Short-term nicotine deprivation alters dorsal anterior cingulate glutamate concentration and concomitant cingulate-cortical functional connectivity.

Most cigarette smokers who wish to quit too often relapse within the first few days of abstinence, primarily due to the aversive aspects of the nicotine withdrawal syndrome (NWS), which remains poorly understood. Considerable research has suggested that the dorsal anterior cingulate cortex (dACC) plays a key role in nicotine dependence, with its functional connections between other brain regions altered as a function of trait addiction and state withdrawal. The flow of information between dACC and fronto-striatal regions is secured through different pathways, the vast majority of which are glutamatergic. As such, we investigated dACC activity using resting state functional connectivity (rsFC) with functional magnetic resonance imaging (fMRI) and glutamate (Glu) concentration with magnetic resonance spectroscopy (MRS). We also investigated the changes in adenosine levels in plasma during withdrawal as a surrogate for brain adenosine, which plays a role in fine-tuning synaptic glutamate transmission. Using a double-blind, placebo-controlled, randomized crossover design, nontreatment seeking smoking participants (N = 30) completed two imaging sessions, one while nicotine sated and another after 36 h nicotine abstinence. We observed reduced dACC Glu (P = 0.029) along with a significant reduction in plasma adenosine (P = 0.03) and adenosine monophosphate (AMP; P < 0.0001) concentrations during nicotine withdrawal in comparison with nicotine sated state. This withdrawal state manipulation also led to an increase in rsFC strength (P < 0.05) between dACC and several frontal cortical regions, including left superior frontal gyrus (LSFG), and right middle frontal gyrus (RMFG). Moreover, the state-trait changes in dACC Glu and rsFC strength between the dACC and both SFG and MFG were positively correlated (P = 0.012, and P = 0.007, respectively). Finally, the change in circuit strength between dACC and LSFG was negatively correlated with the change in withdrawal symptom manifestations as measured by the Wisconsin Smoking Withdrawal Scale (P = 0.04) and Tobacco Craving Questionnaire (P = 0.014). These multimodal imaging-behavioral findings reveal the complex cascade of changes induced by acute nicotine deprivation and call for further investigation into the potential utility of adenosine- and glutamate-signaling as novel therapeutic targets to treat the NWS.

Adenosine signaling in striatal circuits and alcohol use disorders.

Adenosine signaling has been implicated in the pathophysiology of alcohol use disorders and other psychiatric disorders such as anxiety and depression. Numerous studies have indicated a role for A1 receptors (A1R) in acute ethanol-induced motor incoordination, while A2A receptors (A2AR) mainly regulate the rewarding effect of ethanol in mice. Recent findings have demonstrated that dampened A2AR-mediated signaling in the dorsomedial striatum (DMS) promotes ethanol-seeking behaviors. Moreover, decreased A2AR function is associated with decreased CREB activity in the DMS, which enhances goal-oriented behaviors and contributes to excessive ethanol drinking in mice. Interestingly, caffeine, the most commonly used psychoactive substance, is known to inhibit both the A1R and A2AR. This dampened adenosine receptor function may mask some of the acute intoxicating effects of ethanol. Furthermore, based on the fact that A2AR activity plays a role in goal-directed behavior, caffeine may also promote ethanol-seeking behavior. The A2AR is enriched in the striatum and exclusively expressed in striatopallidal neurons, which may be responsible for the regulation of inhibitory behavioral control over drug rewarding processes through the indirect pathway of the basal ganglia circuit. Furthermore, the antagonistic interactions between adenosine and dopamine receptors in the striatum also play an integral role in alcoholism and addiction-related disorders. This review focuses on regulation of adenosine signaling in striatal circuits and the possible implication of caffeine in goal-directed behaviors and addiction.

Adenosine transporter ENT1 regulates the acquisition of goal-directed behavior and ethanol drinking through A2A receptor in the dorsomedial striatum.

Adenosine signaling has been implicated in the pathophysiology of many psychiatric disorders including alcoholism. Striatal adenosine A2A receptors (A2AR) play an essential role in both ethanol drinking and the shift from goal-directed action to habitual behavior. However, direct evidence for a role of striatal A2AR signaling in ethanol drinking and habit development has not been established. In the present study, we found that decreased A2AR-mediated CREB activity in the dorsomedial striatum (DMS) enhanced initial behavioral acquisition of goal-directed behaviors and the vulnerability to progress to excessive ethanol drinking during operant conditioning in mice lacking ethanol-sensitive adenosine transporter ENT1 (ENT1(-/-)). Using mice expressing ß-galactosidase (lacZ) under the control of seven repeated CRE sites in both genotypes (CRE-lacZ/ENT1(+/+) mice and CRE-lacZ/ENT1(-/-) mice) and the dominant-negative form of CREB, we found that reduced CREB activity in the DMS was causally associated with decreased A2AR signaling and increased goal-directed ethanol drinking. Finally, we have demonstrated that the A2AR antagonist ZM241385 dampened protein kinase A activity-mediated signaling in the DMS and promoted excessive ethanol drinking in ENT1(+/+) mice, but not in ENT1(-/-) mice. Our results indicate that A2AR-mediated CREB signaling in the DMS is a key determinant in enhancing the development of goal-directed ethanol drinking in mice.

Exogenous signal-independent nuclear IkappaB kinase activation triggered by Nkx3.2 enables constitutive nuclear degradation of IkappaB-alpha in chondrocytes.

NF-κB is a multifunctional transcription factor involved in diverse biological processes. It has been well documented that NF-κB can be activated in response to various stimuli. While signal-inducible NF-κB activation mechanisms have been extensively characterized, exogenous signal-independent intrinsic NF-κB activation processes remain poorly understood. Here we show that IκB kinase ß (IKKß) can be intrinsically activated in the nucleus by a homeobox protein termed Nkx3.2 in the absence of exogenous IKK-activating signals. We found that ubiquitin chain-dependent, but persistent, interactions between Nkx3.2 and NEMO (also known as IKKγ) can give rise to constitutive IKKß activation in the nucleus. Once the Nkx3.2-NEMO-IKKß complex is formed in the nucleus, IKKß-induced Nkx3.2 phosphorylation at Ser148 and Ser168 allows ßTrCP to be engaged to cause IκB-α ubiquitination independent of IκB-α phosphorylation at Ser32 and Ser36. Taken together, our results provide a novel molecular explanation as to how an intracellular factor such as Nkx3.2 can accomplish persistent nuclear IKK activation to enable intrinsic and constitutive degradation of IκB in the nucleus in the absence of exogenous NF-κB-activating signals, which, in turn, plays a role in chondrocyte viability maintenance.

Type 1 equilibrative nucleoside transporter regulates ethanol drinking through accumbal N-methyl-D-aspartate receptor signaling.

BACKGROUND: Mice lacking type 1 equilibrative nucleoside transporter (ENT1(-/-)) exhibit increased ethanol-preferring behavior compared with wild-type littermates. This phenotype of ENT1(-/-) mice appears to be correlated with increased glutamate levels in the nucleus accumbens (NAc). However, little is known about the downstream consequences of increased glutamate signaling in the NAc. METHODS: To investigate the significance of the deletion of ENT1 and its effect on glutamate signaling in the NAc, we employed microdialysis and iTRAQ proteomics. We validated altered proteins using Western blot analysis. We then examined the pharmacological effects of the inhibition of the N-methyl-D-aspartate (NMDA) glutamate receptor and protein kinase Cγ (PKCγ) on alcohol drinking in wild-type mice. In addition, we investigated in vivo cyclic adenosine monophosphate response element binding activity using cyclic adenosine monophosphate response element-ß-galactosidase mice in an ENT1(-/-) background. RESULTS: We identified that NMDA glutamate receptor-mediated downregulation of intracellular PKCγ-neurogranin-calcium-calmodulin dependent protein kinase type II signaling is correlated with reduced cyclic adenosine monophosphate response element binding activity in ENT1(-/-) mice. Inhibition of PKCγ promotes ethanol drinking in wild-type mice to levels similar to those of ENT1(-/-) mice. In contrast, an NMDA glutamate receptor antagonist reduces ethanol drinking of ENT1(-/-) mice. CONCLUSIONS: These findings demonstrate that the genetic deletion or pharmacological inhibition of ENT1 regulates NMDA glutamate receptor-mediated signaling in the NAc, which provides a molecular basis that underlies the ethanol-preferring behavior of ENT1(-/-) mice.

Functional role of the polymorphic 647 T/C variant of ENT1 (SLC29A1) and its association with alcohol withdrawal seizures.

BACKGROUND: Adenosine is involved in several neurological and behavioral disorders including alcoholism. In cultured cell and animal studies, type 1 equilibrative nucleoside transporter (ENT1, slc29a1), which regulates adenosine levels, is known to regulate ethanol sensitivity and preference. Interestingly, in humans, the ENT1 (SLC29A1) gene contains a non-synonymous single nucleotide polymorphism (647 T/C; rs45573936) that might be involved in the functional change of ENT1. PRINCIPAL FINDINGS: Our functional analysis showed that prolonged ethanol exposure increased adenosine uptake activity of mutant cells (ENT1-216Thr) compared to wild-type (ENT1-216Ile) transfected cells, which might result in reduced extracellular adenosine levels. We found that mice lacking ENT1 displayed increased propensity to ethanol withdrawal seizures compared to wild-type littermates. We further investigated a possible association of the 647C variant with alcoholism and the history of alcohol withdrawal seizures in subjects of European ancestry recruited from two independent sites. Analyses of the combined data set showed an association of the 647C variant and alcohol dependence with withdrawal seizures at the nominally significant level. CONCLUSIONS: Together with the functional data, our findings suggest a potential contribution of a genetic variant of ENT1 to the development of alcoholism with increased risk of alcohol withdrawal-induced seizures in humans.

An essential role for adenosine signaling in alcohol abuse.

In the central nervous system (CNS), adenosine plays an important role in regulating neuronal activity and modulates signaling by other neurotransmitters, including GABA, glutamate, and dopamine. Adenosine suppresses neurotransmitter release, reduces neuronal excitability, and regulates ion channel function through activation of four classes of G protein-coupled receptors, A(1), A(2A), A(2B), and A(3). Central adenosine are largely controlled by nucleoside transporters, which transport adenosine levels across the plasma membrane. Adenosine has been shown to modulate cortical glutamate signaling and ventral-tegmental dopaminergic signaling, which are involved in several aspects of alcohol use disorders. Acute ethanol elevates extracellular adenosine levels by selectively inhibiting the type 1 equilibrative nucleoside transporter, ENT1. Raised adenosine levels mediate the ataxic and sedative/hypnotic effects of ethanol through activation of A(1) receptors in the cerebellum, striatum, and cerebral cortex. Recently, we have shown that pharmacological inhibition or genetic deletion of ENT1 reduces the expression of excitatory amino acid transporter 2 (EAAT2), the primary regulator of extracellular glutamate, in astrocytes. These lines of evidence support a central role for adenosine-mediated glutamate signaling and the involvement of astrocytes in regulating ethanol intoxication and preference. In this paper, we discuss recent findings on the implication of adenosine signaling in alcohol use disorders.

Snail1 is stabilized by O-GlcNAc modification in hyperglycaemic condition.

Protein O-phosphorylation often occurs reciprocally with O-GlcNAc modification and represents a regulatory principle for proteins. O-phosphorylation of serine by glycogen synthase kinase-3ß on Snail1, a transcriptional repressor of E-cadherin and a key regulator of the epithelial-mesenchymal transition (EMT) programme, results in its proteasomal degradation. We show that by suppressing O-phosphorylation-mediated degradation, O-GlcNAc at serine112 stabilizes Snail1 and thus increases its repressor function, which in turn attenuates E-cadherin mRNA expression. Hyperglycaemic condition enhances O-GlcNAc modification and initiates EMT by transcriptional suppression of E-cadherin through Snail1. Thus, dynamic reciprocal O-phosphorylation and O-GlcNAc modification of Snail1 constitute a molecular link between cellular glucose metabolism and the control of EMT.

The CRL4Cdt2 ubiquitin ligase mediates the proteolysis of cyclin-dependent kinase inhibitor Xic1 through a direct association with PCNA.

During DNA polymerase switching, the Xenopus laevis Cip/Kip-type cyclin-dependent kinase inhibitor Xic1 associates with trimeric proliferating cell nuclear antigen (PCNA) and is recruited to chromatin, where it is ubiquitinated and degraded. In this study, we show that the predominant E3 for Xic1 in the egg is the Cul4-DDB1-XCdt2 (Xenopus Cdt2) (CRL4(Cdt2)) ubiquitin ligase. The addition of full-length XCdt2 to the Xenopus extract promotes Xic1 turnover, while the N-terminal domain of XCdt2 (residues 1 to 400) cannot promote Xic1 turnover, despite its ability to bind both Xic1 and DDB1. Further analysis demonstrated that XCdt2 binds directly to PCNA through its C-terminal domain (residues 401 to 710), indicating that this interaction is important for promoting Xic1 turnover. We also identify the cis-acting sequences required for Xic1 binding to Cdt2. Xic1 binds to Cdt2 through two domains (residues 161 to 170 and 179 to 190) directly flanking the Xic1 PCNA binding domain (PIP box) but does not require PIP box sequences (residues 171 to 178). Similarly, human p21 binds to human Cdt2 through residues 156 to 161, adjacent to the p21 PIP box. In addition, we identify five lysine residues (K180, K182, K183, K188, and K193) immediately downstream of the Xic1 PIP box and within the second Cdt2 binding domain as critical sites for Xic1 ubiquitination. Our studies suggest a model in which both the CRL4(Cdt2) E3- and PIP box-containing substrates, like Xic1, are recruited to chromatin through independent direct associations with PCNA.

Reduced effect of NMDA glutamate receptor antagonist on ethanol-induced ataxia and striatal glutamate levels in mice lacking ENT1.

Alcohol-sensitive type 1 equilibrative nucleotide transporter (ENT1) is known to regulate glutamate signaling in the striatum as well as ethanol intoxication. However, it was unclear whether altered extracellular glutamate levels in ENT1(-/-) mice contribute to ethanol-induced behavioral changes. Here we report that altered glutamate signaling in ENT1(-/-) mice is implicated in the ethanol-induced locomotion and ataxia by NMDA receptor antagonist, CGP37849. ENT1(-/-) mice appear less intoxicated following sequential treatment with CGP37849 and ethanol, compared to ENT1(+/+) littermates on the rotarod. These results indicate that inhibiting NMDA glutamate receptors is critical to regulate the response and susceptibility of alcohol related behaviors. Interestingly, a microdialysis experiment showed that the ventral striatum of ENT1(-/-) mice is less sensitive to the glutamate-reducing effect of the NMDA receptor antagonist compared to the dorsal striatum. Our findings suggest that differential glutamate neurotransmission in the striatum regulates ethanol intoxication.

Tyrosylprotein sulfotransferase regulates collagen secretion in Caenorhabditis elegans.

The sulfation of tyrosine residues is an important post-translational modification involved in the regulation of protein function. We examined the activity of worm tyrosylprotein sulfotransferase (TPST-1) on a typical cuticle collagen, ROL-6, in C. elegans. We verified that TPST-1 sulfates three tyrosine residues of ROL-6 in vitro. We found that these tyrosine residues are important for the secretion of ROL-6::GFP. Mutant ROL-6::GFP proteins that contain more than two substitutions of the target tyrosine residues are severely deficient in cuticle localization. Consistently, knock down of tpst-1 blocked the cuticle localization of ROL-6::GFP. Therefore, the sulfation of ROL-6 by TPST-1 is critical for the proper localization of ROL-6. We also confirmed that worm TPST-1 is localized to the trans-Golgi network (TGN). Our results indicate that TPST-1 regulates cuticle organization by promoting the transport of ROL-6 from the TGN to the cuticle.

Altered glutamatergic neurotransmission in the striatum regulates ethanol sensitivity and intake in mice lacking ENT1.

Alcohol-sensitive type 1 equilibrative nucleoside transporter (ENT1) regulates adenosine-mediated glutamate neurotransmission in the brain. Our behavioral studies suggest that the diminished aversive effects of ethanol and the increased resistance to acute ethanol intoxication in mice lacking ENT1, could be related to increased voluntary ethanol self-seeking behavior. In addition, we found that ENT1 null mice were resistant to the ataxic effects of glutamate antagonists when tested on a rotarod. Using microdialysis experiments, we examined glutamate levels in the dorsal and ventral striatum in response to ethanol. In the dorsal striatum of ENT1 null mice, a low intoxicating dose of ethanol (1.5 g/kg) induced a greater increase of glutamate levels, while a higher hypnotic dose of ethanol (3.0 g/kg) decreased to a lesser degree the glutamate levels, compared with that of wild-type mice. In the ventral striatum, however, the low (1.5 g/kg) and the high (3.0 g/kg) ethanol doses altered glutamate levels similarly in both genotypes. Our results suggest that adenosine-regulated glutamatergic signaling contributes to a reduced level of alcohol response, which might be associated with a higher susceptibility for alcoholism in humans.

Novel protective mechanism against irreversible hyperoxidation of peroxiredoxin: Nalpha-terminal acetylation of human peroxiredoxin II.

Peroxiredoxins (Prxs) are a group of peroxidases containing a cysteine thiol at their catalytic site. During peroxidase catalysis, the catalytic cysteine, referred to as the peroxidatic cysteine (C(P)), cycles between thiol (C(P)-SH) and disulfide (-S-S-) states via a sulfenic (C(P)-SOH) intermediate. Hyperoxidation of the C(P) thiol to its sulfinic (C(P)-SO(2)H) derivative has been shown to be reversible, but its sulfonic (C(P)-SO(3)H) derivative is irreversible. Our comparative study of hyperoxidation and regeneration of Prx I and Prx II in HeLa cells revealed that Prx II is more susceptible than Prx I to hyperoxidation and that the majority of the hyperoxidized Prx II formation is reversible. However, the hyperoxidized Prx I showed much less reversibility because of the formation of its irreversible sulfonic derivative, as verified with C(P)-SO(3)H-specific antiserum. In an attempt to identify the multiple hyperoxidized spots of the Prx I on two-dimensional PAGE analysis, an N-acetylated Prx I was identified as part of the total Prx I using anti-acetylated Lys antibody. Using peptidyl-Asp metalloendopeptidase (EC 3.4.24.33) peptide fingerprints, we found that N(alpha)-terminal acetylation (N(alpha)-Ac) occurred exclusively on Prx II after demethionylation. N(alpha)-Ac of Prx II blocks Prx II from irreversible hyperoxidation without altering its affinity for hydrogen peroxide. A comparative study of non-N(alpha)-acetylated and N(alpha)-terminal acetylated Prx II revealed that N(alpha)-Ac of Prx II induces a significant shift in the circular dichroism spectrum and elevation of T(m) from 59.6 to 70.9 degrees C. These findings suggest that the structural maintenance of Prx II by N(alpha)-Ac may be responsible for preventing its hyperoxidation to form C(P)-SO(3)H.

NFkappaB activation is associated with its O-GlcNAcylation state under hyperglycemic conditions.

The transcription factor NFkappaB is activated by phosphorylation and acetylation and plays important roles in inflammatory and immune responses in the cell. Additionally, posttranslational modification of the NFkappaB p65 subunit by O-linked N-acetylglucosamine (O-GlcNAc) has been reported, but the modification site of O-GlcNAc on NFkappaB p65 and its exact function have not been elucidated. In this work, we show that O-GlcNAcylation of NFkappaB p65 decreases binding to IkappaB alpha and increases transcriptional activity under hyperglycemic conditions. Also, we demonstrate that both Thr-322 and Thr-352 of NFkappaB p65 can be modified with O-GlcNAc, but modification on Thr-352, not Thr-322, is important for transcriptional activation. Our findings suggest that site-specific O-GlcNAcylation may be a reason why NFkappaB activity increases continuously under hyperglycemic conditions.

Irreversible oxidation of the active-site cysteine of peroxiredoxin to cysteine sulfonic acid for enhanced molecular chaperone activity.

The thiol (-SH) of the active cysteine residue in peroxiredoxin (Prx) is known to be reversibly hyperoxidized to cysteine sulfinic acid (-SO(2)H), which can be reduced back to thiol by sulfiredoxin/sestrin. However, hyperoxidized Prx of an irreversible nature has not been reported yet. Using an antibody developed against the sulfonylated (-SO(3)H) yeast Prx (Tsa1p) active-site peptide (AFTFVCPTEI), we observed an increase in the immunoblot intensity in proportion to the H(2)O(2) concentrations administered to the yeast cells. We identified two species of hyperoxidized Tsa1p: one can be reduced back (reversible) with sulfiredoxin, and the other cannot (irreversible). Irreversibly hyperoxidized Tsa1p was identified as containing the active-site cysteine sulfonic acid (Tsa1p-SO(3)H) by mass spectrometry. Tsa1p-SO(3)H was not an autoxidation product of Tsa1p-SO(2)H and was maintained in yeast cells even after two doubling cycles. Tsa1p-SO(3)H self-assembled into a ring-shaped multimeric form was shown by electron microscopy. Although the Tsa1p-SO(3)H multimer lost its peroxidase activity, it gained approximately 4-fold higher chaperone activity compared with Tsa1p-SH. In this study, we identify an irreversibly hyperoxidized Prx, Tsa1p-SO(3)H, with enhanced molecular chaperone activity and suggest that Tsa1p-SO(3)H is a marker of cumulative oxidative stress in cells.

Comprehensive royal jelly (RJ) proteomics using one- and two-dimensional proteomics platforms reveals novel RJ proteins and potential phospho/glycoproteins.

Royal jelly (RJ) is an exclusive food for queen honey bee (Apis mellifera L.) that is synthesized and secreted by young worker bees. RJ is also widely used in medical products, cosmetics, and as health foods. However, little is known about RJ functionality and the total protein components, although recent research is attempting to unravel the RJ proteome. We have embarked on a detailed investigation of the RJ proteome, using a modified protein extraction protocol and two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with tandem mass spectrometry. Simultaneously, we examined total soluble protein from RJ collected at 24, 48, and 72 h after honey bee larvae deposition twice (in two flower blooming seasons), to check differences, if any, in RJ proteome therein. Both 1- and 2-D gels stained with silver nitrate revealed similar protein profiles among these three time points. However, we observed a clear difference in two bands (ca. MW of 55 and 75 kDa) on 1-D gel between the first and the second collection of RJ. A similar difference was also observed in the 2-D gel. Except for this difference, the protein profiles were similar at the 3 time points. As the RJ from 48 (or sometimes 72) is commercially used, we selected the RJ sample at 48 h for detailed analysis with the first collection. 1-DGE identified 90 and 15 proteins from the first and second selection, respectively; in total, 47 nonredundant proteins were identified. 2-DGE identified 105 proteins comprising 14 nonredundant proteins. In total, 52 nonredundant proteins were identified in this study, and other than the major royal jelly protein family and some other previously identified proteins, 42 novel proteins were identified. Furthermore, we also report potentially post-translationally modified (phosphorylation and glycosylation) RJ proteins based on the Pro-Q diamond/emerald phosphoprotein/glycoprotein gel stains; MRJP 2p and 7p were suggested as potential phosphoproteins. The 2-DGE data were integrated to develop a 2-D gel reference map, and all data are accessible through RJ proteomics portal (http://foodfunc.agr.ibaraki.ac.jp/RJP.html).

Proteomics of two cultivated mushrooms Sparassis crispa and Hericium erinaceum provides insight into their numerous functional protein components and diversity.

Mushroom can be defined as a macrofungus with a distinctive fruiting body. Mushrooms of class Basidiomycete are primarily wood degradation fungi, but serve as food and a part of traditional medicine used by humans. Although their life cycle is fairly well-established, the information on the molecular components, especially proteins are very limited. Here, we report proteomics analysis of two edible mushrooms (fruiting bodies) Sparassis crispa and Hericium erinaceum using one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) based complementary proteomics approaches. 1-DGE coupled with liquid chromatography and mass spectrometry identified 77 (60 nonredundant proteins) and 121 (88 nonredundant proteins) proteins from S. crispa and H. erinaceum, respectively. 2-DGE analysis revealed 480 and 570 protein spots stained with colloidal coomassie brilliant blue in S. crispa and H. erinaceum, respectively. Of the 71 and 115 selected protein spots from S. crispa and H. erinaceum 2D gel blots on polyvinyldifluoride (PVDF) membranes, respectively, 29 and 35 nonredundant proteins were identified by N-terminal amino acid sequencing. Identified nonredundant proteins from 1- or 2-DGE belonged to 19 functional categories. Twenty-one proteins were found common in both S. crispa and H. erinaceum proteomes, including 14-3-3 protein and septin. Together this study provides evidence for the presence of a large number of functionally diverse proteins, expressed in the fruiting body of two economically important mushrooms, S. crispa and H. erinaceum. Data obtained from 1-DGE and 2-DGE analyses is accessible through the mushroom proteomics portal http://foodfunc.agr.ibaraki.ac.jp/mushprot.html.

Regulation of telomeric repeat binding factor 1 binding to telomeres by casein kinase 2-mediated phosphorylation.

Telomere maintenance is essential for continued cell proliferation and chromosome stability. Telomeres are maintained by telomerase and a collection of associated proteins. The telomeric protein telomeric repeat binding factor 1 (TRF1) negatively regulates telomere length by inhibiting access of telomerase at telomere termini. Here we report that TRF1 interacts with the beta subunit of casein kinase 2 (CK2) and serves as a substrate for CK2. CK2-mediated phosphorylation is required for the efficient telomere binding of TRF1 in vitro and in vivo. Inhibition of CK2 by the CK2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole decreased the ability of TRF1 to bind telomeric DNA. The resulting telomere-unbound form of TRF1 was then ubiquitinated and degraded by the proteasome. Partial knockdown of CK2 by small interfering RNA resulted in removal of TRF1 from telomeres and subsequent degradation of TRF1. Mapping of the CK2 target site identified threonine 122 as a substrate in TRF1. A threonine to alanine change at this position led to a diminished DNA binding due to reduced dimerization of TRF1. In addition, phosphorylation of threonine 122 seemed critical for TRF1-mediated telomere length control. Our findings suggest that CK2-mediated phosphorylation of TRF1 plays an important role in modulating telomere length homeostasis by determining the levels of TRF1 at telomeres.

Systematic investigation of the hemolymph proteome of Manduca sexta at the fifth instar larvae stage using one- and two-dimensional proteomics platforms.

Manduca sexta is an excellent insect model for studying insect physiology, including hemolymph proteins. Larvae stages of this insect are highly damaging to tobacco leaves causing a drastic decrease in crop yield. Investigation on the larval biology should help in controlling its destructive potential, thus increasing crop yields. The hemolymph is the source of its immunity to disease and environmental factors, which invariably involves protein components. To better understand the physiology of M. sexta and the protein components expressed during its life cycle, two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with N-terminal amino acid sequencing and liquid chromatography-mass spectrometry, were employed to analyze the fifth instar larvae hemolymph proteins. These proteomics approaches together identified 123 proteins, which constituted a total of 58 nonredundant proteins and belonged to 10 functional categories. Defense (49%), transport and metabolism (15%), storage (9%), and metamorphosis (7%) categories were highly represented accounting for 80% of the identified proteins. Besides identification of previously reported proteins, 18 novel proteins were identified, which include the lipoprotein-releasing system transmembrane protein lolC, 50S ribosomal protein L24, inducible serine protease inhibitor 1, imaginal disk growth factor, protein disulfide-isomerase-like protein ERp57, etc. The 2-DGE data were integrated to develop a 2-D gel reference map. Data obtained from 1-DGE and 2-DGE analyses are accessible through the M. sexta proteomics portal ( http://foodfunc.agr.ibaraki.ac.jp/mansehemoprot.html). Together, this study provides evidence for the presence of a large number of functionally diverse protein families in the hemolymph of M. sexta. These proteins correlate well with the fifth instar stage, the transition from larvae to pupae.

Exploring novel function of yeast Ssa1/2p by quantitative profiling proteomics using NanoESI-LC-MS/MS.

In the present study, we profiled proteins in ssa1/2 mutant and wild-type using one-dimensional gel electrophoresis coupled with liquid chromatography and mass spectrometry to reveal a total of 322 proteins. Sixty and 84 nonredundant proteins were detected in ssa1/2 and wild-type, respectively, whereas 178 were common. A quantitative profiling proteomic approach using a modified N-terminal isotope tagging method was undertaken to determine quantitative changes in proteins between mutant and wild-type. Out of 210 identified proteins selected for quantification, 103 propionylated proteins were obtained. Eight only D0-propionylated protein (wild-type) and 4 only D5-propionylated proteins (ssa1/2) were detected; 90 proteins were overlapped in the ssa1/2 mutant and wild-type. In the ssa1/2 mutant, 28 proteins were up-regulated and 26 were down-regulated. The expression levels of the rest of 49 proteins were not changed compared with the wild-type. Furthermore, non-correlation between mRNA and protein expressions was found. Among up-regulated proteins, 19 proteins involved in protein synthesis, chromatin condensation, and silencing showed unchanged mRNA expression levels. Among down-regulated proteins, 21 proteins consisting mainly of transcription factors showed unchanged mRNA expressions. Surprisingly, several proteins involved in protein synthesis were also found among the down-regulated proteins. These results suggested that the proteins showing changed protein expressions and unchanged mRNA expressions were affected by the deletion of SSA1 and SSA2 genes at translational efficiency, mRNA degradation, or protein degradation. Moreover, we found the proteins related to chromosomal control were up-regulated in ssa1/2 mutant, a novel finding of this study, suggesting that the Ssa1/2p might contribute to chromosomal control.