IKK phosphorylates Huntingtin and targets it for degradation by the proteasome and lysosome

Expansion of the polyglutamine repeat within the protein Huntingtin (Htt) causes Huntington's disease, a neurodegenerative disease associated with aging and the accumulation of mutant Htt in diseased neurons. Understanding the mechanisms that influence Htt cellular degradation may target treatments designed to activate mutant Htt clearance pathways. We find that Htt is phosphorylated by the inflammatory kinase IKK, enhancing its normal clearance by the proteasome and lysosome. Phosphorylation of Htt regulates additional post-translational modifications, including Htt ubiquitination, SUMOylation, and acetylation, and increases Htt nuclear localization, cleavage, and clearance mediated by lysosomal-associated membrane protein 2A and Hsc70. We propose that IKK activates mutant Htt clearance until an age-related loss of proteasome/lysosome function promotes accumulation of toxic post-translationally modified mutant Htt. Thus, IKK activation may modulate mutant Htt neurotoxicity depending on the cell's ability to degrade the modified species

Altering APP Proteolysis: Increasing sAPPalpha Production by Targeting Dimerization of the APP Ectodomain

One of the events associated with Alzheimer's disease is the dysregulation of α- versus β-cleavage of the amyloid precursor protein (APP). The product of α-cleavage (sAPPα) has neuroprotective properties, while Aβ1-42 peptide, a product of β-cleavage, is neurotoxic. Dimerization of APP has been shown to influence the relative rate of α- and β- cleavage of APP. Thus finding compounds that interfere with dimerization of the APP ectodomain and increase the α-cleavage of APP could lead to the development of new therapies for Alzheimer's disease. Examining the intrinsic fluorescence of a fragment of the ectodomain of APP, which dimerizes through the E2 and Aβ-cognate domains, revealed significant changes in the fluorescence of the fragment upon binding of Aβ oligomers—which bind to dimers of the ectodomain— and Aβ fragments—which destabilize dimers of the ectodomain. This technique was extended to show that RERMS-containing peptides (APP695 328–332), disulfiram, and sulfiram also inhibit dimerization of the ectodomain fragment. This activity was confirmed with small angle x-ray scattering. Analysis of the activity of disulfiram and sulfiram in an AlphaLISA assay indicated that both compounds significantly enhance the production of sAPPα by 7W-CHO and B103 neuroblastoma cells. These observations demonstrate that there is a class of compounds that modulates the conformation of the APP ectodomain and influences the ratio of α- to β-cleavage of APP. These compounds provide a rationale for the development of a new class of therapeutics for Alzheimer's disease

Uptake Mechanism of ApoE-Modified Nanoparticles on Brain Capillary Endothelial Cells as a Blood-Brain Barrier Model

Background: The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. Methodology/Principal Findings: In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. Conclusions/Significance: This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier

Isolation, Characterization, and Stability of Discretely-Sized Nanolipoprotein Particles Assembled with Apolipophorin-III

Background: Nanolipoprotein particles (NLPs) are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. NLPs assembled with human apolipoproteins have been used for myriad biotechnology applications, including membrane protein solubilization, drug delivery, and diagnostic imaging. To expand the repertoire of lipoproteins for these applications, insect apolipophorin-III (apoLp-III) was evaluated for the ability to form discretely-sized, homogeneous, and stable NLPs. Methodology: Four NLP populations distinct with regards to particle diameters (ranging in size from 10 nm to.25 nm) and lipid-to-apoLp-III ratios were readily isolated to high purity by size exclusion chromatography. Remodeling of the purified NLP species over time at 4uC was monitored by native gel electrophoresis, size exclusion chromatography, and atomic force microscopy. Purified 20 nm NLPs displayed no remodeling and remained stable for over 1 year. Purified NLPs with 10 nm and 15 nm diameters ultimately remodeled into 20 nm NLPs over a period of months. Intra-particle chemical cross-linking of apoLp-III stabilized NLPs of all sizes. Conclusions: ApoLp-III-based NLPs can be readily prepared, purified, characterized, and stabilized, suggesting their utilit

Formation and Toxicity of Soluble Polyglutamine Oligomers in Living Cells

Aggregation and cytotoxicity of mutant proteins containing an expanded number of polyglutamine (polyQ) repeats is a hallmark of several diseases, including Huntington's disease (HD). Within cells, mutant Huntingtin (mHtt) and other polyglutamine expansion mutant proteins exist as monomers, soluble oligomers, and insoluble inclusion bodies (IBs). Determining which of these forms constitute a toxic species has proven difficult. Recent studies support a role for IBs as a cellular coping mechanism to sequester levels of potentially toxic soluble monomeric and oligomeric species of mHtt.When fused to a fluorescent reporter (GFP) and expressed in cells, the soluble monomeric and oligomeric polyglutamine species are visually indistinguishable. Here, we describe two complementary biophysical fluorescence microscopy techniques to directly detect soluble polyglutamine oligomers (using Htt exon 1 or Htt(ex1)) and monitor their fates in live cells. Photobleaching analyses revealed a significant reduction in the mobilities of mHtt(ex1) variants consistent with their incorporation into soluble microcomplexes. Similarly, when fused to split-GFP constructs, both wildtype and mHtt(ex1) formed oligomers, as evidenced by the formation of a fluorescent reporter. Only the mHtt(ex1) split-GFP oligomers assembled into IBs. Both FRAP and split-GFP approaches confirmed the ability of mHtt(ex1) to bind and incorporate wildtype Htt into soluble oligomers. We exploited the irreversible binding of split-GFP fragments to forcibly increase levels of soluble oligomeric mHtt(ex1). A corresponding increase in the rate of IBs formation and the number formed was observed. Importantly, higher levels of soluble mHtt(ex1) oligomers significantly correlated with increased mutant cytotoxicity, independent of the presence of IBs.Our study describes powerful and sensitive tools for investigating soluble oligomeric forms of expanded polyglutamine proteins, and their impact on cell viability. Moreover, these methods should be applicable for the detection of soluble oligomers of a wide variety of aggregation prone proteins

Model of biologically active apolipoprotein E bound to dipalmitoylphosphatidylcholine

Apolipoprotein (apo) E plays a critical role in cholesterol transport, through high affinity binding to the low density lipoprotein receptor. This interaction requires apoE to be associated with a lipoprotein particle. To determine the structure of biologically active apoE on a lipoprotein particle, we crystallized dipalmitoylphosphatidylcholine particles containing two apoE molecules and determined the molecular envelope of apoE at 10 angstrom resolution. On the basis of the molecular envelope and supporting biochemical evidence, we propose a model in which each apoE molecule is folded into a helical hairpin with the binding region for the low density lipoprotein receptor at its apex

New Insights into the Heparan Sulfate Proteoglycan binding Activity of Apolipoprotein E4

Journal of Biological Chemistry, 276, 39138-39142Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.Defective binding of apolipoprotein E (apoE) to heparan sulfate proteoglycans (HSPGs) is associated with increased risk of atherosclerosis due to inefficient clearance of lipoprotein remnants by the liver. The interaction of apoE with HSPGs has also been implicated in the pathogenesis of Alzheimer’s disease and may play a role in neuronal repair. To identify which residues in the he parin-binding site of apoE and which structural elements of heparan sulfate interact, we used a variety of approaches, including glycosaminoglycan specificity as says, 13C nuclear magnetic resonance, and heparin affinity chromatography. The formation of the high affinity complex required Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147 from apoE and N- and 6-O-sulfo groups of the glucosamine units from the heparin fragment. As shown by molecular modeling, using a high affinity binding octasaccharide fragment of heparin, these findings are consistent with a binding mode in which five saccharide residues of fully sulfated heparan sulfate lie in a shallow groove of the �-helix that contains the HSPG-binding site (helix 4 of the four-helix bundle of the 22-kDa fragment). This groove is lined with residues Arg-136, Ser-139, His 140, Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147. In the model, all of these residues make direct contact with either the 2-O-sulfo groups of the iduronic acid monosaccharides or the N- and 6-O-sulfo groups of the glucosamine sulfate monosaccharides. This model indicates that apoE has an HSPG-binding site highly complementary to heparan sulfate rich in N- and O-sulfo groups such as that found in the liver and the brain.https://login.libproxy.rpi.edu/login?url=https://doi.org/10.1074/jbc.M10474620