Adaptive microclimatic evolution of the dehydrin 6 gene in wild barley at ��Evolution Canyon��, Israel

Dehydrins are one of the major stress-induced gene families, and the expression of dehydrin 6 (Dhn6) is strictly related to drought in barley. In order to investigate how the evolution of the Dhn6 gene is associated with adaptation to environmental changes, we examined 48 genotypes of wild barley, Hordeum spontaneum, from "Evolution Canyon" at Mount Carmel, Israel. The Dhn6 sequences of the 48 genotypes were identified, and a recent insertion of 342?bp at 5'UTR was found in the sequences of 11 genotypes. Both nucleotide and haplotype diversity of single nucleotide polymorphism in Dhn6 coding regions were higher on the AS ("African" slope or dry slope) than on the ES ("European" slope or humid slope), and the applied Tajima D and Fu-Li test rejected neutrality of SNP diversity. Expression analysis indicated that the 342?bp insertion at 5'UTR was associated with the earlier up-regulation of Dhn6 after dehydration. The genetic divergence of amino acids sequences indicated significant positive selection of Dhn6 among the wild barley populations. The diversity of Dhn6 in microclimatic divergence slopes suggested that Dhn6 has been subjected to natural selection and adaptively associated with drought resistance of wild barley at "Evolution Canyon".     

Functions of MutL��, Replication Protein A (RPA), and HMGB1 in 5��-Directed Mismatch Repair

A purified system comprised of MutSα, MutLα, exonuclease 1 (Exo1), and replication protein A (RPA) (in the absence or presence of HMGB1) supports 5′-directed mismatch-provoked excision that terminates after mismatch removal. MutLα is not essential for this reaction but enhances excision termination, although the basis of this effect has been uncertain. One model attributes the primary termination function in this system to RPA, with MutLα functioning in a secondary capacity by suppressing Exo1 hydrolysis of mismatch-free DNA (Genschel, J., and Modrich, P. (2003) Mol. Cell 12, 1077–1086). A second invokes MutLα as the primary effector of excision termination (Zhang, Y., Yuan, F., Presnell, S. R., Tian, K., Gao, Y., Tomkinson, A. E., Gu, L., and Li, G. M. (2005) Cell 122, 693–705). In the latter model, RPA provides a secondary termination function, but together with HMGB1, also participates in earlier steps of the reaction. To distinguish between these models, we have reanalyzed the functions of MutLα, RPA, and HMGB1 in 5′-directed mismatch-provoked excision using purified components as well as mammalian cell extracts. Analysis of extracts derived from A2780/AD cells, which are devoid of MutLα but nevertheless support 5′-directed mismatch repair, has demonstrated that 5′-directed excision terminates normally in the absence of MutLα. Experiments using purified components confirm a primary role for RPA in terminating excision by MutSα-activated Exo1 but are inconsistent with direct participation of MutLα in this process. While HMGB1 attenuates excision by activated Exo1, this effect is distinct from that mediated by RPA. Assay of extracts derived from HMGB1^+/+ and HMGB1^−/− mouse embryo fibroblast cells indicates that HMGB1 is not essential for mismatch repair.DNA mismatch repair provides several genetic stabilization functions but is best known for its role in the correction of replication errors (reviewed in Refs. 1–5). When triggered by a mismatched base pair, removal of a DNA biosynthetic error by this system is targeted to the newly synthesized strand by secondary signals within the helix. Although the strand signals that direct eukaryotic mismatch repair have not been identified, a strand break in the form of a nick or gap is sufficient to direct repair to the discontinuous strand in cell extracts, and there is evidence that similar signals may function in vivo (6). Analysis of the cell extract reaction has shown that the mammalian repair system possesses a bidirectional capability in the sense that the strand break that directs repair can be located either 3′ or 5′ to the mismatch (7–9).Several purified mammalian systems have been described that support 3′ and/or 5′-directed mismatch-provoked excision or repair (10–13). The simplest of these depends on the mismatch recognition activities MutSα (MSH2·MSH6 heterodimer) or MutSβ (MSH2·MSH3 heterodimer), MutLα (MLH1·PMS2 heterodimer), the single-stranded DNA-binding protein replication protein A (RPA),² and the 5′ to 3′ double-strand hydrolytic activity Exo1 (exonuclease 1). These four activities support a mismatch-provoked excision reaction directed by a 5′ strand break, and excision terminates upon mismatch removal. MutLα is not essential for this excision reaction, but together with RPA has been implicated in excision termination (10, 12). In addition, the non-histone protein HMGB1 has been found to be required for mismatch repair in partially fractionated mammalian nuclear extracts (14). This small DNA binding protein has been postulated to functionally complement RPA in this 5′-directed system (12).Supplementation of MutSα, MutLα, RPA, and Exo1 with the replication clamp PCNA proliferative cell nuclear antigen (PCNA) and the clamp loader replication factor C (RFC) yields a system that supports mismatch-provoked excision directed by a 3′ or 5′ strand break (11). The basis of the bidirectional excision capability of this system was clarified with the demonstration that MutLα is a latent endonuclease that is activated in a mismatch-, MutSα-, RFC-, and ATP-dependent fashion (15). Incision by activated MutLα is restricted to the discontinuous strand of a nicked heteroduplex and tends to occur on the distal side of the mismatch. Thus, for a nicked heteroduplex in which the strand break resides 3′ to the mismatch, activated MutLα introduces an additional strand break 5′ to the mispair. This 5′ strand discontinuity provides the loading site for the 5′ to 3′ excision system described above, which removes the mismatch.MutSα has been shown to activate the 5′ to 3′ hydrolytic function of Exo1 on heteroduplex DNA, rendering the exonuclease highly processive, an effect attributed to physical interaction of the two activities (10). However, there are differences in the literature with respect to the roles of RPA and MutLα in the control of this processive activity that leads to termination of 5′-directed excision (10, 12). One study has ascribed the primary excision termination function to RPA, which was shown to reduce the processive hydrolytic tracts of the MutSα·Exo1 complex from ≈2,000 to ≈250 nucleotides and by binding to gaps, to restrict Exo1 access to 5′ termini in excision intermediates and products (10). Analysis of excision intermediates as a function of nick-mismatch separation distance led to the conclusion that MutSα is able to reload Exo1 at an RPA-filled gap provided that a mismatch remains in the molecule; however, excision is attenuated upon mismatch removal because MutSα is no longer able to assist in this regard. In this mechanism MutLα functions in excision termination by suppressing nonspecific hydrolysis of the mismatch-free product, an effect attributed to its function as a general negative regulator of Exo1 (10, 16). By contrast, a second study has attributed the primary termination function in this 5′-directed excision system to MutLα, with RPA playing a secondary role (12). In this mechanism RPA has several proposed functions. It is postulated to bind to the 5′ strand break where MutSα recruits HMGB1. RPA and HGB1 then partially melt the helix in the vicinity of the strand break leading to recruitment of Exo1, which initiates processive 5′ to 3′ hydrolysis. Binding of RPA to the ensuing gap results in displacement of MutSα and HMGB1 from the DNA and promotes physical interaction of the exonuclease with MutLα. This results in Exo1 inactivation and dissociation of the MutLα·Exo1 complex from the substrate. As in the other mechanism described above, Exo1 is reloaded if a mismatch remains within the DNA.To distinguish between these models, we have reassessed the roles of MutLα, RPA, and HMGB1 in mammalian mismatch repair. We demonstrate that 5′-directed excision terminates normally in extracts of A2780/AD cells, which support 5′-directed mismatch repair despite deficiency of MLH1 and PMS2 (17). Reanalysis of 5′-directed mismatch-provoked excision in the purified system described above has confirmed a direct role for RPA in terminating processive excision by MutSα-activated Exo1 but is inconsistent with direct participation of MutLα in this process. These experiments also indicate that while HMGB1 can attenuate Exo1 excision, this effect is distinct from that mediated by RPA. We also show that in contrast to results obtained with partially fractionated HeLa extracts (14), extracts derived from HMGB1^−/− mouse embryo fibroblast cells are fully proficient in mismatch repair.     

PR55��, a Regulatory Subunit of PP2A, Specifically Regulates PP2A-mediated ��-Catenin Dephosphorylation

A central question in Wnt signaling is the regulation of β-catenin phosphorylation and degradation. Multiple kinases, including CKIα and GSK3, are involved in β-catenin phosphorylation. Protein phosphatases such as PP2A and PP1 have been implicated in the regulation of β-catenin. However, which phosphatase dephosphorylates β-catenin in vivo and how the specificity of β-catenin dephosphorylation is regulated are not clear. In this study, we show that PP2A regulates β-catenin phosphorylation and degradation in vivo. We demonstrate that PP2A is required for Wnt/β-catenin signaling in Drosophila. Moreover, we have identified PR55α as the regulatory subunit of PP2A that controls β-catenin phosphorylation and degradation. PR55α, but not the catalytic subunit, PP2Ac, directly interacts with β-catenin. RNA interference knockdown of PR55α elevates β-catenin phosphorylation and decreases Wnt signaling, whereas overexpressing PR55α enhances Wnt signaling. Taken together, our results suggest that PR55α specifically regulates PP2A-mediated β-catenin dephosphorylation and plays an essential role in Wnt signaling.Wnt/β-catenin signaling plays essential roles in development and tumorigenesis (1–3). Our previous work found that β-catenin is sequentially phosphorylated by CKIα⁴ and GSK3 (4), which creates a binding site for β-Trcp (5), leading to degradation via the ubiquitination/proteasome machinery (3). Mutations in β-catenin or APC genes that prevent β-catenin phosphorylation or ubiquitination/degradation lead ultimately to cancer (1, 2).In addition to the involvement of kinases, protein phosphatases, such as PP1, PP2A, and PP2C, are also implicated in Wnt/β-catenin regulation. PP2C and PP1 may regulate dephosphorylation of Axin and play positive roles in Wnt signaling (6, 7). PP2A is a multisubunit enzyme (8–10); it has been reported to play either positive or negative roles in Wnt signaling likely by targeting different components (11–21). Toward the goal of understanding the mechanism of β-catenin phosphorylation, we carried out siRNA screening targeting several major phosphatases, in which we found that PP2A dephosphorylates β-catenin. This is consistent with a recent study where PP2A is shown to dephosphorylate β-catenin in a cell-free system (18).PP2A consists of a catalytic subunit (PP2Ac), a structure subunit (PR65/A), and variable regulatory B subunits (PR/B, PR/B′, PR/B″, or PR/B‴). The substrate specificity of PP2A is thought to be determined by its B subunit (9). By siRNA screening, we further identified that PR55α, a regulatory subunit of PP2A, specifically regulates β-catenin phosphorylation and degradation. Mechanistically, we found that PR55α directly interacts with β-catenin and regulates PP2A-mediated β-catenin dephosphorylation in Wnt signaling.     

IFN-�� signaling, with the synergistic contribution of TNF-��, mediates cell specific microglial and astroglial activation in experimental models of Parkinson's disease

To through light on the mechanisms underlying the stimulation and persistence of glial cell activation in Parkinsonism, we investigate the function of IFN-γ and TNF-α in experimental models of Parkinson''s disease and analyze their relation with local glial cell activation. It was found that IFN-γ and TNF-α remained higher over the years in the serum and CNS of chronic Parkinsonian macaques than in untreated animals, accompanied by sustained glial activation (microglia and astroglia) in the substantia nigra pars compacta. Importantly, Parkinsonian monkeys showed persistent and increasing levels of IFN-γR signaling in both microglial and astroglial cells. In addition, experiments performed in IFN-γ and TNF-α KO mice treated with MPTP revealed that, even before dopaminergic cell death can be observed, the presence of IFN-γ and TNF-α is crucial for microglial and astroglial activation, and, together, they have an important synergistic role. Both cytokines were necessary for the full level of activation to be attained in both microglial and astroglial cells. These results demonstrate that IFN-γ signaling, together with the contribution of TNF-α, have a critical and cell-specific role in stimulating and maintaining glial cell activation in Parkinsonism.     

Differential effects of the phosphatidylinositol 4-kinases, PI4KII�� and PI4KIII��, on Akt activation and apoptosis

In this study, we investigated the role of PI4P synthesis by the phosphatidylinositol 4-kinases, PI4KIIα and PI4KIIIβ, in epidermal growth factor (EGF)-stimulated phosphoinositide signaling and cell survival. In COS-7 cells, knockdown of either isozyme by RNA interference reduced basal levels of PI4P and PI(4,5)P₂, without affecting receptor activation. Only knockdown of PI4KIIα inhibited EGF-stimulated Akt phosphorylation, indicating that decreased PI(4,5)P₂ synthesis observed by loss of either isoform could not account for this PI4KIIα-specific effect. Phospholipase Cγ activation was also differentially affected by knockdown of either PI4K isozyme. Overexpression of kinase-inactive PI4KIIα, which induces defective endosomal trafficking without reducing PI(4,5)P₂ levels, also reduced Akt activation. Furthermore, PI4KIIα knockdown profoundly inhibited cell proliferation and induced apoptosis as evidenced by the cleavage of caspase-3 and its substrate poly(ADP-ribose) polymerase. However, in MDA-MB-231 breast cancer cells, apoptosis was observed subsequent to knockdown of either PI4KIIα or PI4KIIIβ and this correlated with enhanced proapoptotic Akt phosphorylation. The differential effects of phosphatidylinositol 4-kinase knockdown in the two cell lines lead to the conclusion that phosphoinositide turnover is inhibited through PI4P substrate depletion, whereas impaired antiapoptotic Akt signaling is an indirect consequence of dysfunctional endosomal trafficking.     

A Novel Member of Solute Carrier Family 25 (SLC25A42) Is a Transporter of Coenzyme A and Adenosine 3��,5��-Diphosphate in Human Mitochondria

Mitochondrial carriers are a family of proteins that transport metabolites, nucleotides, and cofactors across the inner mitochondrial membrane thereby connecting cytosolic and matrix functions. The essential cofactor coenzyme A (CoA) is synthesized outside the mitochondrial matrix and therefore must be transported into mitochondria where it is required for a number of fundamental processes. In this work we have functionally identified and characterized SLC25A42, a novel human member of the mitochondrial carrier family. The SLC25A42 gene (Haitina, T., Lindblom, J., Renström, T., and Fredriksson, R., 2006, Genomics 88, 779–790) was overexpressed in Escherichia coli, purified, and reconstituted into phospholipid vesicles. Its transport properties, kinetic parameters, and targeting to mitochondria demonstrate that SLC25A42 protein is a mitochondrial transporter for CoA and adenosine 3′,5′-diphosphate. SLC25A42 catalyzed only a counter-exchange transport, exhibited a high transport affinity for CoA, dephospho-CoA, ADP, and adenosine 3′,5′-diphosphate, was saturable and inhibited by bongkrekic acid and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A42 is to import CoA into mitochondria in exchange for intramitochondrial (deoxy)adenine nucleotides and adenosine 3′,5′-diphosphate. This is the first time that a mitochondrial carrier for CoA and adenosine 3′,5′-diphosphate has been characterized biochemically.The mitochondrial carrier family, or the solute carrier family 25 (SLC25),³ comprises a large group of proteins that transport a variety of substrates across the inner mitochondrial membrane and, in a few cases, across other membranes (1, 2). Common structural features of the mitochondrial carrier family members consist in a tripartite structure (three repeats of ∼100 amino acids), the presence of two transmembrane α-helices separated by hydrophilic loops in each repeat, and the presence of a signature motif at the C terminus of the first helix in each repeat (Ref. 3 and references therein). The SLC25 family is by far the largest of the currently known 43 SLC families. The Saccharomyces cerevisiae genome contains 35 members, that of Arabidopsis thaliana 58, and the human genome at least 48 SLC25 members. Until now, nearly 30 members and isoforms of this family have been identified in humans. These include the uncoupling protein and the carriers for ADP/ATP, phosphate, 2-oxoglutarate/malate, citrate, carnitine/acylcarnitine, dicarboxylates, ornithine and other basic amino acids, oxodicarboxylates, deoxynucleotides and thiamine pyrophosphate, aspartate-glutamate, glutamate, S-adenosylmethionine, ATP-Mg/Pi, pyrimidine nucleotides, and adenine nucleotides in peroxisomes (see Ref. 1 for a review and Refs. 4–8). The present investigation was undertaken to identify the function of SLC25A42, a novel member of the SLC25 family recently found in the human genome (9). SLC25A42 is 318 amino acids long and is highly expressed in virtually all tissues, in most at higher levels than many other SLC25 family members (9).In this study we provide direct evidence that SLC25A42 is a mitochondrial transporter for CoA and PAP. SLC25A42 was overexpressed in Escherichia coli, purified, reconstituted in phospholipid vesicles, and shown to transport CoA, dephospho-CoA, PAP, and (deoxy)adenine nucleotides with high specificity and by a counter-exchange mechanism. The main function of SLC25A42 is probably to catalyze the entry of CoA into the mitochondria in exchange for adenine nucleotides and PAP.