Nello Russo

Potent Inhibition of Mammalian Ribonucleases by 3′,5′-Pyrophosphate-linked Nucleotides

Molecular modeling based on the crystal structure of the complex of bovine pancreatic RNase A with the inhibitor 5′-diphosphoadenosine 3′-phosphate (ppAp) (Leonidas, D. D., Shapiro, R., Irons, L. I., Russo, N., and Acharya, K. R. (1997) Biochemistry 36, 5578–5588) was used to design new inhibitors that extend into unoccupied regions of the enzyme active site. These compounds are dinucleotides that contain an unusual 3′,5′-pyrophosphate linkage and were synthesized in solution by a combined chemical and enzymatic procedure. The most potent of them, 5′-phospho-2′-deoxyuridine 3′-pyrophosphate, P′ → 5′-ester with adenosine 3′-phosphate (pdUppAp), binds to RNase A withK i values of 27 and 220 nm at pH 5.9 and 7, respectively. These values are 6–9-fold lower than those for ppAp and 50-fold lower than that for the transition state analogue, uridine vanadate. pdUppAp has broad specificity; it is an effective inhibitor of at least two other members of the pancreatic RNase superfamily, human RNase-2 (eosinophil-derived neurotoxin) and RNase-4, which share only 36–44% sequence identity with the pancreatic enzyme. The potency of pdUppAp and the other inhibitors described here depends critically on the extended internucleotide linkage; the pyrophosphate group enhances dinucleotide binding to the three RNases by 2.1–2.9 orders of magnitude, as compared with a monophosphate. These data give further insight into the organization of the catalytic centers of the various RNases. Moreover, the new class of inhibitors provides a useful means by which to probe the biological actions of these and other related enzymes.

The two dimeric forms of RNase A

In 1965 Fruchter and Crestfield (J. Biol. Chem. 240, 2868–3874) observed that dimeric RNase A prepared by lyophilization from acetic acid could be separated into two forms. Surprisingly, no other structural or functional differences could be detected between the two forms. In 1998 a structure for dimeric RNase A was determined by X‐ray crystallography by Liu et al. (Proc. Natl. Acad. Sci. USA 95, 3437–3442). We found that the two forms of dimeric RNase A have indeed different structural and functional properties, and suggest that the dimer whose structure was investigated by Liu and coworkers may be identified with the lesser form of dimeric RNase A.

Expression in mammalian cells, purification and characterization of recombinant human pancreatic ribonuclease

A synthetic cDNA coding for human pancreatic RNase, equipped with a secretion signal sequence, was cloned and stably expressed in Chinese hamster ovary cells. The recombinant RNase, secreted into the culture medium, was purified and characterized. It was found to be indistinguishable, by structural and catalytic parameters, from the enzyme isolated from human pancreas. Furthermore, the glycosylated forms were separated from the non‐glycosylated form. Up until now, human RNases have been isolated only in small amounts from autoptic specimens. This has hindered the exploitation of a human RNase for the construction of immunotolerated immunotoxins. On the other hand, the availability of an effective system for the expression of a human RNase may render feasible the transfer, by protein engineering, of the interesting pharmacological actions of non‐human RNase [1993 Trends Cell Biol. 3, 106‐109] to an immunotolerated, human RNase.

Expression of native dimers of bovine seminal ribonuclease in a eukaryotic cell system

Bovine seminal ribonuclease, a uniquely dimeric pancreatic‐like RNase, with its dimeric structure stabilized by two intersubunit disulfides, and endowed with special, i.e. non‐catalytic, actions (antitumor, immunosuppressive, antispermatogenic), was stably expressed in Chinese hamster ovary cells. The recombinant protein, secreted in the culture medium as a correctly folded dimeric enzyme, was purified to homogeneity and found to be fully active both in its catalytic and antitumor activities.

Structural Studies on Angiogenin, a Protein Implicated in Neovascularization during Tumour Growth

Angiogenesis, the formation of new blood vessels, is an essential part of normal physiological processes such as embryonic growth, wound healing, and the cyclical development of the uterine endometrium. It also occurs in a variety of pathological conditions including arthritis, diabetic retinopathy, and tumour growth (Folkman and Cotran, 1976). Early observers had noted a proliferation of blood vessels in the vicinity of such tumours (see Vallee et al., 1985), and it was later proposed by Folkman (1971) that these tumours are totally dependent on angiogenesis for growth beyond a diameter of 1–2 mm. Angiogenesis is also thought to be a prerequisite for the development of metastases since it provides the means whereby cells disseminate from the original primary tumour.