Biology subjects

Cerione, R. A.

Publications and source records attributed to Cerione, R. A..

14 recordsLinked to original sources

bioRxiv2026

Spin-labeling studies implicate a highly dynamic active state for transducin-bound phosphodiesterase-6 in vertebrate phototransduction

In vertebrate phototransduction, the G protein-coupled receptor rhodopsin activates the -subunit of transducin (GT), which, upon binding the {gamma}-subunits of phosphodiesterase-6 (PDE6), stimulates cGMP hydrolysis. We reported a cryoEM structure for a complex containing two constitutively active GT (GT*) subunits coupled by a bivalent antibody bound to PDE6 that demonstrated a striking displacement of both PDE{gamma} subunits from the PDE/PDE{beta} catalytic sites and suggested an alternating-site mechanism for PDE6 activation. Here, we use site-directed spin labeling (SDSL) and double electron-electron resonance spectroscopy (DEER) to probe PDE6 conformational changes upon GT* binding. PDE{gamma} spin-labelled on Cys68 and Ile64Cys demonstrate that PDE{gamma} have highly flexible C-termini that transiently bind to the PDE/PDE{beta} heterodimer. Binding of GT* to PDE6 with the inhibitor udenafil occupying its catalytic sites alters the positions of the PDE{gamma} subunits in agreement with the changes shown in the cryoEM structure for this complex, whereas coupling the GT* subunits to the bivalent antibody does not affect the DEER distributions observed for PDE6 bound to GT*. However, binding of the slow hydrolyzing 8-Br-cGMP substrate in the presence of GT* causes a dramatic increase in the separation and spread of the spin-labelled PDE{gamma} subunits, thereby revealing a previously unobserved conformation of PDE6 associated with catalysis, which is further supported by small angle X-ray scattering (SAXS) analysis. These studies indicate that whereas inhibitors trap GT*-PDE6 complexes in an inactive state as represented by the cryoEM structure, the binding of both substrate and GT* produces a dynamic active state consistent with an alternating-site mechanism.

biochemistry↗

bioRxiv2025

Structure and enzymology of glutaminase mutants that disrupt glutamine-glutamate homeostasis and cause neurological disease

The glutaminase (GLS) isoforms KGA and GAC are expressed in neurons where they hydrolyze glutamine to produce the excitatory neurotransmitter glutamate. Two de novo gain-of-function mutants of GLS, S482C and H461L, were recently identified in patients with developmental delay, epilepsy, and infantile cataract. These patients exhibited high glutamate and low glutamine concentrations in the brain, suggesting that the GLS mutants have abnormal enzymology. Here, we examined the enzymatic properties of these GLS mutants and found that they exhibit a total (S482C) or partial (H461L) loss of glutamate product inhibition, lifting this restriction on glutamate accumulation. The mutant enzymes also no longer require the anionic activator phosphate to stimulate enzymatic activity or induce filament formation. Structural analysis of the S482C GAC mutant shows the mutation shifts the key catalytic residue Y466 into the catalytically competent position and disrupts a key hydrogen bond between it and the glutamate product, explaining how the S482C mutant has enzymatic activity in the absence of phosphate and is insensitive to glutamate product inhibition. These results shed new light on the mechanism of phosphate activation and glutamate product inhibition of GLS and show that loss of these enzymatic properties disrupts glutamate homeostasis in the brain and causes neurological disease.

biochemistry↗

bioRxiv2025

SIRT2 Mediates Integrated Stress Response by Deacetylating and Stabilizing 4EBP1 to Suppress Translation

The ability to alleviate nutrient stress, such as amino acid limitation, is crucial for cell survival. The mTORC1 complex and integrated stress response (ISR) are mechanisms that sense the availability of amino acids and regulate ribosomal protein synthesis. Here we have discovered a new SIRT2-mediated pathway, downstream of ISR, that senses the limitation of amino acids to regulate translation. Under amino acid deprivation, SIRT2 protein level is upregulated translationally by its upstream open reading frame (uORF). SIRT2 in turn suppresses global protein translation, which helps cells to survive amino acid limitation. Mechanistically, we identified eukaryotic translation initiation factor 4E (eIF4E) binding protein 1 (4EBP1), which negatively regulates translation, as a substrate of SIRT2. SIRT2 deacetylates 4EBP1 at Lys69 and stabilizes 4EBP1 by protecting it from proteasomal degradation. Our study reveals a novel role for SIRT2 in regulating protein translation and a new regulatory mechanism of 4EBP1 in cells. Our study provides a better understanding of the intricate regulation of translation and may explain the known non-oncogene addiction role of SIRT2 in cancer cells.

cell biology↗

bioRxiv2025

Revealing Functional Hotspots: Temperature-Dependent Crystallography of K-RAS Highlights Allosteric and Druggable Sites

K-RAS mutations drive oncogenesis in multiple cancers, yet the lack of druggable sites has long hindered therapeutic development. Here, we use multi-temperature X-ray crystallography (MT-XRC) to capture functionally relevant K-RAS conformations across a temperature gradient, spanning cryogenic to physiological and even "fever" conditions, and show how cryogenic conditions may obscure key dynamic states as targets for new drug development. This approach revealed a temperature-dependent conformational landscape of K-RAS, shedding light on the dynamic nature of key regions. We identified significant conformational changes occurring at critical sites, including known allosteric and drug-binding pockets, which were hidden under cryogenic conditions but later discovered to be critically important for drug-protein interactions and inhibitor design. These structural changes align with regions previously highlighted by large-scale mutational studies as functionally significant. However, our MT-XRC analysis provides precise structural snapshots, capturing the exact conformations of these potentially important allosteric sites in unprecedented detail. Our findings underscore the necessity of advancing tools like MT-XRC to visualize conformational transitions that may be important in signal propagation which are missed by standard cryogenic XRC and to address hard-to-drug targets through rational drug design. This approach not only provides unique structural insights into K-RAS signaling events and identifies new potential sites to target with drug candidates but also establishes a powerful framework for discovering therapeutic opportunities against other challenging drug targets.

biochemistry↗

bioRxiv2024

Defining the conformational states that enable transglutaminase 2 to promote cancer cell survival versus cell death

Transglutaminase 2 (TG2) is a GTP-binding/protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that maintain the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotide-bound TG2 adopts a monomeric closed conformation while calcium-bound TG2 assumes an open conformational state that can form higher order oligomers. SAXS analysis also suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time-resolved SAXS to show that LM11 increases the ability of calcium to drive TG2 to an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state. Significance StatementThe multi-functional protein transglutaminase 2 (TG2) undergoes large conformational changes in response to nucleotide and calcium binding, resulting in diverse cellular effects that can differentially promote either cancer cell survival or cell death. Previous biochemical and structural characterizations have revealed that TG2 primarily adopts two conformational states, a closed nucleotide-bound conformation, and an open calcium-bound conformation. In this study, we use advanced structural methods to describe the conformational changes associated with TG2 activation and inhibition and define the mechanism by which small molecule inhibitors maintain TG2 in a structural state that kill cancer cells.

biophysics↗

bioRxiv2023

Inhibiting Glutamine Metabolism Blocks Coronavirus Replication in Mammalian Cells

Developing therapeutic strategies against COVID-19 has gained widespread interest given the likelihood that new viral variants will continue to emerge. Here we describe one potential therapeutic strategy which involves targeting members of the glutaminase family of mitochondrial metabolic enzymes (GLS and GLS2), which catalyze the first step in glutamine metabolism, the hydrolysis of glutamine to glutamate. We show three examples where GLS expression increases during coronavirus infection of host cells, and another in which GLS2 is upregulated. The viruses hijack the metabolic machinery responsible for glutamine metabolism to generate the building blocks for biosynthetic processes and satisfy the bioenergetic requirements demanded by the glutamine addiction of virus-infected host cells. We demonstrate how genetic silencing of glutaminase enzymes reduces coronavirus infection and that newer members of two classes of small molecule allosteric inhibitors targeting these enzymes, designated as SU1, a pan-GLS/GLS2 inhibitor, and UP4, which is specific for GLS, block viral replication in mammalian epithelial cells. Overall, these findings highlight the importance of glutamine metabolism for coronavirus replication in human cells and show that glutaminase inhibitors can block coronavirus infection and thereby may represent a novel class of anti-viral drug candidates. TeaserInhibitors targeting glutaminase enzymes block coronavirus replication and may represent a new class of anti-viral drugs.

molecular biology↗

bioRxiv2023

PTEN loss in glioma cell lines leads to increased extracellular vesicle biogenesis and PD-L1 cargo in a PI3K-dependent manner

Phosphatase and Tensin Homologue (PTEN) is one of the most frequently lost tumor suppressors in cancer and the predominant negative regulator of the PI3K/AKT signaling axis. A growing body of evidence has highlighted the loss of PTEN with immuno-modulatory functions including the upregulation of the programmed death ligand-1 (PD-L1), an altered tumor derived secretome that drives an immunosuppressive tumor immune microenvironment (TIME), and resistance to certain immunotherapies. Given their roles in immunosuppression and tumor growth, we examined whether the loss of PTEN would impact the biogenesis, cargo, and function of extracellular vesicles (EVs) in the context of the anti-tumor associated cytokine interferon-{gamma} (IFN-{gamma}). Through genetic and pharmacological approaches, we show that PD-L1 expression is regulated by JAK/STAT signaling, not PI3K signaling. Instead, we observe that PTEN loss positively upregulates cell surface levels of PD-L1 and enhances the biogenesis of EVs enriched with PD-L1 in a PI3K-dependent manner. We demonstrate that because of these changes, EVs derived from glioma cells lacking PTEN have a greater ability to suppress T cell receptor (TCR) signaling. Taken together, these findings provide important new insights into how the loss of PTEN can contribute to an immunosuppressive TIME, facilitate immune evasion, and highlight a novel role for PI3K signaling in the regulation of EV biogenesis and the cargo they contain.

cancer biology↗

bioRxiv2023

Filament formation by glutaminase enzymes drives catalysis

The glutaminase enzymes GAC and GLS2 catalyze the hydrolysis of glutamine to glutamate, satisfying the glutamine addiction of cancer cells. They are the targets of anti-cancer drugs; however, their mechanisms of activation and catalytic activity have been unclear. Here we demonstrate that the ability of GAC and GLS2 to form filaments is directly coupled to their catalytic activity and present their cryo-EM structures which provide an unprecedented view of the conformational states essential for catalysis. Filament formation guides an activation loop to assume a specific conformation that works together with a lid to close over the active site and position glutamine for nucleophilic attack by an essential serine. Our findings highlight how ankyrin repeats on GLS2 regulate enzymatic activity, while allosteric activators stabilize, and clinically relevant inhibitors block, filament formation that enables glutaminases to catalyze glutaminolysis and support cancer progression.

biophysics↗

bioRxiv2023

Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

The ability of cancer cells to survive microenvironmental stresses is critical for tumor progression and metastasis; however, how they survive these challenges is not fully understood. Here, we describe a novel multiprotein complex (DockTOR) essential for the survival of cancer cells under stress, triggered by the GTPase Cdc42 and a signaling partner Dock7, which includes AKT, mTOR, and the mTOR regulators TSC1, TSC2, and Rheb. DockTOR enables cancer cells to maintain a low but critical mTORC2-dependent phosphorylation of AKT during serum deprivation by preventing AKT dephosphorylation through an interaction between phospho-AKT and the Dock7 DHR1 domain. This activity stimulates a Raptor-independent but Rapamycin-sensitive mTOR/S6K activity necessary for survival. These findings address long-standing questions of how Cdc42 signals result in mTOR activation and demonstrate how cancer cells survive conditions when growth factor-dependent activation of mTORC1 is off. Determining how cancer cells survive stress conditions could identify vulnerabilities that lead to new therapeutic strategies.

cancer biology↗

bioRxiv2022

Proteomic analysis reveals microvesicles containing NAMPT as mediators of radiation resistance in glioma

Glioma is a malignant brain tumor that is highly resistant to radiation and chemotherapy, where patients survive on average only 15 months after diagnosis. Furthering the understanding of mechanisms leading to radiation resistance of glioma is paramount to identify novel therapeutic targets. Previous studies have shown that glioma stem cells (GSCs) play an important role in promoting radiation resistance and disease recurrence. Herein we analyze the proteomic alterations occurring in patient-derived GSCs upon radiation treatment in order to identify molecular drivers of resistance. We show that proteome changes upon radiation accurately predict the resistance status of the cells, whereas resistance to radiation does not correlate with glioma transcriptional subtypes. We further show that the radio-resistant GSC-267 cell line sheds microvesicles (MVs) enriched in the metabolic enzyme nicotinamide phosphoribosyltransferase (NAMPT). These MVs can be transferred to recipient fibroblasts and radio-sensitive GSCs, enhancing their intracellular total NAD+ and NADH level, and their ability to proliferate when cultured in low serum, treated with a radio-mimetic drug or irradiated. The NAMPT enzymatic inhibitor FK-866 blocked the ability of MVs from GSC-267 cells to mediate these effects. Similarly, GSC-267 cells where NAMPT was knocked-down using shRNA, which produced MVs depleted of this enzyme, were unable to promote cell proliferation. Collectively, our findings demonstrates that proteome-level regulation can accurately predict the radio-resistance status of GSCs, and identifies NAMPT transfer via MVs as a mechanism for spreading radiation resistance within the glioma tumor microenvironment. SignificanceThe highly aggressive and deadly brain cancer glioma is commonly resistant to standard chemo- and radio-therapy. We used systems biology approaches to study patient-derived glioma stem cells (GSCs), which are known to be responsible for therapeutic resistance, and cell-to-cell communication mediated by extracellular vesicles (EVs), which plays an important role in tumor progression. Analysis of the proteome of GSCs and of the EVs they release led us to determine that the EV-mediated transfer of the metabolic enzyme nicotinamide phosphorybosyltransferase (NAMPT) from radio-resistant to less aggressive cells confers resistance to radiation. Our findings identify a mechanism of therapy resistance in glioma, and suggest that NAMPT inhibition could enhance the efficacy of radiation for the treatment of glioma.

cancer biology↗

bioRxiv2021

Weakly migratory metastatic breast cancer cells activate fibroblasts via microvesicle-Tg2 to facilitate dissemination and metastasis

Cancer cell migration is highly heterogeneous, and the migratory capability of cancer cells is thought to be an indicator of metastatic potential. It is becoming clear that a cancer cell does not have to be inherently migratory to metastasize, with weakly migratory cancer cells often found to be highly metastatic. However, the mechanism through which weakly migratory cells escape from the primary tumor remains unclear. Here, utilizing phenotypically sorted highly and weakly migratory breast cancer cells, we demonstrate that weakly migratory metastatic cells disseminate from the primary tumor via communication with stromal cells. While highly migratory cells are capable of single cell migration, weakly migratory cells rely on cell-cell signaling with fibroblasts to escape the primary tumor. Weakly migratory cells release microvesicles rich in tissue transglutaminase 2 (Tg2) which activate fibroblasts and lead weakly migratory cancer cell migration in vitro. These microvesicles also induce tumor stiffening and fibroblast activation in vivo and enhance the metastasis of weakly migratory cells. Our results identify microvesicles and Tg2 as potential therapeutic targets for metastasis and reveal a novel aspect of the metastatic cascade in which weakly migratory cells release microvesicles which activate fibroblasts to enhance cancer cell dissemination.

cancer biology↗

bioRxiv2021

New Insights into the Mechanisms Used by Inhibitors Targeting Glutamine Metabolism in Cancer Cells

Many cancer cells become dependent on glutamine metabolism to compensate for glycolysis being uncoupled from the TCA cycle. The mitochondrial enzyme Glutaminase C (GAC) satisfies this glutamine addiction by catalyzing the first step in glutamine metabolism, making it an attractive drug target. Despite one of the allosteric inhibitors (CB-839) being in clinical trials, none of the drugs targeting GAC are approved for cancer treatment and their mechanism of action is not well understood. A major challenge has been the rational design of better drug candidates: standard cryo-cooled X-ray crystal structures of GAC bound to CB-839 and its analogs fail to explain their potency differences. Here, we address this problem by using an emerging technique, serial room temperature crystallography, which enabled us to observe clear differences between the binding conformations of inhibitors with significantly different potencies. A computational model was developed to further elucidate the molecular basis of inhibitor potency. We then corroborated the results from our modeling efforts by using recently established fluorescence assays that directly read-out inhibitor binding to GAC. Together, these findings provide new insights into the mechanisms used by a major class of allosteric GAC inhibitors and for the future rational design of more potent drug candidates.

biochemistry↗

bioRxiv2021

IGF2BP2 Promotes Cancer Progression by Degrading the RNA Transcript Encoding a v-ATPase Subunit

Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) binds to various RNA transcripts and promotes cancer progression, although little is known regarding its regulation. Here we show IGF2BP2 is a substrate of the deacetylase and tumor suppressor sirtuin 1 (SIRT1) and regulates the expression of the vacuolar ATPase subunit ATP6V1A. SIRT1 down-regulation in aggressive cancers leads to increased acetylation of IGF2BP2 which recruits the XRN2 nuclease to degrade the ATP6V1A transcript, decreasing its expression. This impairs lysosomal function and results in the production of a secretome that enhances cancer cell proliferation and metastasis. These findings describe a previously unrecognized role for IGF2BP2 in the degradation of an mRNA transcript essential for lysosomal function and highlight how its sirtuin-regulated acetylation state can have significant biological and disease consequences. One Sentence SummaryAcetylation of the RNA binding protein IGF2BP2, upon down-regulation of SIRT1, leads to degradation of the transcript encoding ATP6V1A and impaired lysosomal function in aggressive cancer cells.

cell biology↗

bioRxiv2020

Structure of the Visual Signaling Complex between Transducin and Phosphodiesterase 6

Heterotrimeric G proteins communicate signals from activated G protein-coupled receptors to downstream effector proteins. In the phototransduction pathway responsible for vertebrate vision, the G protein-effector complex is comprised of the GTP-bound transducin subunit (GT{middle dot}GTP) and the cyclic GMP (cGMP) phosphodiesterase 6 (PDE6), which stimulates cGMP hydrolysis to transmit signals to the optic nerve. Here we report a cryo-electron microscopy (cryoEM) structure of PDE6 complexed to GTP-bound GT. The structure reveals two GT{middle dot}GTP subunits engaging the PDE6 hetero-tetramer at both the PDE6 catalytic core and the PDE{gamma} subunits, driving extensive rearrangements to relieve all inhibitory constraints on enzyme catalysis. Analysis of the conformational ensemble in the cryoEM data highlights the dynamic nature of the contacts between the two GT{middle dot}GTP subunits and PDE6 that support an alternating-site catalytic mechanism.

biophysics↗