Two glycogen synthase isoforms in Saccharomyces cerevisiae are coded by distinct genes that are differentially controlled

In previous work, we identified a Saccharomyces cerevisiae glycogen synthase gene, GSY1, which codes for an 85-kDa polypeptide present in purified yeast glycogen synthase (Farkas, I., Hardy, T.A., DePaoli-Roach, A.A., and Roach, P.J. (1990) J. Biol. Chem. 265, 20879-20886). We have now cloned another gene, GSY2, which encodes a second S. cerevisiae glycogen synthase. The GSY2 sequence predicts a protein of 704 residues, molecular weight 79,963, with 80% identity to the protein encoded by GSY1. Amino acid sequences obtained from a second polypeptide of 77 kDa present in yeast glycogen synthase preparations matched those predicted by GSY2. GSY1 resides on chromosome VI, and GSY2 is located on chromosome XII. Disruption of the GSY1 gene produced a strain retaining about 85% of wild type glycogen synthase activity at stationary phase, while disruption of the GSY2 gene yielded a strain with only about 10% of wild type enzyme activity. The level of glycogen synthase activity in yeast cells disrupted for GSY1 increased in stationary phase, whereas the activity remained at a constant low level in cells disrupted for GSY2. Disruption of both genes resulted in a viable haploid that totally lacked glycogen synthase activity and was defective in glycogen deposition. In conclusion, yeast expresses two forms of glycogen synthase with activity levels that behave differently in the growth cycle. The GSY2 gene product appears to be the predominant glycogen synthase with activity linked to nutrient depletion.     

Distinct MutS DNA-binding modes that are differentially modulated by ATP binding and hydrolysis.

The role of MutS ATPase in mismatch repair is controversial. To clarify further the function of this activity, we have examined adenine nucleotide effects on interactions of Escherichia coli MutS with homoduplex and heteroduplex DNAs. In contrast to previous results with human MutS alpha, we find that a physical block at one end of a linear heteroduplex is sufficient to support stable MutS complex formation in the presence of ATP.Mg(2+). Surface plasmon resonance analysis at low ionic strength indicates that the lifetime of MutS complexes with heteroduplex DNA depends on the nature of the nucleotide present when MutS binds. Whereas complexes prepared in the absence of nucleotide or in the presence of ADP undergo rapid dissociation upon challenge with ATP x Mg(2+), complexes produced in the presence of ATP x Mg(2+), adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) x Mg(2+), or ATP (no Mg(2+)) are resistant to dissociation upon ATP challenge. AMPPNP x Mg(2+) and ATP (no Mg(2+)) reduce MutS affinity for heteroduplex but have little effect on homoduplex affinity, resulting in abolition of specificity for mispaired DNA at physiological salt concentrations. Conversely, the highest mismatch specificity is observed in the absence of nucleotide or in the presence of ADP. ADP has only a limited effect on heteroduplex affinity but reduces MutS affinity for homoduplex DNA.     

Interaction pattern of Arg 62 in the A-pocket of differentially disease-associated HLA-B27 subtypes suggests distinct TCR binding modes

The single amino acid replacement Asp116His distinguishes the two subtypes HLA-B*2705 and HLA-B*2709 which are, respectively, associated and non-associated with Ankylosing Spondylitis, an autoimmune chronic inflammatory disease. The reason for this differential association is so far poorly understood and might be related to subtype-specific HLA:peptide conformations as well as to subtype/peptide-dependent dynamical properties on the nanoscale. Here, we combine functional experiments with extensive molecular dynamics simulations to investigate the molecular dynamics and function of the conserved Arg62 of the α1-helix for both B27 subtypes in complex with the self-peptides pVIPR (RRKWRRWHL) and TIS (RRLPIFSRL), and the viral peptides pLMP2 (RRRWRRLTV) and NPflu (SRYWAIRTR). Simulations of HLA:peptide systems suggest that peptide-stabilizing interactions of the Arg62 residue observed in crystal structures are metastable for both B27 subtypes under physiological conditions, rendering this arginine solvent-exposed and, probably, a key residue for TCR interaction more than peptide-binding. This view is supported by functional experiments with conservative (R62K) and non-conservative (R62A) B*2705 and B*2709 mutants that showed an overall reduction in their capability to present peptides to CD8+ T cells. Moreover, major subtype-dependent differences in the peptide recognition suggest distinct TCR binding modes for the B*2705 versus the B*2709 subtype.     

Generation of Abeta38 and Abeta42 is independently and differentially affected by familial Alzheimer disease-associated presenilin mutations and gamma-secretase modulation

Alzheimer disease amyloid beta-peptide (Abeta) is generated via proteolytic processing of the beta-amyloid precursor protein by beta- and gamma-secretase. Gamma-secretase can be blocked by selective inhibitors but can also be modulated by a subset of non-steroidal anti-inflammatory drugs, including sulindac sulfide. These drugs selectively reduce the generation of the aggregation-prone 42-amino acid Abeta(42) and concomitantly increase the levels of the rather benign Abeta(38). Here we show that Abeta(42) and Abeta(38) generation occur independently from each other. The amount of Abeta(42) produced by cells expressing 10 different familial Alzheimer disease (FAD)-associated mutations in presenilin (PS) 1, the catalytic subunit of gamma-secretase, appeared to correlate with the respective age of onset in patients. However, Abeta(38) levels did not show a negative correlation with the age of onset. Modulation of gamma-secretase activity by sulindac sulfide reduced Abeta(42) in the case of wild type PS1 and two FAD-associated PS1 mutations (M146L and A285V). The remaining eight PS1 FAD mutants showed either no reduction of Abeta(42) or only rather subtle effects. Strikingly, even the mutations that showed no effect on Abeta(42) levels allowed a robust increase of Abeta(38) upon treatment with sulindac sulfide. Similar observations were made for fenofibrate, a compound known to increase Abeta(42) and to decrease Abeta(38). For mutants that predominantly produce Abeta(42), the ability of fenofibrate to further increase Abeta(42) levels became diminished, whereas Abeta(38) levels were altered to varying extents for all mutants analyzed. Thus, we conclude that Abeta(38) and Abeta(42) production do not depend on each other. Using an independent non-steroidal anti-inflammatory drug derivative, we obtained similar results for PS1 as well as for PS2. These in vitro results were confirmed by in vivo experiments in transgenic mice expressing the PS2 N141I FAD mutant. Our findings therefore have strong implications on the selection of transgenic mouse models used for screening of the Abeta(42)-lowering capacity of gamma-secretase modulators. Furthermore, human patients with certain PS mutations may not respond to gamma-secretase modulators.     

The distinct erythropoietin functions that promote cell survival and proliferation are affected by aluminum exposure through mechanisms involving erythropoietin receptor

Erythropoietin (Epo) promotes the development of erythroid progenitors by triggering intracellular signals through the binding to its specific receptor (EpoR). Previous results related to the action of aluminum (Al) on erythropoiesis let us suggest that the metal affects Epo interaction with its target cells. In order to investigate this effect on cell activation by the Epo-EpoR complex, two human cell lines with different dependence on Epo were subjected to Al exposure. In the Epo-independent K562 cells, Al inhibited Epo antiapoptotic action and triggered a simultaneous decrease in protein and mRNA EpoR levels. On the other hand, proliferation of the strongly Epo-dependent UT-7 cells was enhanced by long-term Al treatment, in agreement with the upregulation of EpoR expression during Epo starvation. Results provide some clues to the way by which Epo supports cell survival and growth, and demonstrate that not all the intracellular factors needed to guarantee the different signaling pathways of Epo-cell activation are available or activated in cells expressing EpoR. This study then suggests that at least one of the mechanisms by which Al interfere with erythropoiesis might involve EpoR modulation.     

IS10 transposase mutations that specifically alter target site recognition.

IS10 inserts preferentially into particular hotspots. We describe here mutations of IS10 transposase, called 'ATS' that confer Altered Target Specificity. These mutations yield a general relaxation in target specificity but do not affect other aspects of transposition. Thus, the preference for specific nucleotide sequences at the target site can be cleanly separated from other steps of the transposition reaction. Eleven ATS mutations identified in a genetic screen occur at only two codons in transposase, one in each of two regions of the protein previously implicated in target site interactions (Patch I and Patch II). Genetic analysis suggests that mutations at the two ATS codons affect the same specific function of transposase, thus raising the possibility that Patch I and Patch II interact. For wild-type IS10, insertion specificity is determined in part by a specific 6 bp consensus sequence and in part by the immediately adjacent sequence context of the target DNA. The ATS mutations do not qualitatively alter the hierarchy with which base pairs are recognized in the consensus sequence; instead, sites selected by ATS transposase exhibit a reduction in the degree to which certain base pairs are preferred over others. Models for the basis of this phenotype are discussed.     

Two isoforms of the human cyclin C gene are expressed differentially suggesting that they may have distinct functions

Cyclin C is a highly conserved protein that is involved in divergent cellular processes. The exact roles of its isoforms are presently not very well defined and it is possible that there is a functional divergence amongst them. We therefore sought to assess the expression pattern of cyclin C1 and C2 isoforms in various human tissues and in cell cycle by using real-time PCR experiments. Our findings strongly suggest that the C2 isoform may play a presently unexplored and important role in mammalian testis and probably this isoform is the one that is mainly implicated in cell cycle regulation.     

Rat hippocampal GABAergic molecular markers are differentially affected by ageing

We previously reported that the pharmacological properties of the hippocampal GABAA receptor and the expression of several subunits are modified during normal ageing. However, correlation between these post-synaptic modifications and pre-synaptic deficits were not determined. To address this issue, we have analysed the mRNA levels of several GABAergic molecular markers in young and old rat hippocampus, including glutamic acid decarboxylase enzymes, parvalbumin, calretinin, somatostatin, neuropeptide Y and vasoactive intestinal peptide (VIP). There was a differential age-related decrease in these interneuronal mRNAs that was inversely correlated with up-regulation of the alpha1 GABA receptor subunit. Somatostatin and neuropeptide Y mRNAs were most frequently affected (75% of the animals), then calretinin and VIP mRNAs (50% of the animals), and parvalbumin mRNA (25% of the animals) in the aged hippocampus. This selective vulnerability was well correlated at the protein/cellular level as analysed by immunocytochemistry. Somatostatin interneurones, which mostly innervate principal cell distal dendrites, were more vulnerable than calretinin interneurones, which target other interneurones. Parvalbumin interneurones, which mostly innervate perisomatic domains of principal cells, were preserved. This age-dependent differential reduction of specific hippocampal inteneuronal subpopulations might produce functional alterations in the GABAergic tone which might be compensated, at the post-synaptic level, by up-regulation of the expression of the alpha1 GABAA receptor subunit.     

Mammalian-specific OR37 receptors are differentially activated by distinct odorous fatty aldehydes

The capacity of the mammalian olfactory system to detect an enormous collection of different chemical compounds is based on a large repertoire of odorant receptors (ORs). A small group of these ORs, the OR37 family, is unique due to a variety of special features. Members of this subfamily are exclusively found in mammals, they share a high degree of sequence homology and are highly conserved during evolution. It is still elusive which odorants may activate these atypical receptors. We have reasoned that compounds from skin, hairs, or skin glands might be potential candidates. We have exposed mice to such compounds and monitored activation of glomeruli through the expression of the activity marker c-fos in juxtaglomerular cells surrounding ventrally positioned glomeruli in the olfactory bulb (OB). Employing this methodology it was found that stimulation with long-chain alkanes elicits activation in the ventral part of the OB, however, none of the OR37 glomeruli. Analyses of long-chain hydrocarbon compounds with different functional groups revealed that long-chain aliphatic aldehydes elicited an activation of defined OR37 glomeruli, each of them responding preferentially to an aldehyde with different chain lengths. These results indicate that OR37 receptors may be tuned to distinct fatty aldehydes with a significant degree of ligand specificity.     

Regulatory mechanisms of ROI generation are affected by rice spl mutations

Reactive oxygen intermediates (ROIs) play a pivotal role in the hypersensitive response (HR) in disease resistance. NADPH oxidase is a major source of ROI; however, the mechanisms of its regulation are unclear. Rice spl mutants spontaneously form lesions which resemble those occurring during the HR, suggesting that the mutations affect regulation of the HR. We found that spl2, spl7 and spl11 mutant cells accumulated increased amounts of H(2)O(2) in response to rice blast fungal elicitor. Increased accumulation of ROIs was suppressed by inhibition of NADPH oxidase in the spl cells, and was also observed in the ozone-exposed spl plants. These mutants have sufficient activities of ROI-scavenging enzymes compared with the wild type. In addition, spl7 mutant cells accumulated higher amounts of H(2)O(2) when treated with calyculin A (CA), an inhibitor of protein phosphatase. Furthermore, spl2 mutant plants exhibited accelerated accumulation of H(2)O(2) and increased rates of cell death in response to wounding. These results suggest that the spl2, spl7 and spl11 mutants are defective in the regulation of NADPH oxidase, and the spl7 mutation may give rise to enhancement of the signaling pathway which protein dephosphorylation controls, while the spl2 mutation affects both the pathogen-induced and wound-induced signaling pathways.     

Genome-wide DNA methylation profiling in rheumatoid arthritis identifies disease-associated methylation changes that are distinct to individual T- and B-lymphocyte populations

Changes to the DNA methylome have been described in patients with rheumatoid arthritis (RA). In previous work, we reported genome-wide methylation differences in T-lymphocyte and B-lymphocyte populations from healthy individuals. Now, using HumanMethylation450 BeadChips to interrogate genome-wide DNA methylation, we have determined disease-associated methylation changes in blood-derived T- and B-lymphocyte populations from 12 female patients with seropositive established RA, relative to 12 matched healthy individuals. Array data were analyzed using NIMBL software and bisulfite pyrosequencing was used to validate array candidates. Genome-wide DNA methylation, determined by analysis of LINE-1 sequences, revealed higher methylation in B-lymphocytes compared with T-lymphocytes (P ≤ 0.01), which is consistent with our findings in healthy individuals. Moreover, loci-specific methylation differences that distinguished T-lymphocytes from B-lymphocytes in healthy individuals were also apparent in RA patients. However, disease-associated methylation differences were also identified in RA. In these cases, we identified 509 and 252 CpGs in RA-derived T- and B-lymphocytes, respectively, that showed significant changes in methylation compared with their cognate healthy counterparts. Moreover, this included a restricted set of 32 CpGs in T-lymphocytes and 20 CpGs in B-lymphocytes (representing 15 and 10 genes, respectively, and including two, MGMT and CCS, that were common to both cell types) that displayed more substantial changes in methylation. These changes, apparent as hyper- or hypo-methylation, were independently confirmed by pyrosequencing analysis. Validation by pyrosequencing also revealed additional sites in some candidate genes that also displayed altered methylation in RA. In this first study of genome-wide DNA methylation in individual T- and B-lymphocyte populations in RA patients, we report disease-associated methylation changes that are distinct to each cell type and which support a role for discrete epigenetic regulation in this disease.     

The strength and periodicity of D. melanogaster circadian rhythms are differentially affected by alterations in period gene expression

The per gene of D. melanogaster influences or participates in the generation of biological rhythms. Previous experiments have identified the head as the location from which per exerts its effect on circadian rhythms. To localize further this region and to examine the effects of altered levels and altered spatial expression patterns of the per gene on circadian rhythms of locomotor activity, we have characterized transformed lines containing per gene constructs missing substantial cis-acting regulatory information. The data suggest that wild-type levels of per gene expression are necessary in only a small fraction of the nervous system for near wild-type periods, whereas a larger fraction of per-expressing cells in the brain contributes to the strength of the circadian rhythms.     

Genome-wide DNA methylation profiling in rheumatoid arthritis identifies disease-associated methylation changes that are distinct to individual T- and B-lymphocyte populations

Changes to the DNA methylome have been described in patients with rheumatoid arthritis (RA). In previous work, we reported genome-wide methylation differences in T-lymphocyte and B-lymphocyte populations from healthy individuals. Now, using HumanMethylation450 BeadChips to interrogate genome-wide DNA methylation, we have determined disease-associated methylation changes in blood-derived T- and B-lymphocyte populations from 12 female patients with seropositive established RA, relative to 12 matched healthy individuals. Array data were analyzed using NIMBL software and bisulfite pyrosequencing was used to validate array candidates. Genome-wide DNA methylation, determined by analysis of LINE-1 sequences, revealed higher methylation in B-lymphocytes compared with T-lymphocytes (P ≤ 0.01), which is consistent with our findings in healthy individuals. Moreover, loci-specific methylation differences that distinguished T-lymphocytes from B-lymphocytes in healthy individuals were also apparent in RA patients. However, disease-associated methylation differences were also identified in RA. In these cases, we identified 509 and 252 CpGs in RA-derived T- and B-lymphocytes, respectively, that showed significant changes in methylation compared with their cognate healthy counterparts. Moreover, this included a restricted set of 32 CpGs in T-lymphocytes and 20 CpGs in B-lymphocytes (representing 15 and 10 genes, respectively, and including two, MGMT and CCS, that were common to both cell types) that displayed more substantial changes in methylation. These changes, apparent as hyper- or hypo-methylation, were independently confirmed by pyrosequencing analysis. Validation by pyrosequencing also revealed additional sites in some candidate genes that also displayed altered methylation in RA. In this first study of genome-wide DNA methylation in individual T- and B-lymphocyte populations in RA patients, we report disease-associated methylation changes that are distinct to each cell type and which support a role for discrete epigenetic regulation in this disease.     

GABAA and alpha-amino-3-hydroxy-5-methylsoxazole-4-propionate receptors are differentially affected by aging in the rat hippocampus

We have investigated the age-dependent modifications in the expression of eight different subunits of the gamma-aminobutyric acid, type A (GABA(A)) receptor (alpha1, alpha2, alpha3, alpha5, beta2, beta3, gamma2S, and gamma2L) and all four subunits of the alpha-amino-3-hydroxy-5-methylsoxazole-4-propionate (AMPA) receptor (GluR1-4) in the hippocampus of 24-month-old rats. All aged hippocampi displayed a remarkable increase (aged/adult ratio, 3.53 +/- 0.54) in the mRNA levels of the short version of the gamma2 subunit in parallel with a similar increase in the gamma2 subunit protein (aged/adult ratio, 2.90 +/- 0.62). However, this increase was not observed in the mature receptor. On the other hand, the expression of the different alpha subunit mRNAs increased moderately with aging, displaying a heterogeneous pattern. The most frequent modification consisted in an increase in the expression of the alpha1 subunit mRNA (aged/adult ratio, 1.26 +/- 0.18), in parallel with a similar increase on the alpha1 protein (aged/adult ratio, 1. 27 +/- 0.12) and in the alpha1 incorporated to the assembled GABA(A) receptor (tested by immunoprecipitation; aged/adult ratio, = 1.20 +/- 0.10). However, in the same hippocampal samples, no major modifications were observed on the expression of the AMPA receptor subunits. As a whole, these results indicated the existence of an increased expression of the GABA(A) receptor subunits and a preservation of the AMPA receptor at the hippocampal formation. These modifications could reflect the existence of specific deficiencies (neuronal loss and/or deafferentiation) on the GABAergic system in the aged rats.     

Two activities of cofilin, severing and accelerating directional depolymerization of actin filaments, are affected differentially by mutations around the actin-binding helix

The biochemical activities of cofilin are controversial. We demonstrated that porcine cofilin severs actin filaments and accelerates monomer release at the pointed ends. At pH 7.1, 0.8 microM cofilin cut filaments (2.2 microM actin) about every 290 subunits and increased the depolymerization rate 6.4-fold. A kink in the major alpha-helix of cofilin is thought to constitute a contact site for actin. Side chain hydroxyl groups of Ser119, Ser120 and Tyr82 in cofilin form hydrogen bonds with main chain carbonyl moieties from the helix, causing the kink. We eliminated side chain hydroxyls by Ser-->Ala and/or Tyr-->Phe mutagenesis. Severing and depolymerization-enhancing activities were reduced dramatically in an Ala120 mutant, whereas the latter was decreased in a Phe82 mutant with a relatively small effect on severing, suggesting different structural bases for the two activities of cofilin. The Ala120-equivalent mutation in yeast cofilin affected cell growth, whereas that of the Phe82-equivalent had no effect in yeast. These results indicate the physiological significance of the severing activity of cofilin that is brought about by the kink in the helix.