Androgen receptor activation by G(s) signaling in prostate cancer cells

The androgen receptor (AR) is activated in prostate cancer patients undergoing androgen ablative therapy and mediates growth of androgen-insensitive prostate cancer cells, suggesting it is activated by nonandrogenic factors. We demonstrate that activated alpha subunit of heterotrimeric guanine nucleotide-binding G(s) protein activates the AR in prostate cancer cells and also synergizes with low concentration of androgen to more fully activate the AR. The G alpha(s) activates protein kinase A, which is required for the nuclear partition and activation of AR. These data suggest a role for G alpha(s) and PKA in the transactivation of AR in prostate cancer cells under the environment of reduced androgen levels.     

Function of the SmpB tail in transfer-messenger RNA translation revealed by a nucleus-encoded form

Stalled bacterial ribosomes are freed when they switch to the translation of transfer-messenger RNA (tmRNA). This process requires the tmRNA-binding and ribosome-binding cofactor SmpB, a beta-barrel protein with a protruding C-terminal tail of unresolved structure. Some plastid genomes encode tmRNA, but smpB genes have only been reported from bacteria. Here we identify smpB in the nuclear genomes of both a diatom and a red alga encoding a signal for import into the plastid, where mature SmpB could activate tmRNA. Diatom SmpB was active for tmRNA translation with bacterial components in vivo and in vitro, although less so than Escherichia coli SmpB. The tail-truncated diatom SmpB, the hypothetical product of a misspliced mRNA, was inactive in vivo. Tail-truncated E. coli SmpB was likewise inactive for tmRNA translation but was still able to bind ribosomes, and its affinity for tmRNA was only slightly diminished. This work suggests that SmpB is a universal cofactor of tmRNA. It also reveals a tail-dependent role for SmpB in tmRNA translation that supersedes a simple role of linking tmRNA to the ribosome, which the SmpB body alone could provide.     

Extended DNA binding site in Pot1 broadens sequence specificity to allow recognition of heterogeneous fission yeast telomeres

The Pot1 (protection of telomeres) protein binds to single-stranded telomeric DNA and is essential for the protection of chromosome ends from degradation and end-to-end fusions. The Pot1 amino-terminal DNA binding domain, Pot1N, adopts an oligonucleotide/oligosaccharide binding fold and binds GGTTAC motifs cooperatively and with exceptionally high sequence specificity. We have now examined DNA binding to naturally occurring telomeric substrates based on the analysis of 100 cloned chromosome ends and in the context of the full-length Pot1 protein. Here, we describe several important differences between Pot1 and Pot1N with apparent consequences for chromosome end protection. Specifically, full-length Pot1.DNA complexes are more stable, and the minimal binding site for a Pot1 monomer is extended into two adjacent telomeric repeats. We provide evidence that Pot1 contains a second DNA binding motif that recognizes DNA with reduced sequence specificity compared with the domain present in Pot1N. The two DNA binding motifs cooperate, whereby the amino-terminal oligonucleotide/oligosaccharide binding fold determines the registry of binding, and the internal DNA binding motif stabilizes the complex and expands the protected region toward the 3' -end. Consistent with a role in chromosome end capping, Pot1 prevents access of telomerase to the 3'-end and protects against exonucleolytic degradation.     

X-ray structure of human acid-beta-glucosidase covalently bound to conduritol-B-epoxide. Implications for Gaucher disease

Gaucher disease is an inherited metabolic disorder caused by mutations in the lysosomal enzyme acid-beta-glucosidase (GlcCerase). We recently determined the x-ray structure of GlcCerase to 2.0 A resolution (Dvir, H., Harel, M., McCarthy, A. A., Toker, L., Silman, I., Futerman, A. H., and Sussman, J. L. (2003) EMBO Rep.4, 704-709) and have now solved the structure of Glc-Cerase conjugated with an irreversible inhibitor, conduritol-B-epoxide (CBE). The crystal structure reveals that binding of CBE to the active site does not induce a global conformational change in GlcCerase and confirms that Glu340 is the catalytic nucleophile. However, only one of two alternative conformations of a pair of flexible loops (residues 345-349 and 394-399) located at the entrance to the active site in native GlcCerase is observed in the GlcCerase-CBE structure, a conformation in which the active site is accessible to CBE. Analysis of the dynamics of these two alternative conformations suggests that the two loops act as a lid at the entrance to the active site. This possibility is supported by a cluster of mutations in loop 394-399 that cause Gaucher disease by reducing catalytic activity. Moreover, in silico mutational analysis demonstrates that all these mutations stabilize the conformation that limits access to the active site, thus providing a mechanistic explanation of how mutations in this loop result in Gaucher disease.     

Characterization and purification of Saccharomyces cerevisiae RNase MRP reveals a new unique protein component

In the yeast Saccharomyces cerevisiae, RNase mitochondrial RNA processing (MRP) is an essential endoribonuclease that consists of one RNA component and at least nine protein components. Characterization of the complex is complicated by the fact that eight of the known protein components are shared with a related endoribonuclease, RNase P. To fully characterize the RNase MRP complex, we purified it to apparent homogeneity in a highly active state using tandem affinity purification. In addition to the nine known protein components, both Rpr2 and a protein encoded by the essential gene YLR145w were present in our preparations of RNase MRP. Precipitation of a tagged version of Ylr145w brought with it the RNase MRP RNA, but not the RNase P RNA. A temperature-sensitive ylr145w mutant was generated and found to exhibit a rRNA processing defect identical to that seen in other RNase MRP mutants, whereas no defect in tRNA processing was observed. Homologues of the Ylr145w protein were found in most yeasts, fungi, and Arabidopsis. Based on this evidence, we propose that YLR145w encodes a novel protein component of RNase MRP, but not RNase P. We recommend that this gene be designated RMP1, for RNase MRP protein 1.     

Molecular mechanism for divergent regulation of Cav1.2 Ca2+ channels by calmodulin and Ca2+-binding protein-1

Ca(2+)-binding protein-1 (CaBP1) and calmodulin (CaM) are highly related Ca(2+)-binding proteins that directly interact with, and yet differentially regulate, voltage-gated Ca(2+) channels. Whereas CaM enhances inactivation of Ca(2+) currents through Ca(v)1.2 (L-type) Ca(2+) channels, CaBP1 completely prevents this process. How CaBP1 and CaM mediate such opposing effects on Ca(v)1.2 inactivation is unknown. Here, we identified molecular determinants in the alpha(1)-subunit of Ca(v)1.2 (alpha(1)1.2) that distinguish the effects of CaBP1 and CaM on inactivation. Although both proteins bind to a well characterized IQ-domain in the cytoplasmic C-terminal domain of alpha(1)1.2, mutations of the IQ-domain that significantly weakened CaM and CaBP1 binding abolished the functional effects of CaM, but not CaBP1. Pulldown binding assays revealed Ca(2+)-independent binding of CaBP1 to the N-terminal domain (NT) of alpha(1)1.2, which was in contrast to Ca(2+)-dependent binding of CaM to this region. Deletion of the NT abolished the effects of CaBP1 in prolonging Ca(v)1.2 Ca(2+) currents, but spared Ca(2+)-dependent inactivation due to CaM. We conclude that the NT and IQ-domains of alpha(1)1.2 mediate functionally distinct interactions with CaBP1 and CaM that promote conformational alterations that either stabilize or inhibit inactivation of Ca(v)1.2.     

Maternal embryonic leucine zipper kinase (MELK) regulates multipotent neural progenitor proliferation

Maternal embryonic leucine zipper kinase (MELK) was previously identified in a screen for genes enriched in neural progenitors. Here, we demonstrate expression of MELK by progenitors in developing and adult brain and that MELK serves as a marker for self-renewing multipotent neural progenitors (MNPs) in cultures derived from the developing forebrain and in transgenic mice. Overexpression of MELK enhances (whereas knockdown diminishes) the ability to generate neurospheres from MNPs, indicating a function in self-renewal. MELK down-regulation disrupts the production of neurogenic MNP from glial fibrillary acidic protein (GFAP)-positive progenitors in vitro. MELK expression in MNP is cell cycle regulated and inhibition of MELK expression down-regulates the expression of B-myb, which is shown to also mediate MNP proliferation. These findings indicate that MELK is necessary for proliferation of embryonic and postnatal MNP and suggest that it regulates the transition from GFAP-expressing progenitors to rapid amplifying progenitors in the postnatal brain.     

Compatible solute effects on thermostability of glutamine synthetase and aspartate transcarbamoylase from Methanococcus jannaschii

Methanococcus jannaschii accumulates alpha- and beta-glutamate as osmolytes. The effect of these and other solutes on the thermostability of two multisubunit metabolic enzymes from M. jannaschii, aspartate transcarbamoylase catalytic trimer (ATCase C3) and glutamine synthetase (GS), has been measured and compared to solute effects on bacterial mesophilic counterparts in order to explore if osmolytes accumulated by each organism can preferentially stabilize the proteins to thermal unfolding. For both ATCase enzymes and for the B. subtilis GS, the solutes normally accumulated by the organism were very effective in protecting the enzyme from losing activity at high temperatures, although solute effects on loss of secondary structure did not necessarily correlate with this thermoprotection of activity. The recombinant M. jannaschii GS exhibited quite different behavior. The pure enzyme had a thermal unfolding transition with a midpoint temperature (Tm) less than 60 degrees C, well under the growth temperature of the organism (85 degrees C). None of the small molecule solutes tested (including the K+-glutamate isomers accumulated by M. jannaschii) significantly stabilized the protein to incubation at 85 degrees C. Instead, protein-protein interactions, as illustrated by E. coli GroEL or ribosomal protein L2 stabilization of GS, appeared to be the dominant factor in stabilizing this archaeal enzyme at the growth temperature.     

Zn2+-dependent deoxyribozymes that form natural and unnatural RNA linkages

We report Zn(2+)-dependent deoxyribozymes that ligate RNA. The DNA enzymes were identified by in vitro selection and ligate RNA with k(obs) up to 0.5 min(-)(1) at 1 mM Zn(2+) and 23 degrees C, pH 7.9, which is substantially faster than our previously reported Mg(2+)-dependent deoxyribozymes. Each new Zn(2+)-dependent deoxyribozyme mediates the reaction of a specific nucleophile on one RNA substrate with a 2',3'-cyclic phosphate on a second RNA substrate. Some of the Zn(2+)-dependent deoxyribozymes create native 3'-5' RNA linkages (with k(obs) up to 0.02 min(-)(1)), whereas all of our previous Mg(2+)-dependent deoxyribozymes that use a 2',3'-cyclic phosphate create non-native 2'-5' RNA linkages. On this basis, Zn(2+)-dependent deoxyribozymes have promise for synthesis of native 3'-5'-linked RNA using 2',3'-cyclic phosphate RNA substrates, although these particular Zn(2+)-dependent deoxyribozymes are likely not useful for this practical application. Some of the new Zn(2+)-dependent deoxyribozymes instead create non-native 2'-5' linkages, just like their Mg(2+) counterparts. Unexpectedly, other Zn(2+)-dependent deoxyribozymes synthesize one of three unnatural linkages that are formed upon the reaction of an RNA nucleophile other than a 5'-hydroxyl group. Two of these unnatural linkages are the 3'-2' and 2'-2' linear junctions created when the 2'-hydroxyl of the 5'-terminal guanosine of one RNA substrate attacks the 2',3'-cyclic phosphate of the second RNA substrate. The third unnatural linkage is a branched RNA that results from attack of a specific internal 2'-hydroxyl of one RNA substrate at the 2',3'-cyclic phosphate. When compared with the consistent creation of 2'-5' linkages by Mg(2+)-dependent ligation, formation of this variety of RNA ligation products by Zn(2+)-dependent deoxyribozymes highlights the versatility of transition metals such as Zn(2+) for mediating nucleic acid catalysis.