Specialized Membrane Microdomains Research Articles

Unraveling the Cave: A Seventy-Year Journey into the Caveolar Network, Cellular Signaling, and Human Disease.

In the mid-1950s, a groundbreaking discovery revealed the fascinating presence of caveolae, referred to as flask-shaped invaginations of the plasma membrane, sparking renewed excitement in the field of cell biology. Caveolae are small, flask-shaped invaginations in the cell membrane that play crucial roles in diverse cellular processes, including endocytosis, lipid homeostasis, and signal transduction. The structural stability and functionality of these specialized membrane microdomains are attributed to the coordinated activity of scaffolding proteins, including caveolins and cavins. While caveolae and caveolins have been long appreciated for their integral roles in cellular physiology, the accumulating scientific evidence throughout the years reaffirms their association with a broad spectrum of human disorders. This review article aims to offer a thorough account of the historical advancements in caveolae research, spanning from their initial discovery to the recognition of caveolin family proteins and their intricate contributions to cellular functions. Furthermore, it will examine the consequences of a dysfunctional caveolar network in the development of human diseases.

Structural Basis of the Interaction of the G Proteins, Gαi1, Gβ1γ2 and Gαi1β1γ2, with Membrane Microdomains and Their Relationship to Cell Localization and Activity.

GPCRs receive signals from diverse messengers and activate G proteins that regulate downstream signaling effectors. Efficient signaling is achieved through the organization of these proteins in membranes. Thus, protein-lipid interactions play a critical role in bringing G proteins together in specific membrane microdomains with signaling partners. Significantly, the molecular basis underlying the membrane distribution of each G protein isoform, fundamental to fully understanding subsequent cell signaling, remains largely unclear. We used model membranes with lipid composition resembling different membrane microdomains, and monomeric, dimeric and trimeric Gi proteins with or without single and multiple mutations to investigate the structural bases of G protein-membrane interactions. We demonstrated that cationic amino acids in the N-terminal region of the Gαi1 and C-terminal region of the Gγ2 subunit, as well as their myristoyl, palmitoyl and geranylgeranyl moieties, define the differential G protein form interactions with membranes containing different lipid classes (PC, PS, PE, SM, Cho) and the various microdomains they may form (Lo, Ld, PC bilayer, charged, etc.). These new findings in part explain the molecular basis underlying amphitropic protein translocation to membranes and localization to different membrane microdomains and the role of these interactions in cell signal propagation, pathophysiology and therapies targeted to lipid membranes.

833 Reintroduction of caveolin-1 scaffolding domain improves pathogenesis of psoriasiform dermatitis

Psoriasis is a chronic, inflammatory skin disease characterized by well-defined, erythematous plaques with silvery scale secondary to aberrant keratinocyte proliferation. We have previously identified that a structural membrane protein caveolin-1 (Cav1) is involved in regulation of aberrant keratinocyte proliferation and differentiation, and thus the aim of this study was to elucidate the role of Cav1 in pathogenesis of psoriasis vulgaris. We utilized spatial genomic & proteomic analyses combined with immunohistochemistry (IHC) with patient samples and mouse models. Omics analyses identified caveolae and their structural components (Cav1, -2 and cavin-1) as downregulated in imiquimod (IMQ)-treated mouse model of psoriasis. These observations were validated in patient samples of multiple types of psoriasis stratified by PASI score (plaque, inverse, guttate and nail) by IHC staining and quantified by QuPath image analysis software. Although all biopsy samples exhibited an inverse relationship with levels of Cav1, plaque psoriasis samples were the most pronounced. To assess whether reintroduction of Cav1 can alleviate some of the features associated with IMQ-induced model of psoriasis (increased skin thickness/scaling and upregulation of psoriasis associated markers), C57BL6 and K14cre-Cav1 knockout mice were treated with soluble caveolin scaffolding domain (CSD) peptide, in presence or absence of IMQ. We observed that Cav1ko mice exhibited a trend in increased skin thickness and expression of CD11b, CD14, IL-6, IL-12, IL-17a and TNFa, although to a lesser than IMQ-treated C57BL6 mice (Cav1 wild type). Moreover, topical CSD treatment decreased both skin thickness and scaling, as well as dampened upregulation of aforementioned inflammatory markers. Together, these data support the hypothesis that Cav1 is major pathophysiological target in psoriasis and introduce different approach to localized topical treatment of psoriasis by targeting structural components of the specialized membrane microdomains, such as caveolae.

730 Elevated levels of caveolin-1 in frontal fibrosing alopecia, central centrifugal cicatricial alopecia and lichen planopilaris suggests a conserved pathway in pathogenesis of scarring alopecia

Cicatricial alopecias (CAs) are a disfiguring and difficult to treat types of hair disorder that are associated with distressing scalp symptoms and significant secondary morbidity, marked psychological impact, and significant loss of quality of life. The purpose of our study was to generate a proof-of-principle for a fundamentally innovative strategy for treating CAs by therapeutically targeting caveolin-1 (Cav1), which is the principal structural component of specialized membrane microdomains, using cholesterol depleting agents (cyclodextrins), in full length human ex vivo human hair follicle (HF) organ culture and in vivo mouse models.

Endothelial Cell Behavior Is Determined by Receptor Clustering Induced by Thrombospondin-1.

The thrombospondins (TSPs) are a family of multimeric extracellular matrix proteins that dynamically regulate cellular behavior and response to stimuli. In so doing, the TSPs directly and indirectly affect biological processes such as embryonic development, wound healing, immune response, angiogenesis, and cancer progression. Many of the direct effects of Thrombospondin 1 (TSP-1) result from the engagement of a wide range of cell surface receptors including syndecans, low density lipoprotein receptor-related protein 1 (LRP1), CD36, integrins, and CD47. Different or even opposing outcomes of TSP-1 actions in certain pathologic contexts may occur, depending on the structural/functional domain involved. To expedite response to external stimuli, these receptors, along with vascular endothelial growth factor receptor 2 (VEGFR2) and Src family kinases, are present in specific membrane microdomains, such as lipid rafts or tetraspanin-enriched microdomains. The molecular organization of these membrane microdomains and their constituents is modulated by TSP-1. In this review, we will describe how the presence of TSP-1 at the plasma membrane affects endothelial cell signal transduction and angiogenesis.

A phase I/II, open-label, dose-escalation, and cohort-expansion study evaluating the safety, pharmacokinetics, and therapeutic activity of OBI-999 in patients with advanced solid tumors.

TPS3657 Background: Aberrant glycosylation is a hallmark of cancer. Glycosphingolipids (GSLs), glycans conjugated to a lipid (ceramide) core, are essential for the recruitment of immune-related proteins to specific membrane microdomains. Globo H (GH) is a GSL found on normal cells but highly overexpressed on various epithelial tumors playing a role in tumor development and progression. GH is a promising target for immunotherapy. OBI-999 is an ADC composed of a human recombinant immunoglobulin G (IgG) monoclonal antibody that selectively and specifically binds to GH, attached by a linker to the antimitotic agent monomethyl auristatin E (MMAE). Its mechanism of action is based on tumor-selective delivery of MMAE to GH-expressing tumors with subsequent tumor cell death. Preclinical studies demonstrated that OBI-999 antibody binds specifically to the GH antigen, and antitumor efficacy was noted in breast, gastric, pancreatic, and lung cancer xenograft models. The pharmacokinetics (PK) of OBI-999 were determined in normal and tumor-bearing mice, rats, and monkeys. Exposure of OBI-999 increased proportionally with dose. No sex difference or accumulation was seen. The primary objectives are to determine dose limiting toxicities (DLTs), the maximum tolerated dose (MTD), and the phase 2 recommended dose (P2RD). The secondary objectives are to assess the rates of objective response and clinical benefit, the duration of progression-free survival, the immunogenicity of OBI-999, and the PK and pharmacodynamics (PD) of OBI-999 and MMAE. Methods: In Part 1, a "3+3" dose-escalation part of the study, up to 30 patients with advanced solid tumors refractory to ≥1 line of systemic therapy, who cannot tolerate standard therapy, or for whom no standard treatment is available, regardless of GH status will be treated. OBI-999 will be administered as a 60-minute IV infusion using a dose range of 0.4, 0.8, 1.2, 1.6, and 2.0 mg/kg on day 1 of every 21-day cycle. In Part 2, the cohort-expansion portion of the study, patients will be treated at the MTD or at a lower RP2D as determined by cumulative toxicities and tolerability profile. The study will determine the preliminary clinical activity and safety of OBI-999 in up to 155 patients with advanced solid tumors, pancreatic, gastric, esophageal, and colorectal cancer according to a Simon two-stage phase 2 design. Patients must have GH overexpression defined as an H-score of ≥100 according to an FDA Investigational Device Exempt (IDE) validated IHC assay. Clinical trial information: NCT04084366 .

The clustering of voltage-gated sodium channels in various excitable membranes.

In excitable membranes, the clustering of voltage-gated sodium channels (VGSC) serves to enhance excitability at critical sites. The two most profoundly studied sites of channel clustering are the axon initial segment, where action potentials are generated and the node of Ranvier, where action potentials propagate along myelinated axons. The clustering of VGSC is found, however, in other highly excitable sites such as axonal terminals, postsynaptic membranes of dendrites and muscle fibers, and pre-myelinated axons. In this review, different examples of axonal as well as non-axonal clustering of VGSC are discussed and the underlying mechanisms are compared. Whether the clustering of channels is intrinsically or extrinsically induced, it depends on the submembranous actin-based cytoskeleton that organizes these highly specialized membrane microdomains through specific adaptor proteins.

Quantification of phosphoinositides reveals strong enrichment of PIP2 in HIV-1 compared to producer cell membranes

Human immunodeficiency virus type 1 (HIV-1) acquires its lipid envelope during budding from the plasma membrane of the host cell. Various studies indicated that HIV-1 membranes differ from producer cell plasma membranes, suggesting budding from specialized membrane microdomains. The phosphoinositide PI(4,5)P2 has been of particular interest since PI(4,5)P2 is needed to recruit the viral structural polyprotein Gag to the plasma membrane and thus facilitates viral morphogenesis. While there is evidence for an enrichment of PIP2 in HIV-1, fully quantitative analysis of all phosphoinositides remains technically challenging and therefore has not been reported, yet. Here, we present a comprehensive analysis of the lipid content of HIV-1 and of plasma membranes from infected and non-infected producer cells, resulting in a total of 478 quantified lipid compounds, including molecular species distribution of 25 different lipid classes. Quantitative analyses of phosphoinositides revealed strong enrichment of PIP2, but also of PIP3, in the viral compared to the producer cell plasma membrane. We calculated an average of ca. 8,000 PIP2 molecules per HIV-1 particle, three times more than Gag. We speculate that the high density of PIP2 at the HIV-1 assembly site is mediated by transient interactions with viral Gag polyproteins, facilitating PIP2 concentration in this microdomain. These results are consistent with our previous observation that PIP2 is not only required for recruiting, but also for stably maintaining Gag at the plasma membrane. We believe that this quantitative analysis of the molecular anatomy of the HIV-1 lipid envelope may serve as standard reference for future investigations.

Annexins in Adipose Tissue: Novel Players in Obesity.

Obesity and the associated comorbidities are a growing health threat worldwide. Adipose tissue dysfunction, impaired adipokine activity, and inflammation are central to metabolic diseases related to obesity. In particular, the excess storage of lipids in adipose tissues disturbs cellular homeostasis. Amongst others, organelle function and cell signaling, often related to the altered composition of specialized membrane microdomains (lipid rafts), are affected. Within this context, the conserved family of annexins are well known to associate with membranes in a calcium (Ca2+)- and phospholipid-dependent manner in order to regulate membrane-related events, such as trafficking in endo- and exocytosis and membrane microdomain organization. These multiple activities of annexins are facilitated through their diverse interactions with a plethora of lipids and proteins, often in different cellular locations and with consequences for the activity of receptors, transporters, metabolic enzymes, and signaling complexes. While increasing evidence points at the function of annexins in lipid homeostasis and cell metabolism in various cells and organs, their role in adipose tissue, obesity and related metabolic diseases is still not well understood. Annexin A1 (AnxA1) is a potent pro-resolving mediator affecting the regulation of body weight and metabolic health. Relevant for glucose metabolism and fatty acid uptake in adipose tissue, several studies suggest AnxA2 to contribute to coordinate glucose transporter type 4 (GLUT4) translocation and to associate with the fatty acid transporter CD36. On the other hand, AnxA6 has been linked to the control of adipocyte lipolysis and adiponectin release. In addition, several other annexins are expressed in fat tissues, yet their roles in adipocytes are less well examined. The current review article summarizes studies on the expression of annexins in adipocytes and in obesity. Research efforts investigating the potential role of annexins in fat tissue relevant to health and metabolic disease are discussed.

Cholesterol Depletion Regulates Axonal Growth and Enhances Central and Peripheral Nerve Regeneration.

Axonal growth during normal development and axonal regeneration rely on the action of many receptor signaling systems and complexes, most of them located in specialized raft membrane microdomains with a precise lipid composition. Cholesterol is a component of membrane rafts and the integrity of these structures depends on the concentrations present of this compound. Here we explored the effect of cholesterol depletion in both developing neurons and regenerating axons. First, we show that cholesterol depletion in vitro in developing neurons from the central and peripheral nervous systems increases the size of growth cones, the density of filopodium-like structures and the number of neurite branching points. Next, we demonstrate that cholesterol depletion enhances axonal regeneration after axotomy in vitro both in a microfluidic system using dissociated hippocampal neurons and in a slice-coculture organotypic model of axotomy and regeneration. Finally, using axotomy experiments in the sciatic nerve, we also show that cholesterol depletion favors axonal regeneration in vivo. Importantly, the enhanced regeneration observed in peripheral axons also correlated with earlier electrophysiological responses, thereby indicating functional recovery following the regeneration. Taken together, our results suggest that cholesterol depletion per se is able to promote axonal growth in developing axons and to increase axonal regeneration in vitro and in vivo both in the central and peripheral nervous systems.

Tetraspanins, Another Piece in the HIV-1 Replication Puzzle.

Despite the great research effort placed during the last decades in HIV-1 study, still some aspects of its replication cycle remain unknown. All this powerful research has succeeded in developing different drugs for AIDS treatment, but none of them can completely remove the virus from infected patients, who require life-long medication. The classical approach was focused on the study of virus particles as the main target, but increasing evidence highlights the importance of host cell proteins in HIV-1 cycle. In this context, tetraspanins have emerged as critical players in different steps of the viral infection cycle. Through their association with other molecules, including membrane receptors, cytoskeletal proteins, and signaling molecules, tetraspanins organize specialized membrane microdomains called tetraspanin-enriched microdomains (TEMs). Within these microdomains, several tetraspanins have been described to regulate HIV-1 entry, assembly, and transfer between cells. Interestingly, the importance of tetraspanins CD81 and CD63 in the early steps of viral replication has been recently pointed out. Indeed, CD81 can control the turnover of the HIV-1 restriction factor SAMHD1. This deoxynucleoside triphosphate triphosphohydrolase counteracts HIV-1 reverse transcription (RT) in resting cells via its dual function as dNTPase, catalyzing deoxynucleotide triphosphates into deoxynucleosides and inorganic triphosphate, and as exonuclease able to degrade single-stranded RNAs. SAMHD1 has also been related with the detection of viral nucleic acids, regulating the innate immune response and would promote viral latency. New evidences demonstrating the ability of CD81 to control SAMHD1 expression, and as a consequence, HIV-1 RT activity, highlight the importance of TEMs for viral replication. Here, we will briefly review how tetraspanins modulate HIV-1 infection, focusing on the latest findings that link TEMs to viral replication.

The brain lipidome in neurodegenerative lysosomal storage disorders

Cholesterol, sphingolipids and glycerophospholipids are critical constituents of the brain, subserving neuronal membrane architecture and providing a platform for biochemical processes essential for proper neurodevelopment and function. When lysosomal defects arise in a lipid metabolic pathway, it is therefore easy to imagine that neurological decline will transpire, however for deficits in non-lipid pathways, this becomes harder to envisage. Here we suggest the working hypothesis that neurodegenerative lysosomal storage disorders might manifest as primary and/or secondary disorders of lipid metabolism. Evidence suggests that the mere process of lysosomal substrate accumulation, ubiquitous in all lysosomal storage disorders, impairs lysosome integrity resulting in secondary lipid accumulation. Impaired lysosomal degradation of a specific lipid defines a primary disorder of lipid metabolism and as these lysosomal storage disorders additionally show secondary lipid alterations, all primary disorders can also be considered secondary disorders of lipid metabolism. The outcome is a generalized cellular lipid dyshomeostasis and consequently, the physiological architecture of the lipid-enriched plasma membrane is perturbed, including the lipid composition of specialized membrane microdomains, often termed lipid rafts. Neurotoxicity results from the complex interplay of malfunctioning signaling and vesicular trafficking important for neuronal communication and synaptic plasticity-induced by the imbalance in physiological membrane lipid composition - together with compensatory mechanisms aimed at restoring lipid homeostasis.

Isolation and manipulation of protoplasts from the unicellular green alga Penium margaritaceum

BackgroundThe unicellular charophycean green alga Penium margaritaceum has emerged as an appealing experimental organism in plant cell wall and cell biology research. Innovative practical approaches in the manipulation and maintenance of this unicellular model alga are needed in order to probe the complexities of its subcellular and molecular machinery. Protoplast isolation and manipulation expedites a new range of experimental possibilities for Penium-based studies. These include enhanced means of isolation of subcellular components and macromolecules, application of intracellular probes for high resolution microscopy of live cells, transformation studies and analysis of the fundamental mechanisms of plant cell expansion and wall polymer deposition.ResultsWe present a methodology for enzyme-based digestion of the Penium cell wall and the isolation of protoplasts. The subcellular events associated with this technology are presented using multiple microscopy-based techniques. We also provide protocols for applying an array of intracellular dyes that can be used as markers for specific organelles and membrane microdomains in live cells. Finally, we present a protocol for the purification of a nuclei-rich fraction from protoplasts, which can be used for the isolation of nuclear DNA.ConclusionProtoplast isolation, culturing and manipulation provide valuable means for molecular and cellular studies of Penium. The protocol described here offers a rapid and effective mechanism for fast and effective production of protoplasts. Subsequently, the protoplasts may be used for microscopy-based studies of specific subcellular components and the isolation of organelles and nuclear DNA. These methods offer a new practical methodology for future studies of this model organism in cell and molecular biology.

Retinal de novo lipogenesis coordinates neurotrophic signaling to maintain vision.

Membrane lipid composition is central to the highly specialized functions of neurological tissues. In the retina, abnormal lipid metabolism causes severe forms of blindness, often through poorly understood neuronal cell death. Here, we demonstrate that deleting the de novo lipogenic enzyme fatty acid synthase (FAS) from the neural retina, but not the vascular retina, results in progressive neurodegeneration and blindness with a temporal pattern resembling rodent models of retinitis pigmentosa. Blindness was not rescued by protection from light-evoked activity; by eating a diet enriched in palmitate, the product of the FAS reaction; or by treatment with the PPARα agonist fenofibrate. Vision loss was due to aberrant synaptic structure, blunted responsiveness to glial-derived neurotrophic factor and ciliary neurotrophic factor, and eventual apoptotic cell loss. This progressive neurodegeneration was associated with decreased membrane cholesterol content, as well as loss of discrete n-3 polyunsaturated fatty acid– and saturated fatty acid–containing phospholipid species within specialized membrane microdomains. Neurotrophic signaling was restored by exogenous cholesterol delivery. These findings implicate de novo lipogenesis in neurotrophin-dependent cell survival by maintaining retinal membrane configuration and lipid composition, and they suggest that ongoing lipogenesis may be required to prevent cell death in many forms of retinopathy.

Lipid raft localization of TLR2 and its co-receptors is independent of membrane lipid composition.

BackgroundToll like receptors (TLRs) are an important and evolutionary conserved class of pattern recognition receptors associated with innate immunity. The recognition of Gram-positive cell wall constituents strongly depends on TLR2. In order to be functional, TLR2 predominantly forms a heterodimer with TLR1 or TLR6 within specialized membrane microdomains, the lipid rafts. The membrane lipid composition and the physicochemical properties of lipid rafts are subject to modification by exogenous fatty acids. Previous investigations of our group provide evidence that macrophage enrichment with polyunsaturated fatty acids (PUFA) induces a reordering of lipid rafts and non-rafts based on the incorporation of supplemented PUFA as well as their elongation and desaturation products.MethodsIn the present study we investigated potential constraining effects of membrane microdomain reorganization on the clustering of TLR2 with its co-receptors TLR1 and TLR6 within lipid rafts. To this end, RAW264.7 macrophages were supplemented with either docosahexaenoic acid (DHA) or arachidonic acid (AA) and analyzed for receptor expression and microdomain localization in context of TLR stimulation.Results and ConclusionsOur analyses showed that receptor levels and microdomain localization were unchanged by PUFA supplementation. The TLR2 pathway, in contrast to the TLR4 signaling cascade, is not affected by exogenous PUFA at the membrane level.

The PMCA pumps in genetically determined neuronal pathologies.

Ca2+ signals regulate most aspects of animal cell life. They are of particular importance to the nervous system, in which they regulate specific functions, from neuronal development to synaptic plasticity. The homeostasis of cell Ca2+ must thus be very precisely regulated: in all cells Ca2+ pumps transport it from the cytosol to the extracellular medium (the Plasma Membrane Ca2+ ATPases, hereafter referred to as PMCA pumps) or to the lumen of intracellular organelles (the Sarco/Endoplasmatic Reticulum Ca2+ ATPase and the Secretory Pathway Ca2+ ATPase, hereafter referred to as SERCA and SPCA pumps, respectively). In neurons and other excitable cells a powerful plasma membrane Na+/Ca2+ exchanger (NCX) also exports Ca2+ from cells. Quantitatively, the PMCA pumps are of minor importance to the bulk regulation of neuronal Ca2+. However, they are important in the regulation of Ca2+ in specific sub-plasma membrane microdomains which contain a number of enzymes that are relevant to neuronal function. The PMCA pumps (of which 4 basic isoforms are expressed in animal cells) are P-type ATPases that are characterized by a long C-terminal cytosolic tail which is the site of interaction with most of the regulatory factors of the pump, the most important being calmodulin. In resting neurons, at low intracellular Ca2+the C-terminal tail of the PMCA interacts with the main body of the protein keeping it in an autoinhibited state. Local Ca2+ increase activates calmodulin that removes the C-terminal tail from the inhibitory sites. Dysregulation of the Ca2+ signals are incompatible with healthy neuronal life. A number of genetic mutations of PMCA pumps are associated with pathological phenotypes, those of the neuron-specific PMCA 2 and PMCA 3 being the best characterized. PMCA 2 mutations are associated with deafness and PMCA 3 mutations are linked to cerebellar ataxias. Biochemical analysis of the mutated pumps overexpressed in model cells have revealed their decreased ability to export Ca2+. The defect in the bulk cytosolic Ca2+ homeostasis is minor, in keeping with the role of the PMCA pumps in the local control of Ca2+ in specialized plasma membrane microdomains.

Novel Approaches for HDL-Directed Therapies.

High-density lipoproteins (HDL) are thought to exert a protective role against atherosclerosis. The measurement of the cholesterol mass within HDL (HDL-C) represents a good biomarker of cardiovascular health, but HDL-C appears to be a poor therapeutic target. Here, we discuss new targets for the development of HDL-directed therapies. Among cardio-protective functions of HDL particles, the ability of HDL to remove cholesterol from cells involved in the early stages of atherosclerosis is considered one of the most important functions. This process, termed "HDL biogenesis," is initiated by the formation of highly specialized plasma membrane micro-domains by the ATP-binding cassette transporter A1 (ABCA1) and the binding of apolipoproteins (apo) such as apoA-I, the major protein moiety of HDL, to the micro-domains. Although early strategies aimed at increasing HDL biogenesis by upregulating ABCA1 or apoA-I gene expression have not met with clinical success, recent advances in understanding transcriptional, post-transcriptional, and post-translational regulatory pathways propose new targets for the promotion of HDL biogenesis. We have recently reported that a novel apoA-I-binding protein desmocollin 1 (DSC1) prevents HDL biogenesis and that inhibition of apoA-I-DSC1 interactions promotes HDL biogenesis by stabilizing ABCA1. This new HDL regulation pathway nominates DSC1 as an attractive pharmacological target. In the absence of clinically useful therapy to increase HDL biogenesis, finding novel targets to unlock the therapeutic potential of HDL is highly desired. Modulation of apoA-I-DSC1 interactions may be a viable strategy.

Autoinhibition of ankyrin-B/G membrane target bindings by intrinsically disordered segments from the tail regions.

Ankyrins together with their spectrin partners are the master organizers of micron-scale membrane domains in diverse tissues. The 24 ankyrin (ANK) repeats of ankyrins bind to numerous membrane proteins, linking them to spectrin-based cytoskeletons at specific membrane microdomains. The accessibility of the target binding groove of ANK repeats must be regulated to achieve spatially defined functions of ankyrins/target complexes in different tissues, though little is known in this regard. Here we systemically investigated the autoinhibition mechanism of ankyrin-B/G by combined biochemical, biophysical and structural biology approaches. We discovered that the entire ANK repeats are inhibited by combinatorial and quasi-independent bindings of multiple disordered segments located in the ankyrin-B/G linkers and tails, suggesting a mechanistic basis for differential regulations of membrane target bindings by ankyrins. In addition to elucidating the autoinhibition mechanisms of ankyrins, our study may also shed light on regulations on target bindings by other long repeat-containing proteins.

Desmocollin 1 is abundantly expressed in atherosclerosis and impairs high-density lipoprotein biogenesis.

The biogenesis of high-density lipoprotein (HDL) particles by cholesterol-laden foam cells in atherosclerotic lesions is crucial for the removal of excess cholesterol from the lesions. Impairment in the HDL biogenic process contributes to the progression of atherosclerosis. The aim of this study is to identify novel cellular factors regulating HDL biogenesis. HDL biogenesis is a process of apolipoprotein (apo)-mediated solubilization of specific plasma membrane (PM) microdomains generated in cholesterol-accumulated cells. We established a new method to isolate PM microdomains interacting with the major HDL protein constituent, apoA-I. Lipidomic and proteomic analyses of an isolated PM microdomain revealed that apoA-I binds to cholesterol-rich and desmocollin 1 (DSC1)-containing microdomains. In this novel apoA-I binding microdomain, DSC1 binds and prevents apoA-I from interacting with another PM microdomain created by adenosine triphosphate-binding cassette transporter A1 (ABCA1) for the formation of HDL. Inhibition of apoA-I-DSC1 binding by silencing DSC1 expression or using DSC1 blocking antibodies increases apoA-I accessibility to ABCA1-created microdomains and thus enhances HDL biogenesis. Importantly, DSC1 is abundantly expressed in macrophages and human atherosclerotic lesions, suggesting that DSC1 may contribute to cholesterol accumulation in atherosclerotic lesions by sequestering apoA-I and impairing HDL biogenesis. The binding of apoA-I to two functionally opposing PM microdomains, ABCA1 and DSC1 domains, suggests that HDL biogenesis and PM cholesterol levels may be regulated by the relative abundance of the two domains and that novel HDL biogenic therapies may be developed by targeting DSC1.

P 163 - Bcl-2 modulates ER/SR calcium uptake by interaction with SERCA and heat shock proteins

Bcl-2 is an anti-apoptotic protein, which is involved in the interaction with multiple proteins at various cellular locations, including mitochondria and the ER. We show here, that Bcl-2 interacts efficiently with various isoforms of the sarco/endoplasmic reticulum Ca-ATPase (SERCA), including SERCA1, SERCA2 and SERCA3, which leads to SERCA inactivation, conformational changes, and translocation of SERCA between specific membrane microdomains. These functional effects of Bcl-2 are demonstrated both in vitro, and in cells overexpressing Bcl-2 and SERCA. Co-immunoprecipitation experiments confirm the interaction of Bcl-2 and SERCA in vitro and in cell culture. Amino acids critical for the functional interaction of Bcl-2 with SERCA have been identified ny mutagenesis. One example is the mutation of Gly145 to Glu145, which reduces the ability of Bcl-2 to inactivate SERCA. This mutation had previously been shown by others to lower the anti-apoptotic activity of Bcl-2, providing an interesting parallel between the anti-apoptotic activity of Bcl-2 and its potency to inactivate SERCA. Heat shock proteins prevent the Bcl-2-induced inactivation of SERCA, even when heat shock proteins carry oxidative post-translational modifications such 3-nitrotyrosine and/or 4-hydroxynonenal adducts (i.e., modifications accumulated under conditions of oxidative stress).