␤-Amyloid (A␤) aggregates at low concentrations in vivo, and this may involve covalently modified forms of these peptides. Modification of A␤ by 4-hydroxynonenal (4-HNE) initially increases the hydrophobicity of these peptides and subsequently leads to additional reactions, such as peptide cross-linking. To model these initial events, without confounding effects of subsequent reactions, we modified A␤ at each of its amino groups using a chemically simpler, close analogue of 4-HNE, the octanoyl group: K16-octanoic acid (OA)-A␤, K28-OA-A␤, and N␣-OA-A␤. Octanoylation of these sites on A␤-(1-40) had strikingly different effects on fibril formation. K16-OA-A␤ and K28-OA-A␤, but not N␣-OA-A␤, had increased propensity to aggregate. The type of aggregate (electron microscopic appearance) differed with the site of modification. The ability of octanoyl-A␤ peptides to cross-seed solutions of A␤ was the inverse of their ability to form fibrils on their own (i.e. A␤ ≈ N␣-OA-A␤ Ͼ Ͼ K16-OA-A␤ Ͼ Ͼ K28-OA-A␤). By CD spectroscopy, K16-OA-A␤ and K28-OA-A␤ had increased ␤-sheet propensity compared with A␤-(1-40) or N␣-OA-A␤. K16-OA-A␤ and K28-OA-A␤ were more amphiphilic than A␤-(1-40) or N␣-OA-A␤, as shown by lower "critical micelle concentrations" and higher monolayer collapse pressures. Finally, K16-OA-A␤ and K28-OA-A␤ are much more cytotoxic to N2A cells than A␤-(1-40) or N␣-OA-A␤. The greater cytotoxicity of K16-OA-A␤ and K28-OA-A␤ may reflect their greater amphiphilicity. We conclude that lipidation can make A␤ more prone to aggregation and more cytotoxic, but these effects are highly site-specific.Alzheimer disease, the most prevalent neurodegenerative disorder, leads to progressive memory loss, disability and eventually death (1). It is characterized by the accumulation of extracellular plaques of amyloid ␤ (A␤) 2 (2). Although aggregation of A␤ appears to be important in the pathogenesis of Alzheimer disease, one of the unanswered questions is how A␤ peptides aggregate at the low concentrations at which they exist in the central nervous system. The concentration of A␤ in the cerebrospinal fluid has been estimated as in the nanomolar range or even lower (3-12). This is below the "critical micelle concentration" for A␤ (13) and at the margin of the critical concentration (C r ), at which A␤ solutions can extend fibrillar seeds (14, 15). One recently proposed answer to this question is that an aggregation-prone, covalently modified A␤ could catalyze aggregation of the unmodified peptide. In addition to catalyzing peptide aggregation, aldehyde-modified A␤ could also be cytotoxic to neurons by itself. Oxidative stress, a possible pathogenic factor in Alzheimer disease, can lead to the formation of reactive lipid aldehydes, such as 4-HNE (16 -21) and cholesterol oxides (22), among others. Indeed, 4-HNE immunoreactivity is detected in plaques and cerebrospinal fluid of patients with Alzheimer disease (19,20) and is believed to play a role in many other neurodegenerative diseases, including Parkinson disease (23-26), Lewy body dis...

BackgroundElectrophysiological data suggest that cardiac KATP channels consist of Kir6.2 and SUR2A subunits, but the distribution of these (and other KATP channel subunits) is poorly defined. We examined the localization of each of the KATP channel subunits in the mouse and rat heart.ResultsImmunohistochemistry of cardiac cryosections demonstrate Kir6.1 protein to be expressed in ventricular myocytes, as well as in the smooth muscle and endothelial cells of coronary resistance vessels. Endothelial capillaries also stained positive for Kir6.1 protein. Kir6.2 protein expression was found predominantly in ventricular myocytes and also in endothelial cells, but not in smooth muscle cells. SUR1 subunits are strongly expressed at the sarcolemmal surface of ventricular myocytes (but not in the coronary vasculature), whereas SUR2 protein was found to be localized predominantly in cardiac myocytes and coronary vessels (mostly in smaller vessels). Immunocytochemistry of isolated ventricular myocytes shows co-localization of Kir6.2 and SUR2 proteins in a striated sarcomeric pattern, suggesting t-tubular expression of these proteins. Both Kir6.1 and SUR1 subunits were found to express strongly at the sarcolemma. The role(s) of these subunits in cardiomyocytes remain to be defined and may require a reassessment of the molecular nature of ventricular KATP channels.ConclusionsCollectively, our data demonstrate unique cellular and subcellular KATP channel subunit expression patterns in the heart. These results suggest distinct roles for KATP channel subunits in diverse cardiac structures.

Polyglutamine expansion in the exon 1 domain of huntingtin leads to aggregation into β-sheet-rich insoluble aggregates associated with Huntington's Disease. We assessed eight polyglutamine peptides with different permutations of N-methylation of backbone and side chain amides as potential inhibitors of polyglutamine aggregation. Surprisingly, the most effective inhibitor, 5QMe 2 (Anth-K-Q-Q(Me2)-Q-Q(Me 2 )-Q-CONH 2 , Anth = N-methyl anthranilic acid, Q(Me 2 ) = side chain N-methyl Q) has only side chain methylations at alternate residues, highlighting the importance of side chain interactions in polyglutamine fibrillogenesis. Above a 1:1 stoichiometric ratio, 5QMe 2 can completely prevent fibrillation of a synthetic aggregating peptide YAQ 12 A; it also shows significant inhibition at substoichiometric ratios. Surface plasmon resonance (SPR) measurements show a moderate K d with very fast k on and k off . Sedimentation equilibrium analytical ultracentrifugation indicates that 5QMe 2 is predominantly or entirely monomeric at concentrations up to 1 mM, and that it forms a 1:1 stoichiometric complex with a fibril-forming target, YAQ 12 A. 5QMe 2 inhibits not only nucleation of YAQ 12 A, but also fibril extension, as shown by the fact that it also inhibits seeded fibril growth where the nucleation steps are bypassed. 5QMe 2 acts on its targets only when they are in the PPII-like conformation, but not after they undergo a transition to β-sheets. Thus 5QMe 2 does not disassemble pre-formed YAQ 12 A; this contrasts with our previously described, backbone N-methylated inhibitors of β-amyloid aggregation (16,17). The mode of action of 5QMe 2 is reminiscent of chaperones, since it binds and releases its targets very rapidly, and maintains them in a non-aggregation-prone, monomeric state, in this case, the polyproline II (PPII)-like conformation, as shown by CD spectroscopy.Expanded polyglutamine (polyQ) tracts are responsible for at least nine neurodegenerative diseases, including Huntington's Disease (HD). HD occurs when the polyQ domain of exon † We acknowledge NIH Medical Scientist Training Program Grant (T32 GM07281 JDL), NIH Cardiovascular Pathophysiology Training Grant (HL07237 JDL) and NIH (NS042852 SCM) and the Alzheimer's Association (IIRG-06-27794). * To whom correspondence should be addressed: Department of Pathology, 5841 S. Maryland Ave., Chicago, IL 60637. Tel: 773-702-1267. Fax: 773-834-5251. scmeredi@uchicago.edu . Supporting Information Available The following items are available as supporting information: A derivation of the equations and other details needed for analysis of equilibrium sedimentation analytical ultracentrifugation of two dissimilar interacting species; determination of for YAQ 12 A and 5QMe 2 ; reverse phase HPLC isocratic analysis of YAQ 12 A remaining in solution, in the presence or absence of 5QMe 2 ; experiments showing the effect of added fibril seeds on fibrillation of YAQ 12 A; electron micrograph of fibril seed slurry used in preceding experiments; example of analytical ...

Current evidence suggests that oligomers of the amyloid-β (Aβ) peptide are involved in the cellular toxicity of Alzheimer’s disease, yet their biophysical characterization remains difficult because of lack of experimental control over the aggregation process under relevant physiologic conditions. Here, we show that modification of the Aβ peptide backbone at Gly29 allows for the formation of oligomers but inhibits fibril formation at physiologic temperature and pH. Our results suggest that the putative bend region in Aβ is important for higher-order aggregate formation.

Self-aggregation of proteins and peptides is at the root of many diseases, especially neurodegenerative diseases. These conditions include Alzheimer's disease, Huntington's disease, Parkinson's disease, type-2 diabetes mellitus, and transmissible spongiform encephalopathies, which are associated with self-aggregation of amyloid b -protein (A b ), huntingtin, a -synuclein, islet amyloid polypeptide, and the prion protein, respectively. The list of diseases for which protein/peptide aggregation is the root cause is ever expanding. There does not appear to be a single biochemical mechanism by which proteins and peptides self-associate, or a single pathogenic mechanism to explain all protein-/peptide-aggregation diseases. Nevertheless, inhibition of protein self-aggregation remains a potential target for therapeutic intervention. Beyond therapy, inhibitors of protein self-aggregation can serve as tools to help us understand the mechanisms by which aggregation occurs and harms cells. In this chapter, we examine select examples of inhibitors of protein aggregation. We have divided aggregating proteins/peptides into two types: (1) Proteins that have an unstable tertiary structure, that unfold under cellular stress, or that fail to fold correctly during biosynthesis. This instability leads to persistence of unfolded domains that can act as a nidus for self-association. (2) Peptides or proteins (or protein domains) that cannot fold at all, or fold only in the presence of a bound ligand. Examples of the fi rst group include transthyretin, the mammalian prion protein, and certain point-mutant forms of lysozyme or a 1-antitrypsin. In general, self-aggregation of these proteins results from exposure of normally buried hydrophobic residues to aqueous media. Examples of the second group include A b , islet amyloid polypeptide, and calcitonin. Within the second group, we also include proteins that are "peptide-like" in having domains with no unique, stable