Role of the N Terminus in RNase A Homologues: Differences in Catalytic Activity, Ribonuclease Inhibitor Interaction and Cytotoxicity

Boix, Ester; Wu, You-Neng; Vasandani, Veena M.; Saxena, Shailendra K.; Ardelt, Wojciech; Ladner, Jane E.; Youle, Richard J.

doi:10.1006/jmbi.1996.0218

Cited by 181 publications

(228 citation statements)

References 0 publications

Supporting

Mentioning

218

Contrasting

Order By: Relevance

“…12,29,39 This broad specificity may reflect the role of RI as a sentry to protect cells from all of the pancreatic RNase superfamily enzymes that can pass through the cellular membrane. Therefore, the capability to evade RI may confer cytotoxic properties to the molecule and have potential medical applications.…”

Section: Discussionmentioning

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%

See 1 more Smart Citation

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

et al. 2008

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2008

View full text Add to dashboard Cite

show abstract

“…Like RNase A, human pancreatic RNase [K i ϭ 200 fM for WT hRI (12)] is not inhibited detectably by C408W͞⌬L409͞G410W hRI. Thus, all physiologically relevant binding to the most prevalent human RNase has been eliminated.…”

Section: Discussionmentioning

confidence: 99%

“…The affinity of human RI (hRI), a 50-kDa leucine-rich repeat protein, for its most avid ligand, the 14.1-kDa blood vessel-inducing RNase angiogenin (ANG), is among the highest on record for any protein-protein interaction [K i ϭ 0.5-0.7 fM (8,9)]. Although nonorthologous ligands of RI share no more than Ϸ35% sequence identify with ANG, they all bind with K i values that are still in the femtomolar range (7,8,(10)(11)(12). This broad specificity may reflect the role of RI as a sentry to protect cells from all of the pancreatic RNase superfamily enzymes that inadvertently enter into the cytosol (13,14).…”

mentioning

confidence: 99%

Selective abolition of pancreatic RNase binding to its inhibitor protein

Kumar

Brady

Shapiro

2003

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We have modified RNase inhibitor (RI) protein so that it no longer detectably binds pancreatic RNases but retains near-native affinity for human angiogenin (ANG). The Ki value for RNase A is increased by a factor of >10 8 , from 36 fM to >4 M, and the selectivity factor for ANG is now >10 9 . This dramatic change was achieved by remodeling the human RI loop segment Cys-408 -Leu-409 -Gly-410, which makes minor interactions with pancreatic RNase but does not contact ANG. The modifications selected were designed to sterically hinder docking of the undesired ligand. Three of the variants tested (C408W, G410W, and C408W͞G410W) bind RNase A with almost the same avidity as WT RI. However, combination of the 408͞410 double Trp replacement with deletion of the intervening residue, Leu-409, was sufficient to abolish inhibition of RNase A and human pancreatic RNase. The Ki value for ANG with the deletion variant is 1.1 fM, only 2-fold higher than with WT RI. This variant may have potential utility both as an anticancer drug targeting ANG and as a tool for the investigation of the biological function of ANG. More generally, these findings demonstrate that a protein-protein interaction can be effectively and specifically disrupted by redesigning an interface region that makes no major energetic contribution to complex stability. This finding, in turn, may have implications for the development of small molecules that modulate protein-protein interactions.M ost biological processes critically depend on accurate recognition of proteins by other proteins. Recognition is often highly selective, involving the binding of a single predominant partner by a particular protein surface or domain. However, in many cases, an individual domain must have the capacity to recognize multiple structurally related ligands to fulfill its function. This type of promiscuity can be based on recognition of a common short motif, as for the phosphorylated substrates bound by F-box proteins (1, 2) and Cbl (3). Alternatively, binding can involve extended regions of homologous proteins, as for the interactions of various hormones with different receptors (4), Cdk2 with cyclins (5), and cytosolic RNase inhibitor (RI) with members of the mammalian pancreatic RNase superfamily (see refs. 6 and 7 for reviews).The RI-RNase system represents a particularly remarkable example of natural protein engineering. The affinity of human RI (hRI), a 50-kDa leucine-rich repeat protein, for its most avid ligand, the 14.1-kDa blood vessel-inducing RNase angiogenin (ANG), is among the highest on record for any protein-protein interaction [K i ϭ 0.5-0.7 fM (8, 9)]. Although nonorthologous ligands of RI share no more than Ϸ35% sequence identify with ANG, they all bind with K i values that are still in the femtomolar range (7,8,(10)(11)(12). This broad specificity may reflect the role of RI as a sentry to protect cells from all of the pancreatic RNase superfamily enzymes that inadvertently enter into the cytosol (13,14).In principle, RI might have achieved its relaxed selectivi...

show abstract

“…Because RNases from mammalian sources are silenced due to the tight binding by the intracellular inhibitor protein (RI) and, thus, are not cytotoxic (6), various strategies have been mapped out to accomplish cytotoxicity. Inspired by the cytotoxicity of the amphibian Onconase (5,7,8) and the dimeric bovine seminal RNase (9), tremendous efforts have been made to create RNase derivatives that evade RI binding. While the generation of chimeras (10 -13), the use of chemical modifications (14 -17), or site-directed mutagenesis (5, 6, 18 -21) proved to be strategies of rather varying success, we developed RNase A tandem enzymes (22) in which two RNase A molecules are coupled covalently by a peptide linker.…”

mentioning

confidence: 99%

Endocytotic Internalization as a Crucial Factor for the Cytotoxicity of Ribonucleases

Leich

Stöhr

Rietz

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

The cytotoxic action of ribonucleases (RNases) requires the interaction of the enzyme with the cellular membrane, its internalization, translocation to the cytosol, and the degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the evasion from the intracellular ribonuclease inhibitor (RI) has not yet been fully elucidated. As cytosolic internalization is indispensable for the cytotoxicity of extracellular ribonucleases, we investigated the extent of cytosolic internalization of a cytotoxic, RI-evasive RNase A variant (G88R-RNase A) and of various similarly cytotoxic but RI-sensitive RNase A tandem enzyme variants in comparison to the internalization of the non-cytotoxic and RI-sensitive RNase A. After incubation of K-562 cells with the RNase A variants for 36 h, the internalized amount of RNases was analyzed by rapid cell disruption followed by subcellular fractionation and semiquantitative immunoblotting. The data indicate that an enhanced cellular uptake and an increased entry of the RNases into the cytosol can outweigh the abolishment of catalytic activity by RI. As all RNase A variants proved to be resistant to the proteases present in the different subcellular fractions for more than 100 h, our results suggest that the cytotoxic potency of RNases is determined by an efficient internalization into the cytosol.The ribonucleolytic activity of ribonucleases (RNases) 2 provides the potential to use these enzymes as therapeutics for tumor treatment. Particularly, members of the RNase A and RNase T1 superfamilies have shown promising cytotoxicity to cancer cells (1-3). Among these enzymes, Onconase TM (Alfacell Corp., Bloomfield, NJ), an RNase from the Northern Leopard frog, is farthest along the clinical trials (4). To overcome the disadvantage of the renal toxicity of Onconase and a possible immunogenicity of non-mammalian RNases, RNase A or human pancreatic RNase 1 evolved as targets for the development of antitumor agents (5). Because RNases from mammalian sources are silenced due to the tight binding by the intracellular inhibitor protein (RI) and, thus, are not cytotoxic (6), various strategies have been mapped out to accomplish cytotoxicity. Inspired by the cytotoxicity of the amphibian Onconase (5,7,8) and the dimeric bovine seminal RNase (9), tremendous efforts have been made to create RNase derivatives that evade RI binding. While the generation of chimeras (10 -13), the use of chemical modifications (14 -17), or site-directed mutagenesis (5, 6, 18 -21) proved to be strategies of rather varying success, we developed RNase A tandem enzymes (22) in which two RNase A molecules are coupled covalently by a peptide linker. Due to steric hindrance, a complete binding by RI should be prevented and in contrast to bovine seminal RNase, the RNase A entities of the tandem constructs cannot dissociate, which would result in a subsequent binding by RI. In fact, the RNase A tandem constructs have been found to...

show abstract

Role of the N Terminus in RNase A Homologues: Differences in Catalytic Activity, Ribonuclease Inhibitor Interaction and Cytotoxicity

Cited by 181 publications

References 0 publications

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

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

Selective abolition of pancreatic RNase binding to its inhibitor protein

Endocytotic Internalization as a Crucial Factor for the Cytotoxicity of Ribonucleases

Contact Info

Product

Resources

About