Apolipoprotein (apo) E has a storied history as a lipid transport protein. The integral association between cholesterol homeostasis and lipoprotein clearance from circulation are intimately related to apoE's function as a ligand for cell surface receptors of the low density lipoprotein receptor family. The receptor binding properties of apoE are strongly influenced by isoform specific amino acid differences as well as the lipidation state of the protein. As understanding of apoE as a structural component of circulating plasma lipoproteins has evolved, exciting developments in neurobiology have revitalized interest in apoE. The strong and enduring correlation between the apoE4 isoform and age of onset and increased risk of Alzheimer's disease has catapulted apoE to the forefront of neurobiology. Using genetic tools generated for study of apoE lipoprotein metabolism, transgenic "knock-in" and gene-disrupted mice are now favored models for study of its role in a variety of neurodegenerative diseases. Key structural knowledge of apoE and isoform specific differences is driving research activity designed to elucidate how a single amino acid change can manifest such profoundly significant pathological consequences. This review describes apoE through a lens of structure-based knowledge that leads to hypotheses that attempt to explain the functions of apoE and isoform specific effects relating to disease mechanism.
Phosphorylation of the membrane-cytoskeleton linker protein ezrin has been functionally linked to acid secretion and vesicle recruitment to the apical secretory membrane in gastric parietal cells. Phosphorylation of the conserved T567 residue of ezrin has been shown to alter the N/C oligomerization of ezrin and promote the formation of actin-rich surface projections in other cells. To test the importance of T567 as a regulatory site for ezrin in parietal cell activation, we incorporated wild-type (WT) and mutant forms of ezrin, including the nonphosphorylatable T567A mutation and a mutant mimicking permanent phosphorylation, T567D. All ezrin constructs included C-terminal cyan-fluorescent protein (CFP) and were incorporated into adenoviral constructs for efficient introduction into cultured parietal cells from rabbit stomach. Fluorescence microscopy was used to localize CFP-ezrin and monitor morphological responses. Accumulation of a weak base (aminopyrine) was used to monitor receptor-mediated acid secretory response of the cultured cells. Similar to endogenous ezrin, WT and T567A CFP-ezrin localized heavily to apical membrane vacuoles with considerably lower levels associated with the surrounding basolateral membrane. Interestingly, H,K-ATPase within cytoplasmic tubulovesicles was incorporated into the apical vacuoles along with WT and T567A mutant ezrin. In these parietal cells secretagogue stimulation produced a striking vacuolar expansion associated with HCl secretion and the secretory phenotype. Expression of T567D CFP-ezrin was quite different, being rarely associated with apical vacuoles. T567D was more typically localized to the basolateral membrane, often associated with long spikes and fingerlike projections. Moreover, the cells did not display secretagogue-dependent morphological changes and, to our surprise, H,K-ATPase was recruited to the T567D CFP-ezrin-enriched basolateral projections. We conclude that T567 phosphorylation, which is probably regulated through Rho signaling pathway, may direct ezrin to membrane-cytoskeletal activity at the basolateral membrane and away from apical secretory activity. The large basolateral expansion is predicted to recruit membranes from sources not normally targeted to that surface.
A key feature of Alzheimer’s disease (AD) is deposition of extracellular amyloid plaque comprised chiefly of the amyloid β (Aβ) peptide. Studies of Aβ have shown that it may be catabolized by proteolysis or cleared from brain via members of the low-density lipoprotein receptor family. Alternatively, Aβ can undergo a conformational transition from α-helix to β-sheet, a conformer that displays a propensity to self-associate, oligomerize and form fibrils. Furthermore, β-sheet conformers catalyze conversion of other α-helical Aβ peptides to β-sheet, feeding the oligomer and fibril assembly process. A factor that influences the fate of Aβ in the extracellular space is apolipoprotein (apo) E. Polymorphism at position 112 or 158 in apoE give rise to three major isoforms. One isoform in particular, apoE4 (Arg at 112 and 158), has generated considerable interest since the discovery that it is the major genetic risk factor for development of late onset AD. Despite this striking correlation, the molecular mechanism underlying apoE4’s association with AD remains unclear. A tertiary structural feature distinguishing apoE4 from apoE2 and apoE3, termed domain interaction, is postulated to affect the conformation and orientation of its’ two independently folded domains. This feature has the potential to influence apoE4’s interaction with Aβ, its sensitivity to proteolysis or its lipid accrual and receptor binding activities. Thus, domain interaction may constitute the principal molecular feature of apoE4 that predisposes carriers to late onset AD. By understanding the contribution of apoE4 to AD at the molecular level new therapeutic or prevention strategies will emerge.
Normal and dying cells release various types of membrane-bound vesicles including microvesicles, exosomes, and apoptotic bodies. These vesicles play important roles in intercellular communication and signal transduction. However, their diverse forms and subtypes fluctuate in size and other properties. In result current purification approaches do not fully discriminate between different categories of extracellular vesicles. Here, we present a fluorescence technique that specifically identifies apoptotic bodies in preparations of microvesicles, exosomes, and other extracellular vesicles.The approach exclusively labels the vesicles that contain DNA with 5'PO blunt-ended DNA breaks, such as those produced by the apoptotic CAD nuclease during apoptotic DNA degradation. The technique can be useful in studies of apoptosis involving microvesicles and exosomes.
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