scholarly journals Structural rearrangement of the intracellular gate of the serotonin transporter induced by Thr276 phosphorylation

2021 ◽  
Author(s):  
Matthew Chan ◽  
Erik Procko ◽  
Diwakar Shukla
Keyword(s):  
Serotonin Transporter ◽  
Transmembrane Helix ◽  
Synaptic Cleft ◽  
Structural Rearrangement ◽  
Post Translational Modifications ◽  
State Models ◽  
Dynamics Simulations ◽  
And Function ◽  
A Site

The reuptake of the neurotransmitter serotonin from the synaptic cleft by the serotonin transporter, SERT, is essential for proper neurological signaling. Biochemical studies have shown Thr276 of transmembrane helix 5 is a site of PKG-mediated SERT phosphorylation, which has been proposed to shifts the SERT conformational equlibira to promote inward-facing states, thus enhancing 5HT transport. Recent structural and simulation studies have provided insights into the conformation transitions during substrate transport but have not shed light on SERT regulation via post-translational modifications. Using molecular dynamics simulations and Markov state models, we investigate how Thr276 phosphorylation impacts the SERT mechanism and its role in enhancing transporter stability and function. Our simulations show that Thr276 phosphorylation alters the hydrogen-bonding network involving residues on transmembrane helix 5. This in turn decreases the free energy barriers for SERT to transition to the inward-facing state, thus facilitating 5HT transport. The results provide atomistic insights into in vivo SERT regulation and can be extended to other pharmacologically important transporters in the solute carrier superfamily.

scholarly journals In Vivo Analysis of the Major Exocytosis-sensitive Phosphoprotein in Tetrahymena

1997 ◽  
Vol 139 (5) ◽  
pp. 1197-1207 ◽  
Author(s):  
N. Doane Chilcoat ◽  
Aaron P. Turkewitz
Keyword(s):  
Membrane Fusion ◽  
Protein Function ◽  
Potential Role ◽  
Secretory Granules ◽  
Rabbit Muscle ◽  
Paramecium Tetraurelia ◽  
Post Translational Modifications ◽  
In Vivo Analysis ◽  
And Function

Phosphoglucomutase (PGM) is a ubiquitous highly conserved enzyme involved in carbohydrate metabolism. A number of recently discovered PGM-like proteins in a variety of organisms have been proposed to function in processes other than metabolism. In addition, sequence analysis suggests that several of these may lack PGM enzymatic activity. The best studied PGM-like protein is parafusin, a major phosphoprotein in the ciliate Paramecium tetraurelia that undergoes rapid and massive dephosphorylation when cells undergo synchronous exocytosis of their dense-core secretory granules. Indirect genetic and biochemical evidence also supports a role in regulated exocytotic membrane fusion. To examine this matter directly, we have identified and cloned the parafusin homologue in Tetrahymena thermophila, a ciliate in which protein function can be studied in vivo. The unique T. thermophila gene, called PGM1, encodes a protein that is closely related to parafusin by sequence and by characteristic post-translational modifications. Comparison of deduced protein sequences, taking advantage of the known atomic structure of rabbit muscle PGM, suggests that both ciliate enzymes and all other PGM-like proteins have PGM activity. We evaluated the activity and function of PGM1 through gene disruption. Surprisingly, ΔPGM1 cells displayed no detectable defect in exocytosis, but showed a dramatic decrease in PGM activity. Both our results, and reinterpretation of previous data, suggest that any potential role for PGM-like proteins in regulated exocytosis is unlikely to precede membrane fusion.

scholarly journals Modulation of A2aR Oligomerisation by Conformational State and PIP2 Interactions Revealed by MD Simulations and Markov Models

2020 ◽  
Author(s):  
Wanling Song ◽  
Anna L. Duncan ◽  
Mark S.P. Sansom
Keyword(s):  
Protein Interactions ◽  
Large Scale ◽  
Markov Models ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
Membrane Lipid ◽  
Intracellular Loop ◽  
Gpcr Activation ◽  
State Models

AbstractG protein-coupled receptors (GPCRs) play key roles in cellular signalling. GPCRs are suggested to form dimers and higher order oligomers in response to activation. However, we do not fully understand GPCR activation at larger scales and in an in vivo context. We have characterised oligomeric configurations of the adenosine 2a receptor (A2aR) by combining large-scale molecular dynamics simulations with Markov state models. Receptor activation results in enhanced oligomerisation, more diverse oligomer populations, and a more connected oligomerisation network. The active state conformation of the A2aR shifts protein-protein association interfaces to those involving intracellular loop ICL3 and transmembrane helix TM6. Binding of PIP2 to A2aR stabilises protein-protein interactions via PIP2-mediated association interfaces. These results indicate that A2aR oligomerisation is responsive to the local membrane lipid environment. This in turn suggests a modulatory effect on A2aR whereby a given oligomerisation profile favours the dynamic formation of specific supra-molecular signalling complexes.

scholarly journals Distribution and Function of Laminins in the Neuromuscular System of Developing, Adult, and Mutant Mice

1997 ◽  
Vol 139 (6) ◽  
pp. 1507-1521 ◽  
Author(s):  
Bruce L. Patton ◽  
Jeffrey H. Miner ◽  
Arlene Y. Chiu ◽  
Joshua R. Sanes
Keyword(s):  
Peripheral Nerve ◽  
Hot Spots ◽  
Muscle Cells ◽  
Synaptic Cleft ◽  
The Body ◽  
Motor Axons ◽  
And Function ◽  
Development Proceeds ◽  
Laminin 1

Laminins, heterotrimers of α, β, and γ chains, are prominent constituents of basal laminae (BLs) throughout the body. Previous studies have shown that laminins affect both myogenesis and synaptogenesis in skeletal muscle. Here we have studied the distribution of the 10 known laminin chains in muscle and peripheral nerve, and assayed the ability of several heterotrimers to affect the outgrowth of motor axons. We show that cultured muscle cells express four different α chains (α1, α2, α4, and α5), and that developing muscles incorporate all four into BLs. The portion of the muscle's BL that occupies the synaptic cleft contains at least three α chains and two β chains, but each is regulated differently. Initially, the α2, α4, α5, and β1 chains are present both extrasynaptically and synaptically, whereas β2 is restricted to synaptic BL from its first appearance. As development proceeds, α2 remains broadly distributed, whereas α4 and α5 are lost from extrasynaptic BL and β1 from synaptic BL. In adults, α4 is restricted to primary synaptic clefts whereas α5 is present in both primary and secondary clefts. Thus, adult extrasynaptic BL is rich in laminin 2 (α2β1γ1), and synaptic BL contains laminins 4 (α2β2γ1), 9 (α4β2γ1), and 11 (α5β2γ1). Likewise, in cultured muscle cells, α2 and β1 are broadly distributed but α5 and β2 are concentrated at acetylcholine receptor–rich “hot spots,” even in the absence of nerves. The endoneurial and perineurial BLs of peripheral nerve also contain distinct laminin chains: α2, β1, γ1, and α4, α5, β2, γ1, respectively. Mutation of the laminin α2 or β2 genes in mice not only leads to loss of the respective chains in both nerve and muscle, but also to coordinate loss and compensatory upregulation of other chains. Notably, loss of β2 from synaptic BL in β2−/− “knockout” mice is accompanied by loss of α5, and decreased levels of α2 in dystrophic α2dy/dy mice are accompanied by compensatory retention of α4. Finally, we show that motor axons respond in distinct ways to different laminin heterotrimers: they grow freely between laminin 1 (α1β1γ1) and laminin 2, fail to cross from laminin 4 to laminin 1, and stop upon contacting laminin 11. The ability of laminin 11 to serve as a stop signal for growing axons explains, in part, axonal behaviors observed at developing and regenerating synapses in vivo.

scholarly journals Nitroxyl (HNO) targets phospholamban cysteines 41 and 46 to enhance cardiac function

2019 ◽  
Vol 151 (6) ◽  
pp. 758-770 ◽  
Author(s):  
Gizem Keceli ◽  
Ananya Majumdar ◽  
Chevon N. Thorpe ◽  
Seungho Jun ◽  
Carlo G. Tocchetti ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Myocardial Function ◽  
Transmembrane Domain ◽  
Oxygen Species ◽  
Post Translational Modifications ◽  
Physiological Conditions ◽  
Nmr Method ◽  
Disulfide Formation ◽  
And Function

Nitroxyl (HNO) positively modulates myocardial function by accelerating Ca2+ reuptake into the sarcoplasmic reticulum (SR). HNO-induced enhancement of myocardial Ca2+ cycling and function is due to the modification of cysteines in the transmembrane domain of phospholamban (PLN), which results in activation of SR Ca2+-ATPase (SERCA2a) by functionally uncoupling PLN from SERCA2a. However, which cysteines are modified by HNO, and whether HNO induces reversible disulfides or single cysteine sulfinamides (RS(O)NH2) that are less easily reversed by reductants, remain to be determined. Using an 15N-edited NMR method for sulfinamide detection, we first demonstrate that Cys46 and Cys41 are the main targets of HNO reactivity with PLN. Supporting this conclusion, mutation of PLN cysteines 46 and 41 to alanine reduces the HNO-induced enhancement of SERCA2a activity. Treatment of WT-PLN with HNO leads to sulfinamide formation when the HNO donor is in excess, whereas disulfide formation is expected to dominate when the HNO/thiol stoichiometry approaches a 1:1 ratio that is more similar to that anticipated in vivo under normal, physiological conditions. Thus, 15N-edited NMR spectroscopy detects redox changes on thiols that are unique to HNO, greatly advancing the ability to detect HNO footprints in biological systems, while further differentiating HNO-induced post-translational modifications from those imparted by other reactive nitrogen or oxygen species. The present study confirms the potential of HNO as a signaling molecule in the cardiovascular system.

scholarly journals The guidance and adhesion protein FLRT2 dimerizes in cis via dual Small-X3-Small transmembrane motifs

2020 ◽  
Author(s):  
Verity Jackson ◽  
Julia Hermann ◽  
Christopher J. Tynan ◽  
Daniel J. Rolfe ◽  
Antreas C. Kalli ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transmembrane Helix ◽  
Single Particle Tracking ◽  
Transmembrane Receptors ◽  
Adhesion Protein ◽  
Small X ◽  
In Trans ◽  
Dynamics Simulations ◽  
And Function ◽  
In Cis

AbstractFibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate synapse development and cell guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact in cis remains unknown. Here, we reveal two dimerization motifs in the FLRT2 transmembrane helix. Molecular dynamics simulations and single particle tracking experiments show that these ‘Small-X3-Small’ motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and diffusion patterns of FLRT2 on cells. The results point to a competitive switching mechanism between in cis and in trans interactions which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules.

scholarly journals Markov State Models and Molecular Dynamics Simulations Provide Understanding of the Nucleotide-Dependent Dimerization-Based Activation of LRRK2 ROC Domain

Molecules ◽  
2021 ◽  
Vol 26 (18) ◽  
pp. 5647
Author(s):  
Xinyi Li ◽  
Zengxin Qi ◽  
Duan Ni ◽  
Shaoyong Lu ◽  
Liang Chen ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Structural Rearrangement ◽  
Catalytic Triad ◽  
Gtpase Domain ◽  
Markov State ◽  
Markov State Models ◽  
Activated State ◽  
State Models ◽  
Dynamics Simulations

Mutations in leucine-rich repeat kinase 2 (LRRK2) are recognized as the most frequent cause of Parkinson’s disease (PD). As a multidomain ROCO protein, LRRK2 is characterized by the presence of both a Ras-of-complex (ROC) GTPase domain and a kinase domain connected through the C-terminal of an ROC domain (COR). The bienzymatic ROC–COR–kinase catalytic triad indicated the potential role of GTPase domain in regulating kinase activity. However, as a functional GTPase, the detailed intrinsic regulation of the ROC activation cycle remains poorly understood. Here, combining extensive molecular dynamics simulations and Markov state models, we disclosed the dynamic structural rearrangement of ROC’s homodimer during nucleotide turnover. Our study revealed the coupling between dimerization extent and nucleotide-binding state, indicating a nucleotide-dependent dimerization-based activation scheme adopted by ROC GTPase. Furthermore, inspired by the well-known R1441C/G/H PD-relevant mutations within the ROC domain, we illuminated the potential allosteric molecular mechanism for its pathogenetic effects through enabling faster interconversion between inactive and active states, thus trapping ROC in a prolonged activated state, while the implicated allostery could provide further guidance for identification of regulatory allosteric pockets on the ROC complex. Our investigations illuminated the thermodynamics and kinetics of ROC homodimer during nucleotide-dependent activation for the first time and provided guidance for further exploiting ROC as therapeutic targets for controlling LRRK2 functionality in PD treatment.

scholarly journals Universal protein misfolding intermediates can bypass the proteostasis network and remain soluble and non-functional

2021 ◽  
Author(s):  
Daniel Allen Nissley ◽  
Yang Jiang ◽  
Fabio Trovato ◽  
Ian Sitarik ◽  
Karthik Narayan ◽  
...  
Keyword(s):  
Protein Function ◽  
Protein Misfolding ◽  
Misfolded Proteins ◽  
Control Mechanisms ◽  
Functional Sites ◽  
Synonymous Mutations ◽  
Dynamics Simulations ◽  
And Function ◽  
Kinetic Traps

Misfolded protein conformations with decreased functionality can bypass the proteostasis machinery and remain soluble in vivo. This is an unexpected phenomenon as several cellular quality control mechanisms have evolved to rid cells of misfolded proteins. Three questions, then, are: how is it structurally possible for long-lived, soluble, misfolded proteins to bypass the proteostasis machinery and processes? How widespread are these soluble, misfolded states across the proteome? And how long do they persist for? Here, we address these questions using coarse-grain molecular dynamics simulations of the synthesis, termination, and post-translational dynamics of a representative set of cytosolic E. coli proteins. We find that half of all proteins exhibit subpopulations of misfolded conformations that are likely to bypass molecular chaperones, avoid aggregation, and not be degraded. These misfolded states can persist for months or longer for some proteins. Structurally characterizing these misfolded states, we observe they have a large amount of native structure, but also contain localized misfolded regions from non-native changes in entanglement, in which a protein segment threads through a loop formed by another portion of the protein that is not found in the native state. The surface properties of these misfolded states are native like, allowing them to bypass the proteostasis machinery and processes to remain soluble, while their entanglements make these states long-lived kinetic traps, as disentanglement requires unfolding of already folded portions of the protein. In terms of function, one-third of proteins have subpopulations that misfold into less-functional states that have structurally perturbed functional sites yet remain soluble. These results explain how proteins misfold into soluble, non-functional conformations that bypass cellular quality controls, and indicate that, unexpectedly, this is a wide-spread cellular phenomenon that can lead to reduced protein function across the cytosolic proteome. Such entanglements are observed in many native structures, suggesting the non-native entanglements we observe are plausible. More broadly, these near-native entangled structures suggest a hypothesis for how synonymous mutations can modulate downstream protein structure and function, with these mutations partitioning nascent proteins between these kinetically trapped states.

scholarly journals Genetic deletion of Rgs12 in mice affects serotonin transporter expression and function in vivo and ex vivo

2020 ◽  
Vol 34 (12) ◽  
pp. 1393-1407 ◽  
Author(s):  
Allison N White ◽  
Joshua D Gross ◽  
Shane W Kaski ◽  
Kristen R Trexler ◽  
Kim A Wix ◽  
...  
Keyword(s):  
G Protein ◽  
Serotonin Transporter ◽  
Brain Region ◽  
Ex Vivo ◽  
Brain Regions ◽  
Rgs Proteins ◽  
Protein Signaling ◽  
5 Ht Uptake ◽  
And Function

Background: Regulator of G protein Signaling (RGS) proteins inhibit G protein-coupled receptor (GPCR) signaling, including the signals that arise from neurotransmitter release. We have shown that RGS12 loss diminishes locomotor responses of C57BL/6J mice to dopamine transporter (DAT)-targeting psychostimulants. This diminution resulted from a brain region-specific upregulation of DAT expression and function in RGS12-null mice. This effect on DAT prompted us to investigate whether the serotonin transporter (SERT) exhibits similar alterations upon RGS12 loss in C57BL/6J mice. Aims: Does RGS12 loss affect (a) hyperlocomotion to the preferentially SERT-targeting psychostimulant 3,4-methylenedioxymethamphetamine (MDMA), (b) SERT expression and function in relevant brain regions, and/or (c) serotonergically modulated behaviors? Methods: Open-field and spontaneous home-cage locomotor activities were quantified. 5-HT, 5-HIAA, and SERT levels in brain-region homogenates, as well as SERT expression and function in brain-region tissue preparations, were measured using appropriate biochemical assays. Serotonergically modulated behaviors were assessed using forced swim and tail suspension paradigms, elevated plus and elevated zero maze tests, and social interaction assays. Results: RGS12-null mice displayed no hyperlocomotion to 10 mg/kg MDMA. There were brain region-specific alterations in SERT expression and function associated with RGS12 loss. Drug-naïve RGS12-null mice displayed increases in both anxiety-like and anti-depressive-like behaviors. Conclusion: RGS12 is a critical modulator of serotonergic neurotransmission and serotonergically modulated behavior in mice; lack of hyperlocomotion to low dose MDMA in RGS12-null mice is related to an alteration of steady-state SERT expression and 5-HT uptake.

scholarly journals Dimerization of visual pigments in vivo

2016 ◽  
Vol 113 (32) ◽  
pp. 9093-9098 ◽  
Author(s):  
Tao Zhang ◽  
Li-Hui Cao ◽  
Sandeep Kumar ◽  
Nduka O. Enemchukwu ◽  
Ning Zhang ◽  
...  
Keyword(s):  
Visual Pigment ◽  
Outer Segment ◽  
Transmembrane Helix ◽  
Historical Context ◽  
Convincing Evidence ◽  
Cone Opsin ◽  
Cone Opsins ◽  
And Function

It is a deeply engrained notion that the visual pigment rhodopsin signals light as a monomer, even though many G protein-coupled receptors are now known to exist and function as dimers. Nonetheless, recent studies (albeit all in vitro) have suggested that rhodopsin and its chromophore-free apoprotein, R-opsin, may indeed exist as a homodimer in rod disk membranes. Given the overwhelmingly strong historical context, the crucial remaining question, therefore, is whether pigment dimerization truly exists naturally and what function this dimerization may serve. We addressed this question in vivo with a unique mouse line (S-opsin+Lrat−/−) expressing, transgenically, short-wavelength–sensitive cone opsin (S-opsin) in rods and also lacking chromophore to exploit the fact that cone opsins, but not R-opsin, require chromophore for proper folding and trafficking to the photoreceptor’s outer segment. In R-opsin’s absence, S-opsin in these transgenic rods without chromophore was mislocalized; in R-opsin’s presence, however, S-opsin trafficked normally to the rod outer segment and produced functional S-pigment upon subsequent chromophore restoration. Introducing a competing R-opsin transmembrane helix H1 or helix H8 peptide, but not helix H4 or helix H5 peptide, into these transgenic rods caused mislocalization of R-opsin and S-opsin to the perinuclear endoplasmic reticulum. Importantly, a similar peptide-competition effect was observed even in WT rods. Our work provides convincing evidence for visual pigment dimerization in vivo under physiological conditions and for its role in pigment maturation and targeting. Our work raises new questions regarding a potential mechanistic role of dimerization in rhodopsin signaling.

scholarly journals A structural model of the human serotonin transporter in an outward-occluded state

2019 ◽  
Author(s):  
Eva Hellsberg ◽  
Gerhard F. Ecker ◽  
Anna Stary-Weinzinger ◽  
Lucy R. Forrest
Keyword(s):  
Serotonin Transporter ◽  
Conformational Changes ◽  
Structural Model ◽  
Antidepressant Drugs ◽  
Homology Model ◽  
Synaptic Cleft ◽  
Open Conformation ◽  
Starting Point ◽  
Ligand Interactions ◽  
Dynamics Simulations

AbstractThe human serotonin transporter hSERT facilitates the reuptake of its endogenous substrate serotonin from the synaptic cleft into presynaptic neurons after signaling. Reuptake regulates the availability of this neurotransmitter and therefore hSERT plays an important role in balancing human mood conditions. In 2016, the first 3D structures of this membrane transporter were reported in an inhibitor-bound, outward-open conformation. These structures revealed valuable information about interactions of hSERT with antidepressant drugs. Nevertheless, the question remains how serotonin facilitates the specific conformational changes that open and close pathways from the synapse and to the cytoplasm as required for transport. Here, we present a serotonin-bound homology model of hSERT in an outward-occluded state, a key intermediate in the physiological cycle, in which the interactions with the substrate are likely to be optimal. Our approach uses two template structures and includes careful refinement and comprehensive computational validation. According to microsecond-long molecular dynamics simulations, this model exhibits interactions between the gating residues in the extracellular pathway, and these interactions differ from those in an outward-open conformation of hSERT bound to serotonin. Moreover, we predict several features of this state by monitoring the intracellular gating residues, the extent of hydration, and, most importantly, protein-ligand interactions in the central binding site. The results illustrate common and distinct characteristics of these two transporter states and provide a starting point for future investigations of the transport mechanism in hSERT.

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