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: 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: 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: 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: 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: 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: Scientific reports

Article Title: Discovery of an enzyme and substrate selective inhibitor of ADAM10 using an exosite-binding glycosylated substrate.

doi: 10.1038/s41598-016-0013-4

Figure Lengend Snippet: Figure 1. ADAM10 HTS assay. (A) Structure of glycosylated ADAM10 and ADAM17 uHTS substrate. (B) Pharmacological control (marimastat, structure shown on the graph) study with ADAM10 in 384 and 1536 well plate formats. (C) Scatter plot of ADAM10 and ADAM17 primary uHTS campaign. Green dots–test compounds, blue dots–100% inhibition control (marimastat at 10 µM), red dots–0% inhibition control (wells with uninhibited enzymatic reaction). (D) Examples of structures and dose response curves of HTS hits selected for follow-up studies. Hydroxamate is shown in a blue circle. Y-axis-%inhibition, X-axis–molar concentration of inhibitor.

Article Snippet: MMP-1, MMP-2, MMP-8, MMP-9, MMP-10, MMP-13, MMP-14, ADAM10, and ADAM17 were purchased from R&D Systems (cat # 901-MP, 902-MP, 908-MP, 911-MP, 910-MP, 511-MM, 918-MP, 936-AD, and 930-ADB, respectively).

Techniques: HTS Assay, Control, Inhibition, Concentration Assay