Tài liệu Báo cáo khoa học: The role of electrostatic interactions in the antitumor activity of

The role of electrostatic interactions in the antitumor

activity of dimeric RNases

Eugenio Notomista1

, Jose´ Miguel Manchen˜ o2

, Orlando Crescenzi3

, Alberto Di Donato1

,

Jose´ Gavilanes4 and Giuseppe D’Alessio1

1 Dipartimento di Biologia Strutturale e Funzionale, Universita` di Napoli Federico II, Napoli, Italy

2 Grupo de Cristalografı´a Macromolecular y Biologı´a Estructural, Instituto Rocasolano, Madrid, Spain

3 Dipartimento di Chimica, Universita` di Napoli Federico II, Napoli, Italy

4 Departamento de Bioquı´mica y Biologı´a Molecular I, Universidad Complutense, Madrid, Spain

The superfamily of pancreatic-type RNases [1] includes

several members capable of carrying out ‘special’

actions, i.e., actions other than catalytic, although

strictly dependent on their catalytic, RNA degrading

action [2]. These actions could be linked to physiologi￾cal functions, as in the case of the angiogenic action of

angiogenins [3], or due to the mere reflection in the

laboratory assays, mirrors proposed in the experiment,

of unknown functions, as may be the case of the anti￾fertility action of seminal RNase [4]. Particular atten￾tion has been given to the cytotoxic action of some

RNases, especially because they often appear to be

selective for tumor cells [5].

Many studies have been devoted to the mechanism

of action of these antitumor RNases. However, a con￾clusive understanding of why these RNases kill cells,

especially why some of them selectively kill tumor

cells, has not been obtained. The correlation has been

stressed [6] between the ability of certain RNases to

display a cytotoxic action and their ability to evade

the strong, neutralizing action of the cytosolic RNase

inhibitor (cRI), a 50-kDa protein containing 16 leu￾cine-rich repeat motifs [7,8]. In fact, onconase from

Rana pipiens eggs [9], and seminal RNase from

Bos taurus seminal vesicles [10], among the most stud￾ied natural cytotoxic RNases, do not bind cRI, and

are both totally resistant to its inhibitory action [8].

On the other hand, RNases with no cytotoxic action,

such as bovine or human pancreatic RNase, and a

very high affinity for cRI, acquire the ability to kill

Keywords

antitumor RNases; electrostatic interactions;

electrostatic interaction energy; RNases;

transport through membranes

Correspondence

G. D’Alessio, Dipartimento di Biologia

Strutturale e Funzionale, Universita` di Napoli

Federico II, Via Cinthia, I-80126 Napoli, Italy

Fax: +39 081 679159

Tel: +39 081 679157

E-mail: [email protected]

(Received 12 April 2006, revised 31 May

2006, accepted 12 June 2006)

doi:10.1111/j.1742-4658.2006.05373.x

The cytotoxic action of some ribonucleases homologous to bovine pancre￾atic RNase A, the superfamily prototype, has interested and intrigued

investigators. Their ribonucleolytic activity is essential for their cytotoxic

action, and their target RNA is in the cytosol. It has been proposed that

the cytosolic RNase inhibitor (cRI) plays a major role in determining the

ability of an RNase to be cytotoxic. However, to interact with cRI RNases

must reach the cytosol, and cross intracellular membranes. To investigate

the interactions of cytotoxic RNases with membranes, cytotoxic dimeric

RNases resistant, or considered to be resistant to cRI, were assayed for

their effects on negatively charged membranes. Furthermore, we analyzed

the electrostatic interaction energy of the RNases complexed in silico with

a model membrane. The results of this study suggest that close correlations

can be recognized between the cytotoxic action of a dimeric RNase and its

ability to complex and destabilize negatively charged membranes.

Abbreviations

ANTS, 1,3,6-trisulphonate-8-aminonaphtalene; cRI, cytosolic RNase inhibitor; DMPG, dimyristoylphosphatidylglycerol; DMPS,

dimyristoylphosphatidylserine; DPH, 1,6-diphenyl-1,3,5-hexatriene; DPX, p-xylenebispyridinium bromide; ECP, eosinophil cationic protein;

EDN, eosinophil-derived neurotoxin; EIE, electrostatic interaction energy; HP-RNase, human pancreatic RNase; kT, product of the Boltzmann

constant by absolute temperature in K; PG, phosphatidylglycerol; RET, resonance energy transfer; SVT2, malignant murine fibroblasts.

FEBS Journal 273 (2006) 3687–3697 ª 2006 The Authors Journal compilation ª 2006 FEBS 3687