Biology subjects

Dao, L.

Publications and source records attributed to Dao, L..

3 recordsLinked to original sources

bioRxiv2025

Quantitative Substrate Kinetics Screening: One-Pot LC/MS Based Approach to Map Protease Specificity

Proteases play critical roles in many biological processes and diseases. Proteases tend to have limited and overlapping specificities, which hinders our understanding of their roles in physiology. We designed a naive protease substrate library with the aim of fully utilizing modern mass spectrometry proteomics tools to quantitatively map protease substrate specificity. A library of approximately 200,000 peptides was efficiently produced in E. coli with sufficient library diversity to identify cleavage efficiency of thousands of substrates for most proteases in a single assay. Each experiment results in ample substrate kinetics data to build a positional specificity matrix that quantitatively maps protease specificity. We apply this method to two different proteases, GluC commonly used as a proteomics tool, and Fibroblast Activation Protein Alpha that is overexpressed in activated fibroblasts of epithelial cancers and fibrotic diseases. This research quantitatively maps substrate specificity for GluC and FAP in a single assay and can be applied to map substrate specificity for most proteases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=122 HEIGHT=200 SRC="FIGDIR/small/680748v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1496d11org.highwire.dtl.DTLVardef@1979fc1org.highwire.dtl.DTLVardef@17967e8org.highwire.dtl.DTLVardef@194b741_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

m6A-mediated epi-transcriptomic dysregulation underlies synaptic dysfunction in fragile X syndrome

Fragile X syndrome (FXS), the leading genetic cause of intellectual disability, arises from FMR1 gene silencing and loss of the FMRP protein. N6-methyladenosine (m6A) is a prevalent mRNA modification essential for post-transcriptional regulation. FMRP is known to bind to and regulate the stability of m6A-containing transcripts. However, how loss of FMRP impacts on transcriptome-wide m6A modifications in FXS patients remains unknown. To answer this question, we generated cortical neurons differentiated from induced pluripotent stem cells (iPSC) derived from healthy subjects and FXS patients. In electrophysiology recordings, we validated that synaptic and neuronal network defects in iPSC-derived FXS neurons corresponded to the clinical EEG data of the patients from which the corresponding iPSC line was derived. In analysis of transcriptome-wide methylation, we show that FMRP deficiency led to increased translation of m6A writers, resulting in hypermethylation that primarily affecting synapse-associated transcripts and increased mRNA decay. Conversely, in the presence of an m6A writer inhibitor, synaptic defects in FXS neurons were rescued. Taken together, our findings uncover that an FMRP-dependent epi-transcriptomic mechanism contributes to FXS pathogenesis by disrupting m6A modifications in FXS, suggesting a promising avenue for m6A- targeted therapies.

neuroscience↗

bioRxiv2024

Requirement for Fucosyltransferase 2 in Allergic Airway Hyperreactivity and Mucus Obstruction

Mucus hypersecretion is an important pathological problem in respiratory diseases. Mucus accumulates in the airways of people with asthma, and it contributes to airflow limitation by forming plugs that occlude airways. Current treatments have minimal effects on mucus or its chief components, the polymeric mucin glycoproteins MUC5AC and MUC5B. This treatment gap reflects a poor molecular understanding of mucins that could be used to determine how they contribute to airway obstruction. Due to the prominence of glycosylation as a defining characteristic of mucins, we investigated characteristics of mucin glycans in asthma and in a mouse model of allergic asthma. Mucin fucosylation was observed in asthma, and in healthy mice it was induced as part of a mucous metaplastic response to allergic inflammation. In allergically inflamed mouse airways, mucin fucosylation was dependent on the enzyme fucosyltransferase 2 (Fut2). Fut2 gene deficient mice were protected from asthma-like airway hyperreactivity and mucus plugging. These findings provide mechanistic and translational links between observations in human asthma and a mouse model that may help improve therapeutic targeting of airway mucus.

physiology↗