Biology subjects

Hong, H. J.

Publications and source records attributed to Hong, H. J..

6 recordsLinked to original sources

bioRxiv2025

Heartbeat-like dynamics drives oxygen activation in methane monooxygenase

Soluble methane monooxygenase (sMMO) is an enzyme that hydroxylates methane (CH4), a potent greenhouse gas, at non-heme di-iron active sites under atmospheric conditions. The regulatory component (MMOB) is essential for the catalytic activity of hydroxylase (MMOH) as it induces conformational changes in the active site and facilitating substrate ingress. Recent advances in cryogenic electron microscopy (cryo-EM) have enabled us to elucidate the high resolution picture of sMMO catalytic mechanism. We describe the 2.85 [A] cryo-EM structure of MMOH-MMOB, with one equivalent of MMOB bound to MMOH (H-1B), which is in contrast with previously solved crystal structures. MMOB allosterically regulates the MMOH protomer ({beta}{gamma}) and induces conformational changes that propagate from the surface to the di-iron coordination site. The N-terminal region of the MMOH {beta}-subunit (NT-H{beta}) stabilizes helices essential for iron coordination and oxygen activation. The MMOB-bound protomer (HBA, {beta}{gamma}B) presents the first structural report of a 2.7 [A] Fe{middle dot}{middle dot}{middle dot}Fe distance, while the non-MMOB-bound protomer (HBB, {beta}{gamma}) and MMOH display a 3.1 [A] distance. The coordination of Fe-ligands is maintained by the structural stabilization provided by the {beta}- and {gamma}-subunits of MMOH. This novel cryo-EM structure reveals new coordination environments, offering crucial mechanistic insights into sMMO catalysis.

biochemistry↗

bioRxiv2024

Adaptive thermogenesis is mediated by GDF15 via the GFRAL neuronal axis in mice

Adaptive thermogenesis is a key homeostatic mechanism that primarily occurs in brown adipocytes and enables the maintenance of body temperature. Although this process involves coordinated responses in multiple tissues, including the browning of white adipocytes, the precise inter-organ crosstalk underlying adaptive thermogenesis is unclear. Here, we investigate the pivotal role of the GDNF family receptor alpha-like (GFRAL) neuronal axis in modulating compensatory thermogenic responses in brown and white adipose depots under stress conditions, specifically the mitochondrial unfolded protein response resulting from genetic modification and cold exposure. We employed a mouse model with targeted deletion of Crif1 in the mitoribosomes of brown adipocytes, and cold-exposed mice and immortalized adipocytes, to uncover the mechanism by which mitochondrial stress-induced growth differentiation factor 15 (GDF15) expression affects metabolism and facilitates adaptive thermogenesis. We found that Crif1 deletion resulted in browning of inguinal white adipose depots, increased energy expenditure, reduced food intake, and resistance to weight gain. Retrograde neuronal tracing established that GFRAL-positive neurons in the hindbrain and sympathetic preganglionic neurons in the spinal cord mediated the GDF15-associated browning of inguinal white adipose tissue. Intervention studies using antisense oligonucleotides to inhibit Gfral expression blunted the effect of Crif1 deletion on energy expenditure and food intake, further confirming the essential role the GFRAL axis plays in GDF15-driven thermogenic adaptation in white adipose tissue. Our findings suggest that the GFRAL neuronal axis is key in coordinating the adaptive thermogenic response across multiple tissues and adipose depots, thereby ensuring metabolic homeostasis during mitochondrial stress.

molecular biology↗

bioRxiv2023

Translation initiation factor eIF1.2 promotes Toxoplasma stage conversion by regulating levels of key differentiation factors

AbstractsThe parasite Toxoplasma gondii persists in its hosts by converting from replicating tachyzoites to latent bradyzoites housed in tissue cysts. The molecular mechanisms that mediate T. gondii differentiation remain poorly understood. Through a mutagenesis screen, we identified translation initiation factor eIF1.2 as a critical factor for T. gondii differentiation. A F97L mutation in eIF1.2 or the genetic ablation of eIF1.2 ({Delta}eif1.2) markedly impeded bradyzoite cyst formation in vitro and in vivo. We demonstrated, at single-molecule level, that the eIF1.2 F97L mutation impacts the scanning process of the ribosome preinitiation complex on a model mRNA. RNA sequencing and ribosome profiling experiments unveiled that {Delta}eif1.2 parasites are defective in upregulating bradyzoite induction factors BFD1 and BFD2 during stress-induced differentiation. Forced expression of BFD1 or BFD2 significantly restored differentiation in {Delta}eif1.2 parasites. Together, our findings suggest that eIF1.2 functions by regulating the translation of key differentiation factors necessary to establish chronic toxoplasmosis.

microbiology↗

bioRxiv2023

Single-Molecule Tracking Reveals Dynamic Regulation of Ribosomal Scanning

To initiate protein synthesis, the eukaryotic ribosomal pre-initiation complex must survey a messenger RNA leader sequence to identify the correct start codon.1 This pre-initiation complex motion through the leader, termed scanning, is coordinated by an intricate and highly-dynamic assemblage of translation factors, mRNA, initiator tRNA, and the small ribosomal subunit.2,3,4 Fundamental aspects of scanning dynamics remain poorly understood: estimates of its rate vary widely, and mechanisms that establish and regulate the motion remain largely unknown. Here we show, at the single-molecule level, that the Saccharomyces cerevisiae pre-initiation complex scans a diverse set of mRNA leaders at a rate of 10 - 20 nt s-1. Our data quantitatively support a scanning mechanism in which the mRNA leader is inspected base by base, essentially unidirectionally, and with modest sensitivity to mRNA structure. Unexpectedly, scanning bypasses canonical start sites where the initiator tRNA is present but GTP hydrolysis in the pre-initiation complex is impaired. Conversely, binding of the S. cerevisiae poly(A)-binding protein Pab1p to its own mRNA leader hinders scanning in a concentration-dependent manner. At saturating, physiological concentrations, Pab1p prolongs scanning by more than four-fold, evoking an autoregulation mechanism for translation initiation. Our data provide a real-time mechanistic framework for scanning regulation and energetics.

biophysics↗

bioRxiv2021

Single-Molecule Dynamics of SARS-CoV-2 5' Cap Recognition by Human eIF4F

Coronaviruses initiate translation through recognition of the viral RNA 5 m7GpppAm cap by translation factor eIF4F. eIF4F is a heterotrimeric protein complex with cap-binding, RNA-binding, and RNA helicase activities. Modulating eIF4F function through cellular regulation or small-molecule inhibition impacts coronavirus replication, including for SARS-CoV-2. Translation initiation involves highly coordinated dynamics of translation factors with messenger or viral RNA. However, how the eIF4F subunits coordinate on the initiation timescale to define cap-binding efficiency remains incompletely understood. Here we report that translation supported by the SARS-CoV-2 5-UTR is highly sensitive to eIF4A inhibition by rocaglamide. Through a single-molecule fluorescence approach that reports on eIF4E-cap interaction, we dissect how eIF4F subunits contribute to cap-recognition efficiency on the SARS-CoV-2 5 UTR. We find that free eIF4A enhances cap accessibility for eIF4E binding, but eIF4G alone does not change the kinetics of eIF4E-RNA interaction. Conversely, formation of the full eIF4F complex significantly alters eIF4E-cap interaction, suggesting that coordinated eIF4E and eIF4A activities establish the net eIF4F-cap recognition efficiency. Moreover, the eIF4F complex formed with phosphomimetic eIF4E(S209D) binds the viral UTR more efficiently than with wild-type eIF4E. These results highlight a dynamic interplay of eIF4F subunits and mRNA that determines cap-recognition efficiency.

biophysics↗

bioRxiv2020

De novo design of modular and tunable allosteric biosensors

Naturally occurring allosteric protein switches have been repurposed for developing novel biosensors and reporters for cellular and clinical applications 1, but the number of such switches is limited, and engineering them is often challenging as each is different. Here, we show that a very general class of allosteric protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which binding of a peptide key triggers biological outputs of interest 2. Using broadly applicable design principles, we allosterically couple binding of protein analytes of interest to the reconstitution of luciferase activity and a bioluminescent readout through the association of designed lock and key proteins. Because the sensor is based purely on thermodynamic coupling of analyte binding to switch activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We demonstrate the modularity of this platform by creating biosensors that, with little optimization, sensitively detect the anti-apoptosis protein Bcl-2, the hIgG1 Fc domain, the Her2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac Troponin I and an anti-Hepatitis B virus (HBV) antibody that achieve the sub-nanomolar sensitivity necessary to detect clinically relevant concentrations of these molecules. Given the current need for diagnostic tools for tracking COVID-19 3, we use the approach to design sensors of antibodies against SARS-CoV-2 protein epitopes and of the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. The latter, which incorporates a de novo designed RBD binder, has a limit of detection of 15pM with an up to seventeen fold increase in luminescence upon addition of RBD. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.

synthetic biology↗