Biology subjects

Choi, S. G.

Publications and source records attributed to Choi, S. G..

9 recordsLinked to original sources

bioRxiv2026

α-Synuclein Seeding in Dopaminergic Neurons Transplanted into the Putamen of Parkinson's Disease Patients

Healthy embryonic neurons grafted into the putamen of Parkinson's disease (PD) patients start to develop Lewy pathology 10 years after transplantation. It remains unclear whether this pathology is initiated by the spread of host-derived -synuclein (syn) aggregates which seed aggregation in the grafted cells or arises independently from a hostile PD environment (e.g., chronic inflammation). To distinguish these possibilities, we identified syn seeding sites in the human brain using a multiplex in situ seed immunodetection (isSID) assay. In PD and dementia with Lewy bodies (DLB), isSID labeled a subset of Lewy pathology (LP) as puncta along neurites and within inclusions. In multiple system atrophy (MSA), isSID labeled Papp-Lantos bodies and microglia. In progressive supranuclear palsy (PSP), isSID labeled tufted astrocytes. isSID labeled neuromelanin in the substantia nigra independent of diagnosis. In 16- and 27-year intraputamenal neuronal grafts, isSID labeled within and proximal to Lewy pathology, as well as outside the grafted tissue. Younger grafts (18 months and 4 years) lacked Lewy pathology. In the 18-month graft, isSID labeled grafted neurons; at 4 years, isSID was confined to amorphous deposits in the graft and was absent from tyrosine hydroxylase-positive grafted neurons. In conclusion, syn seeding is detectable in grafted neurons before and during LP formation. Because seeding accompanies melanization, but is not explained by melanization alone, these findings support host-derived seeds as a contributor to graft pathology.

neuroscience↗

bioRxiv2026

Preformed Fibril Seeding Reshapes the Phosphorylated α-Synuclein Proximal Proteome in the Olfactory Bulb

BackgroundAggregated alpha-synuclein (syn) phosphorylated at serine 129 (PS129) accumulates in synucleinopathies, with the olfactory bulb (OB) being severely affected. Non-aggregated physiological PS129 is abundant in the mammalian OB, where it likely modulates syn-protein interactions. The impact of aggregation on the PS129 interactome in the OB remains unclear. We hypothesized that syn aggregation alters the PS129 interactome, shifting canonical synaptic partners (e.g., SNARE proteins) toward the aggregate-associated network. To test this hypothesis, we mapped PS129 interactions in the OB of PFF-injected WT and SNCAA53T/A53T (M83) mice using biotinylation by antibody recognition (BAR) and pretreated with calf-intestine alkaline phosphatase (CIAP) to distinguish physiological from aggregate-associated PS129 interactomes. Seeding and spread were assessed by immunohistochemistry and in situ seeding immunodetection assay (isSID). ResultsFollowing OB-PFF injections, CIAP-resistant aggregates and seeds were detected throughout the neuroaxis of M83 mice (e.g., OB to brainstem) but less so in WT mice. isSID seeding was concentrated near CIAP-resistant aggregates, but the overlap was only partial. BAR-PS129 identified 2,309 proteins in M83 OBs and 990 proteins in WT OBs. Of these, 357 proteins in M83 mice and 247 proteins in WT mice were associated with the CIAP-resistant, aggregate-enriched PS129 fraction. In both models, the CIAP-resistant interactome largely overlapped with the broader PS129 interactome, suggesting that seeded aggregation primarily affects existing PS129 interactions rather than directing the formation of new pathological ones. A conserved 107-protein CIAP-resistant signature shared between M83, and WT mice was enriched for axon-glia adhesion, myelin-associated, axonal/cytoskeletal, proteostatic, and synaptic vesicle-related proteins. ConclusionAggregated PS129 engages a subset of PS129 networks enriched at axon-glial interfaces. CIAP-resistant aggregates were partially associated with seed competency, indicating that CIAP resistance and seed competency are related but not equivalent. These results provide molecular details of syn seeding and spread from the OB.

neuroscience↗

bioRxiv2023

Aggregation inhibits alpha-synuclein dephosphorylation resulting in the observation of pathological enrichment

Alpha-synuclein (syn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of syn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological syn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-minute and 1-hour postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while syn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinsons disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant syn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded syn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that syn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129. Significance StatementPhosphorylated alpha-synuclein (PSER129) was widely regarded as a sensitive, specific marker for pathological aggregates in synucleinopathies until recent data demonstrated that PSER129 is abundant in the healthy mammalian nervous system and results from normal neuronal activity. Differentiating pathological (i.e., aggregated PSER129) and biological (non-aggregated PSER129) has thus become of critical importance to the field. Here, we describe our discovery that aggregated-PSER129 is impervious to enzymatic dephosphorylation. We leverage this discovery to develop a technique (CIAP-PSER129) to detect normal or pathological PSER129 selectively. Our technique allowed us to unambiguously differentiate pathological inclusions in brain regions and mouse models where excessive non-aggregated PSER129 severely limits the sensitivity of aggregate detection. CIAP-PSER129 is nondestructive and compatible with most downstream assays, including mass spectrometry-based peptide identification. These findings have important implications and utility for the synucleinopathy field and may have applicability to other neuropathological proteins (e.g., tau).

neuroscience↗

bioRxiv2023

AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor

Protein-protein interactions (PPIs) offer great opportunities to expand the druggable proteome and therapeutically tackle various diseases, but remain challenging targets for drug discovery. Here, we provide a comprehensive pipeline that combines experimental and computational tools to identify and validate PPI targets and perform early-stage drug discovery. We have developed a machine learning approach that prioritizes interactions by analyzing quantitative data from binary PPI assays and AlphaFold-Multimer predictions. Using the quantitative assay LuTHy together with our machine learning algorithm, we identified high-confidence interactions among SARS-CoV-2 proteins for which we predicted three-dimensional structures using AlphaFold Multimer. We employed VirtualFlow to target the contact interface of the NSP10-NSP16 SARS-CoV-2 methyltransferase complex by ultra-large virtual drug screening. Thereby, we identified a compound that binds to NSP10 and inhibits its interaction with NSP16, while also disrupting the methyltransferase activity of the complex, and SARS-CoV-2 replication. Overall, this pipeline will help to prioritize PPI targets to accelerate the discovery of early-stage drug candidates targeting protein complexes and pathways.

systems biology↗

bioRxiv2022

Expanding the HDAC druggable landscape beyond enzymatic inhibition

Enzymatic pockets such as those of histone deacetylases (HDACs) are among the most favored targets for drug development. However, enzymatic inhibitors often exhibit low selectivity and high toxicity due to targeting multiple enzyme paralogs, which are often involved in distinct multisubunit complexes. Here, we report the discovery and characterization of a non-enzymatic small molecule inhibitor of HDAC transcriptional repression functions with comparable anti-tumor activity to the enzymatic HDAC inhibitor Vorinostat, and anti-psychedelic activity of an HDAC2 knockout in vivo. We highlight that these phenotypes are achieved while modulating the expression of 20- and 80-fold fewer genes than enzymatic and genetic inhibition in the respective models. Thus, by achieving the same biological outcomes as established therapeutics while impacting a dramatically smaller number of genes, inhibitors of protein-protein interactions can offer important advantages in improving the selectivity of epigenetic modulators. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/519454v2_ufig1.gif" ALT="Figure 1"> View larger version (96K): org.highwire.dtl.DTLVardef@3a4e68org.highwire.dtl.DTLVardef@1f1be2corg.highwire.dtl.DTLVardef@1fc5e94org.highwire.dtl.DTLVardef@1a54790_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

Distribution of phosphorylated alpha-synuclein in non-diseased brain implicates olfactory bulb mitral cells in synucleinopathy pathogenesis

Synucleinopathies are neurodegenerative diseases characterized by pathological inclusions called "Lewy pathology" (LP) that consist of aggregated alpha-synuclein predominantly phosphorylated at serine 129 (PSER129). Despite the importance for understanding disease, little is known about the endogenous function of PSER129 or why it accumulates in disease. Here we conducted several observational studies using a sensitive tyramide signal amplification (TSA) technique to determine PSER129 distribution and function in the non-diseased mammalian brain. In wild-type non-diseased mice, PSER129 was detected in the olfactory bulb (OB) and several brain regions across the neuroaxis (i.e., OB to brain stem). In contrast, PSER129 immunoreactivity was not observed in any brain region of alpha-synuclein knockout mice. We found evidence of PSER129 positive structures in OB mitral cells of non-diseased mice, rats, non-human primates, and healthy humans. Using TSA multiplex fluorescent labeling we show that PSER129 positive punctate structures occur within inactive (i.e., cfos negative) T-box transcription factor 21 (TBX21) positive mitral cells and PSER129 within these cells was spatially associated with PK-resistant alpha-synuclein. Ubiquitin was found in PSER129 mitral cells but was not closely associated with PSER129. Biotinylation by antigen recognition (BAR) identified 125 PSER129-interacting proteins in the OB of healthy mice, which were significantly enriched for presynaptic vesicle trafficking/recycling, SNARE, fatty acid oxidation, oxidative phosphorylation, and RNA binding. TSA multiplex labeling confirmed the physical association of BAR identified protein Ywhag with PSER129 in the OB and in other regions across the neuroaxis. We conclude that PSER129 accumulates in mitral cells of the healthy OB as part of alpha-synuclein normal cellular functions. Incidental LP has been reported in the OB, and therefore we speculate that for synucleinopathies either; the disease processes begin locally in OB mitral cells or a systemic disease process is most apparent in the OB because the natural tendency to accumulate PSER129. Significance StatementMultiple lines of evidence have suggested that the disease process in some synucleinopathies begins in the olfactory bulb. Here we demonstrated that disease-associated phosphorylated alpha-synuclein preferentially occurs in mitral cells of the healthy mammalian olfactory bulb. We identified the protein interactome of phosphorylated alpha-synuclein in the healthy mouse olfactory bulb and established phosphorylated alpha-synuclein associates with presynaptic glutamatergic vesicles, SNARE machinery, and RNA metabolism machinery. Our data implicates olfactory bulb mitral cells in synucleinopathy pathogenesis. These findings advance our understanding of synucleinopathy disease origins and set the stage for new experimental models to interrogate the pathogenesis of synucleinopathies.

neuroscience↗

bioRxiv2021

A quantitative mapping approach to identify direct interactions within complexomes

Complementary methods are required to fully characterize all protein complexes, or the complexome, of a cell. Affinity purification coupled to mass-spectrometry (AP-MS) can identify the composition of complexes at proteome-scale. However, information on direct contacts between subunits is often lacking. In contrast, solving the 3D structure of protein complexes can provide this information, but structural biology techniques are not yet scalable for systematic, proteome-wide efforts. Here, we optimally combine two orthogonal high-throughput binary interaction assays, LuTHy and N2H, and demonstrate that their quantitative readouts can be used to differentiate direct interactions from indirect associations within multiprotein complexes. We also show that LuTHy allows accurate distance measurements between proteins in live cells and apply these findings to study the impact of the polyglutamine expansion mutation on the structurally unresolved N-terminal domain of Huntingtin. Thus, we present a new framework based on quantitative interaction assays to complement structural biology and AP-MS techniques, which should help to provide first-approximation contact maps of multiprotein complexes at proteome-scale. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/457734v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1cc7cf1org.highwire.dtl.DTLVardef@1eed9borg.highwire.dtl.DTLVardef@8197b2org.highwire.dtl.DTLVardef@e5cb8_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2019

A reference map of the human protein interactome

Global insights into cellular organization and function require comprehensive understanding of interactome networks. Similar to how a reference genome sequence revolutionized human genetics, a reference map of the human interactome network is critical to fully understand genotype-phenotype relationships. Here we present the first human \"all-by-all\" binary reference interactome map, or \"HuRI\". With ~53,000 high-quality protein-protein interactions (PPIs), HuRI is approximately four times larger than the information curated from small-scale studies available in the literature. Integrating HuRI with genome, transcriptome and proteome data enables the study of cellular function within essentially any physiological or pathological cellular context. We demonstrate the use of HuRI in identifying specific subcellular roles of PPIs and protein function modulation via splicing during brain development. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms underlying tissue-specific phenotypes of Mendelian diseases. HuRI thus represents an unprecedented, systematic reference linking genomic variation to phenotypic outcomes.

systems biology↗

bioRxiv2019

Towards an ″assayome″ for binary interactome mapping

Complementary assays are required to comprehensively map complex biological entities such as genomes, proteomes and interactome networks. However, how various assays can be optimally combined to approach completeness while maintaining high precision often remains unclear. Here, we propose the concept of an "assayome" for binary protein-protein interaction (PPI) mapping as an optimal combination of assays and/or assay versions that maximizes detection of true positive interactions, while avoiding detection of random protein pairs. We engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions differing by protein expression systems and tagging configurations. The resulting N2H assayome recovers as many PPIs as 10 distinct assays combined. Thus, to further improve PPI mapping, developing alternative versions of existing assays might be as productive as designing completely new assays. Our assayome concept should be applicable to systematic mapping of other biological landscapes.

systems biology↗