Strategies for therapeutic amelioration of aberrant plasma Zn2+ handling in thrombotic disease: Targeting fatty acid/serum albumin-mediated effects

Funding: This research was funded by Leverhulme Trust, grant number RPG-2017-214; Bio-technology and Biological Sciences Research Council, grant number BB/J006467/1 and BB/V014684/1; British Heart Foundation, grant number FS/20/3/34956.The initiation, maintenance and regulation of blood coagulation is inexorably linked to the actions of Zn2+ in blood plasma. Zn2+ interacts with a variety of haemostatic proteins in the bloodstream including fibrinogen, histidine-rich glycoprotein (HRG) and high molecular weight kininogen (HMWK) to regulate haemostasis. The availability of Zn2+ to bind such proteins is controlled by human serum albumin (HSA), which binds 70-85% plasma Zn2+ under basal conditions. HSA also binds and transports non-esterified fatty acids (NEFAs). Upon NEFA binding, there is a change in the structure of HSA which leads to a reduction in its affinity for Zn2+. This enables other plasma proteins to better compete for binding of Zn2+. In diseases where elevated plasma NEFA con-centrations are a feature, such as obesity and diabetes, there is a concurrent increase in hyper-coagulability. Evidence indicates that NEFA-induced perturbation of Zn2+-binding by HSA may contribute to the thrombotic complications frequently observed in these pathophysiological conditions. This review highlights potential interventions - both pharmaceutical and non-pharmaceutical - that may be employed to combat this dysregulation. Lifestyle and dietary changes have been shown to reduce plasma NEFA concentrations. Furthermore, drugs that in-fluence NEFA levels such as statins and fibrates may be useful in this context. In severely obese patients more invasive therapies such as bariatric surgery may be useful. Finally, other potential treatments such as chelation therapies, use of cholesteryl transfer protein (CETP) inhibitors, lipase inhibitors, fatty acid inhibitors and other treatments are highlighted, that with additional research and appropriate clinical trials, could prove useful in the treatment and management of thrombotic disease through amelioration of plasma Zn2+ dysregulation in high-risk individuals.Publisher PDFPeer reviewe

Critical role of WASp in germinal center tolerance through regulation of B cell apoptosis and diversification

This project was supported by grant 310030-179251 from the Suisse National Science Foundation (SNF) (to F.C.), funds from the BLACKSWAN Foundation (BSF-005) (to M.D.), and the Wellcome Trust (to A.J.T.).A main feature of Wiskott-Aldrich syndrome (WAS) is increased susceptibility to autoimmunity. A key contribution of B cells to development of these complications has been demonstrated through studies of samples from affected individuals and mouse models of the disease, but the role of the WAS protein (WASp) in controlling peripheral tolerance has not been specifically explored. Here we show that B cell responses remain T cell dependent in constitutive WASp-deficient mice, whereas selective WASp deletion in germinal center B cells (GCBs) is sufficient to induce broad development of self-reactive antibodies and kidney pathology, pointing to loss of germinal center tolerance as a primary cause leading to autoimmunity. Mechanistically, we show that WASp is upregulated in GCBs and regulates apoptosis and plasma cell differentiation in the germinal center and that the somatic hypermutation-derived diversification is the basis of autoantibody development.Publisher PDFPeer reviewe

Critical role of WASp in germinal center tolerance through regulation of B cell apoptosis and diversification

A main feature of Wiskott-Aldrich syndrome (WAS) is increased susceptibility to autoimmunity. A key contribution of B cells to development of these complications has been demonstrated through studies of samples from affected individuals and mouse models of the disease, but the role of the WAS protein (WASp) in controlling peripheral tolerance has not been specifically explored. Here we show that B cell responses remain T cell dependent in constitutive WASp-deficient mice, whereas selective WASp deletion in germinal center B cells (GCBs) is sufficient to induce broad development of self-reactive antibodies and kidney pathology, pointing to loss of germinal center tolerance as a primary cause leading to autoimmunity. Mechanistically, we show that WASp is upregulated in GCBs and regulates apoptosis and plasma cell differentiation in the germinal center and that the somatic hypermutation-derived diversification is the basis of autoantibody development

A splenic IgM memory subset with antibacterial specificities is sustained from persistent mucosal responses

This work was supported by the Ligue contre le Cancer (“Equipe labellisée”), the Fondation Princesse Grace de Monaco, and the European Research Council Advanced grants “Memo-B” (to J.-C. Weill) and “B-response” (to C.-A. Reynaud). L.-H. Thai was supported by a Poste d'Accueil INS ERM.To what extent immune responses against the gut flora are compartmentalized within mucosal tissues in homeostatic conditions remains a much-debated issue. We describe here, based on an inducible AID fate-mapping mouse model, that systemic memory B cell subsets, including mainly IgM+ B cells in spleen, together with IgA+ plasma cells in spleen and bone marrow, are generated in mice in the absence of deliberate immunization. While the IgA component appears dependent on the gut flora, IgM memory B cells are still generated in germ-free mice, albeit to a reduced extent. Clonal relationships and renewal kinetics after anti-CD20 treatment reveal that this long-lasting splenic population is mainly sustained by output of B cell clones persisting in mucosal germinal centers. IgM-secreting hybridomas established from splenic IgM memory B cells showed reactivity against various bacterial isolates and endogenous retroviruses. Ongoing activation of B cells in gut-associated lymphoid tissues thus generates a diversified systemic compartment showing long-lasting clonal persistence and protective capacity against systemic bacterial infections.Publisher PDFPeer reviewe

Shedding light on DNA-protein interactions involved in the nucleotide excision repair pathway

DNA, or deoxyribonucleic acid, contains the molecular blueprint of organisms. Although, very stable, DNA can be damaged by genotoxic agents such as UV-light, reactive oxygen species or chemotherapeutic drugs, amongst others. Organisms have evolved several biological pathways to repair different types of damage and nucleotide excision repair (NER) is one of them. NER repairs mainly lesions inducing structural distortion of the double helix. Briefly, the lesion is detected, unwinding takes place to create a 30 nucleotides long bubble, the damage-containing single strand is then excised; using the opposite strand as a template, the DNA is resynthesized and ligated. Nucleotide Excision Repair is a non-mutagenic repair system.Single-strand DNA binding proteins (SSB) are involved in NER and many other pathways. Their role is to coat and protect the ssDNA from degradation and re-hybridisation. Here, we studied SSBs from Saccharolobus solfataricus and Sulfolobus acidocaldarius as well as a chimeric protein containing DNA binding domains from both organisms. We showed that the chimeric protein had better resistance to extreme conditions of temperature, ionic strength or acidic pH. We also proved that our chimeric SSB can be used as a tool to image single strand DNA or RNA using super resolution microscopy.The first step of NER is damage recognition by XPC. We studied XPC binding to a DNA substrate and showed the possibility of two XPC binding to a damage whilst demonstrating that the presence of a nick on the DNA backbone does not constitute a substrate for XPC. Furthermore, we used single molecule FRET technique to show that XPC could bind a three nucleotides bubble in two different modes, showing a strong preference for one of these binding modes.The second step is the unwinding by the XPD helicase, we studied the impact of the purine/pyrimidine composition on the unwinding activity and investigated a possible damage sensor role of XPD, using Archaean models. To complete our work, we demonstrated a cooperative effect on the distortion of the repair bubble of XPA and RPA

Shedding light on DNA-protein interactions involved in the nucleotide excision repair pathway

DNA, or deoxyribonucleic acid, contains the molecular blueprint of organisms. Although, very stable, DNA can be damaged by genotoxic agents such as UV-light, reactive oxygen species or chemotherapeutic drugs, amongs others. Organisms have evolved several biological pathways to repair different types of damage and nucleotide excision repair (NER) is one of them. NER repairs mainly lesions inducing structural distortion of the double helix. Briefly, the lesion is detected, unwinding takes place to create a 30 nucleotides long bubble, the damage-containing single strand is then excised; using the opposite strand as a template, the DNA is resynthesized and ligated. Nucleotide Excision Repair is a non-mutagenic repair system. Single-strand DNA binding proteins (SSB) are involved in NER and many other pathways. Their role is to coat and protect the ssDNA from degradation and re-hybridisation. Here, we studied SSBs from Saccharolobus solfataricus and Sulfolobus acidocaldarius as well as a chimeric protein containing DNA binding domains from both organisms. We showed that the chimeric protein had better resistance to extreme conditions of temperature, ionic strength or acidic pH. We also proved that our chimeric SSB can be used as a tool to image single strand DNA or RNA using super resolution microscopy. The first step of NER is damage recognition by XPC. We studied XPC binding to a DNA substrate and showed the possibility of two XPC binding to a damage whilst demonstrating that the presence of a nick on the DNA backbone does not constitute a substrate for XPC. Furthermore, we used single molecule FRET technique to show that XPC could bind a three nucleotides bubble in two different modes, showing a strong preference for one of these binding modes. The second step is the unwinding by the XPD helicase, we studied the impact of the purine/pyrimidine composition on the unwinding activity and investigated a possible damage sensor role of XPD, using Archaean models. To complete our work, we demonstrated a cooperative effect on the distortion of the repair bubble of XPA and RPA."This work was supported by a Wellcome Trust ISSF grant (number: 20481/Z/12/Z) as well as a BBSRC grant (number: BBR015570/1)." --Fundin

Targeted removal of the FA2 site on human albumin prevents fatty acid-mediated inhibition of Zn2+-binding

Acknowledgements We thank Dr. Ivan Prokes (University of Warwick) for running the 13C-NMR spectra. Author contributions statement: D.W. and S.J.H. generated the albumin mutants and performed ITC experiments; S.P. performed NMR experiments; M.G., M.M. and K.M. performed molecular dynamics simulations; all authors analyzed and interpreted the results; D.W., S.J.H., M.G., K.M., C.A.B. and A.J.S. wrote the paper; all authors edited the manuscript; M.G., K.M., C.A.B., and A.J.S. designed the research. Competing interests: J.S. is a Co-Founder and CEO at AFFINImeter.Zinc is required for virtually all biological processes. In plasma, Zn2+ is predominantly transported by human serum albumin (HSA), which possesses two Zn2+-binding sites of differing affinities (sites A and B). Fatty acids (FAs) are also transported by HSA, with seven structurally characterized FA-binding sites (named FA1-FA7) known. FA binding inhibits Zn2+-HSA interactions, in a manner that can impact upon hemostasis and cellular zinc uptake, but the degree to which binding at specific FA sites contributes to this inhibition is unclear. Wild-type HSA and H9A, H67A, H247A, and Y150F/R257A/S287A (FA2-KO) mutant albumins were expressed in Pichia pastoris. Isothermal titration calorimetry studies revealed that the Zn2+-binding capacity at the high-affinity Zn2+ site (site A) was reduced in H67A and H247A mutants, with site B less affected. The H9A mutation decreased Zn2+ binding at the lower-affinity site, establishing His9 as a site B ligand. Zn2+ binding to HSA and H9A was compromised by palmitate, consistent with FA binding affecting site A. 13C-NMR experiments confirmed that the FA2-KO mutations prohibited FA binding at site FA2. Zn2+ binding to the FA2-KO mutant was unaffected by myristate, suggesting binding at FA2 is solely responsible for inhibition. Molecular dynamics studies identified the steric obstruction exerted by bound FA in site FA2, which impedes the conformational change from open (FA-loaded) to closed (FA-free) states, required for Zn2+ to bind at site A. The successful targeting of the FA2 site will aid functional studies exploring the interplay between circulating FA levels and plasma Zn2+ speciation in health and disease.Peer reviewe