Muscarinic regulation of dopamine and glutamate transmission in the nucleus accumbens

Cholinergic transmission in the striatum functions as a key modulator of dopamine (DA) transmission and synaptic plasticity, both of which are required for reward and motor learning. Acetylcholine (ACh) can elicit striatal DA release through activation of nicotinic ACh receptors (nAChRs) on DA axonal projections. However, it remains controversial how muscarinic ACh receptors (mAChRs) modulate striatal DA release, with studies reporting both potentiation and depression of striatal DA transmission by mAChR agonists. This study investigates the mAChR-mediated regulation of release from three types of midbrain neurons that project to striatum: DA, DA/glutamate, and glutamate neurons. We found that M₅ mAChRs potentiate DA and glutamate release only from DA and DA/glutamate projections from the midbrain. We also show that M₂/M₄ mAChRs depress the nAChR-dependent mechanism of DA release in the striatum. These results suggest that M₅ receptors on DA neuron terminals enhance DA release, whereas M₂/M₄ autoreceptors on cholinergic terminals inhibit ACh release and subsequent nAChR-dependent DA release. Our findings clarify the mechanisms of mAChR-dependent modulation of DA and glutamate transmission in the striatum.Increases in dopamine (DA) concentration in the nucleus accumbens (NAc) are required for reward seeking, motivation, and motor control (1). DA is released from the axonal projections of midbrain DA neurons to the NAc and the dorsal striatum, where it regulates synaptic plasticity to influence striatal microcircuitry (2). Traditionally, the firing of DA neurons was thought to be the main factor controlling DA release in the striatum. However, DA release can also be evoked by activation of neurons within the striatum. Synchronous activation of striatal cholinergic interneurons (CINs) is sufficient to trigger DA release in the NAc (3) and dorsal striatum (4) through activation of nicotinic acetylcholine receptors (nAChRs) on DA axonal projections. Thus, nAChRs exert powerful control over DA transmission in the striatum.Muscarinic acetylcholine receptors (mAChRs) are also known to modulate striatal DA transmission. Earlier studies show that muscarinic agonists potentiate DA efflux in the striatum (5–8). However, other studies reported that mAChR agonists depress DA transients measured with fast-scan cyclic voltammetry (FSCV) and evoked by electrical stimulation in the striatum (9–11). Thus, the role of mAChRs in modulating striatal DA transmission remains controversial and unclear. In this study, we address this issue by systematically investigating the effect of muscarinic agonists and endogenous acetylcholine (ACh) on DA transmission evoked by classical methods (electrical stimulation), as well as the selective stimulation of DA neuron axonal projections (optogenetic stimulation), using electrochemical and electrophysiological recordings.Different mAChR subtypes have been detected in the striatum (12, 13). G_i/o-coupled M₂ and M₄ receptors are mainly found on presynaptic terminals of CINs (14, 15), and M₂ on glutamatergic terminals (13), where they inhibit neurotransmitter release. G_q/11-coupled M₁ receptors are expressed in spiny projection neurons and have been implicated in endocannabinoid production (16). Interestingly, the G_q/11-coupled M₅ receptors are the only mAChRs subtype found in midbrain DA neurons (12, 17). Although direct anatomic evidence for the presence of M₅ receptors in DA axons is lacking, functional studies implicate M₅ mAChRs in either facilitating (8, 10) or depressing (11) DA release. In the past, studies in this field have been hampered by the lack of selective muscarinic agonists and antagonists (18). To overcome this limitation, this study takes advantage of mutant mouse lines carrying genetic deletions of selected mAChR subtypes (19) to determine their specific role in modulating DA transmission in the striatum. Our findings are highly relevant for the development of novel mAChR subtype-selective drugs to modulate dopaminergic signaling for therapeutic purposes.     

Regional specialization within the human striatum for diverse psychological functions

Decades of animal and human neuroimaging research have identified distinct, but overlapping, striatal zones, which are interconnected with separable corticostriatal circuits, and are crucial for the organization of functional systems. Despite continuous efforts to subdivide the human striatum based on anatomical and resting-state functional connectivity, characterizing the different psychological processes related to each zone remains a work in progress. Using an unbiased, data-driven approach, we analyzed large-scale coactivation data from 5,809 human imaging studies. We (i) identified five distinct striatal zones that exhibited discrete patterns of coactivation with cortical brain regions across distinct psychological processes and (ii) identified the different psychological processes associated with each zone. We found that the reported pattern of cortical activation reliably predicted which striatal zone was most strongly activated. Critically, activation in each functional zone could be associated with distinct psychological processes directly, rather than inferred indirectly from psychological functions attributed to associated cortices. Consistent with well-established findings, we found an association of the ventral striatum (VS) with reward processing. Confirming less well-established findings, the VS and adjacent anterior caudate were associated with evaluating the value of rewards and actions, respectively. Furthermore, our results confirmed a sometimes overlooked specialization of the posterior caudate nucleus for executive functions, often considered the exclusive domain of frontoparietal cortical circuits. Our findings provide a precise functional map of regional specialization within the human striatum, both in terms of the differential cortical regions and psychological functions associated with each striatal zone.In addition to its central role in selecting, planning, and executing motor behavior (1, 2), the human striatum has been reported to be involved in diverse psychological functions, including emotion generation and regulation (3, 4), reward-related processes and decision making (5, 6), and executive functions (7, 8). These discrete functions are thought to map onto distinct functional striatal zones, which participate in separable basal ganglia-thalamocortical circuits (9–12) and are critical for the organization of behavior.Following this logic, recent attempts to parcellate the human striatum using diffusion tensor imaging (DTI) (13, 14) and resting-state functional connectivity (RSFC) (15–17) have relied on patterns of corticostriatal connectivity to identify striatal zones. Although very useful, these studies have been limited to inferring striatal function indirectly via psychological functions of connected cortical regions. In addition, it remains unclear how anatomical connections and RSFC map onto different psychological processes (18). Finally, RSFC is sometimes epiphenomenal (19), and fiber tract reconstruction with DTI is inaccurate for complex axonal projections underlying frontal corticostriatal connectivity (17).A separate body of work has attempted to differentiate striatal contributions directly to behavior empirically. However, these empirical investigations have focused on a restricted set of paradigms that may fail to capture the full range of striatal function, especially in humans. For example, converging evidence suggests a division of labor between the ventral striatum (VS) and dorsal striatum for Pavlovian and instrumental conditioning, respectively (20, 21). Within the dorsal striatum, medial regions support behavioral flexibility and lateral regions support well-learned behavior (22, 23). This work has greatly advanced our understanding of the similarities of striatal function in human and nonhuman animals. However, because of this strong reliance on classic learning paradigms, the integration of ideas about how the striatum is involved in uniquely human psychological functions, such as working memory, planning, and language, remains a work in progress.To generate a comprehensive and precise functional map of the human striatum, in terms of associations with both cortical brain regions and psychological tasks, we simultaneously analyzed corticostriatal coactivation patterns and the frequency of psychological terms in the full text of 5,809 neuroimaging studies (24). In contrast to studies of RSFC, we defined corticostriatal associations based on coactivation in task-related responses across studies. A cortical voxel and a striatal voxel were coactivated if a study reported activation in both voxels. This metric groups voxels that are associated with similar psychological processes. Similar to RSFC, this metric does not imply direct functional coupling of coactivated voxels. In contrast to existing work, we attempted to associate psychological functions with striatal areas directly, rather than inferring them indirectly based on the psychological functions of connected cortical areas. Sampling across a broad spectrum of neuroimaging studies, without regard for the psychological process under investigation, allowed a large-scale comparison of associations of striatal subregions with diverse psychological processes. This data-driven approach allowed us to (i) localize five striatal zones based on their coactivation with cortical brain areas and (ii) simultaneously characterize the association of each zone with psychological states in a relatively unbiased manner, including potential associations overlooked in both individual hypothesis-driven studies and studies of RSFC.     

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.     

Exogenous delivery of chaperonin subunit fragment ApiCCT1 modulates mutant Huntingtin cellular phenotypes

Aggregation of misfolded proteins is characteristic of a number of neurodegenerative diseases, including Huntington disease (HD). The CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring) chaperonin complex can inhibit aggregation and cellular toxicity induced by expanded repeat Huntingtin (mHtt) fragments. The substrate-binding apical domain of CCT/TRiC subunit CCT1, ApiCCT1, is sufficient to inhibit aggregation of expanded repeat mHtt fragments in vitro, providing therapeutic promise for HD. However, a key hurdle in considering ApiCCT1 as a potential treatment is in delivery. Because ApiCCT1 has a region of similarity to the HIV Tat protein cell-transduction domain, we tested whether recombinant ApiCCT1 (ApiCCT1_r) protein could enter cells following exogenous delivery and modulate an established panel of mHtt-mediated cell-based phenotypes. Cell fractionation studies demonstrate that exogenous ApiCCT1_r can penetrate cell membranes and can localize to the nucleus, consistent with a strategy that can target both cytosolic and nuclear pathogenic events in HD. ApiCCT1_r application does indeed modulate HD cellular phenotypes by decreasing formation of visible inclusions, fibrillar oligomers, and insoluble mHtt derived from expression of a truncated mHtt exon 1 fragment. ApiCCT1_r also delays the onset of inclusion body formation as visualized via live imaging. ApiCCT1_r reduces mHtt-mediated toxicity in immortalized striatal cells derived from full-length knock-in HD mice, suggesting that therapeutic benefit may extend beyond effects on aggregation. These studies provide the basis for a potentially robust and unique therapeutic strategy to target mHtt-mediated protein pathogenesis.Huntington disease (HD) is a devastating neurodegenerative disorder that strikes in midlife and is characterized by movement abnormalities, psychiatric symptoms, and cognitive deficits, as well as by the accumulation of pathogenic proteins and peptides (1–3). Currently no disease-modifying therapy is available. The disease is caused by an abnormal CAG repeat expansion in the HD gene, leading to the production of an expanded polyglutamine repeat in the amino terminal domain of the Huntingtin protein (Htt) (2, 3). A hallmark of HD is the propensity for the mutant protein (mHtt) to misfold and aggregate (1). While the connection between large fibrillar deposits and neurodegeneration is not clear, accumulation and aggregation of mHtt is likely to result from a deficit in cellular quality control machinery and can be used as a surrogate outcome measure for disease progression.Molecular chaperones, which are colocalized within mHtt inclusion bodies, suppress neurodegeneration in several animal models of protein misfolding diseases, including HD (4–7). For instance, overexpression of Hsp70 and Hsp40 suppresses neurodegeneration in animal models of polyglutamine diseases (8, 9). In parallel, deletion of Hsp70 markedly worsens pathogenesis in a mouse model of HD (10). Chaperones are thought to suppress the toxicity of disease-associated proteins through direct effects on their misfolding and clearance; however, the mechanisms are not well understood.The 1MDa CCT/TRiC (TCP1-ring complex) chaperonin is an ATP-dependent, ring-shaped hetero-oligomeric chaperone that binds and folds newly translated polypeptides (11). A genome-wide RNAi screen in Caenorhabditis elegans identified six of the eight subunits of CCT (1, 2, 4–7) as suppressors of polyglutamine aggregation (12). CCT/TRiC expression also prevents mHtt aggregation in cell and yeast models of HD (13–16). Further, overexpression of one subunit of the TRiC/CCT complex, CCT1, is sufficient to inhibit aggregation and reduce mHtt-mediated toxicity in mouse N2a neuronal cells (15). CCT/TRiC can also suppress aggregation in vitro, using purified components (15). Reduction to just the 20 kDa substrate-binding apical domain of yeast CCT1 (ApiCCT1) inhibits aggregation of recombinant mHtt in vitro, potentially through its ability to bind to the N-terminal 17 amino acid domain of Htt. This domain is involved in multiple Htt functions and in aggregation kinetics of mHtt (15). However, the efficacy of this apical domain has not yet been tested in HD cell models.Given the potential of ApiCCT1 as a therapeutic for the treatment of HD, we tested if recombinant yeast ApiCCT1 (ApiCCT1_r) protein could alter the aggregation and neurotoxicity of mHtt in selected proof-of-concept assays when purified protein was directly added to the culture media of cellular models of HD. We hypothesized that ApiCCT1_r could be directly taken up by cells given the potential similarity of a positively charged region in Api1 to the protein transduction domain of the HIV protein Tat. We establish here that exogenously delivered ApiCCT1_r can penetrate cell membranes, supporting the idea that ApiCCT1_r has potential therapeutic application without necessitating intracellular expression. Of note, ApiCCT1_r can cofractionate with nuclear fractions, which is significant in the context of nuclear accumulation and toxicity mediated by mHtt (1, 12, 17, 18). Application of just the apical domain of recombinant yeast CCT1 to rat pheochromocytoma-derived PC12 cells stably expressing truncated mutant Htt exon 1 protein (mHttex1p) (14A2.6 cells) decreased the formation of oligomeric and insoluble mHtt and slowed the kinetics of inclusion body formation in 14A2.6 cells. Finally, ApiCCT1_r rescued mHtt-mediated toxicity in mouse striatal STHdh^Q109/Hdh^Q109 cells derived from a knock-in mouse model of HD. Together these results provide a rationale for future testing of extracellular infusion of ApiCCT1_r or cell-based delivery of secreted forms of ApiCCT1 to alleviate HD phenotypes in vivo.     

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.     

Dopamine D2 receptor availability is linked to hippocampal–caudate functional connectivity and episodic memory

D1 and D2 dopamine receptors (D1DRs and D2DRs) may contribute differently to various aspects of memory and cognition. The D1DR system has been linked to functions supported by the prefrontal cortex. By contrast, the role of the D2DR system is less clear, although it has been hypothesized that D2DRs make a specific contribution to hippocampus-based cognitive functions. Here we present results from 181 healthy adults between 64 and 68 y of age who underwent comprehensive assessment of episodic memory, working memory, and processing speed, along with MRI and D2DR assessment with [¹¹C]raclopride and PET. Caudate D2DR availability was positively associated with episodic memory but not with working memory or speed. Whole-brain analyses further revealed a relation between hippocampal D2DR availability and episodic memory. Hippocampal and caudate D2DR availability were interrelated, and functional MRI-based resting-state functional connectivity between the ventral caudate and medial temporal cortex increased as a function of caudate D2DR availability. Collectively, these findings indicate that D2DRs make a specific contribution to hippocampus-based cognition by influencing striatal and hippocampal regions, and their interactions.Dopamine (DA) plays a key role in several cognitive processes (1–4). Reductions of D1 and D2 DA receptors (D1DRs and D2DRs) in aging (5–7) have been linked to age-related cognitive deficits (8, 9). The D1DR system has been related to functions supported by the prefrontal cortex (PFC), such as working memory and executive functions (10–12), which may reflect the relatively high density of D1DRs in the PFC (13). However, the role of D2DRs is far less clear. D2DRs are present in the PFC at very low densities (13), and evidence supporting a role for the D2DR system in working memory and executive functions is elusive (10). Pharmacological (14, 15) and PET studies assessing striatal D2DR availability (or binding potential to nondisplacable tissue uptake; BP_ND) with [¹¹C]raclopride (16, 17) have yielded mixed findings in relation to cognition. It has been hypothesized that D2DRs make a specific contribution to hippocampus-based cognitive functions (10, 18, 19). Supporting these claims, positive links between D2DR BP_ND and episodic memory are commonly observed (20–23). PET imaging of hippocampal D2DR BP_ND also provides support for this hypothesis, although some studies indicate that hippocampal D2DRs may be related to both episodic memory and PFC-based executive functions (22, 23), including verbal working memory (24). Medial temporal lobe regions have been implicated in working memory (25, 26), and D2DR-mediated modulation may be exerted via hippocampal–cortical pathways (27). In addition, a [¹¹C]raclopride task-activation PET study demonstrated contributions of striatal D2DRs to a verbal working-memory task (11).Taken together, the specific role of the D2DR system in cognition remains unclear, likely due to the fact that past studies included small and age-heterogeneous samples and lacked comprehensive test batteries that allowed systematic comparison of the role of D2DRs in different cognitive functions. Here we present results from the Cognition, Brain, and Aging (COBRA) study that include assessment of episodic memory, working memory, and processing speed, in combination with [¹¹C]raclopride PET and MRI of 181 healthy adults between 64 and 68 y of age (28). The main analyses concerned caudate D2DR–cognition associations, as this striatal region has been implicated in cognitive functioning (11, 12, 29, 30). Subsequently, whole-brain analyses were conducted to examine extrastriatal (especially hippocampal) D2DRs in relation to cognition. Finally, resting-state functional connectivity patterns were analyzed in relation to D2DR BP_ND, with special focus on interactions between the ventral caudate (31) and medial temporal cortex regions (32, 33).     

Aberrant calcium signaling by transglutaminase-mediated posttranslational modification of inositol 1,4,5-trisphosphate receptors

The inositol 1,4,5-trisphosphate receptor (IP₃R) in the endoplasmic reticulum mediates calcium signaling that impinges on intracellular processes. IP₃Rs are allosteric proteins comprising four subunits that form an ion channel activated by binding of IP₃ at a distance. Defective allostery in IP₃R is considered crucial to cellular dysfunction, but the specific mechanism remains unknown. Here we demonstrate that a pleiotropic enzyme transglutaminase type 2 targets the allosteric coupling domain of IP₃R type 1 (IP₃R1) and negatively regulates IP₃R1-mediated calcium signaling and autophagy by locking the subunit configurations. The control point of this regulation is the covalent posttranslational modification of the Gln2746 residue that transglutaminase type 2 tethers to the adjacent subunit. Modification of Gln2746 and IP₃R1 function was observed in Huntington disease models, suggesting a pathological role of this modification in the neurodegenerative disease. Our study reveals that cellular signaling is regulated by a new mode of posttranslational modification that chronically and enzymatically blocks allosteric changes in the ligand-gated channels that relate to disease states.Ligand-gated ion channels function by allostery that is the regulation at a distance; the allosteric coupling of ligand binding with channel gating requires reversible changes in subunit configurations and conformations (1). Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are ligand-gated ion channels that release calcium ions (Ca²⁺) from the endoplasmic reticulum (ER) (2, 3). IP₃Rs are allosteric proteins comprising four subunits that assemble a calcium channel with fourfold symmetry about an axis perpendicular to the ER membrane. The subunits of three IP₃R isoforms (IP₃R1, IP₃R2, and IP₃R3) are structurally divided into three domains: the IP₃-binding domain (IBD), the regulatory domain, and the channel domain (3–6). Fitting of the IBD X-ray structures (7, 8) to a cryo-EM map (9) indicates that the IBD activates a remote Ca²⁺ channel by allostery (8); however, the current X-ray structure only spans 5% of each tetramer, such that the mechanism underlying allosteric coupling of the IBD to channel gating remains unknown.The IP₃R in the ER mediates intracellular calcium signaling that impinges on homeostatic control in various subsequent intracellular processes. Deletion of the genes encoding the type 1 IP₃R (IP₃R1) leads to perturbations in long-term potentiation/depression (3, 10, 11) and spinogenesis (12), and the human genetic disease spinocerebellar ataxia 15 is caused by haploinsufficiency of the IP₃R1 gene (13–15). Dysregulation of IP₃R1 is also implicated in neurodegenerative diseases including Huntington disease (HD) (16–18) and Alzheimer’s disease (AD) (19–22). IP₃Rs also control fundamental cellular processes—for example, mitochondrial energy production (23, 24), autophagy regulation (24–27), ER stress (28), hepatic gluconeogenesis (29), pancreatic exocytosis (30), and macrophage inflammasomes (31). On the other hand, excessive IP₃R function promotes cell death processes including apoptosis by activating mitochondrial or calpain pathways (2, 17). Considering these versatile roles of IP₃Rs, appropriate IP₃R structure and function are essential for living systems, and aberrant regulation of IP₃R closely relates to various diseases.Several factors such as cytosolic molecules, interacting proteins, and posttranslational modifications control the IP₃-induced Ca²⁺ release (IICR) through allosteric sites in IP₃Rs. Cytosolic Ca²⁺ concentrations strictly control IICR in a biphasic manner with activation at low concentrations and inhibition at higher concentrations. The critical Ca²⁺ sensor for activation is conserved among the three isoforms of IP₃ and ryanodine receptors, and this sensor is located in the regulatory domain outside the IBD and the channel domain (32). A putative ATP regulatory region is deleted in opisthotonos mice, and IICR is also regulated by this mutation in the regulatory domain (33). Various interacting proteins, such as cytochrome c, Bcl-2-family proteins, ataxin-3, huntingtin (Htt) protein, Htt-associated protein 1A (HAP1A), and G-protein–coupled receptor kinase-interacting protein 1 (GIT1), target allosteric sites in the carboxyl-terminal tail (3–5). The regulatory domain and the carboxyl-terminal tail also undergo phosphorylation by the protein kinases A/G and B/Akt and contain the apoptotic cleavage sites for the protease caspase-3 (4, 5). These factors allosterically regulate IP₃R structure and function to control cellular fates; therefore, understanding the allosteric coupling of the IBD to channel gating will elucidate the regulatory mechanism of these factors.Transglutaminase (TG) catalyses protein cross-linking between a glutamine (Gln) residue and a lysine (Lys) residue via an N^ε-(γ-glutamyl)lysine isopeptide bond (34, 35). TG type 2 (TG2) is a Ca²⁺-dependent enzyme with widespread distribution and is highly inducible by various stimulations such as oxidative stress, cytokines, growth factors, and retinoic acid (RA) (34, 35). TG2 is considered a significant disease-modifying factor in neurodegenerative diseases including HD, AD, and Parkinson’s diseases (PD) (34, 36–45) because TG2 might enzymatically stabilize aberrant aggregates of proteins implicated in these diseases—that is, mutant Htt, β-amyloid, and α-synuclein; however, the causal role of TG2 in Ca²⁺ signaling in brain pathogenesis has been unclear. Ablation of TG2 in HD mouse models is associated with increased lifespan and improved motor function (46, 47). However, TG2 knockout mice do not show impaired Htt aggregation, suggesting that TG2 may play a causal role in these disorders rather than TG2-dependent cross-links in aberrant protein aggregates (47, 48).In this study, we discovered a new mode of chronic and irreversible allosteric regulation in IP₃R1 in which covalent modification of the receptor at Gln2746 is catalyzed by TG2. We demonstrate that up-regulation of TG2 modifies IP₃R1 structure and function in HD models and propose an etiologic role of this modification in the reduction of neuronal signaling and subsequent processes during the prodromal state of the neurodegenerative disease.     

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.     

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).     

Dopamine-associated cached values are not sufficient as the basis for action selection

Phasic dopamine transmission is posited to act as a critical teaching signal that updates the stored (or “cached”) values assigned to reward-predictive stimuli and actions. It is widely hypothesized that these cached values determine the selection among multiple courses of action, a premise that has provided a foundation for contemporary theories of decision making. In the current work we used fast-scan cyclic voltammetry to probe dopamine-associated cached values from cue-evoked dopamine release in the nucleus accumbens of rats performing cost–benefit decision-making paradigms to evaluate critically the relationship between dopamine-associated cached values and preferences. By manipulating the amount of effort required to obtain rewards of different sizes, we were able to bias rats toward preferring an option yielding a high-value reward in some sessions and toward instead preferring an option yielding a low-value reward in others. Therefore, this approach permitted the investigation of dopamine-associated cached values in a context in which reward magnitude and subjective preference were dissociated. We observed greater cue-evoked mesolimbic dopamine release to options yielding the high-value reward even when rats preferred the option yielding the low-value reward. This result identifies a clear mismatch between the ordinal utility of the available options and the rank ordering of their cached values, thereby providing robust evidence that dopamine-associated cached values cannot be the sole determinant of choices in simple economic decision making.In contemporary theories of economic decision making, values are assigned to reward-predictive states in which animals can take action to obtain rewards, and these state-action values are stored (“cached”) for the purpose of guiding future choices based upon their rank order (1–5). It is believed that these cached values are represented as synaptic weights within corticostriatal circuitry, reflected in the activity of subpopulations of striatal projection neurons (6–9), and are updated by dopamine-dependent synaptic plasticity (10–12). Indeed, a wealth of evidence suggests that the phasic activity of dopamine neurons reports instances in which current reward or expectation of future reward differs from current expectations (13–24). This pattern of activity resembles the prediction-error term from temporal-difference reinforcement-learning algorithms, which is considered the critical teaching signal for updating cached values. A notable feature of models that integrate dopamine transmission into this computational framework is that the cached value of an action is explicitly read out by the phasic dopamine response to the unexpected presentation of a cue that designates the transition into a state in which that action yields reward. Therefore, cue-evoked dopamine signaling provides a neural representation of the cached values of available actions, and if these cached values serve as the basis for action selection, then cue-evoked dopamine responses should be rank ordered in a manner that is consistent with animals’ behavioral preferences.Numerous studies that recorded cue-evoked dopamine signaling have reported correlations with the expected utility (subjective value) of actions (24–36). For example, risk-preferring rats demonstrated greater cue-evoked dopamine release for a risky option than for a certain option with equivalent objective expected value (reward magnitude times probability), whereas risk-averse rats showed greater dopamine release for the certain than for the risky option (30). Likewise, the cached values reported by dopamine neurons in macaque monkeys accounted for individual monkeys’ subjective flavor and risk preferences, with each attribute weighted according to its influence on behavioral preferences (31, 32). These observations, which are consistent across measures of dopamine neuronal activity and dopamine release, reinforce the prevailing notion that the dopamine-associated cached values could be the primary determinant of decision making (2–5, 17, 28–32) because the cue-evoked dopamine responses were rank ordered according to the animals’ subjective preferences. However, there have been some reports that other economic attributes, such as effortful response costs (35–38) or the overt aversiveness of an outcome (39), are represented inconsistently by cue-evoked dopamine responses. For example, Gan et al. (35) showed that independent manipulations of two different dimensions (reward magnitude and effort) that had equivalent effects on behavior did not have equivalent effects on dopamine release. Paralleling these findings, a recent report reached a similar conclusion that dopamine transmission preferentially encodes an appetitive dimension but is relatively insensitive to aversiveness (39).Because these cue-evoked dopamine signals represent cached values that are purported to determine action selection, their differential encoding of economic dimensions has potentially problematic implications in the context of decision making. Namely, by extrapolating from these studies (35–39), one might infer that when a decision involves the tradeoff between these economic dimensions, the rank order of the dopamine-associated cached values for each of the available options would not consistently reflect the ordinal utility of these options and therefore these cached values could not, on their own, be the basis of choices. However, this counterintuitive prediction was not tested explicitly by any of these previous studies; thus it remains a provocative notion that merits direct examination, because it is contrary to the prevailing hypothesis described above which is fundamental to contemporary theories of decision making. Therefore, we investigated interactions between dimensions that previously have been shown during independent manipulations to be weakly or strongly incorporated into these cached values. Specifically, we increased the amount of effort required to obtain a large reward so that animals instead preferred a low-effort option yielding a smaller reward, and we used fast-scan cyclic voltammetry to record cue-evoked mesolimbic dopamine release as a neurochemical proxy for each option’s cached value. These conditions permitted us to test whether the cached values reported via cue-evoked dopamine indeed align with animals’ subjective preferences across these mixed cost–benefit attributes.     

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.     

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).     

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.     

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.     

Presynaptic serotonin 2A receptors modulate thalamocortical plasticity and associative learning

Higher-level cognitive processes strongly depend on a complex interplay between mediodorsal thalamus nuclei and the prefrontal cortex (PFC). Alteration of thalamofrontal connectivity has been involved in cognitive deficits of schizophrenia. Prefrontal serotonin (5-HT)_2A receptors play an essential role in cortical network activity, but the mechanism underlying their modulation of glutamatergic transmission and plasticity at thalamocortical synapses remains largely unexplored. Here, we show that 5-HT_2A receptor activation enhances NMDA transmission and gates the induction of temporal-dependent plasticity mediated by NMDA receptors at thalamocortical synapses in acute PFC slices. Expressing 5-HT_2A receptors in the mediodorsal thalamus (presynaptic site) of 5-HT_2A receptor-deficient mice, but not in the PFC (postsynaptic site), using a viral gene-delivery approach, rescued the otherwise absent potentiation of NMDA transmission, induction of temporal plasticity, and deficit in associative memory. These results provide, to our knowledge, the first physiological evidence of a role of presynaptic 5-HT_2A receptors located at thalamocortical synapses in the control of thalamofrontal connectivity and the associated cognitive functions.The prefrontal cortex (PFC) is a brain region critical for many high-level cognitive processes, such as executive functions, attention, and working and contextual memories (1). Pyramidal neurons located in layer V of the PFC integrate excitatory glutamatergic inputs originating from both cortical and subcortical areas. The latter include the mediodorsal thalamus (MD) nuclei, which project densely to the medial PFC (mPFC) and are part of the neuronal network underlying executive control and working memory (2–4). Disruption of this network has been involved in cognitive symptoms of psychiatric disorders, such as schizophrenia (3, 5). These symptoms severely compromise the quality of life of patients and remain poorly controlled by currently available antipsychotics (3, 6).The PFC is densely innervated by serotonin (5-hydroxytryptamine, 5-HT) neurons originating from the dorsal and median raphe nuclei and numerous lines of evidence indicate a critical role of 5-HT in the control of emotional and cognitive functions depending on PFC activity (7, 8). The modulatory action of 5-HT reflects its complex pattern of effects on cortical network activity, depending on the 5-HT receptor subtypes involved, and on receptor localization in pyramidal neurons, GABAergic interneurons or nerve terminals of afferent neurons.Among the 14 5-HT receptor subtypes, the 5-HT_2A receptor is a Gq protein-coupled receptor (9, 10) particularly enriched in the mPFC, with a predominant expression in apical dendrites of layer V pyramidal neurons (11–14). Moreover, a low proportion of 5-HT_2A receptors was detected presynaptically on thalamocortical fibers (12, 15–17).Activation of 5-HT_2A receptors exerts complex effects upon the activity of the PFC network (18). The most prominent one is an increase in pyramidal neuron excitability, which likely results from the inhibition of slow calcium-activated after hyperpolarization current (19). 5-HT_2A receptor stimulation also increases the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons (19–22). The prevailing view is that postsynaptic 5-HT_2A receptors expressed on pyramidal neurons located in layer V are key modulators of glutamatergic PFC network activity (14, 21–24). However, the role of presynaptic 5-HT_2A receptors located on thalamic afferents in the modulation of glutamatergic transmission at thalamocortical synapses remains unexplored.Here, we addressed this issue by combining electrophysiological recordings in acute PFC slices with viral infections to specifically rescue the expression of 5-HT_2A receptors at the presynaptic site (MD) or postsynaptic site (PFC) in 5-HT_2A receptor-deficient (5-HT_2A^−/−) mice (25). We focused our study on NMDA transmission in line with previous findings indicating that many symptoms of schizophrenia might arise from modifications in PFC connectivity involving glutamatergic transmission at NMDA receptors (26, 27). To our knowledge, we provide the first direct evidence that stimulation of presynaptic 5-HT_2A receptors at thalamocortical synapses gates the induction of spike timing-dependent long-term depression (t-LTD) by facilitating the activation of presynaptic NMDA receptors at these synapses. In line with the role of t-LTD in associative learning (28), these studies were extended by behavioral experiments to explore the role of presynaptic 5-HT_2A receptors at thalamocortical synapses in several paradigms of episodic-like memory.     

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.