin concentration (-kobs). The dependence of kobs on MENT concentration in the absence (C) and presence of ds65-mer DNA (D) was plotted to yield the ka value for the reactions.

 
Electrophoretic Gel Mobility Shift Assay—DNA, at a final concentration of 0.5 µM, was used with serpin and cathepsin concentrations varied between 0 and 10 µM in buffer containing 10 mM HEPES, 0.5 mM EDTA, 40 mM NaCl, 0.1% (w/v) Brij-35, pH 7.0 and pH 5.5. The reactions were incubated for 15 min at room temperature before loading onto a 2% agarose I gel.

Measurements of Change in Intrinsic Tryptophan Fluorescence of Cathepsins—Potential cofactor-induced conformational change in cathepsin V was studied by monitoring the intrinsic fluorescence changes of the enzymes in the presence and absence of cofactor. Cathepsin V (200 nM) was incubated with increasing amounts of the cofactor in cathepsin buffer in a final volume of 1 ml. An excitation wave-length of 295 nm was used, and the emission was scanned five times over the range of 300–400 nm at 100 nm/min, using excitation and emission slit widths of 8 nm. The emission spectrum of a buffer control (with or without DNA, as appropriate) was subtracted from the protein emission spectra.

Fluorescence quenching experiments were performed in the presence and absence of 100 nM DNA (ds24mer) in cathepsin buffer using increasing concentrations of acrylamide (0–0.5 M). From the recorded titration spectra, the extent of quenching and the accessibility of tryptophan residues were calculated from Stern-Volmer plots as previously described by Lehrer (27). The absorbance of samples at excitation and emission wavelengths was monitored in all experiments and did not exceed 0.01.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
dsDNA Accelerates the Interaction between Cathepsin V and MENT—A key indicator of the ability of a serpin to inhibit a protease is the association rate constant (ka), which reflects the overall rate of inhibition. MENT inhibits human cathepsins V and L with a ka value on the order of 105-106 M-1 s-1 (15), showing it to be a very effective inhibitor of these enzymes (Fig. 1 and Table 1). The SI value reflects negligible nonproductive inhibitor turnover, with 1.1 molecules of inhibitor required to inhibit one molecule of cathepsin V protease (Table 1).


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  		TABLE 1
Kinetic parameters of MENT, SCCA-1, and cystatin A in the presence of various DNA constructs All assays were performed in triplicate, S.E. values for each value reported were less than 10% of the value.

 
We tested the effect of the presence of several types of DNA on the inhibitory interaction between these molecules (Table 1). The rate of association between MENT and cathepsin L, as well as the SI, has previously been found to be unaffected by DNA (19) (Table 1). In contrast, the presence of double stranded 24- and 65-mer (ds24- and ds65-mer) DNA accelerated the inhibition of cathepsin V by MENT by 10- and 50-fold, respectively, without altering the SI (Fig. 1 and Table 1). This effect was also found to be specific to dsDNA, since it was not observed in the presence of single-stranded oligonucleotides (Table 1).

DNA Accelerates the Interaction between SCCA-1 and Cathepsin V—Since cathepsins V and L are orthologues of cathepsin L molecules in other mammals and 80% identical in sequence (25), it was surprising to find that DNA accelerated the interaction between MENT and human cathepsin V but did not affect the interaction with human cathepsin L. In order to determine whether the acceleration effect was mediated through the protease or the inhibitor, we examined the effect of DNA on the interaction between cathepsin V and the cysteine protease inhibiting serpin, SCCA-1. In contrast to MENT (28), SCCA-1 is an acidic molecule that does not interact with DNA (Fig. 2A). Any observed effect would therefore be mediated via interaction with cathepsin V.


Figure 2
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  		FIGURE 2.
Determination of the binding of serpins and papain-like cysteine proteases to DNA. Gel mobility shift analysis of SCCA-1 (A), cathepsin L (B), and cathepsin V (C and D) incubated with ds65-mer (A–C) and ds24-mer (D) DNA was analyzed by agarose gel electrophoresis. The final concentration of purified protein in each reaction is indicated at the top of each gel panel (µM). The arrows indicate the different species formed: free DNA (F), bound DNA (B).

 


Figure 3
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  		FIGURE 3.
Comparison of electrostatic potential of human cathepsins V and L. CCP4MG (43, 44) electrostatic potential surface of human cathepsin V (A) (Protein Data Bank code 1FH0) and human cathepsin L (B) (Protein Data Bank code 1CS8). The positions of cathepsin V basic residues are indicated.

 
The kinetic experiments revealed that, although SCCA-1 is unable to bind DNA, the ka value for SCCA-1 and cathepsin V was accelerated by ~10-fold in the presence of ds65-mer DNA. The SI of the interaction was unaffected (Table 1). As in the MENT experiments, the interaction between SCCA-1 and cathepsin L was unaltered in the presence of ds65-mer DNA (Table 1). Together, these data suggest strongly that, contrary to our initial expectations, the DNA is exerting a major effect through the cathepsin V enzyme. Further, this effect is specific to cathepsin V, since serpin/protease interactions with cathepsin L were unaffected.

Cathepsin V, but Not Cathepsin L, Interacts with DNA—In order to evaluate the mechanism underlying the increase in ka in the presence of DNA, we evaluated the interaction between DNA and the two cathepsins. In a gel shift analysis, we noted that cathepsin L was unable to interact or even affect the electrophoretic migration of the ds65-mer within agarose (Fig. 2B). However, when cathepsin V was used in a similar experiment, smearing of the ds65-mer was noted when 4 µM protein was added (Fig. 2C). A further experiment with a greater excess of cathepsin V confirmed that it was capable of interacting with the DNA, albeit in an apparent nonspecific manner, since no discrete protein-DNA complexes were noted (Fig. 2C). Gel shifts using ds24-mer showed a similar pattern of cathepsin V-mediated interaction with DNA (Fig. 2D).

The x-ray crystal structure of cathepsin V has been determined (29), and using these data, we investigated whether any prominent positively charged patches, that may be able to interact with DNA, could be identified on the surface of the molecule (Fig. 3A). The structure reveals that the residues Lys-58, Arg-72, and Lys-78 contribute to a large positively charged patch and flank a region that defines one side of the active site cleft at the P1–P3 subsite positions. Analysis of the structure of cathepsin L revealed that this region of the molecule was negatively charged (Fig. 3B).

DNA Promotes a Conformational Change in Cathepsin V—To further investigate DNA binding by cathepsin V and evaluate whether DNA induced any conformational rearrangement in cathepsin V, we examined the changes in the tryptophan fluorescence spectra of cathepsin V in the absence and presence of DNA. The tryptophan fluorescence emission spectra showed a marked difference in the presence and absence of dsDNA (ds24-mer and ds65-mer) with a 15 and 18% reduction, respectively, in the fluorescence emission at 333 nm recorded at the highest concentration of DNA used (Fig. 4A).


Figure 4
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  		FIGURE 4.
The effect of dsDNA on cathepsin V fluorescence. The intrinsic tryptophan fluorescence emission spectra of cathepsin V was scanned over the range of 300–390 nm, after excitation at 295 nm, in the absence (black) and presence of 5.0 (red) and 50 nM (green) ds65-mer DNA and ds24-mer DNA at pH 5.5 (A). B, Stern-Volmer plot ((F0 - F - 1) versus [acrylamide]) for cathepsin V in the presence ({blacksquare}) and absence ({blacktriangleup}) of ds24-mer DNA. Titrations were carried out in the range of 0–0.5 M acrylamide.

 
To determine if these changes in fluorescence emission were indeed due to conformational change in the enzyme and not a result of the DNA shielding the tryptophan residues, fluorescence quenching titrations were performed using acrylamide. The Stern-Volmer plot for both cathepsin V in the absence and presence of DNA was nonlinear. Assuming collisional quenching, this nonlinearity could be explained by at least two populations of tryptophans, with differing levels of accessibility to the quencher (Fig. 4B). The presence of DNA caused an increase in the accessibility of the tryptophan residues of cathepsin V, with the Stern-Volmer plot showing increased quenching. These data indicate that the presence of DNA induces a conformational change in cathepsin V that results in the exposure of at least one tryptophan residue.

The sequence and structure of cathepsin V reveals that the mature protein contains five tryptophan residues. One tryptophan residue, Trp-26, underlies the positively charged patch that we propose may be responsible for coordinating DNA, a region also observed to exhibit some mobility in papain (30). However, the number and distribution of tryptophan residues means it is not possible to ascertain the precise structural effects of DNA binding to cathepsin V through biophysical studies.

DNA Has Effects on the Interaction between Cathepsin V and a Peptide Substrate—The increase in the rate of inhibition brought about by DNA indicated that it might be influencing active site residues or subsites of cathepsin V. We therefore investigated whether the interaction between cathepsin V and a commonly used substrate, Z-Phe-Arg-NHMec (31), was altered in the presence of DNA. The presence of ds65-mer DNA caused a small, nonsignificant decrease in the Km value for the interaction between cathepsin V and the Z-Phe-Arg-NHMec substrate, from 33.1 ± 1.5 to 27.5 ± 0.2 µM. A larger, significant effect on the turnover (kcat) value was observed, with the presence of DNA reducing the value from 93.9 ± 1.5 to 37.7 ± 0.2 s-1. These data indicate that the binding of DNA changes the active site of cathepsin V such that the enzyme interacts with peptide substrates differently.

Involvement of a Templating Mechanism in the Increase of Cathepsin V/MENT Association Rates—Since both cathepsin V and MENT bind DNA, we investigated whether a templating mechanism, brought about by binding of both proteins to one molecule of DNA, was operative in the acceleration of the interaction rate between cathepsin V and MENT. The effect of increasing DNA concentrations on the observed rate of interaction between the molecules was investigated. A clear bell-shaped curve was seen for the relationship between the kobs values and the concentration of ds65-mer DNA (Fig. 5A), indicating that a templating mechanism was indeed involved in the increased rate of interaction in the presence of DNA. This relationship was not seen for ds24-mer DNA (Fig. 5B), indicating that it might have been too short to achieve simultaneous binding to both inhibitor and enzyme (Fig. 5C). In addition, both species of DNA affected the rate of interaction between SCCA-1 and cathepsin V in a monophasic manner (Fig. 5, D and E), indicating that templating was not occurring. This meant that the rate enhancement seen with ds24-mer DNA in cathepsin V-MENT reactions and for both species of DNA in cathepsin V-SCCA-1 reactions could not be explained through a templating effect.

The ds24-mer DNA would span just over two DNA helical turns (one DNA helical turn spans 10.5 bp/34 Å in length) and equates to ~75 Å in length. In contrast, the ds65-mer is over 210 Å in length, spanning over six helical DNA turns. For a templating effect to occur between cathepsin V and MENT, it would be necessary for the DNA to be long enough to effectively reach from the DNA-binding domain of MENT at the bottom of the D-helix and M-loop to the top of the RCL and then beyond to interact with the cathepsin V (Fig. 5C). The length of the MENT molecule from the M-loop to the stumps of the RCL is in excess of 75 Å, indicating that in order to also interact with the cathepsin, the ds24-mer would most likely be insufficient in length to interact with both molecules (Fig. 5C).


Figure 5
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  		FIGURE 5.
Mechanisms of interaction between serpins, human cathepsin V and DNA. A, the relationship between kobs values for interaction between MENT and cathepsin V and the concentration of ds65-mer DNA (0–50 nM) indicates that a templating mechanism is operative. A templating mechanism was not observed for MENT, cathepsin V, and ds24-mer (B) or SCCA-1, cathepsin V, and ds65-mer (D) or ds24-mer (E). The concentration of each dsDNA is indicated at the bottom of each panel (nM). C, schematic diagram showing that to achieve a templating mechanism between MENT and cathepsin V, dsDNA would need to reach from the DNA binding region of MENT, the M-loop/D-helix, to the top of the RCL and then to the cathepsin. The ds24-mer is too short to achieve a template effect, in contrast to the ds65-mer.

 
DNA Does Not Accelerate the Interaction between Cathepsin V and Cystatin A—We investigated whether the interaction between cathepsin V and other classes of inhibitor was influenced by DNA. Cathepsins are strongly inhibited by cystatins in cells and in extracellular fluids (32). We investigated whether DNA influenced the reaction between cathepsin V and cystatin A (33), since the mode of interaction between cathepsins and cystatins is entirely different from that seen with serpins. In particular, cystatins do not interact directly with the catalytic residues of cysteine proteases and instead occlude the active site, preventing access to substrates. Thus, for instance, cystatins are still able to bind the active site of cysteine proteases that have been previously covalently inhibited with molecules such as E-64 (34). It was found that DNA did not affect the rate of association between cystatin A and cathepsin V (Table 1). These data suggest that the acceleration of inhibition is specific to the serpin mode of inhibition, where there is direct interaction between the inhibitor and the active site residues of the protease.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Several recent studies have revealed that cathepsins localize to the nucleus, where they may play key roles, such as regulating DNA conformational changes that are necessary in processes such as transcription (8, 35). Given that numerous intracellular serpins also localize to the nucleus, including PI-6 and MNEI and the cysteine protease inhibitors MENT and SCCA-1 (18, 21, 26, 36–38),9 it is likely that one role for these serpins is the regulation of nuclear proteases (35).

The recent structure of the DNA-binding serpin, MENT, revealed that the RCL of this molecule was partially inserted into the A β-sheet, suggesting that, by analogy with antithrombin, a cofactor may modulate its inhibitory activity by inducing a conformational change. Modulation of plasma serpin activity against serine proteases is relatively common (39, 40). However, in a previous study, dsDNA failed to mediate an effect on the rate of association between MENT and cathepsin L (19).

This study provides evidence for cofactor-mediated modulation of an intracellular serpin/protease interaction; DNA renders cathepsin V susceptible to inhibition by serpins but in a manner independent of serpin conformational change. In particular, the close homologue of cathepsin V, human cathepsin L, is unable to bind DNA and demonstrates no acceleration effect. Thus, the enhanced rate of inhibition was found to be primarily effected through changes in the protease itself and secondarily due to sufficiently long DNA acting as a template for the "docking" inhibitor-enzyme complex. As a result, the DNA-binding serpin MENT and the non-DNA-binding serpin SCCA-1 both demonstrated a monophasic, 10-fold increase in the rate of inhibition with a short length of DNA. With a stretch of DNA capable of spanning the full "docking" complex, however, the inhibition by MENT was accelerated a further 5-fold in a biphasic manner. This templating effect brings about a rate enhancement due to facilitated diffusion of the molecules in solution (41, 42).

Several lines of evidence suggest that changes to the catalytic center of the enzyme are contributing to the effect of DNA on the interaction with serpins. 1) A change in intrinsic tryptophan fluorescence and titration with a fluorescence quenching agent revealed that cathepsin V undergoes a slight conformational shift in the presence of DNA. 2) DNA failed to affect inhibition of cathepsin V by cystatins, whose mode of action is by occluding the active site and therefore independent of proteolysis. 3) DNA alters the kinetics of peptide substrate cleavage by cathepsin V.

There is now strong evidence that cathepsin V is found in the nucleus and also that serpins that can interact with this enzyme are found in this compartment. Bulynko et al. (35) noted that cathepsin inhibition by high doses of a nuclear serpin (MENT or MNEI) may contribute to chromatin rearrangement in mature granulocytes, and it is plausible that other closely related intracellular serpins, such as SCCA-1, SQN-5, and Spi2A, may participate in controlling cathepsin-mediated chromatin regulation in other cell types. This work reveals that DNA should be taken into account when biochemically characterizing nuclear cysteine protease-serpin interactions. Most importantly, it highlights a mechanism by which cathepsin inhibition can be modulated, according to the cellular compartment in which it is localized.


   FOOTNOTES
 
* 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. Back

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

1 Both authors contributed equally to this work. Back

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

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

4 Supported by grants from the Slovene Research Agency. Back

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

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.

8 The abbreviations used are: Z-Phe-Arg-NHMec, N-benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin substrate; SI, stoichiometry of inhibition; dsDNA, double-stranded DNA; ds24-mer and ds65-mer, double-stranded 24- and 65-mer, respectively. Back

9 P. Bird, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Debbie Pike for excellent technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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