Biology subjects

Lupton, C. J.

Publications and source records attributed to Lupton, C. J..

4 recordsLinked to original sources

bioRxiv2024

Acidic pH attenuates immune killing through inactivation of perforin

Cytotoxic lymphocytes are pivotal effectors in our immune system. Their most potent way to delete virus infected or cancerous target cells is by perforin/granzyme dependent killing. Perforin is secreted into the immunological synapse and forms transmembrane pores in the target cell plasma membrane, allowing granzymes to enter the target cell cytosol and trigger apoptosis. The prowess of cytotoxic lymphocytes to efficiently eradicate target cells has been widely harnessed in immunotherapies against haematological cancers. Despite efforts to achieve a similar outcome against solid tumours, the immunosuppressive and acidic tumour microenvironment poses a persistent obstacle. Using different types of effector cells, including therapeutically relevant anti-CD19 CAR T cells, we demonstrate that the acidic pH typically found in solid tumours hinders the efficacy of immune therapies by impeding perforin pore formation within the immunological synapse. Nanometre-scale study of purified recombinant perforin undergoing oligomerization reveals that pore formation is inhibited specifically by preventing the formation of a transmembrane {beta}-barrel. The absence of perforin pore formation directly prevents target cell death. This finding uncovers a novel layer of immune effector inhibition that must be considered in the development of effective immunotherapies for solid tumours. One Sentence SummaryOur study reveals that an acidic environment hinders the efficacy of immune therapies by impeding the action of perforin, a critical protein in immune-mediated cell killing, directly within the immunological synapse.

immunology↗

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

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↗