Conserved amino acid networks modulate discrete functional properties in an enzyme superfamily.

In this work, we applied the sequence-based statistical coupling analysis approach to characterize conserved amino acid networks important for biochemical function in the pancreatic-type ribonuclease (ptRNase) superfamily. This superfamily-wide analysis indicates a decomposition of the RNase tertiary structure into spatially distributed yet physically connected networks of co-evolving amino acids, termed sectors. Comparison of this statistics-based description with new NMR experiments data shows that discrete amino acid networks, termed sectors, control the tuning of distinct functional properties in different enzyme homologs. Further, experimental characterization of evolutionarily distant sequences reveals that sequence variation at sector positions can distinguish homologs with a conserved dynamic pattern and optimal catalytic activity from those with altered dynamics and diminished catalytic activities. Taken together, these results provide important insights into the mechanistic design of the ptRNase superfamily, and presents a structural basis for evolutionary tuning of function in functionally diverse enzyme homologs.

Comparative genomic analysis of eutherian ribonuclease A genes.

The present study attempted to update comprehensive eutherian ribonuclease A gene data sets, using public eutherian genomic sequence data sets and new genomics and molecular evolution tests. Among 448 ribonuclease A potential coding sequences, the present analysis annotated 255 complete coding sequences. The most comprehensive data set of eutherian ribonuclease A genes first characterized 13 major gene clusters, 9 of which showed evidence of differential gene expansions. In addition, the present analysis described common predicted promoter regions of eutherian ribonuclease A genes. The present study also attempted to resolve discrepancies in descriptions of eutherian ribonuclease A genes. Thus, the integrated gene annotations, phylogenetic analysis and protein molecular evolution analysis proposed new classification and nomenclature of eutherian ribonuclease A genes, as new framework of future experiments.

Identification of Apurinic/apyrimidinic endonuclease 1 (APE1) as the endoribonuclease that cleaves c-myc mRNA.

Endonucleolytic cleavage of the coding region determinant (CRD) of c-myc mRNA appears to play a critical role in regulating c-myc mRNA turnover. Using (32)P-labeled c-myc CRD RNA as substrate, we have purified and identified two endoribonucleases from rat liver polysomes that are capable of cleaving the transcript in vitro. A 17-kDa enzyme was identified as RNase1. Apurinic/apyrimidinic (AP) DNA endonuclease 1 (APE1) was identified as the 35-kDa endoribonuclease that preferentially cleaves in between UA and CA dinucleotides of c-myc CRD RNA. APE1 was further confirmed to be the 35-kDa endoribonuclease because: (i) the endoribonuclease activity of the purified 35-kDa native enzyme was specifically immuno-depleted with APE1 monoclonal antibody, and (ii) recombinant human APE1 generated identical RNA cleavage patterns as the native liver enzyme. Studies using E96A and H309N mutants of APE1 suggest that the endoribonuclease activity for c-myc CRD RNA shares the same active center with the AP-DNA endonuclease activity. Transient knockdown of APE1 in HeLa cells led to increased steady-state level of c-myc mRNA and its half-life. We conclude that the ability to cleave RNA dinucleotides is a previously unidentified function of APE1 and it can regulate c-myc mRNA level possibly via its endoribonuclease activity.

RNase A ribonucleases and host defense: an evolving story.

RNase A (bovine pancreatic RNase) is the founding member an extensive family of divergent proteins that share specific elements of sequence homology, a unique disulfide-bonded tertiary structure, and the ability to hydrolyze polymeric RNA. Among the more intriguing and perhaps counterintuitive findings, at the current state of the art, the connection between RNase activity and characterized host defense functions is quite weak; whether this is a scientific reality or more a reflection of what has been chosen for study remains to be determined. Several of the RNase A family RNases are highly cationic and have cytotoxic and bactericidal properties that are clearly (eosinophil cationic protein, leukocyte RNase A-2) or are probably (RNase 7) unrelated to their enzymatic activity. Interestingly, peptides derived from the leukocyte RNase A-2 sequence are nearly as bactericidal as the entire protein, suggesting that among other functions, the RNase A superfamily may be serving as a source of gene scaffolds for the generation of novel cytotoxic peptides. Other RNase A ribonucleases are somewhat less cationic (mouse angiogenin 4, zebrafish RNases) and have moderate bactericidal activities that have not yet been explored mechanistically. Additional host defense functions characterized specifically for the RNase eosinophil-derived neurotoxin include reducing infectivity of RNA viruses for target cells in culture, which does require RNase activity, chemoattraction of immature human dendritic cells via a G-protein-coupled receptor-dependent mechanism, and activation of TLR2. The properties of individual RNase A ribonucleases, recent experimental findings, and important questions for the near and distant future will be reviewed.

RNAse A-like enzymes in serum inhibit the anti-neoplastic activity of siRNA targeting polo-like kinase 1.

Down-modulation of target molecules in tumor cells by small interfering (si) RNAs is a promising anti-cancer strategy. A major challenge of this approach is the loss of silencing activity of the siRNAs in vivo. Our study aimed at investigating the influence of the serum compartment on the anti-tumor activity of siRNA directed against Polo-like kinase 1 (Plk1), a mitosis-associated serine/threonine kinase. The data showed that siRNA-induced suppression of Plk1 expression effectively reduced the viable cell mass and increased apoptosis in several cancer cell lines. Preincubation of the siRNA in human serum led to shortening of the siRNA as well as loss of its Plk1 silencing and anti-tumor activity. This loss of activity was prevented by inhibition of RNAse A-like enzymes. These data indicate that the anti-neoplastic effect of siRNAs declines upon incubation in human serum. This loss of anti-neoplastic activity can be prevented by inhibition of their degradation by RNAse A-like enzymes. This may have important implications for the development of a human therapeutic application of siRNAs.

Subtle functional collective motions in pancreatic-like ribonucleases: from ribonuclease A to angiogenin.

The analysis of the dynamic behavior of enzymes is fundamental to structural biology. A direct relationship between protein flexibility and biological function has been shown for bovine pancreatic ribonuclease (RNase A) (Rasmussen et al., Nature 1992;357:423-424). More recently, crystallographic studies have shown that functional motions in RNase A involve the enzyme beta-sheet regions that move concertedly on substrate binding and release (Vitagliano et al., Proteins 2002;46:97-104). These motions have been shown to correspond to intrinsic dynamic properties of the native enzyme by molecular dynamics (MD) simulations. To unveil the occurrence of these collective motions in other members of pancreatic-like superfamily, we carried out MD simulations on human angiogenin (Ang). Essential dynamics (ED) analyses performed on the trajectories reveal that Ang exhibits collective motions similar to RNase A, despite the limited sequence identity (33%) of the two proteins. Furthermore, we show that these collective motions are also present in ensembles of experimentally determined structures of both Ang and RNase A. Finally, these subtle concerted beta-sheet motions were also observed for other two members of the pancreatic-like superfamily by comparing the ligand-bound and ligand-free structures of these enzymes. Taken together, these findings suggest that pancreatic-like ribonucleases share an evolutionary conserved dynamic behavior consisting of subtle beta-sheet motions, which are essential for substrate binding and release.

Compensating effects on the cytotoxicity of ribonuclease A variants.

Ribonuclease (RNase) A can be endowed with cytotoxic activity by enabling it to evade the cytosolic ribonuclease inhibitor protein (RI). Enhancing its conformational stability can increase further its cytotoxicity. Herein, the A4C/K41R/G88R/V118C variant of RNase A was created to integrate four individual changes that greatly decrease RI affinity (K41R/G88R) and increase conformational stability (A4C/V118C). Yet, the variant suffers a decrease in ribonucleolytic activity and is only as potent a cytotoxin as its precursors. Thus, individual changes that increase cytotoxicity can have offsetting consequences. Overall, cytotoxicity correlates well with the maintenance of ribonucleolytic activity in the presence of RI. The parameter (k(cat)/K(m))(cyto), which reports on the ability of a ribonuclease to manifest its ribonucleolytic activity in the cytosol, is especially useful in predicting the cytotoxicity of an RNase A variant.

Molecular evolution of mammalian ribonucleases 1.

There have been many studies on the chemistry of mammalian pancreatic ribonucleases (ribonucleases 1), but the functional biology of this family of homologous proteins is still largely unknown. Many studies have been performed on the molecular evolution and properties of this enzyme from species belonging to a large number of mammalian taxa, including paralogous gene products resulting from recent gene duplications. Novel ribonuclease 1 sequences were determined for three rodent species (gundi, brush-tailed porcupine, and squirrel), rabbit, a fruit bat, elephant, and aardvark, and the new sequences were used for deriving most parsimonious networks of ribonucleases from different mammalian orders, including earlier determined nucleotide sequences and also a larger set of protein sequences. Weak support for interordinal relationships were obtained, except for an Afrotheria clade containing elephant and aardvark. Results of current analyses and also those obtained 20 years ago on amino acid sequences confirm conclusions derived recently from larger data sets of other molecules. Several examples of recent gene duplications in ribonucleases 1 are discussed, with respect to illustrate the concepts of orthology and paralogy. Previously evidence was presented for extensive parallelism between sequence regions with attached carbohydrate (about one quarter of the molecule) of unrelated species with cecal digestion (pig and guinea pig). These features are also present in the sequences of elephant and fruit bat, species with cecal digestion, but with a very low ribonuclease content in their pancreas.

Rapid diversification of RNase A superfamily ribonucleases from the bullfrog, Rana catesbeiana.

We present sequences of five novel RNase A superfamily ribonuclease genes of the bullfrog, Rana catesbeiana. All five genes encode ribonucleases that are similar to Onconase, a cytotoxic ribonuclease isolated from oocytes of R. pipiens. With amino acid sequence data from 14 ribonucleases from three Rana species (R. catesbeiana, R. japonica, and R. pipiens), we have constructed bootstrap-supported phylogenetic trees that reorganize these ribonucleases into five distinct lineages--the pancreatic ribonucleases (RNases 1), the eosinophil-associated ribonucleases (RNases 2, 3, and 6), the ribonucleases 4, the angiogenins (RNases 5) and the Rana ribonucleases--with the Rana ribonucleases no more closely related to the angiogenins than they are to any of the other ribonuclease lineages shown. Further phylogenetic analysis suggests the division of the Rana ribonucleases into two subclusters (A and B), with positive (Darwinian) selection (dN/dS > 1.0) and an elevated rate of radical nonsynonymous substitution (dR) contributing to the rapid diversification of ribonucleases within each cluster. This pattern of evolution-rapid diversification via positive selection among sequences of a multigene cluster-bears striking resemblance to what we have described for the eosinophil-associated ribonuclease genes of the rodent Mus musculus, a finding that may have implications with respect the physiologic function of this unique family of proteins.

Introduction: the ribonuclease A superfamily.

In this multi-author issue several aspects of the ribonuclease A superfamily are reviewed. This superfamily can be subdivided in a number of mammalian and other vertebrate ribonuclease families. In the introduction chapter the titles of the other contributions are presented. There is little uniformity in the nomenclature of ribonucleases, caused in part by gene duplications, which have occurred independently in several mammalian lineages, and which are nice examples for explaining orthology and paralogy in molecular evolution.

Tissue-specific expression of pancreatic-type RNases and RNase inhibitor in humans.

The tissue-specific expression of five human pancreatic-type RNases and RNase inhibitor was analyzed by Northern hybridization against poly(A)+ RNA prepared from 16 normal tissues. The widespread expression of RNase 1 was observed in almost all of the tissues. RNase 4 and angiogenin showed a similar distribution of expression abundantly present in the liver. This suggested the identity of the cell types producing these two molecules. However, no relativity appeared to be present between the vascularization of the tissues and the angiogenin expression. A narrow range of expression of the eosinophil-derived neurotoxin gene was observed. This localization seems related to the phagocytic cells in the tissues. The undetectable level of the eosinophil cationic protein mRNA in normal tissues suggests that the differentiation of eosinophils, triggered by inflammation and/or atopy, is required. The expression of RNase inhibitor was found to be ubiquitous. The regulatory function of inhibitor against RNases in the cell should be considered in studying the physiological significance of the pancreatic-type RNase family.