Cherlhyun Jeong

MutS switches between two fundamentally distinct clamps during mismatch repair

Single-molecule trajectory analysis has suggested DNA repair proteins may carry out a one-dimensional (1D) search on naked DNA encompassing >10,000 nucleotides. Organized cellular DNA (chromatin) presents substantial barriers to such lengthy searches. Using dynamic single-molecule fluorescence resonance energy transfer, we determined that the mismatch repair (MMR) initiation protein MutS forms a transient clamp that scans duplex DNA for mismatched nucleotides by 1D diffusion for 1 s (~700 base pairs) while in continuous rotational contact with the DNA. Mismatch identification provokes ATP binding (3 s) that induces distinctly different MutS sliding clamps with unusual stability on DNA (~600 s), which may be released by adjacent single-stranded DNA (ssDNA). These observations suggest that ATP transforms short-lived MutS lesion scanning clamps into highly stable MMR signaling clamps that are capable of competing with chromatin and recruiting MMR machinery, yet are recycled by ssDNA excision tracts.

Synergistic nanomedicine by combined gene and photothermal therapy

To date, various nanomaterials with the ability for gene delivery or photothermal effect have been developed in the field of biomedicine. The therapeutic potential of these nanomaterials has raised considerable interests in their use in potential next-generation strategies for effective anticancer therapy. In particular, the advancement of novel nanomedicines utilizing both therapeutic strategies of gene delivery and photothermal effect has generated much optimism regarding the imminent development of effective and successful cancer treatments. In this review, we discuss current research progress with regard to combined gene and photothermal therapy. This review focuses on synergistic therapeutic systems combining gene regulation and photothermal ablation as well as logically designed nano-carriers aimed at enhancing the delivery efficiency of therapeutic genes using the photothermal effect. The examples detailed in this review provide insight to further our understanding of combinatorial gene and photothermal therapy, thus paving the way for the design of promising nanomedicines.

Facile fabrication and application of near-IR light-responsive drug release system based on gold nanorods and phase change material

Near-IR (NIR)-responsive drug delivery systems have received enormous attention because of their good biocompatibility and the high biological penetration of NIR. This work describes the facile fabrication and anticancer effect of a NIR-responsive drug delivery system based on the nano-assembly of mesoporous silica-coated gold nanorods (AuNR@mSiO2) and a phase change material (PCM). The core gold nanorods in AuNR@mSiO2 provided excellent NIR sensitivity, and the mesoporous silica shell provided an increased reservoir of loaded drug. The PCM was applied as both a thermosensitive gatekeeper and a medium for a hydrophobic anticancer drug. The photothermal effect of the AuNRs under NIR irradiation facilitated a rapid release of the drug through the temperature-sensitive phase transition of PCM at just above body temperature. An enhanced anticancer effect was observed against various cell lines, and intracellular release of the drug was easily monitored by live cell imaging. Our system does not require any complicated synthetic methods for fabrication, and PCM is easily capable of solubilizing various hydrophobic drugs. Therefore, this system has significant potential for broad application in the biomedical field as a delivery system for hydrophobic drugs with NIR-responsive behavior.

Beta-Amyloid Oligomers Activate Apoptotic BAK Pore for Cytochrome c Release

In Alzheimers disease, cytochrome c-dependent apoptosis is a crucial pathway in neuronal cell death. Although beta-amyloid (Aβ) oligomers are known to be the neurotoxins responsible for neuronal cell death, the underlying mechanisms remain largely elusive. Here, we report that the oligomeric form of synthetic Aβ of 42 amino acids elicits death of HT-22 cells. But, when expression of a bcl-2 family protein BAK is suppressed by siRNA, Aβ oligomer-induced cell death was reduced. Furthermore, significant reduction of cytochrome c release was observed with mitochondria isolated from BAK siRNA-treated HT-22 cells. Our in vitro experiments demonstrate that Aβ oligomers bind to BAK on the membrane and induce apoptotic BAK pores and cytochrome c release. Thus, the results suggest that Aβ oligomers function as apoptotic ligands and hijack the intrinsic apoptotic pathway to cause unintended neuronal cell death.

Dynamic light scattering analysis of SNARE-driven membrane fusion and the effects of SNARE-binding flavonoids

Soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins generate energy required for membrane fusion. They form a parallelly aligned four-helix bundle called the SNARE complex, whose formation is initiated from the N terminus and proceeds toward the membrane-proximal C terminus. Previously, we have shown that this zippering-like process can be controlled by several flavonoids that bind to the intermediate structures formed during the SNARE zippering. Here, our aim was to test whether the fluorescence resonance energy transfer signals that are observed during the inner leaflet mixing assay indeed represent the hemifused vesicles. We show that changes in vesicle size accompanying the merging of bilayers is a good measure of progression of the membrane fusion. Two merging vesicles with the same size D in diameter exhibited their hydrodynamic diameters 2D + d (d, intermembrane distance), 2D and 2D as membrane fusion progressed from vesicle docking to hemifusion and full fusion, respectively. A dynamic light scattering assay of membrane fusion suggested that myricetin stopped membrane fusion at the hemifusion state, whereas delphinidin and cyanidin prevented the docking of the vesicles. These results are consistent with our previous findings in fluorescence resonance energy transfer assays.

A Chemical Controller of SNARE-Driven Membrane Fusion That Primes Vesicles for Ca2+-Triggered Millisecond Exocytosis

Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca(2+) stimulates fusion-pore formation in a millisecond time scale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca(2+). When 10 μM Ca(2+) was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps.

Structural and biochemical insights into the role of testis-expressed gene 14 (TEX14) in forming the stable intercellular bridges of germ cells

Significance Germ cells possess the inherent ability to inactivate cell abscission through TEX14 (testis-expressed gene 14), and they may provide information on inactivation of the abscission in abnormal cells, including cancer cells. Structural and functional studies of how TEX14 inactivates germ cell abscission reveal that the AxGPPx3YxPP motif of TEX14 competitively binds to CEP55-EABR [endosomal sorting complex required for transport (ESCRT) and ALIX-binding region] to prevent the recruitment of ALIX, which is a component of the ESCRT machinery and which contains the AxGPPx3Y motif. Multiexperiment analyses of CEP55-EABR–TEX14 interactions showed how the TEX14 peptide binds dominantly to CEP55-EABR in the presence of ALIX and safeguards the intercellular bridges of germ cells. Intercellular bridges are a conserved feature of spermatogenesis in mammalian germ cells and derive from arresting cell abscission at the final stage of cytokinesis. However, it remains to be fully understood how germ cell abscission is arrested in the presence of general cytokinesis components. The TEX14 (testis-expressed gene 14) protein is recruited to the midbody and plays a key role in the inactivation of germ cell abscission. To gain insights into the structural organization of TEX14 at the midbody, we have determined the crystal structures of the EABR [endosomal sorting complex required for transport (ESCRT) and ALIX-binding region] of CEP55 bound to the TEX14 peptide (or its chimeric peptides) and performed functional characterization of the CEP55–TEX14 interaction by multiexperiment analyses. We show that TEX14 interacts with CEP55-EABR via its AxGPPx3Y (Ala793, Gly795, Pro796, Pro797, and Tyr801) and PP (Pro803 and Pro804) sequences, which together form the AxGPPx3YxPP motif. TEX14 competitively binds to CEP55-EABR to prevent the recruitment of ALIX, which is a component of the ESCRT machinery with the AxGPPx3Y motif. We also demonstrate that a high affinity and a low dissociation rate of TEX14 to CEP55, and an increase in the local concentration of TEX14, cooperatively prevent ALIX from recruiting ESCRT complexes to the midbody. The action mechanism of TEX14 suggests a scheme of how to inactivate the abscission of abnormal cells, including cancer cells.

Bioreducible guanidinylated polyethylenimine for efficient gene delivery.

Cationic polymers are known to afford efficient gene transfection. However, cytotoxicity remains a problem at the molecular weight for optimal DNA delivery. As such, optimized polymeric gene delivery systems are still a sought‐after research goal. A guanidinylated bioreducible branched polyethylenimine (GBPEI‐SS) was synthesized by using a disulfide bond to crosslink the guanidinylated BPEI (GBPEI). GBPEI‐SS showed sufficient plasmid DNA (pDNA) condensation ability. The physicochemical properties of GBPEI‐SS demonstrate that it has the appropriate size (∼200 nm) and surface potential (∼30 mV) at a nitrogen‐to‐phosphorus ratio of 10. No significant toxicity was observed, possibly due to bioreducibility and to the guanidine group delocalizing the positive charge of the primary amine in BPEI. Compared with the nonguanidinylated analogue, BPEI‐SS, GBPEI‐SS showed enhanced transfection efficiency owing to increased cellular uptake and efficient pDNA release by cleavage of disulfide bonds. This system is very efficient for delivering pDNA into cells, thereby achieving high transfection efficiency and low cytotoxicity.

Ferritin nanocage with intrinsically disordered proteins and affibody: A platform for tumor targeting with extended pharmacokinetics

ABSTRACT Ferritin nanocages are of particular interest as a novel platform for drug and vaccine delivery, diagnosis, biomineralization scaffold and more, due to their perfect and complex symmetry, ideal physical properties, high biocompatibility, low toxicity profiles as well as easy manipulation by genetic or chemical strategies. However, a short half‐life is still a hurdle for the translation of ferritin‐based nanomedicines into the clinic. Here, we developed a series of rationally designed long circulating ferritin nanocages (LCFNs) with ‘Intrinsically Disordered Proteins (IDP)’ as a stealth layer for extending the half‐life of ferritin nanocages. Through predictions with 3D modelling, the LCFNs were designed, generated and their pharmacokinetic parameters including half‐life, clearance rate, mean residence time, and more, were evaluated by qualitative and quantitative analysis. LCFNs have a tenfold increased half‐life and overall improved pharmacokinetic parameters compared to wild‐type ferritin nanocages (wtFN), corresponding to the low binding against bone marrow‐derived macrophages (BMDMs) and endothelial cells. Subsequently, a tumor targeting moiety, epidermal growth factor receptor (EGFR)‐targeting affibody peptide, was fused to LCFNs for evaluating their potential as a theragnostic platform. The tumor targeting‐LCFNs successfully accumulated to the tumor tissue, by efficient targeting via active and passive properties, and also the shielding effect of IDP in vivo. This strategy can be applied to other protein‐based nanocages for further progressing their use in the field of nanomedicine. Graphical abstract Long circulating ferritin nanocages are designed by 3D modelling. Modified by intrinsically disordered protein (IDP) clouds, this novel biocompatible nanocage platform can be applied in the field of nanomedicine. Figure. No caption available.

Structural consequences of aglycosylated IgG Fc variants evolved for FcγRI binding.

In contrast to the glycosylated IgG antibodies secreted by human plasma cells, the aglycosylated IgG antibodies produced by bacteria are unable to bind FcγRs expressed on the surface of immune effector cells and cannot trigger immune effector functions. To avoid glycan heterogeneity problems, elicit novel effector functions, and produce therapeutic antibodies with effector function using a simple bacterial expression system, FcγRI-specific Fc-engineered aglycosylated antibodies, Fc11 (E382V) and Fc (E382V/M428I), containing mutations in the CH3 region, were isolated in a previous study. To elucidate the relationship between FcγRI binding affinity and the structural dynamics of the upper CH2 region of Fc induced by the CH3 mutations, the conformational variation of Fc variants was observed by single-molecule Förster resonance energy transfer (FRET) analysis using alternating-laser excitation (ALEX). In sharp contrast to wild-type Fc, which exhibits a highly dynamic upper CH2 region, the mutations in the CH3 region significantly stabilized the upper CH2 region. The results indicate that conformational plasticity, as well as the openness of the upper CH2 region, is critical for FcγR binding and therapeutic effector functions of IgG antibodies.