Enzymatic specificity of three ribosome-inactivating proteins against fungal ribosomes, and correlation with antifungal activity (original) (raw)
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Fungal Ribotoxins: A Review of Potential Biotechnological Applications
Toxins, 2017
Fungi establish a complex network of biological interactions with other organisms in nature. In many cases, these involve the production of toxins for survival or colonization purposes. Among these toxins, ribotoxins stand out as promising candidates for their use in biotechnological applications. They constitute a group of highly specific extracellular ribonucleases that target a universally conserved sequence of RNA in the ribosome, the sarcin-ricin loop. The detailed molecular study of this family of toxic proteins over the past decades has highlighted their potential in applied research. Remarkable examples would be the recent studies in the field of cancer research with promising results involving ribotoxin-based immunotoxins. On the other hand, some ribotoxin-producer fungi have already been studied in the control of insect pests. The recent role of ribotoxins as insecticides could allow their employment in formulas and even as baculovirus-based biopesticides. Moreover, considering the important role of their target in the ribosome, they can be used as tools to study how ribosome biogenesis is regulated and, eventually, may contribute to a better understanding of some ribosomopathies.
Fungal ribotoxins: molecular dissection of a family of natural killers
FEMS Microbiology Reviews, 2007
RNase T1 is the best known representative of a large family of ribonucleolytic proteins secreted by fungi, mostly Aspergillus and Penicillium species. Ribotoxins stand out among them by their cytotoxic character. They exert their toxic action by first entering the cells and then cleaving a single phosphodiester bond located within a universally conserved sequence of the large rRNA gene, known as the sarcin-ricin loop. This cleavage leads to inhibition of protein biosynthesis, followed by cellular death by apoptosis. Although no protein receptor has been found for ribotoxins, they preferentially kill cells showing altered membrane permeability, such as those that are infected with virus or transformed. Many steps of the cytotoxic process have been elucidated at the molecular level by means of a variety of methodological approaches and the construction and purification of different mutant versions of these ribotoxins. Ribotoxins have been used for the construction of immunotoxins, because of their cytotoxicity. Besides this activity, Aspf1, a ribotoxin produced by Aspergillus fumigatus, has been shown to be one of the major allergens involved in allergic aspergillosis-related pathologies. Protein engineering and peptide synthesis have been used in order to understand the basis of these pathogenic mechanisms as well as to produce hypoallergenic proteins with potential diagnostic and immunotherapeutic applications.
Protein Expression and Purification, 2005
Ribosome-inactivating proteins (RIPs) are toxic proteins synthesized by many plants and some bacteria, that speciWcally depurinate the 28S RNA and thus interrupt protein translation. RIPs hold broad interest because of their potential use as plant defense factors against pathogens. However, study of the activity of type I RIPs has been hampered since their expression in Escherichia coli has typically been toxic to the model system. Mirabilis expansa, an Andean root crop, produces a type I RIP called ME1 in large quantities in its storage roots. In this study, the cDNA sequence of ME1 was used to successfully express the recombinant ME1 protein in E. coli. The production of recombinant ME1 in E. coli was conWrmed by Western blot analysis using anti-ME1 antibodies. The studies with Xuorescence-labeled ME1 showed that ME1 can enter bacteria and be distributed in the cytoplasm uniformly, indicating its ability to access the protein synthesis machinery of the bacteria. The recombinant enzyme was active and depurinated yeast ribosomes. However, both native and recombinant ME1 proteins failed to depurinate the E. coli ribosomes, explaining the non-toxicity of recombinant ME1 to E. coli. Structural modeling of ME1 showed that it has folding patterns similar to other RIPs, indicating that ME1 and PAP, which share a similar folding pattern, can show diVerent substrate speciWcity towards E. coli ribosomes. The results presented here are very signiWcant, as few reports are available in the area of bacterial interaction with type I RIPs.
Febs Letters, 1992
Many plants express enzymes which specifically remove an adenine residue from the skeleton of the 28 S RNA in the major subunit of thccukaryotic ribosomc (ribosome inactivating proleins, RIPS). The site of action of RIPS (A4324 in the rRNA from rdt liver) is in a loop structure whose nucleotidc sequence all around the target adenine is also conserved in those species which are completely or partially insensitive to RIPs. In this paper we identify 3 covalent complex between saporin (the RIP extracted from Saporlrrirr oflcinak) and ribosomal proteins from yeast (Saccha-rorr~yces cereeisiae), by means of chemical crosslinking and immunological or avidin-biotin detection. The main complex (mol. wt. = 60 kDa) is formed only with a protein from the 60 S subunit of yeast ribosomes, and is not detcctcd with ribosomes from E. cdl, 3 resistant spies. This observation supports the hypotesis for 3 molecular recognition mechanism involving one or more ribosomal proteins, which could provide a 'receptor' site for the toxin nnd favour optimal binding of the target adcnine A4324 to the active site of the RIP.
The Therapeutic Potential of Fungal Ribotoxins
Current Pharmaceutical Biotechnology, 2008
Ribotoxins constitute a family of toxic extracellular fungal RNases that exert a highly specific activity on a conserved region of the larger molecule of rRNA, known as the sarcin-ricin loop. This cleavage of a single phosphodiester bond inactivates the ribosome and leads to protein synthesis inhibition and cell death. In addition to this ribonucleolytic activity, ribotoxins can cross lipid membranes in the absence of any known protein receptor. This ability is due to their capacity to interact with acid phospholipid-containing membranes. Both activities together explain their cytotoxic character, being rather specific when assayed against some transformed cell lines. The determination of highresolution structures of some ribotoxins, the characterization of a large number of mutants, and the use of lipid model vesicles and transformed cell lines have been the tools used for the study of their mechanism of action at the molecular level. The present knowledge suggests that wild-type ribotoxins or some modified variants might be used in human therapies. Production of hypoallergenic mutants and immunotoxins designed against specific tumors stand out as feasible alternatives to treat some human pathology in the midterm future.
Molecular Microbiology, 2008
Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga-like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the a-sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic ribosomes. Ricin depurinates the eukaryotic ribosomes more efficiently than the prokaryotic ribosomes, while PAP can depurinate both types of ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the a-sarcin/ ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild-type ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the DP1 and DP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild-type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for ribosome depurination, PAP has different requirements , providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity.
2013
It is reported that some plants contain molecules that inactivate the ribosomes by inhibiting the protein synthesis through their N-glycosidase enzymatic activity. The molecules are identified to be group of proteins namely ribosomal inactivating proteins (RIPs). The RIPs have received a lot of attention in recent biomedical research because of their unique biological and enzymatic activities towards animal and human cells. RIPs can be toxic or non toxic based on their cytotoxicity. The first RIP, Ricin was identified from Ricinus communis seeds (castor beans) and this one was important milestone for plant protein identification. This paper reviews elaborately on recent progress in RIP studies- classification, biological and enzymatic activity and also its potential applications in medical research. Information on structural aspects of RIP compiled from the protein data bank and represented in this review paper will provide insight into researchers who aspire to work on cytotoxic dr...
Minimized natural versions of fungal ribotoxins show improved active site plasticity
Archives of biochemistry and biophysics, 2017
Fungal ribotoxins are highly specific extracellular RNases which cleave a single phosphodiester bond at the ribosomal sarcin-ricin loop, inhibiting protein biosynthesis by interfering with elongation factors. Most ribotoxins show high degree of conservation, with similar sizes and amino acid sequence identities above 85%. Only two exceptions are known: hirsutellin A and anisoplin, produced by the entomopathogenic fungi Hirsutella thompsonii and Metarhizium anisopliae, respectively. Both proteins are similar but smaller than the other known ribotoxins (130 vs 150 amino acids), displaying only about 25% sequence identity with them. They can be considered minimized natural versions of their larger counterparts, best represented by α-sarcin. The conserved α-sarcin active site residue Tyr48 has been replaced by the geometrically equivalent Asp, present in the minimized ribotoxins, to produce and characterize the corresponding mutant. As a control, the inverse anisoplin mutant (D43Y) has ...
Ribosome-inactivating proteins: progress and problems
Cellular and Molecular Life Sciences, 2006
Ribosome-inactivating proteins (RIPs), mostly from plants, are enzymes which depurinate rRNA, thus inhibiting protein synthesis. They also depurinate other polynucleotide substrates. The biological activity of RIPs is not completely clarified, and sometimes independent of the inhibition of protein synthesis. There are differences in the cytotoxicity of RIPs and, consequently, in their toxicity to animals. Some RIPs are potent toxins, the best known being ricin, a potential biological weapon. New toxins have recently been identified. RIPs cause apop-totic and necrotic lesions, and induce production of cytokines causing inflammation. RIPs are potentially useful in agriculture and medicine because (i) they have antiviral activity and (ii) they are used for the preparation of conjugates with antibodies ('immunotoxins') or other carriers, rendering them specifically toxic to the cell target of the carrier, which may be helpful in therapy. The distribution, mechanism of action and role in nature of RIPs are not completely understood, and we can expect several future developments in their practical application.