Biology subjects

Ashkenazi, A.

Publications and source records attributed to Ashkenazi, A..

14 recordsLinked to original sources

bioRxiv2026

Peroxisome quality control is gated by a dynamic ATF6α-ABCD3 tether

The endoplasmic reticulum (ER) coordinates protein folding and lipid biosynthesis, while peroxisomes govern fatty-acid catabolism and redox balance. However, little is known about the mechanisms by which these organelles communicate. Here we show that activating transcription factor 6 (ATF6), a cytoprotective ER-resident sensor of the unfolded protein response (UPR), forms a molecular tether that zippers the ER and peroxisomes together at a 23-nm intermembrane distance. ATF6 achieves this by inserting a short segment of its unstructured N-terminal cytosolic tail into the solvated transmembrane cavity of the peroxisomal ABC-type lipid transporter ABCD3, trapping it in an off-state. This interaction is reinforced by Ceapin, a previously identified small molecule molecular-glue ligand, which promotes ER-peroxisome contact formation. The junctions serve as a scaffold that can initiate peroxisome turnover by p62-triggered autophagy (pexophagy), as enhanced lipid processing resulting from organelle zippering leads to peroxisome damage due to excessive reactive oxygen species (ROS) accumulation. These findings define ATF6 as a non-transcriptional regulator of organelle quality control and provide a template for engineering Ceapin analogues against ATF6/ABCD3-dependent colorectal and hepatocellular cancers and related diseases.

cell biology↗

bioRxiv2026

The selective OX1R antagonist 1-SORA-51 reduces binge-like feeding behavior in male and female mice without detectable changes in dopamine.

Binge eating disorder (BED) is characterized by episodic overconsumption of palatable food and involves dysregulated motivational and arousal processes. The orexin (hypocretin) system, through its widespread projections to mesolimbic circuits, has been implicated in cue-driven reward seeking and escalated intake, raising the possibility that orexin receptor antagonists may modulate binge-like behavior. Here we evaluated the effects of a dual orexin receptor antagonist (DORA-22) and an OX1R-selective antagonist (1-SORA-51) in a cyclic intermittent high-fat access model that generates robust and reproducible binge-like intake in male and female mice. DORA-22 produced no detectable effect on consumption at either early (2 h) or extended (24 h) binge timepoints. In contrast, 1-SORA-51 significantly reduced high-fat intake during the initial 2 hours of access in both sexes, with no effect on 24-hour consumption, indicating a selective attenuation of the early phase of binge intake. Fiber photometry recordings of GRABDA2m fluorescence in the nucleus accumbens revealed that 1-SORA-51 did not alter baseline dopamine signals or the dopamine increase triggered by high-fat pellet delivery, demonstrating that its behavioral effects occur without detectable modulation of mesolimbic dopamine dynamics. Together, these findings identify OX1R antagonism as a strategy for suppressing the initiation of binge-like feeding and highlight the receptor-level specificity of orexin contributions to maladaptive overconsumption.

neuroscience↗

bioRxiv2024

TRAIL induces caspase-dependent cleavage of specific components of the clathrin-mediated endocytosis machinery

It has been reported that caspase activation by the proapoptotic ligand Apo2L/TRAIL disrupts the clathrin-mediated endocytosis machinery (CMEM). To confirm whether TRAIL induces caspase-mediated cleavage of specific CMEM components, we examined the temporal and functional relationship between TRAIL-induced processing of the apoptosis-initiating protease, caspase-8, and the apoptosis-executing protease, caspase-3, versus cleavage of specific adaptin (AP) and clathrin heavy chain (CHC) proteins. TRAIL induced time-dependent proteolytic processing of caspase-8 and caspase-3, which coincided with cleavage of AP2 and CHC. The pan caspase inhibitor zVAD-FMK, which blocked caspase processing in response to TRAIL, also prevented the cleavage AP2. Whereas FADD-deficient or caspase-8-deficient cells showed little or no TRAIL-driven cleavage of either AP2 or CHC, Bax-deficient or caspase-3-deficient cell lines retained AP2 cleavage but failed to cleave CHC in response to TRAIL. The DNA-damaging agent doxorubicin also led to processing of caspase-8 and caspase-3 in conjunction ith cleavage of AP2 and CHC. Together, these results confirm that TRAIL induces caspase-dependent cleavage of AP2 and CHC. Whereas TRAIL-driven cleavage of both AP2 and CHC requires caspase-8, cleavage of CHC, but not of AP2, requires caspase-3. MATERIALS AND METHODS Cell lines and cell cultureAll cell lines were obtained from ATCC or as kind gifts from Dr. Bert Vogelstein (HCT116 Bax-/-) and Dr. John Blenis (Jurkat I9.2 and E1). Cell lines were cultured with standard media as previously described (1). Immunoblot analysisCells were lysed in RIPA lysis buffer (20-188, Millipore) supplemented with Halt protease and phosphatase inhibitor cocktail (ThermoFisher Scientific) and kept on ice for 30 min. Lysates were cleared by centrifugation at 13,600 x g for 15 min at 4 {degrees}C, and protein amount was determined by BCA protein assay (ThermoFisher Scientific). Protein was denatured by adding NuPAGE LDS buffer and DTT reducing buffer (Invitrogen) and incubating the samples at 95 {degrees}C for 5 min. Equal amounts of denatured protein were loaded into each well of NuPAGE pre-cast gels (Invitrogen), resolved by SDS-PAGE, and electro-transferred to nitrocellulose membranes using the iBLOT2 system (Invitrogen). Membranes were blocked in a 5% nonfat milk solution for 1 hr at room temperature and probed with the corresponding primary antibody at 1:1,000 dilution overnight at 4 {degrees}C. This was followed by incubation with the corresponding horseradish peroxidase (HRP)- conjugated secondary antibody at 1:10,000 dilution during 1 hr at room temperature. All secondary antibodies were from Jackson Laboratories. The primary antibodies and secondary HRP-conjugated antibodies are listed respectively in Table 1 and Table 2. {beta}-actin IB was used to verify uniformity of protein loading and electro-transfer. O_TBL View this table: org.highwire.dtl.DTLVardef@ed5a65org.highwire.dtl.DTLVardef@c9aecdorg.highwire.dtl.DTLVardef@f31b4aorg.highwire.dtl.DTLVardef@849deaorg.highwire.dtl.DTLVardef@9b847b_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONPrimary antibodies used for IB analysis C_TABLECAPTION C_TBL O_TBL View this table: org.highwire.dtl.DTLVardef@16c33forg.highwire.dtl.DTLVardef@6c6b81org.highwire.dtl.DTLVardef@5efb4org.highwire.dtl.DTLVardef@1bc354forg.highwire.dtl.DTLVardef@15007aa_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 2.C_FLOATNO O_TABLECAPTIONHorseradish peroxidase conjugated secondary antibodies used for IB analysis C_TABLECAPTION C_TBL Additional reagentsRecombinant soluble non-tagged human TRAIL (Apo2L.0) and Flag-tagged TRAIL were prepared at Genentech. Flag-tagged TRAIL was crosslinked with anti-Flag M2 antibody at a ligand-to-antibody molar ratio of 1:2. zVAD-FMK was purchased from R & D Systems, FMK001. Doxorubicin was purchased from Sigma Aldrich, D1515.

cell biology↗

bioRxiv2023

A nonenzymatic dependency on inositol-requiring enzyme 1 controls cancer cell cycle progression and tumor growth

Endoplasmic-reticulum resident inositol-requiring enzyme 1 (IRE1) supports protein homeostasis via a cytoplasmic kinase-RNase module. Known cancer dependency on IRE1 entails its enzymatic activation of the transcription factor XBP1s and of RNA decay. We discovered that some cancer cells require IRE1 but not its enzymatic activity. IRE1 knockdown, but not enzymatic inhibition or XBP1 disruption, increased DNA damage and chromosome instability while engaging the TP53 pathway and cyclin-dependent kinase inhibitors and attenuating cell cycle progression. IRE1 depletion downregulated factors involved in chromosome replication and segregation and in chromatin remodeling. Immunoelectron microscopy indicated that endogenous IRE1 can localize to the nuclear envelope. Thus, cancer cells can require IRE1 either enzymatically or nonenzymatically, with significant implications for IRE1s biological role and therapeutic targeting.

cancer biology↗

bioRxiv2023

Expression of modified FcγRI enables myeloid cells to elicit robust tumor-specific cytotoxicity

Despite the central role of T cells in tumor immunity, attempts to harness their cytotoxic capacity as a therapy have met limited efficacy, partially as a result of the suppressive microenvironment which limits their migration and activation. In contrast, myeloid cells massively infiltrate tumors and are well adapted to survive these harsh conditions. While they are equipped with cell-killing abilities, they often adopt an immunosuppressive phenotype upon migration to tumors. Therefore, the questions of how to modify their activation programming against cancer, and what signaling cascades should be activated in myeloid cells to elicit their cytotoxicity have remained unclear. Here, we found that activation of IgM-induced signaling in myeloid cells results in secretion of lytic granules and massive tumor cell death. These findings open venues for designing novel immunotherapy by equipping monocytes with chimeric receptors that target tumor antigens and consequently, signal through IgM receptor. Nonetheless, we found that myeloid cells do not express the antibody-derived portion used to recognize the tumor antigen due to the induction of an ER stress response. To overcome this limitation, we designed chimeric receptors that are based on the high-affinity Fc{gamma}RI for IgG. Incubation of macrophages expressing these receptors along with tumor-binding IgG induced massive tumor cell killing and secretion of reactive oxygen species and Granzyme B. Overall, this work highlights the challenges involved in genetically reprogramming the signaling in myeloid cells and provides a framework for endowing myeloid cells with antigen-specific cytotoxicity.

immunology↗

bioRxiv2022

Polyalanine disease mutations impair UBA6-dependent ubiquitination

Expansion mutations in polyalanine stretches are now associated with a growing number of human diseases with common genotypes and similar phenotypes 1-6. These similarities prompted us to query the normal function of physiological polyalanine stretches, and investigate whether a common molecular mechanism is involved in these diseases. Here, we show that UBA6, an E1 ubiquitin-activating enzyme 7, 8, recognizes a polyalanine stretch within its cognate E2 ubiquitin-conjugating enzyme, USE1. Aberrations in this polyalanine stretch reduced ubiquitin transfer to USE1 and downstream target, the E3 ubiquitin ligase, E6AP. Intriguingly, we identified competition for the UBA6-USE1 interaction by various proteins with polyalanine expansion mutations in the disease state. In mouse primary neurons, the deleterious interactions of expanded polyalanine proteins with UBA6, alter the levels and ubiquitination-dependent degradation of E6AP, which in turn affected the levels of the synaptic protein, Arc. These effects could be observed in induced pluripotent stem cell-derived autonomic neurons from patients with polyalanine expansion mutations. Our results suggest a shared mechanism for such mutations, which may contribute to the congenital malformations seen in polyalanine diseases.

cell biology↗

bioRxiv2021

Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers

Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network, termed the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved branch of the UPR. Dimerization and/or higher-order oligomerization of IRE1 are thought to be important for its activation mechanism, yet the actual oligomeric states of inactive, active, and attenuated mammalian IRE1 complexes remained unknown. We developed an automated two-color single-molecule tracking approach to dissect the oligomerization of tagged endogenous human IRE1 in live cells. In contrast to previous models, our data indicate that IRE1 exists as a constitutive homodimer at baseline and assembles into small oligomers upon ER stress. We demonstrate that the formation of inactive dimers and stress-dependent oligomers is fully governed by IRE1s lumenal domain. Phosphorylation of IRE1s kinase domain occurs more slowly than oligomerization and is retained after oligomers disassemble back into dimers. Our findings suggest that assembly of IRE1 dimers into larger oligomers specifically enables trans- autophosphorylation, which in turn drives IRE1s RNase activity.

biophysics↗

bioRxiv2021

Antigen-derived peptides directly engage the unfolded-protein sensor IRE1α to curb cross-presentation by dendritic cells

Dendritic cells (DCs) promote adaptive immunity by cross-presenting antigen-based epitopes to CD8+ T cells. DCs process internalized protein antigens into peptides that enter the endoplasmic reticulum (ER) and upload onto major histocompatibility type I (MHC-I) protein complexes for cell-surface transport and cross-presentation. Perplexingly, DCs often exhibit activation of the ER-stress sensor IRE1 in the absence of classical ER stress--leaving the underlying mechanism unexplained. Here we show that antigen-derived hydrophobic peptides directly engage ER-resident IRE1 by masquerading as unfolded proteins. Furthermore, IRE1 activation depletes MHC-I heavy-chain mRNAs through regulated IRE1-dependent decay (RIDD), thereby curtailing antigen cross-presentation. In tumor-bearing mice, IRE1 disruption increased MHC-I expression on tumor-infiltrating DCs, and enhanced recruitment and activation of CD8+ T cells. Moreover, IRE1 inhibition synergized with anti-PD-L1 antibody treatment to cause tumor regression. Our findings elucidate the mechanism and consequence of antigen-driven IRE1 activation in DCs, yielding a promising combination strategy for cancer immunotherapy.

cell biology↗

bioRxiv2021

Noncanonical mRNA decay by the endoplasmic-reticulum stress sensor IRE1α promotes cancer-cell survival

Eukaryotic IRE1 mitigates endoplasmic-reticulum (ER) stress by orchestrating the unfolded-protein response (UPR). IRE1 spans the ER membrane, and signals through a cytosolic kinase-endoribonuclease module. The endoribonuclease generates the transcription factor XBP1s by intron excision between similar RNA stem-loop endomotifs, and depletes select cellular mRNAs through regulated IRE1-dependent decay (RIDD). Paradoxically, mammalian RIDD seemingly targets only mRNAs with XBP1-like endomotifs, while in flies RIDD exhibits little sequence restriction. By comparing nascent and total IRE1-controlled mRNAs in human breast cancer cells, we discovered not only canonical endomotif-containing RIDD substrates, but also many targets lacking recognizable motifs--degraded by a process we coin RIDDLE, for RIDD lacking endomotif. IRE1 displayed two basic endoribonuclease modalities: endomotif-specific cleavage, minimally requiring dimers; and endomotif-independent promiscuous processing, requiring phospho-oligomers. An oligomer-deficient mutant that did not support RIDDLE failed to rescue cancer-cell viability. These results link IRE1 oligomers, RIDDLE, and cell survival, advancing mechanistic understanding of the UPR.

cancer biology↗

bioRxiv2021

The stress-sensing domain of activated IRE1α forms helical filaments in narrow ER membrane tubes

The signaling network of the unfolded protein response (UPR) adjusts the protein folding capacity of the endoplasmic reticulum (ER) according to need. The most conserved UPR sensor, IRE1, spans the ER membrane and activates through oligomerization. IRE1 oligomers accumulate in dynamic foci. We determined the in-situ structure of IRE1 foci by cryogenic correlated light and electron microscopy (cryo-CLEM), combined with electron cryo-tomography (cryo-ET) and complementary immuno-electron microscopy. IRE1 oligomers localize to a network of narrow anastomosing ER tubes (diameter ~28 nm) with complex branching. The lumen of the tubes contains protein filaments, likely composed of linear arrays of IRE1 lumenal domain dimers, arranged in two intertwined, left-handed helices. Our findings define a previously unrecognized ER subdomain and suggest positive feedback in IRE1 signaling.

cell biology↗

bioRxiv2021

Pumilio protects Xbp1 mRNA from regulated Ire1-dependent decay

The unfolded protein response (UPR) maintains homeostasis of the endoplasmic reticulum(ER). Residing in the ER membrane, the UPR mediator Ire1 deploys its cytoplasmic kinase-endoribonuclease domain to activate the key UPR transcription factor Xbp1 through non-conventional splicing of Xbp1 mRNA. Ire1 also degrades diverse ER-targeted mRNAs through regulated Ire1-dependent decay (RIDD), but how it spares Xbp1 mRNA from this decay is unknown. We identified binding sites for the RNA-binding protein Pumilio in the 3UTR Drosophila Xbp1. In the developing Drosophila eye, Pumilio bound both the Xbp1unspliced and Xbp1spliced mRNAs, but only Xbp1spliced was stabilized by Pumilio. Furthermore, Pumilio displayed Ire1 kinase-dependent phosphorylation during ER stress, which was required for its stabilization of Xbp1spliced. Human IRE1 could directly phosphorylate Pumilio, and phosphorylated Pumilio protected Xbp1spliced mRNA against RIDD. Thus, Ire1-mediated phosphorylation enables Pumilio to shield Xbp1spliced from RIDD. These results uncover an important and unexpected regulatory link between an RNA-binding protein and the UPR.

cell biology↗

bioRxiv2020

Neuronal regulated Ire1-dependent mRNA decay controls germline differentiation in C. elegans

Understanding the molecular events that regulate cell pluripotency versus acquisition of differentiated somatic cell fate is fundamentally important. Studies in C. elegans demonstrate that knockout of the germline-specific translation repressor gld-1, causes germ cells within tumorous gonads to form germline-derived teratoma. Previously we demonstrated that ER stress enhances this phenotype to suppress germline tumor progression (Levi-Ferber M, 2015). Here, we identify a neuronal circuit that non-autonomously suppresses germline differentiation, and show that it communicates with the gonad via the neurotransmitter serotonin to limit somatic differentiation of the tumorous germline. ER stress controls this circuit through regulated IRE-1-dependent mRNA decay of transcripts encoding the neuropeptide FLP-6. Depletion of FLP-6 disrupts the circuits integrity and hence its ability to prevent somatic-fate acquisition by germline tumor cells. Our findings reveal mechanistically how ER stress enhances ectopic germline differentiation, and demonstrate that RIDD can affect animal physiology by controlling a specific neuronal circuit.

cell biology↗

bioRxiv2018

Disruption of IRE1α through its Kinase Domain Attenuates Multiple Myeloma

Multiple myeloma (MM) arises from malignant immunoglobulin-secreting plasma cells and remains an incurable, often lethal disease despite recent therapeutic advances. The unfolded-protein response sensor IRE1 supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may coopt the IRE1-XBP1s pathway; however, the validity of IRE1 as a potential MM therapeutic target is controversial. Here we show that genetic disruption of IRE1 or XBP1s, or pharmacologic IRE1 kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice, and augmented efficacy of two well-established frontline antimyeloma agents, bortezomib or lenalidomide. Mechanistically, IRE1 perturbation inhibited expression of key components of the ER-associated degradation machinery, as well as cytokines and chemokines known to promote MM growth. Selective IRE1 kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells from bone marrow of newly diagnosed MM patients or patients whose disease relapsed after 1 - 4 lines of treatment in both US- and EU-based cohorts. IRE1 inhibition preserved survival and glucose-induced insulin secretion by pancreatic microislets. Together, these results establish a strong therapeutic rationale for targeting IRE1 with kinase-based small-molecule inhibitors in MM.\n\nSignificance statementMultiple myeloma (MM) is a lethal malignancy of plasma cells. MM cells have an expanded endoplasmic reticulum (ER) that is constantly under stress due to immunoglobulin hyperproduction. The ER-resident sensor IRE1 mitigates ER stress by expanding the ERs protein-folding capacity while supporting proteasomal degradation of misfolded ER proteins. IRE1 elaborates these functions by deploying its cytoplasmic kinase-RNase module to activate the transcription factor XBP1s. The validity of IRE1 as a potential therapeutic target in MM has been questioned. Using genetic and pharmacologic disruption in vitro and in vivo, we demonstrate that the IRE1-XBP1s pathway plays a critical role in MM growth. We further show that IRE1s kinase domain is an effective and safe potential small-molecule target for MM therapy.

cancer biology↗

bioRxiv2018

Phospholipid flipping facilitates annexin translocation across membranes

Annexins are phospholipid binding proteins that somehow translocate from the inner leaflet of the plasma membrane to the outer leaflet. For example, Annexin A2 is known to localise to the outer leaflet of the plasma membrane (cell surface) where it is involved in plasminogen activation leading to fibrinolysis and cell migration, among other functions. Despite having well described extracellular functions, the mechanism of annexin transport from the cytoplasmic inner leaflet to the extracellular outer leaflet of the plasma membrane remains unclear. Here, we show that phospholipid flipping activity is crucial for the transport of annexins A2 and A5 across membranes in cells and in liposomes. We identified TMEM16F (anoctamin-6) as a lipid scramblase required for transport of these annexins to the outer leaflet of the plasma membrane. This work reveals a mechanism for annexin translocation across membranes which depends on plasma membrane phospholipid flipping.

cell biology↗