Biology subjects

Park, S. K.

Publications and source records attributed to Park, S. K..

15 recordsLinked to original sources

bioRxiv2025

Functional dissection of SPOP on the amino acid level reveals a comprehensive functional landscape of variants during tumorigenesis

Numerous proteins display pleiotropic functions in different clinical contexts. However, molecular mechanism underlying such effects is rarely understood. Speckle-type POZ protein (SPOP) is a typical example, exhibiting tumor-suppressing or -promoting effects in different tumor types in accordance with different amino acid changes; specifically, two distinct sets of variants in SPOP are commonly found in subsets of prostate cancer and endometrial cancer patients. To comprehensively characterize the functional landscape of SPOP alteration, we performed a deep mutational screening (DMS), elucidating the functionality of 7,933 out of 8,228 possible single amino acid changes (96.4% coverage). Leveraging the observation that overexpression of human SPOP leads to yeast growth arrest, we assessed the functionality of each variant using a yeast proliferation assay. In addition, our approach combined long-read and short-read sequencing. Finally, our DMS model enables a clear distinction of likely-loss-of-function (LoF) variants that are enriched in prostate cancers and reveals their differential characteristics in both protein structure and genetic assessments. These results demonstrate the utility of our approach in high resolution mapping and amino-acid-level interpretation of protein function. Significance statementGenetic mutations often play different roles in cancer, driving or suppressing tumor growth depending on their molecular context. The protein SPOP is a striking example that acts as either a tumor-suppressor or oncogene depending on the specific mutation and tissue. However, understanding how thousands of possible mutations alter its function has been a major challenge. Here, we applied deep mutational scanning to experimentally measure the functional effects of nearly every possible amino acid change in SPOP. This work provides the first comprehensive functional map of SPOP variants, not only advancing basic knowledge of cancer biology but also establishing a framework for interpreting patient mutations in precision medicine.

genetics↗

bioRxiv2025

Structural basis for the evolution of a domesticated group II intron-like reverse transcriptase to function in host cell DNA repair

A previous study found that a domesticated bacterial group II intron-like reverse transcriptase (G2L4 RT) functions in double-strand break repair (DSBR) via microhomology-mediated end joining (MMEJ) and that a mobile group II intron-encoded RT has a basal DSBR activity that uses conserved structural features of non-LTR-retroelement RTs. Here, we determined G2L4 RT apoenzyme and snap-back DNA synthesis structures revealing novel structural adaptations that optimized its cellular function in DSBR. These included a unique RT3a structure that stabilizes the apoenzyme in an inactive conformation until encountering an appropriate substrate; a longer N-terminal extension/RT0-loop with conserved residues that together with a modified active site favors strand annealing; and a conserved dimer interface that localizes G2L4 RT homodimers to DSBR sites with both monomers positioned for MMEJ. Our findings reveal how a non-LTR-retroelement RT evolved a dedicated cellular function and suggest new ways of optimizing these RTs for genome engineering applications.

molecular biology↗

bioRxiv2024

Enhancer activation from transposable elements in extrachromosomal DNA

Extrachromosomal DNA (ecDNA) drives oncogene amplification and intratumoral heterogeneity in aggressive cancers. While transposable element (TE) reactivation is common in cancer, its role on ecDNA remains unexplored. Here, we map the 3D architecture of MYC-amplified ecDNA in colorectal cancer cells and identify 68 ecDNA-interacting elements (EIEs)--genomic loci enriched for TEs that are frequently integrated onto ecDNA. We focus on an L1M4a1#LINE/L1 fragment co-amplified with MYC, which functions only in the ecDNA amplified context. Using CRISPR-CATCH, CRISPR interference, and reporter assays, we confirm its presence on ecDNA, enhancer activity, and essentiality for cancer cell fitness. These findings reveal that repetitive elements can be reactivated and co-opted as functional rather than inactive sequences on ecDNA, potentially driving oncogene expression and tumor evolution. Our study uncovers a mechanism by which ecDNA harnesses repetitive elements to shape cancer phenotypes, with implications for diagnosis and therapy.

genetics↗

bioRxiv2024

Endoplasmic reticulum stress inhibition preserves mitochondrial function and cell survival during early onset of isoniazid-induced oxidative stress

A comprehensive understanding of isoniazid (INH)-mediated hepatotoxic effects is essential for developing strategies to predict and prevent severe liver toxicity in tuberculosis treatment. Our study utilized multi-omics profiling to investigate the toxic effects of INH, revealing significant involvement of endoplasmic reticulum (ER) stress, mitochondrial impairment, redox imbalance, and altered metabolism. Followed-up mechanistic studies revealed that INH triggered the generation of cytosolic reactive oxygen species (ROS) and the activation of the Nrf2 signaling pathway prior to mitochondrial ROS accumulation. Subsequently, INH disrupted mitochondrial function by impairing respiratory complexes I-IV and caused mitochondrial membrane proton leaks without affecting ATP synthase activity, together leading to mitochondrial depolarization and reduced ATP production. These disturbances enhanced mitochondrial fission and mitophagy. While much attention has been given to mitochondrial dysfunction and oxidative stress in INH-induced hepatotoxicity, our findings highlight the potential of inhibiting ER stress during early INH exposure to mitigate cytosolic and mitochondrial oxidative stress. We further revealed the critical role of Nrf2 signaling in protecting liver cells under INH-induced oxidative stress by maintaining redox homeostasis and enabling metabolic reprogramming via regulating the expression of antioxidant genes and cellular lipid abundance. We also identified other antioxidant pathways (e.g., selenocompound metabolism, HIF-1 signaling pathway, and pentose phosphate pathway) as potential alternative mechanisms besides Nrf2 signaling in response to INH-induced oxidative stress. In conclusion, our research emphasizes the importance of ER stress, redox imbalance, metabolic changes, and mitochondrial dysfunction underlying INH-induced hepatotoxicity.

pharmacology and toxicology↗

bioRxiv2024

Placental and Immune Cell DNA Methylation Reference Panel for Bulk Tissue Cell Composition

To distinguish DNA methylation (DNAm) from cell proportion changes in whole placental tissue research, we developed a robust cell type-specific DNAm reference to estimate cell composition. We collated newly collected and existing cell type DNAm profiles quantified via Illumina EPIC or 450k microarrays. To estimate cell composition, we deconvoluted whole placental samples (n=36) with robust partial correlation based on the top 50 hyper- and hypomethylated sites per cell type. To test deconvolution performance, we evaluated RMSE in predicting principal component one of DNAm variation in 204 external placental samples. We analyzed DNAm profiles (n=368,435 sites) from 12 cell types: cytotrophoblasts (n=18), endothelial cells (n=19), Hofbauer cells (n=26), stromal cells (n=21), syncytiotrophoblasts (n=4), six lymphocyte types (n=36), and nucleated red blood cells (n=11). Median cell composition was consistent with placental biology: 60.4% syncytiotrophoblast, 17.1% stromal, 8.8% endothelial, 4.5% cytotrophoblast, 3.9% Hofbauer, 1.7% nucleated red blood cells, and 1.2% neutrophils. Our expanded reference outperformed an existing reference in predicting DNAm variation (15.4% variance explained, IQR=21.61) with cell composition estimates (RMSE:10.51 vs. 11.43, p-value<0.001). This cell type reference can robustly estimate cell composition from whole placental DNAm data to detect important cell types, reveal biological mechanisms, and improve casual inference.

genomics↗

bioRxiv2023

Mechanisms used for cDNA synthesis and site-specific integration of RNA into DNA genomes by a reverse transcriptase-Cas1 fusion protein

Reverse transcriptase-Cas1 (RT-Cas1) fusion proteins found in some CRISPR systems enable spacer acquisition from both RNA and DNA, but the mechanism of RNA spacer acquisition has remained unclear. Here, we found Marinomonas mediterranea RT-Cas1/Cas2 adds short 3-DNA (dN) tails to RNA protospacers enabling their direct integration into CRISPR arrays as 3-dN-RNA/cDNA duplexes or 3-dN-RNAs at rates comparable to similarly configured DNAs. Reverse transcription of RNA protospacers occurs by multiple mechanisms, including recently described de novo initiation, protein priming with any dNTP, and use of short exogenous or synthesized DNA oligomer primers, enabling synthesis of cDNAs from diverse RNAs without fixed sequence requirements. The integration of 3-dN-RNAs or single-stranded (ss) DNAs is favored over duplexes at higher protospacer concentrations, potentially relevant to spacer acquisition from abundant pathogen RNAs or ssDNA fragments generated by phage-defense nucleases. Our findings reveal novel mechanisms for site-specifically integrating RNA into DNA genomes with potential biotechnological applications.

molecular biology↗

bioRxiv2022

Vutiglabridin improves neurodegeneration in MPTP-induced Parkinson's disease mice by targeting mitochondrial paraoxonase-2

Parkinsons disease (PD), characterized by degeneration of dopaminergic neurons, share pathogenic features with obesity, including mitochondrial dysfunction and oxidative stress. Paraoxonase 2 (PON2) is an inner mitochondrial membrane protein that is highly expressed in dopaminergic neurons and is involved in the regulation of mitochondrial oxidative stress. However, no drug targeting PON2 has ever been developed for the treatment of PD. Here, we show that vutiglabridin, a clinical phase 2-stage drug for the treatment of obesity, has therapeutic effects in PD models, targeting mitochondrial PON2. Vutiglabridin penetrates into the brain, binds to PON2, and restores 1-methyl-4-phenylpyridinium (MPP+)-induced mitochondrial dysfunction in SH-SY5Y neuroblastoma cells. Knockdown of PON2 by lentiviral shRNA infection abolished the effects of vutiglabridin on mitochondria. In mice, vutiglabridin significantly alleviated motor impairments and damage to dopaminergic neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model, and these effects were also abolished in PON2-knockdown mice, suggesting that vutiglabridin is neuroprotective via PON2. Extensive in vitro and in vivo assessment of potential neurotoxicity showed vutiglabridin to be safe. Overall, these findings provide support for the clinical development of vutiglabridin as a novel PON2 modulator for the treatment of PD. One Sentence SummaryTargeting paraoxonase-2 by a clinical-stage compound vutiglabridin provides neuroprotective effects in preclinical models of Parkinsons disease.

neuroscience↗

bioRxiv2022

Development and validation of PainFace, a software platform that simplifies and standardizes mouse grimace analyses

Facial grimaces are now commonly used to quantify spontaneous pain in mice and other mammalian species, but scoring remains subjective and relies on humans with very different levels of proficiency. Here, we developed a Mouse Grimace Scale (MGS) for black-coated (C57BL/6) mice consisting of four facial action units (orbitals, nose, ears, whiskers). We used this scale to generate ground truth data from over 70,000 images of black mice in different settings. With this large data set, we developed a deep neural network and cloud-based software platform called PainFace (http://painface.net) that accurately scores facial grimaces of black mice on a 0-8 scale. PainFace generates over two orders of magnitude more MGS data than humans can realistically achieve, and at superhuman speed. By analyzing the frequency distribution of grimace scores, we found that mice spent >7x more time in a high grimace state following laparotomy surgery relative to sham surgery controls. The analgesic carprofen reduced the amount of time animals spent in this high grimace state after surgery. Specific facial action unit score combinations were overrepresented following laparotomy surgery, suggesting that characteristic facial expressions are associated with a high grimace state. We performed validation experiments in two labs located in different countries to demonstrate reproducibility of the PainFace platform. To further enhance rigor and reproducibility, we will invite pain researchers to beta test PainFace and then incorporate their feedback into the software and manuscript prior to peer review. While this study is focused on mice, PainFace was designed to simplify, standardize, and scale up grimace analyses with many other mammalian species, including humans.

neuroscience↗

bioRxiv2022

Npas4-mediated dopaminergic regulation of fear memory states

Amygdala circuitry encodes associations between conditioned stimuli and aversive unconditioned stimuli, and also controls fear expression (Pape and Pare, 2010). However, whether and how irrelevant information for unpaired conditioned stimuli (CS-) is discretely processed, and how it was influenced by stress remain unknown. CS- memory is retrievable immediately after fear conditioning, but then becomes silent after memory consolidation in mice. Synaptic pathway from the lateral to the anterior basal amygdala gates the expression of CS- memory, depending upon Npas4-mediated Drd4 synthesis. The upregulation of Npas4-Drd4 axis, which is precluded by corticosterone, shifts functional states of neural engrams for CS- memory toward silent states and limits its retrievability. In here, we demonstrate the cellular and molecular mechanisms regulating the functional states of neural engrams, which can be switched or maintained, supporting discriminative memory.

neuroscience↗

bioRxiv2022

Group II Intron-Like Reverse TranscriptasesFunction in Double-Strand Break Repair by Microhomology-Mediated End Joining

Bacteria encode free-standing reverse transcriptases (RTs) of unknown function that are closely related to group II intron-encoded RTs. Here, we found that a Pseudomonas aeruginosa group II intron-like RT (G2L4 RT) with YIDD instead of YADD at its active site functions in DNA repair in its native host and when transferred into Escherichia coli. G2L4 RT has biochemical activities strikingly similar to those of human DNA repair polymerase{theta} and uses them for translesion DNA synthesis and double-strand break repair (DSBR) via microhomology-mediated end-joining (MMEJ) in vitro and in vivo. We also found that a group II intron RT can function similarly to G2L4 RT in DNA repair, with reciprocal substitutions at the active site showing an I residue favors MMEJ and an A residue favors primer extension in both enzymes. The DNA repair functions of these enzymes utilize conserved structural features of non-LTR-retroelement RTs, including human LINE-1 and other eukaryotic non-LTR-retrotransposon RTs, suggesting such enzymes may have an inherent ability to function in DSBR in a wide range of organisms.

molecular biology↗

bioRxiv2022

Retinoic acid-induced protein 14 controls dendritic spine dynamics associated with depressive-like behaviors

Dendritic spines are the central postsynaptic machinery that determines synaptic function. The F-actin within dendritic spines regulates their dynamic formation and elimination. Rai14 is an F-actin-regulating protein with a membrane-shaping function. Here, we identified the roles of Rai14 for the regulation of dendritic spine dynamics associated with stress-induced depressive-like behaviors. Rai14-deficient neurons failed to maintain a proper dendritic spine density in the Rai14+/- mouse brain, resulting in impaired functional synaptic activity. Rai14 was protected from degradation by complex formation with Tara, and accumulated in the dendritic spine neck, thereby enhancing spine maintenance. Concurrently, Rai14 deficiency in mice altered gene expression profile relevant to depressive conditions and increased depressive-like behaviors. Moreover, Rai14 expression was reduced in the prefrontal cortex of the mouse stress model, which was blocked by antidepressant treatment. Thus, we propose that Rai14-dependent regulation of dendritic spines may underlie the plastic changes of neuronal connections relevant to depressive-like behaviors.

neuroscience↗

bioRxiv2021

Identification of in vivo CFTR conformations during biogenesis and upon misfolding by covalent protein painting (CPP)

In vivo characterization of protein structures or protein structural changes after perturbation is a major challenge. Therefore, experiments to characterize protein structures are typically performed in vitro and with highly purified proteins or protein complexes. Using a novel low-resolution method named Covalent Protein Painting (CPP) that allows the characterization of protein conformations in vivo, we are the first to report how an ion channel, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), is conformationally changed during biogenesis and channel opening in the cell. Our study led to the identification of a novel opening mechanism for CFTR by revealing that the interaction of the intracellular loop 2 (ICL2) with the nucleotide binding domain 2 (NDB2) of CFTR is needed for channel gating, and this interaction occurs concomitantly with changes to the narrow part of the pore and the walker A lysine in NBD1. However, the ICL2:NBD2 interface, which forms a "ball-in-a-socket" motif, is uncoupled during biogenesis, likely to prevent inadvertent channel activation during transport. In particular, solvent accessibility of lysine 273 (K273) in ICL2 changes with the opening and closing of the channel. Mutation of K273 severely impaired CFTR biogenesis and led to accumulation of CFTR in the Golgi and TGN. CPP further revealed that, even upon treatment with current approved drugs or at permissive temperature, the uncoupled state of ICL2 is a prominent feature of the misfolded CFTR mutants {Delta}F508 and N1303K that cause Cystic Fibrosis (CF), which suggests that stabilization of this interface could produce a more efficient CF drug. CPP is able to characterize a protein in its native environment and measure the effect of complex PTMs and protein interactions on protein structure, making it broadly applicable and invaluable for the development of new therapies.

molecular biology↗

bioRxiv2019

Praziquantel activates a schistosome transient receptor potential channel

The anthelmintic drug praziquantel (PZQ) is used to treat schistosomiasis, a neglected tropical disease that affects over 200 million people. The target of PZQ in the blood fluke responsible for this disease is unknown. Here, we demonstrate that PZQ activates a transient receptor potential (TRP) channel found in parasitic schistosomes and other PZQ-sensitive parasites.

pharmacology and toxicology↗

bioRxiv2018

Evaluating causal associations between previously reported risk factors and epithelial ovarian cancer: a Mendelian randomization analysis

BackgroundPreviously reported observational associations between risk factors and epithelial ovarian cancer (EOC) could reflect residual confounding, reverse causation, or measurement error. Mendelian randomization (MR) uses genetic variants as proxies for modifiable risk factors to strengthen causal inference in observational studies.\n\nMethodsWe used MR to evaluate the causal role of 13 previously reported risk factors in overall and histotype-specific EOC in up to 25,509 case subjects and 40,941 controls in the Ovarian Cancer Association Consortium. Inverse-variance weighted models were employed to generate effect estimates and MR-Egger, weighted median, and weighted mode were performed to examine evidence of horizontal pleiotropy. A Bonferroni-corrected P-value threshold was used to establish \"strong evidence\" (P<0.0038) and \"suggestive evidence\" (0.0038<P<0.05) for associations.\n\nResultsThere was strong or suggestive evidence that 9 of 13 risk factors were causally associated with overall or histotype-specific EOC. Genetic liability to endometriosis was strongly associated with EOC (OR per log odds higher liability: 1.27,95%CI:1.16-1.40;P=6.94x10-7) and lifetime smoking exposure was suggestively associated with EOC (OR per unit increase in smoking score:1.36,95%CI:1.04-1.78;P=0.02). In histotype-stratified analyses, the strongest associations found were between: height and clear cell carcinoma (OR per SD increase:1.36,95%CI:1.15-1.61;P=0.0003); age at natural menopause and endometrioid carcinoma (OR per year later onset:1.09,95% CI:1.02-1.16;P=0.007); and genetic liability to polycystic ovary syndrome and endometrioid carcinoma (OR per log odds higher liability:0.74,95% CI:0.62-0.90;P=0.002). There was little evidence that genetic liability to type 2 diabetes, parity, or circulating levels of 25-hydroxyvitamin D and sex hormone-binding globulin were associated with ovarian cancer or its subtypes.\n\nConclusionsOur comprehensive examination of possible etiological drivers of ovarian carcinogenesis supports a causal role for few of these factors in epithelial ovarian cancer and suggests distinct etiologies across histotypes.

epidemiology↗

bioRxiv2017

Amyloid accumulation drives proteome-wide alterations in mouse models of Alzheimers disease like pathology

Amyloid beta (A{beta}) peptides impair multiple cellular pathways in the brain and play a causative role in Alzheimers disease (AD) pathology, but how the brain proteome is remodeled during this process is unknown. To identify new protein networks associated with AD-like pathology, we performed global quantitative proteomic analysis in three mouse models at pre- and post-symptomatic ages. Our analysis revealed a robust and consistent increase in Apolipoprotein E (ApoE) levels in nearly all transgenic brain regions with increased A{beta} levels. Taken together with prior findings on ApoE driving A{beta} accumulation, this analysis points to a pathological dysregulation of the ApoE-A{beta} axis. We also found dysregulation of protein networks involved in excitatory synaptic transmission consistent with AD pathophysiology. Targeted analysis of the AMPA receptor complex revealed a specific loss of TARP{gamma}-2, a key AMPA receptor trafficking protein. Expression of TARP{gamma}-2 in vivo in hAPP transgenic mice led to a restoration of AMPA currents. This database of proteome alterations represents a unique resource for the identification of protein alterations responsible for AD.\n\nHighlightsO_LIProteomic analysis of mouse brains with AD-like pathology reveals stark remodeling\nC_LIO_LIProteomic evidence points to a dysregulation of ApoE levels associated with A{beta} clearance rather than production\nC_LIO_LICo-expression analysis found distinctly impaired synapse and mitochondria modules\nC_LIO_LIIn-depth analyses of AMPAR complex points to loss of TARP{gamma}-2, which may compromise synapses in AD\nC_LI\n\neTOC BlurbProteome-wide profiling of brain tissue from three mouse models of AD-like pathology reveals A{beta}, brain region, and age dependent alterations of protein levels. This resource provides a new global protein expression atlas for the Alzheimers disease research community.

neuroscience↗