Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.
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PMID:Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus. 292 99

An efficient system for the import of newly synthesized proteins into highly purified rat liver peroxisomes was reconstituted in vitro. 35S-Labeled acyl-CoA oxidase (AOx) was incorporated into peroxisomes in a proteinase K-resistant fashion. This import was specific (did not occur with mitochondria) and was dependent on temperature, time, and peroxisome concentration. Under optimal conditions approximately 30% of [35S]AOx became proteinase resistant. The import of AOx into peroxisomes could be dissociated into two steps: (a) binding occurred at 0 degrees C in the absence of ATP; (b) translocation occurred only at 26 degrees C and required the hydrolysis of ATP. GTP would not substitute for ATP and translocation was not inhibited by carbonylcyanide-m-chlorophenylhydrazone, valinomycin, or other ionophores.
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PMID:Translocation of acyl-CoA oxidase into peroxisomes requires ATP hydrolysis but not a membrane potential. 369 2

We have previously described mutant S. cerevisiae that are defective in peroxisome biogenesis (peb mutants) (Zhang, J. W., Y. Han, and P. B. Lazarow. 1993. J. Cell Biol. 123:1133-1147.). In some mutants, peroxisomes are undetectable. Other mutants contain normal-looking peroxisomes but fail to package subsets of peroxisomal proteins into the organelle (Zhang, J. W., C. Luckey, and P. B. Lazarow. 1993. Mol. Biol. Cell. 4:1351-1359.). In peb1 (pas7) cells, for example, the peroxisomes contain proteins that are targeted by COOH-terminal tripeptides and contain acyl-CoA oxidase (which is probably targeted by internal oligopeptides), but fail to import thiolase (which is targeted by an NH(2)-terminal 16-amino acid sequence). These and other data suggest that there are three branches in the pathway for the import of proteins into peroxisomes, each of which contains a receptor for one type of peroxisomal topogenic information. Here, we report the cloning and characterization of the PEB1 gene, that encodes a 42,320-Da hydrophilic protein with no predicted transmembrane segment. The protein contains six WD repeats, a motif which has been found in 27 proteins involved in diverse cellular functions. The PEB1 gene product was tagged with the hemagglutinin epitope and found to rescue thiolase import in the peb1 null mutant. The epitope-tagged protein was shown to be inside of peroxisomes by immunofluorescence, digitonin permeabilization, equilibrium density centrifugation, immunoelectron microscopy, and proteinase K protection studies. The PEB1 gene product does not cleave the thiolase-targeting sequence. It may function to draw thiolase into peroxisomes.
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PMID:PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intra-peroxisomal protein that is a member of the WD repeat family and is essential for the import of thiolase into peroxisomes. 753 4

Rat liver urate oxidase synthesized from cDNA through coupled transcription and translation was incubated at 26 degrees C for 60 min with purified peroxisomes from rat liver. Urate oxidase was efficiently imported into the peroxisomes, as determined by resistance to externally added proteinase K. The amount of imported urate oxidase increased with time and the import was temperature dependent. A synthetic peptide composed of the C-terminal 10 amino acid residues of acyl-CoA oxidase (the C-terminal tripeptide is Ser-Lys-Leu) inhibited the import of urate oxidase, whereas other peptides, in which the C-terminal Ser-Lys-Leu (SKL) sequence was deleted or mutated, were not effective. Two mutant urate oxidase proteins in which the C-terminal Ser-Arg-Leu (SRL) sequence was deleted or mutated to Ser-Glu-Leu (SEL) were not imported into peroxisomes. With substitution of a lysine residue for arginine in the SRL tripeptide at the C-terminus the import activity was retained. These results show that urate oxidase is important into peroxisomes via a common pathway with acyl-CoA oxidase, and that the C-terminal SRL sequence functions as a peroxisomal-targeting signal.
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PMID:Urate oxidase is imported into peroxisomes recognizing the C-terminal SKL motif of proteins. 803 87

To understand the targeting mechanisms of peroxisomal serine:pyruvate aminotransferase, in vitro import experiments were carried out using this 43 kDa peroxisomal enzyme, which was synthesized in a coupled transcription/translation system. Being different from other peroxisomal enzymes, such as acyl-CoA oxidase and urate oxidase used in previous in vitro import experiment, the soluble form of peroxisomal serine:pyruvate aminotransferase was fairly resistant to proteinase K digestion. However, the enzyme recovered in the peroxisomal fraction was proteinase K sensitive. This indicates that the association of peroxisomal serine:pyruvate aminotransferase with the peroxisomal membrane causes a marked conformational change in the structure of this protein.
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PMID:In vitro association with peroxisomes and conformational change of peroxisomal serine:pyruvate/alanine:glyoxylate aminotransferase in rat and human livers. 892 Sep 16

A non-radioactive in situ hybridization (ISH) protocol for localization of mRNAs encoding peroxisomal proteins in hepatoma cell lines from humans (HepG2) and rats (MH1C1) is presented. In comparison to a similar procedure reported for tissue sections, the cell culture preparations require only brief fixation in 4% paraformaldehyde and their permeabilization is achieved by a very low concentration (1 microg/ml) of proteinase K. The exclusive localization of transcripts in the cytoplasm of hepatoma cells with the absence of nuclear staining and the completely negative sense controls confirm the specificity of the method. The marked differences in signal intensity between the results of albumin and beta-actin mRNAs which are of high abundance in contrast to moderate to low abundance of peroxisomal mRNAs show the high sensitivity and the wide range of applicability of our protocol. This is also confirmed by divergent results of treatment of hepatoma cell lines with clofibrate and cetaben on mRNA levels of catalase and acyl-CoA oxidase. The ISH results of drug treatment of cell lines are confirmed also by slot blot analysis of total RNA extracts using 32P-labeled probes. Thus the protocol presented here provides a sensitive tool for ISH localization of mRNAs encoding peroxisomal proteins. In combination with immunocytochemistry it may be useful to monitor intercellular differences in expression levels of specific mRNAs in correlation with the abundance of structurally divergent forms of peroxisomes (tubular versus spherical) and their importance in the biogenesis of peroxisomes.
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PMID:Localization of mRNAs encoding peroxisomal proteins in cell culture by non-radioactive in situ hybridization. Comparison of rat and human hepatoma cells and their responses to two divergent hypolipidemic drugs. 1145 50

Recently, we isolated CHO cells, termed SK32 cells, that express mutant Pex5p (G432R), and showed mislocalization of catalase in the cytosol, but peroxisomal localization of 3-ketoacyl-CoA thiolase (thiolase) in the mutant cells [Ito, R. et al. (2001) Biochem. Biophys. Res. Commun. 288, 321-327]. While analyzing the mutant cells, we found a novel Pex5p isoform (Pex5pM), which was shorter by seven amino acids than Pex5pL and longer by 30 amino acids than Pex5pS. Similar levels of mRNA syntheses for the PEX5 gene were observed in both the wild type and mutant cells, but the protein levels of Pex5p isoforms were markedly reduced in the mutant cells cultured at 37 degrees C and only slightly discernible at 30 degrees C, suggesting that they could be rapidly degraded. Furthermore, we characterized the peroxisomal localization of thiolase and acyl-CoA oxidase (Aox) in SK32 cells. The proteins in the organelle fraction were protected from proteinase K-digestion in the mutant cells, indicating that they were translocated inside peroxisomes. However, the conversion of Aox from component A to components B and C was completely prevented at both 30 and 37 degrees C, and the precursor form of thiolase was partially processed to the mature one in a temperature-sensitive manner. Transformed SK32 cells stably expressing one of the wild type Pex5p isoforms were isolated, and then the maturation steps for thiolase and Aox were examined. Pex5pM and S restored the processing of the two enzymes, but Pex5pL did not. In addition, Pex5pL prevented the maturation of thiolase observed at 30 degrees C. These results indicate that (i) the novel Pex5pM is functional and (ii) a seven amino acids-insertion, which is present in the L isoform but absent in the M isoform, plays some role in the process of maturation of thiolase and Aox.
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PMID:Identification of Pex5pM, and retarded maturation of 3-ketoacyl-CoA thiolase and acyl-CoA oxidase in CHO cells expressing mutant Pex5p isoforms. 1642 7