Regulated Intramembrane Proteolysis and Degradation of Murine Epithelial Cell Adhesion Molecule mEpCAM

Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-)stem cells, and which plays an important role in the regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM) is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at α-, β-, γ-, and ε-sites to generate soluble ectodomains, soluble Aβ-like-, and intracellular fragments termed mEpEX, mEp-β, and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the α- and β-sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF) are further processed to soluble Aβ-like fragments mEp-β and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the γ-secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified in silico in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent γ-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, γ-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation.     

Alzheimer's Disease

Peptide aldehyde inhibitors of the chymotrypsin-like activity of the proteasome (CLIP) such as N-acetyl-Leu-Leu-Nle-H (or ALLN) have been shown previously to inhibit the secretion of beta-amyloid peptide (A beta) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on A beta formation of a much wider range of peptide-based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide-based inhibitors of beta-amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress A beta formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in A beta formation.     

Molecular characterization of 'Bhut Jolokia' the hottest chilli

The northeast region of India, considered as 'hot spot' of biodiversity, having unique ecological environment with hot and high-humidity conditions, has given rise to the world's hottest chilli, 'Bhut Jolokia', which is at least two times hotter than Red Savina Habanero in terms of Scoville heat units (SHU). This study was undertaken to determine the distinctiveness of 'Bhut Jolokia' from Capsicum frutescens or Capsicum chinense through sequencing of the ribosomal RNA (rRNA) gene-internal transcribed ((ITS) region along with its phylogenetic analysis. Although a compensatory base change (CBC) in the ITS2 region was not observed between the closely related species of C. frutescens and C. chinense when compared with Bhut Jolokia; phylogenetic analysis using ITS1, 5.8S and ITS2 sequences indicated a distinct clade for all the accessions of 'Bhut Joloikia', while C. frutescens and C. chinense occupied discrete lineages. Further, a unique 13-base deletion was observed in all the representative accessions of 'Bhut Jolokia', making it distinct from all other members within the genus and beyond. The degree of genetic variations along with its extreme pungency might be related to ambient environmental factors of northeastern India.     

Studies on the constancy of the blue and green fluorescence yield during the chlorophyll fluorescence induction kinetics (Kautsky effect)

Blue (F 450) and green (F 530) leaf fluorescence were studied together with the red chlorophyll fluorescence (emission maxima F 690 and F 735) during light-induced chlorophyll fluorescence induction kinetics (Kautsky effect) in predarkened leaves of wheat (Triticum aestivum L.) and soybean (Glycine max L.). The intensity of the red chlorophyll fluorescence decreased from maximum fluorescence Fm to steady-state fluorescence Fs, and the fluorescence ratio F 690/F 735 decreased by about 10% from Fm to Fs. However, blue and green fluorescence intensities remained constant throughout the measuring time. Consequently, the ratio of blue to red fluorescence (F 450/F 690) increased during chlorophyll fluorescence induction kinetics, whereas the ratio of blue to green fluorescence (F 450/F 530) remained unchanged within the same period. The knowledge of these ratios will be a prerequisite for the interpretation of remote sensing data from terrestrial vegetation.     

A novel sorting nexin modulates endocytic trafficking and alpha-secretase cleavage of the amyloid precursor protein

Ectodomain shedding of the amyloid precursor protein (APP) by the two proteases alpha- and beta-secretase is a key regulatory event in the generation of the Alzheimer disease amyloid beta peptide (Abeta). beta-Secretase catalyzes the first step in Abeta generation, whereas alpha-secretase cleaves within the Abeta domain, prevents Abeta generation, and generates a secreted form of APP with neuroprotective properties. At present, little is known about the cellular mechanisms that control APP alpha-secretase cleavage and Abeta generation. To explore the contributory pathways, we carried out an expression cloning screen. We identified a novel member of the sorting nexin (SNX) family of endosomal trafficking proteins, called SNX33, as a new activator of APP alpha-secretase cleavage. SNX33 is a homolog of SNX9 and was found to be a ubiquitously expressed phosphoprotein. Exogenous expression of SNX33 in cultured cells increased APP alpha-secretase cleavage 4-fold but surprisingly had little effect on beta-secretase cleavage. This effect was similar to the expression of the dominant negative dynamin-1 mutant K44A. SNX33 bound the endocytic GTPase dynamin and reduced the rate of APP endocytosis in a dynamin-dependent manner. This led to an increase of APP at the plasma membrane, where alpha-secretase cleavage mostly occurs. In summary, our study identifies SNX33 as a new endocytic protein, which modulates APP endocytosis and APP alpha-secretase cleavage, and demonstrates that the rate of APP endocytosis is a major control factor for APP alpha-secretase cleavage.     

Metabolism of sucrose and its five linkage-isomeric alpha-D-glucosyl-D-fructoses by Klebsiella pneumoniae. Participation and properties of sucrose-6-phosphate hydrolase and phospho-alpha-glucosidase

Klebsiella pneumoniae is presently unique among bacterial species in its ability to metabolize not only sucrose but also its five linkage-isomeric alpha-d-glucosyl-d-fructoses: trehalulose, turanose, maltulose, leucrose, and palatinose. Growth on the isomeric compounds induced a protein of molecular mass approximately 50 kDa that was not present in sucrose-grown cells and which we have identified as an NAD(+) and metal ion-dependent 6-phospho-alpha-glucosidase (AglB). The aglB gene has been cloned and sequenced, and AglB (M(r) = 49,256) has been purified from a high expression system using the chromogenic p-nitrophenyl alpha-glucopyranoside 6-phosphate as substrate. Phospho-alpha-glucosidase catalyzed the hydrolysis of a wide variety of 6-phospho-alpha-glucosides including maltose-6'-phosphate, maltitol-6-phosphate, isomaltose-6'-phosphate, and all five 6'-phosphorylated isomers of sucrose (K(m) approximately 1-5 mm) yet did not hydrolyze sucrose-6-phosphate. By contrast, purified sucrose-6-phosphate hydrolase (M(r) approximately 53,000) hydrolyzed only sucrose-6-phosphate (K(m) approximately 80 microm). Differences in molecular shape and lipophilicity potential between sucrose and its isomers may be important determinants for substrate discrimination by the two phosphoglucosyl hydrolases. Phospho-alpha-glucosidase and sucrose-6-phosphate hydrolase exhibit no significant homology, and by sequence-based alignment, the two enzymes are assigned to Families 4 and 32, respectively, of the glycosyl hydrolase superfamily. The phospho-alpha-glucosidase gene (aglB) lies adjacent to a second gene (aglA), which encodes an EII(CB) component of the phosphoenolpyruvate-dependent sugar:phosphotransferase system. We suggest that the products of the two genes facilitate the phosphorylative translocation and subsequent hydrolysis of the five alpha-d-glucosyl-d-fructoses by K. pneumoniae.