Searched for: person:neubet01
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Spns1 is an iron transporter essential for megalin-dependent endocytosis
Beenken, Andrew; Shen, Tian; Jin, Guangchun; Ghotra, Aryan; Xu, Katherine; Nesanir, Kivanc; Sturley, Rachel E; Vijayakumar, Soundarapandian; Kahn, Atlas; Levitman, Abraham; Stauber, Jacob; Chavez, Estefania Y; Robbins-Juarez, Shelief Y; Hao, Luke; Field, Thomas B; Erdjument-Bromage, Hediye; Neubert, Thomas A; Shapiro, Lawrence; Qiu, Andong; Barasch, Jonathan
Proximal tubule endocytosis is essential to produce protein free urine as well as to regulate system wide metabolic pathways, such as the activation of Vitamin D. We have determined that the proximal tubule expresses an endolysosomal membrane protein, protein spinster homolog1 (Spns1), which engenders a novel iron conductance that is indispensable during embryonic development. Conditional knockout of Spns1 with a novel Cre-LoxP construct specific to megalin-expressing cells led to the arrest of megalin receptor-mediated endocytosis as well as dextran pinocytosis in proximal tubules. The endocytic defect was accompanied by changes in megalin phosphorylation as well as enlargement of lysosomes confirming previous findings in Drosophila and Zebrafish. The endocytic defect was also accompanied by iron overload in proximal tubules. Remarkably, iron levels regulated the Spns1 phenotypes, because feeding an iron deficient diet or mating Spns1 knockout with divalent metal transporter1 (DMT1) knockout rescued the phenotypes. Conversely, iron loading wild type mice reproduced the endocytic defect, These data demonstrate a reversible, negative feedback for apical endocytosis, and raise the possibility that regulation of endocytosis, pinocytosis, megalin activation, and organellar size and function is nutrient-responsive.
PMID: 39265081
ISSN: 1522-1466
CID: 5690582
Modulation of GPR133 (ADGRD1) signaling by its intracellular interaction partner extended synaptotagmin 1
Stephan, Gabriele; Haddock, Sara; Wang, Shuai; Erdjument-Bromage, Hediye; Liu, Wenke; Ravn-Boess, Niklas; Frenster, Joshua D; Bready, Devin; Cai, Julia; Ronnen, Rebecca; Sabio-Ortiz, Jonathan; Fenyo, David; Neubert, Thomas A; Placantonakis, Dimitris G
GPR133 (ADGRD1) is an adhesion G-protein-coupled receptor that signals through Gαs/cyclic AMP (cAMP) and is required for the growth of glioblastoma (GBM), an aggressive brain malignancy. The regulation of GPR133 signaling is incompletely understood. Here, we use proximity biotinylation proteomics to identify ESYT1, a Ca2+-dependent mediator of endoplasmic reticulum-plasma membrane bridge formation, as an intracellular interactor of GPR133. ESYT1 knockdown or knockout increases GPR133 signaling, while its overexpression has the opposite effect, without altering GPR133 levels in the plasma membrane. The GPR133-ESYT1 interaction requires the Ca2+-sensing C2C domain of ESYT1. Thapsigargin-mediated increases in cytosolic Ca2+ relieve signaling-suppressive effects of ESYT1 by promoting ESYT1-GPR133 dissociation. ESYT1 knockdown or knockout in GBM slows tumor growth, suggesting tumorigenic functions of ESYT1. Our findings demonstrate a mechanism for the modulation of GPR133 signaling by increased cytosolic Ca2+, which reduces the signaling-suppressive interaction between GPR133 and ESYT1 to raise cAMP levels.
PMID: 38758649
ISSN: 2211-1247
CID: 5663132
Proteomic profiling of interferon-responsive reactive astrocytes in rodent and human
Prakash, Priya; Erdjument-Bromage, Hediye; O'Dea, Michael R; Munson, Christy N; Labib, David; Fossati, Valentina; Neubert, Thomas A; Liddelow, Shane A
Astrocytes are a heterogeneous population of central nervous system glial cells that respond to pathological insults and injury by undergoing a transformation called "reactivity." Reactive astrocytes exhibit distinct and context-dependent cellular, molecular, and functional state changes that can either support or disturb tissue homeostasis. We recently identified a reactive astrocyte sub-state defined by interferon-responsive genes like Igtp, Ifit3, Mx1, and others, called interferon-responsive reactive astrocytes (IRRAs). To further this transcriptomic definition of IRRAs, we wanted to define the proteomic changes that occur in this reactive sub-state. We induced IRRAs in immunopanned rodent astrocytes and human iPSC-differentiated astrocytes using TNF, IL1α, C1Q, and IFNβ and characterized their proteomic profile (both cellular and secreted) using unbiased quantitative proteomics. We identified 2335 unique cellular proteins, including IFIT2/3, IFITM3, OASL1/2, MX1/2/3, and STAT1. We also report that rodent and human IRRAs secrete PAI1, a serine protease inhibitor which may influence reactive states and functions of nearby cells. Finally, we evaluated how IRRAs are distinct from neurotoxic reactive astrocytes (NRAs). While NRAs are described by expression of the complement protein C3, it was not upregulated in IRRAs. Instead, we found ~90 proteins unique to IRRAs not identified in NRAs, including OAS1A, IFIT3, and MX1. Interferon signaling in astrocytes is critical for the antiviral immune response and for regulating synaptic plasticity and glutamate transport mechanisms. How IRRAs contribute to these functions is unknown. This study provides the basis for future experiments to define the functional roles of IRRAs in the context of neurodegenerative disorders.
PMID: 38031883
ISSN: 1098-1136
CID: 5616902
Cardiolipin prolongs the lifetimes of respiratory proteins in Drosophila flight muscle
Ren, Mindong; Xu, Yang; Phoon, Colin K L; Erdjument-Bromage, Hediye; Neubert, Thomas A; Schlame, Michael
Respiratory complexes and cardiolipins have exceptionally long lifetimes. The fact that they co-localize in mitochondrial cristae raises the question of whether their longevities have a common cause and whether the longevity of OXPHOS proteins is dependent on cardiolipin. To address these questions, we developed a method to measure side-by-side the half-lives of proteins and lipids in wild-type Drosophila and cardiolipin-deficient mutants. We fed adult flies with stable isotope-labeled precursors (13C6
PMCID:10622840
PMID: 37690688
ISSN: 1083-351x
CID: 5594302
Lysosomal dysfunction in Down syndrome and Alzheimer mouse models is caused by v-ATPase inhibition by Tyr682-phosphorylated APP βCTF
Im, Eunju; Jiang, Ying; Stavrides, Philip H; Darji, Sandipkumar; Erdjument-Bromage, Hediye; Neubert, Thomas A; Choi, Jun Yong; Wegiel, Jerzy; Lee, Ju-Hyun; Nixon, Ralph A
Lysosome dysfunction arises early and propels Alzheimer's disease (AD). Herein, we show that amyloid precursor protein (APP), linked to early-onset AD in Down syndrome (DS), acts directly via its β-C-terminal fragment (βCTF) to disrupt lysosomal vacuolar (H+)-adenosine triphosphatase (v-ATPase) and acidification. In human DS fibroblasts, the phosphorylated 682YENPTY internalization motif of APP-βCTF binds selectively within a pocket of the v-ATPase V0a1 subunit cytoplasmic domain and competitively inhibits association of the V1 subcomplex of v-ATPase, thereby reducing its activity. Lowering APP-βCTF Tyr682 phosphorylation restores v-ATPase and lysosome function in DS fibroblasts and in vivo in brains of DS model mice. Notably, lowering APP-βCTF Tyr682 phosphorylation below normal constitutive levels boosts v-ATPase assembly and activity, suggesting that v-ATPase may also be modulated tonically by phospho-APP-βCTF. Elevated APP-βCTF Tyr682 phosphorylation in two mouse AD models similarly disrupts v-ATPase function. These findings offer previously unknown insight into the pathogenic mechanism underlying faulty lysosomes in all forms of AD.
PMCID:10371027
PMID: 37494443
ISSN: 2375-2548
CID: 5592302
Systems-level analyses of protein-protein interaction network dysfunctions via epichaperomics identify cancer-specific mechanisms of stress adaptation
Rodina, Anna; Xu, Chao; Digwal, Chander S; Joshi, Suhasini; Patel, Yogita; Santhaseela, Anand R; Bay, Sadik; Merugu, Swathi; Alam, Aftab; Yan, Pengrong; Yang, Chenghua; Roychowdhury, Tanaya; Panchal, Palak; Shrestha, Liza; Kang, Yanlong; Sharma, Sahil; Almodovar, Justina; Corben, Adriana; Alpaugh, Mary L; Modi, Shanu; Guzman, Monica L; Fei, Teng; Taldone, Tony; Ginsberg, Stephen D; Erdjument-Bromage, Hediye; Neubert, Thomas A; Manova-Todorova, Katia; Tsou, Meng-Fu Bryan; Young, Jason C; Wang, Tai; Chiosis, Gabriela
Systems-level assessments of protein-protein interaction (PPI) network dysfunctions are currently out-of-reach because approaches enabling proteome-wide identification, analysis, and modulation of context-specific PPI changes in native (unengineered) cells and tissues are lacking. Herein, we take advantage of chemical binders of maladaptive scaffolding structures termed epichaperomes and develop an epichaperome-based 'omics platform, epichaperomics, to identify PPI alterations in disease. We provide multiple lines of evidence, at both biochemical and functional levels, demonstrating the importance of these probes to identify and study PPI network dysfunctions and provide mechanistically and therapeutically relevant proteome-wide insights. As proof-of-principle, we derive systems-level insight into PPI dysfunctions of cancer cells which enabled the discovery of a context-dependent mechanism by which cancer cells enhance the fitness of mitotic protein networks. Importantly, our systems levels analyses support the use of epichaperome chemical binders as therapeutic strategies aimed at normalizing PPI networks.
PMCID:10290137
PMID: 37353488
ISSN: 2041-1723
CID: 5538522
PTK7 is a positive allosteric modulator of GPR133 signaling in glioblastoma
Frenster, Joshua D; Erdjument-Bromage, Hediye; Stephan, Gabriele; Ravn-Boess, Niklas; Wang, Shuai; Liu, Wenke; Bready, Devin; Wilcox, Jordan; Kieslich, Björn; Jankovic, Manuel; Wilde, Caroline; Horn, Susanne; Sträter, Norbert; Liebscher, Ines; Schöneberg, Torsten; Fenyo, David; Neubert, Thomas A; Placantonakis, Dimitris G
The adhesion G-protein-coupled receptor GPR133 (ADGRD1) supports growth of the brain malignancy glioblastoma. How the extracellular interactome of GPR133 in glioblastoma modulates signaling remains unknown. Here, we use affinity proteomics to identify the transmembrane protein PTK7 as an extracellular binding partner of GPR133 in glioblastoma. PTK7 binds the autoproteolytically generated N-terminal fragment of GPR133 and its expression in trans increases GPR133 signaling. This effect requires the intramolecular cleavage of GPR133 and PTK7's anchoring in the plasma membrane. PTK7's allosteric action on GPR133 signaling is additive with but topographically distinct from orthosteric activation by soluble peptide mimicking the endogenous tethered Stachel agonist. GPR133 and PTK7 are expressed in adjacent cells in glioblastoma, where their knockdown phenocopies each other. We propose that this ligand-receptor interaction is relevant to the pathogenesis of glioblastoma and possibly other physiological processes in healthy tissues.
PMID: 37354459
ISSN: 2211-1247
CID: 5543042
Structures of LRP2 reveal a molecular machine for endocytosis
Beenken, Andrew; Cerutti, Gabriele; Brasch, Julia; Guo, Yicheng; Sheng, Zizhang; Erdjument-Bromage, Hediye; Aziz, Zainab; Robbins-Juarez, Shelief Y; Chavez, Estefania Y; Ahlsen, Goran; Katsamba, Phinikoula S; Neubert, Thomas A; Fitzpatrick, Anthony W P; Barasch, Jonathan; Shapiro, Lawrence
The low-density lipoprotein (LDL) receptor-related protein 2 (LRP2 or megalin) is representative of the phylogenetically conserved subfamily of giant LDL receptor-related proteins, which function in endocytosis and are implicated in diseases of the kidney and brain. Here, we report high-resolution cryoelectron microscopy structures of LRP2 isolated from mouse kidney, at extracellular and endosomal pH. The structures reveal LRP2 to be a molecular machine that adopts a conformation for ligand binding at the cell surface and for ligand shedding in the endosome. LRP2 forms a homodimer, the conformational transformation of which is governed by pH-sensitive sites at both homodimer and intra-protomer interfaces. A subset of LRP2 deleterious missense variants in humans appears to impair homodimer assembly. These observations lay the foundation for further understanding the function and mechanism of LDL receptors and implicate homodimerization as a conserved feature of the LRP receptor subfamily.
PMID: 36750096
ISSN: 1097-4172
CID: 5426892
Proteomic profiling of interferon-responsive reactive astrocytes in rodent and human
Prakash, Priya; Erdjument-Bromage, Hediye; O'Dea, Michael R.; Munson, Christy N.; Labib, David; Fossati, Valentina; Neubert, Thomas A.; Liddelow, Shane A.
Astrocytes are a heterogeneous population of central nervous system glial cells that respond to pathological insults and injury by undergoing a transformation called "reactivity." Reactive astrocytes exhibit distinct and context-dependent cellular, molecular, and functional state changes that can either support or disturb tissue homeostasis. We recently identified a reactive astrocyte sub-state defined by interferon-responsive genes like Igtp, Ifit3, Mx1, and others, called interferon-responsive reactive astrocytes (IRRAs). To further this transcriptomic definition of IRRAs, we wanted to define the proteomic changes that occur in this reactive sub-state. We induced IRRAs in immunopanned rodent astrocytes and human iPSC-differentiated astrocytes using TNF, IL1α, C1Q, and IFNβ and characterized their proteomic profile (both cellular and secreted) using unbiased quantitative proteomics. We identified 2335 unique cellular proteins, including IFIT2/3, IFITM3, OASL1/2, MX1/2/3, and STAT1. We also report that rodent and human IRRAs secrete PAI1, a serine protease inhibitor which may influence reactive states and functions of nearby cells. Finally, we evaluated how IRRAs are distinct from neurotoxic reactive astrocytes (NRAs). While NRAs are described by expression of the complement protein C3, it was not upregulated in IRRAs. Instead, we found ~90 proteins unique to IRRAs not identified in NRAs, including OAS1A, IFIT3, and MX1. Interferon signaling in astrocytes is critical for the antiviral immune response and for regulating synaptic plasticity and glutamate transport mechanisms. How IRRAs contribute to these functions is unknown. This study provides the basis for future experiments to define the functional roles of IRRAs in the context of neurodegenerative disorders.
SCOPUS:85178179989
ISSN: 0894-1491
CID: 5622882
Cocaine perturbs mitovesicle biology in the brain
D'Acunzo, Pasquale; Ungania, Jonathan M; Kim, Yohan; Barreto, Bryana R; DeRosa, Steven; Pawlik, Monika; Canals-Baker, Stefanie; Erdjument-Bromage, Hediye; Hashim, Audrey; Goulbourne, Chris N; Neubert, Thomas A; Saito, Mariko; Sershen, Henry; Levy, Efrat
Cocaine, an addictive psychostimulant, has a broad mechanism of action, including the induction of a wide range of alterations in brain metabolism and mitochondrial homeostasis. Our group recently identified a subpopulation of non-microvesicular, non-exosomal extracellular vesicles of mitochondrial origin (mitovesicles) and developed a method to isolate mitovesicles from brain parenchyma. We hypothesised that the generation and secretion of mitovesicles is affected by mitochondrial abnormalities induced by chronic cocaine exposure. Mitovesicles from the brain extracellular space of cocaine-administered mice were enlarged and more numerous when compared to controls, supporting a model in which mitovesicle biogenesis is enhanced in the presence of mitochondrial alterations. This interrelationship was confirmed in vitro. Moreover, cocaine affected mitovesicle protein composition, causing a functional alteration in mitovesicle ATP production capacity. These data suggest that mitovesicles are previously unidentified players in the biology of cocaine addiction and that target therapies to fine-tune brain mitovesicle functionality may be beneficial to mitigate the effects of chronic cocaine exposure.
PMCID:9871795
PMID: 36691887
ISSN: 2001-3078
CID: 5426532