Biology subjects

Daly, L. A.

Publications and source records attributed to Daly, L. A..

14 recordsLinked to original sources

bioRxiv2026

An integrated mass spectrometry strategy for quantifying the proteoform diversity of the extensively modified O-glycoprotein Osteopontin

O-glycosylation is among the most abundant and structurally diverse post-translational modifications in eukaryotes, yet its heterogeneity renders O-glycoproteins exceptionally difficult to characterize. To overcome these challenges, we have developed an integrated mass spectrometry (MS) strategy to define the proteoform landscape of O-glycoproteins and applied it to human osteopontin (OPN). OPN is a disease-associated extracellular matrix protein subject to extensive modification. By combining native MS with serial exoglycosidase digestions, we directly resolved truncation, phosphorylation, sulfation, and O-glycosylation of OPN. Matched glycoproteomic analyses, using tailored (glyco)protease combinations, allowed us to quantify glycan heterogeneity inaccessible to conventional trypsin-based approaches or protein-centric methods. We integrated these datasets using forward compositional simulations to infer the intact OPN proteoform distribution and benchmarked the resulting models against an experimental intact-mass distribution obtained by proton-transfer charge-reduction MS. This comparison revealed that bottom-up O-glycoproteomics systematically underestimates the true extent of glycan sialylation, whereas assuming (near-)complete sialylation accurately reproduced the experimental intact OPN mass distribution. Together, these results provide a comprehensive, quantitative view of OPN compositional diversity and demonstrate how intact-protein and peptide-level measurements can be reconciled to resolve highly heterogeneous glycoform populations. The workflow establishes a broadly applicable framework for characterizing extensively O-glycosylated and multiply modified proteins.

biophysics↗

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

Pervanadate-induced oxidation relieves autoinhibition of SRC protein tyrosine kinase

Dynamic regulation of protein tyrosine phosphorylation (pTyr) by phosphatases (PTPs) and kinases enables cells to sense and respond to environmental changes. The widely used chemical probe Pervanadate (PV) induces accumulation of high levels of pTyr in cells, an effect primarily attributed to its properties as a PTP inhibitor. This led to the assertion that PTPs are the master gatekeeper of intracellular pTyr homeostasis. Here, we use diverse approaches to reveal that PV disrupts cellular redox homeostasis and directly activates SRC family tyrosine kinases via oxidation of specific cysteine residues. Using mass spectrometry and biophysical approaches, we show that oxidation activates SRC by disrupting autoinhibition and altering phosphopeptide binding by its SH2 domain. We further establish that redox-sensitive cysteine residues are essential for SRC to promote cellular overgrowth. Our findings call for a re-evaluation of PV-based experiments and provide compelling evidence that oxidation is a crucial mechanism in controlling the oncogenic properties of SRC.

molecular biology↗

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↗

bioRxiv2024

PSKH1 kinase activity is differentially modulated via allosteric binding of Ca2+ sensor proteins

Protein Serine Kinase H1 (PSKH1) was recently identified as a crucial factor in kidney development and is overexpressed in prostate, lung and kidney cancers. However, little is known about PSKH1 regulatory mechanisms, leading to its classification as a "dark" kinase. Here, we used biochemistry and mass spectrometry to define PSKH1s consensus substrate motif, protein interactors, and how interactors, including Ca2+ sensor proteins, promote or suppress activity. Intriguingly, despite the absence of a canonical Calmodulin binding motif, Ca2+-Calmodulin activated PSKH1 while, in contrast, the ER-resident Ca2+ sensor of the CREC family, Reticulocalbin-3, suppressed PSKH1 catalytic activity. In addition to antagonistic regulation of the PSKH1 kinase domain by Ca2+ sensing proteins, we identified UNC119B as a protein interactor that activates PSKH1 via direct engagement of the kinase domain. Our findings identify complementary allosteric mechanisms by which regulatory proteins tune PSKH1s catalytic activity, and raise the possibility that different Ca2+ sensors may act more broadly to tune kinase activities by detecting and decoding extremes of intracellular Ca2+ concentrations.

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↗

bioRxiv2023

A bespoke analytical workflow for the confident identification of sulfopeptides and their discrimination from phosphopeptides

Protein tyrosine sulfation (sY) is a post-translational modification (PTM) catalysed by Golgi-resident Tyrosyl Protein SulfoTransferases (TPSTs). Information on protein tyrosine sulfation is currently limited to [~]50 human proteins with only a handful having verified sites of sulfation. The contribution of this chemical moiety for the regulation of biological processes, both inside and outside the cell, remains poorly defined, in large part due to analytical limitations. Mass spectrometry-based proteomics is the method of choice for PTM analysis, but has yet to be applied for the systematic investigation and large-scale analysis of biomolecular sulfation (constituting the sulfome), primarily due to issues associated with discrimination of sY-from phosphotyrosine (pY)-containing peptides. In this study, we developed a mass spectrometry (MS)-based workflow centred on the characterization of sY-peptides, incorporating optimised Zr4+-IMAC and TiO2 enrichment strategies. Extensive characterization of a panel of sY- and pY-peptides using an array of MS fragmentation regimes (CID, HCD, EThcC, ETciD, UVPD) highlights differences in the ability to generate site-determining product ions, which can be exploited to differentiate sulfated peptides from nominally isobaric phosphopeptides based on precursor ion neutral loss at low collision energy. Application of our analytical workflow to a HEK-293 cell extracellular secretome facilitated identification of 21 new sulfotyrosine-containing proteins, several of which we validate enzymatically using in vitro sulfation assays. This study demonstrates the applicability of our strategy for confident, high-throughput, sulfomics studies, and reveals new sY interplay between enzymes relevant to both protein and glycan sulfation.

biochemistry↗

bioRxiv2023

Mechanistic and evolutionary insights into isoform-specific 'supercharging' in DCLK family kinases

Catalytic signaling outputs of protein kinases are dynamically regulated by an array of structural mechanisms, including allosteric interactions mediated by intrinsically disordered segments flanking the conserved catalytic domain. The Doublecortin Like Kinases (DCLKs) are a family of microtubule-associated proteins characterized by a flexible C-terminal autoregulatory tail segment that varies in length across the various human DCLK isoforms. However, the mechanism whereby these isoform-specific variations contribute to unique modes of autoregulation is not well understood. Here, we employ a combination of statistical sequence analysis, molecular dynamics simulations and in vitro mutational analysis to define hallmarks of DCLK family evolutionary divergence, including analysis of splice variants within the DCLK1 sub-family, which arise through alternative codon usage and serve to supercharge the inhibitory potential of the DCLK1 C-tail. We identify co-conserved motifs that readily distinguish DCLKs from all other Calcium Calmodulin Kinases (CAMKs), and a Swiss-army assembly of distinct motifs that tether the C-terminal tail to conserved ATP and substrate-binding regions of the catalytic domain to generate a scaffold for auto-regulation through C-tail dynamics. Consistently, deletions and mutations that alter C-terminal tail length or interfere with co-conserved interactions within the catalytic domain alter intrinsic protein stability, nucleotide/inhibitor-binding, and catalytic activity, suggesting isoform-specific regulation of activity through alternative splicing. Our studies provide a detailed framework for investigating kinome-wide regulation of catalytic output through cis-regulatory events mediated by intrinsically disordered segments, opening new avenues for the design of mechanistically-divergent DCLK1 modulators, stabilizers or degraders.

evolutionary biology↗

bioRxiv2022

Evolutionary and cellular analysis of the dark pseudokinase PSKH2

Pseudokinases, so named because they lack one or more conserved canonical amino acids that define their catalytically-active relatives, have evolved a variety of biological functions in both prokaryotic and eukaryotic organisms. Human PSKH2 is closely related to the canonical kinase PSKH1, which maps to the CAMK family of protein kinases. Primates encode PSKH2 in the form of a pseudokinase, which is predicted to be catalytically inactive due to loss of the invariant catalytic Asp residue. Although the biological role(s) of vertebrate PSKH2s remains unclear, we previously identified species-level adaptions in PSKH2 that have led to the appearance of kinase or pseudokinase variants in vertebrate genomes alongside a canonical PSKH1 paralog. In this paper we confirm that, as predicted, PSKH2 lacks detectable protein phosphotransferase activity, and exploit structural informatics, biochemistry and cellular proteomics to begin to characterise vertebrate PSKH2 orthologues. AlphaFold 2-based structural analysis predicts functional roles for both the PSKH2 N- and C-regions that flank the pseudokinase domain core, and cellular truncation analysis confirms that the N-terminal domain, which contains a conserved myristoylation site, is required for both stable human PSKH2 expression and localisation to a membrane-rich subcellular fraction containing mitochondrial proteins. Using mass spectrometry-based proteomics, we confirm that human PSKH2 is part of a cellular mitochondrial protein network, and that its expression is regulated through client-status within the HSP90/Cdc37 molecular chaperone system. HSP90 interactions are mediated through binding to the PSKH2 C-terminal tail, leading us to predict that this region might act as both a cis and trans regulatory element, driving outputs linked to the PSKH2 pseudokinase domain that are important for functional signalling.

biochemistry↗

bioRxiv2021

Mechanism of glycogen synthase inactivation and interaction with glycogenin

Glycogen is the major glucose reserve in eukaryotes, and defects in glycogen metabolism and structure lead to disease. Glycogenesis involves interaction of glycogenin (GN) with glycogen synthase (GS), where GS is activated by glucose-6-phosphate (G6P) and inactivated by phosphorylation. We describe the 2.6 [A] resolution cryo-EM structure of phosphorylated human GS revealing an autoinhibited GS tetramer flanked by two GN dimers. Phosphorylated N- and C-termini from two GS protomers converge near the G6P-binding pocket and buttress against GS regulatory helices. This keeps GS in an inactive conformation mediated by phospho-Ser641 interactions with a composite "arginine cradle". Structure-guided mutagenesis perturbing interactions with phosphorylated tails led to increased basal/unstimulated GS activity. We propose that multivalent phosphorylation supports GS autoinhibition through interactions from a dynamic "spike" region, allowing a tuneable rheostat for regulating GS activity. This work therefore provides new insights into glycogen synthesis regulation and facilitates studies of glycogen-related diseases.

biochemistry↗

bioRxiv2021

Displacement of PKA catalytic subunit from AKAP signaling islands drives pathology in Cushing's syndrome

Mutations in the catalytic subunit of protein kinase A (PKAc) drive the stress hormone disorder adrenal Cushings syndrome. Here we define mechanisms of action for the PKAc-L205R and W196R variants. Both Cushings mutants are excluded from A kinase anchoring protein (AKAP) signaling islands and consequently diffuse throughout the cell. Kinase-dead experiments show that PKA activity is required for cortisol hypersecretion. However, kinase activation is not sufficient, as only cAMP analog drugs that displace native PKAc from AKAPs enhance cortisol release. Rescue experiments that incorporate mutant PKAc into AKAP signaling islands abolish cortisol overproduction, indicating that kinase anchoring restores normal endocrine function. Phosphoproteomics show that PKAc-L205R and W196R engage different mitogenic signaling pathways. ERK activity is elevated in adrenal-specific PKAc-W196R knock-in mice. Conversely, PKAc-L205R attenuates Hippo signaling, thereby upregulating the YAP/TAZ transcriptional co-activators. Thus, aberrant localization of each Cushings variant promotes the transmission of a distinct downstream pathogenic signal.

molecular biology↗

bioRxiv2020

Oxygen-dependent changes in HIF binding partners and post-translational modifications regulate stability and transcriptional activity

Adaption of cells to low oxygen environments is an essential process mediated in part by the Hypoxia Inducible Factors (HIFs). Like other transcription factors, the stability and transcriptional activity of HIFs, and consequently the hypoxic response, are regulated by post-translational modification (PTM) and changes in biomolecular interactions. However, our current understanding of PTM-mediated regulation of HIFs is primarily based on in vitro protein fragment-based studies, with validation typically having been conducted by in cellulo fragment expression and hypoxia mimicking drugs. Consequently, we still lack an understanding of true oxygen deprivation signaling via HIF. Using an immunoprecipitation-based, mass spectrometry approach, we characterize the regulation of in cellulo expressed full-length HIF-1 and HIF-2, in terms of both PTM and binding partners, in response to normoxia (21% oxygen) and hypoxia (1% oxygen). These studies revealed that a change in oxygen tension significantly alters the complexity and composition of HIF- protein interaction networks, with HIF-2 in particular having an extended hypoxia-induced interactome, most notably with mitochondrial-associated proteins. Both HIF isoforms are heavily covalently modified: we define ~40 different sites of PTM on each of HIF-1 and HIF-2, comprising 13 different PTM types, including multiple cysteine modifications and a highly unusual phosphocysteine. Over 80% of the PTMs identified are novel, and approximately half exhibit oxygen-dependency under these conditions. Combined with domain and evolutionary analysis of >225 vertebrate species, we validate Ser31 phosphorylation on HIF-1 as a regulator of transcription, and propose functional roles for Thr406, Thr528 and Ser581 on HIF-2.

biochemistry↗

bioRxiv2020

Characterisation of protease activity during SARS-CoV-2 infection identifies novel viral cleavage sites and cellular targets for drug repurposing

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, and responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication, and inhibitors targeting proteases have already shown success at inhibiting SARS-CoV-2 in cell culture models. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigenic proteins S and N, which are the main targets for vaccine and antibody testing efforts. We discovered significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases, validating a subset with in vitro assays. We showed that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, showed a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19.

microbiology↗