Biology subjects

Fahlman, R.

Publications and source records attributed to Fahlman, R..

4 recordsLinked to original sources

bioRxiv2025

Optimization of Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) for effective low-disruption labelling of Arabidopsis proteins in vivo

Plants require robust responses in protein synthesis to adapt to variable environmental conditions. Measurement of newly synthesized proteins has been successfully facilitated with Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) across a multitude of organisms. Here, we use non-canonical amino acids (NCAAs) L-azidohomoalanine (AHA) or L-homopropargylglycine (HPG) incorporation in place of methionine residues into the actively translating Arabidopsis proteome, allowing for that subset of proteins to be enriched for mass spectrometry quantification. Although this technique has seen occasional use in plants, optimization of the protocol to maximize functionality while minimizing organismal stress has not yet been established. Here, we provide evidence for successful implementation through the liquid immersion of seedlings in AHA or HPG-containing media that functions with significantly lower concentrations than the literature standard. Our approach splits acute exposure and incorporation phase of labelling to mitigate potential negative impacts of prolonged NCAA exposure without compromising effective enrichment capacity, and demonstrate that this results in an unperturbed growth phenotype for AHA-treated seedlings. Finally, we demonstrate the capacity of this modified approach to enrich newly synthesized proteins from the whole proteome under standard stress conditions. These improvements allow for a broader use of BONCAT technologies in molecular plant research, affording a deeper understanding of the newly synthesized proteome without negatively impacting plant health.

plant biology↗

bioRxiv2025

Hyperacute Response Proteins (HARPs) synthesized on γ-tubulin-FTO-MARK4 translation microdomains upon exposure to stress, regulate stressresponse in cancer.

Compared to normal, cancer cells are particularly resistant to stress, and their immediate response to stress is critical for their subsequent multilayered adaptation programs which pose a major clinical challenge. With unbiased proteomics and transcriptomics analysis, we identified a list of HARPs synthesized from pre-existing mRNAs within 20 min of diverse stresses in A549 cancer cells, despite the known suppressed global translation in stress. HARP mRNAs were translated on microtubule-associated translation microdomains (MATMs) located on {gamma}-tubulin, that host FTO and specialized cytoskeletal ribosomes, structurally and functionally distinct from ER and cytosolic ribosomes. FTO exited the nucleus immediately after stress and was activated by the microtubule-associated stress kinase MARK4 via T6 phosphorylation. Activated FTO demethylated a translation-inhibiting mRNA methylation (m6A) signature, facilitating compartmentalized HARP translation on MATMs, while non-HARP mRNA remained inhibited. FTO or MARK4 inhibition suppressed HARP synthesis and increased apoptosis post various stresses, including chemotherapy. These data were confirmed in 4 additional cancer cell lines and normal fibroblasts. Using the Protein Atlas database, we found that high levels of our identified HARPs had on average a 35% decrease on patient 5-year survival in prevalent and resistant cancers (breast, lung, liver, pancreas). {gamma}-tubulin, FTO and MARK4 are therapeutic targets for many cancers, through their ability to comprehensively promote HARPs translation, a potential Achilles heel for cancers resistance to physiologic or therapeutic stress, offering a new window in stress biology.

cancer biology↗

bioRxiv2023

Fibronectin: a natural barrier against prion infection

A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrPSc, in the central nervous system of prion-affected humans and animals. PrPSc is also found in peripheral tissues, raising concerns about the potential transmission of pathogenic prions through human food supplies and posing a significant risk to public health. Although muscle tissues are considered to contain levels of low prion infectivity, it has been shown that myotubes in culture efficiently propagate PrPSc. Given the high consumption of muscle tissue, it is important to understand what factors could influence the establishment of a prion infection in muscle tissue. Here we used in vitro myotube cultures, differentiated from the C2C12 myoblast cell line (dC2C12), to identify factors affecting prion replication. A range of experimental conditions revealed that PrPSc is tightly associated with proteins found in the systemic extracellular matrix (ECM), mostly fibronectin (FN). The interaction of PrPSc with FN decreased prion infectivity, as determined by standard scrapie cell assay. Interestingly, the prion-resistant reserve cells in dC2C12 cultures displayed a FN-rich ECM while the prion-susceptible myotubes expressed FN at a low level. In agreement with the in vitro results, immunohistopathological analyses of tissues from sheep infected with natural scrapie demonstrated a prion susceptibility phenotype linked to an extracellular matrix with undetectable levels of FN. Conversely, PrPSc deposits were not observed in tissues expressing FN. These data indicate that extracellular FN may act as a natural barrier against prion replication and that the extracellular matrix composition may be a crucial feature determining prion tropism in different tissues. Author summaryPrion diseases are complex fatal neurodegenerative disorders caused by a misfolded form of the cellular protein PrPC (PrPSc). Due to the potential zoonotic transmission of these disorders through animal-based food intake, it is crucial to identify the tissues in which PrPSc can accumulate and what might influence its tropism. Animal muscle (or meat) and related food products are highly consumed, raising concern of the involvement of muscle cells in prion replication. Muscle tissue from prion-affected animals contains low levels of infectivity and PrPSc is mostly associated with nerve structures rather than myofibers, whereas C2C12 myotubes, a muscle-derived cell type, efficiently replicate prions in vitro and generate high levels of infectivity compared with other cell cultures. We demonstrate a fibronectin-mediated interference with prion infection in differentiated C2C12 cultures that correlates with the findings in tissues from naturally scrapie-infected animals. Our results suggest that extracellular matrix composition, specifically regarding the presence of fibronectin, might determine prion tropism and dissemination.

biochemistry↗

bioRxiv2018

Estrogen receptor regulates hormone-induced growth arrest in a luminal A like breast cancer model

Estrogen receptor positive (ER+) breast cancer has been divided into two subtypes, luminal A and luminal B, which differ in their ER expression and response to hormone therapy. The absence of luminal A cell lines means the extensive amount of in vitro work studying the response to hormones in ER+ breast cancers is biased for the luminal B subtype. We have developed a luminal A like cell model by increasing the ER expression in the MCF-7 cell line. Our results show that increased ER expression promotes an anti-proliferative response to estrogen through regulation of genes involved in the G1/S-phase transition of the cell cycle. Furthermore, increased ER expression increases ER-DNA binding in the absence of estrogen and regulates basal gene transcription by promoting DNA looping. These results provide novel evidence that the characteristic increased ER expression of luminal A tumors may promote a novel chromatin configuration that enables growth of these tumors in the absence of estrogen and enables gene repression in the presence of hormones.

pathology↗