15 research outputs found

    Understanding the Mechanism of Translocation of Adenylate Cyclase Toxin across Biological Membranes

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    Adenylate cyclase toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding to the CD11b/CD18 integrin and delivers its N-terminal adenylate cyclase (AC) domain directly to the cell cytosol, where it catalyzes unregulated conversion of cytosolic ATP into cAMP upon activation by binding to cellular calmodulin. High cAMP levels disrupt bactericidal functions of the immune cells, ultimately leading to cell death. In spite of its relevance in the ACT biology, the mechanism by which its ‚Čą400 amino acid-long AC domain is transported through the target plasma membrane, and is released into the target cytosol, remains enigmatic. This article is devoted to refresh our knowledge on the mechanism of AC translocation across biological membranes. Two models, the so-called ‚Äútwo-step model‚ÄĚ and the recently-proposed ‚Äútoroidal pore model‚ÄĚ, will be considered.This study was supported by grants from the Basque Government (Grupos Consolidados IT849-13 and ETORTEK Program KK-2015/0000089). A.E. was recipient of a fellowship from the University of the Basque Country (UPV/EH) and D.G.-B. was recipient of a fellowship from the Bizkaia Biophysics Foundation

    Validation of LDLr Activity as a Tool to Improve Genetic Diagnosis of Familial Hypercholesterolemia: A Retrospective on Functional Characterization of LDLr Variants

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    Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by high blood-cholesterol levels mostly caused by mutations in the low-density lipoprotein receptor (LDLr). With a prevalence as high as 1/200 in some populations, genetic screening for pathogenic LDLr mutations is a cost-effective approach in families classified as definite' or probable' FH and can help to early diagnosis. However, with over 2000 LDLr variants identified, distinguishing pathogenic mutations from benign mutations is a long-standing challenge in the field. In 1998, the World Health Organization (WHO) highlighted the importance of improving the diagnosis and prognosis of FH patients thus, identifying LDLr pathogenic variants is a longstanding challenge to provide an accurate genetic diagnosis and personalized treatments. In recent years, accessible methodologies have been developed to assess LDLr activity in vitro, providing experimental reproducibility between laboratories all over the world that ensures rigorous analysis of all functional studies. In this review we present a broad spectrum of functionally characterized missense LDLr variants identified in patients with FH, which is mandatory for a definite diagnosis of FH.This work was supported by ELKARTEK 2016 and and the Basque Government (Grupos Consolidados IT849-13). A.B.-V. and S.J. were supported by a grant PIF (2014-2015) and (2018-2021), Gobierno Vasco respectively

    Pathophysiology of Type 2 Diabetes Mellitus

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    Type 2 Diabetes Mellitus (T2DM), one of the most common metabolic disorders, is caused by a combination of two primary factors: defective insulin secretion by pancreatic ő≤-cells and the inability of insulin-sensitive tissues to respond appropriately to insulin. Because insulin release and activity are essential processes for glucose homeostasis, the molecular mechanisms involved in the synthesis and release of insulin, as well as in its detection are tightly regulated. Defects in any of the mechanisms involved in these processes can lead to a metabolic imbalance responsible for the development of the disease. This review analyzes the key aspects of T2DM, as well as the molecular mechanisms and pathways implicated in insulin metabolism leading to T2DM and insulin resistance. For that purpose, we summarize the data gathered up until now, focusing especially on insulin synthesis, insulin release, insulin sensing and on the downstream effects on individual insulin-sensitive organs. The review also covers the pathological conditions perpetuating T2DM such as nutritional factors, physical activity, gut dysbiosis and metabolic memory. Additionally, because T2DM is associated with accelerated atherosclerosis development, we review here some of the molecular mechanisms that link T2DM and insulin resistance (IR) as well as cardiovascular risk as one of the most important complications in T2DM.This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). U.G-G. was supported by Fundaci√≥n Biof√≠sica Bizkaia. A.B.-V. was supported by Programa de especializaci.n de Personal Investigador Doctor en la UPV/EHU (2019) 2019‚Äď2020. S.J. and A.L-S. were supported by a grant PIF (2017‚Äď2018) and (2019‚Äď2020), Gobierno Vasco, respectively. A.L.-S. was partially supported by Fundaci√≥n Biof√≠sica Bizkaia

    Statin Treatment-Induced Development of Type 2 Diabetes: From Clinical Evidence to Mechanistic Insights

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    Statins are the gold-standard treatment for the prevention of primary and secondary cardiovascular disease, which is the leading cause of mortality worldwide. Despite the safety and relative tolerability of statins, observational studies, clinical trials and meta-analyses indicate an increased risk of developing new-onset type 2 diabetes mellitus (T2DM) after long-term statin treatment. It has been shown that statins can impair insulin sensitivity and secretion by pancreatic ő≤-cells and increase insulin resistance in peripheral tissues. The mechanisms involved in these processes include, among others, impaired Ca2+ signaling in pancreatic ő≤-cells, down-regulation of GLUT-4 in adipocytes and compromised insulin signaling. In addition, it has also been described that statins‚Äô impact on epigenetics may also contribute to statin-induced T2DM via differential expression of microRNAs. This review focuses on the evidence and mechanisms by which statin therapy is associated with the development of T2DM. This review describes the multifactorial combination of effects that most likely contributes to the diabetogenic effects of statins. Clinically, these findings should encourage clinicians to consider diabetes monitoring in patients receiving statin therapy in order to ensure early diagnosis and appropriate management.This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). U.G.-G. was supported by Fundaci√≥n Biof√≠sica Bizkaia. A.B.-V. was supported by Programa de especializaci√≥n de Personal Investigador Doctor en la UPV/EHU (2019) 2019‚Äď2020. S.J. and A.L.-S. were supported by a grant PIF (2017‚Äď2018) and (2019‚Äď2020), Gobierno Vasco, respectively. A.L.-S. was partially supported by Fundaci√≥n Biof√≠sica Bizkaia

    Cholesterol Efflux Efficiency of Reconstituted HDL Is Affected by Nanoparticle Lipid Composition

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    Cardiovascular disease (CVD), the leading cause of mortality worldwide is primarily caused by atherosclerosis, which is promoted by the accumulation of low-density lipoproteins into the intima of large arteries. Multiple nanoparticles mimicking natural HDL (rHDL) have been designed to remove cholesterol excess in CVD therapy. The goal of this investigation was to assess the cholesterol efflux efficiency of rHDLs with different lipid compositions, mimicking different maturation stages of high-density lipoproteins (HDLs) occurring in vivo. Methods: the cholesterol efflux activity of soybean PC (Soy-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DPPC:Chol:1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPC) and DPPC:18:2 cholesteryl ester (CE):LysoPC rHDLs was determined in several cell models to investigate the contribution of lipid composition to the effectiveness of cholesterol removal. Results: DPPC rHDLs are the most efficient particles, inducing cholesterol efflux in all cellular models and in all conditions the effect was potentiated when the ABCA1 transporter was upregulated. Conclusions: DPPC rHDLs, which resemble nascent HDL, are the most effective particles in inducing cholesterol efflux due to the higher physical binding affinity of cholesterol to the saturated long-chain-length phospholipids and the favored cholesterol transfer from a highly positively curved bilayer, to an accepting planar bilayer such as DPPC rHDLs. The physicochemical characteristics of rHDLs should be taken into consideration to design more efficient nanoparticles to promote cholesterol efflux.This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). U.G.-G. was supported by Fundaci√≥n Biof√≠sica Bizkaia. A.B.-V. was supported by Programa de especializaci√≥n de Personal Investigador Doctor en la UPV/EHU (2019) 2019‚Äď2020. S.J.-B. and A.L.-S. were supported by a grant PIF (2017‚Äď2018) and (2019‚Äď2020), Gobierno Vasco, respectively. A.L.-S. was partially supported by Fundaci√≥n Biof√≠sica Bizkaia

    Replacement of Cysteine at Position 46 in the First Cysteine-Rich Repeat of the LDL Receptor

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    Background and aims Pathogenic mutations in the Low Density Lipoprotein Receptor gene (LDLR) cause Familial Hypercholesterolemia (FH), one of the most common genetic disorders with a prevalence as high as 1 in 200 in some populations. FH is an autosomal dominant disorder of lipoprotein metabolism characterized by high blood cholesterol levels, deposits of cholesterol in peripheral tissues such as tendon xanthomas and accelerated atherosclerosis. To date, 2500 LDLRvariants have been identified in the LDLR gene; however, only a minority of them have been experimentally characterized and proven to be pathogenic. Here we investigated the role of Cys46 located in the first repeat of the LDL receptor binding domain in recognition of apolipoproteins. Methods Activity of the p.(Cys46Gly) LDLR variant was assessed by immunoblotting and flow cytometry in CHO-/d/A7 expressing the receptor variant. Affinity of p.(Cys46Gly) for LDL and VLDL was determined by solid-phase immunoassays and in silico analysis was used to predict mutation effects. Results and conclusion Functional characterization of p.(Cys46Gly) LDLRvariant showed impaired LDL and VLDL binding and uptake activity. Consistent with this, solid-phase immunoassays showed the p. (Cys46Gly) LDLR variant has decreased binding affinity for apolipoproteins. These results indicate the important role of Cys46 in LDL receptor activity and highlight the role of LR1 in LDLr activity modulation. This study reinforces the significance of in vitro functional characterization of LDL receptor activity in developing an accurate approach to FH genetic diagnosis. This is of particular importance because it enables clinicians to tailor personalized treatments for patients' mutation profile.Progenika Biopharma SA, is an Spanish biotech company founded in 2000 with headquarters in Derio, Bizkaia (SPAIN). Progenika Biopharma SA did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries to M.S. and L.P. This work was supported by Gobernio Vasco, ELKARTEK BIOGUNE 2015 (Codigo KK-2015/0000089) and Basque Government (Grupos Consolidados IT849-13). A. B-V. was supported by a grant (PIF 2014/2015) Eusko Jaurlaritza and S.J. was supported by grant (PIF 2018/2019) Eusko Jaurlaritza. The funders did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript

    miR-27b Modulates Insulin Signaling in Hepatocytes by Regulating Insulin Receptor Expression

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    Insulin resistance (IR) is one of the key contributing factors in the development of type 2 diabetes mellitus (T2DM). However, the molecular mechanisms leading to IR are still unclear. The implication of microRNAs (miRNAs) in the pathophysiology of multiple cardiometabolic pathologies, including obesity, atherosclerotic heart failure and IR, has emerged as a major focus of interest in recent years. Indeed, upregulation of several miRNAs has been associated with obesity and IR. Among them, miR-27b is overexpressed in the liver in patients with obesity, but its role in IR has not yet been thoroughly explored. In this study, we investigated the role of miR-27b in regulating insulin signaling in hepatocytes, both in vitro and in vivo. Therefore, assessment of the impact of miR-27b on insulin resistance through the hepatic tissue is of special importance due to the high expression of miR-27b in the liver together with its known role in regulating lipid metabolism. Notably, we found that miR-27b controls post-transcriptional expression of numerous components of the insulin signaling pathway including the insulin receptor (INSR) and insulin receptor substrate 1 (IRS1) in human hepatoma cells. These results were further confirmed in vivo showing that overexpression and inhibition of hepatic miR-27 enhances and suppresses hepatic INSR expression and insulin sensitivity, respectively. This study identified a novel role for miR-27 in regulating insulin signaling, and this finding suggests that elevated miR-27 levels may contribute to early development of hepatic insulin resistance.This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). A.B.-V. was supported by Programa de especializaci√≥n de Personal Investigador Doctor en la UPV/EHU (2019) 2019-2020. U.G-G. was supported by Fundaci√≥n Biof√≠sica Bizkaia. S.J. was supported by a grant PIF (2017‚Äď2018), Gobierno Vasco. We sincerely thank Haziq Siddiqi (Harvard Medical School) for his critical reading and editing of this manuscript

    Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain

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    Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT) is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC) domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active "soluble AC''. The calpain-mediated ACT processing allows trafficking of the "soluble AC'' domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP "pools'', which would play different roles in the cell pathophysiology.This study was supported by grants from the Spanish Ministry of Economy (BFU 2012-36241), the Basque Government (ETORTEK Program), and the University of Basque Country (UPV/EHU, Project UE06/10). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

    Ca2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

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    Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.This study was supported by grants from the Spanish Ministerio de Ciencia y Tecnologia (Project BFU 2007-62062 and Project BFU 2012-36241), the Basque Government (ETORTEK Program), and the University of Basque Country (UPV/EHU, Project UE06/10). KBU and GG were recipients of a fellowship from the Bizkaia Biophysics Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

    Membrane Permeabilization by Pore-Forming RTX Toxins: What Kind of Lesions Do These Toxins Form?

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    Pore-forming toxins (PFTs) form nanoscale pores across target membranes causing cell death. The pore-forming cytolysins of the RTX (repeats in toxin) family belong to a steadily increasing family of proteins characterized by having in their primary sequences a number of glycine- and aspartate-rich nonapeptide repeats. They are secreted by a variety of Gram-negative bacteria and form ion-permeable pores in several cell types, such as immune cells, epithelial cells, or erythrocytes. Pore-formation by RTX-toxins leads to the dissipation of ionic gradients and membrane potential across the cytoplasmic membrane of target cells, which results in cell death. The pores formed in lipid bilayers by the RTX-toxins share some common properties such as cation selectivity and voltage-dependence. Hemolytic and cytolytic RTX-toxins are important virulence factors in the pathogenesis of the producing bacteria. And hence, understanding the function of these proteins at the molecular level is critical to elucidating their role in disease processes. In this review we summarize the current state of knowledge on pore-formation by RTX toxins, and include recent results from our own laboratory regarding the pore-forming activity of adenylate cyclase toxin (ACT or CyaA), a large protein toxin secreted by Bordetella pertussis, the bacterium causative of whooping cough.This study was supported by grant from the Spanish Ministerio de Economia y Competitividad BFU2017-82758-P. D.G.-B. and A.B.G.A. were recipients of a fellowship from the Bizkaia Biophysics Foundation
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