Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis.

The essence of difference between hemostasis and thrombosis is that the clotting reaction is a highly fine-tuned process. Vascular protein disulfide isomerase (PDI) represents a critical mechanism regulating the functions of hemostatic proteins. Herein we show that histidine-rich glycoprotein (HRG) is a substrate of PDI.

Imaging of cell death in malignancy: Targeting pathways or phenotypes?

Cell death is fundamental in health and disease and resisting cell death is a hallmark of cancer. Treatment of malignancy aims to cause cancer cell death, however current clinical imaging of treatment response does not specifically image cancer cell death but assesses this indirectly either by changes in tumor size (using x-ray computed tomography) or metabolic activity (using 2-[F]fluoro-2-deoxy-glucose positron emission tomography). The ability to directly image tumor cell death soon after commencement of therapy would enable personalised response adapted approaches to cancer treatment that is presently not possible with current imaging, which is in many circumstances neither sufficiently accurate nor timely.

Disulfide bond reduction and exchange in C4 domain of von Willebrand factor undermines platelet binding.

Background: The von Willebrand factor (VWF) is a key player in regulating hemostasis through adhesion of platelets to sites of vascular injury. It is a large, multi-domain, mechano-sensitive protein that is stabilized by a net of disulfide bridges. Binding to platelet integrin is achieved by the VWF-C4 domain, which exhibits a fixed fold, even under conditions of severe mechanical stress, but only if critical internal disulfide bonds are closed.

Sulfenylation links oxidative stress to protein disulfide isomerase oxidase activity and thrombus formation.

Background: Oxidative stress contributes to thrombosis in atherosclerosis, inflammation, infection, aging, and malignancy. Oxidant-induced cysteine modifications, including sulfenylation, can act as a redox-sensitive switch that controls protein function. Protein disulfide isomerase (PDI) is a prothrombotic enzyme with exquisitely redox-sensitive active-site cysteines.

Mechano-Redox Control of Mac-1 De-Adhesion by PDI Promotes Directional Movement Under Flow.

Background: Neutrophil migration is critical to the initiation and resolution of inflammation. Macrophage-1 antigen (Mac-1; CD11b/CD18, αMβ2) is a leukocyte integrin essential for firm adhesion to endothelial ICAM-1 (intercellular adhesion molecule 1) and migration of neutrophils in the shear forces of the circulation. PDI (protein disulfide isomerase) has been reported to influence neutrophil adhesion and migration.

Inhibition of mitochondrial translocase SLC25A5 and histone deacetylation is an effective combination therapy in neuroblastoma.

The mitochondrion is a gatekeeper of apoptotic processes, and mediates drug resistance to several chemotherapy agents used to treat cancer. Neuroblastoma is a common solid cancer in young children with poor clinical outcomes following conventional chemotherapy. We sought druggable mitochondrial protein targets in neuroblastoma cells.

Mechano-covalent protection of coagulation factor VIII by von Willebrand factor.

von Willebrand factor (VWF) is the protective carrier of procoagulant factor VIII (FVIII) in the shear forces of the circulation, prolonging its half-life and delivering it to the developing thrombus. Using force spectroscopy, VWF-FVIII complex formation is characterized by catch-bond behavior in which force first decelerates then accelerates bond dissociation. Patients with mutations in VWF at the FVIII binding site phenocopies hemophilia A and the most common mutations are of cysteine residues involving multiple disulfide bonds.

A first-in-human study of [Ga]Ga-CDI: a positron emitting radiopharmaceutical for imaging tumour cell death.

Purpose: This study assesses human biodistribution, radiation dosimetry, safety and tumour uptake of cell death indicator labelled with Ga ([Ga]Ga-CDI), a novel radiopharmaceutical that can image multiple forms of cell death.

Methods: Five participants with at least one extracranial site of solid malignancy > 2 cm and no active cancer treatment in the 8 weeks prior to the study were enrolled. Participants were administered 205 ± 4.

Not one, but many forms of thrombosis proteins.

The disulfide bond is a covalent bond formed between the sulfur atoms of two cysteine residues in proteins. Our understanding of the role of these ubiquitous bonds in protein function has changed dramatically over the past decade. Initially thought to be fully formed and inert in the native protein, we know now that both these assumptions are incorrect for many proteins.

An alternate covalent form of platelet αIIbβ3 integrin that resides in focal adhesions and has altered function.

The αIIbβ3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in ∼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbβ3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin.

Influenza A Virus Hemagglutinin Is Produced in Different Disulfide-Bonded States.

Influenza A virus hemagglutinin (HA) binding to sialic acid on lung epithelial cells triggers membrane fusion and infection. Host thiol isomerases have been shown to play a role in influenza A virus infection, and we hypothesized that this role involved manipulation of disulfide bonds in HA. Analysis of HA crystal structures revealed that three of the six HA disulfides occur in high-energy conformations and four of the six bonds can exist in unformed states, suggesting that the disulfide landscape of HA is generally strained and the bonds may be labile.

Toxicokinetics of the tumour cell mitochondrial toxin, PENAO, in rodents.

PENAO (4-(N-(S-penicillaminylacetyl)amino)phenylarsonous acid) is a second-generation peptide arsenical that inactivates mitochondria in proliferating tumour cells by covalently reacting with mitochondrial inner-membrane adenine nucleotide transferase. The toxicokinetics of PENAO has been investigated in Sprague-Dawley rats to inform route of administration and dosing for human clinical trials. PENAO was well tolerated at 3.

Multiple Disulfide-Bonded States of Native Proteins: Estimate of Number Using Probabilities of Disulfide Bond Formation.

The polypeptide backbone of proteins is held together by two main types of covalent bonds: the peptide bonds that link the amino acid residues and the disulfide bonds that link pairs of cysteine amino acids. Disulfide bonds form as a protein folds in the cell and formation was assumed to be complete when the mature protein emerges. This is not the case for some secreted human blood proteins.

Fibrinogen function achieved through multiple covalent states.

Disulfide bonds link pairs of cysteine amino acids and their formation is assumed to be complete in the mature, functional protein. Here, we test this assumption by quantifying the redox state of disulfide bonds in the blood clotting protein fibrinogen. The disulfide status of fibrinogen from healthy human donor plasma and cultured human hepatocytes are measured using differential cysteine alkylation and mass spectrometry.

Regulation of hepatic insulin signaling and glucose homeostasis by sphingosine kinase 2.

Sphingolipid dysregulation is often associated with insulin resistance, while the enzymes controlling sphingolipid metabolism are emerging as therapeutic targets for improving insulin sensitivity. We report herein that sphingosine kinase 2 (SphK2), a key enzyme in sphingolipid catabolism, plays a critical role in the regulation of hepatic insulin signaling and glucose homeostasis both in vitro and in vivo. Hepatocyte-specific knockout mice exhibit pronounced insulin resistance and glucose intolerance.

The synergistic inhibitory effect of combining therapies targeting EGFR and mitochondria in sarcomas.

Our group previously demonstrated that sarcoma cell lines were insensitive to epidermal growth factor receptor (EGFR) inhibitor gefitinib monotherapy. PENAO, an anti-tumour metabolic compound created in our laboratory, is currently in clinical trials. Considering the positive regulation of tumour energy production by both the EGFR signalling and tumour metabolism pathways, this study aimed to investigate the effect and mechanisms of combination therapy using gefitinib and PENAO in sarcoma cell lines and .

Biodistribution and imaging of an hsp90 ligand labelled with In and Ga for imaging of cell death.

Background: 4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO) when conjugated at the γ-glutamyl residue with fluorophores and radio-isotopes is able to image dead and dying cells in vitro and in vivo by binding to intracellular 90-kDa heat shock proteins (hsp90) when cell membrane integrity is compromised. The ability to image cell death has potential clinical impact especially for early treatment response assessment in oncology. This work aims to assess the biodistribution and tumour uptake of diethylene triamine pentaacetic acid GSAO labelled with In ([In]In-DTPA-GSAO) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid GSAO labelled with Ga ([Ga]Ga-DOTA-GSAO) in a murine subcutaneous tumour xenograft model and estimate dosimetry of [Ga]Ga-DOTA-GSAO.

Reprogramming of human fibroblasts into osteoblasts by insulin-like growth factor-binding protein 7.

The induced pluripotent stem cell (iPSC) is a promising cell source for tissue regeneration. However, the therapeutic value of iPSC technology is limited due to the complexity of induction protocols and potential risks of teratoma formation. A trans-differentiation approach employing natural factors may allow better control over reprogramming and improved safety.

Thiol isomerase ERp57 targets and modulates the lectin pathway of complement activation.

ER protein 57 (ERp57), a thiol isomerase secreted from vascular cells, is essential for complete thrombus formation , but other extracellular ERp57 functions remain unexplored. Here, we employed a kinetic substrate-trapping approach to identify extracellular protein substrates of ERp57 in platelet-rich plasma. MS-based identification with immunochemical confirmation combined with gene ontology enrichment analysis revealed that ERp57 targets, among other substrates, components of the lectin pathway of complement activation: mannose-binding lectin, ficolin-2, ficolin-3, collectin-10, collectin-11, mannose-binding lectin-associated serine protease-1, and mannose-binding lectin-associated serine protease-2.

Allosteric disulfides: Sophisticated molecular structures enabling flexible protein regulation.

Protein disulfide bonds link pairs of cysteine residues in polypeptide chains. Many of these bonds serve a purely structural or energetic role, but a growing subset of cleavable disulfide bonds has been shown to control the function of the mature protein in which they reside. These allosteric disulfides and the factors that cleave these bonds are being identified across biological systems and life forms and have been shown to control hemostasis, the immune response, and viral infection in mammals.

Platelet Protein Disulfide Isomerase Promotes Glycoprotein Ibα-Mediated Platelet-Neutrophil Interactions Under Thromboinflammatory Conditions.

Background: Platelet-neutrophil interactions contribute to vascular occlusion and tissue damage in thromboinflammatory disease. Platelet glycoprotein Ibα (GPIbα), a key receptor for the cell-cell interaction, is believed to be constitutively active for ligand binding. Here, we established the role of platelet-derived protein disulfide isomerase (PDI) in reducing the allosteric disulfide bonds in GPIbα and enhancing the ligand-binding activity under thromboinflammatory conditions.

γ-Crystallin redox-detox in the lens.

In the vertebrate eye, limiting oxidation of proteins and lipids is key to maintaining lens function and avoiding cataract formation. A study by Serebryany identifies a surprising contributor to the eye's oxidative defense in their demonstration that γD-crystallin (HγD) functions as an oxidoreductase and uses disulfide exchange to initiate aggregation of mutant crystallins that mimic oxidative damage. These insights suggest a mechanism by which a dynamic pool of closely packed proteins might avoid oxidation-driven protein-folding traps, providing new avenues to understand the basis of a human disease with global impact.