Biology subjects

Ban, N.

Publications and source records attributed to Ban, N..

At least 19 recordsLinked to original sources

bioRxiv2026

PLCXD2 preferentially hydrolyzes phosphatidylinositol and supports retinal lipid homeostasis and photoreceptor integrity

PLCXD proteins have been linked to phosphoinositide metabolism for more than a decade, and PLCXD2 activity has been associated with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] depletion, yet its preferred direct substrate has remained unresolved. Here, cellular lipidomics showed that catalytically active PLCXD2 reduced the major phosphatidylinositol (PI) 38:4 species, with more modest phosphoinositide changes and increased diacylglycerol (DAG) and phosphatidic acid (PA). Using recombinant PLCXD2 and acyl-chain-matched substrates, we found that PLCXD2 hydrolyzed PI substantially more efficiently than PI(4,5)P2 and most other phosphoinositides tested. In Plcxd2-deficient retina, PI abundance was preserved, whereas DAG and PA were reduced without broad changes in major membrane phospholipid classes. PLCXD2 loss was associated with early structural abnormalities at the photoreceptor-bipolar synaptic interface, followed by outer nuclear layer thinning and reduced electroretinographic a-wave responses. These findings identify PLCXD2 as a PI-preferring mammalian PLC and reveal an endogenous role in retinal lipid homeostasis and outer retinal integrity.

biochemistry↗

bioRxiv2026

C-terminal evolutionary remodelling of isoleucyl-tRNA synthetases is a prokaryote-specific strategy for tuning aminoacylation rate

Aminoacyl-tRNA synthetases are the guardians of translational fidelity. Their complex function is mirrored by an elaborate structure, which includes multiple nested domains. While the evolutionary pressures that promoted the emergence of some domains, such as the editing domain, are clear, the pressures acting on other domains, particularly those at the C-terminus, are not. Here, we use a combination of kinetic analysis, X-ray crystallography, and bioinformatics to unveil the history and evolutionary forces that have shaped isoleucyl-tRNA synthetase (IleRS) domain structure. We find that the traditional classification into IleRS1 and IleRS2, based on the C-terminal tRNA-recognition domains, is incomplete, as it fails to capture features of the synthetic domain. Guided by the crystal structure of the Priestia megaterium IleRS2:tRNA complex, we removed key interactions between IleRS2 and its cognate tRNA and characterised their impact on enzyme activity. We found that D-loop interactions with the IleRS2 C-terminal region are non-essential in prokaryotes, and their loss can even increase catalytic turnover. Further, the zinc-binding domain of IleRS1 recognises the anticodon less stringently than the canonical C-terminal domain of IleRS2. Our data suggest that C-terminal evolutionary remodelling of IleRSs is an ongoing process with a historical precedent, consistent with selection for faster aminoacylation rate.

biochemistry↗

bioRxiv2025

NAC promotes co-translational folding at the ribosomal tunnel exit

The nascent polypeptide-associated complex (NAC) coordinates enzymatic modifications and membrane targeting of nascent chains during translation. While NACs function as a dynamic hub for other factors is well-established, its direct role in co-translational folding is unclear. By proteome-wide profiling NAC co-translational interactions in human cells, we found that NAC recognizes emerging segments enriched in hydrophobicity and -helical propensity, within folded domains of cytonuclear proteins. Single-molecule and structural analyses reveal that NAC, via its {beta}-barrel domain, dynamically interacts with nascent chains at the ribosomal tunnel exit and is capable of promoting on-pathway folding. Compartment-specific nascent chain interactions of NAC further elucidate its role in targeting to the endoplasmic reticulum and mitochondrial membrane protein biogenesis. Together, these findings show that NAC acts as a bona fide co-translational chaperone that facilitates early protein folding at the ribosomal tunnel exit, expanding its functional repertoire in protein biogenesis.

biochemistry↗

bioRxiv2024

An in vitro assay of MCTS1-DENR-dependent re-initiation and ribosome profiling uncover the activity of MCTS2 and distinct function of eIF2D

Ribosomes scanning from the mRNA 5' cap to the start codon may initiate at upstream open reading frames (uORFs), decreasing protein biosynthesis. Termination at a uORF can lead to re-initiation, where the 40S subunit resumes scanning and initiates another translation event downstream. In mammals, the noncanonical translation factors MCTS1-DENR participate in re-initiation at specific uORFs, but knowledge of other trans-acting factors and uORF features influencing re-initiation is limited. Here, we describe a cell-free re-initiation assay using HeLa cell lysates. Comparing in vivo and in vitro re-initiation activities on uORF-containing model reporters, we validate that MCTS1-DENR-dependent re-initiation is accurately recapitulated in vitro. Using this system and ribosome profiling in cultured cells, we found that knockdown of the homolog eIF2D causes widespread gene expression deregulation unrelated to uORF translation, suggesting distinct functions from MCTS1-DENR. Additionally, we identified MCTS2, encoded by a retrogene copy of Mcts1, as an alternative DENR partner that promotes re-initiation in vitro, providing a plausible explanation for the striking clinical differences associated with Denr vs. Mcts1 mutations in humans. Our findings on re-initiation and the new assay provide valuable insights and a powerful tool for future research on uORF features and trans-acting factors.

molecular biology↗

bioRxiv2024

ZNF574 is a Quality Control Factor For Defective Ribosome Biogenesis Intermediates

Eukaryotic ribosome assembly is an intricate process that involves four ribosomal RNAs, 80 ribosomal proteins, and over 200 biogenesis factors that take part in numerous interdependent steps. This complexity creates a large genetic space in which pathogenic mutations can occur. "Dead-end" ribosome intermediates that result from biogenesis errors are rapidly degraded, affirming the existence of quality control pathway(s) that monitor ribosome assembly. However, the factors that differentiate between on-path and dead-end intermediates are unknown. We engineered a system to perturb ribosome assembly in human cells and discovered that faulty ribosomes are degraded via the ubiquitin proteasome system. We identified ZNF574 as a key component of a novel quality control pathway, which we term the Ribosome Assembly Surveillance Pathway (RASP). Loss of ZNF574 results in the accumulation of faulty biogenesis intermediates that interfere with global ribosome production, further emphasizing the role of RASP in protein homeostasis and cellular health.

cell biology↗

bioRxiv2023

Universal features of Nsp1-mediated translational shutdown by coronaviruses

Nonstructural protein 1 (Nsp1) produced by coronaviruses shuts down host protein synthesis in infected cells. The C-terminal domain of SARS-CoV-2 Nsp1 was shown to bind to the small ribosomal subunit to inhibit translation, but it is not clear whether this mechanism is broadly used by coronaviruses, whether the N-terminal domain of Nsp1 binds the ribosome, or how Nsp1 specifically permits translation of viral mRNAs. Here, we investigated Nsp1 from three representative Betacoronaviruses - SARS-CoV-2, MERS-CoV, and Bat-Hp-CoV - using structural, biophysical, and biochemical assays. We revealed a conserved mechanism of host translational shutdown across the three coronaviruses. We further demonstrated that the N-terminal domain of Bat-Hp-CoV Nsp1 binds to the decoding center of the 40S subunit, where it would prevent mRNA and eIF1A binding. Structure-based biochemical experiments identified a conserved role of these inhibitory interactions in all three coronaviruses and showed that the same regions of Nsp1 are responsible for the preferential translation of viral mRNAs. Our results provide a mechanistic framework to understand how Betacoronaviruses overcome translational inhibition to produce viral proteins.

biochemistry↗

bioRxiv2023

Antibiotic hyper-resistance in a class I aminoacyl-tRNA synthetase with altered active site signature motif

Antibiotics target key biological processes that include protein synthesis. Bacteria respond by developing resistance, which increases rapidly due to antibiotics overuse. Mupirocin, a clinically used natural antibiotic, inhibits isoleucyl-tRNA synthetase (IleRS), an enzyme that links isoleucine to its tRNAIle for protein synthesis. Two IleRSs, mupirocin-sensitive IleRS1 and resistant IleRS2, coexist in bacteria. The latter may also be found in resistant Staphylococcus aureus clinical isolates. Here, we describe the structural basis of mupirocin resistance and unravel a mechanism of hyper-resistance evolved by some IleRS2 proteins. We surprisingly find that an up to 103-fold increase in resistance originates from alteration of the HIGH motif, a signature motif of the class I aminoacyl-tRNA synthetases to which IleRSs belong. The structural analysis demonstrates how an altered HIGH motif could be adopted in IleRS2 but not IleRS1, providing insight into an elegant mechanism for coevolution of the key catalytic motif and associated antibiotic resistance.

biochemistry↗

bioRxiv2022

Molecular basis of the TRAP complex function in ER protein biogenesis

The Translocon Associated Protein (TRAP) complex resides in the endoplasmic reticulum (ER) membrane and interacts with the Sec translocon and the translating ribosome to facilitate biogenesis of secretory and membrane proteins1-4. TRAP is essential for the secretion of many hormones, and its key role in the production of the hormone peptide insulin has been particularly well established5,6. The mechanism by which TRAP engages ribosomes and the translocon to facilitate translocation of protein clients in the secretory pathway is not clear. Here, we reveal the molecular architecture of the mammalian TRAP complex and how it engages the translating ribosome associated with Sec61 translocon on the ER membrane. The TRAP complex is anchored to the ribosome via a long tether and its position relative to the ribosome and the translocon is further stabilized by a finger-like loop. This spatial arrangement positions a cradle-like lumenal domain of TRAP below the protein conducting pore of the translocon for interactions with translocated nascent chains. The biological importance of these key interactions is evident by structure-guided TRAP mutations in C. elegans that lead to growth deficits associated with increased ER stress and defects in insulin secretion. Our findings elucidate the molecular basis of the TRAP complex in the biogenesis and translocation of proteins at the ER.

molecular biology↗

bioRxiv2022

Influence of amino acids and glucosinolates in three Brassicaceae vegetable plants on preference and performance of the green peach aphid: Myzus persicae

The green peach aphid, Myzus persicae (Sulzer) is a generalist pest of various host plants, whose feeding preference and growth performance mainly depends on the quantity and quality of nutrients and defensive metabolites in host plants. Here, we studied the preference and performance of M. persicae on three major Brassicaceae vegetables in China and measured nutrient (amino acids) and defensive (glucosinolates) metabolites in these plants. We found that M. persicae preferred and performed better on Chinese cabbage than cabbage and radish, which may be due to the relatively higher concentration of amino acids and lower levels of glucosinolates in their leaves. The glucosinolates level in cabbage leaves was ten times higher than the other two plants, while the amino acid concentration in radish was only half of the cabbage or Chinese cabbage. The higher concentration of glucosinolates in cabbage and lower levels of amino acids in radish may account for the poorer preference and growth of M. persicae on these two plants. These results suggest that both amino acids and glucosinolates in plants are important determinants of the preference and performance of M. persicae, which provide new knowledge for the cultivation and breeding of Brassicaceae vegetables.

ecology↗

bioRxiv2022

A pair of isoleucyl-tRNA synthetases in Bacilli fulfill complementary roles to enhance fitness and provide antibiotic resistance

Isoleucyl-tRNA synthetase (IleRS) is an essential enzyme that covalently couples isoleucine to the corresponding tRNA. Bacterial IleRSs group in two clades, ileS1 and ileS2, the latter bringing resistance to the natural antibiotic mupirocin. Generally, bacteria rely on either ileS1 or ileS2 as a standalone housekeeping gene. However, we have found an exception by noticing that Bacillus species with genomic ileS2 consistently also keep ileS1, which appears mandatory in the family Bacillaceae. Taking Priestia (Bacillus) megaterium as a model organism, we showed that PmIleRS1 is constitutively expressed, while PmIleRS2 is stress-induced. Both enzymes share the same level of the aminoacylation accuracy. Yet, PmIleRS1 exhibited a twofold faster aminoacylation turnover (kcat) than PmIleRS2 and permitted a notably faster cell-free translation. At the same time, PmIleRS2 displayed a 104-fold increase in its Ki for mupirocin, arguing that the aminoacylation turnover in IleRS2 could have been traded-off for antibiotic resistance. As expected, a P. megaterium strain deleted for ileS2 was mupirocin-sensitive. Interestingly, an attempt to construct a mupirocin-resistant strain lacking ileS1, a solution not found among species of the family Bacillaceae in nature, led to a viable but compromised strain. Our data suggests that PmIleRS1 is kept to promote fast translation, whereas PmIleRS2 is maintained to provide antibiotic resistance when needed. This is consistent with an emerging picture in which fast-growing organisms predominantly use IleRS1 for competitive survival.

biochemistry↗

bioRxiv2021

Violet light modulates the central nervous system to regulate memory and mood

Light stimuli from the external environment serves as a signal. Photoreceptors receive photons at the outer nuclear layer of the retina. Non-visual photoreceptors, such as opsin5 (also known as OPN5 or neuropsin), are expressed in the retinal ganglion cells (RGCs) and hypothalamus to regulate the circadian cycle and body temperature. Here, we show that violet light (VL) stimuli received by OPN5-positive RGCs are transmitted to the habenula brain region. VL improves memory in aged mice and simultaneously increases neural architecture-related genes such as oligodendrocyte-related genes in the hippocampus. In addition, VL improves depressive-like behaviors in the social defeat stress model in an OPN5 dependent manner. Following VL exposure, cFos activation is observed at the nucleus accumbens (NAc) and the paraventricular thalamic nucleus (PVT). Taken together, the results indicate that violet light modulates brain function such as memory and mood by transmitting the signal from RGCs to the habenula region in the brain.

neuroscience↗

bioRxiv2021

"XR Mark Test" Reveals Sensorimotor Body Representation in Toddlers

Research on multisensory integration--particularly visuo-proprioceptive integration--has renewed interest in how body representation develops in early childhood. In the past decade, researchers have attempted to adapt body-illusion tasks such as the rubber hand illusion for young children, yet difficulties with verbal reports and inconsistent behavioral responses limit their usefulness for toddlers. At the same time, children naturally begin to recognize their mirror image as self during the early toddler period, suggesting that mirror-based localization may offer a developmentally appropriate window onto emerging body representation. However, the classic mark test is not suitable for repeated measurements. We developed Bodytoypo, a cross-reality body-part localization task that projects a life-size self-image onto a screen and overlays AR markers on multiple body locations using real-time skeleton detection. Toddlers touched the corresponding location on their actual body, allowing quantitative evaluation of localization accuracy. Thirty 2- and 3-year-olds participated. We recorded first-touch outcomes, touch error, reaction times, and hand trajectories. Conventional coding was combined with an automated statistical analysis to estimate factors predicting touch error. Results showed that Bodytoypo is feasible for repeated use with toddlers and provides reliable indices of body-part localization. Automated statistical analyses identified several reaching strategies that appeared to reflect differences in localization precision. These findings suggest age-related changes in how visual information and motor prediction are weighted during visuo-proprioceptive integration. Bodytoypo offers a promising behavioral index for clarifying how sensorimotor information (body schema) and spatial understanding of body parts (body topology) jointly contribute to the early development of body representation.

animal behavior and cognition↗

bioRxiv2021

Stepwise maturation of the peptidyl transferase region of human mitoribosomes

Mitochondrial ribosomes are specialized for the synthesis of membrane proteins responsible for oxidative phosphorylation. Mammalian mitoribosomes diverged considerably from the ancestral bacterial ribosomes and feature dramatically reduced ribosomal RNAs. Structural basis of the mammalian mitochondrial ribosome assembly is currently not understood. Here we present eight distinct assembly intermediates of the human large mitoribosomal subunit involving 7 assembly factors. We discover that NSUN4-MTERF4 dimer plays a critical role in the process by stabilizing the 16S rRNA in a conformation that exposes the functionally important regions of rRNA for modification by MRM2 methyltransferase and quality control interactions with a conserved mitochondrial GTPase MTG2 that contacts the sarcin ricin loop and the immature active site. The successive action of these factors leads to the formation of the peptidyl transferase active site of the mitoribosome and the folding of the surrounding rRNA regions responsible for interactions with tRNAs and the small ribosomal subunit.

biochemistry↗

bioRxiv2021

Release, rescue and recycling: termination of translation in mammalian mitochondria

The mitochondrial translation system originates from a bacterial ancestor but has substantially diverged in the course of evolution. Here, we use single particle cryo-EM as a screening tool to identify mitochondrial translation termination mechanisms and to describe them in molecular detail. We show how mitochondria release factor 1a releases the nascent chain from the ribosome when it encounters the canonical stop codons UAA and UAG. Furthermore, we define how the peptidyl-tRNA hydrolase ICT1 acts as a rescue factor on mitoribosomes that have stalled on truncated messages to recover them for protein synthesis. Finally, we present near-atomic models detailing the process of mitochondrial ribosome recycling, to explain how a dedicated elongation factor, mtEFG2, has specialized for cooperation with the mitochondrial ribosome recycling factor to dissociate the mitoribosomal subunits at the end of the translation process. (134 words)

molecular biology↗

bioRxiv2020

Evolution of a virus-like architecture and packaging mechanism in arepurposed bacterial protein

Viruses are ubiquitous pathogens of global impact. Prompted by the hypothesis that their earliest progenitors recruited host proteins for virion formation, we have used stringent laboratory evolution to convert a bacterial enzyme lacking affinity for nucleic acids into an artificial nucleocapsid that efficiently packages and protects multiple copies of its own encoding mRNA. Revealing remarkable convergence on the molecular hallmarks of natural viruses, the accompanying changes reorganized the protein building blocks into an interlaced 240-subunit icosahedral capsid impermeable to nucleases, while emergence of a robust RNA stem-loop packaging cassette ensured high encapsidation yields and specificity. In addition to evincing a plausible evolutionary pathway for primordial viruses, these findings highlight practical strategies for developing non-viral carriers for diverse vaccine and delivery applications.

biochemistry↗

bioRxiv2020

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

Programmed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.

molecular biology↗

bioRxiv2020

SARS-CoV-2 Nsp1 binds ribosomal mRNA channel to inhibit translation

The non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, is the first viral protein that is synthesized in SARS-CoV-2 infected human cells to suppress host innate immune functions1,2. By combining cryo-electron microscopy and biochemical experiments, we show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes including the 43S pre-initiation complex. The protein inserts its C-terminal domain at the entrance to the mRNA channel where it interferes with mRNA binding. We observe potent translation inhibition in the presence of Nsp1 in lysates from human cells. Based on the high-resolution structure of the 40S-Nsp1 complex, we identify residues of Nsp1 crucial for mediating translation inhibition. We further show that the full-length 5’ untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines inhibition of translation by Nsp1 with efficient translation of the viral mRNA to achieve expression of viral genes3.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2020

SARM1 depletion rescues NMNAT1 dependent photoreceptor cell death and retinal degeneration.

Leber congenital amaurosis type 9 is an autosomal recessive retinopathy caused by mutations of the NAD+ synthesis enzyme NMNAT1. Despite the ubiquitous expression of NMNAT1, patients do not manifest pathologies other than retinal degeneration. Here we demonstrate that widespread NMNAT1 depletion in adult mice mirrors the human pathology, with selective loss of photoreceptors highlighting the exquisite vulnerability of these cells to NMNAT1 loss. Conditional deletion demonstrates that NMNAT1 is required within the photoreceptor. Mechanistically, loss of NMNAT1 activates the NADase SARM1, the central executioner of axon degeneration, to trigger photoreceptor death and vision loss. Hence, the essential function of NMNAT1 in photoreceptors is to inhibit SARM1, highlighting an unexpected shared mechanism between axonal degeneration and photoreceptor neurodegeneration. These results define a novel SARM1-dependent photoreceptor cell death pathway and identifies SARM1 as a therapeutic candidate for retinopathies.

neuroscience↗