• BMB Reports
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• 제42권8호
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• pp.506-510
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• 2009

The Toll signalling pathway in invertebrates is responsible for defense against Gram-positive bacteria and fungi, leading to the expression of antimicrobial peptides via NF-$\kappa$B-like transcription factors. Gram-negative binding protein 3 (GNBP3) detects beta-1,3-glucan, a fungal cell wall component, and activates a three step serine protease cascade for activation of the Toll signalling pathway. Here, we showed that the recombinant N-terminal domain of Tenebrio molitor GNBP3 bound to beta-1,3-glucan, but did not activate down-stream serine protease cascade in vitro. Reversely, the N-terminal domain blocked GNBP3-mediated serine protease cascade activation in vitro and also inhibited beta-1,3-glucan-mediated antimicrobial peptide induction in Tenebrio molitor larvae. These results suggest that the N-terminal GNBP homology domain of GNBP3 functions as a beta-1,3-glucan binding domain and the C-terminal domain of GNBP3 may be required for the recruitment of immediate down-stream serine protease zymogen during Toll signalling pathway activation.

• 약학회지
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• 제33권6호
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• pp.339-344
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• 1989

The alkaline protease produced by Sarcodon aspratus(Berk) S. Ito. was purified from its fruit bodies. The enzyme was purified by using ammonium sulfate fractionation, tris-acryl CM-cellulose column chromtography and chromatofocusing. The protease migrated as one major band with a molecular weight of about 29,000 dalton on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The amino acid sequence of the N-terminal residues(21) of the enzyme was determined by automated sequence analysis. The sequence was Val-Thr-Thr-Lys-Gln-Thr-Asn-Ala-Pro-Trp-Gly-Leu-Gly-Asn-Ile-Ser-Thr-Thr-Asn-Lys-Leu. Comparison of this sequence with the N-terminal sequence of the p-roteinase K from Tritirachium album showed high similarity, i. e. 57.8% identical residues. The protease displayed a relatively high stability in sodium dodecyl sulfate.

• Molecules and Cells
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• 제43권8호
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• pp.694-704
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• 2020

HslUV is a bacterial heat shock protein complex consisting of the AAA+ ATPase component HslU and the protease component HslV. HslV is a threonine (Thr) protease employing the N-terminal Thr residue in the mature protein as the catalytic residue. To date, HslUV from Gram-negative bacteria has been extensively studied. However, the mechanisms of action and activation of HslUV from Gram-positive bacteria, which have an additional N-terminal sequence before the catalytic Thr residue, remain to be revealed. In this study, we determined the crystal structures of HslV from the Gram-positive bacterium Staphylococcus aureus with and without HslU in the crystallization conditions. The structural comparison suggested that a structural transition to the symmetric form of HslV was triggered by ATP-bound HslU. More importantly, the additional N-terminal sequence was cleaved in the presence of HslU and ATP, exposing the Thr9 residue at the N-terminus and activating the ATP-dependent protease activity. Further biochemical studies demonstrated that the exposed N-terminal Thr residue is critical for catalysis with binding to the symmetric HslU hexamer. Since eukaryotic proteasomes have a similar additional N-terminal sequence, our results will improve our understanding of the common molecular mechanisms for the activation of proteasomes.

• BMB Reports
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• 제56권11호
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• pp.606-611
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• 2023

The main protease (Mpro) of SARS-CoV-2 cleaves 11 sites of viral polypeptide chains and generates essential non-structural proteins for viral replication. Mpro is an important drug target against COVID-19. In this study, we developed a real-time fluorometric turn-on assay system to evaluate Mpro proteolytic activity for a substrate peptide between NSP4 and NSP5. It produced reproducible and reliable results suitable for HTS inhibitor assays. Thus far, most inhibitors against Mpro target the active site for substrate binding. Mpro exists as a dimer, which is essential for its activity. We investigated the potential of the Mpro dimer interface to act as a drug target. The dimer interface is formed of domain II and domain III of each protomer, in which N-terminal ten amino acids of the domain I are bound in the middle as a sandwich. The N-terminal part provides approximately 39% of the dimer interface between two protomers. In the real-time fluorometric turn-on assay system, peptides of the N-terminal ten amino acids, N10, can inhibit the Mpro activity. The dimer interface could be a prospective drug target against Mpro. The N-terminal sequence can help develop a potential inhibitor.

• 대한수의학회지
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• 제39권4호
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• pp.772-777
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• 1999

The uncaped genomic RNA of bovine viral diarrhea virus (BVDV) initiates translation by recruitment of eukaryotic translation initiation factors at the internal ribosome entry site (IRES). N-terminal protease ($N^{pro}$) is the first translation product of the open reading frame (ORF). By using the vaccinia virus SP6 RNA polymerase transient expression system, we showed previously that deletion of $N^{pro}$ region reduced translation by 21%. To better understand the biological significance of $N^{pro}$ for translation, we carried out a mutational analysis of the $N^{pro}$ region of BVDV cloned in the intercistronic region of a bicistronic reporter plasmid. We constructed a bicistronic expression vector in which the entire 5 UTR and the mutated $N^{pro}$ region (${\Delta}386-901$, ${\Delta}415-901$ and ${\Delta}657-901$) was cloned between two reporter genes, chloramphenicol acetyltransferase (CAT) and luciferase (LUC). In vivo translation analyses showed that $N^{pro}$ region was dispensible for efficient translation. The results indicate that the $N^{pro}$ region is not essential for BVDV RNA translation and the 3' boundary of BVDV IRES is expanded into $N^{pro}$ region, suggesting that $N^{pro}$ may not play a major role in BVDV replication.

• Journal of Microbiology and Biotechnology
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• 제15권5호
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• pp.1054-1059
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• 2005

An extracellular protease was identified from Bacillus subtilis KCCM 10257 by N-terminal sequencing and mass spectral analysis. The molecular mass of the extracellular protease was estimated to be 28 kDa by SDS-PAGE. Sequencing of the N-terminal of the protease revealed the sequence of A(G,S,R)QXVPYG(A)V(P,L)SQ. The N-terminal sequence exhibited close similarity to the sequence of other proteases from Bacillus sp. A mass list of the monoisotopic peaks in the MALDI-TOF spectrum was searched after peptide fragmentation of the protease. Six peptide sequences exhibiting monoisotopic masses of 1,276.61, 1,513.67, 1,652.81, 1,661.83, 1,252.61, and 1,033.46 were observed from the fragmented protease. These monisotopic masses corresponded to the lytic enzyme L27 from Bacillus subtilis 168, and the Mowse score was found to be 75. A doubly charged Top product (MS) at a m/z of 517.3 exhibiting a molecular mass of 1034.6 was further analyzed by de novo sequencing using a PE Sciex QSTAR Hybrid Quadropole-TOF (MS/MS) mass spectrometer. MS/MS spectra of the Top product (MS) at a m/z of 517.3 obtained from the fragmented peptide mixture of protease with Q-star contained the b-ion series of 114.2, 171.2, 286.2, 357.2, 504.2, 667.4, 830.1, and 887.1 and y-ion series of 147.5, 204.2, 367.2, 530.3, 677.4, 748.4, 863.4, and 920.5. The sequence of analyzed peptide ion was identified as LGDAFYYG from the b- and y-ion series by de novo sequencing and corresponded to the results from the MALDI-TOF spectrum. From these results the extracellular protease from Bacillus subtilis KCCM 10257 was successfully identified with the lytic enzyme L27 from Bacillus subtilis 168.

• 한국식품영양과학회지
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• 제22권6호
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• pp.811-814
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• 1993

Sarcodon aspratus(Berk.) S. Ito에서 분리정제한 단백질가수분해효소의 특성을 조사하였다. 이 효소는 당을 2.1% 함유하고 있었다. Rose bengal, N-bromo succinimide, phenyl methyl sulfonyl fluoride(PMSF)와 같은 화학적 수식 시약에 효소 활성이 저해되었으며, 1차 반응속도론적 불활성 mode를 가지므로 활성 부위는 tryptophan과 serine으로 추정된다. N-말단에서 21번째 잔기까지의 아미노산 배열은 V-T-T-K-Q-T-N-A-P-W-G-L-G-N-I-S-T-T-N-K-L-으로 동정되었다.

• Journal of Microbiology
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• 제33권2호
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• pp.103-108
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• 1995

Streptomyces fradiae protease inhibitor (SFI) was purified effectively by preparative isoelectric focusing and hydroxyapatite chromatography. The molecular weight of SFI was estimated to be 1.7 kDa by SDS-PAGE and 1.8 kDa by molecular sieving HPLC. One hundred and sixty amino acid residues were determined from which molecular weight of SFI was calculated to be 17.054 Da and carbohydrate residue was not detected. SFI was calculated to be 17,064 Da and carbohydrate residue was not detected. SFI was a monomeric protein with two reactive sits, of which isoelectric point was pH 4.1. N-terminal amino acid sequence of SFI had homology with SSI (Streptomyces subsilisin inhibitor) and other protease inhibitors produced by Streptomyces.

• BMB Reports
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• 제37권5호
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• pp.574-581
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• 2004

The full-length cDNA of the lumbrokinase fraction 6 (F6) protease gene of Lumbricus rubellus was amplified using an mRNA template, sequenced and expressed in E. coli cells. The F6 protease gene consisted of pro- and mature sequences by gene sequence analysis, and the protease was translated and modified into active mature polypeptide by N-terminal amino acid sequence analysis of the F6 protease. The pro-region of F6 protease consisted of the 44 residues from methionine-1 to lysine-44, and the mature polypeptide sequence (239 amino acid residues and one stop codon; 720 bp) started from isoleucine-45 and continued to the terminal residue. F6 protease gene clones having pro-mature sequence and mature sequence produced inclusion bodies in E. coli cells. When inclusion bodies were orally administrated rats, generated thrombus weight in the rat' venous was reduced by approximately 60% versus controls. When the inclusion bodies were solubilized in pepsin and/or trypsin solutions, the solubilized enzymes showed hemolytic activity in vitro. It was concluded the F6 protease has hemolytic activity, and that it is composed of pro- and mature regions.

• 한국균학회지
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• 제33권2호
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• pp.69-74
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• 2005

민자주방망이버섯으로부터 분리한 혈전용해효소의 비활성은 22.78 U/mg 이었으며, fibrin를 직접 용해하는 fibrinolytic enzyme 이었다. 15번째까지 N-terminal amino acid 서열 분석 결과, Tyr-Pro-Ser-Pro-Ser-His-Gln-Thr-Ala-Val-Asn-Ala-Ile-Ile-X로 지금까지 발표되지 않은 새로운 효소였다. 분자량은 34 KDa 이고 pH 7.0 부터 pH 9.5의 넓은 영역에서 높은 활성을 나타내는 alkaline protease 였으며, $55^{\circ}C$에서 가장 큰 활성을 보이는 이 효소는 serine protease 저해제인 phenylmethylsulfonyl fluoride를 첨가한 경우 효소의 활성이 전혀 나타나지 않는 것으로 미루어 높은 혈전용해 활성을 갖는 새로운 serine protease로 생각된다. 또한 $Hg^{2+}$의 금속이온을 첨가한 경우에도 효소의 활성은 완전히 사라졌다.