A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiation

Traub, Linton M.

doi:10.7554/elife.41768

Cited by 20 publications

(23 citation statements)

References 125 publications

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“…Another possibility is the activation of a 406 compensatory endocytosis mechanism which could bypass sequential AP-2 activation events so as to 407 ensure slow but steady actin-based endocytosis in HeLa cells (Leyton-Puig et al, 2017). Regardless, all 408 these cases would result in deposition of excess AP-2 and immobile clathrin population on membrane 409 giving rise to structures >0.6 µm 2 as seen in this study as well as in previous studies (Umasankar et al, 410 2014;Ma et al, 2016;Traub., 2019). Since our current investigation is limited to qualitative analyses of 411 endocytosis phenotypes, these possibilities are speculative and require future investigations to delineate 412 precise mechanisms.…”

Section: Degradation Of Bmp2k In Fcho Knockout Cells and Ap-2 Depletesupporting

confidence: 52%

“…Yet, a large pool of AP-2 remains unphosphorylated in FCHO knockout cells. These findings 362 argue that FCHO must be functioning together with EPS15/R to allosterically drive the equilibrium from 363 cytosolic inactive AP-2 close to membrane active AP-2 open / AP-2 open+ (Ma et al, 2016;Traub, 2019). This 364 equilibrium is further driven toward CCP maturation by downstream kinase that phosphorylates open+ 365 state of AP-2 (Wrobel et al, 2019).…”

Section: Functional Fcho-bmp2k Axis In Vivo 302mentioning

confidence: 99%

“…The authenticated and validated parental HeLa SS6 cell line (Umasankar et al, 2014, Traub 2019) was a 623 kind gift from Prof. Linton Traub. HeLa clone #64/1.E (1.E cells) was generated previously by bi-allelic 624 disruption of the FCHO2 and FCHO1 gene loci using TALEN gene-editing technology (Umasankar et 625 al., 2014).…”

Section: Cell Culture and Transfections 622mentioning

confidence: 99%

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Allosteric feed-forward activation of AP-2viaFCHO-BMP2K axis promotes clathrin-mediated endocytosis

et al. 2019

Preprint

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1 Spatio-temporal regulation of central adaptor complex, AP-2 is pivotal for clathrin-mediated endocytosis 2 (CME). We recently discovered that FCHO proteins trigger clathrin-coated pit (CCP) formation by 3 allosterically activating AP-2 on plasma membrane (Umasankar et al., 2014). Here, we demonstrate that 4 this activation promotes AP-2 phosphorylation via recruitment and stabilization of BMP-2 inducible 5 kinase (BMP2K), a bona fide AP-2 kinase leading to CCP maturation. Accordingly, BMP2K mislocalizes 6 and degrades in FCHO knockout/ AP-2 depleted cells. Functional inactivation of kinase impairs AP-2 7 phosphorylation leading to altered lattice morphology and CME phenotypes reminiscent of CCP 8 maturation defects. Reexpression of FCHO rescues AP-2 phosphorylation defects in FCHO knockout 9 cells implying membrane activation of AP-2 is a prerequisite for kinase function. Furthermore, gain-and 10 loss-of function phenotypes of FCHO and BMP2K are analogous and mirror altered AP-2 functions 11 during zebrafish embryogenesis. Together, our findings reveal an in vivo allosteric feed-forward axis for 12 operation of CME. 13

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Section: Degradation Of Bmp2k In Fcho Knockout Cells and Ap-2 Depletesupporting

confidence: 52%

Section: Functional Fcho-bmp2k Axis In Vivo 302mentioning

confidence: 99%

Section: Cell Culture and Transfections 622mentioning

confidence: 99%

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Allosteric feed-forward activation of AP-2viaFCHO-BMP2K axis promotes clathrin-mediated endocytosis

et al. 2019

Preprint

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“…Nanobodies can act against challenging targets such as small molecules and toxins (Wesolowski et al, 2009;Bever et al, 2016), viruses (Wei et al, 2011;Hassiki et al, 2016;Vanlandschoot et al, 2011;Cohen, 2018), enzymes (Muyldermans, 2013), ion channels (Wei et al, 2011;Danquah et al, 2016) and G protein-coupled receptors GPCRs (Cromie et al, 2015). Llama nanobodies have been shown to tether to early endosomes and to mitochondria (Traub, 2019), be used for diagnostics (Shriver-Lake et al, 2018), be used for design of cancer immunotherapeutics (Hussack et al, 2018;Bannas and Koch-Nolte, 2018;Rossotti et al, 2019) and have been approved for passive immunotherapy (Sheridan, 2019). Our current finding, that llama nanobodies can be post-translationally deiminated may shed some light on their observed structural variation which still remains to be fully explained as sequence alignment does not fully elucidate their diversity (Mitchell and Colwell, 2018).…”

Section: Discussionmentioning

confidence: 99%

Deiminated proteins in extracellular vesicles and serum of llama (Lama glama)—Novel insights into camelid immunity

Criscitiello

Kraev

Lange

2020

Molecular Immunology

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A B S T R A C TPeptidylarginine deiminases (PADs) are phylogenetically conserved calcium-dependent enzymes which posttranslationally convert arginine into citrulline in target proteins in an irreversible manner, causing functional and structural changes in target proteins. Protein deimination causes generation of neo-epitopes, affects gene regulation and also allows for protein moonlighting. Furthermore, PADs have been found to be a phylogenetically conserved regulator for extracellular vesicle (EVs) release. EVs are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material. In this study, post-translationally deiminated proteins in serum and serum-EVs are described for the first time in camelids, using the llama (Lama glama L. 1758) as a model animal. We report a poly-dispersed population of llama serum EVs, positive for phylogenetically conserved EV-specific markers and characterised by TEM. In serum, 103 deiminated proteins were overall identified, including key immune and metabolic mediators including complement components, immunoglobulin-based nanobodies, adiponectin and heat shock proteins. In serum, 60 deiminated proteins were identified that were not in EVs, and 25 deiminated proteins were found to be unique to EVs, with 43 shared deiminated protein hits between both serum and EVs. Deiminated histone H3, a marker of neutrophil extracellular trap formation, was also detected in llama serum. PAD homologues were identified in llama serum by Western blotting, via cross reaction with human PAD antibodies, and detected at an expected 70 kDa size. This is the first report of deiminated proteins in serum and EVs of a camelid species, highlighting a hitherto unrecognized post-translational modification in key immune and metabolic proteins in camelids, which may be translatable to and inform a range of human metabolic and inflammatory pathologies.

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“…A target protein has been redirected away from its natural location using an antibody that was tethered to mitochondria using a mitochondrial outer membrane targeting element [61]. Antibodies containing a mitochondrial presequence, as described by Biocca and colleagues [51], correctly form intrachain disulfide bonds, in contrast to those expressed in the cytosol.…”

Section: Mitochondria-targeted Intrabodiesmentioning

confidence: 99%

Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology

et al. 2020

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To interfere with cell function, many scientists rely on methods that target DNA or RNA due to the ease with which they can be applied. Proteins are usually the final executors of function but are targeted only indirectly by these methods. Recent advances in targeted degradation of proteins based on proteolysis-targeting chimaeras (PROTACs), ubiquibodies, deGradFP (degrade Green Fluorescent Protein) and other approaches have demonstrated the potential of interfering directly at the protein level for research and therapy. Proteins can be targeted directly and very specifically by antibodies, but using antibodies inside cells has so far been considered to be challenging. However, it is possible to deliver antibodies or other proteins into the cytosol using standard laboratory equipment. Physical methods such as electroporation have been demonstrated to be efficient and validated thoroughly over time. The expression of intracellular antibodies (intrabodies) inside cells is another way to interfere with intracellular targets at the protein level. Methodological strategies to target the inside of cells with antibodies, including delivered antibodies and expressed antibodies, as well as applications in the research areas of neurobiology, viral infections and oncology, are reviewed here. Antibodies have already been used to interfere with a wide range of intracellular targets. Disease-related targets included proteins associated with neurodegenerative diseases such as Parkinson's disease (α-synuclein), Alzheimer's disease (amyloid-β) or Huntington's disease (mutant huntingtin [mHtt]). The applications of intrabodies in the context of viral infections include targeting proteins associated with HIV (e.g. HIV1-TAT, Rev, Vif, gp41, gp120, gp160) and different oncoviruses such as human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Epstein-Barr virus, and they have been used to interfere with various targets related to different processes in cancer, including oncogenic pathways, proliferation, cell cycle, apoptosis, metastasis, angiogenesis or neo-antigens (e.g. p53, human epidermal growth factor receptor-2 [HER2], signal transducer and activator of transcription 3 [STAT3], RAS-related RHO-GTPase B (RHOB), cortactin, vascular endothelial growth factor receptor 2 [VEGFR2], Ras, Bcr-Abl). Interfering at the protein level allows questions to be addressed that may remain unanswered using alternative methods. This review addresses why direct targeting of proteins allows unique insights, what is currently feasible in vitro, and how this relates to potential therapeutic applications.Therapeutic antibodies are valuable drugs, which mostly act outside of cells.Reaching the numerous drug targets that reside inside cells by antibodies is possible in vitro and allows unique insights compared with other methods.Applying antibodies inside cells for therapeutic purposes has been explored in animal models and promises specific therapeutic benefits in neurobiology, virology and oncology.

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A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiation

Cited by 20 publications

References 125 publications

Allosteric feed-forward activation of AP-2viaFCHO-BMP2K axis promotes clathrin-mediated endocytosis

Allosteric feed-forward activation of AP-2viaFCHO-BMP2K axis promotes clathrin-mediated endocytosis

Deiminated proteins in extracellular vesicles and serum of llama (Lama glama)—Novel insights into camelid immunity

Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology

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