Kepa B. Uribe

Functional characterization of splicing and ligand-binding domain variants in the LDL receptor.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder mostly caused by mutations in the LDLR gene. Although the detection of functional mutations in the LDLR gene provides an unequivocal diagnosis of the FH condition, there are many variants whose pathogenicity is still unknown. The aims of this study were to set up a rapid method to determine the effect of LDLR mutations, thereby providing an accurate diagnosis of FH, and to functionally characterize six LDLR mutations detected at high frequency by the LIPOchip® platform (Progenika Biopharma, Spain) in the Spanish population. LDLR expression and activity were analyzed by one‐single‐step flow cytometry assay and confocal microscopy. Splicing effects were determined by sequencing reverse transcription polymerase chain reaction products. The analysis of three heterozygous variants with a single point mutation within the low‐density lipoprotein binding domain allowed us to classify the c.806G>A variant as nonpathogenic, and c.862G>A and c.895G>A variants as causative of FH. The results obtained for three variants affecting donor splice sites of the LDLR mRNA, c.313+2dupT, c.1186+5G>A, and c.1845+1G>C, demonstrated that these mutations are pathogenic. These results expand our knowledge of mutations responsible for FH, providing an accurate diagnosis and leading to early treatment to reduce the risk of premature cardiovascular events. Hum Mutat 33:232–243, 2012.

Adenylate Cyclase Toxin Promotes Internalisation of Integrins and Raft Components and Decreases Macrophage Adhesion Capacity

Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT) that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin) family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis.

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

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.

Phospholipase A activity of adenylate cyclase toxin mediates translocation of its adenylate cyclase domain

Significance Numerous bacterial toxins can cross cell membranes, penetrating the cytosol of their target cells, but to do so exploits cellular endocytosis or intracellular sorting machineries. Bordetella pertussis adenylate cyclase toxin (ACT) delivers its catalytic domain directly across the cell membrane by an unknown mechanism, and generates cAMP, which subverts the cell signaling. Here, we decipher the fundamentals of the molecular mechanism of ACT transport. We find that AC translocation and, consequently cytotoxicity, are determined by an intrinsic ACT–phospholipase A (PLA) activity, supporting a model in which in situ generation of nonlamellar lysophospholipids by ACT–PLA activity remodels the cell membrane, forming proteolipidic toroidal “holes” through which AC domain transfer may directly take place. PLA-based specific protein transport in cells is unprecedented. Adenylate cyclase toxin (ACT or CyaA) plays a crucial role in respiratory tract colonization and virulence of the whooping cough causative bacterium Bordetella pertussis. Secreted as soluble protein, it targets myeloid cells expressing the CD11b/CD18 integrin and on delivery of its N-terminal adenylate cyclase catalytic domain (AC domain) into the cytosol, generates uncontrolled toxic levels of cAMP that ablates bactericidal capacities of phagocytes. Our study deciphers the fundamentals of the heretofore poorly understood molecular mechanism by which the ACT enzyme domain directly crosses the host cell membrane. By combining molecular biology, biochemistry, and biophysics techniques, we discover that ACT has intrinsic phospholipase A (PLA) activity, and that such activity determines AC translocation. Moreover, we show that elimination of the ACT–PLA activity abrogates ACT toxicity in macrophages, particularly at toxin concentrations close to biological reality of bacterial infection. Our data support a molecular mechanism in which in situ generation of nonlamellar lysophospholipids by ACT–PLA activity into the cell membrane would form, likely in combination with membrane-interacting ACT segments, a proteolipidic toroidal pore through which AC domain transfer could directly take place. Regulation of ACT–PLA activity thus emerges as novel target for therapeutic control of the disease.

Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells

Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of “toxin-coated bacteria” proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or “free” in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca2+-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system.

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

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.

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.

Identification and in vitro characterization of two new PCSK9 Gain of Function variants found in patients with Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by pathogenic variants in genes encoding for LDL receptor (LDLR), Apolipoprotein B and Proprotein convertase subtilisin/kexin type 9 (PCSK9). Among PCSK9 variants, only Gain-of- Function (GOF) variants lead to FH. Greater attention should be paid to the classification of variants as pathogenic. Two hundred sixty nine patients with a clinical suspect of FH were screened for variants in LDLR and the patients without pathogenic variants were screened for variants in PCSK9 and APOB. Functional characterization of PCSK9 variants was performed by assessment of protein secretion, of LDLR activity in presence of PCSK9 variant proteins as well as of the LDLR affinity of the PCSK9 variants. Among 81 patients without pathogenic variants in LDLR, 7 PCSK9 heterozygotes were found, 4 of whom were carriers of variants whose role in FH pathogenesis is still unknown. Functional characterization revealed that two variants (p.(Ser636Arg) and p.(Arg357Cys)) were GOF variants. In Conclusions, we demonstrated a GOF effect of 2 PCSK9 variants that can be considered as FH-causative variants. The study highlights the important role played by functional characterization in integrating diagnostic procedures when the pathogenicity of new variants has not been previously demonstrated.

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved O-glycan sites. Moreover, we found that O-glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11–mediated O-glycosylation on LDLR and related receptor localization and function are unknown. Here, we characterized O-glycosylation of LDLR-related proteins and identified conserved O-glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrate that GalNAc-T11–mediated LDLR and VLDLR O-glycosylation is not required for transport and cell-surface expression and stability of these receptors but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O-glycosylation increased affinity for LDL by ∼5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease.

Replacement of cysteine at position 46 in the first cysteine-rich repeat of the LDL receptor impairs apolipoprotein recognition

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 LDLR variants 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-ldlA7 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) LDLR variant 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.