Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: A. MMP-1/TIMP-1 complex formation abolished MMP-1 protease activity as assessed by fluorescent substrate assay. Each form of MMP-1 (20 nM) was incubated with 200 µM of quenched fluorescent substrate. Fluorescence was measured every 20 seconds for 2 hours. All measurements were controlled for by background subtraction and then the relative fluorescence intensity was averaged across all replicates (n = 3). proMMP-1 (orange), active MMP-1 (blue), and MMP-1/TIMP-1 complex (gray). Inset. SDS PAGE analysis of proMMP-1 under non-reducing conditions. Lane 1, Standards, Lane 2, proMMP-1. B. Purified LRP1 was immobilized on a CM5 sensor chip and 75 nM proMMP-1 was injected in the absence (blue lines) or presence (orange lines) of 3 mM EDTA. C. RAP (1 μM, green) was injected (first arrow) on an LRP1-coated CM5 sensor chip (green) followed by either a co-injection (second arrow) of 75 nM proMMP-1 and 1 μM RAP (orange), a co-injection of buffer and 1 μM RAP (gray), or another injection of 1 μM RAP (green). These traces were compared to an injection of 75 nM proMMP-1 (blue) in the absence of RAP. D-E. Purified LRP1 was immobilized on a CM5 sensor chip and active MMP-1 (D) or MMP-1/TIMP-1 complex (E) were injected in the absence (blue lines) or presence (orange lines) of 3 mM EDTA. The data shown is a representative experiment from three independent experiments that were performed.

Article Snippet: Proteins Human His-tagged proMMP-1 protein (Cat#: 10532-H08H, Uniprot entry P03956) and recombinant human MMP-3 catalytic domain (MMP-3cd) protein (Cat#: 10467-HNAE, Uniprot entry P08254) were purchased from Sino Biological.

Techniques: Activity Assay, Incubation, Fluorescence, SDS Page, Purification, Injection

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: Lysine residues on proMMP-1 or TIMP-1 were alkylated (Alk MMP-1 or Alk TIMP-1) by incubation with a 50-fold molar excess of Sulfo-NHS-acetate at 4°C for 2 hours. The alkylated proteins were then dialyzed into HBS + 1 mM CaCl2. Alk or unmodified proMMP-1 was then activated and complexed with either unmodified TIMP-1 or Alk TIMP-1. The experiment was repeated 3 times. (A) Activity for each MMP-1 species or complex was determined by fluorescent substrate assay using either 20 nM of unmodified or 40 nM of alkylated species or complex. For each replicate, background hydrolysis of the substrate was subtracted and the rate determined from the slope of the linear regression of the data. (B-E) LRP1 was immobilized on a CM5 chip and 75 nM of unmodified (blue) or 100 nM alkylated (orange) MMP-1 or TIMP-1 species or complex were injected over the chip. The data shown is a representative experiment from three independent experiments that were performed.

Article Snippet: Proteins Human His-tagged proMMP-1 protein (Cat#: 10532-H08H, Uniprot entry P03956) and recombinant human MMP-3 catalytic domain (MMP-3cd) protein (Cat#: 10467-HNAE, Uniprot entry P08254) were purchased from Sino Biological.

Techniques: Incubation, Activity Assay, Injection

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: (A) Schematic of the bivalent binding model used to fit the data. In this model, the MMP-1 ligand contains two regions (a-b) that interact with two LDLa ligand binding repeats on LRP1 (AB). The first region on MMP-1 (a) docks into an LDLa repeat (A) to form the initial complex. Then, the second region on MMP-1 (b) docks into the remaining LDLa repeat (B) to form the bivalent complex. (B) Increasing concentrations (4.7, 9.4, 18.7, 37.5, 75, and 150 nM) of proMMP-1 were injected over the LRP1-coated surface. (C) Increasing concentrations (4.7, 9.4, 18.7, 37.5, 75, and 150 nM) of active MMP-1 were injected over the LRP1-coated surface. (D) Increasing concentrations (2.3, 4.7, 9.4, 18.7, 37.5, 75, and 150 nM) of MMP-1/TIMP-1 complex were injected over the LRP1-coated surface. Fits of the experimental data (black lines) to a bivalent binding model are shown as blue lines. The data shown is a representative experiment from three independent experiments that were performed.

Article Snippet: Proteins Human His-tagged proMMP-1 protein (Cat#: 10532-H08H, Uniprot entry P03956) and recombinant human MMP-3 catalytic domain (MMP-3cd) protein (Cat#: 10467-HNAE, Uniprot entry P08254) were purchased from Sino Biological.

Techniques: Binding Assay, Ligand Binding Assay, Injection

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: hAoSMCs were plated at 7.2 × 10⁴ cells per well in a 12-well plate and were incubated with 125I-labeled proMMP-1 (25 nM) for 24 hours at 37°C in the absence or presence of RAP (2.5 μM) or the LRP1-specific polyclonal antibody R2629 (300 mg/mL). Following incubation, the amount of proMMP-1 internalized (A) degraded (B) or located on the cell surface (C) was measured. All data are plotted as mean ± SEM (n=3). Statistical significance was determined by one-way ANOVA with post-hoc Tukey’s test (*p<0.05, **p<0.01, ***p<0.0001).

Article Snippet: Proteins Human His-tagged proMMP-1 protein (Cat#: 10532-H08H, Uniprot entry P03956) and recombinant human MMP-3 catalytic domain (MMP-3cd) protein (Cat#: 10467-HNAE, Uniprot entry P08254) were purchased from Sino Biological.

Techniques: Incubation, Labeling

Journal: Biochemistry

Article Title: High-affinity binding of LDL receptor–related protein 1 to matrix metalloprotease 1 requires protease:inhibitor complex formation

doi: 10.1021/acs.biochem.0c00442

Figure Lengend Snippet: Recombinant LRP1 fragments of either Cluster II, III, or IV were immobilized to three individual flow cells of a CM5 sensor chip using an amine-reactive coupling process. SPR experiments tested binding of proMMP-1 (A) or TIMP-1 (B) to Cluster II (orange), III (blue), and IV (gray) with increasing concentrations of ligand (proMMP-1: 0.29, 0.58, 1.17, 2.34, 4.68, 9.38, 18.75, 37.5, 75, and 150 nM; TIMP-1: 0.58, 1.17, 2.34, 4.68, 9.38, 18.75, 37.5, 75, 150, and 300 nM). At each concentration, the binding association curves were fit to a pseudo-first order process. Req was estimated as the maximum number of response units at equilibrium for each concentration. Shown are the plots of Req versus ligand concentration. Data is plotted as mean ± SEM (n=3). The data were normalized to the amount of cluster coated on the CM5 sensor chip. GraphPad Prism 8.0 software was used to fit the data to the specific binding non-linear regression model to determine the KD of each ligand for each cluster. All calculated KD values are reported in Table 3.

Article Snippet: Proteins Human His-tagged proMMP-1 protein (Cat#: 10532-H08H, Uniprot entry P03956) and recombinant human MMP-3 catalytic domain (MMP-3cd) protein (Cat#: 10467-HNAE, Uniprot entry P08254) were purchased from Sino Biological.

Techniques: Recombinant, Binding Assay, Concentration Assay, Software

Journal: Clinical and Experimental Immunology

Article Title: Chronic exposure of interleukin‐13 suppress the induction of matrix metalloproteinase‐1 by tumour necrosis factor α in normal and scleroderma dermal fibroblasts through protein kinase B/Akt

doi: 10.1111/cei.13045

Figure Lengend Snippet: Dose‐dependent effect of interleukin (IL)‐13 on production of matrix metalloproteinase‐1 (MMP‐1) protein in dermal fibroblasts stimulated with tumour necrosis factor (TNF)‐α. Normal fibroblast cell lines: (a,b,c) and scleroderma lines (d,e) were pretreated with various concentrations of IL‐13 or IL‐4 before stimulation with TNF‐α. Data are expressed as the mean ± standard error of the mean (s.e.m.) and values (MMP‐1/cell) are plotted as a percentage of that seen for cultures stimulated with TNF‐ α. P‐values were determined by analysis of variance (anova) or Student's t‐test where P < 0·05 was considered significant. Experiments were repeated to confirm results.

Article Snippet: Analysis of MMP‐1 protein expression Supernatants from cultured fibroblasts were analysed for MMP‐1 using human MMP‐1 ELISA kit (R&D Systems) and normalized to cell number as determined by CyQuant assay.

Techniques:

Journal: Clinical and Experimental Immunology

Article Title: Chronic exposure of interleukin‐13 suppress the induction of matrix metalloproteinase‐1 by tumour necrosis factor α in normal and scleroderma dermal fibroblasts through protein kinase B/Akt

doi: 10.1111/cei.13045

Figure Lengend Snippet: Suppressive effect of long‐term incubation of interleukin (IL)‐13 on production of matrix metalloproteinase‐1 (MMP‐1) protein in response to tumour necrosis factor (TNF)‐α stimulation in normal dermal fibroblasts. Fibroblast lines (n = 8) were treated with vehicle [Dulbecco's modified Eagle's medium (DMEM) + 10% fetal calf serum (FCS) + phosphate‐buffered saline (PBS) with 0·1% bovine serum albumin (BSA) (vehicle for IL‐13 and TNF‐ α)] or IL‐13 2·5 ng/ml for 14 days prior to stimulation with TNF‐α for enzyme‐linked immunosorbent assay (ELISA) analysis of culture supernatants. Experiments were run in triplicate and expressed as percentage of that seen for cultures stimulated with TNF‐α. P‐values determined by Student's t‐test. P < 0·05 represents significance.

Article Snippet: Analysis of MMP‐1 protein expression Supernatants from cultured fibroblasts were analysed for MMP‐1 using human MMP‐1 ELISA kit (R&D Systems) and normalized to cell number as determined by CyQuant assay.

Techniques: Incubation, Modification, Saline, Enzyme-linked Immunosorbent Assay

Journal: Clinical and Experimental Immunology

Article Title: Chronic exposure of interleukin‐13 suppress the induction of matrix metalloproteinase‐1 by tumour necrosis factor α in normal and scleroderma dermal fibroblasts through protein kinase B/Akt

doi: 10.1111/cei.13045

Figure Lengend Snippet: Expression of matrix metalloproteinase‐1 (MMP‐1). Polymerase chain reaction (PCR) analyses of MMP‐1 mRNA in dermal fibroblasts after a 14‐day culture with recombinant interleukin (IL)‐13 followed by stimulation with tumour necrosis factor (TNF)‐α in normal dermal fibroblast lines 3 (a) and 6 (b). Data are expressed as standard error of the mean (s.e.m.) and plotted as relative MMP‐1. P‐values were determined by analysis of variance (anova) and compared with the value in TNF‐α‐treated cells.

Article Snippet: Analysis of MMP‐1 protein expression Supernatants from cultured fibroblasts were analysed for MMP‐1 using human MMP‐1 ELISA kit (R&D Systems) and normalized to cell number as determined by CyQuant assay.

Techniques: Expressing, Polymerase Chain Reaction, Recombinant

Journal: Clinical and Experimental Immunology

Article Title: Chronic exposure of interleukin‐13 suppress the induction of matrix metalloproteinase‐1 by tumour necrosis factor α in normal and scleroderma dermal fibroblasts through protein kinase B/Akt

doi: 10.1111/cei.13045

Figure Lengend Snippet: Effect of protein kinase B (Akt) inhibitor VIII on interleukin (IL)‐13‐induced alterations of matrix metalloproteinase‐1 (MMP‐1) protein expression in the response to tumour necrosis factor (TNF)‐α in dermal fibroblasts. Fibroblast lines from normal healthy skin (top row); and involved scleroderma skin (bottom row) were pretreated with 5 μM of Akt inhibitor VIII before stimulation with TNF‐α with or without IL‐13 and analysed for MMP‐1 by enzyme‐linked immunosorbent assay (ELISA). Data are expressed as standard error of the mean (s.e.m.) and plotted as a percentage of the value of the TNF‐treated cells. P‐values were determined by analysis of variance (anova). P < 0·05 compared with the value in TNF‐α and IL‐13‐treated cells.

Article Snippet: Analysis of MMP‐1 protein expression Supernatants from cultured fibroblasts were analysed for MMP‐1 using human MMP‐1 ELISA kit (R&D Systems) and normalized to cell number as determined by CyQuant assay.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Clinical and Experimental Immunology

Article Title: Chronic exposure of interleukin‐13 suppress the induction of matrix metalloproteinase‐1 by tumour necrosis factor α in normal and scleroderma dermal fibroblasts through protein kinase B/Akt

doi: 10.1111/cei.13045

Figure Lengend Snippet: Quantitative effect of interleukin (IL)‐13 and protein kinase B (Akt) inhibitor on the expression of matrix metalloproteinase‐1 (MMP‐1) in normal and scleroderma‐involved dermal fibroblasts in the presence of tumour necrosis factor (TNF)‐α. Three normal fibroblast lines and three scleroderma fibroblasts lines from involved skin were either treated alone with IL‐13 or pretreated with Akt‐inh and IL‐13 followed by stimulation with TNF‐α. Supernatants were analysed by enzyme‐linked immunosorbent assay (ELISA) for MMP‐1 production. Data are expressed as stimulation index by dividing MMP‐1 of treated fibroblast by the MMP‐1 of the vehicle treated fibroblast. P‐values were determined by analysis of variance (anova) where P < 0·05 is significant; *P < 0·05 when compared to normal fibroblasts treated with IL‐13 and TNF‐α; **P < 0·05 when compared to systemic sclerosis (SSc)‐involved fibroblasts treated with IL‐13 and TNF‐α.

Article Snippet: Analysis of MMP‐1 protein expression Supernatants from cultured fibroblasts were analysed for MMP‐1 using human MMP‐1 ELISA kit (R&D Systems) and normalized to cell number as determined by CyQuant assay.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay