Biology subjects

Oswald, S. O.

Publications and source records attributed to Oswald, S. O..

4 recordsLinked to original sources

bioRxiv2026

Phosphorylation at S345 Converts HIF-2α from a Transcription Factor to an RNA binding protein

Oxygen sensing and adaptation are vital for metazoan survival. At the cellular level the response to hypoxia is characterised by a switch in transcriptional programming, primarily mediated by hypoxia-inducible factors (HIFs). HIFs comprise an obligatory heterodimer between an oxygen-sensitive HIF- subunit and an oxygen-insensitive HIF-1{beta} subunit, which together regulate gene expression in response to hypoxia. Among the three HIF- proteins in vertebrates (HIF-1, HIF-2, and HIF-3), post-translational modifications (PTMs) play a crucial role in modulating their stability, localisation, and activity. While many modifications have been investigated on HIF-1, our knowledge of HIF-2 regulation is very limited. Here, we investigated the function of the recently identified HIF-2 S345 phosphorylation site and demonstrate that this disrupts the interaction between HIF-2 and HIF-1{beta}, thereby silencing the HIF-2 transcriptional response. Additionally, our findings suggest that this modification redirects HIF-2 from its normal transcription factor function towards a role in mRNA fate control, as indicated by mass spectrometry interactome analysis and RNA-immunoprecipitation. Our discoveries highlight a new regulatory mechanism of HIF-2 activity, where S345 phosphorylation impedes HIF-2/HIF-1{beta} heterodimer formation, creating a switch in HIF-2 function. As HIF-2 is the only HIF- isoform with a clinically approved inhibitor, these insights are crucial for advancing therapeutic strategies targeting hypoxia signalling.

molecular biology↗

bioRxiv2025

Hypoxia-induced regulation of zDHHC23 opens avenues for new biomarkers for NON MYCN-amplified neuroblastoma

Neuroblastoma is a highly metastatic paediatric malignancy with poor prognosis, and remains a leading cause of paediatric cancer mortality. Current risk stratification is disproportionately reliant on MYCN amplification, a feature present in only [~]25% of cases. This narrow focus neglects the majority of patients that have non-MYCN amplified tumours, limiting opportunities for therapeutic innovation. To define molecular drivers of metastasis in non-MYCN amplified neuroblastoma and identify putative prognostic markers, we leveraged our validated in vivo chick embryo xenograft model to profile oxygen-sensitive transcriptional changes, revealing a cohort of [~]400 genes associated with metastatic competence. Integrative survival analysis using two independent patient cohorts identified 59 genes predictive of event-free survival in non-MYCN amplified disease. Among these, the poorly characterized Zinc Finger DHHC-Type Palmitoyltransferase 23 (zDHHC23) emerged as a robust prognostic candidate. Proteomic interrogation of zDHHC23 under varying oxygen conditions uncovered dynamic interactome remodelling, notably hypoxia-attenuated association with the 26S proteasome and the TIM23 mitochondrial import complex. These findings suggest zDHHC23 as a hypoxia-responsive regulator linked to metastatic signalling, and a promising target for biomarker development and therapeutic intervention in high-risk, non-MYCN amplified neuroblastoma.

neuroscience↗

bioRxiv2025

An optimized SP3 sample processing workflow for in-depth and re-producible phosphoproteomics

Protein phosphorylation is a ubiquitous post-translational modification (PTM) found across the kingdoms of life, and is critical for the regulation of protein function in health & disease. Advances in high-throughput mass spectrometry have transformed our ability to interrogate the phosphoproteome. However, sample preparation methodologies optimized for phosphoproteomics have not kept pace, compromising the ability to fully exploit these technological advances. In this study, we present an optimized phosphoproteomics workflow using carboxylated SP3 magnetic beads which have simplified proteomics sample preparation. By employing a washing step with 8 M urea and omitting the conventional C18 SPE clean-up, we demonstrate a significant improvement in phosphopeptide identifications, with application of this refined protocol to HEK-293T cell extracts increasing the number nearly 2-fold compared to standard SP3 techniques (7908 cf. 4129). We also observed a substantial improvement in the detection of multiply phosphorylated peptides. Our findings suggest that the complexity of PTM crosstalk using current peptide-based proteomics workflows is currently under-represented and underscores the necessity of methodological innovations to better capture the intricacies of the phosphoproteome landscape.

biochemistry↗

bioRxiv2023

Redox Regulation of Brain Selective Kinases BRSK1/2: Implications for Dynamic Control of the Eukaryotic AMPK family through Cys-based mechanisms

In eukaryotes, protein kinase signaling is regulated by a diverse array of post-translational modifications (PTMs), including phosphorylation of Ser/Thr residues and oxidation of cysteine (Cys) residues. While regulation by activation segment phosphorylation of Ser/Thr residues is well understood, relatively little is known about how oxidation of cysteine residues modulate catalysis. In this study, we investigate redox regulation of the AMPK-related Brain-selective kinases (BRSK) 1 and 2, and detail how broad catalytic activity is directly regulated through reversible oxidation and reduction of evolutionarily conserved Cys residues within the catalytic domain. We show that redox-dependent control of BRSKs is a dynamic and multilayered process involving oxidative modifications of several Cys residues, including the formation of intra-molecular disulfide bonds involving a pair of Cys residues near the catalytic HRD motif and a highly conserved T-Loop Cys with a BRSK-specific Cys within an unusual CPE motif at the end of the activation segment. Consistently, mutation of the CPE-Cys increases catalytic activity in vitro and drives phosphorylation of the BRSK substrate Tau in cells. Molecular modeling and molecular dynamics simulations indicate that oxidation of the CPE-Cys destabilizes a conserved salt bridge network critical for allosteric activation. The occurrence of spatially proximal Cys amino acids in diverse Ser/Thr protein kinase families suggests that disulfide mediated control of catalytic activity may be a prevalent mechanism for regulation within the broader AMPK family.

biochemistry↗