OptiMo-LDLr: An Integrated In Silico Model with Enhanced Predictive Power for LDL Receptor Variants, Unraveling Hot Spot Pathogenic Residues.

Familial hypercholesterolemia (FH) is an inherited metabolic disease affecting cholesterol metabolism, with 90% of cases caused by mutations in the LDL receptor gene (LDLR), primarily missense mutations. This study aims to integrate six commonly used predictive software to create a new model for predicting LDLR mutation pathogenicity and mapping hot spot residues. Six predictive-software are selected: Polyphen-2, SIFT, MutationTaster, REVEL, VARITY, and MLb-LDLr. Software accuracy is tested with the characterized variants annotated in ClinVar and, by bioinformatic and machine learning techniques all models are integrated into a more accurate one. The resulting optimized model presents a specificity of 96.71% and a sensitivity of 98.36%. Hot spot residues with high potential of pathogenicity appear across all domains except for the signal peptide and the O-linked domain. In addition, translating this information into 3D structure of the LDLr highlights potentially pathogenic clusters within the different domains, which may be related to specific biological function. The results of this work provide a powerful tool to classify LDLR pathogenic variants. Moreover, an open-access guide user interface (OptiMo-LDLr) is provided to the scientific community. This study shows that combination of several predictive software results in a more accurate prediction to help clinicians in FH diagnosis.

Predictive Modeling and Structure Analysis of Genetic Variants in Familial Hypercholesterolemia: Implications for Diagnosis and Protein Interaction Studies.

PURPOSE OF REVIEW: Familial hypercholesterolemia (FH) is a hereditary condition characterized by elevated levels of low-density lipoprotein cholesterol (LDL-C), which increases the risk of cardiovascular disease if left untreated. This review aims to discuss the role of bioinformatics tools in evaluating the pathogenicity of missense variants associated with FH. Specifically, it highlights the use of predictive models based on protein sequence, structure, evolutionary conservation, and other relevant features in identifying genetic variants within LDLR, APOB, and PCSK9 genes that contribute to FH. RECENT FINDINGS: In recent years, various bioinformatics tools have emerged as valuable resources for analyzing missense variants in FH-related genes. Tools such as REVEL, Varity, and CADD use diverse computational approaches to predict the impact of genetic variants on protein function. These tools consider factors such as sequence conservation, structural alterations, and receptor binding to aid in interpreting the pathogenicity of identified missense variants. While these predictive models offer valuable insights, the accuracy of predictions can vary, especially for proteins with unique characteristics that might not be well represented in the databases used for training. This review emphasizes the significance of utilizing bioinformatics tools for assessing the pathogenicity of FH-associated missense variants. Despite their contributions, a definitive diagnosis of a genetic variant necessitates functional validation through in vitro characterization or cascade screening. This step ensures the precise identification of FH-related variants, leading to more accurate diagnoses. Integrating genetic data with reliable bioinformatics predictions and functional validation can enhance our understanding of the genetic basis of FH, enabling improved diagnosis, risk stratification, and personalized treatment for affected individuals. The comprehensive approach outlined in this review promises to advance the management of this inherited disorder, potentially leading to better health outcomes for those affected by FH.

Decellularization of xenografted tumors provides cell-specific in vitro 3D environment.

In vitro cell culture studies are common in the cancer research field, and reliable biomimetic 3D models are needed to ensure physiological relevance. In this manuscript, we hypothesized that decellularized xenograft tumors can serve as an optimal 3D substrate to generate a top-down approach for in vitro tumor modeling. Multiple tumor cell lines were xenografted and the formed solid tumors were recovered for their decellularization by several techniques and further characterization by histology and proteomics techniques. Selected decellularized tumor xenograft samples were seeded with the HCC1806 human triple-negative breast cancer (TNBC) basal-like subtype cell line, and cell behavior was compared among them and with other control 2D and 3D cell culture methods. A soft treatment using Freeze-EDTA-DNAse allows proper decellularization of xenografted tumor samples. Interestingly, proteomic data show that samples decellularized from TNBC basal-like subtype xenograft models had different extracellular matrix (ECM) compositions compared to the rest of the xenograft tumors tested. The in vitro recellularization of decellularized ECM (dECM) yields tumor-type-specific cell behavior in the TNBC context. Data show that dECM derived from xenograft tumors is a feasible substrate for reseeding purposes, thereby promoting tumor-type-specific cell behavior. These data serve as a proof-of-concept for further potential generation of patient-specific in vitro research models.

Leu22_Leu23 Duplication at the Signal Peptide of PCSK9 Promotes Intracellular Degradation of LDLr and Autosomal Dominant Hypercholesterolemia.

BACKGROUND: PCSK9 (Proprotein convertase subtilisin/kexin type 9) regulates LDL-C (low-density lipoprotein cholesterol) metabolism by targeting LDLr (LDL receptor) for lysosomal degradation. PCSK9 gain-of-function variants cause autosomal dominant hypercholesterolemia by reducing LDLr levels, the D374Y variant being the most severe, while loss-of-function variants are associated with low LDL-C levels. Gain-of-function and loss-of-function activities have also been attributed to variants occurring in the PCSK9 signal peptide. Among them, L11 is a very rare PCSK9 variant that seems to increase LDL-C values in a moderate way causing mild hypercholesterolemia. METHODS: Using molecular biology and biophysics methodologies, activities of L8 and L11 variants, both located in the leucine repetition stretch of the signal peptide, have been extensively characterized in vitro. RESULTS: L8 variant is not associated with increased LDLr activity, whereas L11 activity is increased by ≈20% compared with wt PCSK9. The results suggest that the L11 variant reduces LDLr levels intracellularly by a process resulting from impaired cleavage of the signal peptide. This would lead to less efficient LDLr transport to the cell membrane and promote LDLr intracellular degradation. CONCLUSIONS: Deletion of a leucine in the signal peptide in L8 variant does not affect PCSK9 activity, whereas the leucine duplication in the L11 variant enhances LDLr intracellular degradation. These findings highlight the importance of deep in vitro characterization of PCSK9 genetic variants to determine pathogenicity and improve clinical diagnosis and therapy of inherited familial hypercholesterolemia disease.

Boosting Cholesterol Efflux from Foam Cells by Sequential Administration of rHDL to Deliver MicroRNA and to Remove Cholesterol in a Triple-Cell 2D Atherosclerosis Model.

Cardiovascular disease, the leading cause of mortality worldwide, is primarily caused by atherosclerosis, which is characterized by lipid and inflammatory cell accumulation in blood vessels and carotid intima thickening. Although disease management has improved significantly, new therapeutic strategies focused on accelerating atherosclerosis regression must be developed. Atherosclerosis models mimicking in vivo-like conditions provide essential information for research and new advances toward clinical application. New nanotechnology-based therapeutic opportunities have emerged with apoA-I nanoparticles (recombinant/reconstituted high-density lipoproteins, rHDL) as ideal carriers to deliver molecules and the discovery that microRNAs participate in atherosclerosis establishment and progression. Here, a therapeutic strategy to improve cholesterol efflux is developed based on a two-step administration of rHDL consisting of a first dose of antagomiR-33a-loaded rHDLs to induce adenosine triphosphate-binding cassette transporters A1 overexpression, followed by a second dose of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine rHDLs, which efficiently remove cholesterol from foam cells. A triple-cell 2D-atheroma plaque model reflecting the cellular complexity of atherosclerosis is used to improve efficiency of the nanoparticles in promoting cholesterol efflux. The results show that sequential administration of rHDL potentiates cholesterol efflux indicating that this approach may be used in vivo to more efficiently target atherosclerotic lesions and improve prognosis of the disease.

A Systematic Approach to Assess the Activity and Classification of PCSK9 Variants.

BACKGROUND: Gain of function (GOF) mutations of PCSK9 cause autosomal dominant familial hypercholesterolemia as they reduce the abundance of LDL receptor (LDLR) more efficiently than wild-type PCSK9. In contrast, PCSK9 loss of function (LOF) variants are associated with a hypocholesterolemic phenotype. Dozens of PCSK9 variants have been reported, but most remain of unknown significance since their characterization has not been conducted. OBJECTIVE: Our aim was to make the most comprehensive assessment of PCSK9 variants and to determine the simplest approach for the classification of these variants. METHODS: The expression, maturation, secretion, and activity of nine well-established PCSK9 variants were assessed in transiently transfected HEK293 cells by Western blot and flow cytometry. Their extracellular activities were determined in HepG2 cells incubated with the purified recombinant PCSK9 variants. Their binding affinities toward the LDLR were determined by solid-phase immunoassay. RESULTS: LDLR expression increased when cells were transfected with LOF variants and reduced when cells were transfected with GOF variants compared with wild-type PCSK9. Extracellular activities measurements yielded exactly similar results. GOF and LOF variants had increased, respectively reduced, affinities for the LDLR compared with wild-type PCSK9 with the exception of one GOF variant (R218S) that showed complete resistance to inactivation by furin. All variants were expressed at similar levels and underwent normal maturation and secretion patterns except for two LOF and two GOF mutants. CONCLUSIONS: We propose that transient transfections of HEK293 cells with a plasmid encoding a PCSK9 variant followed by LDLR expression assessment by flow cytometry is sufficient to reliably determine its GOF or LOF status. More refined experiments should only be used to determine the underlying mechanism(s) at hand.

Engineered Repeat Protein Hybrids: The New Horizon for Biologic Medicines and Diagnostic Tools.

ConspectusThe last decades have witnessed unprecedented scientific breakthroughs in all the fields of knowledge, from basic sciences to translational research, resulting in the drastic improvement of the lifespan and overall quality of life. However, despite these great advances, the treatment and diagnosis of some diseases remain a challenge. Inspired by nature, scientists have been exploring biomolecules and their derivatives as novel therapeutic/diagnostic agents. Among biomolecules, proteins raise much interest due to their high versatility, biocompatibility, and biodegradability.Protein binders (binders) are proteins that bind other proteins, in certain cases, inhibiting or modulating their action. Given their therapeutic potential, binders are emerging as the next generation of biopharmaceuticals. The most well-known example of binders are antibodies, and inspired by them researchers have developed alternative binders using protein design approaches. Protein design can be based on naturally occurring proteins in which, by means of rational design or combinatorial approaches, new binding interfaces can be engineered to obtain specific functions or based on de novo proteins emerging from state-of-the-art computational methodologies.Among the novel designed proteins, a class of engineered repeat proteins, the consensus tetratricopeptide repeat (CTPR) proteins, stand out due to their stability and robustness. The CTPR unit is a helix-turn-helix motif constituted of 34 amino acids, of which only 8 are essential to ensure correct folding of the structure. The small number of conserved residues of CTPR proteins leaves plenty of freedom for functional mutations, making them a base scaffold that can be easily and reproducibly tailored to endow desired functions to the protein. For example, the introduction of metal-binding residues (e.g., histidines, cysteines) drives the coordination of metal ions and the subsequent formation of nanomaterials. Additionally, the CTPR unit can be conjugated with other peptides/proteins or repeated in tandem to encode larger CTPR proteins with superhelical structures. These properties allow for the design of both binder and nanomaterial-coordination modules as well as their combination within the same molecule, making the CTPR proteins, as we have demonstrated in several recent examples, the ideal platform to develop protein-nanomaterial hybrids. Generally, the fusion of two distinct materials exploits the best properties of each; however, in protein-nanomaterial hybrids, the fusion takes on a new dimension as new properties arise.These hybrids have ushered the use of protein-based nanomaterials as biopharmaceuticals beyond their original therapeutic scope and paved the way for their use as theranostic agents. Despite several reports of protein-stabilized nanomaterials found in the literature, these systems offer limited control in the synthesis and properties of the grown nanomaterials, as the protein acts just as a stabilizing agent with no significant functional contribution. Therefore, the rational design of protein-based nanomaterials as true theranostic agents is still incipient. In this context, CTPR proteins have emerged as promising scaffolds to hold simultaneously therapeutic and diagnostic functions through protein engineering, as it has been recently demonstrated in pioneering in vitro and in vivo examples.

Pre-targeting with ultra-small nanoparticles: boron carbon dots as drug candidates for boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is a promising cancer treatment exploiting the neutron capture capacity and subsequent fission reaction of boron-10. The emergence of nanotechnology has encouraged the development of nanocarriers capable of accumulating boron atoms preferentially in tumour cells. However, a long circulation time, required for high tumour accumulation, is usually accompanied by accumulation of the nanosystem in organs such as the liver and the spleen, which may cause off-target side effects. This could be overcome by using small-sized boron carriers via a pre-targeting strategy. Here, we report the preparation, characterisation and in vivo evaluation of tetrazine-functionalised boron-rich carbon dots, which show very fast clearance and low tumour uptake after intravenous administration in a mouse HER2 (human epidermal growth factor receptor 2)-positive tumour model. Enhanced tumour accumulation was achieved when using a pretargeting approach, which was accomplished by a highly selective biorthogonal reaction at the tumour site with trans-cyclooctene-functionalised Trastuzumab.

Novel PCSK9 (Proprotein Convertase Subtilisin Kexin Type 9) Variants in Patients With Familial Hypercholesterolemia From Cape Town.

OBJECTIVE: Familial hypercholesterolemia (FH) is characterized by elevated low-density lipoprotein-cholesterol and markedly increased cardiovascular risk. In patients with a genetic diagnosis, low-density lipoprotein receptor (LDLR) mutations account for >90% of cases, apolipoprotein B (APOB) mutations for ≈5% of cases, while proprotein convertase subtilisin kexin type 9 (PCSK9) gain of function mutations are rare (<1% of cases). We aimed to evaluate the functional impact of several novel PCSK9 variants in a cohort of patients with FH by genetic cascade screening and in vitro functionality assays. Approach and Results: Patients with clinically diagnosed FH underwent genetic analysis of LDLR, and if negative, sequential testing of APOB and PCSK9. We analyzed cosegregation of hypercholesterolemia with novel PCSK9 variants. Gain of function status was determined by in silico analyses and validated by in vitro functionality assays. Among 1055 persons with clinical FH, we identified nonsynonymous PCSK9 variants in 27 (2.6%) patients and 7 of these carried one of the 4 previously reported gain of function variants. In the remaining 20 patients with FH, we identified 7 novel PCSK9 variants. The G516V variant (c.1547G>T) was found in 5 index patients and cascade screening identified 15 additional carriers. Low-density lipoprotein-cholesterol levels were higher in these 15 carriers compared with the 27 noncarriers (236±73 versus 124±35 mg/dL; P<0.001). In vitro studies demonstrated the pathogenicity of the G516V variant. CONCLUSIONS: In our study, 1.14% of cases with clinical FH were clearly attributable to pathogenic variants in PCSK9. Pathogenicity is established beyond doubt for the G516V variant.

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

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.

Mutation type classification and pathogenicity assignment of sixteen missense variants located in the EGF-precursor homology domain of the LDLR.

The primary genetic cause of familial hypercholesterolemia (FH) is related to mutations in the LDLR gene encoding the Low-density Lipoprotein Receptor. LDLR structure is organized in 5 different domains, including an EGF-precursor homology domain that plays a pivotal role in lipoprotein release and receptor recycling. Mutations in this domain constitute 51.7% of the total missense variants described in LDLR. The aim of the present work was to analyse how clinically significant variants in the EGF-precursor homology domain impact LDLR. The activity of sixteen LDLR variants was functionally characterized by determining LDLR expression by Western blot and LDLR expression, LDL binding capacity and uptake, and LDLR recycling activity by flow cytometry in transfected CHO-ldlA7 cells. Of the analysed variants, we found six non-pathogenic LDLR variants and ten pathogenic variants distributed as follow: three class 3 variants; four class 2 variants; and three class 5 variants. These results can be incorporated into clinical management of patients by helping guide the appropriate level of treatment intensity depending on the extent of loss of LDLR activity. This data can also contribute to cascade-screening for pathogenic FH variants.

Functional Analysis of LDLR (Low-Density Lipoprotein Receptor) Variants in Patient Lymphocytes to Assess the Effect of Evinacumab in Homozygous Familial Hypercholesterolemia Patients With a Spectrum of LDLR Activity.

OBJECTIVE: Homozygous familial hypercholesterolemia is a rare disease usually caused by LDLR (low-density lipoprotein receptor) mutations. Homozygous familial hypercholesterolemia is characterized by markedly elevated LDL-C (low-density lipoprotein cholesterol) levels and an extremely high risk of premature atherosclerotic cardiovascular disease. A phase 2, proof-of-concept study (NCT02265952) demonstrated that evinacumab, a fully human monoclonal antibody to ANGPTL3 (angiopoietin-like 3 protein), reduced LDL-C levels in 9 patients with genotypically confirmed homozygous familial hypercholesterolemia and was well tolerated. The aim of this study was to analyze the effects of evinacumab on LDLR activity in lymphocytes purified from patients in the proof-of-concept study. Approach and Results: LDLR activity was assessed in patient lymphocytes before and after treatment with evinacumab and versus lymphocytes carrying wild-type LDLR, and also in an LDLR-defective Chinese hamster ovary cell line (CHO-ldlA7) transfected with plasmids encoding the LDLR variants. Overall mean peak reduction in LDL-C with evinacumab was -58±18%, occurring between Week 4 and Week 12. Mutations identified in the 9 patients were shown to be pathogenic, with loss of LDLR activity versus wild type. Two of the LDLR variants, p.(Cys681*) and p.(Ala627Profs*38), were class 2 type mutations that are retained in the endoplasmic reticulum. Six variants were class 3 type mutations with impaired LDL-C binding activity: p.(Trp87Gly), occurring in 2 patients, p.(Gln254Pro), p.(Ser177Leu), p.(Gly335Val), and p.(Ser306Leu). Evinacumab had no effect on LDLR activity. CONCLUSIONS: These results suggest that evinacumab is effective for lowering LDL-C in patients with homozygous familial hypercholesterolemia, and the inhibition of ANGPTL3 in humans lowers LDL-C in a mechanism independent of the LDLR.

The Arg499His gain-of-function mutation in the C-terminal domain of PCSK9.

BACKGROUND AND AIMS: Familial hypercholesterolemia (FH) is a monogenic disease characterized by high levels of low-density lipoprotein cholesterol and premature atherosclerotic cardiovascular disease. FH is caused by loss of function mutations in genes encoding LDL receptor (LDLR), and Apolipoprotein B (APOB) or gain of function (GOF) mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9). In this study, we identified a novel variant in PCSK9, p.(Arg499His), located in the C-terminal domain, in two unrelated FH patients from Spain and Italy. METHODS: We studied familial segregation and determined variant activity in vitro. RESULTS: We determined PCSK9 expression, secretion and activity of the variant in transfected HEK293 cells; extracellular activity of the recombinant p.(Arg499His) PCSK9 variant in HEK 293 and HepG2 cells; PCSK9 affinity to the LDL receptor at neutral and acidic pH; the mechanism of action of the p.(Arg499His) PCSK9 variant by co-transfection with a soluble construct of the LDL receptor and by determining total PCSK9 intracellular accumulation when endosomal acidification is impaired and when an excess of soluble LDLr is present in the culture medium. Our results show high LDL-C concentrations and FH phenotype in p.(Arg499His) carriers. In vitro functional characterization shows that p.(Arg499His) PCSK9 variant causes a reduction in LDLr expression and LDL uptake. An intracellular activity for this variant is also shown when blocking the activity of secreted PCSK9 and by inhibiting endosomal acidification. CONCLUSIONS: We demonstrated that p.(Arg499His) PCSK9 variant causes a direct intracellular degradation of LDLr therefore causing FH by reducing LDLr availability.

Membrane Permeabilization by Bordetella Adenylate Cyclase Toxin Involves Pores of Tunable Size.

RTX (Repeats in ToXin) pore-forming toxins constitute an expanding family of exoproteins secreted by many Gram-negative bacteria and involved in infectious diseases caused by said pathogens. Despite the relevance in the host/pathogen interactions, the structure and characteristics of the lesions formed by these toxins remain enigmatic. Here, we capture the first direct nanoscale pictures of lytic pores formed by an RTX toxin, the Adenylate cyclase (ACT), secreted by the whooping cough bacterium Bordetella pertussis. We reveal that ACT associates into growing-size oligomers of variable stoichiometry and heterogeneous architecture (lines, arcs, and rings) that pierce the membrane, and that, depending on the incubation time and the toxin concentration, evolve into large enough "holes" so as to allow the flux of large molecular mass solutes, while vesicle integrity is preserved. We also resolve ACT assemblies of similar variable stoichiometry in the cell membrane of permeabilized target macrophages, proving that our model system recapitulates the process of ACT permeabilization in natural membranes. Based on our data we propose a non-concerted monomer insertion and sequential mechanism of toroidal pore formation by ACT. A size-tunable pore adds a new regulatory element to ACT-mediated cytotoxicity, with different pore sizes being putatively involved in different physiological scenarios or cell types.

Familial Hypercholesterolemia: The Most Frequent Cholesterol Metabolism Disorder Caused Disease.

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.

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.

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.

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.

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

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.

Analysis of LDLR variants from homozygous FH patients carrying multiple mutations in the LDLR gene.

BACKGROUND AND AIMS: Familial hypercholesterolemia (FH) is an autosomal dominant disease with widespread global prevalence that partially accounts for the high prevalence of premature coronary heart disease. Although the majority of research on FH has focused on single heterozygous LDLR mutations, there have been limited reports of double LDLR mutations on the same chromosome. The aim of this study was to gain insight into the clinical consequences of the presence of multiple mutations in the LDLR gene. METHODS: DNA from two clinical homozygous FH patients and their relatives was analysed using targeted exome sequencing and DNA resequencing. Functional characterization of novel variants was performed by Western blot, flow cytometry and confocal microscopy. RESULTS: Proband 1 carried p.Q12X, NTDA (p.N276T and c.892delA) mutations in LDLR, and Proband 2 carried c.971delG, GSDN (p.G77S + D601N). Results showed that p.Q12X, c.892delA, and c.971delG are non-functional LDLR variants. Conversely, N276T and G77S are non-pathogenic variants. Interestingly, while D601N alone only slightly diminishes LDLR activity, its co-presence with the non pathogenic p.G77S mutation results in a more strongly pathogenic variant with LDLR activity reduced by 40%. One of the double mutants, NTDA, is as non functional as c.892delA alone. The other double mutant, GSDN, is more severe than either of the component single mutants. CONCLUSIONS: An early gene screening and laboratory functional verification of LDLR activity is of vital importance to enable a definite FH diagnosis. Functional verification is also necessary for prenatal and postnatal care in patients with FH.