human prommp-1  (Calbiotech)

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: The Journal of Biological Chemistry

Article Title: Discovery of a highly selective chemical inhibitor of matrix metalloproteinase-9 (MMP-9) that allosterically inhibits zymogen activation

doi: 10.1074/jbc.M117.806075

Figure Lengend Snippet: JNJ0966 inhibited activation of proMMP-9 but exhibited no effect on catalytic activity or maturation of other MMPs. A, proMMP-9 activation assay utilizing the fluorescent substrate DQ-gelatin. proMMP-9, catMMP-3, or a combination of the two in the presence or absence of 10 μm JNJ0966 was incubated together then with DQ-gelatin. proMMP-9 activated by catMMP-3 exhibited significant increases in activity as compared with either enzyme alone, and this was significantly inhibited by JNJ0966 (n = 6). RFU, relative fluorescence units; ****, p < 0.0001, one-way ANOVA with Bonferroni multiple-comparison post-test. B, molecular structure of JNJ0966 and molecular weight (MW). C, proMMP-9 activation assay characterizing JNJ0966 concentration response with catMMP-3 activation (IC50 = 440 nm). D and E, activity assay in which concentration responses of JNJ0966 and GM6001 were incubated with catMMP-3 (D) or catMMP-9 (E). GM6001 inhibited catMMP-3 (D, IC50 = 7.2 nm) and catMMP-9 (E, IC50 = 0.5 nm), but JNJ0966 had no effect at any concentration (n = 6). F, activation assay on proMMP-1, proMMP-2, proMMP-3, and proMMP-9 activated by trypsin or catMMP-14, as indicated, in the presence (black bars) or absence (white bars) of 10 μm JNJ0966. JNJ0966 only significantly inhibited the activation of proMMP-9 by trypsin. Data were normalized to 100% for each enzyme maximal activity (n = 6; ****, p < 0.001, two-tailed t test). G, proMMP-9 activation assay characterizing JNJ0966 concentration response with trypsin activation (IC50 = 429 nm). H, HT1080 cellular invasion assay demonstrating the concentration-response curves of doxycycline (green squares; IC50 = 21 μm), GM6001 (blue triangles; IC50 = 1.4 μm), and JNJ0966 (red circle; IC50 = 1.0 μm) inhibiting cellular transmigration across a MatrigelTM layer (n = 4). I, representative images from the bottom side of transwells from the indicated treatments illustrate calcein AM–labeled cells that have migrated through Matrigel to the bottom filter insert layer. In all graphs (A, C, D, E, F, and G), data are presented as means ± S.D. (error bars). All curves are fit by nonlinear regression.

Article Snippet: Human proMMP-1, proMMP-2, proMMP-3, and active MMP-14 were purchased from EMD Millipore (Billerica, MA).

Techniques: Activation Assay, Activity Assay, Incubation, Fluorescence, Molecular Weight, Concentration Assay, Two Tailed Test, Invasion Assay, Transmigration Assay, Labeling