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In bacteria, attenuation of protein-tyrosine phosphorylation occurs during oxidative stress. The main described mechanism behind this effect is the H2O2-triggered conversion of bacterial phospho-tyrosines to protein-bound 3,4-dihydroxyphenylalanine. This disrupts the bacterial tyrosine phosphorylation-based signaling network, which alters the bacterial polysaccharide biosynthesis. Herein, we report an alternative mechanism, in which oxidative stress leads to a direct inhibition of bacterial protein-tyrosine kinases (BY-kinases). We show that DefA, a minor peptide deformylase, inhibits the activity of BY-kinase PtkA when Bacillus subtilis is exposed to oxidative stress. High levels of PtkA activity are known to destabilize B. subtilis pellicle formation, which leads to higher sensitivity to oxidative stress. Interaction with DefA inhibits both PtkA autophosphorylation and phosphorylation of its substrate Ugd, which is involved in exopolysaccharide formation. Inactivation of defA drastically reduces the capacity of B. subtilis to cope with oxidative stress, but it does not affect the major oxidative stress regulons PerR, OhrR, and Spx, indicating that PtkA inhibition is the main pathway for DefA involvement in this stress response. Structural analysis identified DefA residues Asn95, Tyr150, and Glu152 as essential for interaction with PtkA. Inhibition of PtkA depends also on the presence of a C-terminal a-helix of DefA, which resembles PtkA-interacting motifs from known PtkA activators, TkmA, SalA, and MinD. Loss of either the key interacting residues or the inhibitory helix of DefA abolishes inhibition of PtkA in vitro and impairs postoxidative stress recovery in vivo, confirming the involvement of these structural features in the proposed mechanism. [ABSTRACT FROM AUTHOR]

The serotonin transporter (SERT) tightly regulates synaptic serotonin levels and has been the primary target of antidepressants. Binding of inhibitors to the allosteric site of human SERT (hSERT) impedes the dissociation of antidepressants bound at the central site and may enhance the efficacy of such antidepressants to potentially reduce their dosage and side effects. Here, we report the identification of a series of high-affinity allosteric inhibitors of hSERT in a unique scaffold, with the lead compound, Lu AF88273 (3-(1-(2-(1H-indol-3-yl)ethyl)piperidin-4-yl)-6-chloro-1H-indole), having 2.1 nM allosteric potency in inhibiting imipramine dissociation. In addition, we find that Lu AF88273 also inhibits serotonin transport in a noncompetitive manner. The binding pose of Lu AF88273 in the allosteric site of hSERT is determined with extensive molecular dynamics simulations and rigorous absolute binding free energy perturbation (FEP) calculations, which show that a part of the compound occupies a dynamically formed small cavity. The predicted binding location and pose are validated by site-directed mutagenesis and can explain much of the structure–activity relationship of these inhibitors using the relative binding FEP calculations. Together, our findings provide a promising lead compound and the structural basis for the development of allosteric drugs targeting hSERT. Further, they demonstrate that the divergent allosteric sites of neurotransmitter transporters can be selectively targeted. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads efficiently by spike-mediated, direct cell-to-cell transmission. However, the underlying mechanism is poorly understood. Herein, we demonstrate that the tight junction protein occludin (OCLN) is critical to this process. SARS-CoV-2 infection alters OCLN distribution and expression and causes syncytium formation that leads to viral spread. OCLN knockdown fails to alter SARS-CoV-2 binding but significantly lowers internalization, syncytium formation, and transmission. OCLN overexpression also has no effect on virus binding but enhances virus internalization, cell-to-cell transmission, and replication. OCLN directly interacts with the SARS-CoV-2 spike, and the endosomal entry pathway is involved in OCLN-mediated cell-to-cell fusion rather than in the cell surface entry pathway. All SARS-CoV-2 strains tested (prototypic, alpha, beta, gamma, delta, kappa, and omicron) are dependent on OCLN for cell-to-cell transmission, although the extent of syncytium formation differs between strains. We conclude that SARS-CoV-2 utilizes OCLN as an internalization factor for cell-to-cell transmission. [ABSTRACT FROM AUTHOR]

Public goods, ranging from judiciary to sanitation to parkland, permeate daily life. They have been a subject of intense interdisciplinary study, with a traditional focus being on participation levels in isolated public goods games (PGGs) as opposed to a more recent focus on participation in PGGs embedded into complex social networks. We merged the two perspectives by arranging voluntary participants into one of three network configurations, upon which volunteers played a number of iterated PGGs within their network neighborhood. The purpose was to test whether the topology of social networks or a freedom to express preferences for some local public goods over others affect participation. The results show that changes in social networks are of little consequence, yet volunteers significantly increase participation when they freely express preferences. Surprisingly, the increase in participation happens from the very beginning of the game experiment, before any information about how others play can be gathered. Such information does get used later in the game as volunteers seek to correlate contributions with higher returns, thus adding significant value to public goods overall. These results are ascribable to a small number of behavioral phenotypes, and suggest that societies may be better off with bottom-up schemes for public goods provision. [ABSTRACT FROM AUTHOR]

Collective risks permeate society, triggering social dilemmas in which working toward a common goal is impeded by selfish interests. One such dilemma is mitigating runaway climate change. To study the social aspects of climate-change mitigation, we organized an experimental game and asked volunteer groups of three different sizes to invest toward a common mitigation goal. If investments reached a preset target, volunteers would avoid all consequences and convert their remaining capital into monetary payouts. In the opposite case, however, volunteers would lose all their capital with 50% probability. The dilemma was, therefore, whether to invest one's own capital or wait for others to step in. We find that communicating sentiment and outlook helps to resolve the dilemma by a fundamental shift in investment patterns. Groups in which communication is allowed invest persistently and hardly ever give up, even when their current investment deficits are substantial. The improved investment patterns are robust to group size, although larger groups are harder to coordinate, as evidenced by their overall lower success frequencies. A clustering algorithm reveals three behavioral types and shows that communication reduces the abundance of the free-riding type. Climate-change mitigation, however, is achieved mainly by cooperator and altruist types stepping up and increasing contributions as the failure looms. Meanwhile, contributions from free riders remain flat throughout the game. This reveals that the mechanisms behind avoiding collective risks depend on an interaction between behavioral type, communication, and timing. [ABSTRACT FROM AUTHOR]

Tamoxifen, a widely used modulator of the estrogen receptor (ER), targets ER-positive breast cancer preferentially. We used a powerful validation-based insertion mutagenesis method to find that expression of a dominant-negative, truncated form of the histone deacetylase ZIP led to resistance to tamoxifen. Consistently, increased expression of full-length ZIP gives the opposite phenotype, inhibiting the expression of genes whose products mediate resistance. An important example is JAK2. By binding to two specific sequences in the promoter, ZIP suppresses JAK2 expression. Increased expression and activation of JAK2 when ZIP is inhibited lead to increased STAT3 phosphorylation and increased resistance to tamoxifen, both in cell culture experiments and in a mouse xenograft model. Furthermore, data from human tumors are consistent with the conclusion that decreased expression of ZIP leads to resistance to tamoxifen in ERpositive breast cancer. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells play a critical role in controlling malignancies. Susceptibility or resistance to lung cancer, for example, specifically depends on NK cell function. Nevertheless, intrinsic factors that control NK cell-mediated clearance of lung cancer are unknown. Here we report that NK cells exposed to exogenous major histocompatibility class I (MHCI) provide a significant immunologic barrier to the growth and progression of malignancy. Clearance of lung cancer is facilitated by up-regulation of NKG2D, NKp46, and other activating receptors upon exposure to environmental MHCI. Surface expression of the inhibitory receptor Ly49C/I, on the other hand, is down-regulated upon exposure to tumorbearing tissue. We thus demonstrate that NK cells exhibit dynamic plasticity in surface expression of both activating and inhibitory receptors based on the environmental context. Our data suggest that altering the activation state of NK cells may contribute to immunologic control of lung and possibly other cancers. [ABSTRACT FROM AUTHOR]

Normal neural development is essential for the formation of neuronal networks and brain function. Cutaneous T cell lymphoma-associated antigen 5 (cTAGE5)/meningioma expressed antigen 6 (MEA6) plays a critical role in the secretion of proteins. However, its roles in the transport of nonsecretory cellular components and in brain development remain unknown. Here, we show that cTAGE5/MEA6 is important for brain development and function. Conditional knockout of cTAGE5/MEA6 in the brain leads to severe defects in neural development, including deficits in dendrite outgrowth and branching, spine formation and maintenance, astrocyte activation, and abnormal behaviors. We reveal that loss of cTAGE5/MEA6 affects the interaction between the coat protein complex II (COPII) components, SAR1 and SEC23, leading to persistent activation of SAR1 and defects in COPII vesicle formation and transport from the endoplasmic reticulum to the Golgi, as well as disturbed trafficking of membrane components in neurons. These defects affect not only the transport of materials required for the development of dendrites and spines but also the signaling pathways required for neuronal development. Because mutations in cTAGE5/MEA6 have been found in patients with Fahr's disease, our study potentially also provides insight into the pathogenesis of this disorder. [ABSTRACT FROM AUTHOR]

Crystal structures of the neurotransmitter:sodium symporter MhsT revealed occluded inward-facing states with one substrate (Trp) bound in the primary substrate (S1) site and a collapsed extracellular vestibule, which in LeuT contains the second substrate (S2) site. In n-dodecyl-β-D-maltoside, the detergent used to prepare MhsT for crystallization, the substrate-to-protein binding stoichiometry was determined by using scintillation proximity to be 1 Trp:MhsT. Here, using the same experimental approach, as well as equilibrium dialysis, we report that in n-decyl-β-D-maltoside, or after reconstitution in lipid, MhsT, like LeuT, can simultaneously bind two Trp substrate molecules. Trp binding to the S2 site sterically blocks access to a substituted Cys at position 33 in the S2 site, as well as access to the deeper S1 site. Mutation of either the S1 or S2 site disrupts transport, consistent with previous studies in LeuT showing that substrate binding to the S2 site is an essential component of the transport mechanism. [ABSTRACT FROM AUTHOR]

Network reciprocity has been widely advertised in theoretical studies as one of the basic cooperation-promoting mechanisms, but experimental evidence favoring this type of reciprocity was published only recently. When organized in an unchanging network of social contacts, human subjects cooperate provided the following strict condition is satisfied: The benefit of cooperation must outweigh the total cost of cooperating with all neighbors. In an attempt to relax this condition, we perform social dilemma experiments wherein network reciprocity is aided with another theoretically hypothesized cooperation-promoting mechanism--costly punishment. The results reveal how networks promote and stabilize cooperation. This stabilizing effect is stronger in a smaller-size neighborhood, as expected from theory and experiments. Contrary to expectations, punishment diminishes the benefits of network reciprocity by lowering assortment, payoff per round, and award for cooperative behavior. This diminishing effect is stronger in a larger-size neighborhood. An immediate implication is that the psychological effects of enduring punishment override the rational response anticipated in quantitative models of cooperation in networks. [ABSTRACT FROM AUTHOR]

General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABAA receptors. In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. Here we exploit the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, we show that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876,Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, our combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels. [ABSTRACT FROM AUTHOR]

Constitutive proteasomes (c-20S) are ubiquitously expressed cellular proteases that degrade polyubiquitinated proteins and regulate cell functions. An isoform of proteasome, the immunoproteasome (i-20S), is highly expressed in human T cells, dendritic cells (DCs), and B cells, suggesting that it could be a potential target for inflammatory diseases, including those involving autoimmunity and alloimmunity. Here, we describe DPLG3, a rationally designed, noncovalent inhibitor of the immunoproteasome chymotryptic subunit β5i that has thousands-fold selectivity over constitutive β5c. DPLG3 suppressed cytokine release from blood mononuclear cells and the activation of DCs and T cells, diminished accumulation of effector T cells, promoted expression of exhaustion and coinhibitory markers on T cells, and synergized with CTLA4-Ig to promote long-term acceptance of cardiac allografts across a major histocompatibility barrier. These findings demonstrate the potential value of using brief posttransplant immunoproteasome inhibition to entrain a long-term response favorable to allograft survival as part of an immunomodulatory regimen that is neither broadly immunosuppressive nor toxic. [ABSTRACT FROM AUTHOR]

Rapid clearance of adoptively transferred Cd47-null (Cd47-/-) cells in congeneic WT mice suggests a critical self-recognition mechanism, in which CD47 is the ubiquitous marker of self, and its interaction with macrophage signal regulatory protein α (SIRPα) triggers inhibitory signaling through SIRPα cytoplasmic immunoreceptor tyrosinebased inhibition motifs and tyrosine phosphatase SHP-1/2. However, instead of displaying self-destruction phenotypes, Cd47-/- mice manifest no, or only mild, macrophage phagocytosis toward self-cells except under the nonobese diabetic background. Studying our recently established Sirpα-KO (Sirpα-/-) mice, as well as Cd47-/- mice, we reveal additional activation and inhibitory mechanisms besides the CD47-SIRPα axis dominantly controlling macrophage behavior. Sirpα-/- mice and Cd47-/- mice, although being normally healthy, develop severe anemia and splenomegaly under chronic colitis, peritonitis, cytokine treatments, and CFA-/LPS-induced inflammation, owing to splenic macrophages phagocytizing self-red blood cells. Ex vivo phagocytosis assays confirmed general inactivity of macrophages from Sirpα-/- or Cd47-/- mice toward healthy selfcells, whereas they aggressively attack toward bacteria, zymosan, apoptotic, and immune complex-bound cells; however, treating these macrophages with IL-17, LPS, IL-6, IL-1β, and TNFα, but not IFN?, dramatically initiates potent phagocytosis toward self-cells, for which only the Cd47-Sirpα interaction restrains. Even for macrophages from WT mice, phagocytosis toward Cd47-/- cells does not occur without phagocytic activation. Mechanistic studies suggest a PKC-Syk-mediated signaling pathway, towhich IL-10 conversely inhibits, is required for activating macrophage self-targeting, followed by phagocytosis independent of calreticulin. Moreover, we identified spleen red pulp to be one specific tissue that provides stimuli constantly activating macrophage phagocytosis albeit lacking in Cd47-/- or Sirpα-/- mice. [ABSTRACT FROM AUTHOR]

Decarbonization of electricity generation can support climatechange mitigation and presents an opportunity to address pollution resulting from fossil-fuel combustion. Generally, renewable technologies require higher initial investments in infrastructure than fossil-based power systems. To assess the tradeoffs of increased up-front emissions and reduced operational emissions, we present, to our knowledge, the first global, integrated lifecycle assessment (LCA) of long-term, wide-scale implementation of electricity generation from renewable sources (i.e., photovoltaic and solar thermal, wind, and hydropower) and of carbon dioxide capture and storage for fossil power generation. We compare emissions causing particulate matter exposure, freshwater ecotoxicity, freshwater eutrophication, and climate change for the climate-change-mitigation (BLUE Map) and business-as-usual (Baseline) scenarios of the International Energy Agency up to 2050. We use a vintage stock model to conduct an LCA of newly installed capacity year-by-year for each region, thus accounting for changes in the energy mix used to manufacture future power plants. Under the Baseline scenario, emissions of air and water pollutants more than double whereas the low-carbon technologies introduced in the BLUE Map scenario allow a doubling of electricity supply while stabilizing or even reducing pollution. Material requirements per unit generation for low-carbon technologies can be higher than for conventional fossil generation: 11-40 times more copper for photovoltaic systems and 6-14 times more iron for wind power plants. However, only two years of current global copper and one year of iron production will suffice to build a low-carbon energy system capable of supplying the world's electricity needs in 2050. [ABSTRACT FROM AUTHOR]

The dynamic interplay between kinases and substrates is crucial for the formation of catalytically committed complexes that enable phosphoryl transfer. However, a clear understanding on how substrates modulate kinase structural dynamics to control catalytic efficiency is still missing. Here, we used solution NMR spectroscopy to study the conformational dynamics of two complexes of the catalytic subunit of the cAMP-dependent protein kinase A with WT and R14 deletion phospholamban, a lethal human mutant linked to familial dilated cardiomyopathy. Phospholamban is a central regulator of heart muscle contractility, and its phosphorylation by protein kinase A constitutes a primary response to β-adrenergic stimulation. We found that the single deletion of arginine in phospholamban's recognition sequence for the kinase reduces its binding affinity and dramatically reduces phosphorylation kinetics. Structurally, the mutant prevents the enzyme from adopting conformations andmotions committed for catalysis, with concomitant reduction in catalytic efficiency. Overall, these results underscore the importance of a welltuned structural and dynamic interplay between the kinase and its substrates to achieve physiological phosphorylation levels for proper Ca2+ signaling and normal cardiac function. [ABSTRACT FROM AUTHOR]

Water vapor in the upper troposphere strongly regulates the strength of water-vapor feedback, which is the primary process for amplifying the response of the climate system to external radiative forcings. Monitoring changes in upper-tropospheric water vapor and scrutinizing the causes of such changes are therefore of great importance for establishing the credibility of model projections of past and future climates. Here, we use coupled ocean-atmosphere model simulations under different climate-forcing scenarios to investigate satellite-observed changes in global-mean upper-tropospheric water vapor. Our analysis demonstrates that the upper-tropospheric moistening observed over the period 1979-2005 cannot be explained by natural causes and results principally from an anthropogenic warming of the climate. By attributing the observed increase directly to human activities, this study verifies the presence of the largest known feedback mechanism for amplifying anthropogenic climate change. [ABSTRACT FROM AUTHOR]

Maintenance of genomic stability is essential for normal organismal development and is vital to prevent diseases such as cancer. As genetic information is packaged into chromatin, it has become increasingly clear that the chromatin environment plays an important role in DNA damage response. However, how DNA repair is controlled by epigenetic mechanisms is not fully understood. Here we report the identification and characterization of lysine-specific histone demethylase 5B (KDM5B), a member of the JmjC domaincontaining histone demethylases, as an important player in multiple aspects of DNA double-strand break (DSB) response in human cells. We found that KDM5B becomes enriched in DNA-damage sites after ironizing radiation and endonuclease treatment in a poly(ADP ribose) polymerase 1- and histone variant macroH2A1.1- dependent manner. We showed that KDM5B is required for efficient DSB repair and for the recruitment of Ku70 and BRCA1, the essential component of nonhomologous end-joining and homologous recombination, respectively. Significantly, KDM5B deficiency disengages the DNA repair process, promotes spontaneous DNA damage, activates p53 signaling, and sensitizes cells to genotoxic insults. Our results suggest that KDM5B is a bona fide DNA damage response protein and indicate that KDM5B is an important genome caretaker and a critical regulator of genome stability, adding to the understanding of the roles of epigenetics in the maintenance of genetic fidelity. [ABSTRACT FROM AUTHOR]

Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion in the Na1 site organize a connection between their coordinating residues and the extracellular gate of LeuT through a continuous H-bond network. The specific insights from the structures, combined with results from substrate binding studies and molecular dynamics simulations, reveal an anion-dependent occlusion mechanism for NSS and shed light on the functional role of chloride binding. [ABSTRACT FROM AUTHOR]

Noniridescent coloration by the spongy keratin in parrot feather barbs has fascinated scientists. Nonetheless, its ultimate origin remains as yet unanswered, and a quantitative structural and optical description is still lacking. Here we report on structural and optical characterizations and numerical simulations of the blue feather barbs of the scarlet macaw. We found that the sponge in the feather barbs is an amorphous diamond-structured photonic crystal with only short-range order. It possesses an isotropic photonic pseudo-gap that is ultimately responsible for the brilliant noniridescent coloration. We further unravel an ingenious structural optimization for attaining maximum coloration apparently resulting from natural evolution. Upon increasing the material refractive index above the level provided by nature, there is an interesting transition from a photonic pseudogap to a complete bandgap. [ABSTRACT FROM AUTHOR]

Phospholamban (PIN) is a type II membrane protein that inhibits the sarcoplasmic reticulum Ca2+-ATPase (SERCA), thereby regulating calcium homeostasis in cardiac muscle. In membranes, PIN forms pentamers that have been proposed to function either as a storage for active monomers or as ion channels. Here, we report the T-state structure of pentameric PLN solved by a hybrid solution and solid-state NMR method. In lipid bilayers, PLN adopts a pinwheel topology with a narrow hydrophobic pore, which excludes ion transport. In the T state, the cytoplasmic amphipathic helices (domains Ia) are absorbed into the lipid bilayer with the transmembrane domains arranged in a left-handed coiled-coil configuration, crossing the bilayer with a tilt angle of approximately 11° with respect to the membrane normal. The tilt angle difference between the monomer and pentamer is approximately 13°, showing that intramembrane helix-helix association forces dominate over the hydrophobic mismatch, driving the overall topology of the transmembrane assembly. Our data reveal that both topology and function of PIN are shaped by the interactions with lipids, which fine-tune the regulation of SERCA. [ABSTRACT FROM AUTHOR]

It is well-documented that the methylation of histone H3 lysine 4 (H3K4) and of H3K9 are mutually exclusive, an epigenetic phenomenon conserved from yeast to humans. How this opposed methylation modification is accomplished and coordinated in mammalian cells is poorly understood. Here we report that the H3K9 trimethyl demethylase JMJD2B is an integral component of the H3K4-specific methyltransfERαse, the mixed-lineage leukemia (MLL) 2 complex. We show that the JMJD2B/MLL2 complex is copurified with estrogen receptor a (ERα) and is required for ERα-regulated transcription. We demonstrate that H3K9 demethylation and H3K4 methylation are coordinated in ERα-activated transcription such that H3K9 demethylation is a prerequisite for H3K4 methylation. Significantly, depletion of JMJD2B impairs the estrogen-induced G1/S transition of the cell cycle in vitro and inhibits breast tumorigenesis in vivo. Interestingly, JMJD2B itself is an ERα target gene, and forms a feed-forward regulatory loop in regulation of the hormone response. Our results provide a molecular basis for the coordinated H3K4 methylation/H3K9 demethylation in transcription activation, link the trimethyl demethylase JMJD2B to euchromatin functions, and provide a mechanism for JMJD2B in breast carcinogenesis. [ABSTRACT FROM AUTHOR]

Protein kinase A (PKA) is a ubiquitous phosphoryl transferase that mediates hundreds of cell signaling events. During turnover, its catalytic subunit (PKA-C) interconverts between three major conformational states (open, intermediate, and closed) that are dynamically and allosterically activated by nucleotide binding. We show that the structural transitions between these conformational states are minimal and allosteric dynamics encode the motions from one state to the next. NMR and molecular dynamics simulations define the energy landscape of PKA-C, with the substrate allowing the enzyme to adopt a broad distribution of conformations (dynamically committed state) and the inhibitors (high magnesium and pseudosubstrate) locking it into discrete minima (dynamically quenched state), thereby reducing the motions that allow turnover. These results unveil the role of internal dynamics in both kinase function and regulation. [ABSTRACT FROM AUTHOR]

Cortical endoplasmic reticulum (cER) is a permanent feature of yeast cells but occurs transiently in most animal cell types. Ist2p is a transmembrane protein that permanently localizes to the cER in yeast. When Ist2 is expressed in mammalian cells, it induces abundant cER containing Ist2. Ist2 cytoplasmic C-terminal peptide is necessary and sufficient to induce cER. This peptide sequence resembles classic coat protein complex I (COPI) coatomer protein-binding KKXX signals, and indeed the dimerized peptide binds COPI in vitro. Controlled dimerization of this peptide induces cER in cells. RNA interference experiments confirm that coatomer is required for cER induction in vivo, as are microtubules and the microtubule plus-end binding protein EB1. We suggest that Ist2 dimerization triggers coatomer binding and clustering of this protein into domains that traffic at the microtubule growing plus-end to generate the cER beneath the plasma membrane. Sequences similar to the Ist2 lysine-rich tail are found in mammalian STIM proteins that reversibly induce the formation of cER under calcium control. [ABSTRACT FROM AUTHOR]

The p53 tumor suppressor plays a central role in integrating cellular responses to various stresses. Tight regulation of p53 is thus essential for the maintenance of genome integrity and normal cell proliferation. Currently, several ubiquitin ligases, including the single-subunit RING-finger types-MDM2, Pirh2, and COP1-and the HECT-domain type-ARF-BP1-have been reported to target p53 for degradation. Here, we report the identification of a human Kelch domain-containing F-box protein, JFK. We showed that JFK promotes ubiquitination and degradation of p53. But unlike MDM2. Pirh2, COP1, and ARF-BP1, all of which possess an intrinsic ubiquitin ligase activity, JFK destabilizes p53 through the assembly of a Skpl-CuIl-F-box complex. Significantly, JFK inhibits p53- dependent transcription, and depletion of JFK stabilizes p53, pro- motes cell apoptosis, arrests cells in the G1 phase, and sensitizes cells to ionizing radiation-induced cell death. These data indicate that JFK is a critical negative regulator of p53 and represents a pathway for the maintenance of p53 levels in unstressed cells. Our experiments link the Skpl-CuIl-F-box system to p53 regulation. [ABSTRACT FROM AUTHOR]

Phospholamban (PLN) is an essential regulator of cardiac muscle contractility. The homopentameric assembly of PLN is the reservoir for active monomers that, upon deoligomerization form 1:1 complexes with the sarco(endo)plasmic reticulum CaATPase (SERCA), thus modulating the rate of calcium uptake. In lipid bilayers and micelles, monomeric PLN exists in equilibrium between a bent (or resting) T state and a more dynamic (or active) R state. Here, we report the high-resolution structure and topology of the T state of a monomeric PLN mutant in lipid bilayers. using a hybrid of solution and solid-state NMR restraints together with molecular dynamics simulations in explicit lipid environments. Unlike the previous structural ensemble determined in micelles, this approach gives a complete picture of the PLN monomer structure in a lipid bilayer. This hybrid ensemble exemplifies the tilt, rotation, and depth of membrane insertion, revealing the interaction with the lipids for all protein domains. The N-terminal amphipathic helical domain Ia (residues 1-16) rests on the surface of the lipid membrane with the hydrophobic face of domain Ia embedded in the membrane bilayer interior. The helix comprised of domain lb (residues 23-30) and transmembrane domain II (residues 31-52) traverses the bilayer with a tilt angle of ≈24°. The specific interactions between PLN and lipid membranes may represent an additional regulatory element of its inhibitory function. We propose this hybrid method for the simultaneous determination of structure and topology for membrane proteins with compact folds or proteins whose spatial arrangement is dictated by their specific interactions with lipid bilayers. [ABSTRACT FROM AUTHOR]

The first crystal structure of the neurotransmitter/sodium symporter homolog LeuT revealed an occluded binding pocket containing leucine and 2 C Na+; later structures showed tricyclic antidepressants (TCAs) in an extracellular vestibule ≈11 Å above the bound leucine and 2 C Na+. We recently found this region to be a second binding (S2) site and that binding of substrate to this site triggers Na+-coupled substrate symport. Here, we show a profound inhibitory effect of n-octyl-β-d-glucopyranoside (OG), the detergent used for LeuT crystallization, on substrate binding to the S2 site. In parallel, we determined at 2.8 Å the structure of LeuT-E290S, a mutant that, like LeuT-WT, binds 2 substrate molecules. This structure was similar to that of WT and clearly revealed an OG molecule in the S2 site. We also observed electron density at the S2 site in LeuT-WT crystals, and this also was accounted for by an OG molecule in that site. Computational analyses, based on the available crystal structures of LeuT, indicated the nature of structural arrangements in the extracellular region of LeuT that differentiate the actions of substrates from inhibitors bound in the S2 site. We conclude that the current LeuT crystal structures, all of which have been solved in OG, represent functionally blocked forms of the transporter, whereas a substrate bound in the S2 site will promote a different state that is essential for C Na+-coupled symport. [ABSTRACT FROM AUTHOR]

EphA4-dependent growth cone collapse requires reorganization of actin cytoskeleton through coordinated activation of Rho family GTPases. Whereas various guanine exchange factors have recently been identified to be involved in EphA4-mediated regulation of Rho GTPases and growth cone collapse, the functional roles of GTPase-activating proteins in the process are largely unknown. Here we report that EphA4 interacts with α2-chimaerin through its Src homology 2 domain. Activated EphA4 induces a rapid increase of tyrosine phosphorylation of α2-chimaerin and enhances its GTPase-activating protein activity toward Rac1. More importantly, α2-chimaerin regulates the action of EphA4 in growth cone collapse through modulation of Rac1 activity. Our findings have therefore identified a new α2-chimaerin-dependent signaling mechanism through which EphA4 transduces its signals to the actin cytoskeleton and modulates growth cone morphology. [ABSTRACT FROM AUTHOR]

Cytolethal distending toxins (CDTs) are inhibitory cyclomodulins, which block eukaryotic cell proliferation and are produced by a diverse group of Gram-negative bacteria, including Escherichia coli strains associated with intestinal and extraintestinal infections. However, the mode of transmission of the toxin gene clusters among diverse bacterial pathogens is unclear. We found that Cdt-1 produced by enteropathogenic E. coli strains associated with diarrhea is encoded by a lambdoid prophage, which is inducible and infectious. The genome of Cdt-1 converting phage (CDT-1(b) comprises 47,021 nucleotides with 60 predicted ORFs organized into six genomic regions encoding the head and tail, virulence, integrase, unknown functions, regulation, and lysis. The genomic organization of CDT-1Φ is similar to those of SfV, a serotype-converting phage of Shigella flexneri, and UTI89, a prophage identified in uropathogenic E. coll. Besides the cdt1 gene cluster, the virulence region of CDT-1Φ genome contains sequences homologous to a truncated cycle inhibiting factor and a type 3 effector protein. Mutation analysis of susceptible E. coli strain C600 suggested that the outer membrane protein OmpC is a putative receptor for CDT-1Φ. CDT-1Φ genome was also found to integrate into the host bacterial chromosome forming lysogens, which produced biologically active Cdt-1. Furthermore, phage induction appeared to cause enhanced toxigenicity of the E. coli strains carrying lysogenic CDT-1Φ. Our results suggest that CDT-1Φ is the latest member of a growing family of lambdoid phages encoding bacterial cyclomodulins and that the phage may have a role in horizontal transfer of these virulence genes. [ABSTRACT FROM AUTHOR]

Reversible addition of NO to Cys-sulfur in proteins, a modification termed S-nitrosylation, has emerged as a ubiquitous signaling mechanism for regulating diverse cellular processes. A key first-step toward elucidating the mechanism by which S-nitrosylation modulates a protein's function is specification of the targeted Cys (SNO-Cys) residue. To date, S-nitrosylation site specification has been laboriously tackled on a protein-by-protein basis. Here we describe a high-throughput proteomic approach that enables simultaneous identification of SNO-Cys sites and their cognate proteins in complex biological mixtures. The approach, termed SNOSID (SNO Site Identification), is a modification of the biotin-swap technique [Jaffrey, S. R., Erdjument-Bromage, H., Ferris, C. D., Tempst, P. & Snyder, S. H. (2001) Nat. Cell. Biol. 3, 193-197], comprising biotinylation of protein SNO-Cys residues, trypsinolysis, affinity purification of biotinylated-peptides. and amino acid sequencing by liquid chromatography tandem MS. With this approach, 68 SNO-Cys sites were specified on 56 distinct proteins in S-nitrosoglutathione-treated (2-10 µM) rat cerebellum lysates. In addition to enumerating these S-nitrosylation sites, the method revealed endogenous SNO-Cys modification sites on cerebellum proteins, including α-tubulin, β-tubulin, GAPDH, and dihydropyrimidinase-related protein-2. Whereas these endogenous SNO proteins were previously recognized, we extend prior knowledge by specifying the SNO-Cys modification sites. Considering all 68 SNO-Cys sites identified, a machine learning approach failed to reveal a linear Cys-flanking motif that predicts stable transnitrosation by S-nitrosoglutathione under test conditions, suggesting that undefined 3D structural features determine S-nitrosylation specificity. SNOSID provides the first effective tool for unbiased elucidation of the SNO proteome, identifying Cys residues that undergo reversible S-nitrosylation. [ABSTRACT FROM AUTHOR]

Functional crosstalk between G protein-coupled receptors in a homo- or heterodimeric assembly likely involves conformational changes at the dimer interface, but the nature of this interface is not yet established, and the dynamic changes have not yet been identified. We have mapped the homodimer interface in the dopamine D2 receptor over the entire length of the fourth transmembrane segment (TM4) by crosslinking of substituted cysteines. Their susceptibilities to crosslinking are differentially altered by the presence of agonists and inverse agonists. The TM4 dimer interface in the inverse agonist-bound conformation is consistent with the dimer of the inactive form of rhodopsin modeled with constraints from atomic force microscopy. Crosslinking of a different set of cysteines in TM4 was slowed by inverse agonists and accelerated in the presence of agonists; crosslinking of the latter set locks the receptor in an active state. Thus, a conformational change at the TM4 dimer interface is part of the receptor activation mechanism. [ABSTRACT FROM AUTHOR]

The binding site of the dopamine D[SUB2] receptor (D2R), like those of homologous rhodopsin-like G protein-coupled receptors (GPCRs) that bind small molecules, is contained within a water-accessible crevice formed among its seven transmembrane segments (TMs). The high- resolution structure of bovine rhodopsin, however, revealed that the second extracellular loop (E2), which connects TM4 and TM5, folds down into the transmembrane domain and forms part of the ligand-binding surface for retinal. Whether E2 plays a related role in other rhodopsin-like GPCRs is unclear. To address this issue, we have now mutated to cysteine, one at a time, 10 consecutive residues in E2 of D2R. The reaction of five of these mutants with sulfhydryl reagents inhibited antagonist binding, and bound antagonist protected two, N184C and N186C, from reaction. The pattern of accessibility in E2 is consistent with a structure similar to that of bovine rhodopsin, in which the region C-terminal to the conserved disulfide bond is deeper in the binding-site crevice than is the N-terminal part of E2. Thus, E2 likely contributes to the binding site in the D2R and probably in other aminergic GPCRs as well. Knowledge of its detailed positioning and interactions with ligand would benefit GPCR molecular modeling and facilitate the design of novel drugs. [ABSTRACT FROM AUTHOR]

Investifates the structural effects of quinacrine binding in the open channel of the acetylcholine receptor. Quinacrine inhibition of Cys mutants; Quinacrine effects on the reaction of MTSEA with Cys mutants; Quinacrine mustard reaction with Cys mutants.