Selectivity of propeptide-enzyme interaction in cathepsin L-like cysteine proteases

2009 ◽  
Vol 390 (2) ◽  
pp. 167-174 ◽  
Author(s):  
Klaus Schilling ◽  
Alexandra Körner ◽  
Saskia Sehmisch ◽  
Annett Kreusch ◽  
Ramona Kleint ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Tertiary Structure ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Systematic Investigation ◽  
Cross Reactivity ◽  
X Ray ◽  
Chaperone Function ◽  
Inhibitory Function ◽  
Enzyme Unit

Abstract Cathepsin L-like endopeptidases of the papain family are synthesized as proenzymes. N-terminal proregions are essential for folding and latency of the enzyme unit. While selectivity has been reported for the inhibitory function of papain-family propeptides, there is no systematic investigation of the selectivity of their chaperone-like function to date. The chaperone-like cross-reactivity between the cathepsins S, K, and L were thoroughly quantified in trans-experiments, i.e., with isolated propeptides and mature enzymes, and compared to the inhibitory cross-reactivity. The three endopeptidases have been chosen due to only minimal evolutionary distance and nearly identical X-ray structures of their zymogenes. The intramolecular chaperone function of the proregion was found to be more selective than the inhibitory activity and significant differences were found between the selectivity profiles, underlining the assumption that the inhibitory and the chaperone-like propeptide functions are autonomous. Considering the data published on the intramolecular chaperone-like propeptide function within other protease classes as well, our data suggest that intrinsically structured propeptides are more selective than intrinsically unstructured propeptides, i.e., those adopting tertiary structure elements only in complex with their maternal enzyme.

scholarly journals Bioinformatics analysis and characterization of a secretory cystatin from Thelohanellus kitauei

2020 ◽  
Author(s):  
Fengli Zhang ◽  
Yalin Yang ◽  
Chenchen Gao ◽  
Yuanyuan Yao ◽  
Rui Xia ◽  
...  
Keyword(s):  
Binding Energy ◽  
Inhibitory Activity ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Economic Losses ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Multiple Sequence ◽  
Cystatin Gene

Abstract Thelohanellus kitauei , is a group of obligate parasitic Myxozoans, which causes intestinal giant-cystic disease of common carp ( Cyprinus carpio) and has resulted in significant economic losses in carp farms. Cystatin secreted by parasites can regulate the immune response of host to facilitate parasite’s survival. In this study, the secretory TK-cystatin gene, encoding a protein of 120 amino acid residues (13.65 kDa), was cloned from T. kitauei genome. Phylogenetic analysis showed that TK-cystatin gene is closely related to the cystatin-A from Hydra vulgaris . Multiple sequence alignment revealed that TK-cystatin had three conserved motifs: N-terminal G 19 G 20 , Q 73 VVAG 77 , and C-terminal L 102 P 103 . Molecular docking between TK-cystatin and three cysteine proteases showed a lower binding energy (-13 kal/mol) with cathepsin L whereas a higher binding energy (-8.6 kal/mol) with cathepsin B. TK-cystatin gene was expressed in Escherichia coli . Activity assays revealed that TK-cystatin has stronger inhibitory activity on endopeptidases (papain and cathepsin L) and weaker inhibitory activity on exopeptidase (cathepsin B). TK-cystatin was stable under the condition of acidity or alkalinity or below 57 °C. This study laid a foundation for the design and development of the anti-TK-cystatin vaccine in carp culture in the future.

scholarly journals Bioinformatics analysis and characterization of a secretory cystatin from Thelohanellus kitauei

2020 ◽  
Author(s):  
Fengli Zhang ◽  
Yalin Yang ◽  
Chenchen Gao ◽  
Yuanyuan Yao ◽  
Rui Xia ◽  
...  
Keyword(s):  
Binding Energy ◽  
Inhibitory Activity ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Economic Losses ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Multiple Sequence ◽  
Cystatin Gene

Abstract Thelohanellus kitauei, is a group of obligate parasitic Myxozoans, which causes intestinal giant-cystic disease of common carp (Cyprinus carpio) and has resulted in significant economic losses in carp farms. Cystatin secreted by parasites can regulate the immune response of host to facilitate parasite’s survival. In this study, the secretory TK-cystatin gene, encoding a protein of 120 amino acid residues (13.65 kDa), was cloned from T. kitauei genome. Phylogenetic analysis showed that TK-cystatin gene is closely related to the cystatin-A from Hydra vulgaris. Multiple sequence alignment revealed that TK-cystatin had three conserved motifs: N-terminal G19G20, Q73VVAG77, and C-terminal L102P103. Molecular docking between TK-cystatin and three cysteine proteases showed a lower binding energy (-13 kal/mol) with cathepsin L whereas a higher binding energy (-8.6 kal/mol) with cathepsin B. TK-cystatin gene was expressed in Escherichia coli. Activity assays revealed that TK-cystatin has stronger inhibitory activity on endopeptidases (papain and cathepsin L) and weaker inhibitory activity on exopeptidase (cathepsin B). TK-cystatin was stable under the condition of acidity or alkalinity or below 57 °C. This study laid a foundation for the design and development of the anti-TK-cystatin vaccine in carp culture in the future.

1-Methylphenanthro[3,4-b]thiophene: Determination of the tertiary structure in solution and in the crystalline state by NMR spectroscopy and X-ray diffraction

1985 ◽  
Vol 22 (2) ◽  
pp. 545-553 ◽  
Author(s):  
M. J. Musmar ◽  
Gary E. Martin ◽  
Robert T. Gampe ◽  
Vincent M. Lynch ◽  
Stanley H. Simonsen ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Crystalline State ◽  
Tertiary Structure ◽  
X Ray Diffraction ◽  
X Ray ◽  
Structure In Solution

scholarly journals Evolutionary Conservation of Reactions in Translation

2002 ◽  
Vol 66 (3) ◽  
pp. 460-485 ◽  
Author(s):  
M. Clelia Ganoza ◽  
Michael C. Kiel ◽  
Hiroyuki Aoki
Keyword(s):  
Molecular Mechanisms ◽  
Tertiary Structure ◽  
Evolutionary Conservation ◽  
Initiation Factor ◽  
Structural Studies ◽  
X Ray Diffraction ◽  
X Ray ◽  
Common Features ◽  
New Information ◽  
And Function

SUMMARY Current X-ray diffraction and cryoelectron microscopic data of ribosomes of eubacteria have shed considerable light on the molecular mechanisms of translation. Structural studies of the protein factors that activate ribosomes also point to many common features in the primary sequence and tertiary structure of these proteins. The reconstitution of the complex apparatus of translation has also revealed new information important to the mechanisms. Surprisingly, the latter approach has uncovered a number of proteins whose sequence and/or structure and function are conserved in all cells, indicating that the mechanisms are indeed conserved. The possible mechanisms of a new initiation factor and two elongation factors are discussed in this context.

scholarly journals Molecular Characterization of a Novel Cathepsin L in Macrobrachium nipponense and Its Function in Ovary Maturation

2022 ◽  
Vol 12 ◽  
Author(s):  
Sufei Jiang ◽  
Yiwei Xiong ◽  
Wenyi Zhang ◽  
Junpeng Zhu ◽  
Dan Cheng ◽  
...  
Keyword(s):  
Cathepsin L ◽  
Cysteine Proteases ◽  
Macrobrachium Nipponense ◽  
Reading Frame ◽  
Physiological Processes ◽  
Gonad Somatic Index ◽  
Oriental River Prawn ◽  
Ovary Maturation

Cathepsin L genes, which belonged to cysteine proteases, were a series of multifunctional protease and played important roles in a lot of pathological and physiological processes. In this study, we analyzed the characteristics a cathepsin L (named Mn-CL2) in the female oriental river prawn, Macrobrachium nipponense which was involved in ovary maturation. The Mn-CL2 was1,582 bp in length, including a 978 bp open reading frame that encoded 326 amino acids. The Mn-CL2 was classified into the cathepsin L group by phylogenetic analysis. Real-time PCR (qPCR) analysis indicated that Mn-CL2 was highly expressed in the hepatopancreas and ovaries of female prawns. During the different ovarian stages, Mn-CL2 expression in the hepatopancreas and ovaries peaked before ovarian maturation. In situ hybridization studies revealed that Mn-CL2 was localized in the oocyte of the ovary. Injection of Mn-CL2 dsRNA significantly reduced the expression of vitellogenin. Changes in the gonad somatic index also confirmed the inhibitory effects of Mn-CL2 dsRNA on ovary maturation. These results suggest that Mn-CL2 has a key role in promoting ovary maturation.

A possible alternative mechanism of kinin generation in vivo by cathepsin L

2005 ◽  
Vol 386 (7) ◽  
pp. 699-704 ◽  
Author(s):  
Luciano Puzer ◽  
Juliana Vercesi ◽  
Marcio F.M. Alves ◽  
Nilana M.T. Barros ◽  
Mariana S. Araujo ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Enzyme Inhibitor ◽  
Alternative Pathway ◽  
Cathepsin L ◽  
Specific Inhibitor ◽  
Cysteine Proteases ◽  
Hypotensive Effect ◽  
Alternative Mechanism

Abstract We investigated the ability of cathepsin L to induce a hypotensive effect after intravenous injection in rats and correlated this decrease in blood pressure with kinin generation. Simultaneously with blood pressure decrease, we detected plasma kininogen depletion in the treated rats. The effect observed in vivo was abolished by pre-incubation of cathepsin L with the cysteine peptidase-specific inhibitor E-64 (1 μM) or by previous administration of the bradykinin B2 receptor antagonist JE049 (4 mg/kg). A potentiation of the hypotensive effect caused by cathepsin L was observed by previous administration of the angiotensin I-converting enzyme inhibitor captopril (5 mg/kg). In vitro studies indicated that cathepsin L excised bradykinin from the synthetic fluorogenic peptide Abz-MTSVIRRPPGFSPFRAPRV-NH2, based on the Met375–Val393 sequence of rat kininogen (Abz=o-aminobenzoic acid). In conclusion, our data indicate that in vivo cathepsin L releases a kinin-related peptide, and in vitro experiments suggest that the kinin generated is bradykinin. Although it is well known that cysteine proteases are strongly inhibited by kininogen, cathepsin L could represent an alternative pathway for kinin production in pathological processes.

A cookbook for the investigation of coordination polymers by transition metal K-edge XMCD

2021 ◽  
Vol 28 (4) ◽  
Author(s):  
Adama N'Diaye ◽  
Amélie Bordage ◽  
Lucie Nataf ◽  
François Baudelet ◽  
Thierry Moreno ◽  
...  
Keyword(s):  
Transition Metal ◽  
Coordination Polymers ◽  
Magnetic Circular Dichroism ◽  
Systematic Investigation ◽  
Experimental Conditions ◽  
X Ray ◽  
Structural Distortions ◽  
Physical Effects ◽  
Prussian Blue Analogs ◽  
Extended Discussion

In order to disentangle the physical effects at the origin of transition metal K-edge X-ray magnetic circular dichroism (XMCD) in coordination polymers and quantify small structural distortions from the intensity of these signals, a systematic investigation of Prussian blue analogs as model compounds is being conducted. Here the effects of the temperature and of the external magnetic field are tackled; none of these external parameters modify the shape of the XMCD signal but they both critically modify its intensity. The optimized experimental conditions, as well as a reliable and robust normalization procedure, could thus be determined for the study of the intrinsic parameters. Through an extended discussion on measurements on other XMCD-dedicated beamlines and for other coordination compounds, we finally provide new transition metal K-edge XMCD users with useful information to initiate and successfully carry out their projects.

scholarly journals Identification and biochemical characterisation of Acanthamoeba castellanii cysteine protease 3

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Zhixin Wang ◽  
Duo Wu ◽  
Hiroshi Tachibana ◽  
Meng Feng ◽  
Xun-jia Cheng
Keyword(s):  
Cathepsin L ◽  
Mrna Level ◽  
Acanthamoeba Castellanii ◽  
Cysteine Proteases ◽  
Hydrolytic Activity ◽  
Negative Control ◽  
Host Cells ◽  
Signalling Pathways ◽  
Corneal Epithelial ◽  
Biochemical Characterisation

Abstract Background Acanthamoeba spp. are free-living amoeba that are ubiquitously distributed in the environment. This study examines pathogenic Acanthamoeba cysteine proteases (AcCPs) belonging to the cathepsin L-family and explores the mechanism of AcCP3 interaction with host cells. Methods Six AcCP genes were amplified by polymerase chain reaction (PCR). Quantitative real-time PCR was used to analyse the relative mRNA expression of AcCPs during the encystation process and between pre- and post-reactivated trophozoites. To further verify the role of AcCP3 in these processes, AcCP3 recombinant proteins were expressed in Escherichia coli, and the hydrolytic activity of AcCP3 was determined. The influence of the AcCP3 on the hydrolytic activity of trophozoites and the toxicity of trophozoites to human corneal epithelial cells (HCECs) was examined by inhibiting AcCP3 expression using siRNA. Furthermore, the levels of p-Raf and p-Erk were examined in HCECs following coculture with AcCP3 gene knockdown trophozoites by Western blotting. Results During encystation, five out of six AcCPs exhibited decreased expression, and only AcCP6 was substantially up-regulated at the mRNA level, indicating that most AcCPs were not directly correlated to encystation. Furthermore, six AcCPs exhibited increased expression level following trophozoite reactivation with HEp-2 cells, particularly AcCP3, indicating that these AcCPs might be virulent factors. After refolding of recombinant AcCP3 protein, the 27 kDa mature protein from the 34 kDa pro-protein hydrolysed host haemoglobin, collagen and albumin and showed high activity in an acidic environment. After AcCP3 knockdown, the hydrolytic activity of trophozoite crude protein against gelatin was decreased, suggesting that these trophozoites had decreased toxicity. Compared with untreated trophozoites or negative control siRNA-treated trophozoites, AcCP3-knockdown trophozoites were less able to penetrate and damage monolayers of HCECs. Western blot analysis showed that the activation levels of the Ras/Raf/Erk/p53 signalling pathways in HCECs decreased after inhibiting the expression of trophozoite AcCP3. Conclusions AcCP6 was correlated to encystation. Furthermore, AcCP3 was a virulent factor in trophozoites and participated in the activation of the Ras/Raf/Erk/p53 signalling pathways of host cells.

scholarly journals Crystal structure and molecular docking studies of new pyrazole-4-carboxamides

2019 ◽  
Vol 25 (1) ◽  
pp. 66-72 ◽  
Author(s):  
Li Qiao ◽  
Peng-Peng Cai ◽  
Zhong-Hua Shen ◽  
Hong-Ke Wu ◽  
Cheng-Xia Tan ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Docking ◽  
Botrytis Cinerea ◽  
Inhibitory Activity ◽  
Docking Studies ◽  
Active Group ◽  
X Ray Diffraction ◽  
X Ray ◽  
Molecular Docking Studies ◽  
H Nmr

AbstractTwo pyrazol-4-carboxamides, 3-(difluoromethyl)-N-(mesitylcarbamoyl)-1-methyl-1H-pyrazole-4-carboxa-mide (7a) and 3-(difluoromethyl)-N-((3,5-dimethylphenyl) carbamoyl)-1-methyl-1H-pyrazole-4-carboxamide (7b) were synthesized and their structures were confirmed by the aid of 1H NMR and HRMS analyses. The structure of the pyrazole-4-carboxamide, 7a was also determined by X-ray diffraction. The preliminary activity results demonstrate that these two compounds exhibit good inhibitory activity against Botrytis cinerea. Further docking results indicated that the key active group is difluoromethyl pyrazole moiety.

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