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The p90 ribosomal S6 kinases (RSK) family of serine/threonine kinases comprises four isoforms (RSK1-4) that lie downstream of the ERK1/2 mitogen-activated protein kinase pathway. RSKs are implicated in fine tuning of cellular processes such as translation, transcription, proliferation, and motility. Previous work showed that pathogens such as Cardioviruses could hijack any of the four RSK isoforms to inhibit PKR activation or to disrupt cellular nucleocytoplasmic trafficking. In contrast, some reports suggest nonredundant functions for distinct RSK isoforms, whereas Coffin-Lowry syndrome has only been associated with mutations in the gene encoding RSK2. In this work, we used the analog-sensitive kinase strategy to ask whether the cellular substrates of distinct RSK isoforms differ. We compared the substrates of two of the most distant RSK isoforms: RSK1 and RSK4. We identified a series of potential substrates for both RSKs in cells and validated RanBP3, PDCD4, IRS2, and ZC3H11A as substrates of both RSK1 and RSK4, and SORBS2 as an RSK1 substrate. In addition, using mutagenesis and inhibitors, we confirmed analog-sensitive kinase data showing that endogenous RSKs phosphorylate TRIM33 at S1119. Our data thus identify a series of potential RSK substrates and suggest that the substrates of RSK1 and RSK4 largely overlap and that the specificity of the various RSK isoforms likely depends on their cell- or tissuespecific expression pattern. [ABSTRACT FROM AUTHOR]

Cardiac hypertrophy develops in response to hemodynamic overload, which can occur in hypertension and aortic stenosis. Maintenance of these pathological conditions eventually evolves into heart failure (HF) and death. O-GlcNAcylation, a post-translational modification of proteins, has been shown to drastically increase during cardiac hypertrophy development and to mediate maladaptive remodeling implicated in the progression of HF. Using mass spectrometry analysis, our lab recently identified several O-GlcNAcylated cardiac proteins that could be involved in such remodeling. Among them, O-GlcNAcylated protein 1 (OGP-1) looks to be a promising candidate. We investigated the role of OGP-1 in cardiac hypertrophy development. Change in OGP-1 expression was followed in neonatal rat cardiomyocytes (NRVM) treated with the pro-hypertrophic agent phenylephrine (PE) for 24 h. The same was done in vivo in two different hypertrophic mouse models, the first induced by Angiotensin II (Ang II) infusion for 5 to 14 days and the second promoted by transverse aortic constriction (TAC) for 2 to 6 weeks. The expression of OGP-1 was also evaluated in human hearts biopsies from different degree of cardiac remodeling. NRVM size was evaluated using α-actinin staining that labels sarcomeric Z-line. Pro-hypertrophic markers (ANP, BNP and β-MHC) were analyzed by quantitative PCR. The effect of OGP-1 overexpression and siRNA-mediated silencing was finally performed in NRVM. A significant decrease in OGP-1 expression was detected in both in vitro and in vivo rodent hypertrophic models. To evaluate the putative implication of OGP-1 in the regulation of cardiac hypertrophy, we repressed OGP-1 expression using siRNA in NRVM. We showed that OGP-1 knockdown induced a basal increase in cell size, similar to that found in PE-treated control cardiomyocytes, suggesting that OGP-1 repression may be sufficient to induce cardiomyocyte hypertrophy. Unexpectedly, the increase in cell size was not related to classical pro-hypertrophic pathways, ANP, BNP and β-MHC expression being unaffected. The other way round, OGP-1 overexpression prevented PE-induced NRVM hypertrophy. Our results underlie OGP-1 as a potential anti-hypertrophic protein and an interesting future target for the treatment of cardiac hypertrophy. Further investigations aim to identify the precise function of OGP-1 in the regulation of cardiac hypertrophy. [ABSTRACT FROM AUTHOR]

Reference treatment of advanced prostate cancer (PCa) relies on pharmacological or surgical androgen deprivation therapy. However, it is only temporarily efficient as tumor cells inevitably adapt to the low testosterone environment and become hormone-refractory (HRPCa). We observed that androgen removal in HRPCa-derived LNCaP cells causes different alterations in their Ca 2+ homeostasis among which a reduction of ER Ca 2+ content. We show that the decrease in [Ca 2+ ] ER is due to a modest overexpression of type 1 IP3R and a threefold increased phosphorylation of IP3R1 on Ser-1716, a protein kinase A (PKA) consensus site, both implicated in ER Ca 2+ leak. Accordingly, ER Ca 2+ content was restored by siRNA-mediated down-regulation of IP3R1 or by inhibition of its phosphorylation by competition with a permeant TAT-peptide containing the Ser-1716 consensus phosphorylation sequence or by treatment with the PKA inhibitor H89. Moreover, inhibition of the IP3R1 phosphorylation by both methods sensitized the LNCaP cells to androgen deprivation-induced apoptosis. In addition, SERCA2b overexpression precluded the effect of androgen deprivation on ER Ca 2+ store content and reduced resistance to androgen deprivation. Taken together, these results indicate that lowering the ER Ca 2+ -store content by increasing IP3R1 levels and IP3R1 phosphorylation by PKA is a protective mechanism by which HRPCa-derived cells escape cell death in the absence of androgenic stimulation. [ABSTRACT FROM AUTHOR]

We report a new function for Escherichia coli DsbC, a protein best known for disulfide bond isomerization in the periplasm. We found that DsbC regulates the redox state of the single cysteine of the l-arabinose-binding protein AraF. This cysteine, which can be oxidized to a sulfenic acid, mediates the formation of a disulfide-linked homodimer under oxidative stress conditions, preventing l-arabinose binding. DsbC, unlike the homologous protein DsbG, reduces the intermolecular disulfide, restoring AraF binding properties. Thus, our results reveal a new link between oxidative protein folding and the defense mechanisms against oxidative stress. [ABSTRACT FROM AUTHOR]

O-GlcNAcylation, a post-translational modification, has been associated with an improvement of the cell survival and the stress response through a modulation of gene expression. We have previously demonstrated that O-GlcNAc stimulation in different models of sepsis is associated with a reduction in mortality and an improvement of different biological markers. Evaluate how O-GlcNAcylation stimulation improves sepsis outcomes in young rats. Endotoxemic shock was induced in 28 days old rats by lipopolysaccharides injection (O111:B4, 20 mg.kg−1) and compared to control rats (NaCl 0.9%). One hour after lipopolysaccharides injection, rats were randomly assigned to no therapy (LPS), fluidotherapy (NaCl 0.9%, 10 mg.kg−1 -LPS + R) ± NButGT (10 mg.kg−1) to increase O-GlcNAc levels. Physiological functions were evaluated 2 hours later, as well as mortality over 36 hours. Cardiac O-GlcNAcylated proteins were mapped by untargeted mass spectrometry and compared to genes transcription via 3′ SRP analysis. The impact of O-GlcNAc has been evaluated on cardiac tissue and HL1 cells (Thiamet G (O-GlcNAc stimulator): 10-8 to 10-5 M). LPS induced a shock with a decrease in mean arterial pressure (P < 0.05). LPS + R had no beneficial effect while NButGT improves mean arterial pressure and median time of survival (P < 0.05). The mRNA expression was not impacted 2 hours after treatment in LPS + R and NButGT group. However, 33 proteins are differentially O-GlcNAcylated in all groups. Among them, 60% are involved in metabolism and metabolic pathways. ATP-citrate lyase (ACLY) a key enzyme in fatty acid biosynthesis, is the only protein less O-GlcNAcylated in the NButGT group. O-GlcNAc stimulation on HL1 cells increases expression of tetrameric form of ACLY (P < 0.05). Acute O-GlcNAc stimulation improves outcome in young septic rat. Stimulate the O-GlcNAcylation increased cardiac O-GlcNAcylated proteins notably those involved in metabolic pathways. Surprisingly, the transcription is not impacted by the O-GlcNAc stimulation. Study the impact of the O-GlcNAcylation on the activity or the interactome of ACLY is one issue to decipher the involvment of the O-GlcNAcylation during sepsis. [ABSTRACT FROM AUTHOR]

Abstract: Muscle protein synthesis rates decrease during contraction/exercise, but rapidly increase post-exercise. Previous studies mainly focused on signaling pathways that control protein synthesis during post-exercise recovery, such as mTOR and its downstream targets S6K1 and 4E-BP1. In this study, we investigated the effect of high-frequency electrical stimulation on the phosphorylation state of signaling components controlling protein synthesis in rat skeletal muscle. Electrical stimulation increased S6K1 Thr389 phosphorylation, which was unaffected by Torin1, a selective mTOR inhibitor, suggesting that S6K1 phosphorylation by contraction was mTOR-independent. Phosphorylation of eIF4B Ser422 was also increased during electrical stimulation, which was abrogated by inhibition of MEK/ERK/RSK1 activation. Moreover, although phosphorylation of conventional mTOR sites in 4E-BP1 decreased during contraction, mTOR-independent phosphorylation was also apparent, which was associated with the release of 4E-BP1 from eIF4E. The results indicate mTOR-independent phosphorylation of S6K1 and 4E-BP1 and suggest MEK/ERK/RSK1-dependent phosphorylation of eIF4B during skeletal muscle contraction. These phosphorylation events would keep the translation initiation machinery “primed” in an active state so that protein synthesis could quickly resume post-exercise. [Copyright &y& Elsevier]

Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca2+, the purified enzyme specifically hydrolyzed β-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn2+. This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of β-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed β-citrylglutamate in the presence of Ca2+, and acted on both N-acetyl-aspartylglutamate and β-citrylglutamate in the presence of Mn2+, whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze β-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its β-citrylglutamate hydrolase activity. [ABSTRACT FROM AUTHOR]

Abstract: Contraction-induced glucose uptake is only partly mediated by AMPK activation. We examined whether the diacylglycerol-sensitive protein kinase D (PKD; also known as novel PKC isoform μ) is also involved in the regulation of glucose uptake in the contracting heart. As an experimental model, we used suspensions of cardiac myocytes, which were electrically stimulated to contract or treated with the contraction-mimicking agent oligomycin. Induction of contraction at 4 Hz in cardiac myocytes or treatment with 1 μM oligomycin enhanced (i) autophosphorylation of PKD at Ser916 by 5.1- and 3.8-fold, respectively, (ii) phosphorylation of PKD''s downstream target cardiac-troponin-I (cTnI) by 2.9- and 2.1-fold, respectively, and (iii) enzymatic activity of immunoprecipitated PKD towards the substrate peptide syntide-2 each by 1.5-fold. Although AMPK was also activated under these same conditions, in vitro phosphorylation assays and studies with cardiac myocytes from AMPKα2−/− mice indicated that activation of PKD occurs independent of AMPK activation. CaMKKβ, and the cardiac-specific PKC isoforms α, δ, and ε were excluded as upstream kinases for PKD in contraction signaling because none of these kinases were activated by oligomycin. Stimulation of glucose uptake and induction of GLUT4 translocation in cardiac myocytes by contraction and oligomycin each were sensitive to inhibition by the PKC/PKD inhibitors staurosporin and calphostin-C. Together, these data elude to a role of PKD in contraction-induced GLUT4 translocation. Finally, the combined actions of PKD on cTnI phosphorylation and on GLUT4 translocation would efficiently link accelerated contraction mechanics to increased energy production when the heart is forced to increase its contractile activity. [Copyright &y& Elsevier]

Abstract: One of the metabolic fates of 3-deoxyglucosone, a product of protein deglycation and a potent glycating agent, is to be oxidized to 2-keto-3-deoxygluconate, but the enzyme that catalyzes this reaction is presently unknown. Starting from human erythrocytes, which are known to convert 3-deoxyglucosone to 2-keto-3-deoxygluconate, we have purified to near homogeneity a NAD-dependent dehydrogenase that catalyzes this last reaction at neutral pH. Sequencing of a 55kDa band co-eluting with the enzymatic activity in the last step indicated that it corresponded to aldehyde dehydrogenase 1A1 (ALDH1A1), an enzyme known to catalyze the oxidation of retinaldehyde to retinoic acid. Overexpression of human ALDH1A1 in HEK cells led to a more than 20-fold increase in 3-deoxyglucosone dehydrogenase activity. In mouse tissues 3-deoxyglucosone dehydrogenase activity was highest in liver, intermediate in lung and testis, and negligible or undetectable in other tissues, in agreement with the tissue distribution of ALDH1A1 mRNA. 3-Deoxyglucosone dehydrogenase activity was undetectable in tissues from ALDH1A1−/− mice. ALDH1A1 appears therefore to be the major if not the only enzyme responsible for the oxidation of 3-deoxyglucosone to 2-keto-3-deoxygluconate. The urinary excretion of 2-keto-3-deoxygluconate amounted to 16.7μmol/g creatinine in humans, indicating that 3-deoxyglucosone may be quantitatively a more important substrate than retinaldehyde for ALDH1A1. [Copyright &y& Elsevier]

Abstract: A wortmannin-sensitive and insulin-stimulated protein kinase (WISK) that phosphorylates and activates heart 6-phosphofructo-2-kinase (PFK-2) was purified from serum-fed HeLa cells and found to contain protein kinase Cζ (PKCζ). Both WISK and recombinant PKCζ were inhibited by a pseudo-substrate peptide inhibitor of PKCζ. WISK and PKCζ phosphorylated and activated recombinant heart PFK-2 by increasing its V max. The phosphorylation sites in heart PFK-2 for WISK were Ser466 and Thr475, whereas PKCζ phosphorylated only Thr475. In perfused rat hearts, insulin activated protein kinase B (PKB) 16-fold compared with the untreated controls. However in the same experiments, no change in phosphorylation state of the activation loop Thr410 residue of PKCζ was observed. By contrast, in incubations of isolated rat epididymal adipocytes, where insulin activated PKB 30-fold compared with the untreated controls, a 50% increase in PKCζ Thr410 phosphorylation was detected. Lastly in HEK 293T cells transfected with heart PFK-2, co-transfection with a kinase-inactive PKCζ construct failed to prevent insulin-induced PFK-2 activation. Therefore, it is unlikely that PKCζ is required for PFK-2 activation by insulin in heart. [Copyright &y& Elsevier]

Previous studies showed that insulin antagonizes AMP-activated protein kinase activation by ischemia and that protein kinase B might be implicated. Here we investigated whether the direct phosphorylation of AMP-activated protein kinase by protein kinase B might participate in this effect. Protein kinase B phosphorylated recombinant bacterially expressed AMP-activated protein kinase heterotrimers at Ser485 of the α1-subunits. In perfused rat hearts, phosphorylation of the α1/α2 AMP-activated protein kinase subunits on Ser485/Ser491 was increased by insulin and insulin pretreatment decreased the phosphorylation of the α-subunits at Thr172 in a subsequent ischemic episode. It is proposed that the effect of insulin to antagonize AMP-activated protein kinase activation involves a hierarchical mechanism whereby Ser485/Ser491 phosphorylation by protein kinase B reduces subsequent phosphorylation of Thr172 by LKB1 and the resulting activation of AMP-activated protein kinase. [ABSTRACT FROM AUTHOR]

Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxyribonucleoside salvage pathway in mammalian cells and plays a key role in the activation of numerous nucleoside analogues used in anti-cancer and antiviral chemotherapy. Although compelling evidence indicated that dCK activity might be regulated by phosphorylation/dephosphorylation, direct demonstration was lacking. Here we showed that dCK overexpressed in HEK 293T cells was labeled after incubating the cells with [32P]orthophosphate. Sorbitol, which was reported to decrease dCK activity, also decreased the labeling of dCK. These results indicated that dCK may exist as a phosphoprotein in vivo and that its activity can be correlated with its phosphorylation level. After purification of 32p-labeled dCK, digestion by trypsin, and analysis of the radioactive peptides by tandem mass spectrometry, the following four in vivo phosphorylation sites were identified: Thr-3, Set-11, Set-15, and Ser-74, the latter being the major phosphorylation site. Site-directed mutagenesis and use of an anti-phospho-Ser-74 antibody demonstrated that Ser-74 phosphorylation was crucial for dCK activity in HEK 293T cells, whereas phosphorylation of other identified sites did not seem essential. Phosphorylation of Ser-74 was also detected on endogenous dCK in leukemic cells, in which the Ser-74 phosphorylation state was increased by agents that enhanced dCK activity. Our study provided direct evidence that dCK activity can be controlled by phosphorylation in intact cells and highlights the importance of Ser-74 for dCK activity. [ABSTRACT FROM AUTHOR]

Fructosamine-3-kinase (FN3K) phosphorylates fructosamine residues, leading to their destabilization and their shedding from protein. Support for the occurrence of this deglycation mechanism in intact cells has been obtained by showing that hemoglobin is significantly more glycated when human erythrocytes are incubated with an elevated glucose concentration in the presence of 1-deoxy-l-morpholinofructose (DMF), a cell-permeable inhibitor of FN3K, than in its absence. The aim of this work was to identify the fructosamine residues on hemoglobin that are removed as a result of the action of FN3K in intact erythrocytes. Highly glycated hemoglobin derived from intact human erythrocytes incubated for 48 h with 200 mM glucose and DMF was incubated in vitro with FN3K and [γ-32p]ATP. After reduction of fructosamine 3-phosphates with borohydride, the protein was digested with trypsin. Peptides were separated by reversed-phase high-performance liquid chromatography, and the radioactive peaks were analyzed by mass spectrometry. Nine different modified residues were identified. These were Lys-α-16, Lys-α-61, Lys-α-139, Val- β-1, Lys-β-17, Lys-β-59, Lys-β-66, Lys-β-132, and Lys-β- 144. Some (e.g. Lys-α-139) were readily phosphorylated to a maximal extent by FN3K in vitro whereas others (e.g. Val-β-l) were slowly and only very partially phosphorylated. The radiolabeled peptides containing re- duced fructosamine 3-phosphates bound to Lys-α-16, Lys-α-139, and Lys-β-17 were much less abundant if the hemoglobin substrate used for the in vitro phosphorylation with FN3K and [γ-32p]ATP came from erythrocytes incubated with an elevated glucose concentration in the absence of DMF, indicating that these lysine residues had been substantially deglycated in intact cells when FN3K action was unrefrained. Other residues (e.g. Val-β-1, Lys-α-61) seemed to be insignificantly deglycated in intact cells. [ABSTRACT FROM AUTHOR]

Recent studies have demonstrated the importance of protein kinase D (PKD) in cell proliferation and apoptosis. Here, we report that in vitro cleavage of recombinant PKD1 by caspase-3 generates two alternative active PKD fragments. N-terminal sequencing of these fragments revealed two distinct caspase-3 cleavage sites located between the acidic and pleckstrin homology (PH) domains of PKD1. Moreover, we present experimental evidence that PKD1 is an in vitro substrate for both initiator and effector caspases. During doxorubicin-induced apoptosis, a zVAD-sensitive caspase induces cleavage of PKD1 at two sites, generating fragments with the same molecular masses as those determined in vitro. The in vivo caspase-dependent generation of the PKD1 fragments correlates with PKD1 kinase activation. Our results indicate that doxorubicin-mediated apoptosis induces activation of PKD1 through a novel mechanism involving the caspase-mediated proteolysis. [Copyright &y& Elsevier]

Bacterially expressed heterotrimeric (α[sub 1], β[sub 1], and γ[sub 1]) wild-type, catalytically inactive, and constitutively active forms of AMP-activated protein kinase (AMPK) were used to study phosphorylation by an upstream AMPK kinase preparation. Here, we report the identification of two new phosphorylation sites in the α-subunit, viz. Thr[sup 258] and Ser[sup 485] (Ser[sup 491] in the α[sub 2]-subunit) by mass spectrometry, in addition to the previously characterized Thr[sup 172] site. Also, autophosphorylation sites in the β[sub 1]-subunit were identified as Ser[sup 96], Ser[sup 101], and Ser[sup 108]. Mutagenesis of Thr[sup 172], Thr[sup 258], and Ser[sup 485] to acidic residues to mimic phosphorylation in the recombinant proteins indicated that Thr[sup 172] was involved in AMPK activation, whereas Thr[sup 258] and Ser[sup 485] were not. Transfection of the non-phosphorylatable S485A and T258A mutants in CCL13 cells subjected to stresses known to activate AMPK either by increasing the AMP:ATP ratio (slow lysis) or without changing adenine nucleotide concentrations (hyperosmolarity) resulted in no significant differences in AMPK activation. All three sites within the α-subunit were phosphorylated in vivo, as seen in AMPK immunoprecipitated from anoxic rat liver. In transfected CCL13 cells, the level of Ser[sup 485] phosphorylation did not change upon AMPK activation. The newly identified phosphorylation sites could play a subtle role in the regulation of AMPK, e.g. in subcellular localization or substrate recognition. [ABSTRACT FROM AUTHOR]

Many transcripts are targeted by nonsense-mediated decay (NMD), leading to their degradation and the inhibition of their translation. We found that the protein SUZ domain–containing protein 1 (SZRD1) interacts with the key NMD factor up-frameshift 1. When recruited to NMD-sensitive reporter gene transcripts, SZRD1 increased protein production, at least in part, by relieving translational inhibition. The conserved SUZ domain in SZRD1 was required for this effect. The SUZ domain is present in only three other human proteins besides SZRD1: R3H domain–containing protein 1 and 2 (R3HDM1, R3HDM2) and cAMP-regulated phosphoprotein 21 (ARPP21). We found that ARPP21, similarly to SZRD1, can increase protein production from NMD-sensitive reporter transcripts in an SUZ domain–dependent manner. This indicated that the SUZ domain–containing proteins could prevent translational inhibition of transcripts targeted by NMD. Consistent with the idea that SZRD1 mainly prevents translational inhibition, we did not observe a systematic decrease in the abundance of NMD targets when we knocked down SZRD1. Surprisingly, knockdown of SZRD1 in two different cell lines led to reduced levels of the NMD component UPF3B, which was accompanied by increased levels in a subset of NMD targets. This suggests that SZRD1 is required to maintain normal UPF3B levels and indicates that the effect of SZRD1 on NMD targets is not limited to a relief from translational inhibition. Overall, our study reveals that human SUZ domain–containing proteins play a complex role in regulating protein output from transcripts targeted by NMD. [ABSTRACT FROM AUTHOR]

Carnosine (β-alanyl-L-histidine) and homocarnosine (γ-aminobutyryl-L-histidine) are abundant dipeptides in skeletal muscle and brain of most vertebrates and some invertebrates. The formation of both compounds is catalyzed by carnosine synthase, which is thought to convert ATP to AMP and inorganic pyrophosphate, and whose molecular identity is unknown. In the present work, we have purified carnosine synthase from chicken pectoral muscle about 1500-fold until only two major polypeptides of 100 and 90 kDa were present in the preparation. Mass spectrometry analysis of these polypeptides did not yield any meaningful candidate. Carnosine formation catalyzed by the purified enzyme was accompanied by a stoichiometric formation, not of AMP, but of ADP, suggesting that carnosine synthase belongs to the "ATP-grasp family" of ligases. A data base mining approach identified ATPGD1 as a likely candidate. As this protein was absent from chicken protein data bases, we reconstituted its sequence from a PCR-amplified cDNA and found it to fit with the 100-kDa polypeptide of the chicken carnosine synthase preparation. Mouse and human ATPGD1 were expressed in HEK293T cells, purified to homogeneity, and shown to catalyze the formation of carnosine, as confirmed by mass spectrometry, and of homocarnosine. Specificity studies carried out on all three enzymes were in agreement with published data. In particular, they acted with 15-25-fold higher catalytic efficiencies on β-alanine than on γ-aminobutyrate. The identification of the gene encoding carnosine synthase will help for a better understanding of the biological functions of carnosine and related dipeptides, which still remain largely unknown. [ABSTRACT FROM AUTHOR]

Smooth muscle contraction is initiated by a rise in intracellular calcium, leading to activation of smooth muscle myosin light chain kinase (MLCK) via calcium/calmodulin (CaM). Activated MLCK then phosphorylates the regulatory myosin light chains, triggering cross-bridge cycling and contraction. Here, we show that MLCK is a substrate of AMP-activated protein kinase (AMPK). The phosphorylation site in chicken MLCK was identified by mass spectrometry to be located in the CaM-binding domain at Ser815. Phosphorylation by AMPK desensitized MLCK by increasing the concentration of CaM required for half-maximal activation. In primary cultures of rat aortic smooth muscle cells, vasoconstrictors activated AMPK in a calcium-dependent manner via CaM-dependent protein kinase kinase-β, a known upstream kinase of AMPK. Indeed, vasoconstrictor-induced AMPK activation was abrogated by the STO- 609 CaM-dependent protein kinase kinase-β inhibitor. Myosin light chain phosphorylation was increased under these conditions, suggesting that contraction would be potentiated by ablation of AMPK. Indeed, in aortic rings from mice in which α1, the major catalytic subunit isoform in arterial smooth muscle, had been deleted, KCI- or phenylephrine-induced contraction was increased. The findings suggest that AMPK attenuates contraction by phosphorylating and inactivating MLCK. This might contribute to reduced ATP turnover in the tonic phase of smooth muscle contraction. [ABSTRACT FROM AUTHOR]

The molecular identity of mammalian phosphopentomutase has not yet been established unequivocally. That of glucose-1,6-bisphosphate synthase, the enzyme that synthesizes a cofactor for phosphomutases and putative regulator of glycolysis, is completely unknown. In the present work, we have purified phosphopentomutase from human erythrocytes and found it to copurify with a 68-kDa polypeptide that was identified by mass spectrometry as phosphoglucomutase 2 (PGM2), a protein of the α-D-phosphohexomutase family and sharing about 20% identity with mammalian phosphoglucomutase 1. Data base searches indicated that vertebrate genomes contained, in addition to PGM2, a homologue (PGM2L1, for PGM2-like 1) sharing about 60% sequence identity with this protein. Both PGM2 and PGM2L1 were overexpressed in Escherichia coli, purified, and their properties were studied. Using catalytic efficiency as a criterion, PGM2 acted more than 10-fold better as a phosphopentomutase (both on deoxyribose 1-phosphate and on ribose 1-phosphate) than as a phosphoglucomutase. PGM2L1 showed only low (<5%) phosphopentomutase and phosphoglucomutase activities compared with PGM2, but was about 5–20-fold better than the latter enzyme in catalyzing the 1,3-bisphosphoglycerate-dependent synthesis of glucose 1,6-bisphosphate and other aldose-bisphosphates. Furthermore, quantitative real-time PCR analysis indicated that PGM2L1 was mainly expressed in brain where glucose-1,6-bisphosphate synthase activity was previously shown to be particularly high. We conclude that mammalian phosphopentomutase and glucose-1,6-bisphosphate synthase correspond to two closely related proteins, PGM2 and PGM2L1, encoded by two genes that separated early in vertebrate evolution. [ABSTRACT FROM AUTHOR]

Fructosamine-3-kinase (FN3K) is a recently described protein-repair enzyme responsible for the removal of fructosamines, which are the products of a spontaneous reaction of glucose with amines. We show here that, compared with glucose, glucose 6-phosphate (Glu-6-P) reacted 3-6-fold more rapidly with proteins and 8-fold more rapidly with N-α-t-Boc-lysine, being therefore a more significant intracellular glycating agent than glucose in skeletal muscle and heart. Fructosamine 6-phosphates, which result from the reaction of amines with Glu-6-P, were not substrates for FN3K. However, a phosphatase that dephosphorylates protein-bound fructosamine 6-phosphates was found to be present in rat tissues. This enzyme was purified to near homogeneity from skeletal muscle and was identified as magnesium-dependent phosphatase-1 (MDP-1), an enzyme of the haloacid dehalogenase family with a putative protein-tyrosine phosphatase function. Human recombinant MDP-1 acted on protein-bound fructosamine 6-phosphates with a catalytic efficiency >10-fold higher than those observed with its next best substrates (arabinose 5-phosphate and free fructoselysine 6-phosphate) and >100-fold higher than with protein-phosphotyrosine. It had no detectable activity on fructosamine 3-phosphates. MDP-1 dephosphorylated up to ~75% of the fructosamine 6-phosphates that are present on lysozyme after incubation of this protein with Glu-6-P. Furthermore, lysozyme glycated with Glu-6-P was converted by MDP-1 to a substrate for FN3K. We conclude that MDP-1 may act physiologically in conjunction with FN3K to free proteins from the glycation products derived from Glu-6-P. [ABSTRACT FROM AUTHOR]

Protein synthesis, in particular peptide chain elongation, is an energy-consuming biosynthetic process. AMPactivated protein kinase (AMPK) is a key regulatory enzyme involved in cellular energy homeostasis. Therefore, we tested the hypothesis that, as in liver, it could mediate the inhibition of protein synthesis by oxygen deprivation in heart by modulating the phosphorylation of eukaryotic elongation factor-2 (eEF2), which becomes inactive in its phosphorylated form. In anoxic cardiomyocytes, AMPK activation was associated with an inhibition of protein synthesis and an increase in phosphorylation of eEF2. Rapamycin, an inhibitor of the mammalian target of rapamycin (roTOR), did not mimic the effect of oxygen deprivation to inhibit protein synthesis in cardiomyocytes or lead to eEF2 phosphorylation in perfused hearts, suggesting that AMPK activation did not inhibit mTOR/p70 ribosomal protein S6 kinase (p70S6K) signaling. Human recombinant eEF2 kinase (eEF2K) was phosphorylated by AMPK in a timeand AMP-dependent fashion, and phosphorylation led to eEF2K activation, similar to that observed in extracts from ischemic hearts. In contrast, increasing the workload resulted in a dephosphorylation of eEF2, which was rapamycin-insensitive, thus excluding a role for roTOR in this effect, eEF2K activity was unchanged by increasing the workload, suggesting that the decrease in eEF2 phosphorylation could result from the activation of an eEF2 phosphatase. [ABSTRACT FROM AUTHOR]

Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122-Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122-Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation. [ABSTRACT FROM AUTHOR]

The HMG-box protein 1 (HBP1) is a transcriptional regulator and a potential tumor suppressor that controls cell proliferation, differentiation and oncogene-mediated senescence. In a previous study, we showed that AKT activation through the PI3K/AKT/FOXO pathway represses HBP1 expression at the transcriptional level in human fibroblasts as well as in cancer cell lines. In the present study, we investigated whether AKT could also regulate HBP1 directly. First, AKT1 phosphorylated recombinant human HBP1 in vitro on three conserved sites, Ser380, Thr484 and Ser509. In living cells, we confirmed the phosphorylation of HBP1 on residues 380 and 509 using phospho-specific antibodies. HBP1 phosphorylation was induced by growth factors, such as EGF or IGF-1, which activated AKT. Conversely, it was blocked by treatment of cells with an AKT inhibitor (MK-2206) or by AKT knockdown. Next, we observed that HBP1 transcriptional activity was strongly modified by mutating its phosphorylation sites. The regulation of target genes such as DNMT1, P47phox, p16 INK4A and cyclin D1 was also affected. HBP1 had previously been shown to limit glioma cell growth. Accordingly, HBP1 silencing by small-hairpin RNA increased human glioblastoma cell proliferation. Conversely, HBP1 overexpression decreased cell growth and foci formation. This effect was amplified by mutations that prevented phosphorylation by AKT, and blunted by mutations that mimicked phosphorylation. In conclusion, our results suggest that HBP1 phosphorylation by AKT blocks its functions as transcriptional regulator and tumor suppressor. [ABSTRACT FROM AUTHOR]

Proliferating cells depend on glycolysis mainly to supply precursors for macromolecular synthesis. Fructose 2,6-bisphosphate (Fru-2,6-P 2 ) is the most potent positive allosteric effector of 6-phosphofructo-1-kinase (PFK-1), and hence of glycolysis. Mitogen stimulation of rat thymocytes with concanavalin A (ConA) led to time-dependent increases in lactate accumulation (6-fold), Fru-2,6-P 2 content (4-fold), 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase isoenzyme 3 and 4 (PFKFB3 and PFKFB4) protein levels (~ 2-fold and ~ 15-fold, respectively) and rates of cell proliferation (~ 40-fold) and protein synthesis (10-fold) after 68 h of incubation compared with resting cells. After 54 h of ConA stimulation, PFKFB3 mRNA levels were 45-fold higher than those of PFKFB4 mRNA. Although PFKFB3 could be phosphorylated at Ser461 by protein kinase B (PKB) in vitro leading to PFK-2 activation, PFKFB3 Ser461 phosphorylation was barely detectable in resting cells and only increased slightly in ConA-stimulated cells. On the other hand, PFKFB3 and PFKFB4 mRNA levels were decreased (90% and 70%, respectively) by exposure of ConA-stimulated cells to low doses of PKB inhibitor (MK-2206), suggesting control of expression of the two PFKFB isoenzymes by PKB. Incubation of thymocytes with ConA resulted in increased expression and phosphorylation of the translation factors eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) and ribosomal protein S6 (rpS6). Treatment of ConA-stimulated thymocytes with PFK-2 inhibitor (3PO) or MK-2206 led to significant decreases in Fru-2,6-P 2 content, medium lactate accumulation and rates of cell proliferation and protein synthesis. These data were confirmed by using siRNA knockdown of PFKFB3, PFKFB4 and PKB α/β in the more easily transfectable Jurkat E6-1 cell line. The findings suggest that increased PFKFB3 and PFKFB4 expression, but not increased PFKFB3 Ser461 phosphorylation, plays a role in increasing glycolysis in mitogen-stimulated thymocytes and implicate PKB in the upregulation of PFKFB3 and PFKFB4. The results also support a role for Fru-2,6-P 2 in coupling glycolysis to cell proliferation and protein synthesis in this model. [ABSTRACT FROM AUTHOR]

Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen. [ABSTRACT FROM AUTHOR]

Carnosine synthase is the ATP-dependent ligase responsible for carnosine (β-alanyl-histidine) and homocarnosine (Υ-aminobutyryl- histidine) synthesis in skeletal muscle and brain, respectively. This enzyme uses, also at substantial rates, lysine, ornithine, and arginine instead of histidine, yet the resulting dipeptides are virtually absent from muscle or brain, suggesting that they are removed by a "metabolite repair" enzyme. Using a radiolabeled substrate, we found that rat skeletal muscle, heart, and brain contained a cytosolic β-alanyl-lysine dipeptidase activity. This enzyme, which has the characteristics of a metalloenzyme, was purified ≈200-fold from rat skeletal muscle. Mass spectrometry analysis of the fractions obtained at different purification stages indicated parallel enrichment of PM20D2, a peptidase of unknown function belonging to the metallopeptidase 20 family. Western blotting showed coelution of PM20D2 with β-alanyl-lysine dipeptidase activity. Recombinant mouse PM20D2 hydrolyzed β-alanyl-lysine, β-alanyl-ornithine, Υ-aminobutyryl-lysine, and Υ-aminobutyryl-ornithine as its best substrates. It also acted at lower rates on β-alanyl-arginine and Υ-aminobutyryl-arginine but virtually not on carnosine or homocarnosine. Although acting preferentially on basic dipeptides derived from β-alanine or Υ-aminobutyrate, PM20D2 also acted at lower rates on some "classic dipeptides" like β-alanyl-lysine and α-lysyl-lysine. The same activity profile was observed with human PM20D2, yet this enzyme was ~ 100-200-fold less active on all substrates tested than the mouse enzyme. Cotransfection in HEK293T cells of mouse or human PM20D2 together with carnosine synthase prevented the accumulation of abnormal dipeptides (β-alanyl-lysine, β-alanyl-ornithine, Υ-aminobutyryl-lysine), thus favoring the synthesis of carnosine and homocarnosine and confirming the metabolite repair role of PM20D2. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (M. tuberculosis), the pathogen responsible for tuberculosis, detoxifies cytotoxic peroxides produced by activated macrophages. M. tuberculosis expresses alkyl hydroxyperoxide reductase E (AhpE), among other peroxiredoxins. So far the system that reduces AhpE was not known. We identified M. tuberculosis mycoredoxin-1 (MtMrx1) acting in combination with mycothiol and mycothiol disulfide reductase (MR), as a biologically relevant reducing system for MtAhpE. MtMrx1, a glutaredoxin-like, mycothiol-dependent oxidoreductase, directly reduces the oxidized form of MtAhpE, through a protein mixed disulfide with the N-terminal cysteine of MtMrx1 and the sulfenic acid derivative of the peroxidatic cysteine of MtAhpE. This disulfide is then reduced by the C-terminal cysteine in MtMrx1. Accordingly, MtAhpE catalyzes the oxidation of wt MtMrx1 by hydrogen peroxide but not of MtMrx1 lacking the C-terminal cysteine, confirming a dithiolic mechanism. Alternatively, oxidized MtAhpE forms a mixed disulfide with mycothiol, which in turn is reduced by MtMrx1 using a monothiolic mechanism. We demonstrated the H2O2-dependent NADPH oxidation catalyzed by MtAhpE in the presence of MR, Mrx1, and mycothiol. Disulfide formation involving mycothiol probably competes with the direct reduction by MtMrx1 in aqueous intracellular media, where mycothiol is present at millimolar concentrations. However, MtAhpE was found to be associated with the membrane fraction, and since mycothiol is hydrophilic, direct reduction by MtMrx1 might be favored. The results reported herein allow the rationalization of peroxide detoxification actions inferred for mycothiol, and more recently, for Mrx1 in cellular systems. We report the first molecular link between a thiol-dependent peroxidase and the mycothiol/Mrx1 pathway in Mycobacteria. [ABSTRACT FROM AUTHOR]

A limited number of enzymes are known that play a role analogous to DNA proofreading by eliminating non-classical metabolites formed by side activities of enzymes of intermediary metabolism. Because few such "metabolite proofreading enzymes" are known, our purpose was to search for an enzyme able to degrade ethylmalonyl-CoA, a potentially toxic metabolite formed at a low rate from butyryl-CoA by acetyl-CoA carboxylase and propionyl-CoA carboxylase, two major enzymes of lipid metabolism. We show that mammalian tissues contain a previously unknown enzyme that decarboxylates ethylmalonyl-CoA and, at lower rates, methylmalonyl-CoA but that does not act on malonyl-CoA. Ethylmalonyl-CoA decarboxylase is particularly abundant in brown adipose tissue, liver, and kidney in mice, and is essentially cytosolic. Because Escherichia coli methylmalonyl-CoA decarboxylase belongs to the family of enoyl-CoA hydratase (ECH), we searched mammalian databases for proteins of uncharacterized function belonging to the ECH family. Combining this database search approach with sequencing data obtained on a partially purified enzyme preparation, we identified ethylmalonyl-CoA decarboxylase as ECHDC1. We confirmed this identification by showing that recombinant mouse ECHDC1 has a substantial ethylmalonyl-CoA decarboxylase activity and a lower methylmalonyl-CoA decarboxylase activity but no malonyl-CoA decarboxylase or enoyl-CoA hydratase activity. Furthermore, ECHDC1-specific siRNAs decreased the ethylmalonyl-CoA decarboxylase activity in human cells and increased the formation of ethylmalonate, most particularly in cells incubated with butyrate. These findings indicate that ethylmalonyl-CoA decarboxylase may correct a side activity of acetyl-CoA carboxylase and suggest that its mutation may be involved in the development of certain forms of ethylmalonic aciduria. [ABSTRACT FROM AUTHOR]

The reduced forms of NAD and NADP, two major nucleotides playing a central role in metabolism, are continuously damaged by enzymatic or heat-dependent hydration. We report the molecular identification of the eukaryotic dehydratase that repairs these nucleotides and show that this enzyme (Carkd in mammals, YKL151C in yeast) catalyzes the dehydration of the S form of NADHX and NADPHX, at the expense of ATP, which is converted to ADP. Surprisingly, the Escherichia coli homolog, YjeF, a bidomain protein, catalyzes a similar reaction, but using ADP instead of ATP. The latter reaction is ascribable to the C-terminal domain of YjeF. This represents an unprecedented example of orthologous enzymes using either ADP or ATP as phosphoryl donor. We also show that eukaryotic proteins homologous to the N-terminal domain of YjeF (apolipoprotein A-1-binding protein (AIBP) in mammals, YNL200C in yeast) catalyze the epimerization of the S and R forms of NAD(P)HX, thereby allowing, in conjunction with the energy-dependent dehydratase, the repair of both epimers of NAD(P)HX. Both enzymes are very widespread in eukaryotes, prokaryotes, and archaea, which together with the ADP dependence of the dehydratase in some species indicates the ancient origin of this repair system. [ABSTRACT FROM AUTHOR]

The bacterial Rcs phosphorelay is a stress-induced defense mechanism that controls the expression of numerous genes, including those for capsular polysaccharides, motility, and virulence factors. It is a complex multicomponent system that includes the histidine kinase (RcsC) and the response regulator (RcsB) and also auxiliary proteins such as RcsF. RcsF is an outer membrane lipoprotein that transmits signals from the cell surface to RcsC. The physiological signals that activate RcsF and how RcsF interacts with RcsC remain unknown. Here, we report the three-dimensional structure of RcsF. The fold of the protein is characterized by the presence of a central 4-stranded β sheet, which is conserved in several other proteins, including the copper-binding domain of the amyloid precursor protein. RcsF, which contains four conserved cysteine residues, presents two nonconsecutive disulfides between Cys74 and Cys118 and between Cys109 and Cys124, respectively. These two disulfides are not functionally equivalent; the Cys109-Cys124 disulfide is particularly important for the assembly of an active RcsF. Moreover, we show that formation of the nonconsecutive disulfides of RcsF depends on the periplasmic disulfide isomerase DsbC. We trapped RcsF in a mixed disulfide complex with DsbC, and we show that deletion of dsbC prevents the activation of the Rcs phosphorelay by signals that function through RcsF. The three-dimensional structure of RcsF provides the structural basis to understand how this protein triggers the Rcs signaling cascade. [ABSTRACT FROM AUTHOR]

• Celsr3 specifies the directionality and regulates velocity of migration. • Celsr3-deficient neuroblasts exhibit exuberant branching. • Celsr3 regulates the growth and stability of microtubules. • Celsr3 interacts physically with Kif2a and their deficiencies cause similar phenotypes. The tangential migration of immature neurons in the postnatal brain involves consecutive migration cycles and depends on constant remodeling of the cell cytoskeleton, particularly in the leading process (LP). Despite the identification of several proteins with permissive and empowering functions, the mechanisms that specify the direction of migration remain largely unknown. Here, we report that planar cell polarity protein Celsr3 orients neuroblasts migration from the subventricular zone (SVZ) to olfactory bulb (OB). In Celsr3 -forebrain conditional knockout mice, neuroblasts loose directionality and few can reach the OB. Celsr3-deficient neuroblasts exhibit aberrant branching of LP, de novo LP formation, and decreased growth rate of microtubules (MT). Mechanistically, we show that Celsr3 interacts physically with Kif2a, a MT depolymerizing protein and that conditional inactivation of Kif2a in the forebrain recapitulates the Celsr3 knockout phenotype. Our findings provide evidence that Celsr3 and Kif2a cooperatively specify the directionality of neuroblasts tangential migration in the postnatal brain. [ABSTRACT FROM AUTHOR]

Abstract: Earlier crystallographic and spectroscopic studies had shown that horse spleen apoferritin was capable of removing the metal ion from hemin (Fe(III)-protoporphyrin IX) [G. Précigoux, J. Yariv, B. Gallois, A. Dautant, C. Courseille, B. Langlois d’Estaintot, Acta Cryst. D50 (1994) 739–743; R.R. Crichton, J.A. Soruco, F. Roland, M.A. Michaux, B. Gallois, G. Précigoux, J.-P. Mahy, D. Mansuy, Biochemistry 36 (1997) 15049–15054]. We have carried out a detailed re-analysis of this phenomenon using both horse spleen and recombinant horse L-chain apoferritins, by electron paramagnetic resonance spectroscopy (EPR) to unequivocally distinguish between heme and non-heme iron. On the basis of site-directed mutagenesis and chemical modification of carboxyl residues, our results show that the UV–visible difference spectroscopic method that was used to establish the mechanism of demetallation is not representative of hemin demetallation. EPR spectroscopy does establish, as in the initial crystallographic investigation, that hemin demetallation occurs, but it is much slower. The signal at g =4.3 corresponding to high spin non-heme-iron (III) increases while the signal at g =6 corresponding to heme-iron decreases. Demetallation by the mutant protein, while slower than the wild-type, still occurs, suggesting that the mechanism of demetallation does not only involve the cluster of four glutamate residues (Glu 53, 56, 57, 60), proposed in earlier studies. However, the mutant protein had lost its capacity to incorporate iron, as had the native protein in which the four Glu residues had been chemically modified. Interestingly, a signal at g =1.94 is also observed. This signal most likely corresponds to a mixed-valence Fe(II)–Fe(III) cluster suggesting that a redox reaction may also be involved in the mechanism of demetallation. [Copyright &y& Elsevier]