Biology subjects

Bayly-Jones, C.

Publications and source records attributed to Bayly-Jones, C..

7 recordsLinked to original sources

bioRxiv2024

Feed-forward stimulation of CAMK2 by the oncogenic pseudokinase PEAK1 generates a therapeutically "actionable" signalling axis in triple negative breast cancer

The PEAK family of pseudokinases, comprising PEAK1-3, are signalling scaffolds that play oncogenic roles in several poor prognosis human cancers, including triple negative breast cancer (TNBC). However, therapeutic targeting of pseudokinases is challenging due to their lack of catalytic activity. To address this, we screened for PEAK1 effectors by affinity purification and mass spectrometry, identifying calcium/calmodulin-dependent protein kinase 2 (CAMK2)D and CAMK2G. PEAK1 promoted CAMK2D/G activation in TNBC cells via a novel feed-forward mechanism involving PEAK1/PLC{gamma}1/Ca2+ signalling and direct binding via a consensus CAMK2 interaction motif in the PEAK1 N-terminus. In turn, CAMK2 phosphorylated PEAK1 to enhance association with PEAK2, which is critical for PEAK1 oncogenic signalling. To achieve pharmacologic targeting of PEAK1/CAMK2, we repurposed RA306, a second generation CAMK2 inhibitor under pre-clinical development for treatment of cardiovascular disease. RA306 demonstrated on-target activity against CAMK2 in TNBC cells and inhibited PEAK1-enhanced migration and invasion in vitro. Moreover, RA306 significantly attenuated TNBC xenograft growth and blocked metastasis in a manner mirrored by CRISPR-mediated PEAK1 ablation. Overall, these studies establish PEAK1 as a critical cell signalling nexus, identify a novel mechanism for regulation of Ca2+ signalling and its integration with tyrosine kinase signals, and identify CAMK2 as a therapeutically actionable target downstream of PEAK1.

cancer biology↗

bioRxiv2022

The neoepitope of the complement C5b-9 Membrane Attack Complex is formed by proximity of adjacent ancillary regions of C9

The terminal C5b-9 complement complex (TCC) exists in two forms; the soluble sC5b-9 and the solid-phase inserted Membrane Attack Complex (MAC). The MAC is responsible for forming large {beta}-barrel channels in the membranes of pathogens and can target cells such as gram-negative bacteria. In addition, off-target MAC assembly on endogenous tissue is associated with inflammatory diseases and cancer. Accordingly, a human C5b-9 specific antibody, aE11, has been developed that detects a neoepitope exposed in C9 when it is incorporated into the C5b-9 complex and in polyC9, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MACrelated inflammation, and pathophysiology. However, the identity of this C9 neoepitope remains unknown. Herein, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 [A]resolution. The aE11 binding site revealed that the neoepitope was formed by two separate surfaces on the pore periphery, rather than a conformational change in the protein structure, and is therefore a discontinuous quaternary epitope. These surfaces are contributed by portions of the adjacent TSP1, LDLRA and MACPF domains of two neighbouring C9 protomers. By substituting key antibody interacting residues to the murine orthologue, we validated the unusual binding modality of aE11. Furthermore, our observations indicated that aE11 can recognise a partial epitope in purified monomeric C9, albeit binding was two orders of magnitude weaker. Taken together, our results reveal the basis for MAC recognition by aE11 and that the C9 neoepitope can be formed without substantial conformational rearrangements adding insight into how aE11 is used to quantify MAC formation in disease.

biochemistry↗

bioRxiv2022

Helical ultrastructure of the oncogenic metalloprotease meprin α in complex with a small molecule hydroxamate inhibitor

The zinc-dependent metalloprotease meprin is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal tissue homeostasis meprin performs key roles in inflammation, immunity, and extracellular matrix remodelling. The latter activity is furthermore important for driving aggressive metastasis in the context of certain cancers such as colorectal carcinoma. Accordingly, meprin is the target of drug discovery programs. In contrast to meprin {beta}, meprin is secreted into the extracellular space, whereupon it oligomerises to form giant assemblies and is the largest extracellular protease identified to date (~6 MDa). Here, using cryo-electron microscopy, we determine the high-resolution structure of the zymogen and mature form of meprin , as well as the structure of the active form in complex with a prototype small molecule inhibitor and human fetuin-B. Our data reveal that meprin forms a giant, flexible, left-handed helical assembly of roughly 22 nm in diameter. We find that oligomerisation improves proteolytic and thermal stability but does not impact substrate specificity or enzymatic activity. Furthermore, structural comparison with meprin {beta} reveal unique features of the active site of meprin , and helical assembly more broadly.

biochemistry↗

bioRxiv2021

Single-molecule analysis of the entire perfringolysin O poreformation pathway

The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings ([~]35-mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.

biophysics↗

bioRxiv2021

Mining folded proteomes in the era of accurate structure prediction

Protein structure fundamentally underpins the function and processes of numerous biological systems. Fold recognition algorithms offer a sensitive and robust tool to detect structural, and thereby functional, similarities between distantly related homologs. In the era of accurate structure prediction owing to advances in machine learning techniques, previously curated sequence databases have become a rich source of biological information. Here, we use bioinformatic fold recognition algorithms to scan the entire AlphaFold structure database to identify novel protein family members, infer function and group predicted protein structures. As an example of the utility of this approach, we identify novel, previously unknown members of various pore-forming protein families, including MACPFs, GSDMs and aerolysin-like proteins. Further, we explore the use of structure-based mining for functional inference.

bioinformatics↗

bioRxiv2021

The cryo-EM structure of the neurofibromin dimer reveals the molecular basis for von Recklinghausen disease

Neurofibromin (NF1) is a tumour suppressor mutated in neurofibromatosis type 1 (von Recklinghausen disease), one of the most common human genetic diseases(1). NF1 regulates cellular growth through suppressing the Rat Sarcoma (RAS) pathway and, accordingly, mutations in this protein drive numerous cancers, including melanoma, ovarian, breast and brain cancer(2, 3). Currently, however, the molecular basis for NF1 function remains to be understood. Here we address this problem and use cryogenic Electron Microscopy (cryo-EM) to determine the structure of fulllength NF1. The 640 kDa NF1 homodimer forms an extraordinary lemniscate ({infty}) shaped molecule that is ~30 nm in length and ~ 10 nm wide. Each NF1 monomer comprises an N-terminal HEAT-repeat domain (N-HEAT), a guanosine triphosphatase activating protein (GAP)-related domain (GRD), a Sec14 homologous and pleckstrin homologous module (SEC-PH), and a C-terminal HEAT domain (C-HEAT). The core NF1 scaffold is formed via a head-to-tail dimer of the N- and C-HEAT domains. This platform, which is responsible for interacting with more than 10 regulatory binding partners, comprises an extraordinary array of over 150 -helices. Analysis of these EM data revealed that the GRD and SEC-PH domain are highly mobile with respect to the core scaffold and could not initially be accurately placed in electron density. Strikingly, however, using 3D variability analysis we were able to identify a significant subpopulation of NF1 particles and determine the complete NF1 structure to 5.6 [A] resolution. These data revealed that the catalytic GRD and lipid binding SEC-PH domain are positioned against the core scaffold in a closed, autoinhibited conformation. We postulate that interaction with the plasma membrane may release the closed conformation in order to promote RAS inactivation. Our structural data further allow us to map the location of disease-associated NF1 variants and provide a long sought-after structural explanation for the extreme susceptibility of the molecule to loss-of-function mutations. Finally, it is suggested that approaches to combat NF1-linked diseases may include release of the autoinhibited state in order to improve NF1 catalytic efficiency.

biochemistry↗

bioRxiv2019

The structure of Macrophage Expressed Gene-1, a phagolysosome immune effector that is activated upon acidification.

Macrophage Expressed Gene-1 (MPEG-1; also termed Perforin-2) is an endosomal / phagolysosomal perforin-like protein that is conserved across the metazoan kingdom and that functions within the phagolysosome to damage engulfed microbes. Like the Membrane Attack Complex and perforin, MPEG-1 has been postulated to form pores in target membranes, however, its mode of action remains to be established. We used single particle cryo-Electron Microscopy to determine the 2.4 [A] structure of a hexadecameric assembly of MPEG-1 that displays the expected features of a soluble pre-pore complex. We further discovered that the MPEG-1 pre-pore-like assemblies can be induced to perforate membranes through mild acidification, such as would occur within maturing phagolysosomes. We next solved the 3.6 [A] cryo-EM structure of MPEG-1 in complex with liposomes. Remarkably these data revealed that a C-terminal Multi-vesicular body of 12 kDa (MVB12)-associated {beta}-prism (MABP) domain interacts with target membranes in a mode that positions the pore forming machinery of MPEG-1 to point away from the bound membrane. This unexpected mechanism of membrane interaction raises the intriguing possibility that MPEG-1 may be able to remain bound to the phagolysosome membrane while simultaneously forming pores in engulfed bacterial targets.

biochemistry↗