Biology subjects

Lawlor, M.

Publications and source records attributed to Lawlor, M..

4 recordsLinked to original sources

bioRxiv2024

Integrated single cell functional-proteomic profiling of human skeletal muscle reveals a shift in cellular specificity in nemaline myopathy

Skeletal muscle is a complex syncytial arrangement of an array of cell types and, in the case of muscle specific cells (myofibers), sub-types. There exists extensive heterogeneity in skeletal muscle functional behaviour and molecular landscape, at the cell composition, myofiber sub-type and intra-myofiber sub-type level. This heterogeneity highlights limitations in currently applied methodological approaches, which has stagnated our understanding of fundamental skeletal muscle biology in both healthy and myopathic contexts. Here, we developed a novel approach that combines a fluorescence based assay for the biophysical examination of the sarcomeric protein, myosin, coupled with same-myofiber high sensitivity proteome profiling, termed Single Myofiber Protein Function-Omics (SMPFO). Successfully applying this approach to healthy human skeletal muscle tissue, we identify the integrate relationship between myofiber functionality and the underlying proteomic landscape that guides divergent, but physiologically important, behaviour in myofiber sub-types. By applying SMPFO to two forms of human nemaline myopathy (ACTA1 and TNNT1 mutations), we reveal significant reduction in the divergence of myofiber sub-types, across both biophysical and proteomic behaviour. Collectively, we develop SMPFO as a novel approach to study skeletal muscle with greater specificity, accuracy and resolution then currently applied methods, facilitating that advancement in understanding of SkM tissue in both healthy and diseased states.

physiology↗

bioRxiv2023

Characterization of NEB mutations in patients reveals novel nemaline myopathy disease mechanisms and omecamtiv mecarbil force effects

Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Mutations in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking therapies targeting the underlying pathological mechanisms. In this study, we examined a cohort of ten NEM2 patients, each with unique mutations, aiming to understand their impact on mRNA, protein, and functional levels. Results show that truncation mutations affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with splicing mutations which is expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with relatively normal nebulin levels and others with markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, and a positive relation between the reduction in nebulin level and the reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a duplication mutation in nebulin that resulted in a larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type I muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87-318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from splicing mutations. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.

genetics↗

bioRxiv2023

KAT2 paralogs prevent dsRNA accumulation and interferon signaling to maintain intestinal stem cells

Histone acetyltransferases KAT2A and KAT2B are paralogs highly expressed in the intestinal epithelium, but their functions are not well understood. In this study, double knockout of murine Kat2 genes in the intestinal epithelium was lethal, resulting in robust activation of interferon signaling and interferon-associated phenotypes including the loss of intestinal stem cells. Use of pharmacological agents and sterile organoid cultures indicated a cell-intrinsic double-stranded RNA trigger for interferon signaling. Acetyl-proteomics and dsRIP-seq were employed to interrogate the mechanism behind this response, which identified mitochondria-encoded double-stranded RNA as the source of intrinsic interferon signaling. Kat2a and Kat2b therefore play an essential role in regulating mitochondrial functions as well as maintaining intestinal health. Highlights of the workO_LIKat2a and Kat2b double knockout in the murine intestinal epithelium triggers activation of the interferon signaling pathway C_LIO_LIKat2a/Kat2b knockout leads to intestinal stem cell loss and other mucosal phenotypes consistent with interferon activation C_LIO_LIHistone PTM mass spec profiling reveals the first in vivo study showing H3K9ac-specific loss with Kat2a and Kat2b double knockout, yet without correlation to interferon signaling pathway genes C_LIO_LIComprehensive proteomic analysis identifies non-histone acetyl-lysine targets of KAT2 in the mouse intestine in vivo, including mitochondrial proteins C_LIO_LIMitochondrial function is compromised upon Kat2 loss C_LIO_LIdsRIP-seq identifies double-stranded RNA from the mitochondria as a trigger for the intrinsic immune response upon Kat2 double knockout C_LI

molecular biology↗

bioRxiv2021

APOBEC3A drives acquired resistance to targeted therapies in non-small cell lung cancer

Acquired drug resistance to even the most effective anti-cancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified1-6, the underlying molecular mechanisms shaping tumor evolution during treatment are incompletely understood. The extent to which therapy actively drives tumor evolution by promoting mutagenic processes7 or simply provides the selective pressure necessary for the outgrowth of drug-resistant clones8 remains an open question. Here, we report that lung cancer targeted therapies commonly used in the clinic induce the expression of cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Induction of A3A facilitated the formation of double-strand DNA breaks (DSBs) in cycling drug-treated cells, and fully resistant clones that evolved from drug-tolerant intermediates exhibited an elevated burden of chromosomal aberrations such as copy number alterations and structural variations. Preventing therapy-induced A3A mutagenesis either by gene deletion or RNAi-mediated suppression delayed the emergence of drug resistance. Finally, we observed accumulation of A3A mutations in lung cancer patients who developed drug resistance after treatment with sequential targeted therapies. These data suggest that induction of A3A mutagenesis in response to targeted therapy treatment may facilitate the development of acquired resistance in non-small-cell lung cancer. Thus, suppressing expression or enzymatic activity of A3A may represent a potential therapeutic strategy to prevent or delay acquired resistance to lung cancer targeted therapy.

cancer biology↗