Publications by authors named "Ido Livneh"

25 Publications

  • Page 1 of 1

The ubiquitin ligase RNF5 determines acute myeloid leukemia growth and susceptibility to histone deacetylase inhibitors.

Nat Commun 2021 09 13;12(1):5397. Epub 2021 Sep 13.

Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.

Acute myeloid leukemia (AML) remains incurable, largely due to its resistance to conventional treatments. Here, we find that increased abundance of the ubiquitin ligase RNF5 contributes to AML development and survival. High RNF5 expression in AML patient specimens correlates with poor prognosis. RNF5 inhibition decreases AML cell growth in culture, in patient-derived xenograft (PDX) samples and in vivo, and delays development of MLL-AF9-driven leukemogenesis in mice, prolonging their survival. RNF5 inhibition causes transcriptional changes that overlap with those seen upon histone deacetylase (HDAC)1 inhibition. RNF5 induces the formation of K29 ubiquitin chains on the histone-binding protein RBBP4, promoting its recruitment to and subsequent epigenetic regulation of genes involved in AML maintenance. Correspondingly, RNF5 or RBBP4 knockdown enhances AML cell sensitivity to HDAC inhibitors. Notably, low expression of both RNF5 and HDAC coincides with a favorable prognosis. Our studies identify an ERAD-independent role for RNF5, demonstrating that its control of RBBP4 constitutes an epigenetic pathway that drives AML, and highlight RNF5/RBBP4 as markers useful to stratify patients for treatment with HDAC inhibitors.
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http://dx.doi.org/10.1038/s41467-021-25664-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8437979PMC
September 2021

Correction: modulation of ubiquitin chains by -methylated non-proteinogenic cyclic peptides.

RSC Chem Biol 2021 Jun 11;2(3):944. Epub 2021 May 11.

Schulich Faculty of Chemistry, Technion-Israel Institute of Technology Haifa 3200008 Israel

[This corrects the article DOI: 10.1039/D0CB00179A.].
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http://dx.doi.org/10.1039/d1cb90015cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8341468PMC
June 2021

p62-containing, proteolytically active nuclear condensates, increase the efficiency of the ubiquitin-proteasome system.

Proc Natl Acad Sci U S A 2021 Aug;118(33)

Technion Rappaport Integrated Cancer Center, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, 3109601 Haifa, Israel

Degradation of a protein by the ubiquitin-proteasome system (UPS) is a multistep process catalyzed by sequential reactions. Initially, ubiquitin is conjugated to the substrate in a process mediated by concerted activity of three enzymes; the last of them-a ubiquitin ligase (E3)-belongs to a family of several hundred members, each recognizing a few specific substrates. This is followed by repeated addition of ubiquitin moieties to the previously conjugated one to generate a ubiquitin chain that serves as a recognition element for the proteasome, which then degrades the substrate. Ubiquitin is recycled via the activity of deubiquitinating enzymes (DUBs). It stands to reason that efficiency of such a complex process would depend on colocalization of the different components in an assembly that allows the reactions to be carried out sequentially and processively. Here we describe nuclear condensates that are dynamic in their composition. They contain p62 as an essential component. These assemblies are generated by liquid-liquid phase separation (LLPS) and also contain ubiquitinated targets, 26S proteasome, the three conjugating enzymes, and DUBs. Under basal conditions, they serve as efficient centers for proteolysis of nuclear proteins (e.g., c-Myc) and unassembled subunits of the proteasome, suggesting they are involved in cellular protein quality control. Supporting this notion is the finding that such foci are also involved in degradation of misfolded proteins induced by heat and oxidative stresses, following recruitment of heat shock proteins and their associated ubiquitin ligase CHIP.
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http://dx.doi.org/10.1073/pnas.2107321118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8379982PMC
August 2021

In vivo modulation of ubiquitin chains by N-methylated non-proteinogenic cyclic peptides.

RSC Chem Biol 2021 Apr 16;2(2):513-522. Epub 2020 Dec 16.

Schulich Faculty of Chemistry, Technion-Israel Institute of Technology, Haifa 3200008, Israel.

Cancer and other disease states can change the landscape of proteins post-translationally tagged with ubiquitin (Ub) chains. Molecules capable of modulating Ub chains are potential therapeutic agents, but their discovery represents a significant challenge. Recently, it was shown that cyclic peptides, selected from trillion-member random libraries, are capable of binding particular Ub chains. However, these peptides were overwhelmingly proteinogenic, so the prospect of activity was uncertain. Here, we report the discovery of small, non-proteinogenic cyclic peptides, rich in non-canonical features like N-methylation, which can tightly and specifically bind Lys48-linked Ub chains. These peptides engage three Lys48-linked Ub units simultaneously, block the action of deubiquitinases and the proteasome, induce apoptosis , and attenuate tumor growth . This highlights the potential of non-proteinogenic cyclic peptide screening to rapidly find -active leads, and the targeting of ubiquitin chains as a promising anti-cancer mechanism of action.
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http://dx.doi.org/10.1039/d0cb00179aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8232551PMC
April 2021

In-depth characterization of ubiquitin turnover in mammalian cells by fluorescence tracking.

Cell Chem Biol 2021 08 5;28(8):1192-1205.e9. Epub 2021 Mar 5.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 Moscow, Russian Federation; Lomonosov Moscow State University, Leninskie Gory, 119991 Moscow, Russian Federation. Electronic address:

Despite almost 40 years having passed from the initial discovery of ubiquitin (Ub), fundamental questions related to its intracellular metabolism are still enigmatic. Here we utilized fluorescent tracking for monitoring ubiquitin turnover in mammalian cells, resulting in obtaining qualitatively new data. In the present study we report (1) short Ub half-life estimated as 4 h; (2) for a median of six Ub molecules per substrate as a dynamic equilibrium between Ub ligases and deubiquitinated enzymes (DUBs); (3) loss on average of one Ub molecule per four acts of engagement of polyubiquitinated substrate by the proteasome; (4) direct correlation between incorporation of Ub into the distinct type of chains and Ub half-life; and (5) critical influence of the single lysine residue K27 on the stability of the whole Ub molecule. Concluding, our data provide a comprehensive understanding of ubiquitin-proteasome system dynamics on the previously unreachable state of the art.
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http://dx.doi.org/10.1016/j.chembiol.2021.02.009DOI Listing
August 2021

COVID-19-Associated Suspected Myocarditis as the Etiology for Recurrent and Protracted Fever in an Otherwise Healthy Adult.

Am J Med Sci 2021 04 4;361(4):522-525. Epub 2020 Nov 4.

Department of Internal Medicine A, Rambam Health Care Campus, Haifa, Israel; "KETER" - COVID-19 Department, Rambam Health Care Campus, Haifa, Israel; The Rappaport Faculty of Medicine and Research Institute, Technion - Israel Institute of Technology, Haifa, Israel; Infection Control Unit, Rambam Health Care Campus, Haifa, Israel.

Current reports concerning cardiac involvement in the novel corona virus disease (COVID-19) mostly document acute myocardial injury at presentation. Here, we present a healthy young male, with presumed acute myocarditis, presenting 20 days after initial diagnosis of COVID-19 - and after a clinical, and apparent laboratory, resolution of the original episode. His sole substantial clinical finding upon admission was fever, which was followed by a witnessed elevation in troponin-I.
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http://dx.doi.org/10.1016/j.amjms.2020.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7640947PMC
April 2021

SPANX Control of Lamin A/C Modulates Nuclear Architecture and Promotes Melanoma Growth.

Mol Cancer Res 2020 10 22;18(10):1560-1573. Epub 2020 Jun 22.

Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California.

Mechanisms regulating nuclear organization control fundamental cellular processes, including the cell and chromatin organization. Their disorganization, including aberrant nuclear architecture, has been often implicated in cellular transformation. Here, we identify Lamin A, among proteins essential for nuclear architecture, as SPANX (sperm protein associated with the nucleus on the X chromosome), a cancer testis antigen previously linked to invasive tumor phenotypes, interacting protein in melanoma. SPANX interaction with Lamin A was mapped to the immunoglobulin fold-like domain, a region critical for Lamin A function, which is often mutated in laminopathies. SPANX downregulation in melanoma cell lines perturbed nuclear organization, decreased cell viability, and promoted senescence-associated phenotypes. Moreover, SPANX knockdown (KD) in melanoma cells promoted proliferation arrest, a phenotype mediated in part by IRF3/IL1A signaling. SPANX KD in melanoma cells also prompted the secretion of IL1A, which attenuated the proliferation of naïve melanoma cells. Identification of SPANX as a nuclear architecture complex component provides an unexpected insight into the regulation of Lamin A and its importance in melanoma. IMPLICATIONS: SPANX, a testis protein, interacts with LMNA and controls nuclear architecture and melanoma growth.
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http://dx.doi.org/10.1158/1541-7786.MCR-20-0291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541784PMC
October 2020

Intracellular Role for the Matrix-Modifying Enzyme Lox in Regulating Transcription Factor Subcellular Localization and Activity in Muscle Regeneration.

Dev Cell 2020 05 30;53(4):406-417.e5. Epub 2020 Apr 30.

Department of Genetics and Developmental Biology, The Rappaport Faculty of Medicine and Research Institute, Technion - Israel Institute of Technology, Haifa 31096, Israel. Electronic address:

Integration of extracellular matrix (ECM)-derived cues into transcriptional programs is essential primarily in rapidly morphing environments, such as regenerating tissues. Here, we demonstrate that lysyl oxidase (Lox), known for its ECM-modifying activities, primarily collagen crosslinking, also directly regulates transcription factor (TF) localization. Using genetic and pharmacological strategies, we highlight an intracellular role for Lox in myogenic progenitors essential for muscle regeneration. We show that Lox interacts with, and directly oxidizes, vestigial-like 3 (Vgll3), a transcriptional co-activator acting with Mef2 and transcriptional enhancer factor (TEF) TFs. This enzymatic activity is required for Vgll3 cytoplasmic-to-nuclear translocation in regulation of myogenic differentiation. Our work highlights an additional mechanism for TF subcellular localization facilitating integration of ECM organization with transcriptional output during myogenic differentiation. Modulating this integration mechanism could affect the balance between ECM organization and cell differentiation and serve as a basis for novel therapeutic strategies targeting fibrotic pathologies.
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http://dx.doi.org/10.1016/j.devcel.2020.04.002DOI Listing
May 2020

Proteasome phase separation: a novel layer of quality control.

Cell Res 2020 05;30(5):374-375

Technion Integrated Cancer Center, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, 3109602, Haifa, Israel.

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http://dx.doi.org/10.1038/s41422-020-0306-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196124PMC
May 2020

Affinity Maturation of Macrocyclic Peptide Modulators of Lys48-Linked Diubiquitin by a Twofold Strategy.

Chemistry 2020 Jun 8;26(36):8022-8027. Epub 2020 Jun 8.

Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo, 113-0033, Japan.

Messenger RNA display of peptides containing non-proteinogenic amino acids, referred to as RaPID system, has become one of the leading methods to express libraries consisting of more than trillion-members of macrocyclic peptides, which allows for discovering de novo bioactive ligands. Ideal macrocyclic peptides should have dissociation constants (K ) as low as single-digit values in the nanomolar range towards a specific target of interest. Here, a twofold strategy to discover optimized macrocyclic peptides within this affinity regime is described. First, benzyl thioether cyclized peptide libraries were explored to identify tight binding hits. To obtain more insights into critical sequence information, sequence alignment was applied to guide rational mutagenesis for the improvement of their binding affinity. Using this twofold strategy, benzyl thioether macrocyclic peptide binders against Lys48-linked ubiquitin dimer (K48-Ub2) were successfully obtained that display K values in the range 0.3-1.2 nm, which indicate binding two orders of magnitude stronger than those of macrocyclic peptides recently reported. Most importantly, this macrocyclic peptide also showed an improved cellular inhibition of the K48-Ub2 recognition by deubiquitinating enzymes and the 26S proteasome, resulting in the promotion of apoptosis in cancer cells.
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http://dx.doi.org/10.1002/chem.202000273DOI Listing
June 2020

The mA epitranscriptome: transcriptome plasticity in brain development and function.

Nat Rev Neurosci 2020 01 5;21(1):36-51. Epub 2019 Dec 5.

Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

The field of epitranscriptomics examines the recently deciphered form of gene expression regulation that is mediated by type- and site-specific RNA modifications. Similarly to the role played by epigenetic mechanisms - which operate via DNA and histone modifications - epitranscriptomic modifications are involved in the control of the delicate gene expression patterns that are needed for the development and activity of the nervous system and are essential for basic and higher brain functions. Here we describe the mechanisms that are involved in the writing, erasing and reading of N-methyladenosine, the most prevalent internal mRNA modification, and the emerging roles played by N-methyladenosine in the nervous system.
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http://dx.doi.org/10.1038/s41583-019-0244-zDOI Listing
January 2020

Identification of proteins regulated by the proteasome following induction of endoplasmic reticulum stress.

Biochem Biophys Res Commun 2019 09 18;517(2):188-192. Epub 2019 Jul 18.

Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa, 3109601, Israel. Electronic address:

The endoplasmic reticulum (ER) is a major site for protein synthesis, folding and transport, lipid and steroid synthesis, regulating redox potential, as well as calcium storage. It therefore relies on delicate homeostasis, and perturbation of the ER function and induction of ER stress can lead to apoptosis. One cause of disruption of the ER homeostasis is the accumulation of misfolded proteins. To prevent this perturbation, the Endoplasmic Reticulum-Associated Degradation (ERAD) quality control machinery is recruited to remove these proteins in a three-step process: (1) extraction from the ER, (2) ubiquitination, and (3) subsequent proteasomal degradation. However, the identity of the proteins regulated by the proteasome following induction of the ER stress has remained obscure. In the present study, we investigated the role of the proteasome in the modulation of the proteome of HeLa cells after treatment with thapsigargin and tunicamycin, two drugs known to induce ER stress through accumulation of misfolded proteins. Using label-free quantitative proteomics we found that out of the proteins identified to decrease in their level following induction of ER stress, more than 64% are targeted by the proteasome. Among these proteins, key players of the Wnt signaling pathway, such as β-catenin and GSK3, as well as α-catenin which is involved in cell-cell adhesion, were identified as being modulated by the proteasome upon ER stress.
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http://dx.doi.org/10.1016/j.bbrc.2019.07.040DOI Listing
September 2019

De novo macrocyclic peptides that specifically modulate Lys48-linked ubiquitin chains.

Nat Chem 2019 07 10;11(7):644-652. Epub 2019 Jun 10.

Schulich Faculty of Chemistry, Technion-Israel Institute of Technology, Haifa, Israel.

A promising approach in cancer therapy is to find ligands that directly bind ubiquitin (Ub) chains. However, finding molecules capable of tightly and specifically binding Ub chains is challenging given the range of Ub polymer lengths and linkages and their subtle structural differences. Here, we use total chemical synthesis of proteins to generate highly homogeneous Ub chains for screening against trillion-member macrocyclic peptide libraries (RaPID system). De novo cyclic peptides were found that can bind tightly and specifically to K48-linked Ub chains, confirmed by NMR studies. These cyclic peptides protected K48-linked Ub chains from deubiquitinating enzymes and prevented proteasomal degradation of Ub-tagged proteins. The cyclic peptides could enter cells, inhibit growth and induce programmed cell death, opening new opportunities for therapeutic intervention. This highly synthetic approach, with both protein target generation and cyclic peptide discovery performed in vitro, will make other elaborate post-translationally modified targets accessible for drug discovery.
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http://dx.doi.org/10.1038/s41557-019-0278-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341950PMC
July 2019

Comparison of Drosophila melanogaster Embryo and Adult Proteome by SWATH-MS Reveals Differential Regulation of Protein Synthesis, Degradation Machinery, and Metabolism Modules.

J Proteome Res 2019 06 22;18(6):2525-2534. Epub 2019 May 22.

Cambridge Centre for Proteomics, Department of Biochemistry , University of Cambridge , Cambridge CB2 1QR , U.K.

An important area of modern biology consists of understanding the relationship between genotype and phenotype. However, to understand this relationship it is essential to investigate one of the principal links between them: the proteome. With the development of recent mass-spectrometry approaches, it is now possible to quantify entire proteomes and thus relate them to different phenotypes. Here, we present a comparison of the proteome of two extreme developmental states in the well-established model organism Drosophila melanogaster: adult and embryo. Protein modules such as ribosome, proteasome, tricarboxylic acid cycle, glycolysis, or oxidative phosphorylation were found differentially expressed between the two developmental stages. Analysis of post-translation modifications of the proteins identified in this study indicates that they generally follow the same trend as their corresponding protein. Comparison between changes in the proteome and the transcriptome highlighted patterns of post-transcriptional regulation for the subunits of protein complexes such as the ribosome and the proteasome, whereas protein from modules such as TCA cycle, glycolysis, and oxidative phosphorylation seem to be coregulated at the transcriptional level. Finally, the impact of the endosymbiont Wolbachia pipientis on the proteome of both developmental states was also investigated.
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http://dx.doi.org/10.1021/acs.jproteome.9b00076DOI Listing
June 2019

Modulation of the cell cycle regulating transcription factor E2F1 pathway by the proteasome following amino acid starvation.

Biochem Biophys Res Commun 2019 06 13;513(3):721-725. Epub 2019 Apr 13.

Technion Integrated Cancer Center, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa, 3109601, Israel. Electronic address:

The proteasome is one of the main catalytic machineries of eukaryotic cells responsible for protein degradation, and is known to be regulated during several cellular stress conditions. Recent studies suggest that the activity of the proteasome is modulated following mTOR inhibition. However, it is not clear how this process affects the proteome. In the present study, we investigated the role of the proteasome in the modulation of the proteome of HeLa cells following amino acid starvation, a stress known to inhibit mTOR activity. We used label-free quantitative proteomics to identify proteins regulated by the proteasome in starved cells. We found that nearly 50% of the proteins the level of which decreased significantly during starvation stress, could be rescued by addition of the proteasome inhibitor MG132. This suggests a key role for the proteasome in reshaping the proteome under starvation. Importantly, the expression of several of these proteins is known to be dependent on the transcription factor E2F1. Further investigation of E2F1 level showed that this transcription factor along with several other proteins involved in its pathway are regulated by the proteasome upon amino acids starvation.
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http://dx.doi.org/10.1016/j.bbrc.2019.04.066DOI Listing
June 2019

Monitoring stress-induced autophagic engulfment and degradation of the 26S proteasome in mammalian cells.

Methods Enzymol 2019 7;619:337-366. Epub 2019 Mar 7.

Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa, Israel; Department of Biomedical Sciences, Protein Metabolism Medical Research Center, College of Medicine, Seoul National University, Seoul, South Korea.

Almost 70 years after the discovery of the lysosome, and about four decades following the unraveling of ubiquitin as a specific "mark of death," the field of protein turnover-the numerous processes it regulates, the pathologies resulting from its dysregulation, and the drugs that have been developed to target them-is still growing exponentially. Accordingly, the need for new technologies and methods is ever growing. One interesting question in the field is the mechanism(s) by which the "predators become prey". We have reported recently that the 26S proteasome, the catalytic arm of the ubiquitin system, is degraded by the autophagy-lysosome machinery, in a process requiring specific ubiquitination of the proteasome, and subsequent recognition by the shuttle protein p62/SQSTM1. Studying the modification(s), recognition sites, engulfment, and breakdown of the 26S proteasome via such "proteaphagy" has required the use of microscopy, subcellular fractionation, 'classical biochemistry', and proteomics. In this chapter, we present the essentials of these protocols, with emphasis on the refinements we have introduced in order for them to better suit the particular study of proteaphagy.
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http://dx.doi.org/10.1016/bs.mie.2018.12.022DOI Listing
February 2020

Monoubiquitination joins polyubiquitination as an esteemed proteasomal targeting signal.

Bioessays 2017 06 11;39(6). Epub 2017 May 11.

Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa, Israel.

A polyubiquitin chain attached covalently to the target substrate has been recognized for long as the "canonical" proteasomal degradation signal. However, several proteins have been shown to be targeted for degradation following monoubiquitination, indicating that the proteasome can recognize signals other than a ubiquitin polymer. A comprehensive screen aiming at determining the extent of this mode of recognition revealed that ∼40% of mammalian and ∼20% of yeast proteins are degraded following monoubiquitination. Characterization of these proteins showed that on average, the monoubiquitinated proteins are smaller than the polyubiquitinated ones, and in humans, are less disordered. Further, proteins degraded by the two different modes belong to distinct functional groups. These findings along with detailed structural analysis of the proteasome, its ubiquitin receptors and deubiquitinating enzymes, suggest that the ubiquitin signal - its formation, recognition, editing, and removal - is far more complex and diverse than originally assumed. Also see the video abstract here: https://youtu.be/QKpN9c6Rg20.
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http://dx.doi.org/10.1002/bies.201700027DOI Listing
June 2017

Stress-induced polyubiquitination of proteasomal ubiquitin receptors targets the proteolytic complex for autophagic degradation.

Autophagy 2017 Apr 25;13(4):759-760. Epub 2017 Jan 25.

a Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology , Haifa , Israel.

Ubiquitin (Ub) is a small protein (8 kDa) found in all eukaryotic cells, which is conjugated covalently to numerous proteins, tagging them for recognition by a downstream effector. One of the best characterized functions of Ub is targeting proteins for either selective degradation by the proteasome, or for bulk degradation by the autophagy-lysosome system. The executing arm of the UPS is the 26S proteasome, a large multicatalytic complex. While much is known about the synthesis and assembly of the proteasome's subunits, the mechanism(s) underlying its removal has remained obscure, similar to that of many other components of the ubiquitin-proteasome system. Our recent study identified autophagy as the degrading mechanism for the mammalian proteasome, mostly under stress conditions. Amino acid starvation induces specific ubiquitination of certain 19S proteasomal subunits that is essential for its binding to SQSTM1/p62, the protein that shuttles the ubiquitinated proteasome to the autophagic machinery. SQSTM1 delivers ubiquitinated substrates for proteasomal degradation via interaction of its PB1 domain with the 19S proteasomal subunit PSMD4/Rpn10, in situations where the proteasome serves as a "predator." In contrast, we found that the UBA domain of SQSTM1 is essential for its interaction with the ubiquitinated proteasome and its delivery to the autophagosome, rendering the proteasome a "prey."
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http://dx.doi.org/10.1080/15548627.2016.1278327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5388213PMC
April 2017

Identification of UBact, a ubiquitin-like protein, along with other homologous components of a conjugation system and the proteasome in different gram-negative bacteria.

Biochem Biophys Res Commun 2017 02 10;483(3):946-950. Epub 2017 Jan 10.

The Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa 31096, Israel. Electronic address:

Systems analogous to the eukaryotic ubiquitin-proteasome system have been previously identified in Archaea, and Actinobacteria (gram-positive), but not in gram-negative bacteria. Here, we report the bioinformatic identification of a novel prokaryotic ubiquitin-like protein, which we name UBact. The phyletic distribution of UBact covers at least five gram-negative bacterial phyla, including Nitrospirae, Armatimonadetes, Verrucomicroba, Nitrospinae, and Planctomycetes. Additionally, it was identified in seven candidate (uncultured) phyla and one Archaeon. UBact might have been overlooked because only few species in the phyla where it is found have been sequenced. In most of the species where we identified UBact, its neighbors in the genome code for proteins homologous to those involved in conjugation and/or degradation of Pup and Pup-tagged substrates. Among them are PafA-, Dop-, Mpa- and proteasome-homologous proteins. This gene association as well as UBact's size and conserved C-terminal G[E/Q] motif, strongly suggest that UBact is used as a conjugatable tag for degradation. With regard to its C-terminus, UBact differs from ubiquitin and most ubiquitin-like proteins (including the mycobacterial Pup) in that it lacks the characteristic C-terminal di-glycine motif, and it usually ends with the sequence R[T/S]G[E/Q]. The phyla that contain UBact are thought to have diverged over 3000 million years ago, indicating that either this ubiquitin-like conjugation system evolved early in evolution or that its occurrence in distant gram-negative phyla is due to multiple instances of horizontal gene transfer.
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http://dx.doi.org/10.1016/j.bbrc.2017.01.037DOI Listing
February 2017

p62- and ubiquitin-dependent stress-induced autophagy of the mammalian 26S proteasome.

Proc Natl Acad Sci U S A 2016 11 17;113(47):E7490-E7499. Epub 2016 Oct 17.

Technion Integrated Cancer Center, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa 3109602, Israel;

The ubiquitin-proteasome system and autophagy are the two main proteolytic systems involved in, among other functions, the maintenance of cell integrity by eliminating misfolded and damaged proteins and organelles. Both systems remove their targets after their conjugation with ubiquitin. An interesting, yet incompletely understood problem relates to the fate of the components of the two systems. Here we provide evidence that amino acid starvation enhances polyubiquitination on specific sites of the proteasome, a modification essential for its targeting to the autophagic machinery. The uptake of the ubiquitinated proteasome is mediated by its interaction with the ubiquitin-associated domain of p62/SQSTM1, a process that also requires interaction with LC3. Importantly, deletion of the PB1 domain of p62, which is important for the targeting of ubiquitinated substrates to the proteasome, has no effect on stress-induced autophagy of this proteolytic machinery, suggesting that the domain of p62 that binds to the proteasome determines the function of p62 in either targeting substrates to the proteasome or targeting the proteasome to autophagy.
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http://dx.doi.org/10.1073/pnas.1615455113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127335PMC
November 2016

The ubiquitin-proteasome system and autophagy: Coordinated and independent activities.

Int J Biochem Cell Biol 2016 10 20;79:403-418. Epub 2016 Jul 20.

Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa, Israel. Electronic address:

The living cell is an ever changing, responsive, and adaptive environment where proteins play key roles in all processes and functions. While the scientific community focused for a long time on the decoding of the information required for protein synthesis, little attention was paid to the mechanisms by which proteins are removed from the cell. We now realize that the timely and proper activity of proteins is regulated to a large extent by their degradation; that cellular coping with different physiological cues and stress conditions depends on different catabolic pathways; and that many pathological states result from improper protein breakdown. There are two major protein degradation systems in all eukaryotic cells-the ubiquitin- proteasome and the autophagy-lysosome. The two systems are highly regulated, and-via degradation of a broad array of proteins-are responsible for maintenance of protein homeostasis and adaptation to environmental changes. Each is comprised of numerous components responsible for its coordinated function, and together they encompass a considerable fraction of the entire genome. In this review, we shall discuss the common and diverse characteristics of the ubiquitin-proteasome system (UPS) and autophagy-their substructure, mechanisms of action, function and concerted regulation under varying pathophysiological conditions.
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http://dx.doi.org/10.1016/j.biocel.2016.07.019DOI Listing
October 2016

The life cycle of the 26S proteasome: from birth, through regulation and function, and onto its death.

Cell Res 2016 08 22;26(8):869-85. Epub 2016 Jul 22.

Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, Israel.

The 26S proteasome is a large, ∼2.5 MDa, multi-catalytic ATP-dependent protease complex that serves as the degrading arm of the ubiquitin system, which is the major pathway for regulated degradation of cytosolic, nuclear and membrane proteins in all eukaryotic organisms.
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http://dx.doi.org/10.1038/cr.2016.86DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4973335PMC
August 2016

Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination.

Proc Natl Acad Sci U S A 2016 08 6;113(32):E4639-47. Epub 2016 Jul 6.

Technion Integrated Cancer Center, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa, 3109602, Israel; Protein Metabolism Medical Research Center, College of Medicine, Seoul National University, Seoul 110-799, South Korea; Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul 110-799, South Korea;

The "canonical" proteasomal degradation signal is a substrate-anchored polyubiquitin chain. However, a handful of proteins were shown to be targeted following monoubiquitination. In this study, we established-in both human and yeast cells-a systematic approach for the identification of monoubiquitination-dependent proteasomal substrates. The cellular wild-type polymerizable ubiquitin was replaced with ubiquitin that cannot form chains. Using proteomic analysis, we screened for substrates that are nevertheless degraded under these conditions compared with those that are stabilized, and therefore require polyubiquitination for their degradation. For randomly sampled representative substrates, we confirmed that their cellular stability is in agreement with our screening prediction. Importantly, the two groups display unique features: monoubiquitinated substrates are smaller than the polyubiquitinated ones, are enriched in specific pathways, and, in humans, are structurally less disordered. We suggest that monoubiquitination-dependent degradation is more widespread than assumed previously, and plays key roles in various cellular processes.
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http://dx.doi.org/10.1073/pnas.1608644113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987823PMC
August 2016

[Short-and long-term effects of cannabinoids on memory, cognition and mental illness].

Harefuah 2013 Dec;152(12):737-41, 751

Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa, Israel.

Marijuana is considered the most commonly used drug in the world, with estimated millions of users. There is dissent in the medical world about the positive and negative effects of marijuana, and recently, a large research effort has been directed to that domain. The main influencing drug ingredient is THC, which acts on the cannabinoid system and binds to the CB1 receptor. The discovery of the receptor led to the finding of an endogenous ligand, anandamide, and another receptor-CB2. The researchers also discovered that cannabinoids have extensive biological activity, and its short and long-term effects may cause cognitive and emotional deficiencies. Findings show that the short-term effects, such as shortterm memory and verbal Learning, are reversible. However, despite the accumulation of evidence about long-term cognitive damage due to cannabis use, it is difficult to find unequivocal results, arising from the existence of many variables such as large differences between cannabis users, frequency of use, dosage and endogenous brain compensation. Apart from cognitive damage, current studies investigate how marijuana affects mental illness: a high correlation between cannabis use and schizophrenia was found and a high risk to undergo a psychotic attack. Furthermore, patients with schizophrenia who used cannabis showed a selective neuro-psychological disruption, and similar cognitive deficiencies and brain morphological changes were found among healthy cannabis users and schizophrenia patients. In contrast to the negative effects of marijuana including addiction, there are the medical uses: reducing pain, anxiety and nausea, increasing appetite and an anti-inflammatory activity. Medicalization of marijuana encourages frequent use, which may elevate depression.
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December 2013
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