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EVH1 domain interactors of VASP in mature and immature dendritic cells

ABSTRACT: Mass spectrometry study of interactor binding to the EVH1 domain of VASP in mature and immature dendritic cells. We performed mass spectrometry study using a specially designed inhibitor that recognizes this characterized motif with high affinity and analyzed the digested peptides by LC-MS. For both, iDCs and mDCs, two independent experiments were carried out (N=2). As control for the specificity of the identified interactions an additional two independent experiments (N=2) were carried out for both, iDCs and mDCs, where a specific inhibitor of the EVH1 domain was added to outcompete bona fide EVH1 domain interactions.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Dendritic Cell

DISEASE(S): Immune System

SUBMITTER: Heike Stephanowitz

LAB HEAD: Tanja Maritzen

PROVIDER: PXD030882 | Pride | 2022-10-20

REPOSITORIES: Pride

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Project description:The inadequate function of the antigen presenting cells, such as dendritic cells (DCs) in cancer is one of the major factors leading to compromised anti-tumor immune response. Cell-to-cell interactions and cell migration are essential features of the immunological response in cancer. Vascular endothelial growth factor (VEGF) has been shown to affect DCs differentiation and maturation processes, thus leading to impaired DCs function. This study aimed to compare the expression of adherence and cytoskeleton-related genes between mature monocyte-derived dendritic cells (mDCs) and mDCs previously exposed to VEGF (mDCVs). VEGF is known to stimulate cell migration and actin rearrangement, but its association with adhesion molecules has not been largely explored in the context of DCs. Our results show that immature human DCs exposed to VEGF up-regulated CD80 and CD86, typical cell surface markers of DC maturation. However, unlike mDCs, mDCVs showed alterations in cell morphology, with broad changes in the characteristic cytoplasmic processes. DNA microarray analysis revealed that mDCVs up-regulated a significant subset of genes related to cell migration, including cell adhesion molecules and related signaling pathways as well as cytoskeleton-related genes. Our results suggest that VEGF, known to be involved in the inhibition of DC maturation, interferes in cell mechanisms leading to cytoskeleton reorganization and the expression of cell adhesion molecules. Cell morphology is associated with DC maturation and migration, both impaired in pathological conditions such as cancer. Keywords: Gene expression profiling Gene expression profiling was carried out in freshly isolated CD14+ cells prior to cultivation (“D0” samples) and in DCs after 6 days (“D6” samples) and 7 days in culture, with or without VEGF treatment (“D7V” and “D7” samples, respectively). Blood samples were collected on the same day and processed concomitantly. Cell culture, RNA extraction, and microarray hybridization were also carried out simultaneously in an attempt to minimize technical inconsistencies. A total of 14 independent microarray hybridizations were carried out with oligonucleotide microarrays, covering approximately 55,000 human transcripts (Whole Genome CodeLink™ Bioarrays, GE Healthcare). Target preparation and hybridization procedures strictly followed protocols provided by the manufacturer.Arrays were scanned following the recommended scanning procedure and settings for use with CodeLink bioarrays (GE Healthcare) on GenePix 4000B Array Scanner/GenePix Pro 4.1 software (Axon Instruments).

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EVH1 domain interactors of VASP in mature and immature dendritic cells

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