A Nanobody-Based Toolset to Monitor and Modify the Mitochondrial GTPase Miro1

Fagbadebo, Funmilayo O; Kaiser, Philipp; Zittlau, Katharina; Bartlick, Natascha; Wagner, Teresa R.; Froehlich, Theresa; Jarjour, Grace; Nueske, Stefan; Scholz, Armin M.; Traenkle, Bjoern; Maček, Boris; Rothbauer, Ulrich

doi:10.3389/fmolb.2022.835302

Cited by 7 publications

(18 citation statements)

References 80 publications

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“…2A ) and tested whether the selected Nbs recognize specific domains within Drp1 by pull-down assays. To this end, we generated so-called Drp1 nanotraps for which we covalently immobilized D7 and D63 on N-hydroxysuccinimide (NHS)-activated Sepharose beads via primary amino groups of accessible lysine residues (Fagbadebo et al, 2022; Traenkle et al, 2020). For binding analysis, the Drp1 nanotraps were incubated with soluble protein fractions of HEK293 cells transiently expressing the Drp1 deletion constructs.…”

Section: Resultsmentioning

confidence: 99%

“…4A ). Considering that a bivalent format of Nbs can be superior to monovalent versions for imaging purposes (Fagbadebo et al, 2022; Virant et al, 2018), we genetically fused two D7 or two D63 Nbs head-to-tail connected by a flexible Gly-Ser linker [(G 4 S) 4 ] and generated a bivalent format of D7 (bivD7) or of D63 (bivD63), respectively. Additionally, we inserted a sortase-tag for site-directed labelling.…”

Section: Resultsmentioning

confidence: 99%

“…Monovalent Drp1 Nbs were expressed and purified as previously described (Burgstaller et al, 2022). Bivalent Drp1 Nbs were produced using the ExpiCHO system (Thermo Fischer Scientific) according to the manufacturer's instructions (Fagbadebo et al, 2022). Purity of produced Nbs was assessed using SDS-PAGE.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

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Nanobodies as novel tools to monitor the mitochondrial fission factor Drp1

Froehlich,

Jenner,

Cavarischia-Rega

et al. 2023

Preprint

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In cells, mitochondria undergo constant fusion and fission. An essential factor for fission is the mammalian dynamin-related protein 1 (Drp1). Dysregulation of Drp1 has been linked to neurodegenerative diseases including Parkinson’s as well as cardiovascular diseases and cancer. Here, we developed nanobodies (Nbs) for proteomics, advanced microscopy and live cell imaging of Drp1. To specifically enrich endogenous Drp1 with interacting proteins for proteomics, we functionalized high-affinity Nbs as capture matrices. Furthermore, we detected Drp1 by bivalent Nbs combined with site-directed fluorophore labelling in super-resolution STORM microscopy. For real-time imaging of Drp1, we intracellularly expressed fluorescently labelled Nbs, so-called chromobodies (Cbs). To improve the signal-to-noise ratio, we further converted Cbs into a “turnover-accelerated” format. With these imaging probes, we visualized the dynamics of endogenous Drp1 upon compound-induced mitochondrial fission in living cells. Considering the wide range of research applications, the presented Nb toolset will open up new possibilities for advanced functional studies of Drp1 in disease-relevant models.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Nanobodies as novel tools to monitor the mitochondrial fission factor Drp1

Froehlich,

Jenner,

Cavarischia-Rega

et al. 2023

Preprint

Self Cite

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“…In addition to the typical PD-related molecules, damage-related molecules common to some diseases can also be used as therapeutic or diagnostic targets, such as Nbs targeting caspase-3 and Mitochondrial Rho GTPase 1 (Miro1). Nbs against the former can resist cell damage caused by oxidative stress in a variety of brain diseases, while the latter is associated with mitochondrial homeostasis and is a potential biomarker or therapeutic target after visualization or regulation by Nbs ( 103 , 104 ).…”

Section: Diagnostic and Therapeutic Applications Of Nanobodies In Bra...mentioning

confidence: 99%

Applications of nanobodies in brain diseases

Zheng

Pang²,

Li³

et al. 2022

Front. Immunol.

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Nanobodies are antibody fragments derived from camelids, naturally endowed with properties like low molecular weight, high affinity and low immunogenicity, which contribute to their effective use as research tools, but also as diagnostic and therapeutic agents in a wide range of diseases, including brain diseases. Also, with the success of Caplacizumab, the first approved nanobody drug which was established as a first-in-class medication to treat acquired thrombotic thrombocytopenic purpura, nanobody-based therapy has received increasing attention. In the current review, we first briefly introduce the characterization and manufacturing of nanobodies. Then, we discuss the issue of crossing of the brain-blood-barrier (BBB) by nanobodies, making use of natural methods of BBB penetration, including passive diffusion, active efflux carriers (ATP-binding cassette transporters), carrier-mediated influx via solute carriers and transcytosis (including receptor-mediated transport, and adsorptive mediated transport) as well as various physical and chemical methods or even more complicated methods such as genetic methods via viral vectors to deliver nanobodies to the brain. Next, we give an extensive overview of research, diagnostic and therapeutic applications of nanobodies in brain-related diseases, with emphasis on Alzheimer’s disease, Parkinson’s disease, and brain tumors. Thanks to the advance of nanobody engineering and modification technologies, nanobodies can be linked to toxins or conjugated with radionuclides, photosensitizers and nanoparticles, according to different requirements. Finally, we provide several perspectives that may facilitate future studies and whereby the versatile nanobodies offer promising perspectives for advancing our knowledge about brain disorders, as well as hopefully yielding diagnostic and therapeutic solutions.

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“…Intra-VHHs are a new type of biological tools that target proteins present at different sub-cellular compartments [reviewed in ( 5 )], such as the GTP-bound form of RhoA at the inner face of the plasma membrane ( 6 ), or lamin lining the nuclear envelope ( 7 ). They can be used to enhance, block or monitor ( 8 ) protein activation/activity, to track protein trafficking inside the cell when fused to fluorescent probes ( 7 ), to hinder protein/protein interactions, or to direct the antigen protein to degradation pathways ( 9 ). Some of them appear exquisitely sensitive to protein conformational dynamics, and this chaperone-like property has been particularly useful to decipher the conformational complexity of G protein-coupled receptors (GPCR) in their ligand-activated states.…”

Section: Introductionmentioning

confidence: 99%

Intracellular VHHs to monitor and modulate GPCR signaling

et al. 2022

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Single-domain antibody fragments, also known as VHHs or nanobodies, have opened promising avenues in therapeutics and in exploration of intracellular processes. Because of their unique structural properties, they can reach cryptic regions in their cognate antigen. Intracellular VHHs/antibodies primarily directed against cytosolic proteins or transcription factors have been described. In contrast, few of them target membrane proteins and even less recognize G protein-coupled receptors. These receptors are major therapeutic targets, which reflects their involvement in a plethora of physiological responses. Hence, they elicit a tremendous interest in the scientific community and in the industry. Comprehension of their pharmacology has been obscured by their conformational complexity, that has precluded deciphering their structural properties until the early 2010’s. To that respect, intracellular VHHs have been instrumental in stabilizing G protein-coupled receptors in active conformations in order to solve their structure, possibly bound to their primary transducers, G proteins or β-arrestins. In contrast, the modulatory properties of VHHs recognizing the intracellular regions of G protein-coupled receptors on the induced signaling network have been poorly studied. In this review, we will present the advances that the intracellular VHHs have permitted in the field of GPCR signaling and trafficking. We will also discuss the methodological hurdles that linger the discovery of modulatory intracellular VHHs directed against GPCRs, as well as the opportunities they open in drug discovery.

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A Nanobody-Based Toolset to Monitor and Modify the Mitochondrial GTPase Miro1

Cited by 7 publications

References 80 publications

Nanobodies as novel tools to monitor the mitochondrial fission factor Drp1

Nanobodies as novel tools to monitor the mitochondrial fission factor Drp1

Applications of nanobodies in brain diseases

Intracellular VHHs to monitor and modulate GPCR signaling

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