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Initiation of chromosomal replication requires dynamic nucleoprotein complexes. In most eubacteria, the origin oriC contains multiple DnaA box sequences to which the ubiquitous DnaA initiators bind. In Escherichia coli oriC, DnaA boxes sustain construction of higher-order complexes via DnaA-DnaA interactions, promoting the unwinding of the DNA unwinding element (DUE) within oriC and concomitantly binding the single-stranded (ss) DUE to install replication machinery. Despite the significant sequence homologies among DnaA proteins, oriC sequences are highly diverse. The present study investigated the design of oriC (tma-oriC) from Thermotoga maritima, an evolutionarily ancient eubacterium. The minimal tma-oriC sequence includes a DUE and a flanking region containing five DnaA boxes recognized by the cognate DnaA (tmaDnaA). This DUE was comprised of two distinct functional modules, an unwinding module and a tmaDnaA-binding module. Three direct repeats of the trinucleotide TAG within DUE were essential for both unwinding and ssDUE binding by tmaDnaA complexes constructed on the DnaA boxes. Its surrounding AT-rich sequences stimulated only duplex unwinding. Moreover, head-to-tail oligomers of ATP-bound tmaDnaA were constructed within tma-oriC, irrespective of the directions of the DnaA boxes. This binding mode was considered to be induced by flexible swiveling of DnaA domains III and IV, which were responsible for DnaA-DnaA interactions and DnaA box binding, respectively. Phasing of specific tmaDnaA boxes in tma-oriC was also responsible for unwinding. These findings indicate that a ssDUE recruitment mechanism was responsible for unwinding and would enhance understanding of the fundamental molecular nature of the origin sequences present in evolutionarily divergent bacteria. [ABSTRACT FROM AUTHOR]

To ensure efficient photosynthesis, chloroplast proteins need to be flexibly regulated under fluctuating light conditions. Thiol-based redox regulation plays a key role in reductively activating several chloroplast proteins in a light-dependent manner. The ferredoxin (Fd)/thioredoxin (Trx) pathway has long been recognized as the machinery that transfers reducing power generated by photosynthetic electron transport reactions to redox-sensitive target proteins; however, its biological importance remains unclear, because the complete disruption of the Fd/Trx pathway in plants has been unsuccessful to date. Especially, recent identifications of multiple redox-related factors in chloroplasts, as represented by the NADPH-Trx reductase C, have raised a controversial proposal that other redox pathways work redundantly with the Fd/Trx pathway. To address these issues directly, we used CRISPR/Cas9 gene editing to create Arabidopsis mutant plants in which the activity of the Fd/Trx pathway was completely defective. The mutants generated showed severe growth inhibition. Importantly, these mutants almost entirely lost the ability to reduce several redox-sensitive proteins in chloroplast stroma, including four Calvin-Benson cycle enzymes, NADP-malate dehydrogenase, and Rubisco activase, under light conditions. These striking phenotypes were further accompanied by abnormally developed chloroplasts and a drastic decline in photosynthetic efficiency. These results indicate that the Fd/ Trx pathway is indispensable for the light-responsive activation of diverse stromal proteins and photoautotrophic growth of plants. Our data also suggest that the ATP synthase is exceptionally reduced by other pathways in a redundant manner. This study provides an important insight into how the chloroplast redox-regulatory system operates in vivo. [ABSTRACT FROM AUTHOR]

The centromere is an essential chromosomal structure for faithful chromosome segregation during cell division. No protein-coding genes exist at the centromeres, but centromeric DNA is actively transcribed into noncoding RNA (ncRNA). This centromeric transcription and its ncRNA products play important roles in centromere functions. We previously reported that the transcriptional regulator ZFAT (zinc-finger protein with AT hook) plays a pivotal role in ncRNA transcription at the centromere; however, it was unclear how ZFAT involvement was regulated. Here, we show that the death domain-associated protein (DAXX) promotes centromeric localization of ZFAT to regulate ncRNA transcription at the centromere. Coimmunoprecipitation analysis of endogenous proteins clearly reveals that DAXX interacts with ZFAT. In addition, we show that ectopic coexpression of ZFAT with DAXX increases the centromeric levels of both ZFAT and ncRNA, compared with ectopic expression of ZFAT alone. On the other hand, we found that siRNA-mediated depletion of DAXX decreases the centromeric levels of both ZFAT and ncRNA in cells ectopically expressing ZFAT. These results suggest that DAXX promotes the centromeric localization of ZFAT and ZFAT-regulated centromeric ncRNA transcription. Furthermore, we demonstrate that depletion of endogenous DAXX protein is sufficient to cause a decrease in the ncRNA levels at the centromeres of chromosomes 17 and X in which ZFAT regulates the transcription, indicating a physiological significance of DAXX in ZFAT-regulated centromeric ncRNA transcription. Taken together, these results demonstrate that DAXX regulates centromeric ncRNA transcription through ZFAT. [ABSTRACT FROM AUTHOR]

Chloroplast FoF1-ATP synthase (CFoCF1) uses an electrochemical gradient of protons across the thylakoid membrane (ΔμH+) as an energy source in the ATP synthesis reaction. CFoCF1 activity is regulated by the redox state of a Cys pair on its central axis, that is, the γ subunit (CF1-γ). When the ΔμH+ is formed by the photosynthetic electron transfer chain under light conditions, CF1-γ is reduced by thioredoxin (Trx), and the entire CFoCF1 enzyme is activated. The redox regulation of CFoCF1 is a key mechanism underlying the control of ATP synthesis under light conditions. In contrast, the oxidative deactivation process involving CFoCF1 has not been clarified. In the present study, we analyzed the oxidation of CF1-γ by two physiological oxidants in the chloroplast, namely the proteins Trx-like 2 and atypical Cys-His-rich Trx. Using the thylakoid membrane containing the reduced form of CFoCF1, we were able to assess the CF1-γ oxidation ability of these Trx-like proteins. Our kinetic analysis indicated that these proteins oxidized CF1-γ with a higher efficiency than that achieved by a chemical oxidant and typical chloroplast Trxs. Additionally, the CF1-γ oxidation rate due to Trx-like proteins and the affinity between them were changed markedly when ΔμH+ formation across the thylakoid membrane was manipulated artificially. Collectively, these results indicate that the formation status of the ΔμH+ controls the redox regulation of CFoCF1 to prevent energetic disadvantages in plants. [ABSTRACT FROM AUTHOR]

Dynamic actin filaments are required for the formation and internalization of endocytic vesicles. Yeast actin cables serve as a track for the translocation of endocytic vesicles to early endosomes, but the molecular mechanisms regulating the interaction between vesicles and the actin cables remain ambiguous. Previous studies have demonstrated that the yeast Eps15-like protein Pan1p plays an important role in this interaction, and that interaction is not completely lost even after deletion of the Pan1p actin-binding domain, suggesting that additional proteins mediate association of the vesicle with the actin cable. Other candidates for mediating the interaction are endocytic coat proteins Sla2p (yeast Hip1R) and Ent1p/2p (yeast epsins), as these proteins can bind to both the plasma membrane and the actin filament. Here, we investigated the degree of redundancy in the actin-binding activities of Pan1p, Sla2p, and Ent1p/2p involved in the internalization and transport of endocytic vesicles. Expression of the nonphosphorylatable form of Pan1p, Pan1-18TA, caused abnormal accumulation of both actin cables and endocytic vesicles, and this accumulation was additively suppressed by deletion of the actin-binding domains of both Pan1p and Ent1p. Interestingly, deletion of the actin-binding domains of Pan1p and Ent1p in cells lacking the ENT2 gene resulted in severely defective internalization of endocytic vesicles and recruitment of actin cables to the site of endocytosis. These results suggest that Pan1p and Ent1p/2p cooperatively regulate the interaction between the endocytic vesicle and the actin cable. [ABSTRACT FROM AUTHOR]

The centromere is a chromosomal locus that is essential for the accurate segregation of chromosomes during cell division. Transcription of noncoding RNA (ncRNA) at the centromere plays a crucial role in centromere function. The zinc-finger transcriptional regulator ZFAT binds to a specific 8-bp DNA sequence at the centromere, named the ZFAT box, to control ncRNA transcription. However, the precise molecular mecha-nisms by which ZFAT localizes to the centromere remain elusive. Here we show that the centromeric protein CENP-B is required for the centromeric localization of ZFAT to regulate ncRNA transcription. The ectopic expression of CENP-B induces the accumulation of both endogenous and ectopically expressed ZFAT protein at the centromere in human cells, suggesting that the centromeric localization of ZFAT requires the presence of CENP-B. Coimmunoprecipitation analysis reveals that ZFAT interacts with the acidic domain of CENP-B, and depletion of endogenous CENP-B reduces the centromeric levels of ZFAT protein, further supporting that CENP-B is required for the centromeric localization of ZFAT. In addition, knockdown of CENP-B significantly decreased the expression levels of ncRNA at the centromere where ZFAT regulates the transcription, suggesting that CENP-B is involved in the ZFAT-regulated centromeric ncRNA transcription. Thus, we concluded that CENP-B contributes to the establishment of the centromeric localization of ZFAT to regulate ncRNA transcription. [ABSTRACT FROM AUTHOR]

Iron is an essential metal for all living organisms that is absorbed in the intestinal cells as a heme-chelated or free form. It is unclear how important plant-derived chelators, such as nicotianamine (NA), an organic small molecule that is ubiquitous in crops, vegetables, and various other foods, contribute to iron bioavailability in mammals. We performed electrophysiological assays with Xenopus laevis oocytes and radioactive tracer experiments with Caco-2 cells. The findings revealed that the proton-coupled amino acid transporter SLC36A1 (PAT1) transports iron in the form of NA-Fe (II) complex in vitro. Decreased expression of hPAT1 by RNA interference in Caco-2 cells reduced the uptake of NA-59Fe (II) complex. The uptake of inorganic 59Fe (II) was relatively unaffected. These results imply that PAT1 transports iron as a NA-Fe (II) complex. The rate of 59Fe absorption in the spleen, liver, and kidney was higher when mice were orally administered NA-59Fe (II) compared with free 59Fe (II). The profile of sitespecific PAT1 expression in the mouse intestine coincided with those of NA and iron contents, which were the highest in the proximal jejunum. Orally administered NA-59Fe (II) complex in mice was detected in the proximal jejunum by thin layer chromatography. In contrast, much less 59Fe (or NA) was detected in the duodenum, where the divalent metal transporter SLC11A2 (DMT1) absorbs free Fe (II). The collective results revealed the role of PAT1 in NA-Fe (II) absorption in the intestine and potential implication of NA in iron uptake in mammals. [ABSTRACT FROM AUTHOR]

Thiol-based redox regulation is a post-translational protein modification for controlling enzyme activity by switching oxidation/reduction states of Cys residues. In plant cells, numerous proteins involved in a wide range of biological systems have been suggested as the target of redox regulation; however, our knowledge on this issue is still incomplete. Here we report that 3-phosphoglycerate dehydrogenase (PGDH) is a novel redoxregulated protein. PGDH catalyzes the first committed step of Ser biosynthetic pathway in plastids. Using an affinity chromatography- based method, we found that PGDH physically interacts with thioredoxin (Trx), a key factor of redox regulation. The in vitro studies using recombinant proteins fromArabidopsis thaliana showed that a specific PGDH isoform, PGDH1, forms the intramolecular disulfide bond under nonreducing conditions, which lowers PGDH enzyme activity. MS and sitedirected mutagenesis analyses allowed us to identify the redoxactive Cys pair that is mainly involved in disulfide bond formation in PGDH1; this Cys pair is uniquely found in land plant PGDH. Furthermore, we revealed that some plastidial Trx subtypes support the reductive activation of PGDH1. The present data show previously uncharacterized regulatory mechanisms of PGDH and expand our understanding of the Trx-mediated redox-regulatory network in plants. [ABSTRACT FROM AUTHOR]

The immune-regulatory compound histamine is involved in the metabolism of the essential skin component hyaluronan (HA). We previously reported that histamine up-regulates the expression of hyaluronan-binding protein involved in hyaluronan depolymerization (HYBID, aka CEMIP or KIAA1199), which plays a key role in HA degradation. However, no information is available about histamine's effects on HA synthase (HAS) expression, the molecular sizes of the HAs produced, and histamine receptors and their signaling pathways in skin fibroblasts. Moreover, histamine's effects on photoaged skin remain elusive. Here, we show that histamine increases HA degradation by up-regulating HYBID and down-regulating HAS2 in human skin fibroblasts in a dose- and time-dependent manner and thereby decreases the total amounts and sizes of newly produced HA. Histamine H1 blocker abrogated the histamine effects on HYBID up-regulation, HAS2 suppression, and HA degradation. Histamine H1 agonist exhibited effects on HA levels, composition, and breakdown similar to those of histamine. Of note, blockade of protein kinase Cδ (PKCδ) or PI3K-AKT signaling abolished histamine-mediated HYBID stimulation and HAS2 suppression, respectively. Immunohistochemical experiments revealed a significant ~2-fold increase in tryptase-positive mast cells in photoaged skin, where HYBID and HAS2 expression levels were increased and decreased, respectively, compared with photoprotected skin. These results indicate that histamine controls HA metabolism by up-regulating HYBID and down-regulating HAS2 via distinct signaling pathways downstream of histamine receptor H1. They further suggest that histamine may contribute to photoaged skin damage by skewing HA metabolism toward degradation. [ABSTRACT FROM AUTHOR]

In contrast to stage-specific transcription factors, the role of ubiquitous transcription factors in neuronal development remains a matter of scrutiny. Here, we demonstrated that a ubiquitous factor NF-Y is essential for neural progenitor maintenance during brain morphogenesis. Deletion of the NF-YA subunit in neural progenitors by using nestin-cre transgene in mice resulted in significant abnormalities in brain morphology, including a thinner cerebral cortex and loss of striatum during embryogenesis. Detailed analyses revealed a progressive decline in multiple neural progenitors in the cerebral cortex and ganglionic eminences, accompanied by induced apoptotic cell death and reduced cell proliferation. In neural progenitors, the NF-YA short isoform lacking exon 3 is dominant and co-expressed with cell cycle genes. ChIP-seq analysis from the cortex during early corticogenesis revealed preferential binding of NF-Y to the cell cycle genes, some of which were confirmed to be downregulated following NF-YA deletion. Notably, the NF-YA short isoform disappears and is replaced by its long isoform during neuronal differentiation. Forced expression of the NF-YA long isoform in neural progenitors resulted in a significant decline in neuronal count, possibly due to the suppression of cell proliferation. Collectively, we elucidated a critical role of the NF-YA short isoform in maintaining neural progenitors, possibly by regulating cell proliferation and apoptosis. Moreover, we identified an isoform switch in NF-YA within the neuronal lineage in vivo, which may explain the stage-specific role of NF-Y during neuronal development. [ABSTRACT FROM AUTHOR]

ScARP from the bacterium Streptomyces coelicolor belongs to the pierisin family of DNA-targeting ADP-ribosyltransferases (ARTs). These enzymes ADP-ribosylate the N² amino groups of guanine residues in DNA to yield N²-(ADP-ribos-1-yl)-2'-deoxyguanosine. Although the structures of pierisin-1 and Scabin were revealed recently, the substrate recognition mechanisms remain poorly understood because of the lack of a substratebinding structure. Here, we report the apo structure of ScARP and of ScARP bound to NADH and its GDP substrate at 1.50 and 1.57 Å resolutions, respectively. The bound structure revealed that the guanine of GDP is trapped between N-ribose of NADH and Trp-159. Interestingly,N² and N³ of guanine formed hydrogen bonds with the OE1 and NE2 atoms of Gln-162, respectively. Wedirectly observed that the ADP-ribosylating toxin turn-turn (ARTT)-loop, including Trp-159 and Gln-162, plays a key role in the specificity of DNA-targeting, guanine-specific ARTs as well as protein-targeting ARTs such as the C3 exoenzyme. We propose that the ARTT-loop recognition is a common substrate-recognition mechanism in the pierisin family. Furthermore, this complex structure sheds light on similarities and differences among two subclasses that are distinguished by conserved structural motifs: H-Y-E in the ARTD subfamily and R-S-E in the ARTC subfamily. The spatial arrangements of the electrophile and nucleophile were the same, providing the first evidence for a common reaction mechanism in these ARTs. ARTC (including ScARP) uses the ARTT-loop for substrate recognition, whereas ARTD (represented by Arr) uses the C-terminal helix instead of the ARTT-loop. These observations could help inform efforts to improve ART inhibitors. [ABSTRACT FROM AUTHOR]

Photoactivated adenylyl cyclase (PAC) and guanylyl cyclase rhodopsin increase the concentrations of intracellular cyclic nucleotides upon illumination, serving as promising secondgeneration tools in optogenetics. To broaden the arsenal of such tools, it is desirable to have light-activatable enzymes that can decrease cyclic nucleotide concentrations in cells. Here, we report on an unusual microbial rhodopsin that may be able to meet the demand. It is found in the choanoflagellate Salpingoeca rosetta and contains a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. We examined the enzymatic activity of the protein (named Rh-PDE) both in HEK293 membranes and whole cells. Although Rh-PDE was constitutively active in the dark, illumination increased its hydrolytic activity 1.4-fold toward cGMP and 1.6-fold toward cAMP, as measured in isolated crude membranes. Purified full-length Rh-PDE displayed maximal light absorption at 492 nm and formed the M intermediate with the deprotonated Schiff base upon illumination. The M state decayed to the parent spectral state in 7 s, producing long-lasting activation of the enzyme domain with increased activity. We discuss a possible mechanism of the Rh-PDE activation by light. Furthermore, Rh-PDE decreased cAMP concentration in HEK293 cells in a light-dependent manner and could do so repeatedly without losing activity. Thus, Rh-PDE may hold promise as a potential optogenetic tool for light control of intracellular cyclic nucleotides (e.g. to study cyclic nucleotide-associated signal transduction cascades). [ABSTRACT FROM AUTHOR]

Patients with advanced congestive heart failure (CHF) or chronic kidney disease often have increased angiotensin II (Ang II) levels and cachexia. We previously demonstrated that Ang II, via its type 1 receptor, causes muscle protein breakdown and apoptosis and inhibits satellite cell (SC) proliferation and muscle regeneration, likely contributing to cachexia in CHF and chronic kidney disease. In contrast, Ang II, via its type 2 receptor (AT2R) expression, is robustly induced during SC differentiation, and it potentiates muscle regeneration. To understand the mechanisms regulating AT2R expression and its potential role in muscle regeneration in chronic diseases, we used a mouse model of CHF and found that muscle regeneration was markedly reduced and that this was accompanied by blunted increase of AT2R expression. We performed AT2R promoter reporter analysis during satellite cell differentiation and found that the 70 bp upstream of the AT2R transcription start site contain a core promoter region, and regions upstream of 70 bp to 3 kbp are dispensable for AT2R induction. Instead, AT2R intron 2 acts as a transcriptional enhancer during SC differentiation. Further deletion/mutation analysis revealed that multiple transcription factor binding sites in the 286/ 690 region within intron 2 coordinately regulate AT2R transcription. Importantly, 286/690 enhancer activity was suppressed in CHF mouse skeletal muscle, suggesting that AT2R expression is suppressed in CHF via inhibition of AT2R intronic enhancer activity, leading to lowered muscle regeneration. Thus targeting intron 2 enhancer element could lead to the development of a novel intervention to increase AT2R expression in SCs and potentiate skeletal muscle regenerative capacity in chronic diseases. [ABSTRACT FROM AUTHOR]

We recently discovered a biosynthetic system using a novel amino group carrier protein called LysW for lysine biosynthesis via α-aminoadipate (AAA), and revealed that this system is also utilized in the biosynthesis of arginine by Sulfolobus. In the present study, we focused on the biosynthesis of lysine and ornithine in the hyperthermophilic archaeon Thermococcus kodakarensis, and showed that their biosynthesis is accomplished by a single set of metabolic enzymes. We also determined the crystal structure of the LysX family protein from T. kodakarensis, which catalyzes the conjugation of LysW with either AAA or glutamate, in a complex with LysW-γ-AAA. This crystal structure is the first example to show how LysX recognizes AAA as a substrate and provides a structural basis for the bifunctionality of the LysX family protein from T. kodakarensis. Based on comparisons with other LysX family proteins, we propose a mechanism for substrate recognition and its relationship with molecular evolution among LysX family proteins, which have different substrate specificities. [ABSTRACT FROM AUTHOR]

TRAF3IP2 (TRAF3 interacting protein 2; previously known as CIKS or Act1) is a key intermediate in the normal inflammatory response and the pathogenesis of various autoimmune and inflammatory diseases. Induction of TRAF3IP2 activates IKB kinase (I K K) / N F - K B, JNK/AP-1, and c/EBP0 and stimulates the expression of various inflammatory mediators with negative myocardial inotropic effects. To investigate the role of TRAF3IP2 in heart disease, we generated a transgenic mouse model with cardiomyocyte-specific TRAF3IP2 overexpression (TRAF3IP2-Tg). Echocardiography, magnetic resonance imaging, and pressure-volume conductance catheterization revealed impaired cardiac function in 2-month-old male transgenic (Tg) mice as evidenced by decreased ejection fraction, stroke volume, cardiac output, and peak ejection rate. Moreover, the male Tg mice spontaneously developed myocardial hypertrophy (increased heart/body weight ratio, cardiomyocyte cross-sectional area, GATA4 induction, and fetal gene re-expression). Furthermore, TRAF3IP2 overexpression resulted in the activation of I K K / N F - K B, JNK/AP-1, c/EBP0, and p38 MAPK and induction of proinflammatory cytokines, chemokines, and extracellular matrix proteins in the heart. Although myocardial hypertrophy decreased with age, cardiac fibrosis (increased number of myofibroblasts and enhanced expression and deposition of fibrillar collagens) increased progressively. Despite these adverse changes, TRAF3IP2 overexpression did not result in cell death at any time period. Interestingly, despite increased mRNA expression, TRAF3IP2 protein levels and activation of its downstream signaling intermediates remained unchanged in the hearts of female Tg mice. The female Tg mice also failed to develop myocardial hypertrophy. In summary, these results demonstrate that overexpression of TRAF3IP2 in male mice is sufficient to induce myocardial hypertrophy, cardiac fibrosis, and contractile dysfunction. [ABSTRACT FROM AUTHOR]

Type I interferons (IFN-α/β) are essential for immune defense against viruses and induced through the actions of the cytoplasmic helicases, RIG-I and MDA5, and their downstream adaptor molecule IPS-1. TRAF6 and the downstream kinase TAK1 have been shown to be essential for the production of proinflammatory cytokines through the TLR/MyD88/ TRIP pathway. Although binding of TRAF6 with IPS-1 has been demonstrated, the role of the TRAF6 pathway in IFNα/β production has not been fully understood. Here, we demonstrate that TRAF6 is critical for IFN-α/β induction in response to viral infection and intracellular double-stranded RNA, poly(I:C). Activation of NP-KB, JNK, and p38, but not IRF3, was impaired in TRAF6-deficient mouse embryo fibroblasts in response to vesicular stomatitis virus and poly(I:C). However, TAK1 was not required for IFN-β induction in this process, since normal IFN-α/β production was observed in TAK1-deficient mouse embryo fibroblasts. Instead, another MAP3K, MEKK1, was important for the activation of the IFN-β promoter in response to poly(I:C). Forced expression of MEKK1 in combination with IRF3 was sufficient for the induction of IFN-β, whereas suppression of MEKK1 expression by small interfering RNA inhibited the induction of IFN-β by poly(I:C). These data suggest that IPS-1 requires TRAF6 and MEKK1 to activate NP-KB and mitogen-activated protein kinases that are critical for the optimal induction of type I interferons. [ABSTRACT FROM AUTHOR]

Regulation of hyaluronan (HA) synthesis and degradation is essential to maintenance of extracellular matrix homeostasis. We recently reported that HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization), also called KIAA1199, plays a key role in HA depolymerization in skin and arthritic synovial fibroblasts. However, regulation of HA metabolism mediated by HYBID and HA synthases (HASs) under stimulation with growth factors remains obscure. Here we report that TGF-β1, basic FGF, EGF, and PDGF-BB commonly enhance total amount of HA in skin fibroblasts through up-regulation of HAS expression, but molecular size of newly producedHAis dependent on HYBID expression levels. Stimulation of HAS1/2 expression and suppression of HYBID expression by TGF-β1 were abrogated by blockade of theMAPKand/orSmadsignaling and the PI3K-Akt signaling, respectively. In normal human skin, expression of the TGF-β1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-β1 upregulated HAS1/2 expression but exerted only a slight suppressive effect on HYBID expression in synovial fibroblasts from the patients with osteoarthritis or rheumatoid arthritis, resulting in the production of lower molecular weight HA compared with normal skin and synovial fibroblasts. These data demonstrate that although TGF-β1, basic FGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts,TGF-β1mostefficiently contributes to production of high molecular weight HA by HAS up-regulation and HYBID down-regulation and suggests that inefficient downregulation of HYBID by TGF-β1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients. [ABSTRACT FROM AUTHOR]

Cutting-edge technologies such as genome editing and synthetic biology allow us to produce novel foods and functional proteins. However, their toxicity and allergenicity must be accurately evaluated. It is known that specific amino acid sequences in proteins make some proteins allergic, but many of these sequences remain uncharacterized. In this study, we introduce a data-driven approach and a machine-learning method to find undiscovered allergen-specific patterns (ASPs) among amino acid sequences. The proposed method enables an exhaustive search for amino acid subsequences whose frequencies are statistically significantly higher in allergenic proteins. As a proof-of-concept, we created a database containing 21,154 proteins of which the presence or absence of allergic reactions are already known and applied the proposed method to the database. The detected ASPs in this proof-of-concept study were consistent with known biological findings, and the allergenicity prediction performance using the detected ASPs was higher than extant approaches, indicating this method may be useful in evaluating the utility of synthetic foods and proteins. [ABSTRACT FROM AUTHOR]

Redox regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of various functions in chloroplasts. Five Trx subtypes have been reported to reside in chloroplasts, but their functional diversity in the redox regulation of Trx target proteins remains poorly clarified. To directly address this issue, we studied the Trx-dependent redox shifts of several chloroplast thiol-modulated enzymes in vitro and in vivo. In vitro assays using a series of Arabidopsis recombinant proteins provided new insights into Trx selectivity for the redox regulation as well as the underpinning for previous suggestions. Most notably, by combining the discrimination of thiol status with mass spectrometry and activity measurement, we identified an uncharacterized aspect of the reductive activation of NADP-malate dehydrogenase; two redox-active Cys pairs harbored in this enzyme were reduced via distinct utilization of Trxs even within a single polypeptide. In our in vitro assays, Trx-f was effective in reducing all thiol-modulated enzymes analyzed here. We then investigated the in vivo physiological relevance of these in vitro findings, using Arabidopsis wild-type and Trx-f-deficient plants. Photoreduction of fructose-1,6-bisphosphatase was partially impaired in Trx-f-deficient plants, but the global impact of Trx-fdeficiency on the redox behaviors of thiol-modulated enzymes was not as striking as expected from the in vitro data. Our results provide support for the in vivofunctionality of the Trx system and also highlight the complexity and plasticity of the chloroplast redox network. [ABSTRACT FROM AUTHOR]

Subtilisin-like proteases are broadly expressed in organisms ranging from bacteria to mammals. During maturation of these enzymes, N-terminal propeptides function as intramolecular chaperones, assisting the folding of their catalytic domains. However, we have identified an exceptional case, the serine protease from Aeromonas sobria (ASP), that lacks a propeptide. Instead, ORF2, a protein encoded just downstream of asp, appears essential for proper ASP folding. The mechanism by which ORF2 functions remains an open question, because it shares no sequence homology with any known intramolecular propeptide or other protein. Here we report the crystal structure of the ORF2-ASP complex and the solution structure of free ORF2. ORF2 consists of three regions: an N-terminal extension, a central body, and a C-terminal tail. Together, the structure of the central body and the C-terminal tail is similar to that of the intramolecular propeptide. The N-terminal extension, which is not seen in other subtilisin-like enzymes, is intrinsically disordered but forms some degree of secondary structure upon binding ASP.Wealso show that C-terminal (ΔC1 and ΔC5) or N-terminal (ΔN43 and ΔN64) deletion eliminates the ability of ORF2 to function as a chaperone. Characterization of the maturation of ASP with ORF2 showed that folding occurs in the periplasmic space and is followed by translocation into extracellular space and dissociation from ORF2, generating active ASP. Finally, a PSI-BLAST search revealed that operons encoding subtilases and their external chaperones are widely distributed among Gram-negative bacteria, suggesting that ASP and its [ABSTRACT FROM AUTHOR]

In the biosynthesis of lysine by Thermus thermophilus, the metabolite α-ketoglutarate is converted to the intermediate α-aminoadipate (AAA), which is protected by the 54-amino acid acidic protein LysW. In this study, we determined the crystal structure of LysZ from T. thermophilus (TtLysZ), an amino acid kinase that catalyzes the second step in the AAA to lysine conversion, which was in a complex with LysW at a resolution of 1.85 Å. A crystal analysis coupled with isothermal titration calorimetry of the TtLysZ mutants for TtLysW revealed tight interactions between LysZ and the globular and C-terminal extension domains of the LysW protein, which were mainly attributed to electrostatic forces. These results provided structural evidence for LysW acting as a protecting molecule for the α-amino group of AAA and also as a carrier protein to guarantee better recognition by biosynthetic enzymes for the efficient biosynthesis of lysine. [ABSTRACT FROM AUTHOR]

Galnt3, UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetyl-galactosaminyltransferase 3, transfers N-acetyl-D-galactosa-mine to serine and threonine residues, initiating mucin type O-glycosylation of proteins. We searched the target genes of Runx2, which is an essential transcription factor for chondrocyte maturation, in chondrocytes and found that Galnt3 expression was up-regulated by Runx2 and severely reduced in Runx2-/- cartilaginous skeletons. To investigate the function of Galnt3 in chondrocytes, we generated Galnt3-/- mice and chondrocyte-specific Galnt3 transgenic mice under the control of the Col2a1 promoter-enhancer. Galnt3-/- mice showed a delay in endochondral ossification and shortened limbs at embryonic day 16.5, suggesting that Galnt3 is involved in chondrocyte maturation. Galnt3 transgenic mice presented dwarfism, the chondrocyte maturation was retarded, the cell cycle in chondrocytes was accelerated, premature chondrocyte apoptosis occurred, and the growth plates were disorganized. The binding of Vicia villosa agglutinin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), was drastically increased in chondrocytes, and aggrecan (Acan) was highly enriched with Tn antigen. However, safranin O staining, which recognizes glycosamino-glycans (GAGs), and Acan were severely reduced. Chondroitin sulfate was reduced in amount, but the elongation of chondroitin sulfate chains had not been severely disturbed in the isolated GAGs. These findings indicate that overexpression of Galnt3 in chondrocytes caused dwarfism due to the increase of mucin-type O-glycans and the reduction of GAGs, probably through competition with xylosyltransferases, which initiate GAG chains by attaching O-linked xylose to serine residues, suggesting a negative effect ofGalnt family proteins onAcan deposition in addition to the positive effect of Galnt3 on chondrocyte maturation. [ABSTRACT FROM AUTHOR]

Patients with advanced congestive heart failure (CHF) or chronic kidney disease (CKD) often have increased angiotensin II (Ang II) levels and cachexia. Ang II infusion in rodents causes sustained skeletal muscle wasting and decreases muscle regenerative potential through Ang II type 1 receptor (AT1R)-mediated signaling, likely contributing to the development of cachexia in CHF and CKD. However, the potential role of Ang II type 2 receptor (AT2R) signaling in skeletal muscle physiology is unknown. We found that AT2R expression was increased robustly in regenerating skeletal muscle after cardiotoxin (CTX)-induced muscle injury in vivo and differentiating myoblasts in vitro, suggesting that the increase in AT2R played an important role in regulating myoblast differentiation and muscle regeneration. To determine the potential role of AT2R in muscle regeneration, we infused C57BL/6 mice with the AT2R antagonist PD123319 during CTX-induced muscle regeneration. PD123319 reduced the size of regenerating myofibers and expression of the myoblast differentiation markers myogenin and embryonic myosin heavy chain. On the other hand, AT2R agonist CGP42112 infusion potentiated CTX injury-induced myogenin and embryonic myosin heavy chain expression and increased the size of regenerating myofibers. In cultured myoblasts, AT2R knockdown by siRNA suppressed myoblast differentiation marker expression and myoblast differentiation via up-regulation of phos-pho-ERK1/2, and ERK inhibitor treatment completely blocked the effect of AT2R knockdown. These data indicate that AT2R signaling positively regulates myoblast differentiation and potentiates skeletal muscle regenerative potential, providing a new therapeutic target in wasting disorders such as CHF and CKD. [ABSTRACT FROM AUTHOR]

Kruppel-like factor 4 (KLF4) plays an important role in vascular diseases, including atherosclerosis and vascular injury. Although KLF4 is expressed in the heart in addition to vascular cells, the role of KLF4 in cardiac disease has not been fully determined. The goals of this study were to investigate the role of KLF4 in cardiac hypertrophy and to determine the underlying mechanisms. Cardiomyocyte-specific Klf4 knockout (CM Klf4 KO) mice were generated by the Cre/LoxP technique. Cardiac hypertrophy was induced by chronic infusion of the /3-adreno-receptor agonist isoproterenol (ISO). Results showed that ISO-induced cardiac hypertrophy was enhanced in CM Klf4 KO mice compared with control mice. Accelerated cardiac hypertrophy in CM Klf4 KO mice was accompanied by the augmented cellular enlargement of cardiomyocytes as well as the exaggerated expression of fetal cardiac genes, including atrial natriureticfactor (Nppa). Additionally, induction of myocardin, a transcriptional cofactor regulating fetal cardiac genes, was enhanced in CM Klf4 KO mice. Interestingly, KLF4 regulated Nppa expression by modulating the expression and activity of myocardin, providing a mechanical basis for accelerated cardiac hypertrophy in CM Klf4 KO mice. Moreover, we showed that KLF4 mediated the antihypertrophic effect of trichostatin A, a histone deacetylase inhibitor, because ISO-induced cardiac hypertrophy in CM Klf4 KO mice was attenuated by olmesartan, an angiotensin II type 1 antagonist, but not by trichostatin A. These results provide novel evidence that KLF4 is a regulator of cardiac hypertrophy by modulating the expression and the activity of myocardin. [ABSTRACT FROM AUTHOR]

Cachexia is a serious complication of many chronic diseases, such as congestive heart failure (CHF) and chronic kidney disease (CKD). Although patients with advanced CHF or CKD often have increased angiotensin II (Ang II) levels and cachexia and Ang II causes skeletalmuscle wasting in rodents, the potential effects of Ang II onmuscle regeneration are unknown.Muscle regeneration is highly dependent on the ability of a pool of muscle stem cells (satellite cells) to proliferate and to repair damaged myofibers or form new myofibers. Here we show that Ang II reduced skeletal muscle regeneration via inhibition of satellite cell (SC) proliferation. Ang II reduced the number of regenerating myofibers and decreased expression of SC proliferation/differentiation markers (MyoD, myogenin, and active-Notch) after cardiotoxin-induced muscle injury in vivo and in SCs cultured in vitro. Ang II depleted the basal pool of SCs, as detected in Myf5nLacZ/+ mice and by FACS sorting, and this effect was inhibited by Ang II AT1 receptor (AT1R) blockade and inAT1aR-nullmice.AT1Rwas highly expressed in SCs, and Notch activation abrogated the AT1R-mediated antiprolifera- tive effect ofAng II in cultured SCs. Inmice that developed CHF postmyocardial infarction, there was skeletal muscle wasting and reduced SCnumbers that were inhibited byAT1R blockade. Ang II inhibition of skeletal muscle regeneration via AT1 receptor-dependent suppression of SC Notch and MyoD signaling and proliferation is likely to play an important role in mechanisms leading to cachexia in chronic disease states such as CHF and CKD. [ABSTRACT FROM AUTHOR]

Hyperphosphatemia in chronic kidney disease is highly associated with vascular calcification. Previous studies have shown that high phosphate-induced phenotypic switching of vascular smooth muscle cells (SMCs) into osteogenic cells plays an important role in the calcification process. In the present study, we determined whether Krüppel-like factor 4 (Klf4) and phosphorylated Elk-1, transcriptional repressors of SMC differentiation marker genes activated by intimal atherogenic stimuli, contributed to this process. Rat aortic SMCs were cultured in the medium with normal (0.9 mmol/liter) or high (4.5 mmol/liter) phosphate concentration. Results showed that high phosphate concentration induced SMC calcification. Moreover, high phosphate decreased expression of SMC differentiation marker genes including smooth muscle α-actin and SM22α, whereas it increased expression of osteogenic genes, such as Runx2 and osteopontin. High phosphate also induced Klf4 expression, although it did not phosphorylate Elk-1. In response to high phosphate, Klf4 selectively bound to the promoter regions of SMC differentiation marker genes. Of importance, siRNA-mediated knockdown of Klf4 blunted high phosphate-induced suppression of SMC differentiation marker genes, as well as increases in expression of osteogenic genes and calcium deposition. Klf4 was also induced markedly in the calcified aorta of adenine-induced uremic rats. Results provide novel evidence that Klf4 mediates high phosphate-induced conversion of SMCs into osteogenic cells. [ABSTRACT FROM AUTHOR]

For ∼40 years it has been widely accepted that (i) the exercise induced increase in muscle fatty acid oxidation( FAO) is dependent on the increased delivery of circulating fatty acids, and (ii) exercise training-induced FAO up-regulation is largely attributable to muscle mitochondrial biogenesis. These long standing concepts were developed prior to the recent recognition that fatty acid entry into muscle occurs via a regulatable sarcolemmal CD36-mediated mechanism. We examined the role of CD36 in muscle fuel selection under basal conditions, during a metabolic challenge (exercise), and after exercise training. We also investigated whether CD36 overexpression, independent of mitochondrial changes, mimicked exercise training-induced FAO up-regulation. Under basal conditions CD36-KO versus WT mice displayed reduced fatty acid transport (-21%) and oxidation (-25%), intramuscular lipids (less than or equal to -31%), and hepatic glycogen (-20%); but muscle glycogen, VO2max, and mitochondrial content and enzymes did not differ. In acutely exercised (78% VO2max) CD36-KO mice, fatty acid transport (-41%), oxidation (-37%), and exercise duration (-44%) were reduced, whereas muscle and hepatic glycogen depletions were accelerated by 27-55%, revealing 2-fold greater carbohydrate use. Exercise training increased mtDNA and β-hydroxyacyl-CoA dehydrogenase similarly in WT and CD36-KO muscles, but FAO was increased only in WT muscle (+90%). Comparable CD36 increases, induced by exercise training (+44%) or by CD36 overexpression (+41%), increased FAO similarly (84-90%), either when mitochondrial biogenesis and FAO enzymes were up-regulated (exercise training) or when these were unaltered(CD36 overexpression). Thus, sarcolemmal CD36 has a key role in muscle fuel selection, exercise performance, and training-induced muscle FAO adaptation, challenging long held views of mechanisms involved in acute and adaptive regulation of muscle FAO. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is characterized by an impairment of glucose uptake even though blood glucose levels are increased. Methylglyoxal is derived from glycolysis and has been implicated in the development of diabetes mellitus, because methylglyoxal levels in blood and tissues are higher in diabetic patients than in healthy individuals. However, it remains to be elucidated whether such factors are a cause, or consequence, of diabetes. Here, we show that methylglyoxal inhibits the activity of mammalian glucose transporters using recombinant Saccharomyces cerevisiae cells genetically lacking all hexose transporters but carrying cDNA for human GLUT1 or rat GLUT4. We found that methylglyoxal inhibits yeast hexose transporters also. Glucose uptake was reduced in a stepwise manner following treatment with methylglyoxal, i.e. a rapid reduction within 5 min, followed by a slow and gradual reduction. The rapid reduction was due to the inhibitory effect of methylglyoxal on hexose transporters, whereas the slow and gradual reduction seemed due to endocytosis, which leads to a decrease in the amount of hexose transporters on the plasma membrane. We found that Rsp5, a HECT-type ubiquitin ligase, is responsible for the ubiquitination of hexose transporters. Intriguingly, Plc1 (phospholipase C) negatively regulated the endocytosis of hexose transporters in an Rsp5-dependent manner, although the methylglyoxal-induced endocytosis of hexose transporters occurred irrespective of Plc1. Meanwhile, the internalization of hexose transporters following treatment with methylglyoxal was delayed in a mutant defective in protein kinase C. [ABSTRACT FROM AUTHOR]

Mammalian target of rapamycin (mTOR) is a kinase that plays a key role in a wide array of cellular processes and exists in two distinct functional complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Although mTORC2 is primarily activated by growth factors, mTORC1 is regulated by numerous extracellular and intracellular signals such as nutrients, growth factors, and cellular redox. Previous study has shown that cysteine oxidants sufficiently activate mTORC1 activity under amino acid-depleted conditions and that a reducing agent effectively suppresses amino acid-induced mTORC1 activity, thereby raising the possibility that redox-sensitive mechanisms underlie amino acid-dependent mTORC1 regulation. However, the molecular mechanism by which redox regulates mTORC1 activity is not well understood. In this study, we show that the redox-sensitive regulation of mTORC1 occurs via Rheb but not the Rag small GTPase. Enhancing cellular redox potential with cysteine oxidants significantly increases Rheb GTP levels. Importantly, modulation of the cellular redox potential with a cysteine oxidant or reducing agent failed to alter mTORC1 activity in TSC1-/- or TSC2-/- mouse embryonic fibroblast cells. Furthermore, a cysteine oxidant has little effect on mTOR localization but sufficiently activates mTORC1 activity in both p18-/- and control mouse embryonic fibroblast cells, suggesting that the redox-sensitive regulation of mTORC1 occurs independent of the Ragulator·Rag complex. Taken together, our results suggest that the TSC complex plays an important role in redox-sensitive mTORC1 regulation and argues for the activation of mTORC1 in places other than the lysosome upon inhibition of the TSC complex. [ABSTRACT FROM AUTHOR]

The Calvin-Benson-Bassham cycle is responsible for carbon dioxide fixation in all plants, algae, and cyanobacteria. The enzyme that catalyzes the carbon dioxide-fixing reaction is ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk-Rubisco) belongs to the type III group, and shows high activity at high tempera- tures. We have previously found that replacement of the entire α-helix 6 of Tk-Rubisco with the corresponding region of the spinach enzyme (SP6 mutant) results in an improvement of catalytic performance at mesophilic tempera- tures, both in vivo and in vitro, whereas the former and latter half-replacements of the a-helix 6 (SP4 and SP5 mu- tants) do not yield such improvement. We report here the crystal structures of the wild-type Tk-Rubisco and the mutants SP4 and SP6, and discuss the relationships between their structures and enzymatic activities. A comparison among these structures shows the movement and the increase of temperature factors of α-helix 6 induced by four essential factors. We thus supposed that an increase in the flexibility of the α-helix 6 and loop 6 regions was important to increase the catalytic activ- ity of Tk-Rubisco at ambient temperatures. Based on this structural information, we constructed a new mutant, SP5-V330T, which was designed to have significantly greater flexibility in the above region, and it proved to exhibit the highest activity among all mutants examined to date. The thermostability of the SP5- V330T mutant was lower than that of wild-type Tk-Rubisco, providing further support on the relationship between flexibility and activity at ambient temperatures. [ABSTRACT FROM AUTHOR]

Galectin-9, a tandem-repeat-type β-galactoside-specific animal lectin with two carbohydrate recognition domains (CRDs) at the N- and C-terminal ends, is involved in chemoattraction, apoptosis, and the regulation of cell differentiation and has anti-allergic effects. Its ability to recognize carbohydrates is essential for its biological functions. Human galectin-9 (hG9) has high affinity for branched N-glycan-type oligosaccharides (dissociation constants of 0.16-0.70 μm) and linear β1-3-linked poly-N-acetyllactosamines (0.09-8.3 μm) and significant affinity for the α2-3-sialylated oligosaccharides (17-34 μm). Further, its N-terminal CRD (hG9N) and C-terminal CRD (hG9C) differ in specificity. To elucidate this unique feature of hG9, x-ray structures of hG9C in the free form and in complexes with N-acetyllactosamine, the biantennary pyridylaminated oligosaccharide, and α2-3-sialyllactose were determined. They are the first x-ray structural analysis of C-terminal CRD of the tandem-repeat-type galectin. The results clearly revealed the mechanism by which branched and α2-3-sialylated oligosaccharides are recognized and explained the difference in specificity between hG9N and hG9C. Based on structural comparisons with other galectins, we propose that the wide entrance for ligand binding and the shallow binding site of hG9C are favorable for branched oligosaccharides and that Arg221 is responsible for recognizing sialylated oligosaccharides. [ABSTRACT FROM AUTHOR]

Aspartate kinase (AK) is the first and committed enzyme of the biosynthetic pathway producing aspartate family amino acids, lysine, threonine, and methionine. AK from Corynebacterium glutamicum (CgAK), a bacterium used for industrial fermentation of amino acids, including glutamate and lysine, is inhibited by lysine and threonine in a concerted manner. To elucidate the mechanism of this unique regulation in CgAK, we determined the crystal structures in several forms: an inhibitory form complexed with both lysine and threonine, an active form complexed with only threonine, and a feedback inhibition-resistant mutant (S301F) complexed with both lysine and threonine. CgAK has a characteristic α2β2-type heterotetrameric structure made up of two α subunits and two β subunits. Comparison of the crystal structures between inhibitory and active forms revealed that binding inhibitors causes a conformational change to a closed inhibitory form, and the interaction between the catalytic domain in the α subunit and β subunit (regulatory subunit) is a key event for stabilizing the inhibitory form. This study shows not only the first crystal structures of α2β2-type AK but also the mechanism of concerted inhibition in CgAK. [ABSTRACT FROM AUTHOR]

Krüppel-like factor 4 (Klf4) is a transcription factor involved in differentiation and proliferation in multiple tissues. We demonstrated previously that tamoxifen-induced deletion of the Klf4 gene in mice accelerated neointimal formation but delayed down-regulation of smooth muscle cell differentiation markers in carotid arteries following injury. To further determine the role of Klf4 in the cardiovascular system, we herein derived mice deficient for the Klf4 gene in smooth and cardiac muscle using the SM22α promoter (SM22α-CreKI+/Klf4loxP/loxP mice). SM22α-CreKI+/Klf4loxP/loxP mice were born at the expected Mendelian ratio, but they gradually died after birth. Although ~40% of SM22α-CreKI+/Klf4loxP/loxP mice survived beyond postnatal day 28, they exhibited marked growth retardation. In wild-type mice, Klf4 was expressed in the heart from late embryonic development through adulthood, whereas it was not expressed in smooth muscle. No changes were observed in morphology or expression of smooth muscle cell differentiation markers in vessels of SM22αCreKI+/Klf4loxP/loxP mice. Of interest, cardiac output was significantly decreased in SM22α-CreKl+/Klf4loxP/loxP mice, as determined by magnetic resonance imaging. Moreover, a lack of Klf4 in the heart resulted in the reduction in expression of multiple cardiac genes, including Gata4. In vivo chromatin immunoprecipitation assays on the heart revealed that Klf4 bound to the promoter region of the Gata4 gene. Results provide novel evidence that Klf4 plays a key role in late fetal and/or postnatal cardiac development. [ABSTRACT FROM AUTHOR]

Two heptamer rings of chaperonin GroEL undergo opening-closing conformational transition in the reaction cycle with the aid of GroES and ATP. We introduced Cys into the GroEL subunit at Ala-384 and Ser-509, which are very close between adjacent GroEL subunits in the open heptamer ring but far apart in the closed heptamer ring. The open ring-specific inter-subunit cross-linking between these Cys indicated that the number of rings in open conformation in GroEL was two in ATP (GroELoo), one in ADP (GroELo), and none in the absence of nudeotide. ADP showed an inhibitory effect on AT P-induced generation of GroELoo. The isolated GroELo and GroELoo, which lost any bound nucleotide, could bind GroES to form a bulletshaped 1:1 GroEL-GroES complex and a football-shaped 1:2 GroEL-GroES complex, respectively, even without the addition of any nucleotide. Substrate protein was unable to form a stable complex with GroELoo and did not stimulate ATPase activity of GroEL. These results favor a model of the GroEL reaction cycle that includes a football complex as a critical intermediate. [ABSTRACT FROM AUTHOR]

Homologous pairing, an essential process for homologous recombination, is the formation of a heteroduplex joint by an invading single-stranded DNA tail and a complementary sequence within double-stranded DNA (dsDNA). The base rotation of the parental dsDNA, to switch from parental base pairs to heteroduplex ones with the invading single-stranded DNA, sterically requires vertical extension between adjacent base pairs, which inevitably induces untwisting of the dsDNA. RecA is a prototype of the RecA/Rad5l/Dmcl family proteins, which promote ATP-dependent homologous pairing in homologous DNA recombination in vivo, except in mitochondria. As predicted by the requirement for the untwisting, dsDNA bound to RecA is extended and untwisted, and homologous pairing by RecA in vitro is extensively stimulated by the negative supercoils of dsDNA substrates. D-loop formation in negatively supercoiled dsDNA, which serves as an assay for homologous pairing, is also catalyzed in an ATP-independent manner by proteins structurally unrelated to RecA, such as Mhr1. Mhr1 is required for yeast mitochondrial DNA recombination instead of RecA family proteins. Inconsistent with the topological requirements, tests for the effects of negative supercoils revealed that Mhr1 catalyzes homologous pairing with relaxed closed circular dsDNA much more efficiently than with negatively supercoiled dsDNA. Topological analyses indicated that neither the process nor the products of homologous pairing by Mhr1 involve a net topological change of closed circular dsDNA. This would be favorable for homologous recombination in mitochondria, where dsDNA is unlikely to be under topological stress toward unwinding. We propose a novel topological mechanism wherein Mhr1 induces untwisting without net topological change. [ABSTRACT FROM AUTHOR]

Palladin is a widely expressed actin-associated protein localized at stress fibers, focal adhesions, and other actin-based structures, playing a significant role in cell adhesion and cell motility. Knockout of Palladin in mice is embryonic lethal, demonstrating the importance of Palladin in development yet its role in the vasculature is not known. In the present study, smooth muscle cell (SMC) markers, such as myosin, actin, caldesmon, calponin, and LPP, were down-regulated in embryoid bodies (EBs) derived from embryonic stem cells lacking Palladin. Transgenic embryonic stem cell lines were generated that stably expressed a puromycin-resistance gene under the control of a SM α-actin (SMA) promoter. Negative selection was then used to purify SMCs from EBs. Purified SMCs expressing multiple SMC markers were designated APSCs (SMA-puromycin selected cells). Palladin null APSCs express significantly less myosin, actin, calponin, and h-caldesmon. The filamentous (F) to globular (G) actin ratio, known to regulate myocardin family transcription factors, was also decreased. Palladin null APSCs showed increased cell adhesion and decreased cell motility. Importantly, Palladin null APSCs within collagen gels generated less maximum contractile force when stimulated with endothelin-1, sphingosine 1-phosphate (S1P), and thrombin. Myosin light chains (MLC20) were phosphorylated by lysophosphatidic acid to the same extent in Palladin null and wild type APSCs but myosin content/total protein was reduced by >50%, consistent with the observed decreases in contractility. All together, these results suggest that Palladin is essential for expression of the full complement of contractile proteins necessary for optimal force development of SMCs derived from EBs. [ABSTRACT FROM AUTHOR]

We previously reported that the heparan sulfate 3-O-sulfotransferase (3OST)-5 produces a novel component of heparan sulfate, i.e. the tetrasulfated disaccharide (Di-tetraS) unit (Mochizuki, H., Yoshida, K., Gotoh, M., Sugioka, S., Kikuchi, N., Kwon, Y.-D., Tawada, A., Maeyama, K., Inaba, N., Hiruma, T., Kimata, K., and Narimatsu, H. (2003) J. Biol. Chem. 278, 26780-26787). In the present study, we investigated the potential of other 3OST isoforms to produce Di-tetraS with heparan sulfate and heparin as acceptor substrates. 3OST-2, 3OST-3, and 3OST-4 produce Di-tetraS units as a major product from both substrates. 3OST-5 showed the same specificity for heparin, but the production from heparan sulfate was very low. Di-tetraS production by 3OST-1 was negligible. We then investigated the presence of Di-tetraS units in heparan sulfates from various rat tissues. Di-tetraS was detected in all of the tissues analyzed. Liver and spleen contain relatively high levels of Di-tetraS, 1.6 and 0.95%, respectively. However, the content of this unit in heart, large intestine, ileum, and lung is low, less than 0.2%. We further determined the expression levels of 3OST transcripts by quantitative real time PCR. The 3OST-3 transcripts are highly expressed in spleen and liver. The 3OST-2 and -4 are specifically expressed in brain. These results indicate that the Di-tetraS unit is widely distributed throughout the body as a rare and unique component of heparan sulfate and is synthesized by tissue-specific 3OST isoforms specific for Di-tetraS production. [ABSTRACT FROM AUTHOR]

The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists in protein folding with the aid of GroES and ATP. It is believed that GroEL alternates the folding-active rings and that the substrate protein (and GroES) can bind to the open trans-ring only after ATP in the cis-ring is hydrolyzed. However, we found that a substrate protein. prebound to the trans-ring remained bound during the first ATP cycle, and this substrate was assisted by GroEL-GroES when the second cycle began. Moreover, a slow ATP-hydrolyzing GroEL mutant (D398A) in the ATP-bound form bound a substrate protein and GroES to the trans-ring. The apparent discrepancy with the results from an earlier study (Rye, H. S., Roseman, A. M., Chen, S., Furtak, K., Fenton, W. A., Saibil, H. R., and Horwich, A. L. (1999) Cell 97, 325-338) can be explained by the previously unnoticed fact that the ATP-bound form of the D398A mutant exists as a symmetric 1:2 GroEL-GroES complex (the "foot- ball"-shaped complex) and that the substrate protein (and GroES) in the medium is incorporated into the complex only after the slow turnover. In light of these results, the current model of the GroEL-GroES reaction cycle via the asymmetric 1:1 GroEL-GroES complex deserves reexamination. [ABSTRACT FROM AUTHOR]

The cytokine, transforming growth factor-β1 (TGF-β1), converts naive T cells into regulatory T cells that prevent autoimmunity. However, in the presence of interleukin (IL)-6, TGF-β1 has also been found to promote differentiation into IL-17-producing helper T (Th17) cells that are deeply involved in autoimmunity and inflammation. However, it has not been clarified how TGF-β1 and IL-6 determine such a distinct fate. Here we found that a master regulator for Th17, retinoic acid-related orphan receptor γt (RORγt), was rapidly induced by TGF-β1 regardless of the presence of IL-6. IL-6 reduced Foxp3 expression, and overexpression of Foxp3 in a T cell line resulted in a strong reduction of IL-17A expression. We have characterized the IL-17A promoter and found that RORγt binding is sufficient for activation of the minimum promoter in the HEK 293T cells. RORγt-mediated IL-17A promoter activation was suppressed by forced expression of Foxp3. Foxp3 directly interacted with RORγt through exon 2 region of Foxp3. The exon 2 region and forkhead (FKH) domain of Foxp3 were necessary for the suppression of RORγt-mediated IL-17A promoter activation. We propose that induction of Foxp3 is the mechanism for the suppression of Th17 and polarization into inducible Treg. [ABSTRACT FROM AUTHOR]

The coaggregation receptor polysaccharides (RPS) of Streptococcus oralis and related species are recognized by lectin-like adhesins on other members of the oral biofilm community and by RPS-specific antibodies. The former interactions involve β-GalNAc or β-Gal containing host-like motifs in the oligosaccharide repeating units of these polysaccharides, whereas the latter involves features of these molecules that are immunogenic. In the present investigation, the molecular and corresponding structural basis for the serotype specificity of S. oralis ATCC 10557 RPS was determined by engineering the production of this polysaccharide in transformable Streptococcus gordonii 38. This involved the systematic replacement of genes in the rps cluster of strain 38 with different but related genes from S. oralis 10557 and structural characterization of the resulting polysaccharides. The results identify four unique genes in the rps cluster of strain 10557. These include wefI for an α-Gal transferase, wefJ for a GalNAc-1-phosphotransferase that has a unique acceptor specificity, wefK for an acetyl transferase that acts at two positions in the hexasaccharide repeating unit, and a novel wzy associated with the β1-3 linkage between these units. The serotype specificity of engineered polysaccharides correlated with the wefI-dependent presence of α-Gal in these molecules rather than with partial O-acetylation or with the linkage between repeating units. The findings illustrate a direct approach for defining the molecular basis of polysaccharide structure and antigenicity. [ABSTRACT FROM AUTHOR]

The iolABCDEFGHIJ operon of Bacillus subtilis is responsible for rnyo-inositol catabolism involving multiple and stepwise reactions. Previous studies demonstrated that IolG and IolE are the enzymes for the first and second reactions, namely dehydrogenation of myo-inositol to give 2-keto-myo-inositol and the subsequent dehydration to 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione. In the present studies the third reaction was shown to be the hydrolysis of 3D-(3,5/4)-trihydroxycyclohexane-1,2-di-one catalyzed by IolD to yield 5-deoxy-D-glucuronic acid. The fourth reaction was the isomerization of 5-deoxy-D-glucuronic acid by IolB to produce 2-deoxy-5-keto-D-gluconic acid. Next, in the fifth reaction 2-deoxy-5-keto-D-gluconic acid was phosphorylated by IolC kinase to yield 2-deoxy-5-keto-D-gluconic acid 6-phosphate. IolR is known as the repressor controlling transcription of the Iol operon. In this reaction 2-deoxy-5-keto-D-gluconic acid 6-phosphate appeared to be the intermediate acting as inducer by antagonizing DNA binding of IolR. Finally, IolJ turned out to be the specific aldolase for the sixth reaction, the cleavage of 2-deoxy-5-keto-D-gluconic acid 6-phosphate into dihydroxyacetone phosphate and malonic semialdehyde. The former is a known glycolytic intermediate, and the latter was previously shown to be converted to acetyl-CoA and CO2 by a reaction catalyzed by IolA. The net result of the inositol catabolic pathway in B. subtilis is, thus, the conversion of myo-inositol to an equimolar mixture of dihydroxyacetone phosphate, acetyl-CoA, and CO2. [ABSTRACT FROM AUTHOR]

The ϵ subunit of F1-ATPase from the thermophilic Bacillus PS3 (TF1) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the E subunit remains to be determined. To address this question, 11 mutants of the ϵ sub- unit were prepared, in which one of the basic or acidic residues was substituted with alanine. ATP binding to these mutants was tested by gel-filtration chromatography. Among them, four mutants that showed no ATP binding were selected and reconstituted with the α3β3γ complex of TF1. The ATPase activity of the resulting α3β3γϵ complexes was measured, and the extent of inhibition by the mutant ϵ subunits was compared in each case. With one exception, weaker binding of ATP correlated with greater inhibition of ATPase activity. These results clearly indicate that ATP binding to the ϵ subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF1 by fixing the ϵ subunit into the folded conformation. [ABSTRACT FROM AUTHOR]

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH2, a common precursor of various prostanoids, to produce PGD2, an endogenous somnogen and nociceptive modulator, in the brain. L-PGDS is a member of the lipocalin superfamily and binds lipophilic substances, such as retinoids and bile pigments, suggesting that L-PGDS is a dual functional protein acting as a PGD2-synthesizing enzyme and a transporter for lipophilic ligands. In this study we determined by NMR the three-dimensional structure of recombinant mouse L-PGDS with the catalytic residue Cys-65. The structure of L-PGDS exhibited the typical lipocalin fold, consisting of an eight-stranded, antiparallel β-barrel and a long α-helix associated with the outer surface of the barrel. The interior of the barrel formed a hydrophobic cavity opening to the upper end of the barrel, the size of which was larger than those of other lipocalins, and the cavity contained two pockets. Molecular docking studies, based on the result of NMR titration experiments with retinoic acid and PGH2 analog, revealed that PGH2 almost fully occupied the hydrophilic pocket 1, in which Cys-65 was located and all-trans-retinoic acid occupied the hydrophobic pocket 2, in which amino acid residues important for retinoid binding in other lipocalins were well conserved. Mutational and kinetic studies provide the direct evidence for the PGH2 binding mode. These results indicated that the two binding sites for PGH2 and retinoic acid in the large cavity of L-PGDS were responsible for the broad ligand specificity of L-PGDS and the non-competitive inhibition of L-PGDS activity by retinoic acid. [ABSTRACT FROM AUTHOR]

The imidazole 15N signals of histidine 64 (His64), involved in the catalytic function of human carbonic anhydrase II (hCAII), were assigned unambiguously. This was accomplished by incorporating the labeled histidine as probes for solution NMR analysis, with 15N at ring-Nδ1 and Nϵ2, 13C at ring-Cϵ1, 13C and 1SN at all carbon and nitrogen, or 15N at the amide nitrogen and the labeled glycine with 13C at the carbonyl carbon. Using the pH dependence of ring-15N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (KT) of His64 is 1.0, which differs from that of other histidine residues. This unique value characterizes the imidazole nitrogen atoms of His64 as both a general acid (a) and base (b): its ϵ2-nitrogen as (a) releases one proton into the bulk, whereas its δ1-nitrogen as (b) extracts another proton from a water molecule within the water bridge coupling to the zinc-bound water inside the cave. This accelerates the generation of zinc-bound hydroxide to react with the carbon dioxide. Releasing the productive bicarbonate ion from the inside separates the water bridge pathway, in which the next water molecules move into beside zinc ion. A new water molecule is supplied from the bulk to near the δ1-nitrogen of His64. These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His64 can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time- or energy-consuming processes. [ABSTRACT FROM AUTHOR]

Fbs1 is an F-box protein present abundantly in the nervous system. Similar to the ubiquitously expressed Fbs2, Fbs1 recognizes N-glycans at the innermost position as a signal for unfolded glycoproteins, probably in the endoplasmic reticulum-associated degradation pathway. Here, we show that the in vivo majority of Fbs1 is present as Fbs1-Skp1 heterodimers or Fbs1 monomers but not SCFFbs1 complex. The inefficient SCF complex formation of Fbs1 and the restricted presence of SCFFbs1 bound on the endoplasmic reticulum membrane were due to the short linker sequence between the F-box domain and the sugar-binding domain. In vitro, Fbs1 prevented the aggregation of the glycoprotein through the N-terminal unique sequence of Fbs1. Our results suggest that Fbs1 assists clearance of aberrant glycoproteins in neuronal cells by suppressing aggregates formation, independent of ubiquitin ligase activity, and thus functions as a unique chaperone for those proteins. [ABSTRACT FROM AUTHOR]

Vesicular glutamate transporters (VGLUTs) are responsible for the vesicular storage of L-glutamate and play an essential role in glutamatergic signal transmission in the central nervous system. The molecular mechanism of the transport remains unknown. Here, we established a novel in vitro assay procedure, which includes purification of wild and mutant VGLUT2 and their reconstitution with purified bacterial FoF1-ATPase (F-ATPase) into liposomes. Upon the addition of ATP, the proteoliposomes facilitated L-glutamate uptake in a membrane potential (Δψ)-dependent fashion. The ATP-dependent L-glutamate uptake exhibited an absolute requirement for ~4 mM Cl-, was sensitive to Evans blue, but was insensitive to D,L-aspartate. VGLUT2s with mutations in the transmembrane-located residues Arg184, His128, and Glu191 showed a dramatic loss in L-glutamate transport activity, whereas Na+:dependent inorganic phosphate (Pi) uptake remained comparable to that of the wild type. Furthermore, Pi transport did not require Cl- and was not inhibited by Evans blue. Thus, VGLUT2 appears to possess two intrinsic transport machineries that are independent of each other: a Δψ-dependent L-glutamate uptake and a Na+-dependent Pi uptake. [ABSTRACT FROM AUTHOR]

The ompF and ompC genes of Escherichia coli are reciprocally regulated by a single transcription factor, phosphorylated OmpR (OmpR-P), depending upon medium osmolarity. This regulation involves activation of ompF and its repression with concomitant activation of ompC. This occurs through OmpR-P binding to four (F1, F2, F3, and F4) and three (C1, C2, and C3) sites located upstream of the ompF and ompC promoters, respectively, through a novel mechanism, Here we show that there is a distinct OmpR-P binding hierarchy within F1, F2, and F3 sites as well as within C1, C2, and C3 sites. Each of these sites contains two tandem 10-bp OmpR-P-binding subsites, a-site and b-site (from 5′ to 3′ direction). OmpR-P has higher affinity to the downstream b-site than to the upstream a-site in each case. Six OmpR-P molecules bind to F and C sites two-by-two in a discontinuous "galloping" manner. We propose that this tight hierarchical binding of a transcription factor, OmpR, allows distinct stepwise regulation of ompF and ompC transcription, which minimizes their overlapping expression upon changes in the medium osmolarity to achieve the reciprocal expression of ompF and ompC. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests an adaptive response induced by reactive oxygen species and other physiologically existing oxidative stimuli. We have recently reported that a variety of lipid peroxidation products at sublethal concentrations could induce adaptive response and enhance PC12 cell tolerance, although the detailed underlying molecular mechanisms have not been clearly clarified. In the present study, we found that both 7-hydroxycholesterol (7-OHCh) and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) at sublethal concentrations significantly increased the cellular GSH as well as the enzyme activity of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis. Depletion of cellular GSH by buthionine sulfoximine completely abolished the adaptive response. Interestingly, treatment with 15d-PGJ2 significantly increased the gene expression of both subunits of GCL in an NF-E2-related factor 2 (Nrf2)-dependent manner, whereas neither 7-OHCh induced any considerable changes on the GCL gene expression nor did the Nrf2-small interfering RNA treatment exert any appreciable effects on the GSH elevation and subsequent adaptive response induced by 7-OHCh. These results demonstrate that the adaptive response induced by both 7-OHCh and 15d-PGJ2 is mediated similarly through the up-regulation of GSH but via different mechanisms. [ABSTRACT FROM AUTHOR]

The p53 tumor suppressor is activated in the cellular response to genotoxic stress. Transactivation of p53 target genes dictates cell cycle arrest and DNA repair or induction of apoptosis; however, a molecular mechanism responsible for these distinct functions remains unclear. Recent studies revealed that phosphorylation of p53 on Ser46 was associated with induction of p53AIP1 expression, resulting in the commitment of the cell fate into apoptotic cell death. Moreover, upon exposure to genotoxic stress, p53DINP1 was expressed and recruited a kinase(s) to p53 that specifically phosphorylated Ser46. Here, we show that the pro-apoptotic kinase, protein kinase C δ (PKCδ), is involved in phosphorylation of p53 on Ser46. PKCδ-mediated phosphorylation is required for the interaction of PKCδ with p53. The results also demonstrate that p53DINP1 associates with PKCδ upon exposure to genotoxic agents. Consistent with these results, PKCδ potentiates p53-dependent apoptosis by Ser46 phosphorylation in response to genotoxic stress. These findings indicate that PKCδ regulates p53 to induce apoptotic cell death in the cellular response to DNA damage. [ABSTRACT FROM AUTHOR]

ABI1 and ABI2 encode PP2C-type protein phosphatases and are thought to negatively regulate many aspects of abscisic acid (ABA) signaling, including stomatal closure in Arabidopsis. In contrast, SRK2E/OST1/SnRK2.6 encodes an Arabidopsis SnRK2 protein kinase and acts as a positive regulator in the ABA-induced stomatal closure. SRK2E/OST1 is activated by osmotic stress as well as by ABA, but the independence of the two activation processes has not yet been determined. Additionally, interaction between SRK2E/OST1 and PP2C-type phosphatases (ABI1 and ABI2) is not understood. In the present study, we demonstrated that the abi1-1 mutation, but not the abi2-1 mutation, strongly inhibited ABA-dependent SRK2E/OST1 activation. In contrast, osmotic stress activated SRK2E/OST1 even in abi1-1 and aba2-1 plants. The C-terminal regulatory domain of SRK2E/OST1 was required for its activation by both ABA and osmotic stress in Arabidopsis. The C-terminal domain was functionally divided into Domains I and II. Domain II was required only for the ABA-dependent activation of SRK2E/OST1, whereas Domain I was responsible for the ABA-independent activation. Full-length SRK2E/OST1 completely complemented the wilty phenotype of the srk2e mutant, but SRK2E/OST1 lacking Domain II did not. Domain II interacted with the ABI1 protein in a yeast two-hybrid assay. Our results suggested that the direct interaction between SRK2E/OST1 and ABI1 through Domain II plays a critical role in the control of stomatal closure. [ABSTRACT FROM AUTHOR]