Biology subjects

Haugas, M.

Publications and source records attributed to Haugas, M..

4 recordsLinked to original sources

bioRxiv2025

Proximity labelling-based identification of vascular homing peptide receptors

Identifying the receptors of vascular homing peptides (VHPs) is critical for mechanistic understanding and development of peptide-guided precision therapeutics. Conventional receptor discovery methods, such as affinity chromatography, require cell disruption and often expose intracellular proteins, resulting in high background and low specificity. To overcome these limitations, we developed a proximity labelling approach that tags proteins near VHP receptors on intact live cells. Cells were incubated with VHP-horseradish peroxidase (HRP) complexes, which, upon hydrogen peroxide treatment, activate biotin-tyramide to produce short-lived radicals that covalently label nearby membrane proteins. Using the prototypic C-end Rule peptide RPARPAR and its known receptor neuropilin-1 (NRP-1), we validated this method by successful receptor tagging and mass spectrometric identification. Using RPARPAR-HRP conjugates, we achieved selective proximity labeling of membrane proteins in NRP-1-positive PPC1 cells, with a 3- to 5-fold increase in fluorescence intensity over controls by flow cytometry. Affinity purification and Western blotting identified a strong [~]130 kDa band corresponding to NRP-1 exclusively in labeled PPC1 cells. Mass spectrometry analysis revealed a [~]20-fold enrichment of NRP-1 and significant enrichment of integrins (ITGAV, ITGB1, ITGA3), ALCAM, EPHA2, CD109, and PLXNB2 in RPARPAR-labeled samples compared to controls. This approach could be broadly used for molecular mapping of the homing peptide interactome and its spatial proximity in live cells, streamlining the discovery of homing peptide receptors and their associated partners.

cell biology↗

bioRxiv2024

A CYCLIC PEPTIDE TARGETS GLIOBLASTOMA BY BINDING TO ABERRANTLY EXPOSED SNAP25

Disease-specific changes in tumors and other diseased tissues are an important target of research because they provide clues on the pathophysiology of the disease as well as uncovering potentially useful markers for diagnosis and treatment. Here, we report a new cyclic peptide, CESPLLSEC (CES), that specifically accumulated (homed) in intracranial U87MG and the WT-GBM model of glioblastoma from intravenous (IV) injection, associating with the vasculature. Affinity chromatography of U87MG tumor extracts on insolubilized CES peptide identified Synaptosomal Associated Protein 25 (SNAP25) as a candidate target molecule (receptor) for CES. Several results supported the identification of SNAP25 as the CES receptor. IV-injected FAM-CES colocalized with SNAP25 in the tumors, and direct binding studies showed specific CES peptide binding to recombinant human SNAP25. A CES peptide-drug conjugate designed for photodynamic therapy showed selective cytotoxicity to SNAP25+ glioblastoma cell lines. Specific accumulation of systemically injected anti-SNAP25 antibody in U87MG glioblastoma, and labeling of intact U87MG cells with anti-SNAP in flow cytometry showed that SNAP25 is available from the circulation but not in normal tissues and that it is present at the cell surface. Using an array of ECM proteins and surface plasmon resonance revealed that SNAP25 binds moderately to collagen V and strongly to collagen VI. Modeling studies suggested that CES and collagen VI compete for the same binding site on SNAP25. Our results introduce CES as a valuable targeting peptide for drug delivery, and its receptor SNAP25 as a possible molecular marker of interest for glioblastoma.

cancer biology↗

bioRxiv2022

Targeting triple-negative breast cancer with β1-integrin binding aptamer

Targeted therapies have increased the treatment options for triple-negative breast cancer patients. However, the paucity of targetable biomarkers and tumour heterogeneity have limited the ability of precision-guided interventions to live up to their full potential. As affinity targeting ligands, aptamers show high selectivity towards target molecules. Compared to antibodies, aptamers have lower molecular weight, increased stability during transportation, reduced immunogenicity, and increased tissue uptake. Recently, we reported the discovery of GreenB1 aptamer that is internalized in cultured triple-negative MDA-MB-231 human breast cancer cells. We show that the GreenB1 aptamer specifically targets {beta}1-integrin, a protein previously linked to breast cancer cell invasiveness and migration. Aptamer binds to {beta}1-integrin with low nanomolar affinity. GreenB1 homes in the orthotopic 4T1 triple-negative breast cancer lesions modelled in mice. Our findings suggest potential applications for the GreenB1-guided precision agents for the diagnosis and therapy of triple-negative breast cancer.

molecular biology↗

bioRxiv2020

In vivo phage display: identification of organ-specific peptides using deep sequencing and differential profiling across tissues

In vivo phage display is widely used for identification of organ- or disease-specific homing peptides. However, the current in vivo phage biopanning approaches fail to assess biodistribution of specific peptide phages across tissues during the screen, thus necessitating laborious and time-consuming post-screening validation studies on individual peptide phages. Here, we adopted bioinformatics tools used for RNA sequencing for analysis of high throughput sequencing (HTS) data to estimate the representation of individual peptides during biopanning in vivo. The data from in vivo phage screen were analyzed using differential binding - relative representation of each peptide in the target organ vs. in a panel of control organs. Application of this approach in a model study using low-diversity peptide T7 phage library with spiked-in brain homing phage, demonstrated brain-specific differential binding of brain homing phage and resulted in identification of novel lung and brain specific homing peptides. Our study provides a broadly applicable approach to streamline in vivo peptide phage biopanning and to increase its reproducibility and success rate. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/181974v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1853e75org.highwire.dtl.DTLVardef@c7ad8corg.highwire.dtl.DTLVardef@152f4feorg.highwire.dtl.DTLVardef@15261d0_HPS_FORMAT_FIGEXP M_FIG C_FIG In vivo phage display using differential binding approach

molecular biology↗