The GABARAP Co-Secretome Identified by APEX2-GABARAP Proximity Labelling of Extracellular Vesicles

Sanwald, Julia L.; Poschmann, Gereon; Behrends, Christian; Hoffmann, Silke; Willbold, Dieter

doi:10.3390/cells9061468

Cited by 9 publications

(6 citation statements)

References 76 publications

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“…Interestingly, another homologue, GABARAP, is secreted in EVs. GABARAP EV proximity labelling revealed overrepresentation of RNA binding proteins and mitochondrion associated proteins (Sanwald et al., 2020 ) and further supports a role for LC3/GABARAP family proteins in specific cargo sorting.…”

Section: Discussionmentioning

confidence: 80%

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Secretion of pro‐angiogenic extracellular vesicles during hypoxia is dependent on the autophagy‐related protein GABARAPL1

Keulers

Libregts

Beaumont

et al. 2021

J of Extracellular Vesicle

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Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis development and therapy resistance. In response to hypoxia, tumour cells secrete pro‐angiogenic factors to induce blood vessel formation and restore oxygen supply to hypoxic regions. Extracellular vesicles (EVs) are emerging as mediators of intercellular communication in the tumour microenvironment. Here we demonstrate that increased expression of the LC3/GABARAP protein family member GABARAPL1, is required for endosomal maturation, sorting of cargo to endosomes and the secretion of EVs. Silencing GABARAPL1 results in a block in the early endosomal pathway and impaired secretion of EVs with pro‐angiogenic properties. Tumour xenografts of doxycycline inducible GABARAPL1 knockdown cells display impaired vascularisation that results in decreased tumour growth, elevated tumour necrosis and increased therapy efficacy. Moreover, our data show that GABARAPL1 is expressed on the EV surface and targeting GABARAPL1 + EVs with GABARAPL1 targeting antibodies results in blockade of pro‐angiogenic effects in vitro. In summary, we reveal that GABARAPL1 is required for EV cargo loading and secretion. GABARAPL1 + EVs are detectable and targetable and are therefore interesting to pursue as a therapeutic target.

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Section: Discussionmentioning

confidence: 80%

“…After fusion, ILV are secreted as exosomes. Recent data indicates that the autophagy machinery is involved in EV secretion and cargo loading (Guo et al., 2017 ; Leidal et al., 2020 ; Sanwald et al., 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Secretion of pro‐angiogenic extracellular vesicles during hypoxia is dependent on the autophagy‐related protein GABARAPL1

Keulers

Libregts

Beaumont

et al. 2021

J of Extracellular Vesicle

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“…Also, GABARAP was identified in extracellular vesicles using cells expressing APEX2-GABARAP. Interestingly, these extracellular vesicles also contained proteins with which GABARAP was shown to interact inside autophagosomes, further supporting a crosstalk between autophagy and protein secretion [ 139 ].…”

Section: Introductionmentioning

confidence: 99%

Protein complexes and neighborhoods driving autophagy

Sankar

Dengjel

2020

Autophagy

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Autophagy summarizes evolutionarily conserved, intracellular degradation processes targeting cytoplasmic material for lysosomal degradation. These encompass constitutive processes as well as stress responses, which are often found dysregulated in diseases. Autophagy pathways help in the clearance of damaged organelles, protein aggregates and macromolecules, mediating their recycling and maintaining cellular homeostasis. Protein-protein interaction networks contribute to autophagosome biogenesis, substrate loading, vesicular trafficking and fusion, protein translocations across membranes and degradation in lysosomes. Hypothesis-free proteomic approaches tremendously helped in the functional characterization of protein-protein interactions to uncover molecular mechanisms regulating autophagy. In this review, we elaborate on the importance of understanding protein-protein-interactions of varying affinities and on the strengths of mass spectrometry-based proteomic approaches to study these, generating new mechanistic insights into autophagy regulation. We discuss in detail affinity purification approaches and recent developments in proximity labeling coupled to mass spectrometry, which uncovered molecular principles of autophagy mechanisms. Abbreviations : AMPK: AMP-activated protein kinase; AP-MS: affinity purification-mass spectrometry; APEX2: ascorbate peroxidase-2; ATG: autophagy related; BioID: proximity-dependent biotin identification; ER: endoplasmic reticulum; GFP: green fluorescent protein; iTRAQ: isobaric tag for relative and absolute quantification; MS: mass spectrometry; PCA: protein-fragment complementation assay; PL-MS: proximity labeling-mass spectrometry; PtdIns3P: phosphatidylinositol-3-phosphate; PTM: posttranslational modification; PUP-IT: pupylation-based interaction tagging; RFP: red fluorescent protein; SILAC: stable isotope labeling by amino acids in cell culture; TAP: tandem affinity purification; TMT: tandem mass tag.

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“…Notably, another member of the family of sphingomyelinases, the neutral sphingomyelinase 2 (nSMase2), has been linked to LC3, which directly interacts with the nSMase2 regulator FAN (factor associated with nSMase2 activation) to specify cargo loading, e.g., of heterogeneous nuclear ribonucleoprotein K (HNRNPK), into extracellular vesicles (EVs) [ 38 ]. Very recently, we detected HNRNPK also in GABARAP-containing EVs [ 39 ]. Intriguingly, here we identified increased abundances of both SMPDL3B and HNRNPK in the absence of GABARAP-type proteins.…”

Section: Resultsmentioning

confidence: 99%

Lack of GABARAP-Type Proteins Is Accompanied by Altered Golgi Morphology and Surfaceome Composition

Sanwald

Dobner

Simons

et al. 2020

IJMS

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GABARAP (γ-aminobutyric acid type A receptor-associated protein) and its paralogues GABARAPL1 and GABARAPL2 comprise a subfamily of autophagy-related Atg8 proteins. They are studied extensively regarding their roles during autophagy. Originally, however, especially GABARAPL2 was discovered to be involved in intra-Golgi transport and homotypic fusion of post-mitotic Golgi fragments. Recently, a broader function of mammalian Atg8s on membrane trafficking through interaction with various soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) was suggested. By immunostaining and microscopic analysis of the Golgi network, we demonstrate the importance of the presence of individual GABARAP-type proteins on Golgi morphology. Furthermore, triple knockout (TKO) cells lacking the whole GABARAP subfamily showed impaired Golgi-dependent vesicular trafficking as assessed by imaging of fluorescently labelled ceramide. With the Golgi apparatus being central within the secretory pathway, we sought to investigate the role of the GABARAP-type proteins for cell surface protein trafficking. By analysing the surfaceome composition of TKOs, we identified a subset of cell surface proteins with altered plasma membrane localisation. Taken together, we provide novel insights into an underrated aspect of autophagy-independent functions of the GABARAP subfamily and recommend considering the potential impact of GABARAP subfamily proteins on a plethora of processes during experimental analysis of GABARAP-deficient cells not only in the autophagic context.

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The GABARAP Co-Secretome Identified by APEX2-GABARAP Proximity Labelling of Extracellular Vesicles

Cited by 9 publications

References 76 publications

Secretion of pro‐angiogenic extracellular vesicles during hypoxia is dependent on the autophagy‐related protein GABARAPL1

Secretion of pro‐angiogenic extracellular vesicles during hypoxia is dependent on the autophagy‐related protein GABARAPL1

Protein complexes and neighborhoods driving autophagy

Lack of GABARAP-Type Proteins Is Accompanied by Altered Golgi Morphology and Surfaceome Composition

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