scholarly journals Fibroblast and neutrophil collagenases cleave at two sites in the cartilage aggrecan interglobular domain

1993 ◽  
Vol 295 (1) ◽  
pp. 273-276 ◽  
Author(s):  
A J Fosang ◽  
K Last ◽  
V Knäuper ◽  
P J Neame ◽  
G Murphy ◽  
...  
Keyword(s):  
Human Fibroblast ◽  
Catalytic Domain ◽  
Polymorphonuclear Leucocyte ◽  
Gelatinase A ◽  
Cleavage Sites ◽  
Similar Extent ◽  
Human Sequence ◽  
Fibroblast Collagenase ◽  
Gelatinases A And B

The actions of recombinant human fibroblast collagenase (MMP1), purified polymorphonuclear leucocyte collagenase (MMP8) and their N-terminal catalytic domain fragments against cartilage aggrecan and an aggrecan G1-G2 fragment have been investigated in vitro. After activation with recombinant human stromelysin and typsin, both collagenases were able to degrade human and porcine aggrecans to a similar extent. An N-terminal G1-G2 fragment (150 kDa) was used to identify specific cleavage sites occurring within the proteinase-sensitive interglobular domain between G1 and G2. Two specific sites were found; one at an Asn341-Phe342 bond and another at Asp441-Leu442 (human sequence). This specificity of the collagenases for aggrecan G1-G2 was identical with that of the truncated metalloproteinase matrilysin (MMP7), but different from those of stromelysin (MMP3) and the gelatinases (MMP2 or gelatinase A; MMP9 or gelatinase B) which cleave at the Asn-Phe site, but not the Asp-Leu site. In addition, collagenase catalytic fragments lacking C-terminal hemopexin-like domains were tested and shown to exhibit the same specificities for the G1-G2 fragment as the full-length enzymes. Thus the specificity of the collagenases for cartilage aggrecan was not influenced by the presence or absence of the C-terminal domain. Together with our previous findings, the results show that stromelysin-1, matrilysin, gelatinases A and B and fibroblast and neutrophil collagenases cleave at a common, preferred site in the aggrecan interglobular domain, and additionally that both fibroblast and neutrophil collagenases cleave at a second site in the interglobular domain that is not available to stromelysin or gelatinases.

The influence of flavonoid compounds on the in vitro inhibition study of a human fibroblast collagenase catalytic domain expressed in E. coli

2013 ◽  
Vol 52 (1) ◽  
pp. 26-31 ◽  
Author(s):  
Thi Thanh Hanh Nguyen ◽  
Young-Hwan Moon ◽  
Young-Bae Ryu ◽  
Young-Min Kim ◽  
Seung-Hee Nam ◽  
...  
Keyword(s):  
Human Fibroblast ◽  
Catalytic Domain ◽  
Inhibition Study ◽  
E Coli ◽  
Flavonoid Compounds ◽  
Fibroblast Collagenase ◽  
In Vitro Inhibition

scholarly journals Characterization of the Phe-81 and Val-82 Human Fibroblast Collagenase Catalytic Domain Purified fromEscherichia coli

1995 ◽  
Vol 270 (38) ◽  
pp. 22507-22513 ◽  
Author(s):  
Michael R. Gehring ◽  
Brad Condon ◽  
Stephen A. Margosiak ◽  
Chen-Chen Kan
Keyword(s):  
Human Fibroblast ◽  
Catalytic Domain ◽  
Fromescherichia Coli ◽  
Fibroblast Collagenase

Structure of the catalytic domain of human fibroblast collagenase complexed with an inhibitor

1994 ◽  
Vol 1 (2) ◽  
pp. 106-110 ◽  
Author(s):  
N. Borkakoti ◽  
F.K. Winkler ◽  
D.H. Williams ◽  
A. D'Arcy ◽  
M.J. Broadhurst ◽  
...  
Keyword(s):  
Human Fibroblast ◽  
Catalytic Domain ◽  
Fibroblast Collagenase

scholarly journals Quaternary structure and biochemical properties of mycobacterial RNase E/G

2007 ◽  
Vol 403 (1) ◽  
pp. 207-215 ◽  
Author(s):  
Mirijam-Elisabeth Zeller ◽  
Agnes Csanadi ◽  
Andras Miczak ◽  
Thierry Rose ◽  
Thierry Bizebard ◽  
...  
Keyword(s):  
Quaternary Structure ◽  
Catalytic Domain ◽  
Biochemical Properties ◽  
Cleavage Sites ◽  
Rrna Processing ◽  
Rnase E ◽  
Human Pathogens ◽  
E Coli ◽  
Gram Positive Bacteria

The RNase E/G family of endoribonucleases plays the central role in numerous post-transcriptional mechanisms in Escherichia coli and, presumably, in other bacteria, including human pathogens. To learn more about specific properties of RNase E/G homologues from pathogenic Gram-positive bacteria, a polypeptide comprising the catalytic domain of Mycobacterium tuberculosis RNase E/G (MycRne) was purified and characterized in vitro. In the present study, we show that affinity-purified MycRne has a propensity to form dimers and tetramers in solution and possesses an endoribonucleolytic activity, which is dependent on the 5′-phosphorylation status of RNA. Our data also indicate that the cleavage specificities of the M. tuberculosis RNase E/G homologue and its E. coli counterpart are only moderately overlapping, and reveal a number of sequence determinants within MycRne cleavage sites that differentially affect the efficiency of cleavage. Finally, we demonstrate that, similar to E. coli RNase E, MycRne is able to cleave in an intercistronic region of the putative 9S precursor of 5S rRNA, thus suggesting a common function for RNase E/G homologues in rRNA processing.

Structural Analysis of the Catalytic Domain of Human Fibroblast Collagenase

1994 ◽  
Vol 732 (1 Inhibition of) ◽  
pp. 375-378 ◽  
Author(s):  
BRETT LOVEJOY ◽  
ANNE CLEASBY ◽  
ANNE M. HASSELL ◽  
MICHAEL A. LUTHER ◽  
DEBRA WEIGL ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Human Fibroblast ◽  
Catalytic Domain ◽  
Fibroblast Collagenase

Proteomics-Based Characterization of the Effects of MMP14 on the Protein Content of Exosomes from Corneal Fibroblasts

2020 ◽  
Vol 27 (10) ◽  
pp. 979-988
Author(s):  
Kyu-Yeon Han ◽  
Jin-Hong Chang ◽  
Dimitri T. Azar
Keyword(s):  
Protein Content ◽  
Catalytic Domain ◽  
Protein Composition ◽  
Proteomics Analysis ◽  
Knock Out ◽  
Corneal Fibroblast ◽  
Flow Cytometric ◽  
Corneal Fibroblasts ◽  
Liquid Chromatography Tandem Mass

Background: Exosomes secreted by corneal fibroblasts contain matrix metalloproteinase (MMP) 14, which is known to influence pro-MMP2 accumulation on exosomes. Accordingly, we hypothesized that the enzymatic activity of MMP14 may alter the protein content of corneal fibroblast- secreted exosomes. Objective: The aim of this study was to investigate the effects of MMP14 on the composition and biological activity of corneal fibroblast-derived exosomes. Methods: Knock out of the catalytic domain (ΔExon4) of MMP14 in corneal fibroblasts was used to determine the effect of MMP14 expression on the characteristics of fibroblast-secreted exosomes. The amount of secreted proteins and their size distribution were measured using Nano Tracking Analysis. Proteins within exosomes from wild-type (WT) and ΔExon4-deficient fibroblasts were identified by liquid chromatography-tandem mass spectrometry (MS/MS) proteomics analysis. The proteolytic effects of MMP14 were evaluated in vitro via MS identification of eliminated proteins. The biological functions of MMP14-carrying exosomes were investigated via fusion to endothelial cells and flow cytometric assays. Results: Exosomes isolated from WT and ΔExon4-deficient fibroblasts exhibited similar size distributions and morphologies, although WT fibroblasts secreted a greater amount of exosomes. The protein content, however, was higher in ΔExon4-deficient fibroblast-derived exosomes than in WT fibroblast-derived exosomes. Proteomics analysis revealed that WT-derived exosomes included proteins that regulated cell migration, and ΔExon4 fibroblast-derived exosomes contained additional proteins that were cleaved by MMP14. Conclusion: Our findings suggest that MMP14 expression influences the protein composition of exosomes secreted by corneal fibroblasts, and through those biological components, MMP14 in corneal fibroblasts derived-exosomes may regulate corneal angiogenesis.

In Silico and in Vitro Evaluation of Deamidation Effects on the Stability of the Fusion Toxin DAB389IL-2

2019 ◽  
Vol 16 (4) ◽  
pp. 307-313 ◽  
Author(s):  
Nasrin Zarkar ◽  
Mohammad Ali Nasiri Khalili ◽  
Fathollah Ahmadpour ◽  
Sirus Khodadadi ◽  
Mehdi Zeinoddini
Keyword(s):  
In Silico ◽  
Catalytic Domain ◽  
Md Simulations ◽  
Special Importance ◽  
Native Form ◽  
Vitro Experiment ◽  
In Vitro Experiment ◽  
Pharmaceutical Protein ◽  
The Stability

Background: DAB389IL-2 (Denileukin diftitox) as an immunotoxin is a targeted pharmaceutical protein and is the first immunotoxin approved by FDA. It is used for the treatment of various kinds of cancer such as CTCL lymphoma, melanoma, and Leukemia but among all of these, treatment of CTCL has special importance. DAB389IL-2 consists of two distinct parts; the catalytic domain of Diphtheria Toxin (DT) that genetically fused to the whole IL-2. Deamidation is the most important reaction for chemical instability of proteins occurs during manufacture and storage. Deamidation of asparagine residues occurs at a higher rate than glutamine residues. The structure of proteins, temperature and pH are the most important factors that influence the rate of deamidation. Methods: Since there is not any information about deamidation of DAB389IL-2, we studied in silico deamidation by Molecular Dynamic (MD) simulations using GROMACS software. The 3D model of fusion protein DAB389IL-2 was used as a template for deamidation. Then, the stability of deamidated and native form of the drug was calculated. Results: The results of MD simulations were showed that the deamidated form of DAB389IL-2 is more unstable than the normal form. Also, deamidation was carried by incubating DAB389IL-2, 0.3 mg/ml in ammonium hydrogen carbonate for 24 h at 37o C in order to in vitro experiment. Conclusion: The results of in vitro experiment were confirmed outcomes of in silico study. In silico and in vitro experiments were demonstrated that DAB389IL-2 is unstable in deamidated form.

scholarly journals Chemically defined and xeno-free culture condition for human extended pluripotent stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bei Liu ◽  
Shi Chen ◽  
Yaxing Xu ◽  
Yulin Lyu ◽  
Jinlin Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Culture Condition ◽  
Human Fibroblast ◽  
Culture System ◽  
Disease Modeling ◽  
Directed Differentiation

AbstractExtended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xeno-free culture condition has significantly limited their applications. Here, we report a chemically defined and xeno-free culture system for culturing and deriving human EPS cells in vitro. Xeno-free human EPS cells can be long-term and genetically stably maintained in vitro, as well as preserve their embryonic and extraembryonic developmental potentials. Furthermore, the xeno-free culturing system also permits efficient derivation of human EPS cells from human fibroblast through reprogramming. Our study could have broad utility in future applications of human EPS cells in biomedicine.

scholarly journals Chemical Validation of Phosphodiesterase C as a Chemotherapeutic Target in Trypanosoma cruzi, the Etiological Agent of Chagas' Disease

2010 ◽  
Vol 54 (9) ◽  
pp. 3738-3745 ◽  
Author(s):  
Sharon King-Keller ◽  
Minyong Li ◽  
Alyssa Smith ◽  
Shilong Zheng ◽  
Gurpreet Kaur ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Catalytic Domain ◽  
Polypeptide Chain ◽  
Inflammatory Diseases ◽  
New Drugs ◽  
Volume Recovery ◽  
Chronic Pulmonary Diseases ◽  
Potential Inhibitors

ABSTRACT Trypanosoma cruzi phosphodiesterase (PDE) C (TcrPDEC), a novel and rather unusual PDE in which, unlike all other class I PDEs, the catalytic domain is localized in the middle of the polypeptide chain, is able to hydrolyze cyclic GMP (cGMP), although it prefers cyclic AMP (cAMP), and has a FYVE-type domain in its N-terminal region (S. Kunz et al., FEBS J. 272:6412-6422, 2005). TcrPDEC shows homology to the mammalian PDE4 family members. PDE4 inhibitors are currently under development for the treatment of inflammatory diseases, such as asthma, chronic pulmonary diseases, and psoriasis, and for treating depression and serving as cognitive enhancers. We therefore tested a number of compounds originally synthesized as potential PDE4 inhibitors on T. cruzi amastigote growth, and we obtained several useful hits. We then conducted homology modeling of T. cruzi PDEC and identified other compounds as potential inhibitors through virtual screening. Testing of these compounds against amastigote growth and recombinant TcrPDEC activity resulted in several potent inhibitors. The most-potent inhibitors were found to increase the cellular concentration of cAMP. Preincubation of cells in the presence of one of these compounds stimulated volume recovery after hyposmotic stress, in agreement with their TcrPDEC inhibitory activity in vitro, providing chemical validation of this target. The compounds found could be useful tools in the study of osmoregulation in T. cruzi. In addition, their further optimization could result in the development of new drugs against Chagas' disease and other trypanosomiases.

scholarly journals Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan

Microbiology ◽  
2014 ◽  
Vol 160 (10) ◽  
pp. 2157-2169 ◽  
Author(s):  
Sudarson Sundarrajan ◽  
Junjappa Raghupatil ◽  
Aradhana Vipra ◽  
Nagalakshmi Narasimhaswamy ◽  
Sanjeev Saravanan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Mechanism Of Action ◽  
Catalytic Domain ◽  
Antibiotic Sensitivity ◽  
High Sensitivity ◽  
Common Mechanism ◽  
Cross Bridge ◽  
Sensitivity Pattern

P128 is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of resistance to P128, we isolated mutants of Staphylococcus spp., including meticillin-resistant Staphylococcus aureus (MRSA), resistant to P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the mutants tested was found to show reduced virulence in animal models of S. aureus septicaemia suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed that the mutants derived from MRSA strains had become sensitive to meticillin and other β-lactams. Interestingly, the mutant cells were resistant to the lytic action of phage K, although the phage was able to adsorb to these cells. Sequencing of the femA gene of three P128-resistant mutants showed either a truncation or deletion in femA, suggesting that improper cross-bridge formation in S. aureus could be causing resistance to P128. Using glutathione S-transferase (GST) fusion peptides as substrates it was found that both P128 and Lys16 were capable of cleaving a pentaglycine sequence, suggesting that P128 might be killing S. aureus by cleaving the pentaglycine cross-bridge of peptidoglycan. Moreover, peptides corresponding to the reported cross-bridge of Staphylococcus haemolyticus (GGSGG, AGSGG), which were not cleaved by lysostaphin, were cleaved efficiently by P128. This was also reflected in high sensitivity of S. haemolyticus to P128. This showed that in spite of sharing a common mechanism of action with lysostaphin, P128 has unique properties, which allow it to act on certain lysostaphin-resistant Staphylococcus strains.

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