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Examines new research on the role of cholesterol in regulating ion channels and receptors and its effect on health Drawing together and analyzing all the latest research findings, this book explores the role of cholesterol in the regulation of ion channels and receptors, including its pathological effects. It is the first book to comprehensively describe the complex mechanisms by which cholesterol regulates two major classes of membrane proteins. Moreover, it sheds new light on how cholesterol affects essential cellular functions such as the contraction of the heart, propagation of nerve impulses, and regulation of blood pressure and kidney function. Written and edited by leading pioneers in the field, Cholesterol Regulation of Ion Channels and Receptors is divided into three parts: * Part I, Cholesterol Regulation of Membrane Properties, introduces the heterogeneity of cholesterol distribution in biological membranes and the physical and biological implications of the formation of cholesterol-rich membrane domains. * Part II, Cholesterol Regulation of Ion Channels, examines the mechanisms underlying cholesterol sensitivities of ion channels, including the regulation of ion channels by cholesterol as a boundary lipid. * Part III, Cholesterol Regulation of Receptors, explores the latest discoveries concerning how cholesterol regulates distinct types of receptors, including G-protein coupled receptors, LDL and scavenger receptors, and innate immune system receptors. Increased levels of cholesterol represent a major health risk. Understanding cholesterol regulation of ion channels and receptors is essential for facilitating the development of new therapeutic strategies to alleviate the impact of pathological cholesterol conditions. With this book as their guide, readers have access to the most current knowledge in the field.

The myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin proteolipid protein (PLP) is the most abundant protein of compact myelin in the central nervous system (CNS). The integral membrane protein PLP adheres myelin membranes together and enhances the compaction of myelin, having a fundamental role in myelin stability and axonal support. PLP is linked to severe CNS neuropathies, including inherited Pelizaeus-Merzbacher disease and spastic paraplegia type 2, as well as multiple sclerosis. Nevertheless, the structure, lipid interaction properties, and membrane organization mechanisms of PLP have remained unidentified. We expressed, purified, and structurally characterized human PLP and its shorter isoform DM20. Synchrotron radiation circular dichroism spectroscopy and small-angle X-ray and neutron scattering revealed a dimeric, α-helical conformation for both PLP and DM20 in detergent complexes, and pinpoint structural variations between the isoforms and their influence on protein function. In phosphatidylcholine membranes, reconstituted PLP and DM20 spontaneously induced formation of multilamellar myelin-like membrane assemblies. Cholesterol and sphingomyelin enhanced the membrane organization but were not crucial for membrane stacking. Electron cryomicroscopy, atomic force microscopy, and X-ray diffraction experiments for membrane-embedded PLP/DM20 illustrated effective membrane stacking and ordered organization of membrane assemblies with a repeat distance in line with CNS myelin. Our results shed light on the 3D structure of myelin PLP and DM20, their structure–function differences, as well as fundamental protein–lipid interplay in CNS compact myelin.

Dysregulation of integral membrane proteins (IMPs) has been linked to a myriad of diseases, making these proteins an attractive target in drug research. Whilst PROTAC technology has had a significant impact in scientific research, its application to IMPs is still limited. Limitations of the traditional approach of immunoblotting in PROTAC research include the low throughput compared to other methods, as well as a lack of spatial information for the target. Here we compare orthogonal antibody based approaches, i.e. immunoblotting, flow cytometry and immunofluorescence, to measure PROTAC mediated degradation of two established, endogenous targets, epidermal growth factor receptor (EGFR) and hepatocyte growth-factor receptor (c-MET). We discuss advantages and limitations of each methodology for the assessment of PROTAC efficacy on IMPs. Overall, we recommend the use of immunofluorescence and flow cytometry, for an increased accuracy with both a qualitative and quantitative insight into degradation efficacy and a critical distinction between cell membrane-localized and intracellular IMP protein pools.

A single experimental method alone often fails to provide the resolution, accuracy, and coverage needed to model integral membrane proteins (IMPs). Integrating computation with experimental data is a powerful approach to supplement missing structural information with atomic detail. We combine RosettaNMR with experimentally-derived paramagnetic NMR restraints to guide membrane protein structure prediction. We demonstrate this approach using the disulfide bond formation protein B (DsbB), an α-helical IMP. Here, we attached a cyclen-based paramagnetic lanthanide tag to an engineered non-canonical amino acid (ncAA) using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry reaction. Using this tagging strategy, we collected 203 backbone HN pseudocontact shifts (PCSs) for three different labeling sites and used these as input to guide de novo membrane protein structure prediction protocols in Rosetta. We find that this sparse PCS dataset combined with 44 long-range NOEs as restraints in our calculations improves structure prediction of DsbB by enhancements in model accuracy, sampling, and scoring. The inclusion of this PCS dataset improved the Cα-RMSD transmembrane segment values of the best-scoring and best-RMSD models from 9.57 Å and 3.06 Å (no NMR data) to 5.73 Å and 2.18 Å, respectively.

The bimolecular fluorescence complementation (BiFC) assay has been widely used to examine interactions between integral and peripheral proteins within putative plasma membrane (PM) microdomains. In the course of using BiFC assays to examine the co-localization of plasma membrane (PM) targeted receptor-like kinases (RLKs), such as FLS2, with PM micro-domain proteins such as remorins, we unexpectedly observed heterogeneous distribution patterns of fluorescence on the PM of leaf cortical cells. These patterns appeared to co-localize with the endoplasmic reticulum (ER) and with ER-PM contact sites, and closely resembled patterns caused by over-expression of the ER-PM tether protein Synaptotagmin1 (SYT1). Using domain swap experiments with SYT1, we inferred that non-specific dimerization between FLS2-VenusN and VenusC-StRem1.3 could create artificial ER-PM tether proteins analogous to SYT1. The same patterns of ER-PM tethering were produced when a representative set of integral membrane proteins were partnered in BiFC complexes with PM-targeted peripheral membrane proteins, including PtdIns(4)P-binding proteins. We inferred that spontaneous formation of mature fluorescent proteins caused the BiFC complexes to trap the integral membrane proteins in the ER during delivery to the PM, producing a PM-ER tether. This phenomenon could be a useful tool to deliberately manipulate ER-PM tethering or to test protein membrane localization. However, this study also highlights the risk of using the BiFC assay to study membrane protein interactions in plants, due to the possibility of alterations in cellular structures and membrane organization, or misinterpretation of protein-protein interactions. A number of published studies using this approach may therefore need to be revisited.

Herpes simplex virus type 1 nucleocapsids are released from the host nucleus by a budding process through the nuclear envelope called nuclear egress. Two viral proteins, the integral membrane proteins pUL34 and pUL31, form the nuclear egress complex at the inner nuclear membrane, which is critical for this process. The nuclear import of both proteins ensues separately from each other: pUL31 is actively imported through the central pore channel, while pUL34 is transported along the peripheral pore membrane. With this study, we identified a functional bipartite NLS between residues 178 and 194 of pUL34. pUL34 lacking its NLS is mislocalized to the TGN but retargeted to the ER upon insertion of the authentic NLS or a mimic NLS, independent of the insertion site. If co-expressed with pUL31, either of the pUL34-NLS variants is efficiently, although not completely, targeted to the nuclear rim where co-localization with pUL31 and membrane budding seem to occur, comparable to the wild-type. The viral mutant HSV1(17+)Lox-UL34-NLS mt is modestly attenuated but viable and associated with localization of pUL34-NLS mt to both the nuclear periphery and cytoplasm. We propose that targeting of pUL34 to the INM is facilitated by, but not dependent on, the presence of an NLS, thereby supporting NEC formation and viral replication.

α-crystallin is a major protein found in the mammalian eye lens that works as a molecular chaperone by preventing the aggregation of proteins and providing tolerance to stress in the eye lens. These functions of α-crystallin are significant for maintaining lens transparency. However, with age and cataract formation, the concentration of α-crystallin in the eye lens cytoplasm decreases with a corresponding increase in the membrane-bound α-crystallin, accompanied by increased light scattering. The purpose of this review is to summarize previous and recent findings of the role of the: (1) lens membrane components, i.e., the major phospholipids (PLs) and sphingolipids, cholesterol (Chol), cholesterol bilayer domains (CBDs), and the integral membrane proteins aquaporin-0 (AQP0; formally MIP26) and connexins, and (2) α-crystallin mutations and post-translational modifications (PTMs) in the association of α-crystallin to the eye lens’s fiber cell plasma membrane, providing thorough insights into a molecular basis of such an association. Furthermore, this review highlights the current knowledge and need for further studies to understand the fundamental molecular processes involved in the association of α-crystallin to the lens membrane, potentially leading to new avenues for preventing cataract formation and progression.

Mechanosensitive channels (MS) are integral membrane proteins and allow bacteria to survive sudden changes in external osmolarity due to transient opening of their pores. The efflux of cytoplasmic osmolytes reduces the membrane tension and prevents membrane rupture. Therefore these channels serve as emergency valves when experiencing significant environmental stress. The preparation of high quality crystals of integral membrane proteins is a major bottleneck for structure determination by X-ray crystallography. Crystallization chaperones based on various protein scaffolds have emerged as promising tool to increase the crystallization probability of a selected target protein. So far archeal mechanosensitive channels of small conductance have resisted crystallization in our hands. To structurally analyse these channels, we selected nanobodies against an archeal MS channel after immunization of a llama with recombinant expressed, detergent solubilized and purified protein. Here we present the characterization of 23 different binders regarding their interaction with the channel protein using analytical gel filtration, western blotting and surface plasmon resonance. Selected nanobodies bound the target with affinities in the pico- to nanomolar range and some binders had a profound effect on the crystallization of the MS channel. Together with previous data we show that nanobodies are a versatile and valuable tool in structural biology by widening the crystallization space for highly challenging proteins, protein complexes and integral membrane proteins.

Transmembrane proteins comprise ~30% of the mammalian proteome, mediating metabolism, signalling, transport and many other functions required for cellular life. The microenvironment of integral membrane proteins (IMPs) is intrinsically different from that of cytoplasmic proteins, with IMPs solvated by a compositionally and biophysically complex lipid matrix. These solvating lipids affect protein structure and function in a variety of ways, from stereospecific, high-affinity protein–lipid interactions to modulation by bulk membrane properties. Specific examples of functional modulation of IMPs by their solvating membranes have been reported for various transporters, channels and signal receptors; however, generalizable mechanistic principles governing IMP regulation by lipid environments are neither widely appreciated nor completely understood. Here, we review recent insights into the inter-relationships between complex lipidomes of mammalian membranes, the membrane physicochemical properties resulting from such lipid collectives, and the regulation of IMPs by either or both. The recent proliferation of high-resolution methods to study such lipid–protein interactions has led to generalizable insights, which we synthesize into a general framework termed the ‘functional paralipidome’ to understand the mutual regulation between membrane proteins and their surrounding lipid microenvironments.Transmembrane proteins associate with specific subsets of lipids, which create nano-environments with unique properties. Better understanding of how these nano-environments regulate protein dynamics and function will afford means to control activities of transmembrane proteins, many of which serve essential signalling and transport roles.

Roughly one quarter of all genes code for integral membrane proteins that are inserted into the plasma membrane of prokaryotes or the endoplasmic reticulum membrane of eukaryotes. Multiple pathways are used for the targeting and insertion of membrane proteins on the basis of their topological and biophysical characteristics. Multipass membrane proteins span the membrane multiple times and face the additional challenges of intramembrane folding. In many cases, integral membrane proteins require assembly with other proteins to form multi-subunit membrane protein complexes. Recent biochemical and structural analyses have provided considerable clarity regarding the molecular basis of membrane protein targeting and insertion, with tantalizing new insights into the poorly understood processes of multipass membrane protein biogenesis and multi-subunit protein complex assembly.Integral membrane proteins make up around one quarter of the human proteome and are highly diverse in topology, biophysical features, structure and function. Their biogenesis involves multiple pathways for membrane targeting, insertion into the lipid bilayer, folding and assembly with other subunits. Recent biochemical and structural analyses have provided new insights into these mechanisms.