RNase U2 and α-Sarcin: A Study of Relationships

Martínez‐Ruiz, Antonio; Garcı́a-Ortega, Lucı́a; Kao, Richard; Lacadena, Javier; Oñaderra, Mercedes; Mancheño, José M.; Davies, Julian; Pozo, Álvaro Martı́nez del; Gavilanes, José G.

doi:10.1016/s0076-6879(01)41162-1

Cited by 49 publications

(111 citation statements)

References 86 publications

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“…TLC fractionation of the lipid plasma membrane extracts revealed high amounts of phosphatidylcholine and phosphatidylethanolamine (data not shown) in good accordance with the high abundance of these phospholipids in total lipidic extracts of Sf9 cells (Yeh et al, 1997). This pool of insect lipids was used to prepare vesicles as described before (De los Ríos et al, 1998;HerreroGalán et al, 2008;Martínez-Ruiz et al, 2001) by hydrating dry lipid films with 15 mM Tris-HCl, pH 7.0, containing 0.1M NaCl and 1 mM EDTA, for 60 min at 37ºC and in the presence of 12.5 mM ANTS and 45 mM DPX. The lipid suspension was then subjected to five cycles of extrusion through two stacked 0.1 µm (pore diameter) polycarbonate membranes Martínez-Ruiz et al, 2001).…”

Section: Leakage Of Aqueous Contents From Lipid Vesiclessupporting

confidence: 63%

“…Fungal natural wild-type -sarcin and HtA were produced and isolated as reported before Martínez-Ruiz et al, 2001). E. coli BL21 (DE3) cells, previously cotransformed with a thioredoxin-producing plasmid (pT-Trx) and the corresponding plasmid (pINPGαSH137Q), were used to produce the catalytically inactive -sarcin H137Q mutant, also as previously described (García-Ortega et al, 2000;Lacadena et al, 1994Lacadena et al, , 1995Lacadena et al, , 1999.…”

Section: Protein Production and Purificationmentioning

confidence: 99%

“…This mutant retains the structural features of the wild-type protein, as well as its ability to interact with membranes, but lacks the characteristic ribonucleolytic activity of ribotoxins (Lacadena et al, 1995(Lacadena et al, , 1999. SDS-PAGE of proteins, Western blots, protein hydrolysis, amino acid analysis, and spectroscopic characterization were performed according to standardized procedures described before (García-Ortega et al, 2000, 2002Lacadena et al, 1994;Martínez-Ruiz et al, 2001). According to all these criteria, the three proteins used in this study were purified to homogeneity and retained their structural and functional properties.…”

Section: Protein Production and Purificationmentioning

confidence: 99%

“…Fungal ribotoxins are extracellular and highly specific ribonucleases which behave as potent inhibitors of protein biosynthesis by being able to inactivate ribosomes from almost any organism Kao et al, 2001;Lacadena et al, 2007;Martínez-Ruiz et al, 2001). They cleave the larger rRNA component at a single phosphodiester bond located within the universally conserved sarcin/ricin loop (SRL) (Chan et al, 1983;Endo and Wool, 1982;Endo et al, 1983;Schindler and Davies, 1977), leading to complete inactivation of the ribosome and cell death by apoptosis (Olmo et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

“…-Sarcin, restrictocin, and Aspf1 are the most exhaustively characterized ribotoxins (Arruda et al, 1992;García-Ortega et al, 2005;Gasset et al, 1994;Kao et al, 2001;Martínez-Ruiz et al, 2001;Wool, 1997), but many others have been identified and partially characterized within different fungal species (Huang et al,1997;Lin et al, 1995;Martínez-Ruiz et al, 1999a, 1999bParente et al, 1996;Varga and Samson, 2008;Wirth et al, 1997). These studies have shown a high degree of conservation among ribotoxins, as most of those so far characterized display amino acid sequence similarities above 85% (Lacadena et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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Fungal extracellular ribotoxins as insecticidal agents

Olombrada¹,

Herrero-Galán²,

Tello³

et al. 2013

Insect Biochemistry and Molecular Biology

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Fungal ribotoxins were discovered almost 50 years ago as extracellular ribonucleases (RNases) with antitumoral properties. However, the biological function of these toxic proteins has remained elusive. The discovery of the ribotoxin HtA, produced by the invertebrates pathogen H. thompsonii, revived the old proposal that insecticidal activity would be their long searched function. Unfortunately, HtA is rather singular among all ribotoxins known in terms of sequence and structure similarities. Thus, it was intriguing to answer the question of whether HtA is just an exception or, on the contrary, the paradigmatic example of the ribotoxins function. The work presented uses HtA and -sarcin, the most representative member of the ribotoxins family, to show their strong toxic action against insect larvae and cells.

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Section: Leakage Of Aqueous Contents From Lipid Vesiclessupporting

confidence: 63%

Section: Protein Production and Purificationmentioning

confidence: 99%

Section: Protein Production and Purificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Fungal extracellular ribotoxins as insecticidal agents

Olombrada¹,

Herrero-Galán²,

Tello³

et al. 2013

Insect Biochemistry and Molecular Biology

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Fungal Ribotoxins

Garcı́a-Ortega

Palacios-Ortega

Martínez‐del‐Pozo

2018

Encyclopedia of Life Sciences

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Fungal ribotoxins constitute a family of extracellular ribonucleases with exquisite specificity against rRNA (ribonucleic acid). They induce apoptotic death of cells after inhibiting protein translation. Ribosomes become functionally incompetent because ribotoxins cleave one single phosphodiester bond, located at a unique and universally conserved loop, needed for elongation factors function. As secreted proteins, ribotoxins need to cross the membrane of their target cells in order to exert their catalytic activity, and they do it without receptor mediation. Using lipid model systems, it has been shown that they are able to enter cells with membranes enriched in acidic phospholipids. Both membrane‐interacting and ribosomal‐recognition activities are characterised by distinct structural features. Even though the natural function of ribotoxins is not known yet, their production by entomopathogenic fungi has suggested their insecticidal role. After decades of detailed study, the biotechnological potential of ribotoxins in pest control and as antitumour agents is becoming evident. Key Concepts Ribotoxins are extremely specific ribonucleases targeted against ribosomes. Ribotoxins are produced by fungi, some of them entomopathogens. They show a high degree of structural conservation, including the local arrangement of the active site residues. Cleavage of a single rRNA phosphodiester bond leads to cell death by inhibiting translation. Ribotoxins are cyclising RNases because they follow a general acid–base mechanism with production of a 2′,3′‐cyclic intermediate. Ribotoxins must first enter their target cells to exert their lethal action. Cell entrance is possible in cells with membranes enriched in acidic phospholipids and altered permeability. Ribotoxins are optimal candidates to be employed as pest control agents and in antitumour immunotoxins.

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Anomalous electrophoretic behavior of a very acidic protein: Ribonuclease U2

et al. 2005

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Ribonuclease U2 is a low-molecular-weight acidic protein with three disulfide bridges. This protein displays an anomalous electrophoretic behavior on standard SDS-PAGE. The electrophoretic mobility of the nonreduced protein roughly corresponds to its molecular mass while the migration of the reduced protein would be in accordance with the expected molecular mass of the protein dimer. This study reveals that the protein does not bind SDS under the SDS-PAGE conditions, its electrophoretic mobility being only determined by its electrostatic charge and hydrodynamic properties. In addition, the nonreduced protein cannot be blotted to a membrane. Unfolding of the protein upon reduction of its disulfide bridges enables electrotransference to membranes due to a restricted diffusion along the electrophoresis gel.

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RNase U2 and α-Sarcin: A Study of Relationships

Cited by 49 publications

References 86 publications

Fungal extracellular ribotoxins as insecticidal agents

Fungal extracellular ribotoxins as insecticidal agents

Fungal Ribotoxins

Anomalous electrophoretic behavior of a very acidic protein: Ribonuclease U2

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