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Transporters for organic cations (OCs) facilitate exchange of positively charged molecules through the plasma membrane. Substrates for these transporters encompass neurotransmitters, metabolic byproducts, drugs, and xenobiotics. Consequently, these transporters actively contribute to the regulation of neurotransmission, cellular penetration and elimination process for metabolic products, drugs, and xenobiotics. Therefore, these transporters have significant physiological, pharmacological, and toxicological implications. In cells of renal proximal tubules, the vectorial secretion pathways for OCs involve expression of organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs) on basolateral and apical membrane domains, respectively. This review provides an overview of documented regulatory mechanisms governing OCTs and MATEs. Additionally, regulation of these transporters under various pathological conditions is summarized. The expression and functionality of OCTs and MATEs are subject to diverse pre‐ and post‐translational modifications, providing insights into their regulation in various pathological conditions. Typically, in diseases, downregulation of transporter expression is observed, probably as a protective mechanism to prevent additional damage to kidney tissue. This regulation may be attributed to the intricate network of modifications these transporters undergo, shedding light on their dynamic responses in pathological contexts. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus DNA polymerase processivity factor UL44 is transported into the nucleus by importin (IMP) α/β through a classical nuclear localization signal (NLS), and this region is susceptible to cdc2‐mediated phosphorylation at position T427. Whilst phosphorylation within and close to the UL44 NLS regulates nuclear transport, the details remain elusive, due to the paucity of structural information regarding the role of negatively charged cargo phosphate groups. We addressed this issue by studying the effect of UL44 T427 phosphorylation on interaction with several IMPα isoforms by biochemical and structural approaches. Phosphorylation decreased UL44/IMPα affinity 10‐fold, and a comparative structural analysis of UL44 NLS phosphorylated and non‐phosphorylated peptides complexed with mouse IMPα2 revealed the structural rearrangements responsible for phosphorylation‐dependent inhibition of UL44 nuclear import. [ABSTRACT FROM AUTHOR]

RhoGTPases are well known for being controllers of cell cytoskeleton and share common features in the way they act and are controlled. These include their switch from GDP to GTP states, their regulations by different guanine exchange factors (GEFs), GTPase‐activating proteins and guanosine dissociation inhibitors (GDIs), and their similar structure of active sites/membrane anchors. These very similar features often lead to the common consideration that the differences in their biological effects mainly arise from the different types of regulators and specific effectors associated with each GTPase. Focusing on data obtained through biosensors, live cell microscopy and recent optogenetic approaches, we highlight in this review that the regulation of RhoA appears to depart from Cdc42 and Rac1 modes of regulation through its enhanced lability at the plasma membrane. RhoA presents a high dynamic turnover at the membrane that is regulated not only by GDIs but also by GEFs, effectors and a possible soluble conformational state. This peculiarity of RhoA regulation may be important for the specificities of its functions, such as the existence of activity waves or its putative dual role in the initiation of protrusions and contractions. [ABSTRACT FROM AUTHOR]

Thioredoxins (TRXs), a large subclass of ubiquitous oxidoreductases, are involved in thiol redox regulation. Here, we performed a comprehensive analysis of TRXs in the Arabidopsis thaliana genome, revealing 41 genes encoding 18 typical and 23 atypical TRXs, and 6 genes encoding thioredoxin reductases (TRs). The high number of atypical TRXs indicates special functions in plants that mostly await elucidation. We identified an atypical class of thioredoxins called TRX‐c in the genomes of photosynthetic eukaryotes. Localized to the chloroplast, TRX‐c displays atypical CPLC, CHLC and CNLC motifs in the active sites. In silico analysis of the transcriptional regulations of TRXs revealed high expression of TRX‐c in leaves and strong regulation under cold, osmotic, salinity and metal ion stresses. [ABSTRACT FROM AUTHOR]

Some proteins are expressed as a result of a ribosome frameshifting event that is facilitated by a slippery site and downstream secondary structure elements in the mRNA. This review summarizes recent progress in understanding mechanisms of –1 frameshifting in several viral genes, including IBV 1a/1b, HIV‐1 gag‐pol, and SFV 6K, and in Escherichia coli dnaX. The exact frameshifting route depends on the availability of aminoacyl‐tRNAs: the ribosome normally slips into the –1‐frame during tRNA translocation, but can also frameshift during decoding at condition when aminoacyl‐tRNA is in limited supply. Different frameshifting routes and additional slippery sites allow viruses to maintain a constant production of their key proteins. The emerging idea that tRNA pools are important for frameshifting provides new direction for developing antiviral therapies. [ABSTRACT FROM AUTHOR]

Cytoplasmic dynein, a microtubule‐based motor protein, is responsible for many cellular functions ranging from cargo transport to cell division. The various functions are carried out by a single isoform of cytoplasmic dynein, thus requiring different forms of motor regulation. A possible pathway to regulate motor function was revealed in optical trap experiments. Switching motor function from single steps to processive runs could be achieved by changing Mg2+ and ATP concentrations. Here, we confirm by single molecule total internal reflection fluorescence microscopy that a native cytoplasmic dynein dimer is able to switch to processive runs of more than 680 consecutive steps or 5.5 μm. We also identified the ratio of Mg2+‐free ATP to Mg.ATP as the regulating factor and propose a model for dynein processive stepping. [ABSTRACT FROM AUTHOR]

Biological experiments have verified that EIciRNAs (a class of circRNA) produced from pre‐mRNA can regulate gene expression, but the effect of regulation remains unexplored. Here, we refine a mechanistic gene model from experimental facts, in which we assume pre‐mRNA synthesizes EIciRNAs and mRNAs in a probabilistic manner, with the probability called the pathway strength, and the resulting EIciRNAs positively regulate the pre‐mRNA synthesis. We show that there is a critical pathway strength such that the mRNA mean and the mRNA noise reach the highest and lowest levels, respectively. The EIciRNA can induce the unimodal and bimodal mRNA expressions, as well as the transition between them. Our investigation hints that EIciRNA is a non‐negligible factor affecting cell‐to‐cell variability in gene expression. [ABSTRACT FROM AUTHOR]

Nearly all human multiexon genes are subject to alternative splicing in one or more cell types. The splicing machinery therefore has to select between multiple splice sites in a context‐dependent manner, relying on sequence features in cis‐ and trans‐acting splicing regulators that either promote or repress splice site recognition and spliceosome assembly. However, the functional coupling between multiple gene regulatory layers signifies that splicing can also be modulated by transcriptional or epigenetic characteristics. Other, less obvious, aspects of alternative splicing have come to light in recent years, often involving core components of the spliceosome previously thought to perform a basal rather than a regulatory role in splicing. Together this paints a highly dynamic picture of splicing regulation, where the final splice site choice is governed by the entire transcriptional environment of a gene and its cellular context. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are evolutionarily conserved small regulatory RNAs that participate in the adjustment of many, if not all, fundamental biological processes. Molecular mechanisms involved in miRNA biogenesis and mode of action have been elucidated in the past two decades. Similar to many cellular pathways, miRNA processing and function can be globally or specifically regulated at several levels and by numerous proteins and RNAs. Given their role as fine‐tuning molecules, it is essential for miRNA expression to be tightly regulated in order to maintain cellular homeostasis. Here, we review our current knowledge of the first step of their maturation occurring in the nucleus and how it can be specifically and dynamically modulated. [ABSTRACT FROM AUTHOR]

Trichoderma reesei is used to produce saccharifying enzyme cocktails for biofuels. There is limited understanding of the transcription factors (TFs) that regulate genes involved in release and catabolism of l‐arabinose and d‐galactose, as the main TF XYR1 is only partially involved. Here, the T. reesei ortholog of ARA1 from Pyricularia oryzae that regulates l‐arabinose releasing and catabolic genes was deleted and characterized by growth profiling and transcriptomics along with a xyr1 mutant and xyr1/ara1 double mutant. Our results show that in addition to the l‐arabinose‐related role, T. reesei ARA1 is essential for expression of d‐galactose releasing and catabolic genes, while XYR1 is not involved in this process. [ABSTRACT FROM AUTHOR]

Ubiquitination plays a pivotal role in most cellular processes and is critical for protein degradation and signalling. E3 ligases are the matchmakers in the ubiquitination cascade, responsible for substrate recognition and modification with specific polyubiquitin chains. Until recently, it was not clear how the catalytic activity of E3s is modulated, but major recent studies on HECT E3 ligases is filling this void. These enzymes appear to be held in a closed, inactive conformation, which is relieved by biochemical manoeuvres unique to each member, thus ensuring exquisite regulation and specificity of the enzymes. The new advances and their significance to the function of HECT E3s are described here, with a particular focus on the Nedd4 family members. [ABSTRACT FROM AUTHOR]

Numerous biological proteins exhibit intrinsic disorder at their termini, which are associated with multifarious functional roles. Here, we show the surprising result that an increased percentage of terminal short transiently disordered regions with enhanced flexibility (Tst DREF) is associated with accelerated folding rates of globular proteins. Evolutionary conservation of predicted disorder at Tst DREFs and drastic alteration of folding rates upon point-mutations suggest critical regulatory role(s) of Tst DREFs in shaping the folding kinetics. Tst DREFs are associated with long-range intramolecular interactions and the percentage of native secondary structural elements physically contacted by Tst DREFs exhibit another surprising positive correlation with folding kinetics. These results allow us to infer probable molecular mechanisms behind the Tst DREF-mediated regulation of folding kinetics that challenge protein biochemists to assess by direct experimental testing. [ABSTRACT FROM AUTHOR]

In the human brain, pLG72 interacts with the flavoenzyme d-amino acid oxidase (hDAAO), which is involved in catabolism of d-serine, a co-agonist of N-methyl- d-aspartate receptors (NMDAR). Here, we investigated the wild-type pLG72, the R30K variant associated with schizophrenia susceptibility, and the K62E variant. The protein conformation, oligomeric state, ligand-, and hDAAO-binding properties are only slightly modified by the substitutions. All pLG72 variants inhibit hDAAO and lead to an increase in cellular ( d/d+ l)-serine. However, the R30K pLG72 is significantly more prone to degradation than the R30 and the K62E variants in a cell system, thus possessing a lower ability to interact/inhibit hDAAO. This links R30K pLG72 with the hyperactivity of hDAAO, the decreased d-serine level, and NMDAR hypofunction observed in schizophrenia-affected patients. [ABSTRACT FROM AUTHOR]

Clostridium thermocellum efficiently degrades crystalline cellulose by a high molecular weight protein complex, the cellulosome. The bacterium regulates its cellulosomal genes using a unique extracellular biomass-sensing mechanism that involves alternative sigma factors and extracellular carbohydrate-binding modules attached to intracellular anti-sigma domains. In this study, we identified three cellulosomal xylanase genes that are regulated by the σ I6 /RsgI6 system by utilizing sigI6 and rsgI6 knockout mutants together with primer extension analysis. Our results indicate that cellulosomal genes are expressed from both alternative σ I6 and σ A vegetative promoters. [ABSTRACT FROM AUTHOR]

Hox genes encode transcription factors necessary for patterning the major developing anterior to posterior embryonic axis. In addition, during vertebrate evolution, various subsets of this gene family were co-opted along with the emergence of novel body structures, such as the limbs or the external genitalia. The morphogenesis of these axial structures thus relies in part upon the precisely controlled transcription of specific Hox genes, a mechanism involving multiple long-range enhancers. Recently, it was reported that such regulatory mechanisms were largely shared between different developing tissues, though with some specificities, suggesting the recruitment of ancestral regulatory modalities from one tissue to another. The analysis of chromatin architectures at HoxD and HoxA loci revealed the existence of two flanking topologically associating domains (TADs), precisely encompassing the adjacent regulatory landscapes. Here, we discuss the function of these TADs in the control of Hox gene regulation and we speculate about their capacity to serve as structural frameworks for the emergence of novel enhancers. In this view, TADs may have been used as genomic niches to evolve pleiotropic regulations found at many developmental loci. [ABSTRACT FROM AUTHOR]

Intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs) are important constituents of many protein complexes, playing various structural, functional, and regulatory roles. In such disorder-based protein complexes, functional disorder is used both internally (for assembly, movement, and functional regulation of the different parts of a given complex) and externally (for interactions of a complex with its external regulators). In complex assembly, IDPs/IDPRs serve as the molecular glue that cements complexes or as highly flexible scaffolds. Disorder defines the order of complex assembly and the ability of a protein to be involved in polyvalent interactions. It is at the heart of various binding mechanisms and interaction modes ascribed to IDPs. Disorder in protein complexes is related to multifarious applications of induced folding and induced functional unfolding, or defines the entropic chain activities, such as stochastic machines and binding rheostats. This review opens a FEBS Letters Special Issue on Dynamics, Flexibility, and Intrinsic Disorder in protein assemblies and represents a brief overview of intricate roles played by IDPs and IDPRs in various aspects of protein complexes. [ABSTRACT FROM AUTHOR]

The nucleation-growth model has been used extensively for characterizing in vitro amyloid fibril formation kinetics and for simulating the relationship between amyloid and disease. In the majority of studies amyloid has been considered as the dominant, or sole, aggregation end product, with the presence of other competing non-amyloid aggregation processes, for example amorphous aggregate formation, being largely ignored. Here, we examine possible regulatory effects that off-pathway processes might exert on the rate and extent of amyloid formation – in particular their potential for providing false positives and negatives in the evaluation of anti-amyloidogenic agents. Furthermore, we investigate how such competing reactions might influence the standard interpretation of amyloid aggregation as a two-state system. We conclude by discussing our findings in terms of the general concepts of supersaturation and system metastability – providing some mechanistic insight as to how these empirical phenomena may manifest themselves in the amyloid arena. [ABSTRACT FROM AUTHOR]

Abstract: Galactose catabolism in Aspergillus nidulans is regulated by at least two regulators, GalR and GalX. In Aspergillus niger only GalX is present, and its role in d-galactose catabolism in this fungus was investigated. Phenotypic and gene expression analysis of a wild type and a galX disruptant revealed that GalX regulates the d-galactose oxido-reductive pathway, but not the Leloir pathway in A. niger. [Copyright &y& Elsevier]

Yeast species such as Saccharomyces cerevisiae have been exploited by humans for millennia and so it is therefore unsurprising that they are attractive cells to re-engineer for industrial use. Despite many beneficial traits yeast has for synthetic biology, it currently lags behind Escherichia coli in the number of synthetic networks that have been described. While the eukaryotic nature of yeast means that its regulation is not as simple to predict as it is for E. coli , once initial considerations have been made yeast is pleasingly tractable. In this review we provide a loose guide for constructing and implementing synthetic regulatory networks in S. cerevisiae using examples from previous research to highlight available resources, specific considerations and potential future advances. [ABSTRACT FROM AUTHOR]

Abstract: Mdm2 regulates the stability, translation, subcellular localization and transcriptional activity of p53 protein. Mdm2-dependent p53 inhibition is essential in regulating p53 activity during embryonic development and in adult tissues. MdmX, an Mdm2 homolog, is also essential for p53 inhibition in vivo. Recent advances in the field from biochemical and genetic studies have revealed an essential role for the MdmX RING domain in Mdm2-dependent p53 polyubiquitination and degradation. Mdm2 on its own is a monoubiquitin E3 ligase for p53, but is converted to a p53 polyubiquitin E3 ligase by MdmX through their RING–RING domain interactions. MdmX acts as an activator as well as a substrate of Mdm2/MdmX E3 complex. The insufficiency of Mdm2 for p53 polyubiquitination also demands other p53 E3 ligases or E4 factors be incorporated into the p53 degradation arena. Deubiquitinases nullify the effects of E3 actions and reverse the ubiquitination process, which permits a diverse and dynamic pattern of p53 stability control. Unsurprisingly, stress signals target MdmX to disengage the p53/Mdm2 feedback loop for timely and appropriate p53 responses to these stresses. [Copyright &y& Elsevier]

Abstract: DNA polymerase (pol) λ, one of the 15 cellular pols, belongs to the X family. It is a small 575 amino-acid protein containing a polymerase, a dRP-lyase, a proline/serine rich and a BRCT domain. Pol λ shows various enzymatic activities including DNA polymerization, terminal transferase and dRP-lyase. It has been implicated to play a role in several DNA repair pathways, particularly base excision repair (BER), non-homologous end-joining (NHEJ) and translesion DNA synthesis (TLS). Similarly to other DNA repair enzymes, pol λ undergoes posttranslational modifications during the cell cycle that regulate its stability and possibly its subcellular localization. Here we describe our knowledge about ubiquitylation of pol λ and the impact of this modification on its regulation. [Copyright &y& Elsevier]

Abstract: The T box mechanism is widely used in Gram-positive bacteria to regulate expression of aminoacyl-tRNA synthetase genes and genes involved in amino acid biosynthesis and uptake. Binding of a specific uncharged tRNA to a riboswitch element in the nascent transcript causes a structural change in the transcript that promotes expression of the downstream coding sequence. In most cases, this occurs by stabilization of an antiterminator element that competes with formation of a terminator helix. Specific tRNA recognition by the nascent transcript results in increased expression of genes important for tRNA aminoacylation in response to decreased pools of charged tRNA. [Copyright &y& Elsevier]

Abstract: Tryptophanyl-tRNA synthetases (TrpRSs) catalyze the aminoacylation of tRNATrp. Previously, I demonstrated that Zn2+-depleted human TrpRS is enzymatically inactive and that binding of Zn2+ or heme to human TrpRS stimulates its aminoacylation activity. In the present study, bovine and mouse TrpRSs were found to be constitutively active regardless of the presence of Zn2+ or ferriprotoporphyrin IX chloride. Mutagenesis experiments demonstrated that the human H130R mutant is constitutively active and that the bovine R135H, E438A double mutant binds with Zn2+ or heme to enhance its aminoacylation activity as does human wild-type TrpRS. These results provide the first evidence of species-specific regulation of TrpRS activity. [Copyright &y& Elsevier]

Abstract: Systems Biology has a mission that puts it at odds with traditional paradigms of physics and molecular biology, such as the simplicity requested by Occam’s razor and minimum energy/maximal efficiency. By referring to biochemical experiments on control and regulation, and on flux balancing in yeast, we show that these paradigms are inapt. Systems Biology does not quite converge with biology either: Although it certainly requires accurate ‘stamp collecting’, it discovers quantitative laws. Systems Biology is a science of its own, discovering own fundamental principles, some of which we identify here. [Copyright &y& Elsevier]

Abstract: The β-subunit of voltage-gated Ca2+ channels is essential for trafficking the channels to the plasma membrane and regulating their gating. It contains a Src homology 3 (SH3) domain and a guanylate kinase (GK) domain, which interact intramolecularly. We investigated the structural underpinnings of this intramolecular coupling and found that in addition to a previously described SH3 domain β strand, two structural elements are crucial for maintaining a strong and yet potentially modifiable SH3–GK intramolecular coupling: an intrinsically weak SH3–GK interface and a direct connection of the SH3 and GK domains. Alterations of these elements uncouple the two functions of the β-subunit, degrading its ability to regulate gating while leaving its chaperone effect intact. [Copyright &y& Elsevier]

Abstract: Issues remain to be elucidated in the developmental regulation of erythropoiesis. In particular the role of Fas, a member of the tumor necrosis factor family of receptors despite much work remains unclear. During erythropoiesis, Fas is expressed at low levels on erythroblasts. For most cell types, Fas to FasL interaction causes apoptotic cell death via caspase activation. Here, we show that in humans, early erythroid progenitors are refractory to apoptosis triggered through Fas. Further during early human erythropoiesis, Fas triggered caspase activation provides a positive stimulus for erythroid maturation, and does not alter cellular proliferation or trigger apoptosis. [Copyright &y& Elsevier]

Abstract: Although the reversible dissociation of the V1VO holoenzyme into its V1 and VO complexes is a general mechanism for the regulation of V-ATPases, important aspects are still not understood. By analyzing the endogenous nucleotide content of the V1VO holoenzyme and of the V1 complex, both purified from Manduca sexta larval midgut, we found that the V1 complex contained 1.7molec. of ADP, whereas only 0.3molec. of ADP were bound to the V1VO holoenzyme. By contrast, both proteins contained only negligible amounts of ATP. Incubation of the V1VO holoenzyme with various adenine nucleotides revealed that ATP hydrolysis, leading to a state containing tightly bound ADP is necessary for its dissociation. [Copyright &y& Elsevier]

Abstract: Iron uptake in plants is highly regulated in order to supply amounts sufficient for optimal growth while preventing excess accumulation. In response to iron deficiency, plants induce either reduction-based or chelation-based mechanisms to enhance iron uptake from the soil. Genes involved in each mechanism have been identified from various model plants including Arabidopsis and rice. Iron transport within plants is also tightly controlled. New information has emerged on transporters that play a role in xylem loading and phloem loading/unloading of iron, and on the iron chelators involved in iron homeostasis. Some of the components regulating iron deficiency responses also have been elucidated, demonstrating that iron dependent gene regulation occurs at both the transcriptional and post-transcriptional levels. [Copyright &y& Elsevier]

Abstract: The positive strand RNA coronavirus, infectious bronchitis virus (IBV), induces a G2/M phase arrest and reduction in the G1 and G1/S phase transition regulator cyclin D1. Quantitative real-time RT-PCR and Western blot analysis demonstrated that cyclin D1 was reduced post-transcriptionally within infected cells independently of the cell-cycle stage at the time of infection. Confocal microscopy revealed that cyclin D1 decreased in IBV-infected cells as infection progressed and inhibition studies indicated that a population of cyclin D1 could be targeted for degradation by a virus mediated pathway. In contrast to the SARS-coronavirus, IBV nucleocapsid protein did not interact with cyclin D1. [Copyright &y& Elsevier]

Abstract: Regulation of Na+-dependent glutamate transport was studied in isolated luminal and abluminal plasma membranes derived from the bovine blood–brain barrier. Abluminal membranes have Na+-dependent glutamate transporters while luminal membranes have facilitative transporters. This organization allows glutamate to be actively removed from brain. γ-Glutamyl transpeptidase, the first enzyme of the γ-glutamyl cycle (GGC), is on the luminal membrane. Pyroglutamate (oxoproline), an intracellular product of GGC, stimulated Na+-dependent transport of glutamate by 46%, whereas facilitative glutamate uptake in luminal membranes was inhibited. This relationship between GGC and glutamate transporters may be part of a regulatory mechanism that accelerates glutamate removal from brain. [Copyright &y& Elsevier]

Abstract: We discuss the potential for inert biopolymers existing in cells to play a role in regulating the macromolecular crowding effect via their ability to undergo shape changing structural transitions. We have explored this possibility by the use of theory and experiment. The theoretical component utilized Monte-Carlo based simulations to examine the folding of a hypothetical protein in a concentrated environment of hard spheres which are themselves capable of reversible expansion and contraction. The experimental component of the study involved examination of the effect of different sized crowding agents on the thermally induced denaturation of cytochrome c [in phosphate buffered saline solution containing 1.0M guanidinium hydrochloride at pH 7.0]. On the basis of our findings we suggest that in a crowded solution environment the presence of a non-reactive polymer capable of reversible expansion/contraction via folding and unfolding may alter the excluded volume component of the solution. This ability would confer on the non-reactive polymer a novel role in influencing other processes in solution affected by macromolecular crowding. [Copyright &y& Elsevier]

Abstract: Ribosomal proteins play important roles in stabilizing the rRNA structure to facilitate protein synthesis in ribosome. In the present study, we analyzed the potential extraribosomal function of the ribosomal protein L32-2 (RPL32-2), which was expressed by a gene clone isolated from a cDNA library of Schizosaccharomyces pombe (S. pombe). RPL32-2 fused with the GAL4 DNA-bind domain or the GAL4 transcriptional activating domain could, respectively, activate transcriptions of reporter genes in yeast strain AH109. The RPL32-2 mutants with truncation of either the N- or the C-terminal domain resulted in abolishment of this regulatory effect. The DNA binding site for RPL32-2 of S. pombe was identified by using a random oligonucleotide selection strategy and gel motility shift assay and Western blotting confirmed its binding specificity. Moreover, we found RPL32-2 was also able to interact with a to-be-identified AT sequence binding protein. These data suggest that RPL32-2 of S. pombe, besides its ribosomal function, may also act as a potential transcriptional regulator in nucleus. [Copyright &y& Elsevier]

Abstract: Previously, we demonstrated ATP binding to the isolated ε subunit of F1-ATPase from thermophilic Bacillus PS3 [Kato-Yamada Y., Yoshida M. (2003) J. Biol. Chem. 278, 36013]. However, whether it is a general feature of the ε subunit from other sources is yet unclear. Here, using a sensitive method to detect weak interactions between fluorescently labeled ε subunit and nucleotide, it was shown that the ε subunit of F1-ATPase from Bacillus subtilis also bound ATP. The dissociation constant for ATP binding at room temperature was calculated to be 2mM, which may be suitable for sensing cellular ATP concentration in vivo. [Copyright &y& Elsevier]

Abstract: The DnaK system from Thermus thermophilus (DnaKTth) exhibits pronounced differences in organisation and regulation to its mesophile counterpart from Escherichia coli (DnaKEco). While the ATPase cycle of DnaKEco is tightly regulated by the concerted action of the two cofactors DnaJEco and GrpEEco, the DnaKTth system features an imbalance in this cochaperone mediated regulation. GrpETth considerably accelerates the ATP/ADP exchange, but DnaJTth only slightly stimulates ATPase activity, believed to be a key step for chaperone activity of DnaKEco. By in vitro complementation assays, we could not detect significant ATPase-stimulation of orthologous DnaJTth ·DnaKEco or DnaJEco ·DnaKTth-complexes as compared to the DnaKEco system, although they were nevertheless active in luciferase refolding experiments. Assistance of protein recovery by DnaK thus seems to be uncoupled of the magnitude of DnaJ mediated ATPase-stimulation. [Copyright &y& Elsevier]

Abstract: Revealing mechanisms underlying complex diseases poses great challenges to biologists. The traditional linkage and linkage disequilibrium analysis that have been successful in the identification of genes responsible for Mendelian traits, however, have not led to similar success in discovering genes influencing the development of complex diseases. Emerging functional genomic and proteomic (‘omic’) resources and technologies provide great opportunities to develop new methods for systematic identification of genes underlying complex diseases. In this report, we propose a systems biology approach, which integrates omic data, to find genes responsible for complex diseases. This approach consists of five steps: (1) generate a set of candidate genes using gene–gene interaction data sets; (2) reconstruct a genetic network with the set of candidate genes from gene expression data; (3) identify differentially regulated genes between normal and abnormal samples in the network; (4) validate regulatory relationship between the genes in the network by perturbing the network using RNAi and monitoring the response using RT-PCR; and (5) genotype the differentially regulated genes and test their association with the diseases by direct association studies. To prove the concept in principle, the proposed approach is applied to genetic studies of the autoimmune disease scleroderma or systemic sclerosis. [Copyright &y& Elsevier]

Abstract: F0F1-ATP synthase couples ATP synthesis/hydrolysis with transmembrane proton transport. The catalytic mechanism involves rotation of the γεc∼10-subunits complex relative to the rest of the enzyme. In the absence of protonmotive force the enzyme is inactivated by the tight binding of MgADP. Subunit ε also modulates the activity: its conformation can change from a contracted to extended form with C-terminus stretched towards F1. The latter form ihnibits ATP hydrolysis (but not synthesis). We propose that the directionality of the coiled-coil subunit γ rotation determines whether subunit ε is in contracted or extended form. Block of rotation by MgADP presumably induces the extended conformation of subunit ε. This conformation might serve as a safety lock, stabilizing the ADP-inhibited state upon de-energization and preventing spontaneous re-activation and wasteful ATP hydrolysis. The hypothesis merges the known regulatory effects of ADP, protonmotive force and conformational changes of subunit ε into a consistent picture. [Copyright &y& Elsevier]

Abstract: CysA, the ATPase subunit of a putative sulfate ATP-binding cassette transport system of the gram-positive thermoacidophilic bacterium Alicyclobacillus acidocaldarius, was structurally characterized at a resolution of 2.0Å in the absence of nucleotides. In line with previous findings on ABC-ATPases the structures of the two monomers (called CysA-1 and CysA-2) in the asymmetric unit differ substantially in the arrangement of their individual (sub)domains. CysA-2 was found as a physiological dimer composed of two crystallographically related monomers that are arranged in an open state. Interestingly, while the regulatory domain of CysA-2 packs against its opposing domain that of CysA-1 undergoes a conformational change and, in the dimer, would interfere with the opposing monomer thereby preventing solute translocation. Whether this conformational state is used for regulatory purposes will be discussed. [Copyright &y& Elsevier]

Abstract: Expression of several virulence factors in the plant pathogen bacterium Ralstonia solanacearum is controlled by a complex regulatory network, at the center of which is PhcA. We provide genetic evidence that PhcA also represses the expression of hrp genes that code for the Type III protein secretion system, a major pathogenicity determinant in this bacterium. The repression of hrp genes in complete medium is relieved in a phcA mutant and two distinct signals, a quorum-sensing signal and complex nitrogen sources, appear to trigger this PhcA-dependent repression. This control of hrp gene expression by PhcA is realized at the level of the HrpG regulatory protein. [Copyright &y& Elsevier]

Heme is an iron-containing cofactor that aside from serving as the active group of essential proteins is a key element in the control of many molecular and cellular processes. In prokaryotes, the family of Fur (ferric uptake regulator) proteins governs processes essential for the survival of microorganims such as the iron homeostasis. We show that purified recombinant FurA from Anabaena sp. PCC 7120 interacts strongly with heme in the micromolar range and this interaction affects the in vitro ability of FurA to bind DNA, inhibiting that process in a concentration-dependent fashion. Our results provide the first evidence of the possible involvement of heme in the regulatory function of cyanobacterial Fur. [Copyright &y& Elsevier]

The ompX gene of Enterobacter aerogenes was cloned. Its overexpression induced a decrease in the major porin Omp36 production and consequently a β-lactam resistance was noted. Purified outer membrane protein X (OmpX) was reconstituted into artificial membranes and formed ion channels with a conductance of 20 pS in 1 M NaCl and a cationic selectivity. Both MarA expression and high osmolarity induced a noticeable increase of the OmpX synthesis in the E. aerogenes ATCC 13048 strain. In addition, OmpX synthesis increased under conditions in which the expression of the E. aerogenes major non-specific porins, Omp36 and Omp35, decreased. [Copyright &y& Elsevier]

Two main entry points for electrons into the mitochondrial respiratory chain are NADH:ubiquinone oxidoreductase (complex I) and succinate:ubiquinone oxidoreductase (complex II). Metabolic regulation of these two respiratory complexes is not understood in detail. It has been suggested that the Krebs cycle metabolic intermediate oxaloacetate (OAA) inhibits complex II in vivo, whereas complex I undergoes a reversible active/de-active transition. In normoxic and anoxic hearts it has been shown that the proportion of complex I in the active and de-active states is different suggesting a possible mode of regulation of the enzyme by oxygen concentration. In the current studies rapid isolation of mitochondrial membranes in a state that preserves the activity of both complex I and complex II has been achieved using Langendorff perfused rat hearts. The findings indicate that the state of activation of complex I is controlled by the oxygen saturation in the perfusate. In addition, these studies show that complex II is fully active in the mitochondrion and not inhibited by OAA regardless of the oxygen concentration. [Copyright &y& Elsevier]

The potential A1 ATPase genes ahaA, ahaB, ahaC, ahaD, ahaE, ahaF, and ahaG from the anaerobic archaeon Methanosarcina mazei Go¨1 were overexpressed in Escherichia coli DK8 (pTL2). An A1 complex was purified to apparent homogeneity and shown by Western blot and N-terminal sequence analyses to contain subunits A, B, C, D, and F but to be devoid of subunits E and G. Further removal of subunit C was without effect on ATPase activity. The enzyme was most active at pH 5.2 and required bisulfite and acetate for maximal activity. Kinetic studies confirmed three new inhibitors for A1 ATPases (diethylstilbestrol and its derivatives hexestrol and dienestrol) and identified redox modulation as a new type of regulation of archaeal A1 ATPases. [Copyright &y& Elsevier]

We have cloned two genes (rgpH and rgpI) that encode proteins for the formation of the glucose side-chains of the Streptococcus mutans rhamnose-glucose polysaccharide (RGP), which consists of a rhamnan backbone with glucose side-chains. The roles of rgpH and rgpI were evaluated in a rhamnan-synthesizing Escherichia coli. An E. coli strain that harbored rgpH reacted with antiserum directed against complete RGP, whereas the E. coli strain that carried rgpI did not react with this antiserum. Although E. coli:rgpH reacted strongly with rhamnan-specific antiserum, co-transformation of this strain with rgpI increased the number of glucose side-chains and decreased immunoreactivity with the rhamnan-specific antiserum significantly. These results suggest that two genes are involved in side-chain formation during S. mutans RGP synthesis in E. coli: one gene encodes a glucosyltransferase, and the other gene probably controls the frequency of branching. This is the first report to identify a gene that is involved in regulation of branching frequency in polysaccharide synthesis. [Copyright &y& Elsevier]

The helix–loop–helix transcription factor NeuroD1 (also known as Beta2) is involved in β-cell survival during development and insulin gene transcription in adults. Here we show NeuroD1 is primarily cytoplasmic at non-stimulating glucose concentrations (i.e. 3 mM) in MIN6 β-cells and nuclear under stimulating conditions (i.e. 20 mM). Quantification revealed that NeuroD1 was in 40–45% of the nuclei at 3 mM and 80–90% at 20 mM. Treatment with the MEK inhibitor PD98059 or substitution of a serine for an alanine at a potential mitogen-activated protein kinase phosphorylation site (S274) in NeuroD1 significantly increased the cytoplasmic level at 20 mM glucose. The rise in NeuroD1-mediated transcription in response to glucose also correlated with the change in sub-cellular localization, a response attenuated by PD98059. The data strongly suggest that glucose-stimulation of the MEK–ERK signalling pathway influences NeuroD1 activity at least partially through effects on sub-cellular localization. [Copyright &y& Elsevier]