Implication of an Asp residue in the ribonucleolytic activity of hirsutellin A reveals new electrostatic interactions at the active site of ribotoxins

Ribotoxins are fungal extracellular ribonucleases that specifically cleave ribosomes leading to cell-death via apoptosis. α-Sarcin is the ribotoxin studied in deepest detail, and therefore constitutes the referential protein for the whole family. It has been demonstrated that ribotoxin activity depends on a very precise structural microenvironment in which electrostatic interactions among residues in the active site are of the highest importance. Hirsutellin A (HtA) has been recently described as the smallest ribotoxin known to date, encompassing all the abilities of previously characterized members of this family into a shorter sequence. Comparison of HtA and α-sarcin three-dimensional structures suggested that residues presumably forming the catalytic triad of HtA would be His 42, Glu 66, and His 113. Within this same idea, the presence of an Asp residue (Asp 40) in a position equivalent to α-sarcin Tyr 48 is highlighted as a novelty in this field. In this work, substitution mutants H42Q, E66Q and H113Q, as well as double and triple mutants in all possible combinations, are studied regarding their ribonucleolytic activity and cytotoxicity. Implication of these three residues in the ribotoxin activity of HtA is confirmed, though none of them is strictly essential for ribosomal cleavage. Studies with mutants D40N and D40N/E66Q demonstrate an important role for Asp 40 in the activity of HtA and establish a new set of electrostatic interactions different from the one described for already known ribotoxins.     

The insecticidal protein hirsutellin A from the mite fungal pathogen Hirsutella thompsonii is a ribotoxin

The mite fungal pathogen Hirsutella thompsonii produces a single polypeptide chain, insecticidal protein named hirsutellin A (HtA) that is composed of 130 amino acid residues. This protein has been purified from its natural source and produced as a recombinant protein in Escherichia coli. Spectroscopic analysis has determined that the two protein forms are indistinguishable. HtA specifically inactivates ribosomes and produces the alpha-fragment characteristic of ribotoxin activity on rRNA. Behaving as a cyclizing ribonuclease, HtA specifically cleaves oligonucleotides that mimick the sarcin/ricin loop of the ribosome, as well as selected polynucleotides and dinucleosides. HtA interacts with phospholipid membranes as do other ribotoxins. As a consequence of its ribonuclease activity and its ability to interact with cell membranes, HtA exhibits cytotoxic activity on human tumor cells. On the basis of these results, HtA is considered to be a member of the ribotoxin group of proteins, although it is significantly smaller (130 aa) than all known ribotoxins that are composed of 149/150 amino acids. Ribotoxins are members of a larger family of fungal ribonucleases whose members of smaller size (100/110 aa) are not cytotoxic. Thus, the characterization of the fungal ribotoxin HtA represents an important milestone in the study of the diversity and the function of fungal ribonucleases.     

A non-cytotoxic but ribonucleolytically specific ribotoxin variant: implication of tryptophan residues in the cytotoxicity of hirsutellin A

Ribotoxins are a family of toxic proteins that exert a highly specific cleavage at the universally conserved sarcin/ricin loop (SRL) of the larger rRNA molecule. Before this ribonucleolytic action, passage through the cell membrane is a necessary step for ribotoxin internalization and the limiting factor for cytotoxicity. Although extensive knowledge of their ribonucleolytic activity and substrate recognition has been accumulated, little is known about the mechanisms of cell entry of ribotoxins. Hirsutellin A (HtA) is a recently described member of this family, which accommodates the main abilities of previously characterized ribotoxins into a shorter sequence, but exhibits some differences regarding membrane interaction properties. This work investigates the contribution of tryptophan (Trp) residues 71 and 78 to both endoribonucleolytic activity and cellular toxicity of this ribotoxin. Substitution mutants W71F and W78F, as well as the double mutant W71/78F, were obtained and assayed against isolated ribosomes, synthetic SRL, and human tumor cells. The results provide evidence that cell membrane passage and internalization, as well as substrate-specific recognition, require the participation of the region involving both Trp 71 and Trp 78. Additionally, the mutant W71/78F is the first non-cytotoxic but specific ribosome-cleaving ribotoxin mutant obtained to date.     

Fungal extracellular ribotoxins as insecticidal agents

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 Hirsutella 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 α-sarcin, the most representative member of the ribotoxins family, to show their strong toxic action against insect larvae and cells.     

Evidence for the importance of electrostatics in the function of two distinct families of ribosome inactivating toxins

Alpha-sarcin and ricin represent two structurally and mechanistically distinct families of site-specific enzymes that block translation by irreversibly modifying the sarcin/ricin loop (SRL) of 23S-28S rRNA. alpha-Sarcin family enzymes are designated as ribotoxins and act as endonucleases. Ricin family enzymes are designated as ribosome inactivating proteins (RIP) and act as N-glycosidases. Recently, we demonstrated that basic surface residues of the ribotoxin restrictocin promote rapid and specific ribosome targeting by this endonuclease. Here, we report that three RIP: ricin A, saporin, and gypsophilin depurinate the ribosome with strong salt sensitivity and achieve unusually fast kcat/Km approximately 10(9)-10(10) M(-1) s(-1), implying that RIP share with ribotoxins a common mechanism of electrostatically facilitated ribosome targeting. Bioinformatics analysis of RIP revealed that surface charge properties correlate with the presence of the transport chain in the RIP molecule, suggesting a second role for the surface charge in RIP transport. These findings put forward surface electrostatics as an important determinant of RIP activity.     

The electrostatic character of the ribosomal surface enables extraordinarily rapid target location by ribotoxins

Alpha-sarcin ribotoxins comprise a unique family of ribonucleases that cripple the ribosome by catalyzing endoribonucleolytic cleavage of ribosomal RNA at a specific location in the sarcin/ricin loop (SRL). The SRL structure alone is cleaved site-specifically by the ribotoxin, but the ribosomal context enhances the reaction rate by several orders of magnitude. We show that, for the alpha-sarcin-like ribotoxin restrictocin, this catalytic advantage arises from favorable electrostatic interactions with the ribosome. Restrictocin binds at many sites on the ribosomal surface and under certain conditions cleaves the SRL with a second-order rate constant of 1.7 x 10(10) M(-1) s(-1), a value that matches the predicted frequency of random restrictocin-ribosome encounters. The results suggest a mechanism of target location whereby restrictocin encounters ribosomes randomly and diffuses within the ribosomal electrostatic field to the SRL. These studies show a role for electrostatics in protein-ribosome recognition.     

Production and characterization of a noncytotoxic deletion variant of the Aspergillus fumigatus allergen Aspf1 displaying reduced IgE binding

Aspergillus fumigatus is responsible for many allergic respiratory diseases, the most notable of which - due to its severity - is allergic bronchopulmonary aspergillosis. Aspf1 is a major allergen of this fungus: this 149-amino acid protein belongs to the ribotoxin family, whose best characterized member is alpha-sarcin (EC 3.1.27.10). The proteins of this group are cytotoxic ribonucleases that degrade a unique bond in ribosomal RNA impairing protein biosynthesis. Aspf1 and its deletion mutant Aspf1Delta(7-22) have been produced as recombinant proteins; the deleted region corresponds to an exposed beta-hairpin. The conformation of these two proteins has been studied by CD and fluorescence spectroscopy. Their enzymatic activity and cytotoxicity against human rhabdomyosarcoma cells was also measured and their allergenic properties have been studied by using 58 individual sera of patients sensitized to Aspergillus. Aspf1Delta(7-22) lacks cytotoxicity and shows a remarkably reduced IgE reactivity. From these studies it can be concluded that the deleted beta-hairpin is involved in ribosome recognition and is a significant allergenic region.     

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

Asparagine 54 of alpha-sarcin is a conserved residue within the proteins of the ribotoxin family of microbial ribonucleases. It is located in loop 2 of the protein, which lacks repetitive secondary structure elements but exhibits a well-defined conformation. Five mutant variants at this residue have been produced and characterized. The spectroscopic characterization of these proteins indicates that the overall conformation is not changed upon mutation. Activity and denaturation assays show that Asn-54 largely contributes to protein stability, and its presence is a requirement for the highly specific inhibitory activity of these ribotoxins on ribosomes.     

Reduced expression of SRC family kinases decreases PI3K activity in NBS1-/- lymphoblasts

SRC family kinases (SFKs) are involved in the activation of phosphatidylinositol-3-kinase (PI3K). In addition, the activity of this lipid kinase can be regulated by the DNA repair protein NBS1. Here, we describe a disturbed expression of some members of the non-receptor tyrosine kinase family in lymphoblastoid cell lines generated from cells of Nijmegen breakage syndrome (NBS) patients. Especially, only minor amounts of the kinases LCK and HCK are expressed in the NBS1^−/− cell lines as compared to the consanguineous NBS1^+/− cells. We demonstrate that SFK activity is important for a proper activation of PI3K in these cells and that it is reduced in NBS1^−/− cells. We provide evidence that the observed reduced PI3K activity in NBS lymphoblasts is caused by an impaired expression of the SFKs LCK and/or HCK. Thus, our data establish a new function for the NBS1 protein as a regulator of PI3K activity via SFK members.     

Evidence that YycJ is a novel 5′–3′ double-stranded DNA exonuclease acting in Bacillus anthracis mismatch repair

The most important system for correcting replication errors that survive the built in editing system of DNA polymerase is the mismatch repair (MMR) system. We have identified a novel mutator strain yycJ in Bacillus anthracis. Mutations in the yycJ gene result in a spontaneous mutator phenotype with a mutational frequency and specificity comparable to that of MMR-deficient strains such as those with mutations in mutL or mutS. YycJ was annotated as a metallo-β-lactamase (MβL) super family member with unknown activity. In this study we carried out a biochemical characterization of YycJ and demonstrated that a recombinant YycJ protein possesses a 5′–3′ exonuclease activity at the 5′ termini and at nicks of double-stranded DNA. This activity requires a divalent metal cofactor Mn²⁺ and is stimulated by 5′-phosphate ends of duplex DNA. The mutagenesis of conserved amino acid residues revealed that in addition to the five MβL family conserved motifs, YycJ appears to have its specific motifs that can be used to distinguish YycJ from other closely related MβL family members. A phylogenetic survey showed that putative YycJ homologs are present in several bacterial phyla as well as in members of the Methanomicrobiales and Thermoplasmales from Archaea. We propose that YycJ represents a new group of MβL fold exonucleases, which is likely to act in the recognition of MMR entry point and subsequent removal of the mismatched base in certain MutH-less bacterial species.     

A novel homozygous GALC mutation: Very early onset and rapidly progressive Krabbe disease

A clear cut genotype–phenotype correlation for Krabbe disease is not available. Therefore, it is important to identify new mutations and their associated phenotypes to predict the prognosis of the disease. The aim of this study is to identify the causative mutation(s) in a family with Krabbe disease. After a clinical evaluation and suspicion of Krabbe disease galactocerebrosidase activity was analyzed and GALC gene mutation analysis was performed. The galactocerebrosidase enzyme activity was 0.01 nmol/mg/h protein (normal range 0.8–4). For further investigation mutation screening was performed by Sanger sequencing across the 17 exons of GALC gene. A novel homozygous mutation c.727delT (p.S243QfsX7) was found. In this study we present the clinical findings along with a novel GALC mutation in a consanguineous Turkish family. Although the relationship between the various genotypes and phenotypes in Krabbe disease has not been fully elucidated an accurate genetic family study is helpful for genetic counseling follow-up and therapy of Krabbe disease. Also, it is important to identify new mutations in order to clarify their clinical importance, to assess the prognosis of the disease, and to suggest either prenatal diagnosis or preimplantation genetic diagnosis to the effected families.     

Biochemical comparison of two proteolytic enzymes from Carica candamarcensis: Structural motifs underlying resistance to cystatin inhibition

The lattices of Carica candamarcensis and Carica papaya, members of the Caricaceae family, contain isoforms of cysteine proteinases that help protect these plants against injury. In a prior study, we fractionated 14 discrete proteinaceous components from C. candamarcensis, two of them displaying mitogenic activity in mammalian cells. In this study, we compared the kinetic parameters of one of the mitogenic proteinases (CMS2MS2) with one of the isoforms displaying the highest enzyme activity of this group (CMS1MS2). Both enzymes display a similar Km value with either BAPNA (Benzoyl-Arg-pNA) or PFLPNA (Pyr-Phe-Leu-pNA), but the kcat of CMS1MS2 is about 14-fold higher for BAPNA and 129-fold higher with PFLPNA. While both enzymes are inhibited by E-64 and iodoacetamide, chicken cystatin fully inhibits CMS1MS2, but scarcely affects activity of CMS2MS2. Based on the structure of these proteins and other enzymes from the Caricaceae family whose structures have been resolved, it is proposed that Arg¹⁸⁰ located in the cleft at the active site in CMS2MS2 is responsible for its resistance to cystatin.     

Characterization of a Novel β-l-Arabinofuranosidase in Bifidobacterium longum: FUNCTIONAL ELUCIDATION OF A DUF1680 PROTEIN FAMILY MEMBER*

Pfam DUF1680 (PF07944) is an uncharacterized protein family conserved in many species of bacteria, actinomycetes, fungi, and plants. Previously, we cloned and characterized the hypBA2 gene as a β-l-arabinobiosidase in Bifidobacterium longum JCM 1217. In this study, we cloned a DUF1680 family member, the hypBA1 gene, which constitutes a gene cluster with hypBA2. HypBA1 is a novel β-l-arabinofuranosidase that liberates l-arabinose from the l-arabinofuranose (Araf)-β1,2-Araf disaccharide. HypBA1 also transglycosylates 1-alkanols with retention of the anomeric configuration. Mutagenesis and azide rescue experiments indicated that Glu-338 is a critical residue for catalytic activity. This study provides the first characterization of a DUF1680 family member, which defines a new family of glycoside hydrolases, the glycoside hydrolase family 127.     

Exoribonuclease and Endoribonuclease Activities of RNase BN/RNase Z both Function in Vivo

Escherichia coli RNase BN, a member of the RNase Z family of endoribonucleases, differs from other family members in that it also can act as an exoribonuclease in vitro. Here, we examine whether this activity of RNase BN also functions in vivo. Comparison of the x-ray structure of RNase BN with that of Bacillus subtilis RNase Z, which lacks exoribonuclease activity, revealed that RNase BN has a narrower and more rigid channel downstream of the catalytic site. We hypothesized that this difference in the putative RNA exit channel might be responsible for the acquisition of exoribonuclease activity by RNase BN. Accordingly, we generated several mutant RNase BN proteins in which residues within a loop in this channel were converted to the corresponding residues present in B. subtilis RNase Z, thus widening the channel and increasing its flexibility. The resulting mutant RNase BN proteins had reduced or were essentially devoid of exoribonuclease activity in vitro. Substitution of one mutant rbn gene (P142G) for wild type rbn in the E. coli chromosome revealed that the exoribonuclease activity of RNase BN is not required for maturation of phage T4 tRNA precursors, a known specific function of this RNase. On the other hand, removal of the exoribonuclease activity of RNase BN in a cell lacking other processing RNases leads to slower growth and affects maturation of multiple tRNA precursors. These findings help explain how RNase BN can act as both an exo- and an endoribonuclease and also demonstrate that its exoribonuclease activity is capable of functioning in vivo, thus widening the potential role of this enzyme in E. coli.     

Spatiotemporal Regulation of a Legionella pneumophila T4SS Substrate by the Metaeffector SidJ

Modulation of host cell function is vital for intracellular pathogens to survive and replicate within host cells. Most commonly, these pathogens utilize specialized secretion systems to inject substrates (also called effector proteins) that function as toxins within host cells. Since it would be detrimental for an intracellular pathogen to immediately kill its host cell, it is essential that secreted toxins be inactivated or degraded after they have served their purpose. The pathogen Legionella pneumophila represents an ideal system to study interactions between toxins as it survives within host cells for approximately a day and its Dot/Icm type IVB secretion system (T4SS) injects a vast number of toxins. Previously we reported that the Dot/Icm substrates SidE, SdeA, SdeB, and SdeC (known as the SidE family of effectors) are secreted into host cells, where they localize to the cytoplasmic face of the Legionella containing vacuole (LCV) in the early stages of infection. SidJ, another effector that is unrelated to the SidE family, is also encoded in the sdeC-sdeA locus. Interestingly, while over-expression of SidE family proteins in a wild type Legionella strain has no effect, we found that their over-expression in a ∆sidJ mutant completely inhibits intracellular growth of the strain. In addition, we found expression of SidE proteins is toxic in both yeast and mammalian HEK293 cells, but this toxicity can be suppressed by co-expression of SidJ, suggesting that SidJ may modulate the function of SidE family proteins. Finally, we were able to demonstrate both in vivo and in vitro that SidJ acts on SidE proteins to mediate their disappearance from the LCV, thereby preventing lethal intoxication of host cells. Based on these findings, we propose that SidJ acts as a metaeffector to control the activity of other Legionella effectors.     

Role of the basic character of alpha-sarcin's NH2-terminal beta-hairpin in ribosome recognition and phospholipid interaction

Ribotoxins are a family of toxic extracellular fungal RNases that first enter into the cells and then exert a highly specific ribonucleolytic activity on the larger rRNA molecule, leading to protein synthesis inhibition and cell death by apoptosis. α-Sarcin is the best characterized ribotoxin. Previous characterization of a deletion variant of this protein showed that its long NH₂-terminal β-hairpin is essential for its cytotoxicity. Docking, enzymatic, and lipid-protein interaction studies suggested that this β-hairpin establishes specific interactions with ribosomal proteins and that it is a region involved in the interaction with cell membranes. Consequently, in order to assess the influence of the basic character of this NH₂-terminal β-hairpin (there are 1 arginine and 4 lysines along its 16 residues) on the ribotoxins cytotoxic ability, five individual mutants substituting these five basic residues by glutamic acid were produced, purified to homogeneity, and characterized. Regarding ribosomal recognition, all mutants showed a diminished activity in a cell-free reticulocyte lysate, whereas the activity against an oligoribonucleotide mimicking the sarcin/ricin loop rRNA (SRL) or the homopolymer poly(A) remained unaffected, confirming that the mutated basic residues participate in electrostatic interactions with other ribosomal elements apart from this SRL. The study of the interaction with phospholipid vesicles showed that Lys 17, Arg 22, and, most importantly, Lys 14 and Lys 21, are crucial residues in the first stages of the aggregation phenomenon, where protein-vesicle and protein-protein interactions are required. The data obtained reveal that electrostatic interactions involving basic residues of the β-hairpin are required not only for establishing specific interactions with ribosomal regions other than the SRL but also to explain the ability of the protein to interact with acid phospholipid bilayers.     

Characterization of a novel cold active and salt tolerant esterase from Zunongwangia profunda

A novel cold active esterase, EstLiu was cloned from the marine bacterium Zunongwangia profunda, overexpressed in E. coli BL21 (DE3) and purified by glutathione-S transferase (GST) affinity chromatography. The mature esterase EstLiu sequence encodes a protein of 273 amino acids residues, with a predicted molecular weight of 30 KDa and containing the classical pentapeptidase motif from position 156 to 160 with the catalytic triad Ser158-Asp211-His243. Although, EstLiu showed 64% similarity with the hypothetical esterase from Chryseobacterium sp. StRB126 (WP_045498424), phylogenetic analysis showed it had no similarity with any of the established family of lipases/esterases, suggesting that it could be considered as a new family. The purified enzyme showed broad substrate specificity with the highest hydrolytic activity against p-nitrophenyl butyrate (C4). EstLiu showed remarkable activity (75%) at 0 °Cand the optimal activity at pH 8.0 and 30 °C with good thermostability and quickened inactivation above 60 °C. EstLiu retained 81, 103, 67 and 78% of its original activity at 50% (v/v) in ethanol, isopropanol, DMSO and ethylene glycol, respectively. In the presence of Tween 20, Tween 80 and Triton X-100, EstLiu showed 88, 100 and 117% of relative activity. It is also co-factor independent. The high activity at low temperature and desirable stability in organic solvents and salts of this novel family esterase represents a good evidence of novel biocatalyst. Overall, this novel enzyme showed better activity than previously reported esterases in extreme reaction conditions and could promote the reaction in both aqueous and non-aqueous conditions, indicating its great potential for industrial applications.     

Structural and biochemical studies of GH family 12 cellulases: improved thermal stability,and ligand complexes

In this review we will describe how we have gathered structural and biochemical information from several homologous cellulases from one class of glycoside hydrolases (GH family 12), and used this information within the framework of a protein-engineering program for the design of new variants of these enzymes. These variants have been characterized to identify some of the positions and the types of mutations in the enzymes that are responsible for some of the biochemical differences in thermal stability and activity between the homologous enzymes. In this process we have solved the three-dimensional structure of four of these homologous GH 12 cellulases: Three fungal enzymes, Humicola grisea Cel12A, Hypocrea jecorina Cel12A and Hypocrea schweinitzii Cel12A, and one bacterial, Streptomyces sp. 11AG8 Cel12A. We have also determined the three-dimensional structures of the two most stable H. jecorina Cel12A variants. In addition, four ligand-complex structures of the wild-type H. grisea Cel12A enzyme have been solved and have made it possible to characterize some of the interactions between substrate and enzyme. The structural and biochemical studies of these related GH 12 enzymes, and their variants, have provided insight on how specific residues contribute to protein thermal stability and enzyme activity. This knowledge can serve as a structural toolbox for the design of Cel12A enzymes with specific properties and features suited to existing or new applications.