Biology subjects

Forrester, M. T.

Publications and source records attributed to Forrester, M. T..

5 recordsLinked to original sources

bioRxiv2026

Versatile and Cross-Species Alveolar Type II Cell Isolation Using Humanized Monoclonal Antibody Upifitamab

Alveolar type 2 (AT2) cells are primary adult alveolar stem cells that maintain healthy alveoli. AT2 cell dysfunction is broadly linked to many lung pathologies from emphysema to fibrosis. Isolation of AT2 cells with high purity is therefore central to diagnostic studies, proteomics, transcriptomics and ex vivo experiments that can lead to future AT2 cellular therapies. Current markers for AT2 isolation exhibit various limitations from species-restrictions (e.g., mouse vs human) to limited AT2 specificity, which creates challenges for flow cytometric gating/sorting and application to magnetic-activated cell isolation. While underrepresented in the literature, these bottlenecks have made AT2 cell isolation inherently difficult and slowed AT2 cell research. Here we leverage surface protein-restricted transcriptomics to identify the sodium phosphate transporter NaPi2b (SLC34A2) as a highly specific AT2 cell marker optimally detected with the humanized monoclonal antibody upifitamab. Upifitamab-based labeling of AT2 cells is compatible with flow cytometric or magnetic-activated AT2 isolation and applies to both mouse and human AT2 cells, thus streamlining protocols and circumventing species-specific nuances. AT2 cells obtained with upifitamab can be cultured as organoids, in monolayers or differentiated to AT1 cells. This simplified AT2 isolation approach should overcome several current barriers while facilitating ex vivo studies on alveolar biology and related therapeutic developments.

molecular biology↗

bioRxiv2026

A tonic PDGFA-dependent niche organizer maintains alveolar homeostasis

Tissue homeostasis requires mechanisms that preserve quiescence while allowing rapid activation after injury, but how tissues actively restrain injury responses during homeostasis remains poorly understood. Here we identify alveolar type 1 (AT1) cells as organizers of the distal lung niche through tonic expression of PDGFA, which maintains alveolar fibroblast identity and restrains myofibroblast activation. Cell type-specific ablation revealed that loss of AT1s, but not other major alveolar cell populations, was sufficient to evoke spontaneous fibroblast activation and tissue-wide responses that recapitulate injury. In silico ligand-receptor analysis, ex vivo screening, and conditional genetic models identified AT1-derived PDGFA as a key homeostatic signal. Loss of AT1-derived PDGFA caused spontaneous myofibroblast induction and tissue remodeling, whereas increasing PDGFA promoted fibroblast proliferation while suppressing myofibroblast differentiation. Integrated phosphoproteomic, transcriptomic, and chromatin accessibility analyses showed that PDGFA engages SHP2-ERK signaling, suppresses MRTFA-associated SRF programs, and promotes E2F-associated cell-cycle programs, thereby opposing the pro-fibrotic effects of TGF{beta}-driven fibroblast activation. Restoring PDGFA signaling abrogates myofibroblast features from human idiopathic pulmonary fibrotic lungs and attenuated fibrotic remodeling in vivo. These findings define a tonic AT1-derived PDGFA signal that maintains alveolar homeostasis and offers a therapeutic target to prevent or revert lung fibrosis.

cell biology↗

bioRxiv2026

Alveolar Cytochrome P450 Mediates Butylated Hydroxytoluene-Induced Electrophilic Injury and Apoptosis

Butylated hydroxytoluene (BHT) is a synthetic phenolic antioxidant utilized as a preservative in many products from foods to cosmetics. Population-based studies have detected BHT in the vast majority (>90%) of human specimens (serum, urine, fingernails) as well as home dust samples. Thus, BHT constitutes a ubiquitous environmental contaminant without obvious adverse health effects to humans. In contrast, exposure of mice to a single intraperitoneal dose of BHT triggers distal epithelial damage including loss of gas-exchanging alveolar type 1 epithelial cells, thus providing an invaluable tool to study alveolar repair and transient fibrosis. Presently, the molecular basis of BHT lung toxicity remains unknown. To address this, mouse lung single cell transcriptomic data were used to identify cytochrome P450 2B10 (CYP2B10) in AT1 cells as a BHT-activating enzyme. In cell culture systems, expression of CYP2B10 leads to marked BHT sensitization consistent with bioactivation of BHT into a toxic quinone methide. Targeted and proteome-wide experiments identify BHT-induced protein alkylation, DNA damage response, stress kinase activation and intrinsic apoptosis in a CYP2B10-dependent manner. Structure-activity relationship studies reveal the necessity of para-methyl and ortho-t-butyl groups necessary for BHT bioactivation. Our findings elucidate the molecular basis by which BHT is converted from an innocuous antioxidant into a highly toxic quinone methide and identify the first mammalian enzyme known to catalyze BHT bioactivation. Further, CYP2B10-catalyzed BHT bioactivation may provide a strategy for future targeted cell ablation technologies or environmental remediation of BHT.

molecular biology↗

bioRxiv2024

Topology-Driven Discovery of Transmembrane Protein S-Palmitoylation

Protein S-palmitoylation is a reversible lipophilic posttranslational modification regulating a diverse number of signaling pathways. Within transmembrane proteins (TMPs), S-palmitoylation is implicated in conditions from inflammatory disorders to respiratory viral infections. Many small-scale experiments have observed S-palmitoylation at juxtamembrane Cys residues. However, most large-scale S-palmitoyl discovery efforts rely on trypsin-based proteomics within which hydrophobic juxtamembrane regions are likely underrepresented. Machine learning- by virtue of its freedom from experimental constraints - is particularly well suited to address this discovery gap surrounding TMP S-palmitoylation. Utilizing a UniProt-derived feature set, a gradient boosted machine learning tool (TopoPalmTree) was constructed and applied to a holdout dataset of viral S-palmitoylated proteins. Upon application to the mouse TMP proteome, 1591 putative S-palmitoyl sites (i.e. not listed in SwissPalm or UniProt) were identified. Two lung-expressed S-palmitoyl candidates (synaptobrevin Vamp5 and water channel Aquaporin-5) were experimentally assessed. Finally, TopoPalmTree was used for rational design of an S-palmitoyl site on KDEL-Receptor 2. This readily interpretable model aligns the innumerable small-scale experiments observing juxtamembrane S-palmitoylation into a proteomic tool for TMP S-palmitoyl discovery and design, thus facilitating future investigations of this important modification.

molecular biology↗

bioRxiv2024

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap)

Proteins undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 micrograms of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g. H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 micrograms of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamlines the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/586403v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@16d595aorg.highwire.dtl.DTLVardef@55acforg.highwire.dtl.DTLVardef@18cf92eorg.highwire.dtl.DTLVardef@3b5fb4_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗