6 research outputs found
항체광유전학 기술 개발을 통한 세포 내 단백질 기능 연구
No full text학위논문(박사) - 한국과학기술원 : 생명과학과, 2020.2,[v, 99 p. :]Intracellular antibodies, such as nanobodies and single-chain variable fragments (scFv), have become powerful tools for imaging, modulating, and neutralizing endogenous target proteins. Although a variety of antibody engineering techniques have been developed, an optogenetic tool for activating intracellular antibody to precisely control its target protein has not been reported. In chapter 1, I describe an optogenetically activatable intracellular antibody (Optobody) consisting of split antibody fragments and blue light-mediated heterodimerization domains. Blue light stimulation activates the optobody, inducing it to capture its target protein and subsequently inhibit the target. In chapter 2, I expanded this optobody platform by generating various optobodies from previously developed intracellular antibodies. In chapter 3, I demonstrated that photoactivation of a gelsolin (GSN) optobody and 2 adrenergic receptor (2AR) optobody shut down endogenous GSN activity and 2AR signaling, respectively. Applying the novel optogenetic platform to the broad pool of available intracellular antibodies will facilitate optogenetic manipulation of various endogenous proteins and may provide a basis for designing potential inducible drugs.한국과학기술원 :생명과학과
Programmable RNA base editing with photoactivatable CRISPR-Cas13
No full textAbstract CRISPR-Cas13 is widely used for programmable RNA interference, imaging, and editing. In this study, we develop a light-inducible Cas13 system called paCas13 by fusing Magnet with fragment pairs. The most effective split site, N351/C350, was identified and found to exhibit a low background and high inducibility. We observed significant light-induced perturbation of endogenous transcripts by paCas13. We further present a light-inducible base-editing system, herein called the padCas13 editor, by fusing ADAR2 to catalytically inactive paCas13 fragments. The padCas13 editor enabled reversible RNA editing under light and was effective in editing A-to-I and C-to-U RNA bases, targeting disease-relevant transcripts, and fine-tuning endogenous transcripts in mammalian cells in vitro. The padCas13 editor was also used to adjust post-translational modifications and demonstrated the ability to activate target transcripts in a mouse model in vivo. We therefore present a light-inducible RNA-modulating technique based on CRISPR-Cas13 that enables target RNAs to be diversely manipulated in vitro and in vivo, including through RNA degradation and base editing. The approach using the paCas13 system can be broadly applicable to manipulating RNA in various disease states and physiological processes, offering potential additional avenues for research and therapeutic development
Optogenetic activation of intracellular antibodies for direct modulation of endogenous proteins.
No full text© 2019, The Author(s), under exclusive licence to Springer Nature America, Inc.Intracellular antibodies have become powerful tools for imaging, modulating and neutralizing endogenous target proteins. Here, we describe an optogenetically activated intracellular antibody (optobody) consisting of split antibody fragments and blue-light inducible heterodimerization domains. We expanded this optobody platform by generating several optobodies from previously developed intracellular antibodies, and demonstrated that photoactivation of gelsolin and β2-adrenergic receptor (β2AR) optobodies suppressed endogenous gelsolin activity and β2AR signaling, respectively11Nsci
Optogenetic storage and release of protein and mRNA in live cells and animals
No full textCells compartmentalize biomolecules in membraneless structures called biomolecular condensates. While their roles in regulating cellular processes are increasingly understood, tools for their synthetic manipulation remain limited. Here, we introduce RELISR (Reversible Light-Induced Store and Release), an optogenetic condensate system that enables reversible storage and release of proteins or mRNAs. RELISR integrates multivalent scaffolds, optogenetic switches, and cargo-binding domains to trap cargo in the dark and release it upon blue-light exposure. We demonstrate its utility in primary neurons and show that light-triggered release of signaling proteins can modulate fibroblast morphology. Furthermore, light-induced release of cargo mRNA results in protein translation both in vitro and in live mice. RELISR thus provides a versatile platform for spatiotemporal control of protein activity and mRNA translation in complex biological systems, with broad potential for research and therapeutic applications.
Molecular basis for assembly and activation of the Hook3-KIF1C complex-dependent transport machinery
No full textMicrotubule-associated cargo transport, a central process governing the localization and movement of various cellular cargoes, is orchestrated by the coordination of two types of motor proteins (kinesins and dyneins), along with diverse adaptor and accessory proteins. Hook microtubule tethering protein 3 (Hook3) is a cargo adaptor that serves as a scaffold for recruiting kinesin family member 1C (KIF1C) and dynein, thereby regulating bidirectional cargo transport. Herein, we conduct structural and functional analyses of how Hook3 mediates KIF1C-dependent anterograde cargo transport through interaction with KIF1C and PTPN21. We verify the interactions among the three proteins and determine the crystal structure of the Hook3(553-624) - KIF1C(714-809) complex. Subsequent structure-based mutational analysis demonstrates that this complex formation is necessary and sufficient for the interaction between the full-length proteins in HEK293T cells and plays a key role in Hook3- and KIF1C-mediated anterograde transport in RPE1 cells. Thus, this study provides a basis for a comprehensive understanding of how Hook3 cooperates with other components during the initial steps of activation and assembly of the Hook3- and KIF1C-dependent cargo transport machinery.
Mutations in FAM50A suggest that Armfield XLID syndrome is a spliceosomopathy
No full textArmfield X-linked disability (XLID) disorder has previously been linked to a locus in Xq28. Here, the authors report rare missense variants in FAM50A at Xq28, show that FAM50A interacts with the spliceosome, and that mis-splicing is enriched in knockout zebrafish suggesting it is a spliceosomopathy
