Biology subjects

Giladi, M.

Publications and source records attributed to Giladi, M..

11 recordsLinked to original sources

bioRxiv2026

Efficacy of Tumor Treating Fields (TTFields) in triple negative breast cancer cells and synergy with PARP inhibitor talazoparib

Breast cancer is the most diagnosed cancer worldwide. Triple-negative breast cancer (TNBC) is a subtype with a poor prognosis because of its aggressiveness and high likelihood of relapse, making its treatment particularly challenging. We explored the effect of Tumor Treating Fields (TTFields) as a novel treatment option for breast cancer, which can be used together with other therapies, such as PARP inhibitors. TTFields use alternating electric fields to inhibit cell growth and induce cellular damage. Our findings indicate that TTFields increase expression of the universal cell cycle CDK inhibitory protein p21(WAF1)/CDKN1A independently of TP53 mutational status. Moreover, when used together with the PARP inhibitor talazoparib, the combination further upregulates p21 protein. Both TTFields alone and together with talazoparib induce cell death as documented by PARP cleavage while reducing markers of cell growth and anti-apoptotic proteins, including p-STAT3, STAT3, and MCL-1. Additionally, TTFields together with talazoparib induce DNA damage, as indicated by increased {gamma}H2AX expression. The combination of talazoparib plus TTFields treatment decrease PDL-1 levels and given its utility in predicting response to PD-1/PD-L1 by mediating immune evasion, this raises the possibility of future combination for immune modulation in cancer therapy. Our preclinical results suggest TTFields alone or together with PARP inhibitors such as talazoparib as a promising therapeutic approach for further development for the treatment of breast cancer.

cancer biology↗

bioRxiv2025

Mono-allelic p.R37H Dehydrodolichyl Diphosphate Synthase variants lead to protein glycosylation defects, aberrant lipid profiles and interneuron scarcity in a novel mouse model of progressive epileptic encephalopathy.

Developmental delay and seizures with or without movement abnormalities (OMIM 617836) caused by heterozygous pathogenic variants in the DHDDS gene (DHDDS-CDG) is a rare genetic disease that belongs to the progressive encephalopathy spectrum. It results in cognitive delay in affected children, accompanied by myoclonus, seizures, ataxia and tremor, which worsens over time. DHDDS encodes a subunit of a DHDDS/NUS1 cis-prenyltransferase (cis-PTase), a branch point enzyme of the mevalonate pathway essential for N-linked glycosylation. We describe the first mouse model of this disease, DhddsR37H+/-strain, heterozygous for the human recurrent de novo c.110G>A:p.R37H pathogenic variant. DhddsR37H+/- mice present with seizures, myoclonus and memory deficits associated with reduced density or/and maturity of inhibitory interneurons in the cortex. Multiomics analyses of mouse CNS tissues, together with the enzymatic/structural characterization of the R37H DHDDS mutant protein, reveal that the variant produces a catalytically inactive enzyme and results in a brain dolichol deficit, aberrant glycosylation of brain glycoproteins, including those involved in synaptic transmission and major perturbations in the CNS proteome and lipidome. Acetazolamide, a carbonic anhydrase inhibitor clinically approved for treatment of glaucoma, epilepsy, and intracranial hypertension, and successfully used "off-label" to treat genetic movement disorders, reduces seizure susceptibility to pentylenetetrazol in DhddsR37H+/- mice, suggesting potential therapeutic value of using this drug in human DHDDS-CDG patients. Together, our results define cis-PTase as a master regulator of CNS development and function and establish that its monoallelic debilitating variants cause a novel congenital disorder of glycosylation associated with aberrant levels of neuronal proteins and lipids.

neuroscience↗

bioRxiv2025

Structural mechanisms of allosteric regulation in the human cis-prenyltransferase complex

Human cis-prenyltransferase (hcis-PT) synthesizes long-chain isoprenoids essential for N-linked protein glycosylation. This heteromeric complex comprises the catalytic subunit DHDDS and the regulatory Nogo-B receptor (NgBR). Although NgBR dramatically enhances DHDDS activity, the molecular basis for this allosteric regulation remains unclear. Here, we combined crystallography, hydrogen-deuterium exchange mass spectrometry (HDX-MS), molecular dynamics simulations, and network analysis to uncover the structural dynamics and communication pathways within hcis-PT. By solving the apo structure of hcis-PT, we reveal only a localized flexibility at the active site and the NgBR C-terminus. However, HDX-MS demonstrated widespread substrate-induced stabilization, particularly at the NgBR {beta}D-{beta}E loop, highlighting it as an allosteric hub. Functional mutagenesis scanning identified NgBRS249 as critical for enzymatic activity, independent of structural perturbations. Network analysis of MD simulations pinpointed this residue as a central node in inter-subunit communication, with perturbations disrupting downstream allosteric pathways, altering enzymatic activity. Our findings reveal a dynamic regulatory network centered at the inter-subunit interface, wherein specific NgBR residues modulate DHDDS activity through allosteric signaling. This work elucidates a conserved mechanism of subunit coordination in long-chain cis-prenyltransferases and suggests novel avenues for therapeutic targeting of hcis-PT-related disorders.

biochemistry↗

bioRxiv2024

A somatic multiple myeloma mutation unravels a mechanism of oligomerization-mediated product inhibition in GGPPS

Protein prenylation regulates the cellular localization of small GTPases and is pivotal for multiple myeloma (MM) pathology. Geranylgeranyl diphosphate synthase (GGPPS), synthesizing a prenylation moiety, exhibits dimeric or hexameric stoichiometry in different species. However, the functional significance of this divergence remains elusive. Focusing on the hexameric human paralog, formed by trimer-of-dimers, we uncover that GGPPSR235C, expressed in an MM cell line, localizes to the active site lid region at the inter-dimeric interface. Using crystallography and mass spectrometry (MS), we show that GGPPSR235C retains its hexameric stoichiometry but exhibits destabilized inter-dimer interactions. Unexpectedly, this results in increased apparent substrate affinity and product release kinetics. These functional effects are further enhanced in a dimeric mutant, GGPPSY246D. Combining MS and fluorescence spectroscopy, we exposed that reduced lid dynamics and increased active site occupancy by the product are intertwined. Together, our results expose product inhibition as a regulatory mechanism in GGPPS, driven by hexamerization.

biochemistry↗

bioRxiv2023

The SCN8A p.(Gly1625Arg) variant associated with developmental and epileptic encephalopathy causes complex biophysical changes and reduced neuronal firing

BackgroundMutations in the SCN8A gene, encoding the voltage-gated sodium channel NaV1.6, lead to various neurodevelopmental disorders. The SCN8A p.(Gly1625Arg) mutation (NaV1.6G1625R) was identified in a patient diagnosed with developmental epileptic encephalopathy (DEE), presenting with moderate epilepsy and severe developmental delay. MethodsWe performed biophysical and neurophysiological characterizations of NaV1.6G1625R in Neuro-2a cells and cultured hippocampal neurons, followed by computational modeling to determine the impact of its heterozygous expression on cortical neuron function. FindingsVoltage-clamp analyses of NaV1.6G1625R demonstrated a heterogeneous mixture of gain-and loss-of-function properties, including reduced current amplitudes, a marked increase in the time constant of fast voltage-dependent inactivation and a depolarizing shift in the voltage dependence of inactivation. Recordings in transfected cultured neurons showed that these intricate biophysical properties had a minor effect on neuronal excitability when firing relayed on both endogenous and transfected NaV channels. Conversely, there was a marked reduction in the number of action potentials when firing was driven by the transfected mutant NaV1.6 channels. Computational modeling of mature cortical neurons further revealed a mild reduction in neuronal firing when mimicking the patients heterozygous NaV1.6G1625R expression. Structural modeling of NaV1.6G1625R and a double-mutant cycle analysis suggested the possible formation of pathophysiologically-relevant cation-{pi} interaction between R1625 and F1588, affecting the voltage dependence of inactivation. InterpretationOur analyses demonstrate a complex combination of gain and loss-of-function changes resulting in an overall mild reduction in neuronal firing, related to a perturbed interaction network within the voltage sensor domain. FundingISF, DFG, BMBF, The Hartwell Foundation, ICRF, ISCA Research in contextO_ST_ABSEvidence before this studyC_ST_ABSMutations in the SCN8A gene, encoding the voltage-gated sodium channel NaV1.6, result in multiple neurodevelopmental syndromes ranging from benign epilepsy to developmental delay without epilepsy or developmental epileptic encephalopathy (DEE). Recent studies established that most DEE-causing SCN8A mutations result in a gain of function effect. However, several SCN8A mutations that diverge from this pattern were described. Added value of this studyWe performed a multi-tiered study of the SCN8A p.(Gly1625Arg) variant (NaV1.6G1625R), identified in a patient with atypical DEE presentation, featuring moderate epilepsy that is well controlled by the sodium channel blocker Oxcarbazepine, along with profound stagnated developmental delay. This variant is positioned within the S4 segment of domain IV, a critical region for NaV1.6 function, where pathogenic variants were shown to cause either a loss or gain of channel function, but often with mixed biophysical alterations. Our biophysical characterization of NaV1.6G1625R in Neuro-2a cells demonstrated complex gain-and loss-of-function properties, cumulating to reduced firing in cultured hippocampal neurons and computational modeling of mature cortical neurons, demonstrating an overall loss-of-function effect. Implications of all the available evidenceOur results indicate the necessity for combined biophysical and neuronal characterization of individual SCN8A variants, especially those presenting with complex biophysical changes or atypical clinical presentation. Moreover, while sodium channel blockers are the recommended treatment for SCN8A variants associated with gain-of-function, additional considerations may be needed for DEE-causing variants that are associated with mild loss-of-function.

physiology↗

bioRxiv2023

Chloride intracellular channel (CLIC) proteins function as fusogens

Chloride Intracellular Channel (CLIC) family members uniquely transition between soluble and membrane-associated conformations. Despite decades of extensive functional and structural studies, CLICs function as ion channels remains debated, rendering our understanding of their physiological role incomplete. Here, we expose a novel function of CLIC5 as a fusogen. We demonstrate that purified CLIC5 directly interacts with the membrane and induces fusion, as reflected by increased liposomal diameter and lipid and content mixing between liposomes. Moreover, we show that this activity is facilitated by acidic pH, a known trigger for CLICs transition to a membrane-associated conformation, and that increased exposure of the hydrophobic inter-domain interface is crucial for this process. Finally, mutation of a conserved hydrophobic interfacial residue diminishes the fusogenic activity of CLIC5 in vitro and impairs excretory canal extension in C. elegans in vivo. Together, our results unravel the long-sought physiological role of these enigmatic proteins.

biochemistry↗

bioRxiv2021

Structural basis for long-chain isoprenoids synthesis by cis-prenyltransferases

Isoprenoids are the largest group of natural products, found in all living organisms and play an essential role in numerous cellular processes. These compounds are synthesized by prenyltransferases, catalyzing the condensation reaction between an allylic diphosphate primer and a variable number of isopentenyl diphosphate (C5) units. This superfamily of enzymes can be subdivided into trans- or cis-prenyltransferases according to the stereoisomerism of the product. The cis branch can be further classified according to product length. While the active site volume was suggested to determine the final length in enzymes synthesizing short- and medium-chain products (up to C60), long-chain enzymes (up to C120) and rubber synthases (>C10,000) fail to conform to this paradigm. Here, to resolve the structural basis for long-chain isoprenoid synthesis, we focused on the human cis-prenyltransferase complex (hcis-PT). This enzyme, peripheral to the endoplasmic reticulum membrane, produces the precursor for dolichol phosphate, a membrane residing glycosyl carrier. In line with its crucial role in the cellular protein glycosylation machinery, disease-causing mutations in hcis-PT were shown to result in a wide spectrum of clinical phenotypes. The crystallographic structures of hcis-PT in four different substrate/product-bound conformations revealed an outlet enabling product elongation into the bulk solvent. Moreover, hydrogen-deuterium exchange mass spectrometry analysis in solution showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Finally, using a fluorescent substrate analog and a fluorescently-labeled lipid nanodiscs, we show that product elongation and membrane association are closely correlated. Together, our results support directional product synthesis in long-chain enzymes and rubber synthases, with a distinct substrate inlet and product outlet, allowing direct membrane insertion of the elongating isoprenoid during catalysis. This mechanism uncouples active site volume from product length and circumvents the need to expulse hydrophobic product into a polar environment prior to membrane insertion.

biochemistry↗

bioRxiv2021

TTYH family members form tetrameric complexes within the cell membrane

The conserved Tweety homolog (TTYH) family consists of three paralogs in vertebrates, displaying a ubiquitous expression pattern. Although considered as ion channels for almost two decades, recent structural and functional analyses refuted this role. Intriguingly, while all paralogs, studied following detergent solubilization, shared a dimeric stoichiometry, their spatial organization differed. Here, we determined the stoichiometry of intact mouse TTYH (mTTYH) complexes in cells. Using cross-linking and single-molecule fluorescence microscopy, we demonstrated that mTTYH1 and mTTYH3 form tetramers at the plasma membrane. Blue-native PAGE and fluorescence-detection size-exclusion chromatography analyses revealed that detergent solubilization results in the dissolution of tetramers into dimers, suggesting a dimer-of-dimers assembly mode. As cross-linking analysis of the soluble extracellular domains also showed tetrameric stoichiometry, we explored the effect of membrane solubilization and disulfide bridges integrity and established their contribution to tetramer stability. Future studies of the native tetrameric TTYH characterized here may illuminate their long-sought cellular function.

biochemistry↗

bioRxiv2020

Structure of the human heterotetrameric cis-prenyltransferase complex

The human cis-prenyltransferase (hcis-PT) is an enzymatic complex essential for protein N-glycosylation. Synthesizing the precursor of the glycosyl carrier dolichol-phosphate, we reveal here that hcis-PT exhibits a novel heterotetrameric assembly in solution, composed of two catalytic dehydrodolichyl diphosphate synthase (DHDDS) and two inactive Nogo-B receptor (NgBR) subunits. The 2.3 [A] crystal structure of the complex exposes a dimer-of-heterodimers arrangement, with DHDDS C-termini serving as homotypic assembly domains. Furthermore, the structure elucidates the molecular details associated with substrate binding, catalysis, and product length determination. Importantly, the distal C-terminus of NgBR transverses across the heterodimeric interface, directly participating in substrate binding and underlying the allosteric communication between the subunits. Finally, mapping disease-associated hcis-PT mutations involved in blindness, neurological and glycosylation disorders onto the structure reveals their clustering around the active site. Together, our structure of the hcis-PT complex unveils the dolichol synthesis mechanism and its perturbation in disease.

biochemistry↗

bioRxiv2019

Integrative modelling of the full-length human dehydrodolichyl diphosphate synthase using a hybrid computational and experimental approach

Dehydrodolichyl diphosphate synthase (DHDDS) and Nogo-B receptor (NgBR) form the heteromeric human cis-prenyltransferase complex, synthesizing the precursor for the glycosyl carrier involved in N-linked protein glycosylation. In line with the important role of N-glycosylation in protein biogenesis, mutations in DHDDS, the catalytic subunit of the complex, were shown to result in human diseases. Importantly, well-characterized DHDDS homologs function as homodimers and not as heteromeric complexes. Moreover, DHDDS encompasses a C-terminal region, which does not converge with any known conserved domains. Therefore, despite the clinical importance of DHDDS, our understating of its structure-function relations remains poor. Here, we provide a structural model for the full-length human DHDDS using a multidisciplinary experimental and computational approach. Our model suggests that the C-terminal domain of DHDDS forms a helix-loop-helix motif, tightly packed against the core catalytic cis-prenyltransferase domain. This model is consistent with small-angle X-ray scattering data, indicating that the full-length DHDDS maintains a similar conformation in solution. Moreover, hydrogen-deuterium exchange mass-spectrometry experiments show time-dependent deuterium uptake in the C-terminal domain, consistent with its overall folded state. Finally, we provide a model for the DHDDS-NgBR heterodimer, offering a structural framework for future structural and functional studies of the human cis-prenyltransferase complex.

biophysics↗

bioRxiv2019

Structure of KCNH2 Cyclic Nucleotide-Binding Homology Domain Reveals a Functionally vital Salt-Bridge

Human KCNH2 (hKCNH2, Ether-a-go-go (EAG)-Related Gene, hERG) are best known for their role in cardiac action potentials repolarization and have key roles in various pathologies. As other KCNH family members, hKCNH2 contains a unique intracellular complex crucial for channel function, consisting of an N-terminal eag domain and a C-terminal cyclic nucleotide-binding homology domain (CNBHD). Previous studies demonstrated that the CNBHD is occupied by an intrinsic ligand motif (ILM), in a self-liganded conformation, providing a structural mechanism for the lack of KCNH channels regulation by cyclic nucleotides. While significant advancements in structural and functional characterizations of the CNBHD of KCNH channels have been made, a high-resolution structure of the hKCNH2 intracellular complex was missing. Here, we report the 1.5 [A] resolution structure of the hKCNH2 channel CNBHD. The structure reveals the canonical fold shared by other KCNH family members, where the spatial organization of the ILM is preserved within the {beta}-roll region. Moreover, measurements of small-angle X-ray scattering profile in solution, as well as comparison with a recent nuclear magnetic resonance (NMR) analysis of hKCNH2, revealed high agreement with the structure, indicating an overall low flexibility in solution. Importantly, we identified a novel salt-bridge (E807-R863), which was not previously resolved in the NMR and cryogenic electron microscopy (cryo-EM) structures. Strikingly, electrophysiological analysis of charge reversal mutations revealed its crucial role for hKCNH2 function. Moreover, comparison with other KCNH members revealed the structural conservation of this salt-bridge, consistent with its functional significance. Together with the available structure of the mouse KCNH1 intracellular complex, and previous electrophysiological and spectroscopic studies of KCNH family members, we propose that this salt-bridge serves as a strategically positioned linchpin to support both the spatial organization of the ILM and the maintenance of the intracellular complex interface.\n\nSummaryHuman KCNH2 are key channels governing cardiac repolarization. Here, a 1.5 [A] resolution structure of their cyclic nucleotide-binding homology domain is presented. Structural analysis and electrophysiological validation reveal a novel salt-bridge, playing an important role in hKCNH2 functional regulation.

biophysics↗