A dimeric mutant of human pancreatic ribonuclease with selective cytotoxicity toward malignant cells

Abstract

Monomeric human pancreatic RNase, devoid of any biological activity other than its RNA degrading ability, was engineered into a dimeric protein with a cytotoxic action on mouse and human tumor cells, but lacking any appreciable toxicity on mouse and human normal cells. This dimeric variant of human pancreas RNase selectively sensitizes to apoptotic death cells derived from a human thyroid tumor. Because of its selectivity for tumor cells, and because of its human origin, this protein represents a potentially very attractive, novel tool for anticancer therapy.

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

“…16 Monomeric HP-RNase is not cytotoxic and forms with RI a particularly tight complex with a K d value of 2.9 Â 10 À16 M. 29 The dimeric mutant HHP2-RNase, designed to adopt a quaternary structure similar to that of BSRNase, is scarcely prone to swap 35 ; however, its unswapped form is hyperactive as a cytotoxic agent. 34,35 This property has been ascribed to the stability of the interchain disulfides that is significantly larger than that of M¼M-BSRNase. 34,35 Surprisingly, the structural data clearly show that Cys31 and Cys32 have in the two enzymes similar accessible surface area, similar contacts and environments in a range of about 10 Å.…”

“…34,35 This property has been ascribed to the stability of the interchain disulfides that is significantly larger than that of M¼M-BSRNase. 34,35 Surprisingly, the structural data clearly show that Cys31 and Cys32 have in the two enzymes similar accessible surface area, similar contacts and environments in a range of about 10 Å. The only notable structural difference lies in the two hinge peptides, which are disordered in M¼M-BSRNase 19,36 while adopting a well-defined conformation in M¼M-HHP2.…”

“…34,35 Crystals were obtained by sitting drop vapor diffusion method at 277 K. A protein solution of 10 mg/mL in 10 mM tris acetate pH 7, 300 mM NaCl was equilibrated against a solution containing 27% (w/v) polyethylene glycol and 0.2M ammonium sulfate and 100 mM cacodylate buffer pH 6.5. After several weeks, each drop contained long needle crystals.…”

“…17 They differ for the swapping of the N-termini (residues 1-15) within a substantially identical quaternary framework. [18][19][20] In both isoforms, the dimeric interface is formed by the hinge peptides (residues [16][17][18][19][20][21][22] and the helices (residues [23][24][25][26][27][28][29][30][31][32][33][34] that comprise the four cysteines forming the two interchain disulfide bridges (Cys31-Cys32 0 and Cys32-Cys31 0 ) and the side chains of Leu28 and 28 0 that form stabilizing hydrophobic interactions across the molecular twofold axis. 18,20 In the most abundant swapped form, MxM-BSRNase, the dimeric interface also includes the closed interface arising from the swapped elements.…”

“…30 Different approaches have been proposed to confer cytotoxicity to HP-RNase; they include (a) sitedirected mutagenesis to weaken its interaction with RI, 23 (b) conjugation of HP-RNase to protein, peptide, and antibodies, which could enhance the uptake by target cells, 31,32 (c) transformation of the protein into a stable dimer capable to evade RI. 33 Following the latter strategy, Piccoli et al 34 have mutated four residues at the helix-II region according to BSRNase sequence (Gln28!Leu, Arg31!Cys, Arg32!Cys, and Asn34!Lys) and have produced a covalent dimeric variant (HHP-RNase), enzymatically active and selectively cytotoxic for several malignant mouse and human cell lines. The elimination of a negative charge on the HHP-RNase surface (Glu111!Gly) has led to a second dimeric mutant, HHP2-RNase, which is more powerful as a cytotoxic agent than BSRNase and is selective for malignant cells.…”