Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The major excreted protein of transformed mouse fibroblasts (MEP) has recently been identified as the lysosomal cysteine protease, cathepsin L. The synthesis and intracellular trafficking of this protein in mouse fibroblasts are regulated by growth factors and malignant transformation. To further define the basis for this regulation, a cDNA encoding MEP/cathepsin L was isolated from a mouse liver cDNA library and used to compare cathepsin L of normal and Kirsten sarcoma virus-transformed NIH 3T3 fibroblasts. Although cathepsin L message levels were elevated 20-fold in the transformed fibroblasts, normal and transformed cells displayed similar cathepsin L genomic DNA digest patterns and gene copy numbers, and cathepsin L mRNA sequences appeared identical by RNase protection analysis. These findings indicate that (i) cathepsin L is synthesized from the same gene in normal and transformed cells and (ii) cathepsin L polypeptides made by these cells are translated with the same primary sequence. Cathepsin L polypeptides synthesized by quiescent, growing, and transformed cells displayed similar isoelectric focusing patterns, suggesting similar post-translational modification. Site-directed mutagenesis of the mouse liver cDNA and expression in COS monkey cells was used to examine the glycosylation of mouse cathepsin L. The results indicated that only one of the two potential N-linked glycosylation sites (the one at Asn221) is glycosylated. Analysis by ion exchange chromatography on QAE-Sephadex, and affinity chromatography on mannose 6-phosphate receptor-Affi-Gel 10, indicated that the cathepsin L oligosaccharide was phosphorylated similarly in normal and transformed cells. Although several phosphorylated oligosaccharide species were observed, the major species contained two phosphomonoester moieties and bound efficiently to the receptor. These findings suggest that cathepsin L made by normal and transformed mouse fibroblasts are identical and substantiate the hypothesis that trafficking of cathepsin L in these cells is regulated by growth-induced changes in the lysosomal protein transport system.
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PMID:Comparison of cathepsin L synthesized by normal and transformed cells at the gene, message, protein, and oligosaccharide levels. 227 56

The expression of cathepsin B- and L-specific mRNAs as well as active forms of the enzymes was determined in mouse placenta and visceral yolk sac from 7.5 through 17.5 days postconception, a period marked by major anatomic transitions in the mouse conceptus. The level of specific mRNA was determined relative to the 28S ribosomal RNA in a series of multiprobe ribonuclease protection assays using high-specific-activity antisense cathepsin B and L riboprobes. The molecular forms of active cysteine proteases present in the tissues at the time of extraction were detected using a membrane-permeant radiolabeled active site-specific inhibitor, Fmoc-[(125)I(2)]Tyr-Ala-CHN(2). The results of this study show that the expression of active cathepsin L relative to active cathepsin B is significantly higher in visceral yolk sac than in placenta, consistent with a higher proteolytic requirement for the former tissue. Active cathepsin L was highest at Day 9.5 in visceral yolk sac, a stage at which it has been shown that proteolysis in this organ is required for production of amino acids for embryonic protein synthesis. Cathepsin L mRNA was also elevated in the Day 9.5 placenta, but paradoxically this did not result in an increase in cellular active enzyme. An unknown protein, termed p14, highly expressed in placenta, also reacted with the inhibitor. Expression of this protein was highest early during gestation in the ectoplacental cone, suggesting that p14 may be important in the implantation process.
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PMID:Expression of cysteine proteases in extraembryonic tissues during mouse embryogenesis. 1060 Jan 78

In this study, we demonstrate that pGEM-4Z can be used as a mammalian expression vector. Western blotting and Immunocytochemical analyses revealed that transfection of pGEM-4Z-containing human cathepsin L cDNA under T-7 but not under SP-6 promoter into NIH 3T3 cells resulted in a high-level expression of cathepsin L. Expression of proteins using this vector in mammalian cells was further confirmed by using luciferase reporter gene. Furthermore, NIH 3T3 cells after stable or transient transfection with pGEM-4Z containing the first exon, first intron, and rest of the human cathepsin L cDNA downstream to its T-7 promoter synthesized and secreted large quantities of cathepsin L. RNase protection assays and 5' RACE established that the cloned cathepsin L cDNA is transcribed from a cryptic promoter present in the backbone of this vector upstream to T-7 sequence. This promoter was active in cell lines derived from four different mammalian species. In NIH 3T3 cells, this cryptic promoter could transcribe structural part of the genomic DNA into a primary transcript, which was efficiently spliced into mature mRNA and translated into protein. Thus this vector is equally useful for expressing proteins from genomic DNA. This hitherto unknown property of pGEM-4Z may be useful for expression of proteins in mammalian cells besides its use in synthesis of riboprobes, DNA sequencing, and in vitro transcription coupled translation assays.
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PMID:Expression of cloned cDNAs in mammalian cells from a cryptic promoter upstream to T7 in pGEM-4Z cloning vector. 1900 37

The phytophagous stink bug, Nezara viridula (L.) infests multiple plant species and impacts agricultural production worldwide. We analyzed the transcriptomes of N. viridula accessory salivary gland (ASG), principal salivary gland (PSG) and gut, with a focus on putative digestive proteases and nucleases that present a primary obstacle for the stability of protein- or nucleic acid-based stink bug control approaches. We performed high throughput Illumina sequencing followed by de novo transcriptome assemblies. We identified the sequences of 141 unique proteases and 134 nucleases from the N. viridula transcriptomes. Analysis of relative transcript abundance in conjunction with previously reported proteome data (Lomate and Bonning, 2016) supports high levels of serine protease expression in the salivary glands and high cysteine protease expression in the gut. Specifically, trypsin and chymotrypsin transcripts were abundant in the PSG, and cathepsin L-like cysteine protease transcripts were abundant in the gut. Nuclease transcript levels were generally lower than those of the proteases, the exception being abundant transcripts of ribonuclease-C20 in the PSG. The abundance of chymotrypsin, trypsin, and some carboxypeptidase transcripts suggests a significant role for the PSG in production of digestive enzymes. This result is at odds with the premise that the ASG produces watery saliva, which is high in enzymatic activity, while the PSG produces only sheath saliva. We have generated a comprehensive transcriptome sequence dataset from the digestive organs of N. viridula, identified major protease and nuclease genes and confirmed expression of the most abundant enzymes thereby providing greater insight into the digestive physiology of N. viridula.
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PMID:Tissue-specific transcription of proteases and nucleases across the accessory salivary gland, principal salivary gland and gut of Nezara viridula. 3035 60