Identification of a Nuclear Targeting Domain in the Insertion between Helices C and D in Protease Inhibitor-10

Protease inhibitor 10 (PI-10), an intracellular ovalbumin-serpin, contains a series of basic amino acids in the loop between helices C and D that exhibit homology to known nuclear targeting signals. Transfection of HeLa cells with plasmids encoding enhanced green fluorescent protein (EGFP) coupled to PI-10 revealed an intense fluorescence of the nucleus. Immunoblotting demonstrated a single M r 80,000 EGFP·PI-10 complex in isolated nuclei. Mutation of four basic amino acids in the interhelical loop to alanines (i.e. K74A, K75A, R76A, K77A) resulted in the fluorescent complex being confined to the cytoplasm. Further evidence for a nuclear targeting signal in this region was provided by localization of the fluorescent label to the nucleus in cells transfected with a plasmid encoding EGFP fused to the 25 amino acids comprising the interhelical loop of PI-10 (i.e. Arg-63 to Glu-87), whereas a cytoplasmic distribution was noted for the construct encoding EGFP coupled to the mutated interhelical loop. These data raise the possibility that PI-10 may play a role in regulating protease activity within the nucleus, a property unique in the field of serpin biology. Protease inhibitor 10 (PI-10), an intracellular ovalbumin-serpin, contains a series of basic amino acids in the loop between helices C and D that exhibit homology to known nuclear targeting signals. Transfection of HeLa cells with plasmids encoding enhanced green fluorescent protein (EGFP) coupled to PI-10 revealed an intense fluorescence of the nucleus. Immunoblotting demonstrated a single M r 80,000 EGFP·PI-10 complex in isolated nuclei. Mutation of four basic amino acids in the interhelical loop to alanines (i.e. K74A, K75A, R76A, K77A) resulted in the fluorescent complex being confined to the cytoplasm. Further evidence for a nuclear targeting signal in this region was provided by localization of the fluorescent label to the nucleus in cells transfected with a plasmid encoding EGFP fused to the 25 amino acids comprising the interhelical loop of PI-10 (i.e. Arg-63 to Glu-87), whereas a cytoplasmic distribution was noted for the construct encoding EGFP coupled to the mutated interhelical loop. These data raise the possibility that PI-10 may play a role in regulating protease activity within the nucleus, a property unique in the field of serpin biology. Serine proteinase inhibitors (serpins) 1The abbreviations used are: serpin, serine protease inhibitor; EGFP, enhanced green fluorescent protein; ov-serpin, ovalbumin family of serine protease inhibitors; PAI-2, plasminogen activator inhibitor type 2; PCR, polymerase chain reaction; PI-10, protease inhibitor-10; PI-10 A4, protease inhibitor A4 mutant K74A, K75A, R76A, K77A; PAGE, polyacrylamide gel electrophoresis. are a large superfamily of homologous proteins that resemble α1-proteinase inhibitor in overall structure and form stoichiometric 1:1 inhibitory complexes with target proteases that are typically stable to treatment with denaturants (e.g. SDS) (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (833) Google Scholar, 2Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Abstract Full Text PDF PubMed Google Scholar, 3Stein P.E. Carrell R.W. Struct. Biol. 1995; 2: 96-113Crossref PubMed Scopus (394) Google Scholar). Serpins play crucial roles in the neutralization of serine protease activities that are involved in a wide variety of vital processes including blood coagulation, fibrinolysis, complement activation, inflammation, and cell migration (2Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Abstract Full Text PDF PubMed Google Scholar). Within the serpin superfamily, ovalbumin represents the parent prototype of a currently emerging family of structurally related proteins (ov-serpins) (4Remold-O'Donnell E. FEBS Lett. 1993; 315: 105-108Crossref PubMed Scopus (214) Google Scholar). Human members of the ov-serpin family include plasminogen activator inhibitor-2 (PAI-2) (5Ye R.D. Ahern S.M. Le Beau M.M. Lebo R.V. Sadler J.E. J. Biol. Chem. 1989; 264: 5495-5502Abstract Full Text PDF PubMed Google Scholar), an elastase inhibitor isolated from monocyte-like cells (6Remold-O'Donnell E. Chin J. Alberts M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5635-5639Crossref PubMed Scopus (120) Google Scholar), squamous cell carcinoma antigen (7Suminami Y. Kishi F. Sekiguchi K. Kato H. Biochem. Biophys. Res. Commun. 1991; 181: 51-58Crossref PubMed Scopus (195) Google Scholar), cytoplasmic antiproteinase (i.e. protease inhibitor-6) (8Morgenstern K.A. Sprecher C. Holth L. Foster D. Grant F.J. Ching A. Kisiel W. Biochemistry. 1994; 33: 3432-3441Crossref PubMed Scopus (26) Google Scholar,9Coughlin P. Sun J. Cerruti L. Salem H.H. Bird P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9417-9421Crossref PubMed Scopus (75) Google Scholar), and a tumor suppressor called maspin (10Zou Z. Anisowicz A. Hendrix M.J.C. Thor A. Neveu M. Sheng S. Rafidi K. Seftor E. Sager R. Science. 1994; 263: 526-529Crossref PubMed Scopus (829) Google Scholar). Two serpins related to protease inhibitor-6 have been cloned from a placental λgtII library (i.e. protease inhibitors -8 and -9) (11Sprecher C.A. Morgenstern K.A. Mathewes S. Dahlen J.R. Schrader S.K. Foster D.C. Kisiel W. J. Biol. Chem. 1995; 270: 29854-29861Crossref PubMed Scopus (76) Google Scholar), and data of Sunet al. (12Sun J. Bird C.H. Sutton V. McDonald L. Coughlin P.B. De Jong T.A. Trapani J.A. Bird P.I. J. Biol. Chem. 1996; 271: 27802-27809Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) suggest that the latter molecule is an intracellular granzyme B inhibitor that is associated with cytotoxic lymphocytes. During studies investigating the presence of protease inhibitors in hematopoiesis, our group utilized a polymerase chain reaction (PCR)-based homology cloning strategy to identify a novel ov-serpin, which exhibited a high amino acid homology (48%) with PAI-2, protease inhibitor-6, and human leukocyte elastase inhibitor (13Riewald M. Schleef R.R. J. Biol. Chem. 1995; 270: 26754-26757Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The isolated cDNA contains a single large open reading frame that encodes a 397-amino acid protein (13Riewald M. Schleef R.R. J. Biol. Chem. 1995; 270: 26754-26757Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Northern blotting analysis with this cDNA revealed a single 2.3-kilobase transcript that is expressed in human bone marrow cells but was undetectable in all other analyzed human tissues. This molecule was designated bone marrow-associated serpin (bomapin) and assigned the systematic title of protease inhibitor 10 (PI-10) by the Genome Data Base Collaboration (13Riewald M. Schleef R.R. J. Biol. Chem. 1995; 270: 26754-26757Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Recent data obtained in our laboratory (14Riewald M. Chuang T.L. Neubauer A. Schleef R.R. Blood. 1998; 91: 1256-1262Crossref PubMed Google Scholar) revealed that transcripts for this protease inhibitor are elevated in the bone marrow and peripheral blood of patients with acute myeloid leukemia and in chronic myelomonocytic leukemia, suggesting that PI-10 may be expressed preferentially in hematopoietic progenitor cells of monocytic lineage. PI-10 exhibits all the structural features that distinguish ov-serpins from the larger family of serpin proteins (13Riewald M. Schleef R.R. J. Biol. Chem. 1995; 270: 26754-26757Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), which is typified by the absence of an N-terminal signal peptide extension that results in an intracellular distribution for many of these molecules (4Remold-O'Donnell E. FEBS Lett. 1993; 315: 105-108Crossref PubMed Scopus (214) Google Scholar). Another feature that is observed only in ov-serpins is the presence of an insertion between helices C and D that is believed to form a loop between these two helixes (4Remold-O'Donnell E. FEBS Lett. 1993; 315: 105-108Crossref PubMed Scopus (214) Google Scholar). For example, exon 3 in the PAI-2 gene codes for a 33-amino acid residue sequence between helices C/D (5Ye R.D. Ahern S.M. Le Beau M.M. Lebo R.V. Sadler J.E. J. Biol. Chem. 1989; 264: 5495-5502Abstract Full Text PDF PubMed Google Scholar), which contains the substrate sites within PAI-2 for intracellular enzyme transglutaminase (15Jensen P.J. Schuler E. Woodrow G. Richardson M. Goss N. Hejrup P. Petersen T.E. Rasmussen L.K. J. Biol. Chem. 1994; 269: 15394-15398Abstract Full Text PDF PubMed Google Scholar) and a domain responsible for the binding of PAI-2 to several cytosolic proteins (e.g. annexin I) (16Jensen P.H. Jensen T.G. Laug W.E. Hager H. Gliemann J. Pepinsky B. J. Biol. Chem. 1996; 271: 26892-26899Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In comparison, the C/D-interhelical loop of PI-10 contains a series of basic amino acids (KKRK77) (13Riewald M. Schleef R.R. J. Biol. Chem. 1995; 270: 26754-26757Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) that exhibit homology to known nuclear targeting signals (e.g. SV40 antigen, PKKKRKV; underlined amino acids have been shown to be particularly important for signal function; Ref. 17Nigg E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (918) Google Scholar). Hypothesizing that the interhelical loop may direct PI-10 to the nucleus, we examined the role of this region in the cellular distribution of this molecule by generating expression constructs of PI-10 fused to a reporter tag. Transfection experiments using a eukaryotic model cell system (i.e. HeLa cells) revealed that the C/D-interhelical loop contains sufficient and necessary information to target a reporter to the nucleus. The entire coding region for PI-10 was excised from pBluescript SK(+)/PI-10 (13Riewald M. Schleef R.R. J. Biol. Chem. 1995; 270: 26754-26757Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) using the 5′-SacI site and the 3′-ApaI site and subcloned into a SacI/ApaI-digested pEGFP-C3 vector (CLONTECH, Palo Alto, CA). To mutate four basic amino acids in the interhelical loop of PI-10, the 871-nucleotide region between the two EcoRI sites in PI-10 (i.e.nucleotides 234 and 1105) was first removed from pEGFP·PI-10 by digestion with EcoRI, and the plasmid was gel-isolated and religated, thus generating a cDNA construct with a singleEcoRI site immediately downstream of the region encoding the interhelical loop. This construct (i.e. pEGFPC3·PI-10 ▿ 234–1105) was subjected to PCR amplification using 20 pmol ofSacI-containing forward primer 5′-TCAGATCTCGAGCTCCACC-3′ and 20 pmol of mutagenicEcoRI-containing reverse primer 5′-CGCCTTCGAATTCCATTGCCGCTGCTGCTTCACTTTCAGGGTCAC-3′ (underlined sequence indicates mutated region) in combination with conditions described previously (13Riewald M. Schleef R.R. J. Biol. Chem. 1995; 270: 26754-26757Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The PCR product was gel purified, digested with SacI and EcoRI, and ligated into an SacI/EcoRI-digested pEGFP·PI-10 ▿ 234–1105 to generate pEGFP·PI-10 K74A, K75A, R76A, K77A, ▿ 234–1105. In a separate reaction, the 871 bp region between nucleotides 234–1105 was gel-isolated following EcoRI digestion of pEGFP·PI-10 and religated to anEcoRI-digested pEGFP·PI-10 K74A, K75A, R76A, K77A, ▿ 234–1105. The resulting construct (i.e. pEGFP·PI-10 K74A, K75A, R76A, K77A) will be subsequently referred to as pEGFP·PI-10 A4. To subclone the interhelical loop of PI-10 and the PI-10 A4 mutant into pEGFP, primers flanking the cDNA encoding the interhelical loop of PI-10 between Arg-63 to Glu-87 (i.e. forward primer, 5′-TAAGCAGAGCTCAGAGACCAGGGAGTCAAATGTG; reverse primer, 5′-TCCGACGGGCCCTTCCGAGTTGCTCAAGTTGAATTCC) were prepared that contained sites for restriction enzymes to facilitate subcloning (SacI and ApaI, respectively). The interhelical loop of PI-10 and PI-10 mutant A4 were separately amplified, and the PCR products were subcloned in-frame into the pEGFP-C3 vector. Sequencing was performed by the dideoxy termination method as described previously (13Riewald M. Schleef R.R. J. Biol. Chem. 1995; 270: 26754-26757Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The cDNA encoding PAI-2 in pUC8 (i.e. pPAI J7) (18Schleuning W.D. Medcalf R.L. Hession C. Rothenbuhler R. Shaw A. Kruithof E.K.O. Mol. Cell. Biol. 1987; 7: 4564-4567Crossref PubMed Scopus (99) Google Scholar) was provided by E. K. O. Kruithof (University Hospital of Geneva, Geneva, Switzerland). The PAI-2 cDNA was excised usingEcoRI and initially subcloned into anEcoRI-digested pBluescript SK(+). The PAI-2 cDNA was subsequently excised using SalI and BamHI and subcloned into a SalI/BamHI-digested pEGFP-C3. HeLa cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. For transfection studies, HeLa cells were plated into either 8-well cell culture slides (5 × 104/well) or 60-mm diameter culture dishes (5 × 105/dish) in growth media and cultured for 24 h. The cells were washed and transfected with 0.25 μg/ml of a DNA construct by utilizing 2 μg/ml LipofectAMINE according to the manufacturer instructions (Life Technologies, Inc). Transiently transfected cells were washed and fixed, and 200 EGFP-expressing transfected cells were examined for nuclear or cytoplasmic staining using a Leitz Diaplan Data are as the of cells with fluorescent nuclear For of stable cell the transfected cells were washed and in media supplemented with the and were isolated by were analyzed for the of proteins by cells to gel using previously described (14Riewald M. Chuang T.L. Neubauer A. Schleef R.R. Blood. 1998; 91: 1256-1262Crossref PubMed Google Scholar, R.R. E. J. 1989; PubMed Scopus Google Scholar) with the that a to product was the The cDNA of EGFP·PI-10 and EGFP·PI-10 A4 in the vector pBluescript were expressed using a coupled in and system in the presence of and polymerase using conditions previously described (13Riewald M. Schleef R.R. J. Biol. Chem. 1995; 270: 26754-26757Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). For of the reaction in the presence of or A4 were in the absence or presence of were to in the presence of and subjected to and analyzed by of in the were performed by using an and of cells were prepared according to the of and Proc. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). washed cells were in 2 of 0.25 25 and with an of 25 The was with 0.25 and the were washed by the transfected cells were by in a and the were by × 10 as described previously L. Schleef R.R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). The was in the and the examined in a to of the were and washed by × 10 were in by and by × 10 The nuclear were subjected to using previously described (14Riewald M. Chuang T.L. Neubauer A. Schleef R.R. Blood. 1998; 91: 1256-1262Crossref PubMed Google Scholar, R.R. E. J. 1989; PubMed Scopus Google Scholar). to or were with protein The were washed and with of nuclear from The were washed by with and subjected to To information the cellular distribution of PI-10, we prepared eukaryotic expression the cDNA encoding PI-10 fused to a (i.e. enhanced green fluorescent cell (i.e. HeLa cells) was that PI-10 and been shown to be a model system for the expression of ov-serpin (i.e. A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). indicates that of HeLa cells in the presence of LipofectAMINE and pEGFP·PI-10 resulted in intense fluorescence of the nucleus in to the staining of the and the fluorescence of cells with LipofectAMINE in the absence of a plasmid experiments with plasmids encoding EGFP resulted in a staining of the cytoplasm. analysis revealed that of the cells were for nuclear whereas only of the of the cells were To that localization of the complex to the nucleus was by the of a nuclear targeting signal following the of a serpin to EGFP, we subcloned the cDNA encoding the ov-serpin PAI-2 into the vector and prepared Data from a using plasmid are shown in 3 of 2 and that the expressed complex is to the cytoplasm. These transfection experiments have been four with To the localization to the nucleus is a of the transfection we transfected cells with these EGFP-expressing and isolated single cells in the presence of the have been isolated from and intense fluorescence was observed in of in the resulting HeLa that were by transfection with the EGFP·PI-10 whereas cytoplasmic staining (i.e. nuclear was in the stable EGFP vector transfected and in the stable for a nuclear targeting signal in C/D-interhelical loop of nuclear targeting of and of four basic amino acids in the interhelical region of PI-10 cellular HeLa cells were transfected with LipofectAMINE and either pEGFP·PI-10 or pEGFP·PI-10 A4 the cells were and 200 transfected cells were examined for nuclear or cytoplasmic Data are as the of cells with fluorescent nuclear of EGFP·PI-10 in nuclear from transfected were isolated from EGFP·PI-10 transfected and and 3 and by and by were with either or 2 coupled to and analyzed by of complexes between and either EGFP·PI-10 or EGFP·PI-10 A4 of in reaction in the presence of and either or A4 were in the presence of of were subjected to and analyzed by of M complex were performed by and expressed as the of the M EGFP·PI-10 Data from a are an in which EGFP·PI-10 or EGFP·PI-10 A4 was either in the absence or presence of The presence of the fluorescent in the nucleus of cells that either PI-10 contains a nuclear targeting signal or that cytoplasmic PI-10 with molecule that contains the information indicates that proteases are of the nucleus and may play a role in For example, serine proteases have been to be associated with and the of C. Biochem. PubMed Scopus Google Scholar, J. Biochemistry. PubMed Scopus Google Scholar). and J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar) have observed the of a protease with the nuclear and this enzyme to in the of the nuclear treatment with serine proteases have been shown to be within the of cells J.A. M. K.A. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). In of these to for the localization of the fluorescent construct to the nucleus be the between a nuclear protease and EGFP·PI-10 in the and the of this high M r complex to the nucleus. have been in the nucleus Ref. 1998; PubMed Scopus Google Scholar) and in of nuclear enzyme data that proteases are only within the nucleus. al. B. K. E. M. A. Biol. 1996; PubMed Scopus Google Scholar) the of a protease that only be in of carcinoma cells following with and this group that this protease may play a role in the of al. J. M. W. C. Blood. 1998; Google Scholar) this by a serine protease that (i.e. and that this enzyme is to the nucleus of hematopoietic the direct targeting of PI-10 a to proteases that are from cytoplasmic protease the of EGFP·PI-10 in the nucleus was to PI-10 was in the nucleus in either a or an r For this a Proc. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) for the of and was This the of cell and other with the by of by were by and the of EGFP in the were by and by using a to to 2 that to 2 used in are to an M r 80,000 protein in nuclear isolated from cells but from the of the molecule from of cells is in with the of r a acid and r PI-10 In comparison, of nuclear cells revealed EGFP 2 The M r of the system with results were obtained using that were isolated from cells with a by a sequence for nuclear localization be a of basic amino acids been shown to be important for this 17Nigg E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (918) Google and P. H. Biochem. Mol. Biol. 1989; PubMed Scopus (37) Google Scholar). For example, the sequence is in a of proteins (e.g. SV40 the M r and the region of E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (918) Google Scholar, P. H. Biochem. Mol. Biol. 1989; PubMed Scopus (37) Google Scholar). noted that the C/D-interhelical loop of PI-10 contained four basic amino acids that were only to the sequence in SV40 but to the of the basic amino acids in the nuclear targeting signal within human (i.e. P. H. Biochem. Mol. Biol. 1989; PubMed Scopus (37) Google Scholar). To the role of in the of PI-10 in the targeting of this molecule to the nucleus, we mutated four basic amino acids in this region (i.e. indicates mutated region) to using a To that this the molecule to we first important of this mutant that is the of a serpins have revealed that the inhibitory of this superfamily are and structural Ref. 3Stein P.E. Carrell R.W. Struct. Biol. 1995; 2: 96-113Crossref PubMed Scopus (394) Google Scholar). we the of the PI-10 A4 mutant (i.e. pEGFP·PI-10 K74A, K75A, R76A, K77A) and the PI-10 molecule to form high complexes with a protease that are by the For this EGFP·PI-10 and were expressed by in utilizing conditions previously described (13Riewald M. Schleef R.R. J. Biol. Chem. 1995; 270: 26754-26757Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In the absence of information a cytosolic or nuclear protease that with PI-10, was for these experiments to form r complexes PI-10 (13Riewald M. Schleef R.R. J. Biol. Chem. 1995; 270: 26754-26757Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The in B that the of EGFP·PI-10 in EGFP·PI-10 A4 with r a to the of EGFP in the nucleus of cells 2 In of to 2 B that EGFP·PI-10 EGFP·PI-10 A4 r complexes with that are protease of the revealed that r complexes are using EGFP·PI-10 2 or EGFP·PI-10 A4 these data suggest that the PI-10 A4 mutant is but exhibits a as the molecule with to to form a complex with the protease we examined the of the PI-10 A4 to be to the nucleus. 2 indicates that transfection of HeLa cells with EGFP·PI-10 A4 resulted in the distribution of the fluorescent label to be confined to the in with the nuclear localization of type PI-10 coupled to the fluorescent The data shown are of four transfection experiments and have been to include four stable cell using this construct (i.e. nuclear This information indicates that four amino acids in the C/D-interhelical loop of PI-10 are necessary for the targeting of this molecule to the nucleus. These experiments raise the of the C/D-interhelical loop of PI-10 contains sufficient information for targeting this protein to the nucleus or other of the molecule are for this experiments using to the C/D-interhelical loop of the related ov-serpin PAI-2, al. (16Jensen P.H. Jensen T.G. Laug W.E. Hager H. Gliemann J. Pepinsky B. J. Biol. Chem. 1996; 271: 26892-26899Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) that the in ov-serpins protein binding that for To the C/D-interhelical loop of PI-10 represents an domain of targeting a protein to the nucleus, the cDNA encoding this region (i.e. Arg-63 to Glu-87 of was and subcloned into transfection experiments revealed nuclear localization for EGFP·PI-10 and EGFP coupled to the C/D-interhelical loop of PI-10 a the cDNA encoding the interhelical loop of PI-10 A4 (i.e. Arg-63 to Glu-87 with K74A, K75A, R76A, and K77A) was and subcloned into this vector. Transfection experiments revealed that the construct encoding EGFP coupled to the interhelical loop of PI-10 A4 mutant as as EGFP were to the In our data suggest that the C/D-interhelical loop of PI-10 represents an nuclear targeting In to the of proteins with the C/D-interhelical and site a of binding sites for other molecules been within the serpin include the of with molecules in the cell or and the of plasminogen activator inhibitor type with within the (2Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Abstract Full Text PDF PubMed Google P.E. Carrell R.W. Struct. Biol. 1995; 2: 96-113Crossref PubMed Scopus (394) Google Scholar). These binding sites to and the inhibitory activity of this to PI-10, that binding sites PI-10 that to this inhibitor and nuclear proteins in our group is this a wide variety of proteases are known to the nucleus and several processes including and the information in this the possibility that other protease inhibitors be into the nucleus and in and intracellular processes in a but site of within the nucleus.