Biology subjects

Thorpe, C.

Publications and source records attributed to Thorpe, C..

4 recordsLinked to original sources

bioRxiv2025

Structure of the Disulfide-rich Modules of a Striking Tandem Repeat Protein, Avian Cysteine-Rich Eggshell Membrane Protein

Avian eggshell membrane (ESM) is fabricated within the isthmus region of the oviduct and is comprised of three juxtaposed, predominantly proteinaceous layers lying between egg white and the calcified shell. The limiting membrane is less than 0.5 {micro}m in thickness and forms the osmotic barrier for the egg. This first layer provides the foundation for the successive deposition of two mats of protein fibers. Fibers from both inner and outer layers appear to have similar amino acid compositions and are notably disulfide-rich (comprising about 10% Cys). ESM has been utilized in a wide variety of applications, including nutraceutical supplements, tissue engineering, and nanofabrication, and yet fundamental questions concerning protein composition, fiber structure, and membrane assembly remain to be resolved. We previously identified an abundant disulfide-rich structural protein in chicken ESM fibers (Cysteine-rich eggshell membrane protein; CREMP) that contains multiple tandemly repeated modules. In this work, we determine a structural model for four consecutive 2-disulfide containing CREMP modules using a variety of two- and three-dimensional solution NMR experiments. CREMP modules feature an N-terminal loop region positioned above a small beta hairpin that is stabilized by a conserved pattern of disulfide bridges between Cys1-3 and Cys2-4. While the individual CREMP modules are highly ordered, the lack of long-range inter-module restraints suggests an extended structure connected by flexible linkers. Finally, the structural information obtained in this work is considered in the context of full-length CREMP proteins and compared to two other structural proteins that contain multiple tandem repeats of 2-disulfide modules. Statement of Importance and ImpactUnder the shell of an avian egg is a tough, paper-thin, three-layered protein-based structure called the eggshell membrane. This membrane has a range of medical, biomaterial and analytical applications, and yet fundamental questions concerning protein composition and assembly remain unresolved. Here, we address the 3-dimensional structure of part of a major membrane component, cysteine-rich eggshell membrane protein. The work provides new insight into the eggshell membrane and suggests an additional avenue for de-novo protein design.

biochemistry↗

bioRxiv2024

Early regional lymph node activation drives influenza vaccine responses in an ancestrally diverse cohort

Early in vivo dynamics of human immune-cell activation across regionally activated lymphoid tissue sites upon immunisation are poorly characterised in ancestrally diverse individuals. Here, we profiled draining and non-draining axillary lymph nodes (dLNs and ndLNs) by ultrasound-guided fine-needle aspiration (FNA) in 13 Black and Asian ancestry individuals, before and 3-7 days after vaccination with adjuvanted influenza vaccine. Draining but not ndLNs rapidly increased in size post-vaccination, by day 3, with distinct cellular dynamics determined through single cell multiomics. Dissecting LN cellular diversity into 42 lymphoid and non-lymphoid cell states, post-vaccination cell abundance changes were observed across all LNs, but dLNs were specifically characterised by CD4+ T follicular helper (CD4+ Tfh) cell expansion. Gene expression analysis revealed a dLN post-vaccination hub of multicellular activity defined by CD4+ Tfh signalling, cross-compartmental activation, translation, and enhanced antigen-presentation capacity. Thus, robust responses to intramuscular immunisation transcending ancestral inter-individual variation are elicited through temporal, anatomical and cellular lymphatic co-ordination with implications for vaccine design in ancestrally diverse populations. SummaryIn this study of ancestrally diverse young adults, the temporarily co-ordinated response to an adjuvanted influenza vaccine at lymph nodes local to (draining) and distal from (non-draining) the injection site, reveals early regulation of cellular kinetics and anatomical hierarchy of the innate and adaptive immune responses.

immunology↗

bioRxiv2024

Quantifying conformational changes in the TCR:pMHC-I binding interface

T cells form one of the key pillars of adaptive immunity. Using their surface bound T cell antigen receptors (TCRs), these cells screen millions of antigens presented by major histocompatability complex (MHC) or MHC-like molecules. In other protein families, the dynamics of protein-protein interactions have important implications for protein function. Case studies of TCR:class I peptide-MHCs (pMHC-Is) structures have reported mixed results on whether the binding interfaces undergo conformational change during engagement and no robust statistical quantification has been done to generalise these results. It thus remains an open question if movement occurs in the binding interface that enables recognition and activation of T cells. In this work, we quantify the conformational changes in the TCR:pMHC-I binding interface by creating a dataset of 358 structures, comprising 25 TCRs, 20 MHC alleles, and 58 peptide structures in both unbound (apo) and bound (holo) conformations. In support of some case studies, we demonstrate that all complimentary determining region (CDR) loops move to a certain extent but only CDR3 and CDR3{beta} loops modify their shape when binding pMHC-Is. We also map out the contacts between TCRs and pMHC-Is, generating a novel fingerprint of TCRs on MHC molecules and show that the CDR3 tends to bind the N-terminus of the peptide and the CDR3{beta} tends to bind the C-terminus of the peptide. Finally, we show that the presented peptides can undergo conformational changes when engaged by TCRs, as has been reported in past literature, but novelly show these changes depend on how the peptides are anchored in the MHC binding groove. Our work has implications in understanding the behaviour of TCR:pMHC-I interactions and providing insights that can be used for modelling T cell antigen specificity, an ongoing grand challenge in immunology.

immunology↗

bioRxiv2022

Matrix mechanics, not hypoxia, modulate quiescin sulfhydryl oxidase 1 (QSOX1) in pancreatic tumor cells

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related deaths in the U.S., despite only being the 11th most common cancer. The high mortality rates of PDAC can be partially attributed to the tumor microenvironment. Unlike most carcinomas, PDAC is characterized by a strong desmoplastic reaction, or a fibrotic stiffening of the extracellular matrix (ECM) in response to chronic inflammation. The desmoplastic reaction is mediated by cancer-associated fibroblasts that deposit ECM proteins (collagens, laminins, fibronectin, etc.) and secrete matrix-remodeling proteins in the tumor parenchyma. Within the past decade, the enzyme quiescin sulfhydryl oxidase 1 (QSOX1) has gained recognition as a significant contributor to solid tumor pathogenesis, but its biological role remains uncertain. QSOX1 is a disulfide bond-generating catalyst that participates in oxidative protein folding in the mammalian cell. Current studies show that inhibiting or knocking down QSOX1 reduces pancreatic cancer cell migration and invasion, alters ECM deposition and organization, and decreases overall tumor growth in mice. However, it is unclear which features of the tumor microenvironment modulate QSOX1 and cause its overexpression in cancer. In this study, we explored potential regulators of QSOX1 expression and secretion by testing two major features of PDAC: hypoxia and mechanical stiffness. To induce hypoxia, we exposed pancreatic cancer cells to atmospheric (low O2) and chemical (CoCl2) hypoxia for up to 48 hours. QSOX1 gene and protein expression did not change in response to hypoxia. Substratum stiffness was modulated using polyacrylamide gels to represent the dynamic pathological range of elastic moduli found in PDAC tissue. We discovered that QSOX1 levels were decreased on softer surfaces compared to conventional tissue culture plastic. This paper presents new results and challenges prior findings on QSOX1 regulation in pancreatic tumor cells.

bioengineering↗