pH-Dependent conformational stability of the ribotoxin alpha-sarcin and four active site charge substitution variants

Alpha-sarcin is an exquisitely specific ribonuclease that binds and cleaves a single phosphodiester bond in the large rRNA of the eukaryotic ribosome, inactivating it. To better understand this remarkable activity, the contributions of the active site residues (His 50, Glu 96, and His 137) to the conformational stability have been determined as a function of pH using variant proteins containing uncharged substitutes. Wild-type alpha-sarcin and the variants are maximally stable near pH 5.5, coinciding with the pH of optimal activity. A comparison of the stability vs pH profiles determined by thermal denaturation experiments to those calculated on the basis of pKa values shows that the charged forms of Glu 96 and His 137 compromise the enzyme's stability, lowering it. In contrast to barnase, there is little evidence for significant electrostatic interactions in the denatured states of alpha-sarcin or its active site variants between pH 3.5 and pH 8.5. Alpha-sarcin contains a long beta-hairpin and surface loops which are highly positively charged and which play key roles in membrane translocation and in ribosome binding. These positive charges decrease the stability of alpha-sarcin, particularly below pH 5. Hydrogen exchange measurements have been performed at pH 5.5 and reveal that the catalytic residues are firmly anchored in highly stable elements of secondary structure. Significant, though lower, levels of protection are observed for many amide protons in the positively charged beta-hairpin and long loops.     

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.     

Arginine 121 is a crucial residue for the specific cytotoxic activity of the ribotoxin alpha-sarcin.

Alpha-sarcin, a cyclizing ribonuclease secreted by the mould Aspergillus giganteus, is one of the best characterized members of a family of fungal ribotoxins. This protein induces apoptosis in tumour cells due to its highly specific activity on ribosomes. Fungal ribotoxins display a three-dimensional protein fold similar to those of a larger group of microbial noncytotoxic RNases, represented by RNases T1 and U2. This similarity involves the three catalytic residues and also the Arg121 residue, whose counterpart in RNase T1, Arg77, is located in the vicinity of the substrate phosphate moiety although its potential functional role is not known. In this work, Arg121 of alpha-sarcin has been replaced by Gln or Lys. These two mutations do not modify the conformation of the protein but abolish the ribosome-inactivating activity of alpha-sarcin. In addition, the loss of the positive charge at that position produces dramatic changes on the interaction of alpha-sarcin with phospholipid membranes. It is concluded that Arg121 is a crucial residue for the characteristic cytotoxicity of alpha-sarcin and presumably of the other fungal ribotoxins.     

NH2-terminal sequence of calf fetuin

Calf fetuin, one of the 3 major known fetal proteins has been isolated by a two-step purification procedure and characterized by aminoacid composition. The purified glycoprotein, which consisted of a single chain, was submitted to 47 steps of automatic aminoacid sequencing, allowing to determine 44 positions. This section of the molecule was devoid of carbohydrates. Comparison of this sequence with a variety of detectable potentially related protein did not allow to point to any detectable homology.     

Cytotoxic mechanism of the ribotoxin alpha-sarcin. Induction of cell death via apoptosis.

Alpha-sarcin is a ribosome-inactivating protein that has been well characterized in vitro, but little is known about its toxicity in living cells. We have analyzed the mechanism of internalization of alpha-sarcin into human rhabdomyosarcoma cells and the cellular events that result in the induction of cell death. No specific cell surface receptor for alpha-sarcin has been found. The toxin is internalized via endocytosis involving acidic endosomes and the Golgi, as deduced from the ATP requirement and the effects of NH4Cl, monensin and nigericin on its cytotoxicity. Specific cleavage of 28S rRNA in cultured rhabdomyosarcoma cells, associated with protein biosynthesis inhibition, has been detected. alpha-Sarcin kills rhabdomyosarcoma cells via apoptosis: incubation of cells with alpha-sarcin at a concentration below its IC50 induces internucleosomal genomic DNA fragmentation, reversion of membrane asymmetry, activation of caspase-3-like activity and cleavage of poly(ADP-ribose)polymerase. Apoptosis is not a general direct consequence of protein biosynthesis inhibition, as deduced from the comparative analysis of the effects of alpha-sarcin and cycloheximide; the latter does not induce apoptosis even at concentrations far beyond its IC50, where protein biosynthesis is null. Experiments with a catalytically inactive alpha-sarcin mutant, neither toxic nor apoptotic, reveal that induced apoptosis is directly related to the effects of catalytic activity of the toxin on the ribosomes. The caspase inhibitor z-VAD-fmk does not suppress protein synthesis inhibition by alpha-sarcin. Together, these data suggest that alpha-sarcin-induced caspase activation is a pathway downstream of the 28S rRNA catalytic cleavage and consequent protein biosynthesis inhibition.     

Antibodies to an NH2-terminal myristoyl glycine moiety can detect NH2-terminal myristoylated proteins in the retrovirus-infected cells

Novel antibodies were raised against a synthetic NH2-terminal myristoyl glycine moiety which is characteristic of N-myristoyl-proteins. Antisera raised against N-myristoyl-Gly-hemocyanin reacted with N-myristoyl-Gly-[125I]albumin. The immunoreaction was competed for by albumin conjugated with N-myristoyl-glycine, while underivatized albumin had no effect. Of the [3H]myristate-labeled proteins detected, pp60v-src, which is a transforming protein of Rous sarcoma virus, and p19gag and p17gag, which are core proteins in the human T-cell leukemia virus and the human immunodeficiency virus, were identified as N-myristoylated proteins by the radioimmunoprecipitation analyses with the antibody.     

Probing the active site of mitogillin,a fungal ribotoxin

Fungal ribotoxins, such as mitogillin and the related Aspergillus toxins restrictocin and α-sarcin, are highly specific ribonucleases, which inactivate the ribosome enzymatically by cleaving the eukaryotic 28S RNA of the large ribosomal subunit at a single phosphodiester bond. The site of cleavage occurs between G₄₃₂₅ and A₄₃₂₆, which are present in a 14-base sequence (the α-sarcin loop) conserved among the large subunit rRNAs of all living species. The amino acid residues involved in the cytotoxic activities of mitogillin were investigated by introducing point mutations using hydroxylamine into a recombinant Met-mature mitogillin (mitogillin with a Met codon at the N-terminus and no leader sequence) gene constructed from an Aspergillus fumigatus cDNA clone. These constructs were cloned into a yeast expression vector under the control of the GAL1 promoter and transformed into Saccharomyces cerevisiae. Upon induction of mitogillin expression, surviving transformants revealed that substitutions of certain amino acid residues on mitogillin abolished its cytotoxicity. Non-toxic mutant genes were cloned into an Escherichia coli expression vector, the proteins overexpressed and purified to homogeneity and their activities examined by in vitro ribonucleolytic assays. These studies identified the His-49Tyr, Glu-95Lys, Arg-120Lys and His-136Tyr mutations to have a profound impact on the ribonucleolytic activities of mitogillin. We conclude that these residues are key components of the active site contributing to the catalytic activities of mitogillin.     

Peptides with NH2-terminal tryptophan in the adenohypophysis

Summary In the mammalian pituitary formaldehyde-ozone treatment induces strong fluorescence in the cells of the pars intermedia and moderate to strong fluorescence in numerous cells of the pars distalis. Maximum excitation is at 370–375 nm and maximum emission at 495–505 nm. The properties of the cellular fluorescence are indistinguishable from those of tryptamine or peptides with NH₂-terminal tryptophan. From chemical analysis such peptides seem to occur abundantly in the mammalian pituitary. The concentration of these peptides agrees very well with the number and fluorescence intensity of the cells in all species studied. Furthermore, the tryptophyl peptides in the various parts of the pig pituitary have a distribution quite parallel to that of the fluorescent cells. As we have failed to detect tryptamine in the pituitary, we conclude that the formaldehyde-ozone-induced fluorescence in the adenohypophysis reflects the presence of tryptophyl peptides.This study was supported by grants from the Swedish Medical Research Council (04X-1007; 04X-3764), the Ford Foundation, Harald and Greta Jeanssons stiftelse and Riksföreningen mot Cancer (660-K73-01X).For brevity occasionally referred to as tryptophyl peptides.     

NH2-terminal processing of Bacillus subtilis alpha-amylase

Mature alpha-amylase of Bacillus subtilis is known to be formed from its precursor by removal of the NH2-terminal 41-amino acid sequence. To study the mechanism of this processing, the extracellular forms of alpha-amylase were analyzed for B. subtilis N7 alpha-amylase cloned and expressed in B. subtilis. The major form (form N34) isolated from log phase cultures in L-broth had an NH2 terminus corresponding to position 34 from the initiator Met but appeared to be microheterogeneous, as judged by native gel electrophoresis. The major forms from stationary phase cultures had NH2 termini at positions 40 (form N40) or 42 (form N42) and were homogeneous. The conversion of the larger to smaller forms could be achieved in culture supernatants or partially purified samples. The process N34----N40 was inhibited by EDTA; N40----N42 was facilitated by Ca2+. Phenylmethylsulfonyl fluoride inhibited the former but not the latter process. These results suggest that the signal peptidase cleavage site 30 decreases 35 is -Ala-Ala-Ala-Ser-Ala-Glu-Thr- (arrow or further upstream) and that proteolytic maturation occurs after secretion, which involves at least two different processing enzymes.     

Leucine 145 of the ribotoxin alpha-sarcin plays a key role for determining the specificity of the ribosome-inactivating activity of the protein

Secreted fungal RNases, represented by RNase T1, constitute a family of structurally related proteins that includes ribotoxins such as alpha-sarcin. The active site residues of RNase T1 are conserved in all fungal RNases, except for Phe 100 that is not present in the ribotoxins, in which Leu 145 occupies the equivalent position. The mutant Leu145Phe of alpha-sarcin has been recombinantly produced and characterized by spectroscopic methods (circular dichroism, fluorescence spectroscopy, and NMR). These analyses have revealed that the mutant protein retained the overall conformation of the wild-type alpha-sarcin. According to the analyses performed, Leu 145 was shown to be essential to preserve the electrostatic environment of the active site that is required to maintain the anomalous low pKa value reported for the catalytic His 137 of alpha-sarcin. Enzymatic characterization of the mutant protein has revealed that Leu 145 is crucial for the specific activity of alpha-sarcin on ribosomes.     

c-Jun NH2-terminal kinase activation leads to a FADD-dependent but Fas ligand-independent cell death in Jurkat T cells

Persistent c-Jun NH2-terminal kinase (JNK) activation induces cell death. Different mechanisms are ascribed to JNK-induced cell death. Most of the JNK-apoptosis studies employ stress stimuli known to activate kinases other than JNK. Here we used overexpression of mitogen-activated protein kinase kinase 7 (MKK7) to activate selectively JNK in T lymphoma Jurkat cells. Similar to that reported previously, Fas ligand (FasL) expression was up-regulated by JNK activation. Dominant negative-FADD and caspase-8 inhibitor benzyloxycarbonyl-Ile-Glu-Thr-Asp effectively inhibited MKK7-induced cell death, supporting a major involvement of FADD cascade. However, MKK7-induced cell death was not prevented by antagonist antibody ZB4 and Fas-Fc, indicating that Fas-FasL interaction is minimally involved. Confocal microscopy revealed that persistent JNK activation led to clustering of Fas. Our results suggest that, in contrast to that reported previously, JNK alone-induced death in Jurkat cells is FADD-dependent but is not triggered by Fas-FasL interaction.     

Deletion of the first 45 NH2-terminal residues of rabbit skeletal troponin T strengthens binding of troponin to immobilized tropomyosin.

The binding of the NH2-terminal region of troponin T (TnT) to the COOH-terminal region of tropomyosin (Tm) and the head-to-tail overlap between Tm molecules is thought to provide a pivotal link between troponin (Tn) and Tm (White, S.P., Cohen, C., and Phillips, G.N., Jr. (1987) Nature 325, 826-828). To further explore the structure-function relationship of the NH2-terminal region of TnT, we studied the binding of a 26,000-dalton TnT fragment (26K-TnT, Ohtsuki, I., Shiraishi, F., Suenaga, N., Miyata, T., and Tanokura, M.J. (1984) J. Biochem. (Tokyo) 95, 1337-1342) which corresponds to residues 46-259 of TnT2f, the major isoform of TnT in rabbit fast twitch muscle, to immobilized alpha-Tm. Both 26K-TnT and TnT2f were retained by the alpha-Tm affinity column in the presence of 150 mM NaCl. However, upon increasing the NaCl concentration 26K-TnT was eluted from the column at a higher ionic strength than was TnT. When applied alone, the binary complex of TnI and TnC (TnC.TnI) was not retained by the alpha-Tm affinity column. When applied subsequently to prebound TnT2f or 26K-TnT, TnI.TnC was retained by the alpha-Tm affinity column and eluted together with TnT2f or 26K-TnT as ternary troponin complexes. Whether Ca2+ was present or not, Tn containing 26K-TnT was eluted at a higher ionic strength than was Tn containing TnT2f, indicating that removal of the first 45 residues of TnT2f strengthens the binding of Tn to Tm. In the presence of Tm, reconstituted Tn containing 26K-TnT conferred Ca2+ sensitivity on actomyosin-S1 MgATPase, and the steepness of the pCa-ATPase relation was unchanged with respect to the actoS1 ATPase regulated by TnT2f. It is concluded that the first 45 residues of TnT2f are not essential for anchoring the troponin complex to the thin filament and do not play a crucial role in the cooperative response of regulated actoS1 ATPase to Ca2+.     

Mutagenesis of the NH2-terminal domain of elongation factor Tu

Mutagenesis was carried out in the N-terminal domain of elongation factor Tu (EF-Tu) to characterize the structure-function relationships of this model GTP binding protein with respect to stability, the interaction with GTP and GDP, and the catalytic activity. The substitutions were introduced in elements around the guanine nucleotide binding site or in the loops defining this site, in the intact molecule or in the isolated N-terminal domain (G domain). The double substitution Val88----Asp and Leu121----Lys, two residues situated on two vicinal alpha-helices, influences the basic activities of the truncated factor to a limited extent, probably via long-range interactions, and induces a destabilisation of the G domain structure. The functional alterations brought about by substitutions on the consensus sequences 18-24 and 80-83 highlight the importance of these residues for the interaction with GTP/GDP and the GTPase activity. Mutations concerning residues interacting with the guanine base lead to proteins in large part insoluble and inactive. In one case, the mutated protein (EF-TuAsn135----Asp) inhibited the growth of the host cell. This demonstrates the crucial role of the base specificity for the active conformation of EF-Tu. The obtained results are discussed in the light of the three-dimensional structure of EF-Tu.     

Assignment of the contribution of the tryptophan residues to the spectroscopic and functional properties of the ribotoxin alpha-sarcin

alpha-Sarcin, a potent cytotoxic protein from Aspergillus giganteus, contains two tryptophan residues at positions 4 and 51. Two single, W4F and W51F, and the double mutant, W4/51F, have been produced and purified to homogeneity. These two residues are neither required for the highly specific ribonucleolytic activity of the protein on the ribosomes (production of the so called alpha-fragment) nor for its interaction with lipid membranes (aggregation and fusion of vesicles), although the mutant forms involving Trp-51 show a decreased ribonuclease activity. Proton NMR data reveal that no significant changes in the global structure of the enzyme occur upon replacement of Trp-51 by Phe. Substitution of each Trp residue results in a 4 degrees C drop in the thermal denaturation midpoint, and the double mutant's midpoint is 9 degrees C lower. Trp-51 is responsible for most of the near-UV circular dichroism of the protein and also contributes to the overall ellipticity of the protein in the peptide bond region. Trp-51 does not show fluorescence emission. The membrane-bound proteins undergo a thermal denaturation at a lower temperature than the corresponding free forms. The interaction of the protein with phospholipid bilayers promotes a large increase of the quantum yield of Trp-51 and its fluorescence emission is quenched by anthracene incorporated into the hydrophobic region of such bilayers. This indicates that the region around this residue is located in the hydrophobic core of the bilayer following protein-vesicle interaction.