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Endophilin B1/Bif-1 Stimulates BAX Activation Independently from Its Capacity to Produce Large Scale Membrane Morphological Rearrangements

Endophilin B1/BAX-interacting factor 1 (Bif-1) is a protein that cooperates with dynamin-like protein 1 (DLP1/Drp1) to maintain normal mitochondrial outer membrane (MOM) dynamics in healthy cells and also contributes to BAX-driven MOM permeabilization (MOMP), the irreversible commitment point to cell death for the majority of apoptotic stimuli. However, despite its importance, exactly how Bif-1 fulfils its proapoptotic role is unknown. Here, we demonstrate that the stimulatory effect of Bif-1 on BAX-driven MOMP and on BAX conformational activation observed in intact cells during apoptosis can be recapitulated in a simplified system consisting of purified proteins and MOM-like liposomes. In this reconstituted model system the N-BAR domain of Bif-1 reproduced the stimulatory effect of Bif-1 on functional BAX activation. This process was dependent on physical interaction between Bif-1 N-BAR and BAX as well as on the presence of the mitochondrion-specific lipid cardiolipin. Despite that Bif-1 N-BAR produced large scale morphological rearrangements in MOM-like liposomes, this phenomenon could be separated from functional BAX activation. Furthermore, DLP1 also caused global morphological changes in MOM-like liposomes, but DLP1 did not stimulate BAX-permeabilizing function in the absence or presence of Bif-1. Taken together, our findings not only provide direct evidence for a functional interplay between Bif-1, BAX, and cardiolipin during MOMP but also add significantly to the growing body of evidence indicating that components of the mitochondrial morphogenesis machinery possess proapoptotic functions that are independent from their recognized roles in normal mitochondrial dynamics.

BIM and tBID Are Not Mechanistically Equivalent When Assisting BAX to Permeabilize Bilayer Membranes

BIM and tBID are two BCL-2 homology 3 (BH3)-only proteins with a particularly strong capacity to trigger BAX-driven mitochondrial outer membrane permeabilization, a crucial event in mammalian apoptosis. However, the means whereby BIM and tBID fulfill this task is controversial. Here, we used a reconstituted liposomal system bearing physiological relevance to explore systematically how the BAX-permeabilizing function is influenced by interactions of BIM/BID-derived proteins and BH3 motifs with multidomain BCL-2 family members and with membrane lipids. We found that nanomolar dosing of BIM proteins sufficed to reverse completely the inhibition of BAX permeabilizing activity exerted by all antiapoptotic proteins tested (BCL-2, BCL-XL, BCL-W, MCL-1, and A1). This effect was reproducible by a peptide representing the BH3 motif of BIM, whereas an equivalent BID BH3 peptide was less potent and more selective, reversing antiapoptotic inhibition. On the other hand, in the absence of BCL-2-type proteins, BIM proteins and the BIM BH3 peptide were inefficient, directly triggering the BAX-permeabilizing function. In contrast, tBID alone potently assisted BAX to permeabilize membranes at least in part by producing a structural distortion in the lipid bilayer via BH3-independent interaction of tBID with cardiolipin. Together, these results support the notion that BIM and tBID follow different strategies to trigger BAX-driven mitochondrial outer membrane permeabilization with strong potency.

Novel functional APOB mutations outside LDL-binding region causing familial hypercholesterolaemia

Familial hypercholesterolaemia (FH) is characterized by increased circulating low-density lipoprotein (LDL) cholesterol leading to premature atherosclerosis and coronary heart disease. Although FH is usually caused by mutations in LDLR, mutations in APOB and PCSK9 also cause FH but only a few mutations have been reported, APOB p.R3527Q being the most common. However, 30-80% of clinical FH patients do not present an identifiable mutation in any of the described genes. To identify the genetic cause of the hypercholesterolaemia in 65 patients without mutations in LDLR, PCSK9 or in fragments of exon 26 and 29 of APOB currently analysed, we performed whole sequencing of APOB by pyrosequencing. A total of 10 putative mutations in APOB were identified. Flow cytometry with fluorescently labelled LDL from patients and relatives showed that p.Arg1164Thr (exon 22) and p.Gln4494del (exon 29) presented a 40% decrease in internalization in lymphocytes and HepG2 cells, very similar to APOB3527. The proliferation assays with U937 cells showed reduced growth for both cases. The variant p.Tyr1247Cys was found to be neutral and other three alterations were considered polymorphisms. Our results emphasize the need to study the whole APOB in routine protocols to improve patient identification and cardiovascular risk assessment.

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.

The p.Leu167del Mutation in APOE Gene Causes Autosomal Dominant Hypercholesterolemia by Down-regulation of LDL Receptor Expression in Hepatocytes

CONTEXT The p.Leu167del mutation in the APOE gene has been associated with hyperlipidemia. OBJECTIVES Our objective was to determine the frequency of p.Leu167del mutation in APOE gene in subjects with autosomal dominant hypercholesterolemia (ADH) in whom LDLR, APOB, and PCSK9 mutations had been excluded and to identify the mechanisms by which this mutant apo E causes hypercholesterolemia. DESIGN The APOE gene was analyzed in a case-control study. SETTING The study was conducted at a University Hospital Lipid Clinic. PATIENTS OR OTHER PARTICIPANTS Two groups (ADH, 288 patients; control, 220 normolipidemic subjects) were included. INTERVENTION We performed sequencing of APOE gene and proteomic and cellular experiments. MAIN OUTCOME MEASURE To determine the frequency of the p.Leu167del mutation and the mechanism by which it causes hypercholesterolemia. RESULTS In the ADH group, nine subjects (3.1%) were carriers of the APOE c.500_502delTCC, p.Leu167del mutation, cosegregating with hypercholesterolemia in studied families. Proteomic quantification of wild-type and mutant apo E in very low-density lipoprotein (VLDL) from carrier subjects revealed that apo E3 is almost a 5-fold increase compared to mutant apo E. Cultured cell studies revealed that VLDL from mutation carriers had a significantly higher uptake by HepG2 and THP-1 cells compared to VLDL from subjects with E3/E3 or E2/E2 genotypes. Transcriptional down-regulation of LDLR was also confirmed. CONCLUSIONS p.Leu167del mutation in APOE gene is the cause of hypercholesterolemia in the 3.1% of our ADH subjects without LDLR, APOB, and PCSK9 mutations. The mechanism by which this mutation is associated to ADH is that VLDL carrying the mutant apo E produces LDLR down-regulation, thereby raising plasma low-density lipoprotein cholesterol levels.

Functional Characterization and Classification of Frequent Low‐Density Lipoprotein Receptor Variants

Familial hypercholesterolemia (FH) is an autosomal‐dominant disorder mostly caused by mutations in the low‐density lipoprotein receptor (LDLR) gene leading to increased risk for premature cardiovascular diseases. According to functional studies, LDLR mutations may be classified into five classes. The main objective of this study was to characterize seven LDLR variants previously detected in FH patients. Analysis by flow cytometry and confocal microscopy of LDLR activity demonstrate that all the studied variants are pathogenic. Among the mutations located in β‐propeller, p.Trp577Gly and p.Ile624del were classified as class 2, whereas p.Arg416Trp and p.Thr454Asn as class 5. p.Phe800Glyfs*129 (located in the cytoplasmic domain), p.Cys155Tyr (located in the binding domain), and p.Asn825Lys (inside FxNPxY motif) were classified as class 2, 3, and 4, respectively. The results also show that LDLR activity of these class 4 and 5 variants is not completely abolished, showing a milder phenotype. We have also determined that statin response is more efficient lowering total cholesterol in heterozygous patients carrying p.Ile624del (class 2) compared with p.Arg416Trp and p.Thr454Asn (class 5) variants. In conclusion, these findings emphasize the importance of characterizing LDLR pathogenic variants to provide an indisputable FH diagnosis and to gain insight into the statin response depending on the LDLR class mutation.

Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment

Familial hypercholesterolemia (FH) is a common autosomal codominant disease with a frequency of 1∶500 individuals in its heterozygous form. The genetic basis of FH is most commonly mutations within the LDLR gene. Assessing the pathogenicity of LDLR variants is particularly important to give a patient a definitive diagnosis of FH. Current studies of LDLR activity ex vivo are based on the analysis of 125I-labeled lipoproteins (reference method) or fluorescent-labelled LDL. The main purpose of this study was to compare the effectiveness of these two methods to assess LDLR functionality in order to validate a functional assay to analyse LDLR mutations. LDLR activity of different variants has been studied by flow cytometry using FITC-labelled LDL and compared with studies performed previously with 125I-labeled lipoproteins. Flow cytometry results are in full agreement with the data obtained by the 125I methodology. Additionally confocal microscopy allowed the assignment of different class mutation to the variants assayed. Use of fluorescence yielded similar results than 125I-labeled lipoproteins concerning LDLR activity determination, and also allows class mutation classification. The use of FITC-labelled LDL is easier in handling and disposal, cheaper than radioactivity and can be routinely performed by any group doing LDLR functional validations.

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.

Structural analysis of APOB variants, p.(Arg3527Gln), p.(Arg1164Thr) and p.(Gln4494del), causing Familial Hypercholesterolaemia provides novel insights into variant pathogenicity

Familial hypercholesterolaemia (FH) is an inherited autosomal dominant disorder resulting from defects in the low-density lipoprotein receptor (LDLR), in the apolipoprotein B (APOB) or in the proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In the majority of the cases FH is caused by mutations occurring within LDLR, while only few mutations in APOB and PCSK9 have been proved to cause disease. p.(Arg3527Gln) was the first mutation in APOB being identified and characterized. Recently two novel pathogenic APOB variants have been described: p.(Arg1164Thr) and p.(Gln4494del) showing impaired LDLR binding capacity, and diminished LDL uptake. The objective of this work was to analyse the structure of p.(Arg1164Thr) and p.(Gln4494del) variants to gain insight into their pathogenicity. Secondary structure of the human ApoB100 has been investigated by infrared spectroscopy (IR) and LDL particle size both by dynamic light scattering (DLS) and electron microscopy. The results show differences in secondary structure and/or in particle size of p.(Arg1164Thr) and p.(Gln4494del) variants compared with wild type. We conclude that these changes underlie the defective binding and uptake of p.(Arg1164Thr) and p.(Gln4494del) variants. Our study reveals that structural studies on pathogenic variants of APOB may provide very useful information to understand their role in FH disease.

The importance of an integrated analysis of clinical, molecular, and functional data for the genetic diagnosis of familial hypercholesterolemia

Purpose:Familial hypercholesterolemia (FH) is one of the most common monogenic disorders, and the high concentrations of low-density lipoprotein (LDL) cholesterol presented since birth confers on these patients an increased cardiovascular risk. More than 1,600 alterations have been described in the LDL receptor gene (LDLR), but a large number need to be validated as mutations causing disease to establish a diagnosis of FH. This study aims to characterize, both at the phenotypic and genotypic levels, families with a clinical diagnosis of FH and present evidence for the importance of the integration of clinical, molecular, and functional data for the correct diagnosis of patients with FH.Methods:A detailed analysis of the phenotype and genotype presented by 55 families with 13 different alterations in the LDLR was conducted. For eight of these, an extensive functional characterization was performed by flow cytometry, confocal microscopy, and reverse transcriptase polymerase chain reaction.Results:Carriers of neutral alterations presented a significantly lower incidence of premature cardiovascular disease, lower levels of atherogenic lipoproteins and a large number of these individuals had LDL-cholesterol values below the 75th percentile. presented a significantly lower incidence of premature cardiovascular disease, lower levels of atherogenic lipoproteins and a large number of these individuals had LDL-cholesterol values below the 75th percentile However, the functional study was essential to determine the pathogenicity of variants.Conclusion:The data collected illustrate the importance of this integrated analysis for the correct assessment of patients with FH who can otherwise be misdiagnosed.Genet Med 17 12, 980–988.