Suprainduction of p53 by disruption of 40S and 60S ribosome biogenesis leads to the activation of a novel G2/M checkpoint.

Impairment of ribosome biogenesis leads to p53 induction and cell cycle arrest, a checkpoint involved in human disease. Induction of p53 is attributed to the binding and inhibition of human double minute 2 (Hdm2) by a subset of ribosomal proteins (RPs): RPS7, RPL5, RPL11, and RPL23. However, we found that only RPL11 or RPL5, in a mutually dependent manner, elicit this response. We show that depletion of RPS7 or RPL23, like depletion of other RPs, except for RPL11 and RPL5, induces a p53 response and that the effects of RPS7 and RPL23 on p53 induction reported earlier may be ascribed to inhibition of global translation. Moreover, we made the surprising observation that codepletion of two essential RPs, one from each subunit, but not the same subunit, leads to suprainduction of p53. This led to the discovery that the previously proposed RPL11-dependent mechanism of p53 induction, thought to be caused by abrogation of 40S biogenesis and continued 60S biogenesis, is still operating, despite abrogation of 60S biogenesis. This response leads to both a G1 block and a novel G2/M block not observed when disrupting either subunit alone. Thus, induction of p53 is mediated by distinct mechanisms, with the data pointing to an essential role for ribosomal subunits beyond translation.

Trafficking and intracellular ATPase activity of human ecto-nucleotidase NTPDase3 and the effect of ER-targeted NTPDase3 on protein folding.

Ecto-nucleoside triphosphate diphosphohydrolases, NTPDase1 (CD39) and NTPDase3, are integral plasma membrane proteins that hydrolyze extracellular nucleotides, thereby modulating the function of purinergic receptors. During processing in the secretory pathway, the active sites of ecto-nucleotidases are located in the lumen of vesicular compartments, thus raising the question whether the ecto-nucleotidases affect the ATP-dependent processes in these compartments, including protein folding in the endoplasmic reticulum (ER). It has been reported (J. Biol. Chem. (2001) 276, 41518-41525) that CD39 is not active until it reaches the plasma membrane, suggesting that terminal glycosylation in Golgi is critical for its activity. To investigate the subcellular location and the mechanism of ecto-nucleotidase activation, we expressed human NTPDase3 in COS-1 cells and blocked the secretory transport with monensin or brefeldin A, or by targeting to ER with a signal peptide. Cell surface biotinylation, sensitivity to glycosidases, and fluorescence microscopy analyses suggest that, in contrast to the previous report on CD39, NTPDase3 becomes catalytically active in the ER or in the ER-Golgi intermediate compartment, and that terminal glycosylation in Golgi is not essential for activity. Moreover, ER-targeted NTPDase3, but not wild-type NTPDase3 or ER-targeted inactive G221A mutant, significantly diminished the folding efficiency and the transport to the plasma membrane of coexpressed CD39 used as a reporter protein. These data suggest that ER-targeted NTPDase3 significantly depletes ATP in ER, whereas wild-type NTPDase3 is likely to acquire ATPase activity in a post-ER, but pre-Golgi, compartment, thus avoiding unproductive ATP hydrolysis and interference with protein folding in the ER. ER-targeted NTPDase3 may be a useful experimental tool to study the effects of ER ATP depletion on ER function under normal and stress conditions.

Epitope mapping in cell surface proteins by site-directed masking: defining the structural elements of NTPDase3 inhibition by a monoclonal antibody.

We adapted the method of epitope mapping by site-directed masking, which was described for purified soluble antigens [Paus,D. and Winter,G. (2006) Proc. Natl Acad. Sci. USA, 103, 9172-9177.], to map the binding site of an inhibitory monoclonal antibody on the cell surface protein ecto-nucleotidase NTPDase3. Using homology modeling, we built a 3D structure of NTPDase3 and designed 21 single cysteine mutations distributed over the surface of the enzyme. The mutant proteins were expressed in cells, biotinylated with a cysteine-specific reagent, and then extracted with detergent and immobilized on streptavidin-coated plates. Tethering NTPDase3 via cysteine residues located in a surface patch near the active site cleft masked the epitope and blocked antibody binding, as evaluated by enzyme inhibition assay and by ELISA. We then constructed 18 single alanine substitution mutations within the defined patch and found that W403A, D414A, E415A and R419A decreased the inhibitory effect of the antibody, whereas the double mutation W403A/R419A abolished both antibody binding and enzyme inhibition, suggesting the critical role of these residues for interaction with the antibody. Lack of competition between the antibody and a non-hydrolyzable substrate analog AMPPCP, as well as location of the epitope adjacent to the active site, suggest a noncompetitive mechanism of inhibition by steric hindrance. The described technique should be useful for systematic epitope mapping in cell membrane proteins for which either a 3D structure is available, or a sufficiently accurate 3D model can be obtained by homology modeling.

Characterization of a monoclonal antibody as the first specific inhibitor of human NTP diphosphohydrolase-3 : partial characterization of the inhibitory epitope and potential applications.

The study and therapeutic modulation of purinergic signaling is hindered by a lack of specific inhibitors for NTP diphosphohydrolases (NTPDases),which are the terminating enzymes for these processes. In addition, little is known of the NTPDase protein structural elements that affect enzymatic activity and which could be used as targets for inhibitor design. In the present study, we report the first inhibitory monoclonal antibodies specific for an NTPDase, namely human NTPDase3 (EC 3.6.1.5), as assessed by ELISA, western blotting, flow cytometry, immunohistochemistry and inhibition assays. Antibody recognition of NTPDase3 is greatly attenuated by denaturation with SDS, and abolished by reducing agents, indicating the significance of the native conformation and the disulfide bonds for epitope recognition. Using site-directed chemical cleavage, the SDS-resistant parts of the epitope were located in two fragments of the C-terminal lobe ofNTPDase3 (i.e. Leu220-Cys347 and Cys347-Pro485), which are both required for antibody binding. Additional site-directed mutagenesis revealed the importance of Ser297 and the fifth disulfide bond (Cys399-Cys422) for antibody binding, indicating that the discontinuous inhibitory epitope is located on the extracellular C-terminal lobe of NTPDase3. These antibodies inhibit recombinant NTPDase3 by 60-90%, depending on the conditions. More importantly, they also efficiently inhibit the NTPDase3expressed in insulin secreting human pancreatic islet cells in situ. Because insulin secretion is modulated by extracellular ATP and purinergic receptors, this finding suggests the potential application of these inhibitory antibodies for the study and control of insulin secretion.

Characterization of disulfide bonds in human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): implications for NTPDase structural modeling.

Cell-surface nucleotidases (NTPDases) contain 10 invariant cysteine residues in their extracellular regions. To investigate disulfide structure in human NTPDase3, we made single and double mutants of these 10 cysteines, and analyzed their enzymatic activity, glycosylation pattern, trafficking to the cell membrane, and sensitivity to reduction. The mutants constituted five distinct phenotypes, thus, strongly suggesting disulfide bonds between C92-C116 (first bond), C261-C308 (second bond), C289-C334 (third bond), C347-C353 (fourth bond), and C399-C422 (fifth bond). Due to conservation of the 10 cysteines, the identified five disulfide bonds are likely to exist in all cell-surface NTPDases. The third and fifth bonds are also present in the soluble NTPDases and are critical for processing, trafficking, and enzymatic activity. The fourth bond has minimal effect on processing and function, while the first and second bonds are of intermediate importance. Most of the N-linked glycosylation sites in the wild-type enzyme are processed to complex oligosaccharides, but at least one site is high-mannose or hybrid in structure. Interestingly, disruption of the first disulfide bond resulted in some enzyme that lost sensitivity to endoglycosidase H, suggesting that the first disulfide bond in the wild-type enzyme shields some high-mannose glycans from terminal glycosylation. Comparative modeling by threading and homology modeling of the NTPDase3 sequence revealed a high degree of structural fold similarity with a bacterial exopolyphosphatase (PDB ). The resultant theoretical 3-D model of the extracellular portion of NTPDase3, based on homology with this exopolyphosphatase, is consistent with the assignment of the disulfide bonds occurring in regions of good fold similarity between NTPDase3 and the exopolyphosphatase. The 3-D model obtained for NTPDase3 also suggests the structural basis for the importance of several apyrase conserved regions for the nucleotidase activities of the NTPDases.

Identification of cysteine residues responsible for oxidative cross-linking and chemical inhibition of human nucleoside-triphosphate diphosphohydrolase 3.

Cysteine-to-serine mutations were constructed to test the functional and structural significance of the three non-extracellular cysteine residues in ecto-nucleoside-triphosphate diphosphohydrolase 3 (eNTPDase3). None of these cysteines were found to be essential for enzyme activity. However, Cys(10), located on the short N-terminal cytoplasmic tail, was found to be responsible for dimer formation occurring via oxidation during membrane preparation as well as for dimer cross-linking resulting from exogenously added sulfhydryl-specific cross-linking agents. The resistance to further cross-linking of these dimers into higher order oligomers by lysine-specific cross-linkers suggests that this enzyme may form its native tetrameric structure as a "dimer of dimers" with nonequivalent interactions between subunits. Cys(501), located in the hydrophobic C-terminal membrane-spanning domain of eNTPDase3, was found to be the site of chemical modification by a sulfhydryl-specific reagent, p-chloromercuriphenylsulfonic acid (pCMPS), leading to inhibition of enzyme activity. The effect of pCMPS was negligible after dissociation of the enzyme into monomers by Triton X-100, suggesting that the mechanism of inhibition is dependent on the oligomeric structure. Because Cys(501) is accessible for modification by the membrane-impermeant reagent pCMPS, we hypothesize that eNTPDase3 (and possibly other eNTPDases) contains a water-filled crevice allowing access of water and hydrophilic compounds to at least part of the protein's C-terminal membrane-spanning helix.

Bacterial expression and characterization of a novel, soluble, calcium-binding, and calcium-activated human nucleotidase.

A newly discovered human analogue of a bed bug apyrase, which we named hSCAN-1 for human soluble calcium-activated nucleotidase-1, was expressed in bacteria, refolded from inclusion bodies, purified, and characterized. This apyrase, which is distinct from the eNTPDases exemplified by the endothelial CD39 (NTPDase1) apyrase, is a 38 kDa monomeric enzyme capable of hydrolyzing a variety of nucleoside di- and triphosphates, but not monophosphates. Preferred substrates include GDP, UDP, and IDP, with a pH optimum for activity between 6 and 7. The specific activity and substrate preference of the bacterially expressed enzyme closely mimic those of the enzyme expressed in mammalian COS cells, as well as the enzyme synthesized in an in vitro bacterial expression system. This suggests that glycosylation and other posttranslational modifications that do not occur in bacteria are not necessary for nucleotidase activity or proper folding of this human apyrase. hSCAN-1 absolutely requires Ca(2+), but not Mg(2+) or other divalent cations analyzed, for enzymatic activity. Surprisingly, the activity does not increase in a quasi-linear fashion at sub-millimolar Ca(2+) concentrations, as would be expected if Ca(2+) were only used as a cosubstrate for the nucleotide substrate, but rather follows a sigmoidal curve. The intrinsic fluorescence and difference absorption studies of hSCAN-1 in the absence of nucleotides revealed Ca(2+)-induced changes in the environment of tryptophan and tyrosine residues with half-saturation at about 90 microM Ca(2+). NaCl increased the half-saturating Ca(2+) concentration needed for both structural changes detected by optical spectroscopy and enzymatic activation of hSCAN-1 detected by nucleotidase assay. These results suggest that Ca(2+) triggers a conformational change in hSCAN-1, converting the enzymatically inactive protein to the active enzyme, in addition to forming the metal-nucleotide substrate complex necessary for nucleotidase activity.

Bacterial expression, characterization, and disulfide bond determination of soluble human NTPDase6 (CD39L2) nucleotidase: implications for structure and function.

The ectonucleoside triphosphate diphosphohydrolases (NTPDases) control extracellular nucleotide concentrations, thereby modulating many important biological responses, including blood clotting and pain perception. NTPDases1-4 are oligomeric integral membrane proteins, whereas NTPDase5 (CD39L4) and NTPDase6 (CD39L2) are soluble monomeric enzymes, making them more amenable to thorough structural and functional analyses than the membrane-bound forms. Therefore, we report here the bacterial expression, refolding, purification, and biochemical characterization of the soluble portion of human NTPDase6. Consistent with the enzyme expressed in mammalian cells, this recombinant NTPDase6 efficiently hydrolyzes GDP, IDP, and UDP (specific activity of approximately 50000 micromol mg(-1) h(-1)), with slower hydrolysis of CDP, ITP, GTP, CTP, ADP, and UTP and virtually no hydrolysis of ATP. The K(m) for GDP (130 +/- 30 microM) is similar to that determined for the soluble rat NTPDase6 expressed in mammalian cells. The secondary structure of the refolded enzyme was determined by circular dichroism to be 33% alpha-helix, 18% beta-sheet, and 49% random coil, consistent with the secondary structure predicted from the amino acid sequence of soluble NTPDase6. Four of the five cysteine residues in the soluble NTPDase6 are highly conserved among all the NTPDases, while the fifth residue is not. Mutation of this nonconserved cysteine resulted in an enzyme very similar to wild type in its enzymology and secondary structure, indicating that this cysteine exists as a free sulfhydryl and is not essential for structure or function. The disulfide pairing of the other four cysteine residues was determined as Cys(249)-Cys(280) and Cys(340)-Cys(354) by HPLC and mass spectral analysis of tryptic peptides. Due to conservation of these cysteine residues, these two disulfide bonds are likely to exist in all NTPDases. A structural model for NTPDase6, incorporating these and other findings obtained with other NTPDases, is proposed.