The MKC7 gene was isolated as a multicopy suppressor of the cold-sensitive growth phenotype of a yeast kex2 mutant, which lacks the protease that cleaves pro-afactor and other secretory proproteins at pairs of basic residues in a late Golgi compartment in yeast. MKC7 encodes an aspartyl protease most closely related to product of the YAP3 gene, a previously isolated multicopy suppressor of the pro-a-factor processing defect of a kex2 null. Multicopy MKC7 suppressed the a-specific mating defect of a kex2 null as well as multicopy YAP3 MATERIALS AND METHODSYeast Media and Strains. Liquid and solid rich media supplemented with 1% adenine sulfate (YPAD) and synthetic complete medium (SDC) were as described (9). kex2 null mutations were as described (10). kex2 null strains were not cold-sensitive on SDC but were on modified SDC, in which (i) proline (1%) replaced (NH4)2SO4 as a nitrogen source, (ii) KH2PO4 was 1.0 mM, (iii) MgSO4 was 0.5 mM, and (iv) pH was buffered at 6.5 with 50 mM Mes (Calbiochem).All yeast strains were derived from the W303 background (R. Rothstein, Columbia University; ref. 11). Strains: CRY2,

The Alcadeins (Alcs)/calsyntenins and the amyloid ␤-protein precursor (APP) associate with each other in the brain by binding via their cytoplasmic domains to X11L (the X11-like protein). We previously reported that the formation of this APP-X11L-Alc tripartite complex suppresses the metabolic cleavages of APP. We show here that the metabolism of the Alcs markedly resembles that of APP. The Alcs are subjected to a primary cleavage event that releases their extracellular domain. Alcs then undergo a secondary presenilin-dependent ␥-cleavage that leads to the secretion of the amyloid ␤-protein-like peptide and the liberation of an intracellular domain fragment (AlcICD). However, when Alc is in the tripartite complex, it escapes from these cleavages, as does APP. We also found that AlcICD suppressed the FE65-dependent gene transactivation activity of the APP intracellular domain fragment, probably because AlcICD competes with the APP intracellular domain fragment for binding to FE65. We propose that the Alcs and APP are coordinately metabolized in neurons and that their cleaved cytoplasmic fragments are reciprocally involved in the regulation of FE65-dependent gene transactivation. Any imbalance in the metabolism of Alcs and APP may influence the FE65-dependent gene transactivation, which together with increased secretion of amyloid ␤-protein may contribute to neural disorders.The deposition and accumulation of amyloid ␤-protein (A␤) 1 in the human brain are hallmarks of Alzheimer's disease (AD)(1). Amyloid ␤-protein precursor (APP) is the precursor of A␤. It has a receptor-like transmembrane protein structure that consists of an extracellular domain, a transmembrane domain, and a short carboxyl-terminal cytoplasmic domain (2). The cytoplasmic domain of APP controls its metabolism and various physiological functions by interacting with cytoplasmic adaptor proteins (3-8). One of these adaptor proteins is X11L (the X11-like protein), which associates with the cytoplasmic domain of APP and stabilizes APP metabolism (5, 9). During our previous research that aimed to reveal the molecular mechanism by which X11L regulates APP metabolism, we found that the Alcadeins, which form cadherin-related membrane protein family, are X11-and X11L-binding proteins (9). These proteins are also known as calsyntenins, which were originally isolated as postsynaptic Ca 2ϩ -binding membrane proteins, but whose functions were not identified (10, 11). The Alcadeins (Alcs) consist of two Alc␣ isoforms (Alc␣1 and Alc␣2) and Alc␤ and Alc␥, all of which are type I transmembrane proteins and contain a conserved X11L-binding motif in their single cytoplasmic domains, similar to APP (9).Alc does not directly interact with the cytoplasmic domain of APP. Rather, the association between the two molecules is bridged by the phosphotyrosine interaction domain of X11L. This results in the formation of a tripartite complex in the brain (9). The formation of this complex enhances the X11L-mediated stabilization of APP metabolism and suppresses the generation...

Presenilin (PS) is essential for the ␥-cleavage required for the generation of the C terminus of amyloid ␤-protein (A␤). However, the mechanism underlying PS-mediated ␥-cleavage remains unclear. We have identified Herp cDNA by our newly developed screening method for the isolation of cDNAs that increase the degree of ␥-cleavage. Herp was originally identified as a homocysteine-responsive protein, and its expression is up-regulated by endoplasmic reticulum stress. Herp is an endoplasmic reticulum-localized membrane protein that has a ubiquitin-like domain. Here, we report that a high expression of Herp in cells increases the level of A␤ generation, although not in PS-deficient cells. We found that Herp interacts with both PS1 and PS2. Thus, Herp regulates PS-mediated A␤ generation, possibly through its binding to PS. Immunohistochemical analysis of a normal human brain section with an anti-Herp antibody revealed the exclusive staining of neurons and vascular smooth muscle cells. Moreover, the antibody strongly stained activated microglia in senile plaques in the brain of patients with Alzheimer disease. Taken together, Herp could be involved in A␤ accumulation, including the formation of senile plaques and vascular A␤ deposits.

Herp is a stress-response protein localized in the endoplasmic reticulum (ER) membrane. Herp was proposed to improve ER-folding, decrease ER protein load, and participate in ERassociated degradation (ERAD). Intra-muscle-fiber ubiquitinated multiprotein-aggregates containing, among other proteins, either amyloid-b (Ab) or phosphorylated tau are characteristic of sporadic inclusion-body myositis (s-IBM). ER stress and proteasome inhibition appear to play a role in s-IBM pathogenesis. We have now studied Herp in s-IBM muscle fibers and in ER-stress-induced or proteasome-inhibited cultured human muscle fibers. In s-IBM muscle fibers: (i) Herp was strongly immunoreactive in the form of aggregates, which co-localized with Ab, GRP78, and b2 proteasome subunit; (ii) Herp mRNA and protein were increased. In ER-stress-induced cultured human muscle fibers: (i) Herp immunoreactivity was diffusely increased; (ii) Herp mRNA and protein were increased. In proteasome-inhibited cultured human muscle fibers: (i) Herp immunoreactivity was in the form of aggregates; (ii) Herp protein was increased, but its mRNA was not. Accordingly, in s-IBM muscle fibers: (i) increase of Herp might be due to both ER-stress and proteasome inhibition; (ii) co-localization of Herp with Ab, proteasome, and ERchaperone GRP78 could reflect its possible role in processing and degradation of cytotoxic proteins in ER.

The solution conformation of an antibacterial protein sapecin has been detennined by 'H nuclear magnetic resonance (NMR) and dynamical simulated annealing calculations. It has been shown that the polypeptide fold consists of one flexible loop (residues 4-12), one helix (residues 1523), and two extended strands (residues 2431 and 3W). It was found that the tertiary structure of sapecin is completely different from that of rabbit neutrophil defensin NP-5, which is homologous to sapecin in the amino acid sequences and also has the antibacterial activity. The three-dimensional structure determination has revealed that a basic-residue rich region and the hydrophobic surface face each other on the surface of sapecin.