Ribonuclease A variants with potent cytotoxic activity

Leland, Peter A.; Schultz, L. Wayne; Kim, Byung-Moon; Raines, Ronald T.

doi:10.1073/pnas.95.18.10407

Cited by 206 publications

(317 citation statements)

References 56 publications

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“…21,20 In the crystalline hRI·RNase 1 complex, the β4-β5 loop adopts a similar conformation to that of RNase A in the pRI·RNase A complex ( Figure 3). One major difference is with residue 88, as Asn88 of RNase 1 forms a hydrogen bond with Glu264 instead of residing in the pocket formed by Trp261 and Trp263.…”

Section: Recognition Of Rnase 1 By Rimentioning

confidence: 99%

“…Previous studies of the RI·ribonuclease interface have focused on short-range intermolecular contacts between the proteins, thereby raising the dissociation rate. 21,22,17 Diminishing the affinity of RNase 1, the human homologue of RNase A, by modulating short-range interactions has, however, proven to be difficult. 23-25 Although RNase 1 and RNase A share 70% sequence identity, mutagenesis studies have indicated substantial variation in how each is recognized by RI.…”

Section: Introductionmentioning

confidence: 99%

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Inhibition of Human Pancreatic Ribonuclease by the Human Ribonuclease Inhibitor Protein

Johnson

McCoy

Bingman

et al. 2007

Journal of Molecular Biology

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The ribonuclease inhibitor protein (RI) binds to members of the bovine pancreatic ribonuclease (RNase A) superfamily with an affinity in the femtomolar range. Here, we report on structural and energetic aspects of the interaction between human RI (hRI) and human pancreatic ribonuclease (RNase 1). The structure of the crystalline hRI·RNase 1 complex was determined at a resolution of 1.95 Å, revealing the formation of 19 intermolecular hydrogen bonds involving 13 residues of RNase 1. In contrast, only 9 such hydrogen bonds are apparent in the structure of the complex between porcine RI and RNase A. hRI, which is anionic, also appears to use its horseshoe-shaped structure to engender long-range Coulombic interactions with RNase 1, which is cationic. In accordance with the structural data, the hRI·RNase 1 complex was found to be extremely stable (t ½ = 81 days; K d = 2.9 × 10 -16 M). Site-directed mutagenesis experiments enabled the identification of two cationic residues in RNase 1-Arg39 and Arg91-that are especially important for both the formation and stability of the complex, and are thus termed "electrostatic targeting residues". Disturbing the electrostatic attraction between hRI and RNase 1 yielded a variant of RNase 1 that maintained ribonucleolytic activity and conformational stability but had a 2.8 × 10 3 -fold lower association rate for complex formation and 5.9 × 10 9 -fold lower affinity for hRI. This variant of RNase 1, which exhibits the largest decrease in RI affinity of any engineered ribonuclease, is also toxic to human erythroleukemia cells. Together, these results provide new insight into an unusual and important protein-protein interaction, and could expedite the development of human ribonucleases as chemotherapeutic agents.

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Section: Recognition Of Rnase 1 By Rimentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Inhibition of Human Pancreatic Ribonuclease by the Human Ribonuclease Inhibitor Protein

Johnson

McCoy

Bingman

et al. 2007

Journal of Molecular Biology

Self Cite

135

201

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“…It has been proposed that the role of RI in the cell is that of a ''sentry'', to protect its cytosolic RNA from extracellular RNases that may enter that compartment [5]. This hypothesis is based on the following evidence: RI resistant RNases, such as bovine seminal RNase [6] and onconase [7], are cytotoxic; noncytotoxic RNases, strongly inhibited by RI, become cytotoxic when they are engineered into relatively RI-resistant RNases [8]; cells manipulated to increase the RI levels become more resistant to RNase cytotoxicity [5,8,9].…”

Section: Introductionmentioning

confidence: 99%

The ribonuclease/angiogenin inhibitor is also present in mitochondria and nuclei

et al. 2011

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Edited by Vladimir SkulachevKeywords: Ribonuclease/angiogenin inhibitor Reactive oxygen species Reactive oxygen species scavenger Mitochondria Nucleus a b s t r a c tThe data presented here show for the first time that the protein known as ''ribonuclease (RNase) inhibitor'' (RI or RNH1) is present not only in the cell cytosol, but also in mitochondria, the central organelles in cell redox homeostasis. This finding directly correlates with the reported ability of RI to protect the cell from oxidative stress, with its sensitivity to oxidation and reactivity as a reactive oxygen species scavenger. While this study was carried out we also surprisingly discovered the presence of RI in the cell nucleus. We deem that these data open new views in the investigation on the cellular role(s) of the RI.

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“…6,7 This unusual biological activity is critically dependent on the ribonucleolytic activity 8 and on the ability of these molecules to reach the cellular cytosol and to degrade RNA by evading the action of the ribonuclease inhibitor (RI). 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.…”

Section: Introductionmentioning

confidence: 99%

“…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%

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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Ribonuclease A variants with potent cytotoxic activity

Cited by 206 publications

References 56 publications

Inhibition of Human Pancreatic Ribonuclease by the Human Ribonuclease Inhibitor Protein

Inhibition of Human Pancreatic Ribonuclease by the Human Ribonuclease Inhibitor Protein

The ribonuclease/angiogenin inhibitor is also present in mitochondria and nuclei

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

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