X-ray crystal structure of the protease inhibitor domain of Alzheimer's amyloid beta-protein precursor

Alzheimer's amyloid beta-protein precursor contains a Kunitz protease inhibitor domain (APPI) potentially involved in proteolytic events leading to cerebral amyloid deposition. To facilitate the identification of the physiological target of the inhibitor, the crystal structure of APPI has been determined and refined to 1.5-A resolution. Sequences in the inhibitor-protease interface of the correct protease target will reflect the molecular details of the APPI structure. While the overall tertiary fold of APPI is very similar to that of the Kunitz inhibitor BPTI, a significant rearrangement occurs in the backbone conformation of one of the two protease binding loops. A number of Kunitz inhibitors have similar loop sequences, indicating the structural alteration is conserved and potentially an important determinant of inhibitor specificity. In a separate region of the protease binding loops, APPI side chains Met-17 and Phe-34 create an exposed hydrophobic surface in place of Arg-17 and Val-34 in BPTI. The restriction this change places on protease target sequences is seen when the structure of APPI is superimposed on BPTI complexed to serine proteases, where the hydrophobic surface of APPI faces a complementary group of nonpolar side chains on kallikrein A versus polar side chains on trypsin.     

Expression and secretion in Aspergillus nidulans and Aspergillus niger of a cell surface glycoprotein from the cattle tick, Boophilus microplus, by using the fungal amdS promoter system.

A cell surface glycoprotein (Bm86) from cells of the digestive tract of the cattle tick Boophilus microplus, which has been shown to elicit a protective immunological response in vaccinated cattle, was expressed and secreted in the filamentous fungi Aspergillus nidulans and Aspergillus niger by using the fungal amdS promoter system. The cloned gene coded for the Bm86 secretory signal and all of the Bm86 mature polypeptide except for the hydrophobic carboxy-terminal segment. High levels of Bm86 mRNA were detected in the transformed cells. Bm86 polypeptide was secreted from the cells in a soluble form and it was glycosylated, probably to a similar extent to the native glycoprotein. The recombinant product had an apparent molecular mass of 83 to 87 kilodaltons, whereas that predicted from the amino acid sequence was 69 kilodaltons. The Bm86 was expressed at levels of up to 1.8 mg/liter, or approximately 6% of secreted protein under the growth conditions used. No intracellular Bm86 was detected. A general relationship was observed between transformants containing a high number of copies of the expression plasmid and high expression levels.     

A mutation, adjacent to gene amdS, defining the site of action of positive-control gene amdR in Aspergillus nidulans

In Aspergillus nidulans the acetamidase enzyme is inducible by omega-amino acids, sources of acetyl-coenzyme A, and benzoate. The amdR (or intA) gene is a positive-control gene involved in omega-amino acid induction only. A cis-acting mutation amdI93 located in a complex controlling region adjacent to the acetamidase structural gene was found to abolish induction by omega-amino acids but not induction by other sources of induction. As predicted, this mutation was epistatic to constitutive amdR alleles but did not affect the expression of mutations in other regulatory genes.     

Fine-Structure Mapping of the Acetamidase Structural Gene and Its Controlling Region in ASPERGILLUS NIDULANS

A large number of amdS mutants altered in acetamide utilization have been used to construct a fine-structure map of the amdS locus. The mutagen diepoxyoctane generated most of the deletion strains used for mapping. A minimum of 14 sites within the amdS gene were found. Biochemical analysis of amdS mutants defined the extent of the probable coding region. A new mutant, amd-205, which did not produce detectable inactive gene product, was found to be inseparable by recombination from the "up-promoter" mutation amdI18 and was located outside of the apparent amdS coding region. The cis-dominant mutation, amdI9, was also located at this end of the gene. This work, therefore, provides evidence for the separation of a eukaryotic gene into controlling and structural regions.     

Viral transformation of rat myoblasts: effects on fusion and surface properties.

Lines of rat myoblasts infected by avian sarcoma viruses have been isolated, cloned, and used to study the effects of viral transformation on myogenic differentiation and the surface changes associated with differentiation. The lines transformed by sarcoma viruses failed to fuse into myotubes and did not show the increase in myosin synthesis normally associated with fusion. The parental nontransformed line showed, subsequent to fusion, a surface alteration detectable by external labeling methods. This alteration, an increase in the level of an external protein of MW > 200 × 10³, is similar to that observed in fibroblasts arrested in the G₁ phase of the cell cycle. This protein was absent or greatly reduced on the surfaces of the myoblast lines that had been transformed by sarcoma viruses. Therefore, viral transformation causes loss of several properties normally associated with arrest of myoblasts in G₁.     

The location of proteins labeled by the 125I-lactoperoxidase system in the NIL 8 hamster fibroblast.

NIL 8 hamster fibroblast cells were labeled by lactoperoxidase-catalyzed iodination. Their membranes were fractionated by sedimentation-rate and isopycnic zonal centrifugation. All the iodinated proteins except the very prominently labeled high molecular weight protein (greater than 200,000 daltons) were located in a fraction identified enzymically and compositionally as plasma membrane. The high molecular weight protein that was previously shown to be sensitive to virus transformation (Hynes, 1973) is concentrated in a very high density particle (rho equals 1.253-1.259) which contains mainly carbohydrate and protein and very low levels of lipid. 5'-nucleotidase was the only enzyme reproducibly demonstrated in this fraction, and electron micrographs revealed a predominantly amorphous morphology together with a few membraneous structures. The iodine label in this fraction was very sensitive to trypsinization prior to homogenization. All the available evidence indicates that this fraction is derived from the surface coat. Mitochondria, nuclei, and soluble protein were labeled to an insignificant extent. The presence of the iodinated surface proteins associated with the endoplasmic reticulum fraction is discussed in the light of these results.     

The regulation of NADP-linked isocitrate dehydrogenase in Aspergillus nidulans

The regulation of NADP-linked isocitrate dehydrogenase (NADP-IDH) has been studied in wild-type and mutant strains of Aspergillus nidulans. In the wild-type strain studied, the levels of NADP-IDH vary in a similar way to those of acetamidase, acetyl-CoA synthase, isocitrate lyase and malate synthase under all growth conditions used. Similarly, fac mutants, which are altered in the regulation of these enzymes of acetate utilization, are affected in NADP-IDH levels in a parallel fashion, as are cre mutants, which show altered carbon catabolite repression of this group of enzymes. Possible functions of the NADP-IDH enzyme are considered.     

Cell surface and metabolic labelling of the proteins of normal and transformed chicken cells.

We have studied the surface proteins of normal and transformed chick cells using four-labelling techniques with different specificities, (a) lactoperoxidase catalysed iodination (b) galactose oxidase/B3H4 (c) pyridoxal phosphate/B3H4 and (d) periodate/B3H4. All methods labelled a large external transformation-sensitive (LETS) protein, in agreement with previous studies. In addition, using galactose oxidase and periodate labelling techniques, we present evidence which suggests that the transformed cell surface glycoproteins are more sialylated. The LETS protein was also labelled with (14C) glucosamine and after trypsinization a small band of identical molecular weight to LETS remained, possibly representing an internal pool of the protein. In contrast LETS protein labelled with (3H) fucose was completely removed by trypsin, suggesting that the internal pool of the protein is incompletely glycosylated. Evidence is also presented to show that although the level of the protein is drastically reduced at the transformed cell surface, it is still synthesised and shed into the medium.     

Phylogenetic diversity of indigenous Rhizobium trapped from the natural habitat of Pisum sativum L. in eastern and central Algeria

Plant and Soil - The nitrogen-fixing symbiosis fixation between legumes and Rhizobium helps the plant to survive and to compete effectively on nitrogen poor soils. The soil environment attached to...