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Introduction
Yuval Zur currently works at the Department of Biotechnology Engineering , Ben-Gurion University of the Negev. Yuval does research in Molecular Biology, Bone Biology and Cell Biology. Their most recent publication is 'Engineering a monomeric variant of macrophage colony-stimulating factor (M‑CSF) that antagonizes the c-FMS receptor'.
Publications
Publications (35)
Elevated osteoclast (OC) activity is a major contributor to inflammatory bone loss (IBL) during chronic inflammatory diseases. However, the specific OC precursors (OCPs) responding to inflammatory cues and the underlying mechanisms leading to IBL are poorly understood. We identified two distinct OCP subsets: Ly6ChiCD11bhi inflammatory OCPs (iOCPs)...
The repertoire of methods for the detection and chemotherapeutic treatment of prostate cancer (PCa) is currently limited. Prostate-specific membrane antigen (PSMA) is overexpressed in PCa tumors and can be exploited for both imaging and drug-delivery. We developed and characterized four nanobodies that present tight and specific binding and interna...
There is currently a demand for new highly efficient and specific drugs to treat osteoporosis, a chronic bone disease affecting millions of people worldwide. We have developed a combinatorial strategy for engineering bispecific inhibitors that simultaneously target the unique combination of c-FMS and αvβ3 integrin, which act in concert to facilitat...
Amino acid sequences of M-CSFWT, M-CSFRGD libraries, M-CSFc-FMS, M-CSFαvβ3 and the three M-CSFRGD variants that were chosen after an affinity maturation process.
(A) The amino acid sequence of M-CSF with C31, which is required for dimerization, indicated in red. The two flexible loops in the dimerization interface are colored blue (loop 1, residues...
Compatibility of YSD with M-CSFC31S.
YSD M-CSFC31S was analyzed for (A) forward scatter and side scatter and (B) expression using mouse anti-c-myc antibody followed by a secondary PE-labeled anti mouse antibody. (C) The binding of YSD M-CSFC31S to soluble c-FMS-Fc was detected by a goat anti-human Fc-FITC antibody. (D) Cells expressing M-CSFC31S on...
Chemical cross-linking of purified protein variants.
Dimerization of the purified proteins was determined by using increasing concentrations of BS3 cross-linker, denaturation, and analysis on SDS-PAGE. M-CSFWT dimerized at all BS3 concentrations, but the three M-CSFRGD variants, M-CSFc-FMS, and M-CSFαvβ3 did not show any dimerization capability. BS...
Akt and c-FMS phosphorylation without murine M-CSF.
Murine BMMs were seeded for differentiation for 48 h, followed by incubation of purified M-CSFc-FMS, M-CSFαvβ3, and M-CSFRGD variants without the addition of murine M-CSF. Cells were lysed and subjected to SDS-PAGE to test spontaneous activation of (A) c-FMS and (B) Akt. The aspect ratios of the m...
Binding affinities of M-CSFRGD variants to c-FMS and αvβ3 integrin determined by SPR.
M-CSF, macrophage colony-stimulating factor; SPR, surface plasmon resonance.
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Numerical data for generating SPR binding sensorgrams in Fig 3.
SPR, surface plasmon resonance.
(XLSX)
FACS dot plot of M-CSFRGD libraries.
M-CSFRGD (A–D) library 1 and (E–H) library 2 were analyzed for (A and E) FSC/SSC, (B and F) expression, (C and G) 100 nM c-FMS binding, and (D and H) 500 nM αvβ3 integrin binding. FACS, fluorescence-activated cell sorting; FSC, forward scatter; M-CSF, macrophage colony-stimulating factor; RGD, Arginine-Glycine-A...
Numerical data and densitometry results for generating the graphs in Fig 5.
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Numerical data of actin belts quantification in Fig 6.
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Numerical data of individual clones’ flow cytometry binding in S6 Fig.
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Scheme of YSD construct.
The M-CSFRGD library was covalently linked to Aga1p and the yeast cell wall. Binding for c-FMS was determined with c-FMS-Fc recombinant protein and goat anti-human Fc FITC conjugated secondary antibody, and the expression levels were measured with a mouse anti-c-myc primary antibody and PE anti-mouse secondary antibody. For...
Analysis of individual YSD M-CSFRGD clones selected from sorts 4 and 5 for their binding to c-FMS, αvβ3 integrin and other integrins.
Twenty-five different clones from each of sorts 4 (A) and 5 (C) were tested for binding to 20 nM of αvβ3 integrin, normalized to the lowest binder. (B) The best 15 αvβ3 integrin M-CSFRGD binders from sort 4 and the b...
Binding specificity of M-CSFRGD variants for RGD-binding integrins.
(A) αvβ3, (B) α3β1, (C) α4β7, and (D) α5β1 integrins were immobilized on the surface of the chip. Thereafter, the three M-CSFRGD variants 4.22 (green), 4.24 (blue), and 5.6 (red) were allowed to flow over the surface of the chip at a concentration of 1 μM. Source data can be found...
M-CSFRGD/αvβ3
integrin interface seen from different angles (A–D). αv in yellow “surf” presentation, β3 in green, M-CSFRGD in pink, and the mutant QTSRGDSPS loop in red. M-CSF, macrophage colony-stimulating factor; RGD, Arginine-Glycine-Aspartic acid.
(TIF)
Superposition of M-CSFRGD with crystallized cRGD from the 1L5G PDB structure [29].
αv in yellow, β3 in green, RGD from M-CSFRGD in cyan, and RGD from the crystal in pink. cRGD, cyclic RGD; M-CSF, macrophage colony-stimulating factor; RGD, Arginine-Glycine-Aspartic acid.
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Numerical data and analysis of graphs in Fig 7.
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Numerical data of CTX serum levels Fig 9.
CTX, carboxy-terminal telopeptide.
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Numerical data of YSD in S2 Fig.
YSD, yeast surface display.
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FACS FSC and SSC of affinity maturation process.
Yeast-displayed mutant libraries were analyzed, and the living cells population in each sort is represented by a black polygon-shaped gate. The affinity maturation sorting process started with (A) a presorted library followed by (B) sort 1, (C) sort 2, (D) sort 3, (E) sort 4, and (F) sort 5. FACS, fl...
Purification of M-CSFc-FMS, M-CSFαvβ3, and M-CSFRGD variants.
(A) Size exclusion chromatography of nonglycosylated M-CSFRGD clone 4.22 with high molecular weight standards. Variant 4.22 was eluted at the size of 21 kDa. (B) Mass spectrometry of nonglycosylated variant 5.6. (C) CD spectra of nonglycosylated variant 4.22 (red line), nonglycosylated v...
Steady-state equilibrium analysis.
To determine the protein KD,app, the RUs at saturation for each protein concentration were plotted, and a fitted curve was created for (A) c-FMS and (B) αvβ3 integrin. Source data and its analysis can be found in S10 Data. RUs, response units.
(TIF)
Docking model of the M-CSFC31S/c-FMS–αvβ3 integrin complex.
M-CSFC31S is shown in pink, c-FMS in cyan, αv in yellow, and β3 in green. Residues 25–32 of M-CSFC31S are represented in red. M-CSF, macrophage colony-stimulating factor.
(TIF)
Receptor expression levels of MDA-MB-231 and mouse BMMs.
The expression levels of c-FMS and αvβ3 integrin were measured using flow cytometry. The red histograms represent the negative control, and the blue histograms represent receptor expression. Mouse BMMs without differentiation cytokines (t = 0) express c-FMS (A) and αvβ3 integrin (B). MDA-MB-2...
Actin ring formation in mature osteoclasts incubated with M-CSFRGD variants.
Differentiated murine BMMs were incubated for additional 24 h without (positive control) or with inhibitors (5 μM) followed by fixation and F-actin and nuclei staining. Cells were able to form a solid actin ring (white arrowheads), scattered actin ring [44] (white arrows)...
Additional materials and methods information.
(DOCX)
Numerical data and analysis of graphs in Fig 8.
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Numerical data of the protein purification process in S7 Fig.
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Numerical data of steady state equilibrium analysis in S9 Fig.
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Numerical data of different integrins binding using SPR in S10 Fig.
SPR, surface plasmon resonance.
(XLSX)
Enhanced activation of the signaling pathways that mediate the differentiation of mononuclear monocytes into osteoclasts is an underlying cause of several bone diseases and bone metastasis. In particular, dysregulation and over-expression of macrophage colony stimulating factor (M-CSF) and its c-FMS tyrosine kinase receptor, proteins that are essen...
The molecular interactions between macrophage colony stimulating factor (M-CSF) and the tyrosine kinase receptor c-fms play a key role in the immune response, bone metabolism and the development of some cancers. Since no X-ray structure is available for the human M-CSF/c-fms complex, the binding epitope for this complex is largely unknown. Our goal...