Lipoprotein metabolism in familial hypercholesterolemia

Familial hypercholesterolemia (FH) is one of the most common genetic disorders in humans. It is an extremely atherogenic metabolic disorder characterized by lifelong elevations of circulating LDL-C levels often leading to premature cardiovascular events. In this review, we discuss the clinical phenotypes of heterozygous and homozygous FH, the genetic variants in four genes (LDLR/APOB/PCSK9/LDLRAP1) underpinning the FH phenotype as well as the most recent in vitro experimental approaches used to investigate molecular defects affecting the LDL receptor pathway. In addition, we review perturbations in the metabolism of lipoproteins other than LDL in FH, with a major focus on lipoprotein (a). Finally, we discuss the mode of action and efficacy of many of the currently approved hypocholesterolemic agents used to treat patients with FH, with a special emphasis on the treatment of phenotypically more severe forms of FH. © 2021 THE AUTHORS

Coating and Stabilization of Liposomes by Clathrin-Inspired DNA Self-Assembly

The self-assembly of the protein clathrin on biological membranes facilitates essential processes of endocytosis and has provided a source of inspiration for materials design by the highly ordered structural appearance. By mimicking the architecture of the protein building blocks and clathrin self-assemblies to coat liposomes with biomaterials, advanced hybrid carriers can be derived. Here we present a method for fabricating DNA-coated liposomes by hydrophobically anchoring and subsequently connecting DNA-based triskelion structures on the liposome surface inspired by the assembly of the protein clathrin. Dynamic light scattering, ζ-potential, confocal microscopy and cryo-electron microscopy measurements independently demonstrate successful DNA coating. Nanomechanical measurements conducted with atomic force microscopy show that the DNA coating enhances the mechanical stability of the liposomes relative to uncoated ones. Furthermore, we provide the possibility to reverse the coating process by triggering the disassembly of the DNA coats through a toehold-mediated displacement reaction. Our results describe a straightforward, versatile, and reversible approach for coating and stabilizing lipid vesicles through the assembly of rationally designed DNA structures. This method has potential for further development towards the ordered arrangement of tailored functionalities on the surface of liposomes and for applications as hybrid nanocarrier

Molecular Basis of ß-­arrestin Coupling to Formoterol-­Bound ß1-­adrenoceptor

The β1-adrenoceptor (β1AR) is a G-protein-coupled receptor (GPCR) that couples1 to the heterotrimeric G protein Gs. G-protein-mediated signalling is terminated by phosphorylation of the C terminus of the receptor by GPCR kinases (GRKs) and by coupling of β-arrestin 1 (βarr1, also known as arrestin 2), which displaces Gs and induces signalling through the MAP kinase pathway2. The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins-known as biased agonism3-is important in drug development, because the therapeutic effect may arise from only one signalling cascade, whereas the other pathway may mediate undesirable side effects4. To understand the molecular basis for arrestin coupling, here we determined the cryo-electron microscopy structure of the β1AR-βarr1 complex in lipid nanodiscs bound to the biased agonist formoterol5, and the crystal structure of formoterol-bound β1AR coupled to the G-protein-mimetic nanobody6 Nb80. βarr1 couples to β1AR in a manner distinct to that7 of Gs coupling to β2AR-the finger loop of βarr1 occupies a narrower cleft on the intracellular surface, and is closer to transmembrane helix H7 of the receptor when compared with the C-terminal α5 helix of Gs. The conformation of the finger loop in βarr1 is different from that adopted by the finger loop of visual arrestin when it couples to rhodopsin8. β1AR coupled to βarr1 shows considerable differences in structure compared with β1AR coupled to Nb80, including an inward movement of extracellular loop 3 and the cytoplasmic ends of H5 and H6. We observe weakened interactions between formoterol and two serine residues in H5 at the orthosteric binding site of β1AR, and find that formoterol has a lower affinity for the β1AR-βarr1 complex than for the β1AR-Gs complex. The structural differences between these complexes of β1AR provide a foundation for the design of small molecules that could bias signalling in the β-adrenoceptors

The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

The neuronal calcium sensor 1 (NCS-1), an EF-hand Ca binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Gα have revealed how Ric-8A phosphorylation promotes Gα recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Gα subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca signals and phosphorylation events that promote the interaction of Ric-8A with Gα. Our data show that the binding of NCS-1 and Gα to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to casein kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A guanine nucleotide exchange factor (GEF) activity toward Gα when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity

A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated Pits

Multiple proteins act co-operatively in mammalian clathrin-mediated endocytosis (CME) to generate endocytic vesicles from the plasma membrane. The principles controlling the activation and organization of the actin cytoskeleton during mammalian CME are, however, not fully understood. Here, we show that the protein FCHSD2 is a major activator of actin polymerization during CME. FCHSD2 deletion leads to decreased ligand uptake caused by slowed pit maturation. FCHSD2 is recruited to endocytic pits by the scaffold protein intersectin via an unusual SH3-SH3 interaction. Here, its flat F-BAR domain binds to the planar region of the plasma membrane surrounding the developing pit forming an annulus. When bound to the membrane, FCHSD2 activates actin polymerization by a mechanism that combines oligomerization and recruitment of N-WASP to PI(4,5)P2, thus promoting pit maturation. Our data therefore describe a molecular mechanism for linking spatiotemporally the plasma membrane to a force-generating actin platform guiding endocytic vesicle maturation

In vivo DNA assembly using common laboratory bacteria: A re-emerging tool to simplify molecular cloning

12 pags., 5 figs.Molecular cloning is a cornerstone of biomedical, biotechnological, and synthetic biology research. As such, improved cloning methodologies can significantly advance the speed and cost of research projects. Whereas current popular cloning approaches use in vitro assembly of DNA fragments, in vivo cloning offers potential for greater simplification. It is generally assumed that bacterial in vivo cloning requires Escherichia coli strains with enhanced recombination ability; however, this is incorrect. A widely present, bacterial RecA-independent recombination pathway is re-emerging as a powerful tool for molecular cloning and DNA assembly. This poorly understood pathway offers optimal cloning properties (i.e. seamless, directional, and sequence-independent) without requiring in vitro DNA assembly or specialized bacteria, therefore vastly simplifying cloning procedures. Although the use of this pathway to perform DNA assembly was first reported over 25 years ago, it failed to gain popularity, possibly due to both technical and circumstantial reasons. Technical limitations have now been overcome, and recent reports have demonstrated its versatility for DNA manipulation. Here, we summarize the historical trajectory of this approach and collate recent reports to provide a roadmap for its optimal use. Given the simplified protocols and minimal requirements, cloning using in vivo DNA assembly in E. coli has the potential to become widely employed across the molecular biology community.This work is supported by Medical Research Council Grant MC_U105174197 (to J. F. W.); Ministerio de Ciencia, Innovación y Universidades Grant RTI2018-095629-J-I00 (to J. G.-N.); and the Fondo Europeo de Desarrollo Regional (FEDER) (to J. G.-N.)

Algunos Aphidini de la provincia de León (Homoptera, Aphididae)

[ES] Citamos 33 especies de Aphidini de la provincia de León, todas ellas nuevas citas para la misma, y una : Aphis acetosae, nueva cita para la Península Ibérica. Damos además como nuevas plantas hospedadoras para Aphis lichtensteini, A. origani y A. serpylli a Halimium alyssoides, Origanum virens y Thymus mastichina, respectivamente.[FR] On cite 33 espéces d'Aphidini de la Province de León (N.O. de l'Espagne), touttes lesquelles sont nouvelles cites pour la méme et une : Aphis acetosae, nouvelle cite pour la Péninsule Ibérique. Nous apportons aussi trois nouvelles plantes-h6tes : Halintium alyssoides, Origanum virens et Thymus mastichina pour Aphislichtensteini, A. origani et A. serylli, respectivement.Peer reviewe

The structure of Bacillus subtilis SPβ prophage dUTPase and its complexes with two nucleotides.

dUTPases are housekeeping enzymes which catalyse the hydrolysis of dUTP to dUMP in an ion-dependent manner. Bacillus subtilis has both a genomic and an SPβ prophage homotrimeric dUTPase. Here, structure determination of the prophage apoenzyme and of its complexes with dUDP and dUpNHpp-Mg(2+) is described at 1.75, 1.9 and 2.55 Å resolution, respectively. The C-terminal extension, which carries the conserved motif V, is disordered in all three structures. Unlike all other trimeric dUTPases for which structures are available, with the exception of the Bacillus genomic enzyme, the aromatic residue covering the uridine and acting as the Phe-lid is close to motif III in the sequence rather than in motif V. This is in spite of the presence of an aromatic amino acid at the usual Phe-lid position in motif V. The alternative position of the Phe-lid requires a reconsideration of its role in the catalytic cycle of the enzyme. In the dUpNHpp-Mg(2+) complex a water can be seen at the position expected for nucleophilic attack on the α-phosphate, in spite of motif V being disordered. Differences in the active site between the free enzyme and the dUDP and dUpNHpp-Mg(2+) complexes shows that the triphosphate moiety needs to be in the gauche conformation to trigger the conformational changes that can be seen in both B. subtilis dUTPases