Dr. Mehra reports receiving fees for serving as a clinical trial leader from Abbott, Medtronic, and Janssen, and advisory board fees from NuPulseCV, Portola, Bayer, and FineHeart. No other potential conflict of interest relevant to this article was reported.

Amyloid formation has been implicated in a wide range of human diseases, and a diverse set of proteins is involved. There is considerable interest in elucidating the interactions which lead to amyloid formation and which contribute to amyloid fibril stability. Recent attention has been focused upon the potential role of aromatic-aromatic and aromatic-hydrophobic interactions in amyloid formation by short to midsized polypeptides. Here we examine whether aromatic residues are necessary for amyloid formation by islet amyloid polypeptide (IAPP). IAPP is responsible for the formation of islet amyloid in type II diabetes which is thought to play a role in the pathology of the disease. IAPP is 37 residues in length and contains three aromatic residues, Phe-15, Phe-23, and Tyr-37. Structural models of IAPP amyloid fibrils postulate that Tyr-37 is near one of the phenylalanine residues, and it is known that Tyr-37 interacts with one of the phenylalanines during fibrillization; however, it is not known if aromatic-aromatic or aromatic-hydrophobic interactions are absolutely required for amyloid formation. An F15L/F23L/Y37L triple mutant (IAPP-3XL) was prepared, and its ability to form amyloid was tested. CD, thioflavin binding assays, AFM, and TEM measurements all show that the triple leucine mutant readily forms amyloid fibrils. The substitutions do, however, decrease the rate of fibril formation and alter the tendency of fibrils to aggregate. Thus, while aromatic residues are not an absolute requirement for amyloid formation by IAPP, they do play a role in the fibril assembly process.

As a member of the tetraspanin superfamily of proteins, CD81 has been linked to a number of biologic functions including cellular proliferation, differentiation, activation, and degranulation. As a co-receptor for hepatitis C virus, and a requirement for hepatocytes for infectivity of human Plasmodium falciparum and rodent P. yoelii sporozoite infectivity, CD81 may also play a vital role in pathology. Despite the importance of CD81 in multiple cellular functions, the molecular mechanism of action of CD81 in these processes has remained elusive. Here we report an association between CD81 and the epsilon isoform of 14-3-3, a serine/threonine-binding intracellular signaling protein. Furthermore, we provide evidence that in human, this association is influenced by the palmitoylation state of the CD81 cytoplasmic tails. We have generated a series of CD81 cysteine mutants to identify palmitoylated intracellular motifs of CD81, and reveal palmitoylation on the N-and C-terminal tails as well as the intracellular loop between transmembrane domains 2 and 3. One of these mutants lacks all five of its intracellular cysteines and therefore cannot be palmitoylated. This unpalmitoylated version of CD81 shows constitutive association with 14-3-3. Interestingly, we find that under oxidative conditions, CD81 palmitoylation is inhibited and that condition correlates with the association of CD81 and 14-3-3. These finding suggest that CD81 signaling events could be mediated by 14-3-3 adapter proteins, and these signals may be dependent on cellular redox.CD81 is a member of the tetraspanin family of integral membrane proteins. CD81 was first discovered in 1990 as the target of an antibody with reversible antiproliferative effects on a human B cell lymphoma line (1-4). There are currently 26 membrane proteins classified as members of the tetraspanin family. Members must meet the following criteria: four highly conserved hydrophobic transmembrane domains, two extracellular loops, short intracellular amino and carboxyl tails, and three motifs, CCG, PXSC, and EGC, that contain conserved cysteine residues in the major extracellular domain (5). The smaller extracellular loop is between transmembrane 1 and 2, and the larger extracellular loop, which is likely to mediate interactions with other cell surface proteins, lies between transmembrane 3 and 4.Many of the initial discoveries of CD81 function were focused in the immune system. On the surface of B cells, CD81 is associated with the CD19⅐CD21⅐Leu 13 protein complex (6 -8). This complex is involved in regulation of B cell receptor signal transduction through recruitment of phosphatidylinositol 3-kinase (PI3K). The CD21⅐CD19⅐CD81 complex functions by bringing together required signaling molecules to lower the B cell threshold of activation (9, 10). Similarly, on T cells, CD81 associates with the T cell co-receptors CD4 and CD8 and provides a costimulatory signal with the CD3 subunit of the T cell receptor (11). CD81 may play a role in early T cell development in the thymus (12). The roles...