• Journal of Life Science
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• v.25 no.1
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• pp.1-7
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• 2015

Cyclin D is a member of the cyclin protein family, which plays a critical role as a core member of the mammalian cell cycle machinery. D-type cyclins (D1, D2, and D3) bind to and activate the cyclin-dependent kinases 4 and 6, which can then phosphorylate the retinoblastoma tumor suppressor gene products. This phosphorylation in turn leads to release or derepression of E2F transcription factors that promote progression from the G1 to S phase of the cell cycle. Among the D-type cyclins, cyclin D3 encoded by the CCND3 gene is one of the least well studied. In the present study, we have investigated the biochemistry of the proteolytic mechanism that leads to loss of cyclin D3 protein. Treatment of human prostate carcinoma PC-3-M cells with lovastatin and actinomycin D resulted in a loss of cyclin D3 protein that was completely reversible by the peptide aldehyde calpain inhibitor, LLnL. Additionally, using inhibitors for various proteolytic systems, we show that degradation of cyclin D3 protein involves the $Ca^{2+}$-activated neutral protease calpain. Moreover, the half-life of cyclin D3 protein half-life increased by at least 10-fold in PC-3M cells in response to the calpain inhibitor. We have also demonstrated that the transient expression of the calpain inhibitor calpastatin increased cyclin D3 protein in serum-starved NIH 3T3 cells. These data suggested that the function of cyclin D3 is regulated by $Ca^{2+}$-dependent protease calpain.

• Fisheries and Aquatic Sciences
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• v.1 no.2
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• pp.209-215
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• 1998

Trypsin-like enzyme was purified from shrimp hepatopancreas through Q-Sepharose ionic exchange, benzamidine Sepharose-6B affinity, and Superdex 75 gel chromatography. Purity of trypsin-like enzyme was increased 69-fold with $44\%$ yield. The enzyme consisted of a single polypeptide chain with a molecular weight (M.W.) of 32 kDa judged by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was completely inactivated by serine enzyme inhibitors such as soybean trypsin inhibitor (SBTI), tosyl-L­lysine chloromethyl ketone (TLCK), and leupeptin. However, the enzyme was not affected by tosyl-L-phenylalanine chloromethyl ketone (TPCK) which is a chymotrypsin specific inhibitor. The enzyme had no activity against benzoyl-tyrosine ethyl ester (BTEE) which is a chymotrypsin specific substrate. The enzyme showed high activity on the carboxyl terminal of Phe, Tyr. Glu, Arg, and Asp. However. no activity was detected against the carboxyl terminal of Pro, Trp, Cys, Gly, Val, and Ala.

• BMB Reports
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• v.32 no.3
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• pp.239-246
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• 1999

A tripeptidase (PepT) was purified to homogeneity from Bacillus subtilis through four sequential chromatographies including DEAE-Sepharose ion exchange, hydroxylapatite, mono-Q FPLC ion exchange, and Superose-12 FPLC gel filtration. The apparent molecular mass of the enzyme was 49,200 Da and 51,400 Da as determined by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography, respectively, and the enzyme exists in a monomeric form. The physicochemical properties of the enzyme were as follows: optimum pH at 7.5, optimum temperature at $60^{\circ}C$, and pI at 4.9. The $K_m$ and $V_{max}$ values of the enzyme were 4.3 mM and 2.5 mmol/min/mg, respectively, with MetAla-Ser as substrate. The B. subtilis PepT requires $Co^{2+}$ ion(s) for activation, while it is inactivated by EOTA and 1,10-phenanthroline, suggesting that it is a metalloprotein. The enzyme was not inhibited by any of serine protease, aspartic protease, or leucine aminopeptidase inhibitors. The enzyme showed comparable activities towards four different substrates including Met-Ala-Ser, Leu-Gly-Gly, Leu-Ser-Phe, and Leu-Leu-Tyr. The amino terminal sequence of PepT determined by Edman degradation was found to be MKEEIIERFTTYVXV and turned out to be identical to that of PepT deduced from a cloned B. subtilis pepT.

• Korean Journal of Agricultural Science
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• v.36 no.1
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• pp.111-122
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• 2009

When the cattail pollen was identified by using fibrinolytic agents, we found that the fibrinolytic activity was controlled by an enzyme. Therefore, for determining the fibrinolytic activity of cattail pollen, the fibrinolytic enzyme in cattail pollen was purified by gel filtration using DEAE-cellulose, Sephadex G-150 and HPLC. Also, its purity was certified by polyacrylamide gel electrophoresis, and its physico-chemical properties, such as pH and temperature stabilities and effects of metal, inhibitors and substrates, were examined. The specific activity, purification fold, and molecular weight of the enzyme were 38U/mg, 86.4,and 75kDa, respectively. The optimum pH for the purified enzyme was at 4.0 and it was stable at pH 4.0-6.0. The optimum temperature was $55^{\circ}C$ and it was stable at $30-60^{\circ}C$. But the enzyme began to be inactivated at $70^{\circ}C$ and its activity was totally lost at temperatures above $80^{\circ}C$. As for substrate specificity, the enzyme was most effective in dissolving fibrin, followed by whole casein, ${\kappa}$-casein, ${\alpha}$-casein, ${\beta}$-casein, and BSA. With casein as the substrate, Km value was found to be 0.44mM and the enzyme showed a high affinity for casein. As for the metal ions affecting enzyme activity, $K^+$, $Na^+$, and $Mg^{2+}$ had no effect on enzyme reaction while $Zn^{2+}$ and $Fe^{2+}$ showed potent inhibitory activity. Judging from the fact that the purified enzyme was also strongly inhibited by PMSF, iodoacetic acid, and SDA, it assumed to be a serine protease.

• Parasites, Hosts and Diseases
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• v.50 no.1
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• pp.1-6
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• 2012

$Toxoplasma$ $gondii$ penetrates all kinds of nucleated eukaryotic cells but modulates host cells differently for its intracellular survival. In a previous study, we found out that serine protease inhibitors B3 and B4 (SERPIN B3/B4 because of their very high homology) were significantly induced in THP-1-derived macrophages infected with $T.$ $gondii$ through activation of STAT6. In this study, to evaluate the effects of the induced SERPIN B3/B4 on the apoptosis of $T.$ $gondii$-infected THP-1 cells, we designed and tested various small interfering (si-) RNAs of SERPIN B3 or B4 in staurosporine-induced apoptosis of THP-1 cells. Anti-apoptotic characteristics of THP-1 cells after infection with $T.$ $gondii$ disappeared when SERPIN B3/B4 were knock-downed with gene specific si-RNAs transfected into THP-1 cells as detected by the cleaved caspase 3, poly-ADP ribose polymerase and DNA fragmentation. This anti-apoptotic effect was confirmed in SERPIN B3/B4 overexpressed HeLa cells. We also investigated whether inhibition of STAT6 affects the function of SERPIN B3/B4, and vice versa. Inhibition of SERPIN B3/B4 did not influence STAT6 expression but SERPIN B3/B4 expression was inhibited by STAT6 si-RNA transfection, which confirmed that SERPIN B3/B4 was induced under the control of STAT6 activation. These results suggest that $T.$ $gondii$ induces SERPIN B3/B4 expression via STAT6 activation to inhibit the apoptosis of infected THP-1 cells for longer survival of the intracellular parasites themselves.

• Journal of Life Science
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• v.28 no.6
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• pp.753-763
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• 2018

Cysteine cathepsins are lysosomal enzymes that belong to the papain family and can induce the degradation of damaged proteins through the endo-lysosomal pathway. It is highly upregulated in many cancers by regulating gene amplification and transcriptional, translational, and post-transcriptional modifications. Cathepsin S is part of the cysteine cathepsin family. Many studies have demonstrated that cathepsin S not only plays a specific role in MHC class II antigen presentation but also plays a crucial role in cancers. Cathepsin S is more stable at a neutral pH compared to other cysteine cathepsins, which supports the importance of cathepsin S in disease microenvironments. Therefore, the dysregulation of cathepsin S has participated in a variety of pathological processes, including cancer, arthritis, and cardiovascular disease. Furthermore, a decrease or depletion in the expression of cathepsin S has been implicated in the processes of tumor growth, invasion, metastasis, and angiogenesis. Taken together, cathepsin S has been suggested as an attractive therapeutic target for cancer therapy. In this review, the known involvement of cathepsin S in diseases, particularly with respect to recent work indicating its role in cancer therapy, is examined. An overview of current literature on the inhibitors of cathepsin S as a therapeutic target for cancer is also provided.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.798-803
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• 1999

Prolyl endopeptidase [PEP; EC 3.4.21.26], a serine protease which is known to cleave peptide bonds on the carboxy side of a proline residue, plays an important role in the degradation of proline-containing neuropeptides that have been suggested to participate in learning and memory processes. An abnormal increase in the level of PEP, which can lead to generation of $A{\beta}$, is also suggested to be involved in Alzheimer's type senile dementia. In the course of screening PEP inhibitors from Basidiomycetes, the mushroom Polyozellus multiplex exhibited a high inhibitory activity against PEP. Two active compounds were isolated from the ethyl acetate soluble fraction by consecutive purification, using silica gel, Sephadex LH-20, and Lobar RP-18 chromatography. The chemical structures of these compounds were identified as thelephoric acid and 12-acety1-2,3,7,8-tetrahydroxy-[12H]-12-hydroxymethylbenzobis[I.2b,3.4b'] benzofuran-11-one (kynapcin-9) by spectral data including UV, IR, MS, HR-MS, $^1H-,{\;}^{13}C-$, and 2D-NMR. The $IC_{50}$ values of the thelephoric acid and kynapcin-9 were 0.157 ppm (446nM) and 0.087 ppm (212nM) and their inhibitor constants ($K_i$) were 0.73ppm ($2.09{\;}\mu\textrm{m}$) and 0.060 ppm (146 nM), respectively. Furthermore, they were non-competitive with a substrate in Dixon plots.

• The Korean Journal of Pharmacology
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• v.26 no.1
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• pp.51-54
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• 1990

Human leukocyte cathepsin-Gs are active participant in the active phase of inflammations like rheumatoid arthritis, emphysema and glomerular injury. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for treatment of these inflammatory diseases. Mechanism of action of NSAIDs for treatment of inflammatory diseases, especially like rheumatoid arthritis, are known as the inhibitors of prostaglandin synthesis. Inhibitions of the activities of human leukocyte cathepsin-Gs by non-steroidal anti-inflammatory drugs, however, were not same as the known pharmacological effects (inhibition of cyclooxygenase) of these drugs. Among them, especially, sulindac, salicylate, phenylbutazone, oxyphenbutazone, and salicyluric acid inhibited human leukocyte cathepsin-Gs effectively. $IC_{50}s$ of each drug were 4.3mM, 14.3mM, 6.5mM, 11mM and 15mM respectively. The drugs which have same chemical structure and same degree of inhibition effect on cyclooxygenase showed different degree or no effect on inhibition of cathepsin G. These inhibition effect might be, beside of inhibition of cyclooxygenase in the prostaglandin synthesis pathway, another benefitial antiinflammatory effect of NSAIDs by direct protection against tissue destruction in inflammatory diseases.

• Biomolecules & Therapeutics
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• v.21 no.2
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• pp.107-113
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• 2013

Plasminogen activator inhibitor-1 (PAI-1) is a member of serine protease inhibitor family, which regulates the activity of tissue plasminogen activator (tPA). In CNS, tPA/PAI-1 activity is involved in the regulation of a variety of cellular processes such as neuronal development, synaptic plasticity and cell survival. To gain a more insights into the regulatory mechanism modulating tPA/PAI-1 activity in brain, we investigated the effects of proteasome inhibitors on tPA/PAI-1 expression and activity in rat primary astrocytes, the major cell type expressing both tPA and PAI-1. We found that submicromolar concentration of MG132, a cell permeable peptide-aldehyde inhibitor of ubiquitin proteasome pathway selectively upregulates PAI-1 expression. Upregulation of PAI-1 mRNA as well as increased PAI-1 promoter reporter activity suggested that MG132 transcriptionally increased PAI-1 expression. The induction of PAI-1 downregulated tPA activity in rat primary astrocytes. Another proteasome inhibitor lactacystin similarly increased the expression of PAI-1 in rat primary astrocytes. MG132 activated MAPK pathways as well as PI3K/Akt pathways. Inhibitors of these signaling pathways reduced MG132-mediated upregulation of PAI-1 in varying degrees and most prominent effects were observed with SB203580, a p38 MAPK pathway inhibitor. The regulation of tPA/PAI-1 activity by proteasome inhibitor in rat primary astrocytes may underlie the observed CNS effects of MG132 such as neuroprotection.

• Journal of the Korean Society of Food Science and Nutrition
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• v.22 no.4
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• pp.458-464
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• 1993

In the present paper enzymatic properties of the trypsins from the four dark fleshed fish were compared with each other and thermal stabilities of the enzymes were also investigated. The trypsins from the dark fleshed fish showed their activity only in BA-p-NA substrate of the amide substrates such as BA-p-NA and SP-p-NA, and BAEE and TAME of the ester substrate such as ATEE, BAEE, BTEE, and TAME. The enzymes were strongly inhibited by the serine protease inhibitors such as antipain, leupeptin, TLCK, DFP and SBTI, and were also inhibited by such metal ions as Cu$^{2＋}$ and Hg$^{2＋}$, but fairly activated by $Mg^{2＋}$. Denaturation constants of the enzymes were 13.4$\times$10$^{-4}$ sec$^{-1}$ for anchovy trypsin, 47.18$\times$10$^{-4}$ sec$^{-1}$ for mackerel trypsin A, 34.06$\times$10$^{-4}$ sec$^{-1}$ mackerel trypsin B, 42.28$\times$10$^{-4}$ sec$^{-1}$ for yellowfin tuna trypsin and 16.6$\times$10$^{-4}$ sec$^{-1}$ for albacore trypsin at 55$^{\circ}C$. The activation energies of the trypsins at a temperature range of 3$0^{\circ}C$ to 5$0^{\circ}C$ were estimated to be 13.91 ㎉/mole for anchovy trypsin, 11.61㎉/mo1e and 8.43㎉/mole for mackerel trypsin A and for mackerel typsin B, 4.35㎉/mole for yellowfin tuna trypsin, and 3.76㎉/mole for albacore trypsin.