Biology subjects

Li, K. H.

Publications and source records attributed to Li, K. H..

7 recordsLinked to original sources

bioRxiv2025

Glycoengineering of the hepatitis C virus E2 glycoprotein leads to improved biochemical properties and enhanced immunogenicity

An effective vaccine against hepatitis C virus (HCV) must elicit the production of broadly neutralizing antibodies (bnAbs) reproducibly against the E1E2 glycoprotein complex. Little is known about how glycan content affects this process. Ideally, glycans would maximize epitope exposure without compromising antigen stability or exposing new epitopes. However, typical recombinant vaccines contain considerable heterogeneity in glycan content, which can affect the antibody response and neutralization potency. Here we employed glycoengineered Chinese hamster ovary (geCHO) cell lines that impart nearly homogeneous glycosylation as a means to test how specific glycan features influence antigenicity and immunogenicity for the secreted HCV E2 ectodomain (sE2). Specific geCHO antigens exhibited a modest but reproducible increase in affinity for some mAbs relative to CHO- and HEK293-produced sE2. Surprisingly, one geCHO sE2 antigen failed to bind the CD81 receptor, indicating the potential for significant glycan effects on biochemical properties. We immunized mice with the four antigens and found the total antibody response to be the same for all groups. However, sera from one geCHO group exhibited a 7-fold improvement in neutralization against the homologous HCV pseudovirus and had the most mice whose sera exhibited neutralization activity against genotypes 1b, 2a, 2b, and 3. Further analysis identified beneficial and deleterious glycan features, and the glycan that correlated the most with decreased potency was relatively small. However, size was not the sole determinant of glycan-driven effects on the antibody response. In summary, glycan content impacts biochemical properties of antigens to varying degrees and such effects can influence immune response quality and uniformity.

immunology↗

bioRxiv2024

The VGCC auxiliary subunit α2δ1 is an extracellular GluA1 interactor and regulates LTP, spatial memory, and seizure susceptibility

Activity-dependent synaptic accumulation of AMPA receptors (AMPARs) and subsequent long-term synaptic strengthening underlie different forms of learning and memory. The AMPAR subunit GluA1 amino-terminal domain is essential for synaptic docking of AMPAR during LTP, but the precise mechanisms involved are not fully understood. Using unbiased proteomics, we identified the epilepsy and intellectual disability-associated VGCC auxiliary subunit 2{delta}1 as a candidate extracellular AMPAR slot. Presynaptic 2{delta}1 deletion in CA3 affects synaptic AMPAR incorporation during long-term potentiation, but not basal synaptic transmission, at CA1 synapses. Consistently, mice lacking 2{delta}1 in CA3 display a specific impairment in CA1-dependent spatial memory, but not in memory tests involving other cortical regions. Decreased seizure susceptibility in mice lacking 2{delta}1 in CA3 suggests a regulation of circuit excitability by 2{delta}1/AMPAR interactions. Our study sheds light on the regulation of activity-dependent AMPAR trafficking, and highlights the synaptic organizing roles of 2{delta}1. Significance statementActivity-dependent accumulation of AMPA receptors (AMPARs) at excitatory synapses and subsequent synaptic strengthening underlies long-term potentiation (LTP), forms of learning and memory, and some epilepsies. The "slot model" posits that postsynaptic scaffolding contain "slots" for AMPAR complexes, and that increased synaptic activity augments the availability of slots to accommodate more receptors, thereby strengthening synapses and enabling LTP. The presence of the GluA1 AMPAR subunit amino-terminal domain (ATD) has recently emerged as an additional requirement for LTP. Here we identify the auxiliary voltage-gated calcium channel subunit 2{delta}1 as a GluA1 ATD interacting protein and provide evidence supporting a role for 2{delta}1 as an extracellular AMPAR slot regulating activity dependent synaptic AMPAR clustering, excitability, and cognitive function.

neuroscience↗

bioRxiv2024

Tumor localization strategies of multi-cancer early detection tests: a quantitative assessment

IntroductionBlood-based multi-cancer early detection (MCED) tests may expand the number of "screenable" cancers. Defining an optimal approach to diagnostic resolution for individuals with positive MCED test results is critical. Two prospective trials employed distinct diagnostic resolution approaches; one employed a molecular signal to predict tissue of origin (TOO) and the other used an imaging-based diagnostic strategy. Using mathematical modeling, we compared the diagnostic burden of each approach and characterized the risk of excess cancer incidence that may be attributable to radiation exposure associated with a false positive (FP) MCED test result and an imaging-based diagnostic strategy. MethodsA mathematical expression for diagnostic burden was derived using MCED test positive predictive value (PPV), molecular TOO localization accuracy, and the expected number of imaging procedures associated with each diagnostic outcome. Imaging and molecular TOO strategies were compared by estimating diagnostic burden across a wide range of MCED PPVs and TOO accuracies. Organ-specific radiation dose for diagnostic imaging was extracted from the literature and used as input to National Cancer Institute RADRat tool for estimating excess lifetime cancer risk due to radiation exposure. ResultsFor the molecular TOO diagnostic approach, an average of 2.1 procedures are required to reach diagnostic resolution for correctly-localized TPs, 4.4 procedures for incorrectly-localized TPs, and 4 procedures for FPs, vs. an average of 2.75 procedures for TPs and 2.4 for FPs with an imaging-based diagnostic strategy. Across the entire range of possible PPV and localization performance, a molecular TOO strategy resulted in a higher mean diagnostic burden: 3.6 procedures (SD 0.445) vs. 2.6 procedures (SD 0.1) for the imaging strategy. Predicted diagnostic burden was higher for molecular TOO in 95.5% of all possible PPV and TOO accuracy combinations; 79% or higher PPV would be required for a 90% accurate molecular TOO strategy to be less burdensome than imaging. The maximum rate of excess cancer incidence from radiation exposure for FP results from MCED screening between the ages of 50-84 was estimated at 64.6 per 100,000 (annual testing, 99% specificity), 48.5 per 100,000 (biennial testing, 98.5% specificity), and 64.6 per 100,000 (biennial testing, 98% specificity). ConclusionsThis analysis demonstrates that an imaging-based diagnostic strategy is more efficient than a molecular TOO-informed approach across 95.5% of all possible MCED PPV and TOO accuracy combinations. The use of an imaging-based approach for cancer localization can be efficient and low risk compared to a molecular-based approach.

cancer biology↗

bioRxiv2023

Contrasting synaptic roles of MDGA1 and MDGA2

Neurodevelopmental disorders are frequently linked to mutations in synaptic organizing molecules. MAM domain containing glycosylphosphatidylinositol anchor 1 and 2 (MDGA1 and MDGA2) are a family of synaptic organizers suggested to play an unusual role as synaptic repressors, but studies offer conflicting evidence for their localization. Using epitope-tagged MDGA1 and MDGA2 knock-in mice, we found that native MDGAs are expressed throughout the brain, peaking early in postnatal development. Surprisingly, endogenous MDGA1 was enriched at excitatory, but not inhibitory, synapses. Both shRNA knockdown and CRISPR/Cas9 knockout of MDGA1 resulted in cell-autonomous, specific impairment of AMPA receptor- mediated synaptic transmission, without affecting GABAergic transmission. Conversely, MDGA2 knockdown/knockout selectively depressed NMDA receptor-mediated transmission but enhanced inhibitory transmission. Our results establish that MDGA2 acts as a synaptic repressor, but only at inhibitory synapses, whereas both MDGAs are required for excitatory transmission. This nonoverlapping division of labor between two highly conserved synaptic proteins is unprecedented. TeaserMDGAs 1 and 2 independently localize to and modulate excitatory and inhibitory hippocampal synapses by different mechanisms.

neuroscience↗

bioRxiv2022

Ribotoxic collisions on CAG expansions disrupt proteostasis and stress responses in Huntington's Disease

Huntingtons disease (HD) is a devastating neurodegenerative disorder caused by CAG trinucleotide repeat expansions encoding a polyglutamine (polyQ) tract in the Huntingtin (HTT) gene1. Although mutant HTT (mHTT) protein tends to aggregate, the exact causes of neurotoxicity in HD remain unclear2. Here we show that altered elongation kinetics on CAG expansions cause ribosome collisions that trigger ribotoxicity, proteotoxicity and maladaptive stress responses. CAG expansions cause an elongation rate conflict during HTT translation, when ribosomes rapidly decoding the optimal polyQ encounter a flanking slowly-decoded polyproline tract. The ensuing ribosome collisions lead to premature termination and release of aggregation-prone mHTT fragments. Due to the presence of a stress-responsive upstream open reading frame (uORF), HTT translation and aggregation are limited under normal conditions but enhanced under stress, seeding a vicious cycle of dysfunction. mHTT further exacerbates ribotoxicity by progressively sequestering eIF5A, a key regulator of translation elongation, polyamine metabolism and stress responses. eIF5A depletion in HD cells leads to widespread ribosome pausing on eIF5A-dependent sites, impaired cotranslational proteostasis, disrupted polyamine metabolism and maladaptive stress responses. Importantly, drugs that reduce translation initiation attenuate ribosome collisions and mitigate this escalating cascade of ribotoxic stress and dysfunction in HD.

neuroscience↗

bioRxiv2020

Arabidopsis ACINUS is O-glycosylated and regulates transcription and alternative splicing of regulators of reproductive transitions

O-GlcNAc modification plays important roles in metabolic regulation of cellular status. Two homologs of O-GlcNAc transferase, SECRET AGENT (SEC) and SPINDLY (SPY), which have O-GlcNAc and O-fucosyl transferase activities, respectively, are essential in Arabidopsis but have largely unknown cellular targets. Here we show that AtACINUS is O-GlcNAcylated and O-fucosylated and mediates regulation of transcription, alternative splicing (AS), and developmental transitions. Knocking-out both AtACINUS and its distant paralog AtPININ causes severe growth defects including dwarfism, delayed seed germination and flowering, and abscisic acid (ABA) hypersensitivity. Transcriptomic and protein-DNA/RNA interaction analyses demonstrate that AtACINUS represses transcription of the flowering repressor FLC and mediates AS of ABH1 and HAB1, two negative regulators of ABA signaling. Proteomic analyses show AtACINUSs O-GlcNAcylation, O-fucosylation, and association with splicing factors, chromatin remodelers, and transcriptional regulators. Some AtACINUS/AtPININ-dependent AS events are altered in the sec and spy mutants, demonstrating a function of O-glycosylation in regulating alternative RNA splicing.

molecular biology↗

bioRxiv2018

TP53 mutations as potential prognostic markers for specific cancers: Analysis of data from The Cancer Genome Atlas and the International Agency for Research on Cancer TP53 Database

Mutations in the tumor suppressor gene TP53 are associated with a variety of cancers. Therefore, it is important to know the occurrence and the prognostic effects of TP53 mutations in certain cancers. Over 29,000 cases of TP53 mutations were obtained from the April 2016 release of the Internal Agency for Research on Cancer (IARC) TP53 Database, and 7,893 cancer cases were compiled in the cBioPortal for Cancer Genomics from the 33 most recent studies of The Cancer Genome Atlas (TCGA). The data was analyzed, and it was found that the majority of TP53 mutations were missense and the major mutational hotspots were located at codons 248, 273, 175, and 245 in exons 4-8 for somatic mutations with the addition of codon 337 and other mutations in exons 9-10 for germline mutations. Out of 33 TGCA studies, the effects of TP53 mutations were statistically significant in ten cancers (ovarian serous cystadenocarcinoma, lung adenocarcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, acute myeloid leukemia, clear cell renal cell carcinoma (RCC), papillary RCC, chromophobe RCC, uterine endometrial carcinoma, and thymoma) for survival time and in six cancers (ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, hepatocellular carcinoma, chromophobe RCC, acute myeloid leukemia, and thymoma) for disease-free survival time. Also, it was found that the most common p53 mutation in hepatocellular carcinomas (R249S) was a much better indicator for poor prognosis than TP53 mutations as a whole.\n\nAuthor summaryThe TP53 gene codes for the tumor suppressor protein, p53, which is essential for DNA repair, cell cycle arrest, and apoptosis. It is commonly inactivated or partially disabled by mutations, contributing to the development of a variety of human cancers. In this study, over 29,000 cases from the April 2016 release of the International Agency for Research on Cancer TP53 Database (IARC) were analyzed to determine the distribution of mutations in the TP53 gene. Data was also collected from the 33 most recent The Cancer Genome Atlas (TCGA) studies to determine the prevalence of TP53 mutations in cancers and their effects on survival and disease-free survival time. It was found that there were statistically significant differences between cases with and without TP53 mutations in ten cancers when comparing survival time, and in six cancers when comparing disease-free survival time. This indicates that TP53 mutations are potential prognostic markers that can be used to further improve the accuracy of predicting the survival time and disease-free survival time of cancer patients.

cancer biology↗