synaptosome associated protein snap25  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: A CYCLIC PEPTIDE TARGETS GLIOBLASTOMA BY BINDING TO ABERRANTLY EXPOSED SNAP25

doi: 10.1101/2024.09.18.613627

Figure Lengend Snippet: Homing of intravenously administered FAM-CES in mice bearing intracranial U87MG (A) , or in healthy mice (B) . 30 nmoles of FAM-CES were administered intravenously and allowed to circulate for 60 min (n=3). The mice were perfused, and the brains were fixed, sectioned, and stained for FAM (green) and SNAP25 (red). (C) : ELISA binding assay of FAM-CES and FAM-iRGD on plates coated with SNAP25 or Casein. (D): QCM analysis of the mass changes brought about by CES binding to human recombinant SNAP25 and subsequent dissociation. SNAP25 was immobilized on a viscoelastic film deposited on a gold-modified quartz crystal resonating at 5 MHz. The deposition of the protein was accomplished by using layer-by-layer self-assembly on a layer of polyallylamine (PAH) . The figure shows association and dissociation of CES (red) or iRGD (black). The final concentration of peptides was 30µM. CES did not bind to PAH (blue) or to the PAH-BSA multilayer (green). Washes with PBS are denoted by arrows. Scale bars=50µm.

Article Snippet: We excluded cytosolic proteins and focused on synaptosomal-associated protein 25 (SNAP25) as a possible receptor because it had a high CES-eluted/control peptide-eluted ratio, and because it is overrepresented at the RNA level in the U87MG cells (Human Protein Atlas).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Binding Assay, Recombinant, Modification, Concentration Assay

Journal: bioRxiv

Article Title: A CYCLIC PEPTIDE TARGETS GLIOBLASTOMA BY BINDING TO ABERRANTLY EXPOSED SNAP25

doi: 10.1101/2024.09.18.613627

Figure Lengend Snippet: (A): Western blot showing expression of SNAP25 in the different cell lines. (B): Structure of the peptide-drug conjugate CES-V, with the peptide part in red, the linker in green and the drug in blue. (C): Cultured U87MG, LN229, SK-N-AS, or HEK293-T cells were incubated with 3μM of CES-V or CtrlPep-V for 30 min at 37°C, followed by three washes with media. Then 100µL of new media was placed, and the wells were irradiated with a dose of 10 J/cm 2 (using an irradiance of 160 mW/cm 2 ) using a 690nm laser. After irradiation, the cells were cultured for an additional 24 h, and the viability was assessed by MTS assay.

Article Snippet: We excluded cytosolic proteins and focused on synaptosomal-associated protein 25 (SNAP25) as a possible receptor because it had a high CES-eluted/control peptide-eluted ratio, and because it is overrepresented at the RNA level in the U87MG cells (Human Protein Atlas).

Techniques: Western Blot, Expressing, Cell Culture, Incubation, Irradiation, MTS Assay

Journal: bioRxiv

Article Title: A CYCLIC PEPTIDE TARGETS GLIOBLASTOMA BY BINDING TO ABERRANTLY EXPOSED SNAP25

doi: 10.1101/2024.09.18.613627

Figure Lengend Snippet: (A): Rabbit anti-SNAP25 was administered to mice bearing intracranial U87MG tumors and allowed to circulate for 24 hours. The mice were then perfused, and their tissues fixed, sectioned, and stained for rabbit IgG. (B): Administered anti-SNAP25 (red) together with endogenous mouse IgG (green, left panel) or CD31 (green, right panel) in the tumor. (C): SNAP25 and CD31 costaining in U87MG tumor of un-injected mice. Scale bars for A and B represent 100µm, scale bars in C represent 20µm. Representative images from n=3 mice are shown.

Article Snippet: We excluded cytosolic proteins and focused on synaptosomal-associated protein 25 (SNAP25) as a possible receptor because it had a high CES-eluted/control peptide-eluted ratio, and because it is overrepresented at the RNA level in the U87MG cells (Human Protein Atlas).

Techniques: Staining, Injection

Journal: bioRxiv

Article Title: A CYCLIC PEPTIDE TARGETS GLIOBLASTOMA BY BINDING TO ABERRANTLY EXPOSED SNAP25

doi: 10.1101/2024.09.18.613627

Figure Lengend Snippet: (A): Live cell flow cytometry without permeabilization to detect superficial SNAP25. Cultured U87MG or HEK293Tcells were stained for SNAP25 using A488 anti-SNAP25 or its isotype control. (B): Immunofluorescent confocal microscopy of cultured U87MG cells, stained in the absence of any detergent (green: SNAP25, blue: DAPI). (C): Binding of SNAP25 to ECM proteins. Recombinant SNAP25 was added to an ECM array and A488 anti-SNAP25 was used to detect binding. (D): Surface plasmon resonance sensorgram of collagens V and VI on sensors coated either with recombinant SNAP25 or a control protein of similar molecular weight. (E): Optical microscopy shows c ellular morphology on collagen V and VI of cells seeded on either collagen V/VI coated plates or uncoated plates (scale bars= 20μm); the inset shows SNAP25 immunofluorescence of the vesicle-resembling particles (scale bars= 2μm).

Article Snippet: We excluded cytosolic proteins and focused on synaptosomal-associated protein 25 (SNAP25) as a possible receptor because it had a high CES-eluted/control peptide-eluted ratio, and because it is overrepresented at the RNA level in the U87MG cells (Human Protein Atlas).

Techniques: Flow Cytometry, Cell Culture, Staining, Control, Confocal Microscopy, Binding Assay, Recombinant, SPR Assay, Molecular Weight, Microscopy, Immunofluorescence

Journal: bioRxiv

Article Title: A CYCLIC PEPTIDE TARGETS GLIOBLASTOMA BY BINDING TO ABERRANTLY EXPOSED SNAP25

doi: 10.1101/2024.09.18.613627

Figure Lengend Snippet: (A): Representation of SNAP25 and the best CES pose obtained by docking (left panel). Hydrogen bonds formed between GLN53 SNAP25 - GLU2 CES , GLN53 SNAP25 - CYS1 CES , and GLN109 SNAP25 - LEU5 CES (middle panel), and close contacts between SNAP-25 and CES in sphere representation (right panel). (B) : SNAP25-collagen VI complex with CES superimposed in yellow spheres highlights competition for the same binding site. A close-up to the binding site reveals interactions between collagen VI and SNAP25, specifically involving residues Q56 and R59.

Article Snippet: We excluded cytosolic proteins and focused on synaptosomal-associated protein 25 (SNAP25) as a possible receptor because it had a high CES-eluted/control peptide-eluted ratio, and because it is overrepresented at the RNA level in the U87MG cells (Human Protein Atlas).

Techniques: Binding Assay