These modeling data allow us to speculate that the HHpred structural model represents the active state of the PP11 enzyme in which a putative RNA-binding domain can be recognized

These modeling data allow us to speculate that the HHpred structural model represents the active state of the PP11 enzyme in which a putative RNA-binding domain can be recognized. a ribonuclease function to PP11. This newly discovered enzymatic activity places PP11-like proteins in a completely new perspective. PP11 (placental protein 11) is one of the five glycoproteins (together with PP8, PP9, PP10, and PP12) that Lenalidomide-C5-NH2 were first isolated from aqueous extracts of human term placentas in the 1980s. Unlike PP8 and PP9, which were found in relatively high MMP7 concentrations in all tissues analyzed, PP10, PP11, and PP12 were supposed to be specific to the placentas, since they could not be detected in extracts of other human tissues (1,2). Interestingly, it was shown that the placenta-specific proteins were also expressed in several tumor tissues; PP11 was detected in 66.7% of the analyzed mucinous cystadenocarcinomas and in 57.1% of serous cystadenocarcinomas tested, whereas it was not found in normal ovaries (3). Moreover, PP11 was also found in 47% of all breast Lenalidomide-C5-NH2 cancers examined (4) and in 38% of all testicular and gastric cancers studied (5). These results suggested that this protein may be involved in carcinogenesis and, therefore, may represent a suitable molecular marker for tumor diagnosis. A putative function for PP11 was proposed in 1990, when the cDNA was isolated from a placental cDNA library and the encoded protein was expressed inEscherichia coli; two different forms, a precursor of 45 kDa and a mature protein of 42 kDa, were produced. Employing a colorimetric assay on purified placental protein and on periplasmic and cytoplasmic fractions of extracts prepared fromE. coli, containing the 42- and 45-kDa proteins, respectively, a putative serine protease activity was assigned to the placental and to the recombinant 42-kDa proteins (6). Another piece of information, which may contribute to the understanding of PP11 biological role, derives from its subcellular localization; PP11 was exclusively localized in the cytoplasm of syncytiotrophoblast (7). Since then, PP11 has not been subjected to further characterization, validating its protease activity and investigating its natural substrates. Recently, PP11 has been identified as a member of the XendoU family, a novel protein class including several enzymes that share a significant sequence homology with the founding member XendoU (8,9). This enzyme is an amphibian endoribonuclease that participates in the biosynthesis of small nucleolar RNAs, a class of noncoding Lenalidomide-C5-NH2 RNAs involved in ribosome biogenesis (1012). Its viral homolog Nsp15, the second family member characterized, is an endoribonucleolytic activity considered as a major genetic marker of the Nidovirales order, including the coronavirus responsible for the severe acute respiratory syndrome (13,14). Notably, even if the specific substrate of Nsp15 has not yet been identified, an essential role for this protein in virus replication and transcription was established (13,14). Within the XendoU family, PP11 is highly conserved both in vertebrates and in invertebrates (11). The finding that the human protein shares both a high sequence homology (38% identity and 55% similarity) and a predicted secondary structure with XendoU (9,12) did not support its putative serine protease activity: accordingly, no canonical catalytic triad, typical of serine proteases, was found in homology modeling of PP11 (9). To delve into its function, we expressed a recombinant histidine-tagged PP11 (His-PP11) and employed the purified protein to investigate several aspects concerning its enzymatic activity, biochemical properties, and cellular localization. Here we show that PP11 has RNA binding activity, whereas it does not display the predicted serine protease function; it proved to be an endoribonuclease sharing the main biochemical features (i.e.ion requirement, cleavage site specificity, and released products) with the RNases belonging to the same family. Furthermore, homology modeling.