Cholesterol is an essential component of cell barrier formation and signaling transduction involved in many essential physiologic processes. For this reason, cholesterol metabolism must be tightly controlled. Cell cholesterol is mainly acquired from two sources: Dietary cholesterol, which is absorbed in the intestine and, intracellularly synthesized cholesterol that is mainly synthesized in the liver. Once acquired, both are delivered to peripheral tissues in a lipoprotein dependent mechanism. Malfunctioning of cholesterol metabolism is caused by multiple hereditary diseases, including Familial Hypercholesterolemia, Sitosterolemia Type C and Niemann-Pick Type C1. Of these, familial hypercholesterolemia (FH) is a common inherited autosomal co-dominant disorder characterized by high plasma cholesterol levels. Its frequency is estimated to be 1:200 and, if untreated, increases the risk of premature cardiovascular disease. This review aims to summarize the current knowledge on cholesterol metabolism and the relation of FH to cholesterol homeostasis with special focus on the genetics, diagnosis and treatment.

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.

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.

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.

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.

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.