Dynamic PIP2 interactions with voltage sensor elements contribute to KCNQ2 channel gating

The S4 segment and the S4–S5 linker of voltage-gated potassium (Kv) channels are crucial for voltage sensing. Previous studies on the Shaker and Kv1.2 channels have shown that phosphatidylinositol-4,5-bisphosphate (PIP₂) exerts opposing effects on Kv channels, up-regulating the current amplitude, while decreasing the voltage sensitivity. Interactions between PIP₂ and the S4 segment or the S4–S5 linker in the closed state have been highlighted to explain the effects of PIP₂ on voltage sensitivity. Here, we show that PIP₂ preferentially interacts with the S4–S5 linker in the open-state KCNQ2 (Kv7.2) channel, whereas it contacts the S2–S3 loop in the closed state. These interactions are different from the PIP₂–Shaker and PIP₂–Kv1.2 interactions. Consistently, PIP₂ exerts different effects on KCNQ2 relative to the Shaker and Kv1.2 channels; PIP₂ up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel. Disruption of the interaction of PIP₂ with the S4–S5 linker by a single mutation decreases the voltage sensitivity and current amplitude, whereas disruption of the interaction with the S2–S3 loop does not alter voltage sensitivity. These results provide insight into the mechanism of PIP₂ action on KCNQ channels. In the closed state, PIP₂ is anchored at the S2–S3 loop; upon channel activation, PIP₂ interacts with the S4–S5 linker and is involved in channel gating.A series of ion channels, such as inward rectifier K⁺ (Kir) channels, transient receptor potential channels, and voltage-gated channels, are sensitive to the presence of phosphatidylinositol-4,5-bisphosphate (PIP₂) in membranes (1–4). Structural studies on Kir channels (1, 2, 5) demonstrated that PIP₂ directly interacts with the channels. Subsequent studies supported that PIP₂ also interacts directly with voltage-gated potassium (Kv) channels (6–19). Several positive residues that may be critical for PIP₂ activity have been identified (7, 11, 18, 20–24). Previous studies on Kv1.2 and Shaker channels showed that PIP₂ exerts opposing effects on Kv channels, up-regulating the current amplitude, while leading to a decrease in voltage sensitivity (7, 18). The S4 segment and the S4–S5 linker of Kv channels are crucial for voltage sensing. The interactions of PIP₂ with the S4 segments and the S4–S5 linkers of the closed-state Shaker and Kv1.2 channels underlie the loss-of-function effect of PIP₂ on voltage sensitivity (7, 18).The KCNQ (Kv7) family of slowly activated outwardly rectifying potassium channels is one of the Kv channel families that are sensitive to the presence of PIP₂ in the membrane. KCNQ channels have been widely studied because of their important biological and pharmacological functions. Retigabine, a first-in-class K⁺ channel opener used for the treatment of epilepsy, adopts a unique mechanism to enhance the activity of KCNQ channels (25). PIP₂ is important for the functions of KCNQ channels. Reduction of PIP₂ affinity caused by congenic mutations of KCNQ channels is associated with long QT syndrome, suggesting critical physiological implications of PIP₂ on KCNQ channels (23, 26). We reported that PIP₂ also alters the pharmacological selectivity of KCNQ potassium channels (6). Zaydman et al. (27) showed that the coupling of voltage sensing and pore opening in the KCNQ1 channel requires PIP₂ and suggested there is a PIP₂ interaction site at the interface between the voltage-sensing domain (VSD) and the central pore domain (PD). However, the effects and interactions of PIP₂ on KCNQ channels are not well understood.Here, by combining molecular dynamics (MD) simulations, mutagenesis, and electrophysiological determinations, we observed that the effects and interactions of PIP₂ on KCNQ2 are different relative to the Shaker and Kv1.2 channels. PIP₂ up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel. PIP₂ preferentially interacts with the S4–S5 linker of the open-state KCNQ2 channel and does not interact with the S4 segment or S4-S5 linker of the closed state. In the closed state, PIP₂ only interacts with the S2–S3 loop. Furthermore, our electrophysiological experiments suggest that disruption of the interaction of PIP₂ with the S4–S5 linker may decrease the voltage sensitivity and current amplitude, whereas disruption of the interaction with the S2–S3 loop only alters the current amplitude of the channel. These results provide insights into the mechanism of PIP₂ action on Kv channels.     

EFA6 controls Arf1 and Arf6 activation through a negative feedback loop

Guanine nucleotide exchange factors (GEFs) of the exchange factor for Arf6 (EFA6), brefeldin A-resistant Arf guanine nucleotide exchange factor (BRAG), and cytohesin subfamilies activate small GTPases of the Arf family in endocytic events. These ArfGEFs carry a pleckstrin homology (PH) domain in tandem with their catalytic Sec7 domain, which is autoinhibitory and supports a positive feedback loop in cytohesins but not in BRAGs, and has an as-yet unknown role in EFA6 regulation. In this study, we analyzed how EFA6A is regulated by its PH and C terminus (Ct) domains by reconstituting its GDP/GTP exchange activity on membranes. We found that EFA6 has a previously unappreciated high efficiency toward Arf1 on membranes and that, similar to BRAGs, its PH domain is not autoinhibitory and strongly potentiates nucleotide exchange on anionic liposomes. However, in striking contrast to both cytohesins and BRAGs, EFA6 is regulated by a negative feedback loop, which is mediated by an allosteric interaction of Arf6-GTP with the PH-Ct domain of EFA6 and monitors the activation of Arf1 and Arf6 differentially. These observations reveal that EFA6, BRAG, and cytohesins have unanticipated commonalities associated with divergent regulatory regimes. An important implication is that EFA6 and cytohesins may combine in a mixed negative-positive feedback loop. By allowing EFA6 to sustain a pool of dormant Arf6-GTP, such a circuit would fulfill the absolute requirement of cytohesins for activation by Arf-GTP before amplification of their GEF activity by their positive feedback loop.Guanine nucleotide exchange factors (GEFs), which activate small GTPases by stimulating their intrinsically very slow GDP/GTP exchange, are key players in the extraordinary diversity of small GTPases pathways (reviewed in ref. 1). Small GTPases carry little specificity determinants on their own to determine when and where they should be turned on and which pathway they should activate (2), which are instead largely monitored by their GEFs. Thus, understanding how different members of a GEF family activate an individual small GTPase in distinct patterns is a major issue in small GTPase biology in normal cells and in diseases.An important contribution to the functional specificity of GEFs is how they themselves are regulated. Crystallographic studies combined with biochemical studies that reconstituted GEF-stimulated GDP/GTP nucleotide exchange have been instrumental in uncovering a growing complexity of regulatory mechanisms (reviewed in ref. 1). These include autoinhibitory elements outside the catalytic GEF domain that block access to the active site (3–7), large conformational changes that release autoinhibition in response to various stimuli (8–11), positive feedback loops in which freshly produced GTP-bound GTPases stimulate GDP/GTP exchange (10, 12–15), and potentiation of nucleotide exchange by colocalization on membranes (11, 13, 16, 17).These previous studies demonstrated that a wide range of regulatory regimes can be achieved even at the scale of a single GEF family by regulatory mechanisms that combine in multiple ways. GEFs that activate small GTPases of the Arf family (ArfGEFs), which are major regulators of many aspects of membrane traffic and organelle structure in eukaryotic cells (reviewed in refs. 18 and 19), form one of the best-characterized GEF families to date (reviewed in ref. 1), making a comprehensive view of their regulatory repertoire within reach. ArfGEFs comprise two major groups: the BIG/GBF1 group, which functions at the Golgi, and a group composed of the exchange factor for Arf6 (EFA6), brefeldin A-resistant Arf guanine nucleotide exchange factor (BRAG), and cytohesin subfamilies, which activate Arf GTPases at the cell periphery and function in various aspects of endocytosis (reviewed in ref. 20). The actual substrates of these ArfGEFs have been difficult to establish, notably because the most abundant Arf isoform, Arf1, was long believed to be excluded from the plasma membrane where the Arf6 isoform is located. Accordingly, cytohesins and BRAGs have been described as Arf6-specific GEFs in cells but are now recognized as active Arf1-GEFs (16, 21, 22), whereas EFA6 remains the sole ArfGEF considered to be strictly Arf6-specific (23, 24).Members of the EFA6, BRAG, and cytohesin subfamilies have divergent N-terminal domains but a related domain organization in their C terminus comprising a Sec7 domain, which stimulates GDP/GTP exchange, followed by a pleckstrin homology (PH) domain, which has multiple regulatory functions. In cytohesins, the PH domain recognizes signaling phosphoinositides by its canonical lipid-binding site (25), autoinhibits the Sec7 domain by obstructing its Arf-binding site (4), and amplifies nucleotide exchange by a positive feedback loop involving its direct interaction with Arf1-GTP or Arf6-GTP (10, 13, 21). In contrast, the PH domain of BRAG is not autoinhibitory and is not involved in a feedback loop, but instead strongly potentiates nucleotide exchange by binding to polyanionic membranes without marked phosphoinositides preference (16).How members of the EFA6 subfamily are regulated is currently unknown. These ArfGEFs are found predominantly (although not exclusively) in the brain and function in the coordination of endocytosis and actin dynamics (23, 26, 27), in the maintenance of tight junctions (28), in microtubule dynamics in Caenorhabditis elegans embryos (29), and in the formation and maintenance of dendrites (30), although the molecular details of these functions remain largely unknown. Consistent with an important role in the brain, defects in EFA6 functions have been found in neurologic disorders (31) and in human gliomas (32). The PH domain of EFA6 subfamily members drives the localization of EFA6 members to the plasma membrane (26) and it binds to PIP₂ lipids (33). It is followed by a 150-residue C-terminal (Ct) domain predicted to form a coiled coil, which massively induces actin-rich membrane protrusions when expressed with the PH domain (26). The divergence of regulatory mechanisms between Sec7-PH–containing cytohesins and BRAGs prompted us to undertake a quantitative biochemical investigation of EFA6 nucleotide exchange regulation. Our findings reveal an overlooked dual specificity of EFA6 for Arf1 and Arf6 and an unprecedented regulation by a negative feedback loop, with important potential implications for the activation of Arf GTPases in endocytic events.     

The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor SF-1

The signaling phosphatidylinositol lipids PI(4,5)P₂ (PIP₂) and PI(3,4,5)P₃ (PIP₃) bind nuclear receptor 5A family (NR5As), but their regulatory mechanisms remain unknown. Here, the crystal structures of human NR5A1 (steroidogenic factor-1, SF-1) ligand binding domain (LBD) bound to PIP₂ and PIP₃ show the lipid hydrophobic tails sequestered in the hormone pocket, as predicted. However, unlike classic nuclear receptor hormones, the phosphoinositide head groups are fully solvent-exposed and complete the LBD fold by organizing the receptor architecture at the hormone pocket entrance. The highest affinity phosphoinositide ligand PIP₃ stabilizes the coactivator binding groove and increases coactivator peptide recruitment. This receptor-ligand topology defines a previously unidentified regulatory protein-lipid surface on SF-1 with the phosphoinositide head group at its nexus and poised to interact with other proteins. This surface on SF-1 coincides with the predicted binding site of the corepressor DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region on chromosome X), and importantly harbors missense mutations associated with human endocrine disorders. Our data provide the structural basis for this poorly understood cluster of human SF-1 mutations and demonstrates how signaling phosphoinositides function as regulatory ligands for NR5As.The existence of nuclear, nonmembrane pools of signaling phosphorylated derivatives of phosphatidylinositols or phosphoinositides (PIP_n) was reported over two decades ago (1–3). Consistent with these early reports, lipid-modifying enzymes responsible for phosphoinositide metabolism were also found in the nucleus (4–7); however, the function of PIP_n in this cellular compartment remains poorly defined. The nuclear receptors (NRs) steroidogenic factor 1 (SF-1, NR5A1) and liver receptor homolog 1 (LRH-1, NR5A2) bind phosphoinositides as well as other phospholipids in their large hydrophobic pockets (8–13). The ability of NR5As to interact with PIP_n is well-conserved with the Caenorhabditis elegans ortholog nhr-25 able to bind both PIP₂ and PIP₃ (14). That phosphoinositides might serve as endogenous NR5A ligands is suggested by the fact that elevating cellular pools of PIP₃ increases SF-1 activity (15) and that impairing PIP₃ uptake decreases SF-1 activity (12). Further, when purified from mammalian cells, the phosphoinositide PIP₂ is found associated with SF-1 and can be modified by the lipid kinase, IPMK, as well as the lipid phosphatase, PTEN (13). Taken together, these data suggest that signaling phosphoinositides are biologically relevant ligands for SF-1.Phosphoinositide ligands diverge chemically from classic NR hormones in that they contain a long, extended hydrophobic moiety and a prominent hydrophilic head group, which is inherently incompatible with the hydrophobic core of the NR5A ligand-binding pocket. Our previous structural analyses of SF-1 bound to phosphatidylcholine suggest that the acyl tails of phosphoinositides should be sequestered in the hydrophobic core and positioned to fill the hormone-binding pockets of SF-1 and LRH-1 (11). It remains to be determined how the acyl tails and the head groups of phosphoinositides might affect the stability and activity of the NR5A ligand-binding domain (LBD). To date, the only visualized phospholipid head groups in NR5As are those found in the bacterial phospholipids (bPLs), and long and medium chain phosphatidylcholines (PCs) (12, 16). Thus far, neither bPLs nor PCs complete the fold of SF-1 LBD. Indeed, in published ligand-bound SF-1 structures, critical surface loops in the vicinity of the pocket entrance remain poorly ordered. This region is functionally important because it harbors disease-associated mutations in SF-1 (17–19). We hypothesized that phosphoinositide ligands with their charged head groups might anchor this site to render SF-1 fully functional.Here, the crystal structures of SF-1 bound to PIP₂ and PIP₃ were determined, and coactivator peptide-binding studies were performed to gain insights into how signaling phosphoinositides function as NR5A ligands. Based on our results, we suggest that PIP₂ and PIP₃ help organize dynamic loops in the LBD that form part of a previously unidentified regulatory surface on NR5As, similar in function to membrane-bound phosphoinositides.     

Arf6 coordinates actin assembly through the WAVE complex,a mechanism usurped by Salmonella to invade host cells

ADP ribosylation factor (Arf) 6 anchors to the plasma membrane, where it coordinates membrane trafficking and cytoskeleton remodelling, but how it assembles actin filaments is unknown. By reconstituting membrane-associated actin assembly mediated by the WASP family veroprolin homolog (WAVE) regulatory complex (WRC), we recapitulated an Arf6-driven actin polymerization pathway. We show that Arf6 is divergent from other Arf members, as it was incapable of directly recruiting WRC. We demonstrate that Arf6 triggers actin assembly at the membrane indirectly by recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO that activates Arf1 to enable WRC-dependent actin assembly. The pathogen Salmonella usurped Arf6 for host cell invasion by recruiting its canonical GEFs EFA6 and BRAG2. Arf6 and its GEFs facilitated membrane ruffling and pathogen invasion via ARNO, and triggered actin assembly by generating an Arf1–WRC signaling hub at the membrane in vitro and in cells. This study reconstitutes Arf6-dependent actin assembly to reveal a mechanism by which related Arf GTPases orchestrate distinct steps in the WRC cytoskeleton remodelling pathway.ADP ribosylation factor (Arf) GTPases are best known for their roles in vesicle and organelle trafficking (1). Class I and II Arfs (Arf1, Arf3, Arf4, and Arf5) are found predominantly in and around the Golgi apparatus. In contrast, the more divergent Class III Arf (Arf6) operates almost exclusively at the plasma membrane (2). Consistent with its localization, Arf6 has been heavily implicated in trafficking events at the cell surface, including the regulation of endocytosis and exocytosis (1). In particular, Arf6 and its guanine nucleotide exchange factors (GEFs) are believed to be pivotal to the recycling of endosomes and receptors to and from the plasma membrane (3, 4). Arf6 also has a clear role in cortical cytoskeleton rearrangement (5). This is strongly supported by evidence of Arf6 and Rac1 (Ras-related C3 botulinum toxin substrate) interplay (6), exemplified by Arf6 recruitment of the Rac1 GEF Kalirin, Arf6 promotion of Rac1 activation, and lamellipodia formation (7–9).Rac1 is required for generation of lamellipodia that lead to membrane ruffles and macropinocytosis (10). Rac1 is thought to achieve this by activating the WRC, which comprises WAVE (WASP family veroprolin homolog), Abi (abl-interactor 1), Cyfip (cytoplasmic FMR1 interacting protein), Nap1 (NCK-associated protein 1), HSPC300 (heat shock protein C300), or their homologs (10–12). We recently established that Rac1 was not sufficient for WRC recruitment to the membrane and its activation, which instead requires direct binding by Rac1 and an Arf GTPase (13). This in vitro Arf activity could be supplied by multiple isoforms including Arf1 and Arf5, but only Arf1 facilitated WRC-dependent lamellipodia formation and macropinocytosis of the bacterial pathogen Salmonella into human host cells (13–15). Intriguingly, Arf6 also promoted Salmonella invasion (14), and, given the capability of Arfs to modulate WRC, it seems likely that Arf6 also recruits and activates the WRC at the plasma membrane. However, despite mounting evidence that Arf6 remodels the cytoskeleton, a molecular mechanism by which Arf6 drives Arp2/3-dependent actin assembly has not been resolved.     

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.     

Intrinsic regulation of FIC-domain AMP-transferases by oligomerization and automodification

Filamentation induced by cyclic AMP (FIC)-domain enzymes catalyze adenylylation or other posttranslational modifications of target proteins to control their function. Recently, we have shown that Fic enzymes are autoinhibited by an α-helix (α_inh) that partly obstructs the active site. For the single-domain class III Fic proteins, the α_inh is located at the C terminus and its deletion relieves autoinhibition. However, it has remained unclear how activation occurs naturally. Here, we show by structural, biophysical, and enzymatic analyses combined with in vivo data that the class III Fic protein NmFic from Neisseria meningitidis gets autoadenylylated in cis, thereby autonomously relieving autoinhibition and thus allowing subsequent adenylylation of its target, the DNA gyrase subunit GyrB. Furthermore, we show that NmFic activation is antagonized by tetramerization. The combination of autoadenylylation and tetramerization results in nonmonotonic concentration dependence of NmFic activity and a pronounced lag phase in the progress of target adenylylation. Bioinformatic analyses indicate that this elaborate dual-control mechanism is conserved throughout class III Fic proteins.Fic (filamentation induced by cyclic AMP) proteins containing the FIC domain (pfam 02661) are found in all kingdoms of life. FIC domains encode enzymatic activities that modulate target protein function by diverse posttranslational modifications (1, 2). The vast majority of known Fic proteins are AMP-transferases that use ATP to catalyze the transfer of an AMP moiety onto a target hydroxyl side chain (3, 4). This reaction is akin to the situation in protein kinases, which catalyze γ-phosphate transfer onto target side chains.Only a few Fic targets have been identified to date (2). IbpA (4) and VopS (3), two bacterial FIC-domain effectors that get translocated into host cells, catalyze the adenylylation of Rho GTPases, resulting in cytoskeleton collapse. Most recently, we have found that a subset of bacterial Fic proteins adenylylates DNA gyrase and topoisomerase IV, which leads to their inactivation and cellular growth arrest (5). The structure of a FIC-domain/target complex (6) and the catalytic mechanism have been determined (6, 7). However, the biological functions, as well as the molecular mechanism, of the vast majority of Fic proteins have remained elusive. Intriguingly, in vitro automodification has been demonstrated for most Fic proteins that have been described so far (6, 8–15), but its physiological relevance has remained unclear.Recently, we have shown that Fic-mediated adenylylation is tightly regulated (8). In the native state, a helix partly obstructs the ATP binding site with a strictly conserved Glu blocking ATP γ-phosphate binding, thereby preventing productive/competent substrate binding. The inhibitory α-helix (α_inh) is either located on a separate protein that forms a tight toxin/antitoxin complex with the Fic enzyme (16) or at the N- or C-terminal position within the same polypeptide chain. These three possibilities lead to a classification of Fic proteins into classes I, II, and III, respectively (8). Mutation of the inhibitory Glu to Gly relieves autoinhibition, thus boosting both target and autoadenylylation (8, 9). However, the identity of the intrinsic or extrinsic factors that in vivo expulse the inhibitory Glu of α_inh, and thereby relieve autoinhibition, has not been investigated.Fic proteins have evolved in bacteria and have spread by horizontal gene transfer into all domains of life (17). Despite structural conservation of the basic FIC-domain fold, there is significant sequence diversity among class I and II Fic proteins. Additionally, these two classes are frequently combined with other protein domains toward multidomain arrangements, demonstrating a high functional plasticity and adaptability (2). In contrast, class III Fic proteins are highly conserved single-domain proteins even though they are found scattered across all classes of Proteobacteria (8). This conservation suggests that class III Fic proteins are stand-alone autoregulated functional entities that act in a plug-and-play manner upon acquisition by horizontal gene transfer.Here, we dissect the regulatory mechanism of NmFic from Neisseria meningitidis as representative for the class III Fic proteins. First, we identify the B-subunit of DNA gyrase as the main bacterial target of NmFic, as for class I Fic proteins (5). We then reveal the crucial role of autoadenylylation in the activation of class III Fic proteins and the opposing role of oligomerization, resulting in a peculiar and intriguing NmFic concentration dependence of target adenylylation. Ultimately, because the oligomerization interfaces are either highly conserved or covaried, and because the modifiable residue Y183 is strictly conserved in class III Fic proteins, we anticipate that the combination of oligomer dissociation and subsequent cis-autoadenylylation is the major regulatory mechanism of class III Fic proteins.     

Tumor-derived hydrogen sulfide,produced by cystathionine-β-synthase,stimulates bioenergetics,cell proliferation,and angiogenesis in colon cancer

The physiological functions of hydrogen sulfide (H₂S) include vasorelaxation, stimulation of cellular bioenergetics, and promotion of angiogenesis. Analysis of human colon cancer biopsies and patient-matched normal margin mucosa revealed the selective up-regulation of the H₂S-producing enzyme cystathionine-β-synthase (CBS) in colon cancer, resulting in an increased rate of H₂S production. Similarly, colon cancer-derived epithelial cell lines (HCT116, HT-29, LoVo) exhibited selective CBS up-regulation and increased H₂S production, compared with the nonmalignant colonic mucosa cells, NCM356. CBS localized to the cytosol, as well as the mitochondrial outer membrane. ShRNA-mediated silencing of CBS or its pharmacological inhibition with aminooxyacetic acid reduced HCT116 cell proliferation, migration, and invasion; reduced endothelial cell migration in tumor/endothelial cell cocultures; and suppressed mitochondrial function (oxygen consumption, ATP turnover, and respiratory reserve capacity), as well as glycolysis. Treatment of nude mice with aminooxyacetic acid attenuated the growth of patient-derived colon cancer xenografts and reduced tumor blood flow. Similarly, CBS silencing of the tumor cells decreased xenograft growth and suppressed neovessel density, suggesting a role for endogenous H₂S in tumor angiogenesis. In contrast to CBS, silencing of cystathionine-γ-lyase (the expression of which was unchanged in colon cancer) did not affect tumor growth or bioenergetics. In conclusion, H₂S produced from CBS serves to (i) maintain colon cancer cellular bioenergetics, thereby supporting tumor growth and proliferation, and (ii) promote angiogenesis and vasorelaxation, consequently providing the tumor with blood and nutritients. The current findings identify CBS-derived H₂S as a tumor growth factor and anticancer drug target.The endogenous gasotransmitter hydrogen sulfide (H₂S) is a stimulator of vasorelaxation (1–3), angiogenesis (3–5), and cellular bioenergetics (6, 7). H₂S is generated from l-cysteine by two pyridoxal-5′-phospate–dependent enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), and by the combined action of cysteine aminotransferase and 3-mercaptopyruvate sulfurtransferase (3-MST) (8–10). H₂S exerts its cellular actions via multiple mechanisms (1–15), including activation of potassium channels (1–3), stimulation of kinase pathways (4, 11, 12), and inhibition of phosphodiesterases (3, 15).Both ATP generation and angiogenesis are vital factors for the growth and proliferation of tumors (16–19). Using human colon cancer tissues and cancer-derived cell lines, we have now conducted a series of in vitro and in vivo studies to explore whether endogenous, tumor cell-derived H₂S plays a role as a tumor-derived survival factor. The results show that CBS is selectively overexpressed in colon cancer, and that H₂S produced by it serves to maintain the tumor''s cellular bioenergetics and to promote tumor angiogenesis.     

Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation

We compare the antigenicity and conformation of soluble, cleaved vs. uncleaved envelope glycoprotein (Env gp)140 trimers from the subtype A HIV type 1 (HIV-1) strain BG505. The impact of gp120–gp41 cleavage on trimer structure, in the presence or absence of trimer-stabilizing modifications (i.e., a gp120–gp41 disulfide bond and an I559P gp41 change, together designated SOSIP), was assessed. Without SOSIP changes, cleaved trimers disintegrate into their gp120 and gp41-ectodomain (gp41_ECTO) components; when only the disulfide bond is present, they dissociate into gp140 monomers. Uncleaved gp140s remain trimeric whether SOSIP substitutions are present or not. However, negative-stain electron microscopy reveals that only cleaved trimers form homogeneous structures resembling native Env spikes on virus particles. In contrast, uncleaved trimers are highly heterogeneous, adopting a variety of irregular shapes, many of which appear to be gp120 subunits dangling from a central core that is presumably a trimeric form of gp41_ECTO. Antigenicity studies with neutralizing and nonneutralizing antibodies are consistent with the EM images; cleaved, SOSIP-stabilized trimers express quaternary structure-dependent epitopes, whereas uncleaved trimers expose nonneutralizing gp120 and gp41_ECTO epitopes that are occluded on cleaved trimers. These findings have adverse implications for using soluble, uncleaved trimers for structural studies, and the rationale for testing uncleaved trimers as vaccine candidates also needs to be reevaluated.Trimeric envelope glycoprotein (Env gp) spikes on the HIV type 1 (HIV-1) surface mediate entry of the viral genome into the target cell (1, 2). When spikes interact with their cell-surface receptors, a series of conformational changes within the Env culminates in virus–cell membrane fusion. Neutralizing antibodies (NAbs) against various Env epitopes antagonize these events (2, 3). Hence, Env glycoproteins are a focus of vaccine design programs intended to induce NAbs and thereby prevent HIV-1 transmission (3, 4). Env trimers are composed of three gp120 surface glycoprotein subunits and three gp41 transmembrane glycoproteins, the six subunits all associated via noncovalent interactions (5, 6). A critical event in trimer assembly is proteolytic cleavage of the gp160 precursor into its gp120 and gp41 components, a process essential for HIV-1 entry not least because it liberates the fusion peptide (FP) at the gp41 N terminus (5, 6).Trimer-based vaccine strategies involve expressing soluble, recombinant versions of the virion-associated (i.e., native) spikes. To facilitate production and purification, the membrane-spanning and cytoplasmic domains that anchor spikes to the virion, but that are not NAb targets, are eliminated (7–12). However, the resulting proteins, known as gp140s, are highly unstable and disintegrate into their gp120 and gp41-ectodomain (gp41_ECTO) components, making them useless as immunogens. Two fundamentally different protein-engineering strategies have been used to create gp140s that can be produced and purified without falling apart (3, 4, 7–17). The most common method involves eliminating the cleavage site between gp120 and gp41_ECTO, creating uncleaved gp140s (gp140_UNC) where the two subunits remain covalently linked (7–12). Additional trimerization motifs are often added to the gp41_ECTO C terminus (10–12). Our alternative approach is based on the premise that cleavage is a fundamental feature of Env structure and involves stabilizing fully cleaved gp140s. The critical changes are an appropriately positioned disulfide bond (referred to as “SOS”) to link gp120 to gp41_ECTO covalently, and an Ile/Pro (IP) substitution at residue 559 to strengthen inter-gp41_ECTO interactions (13–17). The resulting cleaved trimers are designated SOSIP gp140s (14). Additional modifications have improved their stability, homogeneity, and antigenicity (15–17). Our current design, based on the BG505 subtype A env gene, yields SOSIP.664 trimers that mimic native, virion-associated Env spikes antigenically and when viewed by negative-stain electron microscopy (EM) (17–19).Here we show that cleavage is essential for producing stable, soluble gp140 trimers that resemble native Env spikes. EM studies reveal that purified, trimeric gp140_UNC proteins are heterogeneous and that the irregularly shaped images rarely resemble a native spike; we refer to them as “aberrant configurations” (ACs). In contrast, cleaved SOSIP gp140 trimers are homogeneous and mimic native spikes; we designate them native-like (NL) trimers. The antigenic properties of the cleaved (NL) and uncleaved (AC) trimers, assessed by surface plasmon resonance (SPR) and enzyme-linked immunoabsorbance assays (ELISA), are consistent with the EM images. Nonneutralizing gp120 and gp41_ECTO epitopes are exposed on gp140_UNC trimers but occluded on cleaved ones, whereas quaternary structure-dependent epitopes indicative of proper folding are present only on cleaved trimers. Our findings have substantial implications, because uncleaved trimers are being studied structurally and developed as vaccine candidates (3, 9, 10, 12, 20).     

From the Cover: PNAS Plus: Arl1p regulates spatial membrane organization at the trans-Golgi network through interaction with Arf-GEF Gea2p and flippase Drs2p

ADP ribosylation factors (Arfs) are the central regulators of vesicle trafficking from the Golgi complex. Activated Arfs facilitate vesicle formation through stimulating coat assembly, activating lipid-modifying enzymes and recruiting tethers and other effectors. Lipid translocases (flippases) have been implicated in vesicle formation through the generation of membrane curvature. Although there is no evidence that Arfs directly regulate flippase activity, an Arf-guanine-nucleotide-exchange factor (GEF) Gea2p has been shown to bind to and stimulate the activity of the flippase Drs2p. Here, we provide evidence for the interaction and activation of Drs2p by Arf-like protein Arl1p in yeast. We observed that Arl1p, Drs2p and Gea2p form a complex through direct interaction with each other, and each interaction is necessary for the stability of the complex and is indispensable for flippase activity. Furthermore, we show that this Arl1p-Drs2p-Gea2p complex is specifically required for recruiting golgin Imh1p to the Golgi. Our results demonstrate that activated Arl1p can promote the spatial modulation of membrane organization at the trans-Golgi network through interacting with the effectors Gea2p and Drs2p.ADP ribosylation factors (Arf)/SARs, a subfamily of the Ras small GTP-binding protein superfamily, are critical regulators of vesicular trafficking in eukaryotic cells (1–3). Like Ras, Arf and Arf-like (Arl) proteins cycle between the inactive GDP-bound form and active GTP-bound forms to carry out their functions. The conversion from the GDP-bound to the GTP-bound form is facilitated by Arf guanine-nucleotide-exchange factor (Arf-GEF), whereas GTP hydrolysis is catalyzed by an Arf GTPase-activating protein (Arf-GAP) (4, 5). Activated Arf recruits numerous proteins to the membrane or activates lipid-modifying enzymes to facilitate vesicle formation, including vesicle coat assembly, membrane curvature generation, lipid composition alteration, and final membrane fission (4, 6–8).Arl1p is the best-studied Arl protein and is expressed in organisms ranging from yeast to humans (9, 10). Both yeast Arl1p and human Arl1 localize to the trans-Golgi network (TGN), where they regulate multiple membrane-trafficking pathways (9, 11, 12). Yeast Arl1p participates in three different pathways at the TGN, including glycosylphosphatidylinositol (GPI)-anchored protein transport, clathrin adaptor protein Gga recruitment, and targeting of the GRIP domain–containing protein Imh1p to the Golgi. In addition to Imh1p, the Arf-GEF–like protein, Mon2p (also called Ysl2p), and the clathrin adaptor protein, Gga2p, directly interact with Arl1p and are considered to be Arl1p effectors (13).Arf-GEFs catalyze the GTP binding of Arf/Arl through the conserved Sec7 domain and are upstream regulators of Arf/Arl activities (14, 15). In yeast, the human GBF1 homolog Gea2p, which functions redundantly with Gea1p, displays Arf-GEF activity toward Arf1p and is believed to be involved in ER-Golgi and intra-Golgi transport (16, 17). Recently, Gea2p was shown to directly interact with the TGN-localized aminophospholipid translocase Drs2p and to stimulate its flippase activity (18, 19). Similarly, it was reported that the Arf-GEF–like protein Mon2p interacted with both Arl1p and the Drs2p family protein Neo1p and that the Mon2-Neo1-Arl1 complex cooperated to regulate the Golgi recruitment of Gga2p (13). These results suggest that Arf-GEFs may act with members of the Drs2p subfamily of P-type ATPases to facilitate lipid translocation. However, the role of Gea2p in Drs2p-mediated flippase activity is not clear.Flippases translocate specific phospholipid molecules to establish the asymmetry of membranes (19–22). Therefore, flippases can drive membrane bending toward the cytosol and contribute to vesicle-mediated protein transport by facilitating the generation of membrane curvature that is inherent to this process (23). The inactivation of Drs2p results in defects in the formation of an Arf/clathrin-dependent class of post-Golgi vesicles that carry exocytic cargo (24). Moreover, a mutation in DRS2 was shown to cause synthetic lethality with clathrin heavy chain and ARF1 (25). These observations indicate the importance of flippase activity for vesicle trafficking, and Drs2p is involved in protein transport at the TGN.To explore the possibility that Arl1p might, like Arf proteins, play a role in regulating membrane dynamics, we use an analysis of both physical interaction and functional interaction to demonstrate that Drs2p and Gea2p are effectors of Arl1p. We find that these proteins—Arl1p, Drs2p, and Gea2p—are all required to form a stable complex to stimulate Drs2p flippase activity and complete certain functions of Arl1p. Our study provides insight into the molecular mechanisms of spatial membrane dynamics at the TGN and their regulation by the Arl1p-Gea2p-Drs2p cooperating network.     

Transition paths,diffusive processes,and preequilibria of protein folding

Fundamental relationships between the thermodynamics and kinetics of protein folding were investigated using chain models of natural proteins with diverse folding rates by extensive comparisons between the distribution of conformations in thermodynamic equilibrium and the distribution of conformations sampled along folding trajectories. Consistent with theory and single-molecule experiment, duration of the folding transition paths exhibits only a weak correlation with overall folding time. Conformational distributions of folding trajectories near the overall thermodynamic folding/unfolding barrier show significant deviations from preequilibrium. These deviations, the distribution of transition path times, and the variation of mean transition path time for different proteins can all be rationalized by a diffusive process that we modeled using simple Monte Carlo algorithms with an effective coordinate-independent diffusion coefficient. Conformations in the initial stages of transition paths tend to form more nonlocal contacts than typical conformations with the same number of native contacts. This statistical bias, which is indicative of preferred folding pathways, should be amenable to future single-molecule measurements. We found that the preexponential factor defined in the transition state theory of folding varies from protein to protein and that this variation can be rationalized by our Monte Carlo diffusion model. Thus, protein folding physics is different in certain fundamental respects from the physics envisioned by a simple transition-state picture. Nonetheless, transition state theory can be a useful approximate predictor of cooperative folding speed, because the height of the overall folding barrier is apparently a proxy for related rate-determining physical properties.Protein folding is an intriguing phenomenon at the interface of physics and biology. In the early days of folding kinetics studies, folding was formulated almost exclusively in terms of mass-action rate equations connecting the folded, unfolded, and possibly, one or a few intermediate states (1, 2). With the advent of site-directed mutagenesis, the concept of free energy barriers from transition state theory (TST) (3) was introduced to interpret mutational data (4), and subsequently, it was adopted for the Φ-value analysis (5). Since the 1990s, the availability of more detailed experimental data (6), in conjunction with computational development of coarse-grained chain models, has led to an energy landscape picture of folding (7–15). This perspective emphasizes the diversity of microscopic folding trajectories, and it conceptualizes folding as a diffusive process (16–25) akin to the theory of Kramers (26).For two-state-like folding, the transition path (TP), i.e., the sequence of kinetic events that leads directly from the unfolded state to the folded state (27, 28), constitutes only a tiny fraction of a folding trajectory that spends most of the time diffusing, seemingly unproductively, in the vicinity of the free energy minimum of the unfolded state. The development of ultrafast laser spectroscopy (29, 30) and single-molecule (27, 28, 31) techniques have made it possible to establish upper bounds on the transition path time (t_TP) ranging from <200 and <10 μs by earlier (27) and more recent (28), respectively, direct single-molecule FRET to <2 μs (30) by bulk relaxation measurements. Consistent with these observations, recent extensive atomic simulations have also provided estimated t_TP values of the order of ∼1 μs (32, 33). These advances offer exciting prospects of characterizing the productive events along folding TPs.It is timely, therefore, to further the theoretical investigation of TP-related questions (19). To this end, we used coarse-grained C_α models (14) to perform extensive simulations of the folding trajectories of small proteins with 56- to 86-aa residues. These tractable models are useful, because despite significant progress, current atomic models cannot provide the same degree of sampling coverage for proteins of comparable sizes (32, 33). In addition to structural insights, this study provides previously unexplored vantage points to compare the diffusion and TST pictures of folding. Deviations of folding behaviors from TST predictions are not unexpected, because TST is mostly applicable to simple gas reactions; however, the nature and extent of the deviations have not been much explored. Our explicit-chain simulation data conform well to the diffusion picture but not as well to TST. In particular, the preexponential factors of the simulated folding rates exhibit a small but appreciable variation that depends on native topology. These findings and others reported below underscore the importance of single-molecule measurements (13, 27, 28, 31, 34, 35) in assessing the merits of proposed scenarios and organizing principles of folding (7–25, 36, 37).     

Allosteric activation of M4 muscarinic receptors improve behavioral and physiological alterations in early symptomatic YAC128 mice

Mutations that lead to Huntington’s disease (HD) result in increased transmission at glutamatergic corticostriatal synapses at early presymptomatic stages that have been postulated to set the stage for pathological changes and symptoms that are observed at later ages. Based on this, pharmacological interventions that reverse excessive corticostriatal transmission may provide a novel approach for reducing early physiological changes and motor symptoms observed in HD. We report that activation of the M₄ subtype of muscarinic acetylcholine receptor reduces transmission at corticostriatal synapses and that this effect is dramatically enhanced in presymptomatic YAC128 HD and BACHD relative to wild-type mice. Furthermore, chronic administration of a novel highly selective M₄ positive allosteric modulator (PAM) beginning at presymptomatic ages improves motor and synaptic deficits in 5-mo-old YAC128 mice. These data raise the exciting possibility that selective M₄ PAMs could provide a therapeutic strategy for the treatment of HD.Huntington’s disease (HD) is a rare and fatal neurodegenerative disease caused by an expansion of a CAG triplet repeat in Htt, the gene that encodes for the protein huntingtin (1, 2). HD is characterized by a prediagnostic phase that includes subtle changes in personality, cognition, and motor function, followed by a more severe symptomatic stage initially characterized by hyperkinesia (chorea), motor incoordination, deterioration of cognitive abilities, and psychiatric symptoms. At later stages of disease progression, patients experience dystonia, rigidity, and bradykinesia, and ultimately death (3–7). The cortex and striatum are the most severely affected brain regions in HD and, interestingly, an increasing number of reports suggest that alterations in cortical and striatal physiology are present in prediagnostic individuals and in young HD mice (6–16).Striatal spiny projection neurons (SPNs) receive large glutamatergic inputs from the cortex and thalamus, as well as dopaminergic innervation from the substantia nigra. In the healthy striatum, the interplay of these neurotransmitters coordinates the activity of SPNs and striatal interneurons, regulating motor planning and execution as well as cognition and motivation (17, 18). Htt mutations lead to an early increase in striatal glutamatergic transmission, which begins during the asymptomatic phase of HD (12–14) and could contribute to synaptic changes observed in later stages of HD (19, 20). Based on this, pharmacological agents that reduce excitatory transmission in the striatum could reduce or prevent the progression of alterations in striatal synaptic function and behavior observed in symptomatic stages of HD.Muscarinic acetylcholine receptors (mAChRs), particularly M₄, can inhibit transmission at corticostriatal synapses (21–25). Therefore, it is possible that selective activation of specific mAChR subtypes could normalize excessive corticostriatal transmission in HD. Interestingly, previous studies also suggest that HD is associated with alterations of striatal cholinergic markers, including mAChRs (26–29). We now provide exciting new evidence that M₄-mediated control of corticostriatal transmission is increased in young asymptomatic HD mice and that M₄ positive allosteric modulators (PAMs) may represent a new treatment strategy for normalizing early changes in corticostriatal transmission and reducing the progression of HD.     

Kinesin processivity is gated by phosphate release

Kinesin-1 is a dimeric motor protein, central to intracellular transport, that steps hand-over-hand toward the microtubule (MT) plus-end, hydrolyzing one ATP molecule per step. Its remarkable processivity is critical for ferrying cargo within the cell: over 100 successive steps are taken, on average, before dissociation from the MT. Despite considerable work, it is not understood which features coordinate, or “gate,” the mechanochemical cycles of the two motor heads. Here, we show that kinesin dissociation occurs subsequent to, or concomitant with, phosphate (P_i) release following ATP hydrolysis. In optical trapping experiments, we found that increasing the steady-state population of the posthydrolysis ADP·P_i state (by adding free P_i) nearly doubled the kinesin run length, whereas reducing either the ATP binding rate or hydrolysis rate had no effect. The data suggest that, during processive movement, tethered-head binding occurs subsequent to hydrolysis, rather than immediately after ATP binding, as commonly suggested. The structural change driving motility, thought to be neck linker docking, is therefore completed only upon hydrolysis, and not ATP binding. Our results offer additional insights into gating mechanisms and suggest revisions to prevailing models of the kinesin reaction cycle.Since its discovery nearly 30 years ago (1), kinesin-1—the founding member of the kinesin protein superfamily—has emerged as an important model system for studying biological motors (2, 3). During “hand-over-hand” stepping, kinesin dimers alternate between a two–heads-bound (2-HB) state, with both heads attached to the microtubule (MT), and a one–head-bound (1-HB) state, where a single head, termed the tethered head, remains free of the MT (4, 5). The catalytic cycles of the two heads are maintained out of phase by a series of gating mechanisms, thereby enabling the dimer to complete, on average, over 100 steps before dissociating from the MT (6–8). A key structural element for this coordination is the neck linker (NL), a ∼14-aa segment that connects each catalytic head to a common stalk (9). In the 1-HB state, nucleotide binding is thought to induce a structural reconfiguration of the NL, immobilizing it against the MT-bound catalytic domain (2, 3, 10–17). This transition, called “NL docking,” is believed to promote unidirectional motility by biasing the position of the tethered head toward the next MT binding site (2, 3, 10–17). The completion of an 8.2-nm step (18) entails the binding of this tethered head to the MT, ATP hydrolysis, and detachment of the trailing head, thereby returning the motor to the ATP-waiting state (2, 3, 10–17). Prevailing models of the kinesin mechanochemical cycle (2, 3, 10, 14, 15, 17), which invoke NL docking upon ATP binding, explain the highly directional nature of kinesin motility and offer a compelling outline of the sequence of events following ATP binding. Nevertheless, these abstractions do not speak directly to the branching transitions that determine whether kinesin dissociates from the MT (off-pathway) or continues its processive reaction cycle (on-pathway). The distance moved by an individual motor before dissociating—the run length—is limited by unbinding from the MT. The propensity for a dimer to unbind involves a competition among multiple, force-dependent transitions in the two heads, which are not readily characterized by traditional structural or bulk biochemical approaches. Here, we implemented high-resolution single-molecule optical trapping techniques to determine transitions in the kinesin cycle that govern processivity.     

From the Cover: Why the proportion of transmission during early-stage HIV infection does not predict the long-term impact of treatment on HIV incidence

Antiretroviral therapy (ART) reduces the infectiousness of HIV-infected persons, but only after testing, linkage to care, and successful viral suppression. Thus, a large proportion of HIV transmission during a period of high infectiousness in the first few months after infection (“early transmission”) is perceived as a threat to the impact of HIV “treatment-as-prevention” strategies. We created a mathematical model of a heterosexual HIV epidemic to investigate how the proportion of early transmission affects the impact of ART on reducing HIV incidence. The model includes stages of HIV infection, flexible sexual mixing, and changes in risk behavior over the epidemic. The model was calibrated to HIV prevalence data from South Africa using a Bayesian framework. Immediately after ART was introduced, more early transmission was associated with a smaller reduction in HIV incidence rate—consistent with the concern that a large amount of early transmission reduces the impact of treatment on incidence. However, the proportion of early transmission was not strongly related to the long-term reduction in incidence. This was because more early transmission resulted in a shorter generation time, in which case lower values for the basic reproductive number (R₀) are consistent with observed epidemic growth, and R₀ was negatively correlated with long-term intervention impact. The fraction of early transmission depends on biological factors, behavioral patterns, and epidemic stage and alone does not predict long-term intervention impacts. However, early transmission may be an important determinant in the outcome of short-term trials and evaluation of programs.Recent studies have confirmed that effective antiretroviral therapy (ART) reduces the transmission of HIV among stable heterosexual couples (1–3). This finding has generated interest in understanding the population-level impact of HIV treatment on reducing the rate of new HIV infections in generalized epidemic settings (4). Research, including mathematical modeling (5–10), implementation research (11), and major randomized controlled trials (12–14), are focused on how ART provision might be expanded strategically to maximize its public health benefits (15, 16).One concern is that if a large fraction of HIV transmission occurs shortly after a person becomes infected, before the person can be diagnosed and initiated on ART, this will limit the potential impact of HIV treatment on reducing HIV incidence (9, 17, 18). Data suggest that persons are more infectious during a short period of “early infection” after becoming infected with HIV (19–22), although there is debate about the extent, duration, and determinants of elevated infectiousness (18, 23). The amount of transmission that occurs also will depend on patterns of sexual behavior and sexual networks (17, 24–27). There have been estimates for the contribution of early infection to transmission from mathematical models (7, 17, 21, 24–26) and phylogenetic analyses (28–31), but these vary widely, from 5% to above 50% (23).In this study, we use a mathematical model to quantify how the proportion of transmission that comes from persons who have been infected recently affects the impact of treatment scale-up on HIV incidence. The model is calibrated to longitudinal HIV prevalence data from South Africa using a Bayesian framework. Thus, the model accounts for not only the early epidemic growth rate highlighted in previous research (5, 9, 18), but also the heterogeneity and sexual behavior change to explain the peak and decline in HIV incidence observed in sub-Saharan African HIV epidemics (32, 33).The model calibration allows uncertainty about factors that determine the amount of early transmission, including the relative infectiousness during early infection, heterogeneity in propensity for sexual risk behavior, assortativity in sexual partner selection, reduction in risk propensity over the life course, and population-wide reductions in risk behavior in response to the epidemic (32, 33). This results in multiple combinations of parameter values that are consistent with the observed epidemic and variation in the amount of early transmission. We simulated the impact of a treatment intervention and report how the proportion of early transmission correlates with the reduction in HIV incidence from the intervention over the short- and long-term.     

Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate

cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO₃⁻); it thereby serves as a cellular sensor for HCO₃⁻, carbon dioxide (CO₂), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO₃⁻ binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO₃⁻ activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO₃⁻ sensing and a basis for targeting this system with drugs.The ubiquitous second messenger cAMP regulates diverse physiological processes, from fungal virulence to mammalian brain function (1, 2). In mammals, cAMP can be generated by any of 10 differently expressed and regulated adenylyl cyclases (ACs): nine transmembrane enzymes (tmACs) and one soluble AC (sAC) (3). TmACs reside in the cell membrane, where they mediate cellular responses to hormones acting through G protein-coupled receptors (4). In contrast, sAC functions in various intracellular locations, providing cell-specific spatial and temporal patterns of cAMP (5–7) in response to intracellular signals, including calcium, ATP, and bicarbonate (HCO₃⁻) (3, 8–10). HCO₃⁻ regulation of sAC enzymes is a direct effect on their catalytic domains and is conserved across bacterial, fungal, and animal kingdoms (1, 11–13). Via modulation of sAC, and sAC-like cyclase activities, HCO₃⁻ serves as an evolutionarily conserved signaling molecule mediating cellular responses to HCO₃⁻, CO₂, and pH (3, 14). In mammals, sAC acts as a CO₂/HCO₃⁻/pH sensor in processes such as sperm activation (15), acid-base homeostasis (16), and various metabolic responses (10, 17, 18). sAC has also been implicated in skin and prostate cancer and as a target for male contraceptives (19–21).All mammalian ACs are class III nucleotidyl cyclases sharing homologous catalytic domains. Their catalytic cores are formed through symmetrical or pseudosymmetrical association of two identical or highly similar catalytic domains, C₁ and C₂ (22–24); in mammalian ACs, both domains reside on a single polypeptide chain. Such C₁C₂ pseudoheterodimers form two pseudosymmetrical sites at the dimer interface: the active site and a degenerated, inactive pocket (3, 23). A conserved Lys and an Asp/Thr in the active site recognize the base of the substrate ATP, and two conserved Asp residues bind two divalent cations, normally Mg²⁺ (23). The ions, called ion A and ion B, coordinate the substrate phosphates and support the intramolecular 3′-hydroxyl (3′-OH) attack at the α-phosphorous to form cAMP and pyrophosphate (PP_i) (3). In tmACs, the degenerate site binds forskolin (24), a plant diterpene that activates tmACs but has no effect on sAC (25). The forskolin activation mechanism and the existence of physiological ligands for this site in tmACs or in sAC remain unclear.There are two sAC isoforms known to be generated by alternative splicing (26). Full-length sAC comprises N-terminal catalytic domains along with ∼1,100 residues with a little understood function except for an autoinhibitory motif and a heme-binding domain (3, 27, 28). Exclusion of exon 12 (26) generates a truncated isoform, sAC_t (residues 1–490), which comprises just the two sAC catalytic domains (sAC-cat) (25). sAC_t is widely expressed, and it is the isoform most extensively biochemically characterized (3, 8, 11). It is directly activated by Ca²⁺ and HCO₃⁻; Ca²⁺ supports substrate binding, and HCO₃⁻ increases turnover and relieves substrate inhibition (8), and this regulation is conserved in sAC-like enzymes from Cyanobacteria to humans (3, 13, 29). In a homodimeric, HCO₃⁻-regulated sAC homolog from Spirulina platensis, adenylyl cyclase C (CyaC), HCO₃⁻ appeared to facilitate an active site closure required for catalysis (13), but the HCO₃⁻ binding site and its mechanism of activation remained unknown.Here, we present crystal structures of the human sAC-cat in apo form and in complex with substrate, products, bicarbonate, and a pharmacological inhibitor. The structures reveal insights into binding sites and mechanisms for sAC catalysis and for its regulation by physiological and pharmacological small molecules.     

Electrochemical tuning of vertically aligned MoS2 nanofilms and its application in improving hydrogen evolution reaction

The ability to intercalate guest species into the van der Waals gap of 2D layered materials affords the opportunity to engineer the electronic structures for a variety of applications. Here we demonstrate the continuous tuning of layer vertically aligned MoS₂ nanofilms through electrochemical intercalation of Li⁺ ions. By scanning the Li intercalation potential from high to low, we have gained control of multiple important material properties in a continuous manner, including tuning the oxidation state of Mo, the transition of semiconducting 2H to metallic 1T phase, and expanding the van der Waals gap until exfoliation. Using such nanofilms after different degree of Li intercalation, we show the significant improvement of the hydrogen evolution reaction activity. A strong correlation between such tunable material properties and hydrogen evolution reaction activity is established. This work provides an intriguing and effective approach on tuning electronic structures for optimizing the catalytic activity.Layer-structured 2D materials are an interesting family of materials with strong covalent bonding within molecular layers and weak van der Waals interaction between layers. Beyond intensively studied graphene-related materials (1–4), there has been recent strong interest in other layered materials whose vertical thickness can be thinned down to less than few nanometers and horizontal width can also be reduced to nanoscale (5–9). The strong interest is driven by their interesting physical and chemical properties (2, 10) and their potential applications in transistors, batteries, topological insulators, thermoelectrics, artificial photosynthesis, and catalysis (4, 11–25).One of the unique properties of 2D layered materials is their ability to intercalate guest species into their van der Waals gaps, opening up the opportunities to tune the properties of materials. For example, the spacing between the 2D layers could be increased by intercalation such as lithium (Li) intercalated graphite or molybdenum disulfide (MoS₂) and copper intercalated bismuth selenide (26–29). The electronic structures of the host lattice, such as the charge density, anisotropic transport, oxidation state, and phase transition, may also be changed by different species intercalation (26, 27).As one of the most interesting layered materials, MoS₂ has been extensively studied in a variety of areas such as electrocatalysis (20–22, 30–36). It is known that there is a strong correlation between the electronic structure and catalytic activity of the catalysts (20, 37–41). It is intriguing to continuously tune the morphology and electronic structure of MoS₂ and explore the effects on MoS₂ hydrogen evolution reaction (HER) activity. Very recent studies demonstrated that the monolayered MoS₂ and WS₂ nanosheets with 1T metallic phase synthesized by chemical exfoliation exhibited superior HER catalytic activity to those with 2H semiconducting phase (35, 42), with a possible explanation that the strained 1T phase facilitates the hydrogen binding process during HER (42). However, it only offers two end states of materials and does not offer a continuous tuning. A systematic investigation to correlate the gradually tuned electronic structure, including oxidation state shift and semiconducting–metallic phase transition, and the corresponding HER activity is important but unexplored. We believe that the Li electrochemical intercalation method offers a unique way to tune the catalysts for optimization.In this paper, we demonstrate that the layer spacing, oxidation state, and the ratio of 2H semiconducting to 1T metallic phase of MoS₂ HER catalysts were continuously tuned by Li intercalation to different voltages vs. Li⁺/Li in nanofilms with molecular layers perpendicular to the substrates. Correspondingly, the catalytic activity for HER was observed to be continuously tuned. The lower oxidation state of Mo and 1T metallic phase of MoS₂ turn out to have better HER catalytic activities. The performance of MoS₂ catalyst on both flat and 3D electrodes was dramatically improved when it was discharged to low potentials vs. Li⁺/Li.     

Potentiation of cytotoxic chemotherapy by growth hormone-releasing hormone agonists

The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFβ. Proteomic and gene-expression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and anti-invasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.Glioblastoma multiforme (GBM) is one of the most aggressive human cancers, and the afflicted patients inevitably succumb. The dismal outcome of this malignancy demands great efforts to find improved methods of treatment (1). Many compounds have been synthesized in our laboratory in the past few years that have proven to be effective against diverse malignant tumors (2–14). These are peptide analogs of hypothalamic hormones: luteinizing hormone-releasing hormone (LHRH), growth hormone-releasing hormone (GHRH), somatostatin, and analogs of other neuropeptides such as bombesin and gastrin-releasing peptide. The receptors for these peptides have been found to be widely distributed in the human body, including in many types of cancers (2–14). The regulatory functions of these hypothalamic hormones and other neuropeptides are not confined to the hypothalamo–hypophyseal system or, even more broadly, to the central nervous system (CNS). In particular, GHRH can induce the differentiation of ovarian granulosa cells and other cells in the reproductive system and function as a growth factor in various normal tissues, benign tumors, and malignancies (2–4, 6, 11, 14–18). Previously, we also reported that antagonistic cytototoxic derivatives of some of these neuropeptides are able to inhibit the growth of several malignant cell lines (2–14).Our earlier studies showed that treatment with antagonists of LHRH or GHRH rarely effects complete regression of glioblastoma-derived tumors (5, 7, 10, 11). Previous studies also suggested that growth factors such as EGF or agonistic analogs of LHRH serving as carriers for cytotoxic analogs and functioning as growth factors may sensitize cancer cells to cytotoxic treatments (10, 19) through the activation of maturation processes. We therefore hypothesized that pretreatment with one of our GHRH agonists, such as JI-34 (20), which has shown effects on growth and differentiation in other cell lines (17, 18, 21, 22), might decrease the pluripotency and the adaptability of GBM cells and thereby increase their susceptibility to cytotoxic treatment.In vivo, tumor cells were implanted into athymic nude mice, tumor growth was recorded weekly, and final tumor mass was measured upon autopsy. In vitro, proliferation assays were used for the determination of neoplastic proliferation and cell growth. Changes in stem (nestin) and maturation (GFAP) antigen expression was evaluated with Western blot studies in vivo and with immunocytochemistry in vitro. The production of glial growth factors (FGF basic, TGFβ) was verified by ELISA. Further, using the Human Cancer Pathway Finder real-time quantitative PCR, numerous genes that play a role in the development of cancer were evaluated. We placed particular emphasis on the measurement of apoptosis, using the ApoLive-Glo Multiplex Assay kit and by detection of the expression of the proapoptotic p53 protein. This overall approach permitted the evaluation of the effect of GHRH agonist, JI-34, on the response to chemotherapy with doxorubicin.     

Predictive Value of Brain Natriuretic Peptides in Patients on Peritoneal Dialysis: Results from the ADEMEX Trial

Background and objectives: Natriuretic peptides have been suggested to be of value in risk stratification in dialysis patients. Data in patients on peritoneal dialysis remain limited.Design, setting, participants, & measurements: Patients of the ADEMEX trial (ADEquacy of peritoneal dialysis in MEXico) were randomized to a control group [standard 4 × 2L continuous ambulatory peritoneal dialysis (CAPD); n = 484] and an intervention group (CAPD with a target creatinine clearance ≥60L/wk/1.73 m²; n = 481). Natriuretic peptides were measured at baseline and correlated with other parameters as well as evaluated for effects on patient outcomes.Results: Control group and intervention group were comparable at baseline with respect to all measured parameters. Baseline values of natriuretic peptides were elevated and correlated significantly with levels of residual renal function but not with body size or diabetes. Baseline values of N-terminal fragment of B-type natriuretic peptide (NT-proBNP) but not proANP(1–30), proANP(31–67), or proANP(1–98) were independently highly predictive of overall survival and cardiovascular mortality. Volume removal was also significantly correlated with patient survival.Conclusions. NT-proBNP have a significant predictive value for survival of CAPD patients and may be of value in guiding risk stratification and potentially targeted therapeutic interventions.Plasma levels of cardiac natriuretic peptides are elevated in patients with chronic kidney disease, owing to impairment of renal function, hypertension, hypervolemia, and/or concomitant heart disease (1–7). Atrial natriuretic peptide (ANP) and particularly brain natriuretic peptide (BNP) levels are linked independently to left ventricular mass (3–5,8–16) and function (3,6–17) and predict total and cardiovascular mortality (1,3,8,10,12,18) as well as cardiac events (12,19). ANP and BNP decrease significantly during hemodialysis treatment but increase again during the interdialytic interval (1,2,4,6,7,14,17,20–23). Levels in patients on peritoneal dialysis (PD) have been found to be lower than in patients on hemodialysis (11,24–26), but the correlations with left ventricular function and structure are maintained in both types of dialysis modalities (11,15,27,28).The high mortality of patients on peritoneal dialysis and the failure of dialytic interventions to alter this mortality (29,30) necessitate renewed attention into novel methods of stratification and identification of patients at highest risk to be targeted for specific interventions. Cardiac natriuretic peptides are increasingly considered to fulfill this role in nonrenal patients. Evaluations of cardiac natriuretic peptides in patients on PD have been limited by small numbers (3,9,11,12,15,24–26) and only one study examined correlations between natriuretic peptide levels and outcomes (12). The PD population enrolled in the ADEMEX trial offered us the opportunity to evaluate cardiac natriuretic peptides and their value in predicting outcomes in the largest clinical trial ever performed on PD (29,30). It is hoped that such an evaluation would identify patients at risk even in the absence of overt clinical disease and hence facilitate or encourage interventions with salutary outcomes.     

Major diversification of voltage-gated K+ channels occurred in ancestral parahoxozoans

We examined the origins and functional evolution of the Shaker and KCNQ families of voltage-gated K⁺ channels to better understand how neuronal excitability evolved. In bilaterians, the Shaker family consists of four functionally distinct gene families (Shaker, Shab, Shal, and Shaw) that share a subunit structure consisting of a voltage-gated K⁺ channel motif coupled to a cytoplasmic domain that mediates subfamily-exclusive assembly (T1). We traced the origin of this unique Shaker subunit structure to a common ancestor of ctenophores and parahoxozoans (cnidarians, bilaterians, and placozoans). Thus, the Shaker family is metazoan specific but is likely to have evolved in a basal metazoan. Phylogenetic analysis suggested that the Shaker subfamily could predate the divergence of ctenophores and parahoxozoans, but that the Shab, Shal, and Shaw subfamilies are parahoxozoan specific. In support of this, putative ctenophore Shaker subfamily channel subunits coassembled with cnidarian and mouse Shaker subunits, but not with cnidarian Shab, Shal, or Shaw subunits. The KCNQ family, which has a distinct subunit structure, also appears solely within the parahoxozoan lineage. Functional analysis indicated that the characteristic properties of Shaker, Shab, Shal, Shaw, and KCNQ currents evolved before the divergence of cnidarians and bilaterians. These results show that a major diversification of voltage-gated K⁺ channels occurred in ancestral parahoxozoans and imply that many fundamental mechanisms for the regulation of action potential propagation evolved at this time. Our results further suggest that there are likely to be substantial differences in the regulation of neuronal excitability between ctenophores and parahoxozoans.Voltage-gated K⁺ channels are highly conserved among bilaterian metazoans and play a central role in the regulation of excitation in neurons and muscle. Understanding the functional evolution of these channels may therefore provide important insights into how neuromuscular excitation evolved within the Metazoa. Three major gene families, Shaker, KCNQ, and Ether-a-go-go (EAG) encode all voltage-gated K⁺ channels in bilaterians (1, 2). In this study, we examine the functional evolution and origins of the Shaker and KCNQ gene families. Shaker family channels can be definitively identified by a unique subunit structure that includes both a voltage-gated K⁺ channel core and a family-specific cytoplasmic domain within the N terminus known as the T1 domain. T1 mediates assembly of Shaker family subunits into functional tetrameric channels (3, 4). KCNQ channels are also tetrameric but lack a T1 domain and use a distinct coiled-coil assembly domain in the C terminus (5, 6). KCNQ channels can be identified by the presence of this family-specific assembly motif and high amino acid conservation within the K⁺ channel core. Both channel families are found in cnidarians (1, 7) and thus predate the divergence of cnidarians and bilaterians, but their ultimate evolutionary origins have not yet been defined.Shaker family K⁺ channels serve diverse roles in the regulation of neuronal firing and can be divided into four gene subfamilies based on function and sequence homology: Shaker, Shab, Shal, and Shaw (8, 9). The T1 assembly domain is only compatible between subunits from the same gene subfamily (4, 10) and thus serves to keep the subfamilies functionally segregated. Shaker subfamily channels activate rapidly near action potential threshold and range from rapidly inactivating to noninactivating. Multiple roles for Shaker channels in neurons and muscles have been described, but their most unique and fundamental role may be that of axonal action potential repolarization. Shaker channels are clustered to the axon initial segment and nodes of Ranvier in vertebrate neurons (11–13) and underlie the delayed rectifier in squid giant axons (14). The Shaker subfamily is diverse in cnidarians (15, 16), and the starlet sea anemone Nematostella vectensis has functional orthologs of most identified Shaker current types observed in bilaterians (16).The Shab and Shal gene subfamilies encode somatodendritic delayed rectifiers and A currents, respectively (17–20). Shab channels are important for maintaining sustained firing (21, 22), whereas the Kv4-based A current modulates spike threshold and frequency (17). Shab and Shal channels are present in cnidarians, but cnidarian Shab channels have not been functionally characterized, and the only cnidarian Shal channels expressed to date display atypical voltage dependence and kinetics compared with bilaterian channels (23). Shaw channels are rapid, high-threshold channels specialized for sustaining fast firing in vertebrates (24, 25) but have a low activation threshold and may contribute to resting potential in Drosophila (19, 26, 27). A Caenorhabditis elegans Shaw has slow kinetics but a high activation threshold (28), and a single expressed cnidarian Shaw channel has the opposite: a low activation threshold but relatively fast kinetics (29). Thus, the ancestral properties and function of Shaw channels is not yet understood. Further functional characterization of cnidarian Shab, Shal, and Shaw channels would provide a better understanding of the evolutionary status of the Shaker family in early parahoxozoans.KCNQ family channels underlie the M current in vertebrate neurons (30) that regulates subthreshold excitability (31). The M current provides a fundamental mechanism for regulation of firing threshold through the Gq G-protein pathway because KCNQ channels require phosphatidylinositol 4,5-bisphosphate (PIP₂) for activation (32, 33). PIP₂ hydrolysis and subsequent KCNQ channel closure initiated by Gq-coupled receptors produces slow excitatory postsynaptic potentials, during which the probability of firing is greatly increased (32, 33). The key functional adaptations of KCNQ channels for this physiological role that can be observed in vitro are (i) a requirement for PIP₂ to couple voltage-sensor activation to pore opening (34, 35), and (ii) a hyperpolarized voltage–activation curve that allows channels to open below typical action potential thresholds. Both key features are found in vertebrate (30, 34, 36–38), Drosophila (39), and C. elegans (40) KCNQ channels, suggesting they may have been present in KCNQ channels in a bilaterian ancestor. Evolution of the M current likely represented a major advance in the ability to modulate the activity of neuronal circuits, but it is not yet clear when PIP₂-dependent KCNQ channels first evolved.Here, we examine the origins and functional evolution of the Shaker and KCNQ gene families. If we assume the evolution of neuronal signaling provided a major selective pressure for the functional diversification of voltage-gated K⁺ channels, then we can hypothesize that the appearance of these gene families might accompany the emergence of the first nervous systems or a major event in nervous system evolution. Recent phylogenies that place the divergence of ctenophores near the root of the metazoan tree suggest that the first nervous systems, or at least the capacity to make neurons, may have been present in a basal metazoan ancestor (41–43) (Fig. S1). One hypothesis then is that much of the diversity of metazoan voltage-gated channels should be shared between ctenophores and parahoxozoans [cnidarians, bilaterians, and placozoans (44)]. However, genome analysis indicates that many “typical” neuronal genes are missing in ctenophores and the sponges lack a nervous system, leading to the suggestion that extant nervous systems may have evolved independently in ctenophores and parahoxozoans (42, 45). Thus, a second hypothesis is that important steps in voltage-gated K⁺ channel evolution might have occurred separately in ctenophores and parahoxozoans. We tested these hypotheses by carefully examining the phylogenetic distribution and functional evolution of Shaker and KCNQ family K⁺ channels. Our results support a model in which major innovations in neuromuscular excitability occurred specifically within the parahoxozoan lineage.