Conserved asparagine residue 54 of α-sarcin plays a role in protein stability and enzyme activity

Siemer, Ansgar B.; Masip, Manuel; Carreras, Nelson; Garcı́a-Ortega, Lucı́a; Oñaderra, Mercedes; Bruix, Marta; Pozo, Álvaro Martı́nez del; Gavilanes, José G.

doi:10.1515/bc.2004.150

Cited by 9 publications

(5 citation statements)

References 29 publications

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“…3). It not only contributes to the high stability of ribotoxins, but is also required for their highly specific action on ribosomes, according to the results obtained after the characterization of five different α‐sarcins with mutations at this position (Siemer et al , 2004). These results suggest that Asn54 is involved in local conformational arrangements at the substrate‐binding pocket.…”

Section: Enzymatic Propertiesmentioning

confidence: 99%

“…The results obtained with the α‐sarcin Asn54 mutants are in agreement with the idea that residues 53–56 (52–55 in restrictocin) would form that recognition pocket in ribotoxins (Yang & Moffat, 1996). However, recognition of ribosomes involves a much more complex network of interactions, most of which would not be disturbed by the mutation of Asn54, which would explain why most of the mutants still retained the ability to specifically release the α‐fragment (Siemer et al , 2004). Overall, these results are in perfect agreement with the idea of local conformational rearrangements of the Asn54 and Leu145 mutants' active site, leading to less specific and less cytotoxic enzymes (Masip et al , 2003; Siemer et al , 2004).…”

Section: Enzymatic Propertiesmentioning

confidence: 99%

“…However, recognition of ribosomes involves a much more complex network of interactions, most of which would not be disturbed by the mutation of Asn54, which would explain why most of the mutants still retained the ability to specifically release the α‐fragment (Siemer et al , 2004). Overall, these results are in perfect agreement with the idea of local conformational rearrangements of the Asn54 and Leu145 mutants' active site, leading to less specific and less cytotoxic enzymes (Masip et al , 2003; Siemer et al , 2004). Another important observation was that ribotoxins lack a residue equivalent to RNase T1 Glu46, involved in discriminating guanine from adenine (Gohda et al , 1994).…”

Section: Enzymatic Propertiesmentioning

confidence: 99%

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Fungal ribotoxins: molecular dissection of a family of natural killers

et al. 2007

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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.

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Section: Enzymatic Propertiesmentioning

confidence: 99%

Section: Enzymatic Propertiesmentioning

confidence: 99%

Section: Enzymatic Propertiesmentioning

confidence: 99%

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Fungal ribotoxins: molecular dissection of a family of natural killers

et al. 2007

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“…Escherichia coli RB791 or BL21 (DE3) cells, the latter ones being previously cotransformed with a thioredoxin-producing plasmid (pT-Trx), and the corresponding wildtype or mutant plasmids were used to produce and purify all proteins from the periplasmic soluble fraction, as previously described [35,38,42,[44][45][46]. The only exception was fungal wild-type α-sarcin which was isolated from Aspergillus giganteus MDH18894, its natural source, following the procedure reported before [11].…”

Section: Protein Production and Purificationmentioning

confidence: 99%

Minimized natural versions of fungal ribotoxins show improved active site plasticity

Maestro-López

Olombrada

Garcı́a-Ortega

et al. 2017

Archives of Biochemistry and Biophysics

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12Fungal ribotoxins are highly specific extracellular RNases which cleave a single [23, 24, 32, 35]. Therefore, they can be considered as minimized natural versions 57 of their larger counterparts. This lower sequence identity and size do not however preclude 58 the conservation of the elements of ordered secondary structure as well as the identity and 59 geometric arrangement of the residues configuring the active site (Figure 1). This structural 60 conservation can be even extended to the other non-toxic members of the larger fungal 61 extracellular RNases family, such as RNase T1 and RNase U2, for example [36, 37]. In 74In the work herein presented α-sarcin Tyr 48 has been replaced by an Asp residue to 75 produce and characterize the corresponding Y48D mutant. As a control, the corresponding 76 inverse anisoplin mutant (D43Y) has been also studied. The results shown reveal the key 77 role of these residues not only in maintaining the correct electrostatic environment and 78 active site plasticity in each type of ribotoxins but also in the preservation of their 79 characteristic high thermostability. 80 MATERIALS AND METHODS 81 Mutant cDNA construction 82All materials and reagents were of molecular biology grade. Cloning procedures, PCR-83 based oligonucleotide site-directed mutagenesis, and bacterial manipulations were carried 84 out as previously described [11, 16,[41][42][43]. Mutagenesis constructions were performed 85 using different sets of complementary mutagenic primers (Table S1). Mutations were 86 confirmed by DNA sequencing at the corresponding Complutense University facility. The 87 plasmids used as templates for mutagenesis, containing the cDNA sequence of either wild-88 type α-sarcin or anisoplin, have already been described [35, 38, 42]. 89 Protein production and purification 90Escherichia coli RB791 or BL21 (DE3) cells, the latter ones being previously 91 cotransformed with a thioredoxin-producing plasmid (pT-Trx), and the corresponding wild-92 type or mutant plasmids were used to produce and purify all proteins from the periplasmic 93 soluble fraction, as previously described [35, 38, 42,[44][45][46]. The only exception was fungal 94 wild-type α-sarcin which was isolated from Aspergillus giganteus MDH18894, its natural 95 source, following the procedure reported before [11]. SDS-PAGE of proteins, Western 96 blots, protein hydrolysis, and amino acid analysis were performed according to standard 97 procedures, also as previously described [11, 42]. All four proteins studied were purified to 98 homogeneity according to their SDS-PAGE behavior and amino acid analysis. 99 Spectroscopic characterization 100Spectroscopic characterization was performed following well established procedures [11, 101 22, 38, 41, 44, 47-51]. Absorbance measurements were carried out on a Shimadzu UV- Ribonucleolytic activity assays 118The ribonucleolytic activity of ribotoxins on rabbit ribosomes was followed by detecting 119 the release of a specific 400-nt rRNA fragment, known as the α-fragment, from ...

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“…This highly specific ribonucleolytic activity of ribotoxins has been extensively studied and a wide collection of mutants has been well characterized. Thus, the reaction mechanism, as well as the roles of most of the active site residues, have been elucidated [ 55 , 56 , 57 , 58 , 59 , 60 ].…”

Section: Functional Propertiesmentioning

confidence: 99%

Hirsutellin A: A Paradigmatic Example of the Insecticidal Function of Fungal Ribotoxins

Herrero-Galán

Garcı́a-Ortega

Olombrada

et al. 2013

Insects

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The fungal pathogen Hirsutella thompsonii produces an insecticidal protein named hirsutellin A (HtA), which has been described to be toxic to several species of mites, insect larvae, and cells. On the other hand, on the basis of an extensive biochemical and structural characterization, HtA has been considered to be a member of the ribotoxins family. Ribotoxins are fungal extracellular ribonucleases, which inactivate ribosomes by specifically cleaving a single phosphodiester bond located at the large rRNA. Although ribotoxins were brought to light in the 1960s as antitumor agents, their biological function has remained elusive. Thus, the consideration of hirsutellin A, an insecticidal protein, as a singular ribotoxin recalled the idea of the biological activity of these toxins as insecticidal agents. Further studies have demonstrated that the most representative member of the ribotoxin family, α-sarcin, also shows strong toxic action against insect cells. The determination of high resolution structures, the characterization of a large number of mutants, and the toxicity assays against different cell lines have been the tools used for the study of the mechanism of action of ribotoxins at the molecular level. The aim of this review is to serve as a compilation of the facts that allow identification of HtA as a paradigmatic example of the insecticidal function of fungal ribotoxins.

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Conserved asparagine residue 54 of α-sarcin plays a role in protein stability and enzyme activity

Cited by 9 publications

References 29 publications

Fungal ribotoxins: molecular dissection of a family of natural killers

Fungal ribotoxins: molecular dissection of a family of natural killers

Minimized natural versions of fungal ribotoxins show improved active site plasticity

Hirsutellin A: A Paradigmatic Example of the Insecticidal Function of Fungal Ribotoxins

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