Biology subjects

Schaefbauer, K. J.

Publications and source records attributed to Schaefbauer, K. J..

3 recordsLinked to original sources

bioRxiv2026

Targeting the DNA damage repair protein RAD51 alters fibroblast metabolism and enhances apoptosis in pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease characterized by aberrantly activated, apoptosis-resistant profibrotic lung (myo)fibroblasts. Prior research has demonstrated that lung fibroblasts from patients with IPF exhibit resistance to DNA damage, suggesting that this behavior contributes to their persistent survival and continuous proliferation. We propose that elevated levels of the DNA damage repair protein RAD51 regulate myofibroblast activation and apoptosis and provide a potential therapeutic target to impede fibrosis progression. MethodsHuman lung fibroblasts were transfected with siRNA against RAD51 or treated with RAD51-specific inhibitor B02 and markers of fibrosis, DNA damage, apoptosis, metabolic reprogramming, and mitochondrial dynamics were assessed. The preclinical efficacy of B02 was evaluated in human precision cut lung slices (PCLS) and in a mouse model of pulmonary fibrosis. FindingsRAD51 expression was significantly upregulated in the lungs and lung fibroblasts of IPF patients. Knockdown or inhibition of RAD51 in fibroblasts reduced profibrotic marker expression, suppressed mTORC1 signaling and mitochondrial function, and increased apoptosis susceptibility. Pharmacological inhibition of RAD51 shifted the profibrotic phenotype towards a fibrosis-resolving state in human and mouse PCLS, and in a bleomycin-induced mouse model of lung fibrosis. InterpretationThe inhibition of RAD51 exerts therapeutic benefits in lung fibrosis by promoting apoptosis. Our findings identify that inhibiting RAD51 with B02 in fibroblasts impairs DNA repair and induces metabolic reprogramming, making it a potential therapeutic target. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSPulmonary fibrosis (PF) is characterized by excessive fibroblast activation and subsequent deposition of extracellular matrix (ECM) proteins, which ultimately disrupt normal lung architecture. A significant contributing factor to the pathogenesis of pulmonary fibrosis is the presence of fibroblasts that are resistant to apoptosis, preventing normal wound healing. Recent studies highlight the DNA repair protein RAD51 as effective in protecting fibroblasts from death induced by chemotherapy and ionizing radiation. These finding suggested that RAD51 could have a role in fibroblast activation and apoptosis resistance in pulmonary fibrosis. Added value of this studyWe demonstrated that RAD51 is important for maintaining apoptosis-resistant fibrotic fibroblasts and their metabolic abnormalities. Our findings indicated that TGF{beta}-mediated upregulation of RAD51 reduces DNA damage, activates multiple pathways related to fibroblast activation and proliferation, and induces metabolic reprogramming, ultimately regulating apoptosis. Mechanistically, RAD51 inhibition enhanced p53 acetylation at lysine 120 and upregulated the expression proapoptotic proteins PUMA/BAK in mitochondria, promoting apoptosis. Pharmacological inhibition of RAD51 using the specific inhibitor B02 during the fibrotic phase of experimental lung disease effectively ameliorated pulmonary fibrosis. Implications of all the available evidenceOur findings establish that RAD51 plays an important role in the survival of apoptosis-resistant fibrotic fibroblasts. We propose that reducing RAD51 expression leads to the metabolic reprogramming of activated fibroblasts, resulting in decreased mitochondrial respiration, reduced ATP levels, and diminished glycolysis or glutaminolysis. These observations suggest that targeting energy metabolism through RAD51 inhibition could be a viable strategy to enhance apoptosis, thereby creating a therapeutically targetable pathway in fibrotic cells. These findings highlight the potential of RAD51 as a therapeutic target for the treatment of IPF.

cell biology↗

bioRxiv2022

Targeting Pulmonary Fibrosis by SLC1A5 dependent Glutamine Transport Blockade

The neutral amino acid glutamine plays a central role in TGF-{beta}-induced myofibroblast activation and differentiation. Cells take up glutamine mainly through a transporter expressed on the cell surface known as solute carrier SLC1A5. In this current work, we demonstrated that profibrotic actions of TGF-{beta} are mediated, at least in part, through a metabolic maladaptation of SLC1A5 and targeting SLC1A5 abrogates multiple facets of fibroblast activation. This approach could thus represent a novel therapeutic strategy to treat fibroproliferative diseases. We found that SLC1A5 was highly expressed in fibrotic lung fibroblasts and fibroblasts isolated from IPF lungs. The expression of profibrotic targets, cell migration, and anchorage independent growth by TGF-{beta} required the activity of SLC1A5. Loss or inhibition of SLC1A5 function enhanced fibroblast susceptibility to autophagy, suppressed mTOR, HIF, Myc signaling, and impaired mitochondrial function, ATP production and glycolysis. Pharmacological inhibition of SLC1A5 by small molecule inhibitor V-9302 shifted fibroblast transcriptional profiles from profibrotic to fibrosis resolving, and attenuated fibrosis in a bleomycin treated mouse model of lung fibrosis. This is the first study, to our knowledge, to demonstrate the utility of a pharmacological inhibitor of glutamine transport in fibrosis, laying a framework for new paradigm-shifting therapies targeting cellular metabolism for this devastating disease.

molecular biology↗

bioRxiv2018

Toward Precision Molecular Surgery: Robust, Selective Induction of Microhomology-mediated End Joining in vivo

One key problem in precision genome editing is the resultant unpredictable plurality of sequence outcomes at the site of targeted DNA double-strand breaks (DSBs). This is due to the typical activation of the versatile Non-homologous End Joining (NHEJ) pathway. Such unpredictability limits the utility of somatic gene editing for applications including gene therapy and functional genomics. For germline editing work, the accurate reproduction of identical alleles using NHEJ is a labor intensive process. In this study, we propose inducing Microhomology-mediated End Joining (MMEJ) as a viable solution for improving somatic sequence homogeneity in vivo, capable of generating a single predictable allele at high rates (56% ~ 86% of the entire mutant allele pool). Using a combined dataset from zebrafish (Danio rerio) in vivo and human HeLa cell in vitro as a training dataset, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation and prospectively designed MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated a high level of mutant allele homogeneity at these loci. F0 mutant zebrafish embryos and larvae generated with these gRNAs faithfully recapitulated previously reported, recessive loss-of-function phenotypes. We also provide a novel algorithm MENTHU (http://genesculpt.org/menthu/) for improved prediction of candidate MMEJ loci, suitable for both targeted and genome-wide applications. We believe that this MMEJ-centric approach will have a broad impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of a knockout mutant allele at base pair resolution via the standard NHEJ-based approach, we demonstrate that F0 founders transmitted the identical MMEJ allele of interest at high rates. Most importantly, the ability to directly dictate the reading frame of an endogenous target will have important implications for gene therapy applications in human genetic diseases.\n\nAuthor SummaryNew gene editing tools precisely break DNA at pre-defined genomic locations, but cells repair these lesions using diverse pathways that often lead to unpredictable outcomes in the resulting DNA sequences. This sequence diversity in gene editing outcomes represents an important obstacle to the application of this technology for human therapies. Using a vertebrate animal as a model system, we provide strong evidence that we can overcome this obstacle by selectively directing DNA repair of double-stranded breaks through a lesser-described pathway termed Microhomology-mediated End Joining (MMEJ). Unlike other, better-understood pathways, MMEJ uses recurring short sequence patterns surrounding the site of DNA breakage. This enables the prediction of repair outcomes with improved accuracy. Importantly, we also show that preferential activation of MMEJ is compatible with effective gene editing. Finally, we provide a simple algorithm and software for designing DNA-breaking reagents that have high chance of activating the MMEJ pathway. We believe that the MMEJ-centric approach to be broadly applicable for a variety of gene editing applications both within the laboratory and for human therapies.\n\nAuthor ContributionHA contributed in Conceptualization, Data Curation, Formal Analysis, Investigation, Funding Acquisition, Methodology, Validation, Visualization, Writing - Original draft preparation, and Writing - Review and Editing. TLE contributed in Data Curation, Investigation, Writing - Original draft preparation, and Writing - Review and Editing. GMG contributed in Software, Validation, and Writing. CMM contributed in Software Validation, and Writing. AVD contributed in Investigation, Methodology, Validation, and Writing - Review and Editing. KJS contributed in Investigation and Writing - Review and Editing. ACM contributed in Conceptualization, Data Curation, Investigation, and Writing - Review and Editing. DD contributed in Funding Acquisition, Resources, and Writing - Review and Editing. KJC contributed in Conceptualization, Funding Acquisition, Resources, Supervision, and Writing - Review and Editing. SCE contributed in Conceptualization, Funding Acquisition, Project Administration, Resources, Supervision, Writing - Review and Editing.

molecular biology↗