Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana

Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen. A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical proteins (97% identity). Both proteins are 306 amino acids in length and are very similar (79–80% identity) to the mammalian isotypes of the catalytic subunit of protein phosphatase 2A. Therefore, they have been designated PP2A-1 and PP2A-2. A third cDNA clone, EP7, was isolated and sequenced. The polypeptide encoded (308 amino acids, lacking the initial Met codon) is 80% identical with human phosphatases 2A and was named PP2A-3. The PP2A-3 protein is extremely similar (95% identity) to the predicted protein from a cDNA clone previously found in Brassica napus. Southern blot analysis of genomic DNA using AP-1 and AP-2 probes, as well as probes derived from clones EP7, EP8a and EP14a strongly indicates that at least 6 genes closely related to type 2A phosphatases are present in the genome of A. thaliana. Northern blot analysis using the same set of probes demonstrates that, at the seedling stage, the mRNA levels for PP2A-1, PP2A-3 and the gene containing the AP-1 sequence are much higher than those of PP2A-2 and AP-2. These results demonstrate that a multiplicity of type 2A phosphatases might be differentially expressed in higher plants.     

Substitution of transferrin by FeCl3 in the development of a low foetal calf serum concentration medium for KB-26.5 hybridoma cell line

KB-26.5, a murine hybridoma cell line producing an IgG₃ monoclonal antibody used in blood type determination, primarily adapted to grow at 5% foetal calf serum (FCS) concentration has been adapted to grow at 0.5% FCS, maintaining its ability to produce antibodies at the same level. In the final step of adaptation, the addition of insulin, transferrin, ethanolamine and selenium to the media formulation was studied, using factorial assay techniques to check the effect of the different compounds and to optimize their required level for satisfactory growth and antibody secretion. KB-26.5 cells required only 20 g/ml of transferrin to adapt to 0.5% FCS medium. Furthermore, transferrin could be substituted by FeCl₃, at a relatively low level of 2 g/ml. Maximum cell density decreased by 31.5% in spinner flask test, but the antibody titer was maintained, thus the specific productivity increased. However, inoculum size had to be increased three-fold with 0.5% FCS medium in order to assure cell growth.     

Analysis of the effect of potato carboxypeptidase inhibitor pro-sequence on the folding of the mature protein.

Protein folding can be modulated in vivo by many factors. While chaperones act as folding catalysts and show broad substrate specificity, some pro-peptides specifically facilitate the folding of the mature protein to which they are bound. Potato carboxypeptidase inhibitor (PCI), a 39-residue protein carboxypeptidase inhibitor, is synthesized in vivo as a precursor protein that includes a 27-residue N-terminal and a seven-residue C-terminal pro-regions. In this work the disulfide-coupled folding of mature PCI in vitro has been compared with that of the same protein extended with either the N-terminal pro-sequence (ProNtPCI) or both N- and C-terminal pro-sequences (ProPCI), and also with the N-terminal pro-sequence in trans (ProNt + PCI). No significant differences can be observed in the folding kinetics or efficiencies of all these molecules. In addition, in vivo folding studies in Escherichia coli have been performed using wild-type PCI and three PCI mutant forms with and without the N-terminal pro-sequence, the mutations had been previously reported to affect folding of the PCI mature form. The extent to which the 'native-like' form was secreted to the media by each construction was not affected by the presence of the N-terminal pro-sequence. These results indicate that PCI does not depend on the N-terminal pro-sequence for its folding in both, in vitro and in vivo in E. coli. However, structural analysis by spectroscopy, hydrogen exchange and limited proteolysis by mass spectrometry, indicate the capability of such N-terminal pro-sequence to fold within the precursor form.     

On the conformational dependence of the proton chemical shifts in nucleosides and nucleotides III. Proton chemical shifts of 5′-nucleotides as a function of different conformational parameters

The variation of the chemical shift of the protons of 5′-UMP and 5′-AMP is calculated as a function of χ_CN, ψ and ? torsion angles. The shift of H8 of 5′-AMP and H6 of 5′-UMP is found to be very sensitive to the value of χ_CN. For the anti conformations the shift of these protons is more sensitive to the value of the rotation about CS′-05′ than about C4′-CS′. For the protons of the ribose the calculations show that for the C2′-endo pucker H3′ and H2′ undergo the largest chemical shift variations when ? and ψ vary. The calculated variations are considered in relation with the role of the conformation of the nucleotides in the chemical shift variation between mono and polynucleotides and between the different helical structures of polynucleotides.     

Isolation and re-association of the subunits from the pro-(carboxypeptidase A)–pro-(proteinase E) binary complex from pig pancreas

The component subunits of the pro-(carboxypeptidase A)–pro-(proteinase E) binary complex from pig pancreas were separated with a high recovery (80–95%) of their original potential activity. The isolated subunits and the reconstituted complex have properties similar to those of the corresponding natural species. The tryptic activation course of the pro-(carboxypeptidase A) subunit is substantially modified when bound to pro-(proteinase E), whereas the activation of pro-(proteinase E) is not dependent on this association.     

Architecture and function of metallopeptidase catalytic domains

The cleavage of peptide bonds by metallopeptidases (MPs) is essential for life. These ubiquitous enzymes participate in all major physiological processes, and so their deregulation leads to diseases ranging from cancer and metastasis, inflammation, and microbial infection to neurological insults and cardiovascular disorders. MPs cleave their substrates without a covalent intermediate in a single‐step reaction involving a solvent molecule, a general base/acid, and a mono‐ or dinuclear catalytic metal site. Most monometallic MPs comprise a short metal‐binding motif (HEXXH), which includes two metal‐binding histidines and a general base/acid glutamate, and they are grouped into the zincin tribe of MPs. The latter divides mainly into the gluzincin and metzincin clans. Metzincins consist of globular ～130–270‐residue catalytic domains, which are usually preceded by N‐terminal pro‐segments, typically required for folding and latency maintenance. The catalytic domains are often followed by C‐terminal domains for substrate recognition and other protein–protein interactions, anchoring to membranes, oligomerization, and compartmentalization. Metzincin catalytic domains consist of a structurally conserved N‐terminal subdomain spanning a five‐stranded β‐sheet, a backing helix, and an active‐site helix. The latter contains most of the metal‐binding motif, which is here characteristically extended to HEXXHXXGXX(H,D). Downstream C‐terminal subdomains are generally shorter, differ more among metzincins, and mainly share a conserved loop—the Met‐turn—and a C‐terminal helix. The accumulated structural data from more than 300 deposited structures of the 12 currently characterized metzincin families reviewed here provide detailed knowledge of the molecular features of their catalytic domains, help in our understanding of their working mechanisms, and form the basis for the design of novel drugs.     

Human mitochondrial disease-like symptoms caused by a reduced tRNA aminoacylation activity in flies

The translation of genes encoded in the mitochondrial genome requires specific machinery that functions in the organelle. Among the many mutations linked to human disease that affect mitochondrial translation, several are localized to nuclear genes coding for mitochondrial aminoacyl-transfer RNA synthetases. The molecular significance of these mutations is poorly understood, but it is expected to be similar to that of the mutations affecting mitochondrial transfer RNAs. To better understand the molecular features of diseases caused by these mutations, and to improve their diagnosis and therapeutics, we have constructed a Drosophila melanogaster model disrupting the mitochondrial seryl-tRNA synthetase by RNA interference. At the molecular level, the knockdown generates a reduction in transfer RNA serylation, which correlates with the severity of the phenotype observed. The silencing compromises viability, longevity, motility and tissue development. At the cellular level, the knockdown alters mitochondrial morphology, biogenesis and function, and induces lactic acidosis and reactive oxygen species accumulation. We report that administration of antioxidant compounds has a palliative effect of some of these phenotypes. In conclusion, the fly model generated in this work reproduces typical characteristics of pathologies caused by mutations in the mitochondrial aminoacylation system, and can be useful to assess therapeutic approaches.