Characterization of the Intrinsic Phospholipase A1 Activity of Bordetella pertussis Adenylate Cyclase Toxin

Adenylate cyclase toxin (ACT, CyaA) is one of the important virulence factors secreted by the whooping cough bacterium Bordetella pertussis, and it is essential for the colonization of the human respiratory tract by this bacterium. Cytotoxicity by ACT results from the synergy between toxin's two main activities, production of supraphysiological cAMP levels by its N-terminal adenylate cyclase domain (AC domain), and cell membrane permeabilization, induced by its C-terminal pore-forming domain (hemolysin domain), which debilitate the host defenses. In a previous study we discovered that purified ACT is endowed with intrinsic phospholipase A1 (PLA) activity and that Ser in position 606 of the ACT polypeptide is a catalytic site for such hydrolytic activity, as part of G-X-S-X-G catalytic motif. Recently these findings and our conclusions have been directly questioned by other authors who claim that ACT-PLA activity does not exist. Here we provide new data on ACT phospholipase A1 characteristics. Based on our results we reaffirm our previous conclusions that ACT is endowed with PLA activity; that our purified ACT preparations are devoid of any impurity with phospholipase A activity; that ACT-S606A is a PLA-inactive mutant and thus, that Ser606 is a catalytic site for the toxin hydrolytic activity on phospholipids, and that ACT-PLA activity is involved in AC translocation.This study was supported by grants from the Basque Government (Grupos Consolidados IT849) and grant from the Spanish Ministerio de Economia y Competitividad (BFU2017-82758-P (AEI/FEDER, UE) to H.O

SR-B1, a Key Receptor Involved in the Progression of Cardiovascular Disease: A Perspective from Mice and Human Genetic Studies

High plasma level of low-density lipoprotein (LDL) is the main driver of the initiation and progression of cardiovascular disease (CVD). Nevertheless, high-density lipoprotein (HDL) is considered an anti-atherogenic lipoprotein due to its role in reverse cholesterol transport and its ability to receive cholesterol that effluxes from macrophages in the artery wall. The scavenger receptor B class type 1 (SR-B1) was identified as the high-affinity HDL receptor, which facilitates the selective uptake of cholesterol ester (CE) into the liver via HDL and is also implicated in the plasma clearance of LDL, very low-density lipoprotein (VLDL) and lipoprotein(a) (Lp(a)). Thus, SR-B1 is a multifunctional receptor that plays a main role in the metabolism of different lipoproteins. The aim of this review is to highlight the association between SR-B1 and CVD risk through mice and human genetic studies.This work was supported by grants from Gobierno de Aragón, B14–7R, Spain, and the Spanish Ministry of Science and Innovation PI18/01777, PI19/00694 and CIBERCV. These projects are co-financed by Instituto de Salud Carlos III and the European Regional Development Fund (ERDF) of the European Union ‘‘A way to make Europe’’. IG-R was funded by Sara Borrell fellowship CD19/00245

Translational Bioinformatics for Human Reproductive Biology Research: Examples, Opportunities and Challenges for a Future Reproductive Medicine

Since 1978, with the first IVF (in vitro fertilization) baby birth in Manchester (England), more than eight million IVF babies have been born throughout the world, and many new techniques and discoveries have emerged in reproductive medicine. To summarize the modern technology and progress in reproductive medicine, all scientific papers related to reproductive medicine, especially papers related to reproductive translational medicine, were fully searched, manually curated and reviewed. Results indicated whether male reproductive medicine or female reproductive medicine all have made significant progress, and their markers have experienced the progress from karyotype analysis to single-cell omics. However, due to the lack of comprehensive databases, especially databases collecting risk exposures, disease markers and models, prevention drugs and effective treatment methods, the application of the latest precision medicine technologies and methods in reproductive medicine is limited.This research was funded by Project of Natural Science Foundation of Gansu Province (20JR5RA363); Project of Gansu Provincial Education Department (2020B-003)

Membrane Repair Mechanisms against Permeabilization by Pore-Forming Toxins

Permeabilization of the plasma membrane represents an important threat for any cell, since it compromises its viability by disrupting cell homeostasis. Numerous pathogenic bacteria produce pore-forming toxins that break plasma membrane integrity and cause cell death by colloid-osmotic lysis. Eukaryotic cells, in turn, have developed different ways to cope with the effects of such membrane piercing. Here, we provide a short overview of the general mechanisms currently proposed for plasma membrane repair, focusing more specifically on the cellular responses to membrane permeabilization by pore-forming toxins and presenting new data on the effects and cellular responses to the permeabilization by an RTX (repeats in toxin) toxin, the adenylate cyclase toxin-hemolysin secreted by the whooping cough bacterium Bordetella pertussis, which we have studied in the laboratory.This study was supported by grants from the Basque Government (Grupos Consolidados IT849-13) and grant from the Spanish Ministerio de Economia y Competitividad (BFU2017-82758-PAEI/FEDER, UE)

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

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 ≈400 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 “two-step model” and the recently-proposed “toroidal pore model”, 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

Cholesterol stimulates the lytic activity of Adenylate Cyclase Toxin on lipid membranes by promoting toxin oligomerization and formation of pores with a greater effective size

Several toxins acting on animal cells present different, but specific, interactions with cholesterol. Bordetella pertussis infects the human respiratory tract and causes whooping cough, a highly contagious and resurgent disease. Its virulence factor adenylate cyclase toxin (ACT) plays an important role in the course of infection. ACT is a pore-forming cytolysin belonging to the Repeats in ToXin (RTX) family of leukotoxins/hemolysins and is capable of permeabilizing several cell types and lipid vesicles. Previously, we observed that in the presence of cholesterol ACT induces greater liposome permeabilization. Similarly, recent reports also implicate cholesterol in the cytotoxicity of an increasing number of pore-forming RTX toxins. However, the mechanistic details by which this sterol promotes the lytic activity of ACT or of these other RTX toxins remain largely unexplored and poorly understood. Here, we have applied a combination of biophysical techniques to dissect the role of cholesterol in pore formation by ACT. Our results indicate that cholesterol enhances the lytic potency of ACT by promoting toxin oligomerization, a step which is indispensable for ACT to accomplish membrane permeabilization and cell lysis. Since our experimental design eliminates the possibility that this cholesterol effect derives from toxin accumulation due to lateral lipid phase segregation, we hypothesize that cholesterol facilitates lytic pore formation, by favoring a toxin conformation more prone to protein-protein interactions and oligomerization. Our data shed light on the complex relationship between lipid membranes and protein toxins acting on these membranes. Coupling cholesterol binding, increased oligomerization and increased lytic activity is likely pertinent for other RTX cytolysins.Rocío Alonso is gratefully acknowledged for excellent technical assistance. This study was supported by grants from the Spanish Ministerio de Economia y Competitividad BFU2017-82758-P (H.O.) and of Basque Government (Grupos Consolidados IT1264-19). D.G.B was recipients of a fellowship from the Bizkaia Biophysics Foundation, and JA was recipient of a fellowship from the Basque Government

Phosphatidic Acid Stimulates Lung Cancer Cell Migration through Interaction with the LPA1 Receptor and Subsequent Activation of MAP Kinases and STAT3

Phosphatidic acid (PA) is a key bioactive glycerophospholipid that is implicated in the regulation of vital cell functions such as cell growth, differentiation, and migration, and is involved in a variety of pathologic processes. However, the molecular mechanisms by which PA exerts its pathophysiological actions are incompletely understood. In the present work, we demonstrate that PA stimulates the migration of the human non-small cell lung cancer (NSCLC) A549 adenocarcinoma cells, as determined by the transwell migration assay. PA induced the rapid phosphorylation of mitogen-activated protein kinases (MAPKs) ERK1-2, p38, and JNK, and the pretreatment of cells with selective inhibitors of these kinases blocked the PA-stimulated migration of cancer cells. In addition, the chemotactic effect of PA was inhibited by preincubating the cells with pertussis toxin (PTX), a Gi protein inhibitor, suggesting the implication of a Gi protein-coupled receptor in this action. Noteworthy, a blockade of LPA receptor 1 (LPA1) with the specific LPA1 antagonist AM966, or with the selective LPA1 inhibitors Ki1645 or VPC32193, abolished PA-stimulated cell migration. Moreover, PA stimulated the phosphorylation of the transcription factor STAT3 downstream of JAK2, and inhibitors of either JAK2 or STAT3 blocked PA-stimulated cell migration. It can be concluded that PA stimulates lung adenocarcinoma cell migration through an interaction with the LPA1 receptor and subsequent activation of the MAPKs ERK1-2, p38, and JNK, and that the JAK2/STAT3 pathway is also important in this process. These findings suggest that targeting PA formation and/or the LPA1 receptor may provide new strategies to reduce malignancy in lung cancer.This work was supported by Grant IT1720-22 from “Departamento de Educación, Viceconsejería de Universidades e Investigación del Gobierno Vasco” (GV/EJ, Basque Country, Spain). N. Presa is the recipient of a fellowship from “Departamento de Educación, Universidades e Investigación del Gobierno Vasco” (GV/EJ, Basque Country, Spain)

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

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

Towards a new generation of non-cytotoxic shape memory thermoplastic polyurethanes for biomedical applications

In recent decades, the technology of polymeric materials used in biomedical applications has been greatly improved, replacing the metals that had been used until now. This change has not only meant an improvement in the cost of the raw material and in its processing, but it is also due to the fact that there are applications, such as stents, where the material is required to have a certain flexibility, both during the surgical intervention and during the healing or conditioning the tissue in which the intervention is performed. In this type of application, the so-called shape memory polymers (SMPs) are very interesting, but for this, they must meet the condition of being biocompatible. In this work, new polyurethane materials have been designed in which, in addition to shape memory prevailing, adequate cell proliferation values are obtained for possible use in biomedical applications. Furthermore, during the synthesis, in order to avoid undesired and toxics subproducts, instead of the typical aromatic diisocyanates, an aliphatic 1,6-hexamethylene diisocyanate (HDI) has been selected. Moreover, neither solvents nor catalysts were used, which makes eco-friendly synthesis suitable for scaling at an industrial level. Finally, castor oil (CO) has been used as one of the main synthesis reagents, which is an abundant compound obtained from biological sources. For all this, it can be concluded that the polymers described here have a wide range of application possibilities (biomedicine, food packaging…), and are highly interesting to preserve our Planet.Authors would like to acknowledge the Basque Government funding within the ELKARTEK 2020 AVANSITE (KK-2020/00019). Also, authors gratefully acknowledge funding from the University of the Basque Country (GIU20/075). The authors thank for technical and human support provided by SGIker (UPV/EHU/ ERDF, EU)

Kukutxeztula: "gainditutako" gaitz baten itzulera

Arnasbideetako infekzioek heriotza tasa altuak eragiten dituzte munduan, COVID-19 birusa izanik azken urteetako adibiderik argiena. Hala ere, ez da libre dabilen patogeno bakarra eta berrikuspen honetan Bordetella pertussis bakterio patogenoak eragindako kukutxeztulaz arituko gara. Kukutxeztula arnasbideen infekzio larri eta kutsakorra da, bertako jariakinekin edota listu tantekin kontaktua izatean transmititzen dena. Helduetan arrisku berezirik ez duten sintomak eragiten dituen arren (etengabeko eztul gertakariak, arnasteko zailtasuna edota sukarra, besteak beste), heriotza eragin dezake urte batetik beherako haurretan batez ere. Kukutxeztula fase goiztiar batean gainditu ez duten gaixoek bigarren mailako pneumonia bezalako konplikazioak garatu ditzakete, horixe izaten delarik kukutxeztulaz hiltzen diren gehienen arrazoi nagusia. Lehen txertoa garatu zenetik kontrolpean zegoela uste izan den arren, kalte handiak eragin ditu garapen bidean dauden herrialdeetan batez ere. Herrialde garatuetan ere badu bere inpaktua, eta 2011tik gaur arte Ipar Amerikan eta mendebaldeko Europan epidemia egoera zabaldu da. Berrikuspen honetan kukutxeztulari buruzko informazioaren eguneratze bat aurkezten da, B. pertussisek erabiltzen dituen hainbat mekanismo infekziosotan arreta jarriz. Mekanismo hauen oinarri molekularra ulertzeak lagundu dezake zeluletan gertatzen diren beste hainbat prozesuren ulerkeran, gaixotasunaren aurkako sendagai berrien garapenean edota biologia molekularreko tresna berrien garapenean.; Respiratory infections cause high mortality rates throughout the world, being the COVID-19 virus the clearest example in recent years. However, there are other several pathogens not fully controlled and in this review we will focus on whooping cough, the sickness caused by the pathogenic bacterium Bordetella pertussis.Whooping cough is a severe and contagious respiratory infection that is transmitted by coming into contact with local secretions or with saliva drops. Despite the symptoms are not of special risks for adults (continuous episodes of cough, difficulty breathing or fever), it can cause death especially in children younger than one year of age. Patients who have not overcome cough at an early stage may develop complications such as secondary pneumonia, which is the main cause of most who die with whooping cough. Although since the development of the first vaccine it has been considered to be under control, whooping cough has caused significant damage especially in developing countries. Surprisingly, it has also an impact on developed countries and since 2011 the epidemic has spread in North America and Western Europe. This review presents an update on whooping cough, focusing on the different infective mechanisms used by B.pertussis. Understanding of the molecular basis of these mechanisms may contribute to the understanding of other cellular processes, the development of new anti-disease drugs or the development of new molecular biology instruments