Dopamine release from transplanted neural stem cells in Parkinsonian rat striatum in vivo

Embryonic stem cell-based therapies exhibit great potential for the treatment of Parkinson’s disease (PD) because they can significantly rescue PD-like behaviors. However, whether the transplanted cells themselves release dopamine in vivo remains elusive. We and others have recently induced human embryonic stem cells into primitive neural stem cells (pNSCs) that are self-renewable for massive/transplantable production and can efficiently differentiate into dopamine-like neurons (pNSC–DAn) in culture. Here, we showed that after the striatal transplantation of pNSC–DAn, (i) pNSC–DAn retained tyrosine hydroxylase expression and reduced PD-like asymmetric rotation; (ii) depolarization-evoked dopamine release and reuptake were significantly rescued in the striatum both in vitro (brain slices) and in vivo, as determined jointly by microdialysis-based HPLC and electrochemical carbon fiber electrodes; and (iii) the rescued dopamine was released directly from the grafted pNSC–DAn (and not from injured original cells). Thus, pNSC–DAn grafts release and reuptake dopamine in the striatum in vivo and alleviate PD symptoms in rats, providing proof-of-concept for human clinical translation.Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder characterized by the specific loss of dopaminergic neurons in the substantia nigra pars compacta and their projecting axons, resulting in loss of dopamine (DA) release in the striatum (1). During the last two decades, cell-replacement therapy has proven, at least experimentally, to be a potential treatment for PD patients (2–7) and in animal models (8–15). The basic principle of cell therapy is to restore the DA release by transplanting new DA-like cells. Until recently, obtaining enough transplantable cells was a major bottleneck in the practicability of cell therapy for PD. One possible source is embryonic stem cells (ESCs), which can develop infinitely into self-renewable pluripotent cells with the potential to generate any type of cell, including DA neurons (DAns) (16, 17).Recently, several groups including us have introduced rapid and efficient ways to generate primitive neural stem cells (pNSCs) from human ESCs using small-molecule inhibitors under chemically defined conditions (12, 18, 19). These cells are nonpolarized neuroepithelia and retain plasticity upon treatment with neuronal developmental morphogens. Importantly, pNSCs differentiate into DAns (pNSC–DAn) with high efficiency (∼65%) after patterning by sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) in vitro, providing an immediate and renewable source of DAns for PD treatment. Importantly, the striatal transplantation of human ESC-derived DA-like neurons, including pNSC–DAn, are able to relieve the motor defects in a PD rat model (11–13, 15, 19–23). Before attempting clinical translation of pNSC–DAn, however, there are two fundamental open questions. (i) Can pNSC–DAn functionally restore the striatal DA levels in vivo? (ii) What cells release the restored DA, pNSC–DAn themselves or resident neurons/cells repaired by the transplants?Regarding question 1, a recent study using nafion-coated carbon fiber electrodes (CFEs) reported that the amperometric current is rescued in vivo by ESC (pNSC–DAn-like) therapy (19). Both norepinephrine (NE) and serotonin are present in the striatum (24, 25). However, CFE amperometry/chronoamperometry alone cannot distinguish DA from other monoamines in vivo, such as NE and serotonin (Fig. S1) (see also refs. 26–28). Considering that the compounds released from grafted ESC-derived cells are unknown, the work of Kirkeby et al. was unable to determine whether DA or other monoamines are responsible for the restored amperometric signal. Thus, the key question of whether pNSC–DAn can rescue DA release needs to be reexamined for the identity of the restored amperometric signal in vivo.Regarding question 2, many studies have proposed that DA is probably released from the grafted cells (8, 12, 13, 20), whereas others have proposed that the grafted stem cells might restore striatal DA levels by rescuing injured original cells (29, 30). Thus, whether the grafted cells are actually capable of synthesizing and releasing DA in vivo must be investigated to determine the future cellular targets (residual cells versus pNSC–DAn) of treatment.To address these two mechanistic questions, advanced in vivo methods of DA identification and DA recording at high spatiotemporal resolution are required. Currently, microdialysis-based HPLC (HPLC) (31–33) and CFE amperometric recordings (34, 35) have been used independently by different laboratories to assess evoked DA release from the striatum in vivo. The major advantage of microdialysis-based HPLC is to identify the substances secreted in the cell-grafted striatum (33), but its spatiotemporal resolution is too low to distinguish the DA release site (residual cells or pNSC–DAn). In contrast, the major advantage of CFE-based amperometry is its very high temporal (ms) and spatial (μm) resolution, making it possible to distinguish the DA release site (residual cells or pNSC–DAn) in cultured cells, brain slices, and in vivo (34–39), but it is unable to distinguish between low-level endogenous oxidizable substances (DA versus serotonin and NE) in vivo.In the present study, we developed a challenging experimental paradigm of combining the two in vivo methods, microdialysis-based HPLC and CFE amperometry, to identify the evoked substance as DA and its release site as pNSC–DAn in the striatum of PD rats.     

Kinetics of protein–ligand unbinding: Predicting pathways,rates, and rate-limiting steps

The ability to predict the mechanisms and the associated rate constants of protein–ligand unbinding is of great practical importance in drug design. In this work we demonstrate how a recently introduced metadynamics-based approach allows exploration of the unbinding pathways, estimation of the rates, and determination of the rate-limiting steps in the paradigmatic case of the trypsin–benzamidine system. Protein, ligand, and solvent are described with full atomic resolution. Using metadynamics, multiple unbinding trajectories that start with the ligand in the crystallographic binding pose and end with the ligand in the fully solvated state are generated. The unbinding rate k_off is computed from the mean residence time of the ligand. Using our previously computed binding affinity we also obtain the binding rate k_on. Both rates are in agreement with reported experimental values. We uncover the complex pathways of unbinding trajectories and describe the critical rate-limiting steps with unprecedented detail. Our findings illuminate the role played by the coupling between subtle protein backbone fluctuations and the solvation by water molecules that enter the binding pocket and assist in the breaking of the shielded hydrogen bonds. We expect our approach to be useful in calculating rates for general protein–ligand systems and a valid support for drug design.Understanding the thermodynamics and kinetics of protein–ligand interactions is of paramount relevance in the early stages of drug discovery (1–3). So far the major emphasis has been placed on predicting the most likely binding pose as determined by the highest binding affinity (4, 5). In contrast, it has not been possible to predict the pathways for unbinding and the associated rates. However, it is by now well-recognized that one of the most pertinent factors for sustained drug efficacy and safety is not just its affinity, but possibly even more so, the mean lifetime of the protein–ligand complex (1–3). The latter property is strictly related to the time during which the ligand remains in the binding site (1, 2), and is typically expressed by its inverse, the dissociation rate k_off (2). In principle k_off should be amenable to calculations through all-atom molecular dynamics (MD) simulations. These simulations could give detailed and useful insights into the atomic interactions at work during unbinding, especially in the ephemeral but kinetically most relevant transition state ensemble (TSE) (6, 7). Such information is of great value in designing modifications of the ligand that might improve its pharmaceutical properties.However, despite the potential of MD simulations no such calculation has yet been reported. This is a consequence of the limited timescales of MD simulations. Even with the most modern purpose-built supercomputers or massive distributed computing, one can barely reach the timescale of milliseconds (3). Unfortunately most of the reported ligand–protein dissociation times far exceed this timescale (2). These timescales can be reached either by transition path sampling methods (8, 9), quasi-classical approximations (10), by the construction of Markov state models (11, 12), or through carefully designed enhanced sampling methods (8, 13–30) that make accessible the timescale of seconds and beyond in a controlled and accurate way. The enhanced sampling method we use in this work is based on metadynamics (13–15), which has been widely and successfully applied to a variety of systems including complex protein–ligand systems (25–30), and has been rigorously proven to converge to the correct free-energy surface (31, 32).Recently, we have extended the scope of metadynamics by showing that it can also be used to recover kinetic information (15). Furthermore, we showed that by using an a posteriori statistical analysis (33) one can also establish the reliability of the kinetics thus generated. The use of metadynamics for obtaining kinetic information is still in its infancy, however its usefulness has been tested by us and other groups in a range of systems (15, 33–36).In this work, we demonstrate that the scope of the method reported in ref. 15 can be extended to study protein–ligand dissociation pathways and to determine in an accurate way the ligand unbinding rates. We reach well into the hundreds of milliseconds regime and longer, maintaining at the same time full atomic resolution for protein, ligand, and solvent. Specifically, we study the unbinding of the inhibitor benzamidine from trypsin, a serine protease protein (27, 37, 38) using classical force fields (39, 40). Using our acceleration method (15, 33) we are able to harness 21 independent successful unbinding trajectories in which the ligand goes from the bound to the fully unbound state. We find that one of the most distinctive features of the unbinding process is the role played by the water molecules (41, 42). In particular, the solvent promotes unbinding by assisting in the breakage of shielded hydrogen bonds through the formation of water bridge interactions (41).From the analysis of the unbinding trajectories we find that along the unbinding pathways the ligand rests for times ranging from nanoseconds to milliseconds in a number of intermediate structures. We calculate the rates for all possible transitions between these intermediates and construct a Markov model for the unbinding process (11, 43, 44). The overall escape rate computed from this Markov model is in good agreement with the direct estimation of the mean unbinding time that comes from the metadynamics runs. Reassured by this agreement we use the Markov model to determine the dominant unbinding pathways and rate-limiting steps. To this end, starting from the metadynamics reactive trajectories, we perform a committor analysis and determine the TSE (6). Using the recently computed value of the binding affinity (27) we also estimate the binding rate constant k_on. Our calculated unbinding and binding rates compare reasonably well with the known experimental measurement (37), especially taking into account the margin of error in the experiment and the inaccuracy of the force field used in the simulations (42). Unprecedented structural features of the target are also disclosed. In particular, we find that in its apo state trypsin can exist in two forms. In the first form, loop Val207–Tyr224 (hereafter labeled loop L) oscillates around the crystallographic state. In the other form, a small distortion of this loop is stabilized. The mean lifetime of this distorted state is nearly 0.7 ms and during this time the ligand cannot reach the binding site.We believe that this metadynamics-based strategy is, to our knowledge, the first direct approach for calculating k_off from MD simulations of unbinding. Previous studies have focused on the calculation of k_on and the magnitude of k_off was only indirectly obtained (12, 38). Our strategy should be easily applicable for calculating unbinding pathways and rates for generic protein–ligand systems, thus complementing and extending the role of enhanced sampling-based simulations in drug discovery.     

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.Kinesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1–6). Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7–10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4, 11, 12), consistent with the motor walking in a hand-over-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7–10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head–bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads–bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4, 11, 14–20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7, 19, 21, 22) and single-molecule tracking studies (4, 8–11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11, 12, 14, 15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.Single-molecule microscopy is a powerful tool for studying the kinetics of structural changes in macromolecules (23). Tracking steps and potential substeps for kinesin-1 at saturating ATP has until now been hampered by the high stepping rates of the motor (up to 100 s⁻¹), which necessitates high frame rates, and the small step size (8.2 nm), which necessitates high spatial precision (7). Here, we apply interferometric scattering microscopy (iSCAT), a recently established single-molecule tool with high spatiotemporal resolution (24–27) to directly visualize the structural changes underlying kinesin stepping. By labeling one motor domain in a dimeric motor, we detect a 1HB intermediate state in which the tethered head resides over the bound head for half the duration of the stepping cycle at saturating ATP. We further show that at physiological stepping rates, ATP binding is required to enter this 1HB state and that ATP hydrolysis is required to exit it. This work leads to a significant revision of the sequence and kinetics of mechanochemical transitions that make up the kinesin-1 stepping cycle and provides a framework for understanding functional diversity across the kinesin superfamily.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Microscopic identification of the order parameter governing liquid–liquid transition in a molecular liquid

A liquid–liquid transition (LLT) in a single-component substance is an unconventional phase transition from one liquid to another. LLT has recently attracted considerable attention because of its fundamental importance in our understanding of the liquid state. To access the order parameter governing LLT from a microscopic viewpoint, here we follow the structural evolution during the LLT of an organic molecular liquid, triphenyl phosphite (TPP), by time-resolved small- and wide-angle X-ray scattering measurements. We find that locally favored clusters, whose characteristic size is a few nanometers, are spontaneously formed and their number density monotonically increases during LLT. This strongly suggests that the order parameter of LLT is the number density of locally favored structures and of nonconserved nature. We also show that the locally favored structures are distinct from the crystal structure and these two types of orderings compete with each other. Thus, our study not only experimentally identifies the structural order parameter governing LLT, but also may settle a long-standing debate on the nature of the transition in TPP, i.e., whether the transition is LLT or merely microcrystal formation.Liquid-liquid transition (LLT) is an intriguing phenomenon in which a liquid transforms into another one via a first-order transition. This means that there can be more than two liquid states for a single-component substance. Despite its counterintuitive nature, there have recently been many pieces of experimental and numerical evidence for the existence of LLT, for various liquids such as water (1–5), aqueous solutions (6–8), triphenyl phosphite (9–12), l-butanol (13), phosphorus (14), silicon (15, 16), germanium (17), and Y₂O₃–Al₂O₃ (18, 19). This suggests that the LLT may be rather universally observed for various types of liquids. However, none of the LLTs reported so far is free from criticisms (20, 21), mainly because these LLTs take place under experimentally difficult conditions [e.g., at high temperature and pressure (14, 15, 17–19)] or in a supercooled state below the melting point (1–3, 5–7, 9, 10), where the transition is inevitably contaminated by microcrystal formation. The latter is not limited to experiments but arises in numerical simulations, often causing many controversies [LLT (22–25) vs. crystallization (26–28)]. For ST2 water, however, this issue has recently been settled by an extensive simulation study by Palmer et al. (4).One of the hottest and long-standing debates is on the nature of the transition found in a molecular liquid, triphenyl phosphite (TPP), by Kivelson and his coworkers (29). The transition is very easy to access experimentally, because it takes place at ambient pressure and at a temperature range between 230 and 210 K and the transformation speed is slow enough to follow the kinetics. Since the finding of this transition (29, 30), many researchers thus have been interested in this intriguing phenomenon and there have been hot discussions on the nature of the transition (20, 21). Some people interpreted this as a liquid-associated phenomenon (9, 10, 31, 32), but others interpret it differently. All of the controversies come from the fact that this transition accompanies microcrystal formation and thus the final state, which is called “glacial phase,” often contains microcrystallites. This led many researchers to explain the transition by non-LLT scenarios, which include a defect-ordered phase scenario predicted by a frustration limited domain theory (29, 30, 33, 34), a microcrystallization scenario (35–38), and a liquid-crystal or plastic-crystal phase scenario (39). Each scenario captures a certain feature of the glacial phase, but fails in explaining all of the experimental results in a consistent manner. Similar situations are often seen in other candidates of LLTs, such as l-butanol [LLT (13) vs. microcrystallization (40–43)], confined water [LLT (5) vs. other phenomena (44–46)], and aqueous solutions [LLT (6, 7) vs. microcrystallization (8, 28, 47, 48)]. For TPP, however, some pieces of experimental evidence supportive of the LLT scenario rather than the microcrystallization scenario have recently been reported (11, 12).We propose a two-order-parameter (TOP) model of a liquid to explain LLT (20, 49). The main point of this model is that it is necessary to consider the spatiotemporal hierarchical nature of a liquid to understand LLT. More specifically, we argue that in addition to density order parameter ρ describing a gas–liquid transition, we need an additional scalar order parameter S, which is the number density of locally favored structures (LFS). In this model, LLT is a consequence of the cooperative ordering of the scalar nonconserved order parameter S, i.e., the cooperative formation of LFS. In other words, LLT is regarded as a gas–liquid-like transition of LFS: one liquid is a gas state of LFS (low-S state), and the other is its liquid state (high-S state). Recently, it was proposed by Anisimov and coworkers (50, 51) that the thermodynamic ordering field conjugate to the order parameter is the conversion equilibrium constant, which further characterizes the nature of LLT. We explained our experimental observation of LLT in TPP in terms of this model (9, 10). We also studied the phase transition dynamics and the physical and chemical properties of the second liquid state (liquid II), which were also explained by the model (20, 21).However, we have not had any direct experimental evidence for the formation of such LFS up to now; thus, an open question is, what is the relevant order parameter governing LLT, although the link of the order parameter to the enthalpy (9, 10), the refractive index (or, density) (9, 10, 29, 30), and the polarity associated with local molecular ordering (12) has been suggested for LLT in TPP. There have been structural studies on LLT by X-ray and neutron scattering measurements, focusing on local liquid structures at an inter- and intramolecular scale (36, 38, 52–54) and mesoscopic structures (34, 55). However, there has been no experimental evidence for the presence of locally favored structures, which characterize the liquid state uniquely, or the order parameter has still not been identified from a microscopic viewpoint.Here we study the structural change of TPP during LLT by time-resolved small- and wide-angle X-ray scattering measurements, which cover a length scale from a single molecule size ( ～  1 nm) to more than tens of nanometers. We show, to our knowledge, the first direct evidence for the presence of LFS and the temporal increase upon the liquid I-to-liquid II transformation. Furthermore, we also find an indication of the formation of microcrystallites during LLT. However, we reveal that LFS and microcrystallites have different sizes and growth kinetics, indicating that although they sometimes appear simultaneously during the process of LLT, LLT itself is driven by the formation of LFS and not by that of microcrystallites. We also discover that LFS are destroyed upon crystallization, clearly indicating not only that these two types of orderings are competing with each other but also that LFS is a structure unique to the liquid state. Our findings provide a comprehensive view on the long-standing controversy on the origin of the glacial phase, which was discovered by Kivelson and his coworkers (29, 30), and show that the fraction of LFS may be the relevant order parameter of LLT. This suggests that a liquid can have a spatiotemporal hierarchical structure at a low temperature, contrary to the common picture of a high-temperature liquid where the structure is random and homogeneous beyond the molecular size.     

STEP61 is a substrate of the E3 ligase parkin and is upregulated in Parkinson’s disease

Parkinson’s disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). The loss of SNc dopaminergic neurons affects the plasticity of striatal neurons and leads to significant motor and cognitive disabilities during the progression of the disease. PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in genetic and sporadic PD. Mutations in PARK2 are a major contributing factor in the early onset of autosomal-recessive juvenile parkinsonism (AR-JP), although the mechanisms by which a disruption in parkin function contributes to the pathophysiology of PD remain unclear. Here we demonstrate that parkin is an E3 ligase for STEP₆₁ (striatal-enriched protein tyrosine phosphatase), a protein tyrosine phosphatase implicated in several neuropsychiatric disorders. In cellular models, parkin ubiquitinates STEP₆₁ and thereby regulates its level through the proteasome system, whereas clinically relevant parkin mutants fail to do so. STEP₆₁ protein levels are elevated on acute down-regulation of parkin or in PARK2 KO rat striatum. Relevant to PD, STEP₆₁ accumulates in the striatum of human sporadic PD and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mice. The increase in STEP₆₁ is associated with a decrease in the phosphorylation of its substrate ERK1/2 and the downstream target of ERK1/2, pCREB [phospho-CREB (cAMP response element-binding protein)]. These results indicate that STEP₆₁ is a novel substrate of parkin, although further studies are necessary to determine whether elevated STEP₆₁ levels directly contribute to the pathophysiology of PD.Parkinson’s disease (PD) is a common motor disorder with clinical symptoms that include bradykinesia, resting tremor, rigidity, postural instability, and cognitive deficits (1–3). The pathophysiology of PD includes selective loss of dopaminergic neurons in the substantia nigra, with a progressive depletion of striatal dopamine and the presence of intraneuronal cytoplasmic inclusions known as Lewy bodies. Mutations of several genes are implicated in PD and are responsible for ∼10% of cases; the remaining cases are classified as sporadic PD. Although specific mutations in genes that include PARK2, PINK-1, LRRK2, and DJ-1 are known, the effects these mutations have on intracellular signaling and disease progression are not well understood and form an area of intense investigation (2, 4–6).STEP₆₁ (striatal-enriched protein tyrosine phosphatase) is a brain-specific phosphatase enriched in the striatum and in other regions, including cortex, hippocampus, and substantia nigra (7–9). STEP₆₁ levels are elevated in several disorders, including Alzheimer’s disease, schizophrenia, and fragile X syndrome (10–12). STEP₆₁ levels are normally regulated by the ubiquitin proteasome system, and disruption of this pathway leads to an accumulation of STEP₆₁ in both Alzheimer’s disease and schizophrenia (10, 11).Substrates of STEP₆₁ include ERK1/2, Pyk2, Fyn, the GluN2B subunit of the NMDA receptor, and the GluA2 subunit of the AMPA receptor. The current model of STEP₆₁ function is that it opposes the development of synaptic strengthening by dephosphorylating regulatory tyrosines on these substrates. In the case of the kinases, STEP₆₁-mediated dephosphorylation of the regulatory Tyr within the activation loop inactivates these enzymes (13–16). STEP-mediated dephosphorylation of Tyr residues in the glutamate receptor subunits results in internalization of GluN1/GluN2B and GluA1/GluA2 receptor complexes (17–20). As a result, STEP KO mice have an increase in the basal Tyr phosphorylation of its substrates, including ERK1/2 and its downstream target pCREB (21, 22).Overexpression of STEP disrupts synaptic function, and thereby contributes to cognitive and behavioral deficits (23). Consistent with this hypothesis, genetic or pharmacologic reduction of STEP activity in several disorders in which STEP levels are elevated reverses the biochemical and cognitive deficits that are present (19, 24), and STEP KO mice demonstrate enhanced hippocampal long-term potentiation and enhanced hippocampal- and amygdalar-dependent memory tasks (22, 25).Direct mutations of the E3 ligase parkin (PARK2) result in autosomal recessive juvenile parkinsonism (AR-JP), with early onset of PD symptoms (26, 27); disruption of parkin activity is also implicated in sporadic PD (28–30). Moreover, PD toxins such as MPTP, rotenone, paraquat, and 6-hydroxydopamine alter parkin levels or its ligase activity and result in the accumulation of parkin substrates (31–35). Identification of new parkin substrates and characterization of their role or roles in synaptic function should result in a better understanding the molecular basis of PD.Here we identify parkin as an E3 ligase that ubiquitinates STEP₆₁. STEP₆₁ levels are increased in human PD brains and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD models and are associated with a decrease in the phosphorylation of ERK1/2 and CREB. As an increase in STEP₆₁ expression disrupts synaptic function and contributes to the cognitive deficits in several disorders, these findings suggest that the increase in STEP₆₁ levels in PD may contribute to the pathophysiology of this disorder.     

Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria

F₁F_o-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology.F₁F_o-ATP synthases are ubiquitous, highly conserved energy-converting membrane protein complexes. ATP synthases produce ATP from ADP and inorganic phosphate (P_i) by rotary catalysis (1, 2) using the energy stored in a transmembrane electrochemical gradient. The ∼600-kDa monomer of the mitochondrial ATP synthase is composed of a soluble F₁ subcomplex and a membrane-bound F_o subcomplex (3). The main components of the F₁ subcomplex are the (αβ)₃ hexamer and the central stalk (4). The F_o subcomplex includes a rotor ring of 8–15 hydrophobic c subunits (5), the peripheral stalk, and several small hydrophobic stator subunits. Protons flowing through the membrane part of the F_o subcomplex drive the rotation of the c-ring (6–9). The central stalk transmits the torque generated by c-ring rotation to the catalytic head of the F₁ subcomplex, where it induces conformational changes of the α and β subunits that result in phosphate bond formation and the generation of ATP. The catalytic (αβ)₃ hexamer is held stationary relative to the membrane region by the peripheral stalk (10, 11). Several high-resolution structures of the F₁/rotor ring complexes have been solved by X-ray crystallography (12–16), and the structure of the complete assembly has been determined by cryoelectron microscopy (cryo-EM) (10, 17–20).In mitochondria, the ATP synthase forms dimers in the inner membrane. In fungi, plants, and metazoans, the dimers are V-shaped and associate into rows along the highly curved ridges of lamellar cristae (19–22). F_o subcomplexes of the two monomers in the dimer interact in the lipid bilayer via a number of hydrophobic stator subunits (20, 23–25). Coarse-grained molecular dynamics simulations have suggested that the V-shape of the ATP synthase dimers induces local membrane curvature, which in turn drives the association of ATP synthase dimers into rows (20). The exact role of the dimer rows is unclear, however rows of ATP synthase dimers have been proposed to promote the formation of lamellar cristae in yeast (20, 26).So far, all rows of ATP synthase dimers observed by electron cryotomography have been more or less straight (19–22, 27). However, an earlier deep-etch freeze-fracture study of mitochondria from the ciliate Paramecium multimicronucleatum revealed double rows of interdigitating 10-nm particles on helical tubular cristae (28). These particles were interpreted as ATP synthases, which, if correct, would suggest that the mitochondrial ATP synthase can assemble into rows that differ significantly from the standard geometry found in lamellar cristae (19, 21, 22).To investigate the helical rows in more detail, we performed electron cryotomography of isolated mitochondrial membranes from Paramecium tetraurelia. Using subtomogram averaging, we show that these helical rows do indeed consist of ATP synthase molecules, as suggested by Allen et al. (28). However, unlike the V-shaped dimers of metazoans, the ATP synthase of this species forms U-shaped dimers, which have new and unusual structural features. When assembled into the helical rows, the ATP synthase monomers interdigitate, whereas the U-shaped dimers align side by side. Thus, rows of ATP synthase dimers seem to be a universal feature of all mitochondria. We propose that the particular shape of the P. tetraurelia ATP synthase dimer induces its assembly into helical rows, which in turn cause the formation of the helical tubular cristae of ciliates.     

Novel serologic biomarkers provide accurate estimates of recent Plasmodium falciparum exposure for individuals and communities

Tools to reliably measure Plasmodium falciparum (Pf) exposure in individuals and communities are needed to guide and evaluate malaria control interventions. Serologic assays can potentially produce precise exposure estimates at low cost; however, current approaches based on responses to a few characterized antigens are not designed to estimate exposure in individuals. Pf-specific antibody responses differ by antigen, suggesting that selection of antigens with defined kinetic profiles will improve estimates of Pf exposure. To identify novel serologic biomarkers of malaria exposure, we evaluated responses to 856 Pf antigens by protein microarray in 186 Ugandan children, for whom detailed Pf exposure data were available. Using data-adaptive statistical methods, we identified combinations of antibody responses that maximized information on an individual’s recent exposure. Responses to three novel Pf antigens accurately classified whether an individual had been infected within the last 30, 90, or 365 d (cross-validated area under the curve = 0.86–0.93), whereas responses to six antigens accurately estimated an individual’s malaria incidence in the prior year. Cross-validated incidence predictions for individuals in different communities provided accurate stratification of exposure between populations and suggest that precise estimates of community exposure can be obtained from sampling a small subset of that community. In addition, serologic incidence predictions from cross-sectional samples characterized heterogeneity within a community similarly to 1 y of continuous passive surveillance. Development of simple ELISA-based assays derived from the successful selection strategy outlined here offers the potential to generate rich epidemiologic surveillance data that will be widely accessible to malaria control programs.Many countries have extensive programs to reduce the burden of Plasmodium falciparum (Pf), the parasite responsible for most malaria morbidity and mortality (1). Effectively using limited resources for malaria control or elimination and evaluating interventions require accurate measurements of the risk of being infected with Pf (2–15). To reflect the rate at which individuals are infected with Pf in a useful way, metrics used to estimate exposure in a community need to account for dynamic changes over space and time, especially in response to control interventions (16–18).A variety of metrics can be used to estimate Pf exposure, but tools that are more precise and low cost are needed for population surveillance. Existing metrics have varying intrinsic levels of precision and accuracy and are subject to a variety of extrinsic factors, such as cost, time, and availability of trained personnel (19). For example, entomological measurements provide information on mosquito to human transmission for a community but are expensive, require specially trained staff, and lack standardized procedures, all of which reduce precision and/or make interpretation difficult (19–22). Parasite prevalence can be measured by detecting parasites in the blood of individuals from a cross-sectional sample of a community and is, therefore, relatively simple and inexpensive to perform, but results may be imprecise, especially in areas of low transmission (19, 23), and biased by a number of factors, including immunity and access to antimalarial treatment (5, 6, 19, 23–25). The burden of symptomatic disease in a community can be estimated from routine health systems data; however, such data are frequently unreliable (5, 26–28) and generally underestimate the prevalence of Pf infection in areas of intense transmission. Precise and quantitative information about exposure at an individual level can be reliably obtained from cohort studies by measuring the incidence of asymptomatic and/or symptomatic Pf infection (i.e., by measuring the molecular force of infection) (29–35). Unfortunately, the expense of cohort studies limits their use to research settings. The end result is that most malaria-endemic regions lack reliable, timely data on Pf exposure, limiting the capabilities of malaria control programs to guide and evaluate interventions.Serologic assays offer the potential to provide incidence estimates for symptomatic and asymptomatic Pf infection, which are currently obtained from cohort studies, at the cost of cross-sectional studies (36–38). Although Pf infections are transient, a record of infection remains detectable in an individual’s antibody profile. Thus, appropriately chosen antibody measurements integrated with age can provide information about an individual’s exposure history. Antibodies can be measured by simple ELISAs and obtained from dried blood spots, which are easy to collect, transport, and store (39–41). Serologic responses to Pf antigens have been explored as potential epidemiological tools (42–45), and estimated rates of seroconversion to well-characterized Pf antigens accurately reflect stable rates of exposure in a community, whereas distinct changes in these rates are obtained from successful interventions (22, 39, 41, 46–53). However, current serologic assays are not designed to detect short-term or gradual changes in Pf exposure or measure exposure to infection at an individual level. The ability to calibrate antibody responses to estimates of exposure in individuals could allow for more flexible sampling of a population (e.g., not requiring age stratification), improve accuracy of exposure estimates from small sample sizes, and better characterize heterogeneity in exposure within a community.Different Pf antigens elicit antibody responses with different magnitudes and kinetics, providing a large and diverse set of potential biomarkers for exposure (38, 54–58). We hypothesized that new and more highly informative serologic biomarkers better able to characterize an individual’s recent exposure history could be identified by analyzing antibody responses to a large number of candidate Pf antigens in participants with well-characterized exposure histories. To test this hypothesis, we probed plasma from participants in two cohort studies in Uganda against a protein microarray containing 856 Pf antigens. The primary aim of this analysis was to identify responses to select antigens that were most informative of recent exposure using robust, data-adaptive statistical methods. Each participant’s responses to these selected antigens were used as predictors for two primary outcomes of their recent exposure to Pf: (i) days since last Pf infection and (ii) the incidence of symptomatic malaria in the last year. These individual-level estimates were then aggregated across a population to assess community-level malaria exposure. The selection strategy presented here identified accurate biomarkers of exposure for children living in areas of moderate to high Pf exposure and illustrates the utility of this flexible and broadly applicable approach.     

Convergent evolution toward an improved growth rate and a reduced resistance range in Prochlorococcus strains resistant to phage

Prochlorococcus is an abundant marine cyanobacterium that grows rapidly in the environment and contributes significantly to global primary production. This cyanobacterium coexists with many cyanophages in the oceans, likely aided by resistance to numerous co-occurring phages. Spontaneous resistance occurs frequently in Prochlorococcus and is often accompanied by a pleiotropic fitness cost manifested as either a reduced growth rate or enhanced infection by other phages. Here, we assessed the fate of a number of phage-resistant Prochlorococcus strains, focusing on those with a high fitness cost. We found that phage-resistant strains continued evolving toward an improved growth rate and a narrower resistance range, resulting in lineages with phenotypes intermediate between those of ancestral susceptible wild-type and initial resistant substrains. Changes in growth rate and resistance range often occurred in independent events, leading to a decoupling of the selection pressures acting on these phenotypes. These changes were largely the result of additional, compensatory mutations in noncore genes located in genomic islands, although genetic reversions were also observed. Additionally, a mutator strain was identified. The similarity of the evolutionary pathway followed by multiple independent resistant cultures and clones suggests they undergo a predictable evolutionary pathway. This process serves to increase both genetic diversity and infection permutations in Prochlorococcus populations, further augmenting the complexity of the interaction network between Prochlorococcus and its phages in nature. Last, our findings provide an explanation for the apparent paradox of a multitude of resistant Prochlorococcus cells in nature that are growing close to their maximal intrinsic growth rates.Large bacterial populations are present in the oceans, playing important roles in primary production and the biogeochemical cycling of matter. These bacterial communities are highly diverse (1–4) yet form stable and reproducible bacterial assemblages under similar environmental conditions (5–7).These bacteria are present together with high abundances of viruses (phages) that have the potential to infect and kill them (8–11). Although studied only rarely in marine organisms (12–16), this coexistence is likely to be the result of millions of years of coevolution between these antagonistic interacting partners, as has been well documented for other systems (17–20). From the perspective of the bacteria, survival entails the selection of cells that are resistant to infection, preventing viral production and enabling the continuation of the cell lineage. Resistance mechanisms include passively acquired spontaneous mutations in cell surface molecules that prevent phage entry into the cell and other mechanisms that actively terminate phage infection intracellularly, such as restriction–modification systems and acquired resistance by CRISPR-Cas systems (21, 22). Mutations in the phage can also occur that circumvent these host defenses and enable the phage to infect the recently emerged resistant bacterium (23).Acquisition of resistance by bacteria is often associated with a fitness cost. This cost is frequently, but not always, manifested as a reduction in growth rate (24–27). Recently, an additional type of cost of resistance was identified, that of enhanced infection whereby resistance to one phage leads to greater susceptibility to other phages (14, 15, 28).Over the years, a number of models have been developed to explain coexistence in terms of the above coevolutionary processes and their costs (16, 29–32). In the arms race model, repeated cycles of host mutation and virus countermutation occur, leading to increasing breadths of host resistance and viral infectivity. However, experimental evidence generally indicates that such directional arms race dynamics do not continue indefinitely (25, 33, 34). Therefore, models of negative density-dependent fluctuations due to selective trade-offs, such as kill-the-winner, are often invoked (20, 33, 35, 36). In these models, fluctuations are generally considered to occur between rapidly growing competition specialists that are susceptible to infection and more slowly growing resistant strains that are considered defense specialists. Such negative density-dependent fluctuations are also likely to occur between strains that have differences in viral susceptibility ranges, such as those that would result from enhanced infection (30).The above coevolutionary processes are considered to be among the major mechanisms that have led to and maintain diversity within bacterial communities (32, 35, 37–39). These processes also influence genetic microdiversity within populations of closely related bacteria. This is especially the case for cell surface-related genes that are often localized to genomic islands (14, 40, 41), regions of high gene content, and gene sequence variability among members of a population. As such, populations in nature display an enormous degree of microdiversity in phage susceptibility regions, potentially leading to an assortment of subpopulations with different ranges of susceptibility to coexisting phages (4, 14, 30, 40).Prochlorococcus is a unicellular cyanobacterium that is the numerically dominant photosynthetic organism in vast oligotrophic expanses of the open oceans, where it contributes significantly to primary production (42, 43). Prochlorococcus consists of a number of distinct ecotypes (44–46) that form stable and reproducible population structures (7). These populations coexist in the oceans with tailed double-stranded DNA phage populations that infect them (47–49).Previously, we found that resistance to phage infection occurs frequently in two high-light–adapted Prochlorococcus ecotypes through spontaneous mutations in cell surface-related genes (14). These genes are primarily localized to genomic island 4 (ISL4) that displays a high degree of genetic diversity in environmental populations (14, 40). Although about a third of Prochlorococcus-resistant strains had no detectable associated cost, the others came with a cost manifested as either a slower growth rate or enhanced infection by other phages (14). In nature, Prochlorococcus seems to be growing close to its intrinsic maximal growth rate (50–52). This raises the question as to the fate of emergent resistant Prochlorococcus lineages in the environment, especially when resistance is accompanied with a high growth rate fitness cost.To begin addressing this question, we investigated the phenotype of Prochlorococcus strains with time after the acquisition of resistance. We found that resistant strains evolved toward an improved growth rate and a reduced resistance range. Whole-genome sequencing and PCR screening of many of these strains revealed that these phenotypic changes were largely due to additional, compensatory mutations, leading to increased genetic diversity. These findings suggest that the oceans are populated with rapidly growing Prochlorococcus cells with varying degrees of resistance and provide an explanation for how a multitude of presumably resistant Prochlorococcus cells are growing close to their maximal known growth rate in nature.     

Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

Advances in computational design methods have made possible extensive engineering of soluble proteins, but designed β-barrel membrane proteins await improvements in our understanding of the sequence determinants of folding and stability. A subset of the amino acid residues of membrane proteins interact with the cell membrane, and the design rules that govern this lipid-facing surface are poorly understood. We applied a residue-level depth potential for β-barrel membrane proteins to the complete redesign of the lipid-facing surface of Escherichia coli OmpA. Initial designs failed to fold correctly, but reversion of a small number of mutations indicated by backcross experiments yielded designs with substitutions to up to 60% of the surface that did support folding and membrane insertion.The β-barrel membrane proteins comprise one of the two structural classes of integral membrane proteins. They are found within the outer membranes of bacteria, mitochondria, and chloroplasts, where they perform a range of structural, transport, and catalytic functions (1). In addition to their biological interest they are increasingly relevant to biotechnology, serving as scaffolds for bacterial surface display (2, 3) and atomically precise pores for nanopore-based DNA sequencing. Although the suitability of natural β-barrel membrane proteins for biotechnology has been improved by protein engineering (3–10), the ability to design membrane proteins de novo would deliver tools customized to meet the demands of each application.De novo design provides a stringent test of our understanding of the determinants of protein folding and stability. Protein design software [e.g., Rosetta (11, 12)] has made tremendous strides in addressing the design problem for small water-soluble proteins (13–15), and design of simplified model α-helical membrane proteins including single transmembrane helices and small bundles (16–20) has also been accomplished. In contrast, a designed β-barrel membrane protein has yet to be reported, perhaps as a consequence of the unique design challenges presented by the folding pathway and architecture of these proteins. Unlike the α-helical membrane proteins, nascent β-barrel membrane proteins must transit the periplasm to the outer membrane, where folding and membrane insertion are thought to occur in concert (21, 22). An extensive network of chaperones maintains the solubility of the unfolded barrel and guides membrane insertion. The C-terminal β-strand is known to interact with the BAM chaperone complex (23–25), which assists the folding of all β-barrel membrane proteins. However, despite recent progress (26–30), we do not fully understand how interactions between chaperones and transiting membrane proteins are directed by sequence-encoded information.Further complicating design is the inside-out architecture of β-barrel membrane proteins. In place of a hydrophobic core is either a central water-filled pore or a solid core composed of polar side chains. The lipid bilayer becomes increasingly hydrophobic at greater depths within the membrane (31), and this environmental anisotropy is reflected in the amino acid composition of the barrel surface. Aliphatic side chains are prevalent toward the center of the membrane, and aromatic side chains are common in the lipid head group regions, where they encircle the barrel in external- and periplasmic-side girdles (32).Recently we developed E_zβ, a membrane depth-dependent, residue-level potential calculated from an ensemble of experimentally determined outer membrane protein structures (33, 34). E_zβ can be used to estimate energetics of membrane insertion to predict transmembrane protein orientation within the bilayer, and to detect oligomerization sites on β-barrel surfaces (34). E_zβ and related statistical functions (35, 36) can recapitulate properties of natural outer membrane proteins (37, 38) and predict the effects of mutations on protein stability and oligomerization (39). Similar potentials have driven computational approaches that have fully redesigned α-helical membrane protein surfaces to convert membrane proteins into water-soluble ones (40–42).Here, we considered whether the complete redesign of the lipid-facing surface of an outer membrane protein using a statistical potential such as E_zβ preserves its structure and function. This approach allowed us to investigate whether membrane insertion requires only a lipid-facing surface composed of depth-appropriate hydrophobic residues, or whether folding requires sequence-specific interstrand interactions, chaperone-recruiting sequences, evolutionarily optimized aromatic girdles, folding nucleation sites, or other design features lost during the population averaging inherent in parameter fitting of statistical potentials.Previous studies have explored the sensitivity of the β-barrel fold and its chaperone recognition mechanisms to mutations. The canonical eight-stranded β-barrel membrane protein OmpA tolerates a limited number of mutations to the lipid-facing surface, provided hydrophobicity is maintained (43, 44). More radically, the eight-stranded barrel OmpX has been duplicated to form a 16-stranded barrel capable of membrane insertion (45). However, the lipid-facing residues of transmembrane β-strands are conserved across homologous β-barrel membrane proteins beyond the extent expected from hydrophobicity alone (46, 47), implying a functional role that has yet to be elucidated.To explore the sequence constraints on β-barrel membrane proteins, we extensively redesigned the lipid-facing surface of E. coli OmpA. We created a series of OmpA variants with entirely or partially redesigned lipid-facing surfaces and tested their ability to insert into the outer membrane of E. coli. Our results indicate that the surfaces of β-barrel membrane proteins are amenable to large-scale redesign, provided that energetically destabilizing substitutions are avoided.     

Hyperpolarization-activated,cyclic nucleotide-gated cation channels in Aplysia: Contribution to classical conditioning

Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are critical regulators of neuronal excitability, but less is known about their possible roles in synaptic plasticity and memory circuits. Here, we characterized the HCN gene organization, channel properties, distribution, and involvement in associative and nonassociative forms of learning in Aplysia californica. Aplysia has only one HCN gene, which codes for a channel that has many similarities to the mammalian HCN channel. The cloned acHCN gene was expressed in Xenopus oocytes, which displayed a hyperpolarization-induced inward current that was enhanced by cGMP as well as cAMP. Similarly to its homologs in other animals, acHCN is permeable to K⁺ and Na⁺ ions, and is selectively blocked by Cs⁺ and ZD7288. We found that acHCN is predominantly expressed in inter- and motor neurons, including LFS siphon motor neurons, and therefore tested whether HCN channels are involved in simple forms of learning of the siphon-withdrawal reflex in a semiintact preparation. ZD7288 (100 μM) significantly reduced an associative form of learning (classical conditioning) but had no effect on two nonassociative forms of learning (intermediate-term sensitization and unpaired training) or baseline responses. The HCN current is enhanced by nitric oxide (NO), which may explain the postsynaptic role of NO during conditioning. HCN current in turn enhances the NMDA-like current in the motor neurons, suggesting that HCN channels contribute to conditioning through this pathway.Hyperpolarization-activated, cyclic nucleotide-gated (HCN), cation nonselective ion channels generate hyperpolarization-activated inward currents (I_h) and thus tend to stabilize membrane potential (1–3). In addition, binding of cyclic nucleotides (cAMP and cGMP) to the C-terminal cyclic nucleotide binding domain (CNBD) enhances I_h and thus couples membrane excitability with intracellular signaling pathways (2, 4). HCN channels are widely important for numerous systemic functions such as hormonal regulation, heart contractility, epilepsy, pain, central pattern generation, sensory perception (4–15), and learning and memory (16–24).However, in previous studies it has been difficult to relate the cellular effects of HCN channels directly to their behavioral effects, because of the immense complexity of the mammalian brain. We have therefore investigated the role of HCN channels in Aplysia, which has a numerically simpler nervous system (25). We first identified and characterized an HCN gene in Aplysia, and showed that it codes for a channel that has many similarities to the mammalian HCN channel. We found that the Aplysia HCN channel is predominantly expressed in motor neurons including LFS neurons in the siphon withdrawal reflex circuit (26, 27). We therefore investigated simple forms of learning of that reflex in a semiintact preparation (28–30) and found that HCN current is involved in classical conditioning and enhances the NMDA-like current in the motor neurons. These results provide a direct connection between HCN channels and behavioral learning and suggest a postsynaptic mechanism of that effect. HCN current in turn is enhanced by nitric oxide (NO), a transmitter of facilitatory interneurons, and thus may contribute to the postsynaptic role of NO during conditioning.     

Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl,oxygen-rebound mechanism

Lytic polysaccharide monooxygenases (LPMOs) exhibit a mononuclear copper-containing active site and use dioxygen and a reducing agent to oxidatively cleave glycosidic linkages in polysaccharides. LPMOs represent a unique paradigm in carbohydrate turnover and exhibit synergy with hydrolytic enzymes in biomass depolymerization. To date, several features of copper binding to LPMOs have been elucidated, but the identity of the reactive oxygen species and the key steps in the oxidative mechanism have not been elucidated. Here, density functional theory calculations are used with an enzyme active site model to identify the reactive oxygen species and compare two hypothesized reaction pathways in LPMOs for hydrogen abstraction and polysaccharide hydroxylation; namely, a mechanism that employs a η¹-superoxo intermediate, which abstracts a substrate hydrogen and a hydroperoxo species is responsible for substrate hydroxylation, and a mechanism wherein a copper-oxyl radical abstracts a hydrogen and subsequently hydroxylates the substrate via an oxygen-rebound mechanism. The results predict that oxygen binds end-on (η¹) to copper, and that a copper-oxyl–mediated, oxygen-rebound mechanism is energetically preferred. The N-terminal histidine methylation is also examined, which is thought to modify the structure and reactivity of the enzyme. Density functional theory calculations suggest that this posttranslational modification has only a minor effect on the LPMO active site structure or reactivity for the examined steps. Overall, this study suggests the steps in the LPMO mechanism for oxidative cleavage of glycosidic bonds.Carbohydrates are the most diverse set of biomolecules, and thus, many enzyme classes have evolved to assemble, modify, and depolymerize carbohydrates, including glycosyltransferases, glycoside hydrolases, carbohydrate esterases, and polysaccharide lyases (1). Recently, a new enzymatic paradigm was discovered that employs copper-dependent oxidation to cleave glycosidic bonds in polysaccharides (2–13). These newly classified enzymes, termed lytic polysaccharide monooxygenases (LPMOs), broadly resemble other copper monooxygenases and some hydroxylation catalysts (14–21).The discovery that LPMOs use an oxidative mechanism has attracted interest both because it is a unique paradigm for carbohydrate modification that employs a powerful C–H activation mechanism, and also because LPMOs synergize with hydrolytic enzymes in biomass conversion to sugars because they act directly on the crystalline polysaccharide surface without the requirement for depolymerization (4, 22, 23), making them of interest in biofuels production. LPMOs were originally characterized as Family 61 glycoside hydrolases (GH61s, reclassified as auxiliary activity 9, AA9) or Family 33 carbohydrate-binding modules (CBM33s, reclassified as AA10), which are structurally similar enzymes found in fungi and nonfungal organisms (22), respectively. In 2005, Vaaje-Kolstad et al. described the synergism (24) of a chitin-active CBM33 (chitin-binding protein, CBP21) with hydrolases, but the mechanism was not apparent. Harris et al. demonstrated that a GH61 boosts hydrolytic enzyme activity on lignocellulosic biomass (2). Vaaje-Kolstad et al. subsequently showed that CBP21 employs an oxidative mechanism to cleave glycosidic linkages in chitin (4).Following these initial discoveries, multiple features of LPMOs have been elucidated. LPMOs use copper (5–7) and produce either aldonic acids or 4-keto sugars at oxidized chain ends, believed to result from hydroxylation at the C1 or C4 carbon, respectively. Hydroxylation at the C1 carbon is proposed to spontaneously undergo elimination to a lactone followed by hydrolytic ring opening to an aldonic acid, whereas hydroxylation and elimination at C4 yields a 4-keto sugar at the nonreducing end (5–12). The active site is a mononuclear type(II) copper center ligated by a “histidine brace” (5, 12), comprising a bidentate N-terminal histidine ligand via the amino terminus and an imidazole ring nitrogen atom and another histidine residue also via a ring nitrogen atom. Hemsworth et al. reported a bacterial LPMO structure wherein the active site copper ion was photoreduced to Cu(I) (12), and Aachmann et al. demonstrated that Cu(I) binds with higher affinity than Cu(II) in CBP21 (13). A structural study of a fungal LPMO revealed an N-terminal methylation on a nitrogen atom in the imidazole ring of unknown function (5), but some LPMOs are active without this modification (6, 11). LPMOs require reducing agents for activity such as ascorbate (2–8, 10–12), and cellobiose dehydrogenase (CDH), a common fungal secretome component, can potentiate LPMO activity in lieu of a small-molecule reducing agent (7, 8).Overall, many structural and mechanistic insights have been reported since the discoveries that LPMOs are oxidative enzymes (4–10). However, many questions remain regarding LPMO function (22, 25). Here, we examine the LPMO catalytic mechanism with density functional theory (DFT) calculations on an active site model (ASM) of a fungal LPMO. We seek to (i) understand the identity of the reactive oxygen species (ROS), (ii) compare two hypothesized catalytic mechanisms, and (iii) examine the role of N-terminal methylation in catalysis.     

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.     

Fuxianhuiid ventral nerve cord and early nervous system evolution in Panarthropoda

Panarthropods are typified by disparate grades of neurological organization reflecting a complex evolutionary history. The fossil record offers a unique opportunity to reconstruct early character evolution of the nervous system via exceptional preservation in extinct representatives. Here we describe the neurological architecture of the ventral nerve cord (VNC) in the upper-stem group euarthropod Chengjiangocaris kunmingensis from the early Cambrian Xiaoshiba Lagerstätte (South China). The VNC of C. kunmingensis comprises a homonymous series of condensed ganglia that extend throughout the body, each associated with a pair of biramous limbs. Submillimetric preservation reveals numerous segmental and intersegmental nerve roots emerging from both sides of the VNC, which correspond topologically to the peripheral nerves of extant Priapulida and Onychophora. The fuxianhuiid VNC indicates that ancestral neurological features of Ecdysozoa persisted into derived members of stem-group Euarthropoda but were later lost in crown-group representatives. These findings illuminate the VNC ground pattern in Panarthropoda and suggest the independent secondary loss of cycloneuralian-like neurological characters in Tardigrada and Euarthropoda.The nervous system represents a critical source of phylogenetic information and has been used extensively for exploring the evolutionary relationships of extant Panarthropoda (i.e., Onychophora, Tardigrada, Euarthropoda) (1–7). Identification of fossilized nervous tissues has provided a unique perspective on early euarthropod brain neuroanatomy and suggests that broad patterns of extant neurological diversity were already in place by the Cambrian (8–11). The ventral nerve cord (VNC) reflects fundamental aspects of panarthropod body organization that complement the organization of the brain and together illuminate the evolution of the CNS (1–3, 5, 7, 12–16). The early evolutionary history of the panarthropod postcephalic CNS, however, remains obscure due to the exclusive preservation of brains in most available fossils (8, 10, 11). Moreover, the unresolved phylogenetic relationships within Panarthropoda complicate accurate reconstruction of the CNS ground pattern (16–22). In this study, we demonstrate the exceptional preservation of postcephalic neurological features in the early Cambrian fuxianhuiid Chengjiangocaris kunmingensis, an upper stem-group euarthropod (17) from the Xiaoshiba Lagerstätte, South China (23). These fossils clarify the neurological organization of the VNC in early euarthropod ancestors, thereby polarizing the evolution of the panarthropod CNS.