Immature nicastrin stabilizes APH-1 independent of PEN-2 and presenilin: identification of nicastrin mutants that selectively interact with APH-1

Gamma-secretase is a high molecular mass aspartyl protease complex composed of presenilin (PS1 or PS2), nicastrin (Nct), anterior pharynx-defective-1 (APH-1) and presenilin enhancer-2 (PEN-2). The complex mediates the intramembraneous proteolysis of beta-secretase cleaved beta-amyloid precursor protein (APP) leading to the secretion of the Alzheimer's disease-associated amyloid beta-peptide (Abeta). In order to dissect functionally important domains of Nct required for gamma-secretase complex assembly, maturation, and activity we mutated evolutionary conserved amino acids. The mutant Nct variants were expressed in a cellular background with significantly reduced endogenous Nct. Mutant Nct was functionally investigated by its ability to restore PS, APH-1 and PEN-2 expression as well as by monitoring the accumulation of the APP C-terminal fragments, the immediate substrates of gamma-secretase. We identified three independent mutations within the ectodomain of Nct, which rescued expression of APH-1 but not of PEN-2 or PS and thus failed to restore gamma-secretase activity. Interestingly, these immature Nct variants selectively bound to APH-1, suggesting a stable Nct/APH-1 interaction independent of PS and PEN-2. Consistent with this finding, expression of APH-1 remained largely unaffected in the PS double knock-out and immature Nct co-immunoprecipitated with APH-1 in the absence of PS and PEN-2. Taken together, our findings suggest that immature Nct can stably interact with APH-1 to form a potential scaffold for binding of PS and PEN-2. Moreover, binding of the latter two complex partners critically depends on the integrity of the Nct ectodomain.     

Evaluation of aminolysis of anilinothiazolinones to phenylthiocarbamoyl amino acid methyl amides as an alternative conversion method in protein sequencing.

The aminolysis of products of sequential degradation of proteins and peptides by methylamine is an alternative method of conversion of the unstable 5-alkyl-2-anilino-4-thiazolinones into the stable methyl amides of N alpha-phenylthiocarbamoyl amino acids. The volatility of methylamine permits use in the gas phase during both manual and automatic sequential degradation. Two procedures were studied: (mode A) aminolysis by methylamine in the sequencer reaction chamber after liberation of the thiazolinones by trifluoroacetic acid and (mode B) aminolysis by methylamine vapors passed through a 1-chlorobutane solution of thiazolinones in the conversion flask of the sequencer. The sequencing program was modified for both procedures by making use of the standard sequencer functions. The yields of aminolysis in the conversion flask (mode B) are comparable to those obtained by standard conversion in 25% trifluoracetic acid and the procedure does not affect the repetitive yield. Aminolysis on the glass filter (mode A) requires a major modification of the degradation process, yet gives higher yields of the degraded amino acid derivatives. A disadvantage of both procedures, especially of mode A, is the presence of N-methyl-N'-phenylthiourea in the methyl amide samples. We have not been able to achieve the expected improvement of the yields of degraded hydroxy amino acids. Therefore the replacement of acid conversion of anilinothiazolinones to phenylthiohydantiones by aminolysis for routine degradation cannot be recommended. High yields of methyl amides make aminolysis a promising candidate for the incorporation of fluorescent or other labels in the products of sequencing degradation.     

A new species of Maladera Mulsant & Rey, 1871 from Iran (Coleoptera: Scarabaeidae: Sericini) and a review of the distribution of the genus in Iran

A new species of Maladera Mulsant & Rey, 1871 is described from Iran: M. kermanensis sp. n. The habitus and genitalia of the new species are illustrated. Additional records of Maladera species and a checklist of the species occurring in Iran including a map of their distribution are given.

http://www.zoobank.org/urn:lsid:zoobank.org:pub:812B8D62-2DD5-42CF-8BCD-F576CF3D2A7E     

Concentration of particular high molecular mass phosphoproteins in rat liver nuclei and nuclear matrix decreases following inhibition of RNA synthesis by α-amanitin

Purified liver nuclei were isolated from rats treated with non-lethal doses of α-amanitin, actinomycin D, galactosamine or cycloheximide. The nuclei were incubated in the presence of adenosine 5′-[γ-³²P]triphosphate, and digested with DNAase or DNAase plus high salt concentrations to prepare nuclear residual structures. Using SDS-polyacrylamide gel electrophoresis followed by autoradiography, samples from untreated rats were shown to contain major phosphoproteins in the range 76–260 kDa, with a prominent triplet of bands with 110, 117 and 128 kDa. Treatment of animals with α-amanitin or high doses of actinomycin D and galactosamine caused a significant decrease in the concentration of a few phosphorylated species, including the 110 kDa protein in whole nuclei, and their disappearance from the nuclear matrix or residual ribonucleoprotein structures after 1–3 h. The changes were reversible, complete recovery being observed after 5 h in the case of α-amanitin. No similar results were obtained with nuclei from rats treated with the translation inhibitor cycloheximide. The data are discussed in view of a possible effect of certain high molecular mass phosphoproteins on reactions of the heterogeneous nuclear RNA/mRNA pathway in the cell.     

Comparison between prochymosin and pepsinogen from lamb and calf

1. Prochymosin (EC 3.4.23.4) and pepsinogen A (EC 3.4.23.1) from Mongolian lamb (Ovis platyurea) were purified to homogeneity by salt precipitation, gel filtration and ion-exchange chromatography. 2. Immunoelectrophoresis shows partial immunochemical identity between chymosins and pepsins from lamb and cattle, respectively. 2. Activity determinations, N-terminal amino acid sequences and amino acid compositions also show a close relationship between the proteinases from lamb and cattle. 4. Lamb prochymosin and pepsinogen are both glycosylated.     

Use of zinc ions for fractionation of pig γ-G-globulin and its structural subunits

The different behaviour of the two fractions of pig γ-G-globulin prepared by interaction with zinc ions during oxidative sulphitolysis is described. The γ-G-globulin fraction which is not precipitated by zinc ions is dissociated more readily, as seen from the finding that, unlike the other fraction, it contains practically no incompletely dissociated molecules. Fractions of the light chains, with different molecular weights, were also isolated from this fraction. A technique was elaborated for separation of the component with H antigenic specificity which is present in the light chain preparation. Detailed study of this component showed that it is probably part of the heavy chain. The origin and formation of the component is discussed.     

Identical 110,000 dalton phosphoproteins in nuclei and polyribosomes of a rapidly growing rat hepatoma

Nuclei and free polyribosomes from rat hepatoma tissue were incubated $\text{in vitro}$ in the presence of [³²P] ATP. 0.2 N HCl-soluble nuclear proteins and polyribosomal proteins binding to oligo (dT) cellulose were prepared. A 110 kd phosphoprotein was separated by SDS polyacrylamide gel electrophoresis from each of the samples. The phosphoprotein was analysed using limited digestion with protease V8 in a second SDS gel. The patterns obtained for stained peptides and phosphopeptides were completely identical for both preparations. Thus significant amounts of the 110 kd protein appear to be present, as genuine constituents, in both nuclei and polyribosomes.