s2fieldsofstudy: Biology Medicine Chemistry

text:
Des-γ-carboxy Prothrombin Is a Potential Autologous Growth Factor for Hepatocellular Carcinoma*

Des-γ-carboxyl prothrombin (DCP) is a well recognized tumor marker for hepatocellular carcinoma (HCC). In the present study, we demonstrate that DCP has a mitogenic effect on HCC cell lines. Purified DCP stimulated DNA synthesis of Hep3B and SK-Hep-1 cells in a dose-dependent manner. DCP was found to bind with cell surface receptor Met causing Met autophosphorylation and also to activate STAT3 signaling pathway through Janus kinase 1. Luciferase gene reporter analysis showed that DCP induced STAT3-related transcription. Small interfering RNAs against both STAT3 and Met abrogated DCP-induced cell proliferation. DCP did not affect the mitogen-activated protein kinase pathway, Myc signaling pathway, or phosphoinositide 3-kinase/Akt pathway. Based on these results, we believe that DCP acts as an autologous mitogen for HCC cell lines. The Met-Janus kinase 1-STAT3 signaling pathway may be a major signaling pathway for DCP-induced cell proliferation.

Des-␥-carboxyl prothrombin (DCP) 1 is a well recognized tumor marker for its high sensitivity and specificity in the screening and diagnosis of hepatocellular carcinoma (HCC). Fortyfour to eighty-one percent of HCC patients have elevated serum DCP levels (1)(2)(3)(4)(5). DCP is a prothrombin precursor with no coagulation activity. Prothrombin is synthesized in the liver depending on the presence of vitamin K-dependent ␥-glutamyl carboxylase. The prothrombin precursor has 10 Glu residues in the N terminus that are converted into ␥-carboxy-glutamic acid (Gla) residues by ␥-glutamyl carboxylase. All of these Glu residues must be converted into Gla residues before prothrombin can obtain coagulation activity. In DCP, not all of the 10 Gla residues are transformed. Instead, some remain as Glu residues (6). Reportedly, the vitamin K concentration and enzymatic activity of ␥-glutamyl carboxylase are decreased significantly in HCC tissue (7)(8)(9); however, the precise mechanism underlying DCP production is still controversial.
Many studies consider DCP a prognostic indicator of HCC.
For example, serum and tissue DCP expressions are thought to reflect the biological malignant potential of HCC (10 -14), serum DCP level is used as a clinical parameter for the development of portal venous invasion of HCC (15), and cell proliferation markers have been seen to correlate with tissue DCP expression in clinical pathological studies of HCC (10,16).
In exploring the structure of DCP, there are two kringle domains similar to those of hepatocyte growth factor (HGF), which was originally identified as a potent mitogen for mature hepatocytes (17,18). Kringle domains are mandatory for HGF to bind with Met, and their presence implies that DCP interacts with Met. We hypothesize that DCP stimulates HCC cell proliferation through Met. Met is a membrane-spanning receptor tyrosine kinase that mediates biological responses to various tissues including cell scattering, growth stimulation, and the branching morphogenesis of cells in various tissues (19 -22). Recent studies revealed that the coupling between HGF and Met integrates biological processes, such as the invasive and metastasis progression to cancer cells (23,24).
In this study, we found that DCP stimulated the proliferation of HCC cell lines. During our investigation of the receptor for DCP, DCP was found to bind with Met. The transductional apparatus of DCP was identified to activate Janus kinase 1 (JAK1)/signal transducers and activators of the transcription 3 (STAT3) signaling pathway during cancer proliferation. These findings provide a description of a novel autocrine/paracrine growth stimulatory mechanism behind the development of HCC.
Determination of DCP Levels Produced by HCC Cell Lines-DCP levels produced by HCC cell lines were determined by an electrochemiluminescence immunoassay (Picolumi PIVKA-II TM ; Eisai Co., Ltd., Tokyo, Japan). The electrochemiluminescence immunoassay method uses a mouse monoclonal anti-DCP antibody coated on solid phase beads and a rabbit polyclonal anti-prothrombin that has been ruthenylated. An electrochemically triggered light reaction was quantified by an electrochemiluminescence detection system.
Purification of DCP-The DCP-producing cell line PLC/PRF/5 was cultured in the presence of warfarin sodium (10 g/ml) to enhance DCP production. DCP was purified from the conditioned media by affinity chromatography with an anti-prothrombin antibody (Eisai). The purified DCP was then separated by SDS-PAGE and transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was stained with Blot-FastStain TM protein-staining reagent (Chemicon International, Inc., Temecula, CA) according to the manufacturer's protocol. Purified DCP sample was developed by SDS-PAGE and blotted with anti-DCP antibody (Eisai). DCP could be distinguished from normal prothrombin by high performance liquid chromatography (HPLC) analysis (6). HPLC analysis was performed using a column (TSKgel; DDS-80TS, Tosoh Bioscience, Tokyo, Japan).
Cell Proliferation Assay-The cells were grown to confluence in 12well plastic tissue culture plates. The cells were kept inactive for 24 h and then treated with DCP, prothrombin (Sigma), and apolipoprotein A (Calbiochem, San Diego, CA) at the indicated concentrations. After a 12-h incubation, 5 Ci of [ 3 H]thymidine (Amersham Biosciences) was added to each well and maintained for 8 h. The cells were treated with 5% trichloroacetic acid for 30 min at 4°C. The cells were then harvested, and we used a liquid scintillation counter (Beckman Coulter, Fullerton, CA) to analyze the samples.
Western Blot Analysis-The cells were plated into 6-well plates and grown to confluence. After 24 h of quiescence, the cells were treated with DCP, prothrombin, HGF (PeproTech, Rocky Hill, NJ), or epidermal growth factor (Sigma) at the indicated concentrations for 15 min at 37°C. Cell lysates were resolved by SDS-PAGE and transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore). The membranes were blocked using Tris-buffered saline with Tween 20 (Sigma) (TBS-T) buffer containing 5% bovine serum albumin for 1 h. The membranes were then incubated with anti-phospho-Met The membranes were washed three times with TBS-T and probed with horseradish peroxidase-conjugated secondary antibody before being developed with an ECL Western blotting detection system (Amersham Biosciences) using enhanced chemiluminescence.
Luciferase Gene Reporter Assay-The reporter plasmids, 30 g of pFA2-Elk1, 30 g of pFR-Luc (Path Detect TM Trans-Reporting System, Stratagene, La Jolla, CA), and 7 g of pRL-TK control Renilla luciferase plasmid (Promega, Madison, WI) per 10 5 cells, were cotransfected by electroporation (voltage, 300 V; capacitor, 950 microfarad; cuvette gap, 4 mm) and plated into 6-well plates. For the STAT3 activity reporter assay, 30 g of pSTAT3-Luc (Clontech laboratories, Palo Alto, CA) and 7 g of pRL-TK control Renilla luciferase plasmid (Promega) were co-transfected per 10 5 cells. After 36 h, the media were changed just before the following treatment to avoid the effect of self-produced DCP. The cells were stimulated with DCP (2-200 ng/ml), prothrombin (2-200 ng/ml), and/or epidermal growth factor (10 nM), and HGF (50 ng/ml) and incubated for 12 h. The cells were then lysed, and the activities of firefly and Renilla luciferases were measured sequentially with a luminometer (Berthold Technologies, Bad Wildbad, Germany).
Far Western Blot Analysis-DCP protein was biotinylated using biotinamido hexanoic acid 3-sulfo-N-hydroxysuccinimide ester with an ECL biotinylation module (Sigma). The cell lysates were developed on SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membranes were then blocked with 5% bovine serum albumin in TBS-T and left to react overnight with biotin-labeled DCP in TBS-T containing 1% bovine serum albumin at 4°C. Excess amounts of the probe were removed by four washes in TBS-T. The membranes were incubated with ExtrAvidin TM Peroxidase (Sigma) for detection. After three times washing with TBS-T, the membrane was developed using an ECL detection system (Amersham Biosciences).
Immunoprecipitation-Cell lysates containing the same amount of proteins were immunoprecipitated with anti-Met antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The precipitates were analyzed using 7.5% SDS-PAGE and immunoblotted with an anti-DCP antibody (Eisai), anti-phosphotyrosine 20 (PY-20) antibody (BD Transduction Laboratories, Lexington, KY), and anti-Met antibody.
Inhibitors for Met-JAK-STAT Signaling Pathway-A function-blocking anti-Met antibody (R & D Systems, Minneapolis, MN) was utilized for function blocking assay against Met signaling. The cells were pretreated with function-blocking anti-Met antibody (1 g/ml) for 1 h before DCP stimulation. JAK inhibitor AG490 and STAT3 inhibitor peptide were purchased from Calbiochem.
Gene Silencing with Small Interfering RNAs-siRNA duplexes targeting STAT3 and Met sequences were obtained from Dharmacon SMART TM pool technology (Dharmacon Research, Lafayette, CO). Control (nonsilencing, 5Ј-AATTCTCCGAACGTGTCACGT-3Ј) siRNA duplexes were obtained from Sigma. siRNAs were transfected into cells using the RNAiFect TM transfection reagent (Qiagen). The cells were incubated for 12 h and utilized for the following analysis.
Total RNA was purified from siRNA transfected cells with TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Lamin A/C mRNA expression level was determined by real time PCR using QuantiTect TM gene expression assay system (Qiagen) and Light-Cycler TM (Roche Applied Science) to verify that nonspecific suppression did not occur by siRNA transfection.
The interferences of STAT3 and Met protein expression were confirmed by Western blot analysis using an anti-STAT3 antibody (Cell Signaling Technology) and anti-Met antibody (Santa Cruz Biotechnology). Luciferase gene reporter assays for STAT3 activation and a cell proliferation assay were then performed as described above.
FIG. 1. Purity of DCP extracted from PLC/PRF/5 cells. PLC/PRF/5 cells were cultured in the tissue culture chamber with warfarin sodium (10 g/ml). DCP was purified from the conditioned media by affinity chromatography with an anti-prothrombin antibody (Eisai). A, purified sample and cell lysate were analyzed by 10% SDS-PAGE and stained with Blot-FastStain TM protein staining kit (Chemicon International, Inc.). B, purified sample and prothrombin were analyzed by 10% SDS-PAGE and immunoblotted with anti-DCP antibody (Eisai). C, purified sample were purified from conditioned media of PLC/PRF/5 cells in the presence (lower panel) and absence (upper panel) of warfarin sodium (10 g/ml). HPLC analysis was performed using a column (TSKgel; DDS-80TS, Tosoh).

RESULTS
Purification of DCP from PLC/PRF/5-The purified DCP was analyzed by SDS-PAGE and HPLC analyses. More than 99% was DCP in SDS-PAGE and densitometry analysis (Fig.  1A). The purified DCP was analyzed by Western blot with anti-DCP antibody to demonstrate that the DCP was uncontaminated ( Fig. 1B). Purified DCP from warfarin sodiumtreated PLC/PRF/5 cells contained no detectable level of normal prothrombin in HPLC analysis (Fig. 1C, lower panel). HPLC analysis showed that DCP produced by PLC/PRF/5 cell has 6 -8 Gla residues. This result is compatible with a former report (6).
Self-production of DCP by HCC Cell Lines-The DCP produced by each cell line was determined using an electrochemiluminescence immunoassay. Hep3B and PLC/PRF/5 produced DCP at rates of 0.49 Ϯ 0.04 and 2.33 Ϯ 0.04 (ng/ml/day/10 6 cells), respectively. SK-Hep-1 and HT-29 did not produce a detectable DCP level.
DCP Stimulates the Proliferation of HCC Cell Lines-To investigate the mitogenic effect of DCP, we incorporated [ 3 H]thymidine into DNA as an index of proliferation. As shown in Fig. 2, increasing concentrations of DCP led to increased incorporation of [ 3 H]thymidine in each cell line. DCP stimulated cell proliferation 1.50 Ϯ 0.16-fold at a concentration of 20 ng/ml in the DCP self-producing Hep3B cell, whereas normal prothrombin stimulated cell proliferation 1.15 Ϯ 0.07-fold at the same concentration. This effect reached a plateau with a DCP stimulus of 200 ng/ml (data not shown). In SK-Hep-1 non-DCP-producing cells, DCP showed a significant potential mitogenic effect of 2.10 Ϯ 0.05-fold at a concentration of 20 ng/ml. Normal prothrombin hardly affected proliferation of the SK-Hep-1 cell line. In fact, cell proliferation in SK-Hep-1 cells was not even induced with 100 g/ml of prothrombin, the physiological plasma concentration (data not shown). Moreover, we assessed the effect of DCP in colon cancer cell line HT-29, which does not produce DCP but expresses Met. DCP stimulated cell proliferation 1.91 Ϯ 0.40-fold at a concentration of 20 ng/ml in HT-29 cells, but prothrombin had little effect. Moreover, apolipoprotein A was employed as a control serum protein that contains kringle domains (25). Apolipoprotein A did not affect cell proliferative activity at its physiological serum concentration (280 ng/ml) in HCC cells (1.13 Ϯ 0.17-, 0.90 Ϯ 0.04-, and 0.96 Ϯ 0.03-fold for Hep3B, SK-Hep1, and HT-29, respectively; not significant).
DCP Binds to HGF Receptor Met and Phosphorylates Tyrosine Residues of Met-Met expression in Hep3B, SK-Hep-1, and HT-29 cells was confirmed by Western blot analysis. Far Western blot analysis using a biotin-labeled DCP probe showed that DCP interacted with Met. In all of the cell lysates, a 145-kDa protein was detected by this probe and identified as Met (Fig.  3A). The extracts of Hep3B, SK-Hep-1, and HT-29 cells were immunoprecipitated with anti-Met antibodies and blotted with an anti-DCP antibody. When anti-Met antibody was used for immunoprecipitation, DCP could be visualized with an anti-DCP antibody (Fig. 3B). When Met is activated by its ligand HGF, specific tyrosine residues are reported to be autophosphorylated (26,27). HGF treatment induced the autophosphorylation of Met as detected by the anti-PY-20 antibody in  D). A, cells were grown to confluence in a 10-cm tissue culture dish and lysed with buffer containing sodium vanadate and sodium pyrophosphate. Cell lysates were developed on SDS-PAGE with nonreduced condition and transferred to a polyvinylidene difluoride membrane. The membrane was probed with biotin-labeled DCP, and the Far Western blot was developed with streptavidin-conjugated alkaline phosphatase. Shown is the representative blot of two independent studies. B, cells were grown to confluence in a 10-cm dish and kept quiescent for 24 h. The cells were then stimulated with DCP (20 ng/ml) for 15 min. Met protein was immunoprecipitated with anti-Met antibody (Santa Cruz Biotechnology) from total cell lysates. The immunoprecipitates were analyzed by Western blot using anti-DCP antibody. Shown is the representative blot of two independent studies. IP, immunoprecipitation; IB, immunoblot. C, Hep3B cells were grown to confluence in a 10-cm dish and kept quiescent for 24 h. The cells were stimulated with HGF (50 ng/ml), DCP (20 ng/ml), and prothrombin (20 ng/ml) for 15 min. Met protein was immunoprecipitated from total cell lysates. The precipitates were then analyzed by SDS-PAGE and immunoblotted with anti-PY-20 antibody and anti-Met antibody. Shown are the representative blot of two independent studies. D, Western blots demonstrating the effects of DCP on tyrosine phosphorylation of Met in the Hep3B cell line. The cells were kept quiescent for 24 h and then stimulated with HGF (50 ng/ml), DCP (20 ng/ml), and prothrombin (20 ng/ml) for 15 min. The cell extracts were analyzed using Western blot and anti-phospho-specific Met Tyr 1234/1235 and Tyr 1349 antibodies. Shown are the representative blots of three independent studies. n.t., nontreated control.

FIG. 2. Effect of DCP on DNA synthesis in Hep3B, SK-Hep-1, and HT-29 cells.
The cells were grown to confluence in 12-well dishes and kept quiescent for 24 h. The cells were then treated with DCP or prothrombin for 20 h. A cell proliferation assay was performed as described under "Experimental Procedures" using [ 3 H]thymidine. The data are shown as ratios to untreated control cells and are the means Ϯ S.E. of more than three independent studies. *, p Ͻ 0.05; **, p Ͻ 0.01 (versus nontreatment of DCP); Student's t test.

FIG. 4. JAK1-STAT3 signals induced by DCP stimulation.
A, Hep3B cells were kept quiescent for 24 h and then stimulated with HGF (50 ng/ml), prothrombin (PT, 20 ng/ml), and DCP (20 ng/ml) for 15 min. The cell extracts were analyzed using Western blot with an anti-phosphospecific JAK1 antibody (upper panel) and an anti-phospho-specific STAT3 antibody (lower panel). Shown are the representative blots of more than three independent studies. B, STAT3 activation was determined using the luciferase reporter assay. Hep3B cells were transiently transfected with pSTAT3-Luc reporter plasmid and pRL-TK control Renilla luciferase plasmid. After 36-h of quiescence, the cells were treated with HGF (50 ng/ml), .
Hep3B cells (Fig. 3C). In the phosphorylation analysis using the anti-PY-20 antibody, DCP did not induce phosphorylation of Met as did HGF. Therefore, we conducted further analysis of Met phosphorylation. Activation of the Met receptor results in the autophosphorylation of tyrosines 1234 and 1235 of the tyrosine kinase domain and tyrosines 1349 and 1356 of the multifunctional docking site (27)(28)(29). In this study, DCP phosphorylated only those tyrosine residues located in the kinase activation loop (Tyr 1234/1235 ) and not the C-terminal tail (Tyr 1349 ), whereas HGF phosphorylated residues in both locations in Hep3B (Fig. 3D). Similar results were obtained with SK- Hep-1 cells (data not shown).
DCP Stimulates the JAK-STAT Pathway through Met-To identify the signaling pathway following DCP stimulation, cell lines were treated with DCP, and JAK1 phosphorylation was examined using Western blot analysis. Following exposure of cells with DCP, JAK1 (Tyr 1022/1023 ) was phosphorylated in Hep3B (Fig. 4A). JAK is known as the mediator of phosphorylation of STATs (30 -32). We observed that DCP phosphorylated STAT3 in Hep3B by Western blot analysis (Fig. 4A). Similar results were obtained with SK-Hep-1 cells (data not shown). A luciferase reporter assay was conducted to determine whether DCP induced activation of the STAT3-responsive element. As shown in Fig. 4B, DCP induced a 1.82 Ϯ 0.08-fold increase in luciferase activity in Hep3B cells. Prothrombin phosphorylated STAT3 but induced only a 1.24 Ϯ 0.05-fold increase in luciferase activity in the STAT3-responsive element.
Gene Silencing Using Met siRNA and STAT3 siRNA Abrogates the Mitogenic Effect of DCP-To determine whether Met-STAT3 signaling pathway is a major signaling pathway for DCP-induced mitogenesis, HCC cells were treated with Met siRNA and STAT3 siRNA. Western blot analysis showed that Met protein expressions were reduced by 67% in Hep3B cells and 89% in SK-Hep-1. Lamin A/C mRNA remained constant in both cells (78 Ϯ 13 and 88 Ϯ 19% as ratio to no siRNA transfected cells for Hep3B and SK-Hep-1 cells, respectively). STAT3 siRNA inhibited STAT3 protein expression by 50% in Hep3B cells and 55% in SK-Hep-1 cells. Lamin A/C mRNA remained constant (86 Ϯ 15 and 103 Ϯ 24% as ratio to control siRNA transfected cells for Hep3B and SK-Hep-1 cells, respectively). DCP did not cause the phosphorylation of STAT3 in the Met-silenced Hep3B cells (data not shown). Met siRNA inhibited DCP-induced STAT3 luciferase activity by 67.4% in Hep3B cells and 91.4% in SK-Hep-1 cells (Fig. 5A). RNA interference gene silencing against STAT3 also abrogated DCP-induced luciferase activity in the STAT3-responsive element by 80.3% in Hep3B cells and 100% in SK-Hep-1 cells (Fig. 5A).
When cell proliferative activities were analyzed by [ 3 H]thymidine incorporation assay, Met siRNA transfection decreased DCP-induced incorporation of [ 3 H]thymidine by 39.3% in the Hep3B cell line and 66.6% in the SK-Hep-1 cell line (Fig. 5B). Moreover, STAT3 siRNA transfection decreased DCP-induced incorporation of [ 3 H]thymidine by 56.8% in the Hep3B cell line and by 100% in the SK-Hep-1 cell line (Fig. 5B).
DCP Did Not Affect the Raf-MEK ERK-MAPK Pathway-HCC cell lines were treated with DCP for the indicated period. Raf and ERK-MAPK phosphorylation was analyzed by Western blot analysis. DCP did not phosphorylate either Raf (data not shown) or ERK-MAPK (Fig. 6). In addition, we performed luciferase reporter assays for Elk activation and found that DCP did not raise Elk-dependent luciferase activity in Hep3B cells (Fig. 6). We further investigated whether DCP stimulates prothrombin (20 ng/ml), DCP (20 ng/ml), AG490 (80 M), function-blocking anti-Met antibody (R&D Systems) (1 g/ml), and/or STAT3 inhibitor peptide (100 M) for 12 h. Cellular extracts were collected, and luciferase activities were measured. C, a cell proliferation assay was performed as described under "Experimental Procedures" with DCP (20 ng/ml), function-blocking anti-Met antibody (1 g/ml), AG490 (80 M), and/or STAT3 inhibitor peptide (100 M). The data are shown as the ratios to untreated control cells and are the means Ϯ S.E. of more than three independent studies. **, p Ͻ 0.01 (versus nontreatment of DCP); NS, p Ͼ 0.05; #, p Ͻ 0.05; ##, p Ͻ 0.01 (versus DCP-stimulated cells); Student's t test. n.t., nontreated control.  cell proliferation (B). A, STAT3 activation was determined using a luciferase reporter assay. Met siRNA duplexes (100 nM), STAT3 siRNA duplexes (100 nM), and control (nonsilencing) siRNA duplexes (100 nM) were transfected into cells and incubated for 12 h. The cells were transiently transfected with pSTAT3-Luc reporter plasmid and pRL-TK control Renilla luciferase plasmid. After 36-h of quiescence, the cells were treated with DCP (20 ng/ml) for 12 h. Cellular extracts were collected, and luciferase activities were measured. The STAT3 activity, depending on luciferase activities, was normalized to the constitutive active Renilla luciferase activity. B, after treatment with siRNA duplexes, a cell proliferation assay was performed as described under "Experimental Procedures" using [ 3 H]thymidine. The data are shown as the ratios to DCP untreated control cells and are the means Ϯ S.E. of more than three independent studies. NS, p Ͼ 0.05; *, p Ͻ 0.05; **, p Ͻ 0.01 (versus control nonsilencing siRNA); Student's t test.
the c-Myc and PI3K-Akt signaling pathway by Western blot analysis. DCP did not affect the phosphorylation of c-Myc, p85-PI3K, and Akt (data not shown). Moreover, other Met signaling pathways were screened using an antibody array (MaxArray TM RTK Antibody Array) (Zymed Laboratories Inc. Laboratories, South San Francisco, CA), which contains antibodies for proteins related to receptor tyrosine kinases. Other signaling pathways were not affected significantly by DCP (data not shown). Similar results were obtained with SK-Hep-1 cells (data not shown). DISCUSSION In the current study, we present DCP as a novel autocrine/ paracrine mitogen for HCC cell lines. Clinical studies show a significant correlation between serum DCP level and the clinical malignancy of HCC (11)(12)(13)(14)(15)33); however, the molecular basis for this correlation is still poorly understood. Herein, DCP was demonstrated to stimulate the Met-JAK-STAT signaling pathway in HCC cell lines. To the best of our knowledge, this is the first study that clearly demonstrates a mitogenic effect of DCP in HCC cell lines.
Proto-oncogene c-Met, which encodes the cell surface tyrosine kinase receptor for HGF, is overexpressed in a significant proportion of HCC cases as well as in other human cancers (19, 24, 34 -39). Met ligand HGF is known to be produced in various cancer cells (40,41), and extensive studies suggest that autologous Met activation causes cancer cell growth and invasion (42)(43)(44). ERK-MAPK, STAT, and the PI3K-Akt signaling pathway are elicited by Met activation (45). Through ERK-MAPK, HGF stimulates proliferative activity of the HCC cells (46). In contrast, some studies have shown that HGF has an inhibitory effect or no effect on HCC cell growth (47,48). In keeping with these studies, we found that HGF stimulated neither cell proliferation nor the ERK-MAPK signaling pathway (data not shown) in Hep3B and SK-Hep1 cells. Both DCP and HGF bound to Met and caused its autophosphorylation. HGF induced full phosphorylation of the Met autophosphorylation site, including Tyr 1234/1235 and Try 1349 , whereas DCP only stimulated Tyr 1234/1235 . This partial phosphorylation may cause a different biological effect on cell proliferation. Although the biological response through a single receptor that binds to different ligands is usually indistinguishable, some receptors reportedly can discriminate between ligands and induce different biological responses (49,50). We found, however, that DCP did not activate the ERK-MAPK signaling pathway. Thus, further study is needed to elucidate ligand discrimination in the autophosphorylation and biological response of the Met receptor.
In searching for the downstream signaling pathway of DCP, the JAK-STAT signaling pathway was found to be a promising candidate. A wide variety of extracellular signals activate the STAT class of transcription factors. Many cytokines, lymphokines, and growth factors signal through a related superfamily of cell surface receptor tyrosine kinases that are associated with the JAK-STAT signaling pathway. It was previously reported that HCC cell proliferation is activated by STAT3 signals (51), and STAT3 activation has been reported in various cancer tissues (52,53). The HBx protein from the hepatitis B virus (54) and hepatitis C virus core protein may activate STAT3 (55). This is important because STAT3 signaling is generally considered to be involved in carcinogenesis and cancer development, including HCC. We also show in the present study that STAT3 played a major role in DCP-induced cell proliferation. Both prothrombin and HGF induced phosphorylation of STAT3 in Western blot analysis, but they did not activate STAT3-induced transcription or cell proliferation. This discrepancy may be caused by a Ser 727 -independent STAT3 regulatory mechanism. Although two phosphorylation sites (Tyr 705 and Ser 727 ) are reported to play a major role in STAT3 activation (56 -59), other studies show that Ser 727 phosphorylation causes an inhibitory effect on STAT3-induced transcription (60 -62). The effect of Ser 727 phosphorylation of STAT3 is varied depending on the status of other cells (63,64). RNA interference for STAT3 and STAT3 inhibitor peptide were utilized to confirm whether STAT3 was the key molecule in the DCP signaling pathway. Both STAT3 inhibitor peptide and siRNA against STAT3 abrogated DCP-induced cell proliferation as well as STAT3-induced transcription (Figs. 4 and 5). Thus, the JAK1-STAT3-pathway is considered to be a major signaling pathway for DCP stimulation.
Other Met-related signaling pathways were not affected by DCP stimulation. PI3K-Akt signaling is known to promote survival under apoptotic stress (65), but DCP did not significantly affect this signaling pathway. Furthermore, neither the expression nor phosphorylation of c-Myc was affected by DCP stimulation.
In the current study we demonstrate a novel autocrine/paracrine mechanism of DCP-positive HCC in vitro in which the DCP-Met-STAT3 signaling pathway becomes a capable therapeutic target to inhibit the development of HCC. Further in vivo studies are necessary to validate this mechanism.
FIG. 6. The effect of DCP on Raf-MAPK signaling. Hep3B cells were grown to confluence in a 6-well tissue culture dish and kept quiescent for 24 h. The cells were treated with epidermal growth factor (10 nM), DCP (2-200 ng/ml), and prothrombin (2-200 ng/ml) for 15 min. The cell extracts were analyzed by Western blot using a phospho-specific p44/42 MAPK (Thr 202 /Tyr 204 ) antibody. MAPKrelated transcriptional factor Elk activities were analyzed using a luciferase reporter assay. The cells were transiently transfected with Elk reporter plasmid and then treated with epidermal growth factor, DCP, and prothrombin for 12 h. The cellular extracts were collected, and luciferase activities were measured. The data are shown as the ratios to untreated control cells and are the means Ϯ S.E. of more than three independent studies.


s2fieldsofstudy: Biology Medicine

text:
Coronavirus main proteinase: target for antiviral drug therapy

During the first outbreak of severe acute respiratory syndrome (SARS) in early 2003, almost 8500 people worldwide were diagnosed with the disease, and approximately 800 died of it. The epidemic was contained by July, 2003, but since then, the disease has re-emerged in the Guangdong province in Southern China around the turn of the year 2003, although the number of cases was very limited this time. In addition, there were two cases of SARS caused by improper sample handling in research laboratories in Singapore and Taiwan. A “mini-outbreak” originated from several independent laboratory infections at a research laboratory in Beijing in March and April, 2004, and led to infection chains encompassing three generations.This time, China escaped another epidemic since fortunately, the number of diseased people was limited to nine, although there was one fatality. In any case, these isolated mini-outbreaks remind us of the global threat that SARS still represents. In late March, 2003, a new coronavirus, SARS-CoV, was isolated from affected tissue of SARS patients and identified as the cause of the disease [1, 2]. Coronaviridae are RNA viruses that have been united with the families Arteriviridae and Roniviridae in the order Nidovirales. The phylogenetic relationship of these morphologically unrelated virus families is based on their similar polycistronic genome organization, common transcriptional and post-translational strategies, and a conserved array of homologous domains in the viral polyproteins [3–5]. Coronaviruses are responsible for a number of diseases of man and animals, some of which have major economical impacts, although the human coronaviruses have clearly been neglected before SARS emerged. Transmissible gastroenteritis virus (TGEV) infection is associated with severe and often fatal diarrhoea in young pigs [6, 7]. The human coronaviruses (HCoV) 229E and OC43 are major causes Coronavirus main proteinase: target for antiviral drug therapy

Introduction
During the first outbreak of severe acute respiratory syndrome (SARS) in early 2003, almost 8500 people worldwide were diagnosed with the disease, and approximately 800 died of it. The epidemic was contained by July, 2003, but since then, the disease has re-emerged in the Guangdong province in Southern China around the turn of the year 2003, although the number of cases was very limited this time. In addition, there were two cases of SARS caused by improper sample handling in research laboratories in Singapore and Taiwan. A "mini-outbreak" originated from several independent laboratory infections at a research laboratory in Beijing in March and April, 2004, and led to infection chains encompassing three generations. This time, China escaped another epidemic since fortunately, the number of diseased people was limited to nine, although there was one fatality. In any case, these isolated mini-outbreaks remind us of the global threat that SARS still represents.
In late March, 2003, a new coronavirus, SARS-CoV, was isolated from affected tissue of SARS patients and identified as the cause of the disease [1,2]. Coronaviridae are RNA viruses that have been united with the families Arteriviridae and Roniviridae in the order Nidovirales. The phylogenetic relationship of these morphologically unrelated virus families is based on their similar polycistronic genome organization, common transcriptional and post-translational strategies, and a conserved array of homologous domains in the viral polyproteins [3][4][5]. Coronaviruses are responsible for a number of diseases of man and animals, some of which have major economical impacts, although the human coronaviruses have clearly been neglected before SARS emerged. Transmissible gastroenteritis virus (TGEV) infection is associated with severe and often fatal diarrhoea in young pigs [6,7]. The human coronaviruses (HCoV) 229E and OC43 are major causes Coronavirus main proteinase: target for antiviral drug therapy of upper respiratory tract illness in humans, in particular, common cold-like conditions [8]. To date, only the 229E strain of HCoV has been characterized in detail because until the discovery of SARS-CoV, it used to be the only isolate to grow efficiently in cell culture. In contrast to the relatively harmless disease caused by these human coronaviruses, SARS is characterized by high fever, malaise, rigor, headache, non-productive cough, or dyspnea and may progress to generalized, interstitial infiltrates in the lung, requiring intubation and mechanical ventilation [9]. The infection, however, is not restricted to the lungs. Other affected organs include the kidneys and the liver, as well as the gastrointestinal tract; hence SARS can be considered a systemic disease. Epidemiological evidence suggests that the transmission of this newly emerging pathogen occurs mainly by face-to-face contact, although other routes of transmission cannot be fully excluded.

Coronavirus main proteinase: function and overall structure
Coronaviruses are positive-stranded RNA viruses featuring a large (27-31 kB), single-stranded RNA genome. The replicase gene, encompassing more than 20,000 nucleotides, encodes two overlapping polyproteins, pp1a (replicase 1a,~450 kDa) and pp1ab (replicase 1ab,~750 kDa) [10] that mediate all the functions required for viral replication and transcription [11]. Expression of the 3'-proximal portion of the replicase gene requires (-1) ribosomal frameshifting [10]. The functional non-structural proteins are released from the viral polyproteins by extensive proteolytic processing. The vast majority of cleavages are mediated by the viral main proteinase, M pro [12], a cysteine proteinase which is frequently also called 3C-like proteinase (3CL pro ) to indicate a similarity of its substrate specificity to that observed for picornavirus 3C proteinases (3C pro ) (see Tab. 1), although the structural similarities between the two families of proteinases are limited [13]. The M pro (3CL pro ) cleaves the polyprotein at no less than 11 conserved sites involving Leu-Gln↓ (Ser,Ala,Gly) sequences, a process initiated by the enzyme's own autolytic cleavage from pp1a and pp1ab [14,15]. Importantly, this cleavage pattern appears to be conserved in the M pro from SARS coronavirus (SARS-CoV), as deduced from the genomic sequence [16,17] and shown experimentally for one cleavage site [18]. The SARS-CoV polyproteins have three non-canonical M pro cleavage sites with Phe, Met or Val in the P2 position, but the same cleavage sites are unusual in other coronaviruses as well. The functional importance of M pro in the viral life cycle makes this proteinase an attractive target for the development of drugs directed against SARS and other coronavirus infections.
Our laboratories have determined crystal structures for HCoV strain 229E M pro at 2.54 Å resolution [18], porcine TGEV M pro at 1.96 Å [13], and SARS-CoV M pro (1.9 Å resolution) [19]. In addition, we have also elucidated the structures of substrate-analogous hexapeptidyl chloromethyl ketone inhibitor complexes of the TGEV and SARS-CoV enzymes, at around 2.4 Å resolution [18,19]. The structures of all three enzymes show that the coronavirus main proteinase consists of three domains, the first two of which together distantly resemble chymotrypsin while the third has a unique α-helical fold (Fig. 1). Located between domains I and II, the active site comprises a Cys-His catalytic dyad, rather than the usual triad. Domains I and II (residues 8-99 and 100-183, respectively) are six-stranded antiparallel β-barrels. A long loop (residues 184 to 199) connects domain II to the C-terminal domain (domain III, residues 200-300), which is a globular cluster of five, mostly antiparallel helices.
The interior of the β-barrel of domain I consists entirely of hydrophobic residues. A short α-helix (helix A; residues 53-58) closes the barrel like a   The three M pro structures determined so far are quite similar to one another. The overall r.m.s. deviations for Cα atoms between the structures is below 2 Å for all 300 Cα positions of the molecules that could be located in the electron density maps but the isolated domains exhibit significantly smaller deviations. The most variable domain appears to be the helical domain III, which shows a better spatial overlap between TGEV M pro and HCoV M pro than between each of these and SARS-CoV M pro . To a certain extent, these numbers reflect the degree of sequence identity between the different M pro s. HCoV 229E and TGEV, both being group I coronaviruses, share 61% sequence identity between their main proteinases, whereas SARS-CoV as a distant outlier of group II has 40% and 44%, respectively, of the sequence of its M pro in common with HCoV 229E and TGEV [18]. Interestingly, domains I and II show a higher degree of sequence conservation (42-48% identity) than domain III (36-40%) between SARS-CoV M pro and the coronavirus group I enzymes.

Dimer formation
The crystal structure of the TGEV M pro provided the first indication that coronavirus main proteinases form tight dimers [13]. This has since been confirmed by the X-ray structures of the HCoV 229E and SARS-CoV M pro s (Fig. 2) [18,19]. Dimerisation also occurs in solution: we could show by dynamic light scattering that both HCoV 229E and TGEV M pro exist as a mixture of monomers (~65%) and dimers (~35%) in diluted solutions (1-2 mg proteinase/ml). Lai and coworkers have demonstrated that the enzymatic activity of the SARS-CoV M pro increases linearly with enzyme concentration, suggesting that the enzymatically active species is the dimer and not the monomer [23]. Crystal structures of the SARS-CoV enzyme at different pH values provided an explanation for this observation [19] (see below).
It should be noted that dimer formation as a prerequisite for enzymatic activity makes coronavirus main proteinases unique amongst the proteinases of RNA viruses, and present a clear distinction from the 3C proteinases of picornaviruses. The TGEV M pro X-ray structure showed, and the other coronavirus M pro structures confirmed, that the monomer-monomer contact interface comprises two regions, i.e. i) the N-terminal eight residues of molecule A and domain II of molecule B, and ii) the domains III of each protomer. Although the latter part of the interface is significantly smaller than the former, it is apparently sufficient to hold the isolated domains III together in solution, as has recently been shown for SARS-CoV M pro [24]. The crystal structures show that the two molecules are oriented perpendicular to one another (Fig. 2) [13,18,19]. Since the architecture of the dimers including most details of intermolecular interaction are the same in TGEV M pro -three independent dimers per asymmetric unit -and HCoV 229E as well as SARS-CoV M pro s -both with one dimer per asymmetric unit -, i.e., in completely different crystalline environments, there can be little doubt that dimer formation is of biological relevance in these enzymes.
In the M pro dimer, the N-terminal amino acid residues are squeezed in between domains II and III of the parent monomer -designated "A" in this paragraph -and domain II of the other monomer ("B"), where they make a number of very specific interactions that appear tailor-made to bind this segment with high affinity. These interactions include intermolecular salt bridges between the positively charged amino terminus (residue A1) and GluB165 (all residue numbers given in this section are for TGEV M pro ) of domain II of the neighboring molecule in the dimer, between ArgA4 and GluB286, as well as an intramolecular salt bridge between LysA5 and GluA291 of domain III of the parent molecule. Also, there is an important hydrophobic contact between the semiconserved LeuA3 and a hydrophobic patch on domain III which includes PheA206, AlaA209, PheA287, ValA292, the C β atom of GlnA295, and MetA296. All sequenced members of the coronavirus proteinase family have a hydrophobic residue in position 3 (SARS-CoV has Phe), while glycine is absolutely conserved in position 2.
The latter residue adopts the α L conformation, which is easily accessible only to glycine. This conformation ensures that the N-terminal segment fits into its narrow binding canyon in the dimerization interface.
The existence of a specific binding site for the N-terminal segment of the M pro could be important in the activation of the main proteinase by catalyzing its release from the viral polyprotein. The free N-terminus is created as a consequence of the first cleavage reaction performed by the proteinase, at its N-terminal autocleavage site. In the mature M pro monomer, the first eight residues from the N-terminus are not a part of the globular protein structure. After the autocleavage reaction, they could easily remain bound to the substrate-binding site of the proteinase if an alternative highaffinity binding site was not provided by dimer formation. The existence of such high-affinity binding site ensures that the catalytic site is immediately liberated from the product and can act on other cleavage sites in the polyprotein. However, the exact placement of the amino terminus also has a very important structural role for the mature M pro , and is in fact critical for its catalytic activity, as we will see below.

Catalytic site
In the active site of the M pro , Cys144(145) and His41 (SARS-CoV numbering in brackets where different) form a catalytic dyad. In contrast to serine proteinases and other cysteine proteinases, which have a catalytic triad, there is no third catalytic residue present. HCoV 229E and TGEV M pro s have Val84 in the corresponding sequence position (Cys in SARS-CoV M pro ), with its side chain pointing away from the active site. A buried water molecule is found in the place that would normally be occupied by the third member of the triad; this water is hydrogen-bonded to His41 Nδ1, Gln163 Nε2, and Asp186 Oδ1 in HCoV 229E M pro (His, His, and Asp in both SARS-CoV and TGEV M pro ). Asp186(187) does not act on the active-site His41 through the mentioned water molecule, but rather interacts with conserved Arg40 by forming a strong inter-domain salt bridge.
There has been a long-standing debate on the exact nature of the catalytic centre in cysteine proteinases. Polgár has shown that in papain-like cysteine proteinases, the active species is a thiolate-imidazolium ion pair, with the imidazolium cation held in the correct position by an asparagine residue, the third residue of the catalytic triad [25]. However, such a mechanism does not seem to be predominant in the coronavirus main proteinases; Lai and coworkers have recently shown that if the active-site cysteine residue of SARS-CoV M pro is replaced by serine, some residual enzymatic activity -decreased by a factor of 40 -can still be detected. This can be seen as an indication for the existence of a classical general base mechanism in the wild-type proteinase, involving the non-ionized Cys145 hydrogen bonded to the uncharged imidazole of His41 in the ground state of the free enzyme [26]. We note that in viral cysteine proteinases, both picornaviral 2A and 3C proteinases as well as coronaviral main proteinases, the Sγ of the active-site cysteine residue is exactly in the plane of the histidine imidazole ring, at a distance of between 3.5 and 4.0 Å, while in the papain-like cysteine proteinases, this is not the case. For hydrogen bonding to occur between the two side chains, but not for ion-pair formation, the sulfur has to be more or less in the plane of the imidazole. While this preliminary observation has to be investigated further, it seems that X-ray crystallography can contribute to this important mechanistic question by providing exact geometric details of the ground-states of the free enzymes.

Substrate binding
Comparison of the structures shows that the substrate-binding sites are well conserved among coronavirus main proteinases. This is supported by our experimental finding that recombinant SARS-CoV M pro cleaves a pentadecapeptide corresponding to the N-terminal autocleavage site of the TGEV M pro [18].
To allow structure-based design of drugs directed at coronavirus M pro s, we sought to determine the exact binding mode of M pro substrates. To this end, we synthesized the substrate analog hexapeptidyl chloromethyl ketone inhibitor, Cbz-Val-Asn-Ser-Thr-Leu-Gln-CMK (subsequently named CMK), and soaked it into crystals of TGEV and SARS-CoV M pro [18,19]. The sequence of the inhibitor was derived from the P6-P1 residues of the N-terminal autoprocessing site of TGEV M pro (SARS-CoV M pro and HCoV 229E M pro have Thr-Ser-Ala-Val-Leu-Gln and Tyr-Gly-Ser-Thr-Leu-Gln, respectively, at the corresponding positions). At the outset, we will present here the complex between the TGEV M pro and the inhibitor [18]. In the case of the SARS-CoV enzyme, some unusual binding modes were observed [19], which we will discuss later.
X-ray crystallographic analysis of the TGEV M pro complex at 2.37 Å resolution revealed difference density for all residues (except the benzyloxycarbonyl (Cbz) protective group) of the inhibitor, in two (B and F) out of the six TGEV M pro monomers in the asymmetric unit [18]. In these monomers, there is a covalent bond between the S γ atom of the active-site nucleophile Cys144 and the methylene group of the chloromethyl ketone. There are no significant differences between the structures of the enzyme in the free and in the complexed state. The substrate-analogue inhibitor binds in the shallow substrate-binding site at the surface of the proteinase, between domains I and II (Fig. 3A). The residues Val-Asn-Ser-Thr-Leu-Gln occupy, and thereby define, the subsites S6 to S1 of the proteinase. Residues P5 to P3 form an antiparallel β-sheet with segment 164-167 of the long strand eII on one side, and they also interact with segment 189-191 of the loop linking domains II and III on the other. In the discussion that follows, residue numbers for TGEV M pro are followed by those for the SARS-CoV enzyme in brackets.

S1 specificity site
In coronavirus M pro polyprotein cleavage sites, the P1 position is invariably occupied by Gln. At the very bottom of the M pro S1 subsite, the imidazole of His162(163) is suitably positioned to donate a hydrogen bond from Nε2 to the side-chain carbonyl of the P1 glutamine (Fig. 3A). In order to specifically bind glutamine and not glutamate in the S1 pocket, this histidine residue is required to be uncharged over a broad pH range. This appears to be maintained by two important interactions made by the imidazole ring: i) stacking onto the phenyl ring of Phe139(140), and ii) accepting a hydrogen bond (to Nδ1) from the hydroxyl group of the buried Tyr160(161). In agreement with this structural interpretation, any replacement of His162 completely abolishes the proteolytic activity of HCoV 229E and feline infectious peritonitis virus (FIPV) M pro [27,28]. Furthermore, FIPV M pro Tyr160 mutants have their proteolytic activity reduced by a factor of > 30 [27]. All of these residues are conserved in SARS-CoV M pro and, in fact, in all coronavirus main proteinases. Other elements involved in the S1 pocket of the M pro are the main-chain atoms of Ile51, Leu164(Met165), Glu165(166), and His171(172). The latter two residues form a salt bridge at the outer wall of the S1 specificity pocket in the TGEV and HCoV 229E M pro s, as well as in one conformation of the SARS-CoV M pro . However, the latter displays another conformation which will be discussed below.
In all structures of free coronavirus main proteinases that we determined so far, we find electron density at the bottom of the S1 pocket. We interpreted this as a water molecule or, in the case of TGEV M pro where the density was more dumb-bell-shaped, as two water molecules. However, especially in the latter case, we realized that apart from a possible hydrogen bond between one of these waters and the His162 imidazole, no interactions with the proteins were made by these water molecules. Also, the positions of the two water molecules were in fact too close to one another (about 2.1-2.3 Å) [13]. When we subsequently determined the structure of the complex between the TGEV M pro and the CMK inhibitor, we noted that the oxygen and the nitrogen of the side-chain amide occupied a position exactly matching that of the two water molecules in the free enzyme. It is therefore possible that the free enzyme in fact also contains some residual peptide cleavage product (perhaps from an unidentified E. coli protein). Having realized this possibility, we checked for residual difference electron density in other subsites but could find nothing, with the exception of a patch of relatively strong electron density at the P2 position which we had interpreted and successfully refined as a 2-methyl-2,4-pentandiol (MPD) molecule from the crystallization medium [13]. The MPD does not fully protrude into the S2 pocket but rather occupies a position corresponding to the main chain of the peptide substrate. At this point, we still think the assignment of this density was correct, but studies on other proteinases -e.g. thermolysin -that revealed the presence of cleavage product after years of investigation taught us to be cautious.

S2 specificity site
Apart from a few exceptions, coronavirus M pro cleavage sites have a Leu residue in the P2 position [28]. The hydrophobic S2 subsite of the TGEV enzyme is formed by the side chains of Leu164, Ile51, Thr47, His41, and Tyr53. In addition, residues 186-188 line the S2 subsite with some of their main-chain atoms. The P2 Leu side chain of the inhibitor is well accommodated in this pocket (Fig. 3A).
In the SARS-CoV M pro [19], the S2 pocket is similar to what has been seen in HCoV M pro [18] and TGEV M pro [13]. One wall of the subsite is formed by methionine residues 49 and 165, which adopt positions spatially similar to TGEV and HCoV 229E M pro residues Ile51 and Leu/Ile164, respectively. The longer methionine side chains restrict the S2 pocket in one direction and probably require a slight reorientation of the P2 leucine residue of the substrate, compared to TGEV M pro . On the other side, the S2 pocket appears to be somewhat enlarged in SARS M pro , due to the insertion of Ala46 which is not present in TGEV or HCoV 229E M pro , as well as to many differences in amino acid sequence. In SARS-CoV M pro , the stretch 45-50 forms a short 3 10 -helix, whereas this region has no regular secondary structure in the other coronavirus main proteinases. The net effect of these modifications appears to be a widening of the S2 pocket on this side. This may be required for the efficient binding of a P2 phenylalanine, which is present in the C-terminal autocleavage site of the M pro .

Specificity sites beyond S1 and S2
There is no specificity for any particular side chain at the P3 position of coronavirus M pro cleavage sites. This agrees with the P3 side chain of our Coronavirus main proteinase: target for antiviral drug therapy 183 In the active monomer A of the SARS-CoV M pro crystallized at pH 6.0, the P1 glutamine of the inhibitor protrudes into the S1 pocket, but P2-P5 adopt an unusual binding mode. (C) In the inactive monomer B, the S1 pocket is not accessible, and the P1 glutamine points towards bulk solvent. A: After [18], with kind permission of the American Association for the Advancement of Science (AAAS); B, C: After [19], with kind permission of The National Academy of Sciences of the USA.
substrate analog being oriented towards bulk solvent in the TGEV M pro complex (Fig. 3A). At the P4 position, there has to be a small amino acid residue such as Ser, Thr, Val, or Pro because of the congested cavity formed by the side chains of Leu164, Leu166, and Gln191 as well as the main-chain atoms of Ser189. These residues are conserved or conservatively substituted (Leu164⇒Met165, Ser189⇒Thr190) in SARS-CoV M pro . In the TGEV M pro complex, the P5 Asn side chain interacts with the main chain at Gly167, Ser189, and Gln191 (Pro168, Thr190, Gln192 in the SARS-CoV enzyme), thus involving the loop linking domains II and III, whereas the P6 Val residue is not in contact with the protein (Fig. 3A). Although our CMK inhibitor does not include a P1' residue, it is easily seen that the common small P1' residues (Ser, Ala, or Gly) can be easily accommodated in the S1' subsite of TGEV M pro formed by Leu27, His41, and Thr47, with the latter two residues also being involved in the S2 subsite (Leu, His, and Asp in SARS-CoV M pro ).
Superimposition of the structures of the TGEV M pro -CMK complex and the free HCoV 229E M pro shows that the two substrate-binding sites are basically the same. All residues along the S4-S1 side of the cleft are identical, with the exception of the conservative M190L replacement (Ala191 in SARS-CoV M pro ). In the SARS pathogen, M pro residues 168 and 188 -190 show some substitutions but since these residues contribute to substrate binding with their main-chain atoms only, the identity of the side chains is less important. Indeed, the substrate-binding site of the SARS-CoV M pro matches those of its TGEV and HCoV 229E counterparts quite well (Fig.  3B). And yet, the CMK inhibitor bound to the SARS-CoV M pro in a remarkably different mode (Figs. 3B,C). But before we discuss substrate or inhibitor binding to the SARS-CoV main proteinase, we have to analyze the dynamical nature of the structure of the free enzyme.

pH-dependent conformational changes of the SARS-CoV M pro
The SARS-CoV M pro also forms a dimer in the crystal (and in solutions at concentrations of > 1 mg/ml) with the two protomers oriented almost at right angles to each other [19], in an arrangement that is similar to the HCoV and TGEV M pro structures [13,18]. However, when we first determined the structure of the SARS-CoV enzyme by X-ray analysis of crystals grown at pH 6.0 [19], we were surprised to see that the substrate-binding site of one monomer (denoted "A", Fig. 4A) was in the normal, active conformation described above for the TGEV and HCoV 229E M pro s, whereas the other ("B") had adopted a catalytically incompetent form (Fig. 4B). There were two major conformational changes that led to this inactivation: i) the S1 binding pocket for the glutamine residue at the cleavage site of the substrate had collapsed, and ii) so had the the oxyanion hole (Fig. 4B). We believe that these changes originate from protonation of HisB163, the con-served residue found at the bottom of the S1 pocket, in monomer B. The pH of crystallization of 6.0 is close to the pK of histidine, explaining why the residue appears to be protonated in monomer "B" but not monomer "A" of the SARS-CoV M pro dimer. As a consequence of the protonation, the side chain of PheB140, which stacks onto HisB163 as long as the latter is unprotonated, moves away and reorients towards the A3 of the other monomer. In order to neutralize the positive charge of HisB163 in its relatively hydrophobic environment, GluB166 moves into the S1 pocket and forms a very strong salt bridge of 2.43 Å with HisB163. This leads to breakage of the ion pair between GluB166 and HisB172 as well as of the amino terminus, residue A1, of the other monomer in the dimer. This very amino terminus also interacts with the main chain of residue B140, the phenylalanine that moves away in response to the protonation of HisB163. This interaction, consisting of two hydrogen bonds, between the hydrogen bond with the amino group of A1 and the CO of PheB140 as well as between the CO of A1 and the NH of PheB140, is also lost as part of the conformational rearrangements. As a result, the segment around PheB140 (residues B138 -B143) is no longer fixed. It adopts a different conformation with positional differences from the A chain of up to 5.9 Å and becomes poorly ordered, although we do see continuous electron density for the main chain and for all side chains except PheB140 (Fig. 4B). In fact, the entire loop translates towards the active site. Importantly, residue 140 is part of the oxyanion loop, which stabilizes the transition state of the proteolytic reaction by donating hydrogen bonds from the main-chain amides of Gly143 and Cys145 to the negatively charged oxygen of the tetrahedral intermediate. It is essential that this loop be in the right conformation for the enzyme to exhibit full catalytic activity. However, in the inactive protomer, Gly143 moves by > 3.2 Å towards the active site and leaves no space for the oxyanion to bind.
As mentioned above, the S1 binding site is partly occupied by GluB166 interacting with HisB163. In addition, CysB145 adopts two conformations, each about half occupied, one of which is filling the remainder of the collapsed S1 pocket. The other one is interacting with the catalytic HisB41 as in the active protomer. In summary, protomer "B" in the dimer is inactive because it cannot stabilize the oxyanion transition state and because it is unable to bind the P1 glutamine residue of the substrate. This agrees with the measured activity of the enzyme as dependent on pH, which displays a bell-shaped curve, with a maximum at pH 7.0 and less than 50% activity both at pH 6.0 and at pH 8.5 (Fig. 5) [29].
In a follow-up study, we equilibrated at pH 7.6 and 8.0 crystals of the SARS-CoV M pro that had been grown at pH 6.0, and determined the structures by X-ray analysis [19]. At pH 7.6 ( Fig. 6A), the S1 binding pocket is 186 Kanchan Anand et al. fully formed in both monomers and Phe140 is interacting with the now unprotonated His163. The oxyanion is correctly shaped. Glu166 forms ion pairs with His172 and the N-terminus of the other monomer in the dimer. At pH 8.0 (Fig. 6B), another interesting change takes place. The salt bridge between Glu166 and His172 breaks -but not the one between Glu166 and the amino terminus of the other protomer in the dimer -, resulting in Glu166 becoming more mobile and partly filling the S1 pocket, thereby decreasing the activity of the enzyme. This is presumably due to the deprotonation of His172 at the high pH value. The pK value of histidine residues involved in salt bridges has been shown to be around 8.0 to 8.5, fully consistent with our interpretation of the phenomenon. Thus, we propose that the low-pH and high-pH flanks of the bell-shaped pH-activity curve (Fig. 5) are determined by the protonation and deprotonation of His163 and His172, respectively. In this respect, our interpretation differs from that of Lai and coworkers who proposed that deactivation of the enzyme at high pH is due to deprotonation of the active-site cysteine [26]. In our opinion, the latter would only play a role at even higher pH values of > 9. The different protonation states of His163 and His172 and their influence on the conformation of the substrate-binding site were further explored by molecular-dynamics calculations over 10 ns each (Tan et al., in preparation). The results are in full agreement with the interpretation of the crystal structures put forward here. Furthermore, molecular dynamics calculations have the advantage that they can simulate states of the system under investigation that are not easily accessible to experiment. A dynamics run at pH 5.0, with His163 protonated in both monomers of the dimer, revealed a tendency of the two S1 pockets to collapse, with Glu166 switching between the protonated His163 and His172 residues over the time of the simulation.
It is important to note that none of the conformational differences seen between monomers "A" and "B" of the SARS-CoV M pro when crystallized at pH 6.0 is due to crystal packing artefacts. Symmetry-related contacts between dimers mainly involve domain III of both molecules; the only 188 Kanchan Anand et al. residues in domains I and II to display strong crystal contacts are His A134, Asn B53, Asp B56, Ile B59, Arg B60, His B134. Furthermore, the fact that the conformation of monomer B flips back to the normal, active one within the same crystal lattice after equilibration of the crystals at higher pH values, proves that crystal packing does not play a role here. The amino terminus of the SARS-CoV M pro plays an important role in our structural model, by interacting with essential residues of the substratebinding site of the other monomer in the dimer. Like a finger, the N-terminus docks into its specific binding site (Fig. 7) and flicks on the enzymatic activity of the other monomer. Hydrogen bonding from the "N-finger" to the main chain of Phe140 helps shape the oxyanion loop, and ion-pair formation with Glu166 prevents the latter from blocking the S1 specificity pocket. If a free N-terminus is not available at position 1 of the M pro , such as in the maltose-binding protein (MBP) fusion protein or in the presence of an N-terminal histidine tag, the enzymatic activity should be reduced and this has indeed been observed, at least with the MBP fusion protein [13]. In order to further demonstrate the importance of the N terminus, we deleted residues 1 to 5 in the TGEV M pro and indeed found the enzyme to be almost totally inactive [13]. This observation also solves the mystery of the importance of the "extra" helical domain III of the main proteinase. Its task is to provide defined interaction points for the N-terminal segment and thereby locating the tip of the N-finger to its specific interaction site with Phe140 and Glu166 of the other monomer. Accordingly, deletion of domain III also results in an inactive enzyme [13]. The enzyme is dimeric because of the essential role played by the N-finger, but the N-terminal segment is not essential for dimerization, as shown recently by Shi et al. [24] and also indicated by steered molecular dynamics calculations of a SARS-CoV M pro dimer devoid of the N-terminal residues (Tan et al., personal communication).
A transition between a catalytically competent and an incompetent conformation of the oxyanion-binding site has also been seen in the crystal structure of equine arterivirus main proteinase (nsp4), where the loop structurally equivalent to the M pro 139-143 loop undergoes a peptide flip, resulting in misorientation of the amide corresponding to Gly143 [30]. Also, we have little doubt that the same pH-induced conformational changes that we observed in the SARS-CoV M pro does exist in all other coronavirus main proteinases. We simply failed to detect it earlier because we had crystallized the M pro s of TGEV and HCoV 229E at pH > 8.0, i.e. with both monomers adopting an active conformation [13,18].
In conclusion, SARS-CoV M pro appears to possess a pH-triggered activation switch, which may regulate proteolytic activity in different cellular compartments. Such a pH switch would provide the virus with a mechanism to synchronize the polyprotein processing with the microenvironment. Further mutagenesis and biochemical studies on the details of the switch are clearly desirable.

Self-activation of the main proteinases
The M pro is part of the viral polyprotein itself and has to be activated by autocleavage from this inactive precursor before it can cleave the polyprotein at other cleavage sites. So far, it has been completely unclear how this autoactivation might occur but the crystallographic work on SARS-CoV M pro now provides a clue. The pH-dependent conformational changes seen in the X-ray structures [19] could well correlate with the structural arrangements occurring during self-activation of the enzyme.
Moreover, a comparison with the transition between chymotrypsin and its proenzyme, chymotrypsinogen, reveals a remarkable similarity in the mechanisms used. In mature chymotrypsin, the charged amino terminus, created after cleavage of the proenzyme at the Arg15-Ile16 bond, forms an ion pair with Asp194, a residue involved in forming the wall of the S1 specificity pocket. In chymotrypsinogen, on the other hand, the N-terminal extension of the polypeptide chain by 15 residues causes disruption of this ion pair [31]. The Asp194 side chain changes conformation and so does, along with it, Gly193, whose amide forms part of the oxyanion hole. As a consequence, interaction of Gly193, which corresponds to Gly143 of SARS-CoV M pro , with the tetrahedral intermediate is no longer possible. In SARS-CoV and other coronaviruses, the polyprotein upstream to the Nterminal autocleavage site of the M pro corresponds to the propeptide in chymotrypsinogen. The role of Asp194 of the latter is taken by Glu166 in SARS-CoV M pro and its proenzyme, and the oxyanion-binding region changes in a similar way. The major difference between the two enzymes is that the event is intramolecular in chymotrypsin but intermolecular in SARS-CoV M pro .
It is a matter of debate whether the autocleavage of the M pro s from the polyprotein precursors occur in cis or in trans. In the case of mouse hepatitis (corona)virus (MHV) M pro , autocleavage has exclusively been observed in trans [28]. On the other hand, it has been shown for several picornavirus 3C proteinases, e.g. hepatitis A virus 3C pro [32], that the first step of autocleavage of the enzyme, i.e. processing at the N-terminal cleavage site, occurs in cis, followed by other cleavage reactions in trans. Molecular modeling studies based on the TGEV M pro crystal structure suggested that N-terminal precursor cleavage in cis should be possible, because by introducing a bend around residues 11 to 13, the extended N-terminal chain can be quite easily folded back into the active site of its own molecule, even though the distance between the amino terminus (A1) and CysA145 Sγ is about 34 Å [13]. On the other hand, bringing the N terminus from the position seen in the crystal structure into the catalytic site of the other molecule in the dimer proves quite difficult, even though the distance is only about 11 Å. Thus, the fact that the M pro s are dimers does not necessarily support cleavage in trans. The SARS-CoV M pro structure rather suggests a different role for the dimerization, the mutual activation of the subunits by transition from the proenzyme conformation to that of the active enzyme, triggered by binding of N-fingers to the activation switch. Clearly, asymmetric dimers consisting of one molecule in the mature conformation and one in the proenzyme conformation, as seen in the SARS-CoV M pro crystal structure at pH 6.0, would be important intermediates in this process. Polyprotein processing in SARS-CoV-infected cells could thus be initiated by the occasional dimerization of two membrane-anchored M pro domains within polyproteins and mutual activation through the N-finger tips, or by the rare event of cis-autocleavage of a single M pro domain which then binds to another one within polyprotein, again activating it through the N-finger. The results are highly active M pro dimers which can then cleave other substrates in trans. Such a mechanism could lead to self-acceleration of M pro autoprocessing, after a slow onset triggered by a rare initial event. This mechanism is consistent with the fact that in cells infected with HCoV 229E or MHV, long-lived cleavage intermediates are usually not observed, but polyprotein is processed with increasing speed, once the first cleavage has occurred [28,33].

Complex of the SARS-CoV M pro with a chloromethyl ketone inhibitor
After dissecting the details of conformational dynamics of the SARS-CoV main proteinase, we are now finally in the position to competently discuss the interactions of the enzyme with substrate. To visualize these, the substrate-analoguous hexapeptidyl CMK inhibitor, Cbz-Val-Asn-Ser-Thr-Leu-Gln-CMK, was soaked into the crystals of the SARS-CoV enzyme grown at pH 6.0, and the structure of the complex was determined at 2.5 Å resolution [19]. We expected to see one of two possible cases, i.e. either i) the inhibitor would only bind to the protomer in the active conformation (monomer "A"), or ii) it would bind to the inactive protomer (monomer "B") as well, thereby imposing the active conformation onto the latter by an induced-fit mechanism. Quite unexpectedly, neither assumption turned out to be true. Instead, whereas the inhibitor does bind to both subunits and covalent bonds are being formed between the Sγ of Cys145 and the methylene group of the CMK, the inhibitor exhibits two different modes of binding, each of which is unusual and unexpected (Fig. 3B, C). Protomer "B" remains in the inactive conformation, and the inhibitor does not open the occluded S1 pocket to allow the P1 Gln to enter the specificity site (Fig. 3C). Instead, the P1 Gln points out of the substrate-binding cleft and towards bulk solvent. On the other hand, P2 Leu and P4 Ser bind to the appropriate specificity pockets that have been observed in the structure of TGEV M pro [13], whereas P3 Thr is oriented toward bulk solvent. This result can be interpreted as resulting from the high chemical reactivity of the CMK moiety with obvious concurrent inability of the inhibitor to overcome the protonation of HisB163 at the bottom of the S1 pocket at pH 6.0.
Binding of the CMK inhibitor to protomer A, which exhibits the active conformation, is even less conventional (Fig. 3B). The P1 glutamine resides in the S1 pocket and its side-chain carbonyl accepts a 2.8-Å hydrogen bond from the Nε2 atom of HisA163, whereas its side-chain amide nitrogen donates a 2.8-Å hydrogen bond to the carboxylate of GluA166, ensuring the specificity for glutamine in the S1 subsite. However, the P2 residue fails to penetrate the S2 subsite and becomes partially solvent-accessible. This non-canonical binding results in a frameshift in subsite interaction: P3 Thr occupies the S2 pocket instead and P5 Asn binds at the S4 subsite. We note that the P2 residue specificity of SARS-CoV M pro is less stringent than that of other CoV M pro s, which, with few exceptions, are restricted to leucine [28]. The unexpected binding mode of the substrate-like inhibitor may well be a consequence of the more relaxed specificity requirements of the SARS-CoV M pro , compared to other coronaviral main proteinases. However, before further conclusions are made, it has to be excluded that the binding mode observed is due to an artefact, perhaps because the sequence of the peptidyl CMK inhibitor had been derived from the TGEV M pro and not the SARS-CoV M pro N-terminal autocleavage site. Therefore, we are presently determining the crystal structures of the complexes of SARS-CoV M pro with other inhibitors, both peptidic and nonpeptidic.

Structure-based design of anti-SARS inhibitors
When the genome sequence of the SARS coronavirus was published on the internet on April 13, 2003 [16], we immediately located the segment in orf1a coding for the main proteinase and aligned the amino acid sequence of the enzyme with the M pro s of other coronaviruses. Next, we constructed a homology model of the SARS-CoV M pro , based on this alignment and our crystal structures of the TGEV and HCoV 229E main proteinases [18]. This theoretical model revealed that the substrate-binding site of the SARS-CoV enzyme is very similar to those of the two other viral enzymes. At that time, less than one month after the discovery of the new SARS virus and at the peak of the epidemic in China, this structural model was the best we could get, and it proved in fact very useful (see below). Of course, a theoretical homology model will always be inferior to an experimental crystal structure, but in the absence of the latter, it can provide a reasonably good basis for structure-assisted drug discovery [34,35]. When we had determined the crystal structure of the SARS-CoV M pro another 10 weeks later [19], the overall r.m.s. deviation for all Cα atoms of the protein between theoretical model and experimental structure was found to be as high as 3.8 Å. However, this unreasonably high value is only caused by large deviations of the last four residues at the flexible C-terminus, which deviate by more than 12 Å, and by slight differences of the mutual orientation of the three domains. If the models for the individual domains are compared with the crystal structure, and only this is a fair comparison in view of the large flexibility of the enzyme, the r.m.s. deviation is down to little more than 1 Å.
Whatever the view taken on this issue, the homology model served primarily one purpose, and that was to facilitate the design of anti-SARS inhibitors directed at the main proteinase. With the template of the peptidyl CMK inhibitor -as bound to the TGEV M pro -at hand, we compared its binding mode to those seen for all other inhibitors of cysteine proteinases that occupy the N-terminal half of the substrate binding site (subsites Sn-S1), as long as atomic coordinates were available in the Protein Data Bank (PDB). We found that AG7088, a compound carrying a vinylogous ethyl ester instead of a CMK group and designed to block human rhinovirus (HRV) 3C proteinase [36], interacted in much the same way with its target enzyme than did our CMK inhibitor (Fig. 8) [18].
As mentioned before, the picornavirus 3C proteinases (3C pro ) have a chymotrypsin-related structure, reminiscent of domains I and II of the coronavirus M pro , although several of the secondary-structure elements are arranged differently, making structural alignment difficult with sequence identities < 10%. Also, they completely lack a counterpart to domain III of coronavirus M pro s. Nevertheless, the substrate specificity of picornavirus 3C pro s [32,37] for the P1', P1 and P4 sites is similar to that of the coronavirus M pro s (hence the original designation "3C-like" for the latter; see Tab. 1). Only parts of the two structures, TGEV M pro in complex with the CMK inhibitor and HRV-2 in complex with AG7088, can be spatially superimposed ( Fig. 8; r.m.s. deviation of 2.10 Å for 134 pairs of Cα positions out of the ~180 residues in domains I and II). Both inhibitors, the hexapeptidyl CMK and AG7088, bind to their respective target proteinases through formation of an antiparallel β-sheet with strand eII. However, completely different segments of the polypeptide chain interact with the substrate analogues on the opposite site: residues 188 to 191 of the loop connecting domains II and III in M pro , as opposed to the short β-strand 126 to 128 in HRV 3C pro . As a result, the architectures of the S2 subsites are entirely different between the two enzymes; hence, the different specificities for the P2 residues of the substrates (Leu versus Phe). The inhibitor Figure 8. Superimposed structures of TGEV M pro (blue) in complex with CMK inhibitor (red) and HRV-2 (green) in complex with AG7088 (yellow).
Only the substrate-binding sites are shown. Stereo illustration. From [18], with permission of the American Association for the Advancement of Science (AAAS).
AG7088 has a p-fluorophenylalanine side chain (p-fluorobenzyl) in this position. Based on molecular modeling, we proposed that this side chain might be too long to fit into the S2 pocket of coronavirus M pro , but an unmodified benzyl group would probably fit, as evidenced by Phe occuring in the P2 position of the C-terminal autocleavage site of the SARS coronavirus enzyme (see Tab. 1). Apart from this difference, the superimposition of the two complexes (Fig. 8) suggested that the side chains of AG7088 binding to subsites S1 (lactone derivative of glutamine) and S4 (5-methyl-isoxazole-3-carbonyl) can be easily accommodated by the coronavirus M pro . Thus, we proposed that AG7088 could serve as a starting point for modifications which should quickly lead to an efficient and bioavailable inhibitor for coronavirus main proteinases. In fact, such modifications have been applied (work mostly unpublished so far; a published proposal for modification is found in [38]) and we will see a number of good inhibitors designed on this basis appear in the literature in the next few months.
Another possible approach to inhibiting the SARS-CoV main proteinase could make use of the insight into the importance of the N-finger in activating the enzyme. One could screen for competitve inhibitors that prevent M pro dimerization through binding to the N-finger docking site, but without activating the Glu166/Phe140 switch. Such an approach would open a second front in addition to active site-directed inhibitors, in the urgent search for substances efficiently blocking the SARS coronavirus.
Several groups have proposed to use HIV-1 proteinase inhibitors as starting points for designing inhibitors directed at the SARS-CoV M pro . However, many of these studies remained at the modelling level and failed to prove the usefulness of the HIV-1 proteinase inhibitors experimentally [39,40]. Some of the reports did not even seem to acknowledge the fact that the enzymatic mechanisms of HIV-1 proteinase, an aspartic proteinase, and SARS-CoV main proteinase, a cysteine proteinase, are very different. One of the few studies reporting a clinical usefulness of the application of HIV-1 proteinase inhibitors was published by Peiris et al. [41]. According to their results, a combination of the HIV-1 proteinase inhibitor kaletra and ribavirin was more effective than ribavirin alone, which, however, was basically inactive, as shown later [42]. Based on the present structural knowledge, it is not obvious how HIV-1 proteinase inhibitors should block SARS-CoV.
In conclusion, prospects for discovering and developing inhibitors of the SARS-CoV main proteinase are good, given the structural knowledge that is available for the target today. It should also be emphasized that the crystal structures of the various M pro s allow very efficient virtual screening [43] for existing organic compounds that might bind and inhibit the target. This approach is currently yielding promising inhibitors, which also exhibit anti-SARS activity on virus-infected Vero cells (Jiang, personal communication).

Conclusions
The three-dimensional structures presented here for coronavirus main proteinases provide a solid basis for the design of anti-coronaviral drugs. The binding modes of peptidic inhibitors and substrates have been revealed by the crystal structure of TGEV M pro in complex with a hexapeptidyl chloromethyl ketone, and, within limitations, of the complex with the SARS-CoV M pro and the same inhibitor. The structure of the SARS-CoV enzyme was determined at three different pH values and revealed a pHdependent activation switch for the M pro . The pH-activity profile of the main proteinase was shown to be governed by the protonation and deprotonation of two histidine residues in the substrate-binding site. The conformational changes observed also suggested a mechanism for the self-activation of the M pro by autocleavage from the viral polyprotein. On the basis of structural comparisons between the TGEV M pro in complex with a hexapeptidyl CMK inhibitor and the HRV 3C proteinase in complex with the vinylogous ethyl ester AG7088, it has been proposed that whereas the latter would not be a good inhibitor of the SARS-CoV M pro , it should be a good starting point for designing such inhibitors. Such design studies are being performed at the moment and will start to yield potent anti-SARS compounds in the next few months and years.