Publications by authors named "Hediye Erdjument-Bromage"

166 Publications

Mitovesicles are a novel population of extracellular vesicles of mitochondrial origin altered in Down syndrome.

Sci Adv 2021 Feb 12;7(7). Epub 2021 Feb 12.

Center for Dementia Research, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY 10962, USA.

Mitochondrial dysfunction is an established hallmark of aging and neurodegenerative disorders such as Down syndrome (DS) and Alzheimer's disease (AD). Using a high-resolution density gradient separation of extracellular vesicles (EVs) isolated from murine and human DS and diploid control brains, we identify and characterize a previously unknown population of double-membraned EVs containing multiple mitochondrial proteins distinct from previously described EV subtypes, including microvesicles and exosomes. We term these newly identified mitochondria-derived EVs "mitovesicles." We demonstrate that brain-derived mitovesicles contain a specific subset of mitochondrial constituents and that their levels and cargo are altered during pathophysiological processes where mitochondrial dysfunction occurs, including in DS. The development of a method for the selective isolation of mitovesicles paves the way for the characterization in vivo of biological processes connecting EV biology and mitochondria dynamics and for innovative therapeutic and diagnostic strategies.
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http://dx.doi.org/10.1126/sciadv.abe5085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880603PMC
February 2021

Serine phosphorylation regulates the P-type potassium pump KdpFABC.

Elife 2020 09 21;9. Epub 2020 Sep 21.

Skirball Institute, Dept. of Cell Biology, New York University School of Medicine, New York, United States.

KdpFABC is an ATP-dependent K pump that ensures bacterial survival in K-deficient environments. Whereas transcriptional activation of kdpFABC expression is well studied, a mechanism for down-regulation when K levels are restored has not been described. Here, we show that KdpFABC is inhibited when cells return to a K-rich environment. The mechanism of inhibition involves phosphorylation of Ser162 on KdpB, which can be reversed in vitro by treatment with serine phosphatase. Mutating Ser162 to Alanine produces constitutive activity, whereas the phosphomimetic Ser162Asp mutation inactivates the pump. Analyses of the transport cycle show that serine phosphorylation abolishes the K-dependence of ATP hydrolysis and blocks the catalytic cycle after formation of the aspartyl phosphate intermediate (E1~P). This regulatory mechanism is unique amongst P-type pumps and this study furthers our understanding of how bacteria control potassium homeostasis to maintain cell volume and osmotic potential.
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http://dx.doi.org/10.7554/eLife.55480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7535926PMC
September 2020

Molecular Stressors Engender Protein Connectivity Dysfunction through Aberrant N-Glycosylation of a Chaperone.

Cell Rep 2020 06;31(13):107840

Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Stresses associated with disease may pathologically remodel the proteome by both increasing interaction strength and altering interaction partners, resulting in proteome-wide connectivity dysfunctions. Chaperones play an important role in these alterations, but how these changes are executed remains largely unknown. Our study unveils a specific N-glycosylation pattern used by a chaperone, Glucose-regulated protein 94 (GRP94), to alter its conformational fitness and stabilize a state most permissive for stable interactions with proteins at the plasma membrane. This "protein assembly mutation' remodels protein networks and properties of the cell. We show in cells, human specimens, and mouse xenografts that proteome connectivity is restorable by inhibition of the N-glycosylated GRP94 variant. In summary, we provide biochemical evidence for stressor-induced chaperone-mediated protein mis-assemblies and demonstrate how these alterations are actionable in disease.
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http://dx.doi.org/10.1016/j.celrep.2020.107840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372946PMC
June 2020

Molecular basis for receptor tyrosine kinase A-loop tyrosine transphosphorylation.

Nat Chem Biol 2020 03 20;16(3):267-277. Epub 2020 Jan 20.

Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, USA.

A long-standing mystery shrouds the mechanism by which catalytically repressed receptor tyrosine kinase domains accomplish transphosphorylation of activation loop (A-loop) tyrosines. Here we show that this reaction proceeds via an asymmetric complex that is thermodynamically disadvantaged because of an electrostatic repulsion between enzyme and substrate kinases. Under physiological conditions, the energetic gain resulting from ligand-induced dimerization of extracellular domains overcomes this opposing clash, stabilizing the A-loop-transphosphorylating dimer. A unique pathogenic fibroblast growth factor receptor gain-of-function mutation promotes formation of the complex responsible for phosphorylation of A-loop tyrosines by eliminating this repulsive force. We show that asymmetric complex formation induces a more phosphorylatable A-loop conformation in the substrate kinase, which in turn promotes the active state of the enzyme kinase. This explains how quantitative differences in the stability of ligand-induced extracellular dimerization promotes formation of the intracellular A-loop-transphosphorylating asymmetric complex to varying extents, thereby modulating intracellular kinase activity and signaling intensity.
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http://dx.doi.org/10.1038/s41589-019-0455-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040854PMC
March 2020

The epichaperome is a mediator of toxic hippocampal stress and leads to protein connectivity-based dysfunction.

Nat Commun 2020 01 16;11(1):319. Epub 2020 Jan 16.

Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.

Optimal functioning of neuronal networks is critical to the complex cognitive processes of memory and executive function that deteriorate in Alzheimer's disease (AD). Here we use cellular and animal models as well as human biospecimens to show that AD-related stressors mediate global disturbances in dynamic intra- and inter-neuronal networks through pathologic rewiring of the chaperome system into epichaperomes. These structures provide the backbone upon which proteome-wide connectivity, and in turn, protein networks become disturbed and ultimately dysfunctional. We introduce the term protein connectivity-based dysfunction (PCBD) to define this mechanism. Among most sensitive to PCBD are pathways with key roles in synaptic plasticity. We show at cellular and target organ levels that network connectivity and functional imbalances revert to normal levels upon epichaperome inhibition. In conclusion, we provide proof-of-principle to propose AD is a PCBDopathy, a disease of proteome-wide connectivity defects mediated by maladaptive epichaperomes.
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http://dx.doi.org/10.1038/s41467-019-14082-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965647PMC
January 2020

Sam68 Enables Metabotropic Glutamate Receptor-Dependent LTD in Distal Dendritic Regions of CA1 Hippocampal Neurons.

Cell Rep 2019 11;29(7):1789-1799.e6

Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA; Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA. Electronic address:

The transport and translation of dendritic mRNAs by RNA-binding proteins (RBPs) allows for spatially restricted gene expression in neuronal processes. Although local translation in neuronal dendrites is now well documented, there is little evidence for corresponding effects on local synaptic function. Here, we report that the RBP Sam68 promotes the localization and translation of Arc mRNA preferentially in distal dendrites of rodent hippocampal CA1 pyramidal neurons. Consistent with Arc function in translation-dependent synaptic plasticity, we find that Sam68 knockout (KO) mice display impaired metabotropic glutamate-receptor-dependent long-term depression (mGluR-LTD) and impaired structural plasticity exclusively at distal Schaffer-collateral synapses. Moreover, by using quantitative proteomics, we find that the Sam68 interactome contains numerous regulators of mRNA translation and synaptic function. This work identifies an important player in Arc expression, provides a general framework for Sam68 regulation of protein synthesis, and uncovers a mechanism that enables the precise spatiotemporal expression of long-term plasticity throughout neurons.
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http://dx.doi.org/10.1016/j.celrep.2019.10.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6871770PMC
November 2019

Lipidome-wide C flux analysis: a novel tool to estimate the turnover of lipids in organisms and cultures.

J Lipid Res 2020 01 11;61(1):95-104. Epub 2019 Nov 11.

Departments of Anesthesiology, New York University School of Medicine, New York, NY 10016.

Lipid metabolism plays an important role in the regulation of cellular homeostasis. However, because it is difficult to measure the actual rates of synthesis and degradation of individual lipid species, lipid compositions are often used as a surrogate to evaluate lipid metabolism even though they provide only static snapshots of the lipodome. Here, we designed a simple method to determine the turnover rate of phospholipid and acylglycerol species based on the incorporation of C-glucose combined with LC-MS/MS. We labeled adult with C-glucose that incorporates into the entire lipidome, derived kinetic parameters from mass spectra, and studied effects of deletion of CG6718, the fly homolog of the calcium-independent phospholipase A2β, on lipid metabolism. Although C-glucose gave rise to a complex pattern of C incorporation, we were able to identify discrete isotopomers in which C atoms were confined to the glycerol group. With these isotopomers, we calculated turnover rate constants, half-life times, and fluxes of the glycerol backbone of multiple lipid species. To perform these calculations, we estimated the fraction of labeled molecules in glycerol-3-phosphate, the lipid precursor, by mass isotopomer distribution analysis of the spectra of phosphatidylglycerol. When we applied this method to , we found a range of lipid half-lives from 2 to 200 days, demonstrated tissue-specific fluxes of individual lipid species, and identified a novel function of CG6718 in triacylglycerol metabolism. This method provides fluxomics-type data with significant potential to improve the understanding of complex lipid regulation in a variety of research models.
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http://dx.doi.org/10.1194/jlr.D119000318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939592PMC
January 2020

A glucose-sensing neuron pair regulates insulin and glucagon in Drosophila.

Nature 2019 10 23;574(7779):559-564. Epub 2019 Oct 23.

Skirball Institute of Biomolecular Medicine, Department of Cell Biology, Neuroscience Institute, New York University School of Medicine, New York, NY, USA.

Although glucose-sensing neurons were identified more than 50 years ago, the physiological role of glucose sensing in metazoans remains unclear. Here we identify a pair of glucose-sensing neurons with bifurcated axons in the brain of Drosophila. One axon branch projects to insulin-producing cells to trigger the release of Drosophila insulin-like peptide 2 (dilp2) and the other extends to adipokinetic hormone (AKH)-producing cells to inhibit secretion of AKH, the fly analogue of glucagon. These axonal branches undergo synaptic remodelling in response to changes in their internal energy status. Silencing of these glucose-sensing neurons largely disabled the response of insulin-producing cells to glucose and dilp2 secretion, disinhibited AKH secretion in corpora cardiaca and caused hyperglycaemia, a hallmark feature of diabetes mellitus. We propose that these glucose-sensing neurons maintain glucose homeostasis by promoting the secretion of dilp2 and suppressing the release of AKH when haemolymph glucose levels are high.
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http://dx.doi.org/10.1038/s41586-019-1675-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857815PMC
October 2019

Haploinsufficiency in the ANKS1B gene encoding AIDA-1 leads to a neurodevelopmental syndrome.

Nat Commun 2019 08 6;10(1):3529. Epub 2019 Aug 6.

Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, 10461, NY, USA.

Neurodevelopmental disorders, including autism spectrum disorder, have complex polygenic etiologies. Single-gene mutations in patients can help define genetic factors and molecular mechanisms underlying neurodevelopmental disorders. Here we describe individuals with monogenic heterozygous microdeletions in ANKS1B, a predicted risk gene for autism and neuropsychiatric diseases. Affected individuals present with a spectrum of neurodevelopmental phenotypes, including autism, attention-deficit hyperactivity disorder, and speech and motor deficits. Neurons generated from patient-derived induced pluripotent stem cells demonstrate loss of the ANKS1B-encoded protein AIDA-1, a brain-specific protein highly enriched at neuronal synapses. A transgenic mouse model of Anks1b haploinsufficiency recapitulates a range of patient phenotypes, including social deficits, hyperactivity, and sensorimotor dysfunction. Identification of the AIDA-1 interactome using quantitative proteomics reveals protein networks involved in synaptic function and the etiology of neurodevelopmental disorders. Our findings formalize a link between the synaptic protein AIDA-1 and a rare, previously undefined genetic disease we term ANKS1B haploinsufficiency syndrome.
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http://dx.doi.org/10.1038/s41467-019-11437-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684583PMC
August 2019

PINK1 Content in Mitochondria is Regulated by ER-Associated Degradation.

J Neurosci 2019 09 12;39(36):7074-7085. Epub 2019 Jul 12.

Departments of Pathology & Cell Biology,

Maintaining a pool of functional mitochondria requires degradation of damaged ones within the cell. PINK1 is critical in this quality-control process: loss of mitochondrial membrane potential causes PINK1 to accumulate on the mitochondrial surface, triggering mitophagy. However, little is known about how PINK1 is regulated. Recently, we showed that PINK1 content is kept low in healthy mitochondria by continuous ubiquitination and proteasomal degradation of its mature form via a mechanism inconsistent with the proposed N-end rule process. Using both human female and monkey cell lines, we now demonstrate that once generated within the mitochondria, 52 kDa PINK1 adopts a mitochondrial topology most consistent with it being at the mitochondrial-endoplasmic reticulum (ER) interface. From this particular submitochondrial location, PINK1 interacts with components of the ER-associated degradation pathway, such as the E3 ligases gp78 and HRD1, which cooperate to catalyze PINK1 ubiquitination. The valosin-containing protein and its cofactor, UFD1, then target ubiquitinated PINK1 for proteasomal degradation. Our data show that PINK1 in healthy mitochondria is negatively regulated via an interplay between mitochondria and ER, and shed light on how this mitochondrial protein gains access to the proteasome. Regulation of mitochondrial content of PINK1, a contributor to mitophagy, is an important area of research. Recently, we found that PINK1 content is kept low in healthy mitochondria by continuous ubiquitination and proteasomal degradation. We now extend and refine this novel finding by showing that PINK1 localizes at the mitochondrial-endoplasmic reticulum (ER) interface, from where it interacts with the ER-associated degradation machinery, which catalyzes its ubiquitination and transfer to the proteasome. Thus, these data show that PINK1 in healthy mitochondria is negatively regulated via a mitochondria and ER interplay, and how this mitochondrial protein gains access to the proteasome.
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http://dx.doi.org/10.1523/JNEUROSCI.1691-18.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6733537PMC
September 2019

Extramitochondrial cardiolipin suggests a novel function of mitochondria in spermatogenesis.

J Cell Biol 2019 05 26;218(5):1491-1502. Epub 2019 Mar 26.

Department of Anesthesiology, New York University School of Medicine, New York, NY

Mitochondria contain cardiolipin (CL), an organelle-specific phospholipid that carries four fatty acids with a strong preference for unsaturated chains. Unsaturation is essential for the stability and for the function of mitochondrial CL. Surprisingly, we found tetrapalmitoyl-CL (TPCL), a fully saturated species, in the testes of humans and mice. TPCL was absent from other mouse tissues but was the most abundant CL species in testicular germ cells. Most intriguingly, TPCL was not localized in mitochondria but was in other cellular membranes even though mitochondrial CL was the substrate from which TPCL was synthesized. During spermiogenesis, TPCL became associated with the acrosome, a sperm-specific organelle, along with a subset of authentic mitochondrial proteins, including Ant4, Suox, and Spata18. Our data suggest that mitochondria-derived membranes are assembled into the acrosome, challenging the concept that this organelle is strictly derived from the Golgi apparatus and revealing a novel function of mitochondria.
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http://dx.doi.org/10.1083/jcb.201808131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504895PMC
May 2019

HSP90-incorporating chaperome networks as biosensor for disease-related pathways in patient-specific midbrain dopamine neurons.

Nat Commun 2018 10 19;9(1):4345. Epub 2018 Oct 19.

The Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box 256, New York, NY, 10065, USA.

Environmental and genetic risk factors contribute to Parkinson's Disease (PD) pathogenesis and the associated midbrain dopamine (mDA) neuron loss. Here, we identify early PD pathogenic events by developing methodology that utilizes recent innovations in human pluripotent stem cells (hPSC) and chemical sensors of HSP90-incorporating chaperome networks. We show that events triggered by PD-related genetic or toxic stimuli alter the neuronal proteome, thereby altering the stress-specific chaperome networks, which produce changes detected by chemical sensors. Through this method we identify STAT3 and NF-κB signaling activation as examples of genetic stress, and phospho-tyrosine hydroxylase (TH) activation as an example of toxic stress-induced pathways in PD neurons. Importantly, pharmacological inhibition of the stress chaperome network reversed abnormal phospho-STAT3 signaling and phospho-TH-related dopamine levels and rescued PD neuron viability. The use of chemical sensors of chaperome networks on hPSC-derived lineages may present a general strategy to identify molecular events associated with neurodegenerative diseases.
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http://dx.doi.org/10.1038/s41467-018-06486-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195591PMC
October 2018

Quantitative Comparison of Proteomes Using SILAC.

Curr Protoc Protein Sci 2019 02 20;95(1):e74. Epub 2018 Sep 20.

Kimmel Center for Biology and Medicine at the Skirball Institute and Department of Cell Biology, New York University School of Medicine, New York, New York.

Stable isotope labeling by amino acids in cell culture (SILAC) has become very popular as a quantitative proteomic method since it was firstly introduced by Matthias Mann's group in 2002. It is a metabolic labeling strategy in which isotope-labeled amino acids are metabolically incorporated in vivo into proteins during translation. After natural (light) or heavy amino acid incorporation, differentially labeled samples are mixed immediately after cell lysis and before any further processing, which minimizes quantitative errors caused by handling different samples in parallel. In this unit, we describe protocols for basic duplex SILAC, triplex SILAC for use in nondividing cells such as neurons, and for measuring amounts of newly synthesized proteins. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpps.74DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6342620PMC
February 2019

Sample Preparation for Relative Quantitation of Proteins Using Tandem Mass Tags (TMT) and Mass Spectrometry (MS).

Methods Mol Biol 2018 ;1741:135-149

Department of Cell Biology, Kimmel Center for Biology and Medicine at the Skirball Institute, New York University School of Medicine, New York, NY 10016, USA.

Quantitative proteome analysis allows comparisons of protein or phosphoprotein levels across multiple cell types or conditions. A number of experimental approaches have been described toward quantitative proteomics. In this chapter, we focus on Tandem Mass Tag (TMT) isobaric labeling of peptides for global, relative quantitation of proteins and phosphopeptides. To date, there has been no published protocol describing chemical labeling of small amounts of peptides specifically extracted from small tumor samples, for which rigorous sample preparation is necessary to ensure reproducible TMT labeling.
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http://dx.doi.org/10.1007/978-1-4939-7659-1_11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6472261PMC
December 2018

EGFR feedback-inhibition by Ran-binding protein 6 is disrupted in cancer.

Nat Commun 2017 12 11;8(1):2035. Epub 2017 Dec 11.

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.

Transport of macromolecules through the nuclear pore by importins and exportins plays a critical role in the spatial regulation of protein activity. How cancer cells co-opt this process to promote tumorigenesis remains unclear. The epidermal growth factor receptor (EGFR) plays a critical role in normal development and in human cancer. Here we describe a mechanism of EGFR regulation through the importin β family member RAN-binding protein 6 (RanBP6), a protein of hitherto unknown functions. We show that RanBP6 silencing impairs nuclear translocation of signal transducer and activator of transcription 3 (STAT3), reduces STAT3 binding to the EGFR promoter, results in transcriptional derepression of EGFR, and increased EGFR pathway output. Focal deletions of the RanBP6 locus on chromosome 9p were found in a subset of glioblastoma (GBM) and silencing of RanBP6 promoted glioma growth in vivo. Our results provide an example of EGFR deregulation in cancer through silencing of components of the nuclear import pathway.
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http://dx.doi.org/10.1038/s41467-017-02185-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5725448PMC
December 2017

Sex-Specific Differences in Oxytocin Receptor Expression and Function for Parental Behavior.

Gend Genome 2017 Dec;1(4):142-166

Skirball Institute for Biomolecular Medicine, New York University School of Medicine, New York, New York.

Parental care is among the most profound behavior expressed by humans and other animals. Despite intense interest in understanding the biological basis of parental behaviors, it remains unknown how much of parenting is encoded by the genome and which abilities instead are learned or can be refined by experience. One critical factor at the intersection between innate behaviors and experience-dependent learning is oxytocin, a neurohormone important for maternal physiology and neuroplasticity. Oxytocin acts throughout the body and brain to promote prosocial and maternal behaviors and modulates synaptic transmission to affect neural circuit dynamics. Recently we developed specific antibodies to mouse oxytocin receptors, found that oxytocin receptors are left lateralized in female auditory cortex, and examined how oxytocin enables maternal behavior by sensitizing the cortex to infant distress sounds. In this study we compare oxytocin receptor expression and function in male and female mice. Receptor expression is higher in adult female left auditory cortex than in right auditory cortex or males. Developmental profiles and mRNA expression were comparable between males and females. Behaviorally, male and female mice began expressing parental behavior similarly after cohousing with experienced females; however, oxytocin enhanced parental behavior onset in females but not males. This suggests that left lateralization of oxytocin receptor expression in females provides a mechanism for accelerating maternal behavior onset, although male mice can also effectively co-parent after experience with infants. The sex-specific pattern of oxytocin receptor expression might genetically predispose female cortex to respond to infant cues, which both males and females can also rapidly learn.
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http://dx.doi.org/10.1089/gg.2017.0017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500123PMC
December 2017

A novel requirement for DROSHA in maintenance of mammalian CG methylation.

Nucleic Acids Res 2017 Sep;45(16):9398-9412

Department of Cancer Biology, UCL Cancer Institute, London WC1E 6BT, UK.

In mammals, faithful inheritance of genomic methylation patterns ensures proper gene regulation and cell behaviour, impacting normal development and fertility. Following establishment, genomic methylation patterns are transmitted through S-phase by the maintenance methyltransferase Dnmt1. Using a protein interaction screen, we identify Microprocessor component DROSHA as a novel DNMT1-interactor. Drosha-deficient embryonic stem (ES) cells display genomic hypomethylation that is not accounted for by changes in the levels of DNMT proteins. DNMT1-mediated methyltransferase activity is also reduced in these cells. We identify two transcripts that are specifically upregulated in Drosha- but not Dicer-deficient ES cells. Regions within these transcripts predicted to form stem-loop structures are processed by Microprocessor and can inhibit DNMT1-mediated methylation in vitro. Our results highlight DROSHA as a novel regulator of mammalian DNA methylation and we propose that DROSHA-mediated processing of RNA is necessary to ensure full DNMT1 activity. This adds to the DROSHA repertoire of non-miRNA dependent functions as well as implicating RNA in regulating DNMT1 activity and correct levels of genomic methylation.
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http://dx.doi.org/10.1093/nar/gkx695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766157PMC
September 2017

The Ubiquitination of PINK1 Is Restricted to Its Mature 52-kDa Form.

Cell Rep 2017 07;20(1):30-39

Departments of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA; Department of Neurology, Columbia University, New York, NY 10032, USA; Center for Motor Neuron Biology and Diseases, Columbia University, New York, NY 10032, USA. Electronic address:

Along with Parkin, PINK1 plays a critical role in maintaining mitochondrial quality control. Although PINK1 is expressed constitutively, its level is kept low in healthy mitochondria by polyubiquitination and ensuing proteasomal degradation of its mature, 52 kDa, form. We show here that the target of PINK1 polyubiquitination is the mature form and is mediated by ubiquitination of a conserved lysine at position 137. Notably, the full-length protein also contains Lys-137 but is not ubiquitinated. On the basis of our data, we propose that cleavage of full-length PINK1 at Phe-104 disrupts the major hydrophobic membrane-spanning domain in the protein, inducing a conformation change in the resultant mature form that exposes Lys-137 to the cytosol for subsequent modification by the ubiquitination machinery. Thus, the balance between the full-length and mature PINK1 allows its levels to be regulated via ubiquitination of the mature form and ensures that PINK1 functions as a mitochondrial quality control factor.
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http://dx.doi.org/10.1016/j.celrep.2017.06.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5745057PMC
July 2017

Inhibition of Hsp90 Suppresses PI3K/AKT/mTOR Signaling and Has Antitumor Activity in Burkitt Lymphoma.

Mol Cancer Ther 2017 09 15;16(9):1779-1790. Epub 2017 Jun 15.

Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, New York.

Hsp90 is a molecular chaperone that protects proteins, including oncogenic signaling complexes, from proteolytic degradation. PU-H71 is a next-generation Hsp90 inhibitor that preferentially targets the functionally distinct pool of Hsp90 present in tumor cells. Tumors that are driven by the MYC oncoprotein may be particularly sensitive to PU-H71 due to the essential role of Hsp90 in the epichaperome, which maintains the malignant phenotype in the setting of MYC. Burkitt lymphoma (BL) is an aggressive B-cell lymphoma characterized by MYC dysregulation. In this study, we evaluated Hsp90 as a potential therapeutic target in BL. We found that primary BL tumors overexpress Hsp90 and that Hsp90 inhibition has antitumor activity and , including potent activity in a patient-derived xenograft model of BL. To evaluate the targets of PU-H71 in BL, we performed high-affinity capture followed by proteomic analysis using mass spectrometry. We found that Hsp90 inhibition targets multiple components of PI3K/AKT/mTOR signaling, highlighting the importance of this pathway in BL. Finally, we found that the anti-lymphoma activity of PU-H71 is synergistic with dual PI3K/mTOR inhibition and Overall, this work provides support for Hsp90 as a therapeutic target in BL and suggests the potential for combination therapy with PU-H71 and inhibitors of PI3K/mTOR. .
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587381PMC
September 2017

Unique Transcriptional Programs Identify Subtypes of AKI.

J Am Soc Nephrol 2017 Jun 27;28(6):1729-1740. Epub 2016 Dec 27.

Departments of *Medicine, Division of Nephrology,

Two metrics, a rise in serum creatinine concentration and a decrease in urine output, are considered tantamount to the injury of the kidney tubule and the epithelial cells thereof (AKI). Yet neither criterion emphasizes the etiology or the pathogenetic heterogeneity of acute decreases in kidney excretory function. In fact, whether decreased excretory function due to contraction of the extracellular fluid volume (vAKI) or due to intrinsic kidney injury (iAKI) actually share pathogenesis and should be aggregated in the same diagnostic group remains an open question. To examine this possibility, we created mouse models of iAKI and vAKI that induced a similar increase in serum creatinine concentration. Using laser microdissection to isolate specific domains of the kidney, followed by RNA sequencing, we found that thousands of genes responded specifically to iAKI or to vAKI, but very few responded to both stimuli. In fact, the activated gene sets comprised different, functionally unrelated signal transduction pathways and were expressed in different regions of the kidney. Moreover, we identified distinctive gene expression patterns in human urine as potential biomarkers of either iAKI or vAKI, but not both. Hence, iAKI and vAKI are biologically unrelated, suggesting that molecular analysis should clarify our current definitions of acute changes in kidney excretory function.
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http://dx.doi.org/10.1681/ASN.2016090974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461802PMC
June 2017

The epichaperome is an integrated chaperome network that facilitates tumour survival.

Nature 2016 Oct 5;538(7625):397-401. Epub 2016 Oct 5.

Program in Chemical Biology, Sloan Kettering Institute, New York, New York 10065, USA.

Transient, multi-protein complexes are important facilitators of cellular functions. This includes the chaperome, an abundant protein family comprising chaperones, co-chaperones, adaptors, and folding enzymes-dynamic complexes of which regulate cellular homeostasis together with the protein degradation machinery. Numerous studies have addressed the role of chaperome members in isolation, yet little is known about their relationships regarding how they interact and function together in malignancy. As function is probably highly dependent on endogenous conditions found in native tumours, chaperomes have resisted investigation, mainly due to the limitations of methods needed to disrupt or engineer the cellular environment to facilitate analysis. Such limitations have led to a bottleneck in our understanding of chaperome-related disease biology and in the development of chaperome-targeted cancer treatment. Here we examined the chaperome complexes in a large set of tumour specimens. The methods used maintained the endogenous native state of tumours and we exploited this to investigate the molecular characteristics and composition of the chaperome in cancer, the molecular factors that drive chaperome networks to crosstalk in tumours, the distinguishing factors of the chaperome in tumours sensitive to pharmacologic inhibition, and the characteristics of tumours that may benefit from chaperome therapy. We find that under conditions of stress, such as malignant transformation fuelled by MYC, the chaperome becomes biochemically 'rewired' to form a network of stable, survival-facilitating, high-molecular-weight complexes. The chaperones heat shock protein 90 (HSP90) and heat shock cognate protein 70 (HSC70) are nucleating sites for these physically and functionally integrated complexes. The results indicate that these tightly integrated chaperome units, here termed the epichaperome, can function as a network to enhance cellular survival, irrespective of tissue of origin or genetic background. The epichaperome, present in over half of all cancers tested, has implications for diagnostics and also provides potential vulnerability as a target for drug intervention.
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http://dx.doi.org/10.1038/nature19807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5283383PMC
October 2016

N-Terminal Amino Acid Sequence Determination of Proteins by N-Terminal Dimethyl Labeling: Pitfalls and Advantages When Compared with Edman Degradation Sequence Analysis.

J Biomol Tech 2016 07 7;27(2):61-74. Epub 2016 Mar 7.

1 Microchemistry and Proteomics Core Laboratory, 2 Structural Biology and 3 Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

In recent history, alternative approaches to Edman sequencing have been investigated, and to this end, the Association of Biomolecular Resource Facilities (ABRF) Protein Sequencing Research Group (PSRG) initiated studies in 2014 and 2015, looking into bottom-up and top-down N-terminal (Nt) dimethyl derivatization of standard quantities of intact proteins with the aim to determine Nt sequence information. We have expanded this initiative and used low picomole amounts of myoglobin to determine the efficiency of Nt-dimethylation. Application of this approach on protein domains, generated by limited proteolysis of overexpressed proteins, confirms that it is a universal labeling technique and is very sensitive when compared with Edman sequencing. Finally, we compared Edman sequencing and Nt-dimethylation of the same polypeptide fragments; results confirm that there is agreement in the identity of the Nt amino acid sequence between these 2 methods.
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http://dx.doi.org/10.7171/jbt.16-2702-002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4802820PMC
July 2016

Merlin/NF2 loss-driven tumorigenesis linked to CRL4(DCAF1)-mediated inhibition of the hippo pathway kinases Lats1 and 2 in the nucleus.

Cancer Cell 2014 Jul;26(1):48-60

Cell Biology Program, Sloan Kettering Institute for Cancer Research, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

It is currently unclear whether Merlin/NF2 suppresses tumorigenesis by activating upstream components of the Hippo pathway at the plasma membrane or by inhibiting the E3 ubiquitin ligase CRL4(DCAF1) in the nucleus. We found that derepressed CRL4(DCAF1) promotes YAP- and TEAD-dependent transcription by ubiquitylating and, thereby, inhibiting Lats1 and 2 in the nucleus. Genetic epistasis experiments and analysis of tumor-derived missense mutations indicate that this signaling connection sustains the oncogenicity of Merlin-deficient tumor cells. Analysis of clinical samples confirms that this pathway operates in NF2-mutant tumors. We conclude that derepressed CRL4(DCAF1) promotes activation of YAP by inhibiting Lats1 and 2 in the nucleus.
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http://dx.doi.org/10.1016/j.ccr.2014.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4126592PMC
July 2014

Affinity purification probes of potential use to investigate the endogenous Hsp70 interactome in cancer.

ACS Chem Biol 2014 Aug 17;9(8):1698-705. Epub 2014 Jun 17.

Program in Molecular Pharmacology and Chemistry and Department of Medicine and §Program in Molecular Biology, Proteomics Core, Memorial Sloan-Kettering Cancer Center , New York, New York 10021, United States.

Heat shock protein 70 (Hsp70) is a family of proteins with key roles in regulating malignancy. Cancer cells rely on Hsp70 to inhibit apoptosis, regulate senescence and autophagy, and maintain the stability of numerous onco-proteins. Despite these important biological functions in cancer, robust chemical tools that enable the analysis of the Hsp70-regulated proteome in a tumor-by-tumor manner are yet unavailable. Here we take advantage of a recently reported Hsp70 ligand to design and develop an affinity purification chemical toolset for potential use in the investigation of the endogenous Hsp70-interacting proteome in cancer. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins.
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http://dx.doi.org/10.1021/cb500256uDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134716PMC
August 2014

The histone variant MacroH2A1 regulates target gene expression in part by recruiting the transcriptional coregulator PELP1.

Mol Cell Biol 2014 Jul 21;34(13):2437-49. Epub 2014 Apr 21.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA Graduate Field of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York, USA Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, and Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

MacroH2A1 is a histone variant harboring an ∼25-kDa carboxyl-terminal macrodomain. Due to its enrichment on the inactive X chromosome, macroH2A1 was thought to play a role in transcriptional repression. However, recent studies have shown that macroH2A1 occupies autosomal chromatin and regulates genes in a context-specific manner. The macrodomain may play a role in the modulation of gene expression outcomes via physical interactions with effector proteins, which may depend on the ability of the macrodomain to bind NAD(+) metabolite ligands. Here, we identify proline, glutamic acid, and leucine-rich protein 1 (PELP1), a chromatin-associated factor and transcriptional coregulator, as a ligand-independent macrodomain-interacting factor. We used chromatin immunoprecipitation coupled with tiling microarrays (ChIP-chip) to determine the genomic localization of PELP1 in MCF-7 human breast cancer cells. We find that PELP1 genomic localization is highly correlated with that of macroH2A1. Additionally, PELP1 positively correlates with heterochromatic chromatin marks and negatively correlates with active transcription marks, much like macroH2A1. MacroH2A1 specifically recruits PELP1 to the promoters of macroH2A1 target genes, but macroH2A1 occupancy occurs independent of PELP1. This recruitment allows macroH2A1 and PELP1 to cooperatively regulate gene expression outcomes.
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http://dx.doi.org/10.1128/MCB.01315-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054320PMC
July 2014

Aminopeptidase activities as prospective urinary biomarkers for bladder cancer.

Proteomics Clin Appl 2014 Jun 31;8(5-6):317-26. Epub 2014 Mar 31.

Protein Center, Memorial Sloan-Kettering Cancer Center, New York, NY, USA; Department of Surgery, Urology Service, Memorial Sloan-Kettering Cancer Center, New York, NY, USA; Department of Urology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.

Purpose: Proteases have been implicated in cancer progression and invasiveness. We have investigated the activities, as opposed to simple protein levels, of selected aminopeptidases in urine specimens to serve as potential novel biomarkers for urothelial cancer.

Experimental Design: The unique urinary proteomes of males and females were profiled to establish the presence of a gender-independent set of aminopeptidases. Samples were also collected from patients with urothelial cancer and matched controls. A SOP for urine processing was developed taking into account hydration variation. Five specific aminopeptidase activity assays, using fluorophoric substrates, were optimized for evaluation of marker potential.

Results: Nineteen exopeptidases and 21 other proteases were identified in urine and the top-five most abundant aminopeptidases, identical in both genders, selected for functional studies. Depending on the enzyme, activities were consistently lower (p ≤ 0.05), higher or unchanged in the cancer samples as compared to controls. Two selected aminopeptidase activities used as a binary classifier resulted in a ROC curve with an AUC = 0.898.

Conclusion And Clinical Relevance: We have developed functional assays that characterize aminopeptidase activities in urine specimens with adequate technical and intraindividual reproducibility. With further testing, it could yield a reliable biomarker test for bladder cancer detection or prognostication.
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http://dx.doi.org/10.1002/prca.201300118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4059539PMC
June 2014

PRMT4 blocks myeloid differentiation by assembling a methyl-RUNX1-dependent repressor complex.

Cell Rep 2013 Dec 12;5(6):1625-38. Epub 2013 Dec 12.

Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA; Gerstner Sloan-Kettering Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA; Department of Biochemistry and Molecular Biology, Sylvester Comprehensive Cancer Center, University of Miami, Miller School of Medicine, Miami, FL 33136, USA; Department of Medicine, Sylvester Comprehensive Cancer Center, University of Miami, Miller School of Medicine, Miami, FL 33136, USA. Electronic address:

Defining the role of epigenetic regulators in hematopoiesis has become critically important, because recurrent mutations or aberrant expression of these genes has been identified in both myeloid and lymphoid hematological malignancies. We found that PRMT4, a type I arginine methyltransferase whose function in normal and malignant hematopoiesis is unknown, is overexpressed in acute myelogenous leukemia patient samples. Overexpression of PRMT4 blocks the myeloid differentiation of human stem/progenitor cells (HSPCs), whereas its knockdown is sufficient to induce myeloid differentiation of HSPCs. We demonstrated that PRMT4 represses the expression of miR-223 in HSPCs via the methylation of RUNX1, which triggers the assembly of a multiprotein repressor complex that includes DPF2. As part of the feedback loop, PRMT4 expression is repressed posttranscriptionally by miR-223. Depletion of PRMT4 results in differentiation of myeloid leukemia cells in vitro and their decreased proliferation in vivo. Thus, targeting PRMT4 holds potential as a novel therapy for acute myelogenous leukemia.
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http://dx.doi.org/10.1016/j.celrep.2013.11.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073674PMC
December 2013

Identification of an allosteric pocket on human hsp70 reveals a mode of inhibition of this therapeutically important protein.

Chem Biol 2013 Dec 14;20(12):1469-80. Epub 2013 Nov 14.

Program in Molecular Pharmacology and Chemistry and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. Electronic address:

Hsp70s are important cancer chaperones that act upstream of Hsp90 and exhibit independent anti-apoptotic activities. To develop chemical tools for the study of human Hsp70, we developed a homology model that unveils a previously unknown allosteric site located in the nucleotide binding domain of Hsp70. Combining structure-based design and phenotypic testing, we discovered a previously unknown inhibitor of this site, YK5. In cancer cells, this compound is a potent and selective binder of the cytosolic but not the organellar human Hsp70s and has biological activity partly by interfering with the formation of active oncogenic Hsp70/Hsp90/client protein complexes. YK5 is a small molecule inhibitor rationally designed to interact with an allosteric pocket of Hsp70 and represents a previously unknown chemical tool to investigate cellular mechanisms associated with Hsp70.
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http://dx.doi.org/10.1016/j.chembiol.2013.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985611PMC
December 2013