Inhibition of Human Pancreatic Ribonuclease by the Human Ribonuclease Inhibitor Protein
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RNase PH
Pancreatic ribonuclease
RNase H
Bovine pancreatic ribonuclease
S-tag
RNase MRP
Bovine seminal ribonuclease (BS-RNase), a dimeric homolog of bovine pancreatic ribonuclease A (RNase A), is toxic to mammalian cells. In contrast to dimeric BS-RNase, monomeric BS-RNase and RNase A are not cytotoxic and are bound tightly by cytosolic ribonuclease inhibitor. To elucidate the mechanism of ribonuclease cytotoxicity, we constructed a series of hybrid and semisynthetic enzymes and examined their properties. In five hybrid enzymes, divergent residues in BS-RNase were replaced with the analogous residues of RNase A so as to diminish an interaction with a putative cellular receptor. In a semisynthetic enzyme, the disulfide bonds that cross-link the monomeric subunits of dimeric BS-RNase were replaced with thioether bonds, which can withstand the reducing environment of the cytosol. Each hybrid and semisynthetic enzyme had ribonucleolytic and cytotoxic activities comparable with those of wild-type BS-RNase. These results suggest that dimeric BS-RNase (pI = 10.3) enters cells by adsorptive rather than receptor-mediated endocytosis and then evades cytosolic ribonuclease inhibitor so as to degrade cellular RNA. This mechanism accounts for the need for a cytosolic ribonuclease inhibitor and for the cytotoxicity of other homologs of RNase A. Bovine seminal ribonuclease (BS-RNase), a dimeric homolog of bovine pancreatic ribonuclease A (RNase A), is toxic to mammalian cells. In contrast to dimeric BS-RNase, monomeric BS-RNase and RNase A are not cytotoxic and are bound tightly by cytosolic ribonuclease inhibitor. To elucidate the mechanism of ribonuclease cytotoxicity, we constructed a series of hybrid and semisynthetic enzymes and examined their properties. In five hybrid enzymes, divergent residues in BS-RNase were replaced with the analogous residues of RNase A so as to diminish an interaction with a putative cellular receptor. In a semisynthetic enzyme, the disulfide bonds that cross-link the monomeric subunits of dimeric BS-RNase were replaced with thioether bonds, which can withstand the reducing environment of the cytosol. Each hybrid and semisynthetic enzyme had ribonucleolytic and cytotoxic activities comparable with those of wild-type BS-RNase. These results suggest that dimeric BS-RNase (pI = 10.3) enters cells by adsorptive rather than receptor-mediated endocytosis and then evades cytosolic ribonuclease inhibitor so as to degrade cellular RNA. This mechanism accounts for the need for a cytosolic ribonuclease inhibitor and for the cytotoxicity of other homologs of RNase A.
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The primary structure of a non-secretory ribonuclease from bovine kidney (RNase K2) was determined. The sequence determined was VPKGLTKARWFEIQHIQPRLLQCNKAMSGV NNYTQHCKPENTFLHNVFQDVTAVCDMPNIICKNGRHNCHQSPKPVNLTQCNFIAGRYPDC RYHDDAQYKFFIVACDPPQKTDPPYHLVPVHLDKYF. The sequence homology with human non-secretory RNase, bovine pancreatic RNase, and human secretory RNase are 46, 34.6, and 32.3%, respectively. The bovine kidney RNase has two inserted sequences, a tripeptide at the N-terminus and a heptapeptide between the 113th and 114th position of bovine pancreatic RNase; on the other hand, it is deleted of the hexapeptide consisting of the 17th to the 22nd amino acid residue of RNase A. The amino acid residues assumed to be the constituents of the bovine pancreatic RNase active site are all conserved except F120 (L in RNase K2).
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Bovine pancreatic ribonuclease
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Pancreatic ribonuclease
Tripeptide
Protein primary structure
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RNase PH
RNase MRP
Pancreatic ribonuclease
S-tag
Ribonuclease III
RNase H
Bovine pancreatic ribonuclease
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Bovine pancreatic ribonuclease
S-tag
Pancreatic ribonuclease
RNase PH
Alanine
Ribonuclease III
Aspartic acid
Angiogenin
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A cDNA coding for bovine pancreatic RNase A was mutagenized to insert a proline, a leucine, and 2 cysteine residues, i.e. the residues present at corresponding positions in the subunit of seminal RNase, the only dimeric RNase of the pancreatic-type superfamily. The mutant, expressed in Escherichia coli, eventually aggregated into catalytically active dimers. Like naturally dimeric seminal RNase, at equilibrium the mutant dimeric RNase A adopted two quaternary structures (one with an exchange of the N-terminal segments between partner subunits, the other with no exchange) and displayed a selective toxicity for malignant cells, absent in the monomeric, parent protein.
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Pancreatic ribonuclease
Bovine pancreatic ribonuclease
RNase PH
RNase MRP
Protein quaternary structure
Ribonuclease III
RNase H
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A ribonuclease (RNase) that cleaves specifically on the 3′ side of uridine [Shapiro, R., Fett, J. W., Strydom, D. J. & Vallee, B. L. (1986a) Biochemistry 25 , 7255–7264] was purified from human plasma and its amino acid sequence was determined. This protein is a 119‐residue single‐chain polypeptide cross‐linked by four disulfide bonds and has an amino‐terminal pyroglutaminyl residue. No post‐translational modifications were observed during extensive sequence studies on peptide fragments, except for the amino‐terminal pyroglutamic acid and a possible deamidation of Asn66. The protein is homologous to the pancreatic ribonucleases and angiogenin, but differs substantially from both of these proteins; the protein sequence has 43% identity with human pancreatic ribonuclease and 39% identity with human angiogenin, as compared to 35% identity between human angiogenin and pancreatic ribonuclease. It is referred to as RNase 4, based on the nomenclature currently used for the genes of pancreatic RNase (RNase 1) and the eosinophil‐derived RNases (RNase 2 and RNase 3). Virtually all of the RNase active‐site components, including the catalytic residues His12, His119 and Lys41, are preserved. However, some invariant residues of RNase 1 are replaced, e.g. Lys7 by arginine, Asp14 by histidine, and Pro42 by arginine. RNase 4 contains a unique two‐residue deletion at the position corresponding to amino acids 77 and 78 of pancreatic RNase, and its carboxy‐terminal sequence is truncated at position 122. The deletion in angiogenin at position 21 is also found in RNase 4. RNase 4 is very similar to two RNases isolated from bovine and porcine liver, and together they form a new family in the RNase superfamily. The degree of inter‐species similarity (90%) is much greater than within the pancreatic RNase and angiogenin families, which suggests that this ribonuclease could possess a physiologically important function other than general RNA catabolism.
Angiogenin
Pancreatic ribonuclease
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Ribonuclease III
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RNase MRP
Bovine pancreatic ribonuclease
RNase H
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RNase MRP
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Pancreatic ribonuclease
Bovine pancreatic ribonuclease
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RNase H
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Mammalian ribonucleases constitute one of the fastest evolving protein families in nature. The addition of a four‐residue carboxyl‐terminal tail: Glu‐Asp‐Ser‐Thr (EDST) in human pancreatic ribonuclease (HPR) in comparison with bovine pancreatic RNase (RNase A) could have adaptive significance in humans. We have cloned and expressed human pancreatic ribonuclease in Escherichia coli to probe the influence of the four‐residue extension and neighboring C‐terminal residues on the biochemical properties of the enzyme. Removal of the C‐terminal extension from HPR yielded an enzyme, HPR‐(1–124)‐peptide, with enhanced ability to cleave poly(C). HPR‐(1–124)‐peptide also exhibited a steep increase in thermal stability mimicking that known for RNase A. Wild‐type HPR had significantly low thermal stability compared to RNase A. The study identifies the C‐terminal boundary in the human pancreatic ribonuclease required for efficient catalysis.
Bovine pancreatic ribonuclease
Pancreatic ribonuclease
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RNase PH
Cleave
RNase H
Ribonuclease III
Residue (chemistry)
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RNase PH
Pancreatic ribonuclease
RNase H
Bovine pancreatic ribonuclease
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RNase MRP
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Citations (142)
A variant of bovine pancreatic ribonuclease A has been prepared with seven amino acid substitutions (Q55K, N62K, A64T, Y76K, S80R, E111G, N113K). These substitutions recreate in RNase A the basic surface found in bovine seminal RNase, a homologue of pancreatic RNase that diverged some 35 million years ago. Substitution of a portion of this basic surface (positions 55, 62, 64, 111 and 113) enhances the immunosuppressive activity of the RNase variant, activity found in native seminal RNase, while substitution of another portion (positions 76 and 80) attenuates the activity. Further, introduction of Gly at position 111 has been shown to increase the catalytic activity of RNase against double‐stranded RNA. The variant and the wild‐type (recombinant) protein were crystallized and their structures determined to a resolution of 2.0 Å. Each of the mutated amino acids is seen in the electron density map. The main change observed in the mutant structure compared with the wild‐type is the region encompassing residues 16–22, where the structure is more disordered. This loop is the region where the polypeptide chain of RNase A is cleaved by subtilisin to form RNase S, and undergoes conformational change to allow residues 1–20 of the RNase to swap between subunits in the covalent seminal RNase dimer.
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Bovine pancreatic ribonuclease
RNase PH
Pancreatic ribonuclease
RNase MRP
Ribonuclease III
RNase H
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