Biology subjects

Yu, C.

Publications and source records attributed to Yu, C..

At least 145 records · Page 8Linked to original sources

bioRxiv2023

Defective transfer of parental histone decreases frequency of homologous recombination in budding yeast

Recycling of parental histones is an important step in epigenetic inheritance. During DNA replication, DNA polymerase epsilon subunit DPB3/DPB4 and DNA replication helicase subunit MCM2 are involved in the transfer of parental histones to the leading and lagging DNA strands, respectively. Single Dpb3 deletion (dpb3{Delta}) or Mcm2 mutation (mcm2-3A), which each disrupt one parental histone transfer pathway, leads to the others predominance. However, the impact of the two histone transfer pathways on chromatin structure and DNA repair remains elusive. In this study, we used budding yeast Saccharomyces cerevisiae to determine the genetic and epigenetic outcomes from disruption of parental histone H3-H4 tetramer transfer. We found that a dpb3{Delta}/mcm2-3A double mutant did not exhibit the single dpb3{Delta} and mcm2-3A mutants asymmetric parental histone patterns, suggesting that the processes by which parental histones are transferred to the leading and lagging strands are independent. Surprisingly, the frequency of homologous recombination was significantly lower in dpb3{Delta}, mcm2-3A, and dpb3{Delta}/mcm2-3A mutants relative to the wild-type strain, likely due to the elevated levels of free histones detected in the mutant cells. Together, these findings indicate that proper transfer of parental histones to the leading and lagging strands during DNA replication is essential for maintaining chromatin structure and that high levels of free histones due to parental histone transfer defects are detrimental to cells.

molecular biology↗

bioRxiv2022

Monkeypox virus quadrivalent mRNA vaccine induces antibody responses and cellular immunity and protects mice against Vaccinia virus

There is an urgent need for efficient and safe vaccines against the monkeypox virus (MPXV) in response to the rapidly spreading monkeypox epidemic. In the age of COVID-19, mRNA vaccines have been highly successful and emerged as platforms enabling rapid development and large-scale preparation. Here, we have developed two MPXV quadrivalent mRNA vaccines, named mRNA-A-LNP and mRNA-B-LNP, based on two IMVs (A29L and M1R) and two EEVs (A35R and B6R). By administering mRNA-A-LNP and mRNA-B-LNP intramuscularly twice, mice have induced MPXV-specific IgG antibodies and potent Vaccinia virus (VACV)-specific neutralizing antibodies. Additionally, it elicited durable MPXV-specific killer memory T-cell immunity as well as memory B-cell immunity in mice. Furthermore, the passive transfer of sera from mRNA-A-LNP and mRNA-B-LNP-immunized mice protected nude mice against the VACV challenge. In addition, two doses of mRNA-A-LNP and mRNA-B-LNP were also protective against the VACV challenge in mice. Overall, our results demonstrated that mRNA-A-LNP and mRNA-B-LNP appear to be safe and effective vaccine candidates against monkeypox epidemics, as well as against outbreaks caused by other orthopoxviruses, including the smallpox virus.

immunology↗

bioRxiv2022

Acyl-CoA dehydrogenase substrate promiscuity limits the potential for development of substrate reduction therapy in disorders of valine and isoleucine metabolism

Toxicity of accumulating substrates is a significant problem in several disorders of valine and isoleucine degradation notably short-chain enoyl-CoA hydratase (ECHS1 or crotonase) deficiency, 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency, propionic acidemia (PA) and methylmalonic aciduria (MMA). Isobutyryl-CoA dehydrogenase (ACAD8) and short/branched-chain acyl-CoA dehydrogenase (SBCAD, ACADSB) function in the valine and isoleucine degradation pathways, respectively. Deficiencies of these acyl-CoA dehydrogenase (ACAD) enzymes are considered biochemical abnormalities with limited or no clinical consequences. We investigated whether substrate reduction therapy through inhibition of ACAD8 and SBCAD can limit the accumulation of toxic metabolic intermediates in disorders of valine and isoleucine metabolism. Using analysis of acylcarnitine isomers, we show that 2-methylenecyclopropaneacetic acid (MCPA) inhibited SBCAD, isovaleryl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase and medium-chain acyl-CoA dehydrogenase, but not ACAD8. MCPA treatment of wild-type and PA HEK-293 cells caused a pronounced decrease in C3-carnitine. Furthermore, deletion of ACADSB in HEK-293 cells led to an equally strong decrease in C3-carnitine when compared to wild-type cells. Deletion of ECHS1 in HEK-293 cells caused a defect in lipoylation of the E2 component of the pyruvate dehydrogenase complex, which was not rescued by ACAD8 deletion. MCPA was able to rescue lipoylation in ECHS1 KO cells, but only in cells with prior ACAD8 deletion. SBCAD was not the sole ACAD responsible for this compensation, which indicates substantial promiscuity of ACADs in HEK-293 cells for the isobutyryl-CoA substrate. Substrate promiscuity appeared less prominent for 2-methylbutyryl-CoA at least in HEK-293 cells. We suggest that pharmacological inhibition of SBCAD to treat PA should be investigated further.

biochemistry↗

bioRxiv2022

Structural surfaceomics reveals an AML-specific conformation of Integrin-β2 as a CAR-T therapy target

Safely expanding indications for cellular therapies has been challenging given a lack of highly cancer-specific surface markers. Here, we explore the hypothesis that tumor cells express cancer-specific surface protein conformations, invisible to standard target discovery pipelines evaluating gene or protein expression, that can be identified and immunotherapeutically targeted. We term this strategy, integrating cross-linking mass spectrometry (XL-MS) with glycoprotein surface capture, "structural surfaceomics". As a proof of principle, we apply this technology to acute myeloid leukemia, a hematologic malignancy with dismal outcomes and no known optimal immunotherapy target. We identify the activated conformation of integrin-{beta}2 as a structurally-defined, widely-expressed, AML-specific target. We develop and characterize recombinant antibodies to this protein conformation, and show that chimeric antigen receptor (CAR) T-cells eliminate AML cells and patient-derived xenografts without notable toxicity versus normal hematopoietic cells. Our findings validate an AML conformation-specific target antigen while demonstrating a toolkit for applying these strategies more broadly.

cancer biology↗

bioRxiv2022

The differential impacts of dataset imbalance in single-cell data integration

Single-cell transcriptomic data measured across distinct samples has led to a surge in computational methods for data integration. Few studies have explicitly examined the common case of cell-type imbalance between datasets to be integrated, and none have characterized its impact on downstream analyses. To address this gap, we developed the Iniquitate pipeline for assessing the stability of single-cell RNA sequencing (scRNA-seq) integration results after perturbing the degree of imbalance between datasets. Through benchmarking 5 state-of-the-art scRNA-seq integration techniques in 1600 perturbed integration scenarios for a multi-sample peripheral blood mononuclear cell (PBMC) dataset, our results indicate that sample imbalance has significant impacts on downstream analyses and the biological interpretation of integration results. We observed significant variation in clustering, cell-type classification, marker gene-based annotation, and query-to-reference mapping in imbalanced settings. Two key factors were found to lead to quantitation differences after scRNA-seq integration - the cell-type imbalance within and between samples (relative cell-type support) and the relatedness of cell-types across samples (minimum cell-type center distance). To account for evaluation gaps in imbalanced contexts, we developed novel clustering metrics robust to sample imbalance, including the balanced Adjusted Rand Index (bARI) and balanced Adjusted Mutual Information (bAMI). Our analysis quantifies biologically-relevant effects of dataset imbalance in integration scenarios and introduces guidelines and novel metrics for integration of disparate datasets. The Iniquitate pipeline and balanced clustering metrics are available at https://github.com/hsmaan/Iniquitate and https://github.com/hsmaan/balanced-clustering, respectively.

bioinformatics↗

bioRxiv2022

Comprehensive structural analysis reveals broad-spectrum neutralizing antibodies against Omicron

The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread around the world. Mutant strains of SARS-CoV-2 are constantly emerging. At present, Omicron variants have become mainstream. In this work, we carried out a systematic and comprehensive analysis of the reported spike protein antibodies, counting the antibodies epitopes and genotypes. We further comprehensively analyzed the impact of Omicron mutations on antibody epitopes and classified these antibodies according to their binding patterns. We found that the epitopes of one class of antibodies were significantly less affected by Omicron mutations than other classes. Binding and virus neutralization experiments show that such antibodies can effectively inhibit the immune escape of Omicron. Cryo-EM results show that this class of antibodies utilizes a conserved mechanism to neutralize SARS-CoV-2. Our results greatly help us deeply understand the impact of Omicron mutations. At the same time, it also provides guidance and insights for developing Omicron antibodies and vaccines.

microbiology↗

bioRxiv2022

Structural basis for intrinsic transcription termination

Efficient and accurate termination is required for gene transcription in all living organisms. Cellular RNA polymerases (RNAP) in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway (called intrinsic termination transcription in bacteria), in which RNAP recognizes terminator sequences, stops nucleotide addition, and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-EM structures of E. coli transcription intrinsic termination complexes representing key intermediate states of the event. The structures show how RNAP pauses at terminator sequences, how the terminator RNA hairpin folds inside RNAP, and how RNAP rewinds the transcription bubble to release RNA and then DNA. These macromolecular snapshots define a structural mechanism for bacterial intrinsic termination and a pathway for RNA release and DNA collapse relevant for factor-independent termination by all RNA polymerases.

molecular biology↗

bioRxiv2022

Discovery, Structure, and Function of Filamentous 3-Methylcrotonyl-CoA Carboxylase

3-methylcrotonyl-CoA carboxylase (MCC) is a biotin-dependent enzyme necessary for leucine catabolism in most organisms. While the crystal structure of recombinant bacterial MCC has been characterized, the structure and potential polymerization of native MCC remain elusive. Here, we discovered that native MCC from Leishmania tarentolae (LtMCC) forms filaments and determined its structure at near-atomic resolution using cryoEM. 6{beta}6 LtMCC dodecamers assemble in a twisted-stacks architecture, manifesting as supramolecular rods extending up to approximately 400 nanometers. LtMCCs in filaments bind biotin but are not covalently biotinylated and lack coenzyme A. Filaments elongate by stacking 6{beta}6 LtMCCs onto the exterior -trimer of the terminal 6{beta}6 dodecamer. This stacking immobilizes the biotin carboxylase domains, sequestering the enzyme in an inactive state within the mitochondrial matrix. Our results support a new model for LtMCC catalysis, termed the dual-swinging-domains model, and cast new light on the functional significance of polymerization in the carboxylase superfamily and beyond.

molecular biology↗

bioRxiv2022

Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome

Mapping the subcellular organization of proteins is crucial for understanding their biological functions. Herein, we report a reactive oxygen species induced protein labeling and identification (RinID) method for profiling subcellular proteome in the context of living cells. Our method capitalizes on a genetically encoded photocatalyst, miniSOG, to locally generate singlet oxygen that reacts with proximal proteins. Labeled proteins are conjugated in situ with an exogenously supplied nucleophilic probe, which serves as a functional handle for subsequent affinity enrichment and mass spectrometry-based protein identification. From a panel of nucleophilic compounds, we identify biotin-conjugated aniline and propargyl amine as highly reactive probes. As a demonstration of the spatial specificity and depth of coverage in mammalian cells, we apply RinID in the mitochondrial matrix, capturing 394 mitochondrial proteins with 97% specificity. We further demonstrate the broad applicability of RinID in various subcellular compartments, including the nucleus and the endoplasmic reticulum.

biochemistry↗

bioRxiv2022

ADOLESCENT EXPOSURE TO LOW-DOSE THC DISRUPTS ENERGY BALANCE AND ADIPOSE ORGAN HOMEOSTASIS IN ADULTHOOD

One of cannabis most iconic effects is the stimulation of hedonic high-calorie eating - the munchies - yet habitual cannabis users are on average leaner than non-users. We asked whether this unexpected phenotype might result from lasting changes in energy balance established during adolescence, when habitual use of the drug often begins. We found that daily low-dose administration of cannabis intoxicating constituent, {Delta}9-tetrahydrocannabinol (THC), to adolescent mice causes an adult metabolic phenotype characterized by reduced fat mass, increased lean mass and utilization of fat as fuel, partial resistance to diet-induced obesity and dyslipidemia, and enhanced thermogenesis. Multi-omics analyses revealed that this phenotype is associated with multiple molecular anomalies in the adipose organ, which include ectopic overexpression of muscle-associated proteins and heightened anabolic processing. Thus, adolescent exposure to THC may promote an enduring pseudo-lean state that superficially resembles healthy leanness but might in fact be rooted in adipose organ dysfunction.

pharmacology and toxicology↗

bioRxiv2022

Accurate and efficient protein sequence designthrough learning concise local environment of residues

Protein sequence design has been widely applied in rational protein engineering and increasing the design accuracy and efficiency is highly desired. Here we present ProDESIGN-LE, an accurate and efficient design approach, which adopts a concise but informative representation of residues local environment and trains a transformer to select an appropriate residue at a position from its local environment. ProDESIGN-LE iteratively applies the transformer on the positions in the target structure, eventually acquiring a designed sequence with all residues fitting well with their local environments. ProDESIGN-LE designed sequences for 68 naturally occurring and 129 hallucinated proteins within 20 seconds per protein on average, and the predicted structures from the designed sequences perfectly resemble the target structures with state-of-the-art average TM-score exceeding 0.80. We further experimentally validated ProDESIGN-LE by designing five sequences for an enzyme, chloramphenicol O-acetyltransferase type III (CAT III), and recombinantly expressing the proteins in E. coli. Of these proteins, three exhibited excellent solubility, and one yielded monomeric species with circular dichroism spectra consistent with the natural CAT III protein.

bioinformatics↗

bioRxiv2022

Proteomic Dynamics of Breast Cancers Identifies Potential Therapeutic Protein Targets

Treatment and relevant targets for breast cancer (BC) remain limited, especially for triple-negative BC (TNBC). We quantified the proteomes of 76 human BC cell lines using data independent acquisition (DIA) based proteomics, identifying 6091 proteins. We then established a 24-protein panel distinguishing TNBC from other BC types. Integrating prior multi-omics datasets with the present proteomic results to predict the sensitivity of 90 drugs, we found that proteomics data improved drug sensitivity predictions. The sensitivity of the 90 drugs was mainly associated with cell cytoskeleton, signal transduction and mitochondrial function. We next profiled the proteome changes of nine cell lines (five TNBC cell lines, four non-TNBC cell lines) perturbated by EGFR/AKT/mTOR inhibitors. In the TNBC cell lines, metabolism pathways were dysregulated after EGFR/mTOR inhibitors treatment, while RNA modification and cell cycle pathways were dysregulated after AKT inhibitor treatment. Our study presents a systematic multi-omics and in-depth analysis of the proteome of BC cells. This work aims to aid in prioritization of potential therapeutic targets for TNBC as well as to provide insight into adaptive drug resistance in TNBC.

systems biology↗

bioRxiv2022

Characterization and structure of the human lysine-2-oxoglutarate reductase domain, a novel therapeutic target for treatment of glutaric aciduria type 1

In humans, a single enzyme 2-aminoadipic semialdehyde synthase (AASS) catalyzes the initial two critical reactions in the lysine degradation pathway. This enzyme evolved to be a bifunctional enzyme with both lysine 2-oxoglutarate reductase (LOR) and saccharopine dehydrogenase domains (SDH). Moreover, AASS is a unique drug target for metabolic genetic diseases such as glutaric aciduria type 1 that arise from deficiencies downstream in the lysine degradation pathway. While work has been done to elucidate the SDH domain structurally and to develop inhibitors, neither has been done for the LOR domain. Here, we purify and characterize LOR, show that AASS is rate-limiting upon high lysine exposure of mice, and present the crystal structure of the human LOR domain, which should enable future efforts to identify inhibitors of this novel drug target.

biochemistry↗

bioRxiv2022

Cryo-EM structures reveal a dynamic transformation process of human alpha-2-macroglobulin working as a protease inhibitor

Human alpha-2-macroglobulin is a well-known proteases inhibitor against a broad spectrum of proteases. It also plays important roles in immunity, inflammation, and infections. Here, we report cryo-EM structures of human alpha-2-macroglobulin of the native state, the transformed state induced by its authentic substrate, human trypsin, and serial intermediate states between the native and the fully induced state. These structures exhibit distinct conformations, which reveal a dynamic transformation process of alpha-2-macroglobulin acting as a protease inhibitor. The results shed light on the molecular mechanism of alpha-2-macroglobulin entrapping substrates, and help to understand how alpha-2-macroglobulin possesses variant physiological functions.

biochemistry↗

bioRxiv2022

The multi-BRCT domain protein DDRM2 is required for homologous recombination in plants

DNA double-strand breaks (DSBs) are the most toxic DNA damage for cells. Homologous recombination (HR) is a precise DSB repair mechanism as well as a basis for gene targeting using genome-editing techniques. Despite the importance of HR, the HR mechanism in plants is poorly understood. In a genetic screen for DNA Damage Response Mutants (DDRMs), we find that the Arabidopsis ddrm2 mutant is hypersensitive to DSB-inducing reagents. DDRM2 encodes a protein with four BRCA1 C-terminal (BRCT) domains and is highly conserved in plants including the earliest land plant linage, bryophytes. The plant-specific transcription factor SOG1 binds to the promoter of DDRM2 and activates its expression, suggesting that DDRM2 is a direct target of SOG1. In consistence, the expression of DDRM2 is induced by DSBs in a SOG1-dependent manner. Epistasis analysis indicates that DDRM2 functions downstream of SOG1. Similar to the sog1 mutant, the ddrm2 mutant shows dramatically reduced HR efficiency. Our study suggests that the SOG1-DDRM2 module is required for HR, providing new insights into the HR mechanisms in plants and a potential target for improving the efficiency of gene targeting. One Sentence SummaryA genetic screen in Arabidopsis reveals that the multi-BRCT domain protein DDRM2 is required for homologous recombination and is targeted by the master DNA damage response regulator SOG1.

plant biology↗

bioRxiv2022

Identification and characterization of the HERV-K (HML-8) group of human endogenous retroviruses in the genome

Human endogenous retroviruses (HERV) could vertically transmit in a Mendelian fashion and stable maintenance in the human genome which are estimated to comprise about 8%. HERVs affect human physiology and pathology based on the effect of the provirus-encoded protein or LTR elements. The characterization of the genomic distribution is an essential step to understanding the relationship between endogenous retrovirus expression and diseases. However, the poorly characterization of HML-8 hinders a detailed understanding of the expression regulation of this family in human health and its actual impact on host genomes. In the light of this, the definition of a precise and updated HERV-K HML-8 genomic map is urgently needed. Here we report a comprehensive analysis of HERV-K HML-8 sequences presence and distribution within the human genome, with a detailed description of the different structural and phylogenetic aspects characterizing the group. A total of 40 proviruses and 5 solo LTR elements were characterized with a detailed description of provirus structure, integration time, potentially regulated genes, transcription factor binding sites, and primer binding site feature. The integration time results showed that the HML-8 elements found in the human genome have been integrated in the primate lineage between 23.5 and 52 million years ago (mya). Overall, the results have finally clarified the composition of HML-8, providing an exhaustive background for subsequent functional studies. Highlights[tpltrtarr] A comprehensive characterization of the HERV-K (HML-8) in human genome. [tpltrtarr]There is an apparent preference of HML-8 into intergenic regions and introns. [tpltrtarr]There are two distinct clusters for the env region of the HML-8 elements. [tpltrtarr]The average time of HML-8 integration in human is 37.1 mya.

microbiology↗

bioRxiv2022

The Chromosome-level Genome of Dracaena cochinchinesis Provides Insights into its Biological Features and the Mechanism of Dragon's Blood Formation

Dracaena, a remarkably long-lived and slowly maturing species, is famous all over the world for the production of dragons blood, a precious traditional medicine used by different cultures since ancient times. However, lacking a high-quality genome,the molecular mechanisms underlying these traits are largely unknown and that greatly restricts the protection and regeneration of the rare and endangered plant resources. Here, we sequenced and assembled a chromosome-level genome of the Dracaena cochinchinensis, the first to be sequenced of Dracaena Vand. ex L. The D. cochinchinensis genome covering 1.21 Gb with a scaffold N50 of 50.06 Mb, and encodes 31,619 predicted protein-coding genes. We found D. cochinchinensis has undergone two whole genome duplications (WGDs) and two long terminal repeats (LTRs) insertion burst events. The expansion of cis-zeatin O-glucosyltransferase (cZOGT) and small auxin up-regulated RNA (SAUR) genes is account for its longevity and slow growth. In flavonoids biosynthesis pathway, transcription factors bHLH and MYB were predicted as the core regulators, and ROS as the specific signal molecule during the process of injury-induced dragons blood formation. Our study not only provides high-quality genomic knowledge of D. cochinchinensis, but also deciphered the mystery of its longevity, and preliminarily elucidated the molecular mechanism of dragons blood formation, which will facilitate the resource protection and sustainable utilization of Dracaena. SHORT SUMMARYThis study reports a chromosome-level genome assembly for D. cochinchinensis, the first genome of Dracaena Vand. ex L. It 29 provides valuable genetic resources and creates a large scope for studying Dracaena. We found the significant expansion of genes associated with its longevity and slow growth, and genes in flavonoids biosynthesis were first completely identified. Moreover, transcription factors bHLH and MYB as the core regulator of flavonoids biosynthesis, and ROS as the specific signal molecule during the process of injury-induced dragons blood formation were also identified. These results not only deciphered the mystery of its longevity, but also elucidated the molecular mechanism of dragon[s blood formation preliminarily, which will facilitate the resource protection and sustainable utilization of Dracaena.

genomics↗

bioRxiv2022

Genomic perspectives on the emerging SARS-CoV-2 Omicron variant

A new variant of concern for SARS-CoV-2, Omicron (B.1.1.529), was designated by the World Health Organization on November 26, 2021. This study analyzed the viral genome sequencing data of 108 samples collected from patients infected with Omicron. First, we found that the enrichment efficiency of viral nucleic acids was reduced due to mutations in the region where the primers anneal to. Second, the Omicron variant possesses an excessive number of mutations compared to other variants circulating at the same time (62 vs. 45), especially in the Spike gene. Mutations in the Spike gene confer alterations in 32 amino acid residues, which was more than those observed in other SARS-CoV-2 variants. Moreover, a large number of nonsynonymous mutations occur in the codons for the amino acid residues located on the surface of the Spike protein, which could potentially affect the replication, infectivity, and antigenicity of SARS-CoV-2. Third, there are 53 mutations between the Omicron variant and its closest sequences available in public databases. Many of those mutations were rarely observed in the public database and had a low mutation rate. In addition, the linkage disequilibrium between these mutations were low, with a limited number of mutations (6) concurrently observed in the same genome, suggesting that the Omicron variant would be in a different evolutionary branch from the currently prevalent variants. To improve our ability to detect and track the source of new variants rapidly, it is imperative to further strengthen genomic surveillance and data sharing globally in a timely manner.

genomics↗