Publications by authors named "Miriam Eisenstein"

86 Publications

The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility.

EMBO J 2021 Feb 23:e104683. Epub 2021 Feb 23.

Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel.

Regulatory switches are wide spread in many biological systems. Uniquely among them, the switch of the bacterial flagellar motor is not an on/off switch but rather controls the motor's direction of rotation in response to binding of the signaling protein CheY. Despite its extensive study, the molecular mechanism underlying this switch has remained largely unclear. Here, we resolved the functions of each of the three CheY-binding sites at the switch in E. coli, as well as their different dependencies on phosphorylation and acetylation of CheY. Based on this, we propose that CheY motor switching activity is potentiated upon binding to the first site. Binding of potentiated CheY to the second site produces unstable switching and at the same time enables CheY binding to the third site, an event that stabilizes the switched state. Thereby, this mechanism exemplifies a unique combination of tight motor regulation with inherent switching flexibility.
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http://dx.doi.org/10.15252/embj.2020104683DOI Listing
February 2021

A piggybacking mechanism enables peroxisomal localization of the glyoxylate cycle enzyme Mdh2 in yeast.

J Cell Sci 2020 Dec 17;133(24). Epub 2020 Dec 17.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel

Eukaryotic cells have evolved organelles that allow the compartmentalization and regulation of metabolic processes. Knowledge of molecular mechanisms that allow temporal and spatial organization of enzymes within organelles is therefore crucial for understanding eukaryotic metabolism. Here, we show that the yeast malate dehydrogenase 2 (Mdh2) is dually localized to the cytosol and to peroxisomes and is targeted to peroxisomes via association with Mdh3 and a Pex5-dependent piggybacking mechanism. This dual localization of Mdh2 contributes to our understanding of the glyoxylate cycle and provides a new perspective on compartmentalization of cellular metabolism, which is critical for the perception of metabolic disorders and aging.
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http://dx.doi.org/10.1242/jcs.244376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758625PMC
December 2020

Blind prediction of homo- and hetero-protein complexes: The CASP13-CAPRI experiment.

Authors:
Marc F Lensink Guillaume Brysbaert Nurul Nadzirin Sameer Velankar Raphaël A G Chaleil Tereza Gerguri Paul A Bates Elodie Laine Alessandra Carbone Sergei Grudinin Ren Kong Ran-Ran Liu Xi-Ming Xu Hang Shi Shan Chang Miriam Eisenstein Agnieszka Karczynska Cezary Czaplewski Emilia Lubecka Agnieszka Lipska Paweł Krupa Magdalena Mozolewska Łukasz Golon Sergey Samsonov Adam Liwo Silvia Crivelli Guillaume Pagès Mikhail Karasikov Maria Kadukova Yumeng Yan Sheng-You Huang Mireia Rosell Luis A Rodríguez-Lumbreras Miguel Romero-Durana Lucía Díaz-Bueno Juan Fernandez-Recio Charles Christoffer Genki Terashi Woong-Hee Shin Tunde Aderinwale Sai Raghavendra Maddhuri Venkata Subraman Daisuke Kihara Dima Kozakov Sandor Vajda Kathryn Porter Dzmitry Padhorny Israel Desta Dmitri Beglov Mikhail Ignatov Sergey Kotelnikov Iain H Moal David W Ritchie Isaure Chauvot de Beauchêne Bernard Maigret Marie-Dominique Devignes Maria E Ruiz Echartea Didier Barradas-Bautista Zhen Cao Luigi Cavallo Romina Oliva Yue Cao Yang Shen Minkyung Baek Taeyong Park Hyeonuk Woo Chaok Seok Merav Braitbard Lirane Bitton Dina Scheidman-Duhovny Justas Dapkūnas Kliment Olechnovič Česlovas Venclovas Petras J Kundrotas Saveliy Belkin Devlina Chakravarty Varsha D Badal Ilya A Vakser Thom Vreven Sweta Vangaveti Tyler Borrman Zhiping Weng Johnathan D Guest Ragul Gowthaman Brian G Pierce Xianjin Xu Rui Duan Liming Qiu Jie Hou Benjamin Ryan Merideth Zhiwei Ma Jianlin Cheng Xiaoqin Zou Panagiotis I Koukos Jorge Roel-Touris Francesco Ambrosetti Cunliang Geng Jörg Schaarschmidt Mikael E Trellet Adrien S J Melquiond Li Xue Brian Jiménez-García Charlotte W van Noort Rodrigo V Honorato Alexandre M J J Bonvin Shoshana J Wodak

Proteins 2019 12 25;87(12):1200-1221. Epub 2019 Oct 25.

VIB Structural Biology Research Center, VUB, Brussels, Belgium.

We present the results for CAPRI Round 46, the third joint CASP-CAPRI protein assembly prediction challenge. The Round comprised a total of 20 targets including 14 homo-oligomers and 6 heterocomplexes. Eight of the homo-oligomer targets and one heterodimer comprised proteins that could be readily modeled using templates from the Protein Data Bank, often available for the full assembly. The remaining 11 targets comprised 5 homodimers, 3 heterodimers, and two higher-order assemblies. These were more difficult to model, as their prediction mainly involved "ab-initio" docking of subunit models derived from distantly related templates. A total of ~30 CAPRI groups, including 9 automatic servers, submitted on average ~2000 models per target. About 17 groups participated in the CAPRI scoring rounds, offered for most targets, submitting ~170 models per target. The prediction performance, measured by the fraction of models of acceptable quality or higher submitted across all predictors groups, was very good to excellent for the nine easy targets. Poorer performance was achieved by predictors for the 11 difficult targets, with medium and high quality models submitted for only 3 of these targets. A similar performance "gap" was displayed by scorer groups, highlighting yet again the unmet challenge of modeling the conformational changes of the protein components that occur upon binding or that must be accounted for in template-based modeling. Our analysis also indicates that residues in binding interfaces were less well predicted in this set of targets than in previous Rounds, providing useful insights for directions of future improvements.
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http://dx.doi.org/10.1002/prot.25838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274794PMC
December 2019

A cancer associated somatic mutation in LC3B attenuates its binding to E1-like ATG7 protein and subsequent lipidation.

Autophagy 2019 03 8;15(3):438-452. Epub 2018 Oct 8.

a Departments of Molecular Genetics , Weizmann Institute of Science , Rehovot , Israel.

Macroautophagy/autophagy is a conserved catabolic process that maintains cellular homeostasis under basal growth and stress conditions. In cancer, autophagy can either prevent or promote tumor growth, at early or advanced stages, respectively. We screened public databases to identify autophagy-related somatic mutations in cancer, using a computational approach to identify cancer mutational target sites, employing exact statistics. The top significant hit was a missense mutation (Y113C) in the MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) protein, which occurred at a significant frequency in cancer, and was detected in early stages in primary tumors of patients with known tumor lineage. The mutation reduced the formation of GFP-LC3B puncta and attenuated LC3B lipidation during Torin1-induced autophagy. Its effect on the direct physical interaction of LC3B with each of the 4 proteins that control its maturation or lipidation was tested by applying a protein-fragment complementation assay and co-immunoprecipitation experiments. Interactions with ATG4A and ATG4B proteases were reduced, yet without perturbing the cleavage of mutant LC3B. Most importantly, the mutation significantly reduced the interaction with the E1-like enzyme ATG7, but not the direct interaction with the E2-like enzyme ATG3, suggesting a selective perturbation in the binding of LC3B to some of its partner proteins. Structure analysis and molecular dynamics simulations of LC3B protein and its mutant suggest that the mutation changes the conformation of a loop that has several contact sites with ATG4B and the ATG7 homodimer. We suggest that this loss-of-function mutation, which attenuates autophagy, may promote early stages of cancer development.
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http://dx.doi.org/10.1080/15548627.2018.1525476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6351123PMC
March 2019

Helminths-based bi-functional molecule, tuftsin-phosphorylcholine (TPC), ameliorates an established murine arthritis.

PLoS One 2018 8;13(8):e0200615. Epub 2018 Aug 8.

Zabludowicz Center for Autoimmune Diseases, Sheba Medical Center, affiliated with Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

A novel small molecule named tuftsin-phosphorylcholine (TPC), which is linked to the biological activity of helminths, was constructed. The current study address the effect of TPC treatment in established collagen-induced arthritis (CIA) mice and propose TPC bi-functional activity. TPC treatment was initiated when clinical score was 2 to 4. Arthritis scores in TPC treated mice were lower compared to mice treated with vehicle (P < 0.001). Joint staining showed normal joint structure in TPC-treated mice compared to control groups treated with phosphate buffered saline (PBS), phosphorylcholine, or tuftsin, which exhibited severely inflamed joints. TPC enhanced anti-inflammatory response due to increased IL-10 secretion, and reduced pro-inflammatory cytokine secretion (IL-1-β, IL-6, TNF-αP < 0.001). Furthermore, TPC therapy increased expansion of CD4+CD25+FOXP3+T regulatory cells and IL-10+CD5+CD1d+B regulatory cells. We propose that the immunomodulatory activity of TPC can be a result of a bi-specific activity of TPC: (a) The tuftsin part of the TPC shifts RAW macrophage cells from pro-inflammatory macrophages M1 to anti-inflammatory M2-secreting IL-10 (P < 0.001) through neuropilin-1 and (b) TPC significantly reduce mouse TLR4 expression via NFkB pathway by HEKTM cells (P < 0.02) via the phosphorylcholine site of the molecule. Our results indicate that TPC, significantly ameliorated established CIA by its immunomodulatory activity. These data could lead to a novel self bi-functional small molecule for treating patients with progressive RA.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0200615PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082512PMC
January 2019

Non-canonical activation of DAPK2 by AMPK constitutes a new pathway linking metabolic stress to autophagy.

Nat Commun 2018 05 1;9(1):1759. Epub 2018 May 1.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, 76100, Israel.

Autophagy is an intracellular degradation process essential for adaptation to metabolic stress. DAPK2 is a calmodulin-regulated protein kinase, which has been implicated in autophagy regulation, though the mechanism is unclear. Here, we show that the central metabolic sensor, AMPK, phosphorylates DAPK2 at a critical site in the protein structure, between the catalytic and the calmodulin-binding domains. This phosphorylation activates DAPK2 by functionally mimicking calmodulin binding and mitigating an inhibitory autophosphorylation, providing a novel, alternative mechanism for DAPK2 activation during metabolic stress. In addition, we show that DAPK2 phosphorylates the core autophagic machinery protein, Beclin-1, leading to dissociation of its inhibitor, Bcl-X. Importantly, phosphorylation of DAPK2 by AMPK enhances DAPK2's ability to phosphorylate Beclin-1, and depletion of DAPK2 reduces autophagy in response to AMPK activation. Our study reveals a unique calmodulin-independent mechanism for DAPK2 activation, critical to its function as a novel downstream effector of AMPK in autophagy.
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http://dx.doi.org/10.1038/s41467-018-03907-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931534PMC
May 2018

Conformational states during vinculin unlocking differentially regulate focal adhesion properties.

Sci Rep 2018 02 9;8(1):2693. Epub 2018 Feb 9.

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, 7610001, Israel.

Focal adhesions (FAs) are multi-protein complexes that connect the actin cytoskeleton to the extracellular matrix, via integrin receptors. The growth, stability and adhesive functionality of these structures are tightly regulated by mechanical stress, yet, despite the extensive characterization of the integrin adhesome, the detailed molecular mechanisms underlying FA mechanosensitivity are still unclear. Besides talin, another key candidate for regulating FA-associated mechanosensing, is vinculin, a prominent FA component, which possesses either closed ("auto-inhibited") or open ("active") conformation. A direct experimental demonstration, however, of the conformational transition between the two states is still absent. In this study, we combined multiple structural and biological approaches to probe the transition from the auto-inhibited to the active conformation, and determine its effects on FA structure and dynamics. We further show that the transition from a closed to an open conformation requires two sequential steps that can differentially regulate FA growth and stability.
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http://dx.doi.org/10.1038/s41598-018-21006-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807537PMC
February 2018

A unique type of GSK-3 inhibitor brings new opportunities to the clinic.

Sci Signal 2016 11 15;9(454):ra110. Epub 2016 Nov 15.

Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.

Development of protein kinase inhibitors is a focus of many drug discovery programs. A major problem, however, is the limited specificity of the commonly used adenosine triphosphate-competitive inhibitors and the weak inhibition of the more selective substrate-competitive inhibitors. Glycogen synthase kinase-3 (GSK-3) is a promising drug target for treating neurodegenerative disorders, including Alzheimer's disease (AD), but most GSK-3 inhibitors have not reached the clinic. We describe a new type of GSK-3 inhibitor, L807mts, that acts through a substrate-to-inhibitor conversion mechanism that occurs within the catalytic site of the enzyme. We determined that L807mts was a potent and highly selective GSK-3 inhibitor with reasonable pharmacological and safety properties when tested in rodents. Treatment with L807mts enhanced the clearance of β-amyloid loads, reduced inflammation, enhanced autophagic flux, and improved cognitive and social skills in the 5XFAD AD mouse model. This new modality of GSK-3 inhibition may be therapeutic in patients with AD or other central nervous system disorders associated with dysregulated GSK-3.
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http://dx.doi.org/10.1126/scisignal.aah7102DOI Listing
November 2016

Prediction of homoprotein and heteroprotein complexes by protein docking and template-based modeling: A CASP-CAPRI experiment.

Authors:
Marc F Lensink Sameer Velankar Andriy Kryshtafovych Shen-You Huang Dina Schneidman-Duhovny Andrej Sali Joan Segura Narcis Fernandez-Fuentes Shruthi Viswanath Ron Elber Sergei Grudinin Petr Popov Emilie Neveu Hasup Lee Minkyung Baek Sangwoo Park Lim Heo Gyu Rie Lee Chaok Seok Sanbo Qin Huan-Xiang Zhou David W Ritchie Bernard Maigret Marie-Dominique Devignes Anisah Ghoorah Mieczyslaw Torchala Raphaël A G Chaleil Paul A Bates Efrat Ben-Zeev Miriam Eisenstein Surendra S Negi Zhiping Weng Thom Vreven Brian G Pierce Tyler M Borrman Jinchao Yu Françoise Ochsenbein Raphaël Guerois Anna Vangone João P G L M Rodrigues Gydo van Zundert Mehdi Nellen Li Xue Ezgi Karaca Adrien S J Melquiond Koen Visscher Panagiotis L Kastritis Alexandre M J J Bonvin Xianjin Xu Liming Qiu Chengfei Yan Jilong Li Zhiwei Ma Jianlin Cheng Xiaoqin Zou Yang Shen Lenna X Peterson Hyung-Rae Kim Amit Roy Xusi Han Juan Esquivel-Rodriguez Daisuke Kihara Xiaofeng Yu Neil J Bruce Jonathan C Fuller Rebecca C Wade Ivan Anishchenko Petras J Kundrotas Ilya A Vakser Kenichiro Imai Kazunori Yamada Toshiyuki Oda Tsukasa Nakamura Kentaro Tomii Chiara Pallara Miguel Romero-Durana Brian Jiménez-García Iain H Moal Juan Férnandez-Recio Jong Young Joung Jong Yun Kim Keehyoung Joo Jooyoung Lee Dima Kozakov Sandor Vajda Scott Mottarella David R Hall Dmitri Beglov Artem Mamonov Bing Xia Tanggis Bohnuud Carlos A Del Carpio Eichiro Ichiishi Nicholas Marze Daisuke Kuroda Shourya S Roy Burman Jeffrey J Gray Edrisse Chermak Luigi Cavallo Romina Oliva Andrey Tovchigrechko Shoshana J Wodak

Proteins 2016 09 1;84 Suppl 1:323-48. Epub 2016 Jun 1.

Departments of Biochemistry and Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

We present the results for CAPRI Round 30, the first joint CASP-CAPRI experiment, which brought together experts from the protein structure prediction and protein-protein docking communities. The Round comprised 25 targets from amongst those submitted for the CASP11 prediction experiment of 2014. The targets included mostly homodimers, a few homotetramers, and two heterodimers, and comprised protein chains that could readily be modeled using templates from the Protein Data Bank. On average 24 CAPRI groups and 7 CASP groups submitted docking predictions for each target, and 12 CAPRI groups per target participated in the CAPRI scoring experiment. In total more than 9500 models were assessed against the 3D structures of the corresponding target complexes. Results show that the prediction of homodimer assemblies by homology modeling techniques and docking calculations is quite successful for targets featuring large enough subunit interfaces to represent stable associations. Targets with ambiguous or inaccurate oligomeric state assignments, often featuring crystal contact-sized interfaces, represented a confounding factor. For those, a much poorer prediction performance was achieved, while nonetheless often providing helpful clues on the correct oligomeric state of the protein. The prediction performance was very poor for genuine tetrameric targets, where the inaccuracy of the homology-built subunit models and the smaller pair-wise interfaces severely limited the ability to derive the correct assembly mode. Our analysis also shows that docking procedures tend to perform better than standard homology modeling techniques and that highly accurate models of the protein components are not always required to identify their association modes with acceptable accuracy. Proteins 2016; 84(Suppl 1):323-348. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/prot.25007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5030136PMC
September 2016

CSNAP Is a Stoichiometric Subunit of the COP9 Signalosome.

Cell Rep 2015 Oct 8;13(3):585-598. Epub 2015 Oct 8.

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 7610001, Israel. Electronic address:

The highly conserved COP9 signalosome (CSN) complex is a key regulator of all cullin-RING-ubiquitin ligases (CRLs), the largest family of E3 ubiquitin ligases. Until now, it was accepted that the CSN is composed of eight canonical components. Here, we report the discovery of an additional integral and stoichiometric subunit that had thus far evaded detection, and we named it CSNAP (CSN acidic protein). We show that CSNAP binds CSN3, CSN5, and CSN6, and its incorporation into the CSN complex is mediated through the C-terminal region involving conserved aromatic residues. Moreover, depletion of this small protein leads to reduced proliferation and a flattened and enlarged morphology. Finally, on the basis of sequence and structural properties shared by both CSNAP and DSS1, a component of the related 19S lid proteasome complex, we propose that CSNAP, the ninth CSN subunit, is the missing paralogous subunit of DSS1.
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http://dx.doi.org/10.1016/j.celrep.2015.09.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5724754PMC
October 2015

Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase.

J Biol Chem 2015 Nov 1;290(48):28746-59. Epub 2015 Oct 1.

From the Department of Biological Chemistry.

Phospholemman (FXYD1) is a single-transmembrane protein regulator of Na,K-ATPase, expressed strongly in heart, skeletal muscle, and brain and phosphorylated by protein kinases A and C at Ser-68 and Ser-63, respectively. Binding of FXYD1 reduces Na,K-ATPase activity, and phosphorylation at Ser-68 or Ser-63 relieves the inhibition. Despite the accumulated information on physiological effects, whole cell studies provide only limited information on molecular mechanisms. As a complementary approach, we utilized purified human Na,K-ATPase (α1β1 and α2β1) reconstituted with FXYD1 or mutants S63E, S68E, and S63E,S68E that mimic phosphorylation at Ser-63 and Ser-68. Compared with control α1β1, FXYD1 reduces Vmax and turnover rate and raises K0.5Na. The phosphomimetic mutants reverse these effects and reduce K0.5Na below control K0.5Na. Effects on α2β1 are similar but smaller. Experiments in proteoliposomes reconstituted with α1β1 show analogous effects of FXYD1 on K0.5Na, which are abolished by phosphomimetic mutants and also by increasing mole fractions of DOPS in the proteoliposomes. Stopped-flow experiments using the dye RH421 show that FXYD1 slows the conformational transition E2(2K)ATP → E1(3Na)ATP but does not affect 3NaE1P → E2P3Na. This regulatory effect is explained simply by molecular modeling, which indicates that a cytoplasmic helix (residues 60-70) docks between the αN and αP domains in the E2 conformation, but docking is weaker in E1 (also for phosphomimetic mutants). Taken together with previous work showing that FXYD1 also raises binding affinity for the Na(+)-selective site III, these results provide a rather comprehensive picture of the regulatory mechanism of FXYD1 that complements the physiological studies.
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http://dx.doi.org/10.1074/jbc.M115.687913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4661392PMC
November 2015

Protein recognition by bivalent, 'turn-on' fluorescent molecular probes.

Chem Sci 2015 Oct 12;6(10):5419-5425. Epub 2015 Jun 12.

Department of Organic Chemistry , Weizmann Institute of Science , 76100 Rehovot , Israel . Email:

We show that the conversion of a known intercalating dye (, thiazole orange) into a bivalent protein binder could lead to the realization of a novel class of 'turn-on' fluorescent molecular probes that detect proteins with high affinity, selectivity, and a high signal-to-noise (/) ratio. The feasibility of the approach is demonstrated with monomolecular probes that light-up in the presence of three different proteins: acetylcholinesterase (AChE), glutathione--transferase (GST), or avidin (Av) at low concentrations and with minimal background signal. The way by which such probes can be used to detect individual protein isoforms and be applied in inhibitor screening, cell imaging, and biomarker detection is described.
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http://dx.doi.org/10.1039/c5sc01038aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5502391PMC
October 2015

Lipid Droplets Are Essential for Efficient Clearance of Cytosolic Inclusion Bodies.

Dev Cell 2015 Jun 21;33(5):603-10. Epub 2015 May 21.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel. Electronic address:

Exposing cells to folding stress causes a subset of their proteins to misfold and accumulate in inclusion bodies (IBs). IB formation and clearance are both active processes, but little is known about their mechanism. To shed light on this issue, we performed a screen with over 4,000 fluorescently tagged yeast proteins for co-localization with a model misfolded protein that marks IBs during folding stress. We identified 13 proteins that co-localize to IBs. Remarkably, one of these IB proteins, the uncharacterized and conserved protein Iml2, exhibited strong physical interactions with lipid droplet (LD) proteins. Indeed, we here show that IBs and LDs are spatially and functionally linked. We further demonstrate a mechanism for IB clearance via a sterol-based metabolite emanating from LDs. Our findings therefore uncover a function for Iml2 and LDs in regulating a critical stage of cellular proteostasis.
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http://dx.doi.org/10.1016/j.devcel.2015.04.015DOI Listing
June 2015

Role of a Novel Human Leukocyte Antigen-DQA1*01:02;DRB1*15:01 Mixed Isotype Heterodimer in the Pathogenesis of "Humanized" Multiple Sclerosis-like Disease.

J Biol Chem 2015 Jun 24;290(24):15260-78. Epub 2015 Apr 24.

From the Departments of Immunology and

Gene-wide association and candidate gene studies indicate that the greatest effect on multiple sclerosis (MS) risk is driven by the HLA-DRB1*15:01 allele within the HLA-DR15 haplotype (HLA-DRB1*15:01-DQA1*01:02-DQB1*0602-DRB5*01:01). Nevertheless, linkage disequilibrium makes it difficult to define, without functional studies, whether the functionally relevant effect derives from DRB1*15:01 only, from its neighboring DQA1*01:02-DQB1*06:02 or DRB5*01:01 genes of HLA-DR15 haplotype, or from their combinations or epistatic interactions. Here, we analyzed the impact of the different HLA-DR15 haplotype alleles on disease susceptibility in a new "humanized" model of MS induced in HLA-transgenic (Tg) mice by human oligodendrocyte-specific protein (OSP)/claudin-11 (hOSP), one of the bona fide potential primary target antigens in MS. We show that the hOSP-associated MS-like disease is dominated by the DRB1*15:01 allele not only as the DRA1*01:01;DRB1*15:01 isotypic heterodimer but also, unexpectedly, as a functional DQA1*01:02;DRB1*15:01 mixed isotype heterodimer. The contribution of HLA-DQA1/DRB1 mixed isotype heterodimer to OSP pathogenesis was revealed in (DRB1*1501xDQB1*0602)F1 double-Tg mice immunized with hOSP(142-161) peptide, where the encephalitogenic potential of prevalent DRB1*1501/hOSP(142-161)-reactive Th1/Th17 cells is hindered due to a single amino acid difference in the OSP(142-161) region between humans and mice; this impedes binding of DRB1*1501 to the mouse OSP(142-161) epitope in the mouse CNS while exposing functional binding of mouse OSP(142-161) to DQA1*01:02;DRB1*15:01 mixed isotype heterodimer. This study, which shows for the first time a functional HLA-DQA1/DRB1 mixed isotype heterodimer and its potential association with disease susceptibility, provides a rationale for a potential effect on MS risk from DQA1*01:02 through functional DQA1*01:02;DRB1*15:01 antigen presentation. Furthermore, it highlights a potential contribution to MS risk also from interisotypic combination between products of neighboring HLA-DR15 haplotype alleles, in this case the DQA1/DRB1 combination.
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http://dx.doi.org/10.1074/jbc.M115.641209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4463466PMC
June 2015

Systemic lupus erythematosus-associated neutrophil cytosolic factor 2 mutation affects the structure of NADPH oxidase complex.

J Biol Chem 2015 May 20;290(20):12595-602. Epub 2015 Mar 20.

From the Lupus Genetic Group, Department of Medicine, University of Southern California, Los Angeles, California 90089,

In a case-control association study with 3716 North Americans of Hispanic descent and 4867 North Americans of European descent, we show that the associations of rs17849502 (NCF2 His-389 → Gln) and rs13306575 (NCF2 Arg-395 → Trp) with systemic lupus erythematosus are independent. We have shown that His-389 → Gln disrupts the binding of NCF2 to the ZF domain of VAV1, resulting in decreased NADPH oxidase activity. With respect to Arg-395 → Trp, using protein docking and structure analyses, we provide a model for the involvement of this mutation in the structure and function of the NADPH oxidase complex. This model assigns a central role to Arg-395 in the structure and stability of the quaternary NCF2/NCF4/VAV1/RAC1 NADPH oxidase complex. Arg-395 stabilizes the C-terminal tail of NCF4 and the conformation of NCF2 loop 395-402, which in turn stabilize the evolutionarily conserved interactions of NCF2/NCF4 with the DH domain of VAV1 and RAC1 region 120-137. Our findings are consistent with the high levels of conservation of all of the residues involved in these interactions.
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http://dx.doi.org/10.1074/jbc.M115.639021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4432280PMC
May 2015

Bispecificity for myelin and neuronal self-antigens is a common feature of CD4 T cells in C57BL/6 mice.

J Immunol 2014 Oct 18;193(7):3267-77. Epub 2014 Aug 18.

INSERM, U1043, Toulouse F-31300, France; Centre National de la Recherche Scientifique, U5282, Toulouse F-31300, France; Centre de Physiopathologie Toulouse-Purpan, Université Toulouse 3, Toulouse F-31300, France; Département d'Immunologie, Centre Hospitalier Universitaire Toulouse, Hôpital Purpan, Toulouse F-31300, France

The recognition of multiple ligands by a single TCR is an intrinsic feature of T cell biology, with important consequences for physiological and pathological processes. Polyspecific T cells targeting distinct self-antigens have been identified in healthy individuals as well as in the context of autoimmunity. We have previously shown that the 2D2 TCR recognizes the myelin oligodendrocyte glycoprotein epitope (MOG)35-55 as well as an epitope within the axonal protein neurofilament medium (NF-M15-35) in H-2(b) mice. In this study, we assess whether this cross-reactivity is a common feature of the MOG35-55-specific T cell response. To this end, we analyzed the CD4 T cell response of MOG35-55-immunized C57BL/6 mice for cross-reactivity with NF-M15-35. Using Ag recall responses, we established that an important proportion of MOG35-55-specific CD4 T cells also responded to NF-M15-35 in all mice tested. To study the clonality of this response, we analyzed 22 MOG35-55-specific T cell hybridomas expressing distinct TCR. Seven hybridomas were found to cross-react with NF-M15-35. Using an alanine scan of NF-M18-30 and an in silico predictive model, we dissected the molecular basis of cross-reactivity between MOG35-55 and NF-M15-35. We established that NF-M F24, R26, and V27 proved important TCR contacts. Strikingly, the identified TCR contacts are conserved within MOG38-50. Our data indicate that due to linear sequence homology, part of the MOG35-55-specific T cell repertoire of all C57BL/6 mice also recognizes NF-M15-35, with potential implications for CNS autoimmunity.
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http://dx.doi.org/10.4049/jimmunol.1400523DOI Listing
October 2014

Blind prediction of interfacial water positions in CAPRI.

Proteins 2014 Apr 23;82(4):620-32. Epub 2013 Nov 23.

Interdisciplinary Research Institute USR3078 CNRS, University Lille North of France, Villeneuve d'Ascq, France.

We report the first assessment of blind predictions of water positions at protein-protein interfaces, performed as part of the critical assessment of predicted interactions (CAPRI) community-wide experiment. Groups submitting docking predictions for the complex of the DNase domain of colicin E2 and Im2 immunity protein (CAPRI Target 47), were invited to predict the positions of interfacial water molecules using the method of their choice. The predictions-20 groups submitted a total of 195 models-were assessed by measuring the recall fraction of water-mediated protein contacts. Of the 176 high- or medium-quality docking models-a very good docking performance per se-only 44% had a recall fraction above 0.3, and a mere 6% above 0.5. The actual water positions were in general predicted to an accuracy level no better than 1.5 Å, and even in good models about half of the contacts represented false positives. This notwithstanding, three hotspot interface water positions were quite well predicted, and so was one of the water positions that is believed to stabilize the loop that confers specificity in these complexes. Overall the best interface water predictions was achieved by groups that also produced high-quality docking models, indicating that accurate modelling of the protein portion is a determinant factor. The use of established molecular mechanics force fields, coupled to sampling and optimization procedures also seemed to confer an advantage. Insights gained from this analysis should help improve the prediction of protein-water interactions and their role in stabilizing protein complexes.
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http://dx.doi.org/10.1002/prot.24439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4582081PMC
April 2014

The topology, in model membranes, of the core peptide derived from the T-cell receptor transmembrane domain.

Chembiochem 2013 Sep 24;14(14):1867-75. Epub 2013 Jul 24.

Department of Chemical Physics, Weizmann Institute of Science, 234 Hertzl St, Rehovot, 7632700 (Israel).

The T-cell receptor-CD3 complex (TCR-CD3) serves a critical role in protecting organisms from infectious agents. The TCR is a heterodimer composed of α- and β-chains, which are responsible for antigen recognition. Within the transmembrane domain of the α-subunit, a region has been identified to be crucial for the assembly and function of the TCR. This region, termed core peptide (CP), consists of nine amino acids (GLRILLLKV), two of which are charged (lysine and arginine) and are crucial for the interaction with CD3. Earlier studies have shown that a synthetic peptide corresponding to the CP sequence can suppress the immune response in animal models of T-cell-mediated inflammation, by disrupting proper assembly of the TCR. As a step towards the understanding of the source of the CP activity, we focused on CP in egg phosphatidylcholine/cholesterol (9:1, mol/mol) model membranes and determined its secondary structure, oligomerization state, and orientation with respect to the membrane. To achieve this goal, 15-residue segments of TCRα, containing the CP, were synthesized and spin-labeled at different locations with a nitroxide derivative. Electron spin-echo envelope modulation spectroscopy was used to probe the position and orientation of the peptides within the membrane, and double electron-electron resonance measurements were used to probe its conformation and oligomerization state. We found that the peptide is predominantly helical in a membrane environment and tends to form oligomers (mostly dimers) that are parallel to the membrane plane.
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http://dx.doi.org/10.1002/cbic.201300191DOI Listing
September 2013

Variable internal flexibility characterizes the helical capsid formed by agrobacterium VirE2 protein on single-stranded DNA.

Structure 2013 Jul 13;21(7):1158-67. Epub 2013 Jun 13.

Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel.

Agrobacterium is known for gene transfer to plants. In addition to a linear ssDNA oligonucleotide, Agrobacterium tumefaciens secretes an abundant ssDNA-binding effector, VirE2. In many ways VirE2 adapts the conjugation mechanism to transform the eukaryotic host. The crystal structure of VirE2 shows two compact domains joined by a flexible linker. Bound to ssDNA, VirE2 forms an ordered solenoidal shell, or capsid known as the T-complex. Here, we present a three-dimensional reconstruction of the VirE2-ssDNA complex using cryo-electron microscopy and iterative helical real-space reconstruction. High-resolution refinement was not possible due to inherent heterogeneity in the protein structure. By a combination of computational modeling, chemical modifications, mass spectroscopy, and electron paramagnetic resonance, we found that the N-terminal domain is tightly constrained by both tangential and longitudinal links, while the C terminus is weakly constrained. The quaternary structure is thus rigidly assembled while remaining locally flexible. This flexibility may be important in accommodating substrates without sequence specificity.
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http://dx.doi.org/10.1016/j.str.2013.04.027DOI Listing
July 2013

Water-induced pyroelectricity from nonpolar crystals of amino acids.

Angew Chem Int Ed Engl 2013 Jun 6;52(25):6513-6. Epub 2013 May 6.

Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, 76100, Israel.

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http://dx.doi.org/10.1002/anie.201301836DOI Listing
June 2013

Inhibition of inositol monophosphatase (IMPase) at the calbindin-D28k binding site: molecular and behavioral aspects.

Eur Neuropsychopharmacol 2013 Dec 22;23(12):1806-15. Epub 2013 Apr 22.

Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel; Psychiatry Research Unit, Mental Health Center, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.

Bipolar-disorder (manic-depressive illness) is a severe chronic illness affecting ∼1% of the adult population. It is treated with mood-stabilizers, the prototypic one being lithium-salts (lithium), but it has life threatening side-effects and a significant number of patients fail to respond. The lithium-inhibitable enzyme inositol-monophosphatase (IMPase) is one of the viable targets for lithium's mechanism of action. Calbindin-D28k (calbindin) up-regulates IMPase activity. The IMPase-calbindincomplex was modeled using the program MolFit. The in-silico model indicated that the 55-66 amino-acid segment of IMPase anchors calbindin via Lys59 and Lys61 with a glutamate in between (Lys-Glu-Lys motif) and that the motif interacts with residues Asp24 and Asp26 of calbindin. We found that differently from wildtype calbindin, IMPase was not activated by mutated calbindin in which Asp24 and Asp26 were replaced by alanine. Calbindin's effect was significantly reduced by a linear peptide with the sequence of amino acids 58-63 of IMPase (peptide 1) and by six amino-acid linear peptides including at least part of the Lys-Glu-Lys motif. The three amino-acid peptide Lys-Glu-Lys or five amino-acid linear peptides containing this motif were ineffective. Mice administered peptide 1 intracerebroventricularly exhibited a significant anti-depressant-like reduced immobility in the forced-swim test. Based on the sequence of peptide 1, and to potentially increase the peptide's stability, cyclic and linear pre-cyclic analog peptides were synthesized. One cyclic peptide and one linear pre-cyclic analog peptide inhibited calbindin-activated brain IMPase activity in-vitro. Our findings may lead to the development of molecules capable of inhibiting IMPase activity at an alternative site than that of lithium.
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http://dx.doi.org/10.1016/j.euroneuro.2013.02.004DOI Listing
December 2013

GSK-3 inhibition: achieving moderate efficacy with high selectivity.

Biochim Biophys Acta 2013 Jul 29;1834(7):1410-4. Epub 2013 Jan 29.

Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

Inhibiting glycogen synthase kinase-3 (GSK-3) activity has become an attractive approach for treatment of neurodegenerative and psychiatric disorders. Diverse GSK-3 inhibitors have been reported and used in cellular and in vivo models. A major challenge, however, is achieving selectivity. In addition, it is increasingly recognized that a moderate inhibition of a cellular target, particularly for long-term treatment, provides more favorable outcome than complete inhibition. Substrate competitive inhibitors can fulfill the requirement for selectivity and allow fine tuning of the degree of inhibition. Here we describe the therapeutic potential of GSK-3 inhibitors and highlight our progress in the development of substrate competitive inhibitors. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).
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http://dx.doi.org/10.1016/j.bbapap.2013.01.016DOI Listing
July 2013

Topology of the trans-membrane peptide WALP23 in model membranes under negative mismatch conditions.

J Phys Chem B 2013 Feb 14;117(8):2280-93. Epub 2013 Feb 14.

Department of Chemical Physics, Weizmann Institute of Science, Rehovot, Israel 76100.

The organization and orientation of membrane-inserted helices is important for better understanding the mode of action of membrane-active peptides and of protein-membrane interactions. Here we report on the application of ESEEM (electron spin-echo envelope modulation) and DEER (double electron-electron resonance) techniques to probe the orientation and oligomeric state of an α-helical trans-membrane model peptide, WALP23, under conditions of negative mismatch between the hydrophobic cores of the model membrane and the peptide. Using ESEEM, we measured weak dipolar interactions between spin-labeled WALP23 and (2)H nuclei of either the solvent (D2O) or of lipids specifically deuterated at the choline group. The ESEEM data obtained from the deuterated lipids were fitted using a model that provided the spin label average distance from a layer of (2)H nuclei in the hydrophilic region of the membrane and the density of the (2)H nuclei in the layer. DEER was used to probe oligomerization through the dipolar interaction between two spin-labels on different peptides. We observed that the center of WALP23 does not coincide with the bilayer midplane and its N-terminus is more buried than the C-terminus. In addition, the ESEEM data fitting yielded a (2)H layer density that was much lower than expected. The DEER experiments revealed the presence of oligomers, the presence of which was attributable to the negative mismatch and the electrostatic dipole of the peptide. A discussion of a possible arrangement of the individual helices in the oligomers that is consistent with the ESEEM and DEER data is presented.
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http://dx.doi.org/10.1021/jp310056hDOI Listing
February 2013

Interactions of subunit CCT3 in the yeast chaperonin CCT/TRiC with Q/N-rich proteins revealed by high-throughput microscopy analysis.

Proc Natl Acad Sci U S A 2012 Nov 29;109(46):18833-8. Epub 2012 Oct 29.

Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.

The eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) is an ATP-fueled machine that assists protein folding. It consists of two back-to-back stacked rings formed by eight different subunits that are arranged in a fixed permutation. The different subunits of CCT are believed to possess unique substrate binding specificities that are still mostly unknown. Here, we used high-throughput microscopy analysis of yeast cells to determine changes in protein levels and localization as a result of a Glu to Asp mutation in the ATP binding site of subunits 3 (CCT3) or 6 (CCT6). The mutation in subunit CCT3 was found to induce cytoplasmic foci termed P-bodies where mRNAs, which are not translated, accumulate and can be degraded. Analysis of the changes in protein levels and structural modeling indicate that P-body formation in cells with the mutation in CCT3 is linked to the specific interaction of this subunit with Gln/Asn-rich segments that are enriched in many P-body proteins. An in vitro gel-shift analysis was used to show that the mutation in subunit CCT3 interferes with the ability of CCT to bind a Gln/Asn-rich protein aggregate. More generally, the strategy used in this work can be used to unravel the substrate specificities of other chaperone systems.
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http://dx.doi.org/10.1073/pnas.1209277109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503220PMC
November 2012

Anti-GalNAcβ: a novel anti-glycan autoantibody associated with pregnancy loss in women with antiphospholipid syndrome and in a mouse experimental model.

J Autoimmun 2012 Dec 1;39(4):420-7. Epub 2012 Aug 1.

Zabludowicz Center for Autoimmune Diseases, Sheba Medical Center, Tel-Hashomer, Affiliated to Sackler Faculty of Medicine Tel-Aviv University, Israel.

Objectives: We evaluated the presence of anti-glycan antibodies (aGA) in patients with antiphospholipid syndrome (APS), and associations between aGA and clinical features of the disease.

Methods: Sera from APS patients and healthy controls were analyzed for aGA levels by ELISA. Analysis of the association of specific aGA with clinical manifestations of APS was performed. Selected aGA were affinity-purified and injected intravenously into naive mice which were tested for fetal loss. Matrigel invasion assay was performed for detection of choriocarcinoma cells (JAR) invasion and proliferation in the presence of selected aGA. Culture fluid of JAR invasion assays was analyzed for the presence of MMP2 and MMP9.

Results: High levels of several aGA were found in APS sera, of which anti-GalNAc-β was significantly associated with recurrent pregnancy loss. Naive mice infused intravenously with anti-GalNAc-β developed increased fetal loss. Anti-GalNAc-β significantly inhibited the in-vitro percentage of JAR invasiveness and the secretion of MMP2 and MMP9 by human JAR cells.

Conclusions: APS sera contain significant levels of aGA directed against several glycans. Anti-GalNAc-β Ab is specifically associated with recurrent pregnancy loss both in human patients and experimental mouse model. The pathogenic effects of anti-GalNAc-β include inhibition of JAR cells invasiveness accompanied by decreased MMP2 and MMP9 secretion.
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http://dx.doi.org/10.1016/j.jaut.2012.07.002DOI Listing
December 2012

A unique spatial arrangement of the snRNPs within the native spliceosome emerges from in silico studies.

Structure 2012 Jun 10;20(6):1097-106. Epub 2012 May 10.

Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.

The spliceosome is a mega-Dalton ribonucleoprotein (RNP) assembly that processes primary RNA transcripts, producing functional mRNA. The electron microscopy structures of the native spliceosome and of several spliceosomal subcomplexes are available; however, the spatial arrangement of the latter within the native spliceosome is not known. We designed a computational procedure to efficiently fit thousands of conformers into the spliceosome envelope. Despite the low resolution limitations, we obtained only one model that complies with the available biochemical data. Our model localizes the five small nuclear RNPs (snRNPs) mostly within the large subunit of the native spliceosome, requiring only minor conformation changes. The remaining free volume presumably accommodates additional spliceosomal components. The constituents of the active core of the spliceosome are juxtaposed, forming a continuous surface deep within the large spliceosomal cavity, which provides a sheltered environment for the splicing reaction.
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http://dx.doi.org/10.1016/j.str.2012.03.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3372696PMC
June 2012

Lupus-associated causal mutation in neutrophil cytosolic factor 2 (NCF2) brings unique insights to the structure and function of NADPH oxidase.

Proc Natl Acad Sci U S A 2012 Jan 27;109(2):E59-67. Epub 2011 Dec 27.

The Lupus Genetic Group, Department of Medicine, University of Southern California, Los Angeles, CA 90089, USA.

Systemic lupus erythematosus (SLE), the prototypic systemic autoimmune disease, is a debilitating multisystem autoimmune disorder characterized by chronic inflammation and extensive immune dysregulation in multiple organ systems, resulting in significant morbidity and mortality. Here, we present a multidisciplinary approach resulting in the identification of neutrophil cytosolic factor 2 (NCF2) as an important risk factor for SLE and the detailed characterization of its causal variant. We show that NCF2 is strongly associated with increased SLE risk in two independent populations: childhood-onset SLE and adult-onset SLE. The association between NCF2 and SLE can be attributed to a single nonsynonymous coding mutation in exon 12, the effect of which is the substitution of histidine-389 with glutamine (H389Q) in the PB1 domain of the NCF2 protein, with glutamine being the risk allele. Computational modeling suggests that the NCF2 H389Q mutation reduces the binding efficiency of NCF2 with the guanine nucleotide exchange factor Vav1. The model predicts that NCF2/H389 residue interacts with Vav1 residues E509, N510, E556, and G559 in the ZF domain of Vav1. Furthermore, replacing H389 with Q results in 1.5 kcal/mol weaker binding. To examine the effect of the NCF2 H389Q mutation on NADPH oxidase function, site-specific mutations at the 389 position in NCF2 were tested. Results show that an H389Q mutation causes a twofold decrease in reactive oxygen species production induced by the activation of the Vav-dependent Fcγ receptor-elicited NADPH oxidase activity. Our study completes the chain of evidence from genetic association to specific molecular function.
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http://dx.doi.org/10.1073/pnas.1113251108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258621PMC
January 2012

Antibodies targeting the catalytic zinc complex of activated matrix metalloproteinases show therapeutic potential.

Nat Med 2011 Dec 25;18(1):143-7. Epub 2011 Dec 25.

Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.

Endogenous tissue inhibitors of metalloproteinases (TIMPs) have key roles in regulating physiological and pathological cellular processes. Imitating the inhibitory molecular mechanisms of TIMPs while increasing selectivity has been a challenging but desired approach for antibody-based therapy. TIMPs use hybrid protein-protein interactions to form an energetic bond with the catalytic metal ion, as well as with enzyme surface residues. We used an innovative immunization strategy that exploits aspects of molecular mimicry to produce inhibitory antibodies that show TIMP-like binding mechanisms toward the activated forms of gelatinases (matrix metalloproteinases 2 and 9). Specifically, we immunized mice with a synthetic molecule that mimics the conserved structure of the metalloenzyme catalytic zinc-histidine complex residing within the enzyme active site. This immunization procedure yielded selective function-blocking monoclonal antibodies directed against the catalytic zinc-protein complex and enzyme surface conformational epitopes of endogenous gelatinases. The therapeutic potential of these antibodies has been demonstrated with relevant mouse models of inflammatory bowel disease. Here we propose a general experimental strategy for generating inhibitory antibodies that effectively target the in vivo activity of dysregulated metalloproteinases by mimicking the mechanism employed by TIMPs.
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http://dx.doi.org/10.1038/nm.2582DOI Listing
December 2011

The autophagy protein Atg12 associates with antiapoptotic Bcl-2 family members to promote mitochondrial apoptosis.

Mol Cell 2011 Dec;44(5):698-709

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

Autophagy and apoptosis constitute important determinants of cell fate and engage in a complex interplay in both physiological and pathological settings. The molecular basis of this crosstalk is poorly understood and relies, in part, on "dual-function" proteins that operate in both processes. Here, we identify the essential autophagy protein Atg12 as a positive mediator of mitochondrial apoptosis and show that Atg12 directly regulates the apoptotic pathway by binding and inactivating prosurvival Bcl-2 family members, including Bcl-2 and Mcl-1. The binding occurs independently of Atg5 or Atg3 and requires a unique BH3-like motif in Atg12, characterized by interaction studies and computational docking. In apoptotic cells, knockdown of Atg12 inhibited Bax activation and cytochrome c release, while ectopic expression of Atg12 antagonized the antiapoptotic activity of Mcl-1. The interaction between Atg12 and Bcl-2 family members may thus constitute an important point of convergence between autophagy and apoptosis in response to specific signals.
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http://dx.doi.org/10.1016/j.molcel.2011.10.014DOI Listing
December 2011

Removing allosteric feedback inhibition of tomato 4-coumarate:CoA ligase by directed evolution.

Plant J 2012 Jan 17;69(1):57-69. Epub 2011 Oct 17.

Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food, and Environment, Hebrew University of Jerusalem, Rehovot 76100, Israel.

Plant secondary metabolites, such as those derived from the phenylpropanoid pathway, have a beneficial effect on human health. Manipulation of metabolic flux in the phenylpropanoid pathway is important for achieving enhanced production of compounds such as anthocyanins, flavonoids and isoflavonoids. Here, we describe the development of a high-throughput molecular evolution approach that can be used for catalytic improvement of at least four key phenylpropanoid pathway enzymes, within the context of the metabolic pathway. This method uses yeast cells that express plant phenylpropanoid pathway enzymes, leading to formation of a colored intermediate that can be used as a readout in high-throughput screening. Here we report the identification of improved tomato peel 4-coumarate:CoA ligase variants using this approach. We found that the wild-type enzyme is strongly allosterically inhibited by naringenin, a downstream product of the pathway. Surprisingly, at least two of the improved variants are completely insensitive to feedback inhibition by naringenin. We suggest that this inhibition is exerted through a unique and previously unrecognized allosteric domain.
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http://dx.doi.org/10.1111/j.1365-313X.2011.04770.xDOI Listing
January 2012