Biology subjects

Lee, M.

Publications and source records attributed to Lee, M..

At least 91 records · Page 5Linked to original sources

bioRxiv2025

SIRT6 activator fucoidan extends healthspan and lifespan in aged wild-type mice

SIRT6 is a protein deacylase, deacetylase, and mono-ADP-ribosylase (mADPr) regulating biological pathways important for longevity including DNA repair and silencing of LINE1 retrotransposons. SIRT6 knockout mice die by 30 days of age, whereas SIRT6 overexpression increases lifespan in male mice. Finding safe pharmacological activators of SIRT6 would have clinical benefits. Fucoidan, a polysaccharide purified from brown seaweed, has been identified as an activator of SIRT6 deacetylation activity. Here, we show that fucoidan also activates SIRT6 mADPr activity, which was shown to be elevated in certain human centenarians. Administering fucoidan to aged mice led to a significant increase in median lifespan in male mice. Both male and female mice demonstrated a marked reduction in frailty and epigenetic age. Fucoidan-treated mice showed repression of LINE1 elements suggesting that the beneficial effects of fucoidan are mediated, at least in part, by SIRT6. As brown seaweed rich in fucoidan is a popular food item in South Korea and Japan, countries with the highest life expectancy, we propose that fucoidan supplementation should be explored as a safe strategy for activating SIRT6 and improving human healthspan and lifespan.

molecular biology↗

bioRxiv2025

A human electrophysiological biomarker of Fragile X Syndrome is shared in V1 of Fmr1 KO mice and caused by loss of FMRP in cortical excitatory neurons

Predicting clinical therapeutic outcomes from preclinical animal studies remains an obstacle to developing treatments for neuropsychiatric disorders. Electrophysiological biomarkers analyzed consistently across species could bridge this divide. In humans, alpha oscillations in the resting state electroencephalogram (rsEEG) are altered in many disorders, but these disruptions have not yet been characterized in animal models. Here, we employ a uniform analytical method to show in males with fragile X syndrome (FXS) that the slowed alpha oscillations observed in adults are also present in children and in visual cortex of adult and juvenile Fmr1-/y mice. We find that alpha-like oscillations in mice reflect the differential activity of two classes of inhibitory interneurons, but the phenotype is caused by deletion of Fmr1 specifically in cortical excitatory neurons. These results provide a framework for studying alpha oscillation disruptions across species, advance understanding of a critical rsEEG signature in the human brain and inform the cellular basis for a putative biomarker of FXS.

neuroscience↗

bioRxiv2025

GLYCO-2: a Tool to Quantify Glycan Shielding of Glycosylated Proteins with Improved Data Processing and Computation Speed

MotivationGlycans bound to glycoproteins mediate immune response, including antibody recognition and immune evasion. Previously, we developed an in silico tool GLYCO (GLYcan COverage) to quantify the glycan shielding of a protein surface, applying it to various studies. However, GLYCO lacks sufficient computational efficiency when analyzing larger datasets. ResultsHere we introduce GLYCO-2 which improves the computational speed by [~]4- fold compared to GLYCO by adopting a new analytical cylinder method with k-d trees. GLYCO-2 can calculate glycan shielding from a single coordinate file or from multiple frames derived from molecular dynamics simulations accounting for the inherent flexibility of oligosaccharides. We applied GLYCO-2 to quantify glycan shielding of influenza hemagglutinin (HA) proteins across diverse subtypes that infect humans, revealing an increasing trend in glycan shielding over time within each subtype, likely contributing to immune evasion. Overall, the enhanced computational efficiency of GLYCO-2 allows for faster and easier quantification of glycans, which contributes to the understand of glycan shielding effects in fields such as immunology and vaccine design. Availability and implementationGLYCO-2 is freely available at https://github.com/meteosR/GLYCO-2/ Contactmyungjin.lee@nih.gov or reda.rawi@nih.gov Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2025

Sequencing by Expansion (SBX) -- a novel, high-throughput single-molecule sequencing technology

Remarkable advances in high-throughput sequencing have enabled major biological discoveries and clinical applications, but achieving wider distribution and use depends critically on further improvements in scale and cost reduction. Nanopore sequencing has long held the promise for such progress, but has had limited market penetration. This is because efficient and accurate nanopore sequencing of nucleic acids has been challenged by fundamental signal-to-noise limitations resulting from the poor spatial resolution and molecular distinction of nucleobases. Here, we describe Sequencing by Expansion (SBX), a single-molecule sequencing technology that overcomes these limitations by using a biochemical conversion process to encode the sequence of a target nucleic acid molecule into an Xpandomer, a highly measurable surrogate polymer. Expanding over 50 times longer than the parent DNA templates, Xpandomers are engineered with high signal-to-noise reporter codes to enable facile, high-accuracy nanopore sequencing. We demonstrate the performance of SBX and present the specialized molecular structures, chemistries, enzymes and methods that enable it. The innovative molecular and systems engineering in SBX create a transformative technology to address the needs of existing and emerging sequencing applications.

genomics↗

bioRxiv2025

From Mechanistic Interpretability to Mechanistic Biology: Training, Evaluating, and Interpreting Sparse Autoencoders on Protein Language Models

Protein language models (pLMs) are powerful predictors of protein structure and function, learning through unsupervised training on millions of protein sequences. pLMs are thought to capture common motifs in protein sequences, but the specifics of pLM features are not well understood. Identifying these features would not only shed light on how pLMs work, but potentially uncover novel protein biology--studying the model to study the biology. Motivated by this, we train sparse autoencoders (SAEs) on the residual stream of a pLM, ESM-2. By characterizing SAE features, we determine that pLMs use a combination of generic features and family-specific features to represent a protein. In addition, we demonstrate how known sequence determinants of properties such as thermostability and subcellular localization can be identified by linear probing of SAE features. For predictive features without known functional associations, we hypothesize their role in unknown mechanisms and provide visualization tools to aid their interpretation. Our study gives a better understanding of the limitations of pLMs, and demonstrates how SAE features can be used to help generate hypotheses for biological mechanisms. We release our code, model weights and feature visualizer.

bioinformatics↗

bioRxiv2025

Spatial transcriptomics reveals heterogeneous cell-cell interactions among brain regions in a cuprizone model consistent with multiple sclerosis lesions

The cuprizone (CPZ) model is widely used for modeling demyelination in multiple sclerosis (MS) and for testing potential remyelination therapies. We integrated single-cell and spatial transcriptomics (ST) to fine map the spatial cellular and molecular responses during de and remyelination. ST revealed global demyelination and neuroinflammation in the brain beyond the corpus callosum, with region-specific differences. We identified oligodendroglia and microglia as two major cell types with significant transcriptomic changes in the model. Ligand receptor pairing analyses predicted growth factor and phagocytic pathway enrichment during demyelination, which is consistent with changes in MS lesions. During remyelination, while mature oligodendrocytes nearly reversed their phenotype back to the control state, microglia remained associated with the demyelination phenotype. Finally, astrocytes in the CPZ model had the greatest preservation of disease-associated modules to MS lesions, while the MOL, OPC, and microglia showed moderate to low preservation, which overall suggested that the CPZ model had moderate translatability to chronically active MS lesions.

neuroscience↗

bioRxiv2025

A nonhuman primate model mirrors human congenital cytomegalovirus infection and reveals a spectrum of vertical transmission outcomes

Congenital cytomegalovirus (cCMV) is the leading infectious cause of birth defects worldwide, yet immune determinants of protection to inform design of a maternal vaccine remain elusive. Here, we characterized the outcome of primary rhesus CMV (RhCMV) infection during pregnancy in an immune competent nonhuman primate (NHP) model. RhCMV DNA was detected in amniotic fluid and/or fetal tissues in six of 12 (50% placental transmission) CMV-naive rhesus macaque dams inoculated intravenously with RhCMV in early second trimester gestation. Widespread tissue dissemination dominated by one of two inoculated RhCMV strains was present in one fetus (8.3% cCMV disease). Placental RhCMV transmission was associated with elevated fetal and maternal plasma TNF-alpha and reduced maternal brain-derived neurotrophic factor and IL-10 levels. CMV exposure during pregnancy had a broad impact on the placenta and fetus even in the absence of congenital infection as evidenced by RhCMV infection at the maternal-fetal interface in all 12 dams, along with significantly reduced placental efficiency and fetal growth metrics compared to gestation-matched control pregnancies. This NHP model recapitulates key aspects of human cCMV and provides new insight into barriers and biomarkers of successful vertical transmission. One sentence summary: The nonhuman primate model mirrors the epidemiology of human congenital CMV (cCMV) after primary infection and reveals its transmission bottlenecks.

immunology↗

bioRxiv2025

Complex roles for proliferating cell nuclear antigen in human cytomegalovirus replication

DNA viruses at once elicit and commandeer host pathways, including DNA repair pathways for virus replication. Despite encoding its own DNA polymerase and processivity factor, human cytomegalovirus (HCMV) recruits the cellular processivity factor, proliferating cell nuclear antigen (PCNA) and specialized host DNA polymerases involved in translesion synthesis (TLS) to replication compartments (RCs) where viral DNA (vDNA) is synthesized. While the recruitment of TLS polymerases is important for viral genome stability, the role of PCNA is poorly understood. PCNA function in DNA repair is regulated by monoubiquitination (mUb) or SUMOylation of PCNA at lysine 164 (K164). We find that mUb-PCNA increases over the course of infection, and modification of K164 is required for PCNA-mediated restriction of virus replication. mUb-PCNA plays important known roles in recruiting TLS polymerases to DNA, which we have shown are important for viral genome integrity and diversity, represented by novel junctions and single nucleotide variants (SNVs), respectively. We find that PCNA drives SNVs on vDNA similar to Y-family TLS polymerases, but that this did not require modification at K164. Unlike TLS polymerases, PCNA was dispensable for preventing large scale rearrangements on vDNA. These striking results suggest separable PCNA-dependent and - independent functions of TLS polymerases on vDNA. By extension, these results imply roles for TLS polymerase beyond their canonical function in TLS in host biology. These findings highlight PCNA as a complex restriction factor for HCMV infection, likely with multiple distinct roles, and provides new insights into the PCNA-mediated regulation of DNA synthesis and repair in viral infection. IMPORTANCEGenome synthesis is a critical step of virus life cycles and a major target of antiviral drugs. Human cytomegalovirus, like other herpesviruses, encodes machinery sufficient for viral DNA synthesis and relies on host factors for efficient replication. We have shown that host DNA repair factors play important roles in HCMV replication, but our understanding of this is incomplete. Building on previous findings that specialized host DNA polymerases contribute to HCMV genome integrity and diversity, we sought to determine the importance of PCNA, the central polymerase regulator. PCNA associates with nascent viral DNA and restricts HCMV replication. While PCNA is dispensable for genome integrity, it contributes to genome diversity. Our findings suggest that host polymerases function on viral genomes by separable PCNA-dependent and - independent mechanisms. Through revealing complex roles for PCNA in HCMV replication, this study expands the repertoire of host DNA synthesis and repair proteins hijacked by this ubiquitous herpesvirus.

microbiology↗

bioRxiv2025

Cycling Molecular Assemblies for Selective Cancer Cell Golgi Disruption

The Golgi apparatus is a critical organelle responsible for intracellular trafficking and signaling, orchestrating essential processes such as protein and lipid sorting1-5. Dysregulation of its function has been implicated in various pathologies, including obesity, diabetes, and cancer, highlighting its importance as a potential therapeutic target. Despite this, the development of tools to selectively target the Golgi in specific cell types remain a significant unmet challenge in imaging and drug discovery. Golgi-specific enzyme activities, such as those mediated by protein acyltransferases and thioesterases6, offer an untapped opportunity to develop subcellularly localized therapeutics. Current approaches predominantly rely on direct protein binding but lack the necessary cell selectivity7, underscoring the unmet need for innovative strategies to selectively disrupt Golgi function in cancer cells. Here, we report the development of cycling molecular assemblies (CyMA), a novel class of small peptide derivatives (e.g., dipeptides), which exploit the unique enzymatic environment of the Golgi to establish futile cycles of reversible S-acylation. These assemblies selectively accumulate in cancer cell Golgi, interfering with protein S-acylation cycles and disrupting organelle homeostasis. CyMA impair key Golgi functions, including protein trafficking, glycosylation, and secretion, while demonstrating selective sparing hepatocytes and immune cells such as M1 macrophages. This selective activity represents a paradigm shift, utilizing an enzyme switch and leveraging intracellular environment rather than direct protein binding. Unlike conventional approaches, CyMA reduce tumor growth, drug resistance, and metastasis by pleiotropically disrupting Golgi related functions. By demonstrating the potential of futile cycles as a therapeutic strategy8, this study introduces a generalizable method for targeting organelle-specific enzyme activities. These findings not only underscore the therapeutic potential of CyMA in cancer but also pave the way for future applications in other Golgi-associated diseases.

biochemistry↗

bioRxiv2024

The NPxY motifs in the β integrin regulate serotonin response and mechanosensation in C. elegans

BackgroundIntegrin is an {beta} heterodimeric receptor to the extracellular matrix; its binding to the matrix recruits focal adhesions to two NPxY motifs, the tyrosine phosphorylation sites in the cytoplasmic domain. Studies found that replacing tyrosines (Y) with phenylalanines (F) in the motif of {beta}1 integrin displayed little developmental or behavioral defects. However, the tyrosine-to-alanine (A) caused embryonic lethality. ResultsHere we report novel functions of the NPxY motifs in C. elegans pat-3 {beta} integrin. The membrane-proximal non-phosphorylation pat-3(Y792F) mutation caused hypersensitive egg laying in serotonin, which is more prominent than the membrane-distal NPxY804. The double non-phosphorylatable pat-3(YYFF) mutant exhibited serotonin hypersensitivity and defective egg retention. The phosphomimetic NPxY, pat-3(Y804E), mutant displayed a reduced egg laying in response to serotonin and fluoxetine, suggesting that the NPxY phosphorylation is associated with vulval contraction and serotonin sensitivity. Additionally, pat-3(Y792A), pat-3(Y792F), pat-3(Y804E), and pat-3(YYFF) mutants exhibited mechanosensation defects, demonstrating that NPxY phosphorylation regulates sensory neuron activity. Further revealed that exogenous serotonin reduced mechanosensation, while blocking serotonin secretion rescued the mechanosensation of pat-3 NPxY mutants, suggesting that integrin NPxY modulates serotonin levels in C. elegans. ConclusionOur results underscore the functional importance of pat-3 NPxY motifs in muscle and neurons, potentially linking integrin NPxY motifs to neurotransmitter response and mechanosensory functions.

cell biology↗

bioRxiv2024

SOX2-induced IL-1alpha-mediated immune suppression drives epithelial dysplasia malignant transformation

Squamous cell carcinomas (SCC) are often preceded by potentially malignant precursor lesions, most of which remain benign. The terminal exhaustion phenotypes of effector T-cells and the accumulation of myeloid-derived suppressor cells (MDSC) have been thoroughly characterized in established SCC. However, it is unclear what precancerous lesions harbor a bona fide high risk for malignant transformation and how precancerous epithelial dysplasia drives the immune system to the point of no return. Here we show that expression of SRY-box transcription factor 2 (SOX2) in precancerous lesions imparts an irreversible risk that recruits suppressive myeloid cells by promoting the release of CCL2. We developed a unique genetically engineered mouse model (GEMM) to recapitulate the malignant transformation of epithelial dysplasia to SCC in the oral mucosa with high histologic and phenotypic fidelity. Using a combination of longitudinal human specimens and the Sox2-GEMM, we found that the myeloid cells in precancerous epithelial dysplasia exhibit a distinctive dichotomous profile featuring high levels of IL-1-SLC2A1 and low levels of type-I interferon (IFN-I) signatures, which occurs before SCC emerges histologically. Brief priming of myeloid cells with IL-1 desensitizes them to IFN-I agonists and makes myeloid-derived suppressor cells (MDSC) even more suppressive of T-cell activation. Mechanistically, IL-1 activation represses the expression of DHHC3/7 enzymes, which are responsible for the palmitoylation of stimulator of interferon genes (STING). Early blockade of IL1 signaling using pharmacologic and genetic approaches similarly reduces MDSC and SLC2A1high myeloid cells, suppresses epithelial dysplasia transformation, and extends survival. This work establishes a previously unrecognized SOX2-CCL2-IL1 pathway that leads to irreversible immune escape when precancerous epithelial lesions transform.

cancer biology↗

bioRxiv2024

The C. elegans LON-1 protein requires its CAP domain for function in regulating body size and BMP signaling

The CAP (Cysteine-rich secretory proteins, Antigen-5, Pathogenesis-Related) proteins are widely expressed and have been implicated to play diverse roles ranging from mammalian reproduction to plant immune response. Increasing evidence supports a role of CAP proteins in lipid binding. The C. elegans CAP protein LON-1 is known to regulate body size and Bone Morphogenetic Protein (BMP) signaling. LON-1 is a secreted protein with a conserved CAP domain and a C-terminal unstructured domain with no homology to other proteins. In this study, we report that the C-Terminal Domain (CTD) of LON-1 is dispensable for its function. Instead, key conserved residues located in the CAP domain are critical for LON-1 function in vivo. We further showed that LON-1 is capable of binding sterol, but not fatty acid, in vitro, and that certain key residues implicated in LON-1 function in vivo are also important for LON-1 sterol binding in vitro. These findings suggest a role of LON-1 in regulating body size and BMP signaling via sterol binding. ARTICLE SUMMARYThe C. elegans LON-1 protein is known to regulate body size and Bone Morphogenetic Protein (BMP) signaling. However, its molecular mode of action remains elusive. This study shows that LON-1 can bind sterol, but not fatty acid, in vitro. Furthermore, key conserved residues in the CAP domain of LON-1 are required for LON-1 function in vivo. These findings suggest a role of LON-1 in regulating body size and BMP signaling via sterol binding.

genetics↗

bioRxiv2024

Learning Impairments in Fmr1-/- mice on an Audio-Visual Temporal Pattern Discrimination Task.

Estimating time and making predictions is integral to our experience of the world. Given the importance of timing to most behaviors, disruptions in temporal processing and timed performance are reported in a number of neuropsychiatric disorders such as Schizophrenia, Autism Spectrum Disorder (ASD), Fragile X Syndrome (FXS), and Attention-deficit Hyperactivity Disorder (ADHD). Symptoms that implicitly include disruption in timing are atypical turn-taking during social interactions, unusual verbal intonations, poor reading, speech and language skills, inattention, delays in learning, and difficulties making predictions. Currently, there are no viable treatments for these symptoms, the reason being the underlying neural dysfunction that contributes to timing deficits in neuropsychiatric disorders is unknown. To address this unknown, we have designed a novel Temporal Pattern Discrimination Task (TPSD) for awake-behaving mice. Stimuli consist of audiovisual stimuli that differ in duration. Compared to Wild-Type (WT) mice, Fmr1-/- mice, a well-established mouse model of FXS, showed significant impairment in learning the TPSD task, as evidenced by reduced discriminability indices and atypical licking patterns. Often sensory information is multimodal and indeed studies show that learning in humans and rodents improves with multimodal stimuli than with unimodal stimuli. To test how the multimodal nature of stimuli impacted performance of Fmr1-/- mice, following training on the audiovisual stimuli, we tested mice on audio-only or visual-only stimuli. While WT mice showed significant disruption in performance when tested on unimodal stimuli, Fmr1-/- mice displayed equivalent performance on visual-only stimuli when compared to the multimodal task. Our novel task captures timing difficulties and multisensory integration issues in Fmr1-/- mice and provides an assay to examine the associated neural dysfunction.

animal behavior and cognition↗

bioRxiv2024

BRCA2 C-terminal clamp restructures RAD51 dimers to bind B-DNA for replication fork stability

Tumor suppressor protein BRCA2 acts with RAD51 in replication-fork protection (FP) and homology-directed DNA break repair (HDR). Critical for cancer etiology and therapy resistance, BRCA2 C-terminus was thought to stabilize RAD51-filaments after they assemble on single-stranded (ss)DNA. Here we determined the detailed crystal structure for BRCA2 C-terminal interaction-domain (TR2i) with ATP-bound RAD51 prior to DNA binding. In contrast to recombinogenic RAD51-filaments comprising extended ATP-bound RAD51 dimers, TR2i unexpectedly reshapes ATP-RAD51 into a unique dimer conformation accommodating double-stranded B-DNA binding unsuited for HDR initiation. Structural, biochemical, and molecular results with interface-guided mutations uncover TR2is FP mechanism. Proline-driven secondary-structure stabilizes residue triads and spans the RAD51 dimer engaging pivotal interactions of RAD51 M210 and BRCA2 S3291/P3292, the cyclin-dependent kinase (CDK) phosphorylation site that toggles between FP during S-phase and HDR in G2. TR2i evidently acts as an allosteric clamp switching RAD51 from ssDNA to double-stranded and B-DNA binding enforcing FP over HDR.

molecular biology↗

bioRxiv2024

Patterned wireless transcranial optogenetics generates artificial perception

Synthesizing perceivable artificial neural inputs independent of typical sensory channels remains a fundamental challenge in the development of next-generation brain-machine interfaces. Establishing a minimally invasive, wirelessly effective, and miniaturized platform with long-term stability is crucial for creating a clinically meaningful interface capable of mediating artificial perceptual feedback. In this study, we demonstrate a miniaturized fully implantable wireless transcranial optogenetic encoder designed to generate artificial perceptions through digitized optogenetic manipulation of large cortical ensembles. This platform enables the spatiotemporal orchestration of large-scale cortical activity for remote perception genesis via real-time wireless communication and control, with optimized device performance achieved by simulation-guided methods addressing light and heat propagation during operation. Cue discrimination during operant learning demonstrates the wireless genesis of artificial percepts sensed by mice, where spatial distance across large cortical networks and sequential order-based analyses of discrimination performance reveal principles that adhere to general perceptual rules. These conceptual and technical advancements expand our understanding of artificial neural syntax and its perception by the brain, guiding the evolution of next-generation brain-machine communication.

neuroscience↗

bioRxiv2024

Synaptonemal complex protects double-Holliday junctions during meiosis

Chromosomal linkages formed through crossover recombination are essential for accurate segregation of homologous chromosomes during meiosis1. The DNA events of recombination are linked to structural components of meiotic chromosomes2. Imperatively, the biased resolution of double-Holliday junction intermediates (dHJs) into crossovers3,4 occurs within the synaptonemal complex (SC), the meiosis-specific structure that mediates end-to-end synapsis of homologs during the pachytene stage5,6. However, the SCs roles in crossover-specific dHJ resolution remains unclear. Here, we show that key SC components function through dependent and interdependent relationships to protect dHJs from aberrant "dissolution" into noncrossover products. Conditional ablation experiments reveal that cohesin, the core of SC lateral elements, is required to maintain both synapsis and dHJ-associated crossover recombination complexes (CRCs) during pachytene. The SC central region transverse-filament protein is also required to maintain CRCs. Reciprocally, stability of the SC central region requires the continuous presence of CRCs, thereby coupling synapsis and desynapsis to dHJ formation and resolution. However, dHJ protection and maintenance of CRCs can occur without end-to-end homolog synapsis mediated by the central element of the SC central region. We conclude that local ensembles of SC components are sufficient to enable crossover-specific dHJ resolution and thereby ensure the linkage and segregation of homologous chromosomes.

cell biology↗

bioRxiv2024

Single-cell analysis of the somatic mutational landscape in human chondrocytes during aging and in osteoarthritis

Somatic mutation is now recognized as a cause of multiple human diseases other than cancer. Osteoarthritis (OA), a highly prevalent age-related disease, has been associated with increased chromosomal abnormalities in articular cartilage of OA patients. Thus far no systematic attempt has been made to characterize the somatic mutational landscape of chondrocytes during normal aging and in affected cartilage of OA patients. Here we used single-cell whole genome sequencing to quantitatively analyze single-nucleotide variants (SNVs) and small insertions and deletions (InDels) in 100 single chondrocytes isolated from the cartilage of hip femoral heads of 17 subjects aged from 26 to 90 years, including 9 OA patients and 8 non-OA donors. Both SNVs and InDels were found to accumulate with age in chondrocytes with a clock-like mutational signature. Surprisingly, the age-related accumulation rate of these mutations was found to be lower in OA chondrocytes compared with chondrocytes from non-OA control subjects.

genomics↗

bioRxiv2024

Context-specific inhibition of mitochondrial ribosomes by phenicol and oxazolidinone antibiotics

The antibiotics chloramphenicol (CHL) and oxazolidinones including linezolid (LZD) are known to inhibit mitochondrial translation. This can result in serious, potentially deadly, side effects when used therapeutically. Although the mechanism by which CHL and LZD inhibit bacterial ribosomes has been elucidated in detail, their mechanism of action against mitochondrial ribosomes has yet to be explored. CHL and oxazolidinones bind to the ribosomal peptidyl transfer center (PTC) of the bacterial ribosome and prevent incorporation of incoming amino acids under specific sequence contexts, causing ribosomes to stall only at certain sequences. Through mitoribosome profiling, we show that inhibition of mitochondrial ribosomes is similarly context-specific - CHL and LZD lead to mitoribosome stalling primarily when there is an alanine, serine, or threonine in the penultimate position of the nascent peptide chain. We further validate context-specific stalling through in vitro translation assays. A high resolution cryo-EM structure of LZD bound to the PTC of the human mitoribosome shows extensive similarity to the mode of bacterial inhibition and also suggests potential avenues for altering selectivity. Our findings could help inform the rational development of future, less mitotoxic, antibiotics, which are critically needed in the current era of increasing antimicrobial resistance.

biochemistry↗