The two dimeric forms of RNase A

Sorrentino, Salvatore; Barone, Roberto; Bucci, Enrico; Gotte, Giovanni; Russo, Aniello; Libonati, Massimo; D’Alessio, Giuseppe

doi:10.1016/s0014-5793(99)01742-1

Cited by 28 publications

(41 citation statements)

References 24 publications

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“…These signals can reasonably be attributed to the phenolic rings of tyrosine residues (Simons and Blout 1968;Grandi et al 1979). It has already been reported (Sorrentino et al 2000) that in going from the monomer to the minor and to the major dimer, the CD signals become more positive at 242 nm, and less negative at 278 nm. This finding has been reported as being the evidence of a progressively higher exposure of the phenolic rings of tyrosine residues (Grandi et al 1979).…”

Section: Circular Dichroism Analysesmentioning

confidence: 80%

“…The area of each peak was measured and expressed as a percentage of the area of the corresponding input (trimers or tetramers at zero time). The results were (1) that both more basic conformers (T 2 and TT 2 ) are more labile than their less basic counterparts, a property also shared by dimeric RNase A (Sorrentino et al 2000), and (2) that both trimers are remarkably more resistant than the two tetramers. In fact, although after 2 h at 35°C about 40% of TT 1 and 60% of TT 2 were already dissociated, almost no dissociation could be detected for both T 1 and T 2 .…”

Section: Kinetics Of Thermal Dissociation Of Trimeric and Tetrameric mentioning

confidence: 99%

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Structural properties of trimers and tetramers of ribonuclease A

et al. 2001

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Ribonuclease A aggregates (dimers, trimers, tetramers, pentamers) can be obtained by lyophilization from 40% acetic acid solutions. Each aggregate forms two conformational isomers distinguishable by different basic net charge. The crystal structure of the two dimers has recently been determined; the structure of the higher oligomers is unknown. The results of the study of the two trimeric and tetrameric conformers can be summarized as follows: (1) RNase A trimers and tetramers form by a 3D domain-swapping mechanism. N-terminal and C-terminal types of domain swapping could coexist; (2) the secondary structures of the trimeric and tetrameric conformers do not show significant differences if compared with the secondary structure of monomeric RNase A or its two dimers; (3) a different exposure of tyrosine residues indicates that in the aggregates they have different microenvironments; (4) the two trimeric and tetrameric conformers show different susceptibility to digestion by subtilisin; (5) dimers, trimers, and tetramers of RNase A show unwinding activity on double-helical poly(dA-dT) · poly(dA-dT), that increases as a function of the size of the oligomers; (6) the less basic conformers are more stable than the more basic ones, and a low concentration in solution of trimers and tetramers favors their stability, which is definitely increased by the interaction of the aggregates with poly(dA-dT) · poly(dA-dT); (7) the products of thermal dissociation of the two trimers indicate that their structures could be remarkably different. The dissociation products of the two tetramers allow the proposal of two models for their putative structures.Keywords: RNase A oligomers; trimers and tetramers of RNase A; properties of trimeric and tetrameric RNase A; RNase A aggregates higher than dimersThe study of the manner in which proteins aggregate in vitro can help in understanding the process of protein aggregation in vivo, and, therefore, also the origin of pathologic proteins responsible for several severe diseases.Although it has recently been reported that native ribonuclease A can dimerize at neutral pH (Park and Raines 2000), it is known that by lyophilization from 30-50% acetic acid solutions RNase A gives rise to oligomers (Crestfield et al. 1962) ranging from dimers to pentamers and possibly higher aggregates, each oligomeric species existing in the form of at least two conformational isomers (Gotte et al. 1999).Dimeric RNase A obtained by lyophilization consists of a minor and a major component that are in the ratio of about 1:3-1:4. Their crystal structures have been determined (Liu et al. 1998(Liu et al. , 2001, and the two dimeric conformers are 3D domain-swapping dimers formed by the exchange of the N-terminal ␣-helix of each monomeric subunit in the case of the minor dimer, of the C-terminal ␤-strand, instead, for the major RNase A dimer. This unique behavior of RNase A, in the dimeric aggregates of which the two known mechanisms of protein aggregation by domain swapping coexist, expands the range of possibi...

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Section: Circular Dichroism Analysesmentioning

confidence: 80%

Section: Kinetics Of Thermal Dissociation Of Trimeric and Tetrameric mentioning

confidence: 99%

Structural properties of trimers and tetramers of ribonuclease A

et al. 2001

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“…Accordingly, the latter shows in vitro a lower antitumor activity than that displayed by the CNC-tetramer. However, it has to be taken into account that, although, as mentioned above, under all experimental conditions tested the stability of the RNase A oligomers undoubtedly decreases in going from dimers to tetramers, for each oligomeric species the stability of one of the two conformers relative to that of the other appears to be greatly influenced by the environmental conditions (3,49). 2 In conclusion, however, the activity patterns shown in Figs.…”

Section: Mice Embryos After 72 Hours Incubation With Rnase a Oligomermentioning

confidence: 81%

“…Another point to be considered in relation to the biological activities reported here is the survival of the various RNase A oligomers in solution and in in vitro or in vivo experiments. The stability of dimers, trimers and tetramers, dissolved in different buffers at neutral pH, was studied as a function of temperature, and the kinetics of the dissociation of trimers and tetramers at 35°C was measured (3,49). The results consistently indicated that dimers are relatively more stable than trimers and tetramers, the latter being the least stable oligomers.…”

Section: Mice Embryos After 72 Hours Incubation With Rnase a Oligomermentioning

confidence: 96%

Antitumor Activity and Other Biological Actions of Oligomers of Ribonuclease A

Matoušek

Gotte

Poučková

et al. 2003

Journal of Biological Chemistry

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Dimers, trimers, and tetramers of bovine ribonuclease A, obtained by lyophilization of the enzyme from 40% acetic acid solutions, were purified and isolated by cation exchange chromatography. The two conformers constituting each aggregated species were assayed for their antitumor, aspermatogenic, or embryotoxic activities in comparison with monomeric RNase A and bovine seminal RNase, which is dimeric in nature. The antitumor action was tested in vitro on ML-2 (human myeloid leukemia) and HL-60 (human myeloid cell line) cells and in vivo on the growth of human non-pigmented melanoma (line UB900518) transplanted subcutaneously in nude mice. RNase A oligomers display a definite antitumor activity that increases as a function of the size of the oligomers. On ML-2 and HL-60 cells, dimers and trimers generally show a lower activity than bovine seminal RNase; the activity of tetramers, instead, is similar to or higher than that of the seminal enzyme. The growth of human melanoma in nude mice is inhibited by RNase A oligomers in the order dimers < trimers < tetramers. The action of the two tetramers is very strong, blocking almost completely the growth of melanoma. RNase A dimers, trimers, and tetramers display aspermatogenic effects similar to those of bovine seminal RNase, but, contrarily, they do not show any embryotoxic activity.Bovine ribonuclease A oligomerizes in the forms of dimers (1), trimers, tetramers, and higher order oligomers (2) during lyophilization from 40% acetic acid solutions. Each oligomer consists of two conformational isomers, which can be separated by cation exchange chromatography into a less basic and a more basic species (2, 3). The molecular structures of the two dimers have been solved (4, 5). They form by a three-dimensional domain-swapping mechanism (6); the less basic dimer, formerly named minor because of its ratio of 1:4 to the more basic dimer (2,3,5), is formed by the swapping of the Nterminal ␣-helix (residues 1-15) of each monomeric subunit, and the more basic or major dimer (2, 3, 5) is formed by the swapping of the C-terminal ␤-strand (residues 116 -124) of each monomer. On this basis, the two dimers will be called N-dimer and C-dimer, respectively. The structure of the more basic or minor trimer (2, 3) has also been solved; it is formed by three monomers linked to each other by swapping their Cterminal ␤-strands, thereby forming a circular structure that looks like a propeller (7). It will be called the C-trimer in this paper. On the basis of its dissociation products (3, 7), a plausible linear model was proposed for the less basic, major trimer (its abundance is 1.5 times that of the more basic, minor trimer). In this linear model, two monomers are linked through swapping of their N termini, and a third monomer is bound to one of them by C-terminal domain swapping (5, 7). It will be called the NC-trimer. Two linear structures for the two tetramers, the less basic minor and the more basic major (ratio, 1:1.6), have also been proposed on the basis of their dissociation products (...

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“…Beside BS-RNase, other ribonucleases, such as RNase A (25,26) and human pancreatic ribonuclease (HP-RNase) (27), are able to form in vitro non-covalent dimers through the swapping of a structural element among subunits. In particular, under well defined experimental conditions, monomeric RNase A produces two different non-covalent dimeric forms (26), both studied crystallographically: ND-RNase A, where the N-terminal ␣-helices are interchanged (28) and CD-RNase A, where the swapping involves the C-terminal ␤-strands (29).…”

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confidence: 99%

Structure and Stability of the Non-covalent Swapped Dimer of Bovine Seminal Ribonuclease

Sica

Fiore

Merlino

et al. 2004

Journal of Biological Chemistry

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A growing number of pancreatic-type ribonucleases (RNases) present cytotoxic activity against malignant cells. The cytoxicity of these enzymes is related to their resistance to the ribonuclease protein inhibitor (RI). In particular, bovine seminal ribonuclease (BS-RNase) is toxic to tumor cells both in vitro and in vivo. BS-RNase is a covalent dimer with two intersubunit disulfide bridges between Cys 31 of one chain and Cys 32 of the second and vice versa. The native enzyme is an equilibrium mixture of two isomers, MxM and M‫؍‬M. In the former the two subunits swap their N-terminal helices. The cytotoxic action is a peculiar property of MxM. In the reducing environment of cytosol, M‫؍‬M dissociates into monomers, which are strongly inhibited by RI, whereas MxM remains as a non-covalent dimer (NCD), which evades RI. We have solved the crystal structure of NCD, carboxyamidomethylated at residues Cys 31 and Cys 32 (NCD-CAM), in a complex with 2-deoxycitidylyl(3-5)-2-deoxyadenosine. The molecule reveals a quaternary structural organization much closer to MxM than to other N-terminal-swapped non-covalent dimeric forms of RNases. Model building of the complexes between these non-covalent dimers and RI reveals that NCD-CAM is the only dimer equipped with a quaternary organization capable of interfering seriously with the binding of the inhibitor. Moreover, a detailed comparative structural analysis of the dimers has highlighted the residues, which are mostly important in driving the quaternary structure toward that found in NCD-CAM.The RNA population in cells is controlled post-transcriptionally by ribonucleases (RNases) of varying specificity. These secretory enzymes operate at the cross-roads of transcription and translation by catalyzing RNA degradation (1, 2). RNases exhibit diverse biological activities, including specific toxicity to cancer cells (3). RNase activity in serum and cell extracts is elevated in a variety of cancers and infectious diseases. The antitumor effect of ribonucleases has been studied using pancreatic RNase (RNase A) and seminal RNase (BS-RNase) 1 from Bos taurus, and onconase and RC-RNase from amphibians (2, 3-6). RNase A does not display appreciable cytotoxic effects toward tumor cells, whereas BS-RNase (6 -9), onconase (6, 10 -12), and RC-RNase (13) are effective cytotoxic agents both in vitro and in vivo. It is generally accepted that the cytoxicity of these RNases is mainly related to their ability to evade the cytoplasmic RNase inhibitor (RI) and to degrade cellular RNA (5, 14). The antitumor activity exhibited by cytotoxic RNases varies depending on the type of tumor. Factors that could determine susceptibility are the ability of RNases to bind to the cell surface and the efficiency of their internalization (15).In the vertebrate RNase family, for which RNase A is the prototype, BS-RNase holds a unique position, as it is the only native dimeric member, and the two subunits are cross-linked by disulfide bonds between Cys 31 of one chain and Cys 32 of the other and vice versa (16). Anot...

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The two dimeric forms of RNase A

Cited by 28 publications

References 24 publications

Structural properties of trimers and tetramers of ribonuclease A

Structural properties of trimers and tetramers of ribonuclease A

Antitumor Activity and Other Biological Actions of Oligomers of Ribonuclease A

Structure and Stability of the Non-covalent Swapped Dimer of Bovine Seminal Ribonuclease

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