Biology subjects

Eliezer, D.

Publications and source records attributed to Eliezer, D..

15 recordsLinked to original sources

bioRxiv2025

Genetic Code Expansion and Enzymatic Modifications as Accessible Methods for Studying Site-Specific Post-Translational Modifications of Alpha-Synuclein and Tau

Alpha-synuclein (S) and tau play important roles in the pathology of Parkinsons Disease and Alzheimers Disease, respectively, as well as numerous other neurodegenerative diseases. Both proteins are classified as intrinsically disordered proteins (IDPs), as they have no stable structure that underlies their function in healthy tissue, and both proteins are prone to aggregation in disease states. There is substantial interest in understanding the roles that post-translational modifications (PTMs) play in regulating the structural dynamics and function of S and tau monomers, as well as their propensity to aggregate. While there have been many valuable insights into site-specific effects of PTMs garnered through chemical synthesis and semi-synthesis, these techniques are often outside of the expertise of biochemistry and biophysics laboratories wishing to study S and tau. Therefore, we have assembled a primer on genetic code expansion and enzymatic modification approaches to installing PTMs into S and tau site-specifically, including isotopic labeling for NMR and fluorescent labeling for biophysics and microscopy experiments. These methods should be enabling for those wishing to study authentic PTMs in S or tau as well as the broader field of IDPs and aggregating proteins.

biochemistry↗

bioRxiv2025

Structural Diversity of Metabotropic Glutamate Receptor/Beta-Arrestin Coupling

Beta-arrestins ({beta}-arrs) are cytosolic proteins which mediate G protein-coupled receptor (GPCR) desensitization, endocytosis, and signaling. Despite the widespread physiological roles of {beta}-arr coupling, the molecular basis of GPCR/{beta}-arr interaction has been studied primarily in monomeric family A GPCRs. Here we take an integrative biophysical and structural approach to uncover molecular diversity in {beta}-arr coupling to the neuromodulatory metabotropic glutamate receptors (mGluRs), prototypical, dimeric family C GPCRs. We find, using a new single molecule pulldown assay, that mGluRs couple to {beta}-arrs with a 2:1 or 2:2 stoichiometry via a combination of "tail" and "core" interactions. Using single molecule FRET analysis, we also find that {beta}-arr1 stabilizes active conformations of mGluR8. Cryo-EM structures of mGluR8 alone or with either G proteins or {beta}-arr1 reveal transducer-specific mGluR8 active states and, in combination with molecular dynamics simulations, define the positioning of mGluR8-bound {beta}-arr1, supporting a steric mechanism of mGluR desensitization involving interactions with both subunits and the lipid bilayer. Finally, combinatorial mutagenesis enables the identification of a landscape of homo- and hetero-dimeric mGluR/{beta}-arr complexes, including mGluR/{beta}-arr1/{beta}-arr2 megacomplexes, providing a framework for family C GPCR/{beta}-arr coupling and expanding the known range of GPCR/transducer coupling modes.

biophysics↗

bioRxiv2025

Investigation of All Disease-Relevant Lysine Acetylation Sites in α-Synuclein Enabled by Non-canonical Amino Acid Mutagenesis

Aggregates of -synuclein (S) are hallmarks of synucleinopathies, including Parkinsons Disease (PD) and Multiple System Atrophy (MSA). We have recently shown that S lysine acetylation in the soluble monomer pool varies between healthy controls, PD, and MSA patients. We used non-canonical amino acid (ncAA) mutagenesis to express and purify all 12 currently known disease-relevant variants of S and studied their binding to membranes as well as their aggregation propensities. We found that acetylation of lysine 12, 43, and 80 had particularly strong effects. To understand the implications for acetylation of monomeric S found in healthy cells, we performed NMR experiments to study protein conformation and fluorescence correlation spectroscopy experiments to quantify lipid binding. We also investigated the effects of acetylation at lysine 12, 43, and 80 on fibril seeding in neurons. Collectively, our biochemical and cell biological investigations indicated that acetylation of K80 could inhibit aggregation without conferring negative effects on monomer function in healthy cells. Therefore, we studied the structures of fibrils with K80 acetylation through cryo-electron microscopy to uncover the structural basis for these effects. Finally, we identified inhibition of HDAC8 as a way of potentially increasing acetylation at K80 and other inhibitory sites for therapeutic benefit.

biochemistry↗

bioRxiv2024

Amyloid fibril structures link CHCHD10 and CHCHD2 to neurodegeneration.

CHCHD10 is mutated in rare cases of FTD and ALS and aggregates in mouse models of disease. Here we show that the disordered N-terminal domain of CHCHD10 forms amyloid fibrils and report their cryoEM structure. Disease-associated mutations cannot be accommodated by the WT fibril structure, while sequence differences between CHCHD10 and CHCHD2 are tolerated, explaining the co-aggregation of the two proteins and linking CHCHD10 and CHCHD2 amyloid fibrils to neurodegeneration.

biophysics↗

bioRxiv2023

Phosphoglycerate kinase 1 is a central leverage point in Parkinson's disease driven neuronal metabolic deficits

Phosphoglycerate kinase 1 (PGK1), the first ATP producing glycolytic enzyme, has emerged as a therapeutic target for Parkinsons Disease (PD), since a potential enhancer of its activity was reported to significantly lower PD risk. We carried out a suppressor screen of hypometabolic synaptic deficits and demonstrated that PGK1 is a rate limiting enzyme in nerve terminal ATP production. Increasing PGK1 expression in mid-brain dopamine neurons protected against hydroxy-dopamine driven striatal dopamine nerve terminal dysfunction in-vivo and modest changes in PGK1 activity dramatically suppressed hypometabolic synapse dysfunction in vitro. Furthermore, PGK1 is cross-regulated by PARK7 (DJ-1), a PD associated molecular chaperone, and synaptic deficits driven by PARK20 (Synaptojanin-1) can be reversed by increasing local synaptic PGK1 activity. These data indicate that nerve terminal bioenergetic deficits may underly a spectrum of PD susceptibilities and the identification of PGK1 as the limiting enzyme in axonal glycolysis provides a mechanistic underpinning for therapeutic protection. O_LISuppressor screen identifies PGK1 as the rate limiting enzyme in axon metabolism C_LIO_LIPGK1 expression in-vivo protects against striatal dopaminergic axon degeneration C_LIO_LILoss of DJ-1 impairs axonal ATP production C_LIO_LIPGK1 function is regulated by PARK7/DJ-1, and can reverse PARK20 synaptic deficits C_LI

neuroscience↗

bioRxiv2023

Control of G protein-coupled receptor function via membrane-interacting intrinsically disordered C-terminal domains

G protein-coupled receptors (GPCRs) control intracellular signaling cascades via agonist-dependent coupling to intracellular transducers including heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. In addition to their critical interactions with the transmembrane core of active GPCRs, all three classes of transducers have also been reported to interact with receptor C-terminal domains (CTDs). An underexplored aspect of GPCR CTDs is their possible role as lipid sensors given their proximity to the membrane. CTD-membrane interactions have the potential to control the accessibility of key regulatory CTD residues to downstream effectors and transducers. Here we report that the CTDs of two closely related family C GPCRs, metabotropic glutamate receptor 2 (mGluR2) and mGluR3, bind to membranes and that this interaction can regulate receptor function. We first characterize CTD structure with NMR spectroscopy, revealing lipid composition-dependent modes of membrane binding. Using molecular dynamics simulations and structure-guided mutagenesis, we then identify key conserved residues and cancer-associated mutations that modulate CTD-membrane binding. Finally, we provide evidence that mGluR3 transducer coupling is controlled by CTD-membrane interactions in live cells, which may be subject to regulation by CTD phosphorylation and changes in membrane composition. This work reveals a novel mechanism of GPCR modulation, suggesting that CTD-membrane binding may be a general regulatory mode throughout the broad GPCR superfamily. Significance StatementG protein-coupled receptors (GPCRs) allow cells to sense and respond to their environment and constitute the largest class of targets for approved therapeutic drugs. Temporally precise GPCR signaling is achieved by coupling the binding of extracellular ligands to the binding of intracellular signal transducers (e.g. heterotrimeric G proteins) and regulators (e.g. {beta}-arrestins). The C-terminal domains (CTDs) of GPCRs are targets of various post-translational modifications and play a critical role in transducer and regulator recruitment. Here we report novel interactions of the CTDs of two GPCRs of the metabotropic glutamate receptor family with cellular membranes. These interactions serve to regulate CTD accessibility and thus, mGluR coupling to transducers and regulators. We propose that dynamic CTD-membrane interaction constitutes a general mechanism for regulating GPCR function.

biophysics↗

bioRxiv2023

Alpha Synuclein Modulates Mitochondrial Ca2+ Uptake from ER During Cell Stimulation and Under Stress Conditions

Alpha synuclein (a-syn) is an intrinsically disordered protein prevalent in neurons, and aggregated forms are associated with synucleinopathies including Parkinson disease (PD). Despite the biomedical importance and extensive studies, the physiological role of a-syn and its participation in etiology of PD remain uncertain. We showed previously in model RBL cells that a-syn colocalizes with mitochondrial membranes, depending on formation of N-terminal helices and increasing with mitochondrial stress.1 We have now characterized this colocalization and functional correlates in RBL, HEK293, and N2a cells. We find that expression of a-syn enhances stimulated mitochondrial uptake of Ca2+ from the ER, depending on formation of its N-terminal helices but not on its disordered C-terminal tail. Our results are consistent with a-syn acting as a tether between mitochondria and ER, and we show increased contacts between these two organelles using structured illumination microscopy. We tested mitochondrial stress caused by toxins related to PD, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and found that a-syn prevents recovery of stimulated mitochondrial Ca2+ uptake. The C-terminal tail, and not N-terminal helices, is involved in this inhibitory activity, which is abrogated when phosphorylation site serine-129 is mutated (S129A). Correspondingly, we find that MPTP/MPP+ and CCCP stress is accompanied by both phosphorylation (pS129) and aggregation of a-syn. Overall, our results indicate that a-syn can participate as a tethering protein to modulate Ca2+ flux between ER and mitochondria, with potential physiological significance. A-syn can also prevent cellular recovery from toxin-induced mitochondrial dysfunction, which may represent a pathological role of a-syn in the etiology of PD.

cell biology↗

bioRxiv2023

Structure and DNA bridging activity of the essential Rec114-Mei4 trimer interface

The DNA double-strand breaks (DSBs) that initiate meiotic recombination are formed by an evolutionarily conserved suite of factors that includes Rec114 and Mei4 (RM), which regulate DSB formation both spatially and temporally. In vivo, these proteins form large immunostaining foci that are integrated with higher order chromosome structures. In vitro, they form a 2:1 heterotrimeric complex that binds cooperatively to DNA to form large, dynamic condensates. However, understanding of the atomic structures and dynamic DNA binding properties of RM complexes is lacking. Here, we report a structural model of a heterotrimeric complex of the C-terminus of Rec114 with the N-terminus of Mei4, supported by nuclear magnetic resonance experiments. This minimal complex, which lacks the predicted intrinsically disordered region of Rec114, is sufficient to bind DNA and form condensates. Single-molecule experiments reveal that the minimal complex can bridge two or more DNA duplexes and can generate force to condense DNA through long-range interactions. AlphaFold2 predicts similar structural models for RM orthologs across diverse taxa despite their low degree of sequence similarity. These findings provide insight into the conserved networks of protein-protein and protein-DNA interactions that enable condensate formation and promote formation of meiotic DSBs.

biochemistry↗

bioRxiv2022

An environmentally ultrasensitive fluorine-19 NMR probe for monitoring protein conformational equilibria

Limited chemical shift dispersion represents a significant barrier to studying multi-state equilibria of large membrane proteins by 19F NMR. We describe a novel monofluoroethyl 19F probe that dramatically increases the chemical shift dispersion. The improved conformational sensitivity and line shape enable the detection of previously unresolved states in 1D NMR spectra of a 134 kDa membrane transporter. Changes in the populations of these states in response to ligand binding, mutations, and temperature correlate with population changes of distinct conformations in structural ensembles determined by single-particle cryo-electron microscopy. Thus, 19F NMR can guide sample preparation to discover and visualize novel conformational states and facilitate image analysis and 3D classification.

biophysics↗

bioRxiv2020

BETA- AND GAMMA-SYNUCLEINS MODULATE SYNAPTIC VESICLE-BINDING OF ALPHA-SYNUCLEIN

-Synuclein (Syn), {beta}-synuclein ({beta}Syn), and {gamma}-synuclein ({gamma}Syn) are abundantly expressed in the vertebrate nervous system. Syn functions in neurotransmitter release via binding to and clustering synaptic vesicles and chaperoning of SNARE-complex assembly. The functions of {beta}Syn and {gamma}Syn are unknown. Functional redundancy of the three synucleins and mutual compensation when one synuclein is deleted have been proposed, but with conflicting evidence. Here, we demonstrate that {beta}Syn and {gamma}Syn have a reduced affinity towards membranes compared to Syn, and that direct interaction of {beta}Syn or {gamma}Syn with Syn results in reduced membrane binding of Syn. Our data suggest that all three synucleins affect synapse function, but only Syn mediates the downstream function of vesicle clustering and SNARE-complex assembly, while {beta}Syn and {gamma}Syn modulate the activity of Syn through regulating its binding to synaptic vesicles.

neuroscience↗

bioRxiv2020

Phosphorylation of the overlooked tyrosine 310 regulates the structure, aggregation, and microtubule- and lipid-binding properties of Tau

The microtubule-associated protein Tau is implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimers disease. Increasing evidence suggests that post-translational modifications play critical roles in regulating Tau normal functions and its pathogenic properties in Tauopathies. Very little is known about how phosphorylation of tyrosine residues influences the structure, aggregation, and microtubule- and lipid-binding properties of Tau. In this work, we aimed to address this knowledge gap and determine the relative contribution of phosphorylation of one or several of the five tyrosine residues in Tau (Y18, Y29, Y197, Y310 and Y394) to the regulation of its biophysical, aggregation and functional properties. Towards this goal, we used a combination of site-specific mutagenesis and in vitro phosphorylation by c-Abl kinase to generate Tau species phosphorylated at all tyrosine residues, all tyrosine residues except Y310 or Y394 (pTau-Y310F, pTau-Y394F) and Tau phosphorylated only at Y310 or Y394 (4F\pY310 or 4F\pY394). Our results show that phosphorylation at all five tyrosine residues, multiple N-terminal tyrosine residues (Y18, Y29 and Y197) or site-specific phosphorylation at residue Y310, itself located in the microtubule-binding and aggregation-prone domain of Tau, was sufficient to abolish Tau aggregation and inhibit its microtubule- and lipid-binding properties. NMR studies demonstrated that these effects were mediated by a local decrease in {beta}-sheet propensity of the PHF6 domain. Our findings underscore the unique role of Y310 phosphorylation in the regulation of Tau aggregation, microtubule and lipid interactions and highlight the importance of conducting further studies to elucidate its role in the regulation of Tau normal functions and its pathogenic properties.

biochemistry↗

bioRxiv2019

Monitoring Dynamics of Large Membrane Proteins by 19F Paramagnetic Longitudinal Relaxation: Domain Movement in a Glutamate Transporter Homolog

In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homologue with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified two outward- and one inward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by Cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.

biophysics↗

bioRxiv2019

Succinylation Links Metabolic Reductions to Amyloid and Tau Pathology

Abnormalities in glucose metabolism and misfolded protein deposits composed of the amyloid-{beta} peptide (A{beta}) and tau are the three most common neuropathological hallmarks of Alzheimers disease (AD), but their relationship(s) to the disease process or to each other largely remains unclear. In this report, the first human brain quantitative lysine succinylome together with a global proteome analysis from controls and patients reveals that lysine succinylation contributes to these three key AD-related pathologies. Succinylation, a newly discovered protein post-translational modification (PTM), of multiple proteins, particularly mitochondrial proteins, declines with the progression of AD. In contrast, amyloid precursor protein (APP) and tau consistently exhibit the largest AD-related increases in succinylation, occurring at specific sites in AD brains but never in controls. Transgenic mouse studies demonstrate that succinylated APP and succinylated tau are detectable in the hippocampus concurrent with A{beta} assemblies in the oligomer and insoluble fiber assembly states. Multiple biochemical approaches revealed that succinylation of APP alters APP processing so as to promote A{beta} accumulation, while succinylation of tau promotes its aggregation and impairs its microtubule binding ability. Succinylation, therefore, is the first single PTM that can be added in parallel to multiple substrates, thereby promoting amyloidosis, tauopathy, and glucose hypometabolism. These data raise the possibility that, in order to show meaningful clinical benefit, any therapeutic and/or preventative measures destined for success must have an activity to either prevent or reverse the molecular pathologies attributable to excess succinylation.

neuroscience↗

bioRxiv2019

Water for sterol: an unusual mechanism of sterol egress from a StARkin domain

Previously we identified a new family of endoplasmic reticulum membrane proteins that possess sterol-binding StARkin domains (Gatta et al. eLife 2015). These Lam/GramD1 proteins are implicated in intracellular sterol homeostasis, a function that requires them to be able to bind sterols. Here we show how these proteins exchange sterol molecules with membranes. An aperture at one end of the StARkin domain enables sterol to enter/exit the binding pocket. Strikingly, the wall of the pocket is fractured along its length, exposing bound sterol to solvent. We considered whether hydration of the pocket could mediate sterol entry/exit. Large-scale atomistic molecular dynamics simulations reveal that sterol egress involves widening of the fracture, penetration of water into the cavity and consequent destabilization of the bound sterol. The simulations also identify polar residues along the fracture that are important for sterol release. Their replacement with alanine affects the ability of the StARkin domain to bind sterol, catalyze inter-vesicular sterol exchange and alleviate the nystatin-sensitivity of lam2{Delta} yeast cells. These data suggest an unprecedented, water-controlled mechanism of sterol acquisition and discharge from a StARkin domain.

biochemistry↗

bioRxiv2018

Regulation of Exocytosis and Mitochondrial Relocalization by Alpha-Synuclein in a Mammalian Cell Model

We characterized phenotypes in RBL-2H3 mast cells transfected with human alpha synuclein (a-syn) using stimulated exocytosis of recycling endosomes as a proxy for similar activities of synaptic vesicles in neurons. We found that low expression of a-syn inhibits stimulated exocytosis and that higher expression causes slight enhancement. NMR measurements of membrane interactions correlate with these functional effects: they are eliminated differentially by mutants that perturb helical structure in the helix 1 (A30P) or NAC/helix-2 (V70P) regions of membrane-bound a-syn, but not by other PD-associated mutants or C-terminal truncation. We further found that a-syn (but not A30P or V70P mutants) associates weakly with mitochondria, but this association increases markedly under conditions of cellular stress. These results highlight the importance of specific structural features of a-syn in regulating vesicle release, and point to a potential role for a-syn in perturbing mitochondrial function under pathological conditions.

cell biology↗