Centromere proteins CENP-C and CAL1 functionally interact in meiosis for centromere clustering,pairing, and chromosome segregation

Meiotic chromosome segregation involves pairing and segregation of homologous chromosomes in the first division and segregation of sister chromatids in the second division. Although it is known that the centromere and kinetochore are responsible for chromosome movement in meiosis as in mitosis, potential specialized meiotic functions are being uncovered. Centromere pairing early in meiosis I, even between nonhomologous chromosomes, and clustering of centromeres can promote proper homolog associations in meiosis I in yeast, plants, and Drosophila. It was not known, however, whether centromere proteins are required for this clustering. We exploited Drosophila mutants for the centromere proteins centromere protein-C (CENP-C) and chromosome alignment 1 (CAL1) to demonstrate that a functional centromere is needed for centromere clustering and pairing. The cenp-C and cal1 mutations result in C-terminal truncations, removing the domains through which these two proteins interact. The mutants show striking genetic interactions, failing to complement as double heterozygotes, resulting in disrupted centromere clustering and meiotic nondisjunction. The cluster of meiotic centromeres localizes to the nucleolus, and this association requires centromere function. In Drosophila, synaptonemal complex (SC) formation can initiate from the centromere, and the SC is retained at the centromere after it disassembles from the chromosome arms. Although functional CENP-C and CAL1 are dispensable for assembly of the SC, they are required for subsequent retention of the SC at the centromere. These results show that integral centromere proteins are required for nuclear position and intercentromere associations in meiosis.Centromeres are the control centers for chromosomes, and thus are essential for accurate segregation in cell division. Centromeres are the DNA regions with a specialized chromatin structure upon which the kinetochore is built. The kinetochore is a complex of at least 100 proteins that contains the proteins to bind microtubules, motors to move on or destabilize microtubules, as well as checkpoint proteins monitoring kinetochore–microtubule attachment (1). The ability of the kinetochore to control microtubule binding and chromosome movement is essential for proper segregation in both mitosis and meiosis. In meiosis, additional constraints are placed on kinetochore function to ensure that homologs segregate in the first division and that segregation of sister chromatids is deferred until the second division (2). Recent studies indicate that in addition, the centromere itself may influence homolog segregation by controlling homolog pairing and formation of the synaptonemal complex (SC) (3).In prophase of meiosis I, the homologs must pair and ultimately become attached, usually by recombination and crossing-over. By quantifying centromere number through prophase I, it has been observed that centromeres pair in yeast, plants, and Drosophila (3). Perhaps unexpectedly, this pairing can be between nonhomologous centromeres; in yeast, this has been proposed as a mechanism to prevent recombination around the centromere, as centromere pairing resolves from initially being nonhomologous to being homologous (4, 5). Homologous centromere pairing may play a critical role in ensuring segregation of chromosomes that do not undergo crossing-over, possibly by affecting orientation of the kinetochores (3, 6–8).The centromere also regulates synapsis via the formation of the SC. SC formation initiates at the centromere and sites of cross-over formation in yeast, and the centromere is the first site for SC formation in Drosophila prophase I (9, 10). In addition, the SC persists at the centromere in yeast and Drosophila after the SC present along the chromosome arms has disassembled late in prophase I (7, 9, 11). Although SC assembly does not begin at centromeres in mouse meiosis, it persists at the centromeres and appears to promote proper segregation (12, 13).Another centromere property has been observed in Drosophila oocytes. In most organisms, the centromeres are clustered together at one site at the onset of meiosis, likely a remnant of their configuration in mitosis, but this clustering breaks down as centromeres arrange in pairs (3, 4). In Drosophila, however, the centromeres remain clustered until exit from prophase I at oocyte maturation (9, 10, 14). Although an essential role for centromere clustering has not been demonstrated, it may facilitate homolog pairing, synapsis, or accurate segregation, particularly given that the homologous telomeres do not pair into a bouquet formation in Drosophila meiosis (15, 16). Components of the SC are necessary for centromere clustering, as is the cohesion protein ORD (9, 14).The studies on centromere pairing and clustering define centromere geography within the meiotic nucleus, but they did not test whether centromere structure or function was involved. Centromeres have specialized nucleosomes with a histone H3 variant, centromere protein-A (CENP-A) (17). Incorporation of CENP-A into centromere chromatin is regulated precisely, although it occurs at distinct cell cycle times in different cell types, varying between late mitosis and G1 (17). In vertebrates, a complex of 15 proteins, the constitutive centromere-associated network (CCAN), is present on the CENP-A chromatin throughout the cell cycle and is crucial for assembling kinetochore proteins (1). In Drosophila, the entire CCAN complex has not been identified, although the CENP-C protein is present (18). Another Drosophila protein, CAL1, binds to CENP-A (called CID in Drosophila) in a prenucleosomal complex, and CAL1 is required for loading CID (19–22). CAL1 interacts with both CID and CENP-C, and all three proteins show interdependency for centromere localization (21, 23).Little is known about the activities of these centromere proteins in meiosis. In fission yeast, CENP-C has been demonstrated to be critical for kinetochore–microtubule binding in meiosis and also to control kinetochore orientation in meiosis I (24). The timing of assembly of kinetochore and centromere proteins onto meiotic chromosomes has been examined in mouse spermatocytes (25) and in Drosophila spermatocytes and sperm (26, 27). RNAi studies have shown that CAL1 and CENP-C (the latter to a lesser extent) are needed for CID localization in Drosophila male meiosis, with reduction in the levels of any of these three proteins being associated with meiotic segregation errors (26). Drosophila males differ from most organisms in not undergoing recombination or forming an SC, and centromere clustering does not occur (28). A question of particular interest that has yet to be addressed is whether centromere architecture and function are required for centromere clustering and pairing in meiosis.     

Hydroxylamine as an intermediate in ammonia oxidation by globally abundant marine archaea

The ammonia-oxidizing archaea have recently been recognized as a significant component of many microbial communities in the biosphere. Although the overall stoichiometry of archaeal chemoautotrophic growth via ammonia (NH₃) oxidation to nitrite (NO₂⁻) is superficially similar to the ammonia-oxidizing bacteria, genome sequence analyses point to a completely unique biochemistry. The only genomic signature linking the bacterial and archaeal biochemistries of NH₃ oxidation is a highly divergent homolog of the ammonia monooxygenase (AMO). Although the presumptive product of the putative AMO is hydroxylamine (NH₂OH), the absence of genes encoding a recognizable ammonia-oxidizing bacteria-like hydroxylamine oxidoreductase complex necessitates either a novel enzyme for the oxidation of NH₂OH or an initial oxidation product other than NH₂OH. We now show through combined physiological and stable isotope tracer analyses that NH₂OH is both produced and consumed during the oxidation of NH₃ to NO₂⁻ by Nitrosopumilus maritimus, that consumption is coupled to energy conversion, and that NH₂OH is the most probable product of the archaeal AMO homolog. Thus, despite their deep phylogenetic divergence, initial oxidation of NH₃ by bacteria and archaea appears mechanistically similar. They however diverge biochemically at the point of oxidation of NH₂OH, the archaea possibly catalyzing NH₂OH oxidation using a novel enzyme complex.Microbial oxidation of ammonia (NH₃) to nitrite (NO₂⁻), the first step in nitrification, plays a central role in the global cycling of nitrogen. Recent studies have established that marine and terrestrial representatives of an abundant group of archaea, now classified as Thaumarchaeota, are autotrophic NH₃ oxidizers (1–5). Despite increasing evidence that ammonia-oxidizing archaea (AOA) generally outnumber ammonia-oxidizing bacteria (AOB), and likely nitrify in most natural environments, very little is known about their physiology or supporting biochemistry (6, 7). Genome sequence analyses have pointed to a unique pathway for NH₃ oxidation, likely using copper as a major redox active metal, and coupled to a variant of the hydroxypropionate/hydroxybutyrate cycle (8). However, the only genome sequence feature that associates the archaeal pathway for NH₃ oxidation with that of the better characterized AOB is a divergent variant of the ammonia monooxygenase (AMO), which may or may not be a functional equivalent of the bacterial AMO. Thus, the supporting biochemistry of a biogeochemically significant group of microorganisms remains unresolved (8, 9).Among the AOB, as represented by the model organism Nitrosomonas europaea, NH₃ is first oxidized to hydroxylamine (NH₂OH) by AMO, an enzyme composed of three subunits encoded by amoC, amoA, and amoB genes (7). NH₂OH is subsequently oxidized to NO₂⁻ by the hydroxylamine oxidoreductase (HAO) (7), a heme-rich enzyme encoded by the hao gene (7). Of the four electrons released from the oxidation of NH₂OH to NO₂⁻, two are transferred to the terminal oxidase for respiratory purposes and two are transferred to AMO for further oxidation of NH₃ (7). Although all available genome sequences for the AOA contain homologs of the bacterial AMO (amoB, amoC, and amoA), there are no obvious homologs of AOB-like HAO, or cytochromes c554 and c_M552 critical for energy conversion in AOB (8–15). Thus, either the product of NH₃ oxidation is not NH₂OH or, alternatively, these phylogenetically deeply branching thaumarchaea use a novel biochemistry for NH₂OH oxidation and electron transfer (8).In an attempt to gain further insights into the biochemistry and physiology of these unique archaeal nitrifiers, we here investigated the role of NH₂OH in Nitrosopumilus maritimus metabolism. These studies were complicated by the extremely oligotrophic character of this organism contributing to very low cell densities in culture (16). To overcome the challenge of working with low cell density cultures of N. maritimus, we established a method to concentrate cells on nylon membrane filters such that the cells remained competent for NH₃-dependent NO₂⁻ formation and oxygen (O₂) uptake. This method enabled us to carry out relatively short duration physiological studies and stable isotope tracer experiments directed at determining if N. maritimus can oxidize exogenous NH₂OH to NO₂⁻ while consuming O₂ and producing ATP, and if NH₂OH is an intermediate in NH₃ oxidation pathway of N. maritimus.     

Luminescence color switching of supramolecular assemblies of discrete molecular decanuclear gold(I) sulfido complexes

A series of discrete decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of various lengths on the aminodiphosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂, has been synthesized and characterized. These complexes have been shown to form supramolecular nanoaggregate assemblies upon solvent modulation. The photoluminescence (PL) colors of the nanoaggregates can be switched from green to yellow to red by varying the solvent systems from which they are formed. The PL color variation was investigated and correlated with the nanostructured morphological transformation from the spherical shape to the cube as observed by transmission electron microscopy and scanning electron microscopy. Such variations in PL colors have not been observed in their analogous complexes with short alkyl chains, suggesting that the long alkyl chains would play a key role in governing the supramolecular nanoaggregate assembly and the emission properties of the decanuclear gold(I) sulfido complexes. The long hydrophobic alkyl chains are believed to induce the formation of supramolecular nanoaggregate assemblies with different morphologies and packing densities under different solvent systems, leading to a change in the extent of Au(I)–Au(I) interactions, rigidity, and emission properties.Gold(I) complexes are one of the fascinating classes of complexes that reveal photophysical properties that are highly sensitive to the nuclearity of the metal centers and the metal–metal distances (1–59). In a certain sense, they bear an analogy or resemblance to the interesting classes of metal nanoparticles (NPs) (60–69) and quantum dots (QDs) (70–76) in that the properties of the nanostructured materials also show a strong dependence on their sizes and shapes. Interestingly, while the optical and spectroscopic properties of metal NPs and QDs show a strong dependence on the interparticle distances, those of polynuclear gold(I) complexes are known to mainly depend on the nuclearity and the internuclear separations of gold(I) centers within the individual molecular complexes or clusters, with influence of the intermolecular interactions between discrete polynuclear molecular complexes relatively less explored (34–38), and those of polynuclear gold(I) clusters not reported. Moreover, while studies on polynuclear gold(I) complexes or clusters are known (34–54), less is explored of their hierarchical assembly and nanostructures as well as the influence of intercluster aggregation on the optical properties (34–38). Among the gold(I) complexes, polynuclear gold(I) chalcogenido complexes represent an important and interesting class (44–51). While directed supramolecular assembly of discrete Au₁₂ (52), Au₁₆ (53), Au₁₈ (51), and Au₃₆ (54) metallomacrocycles as well as trinuclear gold(I) columnar stacks (34–38) have been reported, there have been no corresponding studies on the supramolecular hierarchical assembly of polynuclear gold(I) chalcogenido clusters.Based on our interests and experience in the study of gold(I) chalcogenido clusters (44–46, 51), it is believed that nanoaggegrates with interesting luminescence properties and morphology could be prepared by the judicious design of the gold(I) chalcogenido clusters. As demonstrated by our previous studies on the aggregation behavior of square-planar platinum(II) complexes (77–80) where an enhancement of the solubility of the metal complexes via introduction of solubilizing groups on the ligands and the fine control between solvophobicity and solvophilicity of the complexes would have a crucial influence on the factors governing supramolecular assembly and the formation of aggregates (80), introduction of long alkyl chains as solubilizing groups in the gold(I) sulfido clusters may serve as an effective way to enhance the solubility of the gold(I) clusters for the construction of supramolecular assemblies of novel luminescent nanoaggegrates.Herein, we report the preparation and tunable spectroscopic properties of a series of decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of different lengths on the aminophosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂ [n = 8 (1), 12 (2), 14 (3), 18 (4)] and their supramolecular assembly to form nanoaggregates. The emission colors of the nanoaggregates of 2−4 can be switched from green to yellow to red by varying the solvent systems from which they are formed. These results have been compared with their short alkyl chain-containing counterparts, 1 and a related [Au₁₀{Ph₂PN(C₃H₇)PPh₂}₄(μ₃-S)₄](ClO₄)₂ (45). The present work demonstrates that polynuclear gold(I) chalcogenides, with the introduction of appropriate functional groups, can serve as building blocks for the construction of novel hierarchical nanostructured materials with environment-responsive properties, and it represents a rare example in which nanoaggregates have been assembled with the use of discrete molecular metal clusters as building blocks.     

High-pressure study of lithium amidoborane using Raman spectroscopy and insight into dihydrogen bonding absence

One of the major obstacles to the use of hydrogen as an energy carrier is the lack of proper hydrogen storage material. Lithium amidoborane has attracted significant attention as hydrogen storage material. It releases ∼10.9 wt% hydrogen, which is beyond the Department of Energy target, at remarkably low temperature (∼90 °C) without borazine emission. It is essential to study the bonding behavior of this potential material to improve its dehydrogenation behavior further and also to make rehydrogenation possible. We have studied the high-pressure behavior of lithium amidoborane in a diamond anvil cell using in situ Raman spectroscopy. We have discovered that there is no dihydrogen bonding in this material, as the N—H stretching modes do not show redshift with pressure. The absence of the dihydrogen bonding in this material is an interesting phenomenon, as the dihydrogen bonding is the dominant bonding feature in its parent compound ammonia borane. This observation may provide guidance to the improvement of the hydrogen storage properties of this potential material and to design new material for hydrogen storage application. Also two phase transitions were found at high pressure at 3.9 and 12.7 GPa, which are characterized by sequential changes of Raman modes.Hydrogen economy has been considered as potentially efficient and environmental friendly alternative energy solution (1). However, one of the most important scientific and technical challenges facing the “hydrogen economy” is the development of safe and economically viable on-board hydrogen storage for fuel cell applications, especially to the transportation sector. Ammonia borane (BH₃NH₃), a solid state hydrogen storage material, possesses exceptionally high hydrogen content (19.6 wt%) and in particular, it contains a unique combination of protonic and hydridic hydrogen, and on this basis, offers new opportunities for developing a practical source for generating molecular dihydrogen (2–5). Stepwise release of H₂ takes place through thermolysis of ammonia borane, yielding one-third of its total hydrogen content (6.5 wt%) in each heating step, along with emission of toxic borazine (6–8). Recently, research interests are focusing on how to improve discharge of H₂ from ammonia borane, including lowering the dehydrogenation temperature and enhancing hydrogen release rate using different techniques, e.g., nanoscaffolds (9), ionic liquids (10), acid catalysis (11), base metal catalyst (12), or transition metal catalysts (13, 14). More recently, significant attention is given to chemical modification of ammonia borane through substitution of one of the protonic hydrogen atoms with an alkali or alkaline–earth element (15–21). Lithium amidoborane (LiNH₂BH₃) has been successfully synthesized by ball milling LiH with NH₃BH₃ (15–18). One of the driving forces suggested for the formation of LiNH₂BH₃ is the chemical potential of the protonic H^δ+ in NH₃ and the hydridic H^δ− in alkali metal hydrides making them tend to combine, producing H₂ + LiNH₂BH₃. LiNH₂BH₃ exhibits significantly different and improved dehydrogenation characteristics from its parent compound ammonia borane. It releases more than 10 wt% of hydrogen at around 90 °C without borazine emission. Also, the dehydrogenation process of lithium amidoborane is much less exothermic (∼3–5 kJmole⁻¹ H₂) (15–17) than that of NH₃BH₃ (∼22.5 kJmole⁻¹ H₂) (6–8), which greatly enhances the search for suitable regeneration routes (prerequisite for a hydrogen storage material). Although the rationale behind the improved dehydrogenation behavior is still unclear, these improved property modifications evidently originate from the substitution of one H in the NH₃ group by the more electron-donating Li, which exerts influences on the bonding characteristics, especially on the dihydrogen bonding, which is one of the characteristic bonds of ammonia borane (15). So, it is essential to understand details about the bonding behavior of this potential material.High-pressure study of molecular crystals can provide unique insight into the intermolecular bonding forces, such as hydrogen bonding and phase stability in hydrogen storage materials and thus provides insight into the improvement of design (22–30). For instance, Raman spectroscopic study of ammonia borane at high pressure provided insight about its phase transition behavior and the presence of dihydrogen bonding in its structure (25–30). We have investigated LiNH₂BH₃ at high pressure using Raman spectroscopy. We have found that, other than in NH₃BH₃, dihydrogen bonding is absent in lithium amidoborane structure and LiNH₂BH₃ shows two phase transitions at high pressure.     

Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors

Membrane recruitment of cytohesin family Arf guanine nucleotide exchange factors depends on interactions with phosphoinositides and active Arf GTPases that, in turn, relieve autoinhibition of the catalytic Sec7 domain through an unknown structural mechanism. Here, we show that Arf6-GTP relieves autoinhibition by binding to an allosteric site that includes the autoinhibitory elements in addition to the PH domain. The crystal structure of a cytohesin-3 construct encompassing the allosteric site in complex with the head group of phosphatidyl inositol 3,4,5-trisphosphate and N-terminally truncated Arf6-GTP reveals a large conformational rearrangement, whereby autoinhibition can be relieved by competitive sequestration of the autoinhibitory elements in grooves at the Arf6/PH domain interface. Disposition of the known membrane targeting determinants on a common surface is compatible with multivalent membrane docking and subsequent activation of Arf substrates, suggesting a plausible model through which membrane recruitment and allosteric activation could be structurally integrated.Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing exchange of GDP for GTP (1). Because many GEFs are recruited to membranes through interactions with phospholipids, active GTPases, or other membrane-associated proteins (1–5), GTPase activation can be restricted or amplified by spatial–temporal overlap of GEFs with binding partners. GEF activity can also be controlled by autoregulatory mechanisms, which may depend on membrane recruitment (6–11). Structural relationships between these mechanisms are poorly understood.Arf GTPases function in trafficking and cytoskeletal dynamics (5, 12, 13). Membrane partitioning of a myristoylated (myr) N-terminal amphipathic helix primes Arfs for activation by Sec7 domain GEFs (14–17). Cytohesins comprise a metazoan Arf GEF family that includes the mammalian proteins cytohesin-1 (Cyth1), ARNO (Cyth2), and Grp1 (Cyth3). The Drosophila homolog steppke functions in insulin-like growth factor signaling, whereas Cyth1 and Grp1 have been implicated in insulin signaling and Glut4 trafficking, respectively (18–20). Cytohesins share a modular architecture consisting of heptad repeats, a Sec7 domain with exchange activity for Arf1 and Arf6, a PH domain that binds phosphatidyl inositol (PI) polyphosphates, and a C-terminal helix (CtH) that overlaps with a polybasic region (PBR) (21–28). The overlapping CtH and PBR will be referred to as the CtH/PBR. The phosphoinositide specificity of the PH domain is influenced by alternative splicing, which generates diglycine (2G) and triglycine (3G) variants differing by insertion of a glycine residue in the β1/β2 loop (29). Despite similar PI(4,5)P₂ (PIP₂) affinities, the 2G variant has 30-fold higher affinity for PI(3,4,5)P₃ (PIP₃) (30). In both cases, PIP₃ is required for plasma membrane (PM) recruitment (23, 26, 31–33), which is promoted by expression of constitutively active Arf6 or Arl4d and impaired by PH domain mutations that disrupt PIP₃ or Arf6 binding, or by CtH/PBR mutations (8, 34–36).Cytohesins are autoinhibited by the Sec7-PH linker and CtH/PBR, which obstruct substrate binding (8). Autoinhibition can be relieved by Arf6-GTP binding in the presence of the PIP₃ head group (8). Active myr-Arf1 and myr-Arf6 also stimulate exchange activity on PIP₂-containing liposomes (37). Whether this effect is due to relief of autoinhibition per se or enhanced membrane recruitment is not yet clear. Phosphoinositide recognition by PH domains, catalysis of nucleotide exchange by Sec7 domains, and autoinhibition in cytohesins are well characterized (8, 16, 17, 30, 38–43). How Arf-GTP binding relieves autoinhibition and promotes membrane recruitment is unknown. Here, we determine the structural basis for relief of autoinhibition and investigate potential mechanistic relationships between allosteric regulation, phosphoinositide binding, and membrane targeting.     

Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease

Inflammatory bowel disease (IBD) arises from inappropriate activation of the mucosal immune system resulting in a state of chronic inflammation with causal links to colon cancer. Helicobacter hepaticus-infected Rag2^−/− mice emulate many aspects of human IBD, and our recent work using this experimental model highlights the importance of neutrophils in the pathology of colitis. To define molecular mechanisms linking colitis to the identity of disease biomarkers, we performed a translational comparison of protein expression and protein damage products in tissues of mice and human IBD patients. Analysis in inflamed mouse colons identified the neutrophil- and macrophage-derived damage products 3-chlorotyrosine (Cl-Tyr) and 3-nitrotyrosine, both of which increased with disease duration. Analysis also revealed higher Cl-Tyr levels in colon relative to serum in patients with ulcerative colitis and Crohn disease. The DNA chlorination damage product, 5-chloro-2′-deoxycytidine, was quantified in diseased human colon samples and found to be present at levels similar to those in inflamed mouse colons. Multivariate analysis of these markers, together with serum proteins and cytokines, revealed a general signature of activated innate immunity in human IBD. Signatures in ulcerative colitis sera were strongly suggestive of neutrophil activity, and those in Crohn disease and mouse sera were suggestive of both macrophage and neutrophil activity. These data point to innate immunity as a major determinant of serum and tissue profiles and provide insight into IBD disease processes.Inflammatory bowel disease (IBD) is a chronic and relapsing intestinal inflammatory disease that arises through unknown genetic, environmental, and bacterial origins (1, 2). Ulcerative colitis (UC) and Crohn disease (CD) are the two main forms of IBD, and their incidence is increasing in industrialized countries (3). Furthermore, IBD is a risk factor for the development of colon cancer (4). Although the specific determinants remain elusive, persistent inflammation is believed to play a significant role in colon cancer development (5).Neutrophil recruitment and activation are key steps in the intestinal innate immune response observed in IBD (6–8), and studies with animal models of colitis highlight the relationship between neutrophil infiltration and disease severity (9–11). We recently reported results of a comprehensive analysis of histopathology, changes in gene expression, and nucleic acid damage occurring during progression of lower bowel disease in Rag2^−/− mice infected with Helicobacter hepaticus (Hh) (10). This mouse model emulates many aspects of human IBD, and infected mice develop severe colitis that progress into colon carcinoma, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages (12, 13).Phagocytes produce strong oxidants and radicals that damage cellular macromolecules and promote tissue damage at sites of inflammation (14–16). Myeloperoxidase (MPO) is an abundant enzyme in neutrophils that produces hypochlorous acid (HOCl) from hydrogen peroxide (H₂O₂) and chloride ion (17, 18). HOCl can oxidize and chlorinate DNA, proteins, and lipids (19, 20). A prominent target of HOCl is tyrosine, which leads to the formation of the stable aromatic residue, 3-chlorotyrosine (Cl-Tyr) (21, 22). MPO also produces chlorinating species that react with DNA to form chlorinated adducts such as 5-chloro-2′-deoxycytidine (5-Cl-dC) (23), the presence of which was identified in colon tissue of H. hepaticus-infected Rag2^−/− mice (10). This modification of DNA may provide a mechanistic link between neutrophil activity and colitis-associated carcinoma (10, 24, 25).Macrophages also contribute to the array of oxidants and radicals at sites of inflammation through release of nitric oxide (NO) generated by the inducible NO synthase (iNOS) enzyme. NO reacts with superoxide anion (O₂^−•) at diffusion-controlled rates to yield highly reactive peroxynitrite (ONOO⁻) (26, 27). MPO also reacts H₂O₂ with nitrite (NO₂⁻, the endpoint of cellular NO oxidation) to produce the strong nitrating agent, nitrogen dioxide radical (NO₂^•) (28). Both NO₂^• and ONOO⁻ can react with tyrosine residues to generate the stable tyrosine nitration product, 3-nitrotyrosine (Nitro-Tyr) (29, 30).Multiple MS methods have been applied for determination of Cl-Tyr and Nitro-Tyr levels in biological systems (10, 31–38), and both have been detected in inflamed tissues from animals and humans (11, 39). The presence of Nitro-Tyr has been demonstrated in colon tissue of IBD patients by immunohistochemistry, and levels were reported to correlate with disease activity (40, 41). We undertook the present study to test the null hypothesis that the H. hepaticus-infected mouse model of colitis and colitis-associated carcinoma represents a useful surrogate of human IBD. To examine this hypothesis, we first quantified levels of Nitro-Tyr and Cl-Tyr in proteins and 5-Cl-dC in DNA of colon tissues of IBD patients. Comparison of these data with our previous findings (10) further assessed the validity of this animal model. We then tested the hypothesis that inflammation-induced damage in the colon would be reflected in changes in serum constituents, and would therefore serve as a noninvasive measure of IBD activity. For this purpose, we determined levels of protein chlorination and nitration products, acute-phase proteins, cytokines, and chemokines in human and mouse sera. In addition, gene expression of several inflammatory signaling molecules was monitored in mice colons to determine whether colonic inflammation was directly associated with serum cytokine levels. We then used multivariate analysis to determine which systemic inflammatory markers in serum were most closely associated with disease activity and were also common to human IBD and H. hepaticus-associated colitis in Rag2^−/− mice.     

Direct observation of electrogenic NH4+ transport in ammonium transport (Amt) proteins

Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH₄⁺ scavengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH₄⁺/NH₃ transport is used instead in acid–base and pH homeostasis in kidney or NH₄⁺/NH₃ (and eventually CO₂) detoxification in erythrocytes. Crystal structures and variant proteins are available, but the inherent challenges associated with the unambiguous identification of substrate and monitoring of transport events severely inhibit further progress in the field. Here we report a reliable in vitro assay that allows us to quantify the electrogenic capacity of Amt proteins. Using solid-supported membrane (SSM)-based electrophysiology, we have investigated the three Amt orthologs from the euryarchaeon Archaeoglobus fulgidus. Af-Amt1 and Af-Amt3 are electrogenic and transport the ammonium and methylammonium cation with high specificity. Transport is pH-dependent, with a steep decline at pH values of ∼5.0. Despite significant sequence homologies, functional differences between the three proteins became apparent. SSM electrophysiology provides a long-sought-after functional assay for the ubiquitous ammonium transporters.Ammonium transport (Amt) proteins are a class of trimeric, integral membrane proteins found throughout all domains of life. Despite moderate primary sequence homologies, distinct family members from bacteria, archaea, and eukarya (including humans) share conserved structural features and a high number of conserved amino acid residues that are considered functionally relevant (1–4). Although the involvement of all Amt proteins in transporting NH₄⁺/NH₃ across biological membranes is undisputed, their functional context is diverse. Prokaryotes and plants use Amt proteins to scavenge NH₄⁺/NH₃—a preferred nitrogen source for cell growth—from their environment, whereas mammals use Amt orthologs, the Rhesus proteins, for detoxification and ion homeostasis in erythrocytes and in the kidney and liver tissues (1, 5, 6).Three decades ago, Kleiner and coworkers suggested that Amt proteins are secondary active and electrogenic transporters for ammonium (7–9). Various groups have subsequently confirmed this finding by two-electrode voltage-clamp experiments with protein produced recombinantly from RNA injected into Xenopus laevis oocytes. Here, plant Amt and Rhesus proteins were the main object of study, but some mechanistic details remained unclear, in particular the distinction between electrogenic NH₄⁺ uniport (10–13), NH₃/H⁺ symport (11, 12), or electroneutral NH₄⁺/H⁺ antiport (14, 15). In contrast, bacterial Amt proteins were described as passive channels for the uncharged gas ammonia (NH₃) (16). The first crystal structure for an Amt family member, AmtB from Escherichia coli (17), was interpreted to support this hypothesis, and an ongoing controversy concerning the transported species has persisted in the field ever since. Several points have been raised to challenge the possibility of gas channeling, the most critical of which seems to be that at physiological pH the protonation equilibrium of NH₃—with a pK_a of 9.4—would be >99% on the side of charged NH₄⁺. This point implies that the import of neutral ammonia gas must be preceded by extracellular deprotonation and followed immediately by intracellular protonation. In summary, the import of NH₃ would thus result in a net NH₄⁺/H⁺ antiport. Such a mechanism would be electroneutral, but it would be secondary active in the presence of a proton motive force, resulting in a vectorial pumping of ammonium out of the cell—which is, of course, physiologically unreasonable. A second point is that biological membranes are themselves highly permeable for uncharged ammonia, with a permeability coefficient, P_d = 10⁻³ cm·s⁻¹, similar to that of water (18), such that a dedicated transport protein would hardly be required. Westerhoff and coworkers have argued that active Amt transport thus is imperative and that cells must be able to quickly block Amt transport upon intracellular accumulation of ammonium to avoid uncoupling of the proton gradient through back-diffusion of NH₃ (19). In prokaryotes and some plants, this blocking is the task of regulatory GlnK proteins belonging to the signal transducing P_II family that bind to corresponding ammonium transporters when their regulatory ligand 2-oxoglutarate, the primary metabolic acceptor for NH₄⁺ during nitrogen assimilation, is depleted (20).The high expectations to understand the mechanism of Amt transport from 3D structures have not been met to date. The available structures of E. coli AmtB (17, 21) and its complex with GlnK (22, 23) of A. fulgidus Amt-1 (24), Nitrosomonas europaea Rh50 (25, 26), and human RhCG (27) all show the same, inward-facing state of the protein. Such apparent structural rigidity would match the picture of a fast channel, whereas active transport is generally considered to involve conformational changes that expose a binding site for the cargo molecule(s) alternatingly to either side of the membrane (28). In addition, the difficulties to detect NH₄⁺/NH₃ and to assay Amt transport led to a lack of functional studies carried out in vitro on well-defined systems. An uptake assay with AmtB reconstituted in proteoliposomes was described to provide evidence for passive gas channeling (17), but the methodology was later contested (2). Assays based on the detection of radioactive methylammonium (MA) uptake were only carried out in whole cells of E. coli, and studies with voltage-clamp electrophysiology using Amt-1 reconstituted in planar lipid bilayers did not yield conclusive results (our work). A series of potentially important variants have been produced (29–39), but the lack of an adequate functional assay has precluded definite conclusions.The debate concerning the transport mechanism of Amt proteins has not been settled to date, necessitating a reliable functional in vitro assay. The finding that electrogenic transport was observed in X. laevis oocytes, but not in the far smaller membrane patch of a planar lipid bilayer setup, suggested that the transport rate of Amt proteins was possibly too low to lead to a detectable current response, unless a larger number of protein units were incorporated into the bilayer. We have therefore focused on a controlled method of in vitro electrophysiology that allows the simultaneous activation of >10⁸ protein units, the solid-supported membrane (SSM) electrophysiology (40). With this approach, pioneered by Fendler and coworkers, we were able to detect robust ion currents from isolated and reconstituted Amt proteins.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage

We report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5′-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg²⁺ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand.Argonaute (Ago) proteins, critical components of the RNA-induced silencing complex, play a key role in guide strand-mediated target RNA recognition, cleavage, and product release (reviewed in refs. 1–3). Ago proteins adopt a bilobal scaffold composed of an amino terminal PAZ-containing lobe (N and PAZ domains), a carboxyl-terminal PIWI-containing lobe (Mid and PIWI domains), and connecting linkers L1 and L2. Ago proteins bind guide strands whose 5′-phosphorylated and 3′-hydroxyl ends are anchored within Mid and PAZ pockets, respectively (4–7), with the anchored guide strand then serving as a template for pairing with the target strand (8, 9). The cleavage activity of Ago resides in the RNase H fold adopted by the PIWI domain (10, 11), whereby the enzyme’s Asp-Asp-Asp/His catalytic triad (12–15) initially processes loaded double-stranded siRNAs by cleaving the passenger strand and subsequently processes guide-target RNA duplexes by cleaving the target strand (reviewed in refs. 16–18). Such Mg²⁺ cation-mediated endonucleolytic cleavage of the target RNA strand (19, 20) resulting in 3′-OH and 5′-phosphate ends (21) requires Watson–Crick pairing of the guide and target strands spanning the seed segment (positions 2–2′ to 8–8′) and the cleavage site (10′–11′ step on the target strand) (9). Insights into target RNA recognition and cleavage have emerged from structural (9), chemical (22), and biophysical (23) experiments.Notably, bacterial and archaeal Ago proteins have recently been shown to preferentially bind 5′-phosphoryated guide DNA (14, 15) and use an activated water molecule as the nucleophile (reviewed in ref. 24) to cleave both RNA and DNA target strands (9). Structural studies have been undertaken on bacterial and archaeal Ago proteins in the free state (10, 15) and bound to a 5′-phosphorylated guide DNA strand (4) and added target RNA strand (8, 9). The structural studies of Thermus thermophilus Ago (TtAgo) ternary complexes have provided insights into the nucleation, propagation, and cleavage steps of target RNA silencing in a bacterial system (9). These studies have highlighted the conformational transitions on proceeding from Ago in the free state to the binary complex (4) to the ternary complexes (8, 9) and have emphasized the requirement for a precisely aligned Asp-Asp-Asp triad and a pair of Mg²⁺ cations for cleavage chemistry (9), typical of RNase H fold-mediated enzymes (24, 25). Structural studies have also been extended to binary complexes of both human (5, 6) and yeast (7) Agos bound to 5′-phosphorylated guide RNA strands.Despite these singular advances in the structural biology of RNA silencing, further progress was hampered by the modest resolution (2.8- to 3.0-Å resolution) of TtAgo ternary complexes with guide DNA (4) and added target RNAs (8, 9). This precluded identification of water molecules coordinated with the pair of Mg²⁺ cations, including the key water that acts as a nucleophile and targets the cleavable phosphate between positions 10′-11′ on the target strand. We have now extended our research to TtAgo ternary complexes with guide DNA and target DNA strands, which has permitted us to grow crystals of ternary complexes that diffract to higher (2.2–2.3 Å) resolution in the cleavage-incompatible, cleavage-compatible, and postcleavage steps. These high-resolution structures of TtAgo ternary complexes provide snapshots of distinct key steps in the catalytic cleavage pathway, opening opportunities for experimental probing into DNA target cleavage as a defense mechanism against plasmids and possibly other mobile elements (26, 27).     

Impaired excitability of somatostatin- and parvalbumin-expressing cortical interneurons in a mouse model of Dravet syndrome

Haploinsufficiency of the voltage-gated sodium channel Na_V1.1 causes Dravet syndrome, an intractable developmental epilepsy syndrome with seizure onset in the first year of life. Specific heterozygous deletion of Na_V1.1 in forebrain GABAergic-inhibitory neurons is sufficient to cause all the manifestations of Dravet syndrome in mice, but the physiological roles of specific subtypes of GABAergic interneurons in the cerebral cortex in this disease are unknown. Voltage-clamp studies of dissociated interneurons from cerebral cortex did not detect a significant effect of the Dravet syndrome mutation on sodium currents in cell bodies. However, current-clamp recordings of intact interneurons in layer V of neocortical slices from mice with haploinsufficiency in the gene encoding the Na_V1.1 sodium channel, Scn1a, revealed substantial reduction of excitability in fast-spiking, parvalbumin-expressing interneurons and somatostatin-expressing interneurons. The threshold and rheobase for action potential generation were increased, the frequency of action potentials within trains was decreased, and action-potential firing within trains failed more frequently. Furthermore, the deficit in excitability of somatostatin-expressing interneurons caused significant reduction in frequency-dependent disynaptic inhibition between neighboring layer V pyramidal neurons mediated by somatostatin-expressing Martinotti cells, which would lead to substantial disinhibition of the output of cortical circuits. In contrast to these deficits in interneurons, pyramidal cells showed no differences in excitability. These results reveal that the two major subtypes of interneurons in layer V of the neocortex, parvalbumin-expressing and somatostatin-expressing, both have impaired excitability, resulting in disinhibition of the cortical network. These major functional deficits are likely to contribute synergistically to the pathophysiology of Dravet syndrome.Dravet syndrome (DS), also referred to as “severe myoclonic epilepsy in infancy,” is a rare genetic epileptic encephalopathy characterized by frequent intractable seizures, severe cognitive deficits, and premature death (1–3). DS is caused by loss-of-function mutations in SCN1A, the gene encoding type I voltage-gated sodium channel Na_V1.1, which usually arise de novo in the affected individuals (4–7). Like DS patients, mice with heterozygous loss-of-function mutations in Scn1a exhibit ataxia, sleep disorder, cognitive deficit, autistic-like behavior, and premature death (8–14). Like DS patients, DS mice first become susceptible to seizures caused by elevation of body temperature and subsequently experience spontaneous myoclonic and generalized tonic-clonic seizures (11). Global deletion of Na_V1.1 impairs Na⁺ currents and action potential (AP) firing in GABAergic-inhibitory interneurons (8–10), and specific deletion of Na_V1.1 in forebrain interneurons is sufficient to cause DS in mice (13, 15). These data suggest that the loss of interneuron excitability and resulting disinhibition of neural circuits cause DS, but the functional role of different subtypes of interneurons in the cerebral cortex in DS remains unknown.Neocortical GABAergic interneurons shape cortical output and display great diversity in morphology and function (16, 17). The expression of parvalbumin (PV) and somatostatin (SST) defines two large, nonoverlapping groups of interneurons (16, 18, 19). In layer V of the cerebral cortex, PV-expressing fast-spiking interneurons and SST-expressing Martinotti cells each account for ∼40% of interneurons, and these interneurons are the major inhibitory regulators of cortical network activity (17, 20). Layer V PV interneurons make synapses on the soma and proximal dendrites of pyramidal neurons (18, 19), where they mediate fast and powerful inhibition (21, 22). Selective heterozygous deletion of Scn1a in neocortical PV interneurons increases susceptibility to chemically induced seizures (23), spontaneous seizures, and premature death (24), indicating that this cell type may contribute to Scn1a deficits. However, selective deletion of Scn1a in neocortical PV interneurons failed to reproduce the effects of DS fully, suggesting the involvement of other subtypes of interneurons in this disease (23, 24). Layer V Martinotti cells have ascending axons that arborize in layer I and spread horizontally to neighboring cortical columns, making synapses on apical dendrites of pyramidal neurons (17, 25, 26). They generate frequency-dependent disynaptic inhibition (FDDI) that dampens excitability of neighboring layer V pyramidal cells (27–29), contributing to maintenance of the balance of excitation and inhibition in the neocortex. However, the functional roles of Martinotti cells and FDDI in DS are unknown.Because layer V forms the principal output pathway of the neocortex, reduction in inhibitory input to layer V pyramidal cells would have major functional consequences by increasing excitatory output from all cortical circuits. However, the effects of the DS mutation on interneurons and neural circuits that provide inhibitory input to layer V pyramidal cells have not been determined. Here we show that the intrinsic excitability of layer V fast-spiking PV interneurons and SST Martinotti cells and the FDDI mediated by Martinotti cells are reduced dramatically in DS mice, leading to an imbalance in the excitation/inhibition ratio. Our results suggest that loss of Na_V1.1 in these two major types of interneurons may contribute synergistically to increased cortical excitability, epileptogenesis, and cognitive deficits in DS.     

Reconstitution of long and short patch mismatch repair reactions using Saccharomyces cerevisiae proteins

A problem in understanding eukaryotic DNA mismatch repair (MMR) mechanisms is linking insights into MMR mechanisms from genetics and cell-biology studies with those from biochemical studies of MMR proteins and reconstituted MMR reactions. This type of analysis has proven difficult because reconstitution approaches have been most successful for human MMR whereas analysis of MMR in vivo has been most advanced in the yeast Saccharomyces cerevisiae. Here, we describe the reconstitution of MMR reactions using purified S. cerevisiae proteins and mispair-containing DNA substrates. A mixture of MutS homolog 2 (Msh2)–MutS homolog 6, Exonuclease 1, replication protein A, replication factor C-Δ1N, proliferating cell nuclear antigen and DNA polymerase δ was found to repair substrates containing TG, CC, +1 (+T), +2 (+GC), and +4 (+ACGA) mispairs and either a 5′ or 3′ strand interruption with different efficiencies. The Msh2–MutS homolog 3 mispair recognition protein could substitute for the Msh2–Msh6 mispair recognition protein and showed a different specificity of repair of the different mispairs whereas addition of MutL homolog 1–postmeiotic segregation 1 had no affect on MMR. Repair was catalytic, with as many as 11 substrates repaired per molecule of Exo1. Repair of the substrates containing either a 5′ or 3′ strand interruption occurred by mispair binding-dependent 5′ excision and subsequent resynthesis with excision tracts of up to ∼2.9 kb occurring during the repair of the substrate with a 3′ strand interruption. The availability of this reconstituted MMR reaction now makes possible detailed biochemical studies of the wealth of mutations identified that affect S. cerevisiae MMR.DNA mismatch repair (MMR) is a critical DNA repair pathway that is coupled to DNA replication in eukaryotes where it corrects misincorporation errors made during DNA replication (1–9). This pathway prevents mutations and acts to prevent the development of cancer (10, 11). MMR also contributes to gene conversion by repairing mispaired bases that occur during the formation of recombination intermediates (3, 4, 12). Finally, MMR acts to suppress recombination between divergent but homologous DNA sequences, thereby preventing the formation of genome rearrangements that can result from nonallelic homologous recombination (4, 13–15).Our knowledge of the mechanism of eukaryotic MMR comes from several general lines of investigation (3–9). Studies of bacterial MMR have provided a basic mechanistic framework for comparative studies (5). Genetic and cell-biology studies, primarily in Saccharomyces cerevisiae, have identified eukaryotic MMR genes, provided models for how their gene products define MMR pathways, and elucidated some of the details of how MMR pathways interact with replication (1–4). Reconstitution studies, primarily in human systems, have identified some of the catalytic features of eukaryotic MMR (7–9, 16, 17). Biochemical and structural studies of S. cerevisiae and human MMR proteins have provided information about the function of individual MMR proteins (6–9).In eukaryotic MMR, mispairs are bound by MutS homolog 2 (Msh2)–MutS homolog 6 (Msh6) and Msh2–MutS homolog 3 (Msh3), two partially redundant complexes of MutS-related proteins (3, 4, 18, 19). These complexes recruit a MutL-related complex, called MutL homoloh 1 (Mlh1)–postmeiotic segregation 1 (Pms1) in S. cerevisiae and Mlh1–postmeiotic segregation 2 (Pms2) in human and mouse (3, 4, 20–23). The Mlh1–Pms1/Pms2 complex has an endonuclease activity suggested to play a role in the initiation of the excision step of MMR (24, 25). Downstream of mismatch recognition is a mispair excision step that can be catalyzed by Exonuclease 1 (Exo1) (26–28); however, defects in both S. cerevisiae and mouse Exo1 result in only a partial MMR deficiency, suggesting the existence of additional excision mechanisms (26, 27, 29). DNA polymerase δ, the single-strand DNA binding protein replication protein A (RPA), the sliding clamp proliferating cell nuclear antigen (PCNA), and the clamp loader replication factor C (RFC) are also required for MMR at different steps, including activation of Mlh1–Pms1/Pms2, stimulation of Exo1, potentially in Exo1-independent mispair excision, and in the gap-filling resynthesis steps of MMR (3, 16, 17, 24, 27, 30–36). Although much is known about these core MMR proteins, it is not well understood how eukaryotic MMR is coupled to DNA replication (1, 2), how excision is targeted to the newly replicated strand (1, 25, 37–39), or how different MMR mechanisms such as Exo1-dependent and -independent subpathways are selected or how many such subpathways exist (1, 24, 27, 29).S. cerevisiae has provided a number of tools for studying MMR, including forward genetic screens for mutations affecting MMR, including dominant and separation-of-function mutations, the ability to evaluate structure-based mutations in vivo, cell biological tools for visualizing and analyzing MMR proteins in vivo, and overproduction of individual MMR proteins for biochemical analysis. However, linking these tools with biochemical systems that catalyze MMR reactions in vitro for mechanistic studies has not yet been possible. Here, we describe the development of MMR reactions reconstituted using purified proteins for the analysis of MMR mechanisms.     

Residue-level resolution of alphavirus envelope protein interactions in pH-dependent fusion

Alphavirus envelope proteins, organized as trimers of E2–E1 heterodimers on the surface of the pathogenic alphavirus, mediate the low pH-triggered fusion of viral and endosomal membranes in human cells. The lack of specific treatment for alphaviral infections motivates our exploration of potential antiviral approaches by inhibiting one or more fusion steps in the common endocytic viral entry pathway. In this work, we performed constant pH molecular dynamics based on an atomic model of the alphavirus envelope with icosahedral symmetry. We have identified pH-sensitive residues that cause the largest shifts in thermodynamic driving forces under neutral and acidic pH conditions for various fusion steps. A series of conserved interdomain His residues is identified to be responsible for the pH-dependent conformational changes in the fusion process, and ligand binding sites in their vicinity are anticipated to be potential drug targets aimed at inhibiting viral infections.Alphaviruses, mosquito-borne human pathogens causing severe inflammations and fatal fevers, have infected many millions of people in recent outbreaks worldwide since 2005 (1–3). The lack of a vaccine or specific treatment prompts investigations of the fundamental mechanisms of the alphaviral lifecycle to facilitate the development of effective antiviral therapies (4). Alphaviruses have been reported to enter the cell through receptor-mediated endocytosis. Here, alphaviruses are ferried toward the perinuclear space of the host cell inside vesicles towed by molecular motors and delivered to specific locations for productive replication (5–11). Even when direct entry into the cytoplasm is possible (11–15), the endocytic entry pathway facilitates the transportation of viruses across the crowded cytoplasmic space and delays detection by the immune system without leaving empty capsid or envelope as obvious evidence of the viral infection exposed outside the host cell (10, 11). Before the delivery of its viral genome into the cytoplasm of a host cell, the alphavirus must undergo a critical step of low pH-triggered membrane fusion, which is a common mechanism in the endocytic viral entry pathway among many different viruses. Understanding the mechanism of the low pH-triggered alphaviral membrane fusion is essential for the development of therapies against alphavirus as well as other viruses using similar endocytic entry mechanisms.Recent studies of the lifecycle of alphavirus reveal that a precursor, p62, is first synthesized as a chaperon forming a heterodimer with E1, which is essential for viral budding (16); p62 protects the E1 protein in the low-pH environment of the secretory pathway before being cleaved by cellular furin to produce mature E2-E1 and a smaller fragment, E3 (17–21). After the virus buds from the cytoplasmic membrane, E3 is released from the virus particle under neutral pH conditions outside the host cell (13, 22–24).On the surface of a mature alphavirus, 80 (E2–E1)₃ viral spikes, organized in T = 4 icosahedral symmetry on the viral lipid membrane, enclose the viral capsid and genome (25–43). On internalization of the mature virus in the endosome of the host cell in a new round of infection cycle, the increasingly acidified endosomal environment triggers a series of conformational changes in the alphaviral spike (E2–E1)₃ (38), including the dissociation of E2 (42, 44, 45), release of a fusion loop on E1 (46, 47), and trimerization of E1 (48). The fusion loop, roughly residues 83–100 on the cd loop of each E1 protein (13, 49, 50), in the newly formed E1 homotrimer (HT), inserts into the endosomal membrane. Then, the E1 proteins fold back, pulling the viral and endosomal membranes together and thus, promoting membrane fusion (13, 24).Recently solved high-resolution structures of the alphavirus envelope proteins E2–E1 fitted into cryo-EM data representing the intact virus under both acidic and neutral pH conditions (43, 51, 52) provide excellent atomic models for studies of the low pH-triggered fusion process. The structure of Chikungunya virus (CHIKV) obtained at pH 8.0 represents the initial mature state (M state) of the (E2–E1)₃ viral spike before the fusion process (51). Under pH 5.6, domain B (DB) of E2, which protects the E1 fusion loop, is observed to be disordered in Sindbis virus (52). The rest of the domains of the (E2–E1)₃ spike show moderate conformational differences with an rmsd = 4.0 Å among C_α atoms compared with the structures obtained at pH 8.0 for CHIKV (43, 51). The structure of the envelope proteins in acidic conditions most likely depicts a fusion intermediate (FI) state (52) before E2 dissociation during the low pH-triggered fusion process. In addition, the crystal structure of the folded-back E1 HT (53) is a good model to describe the postfusion state.Based on these atomic models of the E2 and E1 envelope proteins and our previously developed constant pH molecular dynamics (CPHMD) method (54–58), we simulated the envelope proteins with icosahedral symmetry under various pH conditions covering pH 2.0–9.0. We used pH replica exchange in CPHMD and calculated pK_a values using pH titration fitting, which has been shown as a reliable and accurate approach to capture pK_a values of protein residues in various systems (59–64). Through the CPHMD modeling, we calculated the pK_a of the possible pH-sensitive residues (Asp, Glu, and His) in the M, FI, dissociated E2 (Dis), and HT states. We, therefore, derive the shifts in the thermodynamic stabilities originating from each titrating residue for the steps from the M to the FI state (M→FI) of (E2–E1)₃, from the FI to the Dis state (FI→Dis) of E2 proteins, and from the FI to the HT state (FI→HT) of E1 proteins as shown in Fig. 1D. For these processes, we assume that the virus is in the endosomal environment, and we do not consider possible receptor-induced conformational changes. Our residue-level resolution simulations and analyses allow us to identify the critical functional residues with significant pK_a shifts and changes in thermodynamic stability in the low pH-triggered fusion activation. Our results suggest that the most pH-sensitive residues are highly conserved among different alphaviral species and that these critical residues control the pH threshold of fusion activities, provide guidance to further mutagenesis experiments, and lead to more fundamental understanding of low pH-triggered alphaviral membrane fusion.Open in a separate windowFig. 1.Structure and organization of alphaviral envelope proteins. (A) The alphaviral envelope modeled in our simulations. (B) The alphaviral envelope proteins in an MAU. (C) The heterodimer of E2 (DA–DB–DC) and E1 (DI–DII–DIII). (D) Structures of a viral spike in different conformational states simulated for shifts in pK_a values and thermodynamic stabilities. E1 proteins are shown in blue, cyan, and light blue. E2 proteins are shown in red, magenta, and pink.