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The soil-dwelling filamentous bacteria, Streptomyces, is widely known for its ability to produce numerous bioactive natural products. Despite many efforts toward their overproduction and reconstitution, our limited understanding of the relationship between the host's chromosome three dimension (3D) structure and the yield of the natural products escaped notice. Here, we report the 3D chromosome organization and its dynamics of the model strain, Streptomyces coelicolor, during the different growth phases. The chromosome undergoes a dramatic global structural change from primary to secondary metabolism, while some biosynthetic gene clusters (BGCs) form special local structures when highly expressed. Strikingly, transcription levels of endogenous genes are found to be highly correlated to the local chromosomal interaction frequency as defined by the value of the frequently interacting regions (FIREs). Following the criterion, an exogenous single reporter gene and even complex BGC can achieve a higher expression after being integrated into the chosen loci, which may represent a unique strategy to activate or enhance the production of natural products based on the local chromosomal 3D organization. [ABSTRACT FROM AUTHOR]

Proteins achieve their complex functions, such as molecular recognition with high affinity and specificity, through intricate threedimensional geometries in functional sites. To engineer new protein functions, accurate positioning of amino acid functional groups is therefore critical but has remained difficult to achieve by computational methods because of current limitations in the design of new conformations with arbitrary user-defined geometries. Here, we introduce two computational methods capable of generating and predicting new local protein geometries: fragment kinematic closure (FKIC) and loophash kinematic closure (LHKIC). FKIC and LHKIC integrate two approaches: robotics-inspired kinematics of protein conformations and insertion of peptide fragments. We show that FKIC and LHKIC predict native-like conformations at atomic accuracy and with up to 140-fold improvements in sampling efficiency over previous approaches. We then use these methods to create a design protocol, pull into place (PIP), to position functionally important side chains via design of backbone conformations. We validate PIP by remodeling a sizeable active site region in an enzyme and confirming the engineered new conformations of two designs with crystal structures. The described methods can be applied broadly to the design of userdefined geometries for new protein functions. [ABSTRACT FROM AUTHOR]

Skeletal muscle atrophy is caused by various conditions, including aging, disuse related to a sedentary lifestyle and lack of physical activity, and cachexia. Our insufficient understanding of the molecular mechanism underlying muscle atrophy limits the targets for the development of effective pharmacologic treatments and preventions. Here, we identified Krüppel-like factor 5 (KLF5), a zinc-finger transcription factor, as a key mediator of the early muscle atrophy program. KLF5 was up-regulated in atrophying myotubes as an early response to dexamethasone or simulated microgravity in vitro. Skeletal muscle-selective deletion of Klf5 significantly attenuated muscle atrophy induced by mechanical unloading in mice. Transcriptomeand genome-wide chromatin accessibility analyses revealed that KLF5 regulates atrophy-related programs, including metabolic changes and E3-ubiquitin ligase-mediated proteolysis, in coordination with Foxo1. The synthetic retinoic acid receptor agonist Am80, a KLF5 inhibitor, suppressed both dexamethasone- and microgravityinduced muscle atrophy in vitro and oral Am80 ameliorated disuse- and dexamethasone-induced atrophy in mice. Moreover, in three independent sets of transcriptomic data from human skeletal muscle, KLF5 expression significantly increased with age and the presence of sarcopenia and correlated positively with the expression of the atrophy-related ubiquitin ligase genes FBXO32 and TRIM63. These findings demonstrate that KLF5 is a key transcriptional regulator mediating muscle atrophy and that pharmacological intervention with Am80 is a potentially preventive treatment. [ABSTRACT FROM AUTHOR]

Two cytochrome P450 enzymes, CYP97A3 and CYP97C1, catalyze hydroxylations of the β- and ε-rings of α-carotene to produce lutein. Chirality is introduced at the C-3 atom of both rings, and the reactions are both pro-3R-stereospecific. We determined the crystal structures of CYP97A3 in substrate-free and complex forms with a nonnatural substrate and the structure of CYP97C1 in a detergent-bound form. The structures of CYP97A3 in different states show the substrate channel and the structure of CYP97C1 bound with octylthioglucoside confirms the binding site for the carotenoid substrate. Biochemical assays confirm that the ferredoxin-NADP+ reductase (FNR)-ferredoxin pair is used as the redox partner. Details of the pro-3R stereospecificity are revealed in the retinal-bound CYP97A3 structure. Further analysis indicates that the CYP97B clan bears similarity to the β-ring-specific CYP97A clan. Overall, our research describes the molecular basis for the last steps of lutein biosynthesis. [ABSTRACT FROM AUTHOR]

Lysosomal storage diseases (LSDs) are typically caused by a deficiency in a soluble acid hydrolase and are characterized by the accumulation of undegraded substrates in the lysosome. Determining the role of specific cell types in the pathogenesis of LSDs is a major challenge due to the secretion and subsequent uptake of lysosomal hydrolases by adjacent cells, often referred to as "cross-correction." Here we create and validate a conditional mouse model for cell-autonomous expression of galactocerebrosidase (GALC), the lysosomal enzyme deficient in Krabbe disease. We show that lysosomal membrane-tethered GALC (GALCLAMP1) retains enzyme activity, is able to cleave galactosylsphingosine, and is unable to cross-correct. Ubiquitous expression of GALCLAMP1 fully rescues the phenotype of the GALC-deficient mouse (Twitcher), and widespread deletion of GALCLAMP1 recapitulates the Twitcher phenotype. We demonstrate the utility of this model by deleting GALCLAMP1 specifically in myelinating Schwann cells in order to characterize the peripheral neuropathy seen in Krabbe disease. [ABSTRACT FROM AUTHOR]

The reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) is the penultimate step of chlorophyll biosynthesis. In oxygenic photosynthetic bacteria, algae, and plants, this reaction can be catalyzed by the light-dependent Pchlide oxidoreductase (LPOR), a member of the short-chain dehydrogenase superfamily sharing a conserved Rossmann fold for NAD(P)H binding and the catalytic activity. Whereas modeling and simulation approaches have been used to study the catalytic mechanism of this light-driven reaction, key details of the LPOR structure remain unclear. We determined the crystal structures of LPOR from two cyanobacteria, Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus. Structural analysis defines the LPOR core fold, outlines the LPOR-NADPH interaction network, identifies the residues forming the substrate cavity and the proton-relay path, and reveals the role of the LPOR-specific loop. These findings provide a basis for understanding the structurefunction relationships of the light-driven Pchlide reduction. [ABSTRACT FROM AUTHOR]

High-income countries often outsource material demands to poorer countries along with the associated environmental damage. This phenomenon can also occur within (large) countries, such as China, which was responsible for 24 to 30% of the global material footprint (MF) between 2007 and 2010. Understanding the distribution and development of China's MF is hence critical for resource efficiency and circular economy ambitions globally. Here we present a comprehensive analysis of China's MF at the provincial and sectoral levels. We combine provincial-level input-output data with sector- and province-specific trade data, detailed material extraction data, and the global input-output database EXIOBASE. We find that some provinces haveMFs equivalent to medium-sized, highincome countries and limited evidence of material decoupling. Lower-income regions with high levels of material extraction can have an MF per capita as large as developed provinces due to much higher material intensities. The higher-income south-coastal provinces have lower MF per capita than equally developed provinces. This finding relates partly to differences in economic structure but indicates the potential for improvement across provinces. Investment via capital formation is up to 4 times more resource-intensive than consumption and drives 49 to 86% of provincial-level MFs (the Organisation for Economic Co-operation and Development average is 37%). Resource-efficient production, efficient use of capital goods/infrastructure, and circular design are essential for reductions in China's MF. Policy efforts to shift to a high-quality development model may reduce material intensities, preferably while avoiding the further outsourcing of high-intensity activities to other provinces or lower-income countries. [ABSTRACT FROM AUTHOR]

The cytoplasmic coat protein complex-II (COPII) is evolutionarily conserved machinery that is essential for efficient trafficking of protein and lipid cargos. How the COPII machinery is regulated to meet the metabolic demand in response to alterations of the nutritional state remains largely unexplored, however. Here, we show that dynamic changes of COPII vesicle trafficking parallel the activation of transcription factor X-box binding protein 1 (XBP1s), a critical transcription factor in handling cellular endoplasmic reticulum (ER) stress in both live cells and mouse livers upon physiological fluctuations of nutrient availability. Using live-cell imaging approaches, we demonstrate that XBP1s is sufficient to promote COPII-dependent trafficking, mediating the nutrient stimulatory effects. Chromatin immunoprecipitation (ChIP) coupled with high-throughput DNA sequencing (ChIP-seq) and RNA-sequencing analyses reveal that nutritional signals induce dynamic XBP1s occupancy of promoters of COPII traffic-related genes, thereby driving the COPII-mediated trafficking process. Liver-specific disruption of the inositol-requiring enzyme 1α (IRE1α)-XBP1s signaling branch results in diminished COPII vesicle trafficking. Reactivation of XBP1s in mice lacking hepatic IRE1α restores COPII-mediated lipoprotein secretion and reverses the fatty liver and hypolipidemia phenotypes. Thus, our results demonstrate a previously unappreciated mechanism in the metabolic control of liver protein and lipid trafficking: The IRE1α-XBP1s axis functions as a nutrient-sensing regulatory nexus that integrates nutritional states and the COPII vesicle trafficking. [ABSTRACT FROM AUTHOR]

RNA polymerase is the only known protein partner of the transcriptional regulator DksA. Herein, we demonstrate that the chaperone DnaJ establishes direct, redox-based interactions with oxidized DksA. Cysteine residues in the zinc finger of DksA become oxidized in Salmonella exposed to low concentrations of hydrogen peroxide (H2O2). The resulting disulfide bonds unfold the globular domain of DksA, signaling high-affinity interaction of the C-terminal α-helix to DnaJ. Oxidoreductase and chaperone activities of DnaJ reduce the disulfide bonds of its client and promote productive interactions between DksA and RNA polymerase. Simultaneously, guanosine tetraphosphate (ppGpp), which is synthesized by RelA in response to low concentrations of H2O2, binds at site 2 formed at the interface of DksA and RNA polymerase and synergizes with the DksA/DnaJ redox couple, thus activating the transcription of genes involved in amino acid biosynthesis and transport. However, the high concentrations of ppGpp produced by Salmonella experiencing oxidative stress oppose DksA/DnaJ-dependent transcription. Cumulatively, the interplay of DksA, DnaJ, and ppGpp on RNA polymerase protects Salmonella from the antimicrobial activity of the NADPH phagocyte oxidase. Our research has identified redox-based signaling that activates the transcriptional activity of the RNA polymerase regulator DksA. [ABSTRACT FROM AUTHOR]

The glycosyltransferases of the mammalian Golgi complex must recycle between the stacked cisternae of that organelle to maintain their proper steady-state localization. This trafficking is mediated by COPI-coated vesicles, but how the glycosyltransferases are incorporated into these transport vesicles is poorly understood. Here we show that the N-terminal cytoplasmic tails (N-tails) of a number of cis Golgi glycosyltransferases which share a ϕ-(K/R)-X-L-X-(K/R) sequence bind directly to the δ- and ζ-subunits of COPI. Mutations of this N-tail motif impair binding to the COPI subunits, leading to mislocalization of the transferases to lysosomes. The physiological importance of these interactions is illustrated by mucolipidosis III patients with missense mutations in the N-tail of GlcNAc-1-phosphotransferase that cause the transferase to be rapidly degraded in lysosomes. These studies establish that direct binding of the N-tails of mammalian cis Golgi glycosyltransferases with COPI subunits is essential for recycling within the Golgi. [ABSTRACT FROM AUTHOR]

The 3' end methylation catalyzed by HUA Enhancer 1 (HEN1) is a crucial step of small RNA stabilization in plants, yet how unmethylated small RNAs undergo degradation remains largely unknown. Using a reverse genetic approach, we here show that Atrimmer 2 (ATRM2), a DEDDy-type 3' to 5' exoribonuclease, acts in the degradation of unmethylated miRNAs and miRNA*s in Arabidopsis. Lossof- function mutations in ATRM2 partially suppress the morphological defects caused by HEN1 malfunction, with restored levels of a subset of miRNAs and receded expression of corresponding miRNA targets. Dysfunction of ATRM2 has negligible effect on miRNA trimming, and further increase the fertility of hen1 heso1 urt1, a mutant with an almost complete abolishment of miRNA uridylation, indicating that ATRM2 may neither be involved in 3' to 5' trimming nor be the enzyme that specifically degrades uridylated miRNAs. Notably, the fold changes of miRNAs and their corresponding miRNA*s were significantly correlated in hen1 atrm2 versus hen1. Unexpectedly, we observed a marked increase of 3' to 5' trimming of several miRNA*s but not miRNAs in ATRM2 compromised backgrounds. These data suggest an action of ATRM2 on miRNA/miRNA* duplexes, and the existence of an unknown exoribonuclease for specific trimming of miRNA*. This asymmetric effect on miRNA/miRNA* is likely related to Argonaute (AGO) proteins, which can distinguish miRNAs from miRNA*s. Finally, we show that ATRM2 colocalizes and physically interacts with Argonaute 1 (AGO1). Taken together, our results suggest that ATRM2 may be involved in the surveillance of unmethylated miRNA/miRNA* duplexes during the initiation step of RNA-induced silencing complex assembly. [ABSTRACT FROM AUTHOR]

Asn-linked oligosaccharides are extensively modified during transit through the secretory pathway, first by trimming of the nascent glycan chains and subsequently by initiating and extending multiple oligosaccharide branches from the trimannosyl glycan core. Trimming and branching pathway steps are highly ordered and hierarchal based on the precise substrate specificities of the individual biosynthetic enzymes. A key committed step in the synthesis of complex-type glycans is catalyzed by N-acetylglucosaminyltransferase II (MGAT2), an enzyme that generates the second GlcNAcβ1,2- branch from the trimannosyl glycan core using UDPGlcNAc as the sugar donor. We determined the structure of human MGAT2 as a Mn2+-UDP donor analog complex and as a GlcNAc- Man3GlcNAc2-Asn acceptor complex to reveal the structural basis for substrate recognition and catalysis. The enzyme exhibits a GTA Rossmann-like fold that employs conserved divalent cationdependent substrate interactions with the UDP-GlcNAc donor. MGAT2 interactions with the extended glycan acceptor are distinct from other related glycosyltransferases. These interactions are composed of a catalytic subsite that binds the Man-α1,6- monosaccharide acceptor and a distal exosite pocket that binds the GlcNAc-β1,2Man-α1,3Manβ- substrate "recognition arm." Recognition arm interactions are similar to the enzyme-substrate interactions for Golgi α-mannosidase II, a glycoside hydrolase that acts just before MGAT2 in the Asn-linked glycan biosynthetic pathway. These data suggest that substrate binding by MGAT2 employs both conserved and convergent catalytic subsite modules to provide substrate selectivity and catalysis. More broadly, the MGAT2 active-site architecture demonstrates how glycosyltransferases create complementary modular templates for regiospecific extension of glycan structures in mammalian cells. [ABSTRACT FROM AUTHOR]

Despite mammalian glycans typically having highly complex asymmetrical multiantennary architectures, chemical and chemoenzymatic synthesis has almost exclusively focused on the preparation of simpler symmetrical structures. This deficiency hampers investigations into the biology of glycan-binding proteins, which in turn complicates the biomedical use of this class of biomolecules. Herein, we describe an enzymatic strategy, using a limited number of human glycosyltransferases, to access a collection of 60 asymmetric, multiantennary human milk oligosaccharides (HMOs), which were used to develop a glycan microarray. Probing the array with several glycan-binding proteins uncovered that not only terminal glycoepitopes but also complex architectures of glycans can influence binding selectivity in unanticipated manners. N- and O-linked glycans express structural elements of HMOs and thus, the reported synthetic principles will find broad applicability. [ABSTRACT FROM AUTHOR]

X-ray diffraction has the potential to provide rich information about the structural dynamics of macromolecules. To realize this potential, both Bragg scattering, which is currently used to derive macromolecular structures, and diffuse scattering, which reports on correlations in charge density variations, must be measured. Until now, measurement of diffuse scattering from protein crystals has been scarce because of the extra effort of collecting diffuse data. Here, we present 3D measurements of diffuse intensity collected from crystals of the enzymes cyclophilin A and trypsin. The measurements were obtained from the same X-ray diffraction images as the Bragg data, using best practices for standard data collection. To model the underlying dynamics in a practical way that could be used during structure refinement, we tested translation-libration-screw (TLS), liquid- like motions (LLM), and coarse-grained normal-modes (NM) models of protein motions. The LLM model provides a global picture of motions and was refined against the diffuse data, whereas the TLS and NM models provide more detailed and distinct descriptions of atom displacements, and only used information from the Bragg data. Whereas different TLS groupings yielded similar Bragg intensities, they yielded different diffuse intensities, none of which agreed well with the data. In contrast, both the LLM and NM models agreed substantially with the diffuse data. These results demonstrate a realistic path to increase the number of diffuse datasets available to the wider biosciences community and indicate that dynamics-inspired NM structural models can simultaneously agree with both Bragg and diffuse scattering. [ABSTRACT FROM AUTHOR]

Inhibition of Mek/Erk signaling by pharmacological Mek inhibitors promotes self-renewal and pluripotency of mouse embryonic stem cells (ESCs). Intriguingly, Erk signaling is essential for human ESC self-renewal. Here we demonstrate that Erk signaling is critical for mouse ESC self-renewal and genomic stability. Erk-depleted ESCs cannot be maintained. Lack of Erk leads to rapid telomere shortening and genomic instability, in association with misregulated expression of pluripotency genes, reduced cell proliferation, G1 cell-cycle arrest, and increased apoptosis. Erk signaling is also required for the activation of differentiation genes but not for the repression of pluripotency genes during ESC differentiation. Furthermore, we find an Erk-independent function of Mek, which may explain the diverse effects of Mek inhibition and Erk knockout on ESC self-renewal. Together, in contrast to the prevailing view, Erk signaling is required for telomere maintenance, genomic stability, and self-renewal of mouse ESCs. [ABSTRACT FROM AUTHOR]

Tetrapyrrole biosynthesis in plants, algae, and most bacteria starts from the NADPH-dependent reduction of glutamyl-tRNA by glutamyl-tRNA reductase (GluTR). The GluTR-catalyzed reaction is the rate-limiting step, and GluTR is the target of multiple posttranslational regulations, such as heme feedback inhibition, for the tetrapyrrole biosynthetic pathway. A recently identified GluTR regulator, GluTR binding protein (GluBP), has been shown to spatially organize tetrapyrrole synthesis by distributing GluTR into different suborganellar locations. Here we report the complex structure of GluTR-GluBP from Arabidopsis thaliana. The dimeric GluBP binds symmetrically to the catalytic domains of the V-shaped GluTR dimer via its C-terminal domain. A substantial conformational change of the GluTR NADPH-binding domain is observed, confirming the postulated rotation of the NADPH-binding domain for hydride transfer from NADPH to the substrate. Arg146, "guarding the door" for metabolic channeling, adopts alternative conformations, which may represent steps involved in substrate recognition and product release. A coupled enzyme assay shows that GluBP stimulates GluTR catalytic efficiency with an approximate threefold increase of the 5-aminolevulinic acid formation rate. In addition, the GluTR activity can be inhibited by heme in a concentration-dependent way regardless of the presence of GluBP. A structural alignment indicates that GluBP belongs to a heme-binding family involved in heme metabolism. We propose a catalytic mechanism model for GluTR, through which photosynthetic organisms can achieve precise regulation of tetrapyrrole biosynthesis. [ABSTRACT FROM AUTHOR]

RNA editing is a posttranscriptional process that covalently alters the sequence of RNA molecules and plays important biological roles in both animals and land plants. In flowering plants, RNA editing converts specific cytidine residues to uridine in both plastid and mitochondrial transcripts. Previous studies identified pentatricopeptide repeat (PPR) motif-containing proteins as site-specific recognition factors for cytidine targets in RNA sequences. However, the regulatory mechanism underlying RNA editing was largely unknown. Here, we report that protoporphyrinogen IX oxidase 1 (PPO1), an enzyme that catalyzes protoporphyrinogen IX into protoporphyrin IX in the tetrapyrrole biosynthetic pathway, plays an unexpected role in editing multiple sites of plastid RNA transcripts, most of which encode subunits of the NADH dehydrogenase-like complex (NDH), in the reference plant Arabidopsis thaliana. We identified multiple organellar RNA editing factors (MORFs), including MORF2, MORF8, and MORF9, that interact with PPO1. We found that two conserved motifs within the 22-aa region at the N terminus of PPO1 are essential for its interaction with MORFs, its RNA editing function, and subsequently, its effect on NDH activity. However, transgenic plants lacking key domains for the tetrapyrrole biosynthetic activity of PPO1 exhibit normal RNA editing. Furthermore, MORF2 and MORF9 interact with three PPRs or related proteins required for editing of ndhB and ndhD sites. These results reveal that the tetrapyrrole biosynthetic enzyme PPO1 is required for plastid RNA editing, acting as a regulator that promotes the stability of MORF proteins through physical interaction. [ABSTRACT FROM AUTHOR]

Although it has long been established that the amino acid sequence encodes the fold of a protein, how individual proteins arrive at their final conformation is still difficult to predict, especially for oligomeric structures. Here, we present a comprehensive characterization of oligomeric species of cyanovirin-N that all are formed by a polypeptide chain with the identical amino acid sequence. Structures of the oligomers were determined by X-ray crystallography, and each one exhibits 3D domain swapping. One unique 3D domain-swapped structure is observed for the trimer, while for both dimer and tetramer, two different 3D domain-swapped structures were obtained. In addition to the previously identified hinge-loop region of the 3D domain-swapped dimer, which resides between strands β5 and β6 in the middle of the polypeptide sequence, another hinge-loop region is observed between strands β7 and β8 in the structures. Plasticity in these two regions allows for variability in dihedral angles and concomitant differences in chain conformation that results in the differently 3D domain-swapped multimers. Based on all of the different structures, we propose possible folding pathways for this protein. Altogether, our results illuminate the amazing ability of cyanovirin-N to proceed down different folding paths and provide general insights into oligomer formation via 3D domain swapping. [ABSTRACT FROM AUTHOR]

A cascade of 20-hydroxyecdysone-mediated gene expression and repression initiates larva-to-pupa metamorphosis. We recently showed that two transcription factors, BmPOUM2 and BmβFTZ-F1, bind to the os-regulatory elements in the promoter of the gene coding for cuticle protein, BmWCP4, and regulate its expression during Bombyx mori metamorphosis. Here we show that downregulation of BmPOUM2 expression by RNA interference during the wandering stage resulted in failure to complete metamorphosis. The thorax epidermis of RNA interference-treated larvae became transparent, wing disc growth and differentiation were arrested, and the larvae failed to spin cocoons. Quantitative real-time PCR analysis showed that expression of the genes coding for pupal-specific wing cuticle proteins BmWCPI, BmWCP2, BmWCP3, BmWCP4, BmWCP5, BmWCP6, BmWCP8, and BmWCP9 were down-regulated in BmPOUM2 dsRNA-treated animals, whereas overexpression of BmPOUM2 protein increased the expression of BmWCP4, BmWCP5, BmWCP6, BmWCP7, and BmWCP8. Pull-down assays, far-Western blot, and electrophoretic mobility shift assay showed that the BmPOUM2 protein interacted with another homeodomain transcription factor, BmAbd-A, to induce the expression of BmWCP4. Immunohistochemical localization of BmPOUM2, BmAbd-A, and BmWCP4 proteins revealed that BmAbd-A and BmPOUM2 proteins are colocalized in the wing disc cell nuclei, whereas BmWCP4 protein is localized in the cytoplasm. Together these data suggest that BmPOUM2 interacts with the homeodomain transcription factor BmAbd-A and regulates the expression of BmWCP4 and probably other BmWCPs to complete the larva-topupa transformation. Although homeodomain proteins are known to regulate embryonic development, this study showed that these proteins also regulate metamorphosis. [ABSTRACT FROM AUTHOR]

During coevolution with the host, HIV-1 developed the ability to hijack the cellular ubiquitin/proteasome degradation pathway to counteract the antiviral activity of APOBEC3G (A3G). a host cytidine deaminase that can block HIV-1 replication. Abrogation of A3G function involves the HIV-1 Vif protein, which binds A3G and serves as an adapter molecule to recruit A3G to a Cullin5-based E3 ubiquitin ligase complex. Structure-guided mutagenesis of A3G focused on the 14 most surface-exposed Lys residues allowed us to identify four Lys residues (Lys-297, 301. 303, and 334) that are required for Vif-mediated A3G ubiquitination and degradation. Substitution of Arg for these residues confers Vif resistance and restores A3G's antiviral activity in the presence of Vif. In our model, the critical four Lys residues cluster at the C terminus, opposite to the known N-terminal Vif-interaction region in the protein. Thus, spatial constraints imposed by the E3 ligase complex may be an important determinant in Vif-dependent A3G ubiquitination. [ABSTRACT FROM AUTHOR]

Two proteins, STIM1 in the endoplasmic reticulum and Orail in the plasma membrane, are required for the activation of Ca[sup2+] release-activated Ca[sup2+] (CRAC) channels at the cell surface. How these proteins interact to assemble functional CRAC channels has remained uncertain. Here, we determine how many Orail and STIM1 molecules are required to form a functional CRAC channel. We engineered several genetically expressed fluorescent Orail tandem multimers and a. fluorescent, constitutively active STIM1 mutant. The tandem multimers assembled into CRAC channels, as seen by rectifying inward currents and by cytoplasmic calcium elevations. CRAC channels were visualized as fluorescent puncta in total internal reflection microscopy. With single-molecule imaging techniques, it was possible to observe photo-bleaching of individual fluorophores and to count the steps of bleaching as a measure of the stoichiometry of each CRAC channel complex. We conclude that the subunit stoichiometry in an active CRAC channel is four Orail molecules and two STIM1 molecules. Fluorescence resonance energy transfer experiments also showed that four Orail subunits form the assembled channel. From the fluorescence intensity of single fluorophores, we could estimate that our transfected HEK293 cells had almost 400,000 CRAC channels and that, when intracellular Ca[sup2+] stores were depleted, the channels clustered in aggregates containing ≈1,3O0 channels, amplifying the local Ca[sup2+] entry. [ABSTRACT FROM AUTHOR]

The tumor suppressor PTEN is mutated or deleted in many tumors, causing the activation of the PI3K pathway. Here, we show that the loss of PTEN increases the transcriptional activity of hypoxia-inducible factor 1 (HIF-1) through the inactivation of Forkhead transcription factors (FOXO) in PTEN-null cells. Reintroduction of PTEN into the nucleus, overexpression of a nonphosphorylatable FOXO3a. which accumulates in the nucleus, or inhibition of nuclear export of FOXO3a by leptomycin B represses HIF-1 transcriptional activity in PTEN-null cells. HIF-1 transcriptional activity increases in PTEN-positive cells depleted of FOXO3a with sIRNA. PTEN and FOXO3a regulate the transactivation domain of HIF-1α. Chromatin immunoprecipitation indicates that FOXO3a complexes with HIF-1α and p300 on the Glut-1 promoter, a HIF-1 target gene. Overexpression of p300 reverses FOXO3a-mediated repression of HIF-1 transcriptional activity. Coimmunoprecipitation and GAL4-HIF-1α transactivation assays reveal that FOXO3a interferes with p300-dependent HIF-1 transcriptional activity. Thus, FOXO3a negatively regulates HIF-1 transcriptional activity. [ABSTRACT FROM AUTHOR]

Many multifunctional tumor suppressor proteins have low stability, a property linked to cancer development. The von Hippel-Lindau tumor suppressor protein (pVHL) is one of these proteins. pVHL forms part of the E3 ubiquitin ligase complex that regulates the degradation of the hypoxia-inducible factor (HIF). Under native conditions, free pVHL is a molten globule, but it is stabilized in the E3 complex. By using molecular dynamics simulations, we observed that the interface between the two pVHL domains is the least stable region in unbound pVHL. We designed five stable mutants: one with a mutation at the interdomain interface and the others in the α- or β-domains. Experimentally, type ZB pVHL disease mutant Y98N at the HIF binding site was shown to destabilize pVHL and decrease its binding affinity to HIF. Our simulations showed that the decrease in pVHL stability and binding affinity are allosterically regulated. The mutations designed to stabilize unbound wild-type pVHL which are away from the elongin C and HIF binding sites, successfully stabilized the Y98N pVHL-elongin C complex and lowered the binding free energy of pVHL with HIF. Our results indicated both the enthalpic and dynamic allosteric components between the elongin C and HIF binding sites in pVHL in the a- and p-domains, respectively, mediated by the interdomain interface and linker. Drugs mimicking the allosteric effects of these mutants may rescue pVHL function in von Hippel-Lindau disease. [ABSTRACT FROM AUTHOR]

The generation of plasmin by plasminogen (Pg) activators (PAs) is a physiologic process in animals that dissolves blood clots and promotes wound healing, blood vessel growth, and the migration of normal and cancerous cells. Pathogenic bacteria have evolved PAs [e.g., streptokinase (SK) and staphylokinase] that exploit the Pg system to infect animals. Animal PAs have a conserved ability to cleave a wide spectrum of animal Pgs, but the ability of bacterial PAs to cleave different animal Pgs is surprisingly restricted. We show that the spectrum of activity of an archetypal bacterial PA (SK) with animal Pgs can be profoundly altered by mutations that affect intermolecular complementarity at sites that participate in complex formation or substrate binding. Comparative sequence analysis of animal plasmins vs. close structural homologues (trypsin and chymotrypsin) that are not molecular targets for invading bacteria indicates that the sites in plasmin that interact with SK are preferentially targeted for mutation. Conversely, intermolecular contact sites in SKs that activate human Pg are more highly conserved than other loci in the molecule or than the same sites in other SKs that activate non-human Pgs. We propose that active modulation of intermolecular complementarity at sites of contact between SK and Pg may represent a competitive evolutionary strategy in a survival battle, whereby animals seek to evade bacterial invasion, and bacteria endeavor to invade their animal hosts. [ABSTRACT FROM AUTHOR]