The antitumor action of seminal ribonuclease and its quaternary conformations

Cafaro, Valeria; Lorenzo, Claudia De; Piccoli, Renata; Bracale, Aurora; Mastronicola, Maria Rosaria; Donato, Alberto Di; D’Alessio, Giuseppe

doi:10.1016/0014-5793(94)01450-f

Cited by 76 publications

(67 citation statements)

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“…9,10 It is currently accepted that RI, which binds most RNase A family members with femtomolar affinities, plays an important role in the protection of host cells from endogenous RNases. [9][10][11] Only frog RNases such as Onconase TM (ONC) 12 and BSRNase [13][14][15] are resistant to human RI. The former, which is in Phase III clinical trials as an antitumor agent against malignant mesothelioma, 3 escapes RI because it lacks many of the residues involved in the RNase A-RI recognition.…”

Section: Introductionmentioning

confidence: 99%

“…17 Although the two dimers present an almost undistinguishable external shape, 21 the cytotoxic action is a peculiar property of MxM-BSRNase. [13][14][15] Following the experimental results obtained in vitro, it is believed that in the reducing cytosolic compartment M¼M-BSRNase dissociates into monomers, which are strongly inhibited by RI, whereas MxM-BSRNase survives as NCD-BSRNase. This dimer presents an overall structure highly reminiscent of the covalent forms, with the cysteine residues involved in the intersubunit linkages not far away from each other.…”

Section: Introductionmentioning

confidence: 99%

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Structural features for the mechanism of antitumor action of a dimeric human pancreatic ribonuclease variant

et al. 2008

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A specialized class of RNases shows a high cytotoxicity toward tumor cell lines, which is critically dependent on their ability to reach the cytosol and to evade the action of the ribonuclease inhibitor (RI). The cytotoxicity and antitumor activity of bovine seminal ribonuclease (BSRNase), which exists in the native state as an equilibrium mixture of a swapped and an unswapped dimer, are peculiar properties of the swapped form. A dimeric variant (HHP2-RNase) of human pancreatic RNase, in which the enzyme has been engineered to reproduce the sequence of BSRNase helix-II (Gln28fiLeu, Arg31fiCys, Arg32fiCys, and Asn34fiLys) and to eliminate a negative charge on the surface (Glu111fiGly), is also extremely cytotoxic. Surprisingly, this activity is associated also to the unswapped form of the protein. The crystal structure reveals that on this molecule the hinge regions, which are highly disordered in the unswapped form of BSRNase, adopt a very well-defined conformation in both subunits. The results suggest that the two hinge peptides and the two Leu28 side chains may provide an anchorage to a transient noncovalent dimer, which maintains Cys31 and Cys32 of the two subunits in proximity, thus stabilizing a quaternary structure, similar to that found for the noncovalent swapped dimer of BSRNase, that allows the molecule to escape RI and/or to enhance the formation of the interchain disulfides.Keywords: crystal structure; antitumor agents; ribonuclease; 3D-domain swapping; dimers Abbreviations: BSRNase, bovine seminal ribonuclease; des(1-7)HP-RNase, human pancreatic ribonuclease in which the first seven residues have been deleted; HHP-RNase, covalent dimeric variant of human pancreatic ribonuclease in which four residues at the helix-II region are mutated according to BSRNase sequence (Gln28!Leu, Arg31!Cys, Arg32!Cys, and Asn34!Lys); HHP2-RNase, HHP-RNase with an additional mutation (Glu111!Gly); HP-RI, structure of human pancreatic ribonuclease complexed with ribonuclease inhibitor; HP-RNase, human pancreatic ribonuclease; MxM-BSRNase, dimeric form of BSRNase in which the chains swap their N-terminal tails; M¼M-BSRNase, unswapped dimer of BSRNase; M¼M-HHP2, unswapped dimer of HHP2-RNase; NCD-BSRNase, noncovalent swapped form of BSRNase; ONC, Onconase; PDB, Protein Data Bank; PM7, human pancreatic ribonuclease in which residues 1-21 are replaced by the corresponding residues of BSRNase; PM8, N-terminal swapped dimer of a variant of human pancreatic ribonuclease; RI, ribonuclease inhibitor; RMSD, root-mean-square deviation.Grant sponsor: Ministero dell'Istruzione, dell'Università e della Ricerca; Grant number: FIRB RBNE03B8KK and PRIN2007.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Structural features for the mechanism of antitumor action of a dimeric human pancreatic ribonuclease variant

et al. 2008

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“…In fact, although dimeric BS-RNase has lower activity, it displays mixed cooperativity, in contrast to monomeric BS-RNase and RNase A (Piccoli et al, 1988). In addition, domain-swapped dimeric BS-RNase displays selective toxicity for tumor cells, whereas monomeric BS-RNase and RNase A do not (Cafaro et al, 1995). Thus, domain-swapped BS-RNase differs both in structure and function from monomeric BS-RNase, even though both are formed from the same protein chain.…”

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confidence: 99%

3D domain swapping: A mechanism for oligomer assembly

1995

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Abstract3D domain swapping is a mechanism for forming oligomeric proteins from their monomers. In 3D domain swapping, one domain of a monomeric protein is replaced by the same domain from an identical protein chain. The result is an intertwined dimer or higher oligomer, with one domain of each subunit replaced by the identical domain from another subunit. The swapped "domain" can be as large as an entire tertiary globular domain, or as small as an a-helix or a strand of a P-sheet. Examples of 3D domain swapping are reviewed that suggest domain swapping can serve as a mechanism for functional interconversion between monomers and oligomers, and that domain swapping may serve as a mechanism for evolution of some oligomeric proteins. Domain-swapped proteins present examples of a single protein chain folding into two distinct structures.Keywords: aggregation; complementation; oligomer evolution; protein dimerization Since Svedberg's discovery of functional molecules composed of two or more identical protein chains, much effort has been expended in studying their metabolic regulation (Monod et al., 1965;Koshland et al., 1966) and their assembly and disassembly (Kikuchi & King, 1975;Caspar, 1980;Jaenicke, 1995).Despite this progress, understanding the assembly of oligomeric proteins from monomers remains a challenge. A common observation is that disassembly of an oligomeric protein into its monomeric subunits is accompanied by irreversible unfolding and aggregation. This observation is often interpreted in terms of exposing apolar patches on the monomer surface that are covered in the oligomer, thereby providing binding energy from a hydrophobic interaction. Thus, the question remains of how the oligomer could have been assembled in the first place. We propose an answer to this question for some oligomers based on a mode of association that we have noticed in several proteins of Reprint requests to: David Eisenberg, Molecular Biology Institute, Department of Chemistry and Biochemistry, and UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, University of California-Los Angeles, Los Angeles, California 90095-1570; e-mail: david@pauling.rnbi.ucla.edu.Abbreviations: BS-RNase, bovine seminal ribonuclease; DT, diphtheria toxin; GM-CSF, granulocyte-macrophage colony-stimulating factor; GST, glutathione S-transferase; IF, interferon; IL, interleukin; RNase, ribonuclease. known structure. We term this mode of association 3 0 domain swapping, because oligomers are formed from stable monomers by exchanging domains.A problem related to the formation of oligomeric proteins in a cell is the problem of how oligomeric proteins evolved from monomeric precursor proteins. For an oligomer to evolve, random mutations must change the surface of the monomer so that sufficient free energy is released upon oligomerization to overcome the accompanying entropy loss of immobilizing the monomers. As we discuss in this review, single amino acid replacements must be fortuitous to provide an adequate free energy of interaction. But an ...

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“…The cytosol of mammalian cells contains a ribonuclease inhibitor (RI) 1 protein that binds tightly to mammalian secretory ribonucleases (19,20). The ability of secretory ribonucleases from human (21), cow (22)(23)(24), bull (25)(26)(27), and frog (28) to evade RI endows them with toxicity for mammalian cells. These secretory ribonucleases from vertebrates are not homologous to microbial ribonucleases.…”

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confidence: 99%