Biology subjects

Hogg, P. J.

Publications and source records attributed to Hogg, P. J..

4 recordsLinked to original sources

bioRxiv2026

Allosteric disulfide control of ligand binding and endocytosis of the natural killer cell receptor for HLA-G

Human Leukocyte Antigen (HLA)-G is selectively expressed by fetal trophoblast cells that invade maternal tissue and encounter maternal natural killer (NK) cells early in pregnancy. In NK cells, the endosomal receptor KIR2DL4 responds to soluble HLA-G by inducing a broad transcriptional program to support placental development. Structural features of KIR2DL4 that control ligand binding and endocytosis are unclear. Random mutagenesis revealed that three cysteines in the first immunoglobulin domain of KIR2DL4 regulate endocytosis and uptake of HLA-G. We found that the atypical Cys10-Cys28 disulfide bond observed in the KIR2DL4 crystal structure is an allosteric disulfide with potential to switch to a conventional Cys28-Cys74 bond. KIR2DL4 in human cells exists in both disulfide-bonded states, as shown by mass spectrometry analysis. The Cys10-Cys28 bond in a Cys74Ser KIR2DL4 mutant was reduced by protein disulfide isomerase (PDI) in vitro. Inhibition of PDI prevented HLA-G uptake by KIR2DL4 in both transfected 293T cells and primary NK cells. Mutants in the Cys10-Cys28 configuration endocytosed spontaneously but did not bind HLA-G. Conversely, KIR2DL4 with a Cys28-Cys74 bond was at the plasma membrane and responded to soluble HLA-G by endocytosis and transcription of the interferon stimulated gene IFI44L, like wild-type cells. A purified Cys10Leu KIR2DL4 mutant, which exhibited a reduced tendency to oligomerize, bound HLA-G in a peptide dependent manner with an affinity in the low micromolar range. Disulfide switching from the Cys10-Cys28 to the Cys28-Cys74 form correlated with a distant allosteric change, as predicted by AlphaFold, that could reorient a D0 domain loop to facilitate HLA-G binding. Thus, conversion from an inactive state to an HLA-G binding form regulates KIR2DL4 cellular localization and function to promote fetal development.

immunology↗

bioRxiv2022

Mechano-redox control of Mac-1 de-adhesion from ICAM-1 by protein disulfide isomerase promotes directional movement of neutrophils under flow

Macrophage-1 antigen or Mac-1 (CD11b/CD18, M{beta}2) is a leukocyte integrin essential for firm adhesion of neutrophils, lymphocytes and monocytes against flow when recruited to the endothelium. To migrate to the site of inflammation, leukocytes require coordinated adhesion and de-adhesion for directional movement. The vascular thiol isomerase, protein disulfide isomerase (PDI), was found by fluorescence microscopy to colocalize with high affinity Mac-1 at the trailing edge of stimulated neutrophils when adhered to ICAM-1 under fluid shear. From differential cysteine alkylation and mass spectrometry studies, PDI cleaves two allosteric disulfide bonds, C169-C176 and C224-C264, in the {beta}I domain of the {beta}2 subunit, and in mutagenesis and cell transfection studies, cleavage of the C224-C264 disulfide bond was shown to selectively control Mac-1 dis-engagement from ICAM-1 under fluid shear. Molecular dynamics simulations and binding of conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the {beta}I domain that allosterically alters exposure of an I domain epitope and shifts Mac-1 to a lower affinity state. From studies of neutrophil adherence to ICAM-1 under fluid shear, these molecular events promote neutrophil motility in the direction of flow at high shear stress. In summary, shear-dependent PDI cleavage of neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.

biochemistry↗

bioRxiv2019

Protein function achieved through multiple covalent states

The structure of proteins is defined by two main types of covalent bonds; the peptide bonds that link the amino acid residues and disulfide bonds that link pairs of cysteine amino acids. Disulfide bonds are introduced during protein folding and their formation is assumed to be complete in the mature, functional protein. We tested this assumption by quantifying the redox state of disulfide bonds in human blood proteins in their native environment. Using a differential cysteine alkylation and mass spectrometry method, we measured the redox state of disulfide bonds in circulating fibrinogen and von Willebrand factor. There is an extraordinary disulfide lability in the proteins, with 27 bonds in the two proteins ranging from 3 to 50% reduced in healthy human donors. Modelling of the data indicates that the proteins exist in hundreds of different disulfide-bonded states in the circulation. Different covalent states of fibrinogen are associated with different binding activities and their distribution is changed by fluid shear forces and altered in patients with cardiovascular disease, indicating that the different states have different functions and are dynamic. These findings have implications for protein function generally and how proteins are targeted in experimental settings and for therapeutic purposes.

biochemistry↗

bioRxiv2019

Evaluation of a potential redox switch in blood coagulation tenase

Blood coagulation factor IXa (FIXa) activates factor X that leads to thrombin and fibrin formation and a stable thrombus. FIXa catalytic efficiency is markedly enhanced when bound to the co-factor, factor VIII (FVIII), and a negatively charged phospholipid surface in the tenase complex. Small redox active peptides and protein oxidoreductases have been shown previously to have some FIXa co-factor activity and thiol modifying agents have been reported to influence FVIII activity. These observations suggested that FIXa might contain an allosteric disulfide that is regulated by FVIII. This idea was tested by measuring the influence of FVIII on the redox state of FIXa disulfide bonds and the effect of plasma oxidoreductases on FIXa activity. The redox state of nine of the 11 disulfide bonds in FIXa was measured using differential cysteine labelling and mass spectrometry and all were oxidized in the protein, and this did not change upon binding of the enzyme to FVIII. All eight disulfide bonds in FVIII were also predominantly oxidized and this did not appreciably change upon FIXa binding. In addition, relevant protein reductants in the circulation inhibited rather than activated FIXa activity. In conclusion, we found no evidence that the co-factor function of FVIII involves a change in the redox state of one or more FIXa disulfide bonds.

biochemistry↗