Arrestin‐Domain Containing Protein 1 (Arrdc1) Regulates the Protein Cargo and Release of Extracellular Vesicles

Abstract

Extracellular vesicles (EVs) are lipid-bilayered vesicles that are released by multiple cell types and contain nucleic acids and proteins. Very little is known about how the cargo is packaged into EVs. Ubiquitination of proteins is a key posttranslational modification that regulates protein stability and trafficking to subcellular compartments including EVs. Recently, arrestin-domain containing protein 1 (Arrdc1), an adaptor for the Nedd4 family of ubiquitin ligases, has been implicated in the release of ectosomes, a subtype of EV that buds from the plasma membrane. However, it is currently unknown whether Arrdc1 can regulate the release of exosomes, a class of EVs that are derived endocytically. Furthermore, it is unclear whether Arrdc1 can regulate the sorting of protein cargo into the EVs. Exosomes and ectosomes are isolated from mouse embryonic fibroblasts isolated from wild type and Arrdc1-deficient (Arrdc1 ) mice. Nanoparticle tracking analysis-based EV quantitation shows that Arrdc1 regulates the release of both exosomes and ectosomes. Proteomic analysis highlights the change in protein cargo in EVs upon deletion of Arrdc1. Functional enrichment analysis reveals the enrichment of mitochondrial proteins in ectosomes, while proteins implicated in apoptotic cleavage of cell adhesion proteins and formation of cornified envelope are significantly depleted in exosomes upon knockout of Arrdc1.

Arrdc4‐dependent extracellular vesicle biogenesis is required for sperm maturation

“…We and others have identified specific members of the α‐arrestin protein family as key regulators of EV release, namely Arrdc1 (Anand et al., 2018; Nabhan et al., 2012) and Arrdc4 (Mackenzie et al., 2016). α‐arrestins act as adaptors for ubiquitin ligases of the Nedd4 family (Dores et al., 2015; Foot et al., 2017; Han et al., 2013; Nabhan et al., 2010, Shah and Kumar, 2021; Shea et al., 2012).…”

Exosomes from N‐Myc amplified neuroblastoma cells induce migration and confer chemoresistance to non‐N‐Myc amplified cells: implications of intra‐tumour heterogeneity

“…The relative protein abundance between exosomal samples was obtained by estimating the ratio of normalized spectral counts (Rsc) as previously described [14]. When RSc is less than 1, the negative inverse RSc value was used …”

Extracellular vesicles containing oncogenic mutant β‐catenin activate Wnt signalling pathway in the recipient cells

“…A constant voltage of 150 V was applied, and proteins were visualised using a Coomassie stain (Bio‐Rad) for 1 h. Gels were destained using 20% methanol and 7.5% acetic acid in Milli‐Q water overnight. Protein bands (20) were excised and subjected to in gel‐digestion as described previously [23,24]. Briefly, the excised bands were reduced using 10 mM DTT (Bio‐Rad) for 30 min, followed by 20 min alkylation using 25 mM iodoacetamide (Sigma®).…”