Biology subjects

Chen, P.-J.

Publications and source records attributed to Chen, P.-J..

10 recordsLinked to original sources

bioRxiv2025

Native epitranscriptome sequencing reveals Hepatitis B virus RNA stability protected by heavy m6A modification

Although the RNA of Hepatitis B virus (HBV) is known to be regulated by the epitranscriptomic modification N6-methyladenosine (m6A), it remains controversial whether if m6A is beneficial or detrimental for viral replication. Here, we employ Nanopore direct RNA sequencing (DRS) to analyze the HBV m6A epitranscriptome on a single molecule level and elucidate how m6A regulates the HBV replication cycle. We found more than 60% of all HBV RNA in cells are m6A methylated, [~]3x more methylated than host mRNAs. Previously unreported m6A sites were found in pol gene, which when mutated reduced viral production. A sizable proportion of HBV RNAs carry poly(A) tails at least twice as long (>120nt) as cellular mRNAs, with the shorter-tailed population more pronounced among m6A-free transcripts. In contrast, encapsidated RNAs have shorter poly(A) tails and less m6A. Concordantly, pharmacological reduction of viral m6A using the m6A methyltransferase inhibitor STM2457, shortens viral poly(A) tails and reduces viral RNA stability. Our findings thus suggest that the abundance of m6A on viral RNAs protects the surprisingly long viral poly(A) tails and thus enhances RNA stability. Overall, m6A strongly enhances HBV viral replication, in support of RNA modification machinery as a promising class of antiviral targets.

microbiology↗

bioRxiv2024

Hippocampal γCaMKII dopaminylation promotes synaptic-to-nuclear signaling and memory formation

Protein monoaminylation is a class of posttranslational modification (PTM) that contributes to transcription, physiology and behavior. While recent analyses have focused on histones as critical substrates of monoaminylation, the broader repertoire of monoaminylated proteins in brain remains unclear. Here, we report the development/implementation of a chemical probe for the bioorthogonal labeling, enrichment and proteomics-based detection of dopaminylated proteins in brain. We identified 1,557 dopaminylated proteins - many synaptic - including {gamma}CaMKII, which mediates Ca2+-dependent cellular signaling and hippocampal-dependent memory. We found that {gamma}CaMKII dopaminylation is largely synaptic and mediates synaptic-to-nuclear signaling, neuronal gene expression and intrinsic excitability, and contextual memory. These results indicate a critical role for synaptic dopaminylation in adaptive brain plasticity, and may suggest roles for these phenomena in pathologies associated with altered monoaminergic signaling.

neuroscience↗

bioRxiv2023

Antagonistic pleiotropy plays an important role in governing the evolution and genetic diversity of SARS-CoV-2

Analyses of the genomic diversity of SARS-CoV-2 found that some sites across the genome appear to have mutated independently multiple times with frequency significantly higher than four-fold sites, which can be either due to mutational bias, i.e., elevated mutation rate in some sites of the genome, or selection of the variants due to antagonistic pleiotropy, a condition where mutations increase some components of fitness at a cost to others. To examine how different forces shaped evolution of SARS-CoV-2 in 2020-2021, we analyzed a large set of genome sequences (~ 2 million). Here we show that while evolution of SARS-CoV-2 during the pandemic was largely mutation-driven, a group of nonsynonymous changes is probably maintained by antagonistic pleiotropy. To test this hypothesis, we studied the function of one such mutation, spike M1237I. Spike I1237 increases viral assembly and secretion, but decreases efficiency of transmission in vitro. Therefore, while the frequency of spike M1237I may increase within hosts, viruses carrying this mutation would be outcompeted at the population level. We also discuss how the antagonistic pleiotropy might facilitate positive epistasis to promote virus adaptation and reconcile discordant estimates of SARS-CoV-2 transmission bottleneck sizes in previous studies.

evolutionary biology↗

bioRxiv2022

Spatial and temporal origin of the third SARS-CoV-2 Outbreak in Taiwan

Since the first report of SARS-CoV-2 in December 2019, Taiwan has gone through three local outbreaks. Unlike the first two outbreaks, the spatial and temporal origin of the third outbreak (April 20 to November 5, 2021) is still unclear. We assembled and analyzed a data set of more than 6,000 SARS-CoV-2 genomes, including 300 from Taiwan and 5812 related sequences downloaded from GISAID as of 2021/12/08. We found that the third outbreak in Taiwan was caused by a single virus lineage belonging to Alpha (B.1.1.7) strain. This lineage, T-III (the third outbreak in Taiwan), carries a distinct genetic fingerprint, consisting of spike M1237I (S-M1237I) and three silent mutations, C5812T, C15895T, and T27869C. The T-III is closest to the sequences derived from Turkey on February 8, 2021. The estimated age of the most recent common ancestor (TMRCA) of T-III is March 23, 2021 (95% highest posterior density [HPD] February 24 - April 13, 2021), almost one month before the first three confirmed cases on April 20, 2021. The effective population size of the T-III showed approximately 20-fold increase after the onset of the outbreak and reached a plateau in early June 2021. Our results reconcile several unresolved observations, including the occurrence of two infection clusters at the same time without traceable connection and several airline pilots who were PCR negative but serum IgM-/IgG+ for SARS-CoV-2 in late April. Therefore, in contrast to the general notion that the third SARS-CoV-2 outbreak in Taiwan was sparked by two imported cases from USA on April 20, 2021, which, in turn, was caused by the partial relaxation of entry quarantine measures in early April 2021, our comprehensive analyses demonstrated that the outbreak was most likely originated from Europe in February 2021.

evolutionary 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↗

bioRxiv2021

Signatures of adaptive evolution during human to mink SARS CoV2 cross species transmission inform estimates of the COVID19 pandemic timing

One of the unique features of SARS-CoV-2 is that it mainly evolved neutrally or under purifying selection during the early pandemic. This contrasts with the preceding epidemics of the closely related SARS-CoV and MERS-CoV, both of which evolved adaptively. It is possible that the SARS-CoV-2 exhibits a unique or adaptive feature which deviates from other coronaviruses. Alternatively, the virus may have been cryptically circulating in humans for a sufficient time to have acquired adaptive changes for efficient transmission before the onset of the current pandemic. In order to test the above scenarios, we analyzed the SARS-CoV-2 sequences from minks (Neovision vision) and parenteral human strains. In the early phase of the mink epidemic (April to May 2020), nonsynonymous to synonymous mutation ratios per site within the spike protein was 2.93, indicating a selection process favoring adaptive amino acid changes. In addition, mutations within this protein concentrated within its receptor binding domain and receptor binding motif. Positive selection also left a trace on linked neutral variation. An excess of high frequency derived variants produced by genetic hitchhiking was found during middle (June to July 2020) and early late (August to September 2020) phases of the mink epidemic, but quickly diminished in October and November 2020. Strong positive selection found in SARS-CoV-2 from minks implies that the virus may be not unique in super-adapting to a wide range of new hosts. The mink study suggests that SARS-CoV-2 already went through adaptive evolution in humans, and likely been circulating in humans at least six months before the first case found in Wuhan, China. We also discuss circumstances under which the virus can be well-adapted to its host but fail to induce an outbreak.

evolutionary biology↗

bioRxiv2021

Dopamine D3 Receptor Ligand Suppresses the Expression of Levodopa-Induced Dyskinesia in Nonhuman Primate Model of Parkinson's Disease

Parkinsons disease (PD) is a complex multisystem, chronic and so far incurable disease with significant unmet medical needs. The incidence of PD increases with aging and the expected burden will continue to escalate with our aging population. Since its discovery in the 1961 levodopa remains the gold standard pharmacotherapy for PD. However, the progressive nature of the neurodegenerative process in and beyond the nigrostriatal system causes a multitude of side effects, including levodopa-induced dyskinesia within 5 years of therapy. Attenuating dyskinesia has been a significant challenge in the clinical management of PD. We report on a small molecule that eliminates the expression of levodopa-induced dyskinesia and significantly improves PD-like symptoms. The lead compound PD13R we discovered is a dopamine D3 receptor partial agonist with high affinity and selectivity, orally active and with desirable drug-like properties. Future studies are aimed at developing this lead compound for treating PD patients with dyskinesia.

neuroscience↗

bioRxiv2021

D614G Substitution of SARS-CoV-2 Spike Protein Increases Syncytium Formation and Viral Transmission via Enhanced Furin-mediated Spike Cleavage

Since the D614G substitution in the spike (S) of SARS-CoV-2 emerged, the variant strain underwent rapid expansion to become the most abundant strain worldwide. Therefore, this substitution may provide an advantage of viral spreading. To explore the mechanism, we analyzed 18 viral isolates containing S proteins with either G614 or D614. Both the virus titer and syncytial phenotype were significantly increased in S-G614 than in S-D614 isolates. We further showed increased cleavage of S at the furin substrate site, a key event that promotes syncytium, in S-G614 isolates. These functions of the D614G substitution were validated in cells expressing S protein. The effect on syncytium was abolished by furin inhibitor treatment and mutation of the furin-cleavage site, suggesting its dependence on cleavage by furin. Our study provides a mechanistic explanation for the increased transmissibility of S-G614 containing SARS-CoV-2 through enhanced furin-mediated S cleavage, which increases membrane fusion and virus infectivity.

microbiology↗

bioRxiv2020

The origin and underlying driving forces of the SARS-CoV-2 outbreak

The spread of SARS-CoV-2 since December 2019 has become a pandemic and impacted many aspects of human society. Here, we analyzed genetic variation of SARS-CoV-2 and its related coronavirus and found the evidence of intergenomic recombination. After correction for mutational bias, analysis of 137 SARS-CoV-2 genomes as of 2/23/2020 revealed the excess of low frequency mutations on both synonymous and nonsynonymous sites which is consistent with recent origin of the virus. In contrast to adaptive evolution previously reported for SARS-CoV in its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation of selection. The sequence similarity of the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression. Therefore, SARS-CoV-2 might have cryptically circulated within humans for years before being recently noticed. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and reveal critical steps required for effective spreading. Two mutations, 84S in orf8 protein and 251V in orf3 protein, occurred coincidentally with human intervention. The 84S first appeared on 1/5/2020 and reached a plateau around 1/23/2020, the lockdown of Wuhan. 251V emerged on 1/21/2020 and rapidly increased its frequency. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China was two time higher than those derived from the rest of the world. In addition, in network analysis, haplotypes collected from Wuhan city were at interior and have more mutational connections, both of which are consistent with the observation that the outbreak of cov-19 was originated from China. SUMMARYIn contrast to adaptive evolution previously reported for SARS-CoV in its brief epidemic, our analysis of SARS-CoV-2 genomes shows signs of relaxation of selection. The sequence similarity of the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression. Therefore, SARS-CoV-2 might have cryptically circulated within humans for years before being recently noticed. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and reveal critical steps required for effective spreading. Two mutations, 84S in orf8 protein and 251V in orf3 protein, occurred coincidentally with human intervention. The 84S first appeared on 1/5/2020 and reached a plateau around 1/23/2020, the lockdown of Wuhan. 251V emerged on 1/21/2020 and rapidly increased its frequency. Thus, the roles of these mutations on infectivity need to be elucidated.

evolutionary biology↗

bioRxiv2018

Targeting redox regulatory site of protein kinase B impedes neutrophilic inflammation

Neutrophil activation has a pathogenic effect in inflammatory diseases. Protein kinase B (PKB)/AKT regulates diverse cellular responses. However, the significance of AKT in neutrophilic inflammation is still not well understood. Here, we identified CLLV-1 as a novel AKT inhibitor. CLLV-1 inhibited respiratory burst, degranulation, chemotaxis, and AKT phosphorylation in activated human neutrophils and dHL-60 cells. Significantly, CLLV-1 blocked AKT activity and covalently reacted with AKT Cys310 in vitro. The AKT309-313 peptide-CLLV-1 adducts were determined by NMR or mass spectrometry assay. The alkylation agent-conjugated AKT (reduced form) level was also inhibited by CLLV-1. Additionally, CLLV-1 ameliorated lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. CLLV-1 acts as a covalent allosteric AKT inhibitor by targeting AKT Cys310 to restrain inflammatory responses in human neutrophils and LPS-induced ALI in vivo. Our findings provide a mechanistic framework for redox modification of AKT that may serve as a novel pharmacological target to alleviate neutrophilic inflammation.

pharmacology and toxicology↗