Biomedical subjects

T Diefenthal

Publications and source records attributed to T Diefenthal.

7 recordsLinked to original sources

PubMed1997

A homolog of the mammalian GTPase Rab2 is present in Arabidopsis and is expressed predominantly in pollen grains and seedlings.

Vesicle traffic between the endoplasmic reticulum and the Golgi apparatus in mammals requires the small GTP-binding protein Rab2, but Saccharomyces cerevisiae appears not to have a Rab2 homolog. Here it is shown that the higher plant, Arabidopsis thaliana, contains a gene, At-RAB2, whose predicted product shares 79% identity with human Rab2 protein. Transgenic plants containing fusions between beta-glucuronidase and sequences upstream of At-RAB2 demonstrated histochemical staining predominantly in maturing pollen and rapidly growing organs of germinating seedlings. beta-glucuronidase activity in pollen is first detectable at microspore mitosis and increases thereafter. In this respect, the promoter of At-RAB2 behaves like those of class II pollen-specific genes, whose products are often required after germination for pollen tube growth. Seedling germination and pollen tube growth are notable for their unusually high rates of cell wall and membrane biosynthesis. These results are consistent with a role for At-RAB2 in secretory activity.

Amino Acid Sequence↗

PubMed1996

Substrates containing phosphorylated residues adjacent to proline decrease the cleavage by proline-specific peptidases.

Thirteen dipeptide rho-nitroanilides of the common structure H-Xaa-Pro-4-NA (Xaa = serine, threonine and tyrosine) and seven tripeptide rho-nitroanilides of the common structure H-Gly-Xaa-Pro-4-NA (Xaa = serine or threonine) were prepared and analyzed as substrates of the proline-specific peptidases dipeptidyl peptidase IV and prolyl endopeptidase, respectively. The side chains of the hydroxy amino acids were synthetically modified by various acyl-, benzyl- and phosphate residues. The presence of aliphatic or aromatic residues attached to the side chain of the P2-hydroxy amino acids resulted in no significant change of the specificity constants of the enzyme-catalyzed substrate hydrolysis. In some cases, however, substrate inhibition was observed. In contrast, the reactivity of dipeptidyl peptidase IV and prolyl endopeptidase decreases more than two orders of magnitude towards the phosphorylated di- and tripeptide substrates compared to the hydrolysis of unmodified substrates. The kinetic data obtained with the model compounds suggest that side-chain modification of proline-containing peptide substrates may influence their resistance towards the hydrolytic activity of proline-specific hydrolases. Additionally, the results support that structural changes of the substrate during enzyme-hydrolysis may be involved in the mechanism of action of proline-specific serine peptidases. From this result we speculate that posttranslational phosphorylation of peptide sequences found in protein kinase recognition motifs such as -Xaa-Ser/Thr-Pro-Yaa- and -Xaa-Pro-Ser/Thr-Yaa- may serve as structural determinants that modulate their proteolytic stability.

Amino Acid Sequence↗

PubMed1994

Heterologous expression of the clostripain gene from Clostridium histolyticum in Escherichia coli and Bacillus subtilis: maturation of the clostripain precursor is coupled with self-activation.

Clostripain-specific antibodies were used to analyse the maturation of clostripain prepro-enzyme and core protein heterologously synthesized in Escherichia coli and Bacillus subtilis. Core protein purified from E. coli cells harbouring plasmid pHM3-23 underwent calcium-dependent, self-triggered maturation. Concomitantly, the inactive form of the enzyme was converted into an active form, demonstrating the self-activation capacity of the clostripain core protein. As judged from Western blot analysis, the major portion of the protein in E. coli was degraded, presumably by the activated clostripain. The enzyme was not exported to the E. coli periplasm, either by use of the putative Clostridium histolyticum signal peptide or by use of the E. coli OmpA signal peptide. Therefore, the Gram-positive micro-organism B. subtilis was chosen as an alternative host for the expression of the prepro-enzyme and the core protein. BR 151 cells harbouring pHM7-10B secreted clostripain precursor to the growth medium and matured subsequently to the active enzyme. As only a small amount of activity was detected intracellularly, the putative C. histolyticum signal peptide was efficiently recognized by the B. subtilis secretion apparatus. Under optimized conditions, a level of 4500 U I-1 could be obtained in batch cultures.

Bacillus subtilis↗

PubMed1993

Cloning of proline-specific endopeptidase gene from Flavobacterium meningosepticum: expression in Escherichia coli and purification of the heterologous protein.

Proline-specific endopeptidase (PSE) (EC 3.4.21.26) from Flavobacterium meningosepticum was subjected to partial amino acid sequencing. According to the peptide sequences obtained, oligonucleotides were used to amplify a PSE-specific DNA fragment of 930 bp from F. meningosepticum genomic DNA, employing the polymerase chain reaction technique. This fragment served as a molecular probe to isolate the respective gene. DNA sequencing revealed that the PSE gene consists of 2118 bp coding for a 78,634 Da protein of 705 amino acids. The coding region was cloned in different expression vectors of Escherichia coli. Transformed E. coli cells overproduce an active prolyl endopeptidase of 75,000 relative molecular mass, which is delivered to the bacterial periplasmic space. Up to 1.6 units of active prolyl endopeptidase were obtained from 1 mg E. coli cells. Furthermore, the efficient purification of active prolyl endopeptidase from the periplasm of recombinant E. coli cells is described.

Amino Acid Sequence↗

PubMed1993

The heterodimeric protease clostripain from Clostridium histolyticum is encoded by a single gene.

Clostripain (EC 3.4.22.8) is a heterodimeric cysteine endopeptidase with strict specificity for Arg-Xaa peptidyl bonds. It is secreted by Clostridium histolyticum strains. For the first time we present evidence that both polypeptide chains of native clostripain are encoded by a single gene. DNA sequencing of two overlapping genomic DNA fragments revealed a single open reading frame (ORF) of 1581 nucleotides encoding a polypeptide of 526 amino acid residues. The ORF is preceded by canonical transcription signals and both chains of the clostripain heterodimer are completely represented by the deduced coding sequence. Most interestingly, the sequences coding for the light and the heavy chain are joined by a DNA stretch coding for a linker nonapeptide that is preceded by the C-terminal arginyl residue of the light chain and also ends with an arginyl residue. Heterologous expression of the gene in Escherichia coli yielded an enzyme capable of hydrolyzing the clostripain substrates N alpha-benzoyl-L-arginine ethyl ester (BAEE) and N-carbobenzoxy-L-arginine p-nitroanilide (Z-Arg-pNA).

Amino Acid Sequence↗

PubMed1992

Molecular cloning and structural analysis of genes from Zea mays (L.) coding for members of the ras-related ypt gene family.

We have isolated, cloned, and characterized two cDNAs from Zea mays (L.), denoted yptm1 and yptm2, encoding proteins related to the ypt protein family. Amino acid similarity scores with YPT1 from yeast and ypt from mouse are in the range of 70% for yptm1 and 74% for yptm2, respectively, whereas similarities with p21 ras and other ras-related proteins are less than 40%. Most amino acid residues showing identity are clustered in the GTP/GDP binding domain. In addition, two cysteine residues close to the C-terminal ends, known to be palmitoylated and necessary for membrane binding in all eukaryotic ras-related proteins that have been characterized so far, are conserved in the maize genes as well. Northern blot hybridization analysis of poly(A)+ mRNA from etiolated maize coleoptiles revealed single mRNA species of approximately the same size as the isolated cDNAs. The gene for yptm1 is expressed at very low levels in maize coleoptiles and tissue culture cells. The gene for yptm2 is expressed at higher levels and is differentially represented in RNAs isolated from various organs of maize plants, with its highest level in leaves and flowers. The structural similarity of the genes identified suggests that they could be involved in the control of secretory processes.

Amino Acid Sequence↗

PubMed1992

The Ncypt1 gene from Neurospora crassa is located on chromosome 2: molecular cloning and structural analysis.

Small GTP-binding proteins are encoded by ras-like genes and play a central role in cell differentiation and membrane vesicle transport. By screening genomic and cDNA libraries of the Ascomycete fungus Neurospora crassa with Zmypt genes from Zea mays we have isolated a member of the ypt gene family, Ncypt1. The gene resides on a 4 kb fragment of genomic DNA and contains four introns, which interrupt the coding sequence of a protein of 203 amino acid residues. The Ncytp1 gene was assigned to a single-copy gene encoding a transcript of 1.5 kb and a protein of 26,000 daltons. The gene maps on linkage group IIR between DB0001 and ccg-2 close to the Fsr-3 locus. Analysis of the nucleotide sequence and the deduced protein sequence revealed a striking homology to yeast, mouse and human genes encoding small GTP-binding proteins that are related to the ras supergene family. Homology was most significant to ypt1 from Schizosaccharomyces pombe, Mus musculus and Homo sapiens sharing 84.8%, 82.3%, and 82.3% identity, respectively. Common domains present in other small GTP-binding proteins were identified in the predicted sequence of the NCYPT1 protein, and the arrangement of peptide motifs sharing similarity with well characterized, small GTP-binding proteins suggests that the NCYPT1 protein is a GTPase. The C-terminal region extending from amino acid residues 175 to 199 shares only weak amino acid sequence similarity with other eukaryotic GTPases. Like other RAS proteins the NCYPT1 protein contains two conserved C-terminal cysteine residues, suggesting post-translational modification(s) by fatty acylation required for membrane anchoring. The high degree of homology between the NCYPT1 protein and eukaryotic YPT proteins suggests that NCYPT1 could be involved in the control of secretory processes.

Amino Acid Sequence↗