Biology subjects

Huang, C.-F.

Publications and source records attributed to Huang, C.-F..

8 recordsLinked to original sources

bioRxiv2025

Top-Down Individual Ion Mass Spectrometry Reveals 85-110 kDa Catenin Phospho-Proteoforms Regulated by Actomyosin Contractility

A central challenge in top-down proteomics is the characterization of large proteoforms (>70 kDa) due to their high spectral complexity in mass spectrometers. Here, we advance individual ion mass spectrometry (I2MS) for intact mass and fragmentation analysis of {beta}- and -catenins (85-110 kDa), key components of adherens junctions. Using denatured I2MS, we resolved discrete phosphorylation states of catenins isolated from HEK cells subjected to differential actomyosin tension. Up to 10 phosphorylations were detected on {beta}-catenin and 7 on -catenin, with site-specific changes corresponding to actomyosin contractility. Notably, phosphorylation at -catenin S641 was constitutive, while other sites in the P-linker and actin-binding domains as well as {beta}-catenin S675 and S552 were sensitive to actomyosin perturbation. Application of I2MS for fragment ion detection (I2MS2) also enabled 25-30% sequence coverage for these exceptionally large proteoforms, compared to <1% using conventional methods for top-down mass spectrometry. Our results support a "catenin phospho-code" model, wherein combinatorial phosphorylation patterns encode mechano-transductive signals regulating cell-cell adhesion. This work establishes top-down I2MS as a viable approach for probing complex post-translational modification landscapes in high-mass proteins and highlights proteoforms as functional units in cellular regulation.

biochemistry↗

bioRxiv2025

Replication stress alters CENP-A nucleosome stability during S phase

1.The maintenance of centromere identity is essential for the proper segregation of chromosomes during cell division. Centromere identity is epigenetically specified by centromeric histone H3 variant CENP-A, and its retention during DNA replication is facilitated by HJURP chaperone. Replication stress disrupts replication fork progression and can negatively influence the interactions between histone chaperone network necessary for retention and deposition of parental and new histones, respectively. In this study we investigate the role of replication stress response on centromere inheritance. We define changes in centromere-associated proteins that govern stability of centromeric and canonical nucleosomes through proximity labeling coupled with affinity purification mass spectrometry. We identified that under replication stress, CENP-A-containing chromatin strongly enriches for SWI/SNF chromatin remodeling proteins ATRX. We show that depletion of ATRX and its associated histone H3.3 chaperone DAXX results in the loss CENP-A retention in S-phase and loss persists into the subsequent cell cycle. Altogether our findings provide insight into how replication stress negatively influences centromeric chromatin instability and delineates a function of DAXX-ATRX complex in maintaining centromere inheritance during DNA replication.

biochemistry↗

bioRxiv2024

α-catenin phosphorylation is elevated during mitosis to resist apical rounding and epithelial barrier leak

Epithelial cell cohesion and barrier function critically depend on -catenin, an actin-binding protein and essential constituent of cadherin-catenin-based adherens junctions. -catenin undergoes actomyosin force-dependent unfolding of both actin-binding and middle domains to strongly engage actin filaments and its various effectors, where this mechanosensitivity is critical for adherens junction function. We previously showed that -catenin is highly phosphorylated in an unstructured region that links mechanosensitive middle- and actin-binding domains (known as the P-linker region), but the cellular processes that promote -catenin phosphorylation have remained elusive. Here, we leverage a previously published phospho-proteomic data set to show that the -catenin P-linker region is maximally phosphorylated during mitosis. By reconstituting -catenin Crispr KO MDCK with wild-type, phospho-mutant and mimic forms of -catenin, we show that full phosphorylation restrains mitotic cell rounding in the apical direction, strengthening interactions between dividing and non-dividing neighbors to limit epithelial barrier leak. Since major scaffold components of adherens junctions, tight junctions and desmosomes are also differentially phosphorylated during mitosis, we reason that epithelial cell division may be a tractable system to understand how junction complexes are coordinately regulated to sustain barrier function under tension-generating morphogenetic processes.

cell biology↗

bioRxiv2024

Deep Profiling of Plasma Proteoforms with Engineered Nanoparticles for Top-down Proteomics

The dynamic range challenge for detection of proteins and their proteoforms in human plasma has been well documented. Here, we use the nanoparticle protein corona approach to enrich low-abundant proteins selectively and reproducibly from human plasma and use top-down proteomics to quantify differential enrichment for the 2841 detected proteoforms from 114 proteins. Furthermore, nanoparticle enrichment allowed top-down detection of proteoforms between [~]1 {micro}g/mL and [~]10 pg/mL in absolute abundance, providing up to 105-fold increase in proteome depth over neat plasma in which only proteoforms from abundant proteins (>1 {micro}g/mL) were detected. The ability to monitor medium and some low abundant proteoforms through reproducible enrichment significantly extends the applicability of proteoform research by adding depth beyond albumin, immunoglobins and apolipoproteins to uncover many involved in immunity and cell signaling. As proteoforms carry unique information content relative to peptides, this report opens the door to deeper proteoform sequencing in clinical proteomics of disease or aging cohorts.

biochemistry↗

bioRxiv2024

Top-Down Proteomics Identifies Plasma Proteoform Signatures of Liver Cirrhosis Progression

Cirrhosis, advanced liver disease, affects 2-5 million Americans. While most patients have compensated cirrhosis and may be fairly asymptomatic, many decompensate and experience life-threatening complications such as gastrointestinal bleeding, confusion (hepatic encephalopathy), and ascites, reducing life expectancy from 12 to less than 2 years. Among patients with compensated cirrhosis, identifying patients at high risk of decompensation is critical to optimize care and reduce morbidity and mortality. Therefore, it is important to preferentially direct them towards specialty care which cannot be provided to all patients with cirrhosis. We used discovery Top-down Proteomics (TDP) to identify differentially expressed proteoforms (DEPs) in the plasma of patients with progressive stages of liver cirrhosis with the ultimate goal to identify candidate biomarkers of disease progression. In this pilot study, we identified 209 DEPs across three stages of cirrhosis (compensated, compensated with portal hypertension, and decompensated), of which 115 derived from proteins enriched in the liver at a transcriptional level and discriminated the three stages of cirrhosis. Enrichment analyses demonstrated DEPs are involved in several metabolic and immunological processes known to be impacted by cirrhosis progression. We have preliminarily defined the plasma proteoform signatures of cirrhosis patients, setting the stage for ongoing discovery and validation of biomarkers for early diagnosis, risk stratification, and disease monitoring. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/599662v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@797e27org.highwire.dtl.DTLVardef@495bb7org.highwire.dtl.DTLVardef@2478a9org.highwire.dtl.DTLVardef@6dbc0a_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPerformed a pilot top-down LC-MS/MS analysis to identify proteoforms (PFRs) in the plasma of patients with 3 progressive stages of liver cirrhosis. C_LIO_LIIdentified 2867 proteoforms (PFRs) and 209 differentially regulated proteoforms (DRPs) in the different stages of the disease. C_LIO_LIIdentified DRP profiles able to potentially distinguish early from late stages of the disease, including 115 liver-derived DRPs. C_LIO_LIFibrinogen alpha chain, haptoglobin, and Apo A-I are the proteins with the highest number of DRPs and represent potential candidate biomarkers of liver cirrhosis progression. C_LI

molecular biology↗

bioRxiv2024

The RAE1-STOP1 Module Reduces Sensitivity to Exogenous ABA Treatment by Regulating ABI5 in Arabidopsis

The SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) transcription factor plays a pivotal role in maintaining cellular ion balance and governing aluminum tolerance in plants. Abscisic acid (ABA) participates in aluminum tolerance by inducing the expression of several genes that are STOP1 targets. However, the mechanisms underlying ABA signaling and STOP1-mediated gene expression remain poorly understood. The F-box protein RAE1, an SCF-type E3 ligase component, recognizes STOP1 and controls its ubiquitination and degradation. This study revealed that exogenous ABA supplementation reduced STOP1 levels by promoting the expression of RAE1. Notably, both RAE1 loss-of-function mutants and STOP1 overexpressing lines showed enhanced sensitivity to exogenous ABA treatment, which correlated with early stage post-transcriptional upregulation of ABSCISIC ACID INSENSITIVE5 (ABI5). Our observations strongly suggest that RAE1 operates as an ABA-responsive factor, exerting control over STOP1 homeostasis, and thus establishing a negative feedback loop that controls ABA responses in Arabidopsis. Thus, our study revealed a novel function of the RAE1-STOP1 module in ABA signaling, highlighting its role in reducing ABA sensitivity by preventing ABI5 increase. Single Sentence SummaryF-box protein RAE1 functions as an exogenous ABA responsive mediator to reduce STOP1-mediated ABA sensitivity.

plant biology↗

bioRxiv2019

Development of a Genomic DNA Reference Material Panel for Thalassemia Genetic Testing

Thalassemia is one of the most common autosomal recessive inherited diseases worldwide, and it is also highly prevalent and variable in Southern China. Various types of genetic testing technologies have been developed for diagnosis and screening of thalassemia. Characterized genomic DNA reference materials are necessary for assay development, validation, proficiency testing and quality assurance. However, there is no publicly available reference materials for thalassemia genetic testing as yet. To address the need for these materials, the National Institutes for Food and Drug Control and the China National Gene Bank established 31 new cell lines with 2 wild genotypes and 29 distinct genotypes of thalassemia which account for approximately 90% thalassemia carriers in China. The genomic DNA of 31 cell lines were characterized by four clinical genetic testing laboratories using different genetic testing methods and technology platforms. The genotyping results were concordant among four laboratories. In addition, the results of stability test demonstrated that the genotypes of these DNA samples were not influenced by preanalytical conditions such as long-term exposure to high temperature(37{square}) environment and repeated freeze-thawing. In conclusion, we developed the first national panel of 31 genomic DNA reference materials which are renewable and publicly available for the quality assurance of various genetic testing methods and will facilitate research and development in thalassemia genetic testing.

genetics↗

bioRxiv2018

The efficacy of a 14-day modified quadruple therapy containing amoxicillin, tetracycline and high dose metronidazole and proton-pump inhibitors as an empirical third line helicobacter pylori eradication treatment in Taiwan

The antibiotics resistances to amoxicilln, tetracycline was low in Taiwan even after multiple H. pylori treatment failures and high dose metronidazole could overcome antibiotics resistance. In real world practice, susceptibility-guided treatments are not widely available. Therefore, we assessed the efficacy of 14-day modified quadruple therapy containing amoxicillin, tetracycline and high dose metronidazole and PPI as an empirical third-line rescue H. pylori treatment. This study was conducted by analyzing 70 consecutive prospectively registered patients who failed two times H. pylori eradication. All of them received endoscopy for H. pylori culture. Seven patients were lost to follow up. They were then treated according to the antibiotic susceptibility testing reports (Cultured group, n=39). Those who failed H. pylori culture were prescribed with a modified 14-day quadruple therapy containing esomeprazole 40 mg twice daily, amoxicillin 1 g twice daily, tetracycline 500 mg four times daily and metronidazole 500 mg three times daily (empirical group, n=24). Follow-up urea breath test was performed 8 weeks later. The eradication rates attained by Cultured group and empirical group were 89.7% (95% confidence interval [CI] = 72.72%-97.11%) and 58.3% (95% CI=36.61%-77.86%), in the per protocol analysis (p=0.004); 81.4%(95% CI=66.60%-91.61%) and 51.8% (95% CI=31.9%-71.29%), in the intention-to-treat analysis (p=0.014). Culture-guided therapy was the clinical factors influencing the efficacy of H. pylori eradication (OR: 0.16; 95% CI: 0.04-0.60, p=0.006). In conclusion, empirical 14-day modified 3 quadruple therapy is not acceptable as an alternative third-line rescue H. pylori treatment arobably but the success rate of the third-line susceptibility-guided treatment was only moderate (<90%).

microbiology↗