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Biology subjects

Park, C. H.

Publications and source records attributed to Park, C. H..

12 recordsLinked to original sources

DAPK1-mediated parkin inactivation enhances neurotoxicity via MITOL-dependent degradation

Parkinsons disease (PD) is characterized by progressive neurodegeneration and is marked by the formation of Lewy bodies, which are intracellular aggregates primarily composed of -synuclein. Mitochondrial dysfunction and impaired protein degradation pathways play critical roles in the progression of PD, contributing to the loss of dopaminergic neurons in the substantia nigra. Phosphorylation of -synuclein promotes its aggregation, underscoring its role in disease progression. Parkin, an E3 ubiquitin ligase, is considered to be a pleiotropic, neuroprotective protein that modulates the mitochondrial quality control as well as metabolic turnover and the accumulation of -synuclein. Death-associated protein kinase 1 (DAPK1), involved in controlling apoptosis and autophagy, has recently emerged as an important factor in neurodegeneration. While DAPK1 is implicated in Alzheimers disease through its role in tau aggregation and amyloid-{beta} production, we demonstrate that DAPK1 plays a role in PD by phosphorylating parkin at Ser136 and Ser198. This phosphorylation causes the mitochondrial transport of parkin, enhancing interaction with mitochondria-localized E3 ubiquitin ligase MITOL and consequently leading to the degradation of parkin. As parkin is critical for neuroprotection, its degradation exacerbates the toxic effect of 6-hydroxydopamine, further compromising neuronal survival. These results indicate that DAPK1 acts as a previously unrecognized modulator of parkin and a key contributor to PD pathogenesis, bridging pathways of mitochondrial dysfunction, -synuclein aggregation, and neuronal cell death.

cell biology↗

THOC1 complexes with SIN3A to regulate R-loops and promote glioblastoma progression

ABSTRACTGlioblastoma (GBM), the most common and aggressive malignant brain tumor in adults, has a median survival of 21 months. To identify drivers of GBM proliferation, we conducted a CRISPR-knockout screen, which revealed THO Complex 1 (THOC1) as a key driver. Knocking down THOC1 significantly reduced GBM cell viability across patient-derived xenograft (PDX) lines, enhancing survival (p<0.01) in primary PDX models. Conversely, overexpressing THOC1 in non-cancerous cells bolstered viability, decreasing survival and causing tumor engraftment in vivo (p<0.01). Further investigation revealed THOC1s robust interaction with SIN3A, a histone deacetylase complex. Histone deacetylation has been previously shown to prevent the buildup of R-loops, structures that form normally during transcription but can be lethal in excess. We found that THOC1-knockdown leads to elevated R-loop levels and reduced histone deacetylation levels. Next, to understand the networks specifically regulated by THOC1-mediated R-loop prevention, we conducted unbiased RNA-sequencing on control and THOC1-knockdown GBM cells. We found that THOC1s role in R-loop prevention primarily affects telomeres, critical regions for cell replication. We further show that THOC1-knockdown results in significantly increased telomeric R-loop levels and shortened telomeres. Ultimately, this study suggests that targeting THOC1 shows promise as a therapeutic strategy to disrupt the delicate R-loop landscape and undermine GBMs replicative potential.

cancer biology↗

ADPKD-Causing Missense Variants in Polycystin-1 Disrupt Cell Surface Localization or Polycystin Channel Function

Autosomal dominant polycystic kidney disease (ADPKD) is the leading monogenic cause of kidney failure and affects millions of people worldwide. Despite the prevalence of this monogenic disorder, our limited mechanistic understanding of ADPKD has hindered therapeutic development. Here, we successfully developed bioassays that functionally classify missense variants in polycystin-1 (PC1). Strikingly, ADPKD pathogenic missense variants cluster into two major categories: 1) those that disrupt polycystin cell surface localization or 2) those that attenuate polycystin ion channel activity. We found that polycystin channels with defective surface localization could be rescued with a small molecule. We propose that small-molecule-based strategies to improve polycystin cell surface localization and channel function will be effective therapies for ADPKD patients.

cell biology↗

Real-Time Spatiotemporal Measurement of Extracellular Signaling Molecules Using an Aptamer Switch-Conjugated Hydrogel Matrix

Cells rely on secreted signaling molecules to coordinate essential biological functions including development, metabolism, and immunity. Unfortunately, such signaling processes remain difficult to measure with sufficient chemical specificity and temporal resolution. To address this need, we have developed an aptamer-conjugated hydrogel matrix that enables continuous fluorescent measurement of specific secreted analytes - in two dimensions, in real-time. As a proof of concept, we performed real-time imaging of Dictyostelium discoideum cells, a well-studied amoeba model wherein inter-cellular communication is performed though cAMP signaling. We engineered a set of aptamer switches that generate a rapid and reversible change in fluorescence in response to cAMP signals. By combining multiple switches with different dynamic ranges, we can measure cAMP concentrations spanning three orders of magnitude in a single experiment. These sensors are embedded within a biocompatible hydrogel on which cells are cultured and their cAMP secretions can be imaged using fluorescent microscopy. Using this aptamer-hydrogel material system, we achieved the first direct measurements of oscillatory cAMP signaling that correlate closely with previous indirect measurements. Using different aptamer switches, this approach could be generalized for measuring other secreted molecules to directly visualize diverse extracellular signaling processes and the biological effects that they trigger in recipient cells.

bioengineering↗

Regulation of adaptive growth decisions via phosphorylation of the TRAPPII complex in Arabidopsis

Plants often adapt to adverse or stress conditions via differential growth. The trans-Golgi Network (TGN) has been implicated in stress responses, but it is not clear in what capacity it mediates adaptive growth decisions. In this study, we assess the role of the TGN in stress responses by exploring the interactome of the Transport Protein Particle II (TRAPPII) complex, required for TGN structure and function. We identified physical and genetic interactions between TRAPPII and shaggy-like kinases (GSK3/AtSKs). Kinase assays and pharmacological inhibition provided in vitro and in vivo evidence that AtSKs target the TRAPPII-specific subunit AtTRS120/TRAPPC9. GSK3/AtSK phosphorylation sites in AtTRS120/TRAPPC9 were mutated, and the resulting AtTRS120 phosphovariants subjected to a variety of single and multiple stress conditions in planta. The non-phosphorylatable TRS120 mutant exhibited enhanced adaptation to multiple stress conditions and to osmotic stress whereas the phosphomimetic version was less resilient. Higher order inducible trappii atsk mutants had a synthetically enhanced defect in root gravitropism. Our results suggest that the TRAPPII phosphostatus mediates adaptive responses to abiotic cues. AtSKs are multifunctional kinases that integrate a broad range of signals. Similarly, the TRAPPII interactome is vast and considerably enriched in signaling components. An AtSK-TRAPPII interaction would integrate all levels of cellular organization and instruct the TGN, a central and highly discriminate cellular hub, as to how to mobilize and allocate resources to optimize growth and survival under limiting or adverse conditions.

plant biology↗

NULISA: a novel proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range across the proteome. We report a novel proteomic technology - NUcleic acid Linked Immuno-Sandwich Assay (NULISA) - that incorporates a dual capture and release mechanism to suppress the assay background and improves the sensitivity of the proximity ligation assay by over 10,000-fold to the attomolar level. It utilizes pairs of antibodies conjugated to DNA oligonucleotides that enable immunocomplex purification and generate reporter DNA containing target- and sample-specific barcodes for a next-generation sequencing-based, highly multiplexed readout. A 200-plex NULISA targeting 124 cytokines and chemokines and 80 other immune response-related proteins demonstrated superior sensitivity for detecting low-abundance proteins and high concordance with other immunoassays. The ultrahigh sensitivity allowed the detection of previously difficult-to-detect, but biologically important, low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA addresses longstanding challenges in proteomic analysis of liquid biopsies and makes broad and in-depth proteomic analysis accessible to the general research community and future diagnostic applications.

genomics↗

Tailoring electrode surface charge to achieve discrimination and quantification of chemically similar small molecules with electrochemical aptamers

Electrochemical biosensors based on structure-switching aptamers offer many advantages because they can operate directly in complex samples and offer the potential to integrate with miniaturized electronics. Unfortunately, these biosensors often suffer from cross-reactivity problems when measuring a target in samples containing other chemically similar molecules, such as precursors or metabolites. While some progress has been made in selecting highly specific aptamers, the discovery of these reagents remains slow and costly. In this work, we demonstrate a novel strategy to distinguish molecules with miniscule difference in chemical composition (such as a single hydroxyl group) - with cross reactive aptamer probes - by tuning the charge state of the surface on which the aptamer probes are immobilized. As an exemplar, we show that our strategy can distinguish between DOX and many structurally similar analytes, including its primary metabolite doxorubicinol (DOXol). We then demonstrate the ability to accurately quantify mixtures of these two molecules based on their differential response to sensors with different surface-charge properties. We believe this methodology is general and can be extended to a broad range of applications.

bioengineering↗

A Spatiotemporal Molecular Switch Governs Plant Asymmetric Cell Division

Asymmetric cell division (ACD) often requires protein polarization in the mother cell to produce daughter cells with distinct identities ("cell-fate asymmetry"). Here, we define a previously undocumented mechanism for establishing cell-fate asymmetry in Arabidopsis stomatal stem cells. In particular, we show that polarization of BSL protein phosphatases promotes stomatal ACD by establishing a "kinase-based signaling asymmetry" in the two daughter cells. BSL polarization in the stomatal ACD mother cell is triggered upon commitment to cell division. Polarized BSL is inherited by the differentiating daughter cell where it suppresses cell division and promotes cell-fate determination. Plants lacking BSL exhibit stomatal over-proliferation, demonstrating BSL plays an essential role in stomatal development. Our findings establish that BSL polarization provides a spatiotemporal molecular switch that enables cell-fate asymmetry in stomatal ACD daughter cells. We propose BSL polarization is triggered by an ACD "checkpoint" in the mother cell that monitors establishment of division-plane asymmetry.

plant biology↗

Derivation and characterization of chimera-competent eXtra-Embryonic eNdoderm 1 (XEN) cells from pig blastocysts

In this article, we report for the first time the derivation and characterization of extra-embryonic endoderm (XEN) cells from primitive endoderm (PrE) of porcine (p) embryos. The pXEN cells can be reliably and reproducibly generated from parthenote, in vitro and in vivo derived embryos. The pXEN cells retained all the hallmarks of PrE including expression of canonical PrE and XEN cell markers (GATA4, GATA6, SOX17, SALL4, FOXA2, and HNF4A). Transcriptome analysis further confirmed their XEN cell origin. The pXEN cells when introduced into blastocyst stage embryo contributed to wide-spread chimerism including visceral yolk sac, chorion, as well as embryonic gut and liver primordium in the fetus. The pXEN cells were shown to be an efficient nuclear donor for generating cloned offspring. Taken together, pXEN cells fulfil a longstanding need for a stable, chimera-competent, and nuclear transfer-compatible porcine embryonic cells with applications for agriculture and medicine. Significance StatementWe report for the first time, the derivation and characterization of extraembryonic endoderm (XEN) stem cells from porcine (p) embryos. The pXEN cells can be reliably and reproducibly derived from primitive endoderm precursors. When injected into blastocyst-stage embryos, the pXEN cells have contributed to wide-spread chimerism including visceral yolk sac, chorion of the extraembryonic membranes, as well as definitive endoderm of the fetus, primarily the embryonic gut and liver primordium. Additionally, these XEN cells have proven to be an efficient nuclear donor for generating cloned offspring. These newly discovered stem cells provide a novel model for studying lineage segregation, as well as a source for interspecies chimeras for generating endodermal organs, and for genome editing in livestock.

developmental biology↗

BSU1 family phosphatases mediate Flagellin-FLS2 signaling through a specific phosphocode.

Hundreds of leucine-rich repeat receptor kinases (LRR-RKs) have evolved to control diverse processes of growth, development, and immunity in plants; the mechanisms that link LRR-RKs to distinct cellular responses are not understood. Here we show that two LRR-RKs, the brassinosteroid hormone receptor BRI1 (BRASSINOSTEROID INSENSITIVE 1) and the flagellin receptor FLS2 (FLAGELLIN SENSING 2), regulate downstream glycogen synthase kinase 3 (GSK3) and mitogen-activated protein (MAP) kinases, respectively, through phosphocoding of the BRI1-SUPPRESSOR1 (BSU1) phosphatase. BSU1 was previously identified as a component that inactivates GSK3s in the BRI1 pathway. We found surprisingly that loss of the BSU1 family phosphatases activates effector-triggered immunity (ETI) and impairs flagellin-triggered MAP kinase activation and immunity. The flagellin-activated BOTRYTIS-INDUCED KINASE 1 (BIK1) phosphorylates BSU1 at serine-251. Mutation of serine-251 reduces the ability of BSU1 to mediate flagellin-induced MAP kinase activation and immunity, but not its abilities to suppress ETI and interact with GSK3, which is enhanced through the phosphorylation of BSU1 at serine-764 upon brassinosteroid signaling. These results demonstrate that BSU1 plays an essential role in immunity and transduces brassinosteroid-BRI1 and flagellin-FLS2 signals using different phosphorylation sites. Our study illustrates that phosphocoding in shared downstream components provides signaling specificities for diverse plant receptor kinases.

plant biology↗

Application of TurboID-mediated proximity labeling for mapping a GSK3 kinase signaling network in Arabidopsis

Transient protein-protein interactions (PPIs), such as those between posttranslational modifying enzymes and their substrates, play key roles in cellular regulation, but are difficult to identify. Here we demonstrate the application of enzyme-catalyzed proximity labeling (PL), using the engineered promiscuous biotin ligase TurboID, as a sensitive method for characterizing PPIs in signaling networks. We show that TurboID fused with the GSK3-like kinase BIN2 or a PP2A phosphatase biotinylates their known substrate, the BZR1 transcription factor, with high specificity and efficiency. We optimized the protocol of biotin labeling and affinity purification in transgenic Arabidopsis expressing a BIN2-TurboID fusion protein. Subsequent quantitative mass spectrometry (MS) analysis identified about three hundred proteins biotinylated by BIN2-TurboID more efficiently than the YFP-TurboID control. These include a significant subset of previously proven BIN2 interactors and a large number of new BIN2-proximal proteins that uncover a broad BIN2 signaling network. Our study illustrates that PL-MS using TurboID is a powerful tool for mapping signaling networks, and reveals broad roles of BIN2 kinase in cellular signaling and regulation in plants.\n\nImpact StatementTurboID-mediated proximity labeling is a powerful tool for protein interactomics in plants.

plant biology↗

Interleukin-8/CXCR2 signaling regulates therapy-induced plasticity and enhances tumorigenicity in glioblastoma

Glioblastoma (GBM) remains one of the least treatable types of cancer. Recent work highlights two key factors contributing to this resistant phenotype--cellular plasticity, the ability of GBM cells to adopt many phenotypes, and the microenvironment. Here, we provide evidence that Interleukin-8 (IL-8) plays a vital role in promoting cellular plasticity and cancer initiating cells (CICs) niche during anti-glioma chemotherapy. IL-8 expression is significantly elevated during chemotherapy, and immunohistochemical analysis of matched primary and recurrent patient GBM tissues revealed about 60% of recurrent tissues IL-8 expression is upregulated. In silico analysis of the TCGA data indicated that IL-8 signaling could promote epigenetic plasticity by altering the polycomb repressor complex activity. We are proposing that such regulation my promote epigenetic plasticity, which allows the GBM cells to adapt therapy and may promote therapeutic resistance. Our data show that IL-8 is a crucial microenvironmental factor involved in developing therapeutic adaptation and can be targeted in combination with conventional chemo-and radiotherapy to prevent disease recurrence.\n\nABSTRACTEmerging evidence reveals enrichment of glioma initiating cells (GICs) following therapeutic intervention. This enrichment occurs partly by dedifferentiation of non-GICs to GICs within the tumor, which may contribute to therapeutic resistance and the generation of lethal recurrent tumors. To elucidate the molecular mechanisms governing therapy-induced cellular plasticity, we performed genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq) and gene expression analysis (gene microarray analysis) during treatment with standard of care temozolomide (TMZ) chemotherapy. Analysis revealed significant enhancement of open chromatin marks in known astrocytic enhancers for Interleukin-8 (IL-8) loci as well as elevated expression during anti-glioma chemotherapy. The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project data demonstrated that IL-8 transcript expression is negatively correlated with GBM patient survival (p=0.001) and positively correlated with that of genes associated with the CIC phenotype such as KLF4, c-Myc and HIF2 (p<0.001). Immunohistochemical analysis of patient samples demonstrated elevated IL-8 expression in about 60% of recurrent GBM tumors relative to matched primary tumors and this expression also positively correlates with time to recurrence. Exposure to IL-8 significantly enhanced the self-renewing capacity of patient-derived xenograft (PDX) GBM (average 3-fold, p<0.0005). Furthermore, IL-8 knockdown significantly delayed PDX GBM tumor growth in vivo (p<0.0005). Finally, guided by in silico analysis of TCGA data, we examined the effect of therapy-induced IL-8 expression on the epigenomic landscape of GBM cells and observed increased trimethylation of H3K9 and H3K27. Our results show that IL-8 alters cellular plasticity and mediates alterations in histone status. These finding suggest that IL-8 signaling participates in regulating GBM adaptation to therapeutic stress and therefore represents a promising target for combination with conventional chemotherapy in order to limit GBM recurrence.

cancer biology↗