Mini Reviews in Medicinal Chemistry - Volume 4, Issue 5, 2004

To be invited to edit a special issue on a field of the editor's choice was an honour, and an even greater one came when some of the best scientists working on ribosome-inactivating proteins consented to contribute. But perhaps the greatest satisfaction for me was to realize how much progress has been done in a field to which our laboratory contributed from the very beginning of the modern studies, in the seventies. Scientists should always look to the future, but I must admit that when we started working on ricin I could not possibly dream of such a development. In thirty years we saw that the number of potent toxins grew from two, ricin and abrin, to six, that single-chain ribosomeinactivating proteins (RIPs) were found, and that non-toxic two-chain RIPs were identified. In other words, a whole class of proteins was defined. We also saw the development of research aimed to find useful applications of RIPs. The bulk of this was on the conjugates of RIPs with carriers, mainly antibodies to form “immunotoxins”, which could deliver them in a selective manner to cells to be eliminated. The “magic bullet” envisaged several decades before by the genius of Paul Ehrlich seemed at hand, and in a sense it was obtained, in that immunotoxins have been done and work, although their use in medicine is not yet practicable. They are, however, excellent experimental tools, as it is demonstrated by their use in neurology, although their potential has not yet been fully realised. Also interesting are the possible applications of RIPs for their antiviral properties. Attempts have been, and still are done, to utilise these proteins not only to improve plant resistance to viral infection, but also to combat human viral diseases. So far full success has not been achieved, but the results are promising. Sadly, we also heard of the possible use of ricin as a biological weapon, but fortunately the fears did not materialize. However, the greatest importance of RIPs is their very existence, and a great challenge will be to understand the role in Nature of these proteins. Proteins are expensive to make, and the widespread conservation of RIPs in plants, in at least one bacterium, and possibly in animals suggests they must have an important role in the producing organisms. Finally, one wonders what have been the economical consequences of the discovery of RIPs. They are definitely minuscule as compared to those of mass-produced goods, however, they are in the catalogues of several suppliers, an industry was created and people were employed. In a time when the emphasis is on research giving immediate economic returns, this is another example, although modest, of the consequences of “basic” research, born out of the “scientific curiosity” of few scientists. Last but not least, I wish to express my gratitude to the Editors who assigned me the task of editing this special issue, and to all authors of the reviews. It was a great pleasure to renew long-time friendships and to make new ones.

Ribosome-Inactivating Proteins (RIPs) are enzymes that trigger the catalytic inactivation of ribosomes and other substrates. They are present in a large number of plants and have been found also in fungi, algae and bacteria. RIPs are currently classified as type 1, those formed by a single polypeptide chain with the enzymatic activity, and type 2, those formed by 2 types of chains, i.e. A chains equivalent to a type 1 RIPs and B chains with lectin activity. Type 2 RIPs usually contain the formulae A-B, (A-B)2 and less frequent (A-B)4 and polymeric forms of type 2 RIPs lectins. RIPs are broadly distributed in plants, and are present also in fungi, bacteria, at least in one alga; recently RIP-type activity has been described in mammalian tissues. The highest number of RIPs has been found in Caryophyllaceae, Sambucaceae, Cucurbitaceae, Euphorbiaceae, Phytolaccaceae and Poaceae. However there are no systematic screening studies to allow generalisations about occurrence. The most known activity of RIPs is the translational inhibitory activity, which seems a consequence of a N-glycosidase on the 28 S rRNA of the eukaryotic ribosome that triggers the split of the A4324 (or an equivalent base in other ribosomes), which is key for translation. This activity seems to be part of a general adenine polynucleotide glycosylase able to act on several substrates other than ribosomes, such as tRNA, mRNA, viral RNA and DNA. Other enzymatic activities found in RIPs are lipase, chitinase and superoxide dismutase. RIPs are phylogenetically related. In general RIPs from close families share good amino acid homologies. Type 1 RIPs and the A chains of type 2 RIPs from Magnoliopsida (dicotyledons) are closely related. RIPs from Liliopsida (monocotyledons) are at the same time closely related and distant from Magnoliopsida. Concerning the biological roles played by RIPs there are several hypotheses, but the current belief is that they could play significant roles in the antipathogenic (viruses and fungi), stress and senescence responses. In addition, roles as antifeedant and storage proteins have been also proposed. Future research will approach the potential biological roles played by RIPs and their use as toxic effectors in the construction of immunotoxins and conjugates for target therapy.

Ribosome Inactivating Proteins, RIPs, depurinate an invariant adenine from the 28S rRNA of eukaryotic ribosomes; they have evolved to near enzymatic perfection for this task. The N-glycosidase fold is conserved in plant and bacterial enzymes. RIPs can form complexes with cell surface recognition proteins that dramatically increase the cytotoxicity of the molecule.

Ribosome-inactivating proteins (RIPs) are a heterogeneous group of enzymes found mainly in plants and a few bacteria that possess N-glycosidase activity on ribosomes and a related polynucleotide adenosine glycosidase activity on naked nucleic acids. They encompass single enzymatic chains, heterodimeric toxic lectins and related agglutinins. Plants commonly produce several RIP isoforms encoded by multi-gene families. The toxic lectins possess adaptations related to their cytotoxic role.

Plants contain proteins that are capable of inactivating ribosomes, commonly referred to as Ribosome Inactivating Proteins (RIPs). These particular plant proteins have received attention in biological and biomedical research because of their unique biological activities towards animals and human cells as cellkilling agents. Some of the best-characterised RIPs have been isolated from exotic plants, but they have also been found in cereals and other food crops. Cereals contain, in general, RIPs in the endosperm protein pool: they share a high similarity with all the other RIPs retaining, however, characteristic features forming a distinct class which diversified significantly during evolution. They appear to be involved in quite different physiological roles, such as defence against pathogens and / or involved in regulatory and developmental processes. This review aims to provide a critical assessment to work related to cereal RIP with particular emphasis to the maize RIPs.

To catalytically-modify ribosomes in vivo, ribosome-inactivating proteins produced by plants must enter susceptible mammalian cells in order to reach their substrates in the cytosol. This review primarily focuses on the biosynthesis, mechanism of cell entry and intracellular trafficking of ricin, the most thoroughly studied ribosome-inactivating protein in this respect.

The toxicity to cells and animals of type 1 and toxic and non-toxic type 2 Ribosome-Inactivating Proteins (RIP) is discussed in correlation with their catalytic activity, resulting in ribosome inactivation and apoptosis. The symptoms and histopathological lesions induced by RIP to animals and humans is also reviewed.

Pokeweed antiviral protein and several other ribosome inactivating proteins are effective against a broad range of viruses. Recent results have shown that their enzymatic activity is not limited to depurination of the large rRNA, they can depurinate other nucleic acids, including viral RNAs. Antiviral activity of RIPs is summarized here in light of their novel activities and recent developments in the field.

Targeted toxins represent an invaluable tool offering a wide range of potential applications, both in experimental models and in the clinics. Here we will review several aspects related to the preparation and properties of carrier molecule-toxin heteroconjugates and fusion toxins.

A wide variety of conjugates containing RIPs, of either chemical or recombinant type, have been made and tested against dangerous cells in vitro and in animal models. Many of these pre-clinical studies will be reviewed here dividing them on the basis of the target cell and the surface molecule specifically recognized.

The use of targeted toxins in research applications has recently grown considerably. The ability to remove a few specific cells, even when surrounded by different populations, has given scientists a powerful tool for the understanding of systems biology. The use of targeted toxins in research is rich and varied; here we limit ourselves to describe some of those exciting results that researchers have made in the neurosciences.