Biology subjects

Kim, S. E.

Publications and source records attributed to Kim, S. E..

11 recordsLinked to original sources

bioRxiv2026

Activity and specificity trade-offs in adenine base editors

Adenine base editors (ABEs) are CRISPR effectors that introduce A-T to G-C transitions in the genome using a nucleotide deaminase fused to a Cas protein. ABEs have been evolved to have very high editing efficiency, but off-target editing effects compromise their precision and hinder their applications. Here we explore the activity and specificity relationship of ABEs using a combination of machine learning-guided design and high-throughput screening. We designed a diverse library of 12,000 variants and built quantitative bacterial selection systems that allowed us to simultaneously measure their on-target and off-target editing. We found that the ABEs were fully described by the single dimension of intrinsic deaminase activity with no evidence for independent specialization with respect to local sequence context, editing window width, RNA editing, or genotoxicity. These results were supported by in vitro studies and consistent with editing experiments in mammalian cells. Finally, the activity and specificity trade-offs were recapitulated among previously reported engineered variants and a selection of library variants spanning the activity spectrum. Our results suggest that fundamental architectural improvements will be necessary to transcend the activity and specificity limitations for the next generation of ABEs.

molecular biology↗

bioRxiv2025

Identification of novel interacting proteins of FUZ and GPR161.

Proteins modulate signaling pathways through dynamic protein-protein interactions, which provide critical insights into the underlying mechanisms of cellular processes leading to human diseases. Our previous study revealed the biochemical and genetic interactions between FUZ and GPR161 that regulate sonic hedgehog signaling during spinal neural tube development. This study aimed to identify novel co-interacting proteins of FUZ and GPR161 and explore their biochemical and functional associations. We performed affinity-based liquid chromatography-tandem mass spectrometry on immunoprecipitated FUZ and GPR161 proteins. Using this approach, we identified 159 co-interacting proteins, of which 289 proteins that interacted exclusively with FUZ and 617 proteins that interacted exclusively with GPR161. Gene Ontology (GO) analysis of the FUZ and GPR161 co-interactome revealed an enrichment of proteins associated with proteasomal catabolic processes and trafficking. GO analysis of the exclusive FUZ and GPR161 interactomes identified cell cycle progression, mitochondrial membrane, RNA metabolism, receptor complex, and Endoplasmic Reticulum-Golgi transport. These findings were further validated using STRING network analysis. Among the identified proteins, we prioritized FKBP8 and confirmed its biochemical interactions with both FUZ and GPR161 exclusively. In summary, our proteomic profiling uncovered the protein network of FUZ and GPR161, revealing their individual and cooperative functions in multiple cellular processes. Statement of Significance- The functional link between FUZ and GPR161 remains unknown, while our previous study showed their genetic and biochemical link in regulating sonic hedgehog signaling during spinal neural tube development. - This study identified the protein network involving FUZ and GPR161 using affinity- based mass spectrometry combining with gene ontology and STRING analysis, providing the novel molecular regulatory role of FUZ and GPR161. - We prioritized the proteins, FKBP8, as a co-interacting partners of FUZ and GPR161, and validated their biochemical interaction. We identified the FUZ-GPR161-FKBP8 forms protein complex, and their interaction was not exclusive, suggesting those three proteins located in the same signaling pathway potentially regulating spinal neural tube development.

biochemistry↗

bioRxiv2025

Wheat fiber-induced peripheral regulatory T-cells suppress development of colitis

Reduced dietary fiber intake is associated with, and may have contributed to, the post-mid-20th century increase in immune-mediated chronic inflammatory diseases, including inflammatory bowel disease. Reduced fiber intake has resulted, in part, from highly refined foods. For example, modern methods of producing bread removes much of the fiber naturally present in wheat kernels. Accordingly, we hypothesized that wheat fiber might protect against chronic inflammatory diseases. We tested this notion in a murine T-cell transfer colitis model. Rag1-/- mice were fed open-source low-fiber diets enriched, or not with wheat fiber (WF) and then administered CD45Rbhi T-cells. WF conferred robust protection in this colitis model as assessed by an array of clinical, histopathologic, morphologic, and immune-related parameters. WFs protection against colitis associated with a microbiota-dependent increase in Foxp3+ T-cell (Tregs), which could be recapitulated in vitro. WF did not induce Tregs in CNS1-/- mice nor did WF protect against T cell transfer colitis driven by transplant of colitogenic T-cells from CNS1-/- mice. Thus, enriching diet with WF has potential to promote microbiota-dependent peripheral Treg development and, consequently, protect against chronic inflammatory diseases.

immunology↗

bioRxiv2024

The novel linkage between Fuz and Gpr161 genes regulates sonic hedgehog signaling during mouse embryonic development.

Sonic hedgehog (Shh) signaling regulates embryonic morphogenesis utilizing primary cilia, the cell antenna acting as a signaling hub. Fuz, an effector of planar cell polarity (PCP) signaling, involves Shh signaling via cilia formation, while the G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling. The range of phenotypic malformations observed in mice bearing mutations in either of these two genes is similar; however, their functional relations have not been previously explored. This study identified the genetic and biochemical link between Fuz and Gpr161 in mouse embryonic development. Fuz was genetically epistatic to Gpr161 via Shh signaling during mouse embryonic development. The FUZ biochemically interacted with GPR161, and Fuz regulated Gpr161 ciliary trafficking via {beta}-arrestin2. Our study suggested the novel Gpr161-Fuz axis that regulates Shh signaling during mouse embryonic development. Summary statementThis study illuminates the novel genetic and biochemical linkages between Fuz and Gpr161 to regulate sonic hedgehog signaling during mouse embryonic development.

genetics↗

bioRxiv2023

Pax3 lineage-specific deletion of Gpr161 is associated with spinal neural tube and craniofacial malformations during embryonic development.

Shh signaling is the morphogen signaling that regulates embryonic craniofacial and neural tube development. G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling, and its inactivation in mice results in embryo lethality with craniofacial and neural tube defects (NTDs). However, the structural defects of later embryonic stages in Gpr161 null mice and cell lineages underlying abnormalities were not well characterized due to their limited lifespan. We found the Pax3 lineage-specific deletion of Gpr161 in mice presented with tectal hypertrophy (anterior dorsal neuroepithelium), cranial vault and facial bone hypoplasia (cranial neural crest (CNC)), vertebral abnormalities (somite), and the closed form of spina bifida (posterior dorsal neuroepithelium). In particular, the closed form of spina bifida is partly due to the reduced Pax3 and Cdx4 gene expression of the posterior dorsal neural tubes of Gpr161 mutant embryos involving decreased Wnt signaling whereas Shh signaling was increased. This study provides the novel role of Gpr161 in the posterior neural tube development and confirms its role on CNC- and somite-derived skeletogenesis and midbrain morphogenesis in mice.

genetics↗

bioRxiv2023

Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs

Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here we tested the ability of cell penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low-endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.

bioengineering↗

bioRxiv2022

Two opposing effects of monovalent cations on the stability of i-motif structure

At acidic pH, cytosine-rich single-stranded DNA can be folded into a tetraplex structure called i-motif (iM). In recent studies, the effect of monovalent cations on the stability of iM structures has been addressed, but a consensus about the issue has not been reached yet. Thus, we investigated the effects of various factors on the stability of iM structures using fluorescence resonance energy transfer (FRET)-based analysis for three types of iM derived from human telomere sequences. We confirmed that the iM structure is destabilized as the concentration of monovalent cations (Li+, Na+, K+) increases and that Li+ has the greatest tendency of destabilization. This cation-induced destabilization is rather unexpected and specific to the iM structure, considering the cations electrostatic effect of supporting DNA folding. Monovalent cations of different kinds promote the flexibility of single-stranded DNA (ssDNA) and the stability of folded DNA structures to different degrees, suggesting that the size of cation be a key factor in its function. All taken together, we conclude that the stability of iM structures is controlled by the subtle balance of the two counteractive effects of monovalent cations, electrostatic screening and disruption of cytosine base pairing.

biophysics↗

bioRxiv2021

Distinct representation of cue-outcome association by D1 and D2 neurons in the olfactory striatum

Positive and negative associations acquired through olfactory experience are thought to be especially strong and long-lasting. The conserved direct olfactory sensory input to the ventral striatal olfactory tubercle (OT) and its convergence with dense dopaminergic input to the OT could underlie this privileged form of associative memory, but how this process occurs is not well understood. We imaged the activity of the two canonical types of striatal neurons, expressing D1 or D2 type dopamine receptors, in the OT at cellular resolution while mice learned odor-outcome associations ranging from aversive to rewarding. D1 and D2 neurons both responded to rewarding and aversive odors. D1 neurons in the OT robustly and bidirectionally represented odor valence, responding similarly to odors predicting similar outcomes regardless of odor identity. This valence representation persisted even in the absence of a licking response to the odors and in the absence of the outcomes, indicating a true transformation of odor sensory information by D1 OT neurons. In contrast, D2 neuronal representation of the odor-outcome associations was weaker, contingent on a licking response by the mouse, and D2 neurons were more selective for odor identity than valence. Stimulus valence coding in the OT was modality-sensitive, with separate sets of D1 neurons responding to odors and sounds predicting the same outcomes, suggesting that integration of multimodal valence information happens downstream of the OT. Our results point to distinct representation of identity and valence of odor stimuli by D1 and D2 neurons in the OT.

neuroscience↗

bioRxiv2021

Engineering improved Cas13 effectors for targeted post-transcriptional regulation of gene expression

Cas13 is a family of unique RNA-targeting CRISPR-Cas effectors, making it an appealing tool for probing and perturbing RNA function. However only a few Cas13 homologs have been shown to mediate robust RNA targeting in human cells, suggesting that unknown elements may be limiting their efficacy. Furthermore, many Cas13 enzymes show high degrees of toxicity upon targeting and have not been shown to mediate specific knockdown in other cell types such as E. coli. Here, we show that catalytically inactive Cas13 enzymes can be repurposed for efficient translational repression in bacteria with no associated growth defects. To achieve this advance, we carried out a directed evolution screen to engineer functional Cas13a variants, and identified a number of stabilizing mutations, which enabled efficient post transcriptional knockdown of gene expression. In vitro characterization of the resulting engineered Lbu Cas13a mutant, termed eLbu, revealed both stabilization and altered cleavage kinetics. Finally, we show that eLbu can be used for efficient exon skipping in human cells. This work represents the first demonstration of targeted translational repression in E. coli using a CRISPR enzyme, as well as the first directed evolution of a Cas13 enzyme. Such a platform could allow for engineering other aspects of this protein family to obtain more robust RNA targeting tools.

bioengineering↗

bioRxiv2021

Targeting tau mitigates mitochondrial fragmentation and oxidative stress in amyotrophic lateral sclerosis

Understanding the mechanisms underlying amyotrophic lateral sclerosis (ALS) is crucial for the development of new therapies. Recent evidence suggest that tau may be involved in ALS pathogenesis. Here, we demonstrated that hyperphosphorylated tau (pTau-S396) is mis-localized to synapses in human post-mortem motor cortex (mCTX) across ALS subtypes. Treatment with ALS synaptoneurosomes (SNs) derived from post-mortem mCTX, enriched in pTau-S396, increased oxidative stress, induced mitochondrial fragmentation, and altered mitochondrial connectivity in vitro. Furthermore, our findings revealed that pTau-S396 interacts with the pro-fission dynamin-related protein (DRP1), and similar to pTau-S396, DRP1 accumulated in ALS SNs across ALS subtypes. Lastly, reducing tau with a specific bifunctional degrader, QC-01-175, prevented ALS SNs-induced mitochondrial fragmentation and oxidative stress in vitro. Collectively, our findings suggest that increases in pTau-S396 may lead to mitochondrial fragmentation and oxidative stress in ALS and decreasing tau may provide a novel strategy to mitigate mitochondrial dysfunction in ALS. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/436505v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1d805baorg.highwire.dtl.DTLVardef@1601f49org.highwire.dtl.DTLVardef@1a62abforg.highwire.dtl.DTLVardef@40699f_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIpTau-S396 mis-localizes to synapses in ALS. C_LIO_LIALS synaptoneurosomes (SNs), enriched in pTau-S396, increase oxidative stress and induce mitochondrial fragmentation in vitro. C_LIO_LIpTau-S396 interacts with the pro-fission GTPase DRP1 in ALS. C_LIO_LIReducing tau with a specific degrader, QC-01-175, mitigates ALS SNs-induced mitochondrial fragmentation and increases in oxidative stress in vitro. C_LI

neuroscience↗

bioRxiv2020

UDP-glucose pyrophosphorylase 2, a regulator of glycosylation and glycogen, is essential for pancreatic cancer growth

Pancreatic ductal adenocarcinomas (PDACs) have enhanced nutrient uptake requirements and rapid metabolic processing. The enzyme UDP-glucose pyrophosphorylase 2 (UGP2) rests at the convergence of multiple metabolic pathways, however the role of UGP2 in tumor maintenance and cancer metabolism remains unclear. Here, we identify an essential role for UGP2 in the maintenance of PDAC growth in both in vitro and in vivo tumor models. Transcription of UGP2 is directly regulated by the YAP/TEAD complex. Loss of UGP2 leads to decreased intracellular glycogen and defects in N-glycosylation targets important for cell growth including epidermal growth factor receptor (EGFR). In murine xenograft models, knockdown of UGP2 halted tumor growth and repressed expression of EGFR. The critical roles of UGP2 in cancer maintenance, metabolism, and protein glycosylation may offer new avenues of therapy for otherwise intractable PDACs. Impact StatementConvergent findings reveal that UDP-glucose pyrophosphorylase 2 has a central role in growth and metabolism of pancreatic ductal adenocarcinomas, highlighting novel therapeutic possibilities for this deadly cancer.

cancer biology↗