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: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

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: SPR experiments examined binding of TIMP-1 (A), TIMP-2 (B), TIMP-3 (C), or TIMP-4 (D) to full length LRP1. Concentrations used were: 0.58, 1.17, 2.34, 4.68, 9.38, 18.75, 37.5, 75, and 150 nM for TIMP-1; 4.68, 9.38, 18.75, 37.5, 75, and 150 nM for TIMP-2; 0.29, 0.58, 1.17, 2.34, 4.68, 9.38, 18.75, 37.5, 75, and 150 nM for TIMP-3 and TIMP-4. At each concentration, the binding association curves were fit to a pseudo-first order process to determine Req. Req values were then plotted versus concentration, and the data fit to a binding isotherm using GraphPad Prism 8.0 software. To normalize the data from different experiments, Req/Rmax was plotted versus ligand concentrations, and the data plotted shows mean ± SEM (n=3).

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Binding Assay, Concentration Assay, Software

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: Equilibrium binding constants for the interaction of TIMP-1, TIMP-2, TIMP-3, and TIMP-4 with LRP1. Equilibrium binding constants were calculated from equilibrium SPR measurements, in which Req was determined by fitting the association data to a pseudo-first-order process. Req was then plotted versus concentration and analyzed by non-linear regression analysis to determine the K D using GraphPad 8.0 software.

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Binding Assay, Concentration Assay, Software

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: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

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: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

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: Kinetic and equilibrium constants for the binding of proMMP-1, proMMP-9, MMP-1/TIMP-1 complexes, and proMMP-9/TIMP-1 complexes to LRP1.

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Binding Assay

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: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

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: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Recombinant, Binding Assay, Concentration Assay, Software

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: Equilibrium binding constants for the interaction of proMMP-1 and TIMP-1 with LRP1 clusters.

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Binding Assay

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: Kinetic and equilibrium constants for the binding of MMP-1/TIMP-1 complexes to LRP1 ligand binding clusters.

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Binding Assay, Ligand Binding Assay

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) Domain structure of MMP-9 with schematic representation of intact 92 kDa proMMP-9 and predicted fragment sizes by activation with MMP-3cd and APMA indicated. Purified TIMP-free MMP-9 (75 μg) was treated with either the MMP-3cd at 100 μM (B) or 2 mM APMA (C) for the indicated times. At each time point, 15 μg was removed for analysis by Coomassie protein staining or receptor blotting as indicated. Receptor binding was detected using monoclonal anti-LRP1 IgG 8G1 and goat anti-mouse IgG conjugated to HRP and visualized using chemiluminescence. MMP-9 is a 92 kDa zymogen. In the presence of either MMP-3cd or APMA, N-terminal cleavage of MMP-9 occurs yielding 86 and 82 kDa enzyme species (bracket). In the presence of APMA, the 82 kDa species undergoes C-terminal processing yielding a 68 kDa enzyme species (*).

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Activation Assay, Purification, Staining, Binding Assay

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: Full length LRP1 was immobilized on a CM5 sensor chip using an amine-reactive coupling process. (A) proMMP-9 was injected over the chip in increasing concentrations of ligand (11.1, 33.3, 100, and 300 nM). (B) proMMP-9/TIMP-1 complexes were injected over the chip in increasing concentrations of ligand (3.7, 11.1, 33.3, 100, and 300 nM). 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 two independent experiments that were performed.

Article Snippet: Recombinant human LRP1 cluster II, III, and IV Fc chimera proteins were obtained from Molecular Innovations.

Techniques: Injection, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Carboxyl-terminal Tail-mediated Homodimerizations of Sphingomyelin Synthases Are Responsible for Efficient Export from the Endoplasmic Reticulum *

doi: 10.1074/jbc.M116.746602

Figure Lengend Snippet: The two N-terminal tails, the N- and C-terminal tails, and the two C-terminal tails in the SMS homodimers are in close proximity. A, schematic of the chimeric proteins used for BiFC assays. The chimeras included VN (N-terminal residues 1–173 of the Venus protein) and V5 epitope or VC (C-terminal residues 155–238 of the Venus protein) and FLAG epitope. B–D, COS7 cells were transfected with a plasmid encoding V5-tagged SMS2-VN and FLAG-tagged SMS2-VC or V5-tagged SMS2-VN and FLAG-tagged VC-GlcT. 24 h post-transfection, the cells were fixed and permeabilized with 0.1% Triton X-100. For confocal microscopy (B), the cells were stained with anti-V5 and anti-FLAG antibodies, followed by appropriate Alexa Fluor-conjugated secondary antibodies. For flow cytometry (C and D), the cells were stained with anti-V5 antibody, followed by PerCP/Cy5.5-conjugated secondary antibody and APC-conjugated anti-FLAG antibody. B, SMS2-VN, blue; SMS2-VC or VC-GlcT, red; Venus, green. B, a close-up view of the plasma membrane in the squared region in a. Scale bar = 10 μm. C, representative dot plot from cells expressing SMS2-VN/SMS2-VC (left panel) and SMS2-VN/VC-GlcT (right panel). The gating of the double-positive cell population expressing both VN- and VC-fused proteins is shown as a bold square. D, FACS histogram representing the intensities of Venus fluorescence detected by the FITC channel in double-positive cells (PerCP-Cy5.5/APC). Histograms represent the extent of FITC quantified in cells expressing SMS2-VN/SMS2-VC (filled) or SMS2-VN/VC-GlcT (open). E and F, the intensity of Venus fluorescence in COS7 cells co-expressing the indicated combinations of VN- or VC-fused SMS1 (E) or SMS2 (F) was quantified by flow cytometry. Individual data points are shown as a scatterplot. Values represent the mean ± S.D. from at least three independent experiments. *, p < 0.01; **, p < 0.05. G and H, schematics of SMS homodimers, viewed as a section through the Golgi or plasma membrane (G) and viewed from the cytosolic space (H). Individual monomers of transmembrane segments in SMSs are shown in black and gray. N- and C-terminal tails are represented by blue and red lines, respectively. 1–6 and I-VI indicated the transmembrane helices. H, SMS1 and SMS2 have the following intermolecular interactions: between the N-terminal tails, between the N-terminal tail and C-terminal tail, and between the C-terminal tails, shown as dashed arrow lines.

Article Snippet: Co-immunoprecipitation was performed with anti-FLAG M2 beads at 4 °C for 4 h. Then the beads were washed four times with lysis buffer and eluted in SDS sample buffer by incubation at 65 °C for 4 min. Co-immunoprecipitated proteins were subjected to SDS-PAGE with Dr. Western (Oriental Yeast) or WIDE-VIEW TM Prestained Protein Size Marker III (Wako).

Techniques: FLAG-tag, Transfection, Plasmid Preparation, Confocal Microscopy, Staining, Flow Cytometry, Expressing, Fluorescence

Journal: Computational and Structural Biotechnology Journal

Article Title: PROMISed: A novel web-based tool to facilitate analysis and visualization of the molecular interaction networks from co-fractionation mass spectrometry (CF-MS) experiments

doi: 10.1016/j.csbj.2021.08.042

Figure Lengend Snippet: Published datasets derived from co-fractionation-based methods. CN-PAGE: clear native PAGE, IEF: isoelectric focusing, IEX: ion-exchange chromatography, SEC: size-exclusion chromatography, SDG: sucrose density gradient. *1) the nine model species include: Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, Strongylocentrotus purpuratus, Homo sapiens, Xenopus laevis, Nematostella vectensis, Dictyostelium discoideum and Saccharomyces cerevisiae ; *2) plant species studied: Arabidopsis thaliana, Brassica oleracea, Glycine max , Cannabis sativa, Solanum lycopersicum, Chenopodium quinoa, Zea mays, Oryza sativa ssp. japonica, Triticum aestivum, Cocos nucifera, Ceratopteris richardii, Selaginella moellendorf and Chlamydomonas reinhardtii.

Article Snippet: By expanding co-fractionation-based methods to protein–metabolite interactions, PROMIS has delineated putative protein interactors for over 140 metabolites in A. thaliana , and validated 225 previously predicted PMIs in yeast .

Techniques: Derivative Assay, Clear Native PAGE, Ion Exchange Chromatography, Size-exclusion Chromatography, Cannabis, Cell Culture, Drug discovery, Suspension, Membrane, Activity Assay

Journal: STAR Protocols

Article Title: scGR-seq: Integrated analysis of glycan and RNA in single cells

doi: 10.1016/j.xpro.2022.101179

Figure Lengend Snippet:

Article Snippet: WIDE-VIEW Prestained Protein Size Marker , Fujifilm Wako Pure Chemical Co. , Cat#230-02221.

Techniques: Recombinant, Marker, Gentle, Plasmid Preparation, Silver Staining, Bradford Protein Assay, SYBR Green Assay, Labeling, DNA Library Preparation, Software, Binding Assay, Chromatography