• IMMUNE NETWORK
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• 제22권3호
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• pp.28.1-28.11
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• 2022

The 26S proteasome irreversibly hydrolyzes polyubiquitylated substrates to maintain protein homeostasis; it also regulates immune responses by generating antigenic peptides. An alternative form of the 26S proteasome is the immunoproteasome, which contains substituted catalytic subunits (β1i/PSMB9, β2i/PSMB10, and β5i/PSMB8) instead of constitutively expressed counterparts (β1/PSMB6, β2/PSMB7, and β5/PSMB5). The immunoproteasome expands the peptide repertoire presented on MHC class I molecules. However, how its activity changes in this context is largely elusive, possibly due to the lack of a standardized methodology to evaluate its specific activity. Here, we describe an assay protocol that measures the immunoproteasome activity of whole-cell lysates using commercially available fluorogenic peptide substrates. Our results showed that the most accurate assessment of immunoproteasome activity could be achieved by combining β5i-targeting substrate Ac-ANW-AMC and immunoproteasome inhibitor ONX-0914. This simple and reliable protocol may contribute to future studies of immunoproteasomes and their pathophysiological roles during viral infection, inflammation, and tumorigenesis.

• BMB Reports
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• 제34권3호
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• pp.219-224
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• 2001

To identify the antioxidative peptides in the gelatin hydrolysate of bovine skin, the gelatin was hydrolyzed with serial digestions in the order of Alcalase, pronase E, and collagenase using a three-step recycling membrane reactor. The second enzymatic hydrolysate (hydrolyzed with pronase E) was composed of peptides ranging from 1.5 to 4.5 kDa, and showed the highest antioxidative activity, as determined by the thiobarbituric acid method. Three different peptides were purified from the second hydrolysate using consecutive chromatographic methods. This included gel filtration on a Sephadex G-25 column, ion-exchange chromatography on a SP-Sephadex C-25 column, and high-performance liquid chromatography on an octadecylsilane chloride column. The isolated peptides were composed of 9 or 10 amino acid residues. They are: Gly-Glu-Hyp-Gly-Pro-Hyp-Gly-Ala-Hyp (PI), Gly-ProHyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly (PII), and Gly-ProHyp-Gly-Pro-Hyp-Gly-Pro-Hyp (PIII), as characterized by Edman degradation and fast-atom bombardment mass spectrometry. The antioxidative activities of the purified peptides were measured using the thiobarbituric acid method, and the cell viability with a methylthiazol tetrazolium assay The results showed that PII had potent antioxidative activity on peroxidation of linoleic acid. Moreover, the cell viability of cultured liver cells was significantly enhanced by the addition of the peptide. These results suggest that the purified peptide, PII, from the gelatin hydrolysate of bovine skin is a natural antioxidant, which has potent antioxidative activity.

• Journal of Animal Science and Technology
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• 제64권6호
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• pp.1132-1143
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• 2022

Insects are a valuable natural source that can produce a variety of bioactive compounds due to their increasing species diversity. CopA3 is an antimicrobial peptide derived from Copris tripartitus (i.e., the dung beetle). It is known to increase the proliferation of colonic epithelial and neuronal stem cells by regulating cell cycle. This research hypothesized that CopA3 can promote the proliferation of porcine muscle satellite cells (MSCs). The effects of CopA3 on porcine MSCs, which are important for muscle growth and regeneration, remain unclear. Here, we investigated the effects of CopA3 on porcine MSCs. According to viability results, we designed four groups: control (without CopA3) and three treatment groups (treated with 5,10, and 25 ㎍/mL of CopA3). At a CopA3 concentration of 5 ㎍/mL and 10 ㎍/mL, the proliferation of MSCs increased more than that observed in the control group. Furthermore, compared to that in the control, CopA3 treatment increased the S phase but decreased the G0/G1 phase ratio. Additionally, early and late apoptotic cells were found to be decreased in the 5 ㎍/mL group. The expressions of the myogenesis-related transcription factor PAX7 and MYOD proteins were significantly upregulated in the 5 ㎍/mL and 10 ㎍/mL groups, whereas the MYOG protein remained undetected in all group. This study suggested that CopA3 promotes muscle cell proliferation by regulating the cell cycle of MSCs and can regulate the activity of MSCs by increasing the expressions of PAX7 and MYOD.

• Journal of Microbiology and Biotechnology
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• 제34권2호
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• pp.415-424
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• 2024

This study reveals that low-molecular-weight collagen peptide (LMWCP) can stimulate the differentiation and the mineralization of MC3T3-E1 cells in vitro and attenuate the bone remodeling process in ovariectomized (OVX) Sprague-Dawley rats in vivo. Moreover, the assessed LMWCP increased the activity of alkaline phosphatase (ALP), synthesis of collagen, and mineralization in MC3T3-E1 cells. Additionally, mRNA levels of bone metabolism-related factors such as the collagen type I alpha 1 chain, osteocalcin (OCN), osterix, bone sialoprotein, and the Runt family-associated transcription factor 2 were increased in cells treated with 1,000 ㎍/ml of LMWCP. Furthermore, we demonstrated that critical bone morphometric parameters exhibited significant differences between the LMWCP (400 mg/kg)-receiving and vehicle-treated rat groups. Moreover, the expression of type I collagen and the activity of ALP were found to be higher in both the femur and lumbar vertebrae of OVX rats treated with LMWCP. Finally, the administration of LMWCP managed to alleviate osteogenic parameters such as the ALP activity and the levels of the bone alkaline phosphatase, the OCN, and the procollagen type 1 N-terminal propeptide in OVX rats. Thus, our findings suggest that LMWCP is a promising candidate for the development of food-based prevention strategies against osteoporosis.

• Journal of Microbiology and Biotechnology
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• 제20권1호
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• pp.132-137
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• 2010

The skc gene encoding streptokinase (SK) with a molecular mass of approximately 47.4 kDa was cloned from Streptococcus equisimilis ATCC 9542 and heterologously overexpressed in Streptomyces lividans TK24 and E. coli using various strong promoters. When the promoter for sprT [Streptomyces griseus trypsin (SGT)] was used in the host S. lividans TK24, a 47.4-kDa protein was detected along with a smaller hydrolyzed protein (44 kDa), suggesting that posttranslational hydrolysis had occurred as has been reported in other expression systems. The casein/plasminogen plate assay revealed that the plasmid construct containing the SGT signal peptide was superior to that containing the SK signal peptide in terms of SK production. Maximal production of SK was calculated to be about 0.25 unit/ml of culture broth, a value that was five times higher than that obtained with other expression systems using ermE and tipA promoters in the same host. When the skc gene was expressed in E. coli BL21(${\Delta}DE3$)pLys under the control of the T7 promoter, a relatively large amount of SK was expressed in soluble form without hydrolysis. SK activity in E. coli/pET28a-$T7_pSK_m$ was more than 2 units/ml of culture broth, even though about half of the expressed protein formed an inactive inclusion body.

• Molecules and Cells
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• 제41권3호
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• pp.234-243
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• 2018

In recent years, the interest towards the relationship between incretins and bone has been increasing. Previous studies have suggested that glucagon-like peptide-1 (GLP-1) and its receptor agonists exert beneficial anabolic influence on skeletal metabolism, such as promoting proliferation and differentiation of osteoblasts via entero-osseous-axis. However, little is known regarding the effects of GLP-1 on osteoblast apoptosis and the underlying mechanisms involved. Thus, in the present study, we investigated the effects of liraglutide, a glucagon-like peptide-1 receptor agonist, on apoptosis of murine MC3T3-E1 osteoblastic cells. We confirmed the presence of GLP-1 receptor (GLP-1R) in MC3T3-E1 cells. Our data demonstrated that liraglutide inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as detected by Annexin V/PI and Hoechst 33258 staining and ELISA assays. Moreover, liraglutide upregulated Bcl-2 expression and downregulated Bax expression and caspase-3 activity at intermediate concentration (100 nM) for maximum effect. Further study suggested that liraglutide stimulated the phosphorylation of AKT and enhanced cAMP level, along with decreased phosphorylation of $GSK3{\beta}$, increased ${\beta}-catenin$ phosphorylation at Ser675 site and upregulated nuclear ${\beta}-catenin$ content and transcriptional activity. Pretreatment of cells with the PI3K inhibitor LY294002, PKA inhibitor H89, and siRNAs GLP-1R, ${\beta}-catenin$ abrogated the liraglutide-induced activation of cAMP, AKT, ${\beta}-catenin$, respectively. In conclusion, these findings illustrate that activation of GLP-1 receptor by liraglutide inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation through $cAMP/PKA/{\beta}-catenin$ and $PI3K/Akt/GSK3{\beta}$ signaling pathways.

• Journal of Plant Biotechnology
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• 제31권4호
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• pp.319-323
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• 2004

Euphorbia lathyris의 유관세포로부터 분비되는 수용성 latex단백질을 10% SDS-polyacrylamide 전기영동으로 분리하여 특징적으로 잘 나타나고 있는 ELp65, ELp55, ELp43, ELp32 그리고 ELp23 등의 주요단백질을 확인하였다. 이들 latex 주요단백질들 중에서 ELp65는 ammonium sulfate 침전, gel permeation chromatography 그리고 ion exchange chromatography 등의 방법으로 순수 분리하였는데 ELp65의 N-terminal amino acid 서열분석에 의하면 이는 토마토의 p69a subtilisin-like pretense의 mature peptide의 앞부분과 강한 유사성을 지니고 있었으며 식물방어와 관련된 기능이 제시되었다. PCR증폭에 의하여 클로닝된 토마토의 p69a DNA를 probe로 이용하여 Southern blot hybridization을 수행한 결과 E. lathyris genome은 토마토의 subtilisin-like proteases와 유사한 정보를 지닐 수도 있는 3-5 유전자들로 구성된 gene family가 분석되었다.

• BMB Reports
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• 제39권1호
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• pp.16-21
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• 2006

The coat protein (CP) of Papaya ringspot virus (PRSV) was analyzed for presentation of the antigenic peptide of animal virus, Canine parvovirus (CPV), in Escherichia coli (E. coli). The 45 nucleotides fragment coding for the 15-aa peptide epitope of the CPV-VP2 protein was either inserted into the PRSV-cp gene at the 5', 3' ends, both 5' and 3' ends or substituted into the 3' end of the PRSV cp gene. Each of the chimeric PRSV cp genes was cloned into the pRSET B vector under the control of the T7 promoter and transformed into E. coli. The recombinant coat proteins expressed from different chimeric PRSV-cp genes were purified and intraperitoneally injected into mice. All of the recombinant coat proteins showed strong immunogenicity and stimulate mice immune response. The recombinant coat proteins containing the CPV epitope insertion at the C terminus and at both N and C termini elicited ten times higher specific antisera in immunized mice compared with the other two recombinant coat proteins which contain the CPV epitope insertion at the N terminus and substitution at the C terminus.

• Journal of Microbiology and Biotechnology
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• 제13권5호
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• pp.660-665
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• 2003

High-level expression of Thermus caldophilus GK24 alkaline phosphatase (Tca APase) was achieved in Escherichia coli using the pET-based expression plasmids, pEAP1 and pEAP2. In the case of plasmid pEAP2, the signal peptide region of Tca APase was replaced by the PelB leader peptide of expression vector pET-22b(+). Furthermore, the expression level was somewhat higher than that of plasmid pEAPl. A rapid purification procedure of Tca APase overproduced in E. coli was developed which involved heating to denature E. coli proteins followed by HiTrap Heparin HP column chromatography. Optimal temperature and pH and $Mg^{2+}$ dependence of the recombinant Tca APase were similar to those of native enzyme isolated from T. caldophilus GK24.

• Food Science and Biotechnology
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• 제16권4호
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• pp.590-593
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• 2007

The textural properties of doughs mixed with L-ascorbic acid (AA), trypsin hydrolyzed gluten peptide (THGP), and a mixture of AA-THGP were investigated using texture analyzer under the fermentation of the full formula and the freezing process. The full formula dough (FFD) required a shorter mixing time than the flour and water formula dough (FWD). The maximum resistance (Rmax) values of both the unfrozen and frozen doughs were lower for the FFD. The effects of AA and THGP additions were not significant (p<0.01) in FFD, however, they were significant in FWD. The freezing effect was significant (p<0.0001) for FFD, indicating that yeast fermented dough was much more sensitive to damage from freezing, which subsequently affected dough strength. Additions of AA (p=0.0026) and THGP (p=0.0097) had a significant effect on the extensibility (E-value) of unfrozen FWD, where THGP increased and AA decreased the E-value. However, freezing did not significantly effect the extensibilities of FWD (p=0.64) or FFD (p=0.21). The area of FFD was lower than the area of FWD for both the unfrozen and frozen doughs. However, the frozen dough mixed with THGP alone had the largest area overall. The addition of additives did not result in significantly different (p<0.01) areas under the curve, except in the frozen FFD. Freezing caused a statistically significant difference in the area of FWD (p=0.0045).