Solubilization of membranes by detergents

The renin-angiotensin system is a central component of the physiological and pathological responses of cardiovascular system. Its primary effector hormone, angiotensin II (ANG II), not only mediates immediate physiological effects of vasoconstriction and blood pressure regulation, but is also implicated in inflammation, endothelial dysfunction, atherosclerosis, hypertension, and congestive heart failure. The myriad effects of ANG II depend on time (acute vs. chronic) and on the cells/tissues upon which it acts. In addition to inducing G protein- and non-G protein-related signaling pathways, ANG II, via AT(1) receptors, carries out its functions via MAP kinases (ERK 1/2, JNK, p38MAPK), receptor tyrosine kinases [PDGF, EGFR, insulin receptor], and nonreceptor tyrosine kinases [Src, JAK/STAT, focal adhesion kinase (FAK)]. AT(1)R-mediated NAD(P)H oxidase activation leads to generation of reactive oxygen species, widely implicated in vascular inflammation and fibrosis. ANG II also promotes the association of scaffolding proteins, such as paxillin, talin, and p130Cas, leading to focal adhesion and extracellular matrix formation. These signaling cascades lead to contraction, smooth muscle cell growth, hypertrophy, and cell migration, events that contribute to normal vascular function, and to disease progression. This review focuses on the structure and function of AT(1) receptors and the major signaling mechanisms by which angiotensin influences cardiovascular physiology and pathology.

Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system

Although it was thought that apoptotic cells, when rapidly phagocytosed, underwent a silent death that did not trigger an immune response, in recent years a new concept of immunogenic cell death (ICD) has emerged. The immunogenic characteristics of ICD are mainly mediated by damage-associated molecular patterns (DAMPs), which include surface-exposed calreticulin (CRT), secreted ATP and released high mobility group protein B1 (HMGB1). Most DAMPs can be recognized by pattern recognition receptors (PRRs). In this Review, we discuss the role of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) in regulating the immunogenicity of dying cancer cells and the effect of therapy-resistant cancer microevolution on ICD.

Immunogenic cell death and DAMPs in cancer therapy.

Zinc coordination, function, and structure of zinc enzymes and other proteins

Protein–protein interactions have a key role in most biological processes, and offer attractive opportunities for therapeutic intervention. Developing small molecules that modulate protein–protein interactions is difficult, owing to issues such as the lack of well-defined binding pockets. Nevertheless, there has been important progress in this endeavour in recent years. Here, we use illustrative examples to discuss general strategies for addressing the challenges inherent in the discovery and characterization of small-molecule inhibitors of protein–protein interactions.

Small-molecule inhibitors of protein–protein interactions: progressing towards the dream

Membrane formation by the adenosine triphosphatase of sarcoplasmic reticulum.

The photoreactive insulin derivatives N epsilon B29-(azidobenzoyl)insulin (MAB-insulin) and N alpha A1, N epsilon B29-di(azidobenzoyl)insulin (DAB-insulin) were synthesized by reacting bovine insulin with the N-hydroxysuccinimide ester of 4-azidobenzoic acid. These derivatives were purified by ion exchange chromatography on SP-Sephadex, and their identities were established by polyacrylamide gel electrophoresis, amino acid analysis, and end-group determination. Their biological activities were measured by receptor binding assay and fat cell assay. The photoreactivity of these two derivatives was demonstrated by spectral changes and by the formation of covalent polymers of high molecular weight when exposed to light. Radioactive MAB-insulin and DAB-insulin were prepared by iodination with [125I]iodine. These radioactive derivatives were characterized for their photoreactivity, immunoreactivity, and receptor binding to liver plasma membrane. Liver plasma membrane preparations of rat, mouse, and guinea pig were incubated with these radioactive insulin derivatives and irradiated with light. Sodium dodecyl sulfate gel electrophoresis of these plasma membrane preparations after solubilization and reduction showed that two proteins were specifically labeled. The molecular weights of the two radioactive bands were estimated to be about 130 000 and 90 000 in all three species of animals.

Photoaffinity labeling of insulin receptor proteins of liver plasma membrane preparations.

Isolation of Sarcoplasmic Reticulum Proteins

Transmembrane signaling via receptor tyrosine kinases generally requires oligomerization of receptor monomers, with the formation of ligand-induced dimers or higher multimers of the extracellular domains of the receptors. Such formations are expected to juxtapose the intracellular kinase domains at the correct distances and orientations for transphosphorylation. For receptors of the insulin receptor family that are constitutively dimeric, or those that form noncovalent dimers without ligands, the mechanism must be more complex. For these, the conformation must be changed by the ligand from one that prevents activation to one that is permissive for kinase phosphorylation. How the insulin ligand accomplishes this action has remained a puzzle since the discovery of the insulin receptor over 2 decades ago, primarily because membrane proteins in general have been refractory to structure determination by crystallography. However, high-resolution structural evidence on individual separate subdomains of the insulin receptor and of analogous proteins has been obtained. The recently solved quaternary structure of the complete dimeric insulin receptor in the presence of insulin has now served as the structural envelope into which such individual domains were fitted. The combined structure has provided answers on the details of insulin/receptor interactions in the binding site and on the mechanism of transmembrane signaling of this covalent dimer. The structure explains many observations on the behavior of the receptor, from greater or lesser binding of insulin and its variants, point and deletion mutants of the receptor, to antibody-binding patterns, and to the effects on basal and insulin-stimulated autophosphorylation under mild reducing conditions.

Mechanism of Transmembrane Signaling: Insulin Binding and the Insulin Receptor†

The three-dimensional (3D) structure of the intrinsically dimeric insulin receptor bound to its ligand, insulin, was determined by electron cryomicroscopy. Gold-labeled insulin served to locate the insulin-binding domain. The 3D structure was then fitted with available known high-resolution domain substructures to obtain a detailed contiguous model for this heterotetrameric transmembrane receptor. The 3D reconstruction indicates that the two alpha subunits jointly participate in insulin binding and that the kinase domains in the two beta subunits are in a juxtaposition that permits autophosphorylation of tyrosine residues in the first step of insulin receptor activation.

https://science.sciencemag.org/content/sci/285/5430/1077.full.pdf

Cecil C. Yip

Papers

Membrane formation by the adenosine triphosphatase of sarcoplasmic reticulum.

Photoaffinity labeling of insulin receptor proteins of liver plasma membrane preparations.

Isolation of Sarcoplasmic Reticulum Proteins

Mechanism of Transmembrane Signaling: Insulin Binding and the Insulin Receptor†

Quaternary structure of the insulin-insulin receptor complex.