129 results on '"Ashraf Brik"'
Search Results
2. Highly Efficient Cyclization Approach of Propargylated Peptides via Gold(I)-Mediated Sequential C–N, C–O, and C–C Bond Formation
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Ganga B. Vamisetti, Ashraf Brik, Emad Eid, Rajeshwer Vanjari, and Shaswati Mandal
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Chemistry ,010405 organic chemistry ,Stereochemistry ,General Chemical Engineering ,General Chemistry ,Bond formation ,010402 general chemistry ,QD1-999 ,01 natural sciences ,Research Article ,0104 chemical sciences - Abstract
A rapid and efficient cyclization of unprotected N-propargylated peptides using the Au(I) organometallic complex is reported. The method relies on the activation of the propargyl functionality using gold(I) to produce a new linkage with the N-terminus amine at the cyclization site. The presented method features a fast reaction rate (within 20 min), mild conditions, chemoselectivity, wide sequence scope, and high yields (up to 87%). The strategy was successfully tested on a wide variety of 30 unprotected peptides having various sequences and lengths, thus providing access to structurally distinct cyclic peptides. The practical usefulness of this method was demonstrated in producing peptides that bind efficiently to Lys48-linked di- and tetra-ubiquitin chains. The new cyclic peptide modulators exhibited high permeability to living cells and promoted apoptosis via binding with the endogenous Lys48-linked ubiquitin chains., A rapid and efficient cyclization of unprotected N-propargylated peptides using (JohnPhos)Au(ACN)SbF6 complex is reported and features a fast reaction rate (within 20 min) and chemoselectivity.
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- 2021
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3. Chemical Synthesis of Ubiquitinated Proteins for Biochemical Studies
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Ashraf Brik, Gandhesiri Satish, and Ganga B. Vamisetti
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Ubiquitinated Proteins ,Ubiquitin ,biology ,Biochemistry ,Chemistry ,Posttranslational modification ,biology.protein ,Chemical synthesis - Published
- 2021
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4. The Development of a Fluorescence‐Based Competitive Assay Enabled the Discovery of Dimeric Cyclic Peptide Modulators of Ubiquitin Chains
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Ashraf Brik, Roman Meledin, Ganga B. Vamisetti, Mickal Nawatha, and Hiroaki Suga
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High-throughput screening ,Peptides | Hot Paper ,Peptide ,010402 general chemistry ,Ligands ,01 natural sciences ,high-throughput screening ,Peptides, Cyclic ,Catalysis ,Fluorescence ,Ubiquitin ,Drug Development ,In vivo ,Protein biosynthesis ,Humans ,chemistry.chemical_classification ,fluorescence-based assay ,biology ,Cell Death ,Dose-Response Relationship, Drug ,010405 organic chemistry ,Communication ,dimeric peptides ,cyclic peptides ,General Medicine ,General Chemistry ,Combinatorial chemistry ,Cyclic peptide ,Communications ,0104 chemical sciences ,High-Throughput Screening Assays ,chemistry ,biology.protein ,peptide engineering ,Conjugate ,HeLa Cells - Abstract
Development of modulators targeting specific interactions of ubiquitin‐based conjugates with their partners is a formidable task since it requires a suitable screening assay and homogeneous ubiquitin conjugates. We developed a novel high‐throughput strategy for screening ligands for Lys48‐linked tetraubiquitin chain in a relatively simple, fast, and affordable manner. This approach combined with a state‐of‐the‐art toolbox of chemical protein synthesis and a specially optimized Cys deprotection protocol enabled us to design highly potent, Lys48‐linked tetraubiquitin chain selective “next generation” dimeric peptide modulators. The dimeric peptide exhibited cancer cell permeability and induced cell death with higher efficiency compared to its monocyclic analogue. These features make our dimeric peptide a promising candidate for further studies using in vivo models. Our assay can be adopted for other various ubiquitin chains in their free or anchored forms as well as conjugates for Ub‐like modifiers., A fluorescence‐based competitive assay was developed, which enabled the high‐throughput screening of various cyclic peptides targeting the Lys48‐linked tetraubiquitin chain. This assay combined with chemical tools enabled the design of a potent dimeric cyclic peptide, which induced cell death with higher efficiency compared to its monocyclic analogue.
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- 2021
5. General synthetic strategy for regioselective ultrafast formation of disulfide bonds in peptides and proteins
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Ashraf Brik, Shay Laps, Hao Sun, Fatima Atamleh, and Guy Kamnesky
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Spectrometry, Mass, Electrospray Ionization ,Photochemistry ,Science ,General Physics and Astronomy ,Synthetic chemistry methodology ,010402 general chemistry ,01 natural sciences ,Article ,General Biochemistry, Genetics and Molecular Biology ,medicine ,Chemical synthesis ,Disulfides ,Conotoxin ,Chromatography, High Pressure Liquid ,Multidisciplinary ,010405 organic chemistry ,Drug discovery ,Chemistry ,Disulfide bond ,Proteins ,Regioselectivity ,Stereoisomerism ,General Chemistry ,Plectasin ,Small molecule ,Combinatorial chemistry ,0104 chemical sciences ,Peptides ,medicine.drug ,Cysteine - Abstract
Despite six decades of efforts to synthesize peptides and proteins bearing multiple disulfide bonds, this synthetic challenge remains an unsolved problem in most targets (e.g., knotted mini proteins). Here we show a de novo general synthetic strategy for the ultrafast, high-yielding formation of two and three disulfide bonds in peptides and proteins. We develop an approach based on the combination of a small molecule, ultraviolet-light, and palladium for chemo- and regio-selective activation of cysteine, which enables the one-pot formation of multiple disulfide bonds in various peptides and proteins. We prepare bioactive targets of high therapeutic potential, including conotoxin, RANTES, EETI-II, and plectasin peptides and the linaclotide drug. We anticipate that this strategy will be a game-changer in preparing millions of inaccessible targets for drug discovery., Synthesis of peptides and proteins containing multiple disulfide bonds is challenging, limiting the elucidation of their biological functions. Here, the authors report a general synthetic strategy for fast formation of two and three disulfide bonds in peptides and proteins, and apply it to prepare several therapeutically important peptides.
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- 2021
6. Gold(I)-Mediated Decaging or Cleavage of Propargylated Peptide Bond in Aqueous Conditions for Protein Synthesis and Manipulation
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Ashraf Brik, Muhammad Jbara, and Emad Eid
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Aqueous solution ,Molecular Structure ,NEDD8 Protein ,Chemistry ,Stereochemistry ,Hydrogen bond ,Water ,Hydrogen Bonding ,Dipeptides ,General Chemistry ,010402 general chemistry ,Cleavage (embryo) ,01 natural sciences ,Biochemistry ,Catalysis ,0104 chemical sciences ,Colloid and Surface Chemistry ,Protein biosynthesis ,Humans ,Peptide bond ,Gold ,sense organs ,skin and connective tissue diseases - Abstract
Chemists have been interested in the N-alkylation of a peptide bond because such a modification alters the conformation of the amide bond, interferes with hydrogen bond formation, and changes other properties of the peptide (e.g., solubility). This modification also opens the door for attaching functional groups for various applications. Nonetheless, the irreversibility of some of these modifications and the harsh conditions required for their removal currently limits the wide utility of this approach. Herein, we report applying a propargyl group for peptide bond modification at diverse junctions, which can be removed under mild and aqueous conditions via treatment with gold(I). Considering the straightforward conditions for both the installation and removal of this group, the propargyl group provides access to the benefits of backbone N-alkylation, while preserving the ability for on-demand depropargylation and full recovery of the native amide bond. This reversible modification was found to improve solid-phase peptide synthesis as demonstrated in the chemical synthesis of NEDD8 protein, without the use of special dipeptide analogues. Also, the reported approach was found to be useful in decaging a broad range of propargyl-based protecting groups used in chemical protein synthesis. Remarkably, reversing the order of the two residues in the propargylation site resulted in rapid amide bond cleavage, which extends the applicability of this approach beyond a removable backbone modification to a cleavable linker. The easy attach/detach of this functionality was also examined in loading and releasing of biotinylated peptides from streptavidin beads.
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- 2020
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7. The 20S as a stand-alone proteasome in cells can degrade the ubiquitin tag
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Cong Xu, Indrajit Sahu, Andrey Rozenberg, Roni Morag, Sumeet K. Singh, Yao Cong, Manisha Priyadarsini Sahoo, Sachitanand M. Mali, Michael H. Glickman, Zhanyu Ding, Sharleen Day, Prasad Sulkshane, Ashraf Brik, Oded Kleifeld, and Yifan Wang
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Proteasome Endopeptidase Complex ,Ubiquitylation ,Cell Survival ,Protein Conformation ,Science ,Proteolysis ,medicine.medical_treatment ,General Physics and Astronomy ,Peptide ,General Biochemistry, Genetics and Molecular Biology ,Article ,Substrate Specificity ,Ubiquitylated proteins ,Ubiquitin ,Cryoelectron microscopy ,medicine ,Humans ,30S ,chemistry.chemical_classification ,Heart Failure ,Multidisciplinary ,Protease ,biology ,medicine.diagnostic_test ,Proteasome ,Chemistry ,Ubiquitination ,Substrate (chemistry) ,General Chemistry ,Ubiquitinated Proteins ,Cell Hypoxia ,Cell biology ,biology.protein ,Peptides ,Intracellular - Abstract
The proteasome, the primary protease for ubiquitin-dependent proteolysis in eukaryotes, is usually found as a mixture of 30S, 26S, and 20S complexes. These complexes have common catalytic sites, which makes it challenging to determine their distinctive roles in intracellular proteolysis. Here, we chemically synthesize a panel of homogenous ubiquitinated proteins, and use them to compare 20S and 26S proteasomes with respect to substrate selection and peptide-product generation. We show that 20S proteasomes can degrade the ubiquitin tag along with the conjugated substrate. Ubiquitin remnants on branched peptide products identified by LC-MS/MS, and flexibility in the 20S gate observed by cryo-EM, reflect the ability of the 20S proteasome to proteolyze an isopeptide-linked ubiquitin-conjugate. Peptidomics identifies proteasome-trapped ubiquitin-derived peptides and peptides of potential 20S substrates in Hi20S cells, hypoxic cells, and human failing-heart. Moreover, elevated levels of 20S proteasomes appear to contribute to cell survival under stress associated with damaged proteins., The 20S particle is part of the 26S proteasome, but also exists as a free complex. Here, the authors outline signature activities of the 20S and combine chemical, structural, functional and proteomic assays to show that the 20S can degrade ubiquitin tags along with conjugated substrates.
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- 2021
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8. Synthesis and Evaluation of Ubiquitin-Dioxetane Conjugate as a Chemiluminescent Probe for Monitoring Deubiquitinase Activity
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satish gandhesiri, Ashraf Brik, Sara Gutkin, and Doron Shabat
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Biomedical Engineering ,Pharmaceutical Science ,Bioengineering ,Deubiquitinating enzyme ,law.invention ,chemistry.chemical_compound ,Deubiquitinase activity ,Ubiquitin ,law ,Endopeptidases ,Humans ,Chemiluminescence ,Pharmacology ,chemistry.chemical_classification ,biology ,Communication ,Organic Chemistry ,Combinatorial chemistry ,Dioxetane ,Enzyme ,chemistry ,biology.protein ,Protein Processing, Post-Translational ,Function (biology) ,Biotechnology ,Conjugate - Abstract
The removal of ubiquitin (Ub) from a modified protein or Ub chain is a process that occurs regularly by the ubiquitin-proteasome system. This process is known to be mediated by various deubiquitinating enzymes (DUBs) in order to control the protein's half-life and its expression levels among many other signaling processes. Since the function of DUBs is also involved in numerous human diseases, such as cancer, there is an obvious need for an effective diagnostic probe that can monitor the activity of these enzymes. We have developed the first chemiluminescence probe for detection of DUBs activity. The probe was prepared by conjugation of the chemically synthesized C-terminally activated Ub(1-75) with a Gly-enolether precursor. Subsequent oxidation, under aqueous conditions, of the enolether conjuagate with singlet-oxygen furnished the dioxetane probe Ub-CL. This synthesis provides the first example of a dioxetane-luminophore protein conjugate. The probe's ability to detect deubiquitinating activity was successfully validated with three different DUBs. In order to demonstrate the advantage of our new probe, comparison measurements for detection of DUB UCH-L3 activity were performed between the chemiluminescent probe Ub-CL and the well-known Ub-AMC probe. The obtained data showed significantly higher S/N, for probe Ub-CL (>93-fold) in comparison to that observed for Ub-AMC (1.5-fold). We anticipate that the successful design and synthesis of the turn-ON protein-dioxetane conjugate probe, demonstrated in this work, will provide the insight and motivation for preparation of other relevant protein-dioxetane conjugates.
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- 2021
9. Palladium‐Mediated Cleavage of Proteins with Thiazolidine‐Modified Backbone in Live Cells
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Ashraf Brik, Ganga B. Vamisetti, Guy Mann, Roman Meledin, and Gandhesiri Satish
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010405 organic chemistry ,Thiazolidine ,Proteins ,chemistry.chemical_element ,Total synthesis ,General Chemistry ,General Medicine ,010402 general chemistry ,Cell delivery ,Cleavage (embryo) ,01 natural sciences ,Semisynthesis ,Catalysis ,In vitro ,0104 chemical sciences ,chemistry.chemical_compound ,chemistry ,Biochemistry ,Protein biosynthesis ,Thiazolidines ,Palladium - Abstract
Chemical protein synthesis and biorthogonal modification chemistries allow production of unique proteins for a range of biological studies. Bond-forming reactions for site-selective protein labeling are commonly used in these endeavors. Selective bond-cleavage reactions, however, are much less explored and still pose a great challenge. In addition, most of studies with modified proteins prepared by either total synthesis or semisynthesis have been applied mainly for in vitro experiments with very limited extension to live cells. Reported here is an approach for studying uniquely modified proteins containing a traceless cell delivery unit and palladium-based cleavable element for chemical activation, and monitoring the effect of these proteins in live cells. This approach is demonstrated for the synthesis of a caged ubiquitin-aldehyde, which was decaged for the inhibition of deubiquitinases in live cells.
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- 2019
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10. De novo macrocyclic peptides that specifically modulate Lys48-linked ubiquitin chains
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Aaron Ciechanover, Yichao Huang, Sachitanand M. Mali, Steven M. Bonn, Ganga B. Vamisetti, Ashraf Brik, Joseph M. Rogers, Hiroaki Suga, Beatrice Bercovich, Betsegaw Lemma, Ido Livneh, Mickal Nawatha, Hao Sun, and David Fushman
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Proteasome Endopeptidase Complex ,Programmed cell death ,Cell Survival ,General Chemical Engineering ,Antineoplastic Agents ,Apoptosis ,Plasma protein binding ,Cysteine Proteinase Inhibitors ,010402 general chemistry ,Peptides, Cyclic ,01 natural sciences ,Chemical synthesis ,Article ,Deubiquitinating enzyme ,Small Molecule Libraries ,Ubiquitin ,Humans ,Ubiquitins ,chemistry.chemical_classification ,Deubiquitinating Enzymes ,Molecular Structure ,biology ,010405 organic chemistry ,Chemistry ,Drug discovery ,Lysine ,General Chemistry ,In vitro ,Cyclic peptide ,0104 chemical sciences ,Cysteine Endopeptidases ,Biochemistry ,biology.protein ,Proteasome Inhibitors ,HeLa Cells ,Protein Binding - Abstract
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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- 2019
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11. Examining Several Strategies for the Chemical Synthesis of Phosphorylated Histone H3 Reveals the Effectiveness of the Convergent Approach
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Muhammad Jbara, Ashraf Brik, and Suman Kumar Maity
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Histone H3 ,chemistry ,Biochemistry ,Organic Chemistry ,Convergent synthesis ,chemistry.chemical_element ,Phosphorylation ,Physical and Theoretical Chemistry ,Chemical synthesis ,Palladium - Published
- 2019
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12. Diverse fate of ubiquitin chain moieties: The proximal is degraded with the target, and the distal protects the proximal from removal and recycles
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Ashraf Brik, Sumeet K. Singh, Aaron Ciechanover, Hao Sun, Roman Meledin, Sachitanand M. Mali, Yelena Kravtsova-Ivantsiv, Beatrice Bercovich, and Yong Tae Kwon
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0301 basic medicine ,Multidisciplinary ,biology ,Chemistry ,Protein degradation ,010402 general chemistry ,01 natural sciences ,0104 chemical sciences ,Cell biology ,Deubiquitinating enzyme ,03 medical and health sciences ,030104 developmental biology ,Ubiquitin ,Proteasome ,Commentaries ,biology.protein ,Moiety ,Globin ,Receptor ,Deubiquitination - Abstract
One of the enigmas in the ubiquitin (Ub) field is the requirement for a poly-Ub chain as a proteasomal targeting signal. The canonical chain appears to be longer than the distance between the two Ub-binding proteasomal receptors. Furthermore, genetic manipulation has shown that one receptor subunit is sufficient, which suggests that a single Ub can serve as a degradation signal. To shed light on this mystery, we chemically synthesized tetra-Ub, di-Ub (K48-based), and mono-Ub adducts of HA-α-globin, where the distal or proximal Ub moieties were tagged differentially with either Myc or Flag. When incubated in a crude cell extract, the distal Ub moiety in the tetra-Ub adduct was mostly removed by deubiquitinating enzymes (DUBs) and reconjugated to other substrates in the extract. In contrast, the proximal moiety was most likely degraded with the substrate. The efficacy of degradation was proportionate to the chain length; while tetra-Ub globin was an efficient substrate, with mono-Ub globin, we observed rapid removal of the Ub moiety with almost no degradation of the free globin. Taken together, these findings suggest that the proximal moieties are necessary for securing the association of the substrate with the proteasome along the proteolytic process, whereas the distal moieties are important in protecting the proximal moieties from premature deubiquitination. Interestingly, when the same experiment was carried out using purified 26S proteasome, mono- and tetra-Ub globin were similarly degraded, highlighting the roles of the entire repertoire of cellular DUBs in regulating the degradation of proteasomal substrates.
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- 2019
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13. Chemical Biology: Powerful Synergy Between Two Cultures
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Ashraf Brik and Ehud Keinan
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Chemistry ,Chemical biology ,General Chemistry ,Computational biology - Published
- 2019
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14. Insight on the Order of Regioselective Ultrafast Formation of Disulfide Bonds in (Antimicrobial) Peptides and Miniproteins
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Guy Kamnesky, Fatima Atamleh, Shaked Uzi, Michael M. Meijler, Shay Laps, and Ashraf Brik
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chemistry.chemical_classification ,Bioactive molecules ,Antimicrobial peptides ,Disulfide bond ,Regioselectivity ,Peptide ,General Medicine ,General Chemistry ,Plectasin ,Combinatorial chemistry ,Catalysis ,chemistry.chemical_compound ,Cucurbitaceae ,Order (biology) ,chemistry ,Peptide synthesis ,medicine ,Humans ,Disulfides ,Antimicrobial Peptides ,medicine.drug ,Plant Proteins - Abstract
Disulfide-rich peptides and proteins are among the most fascinating bioactive molecules. The difficulties associated with the preparation of these targets prompted the development of various chemical strategies. Nevertheless, the production of these targets remains very challenging or elusive. Recently, we introduced a strategy for one-pot disulfide bonds formation, tackling most of the previous limitations. Nevertheless, the effect of the order of oxidation remains an underexplored issue. Here we report on the synthetic flexibility and the full rainbow of oxidation orders of three disulfide bonds in targets that lack the knot motif. In contrast, our study reveals an essential order of disulfide bond formation in the EETI-II knotted miniprotein. This synthetic strategy was straightforwardly applied for the synthesis of novel analogues of the plectasin antimicrobial peptide with enhanced activities against methicillin-resistantn Staphylococcus n aureus (MRSA) , a notorious human pathogen. .
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- 2021
15. On-Demand Detachment of Succinimides on Cysteine to Facilitate (Semi)Synthesis of Challenging Proteins
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Gandhesiri Satish, Guy Mann, Prasad Sulkshane, Michael H. Glickman, Ashraf Brik, and Ganga B. Vamisetti
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Succinimides ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Chemical synthesis ,Article ,Catalysis ,Inteins ,Maleimides ,chemistry.chemical_compound ,Thioredoxins ,Colloid and Surface Chemistry ,Succinimide ,Coordination Complexes ,Solid-Phase Synthesis Techniques ,Cysteine ,Disulfides ,Maleimide ,Bioconjugation ,Cycloaddition Reaction ,Ubiquitin ,Proteins ,General Chemistry ,Combinatorial chemistry ,Globins ,0104 chemical sciences ,chemistry ,Thiazolidines ,Chemical ligation ,Peptides ,Intein ,Ubiquitin Thiolesterase ,Palladium - Abstract
The maleimide group is a widely used reagent for bioconjugation of peptides, proteins, and oligonucleotides employing Michael addition and Diels−Alder cycloaddition reactions. However, the utility of this functionality in chemical synthesis of peptides and proteins remains unexplored. We report, for the first time that PdII complexes can mediate the efficient removal of various succinimide derivatives in aqueous conditions. Succinimide removal by PdII was applied for the synthesis of two ubiquitin activity-based probes (Ub-ABPs) employing solid phase chemical ligation (SPCL). SPCL was achieved through a sequential three segment ligation on a polymer support via a maleimide anchor. The obtained probes successfully formed the expected covalent complexes with deubiquitinating enzymes (DUBs) USP2 and USP7, highlighting the use of our new method for efficient preparation of unique synthetic proteins. Importantly, we demonstrate the advantages of our newly developed method for the protection and deprotection of native cysteine with a succinimide group in a peptide fragment derived from thioredoxin-1 (Trx-1) obtained via intein based expression to enable ligation/desulfurization and subsequent disulfide bond formation in a one-pot process.
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- 2020
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16. De Novo Synthetic Design for Ultrafast Formation of Disulfide Bonds in Peptides and Proteins .pdf
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Hao Sun, Ashraf Brik, Guy Kamnesky, Shay Laps, and Fatima Atamleh
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Chemistry ,Drug discovery ,medicine ,Protein biosynthesis ,Disulfide bond ,Regioselectivity ,Conotoxin ,Plectasin ,Small molecule ,Combinatorial chemistry ,medicine.drug - Abstract
Despite six decades of efforts to synthesize peptides and proteins bearing multiple disulfide bonds, this synthetic challenge remains an unsolved problem in most targets (e.g. knotted mini proteins). Here we show a de novo general synthetic strategy for the ultrafast, high-yielding formation of two and three disulfide bonds in peptides and proteins. We developed an approach based on the combination of a small molecule, UV-light, and palladium for chemo- and regio-selective activation of Cys, which enables the one-pot formation of multiple disulfide bonds in various peptides and proteins. We prepared bioactive targets of high therapeutic potential, including conotoxin, RANTES, EETI-II, and plectasin peptides and the linaclotide drug. We anticipate that this strategy will be a game-changer in preparing millions of inaccessible targets for drug discovery.
- Published
- 2020
- Full Text
- View/download PDF
17. Harnessing the power of transition metals in solid-phase peptide synthesis and key steps in the (semi)synthesis of proteins
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Ashraf Brik, Gandhesiri Satish, and Shay Laps
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Surface Properties ,Context (language use) ,Peptide ,010402 general chemistry ,01 natural sciences ,Chemical synthesis ,Catalysis ,chemistry.chemical_compound ,Transition metal ,Phase (matter) ,Transition Elements ,Peptide synthesis ,Humans ,Amino Acid Sequence ,Disulfides ,Solid-Phase Synthesis Techniques ,chemistry.chemical_classification ,010405 organic chemistry ,Proteins ,Regioselectivity ,General Chemistry ,Native chemical ligation ,Combinatorial chemistry ,0104 chemical sciences ,chemistry ,Cyclization ,Metals ,Solvents ,Peptides - Abstract
Peptides and proteins can be either synthesized using solid-phase peptide synthesis (SPPS) or by applying a combination of SPPS and ligation approaches to address fundamental questions related to human health and disease, among others. The demand for their production either by chemical or biological methods continues to raise significant interests from the synthetic community. In this context, transition metals such as Pd, Ag, Hg, Tl, Au, Zn, Ni, and Cu have also contributed to the field of peptide and protein synthesis such as in peptide conjugation, extending native chemical ligation (NCL), and for regioselective disulfide bonds formation. In this review, we highlight, summarize, and evaluate the use of various transition metals in the chemical synthesis of peptides and proteins with emphasis on recent developments in this exciting research area.
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- 2020
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18. On-Demand Detachment of Maleimide Derivatives on Cysteine to Facilitate (Semi)Synthesis of Challenging Proteins
- Author
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satish gandhesiri, Prasad Sulkshane, Michael H. Glickman, Guy Mann, Ganga B. Vamisetti, and Ashraf Brik
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chemistry.chemical_compound ,biology ,chemistry ,Oligonucleotide ,Covalent bond ,biology.protein ,Chemical ligation ,Intein ,Combinatorial chemistry ,Maleimide ,Chemical synthesis ,Cysteine ,Deubiquitinating enzyme - Abstract
The maleimide group is a widely used reagent for bio-conjugation of peptides, proteins and oligonucleotides employing Michael addition and Diels-Alder cycloaddition reactions. However, the utility of this functionality in chemical synthesis of peptides and proteins remains unexplored. We report, for the first time that PdII complexes can mediate the efficient removal of various maleimide derivatives in aqueous conditions. Maleimide removal by PdII was applied for the synthesis of two ubiquitin activity-based probes (Ub-ABPs) employing solid phase chemical ligation (SPCL). SPCL was achieved through a sequential three segments ligation on a polymer support via a maleimide anchor. The obtained probes successfully formed the expected covalent complexes with deubiquitinating enzymes (DUBs) USP2 and USP7, highlighting the use of our new method for efficient preparation of unique synthetic proteins. Importantly, we demonstrate the advantages of our newly developed method for the protection and deprotection of native cysteine with a maleimide group in a peptide fragment derived from thioredoxin-1 (Trx-1) obtained via intein based expression to enable ligation/desulfurization and subsequent disulfide bond formation in a one-pot process.
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- 2020
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19. Total Chemical Synthesis of ISGylated-Ubiquitin Hybrid Chain Assisted by Acetamidomethyl Derivatives with Dual Functions
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Ashraf Brik, Hao Sun, Gábor N. Boross, Sumeet K. Singh, Muna Msallam, and Emad Eid
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Models, Molecular ,Protein Conformation ,Stereochemistry ,Biomedical Engineering ,Pharmaceutical Science ,Bioengineering ,Peptide ,Chemistry Techniques, Synthetic ,02 engineering and technology ,01 natural sciences ,Chemical synthesis ,Article ,Protein structure ,Ubiquitin ,Chain (algebraic topology) ,Amino Acid Sequence ,Ubiquitins ,Peptide sequence ,Pharmacology ,chemistry.chemical_classification ,biology ,010405 organic chemistry ,Organic Chemistry ,Substrate (chemistry) ,021001 nanoscience & nanotechnology ,ISG15 ,0104 chemical sciences ,Solubility ,chemistry ,biology.protein ,0210 nano-technology ,Biotechnology - Abstract
Interferon-stimulated gene 15 (ISG15) is a member of the ubiquitin-like modifiers (ULM) family, which adopts a β- grasp fold domain(s) similar to ubiquitin (Ub) with only minor sequence homology. ISG15 consists of two Ub-like domains and aids the immune system in neutralizing infections by numerous pathogens and plays an important role in defending cells against many viruses including influenza A. Recently, Ub was found to be a substrate for ISG15, which can be ISGylated on Lys29 and Lys48, while the former is more dominant. The discovery of such hybrid ISG15-Ub chains brought forward various fundamental questions regarding the nature and effect of this conjugation. To further investigate the role of hybrid ISG15-Ub chains, the pure homogeneous material of these chains is needed in workable quantities. By applying advanced chemical strategies for protein synthesis, we report the total chemical synthesis of a 231-residue ISG15-Lys29-Ub hybrid chain. During the synthesis we encountered insoluble peptide fragments, and therefore we developed a new reversible Acm based solubilizing tag to efficiently tackle this hurdle. This new Acm tag was compared with the known Arg based Acm solubilizing tag and was found to be more reliable in terms of incorporation and efficiency as demonstrated in the synthesis of the native ISG15-Ub hybrid chain.
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- 2020
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20. Affinity Maturation of Macrocyclic Peptide Modulators of Lys48-Linked Diubiquitin by a Twofold Strategy
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Ashraf Brik, Hiroaki Suga, Aaron Ciechanover, Ido Livneh, Mickal Nawatha, Yichao Huang, Joseph M. Rogers, Hao Sun, and Sumeet K. Singh
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Proteasome Endopeptidase Complex ,Stereochemistry ,Sequence alignment ,Peptide ,010402 general chemistry ,01 natural sciences ,Catalysis ,Deubiquitinating enzyme ,Affinity maturation ,chemistry.chemical_compound ,Thioether ,Ubiquitin ,Peptide Library ,Humans ,Amino Acids ,Ubiquitins ,chemistry.chemical_classification ,biology ,010405 organic chemistry ,Organic Chemistry ,General Chemistry ,0104 chemical sciences ,Amino acid ,Proteasome ,chemistry ,biology.protein ,Peptides ,Sequence Alignment - Abstract
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 (KD ) 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 KD 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.
- Published
- 2020
21. Examining the role of phosphorylation of p19 INK4d in its stability and ubiquitination using chemical protein synthesis
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Hao Sun, Pauline Franz, Ashraf Brik, Muna Msallam, and Roman Meledin
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inorganic chemicals ,0303 health sciences ,Circular dichroism ,biology ,Chemistry ,Kinase ,Retinoblastoma protein ,macromolecular substances ,General Chemistry ,Cell cycle ,010402 general chemistry ,environment and public health ,01 natural sciences ,Chemical synthesis ,0104 chemical sciences ,Cell biology ,enzymes and coenzymes (carbohydrates) ,03 medical and health sciences ,Ubiquitin ,biology.protein ,Protein biosynthesis ,bacteria ,Phosphorylation ,030304 developmental biology - Abstract
p19INK4d plays an important role in the regulation of the cell cycle by inhibiting the function of cyclin-dependent kinases 4/6 that is responsible for the phosphorylation and deactivation of the retinoblastoma protein (pRb) tumour suppressor. Recently, it was reported that phosphorylation of p19INK4d at Ser76 and Ser66 causes structural changes, which lead to its ubiquitination and degradation. Yet the exact contribution of each phosphorylation site remains unclear. To shed light on the role of these sites, we developed the chemical synthesis of unmodified, mono- and doubly phosphorylated p19INK4d using state of the art methods for chemical protein synthesis. The synthesized proteins were characterized by circular dichroism and biochemical methods to examine the effect of phosphorylation on the thermal stability and ubiquitination, respectively. Our results provide clear determination of p19INK4d stability upon phosphorylation at different sites and reveal that phosphorylation of both Ser residues might be necessary for promoting ubiquitination of p19INK4d.
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- 2020
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22. Chemical chromatin ubiquitylation
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Guy Kamnesky, Hao Sun, Ashraf Brik, and Muhammad Jbara
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Models, Molecular ,0301 basic medicine ,Chemistry Techniques, Synthetic ,Biochemistry ,Epigenesis, Genetic ,Analytical Chemistry ,Histones ,03 medical and health sciences ,chemistry.chemical_compound ,Ubiquitin ,Protein biosynthesis ,Animals ,Humans ,Nucleosome ,Epigenetics ,biology ,Ubiquitination ,Chromatin ,Nucleosomes ,Cell biology ,030104 developmental biology ,Histone ,chemistry ,biology.protein ,Function (biology) ,DNA - Abstract
Histone modifications dynamically regulate chromatin structure and function, thereby mediating many processes that require access to DNA. Chemical protein synthesis has emerged as a powerful approach for generating homogeneously modified histone analogues in workable amounts for subsequent incorporation into nucleosome arrays for biochemical, functional and structural studies. This short review focuses on the strength of total chemical protein synthesis and semisynthetic approaches to generate ubiquitylated histones in their native or non-native forms and the utility of these analogues to decode the role of ubiquitylation in epigenetics.
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- 2018
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23. Activity-Based Probes Developed by Applying a Sequential Dehydroalanine Formation Strategy to Expressed Proteins Reveal a Potential α-Globin-Modulating Deubiquitinase
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Ashraf Brik, Oded Kleifeld, Sachitanand M. Mali, and Roman Meledin
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Alanine ,Deubiquitinating Enzymes ,Molecular Structure ,biology ,010405 organic chemistry ,Chemistry ,General Medicine ,General Chemistry ,010402 general chemistry ,01 natural sciences ,Catalysis ,α globin ,0104 chemical sciences ,Cell biology ,Deubiquitinating enzyme ,chemistry.chemical_compound ,alpha-Globins ,Ubiquitin ,Dehydroalanine ,Molecular Probes ,hemic and lymphatic diseases ,biology.protein ,Humans ,Deubiquitination - Abstract
We report a general and novel semisynthetic strategy for the preparation of ubiquitinated protein-activity-based probes on the basis of sequential dehydroalanine formation on expressed proteins. We applied this approach to construct a physiologically and therapeutically relevant ubiquitinated α-globin probe, which was used for the enrichment and proteomic identification of α-globin-modulating deubiquitinases. We found USP15 as a potential deubiquitinase for the modulation of α-globin, an excess of which aggravates β-thalassemia symptoms. This development opens new opportunities for activity-based-probe design to shed light on the important aspects underlying ubiquitination and deubiquitination in health and disease.
- Published
- 2018
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24. Palladium mediated deallylation in fully aqueous conditions for native chemical ligation at aspartic and glutamic acid sites
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Muhammad Jbara, Emad Eid, and Ashraf Brik
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chemistry.chemical_classification ,Aqueous solution ,010405 organic chemistry ,Stereochemistry ,organic chemicals ,Organic Chemistry ,food and beverages ,chemistry.chemical_element ,Peptide ,Glutamic acid ,010402 general chemistry ,Hydrazide ,Native chemical ligation ,Thioester ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,chemistry.chemical_compound ,chemistry ,Side chain ,Physical and Theoretical Chemistry ,Palladium - Abstract
An efficient native chemical ligation approach at Asp and Glu sites is reported applying a hydrazide precursor, as a peptide thioester, and allyl protection at the side chain of Asp and Glu. The allyl protection was efficiently removed, after the ligation step, using the water-soluble palladium complex [Pd(allyl)Cl]2 and glutathione within a few minutes under fully aqueous conditions.
- Published
- 2018
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25. Total chemical synthesis of ester-linked ubiquitinated proteins unravels their behavior with deubiquitinases
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Roman Meledin, Ashraf Brik, Hao Sun, and Sachitanand M. Mali
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0301 basic medicine ,biology ,Chemistry ,Substrate (chemistry) ,General Chemistry ,010402 general chemistry ,01 natural sciences ,Chemical synthesis ,0104 chemical sciences ,03 medical and health sciences ,Ester bond ,030104 developmental biology ,Ubiquitin ,Biochemistry ,Ubiquitinated Proteins ,Cleave ,biology.protein - Abstract
Ester-linked ubiquitinated proteins have been reported by several groups to be involved in ubiquitin signalling. However, due to the lack of the suitable tools to homogeneously produce such conjugates, their exact physiological roles and biochemical behavior remain enigmatic. Here, we report for the first time on the development of a novel synthetic strategy based on total chemical synthesis of proteins to construct ubiquitinated proteins, where ubiquitin is linked to the substrate via an ester bond. In this study, we prepared ester- and isopeptide-linked ubiquitinated α-globin and examined their relative behaviors with various deubiquitinases. We found that deubiquitinases are able to cleave the ester linkage with different efficiency relative to the isopeptide-linked substrate. These results may indicate that ester-linked ubiquitinated proteins are natural substrates for deubiquitinases.
- Published
- 2018
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26. Palladium in der chemischen Synthese und Modifizierung von Proteinen
- Author
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Muhammad Jbara, Ashraf Brik, and Suman Kumar Maity
- Subjects
inorganic chemicals ,010405 organic chemistry ,chemistry.chemical_element ,Context (language use) ,General Medicine ,010402 general chemistry ,01 natural sciences ,Small molecule ,Combinatorial chemistry ,Coupling reaction ,0104 chemical sciences ,Catalysis ,chemistry ,Protein biosynthesis ,Organic chemistry ,Bioorthogonal chemistry ,Bond cleavage ,Palladium - Abstract
The field of site-specific modification of proteins has drawn significant attentions in recent years owing to its high importance in various research areas such as the development of novel therapeutics and understanding the biochemical and cellular behaviors of proteins. The presence of a large number of reactive functional groups in the protein of interest and in the cellular environment renders the particular modification at a specific site a highly challenging task. However, with the development of sophisticated chemical methodologies it is now possible to target a specific site of a protein with a desired modification despite many challenges remain to be solved. In this context, transition metals in particular palladium mediated C-C bond forming and C-O bond cleavage reactions gained great interest owing to the unique catalytic properties of palladium. Palladium chemistry is being significantly explored for protein modifications in vitro, on cell surface and in cellular context. Very recently, palladium complexes have been also applied for the rapid deprotection of several widely utilized cysteine-protecting groups as well as in the removal of solubilizing tags to facilitate chemical protein synthesis. This minireview highlights these advances and how the accumulated knowledge of palladium chemistry in small molecules is being impressively transferred to the area of chemical protein synthesis and modification.
- Published
- 2017
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27. Palladium in the Chemical Synthesis and Modification of Proteins
- Author
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Ashraf Brik, Muhammad Jbara, and Suman Kumar Maity
- Subjects
Models, Molecular ,inorganic chemicals ,010405 organic chemistry ,Research areas ,Molecular Conformation ,Proteins ,chemistry.chemical_element ,Context (language use) ,Nanotechnology ,General Chemistry ,010402 general chemistry ,01 natural sciences ,Chemical synthesis ,Small molecule ,Catalysis ,0104 chemical sciences ,Small Molecule Libraries ,chemistry ,Organometallic Compounds ,Protein biosynthesis ,Organic chemistry ,Bioorthogonal chemistry ,Palladium - Abstract
The field of site-specific modification of proteins has drawn significant attention in recent years owing to its importance in various research areas such as the development of novel therapeutics and understanding the biochemical and cellular behaviors of proteins. The presence of a large number of reactive functional groups in the protein of interest and in the cellular environment renders modification at a specific site a highly challenging task. With the development of sophisticated chemical methodologies it is now possible to target a specific site of a protein with a desired modification, however, many challenges remain to be solved. In this context, transition metals in particular palladium-mediated C-C bond-forming and C-O bond-cleavage reactions gained great interest owing to the unique catalytic properties of palladium. Palladium chemistry is being explored for protein modifications in vitro, on the cell surface, and within the cell. Very recently, palladium complexes have been applied for the rapid deprotection of several widely utilized cysteine protecting groups as well as in the removal of solubilizing tags to facilitate chemical protein synthesis. This Minireview highlights these advances and how the accumulated knowledge of palladium chemistry for small molecules is being impressively transferred to synthesis and modification of chemical proteins.
- Published
- 2017
- Full Text
- View/download PDF
28. Switching Futilepara-Quinone to Efficient Reactive Oxygen Species Generator: Ubiquitin-Specific Protease-2 Inhibition, Electrocatalysis, and Quantification
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Doron Shabat, Pushparathinam Gopinath, Shimrit Ohayon, Tal Eilon-Shaffer, Zeev Gross, Mickal Nawatha, Ashraf Brik, and Atif Mahammed
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0301 basic medicine ,010402 general chemistry ,Electrocatalyst ,01 natural sciences ,Biochemistry ,Catalysis ,Structure-Activity Relationship ,03 medical and health sciences ,Ubiquitin ,Cell Line, Tumor ,Humans ,Mode of action ,Molecular Biology ,chemistry.chemical_classification ,Reactive oxygen species ,biology ,Bicyclic molecule ,Chemistry ,Mechanism (biology) ,Organic Chemistry ,Quinones ,Para-quinone ,Electrochemical Techniques ,0104 chemical sciences ,030104 developmental biology ,Enzyme ,Drug Design ,Luminescent Measurements ,biology.protein ,Molecular Medicine ,Ubiquitin-Specific Proteases ,Reactive Oxygen Species - Abstract
Understanding the correlation between structural features of small-molecule drugs and their mode of action is a fascinating topic and crucial for the drug-discovery process. However, in many cases, knowledge of the exact parameters that dictate the mode of action is still lacking. Following a large screening for ubiquitin specific protease 2 (USP2) inhibition, an effective para-quinone-based inhibitor with an unclear mode of action was identified. To gain a deeper understanding of the mechanism of inhibition, a set of para-quinones were prepared and studied for USP2 inhibition, electrocatalysis, and reactive oxygen species (ROS) quantification. The excellent correlation obtained from the above-mentioned studies disclosed a distinct pattern of "N-C=O-N" in the bicyclic para-quinones to be a crucial factor for ROS generation, and demonstrated that minor changes in such a skeleton drastically altered the ROS-generating ability. The knowledge acquired herein would serve as an important guideline for future medicinal chemistry optimization of related structures to select the preferred mode of action.
- Published
- 2017
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29. Examining the role of phosphorylation of p19
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Muna, Msallam, Hao, Sun, Roman, Meledin, Pauline, Franz, and Ashraf, Brik
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inorganic chemicals ,enzymes and coenzymes (carbohydrates) ,Chemistry ,bacteria ,macromolecular substances ,environment and public health - Abstract
This study describes the chemical synthesis of unmodified and phosphorylated p19INK4d that were characterized by circular dichroism and biochemical methods to examine the effect of phosphorylation on the thermal stability and ubiquitination., p19INK4d plays an important role in the regulation of the cell cycle by inhibiting the function of cyclin-dependent kinases 4/6 that is responsible for the phosphorylation and deactivation of the retinoblastoma protein (pRb) tumour suppressor. Recently, it was reported that phosphorylation of p19INK4d at Ser76 and Ser66 causes structural changes, which lead to its ubiquitination and degradation. Yet the exact contribution of each phosphorylation site remains unclear. To shed light on the role of these sites, we developed the chemical synthesis of unmodified, mono- and doubly phosphorylated p19INK4d using state of the art methods for chemical protein synthesis. The synthesized proteins were characterized by circular dichroism and biochemical methods to examine the effect of phosphorylation on the thermal stability and ubiquitination, respectively. Our results provide clear determination of p19INK4d stability upon phosphorylation at different sites and reveal that phosphorylation of both Ser residues might be necessary for promoting ubiquitination of p19INK4d.
- Published
- 2019
30. The Journey for the Total Chemical Synthesis of a 53 kDa Protein
- Author
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Hao Sun and Ashraf Brik
- Subjects
Molecular Weight ,Biochemistry ,010405 organic chemistry ,Chemistry ,Protein biosynthesis ,Proteins ,General Medicine ,General Chemistry ,Chemistry Techniques, Synthetic ,010402 general chemistry ,01 natural sciences ,Chemical synthesis ,0104 chemical sciences - Abstract
Chemical protein synthesis has been proved as an efficient way to afford medium-sized proteins with high homogeneity in workable quantities for various biochemical, structural, and functional studies. In particular, chemical protein synthesis has enabled access to proteins that are difficult or impossible to prepare by molecular biology approaches, such as those with post-translational modifications and mirror-image proteins. One prominent example is related to ubiquitination, a well-known modification that mediates a variety of cellular processes (e.g., proteasomal degradation). Ubiquitination is considered as a modification that is difficult to introduce into proteins in a test tube to generate ubiquitin (Ub) conjugates with high homogeneity with respect to the chain length and the anchored Lys residue in workable quantities to perform the biochemical and biophysical studies. Chemical protein synthesis has emerged as a powerful approach to prepare Ub conjugates for studies aiming to understand ubiquitination in great detail and decipher its roles in cell processes. Nevertheless, in order to answer more challenging questions in this field, it has been clear that researchers must also prepare Ub conjugates with increased size and complexity. Employing solid-phase peptide synthesis and chemoselective ligation, chemical protein synthesis offers a powerful way to furnish polypeptides composed of 100-200 residues. However, to synthesize larger proteins such as Ub conjugates, longer and more segments are required. This on the other hand leads to difficulties related to solubility, purification, ligation, and late-stage modifications. These challenges have encouraged us to explore more practical synthetic tools to facilitate the synthesis of complex Ub conjugates. In this Account, we summarize the synthetic tools that we have developed to achieve these goals. These include (1) δ-mercaptolysine-mediated isopeptide chemical ligation, (2) chemical synthesis of Ub building blocks, (3) palladium-mediated deprotection of key side chains during protein synthesis, (4) one-pot ligation and desulfurization, and (5) improving the solubility of peptide segments. The developed chemical toolbox has been a key for our successes in the synthesis of diverse and complex Ub conjugates. In this Account, we describe our approaches for generating various Ub conjugates, including (1) the K48 tetra-Ub chain composed of 304 amino acids, (2) the ubiquitinated histones and their analogues made of200 amino acids, (3) the di-Ub-SUMO-2 hybrid chain composed of 245 amino acids, and (4) the 53 kDa tetra-Ub-α-globin composed of 472 amino acids, which represents the largest protein composed of natural amino acids ever made using chemical protein synthesis. The last target, Flag-Ub-Ub-Ub-Myc-Ub-(HA-α-globin), was prepared in the labeled form where the proximal Ub and distal Ub in the chain were labeled with Myc and Flag tags, respectively, while the α-globin was labeled with the HA tag. Applying the tetra-Ub-α-globin in proteasomal degradation studies assisted us to shed light on the proteolytic signal and the fates of the Ub moieties in the chains. Although these developments have contributed to the synthesis of interesting and challenging targets related to Ub signaling, several other targets may enforce new synthetic challenges. Hence, there is still a need to optimize the current synthetic tools and explore novel synthetic approaches to facilitate this process.
- Published
- 2019
31. Semisynthesis of ubiquitinated histone H2B with a native or nonhydrolyzable linkage
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Muhammad Jbara, Ashraf Brik, Cynthia Wolberger, and Michael T. Morgan
- Subjects
Models, Molecular ,chemistry.chemical_classification ,Isopeptide bond ,biology ,Histone ubiquitination ,Ubiquitin ,Chemistry ,Hydrolysis ,Lysine ,Ubiquitination ,Xenopus Proteins ,Native chemical ligation ,Article ,Histones ,Xenopus laevis ,Histone ,Biochemistry ,biology.protein ,Histone H2B ,Animals ,Monoubiquitination ,Nucleosome - Abstract
Post-translational modifications of histone proteins regulate all biological processes requiring access to DNA. Monoubiquitination of histone H2B is a mark of actively transcribed genes in all eukaryotes that also plays a role in DNA replication and repair. Solution and structural studies of the mechanism by which histone ubiquitination modulates these processes depend on the ability to generate homogeneous preparations of nucleosomes containing ubiquitin conjugated to a specific lysine residue. We describe here methods for generating milligram quantities of histone H2B with ubiquitin (Ub) conjugated to Lys 120 via either a non-hydrolyzable, dichloroacetone linkage or a cleavable isopeptide bond. H2B-Ub with an isopeptide linkage is generated by a combination of intein-fusion protein derivatization and native chemical ligation, yielding a fully native ubiquitinated lysine that can be cleaved by Ub isopeptidases. We also describe how to reconstitute nucleosomes containing ubiquitinated H2B.
- Published
- 2019
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32. Signature activities of 20S proteasome include degradation of the ubiquitin-tag with the protein under hypoxia
- Author
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Zhanyu Ding, Cong Xu, Indrajit Sahu, Sumeet K. Singh, Andrey Rozenberg, Manisha Priyadarsini Sahoo, Yifan Wang, Prasad Sulkshane, Sachitanand M. Mali, Roni Morag, Yao Cong, Michael H. Glickman, Sharleen Day, Ashraf Brik, and Oded Kleifeld
- Subjects
0303 health sciences ,Protease ,biology ,medicine.diagnostic_test ,Chemistry ,Proteolysis ,medicine.medical_treatment ,Substrate (chemistry) ,Protein degradation ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,Ubiquitin ,biology.protein ,medicine ,Target protein ,030217 neurology & neurosurgery ,Function (biology) ,Intracellular ,030304 developmental biology - Abstract
Careful removal of unwanted proteins is necessary for cell survival. The primary constitutive intracellular protease is the 26S proteasome complex, often found in equilibrium with its free catalytic subcomplex– the 20S core particle. Protein degradation by 26S is tightly regulated by prior ubiquitination of substrates, whereas 20S is amenable to substrates with an unstructured segment. Differentiating their contributions to intracellular proteolysis is challenging due to their common catalytic sites. Here, by chemically synthesizing a synoptic set of homogenous ubiquitinated proteins, we ascribe signature features to 20S function and demonstrate a unique property: degrading the ubiquitin-tag along with the target protein. Cryo-EM confirms that a ubiquitinated substrate can induce asymmetric conformational changes to 20S. Mass-spectrometry of intracellular peptidome under hypoxia and in human failing heart identifies the signature properties of 20S in cells. Moreover, the ability of 20S proteasome to clear toxic proteins rapidly, contributes to better survival under these conditions.
- Published
- 2019
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33. Correction: In vivo modulation of ubiquitin chains by N-methylated non-proteinogenic cyclic peptides
- Author
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Betsegaw Lemma, Uri Barash, Joseph M. Rogers, Ido Livneh, Hiroaki Suga, David Fushman, Aaron Jehuda Ciechanover, Israel Vlodavsky, Ganga B. Vamisetti, Mickal Nawatha, and Ashraf Brik
- Subjects
chemistry.chemical_classification ,Ubiquitin ,biology ,chemistry ,Chemistry (miscellaneous) ,In vivo ,Modulation ,biology.protein ,Biophysics ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Molecular Biology ,Biochemistry ,Cyclic peptide - Abstract
Correction for ‘In vivo modulation of ubiquitin chains by N-methylated non-proteinogenic cyclic peptides’ by Joseph M. Rogers et al., RSC Chem. Biol., 2021, 2, 513–522, DOI: 10.1039/D0CB00179A.
- Published
- 2021
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34. Synthetic Uncleavable Ubiquitinated Proteins Dissect Proteasome Deubiquitination and Degradation, and Highlight Distinctive Fate of Tetraubiquitin
- Author
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Sumeet K. Singh, Ashraf Brik, Michael H. Glickman, Oded Kleifeld, Sachitanand M. Mali, Hosahalli P. Hemantha, and Indrajit Sahu
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,Proteasome Endopeptidase Complex ,Isopeptide bond ,Deubiquitinating Enzymes ,Molecular Structure ,Ubiquitin ,Chemistry ,Substrate (chemistry) ,General Chemistry ,Cleavage (embryo) ,Ubiquitinated Proteins ,Biochemistry ,Catalysis ,Cell biology ,03 medical and health sciences ,Chain length ,030104 developmental biology ,Colloid and Surface Chemistry ,Proteasome ,Humans ,Degradation (geology) ,Deubiquitination - Abstract
Various hypotheses have been proposed regarding how chain length, linkage type, position on substrate, and susceptibility to deubiquitinases (DUBs) affect processing of different substrates by proteasome. Here we report a new strategy for the chemical synthesis of ubiquitinated proteins to generate a set of well-defined conjugates bearing an oxime bond between the chain and the substrate. We confirmed that this isopeptide replacement is resistant to DUBs and to shaving by proteasome. Analyzing products generated by proteasomes ranked how chain length governed degradation outcome. Our results support that (1) the cleavage of the proximal isopeptide bond is not a prerequisite for proteasomal degradation, (2) by overcoming trimming at the proteasome, tetraUb is a fundamentally different signal than shorter chains, and (3) the tetra-ubiquitin chain can be degraded with the substrate. Together these results highlight the usefulness of chemistry to dissect the contribution of proteasome-associated DUBs and the complexity of the degradation process.
- Published
- 2016
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35. Palladium-Assisted Cleavage of Peptides and Proteins Containing a Backbone with Thiazolidine Linkage
- Author
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Shay Laps, Ashraf Brik, Muhammad Jbara, and Suman Kumar Maity
- Subjects
Thiazolidine ,Chemical biology ,Peptide ,010402 general chemistry ,Cleavage (embryo) ,01 natural sciences ,Catalysis ,chemistry.chemical_compound ,Ubiquitin ,chemistry.chemical_classification ,biology ,010405 organic chemistry ,Chemistry ,Biomolecule ,Organic Chemistry ,Proteins ,Cell Biology ,General Chemistry ,Combinatorial chemistry ,Protein tertiary structure ,0104 chemical sciences ,biology.protein ,Thiazolidines ,Peptides ,Palladium ,Macromolecule - Abstract
The design and synthesis of biomolecules that are responsive to external stimuli is of great interest in various research areas, such as in the preparation of smart biomaterial and chemical biology. Polypeptide backbone disassembly as a response to a particular stimulus is of interest, as it leads to a complete loss of the protein tertiary structure and, as a result, to a loss of function. In this study, a strategy based on palladium-assisted efficient cleavage of backbone thiazolidine linkage in peptides and proteins was developed. Using a fluorescence-based assay, encompassing ubiquitinated peptide with a quenching florescence pair, it was possible to optimize the cleavage step after rapid screening of various conditions, such as the type of metal complexes and reaction additives. The optimized conditions prompted fast cleavage of the thiazolidine linkage. The straightforward introduction of a backbone thiazolidine linkage in peptide and proteins coupled with the chemical methods used offers new opportunities in controlling macromolecule function and might, with the aid of cellular protein delivery methods, be applied in cellular settings.
- Published
- 2016
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36. Efficient Palladium-Assisted One-Pot Deprotection of (Acetamidomethyl)Cysteine Following Native Chemical Ligation and/or Desulfurization To Expedite Chemical Protein Synthesis
- Author
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Shay Laps, Muhammad Jbara, Ashraf Brik, and Suman Kumar Maity
- Subjects
Peptide ,010402 general chemistry ,01 natural sciences ,Chemical synthesis ,Catalysis ,Combinatorial Chemistry Techniques ,Humans ,Moiety ,Organic chemistry ,Amino Acid Sequence ,Cysteine ,Eye Proteins ,Protecting group ,Ubiquitins ,chemistry.chemical_classification ,010405 organic chemistry ,Proteins ,General Medicine ,General Chemistry ,Native chemical ligation ,Combinatorial chemistry ,Semisynthesis ,0104 chemical sciences ,chemistry ,Chemical ligation ,Palladium ,Sulfur - Abstract
The acetamidomethyl (Acm) moiety is a widely used cysteine protecting group for the chemical synthesis and semisynthesis of peptide and proteins. However, its removal is not straightforward and requires harsh reaction conditions and additional purification steps before and after the removal step, which extends the synthetic process and reduces the overall yield. To overcome these shortcomings, a method for rapid and efficient Acm removal using Pd(II) complexes in aqueous medium is reported. We show, for the first time, the assembly of three peptide fragments in a one-pot fashion by native chemical ligation where the Acm moiety was used to protect the N-terminal Cys of the middle fragment. Importantly, an efficient synthesis of the ubiquitin-like protein UBL-5, which contains two native Cys residues, was accomplished through the one-pot operation of three key steps, namely ligation, desulfurization, and Acm deprotection, highlighting the great utility of the new approach in protein synthesis.
- Published
- 2016
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37. Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins
- Author
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Muhammad Jbara, Somasekhar Bondalapati, and Ashraf Brik
- Subjects
chemistry.chemical_classification ,Chemical substance ,010405 organic chemistry ,Chemistry ,General Chemical Engineering ,Chemoselective ligation ,Cellular functions ,Proteins ,Peptide ,General Chemistry ,Computational biology ,Protein Engineering ,010402 general chemistry ,Native chemical ligation ,01 natural sciences ,Chemical synthesis ,0104 chemical sciences ,Protein structure ,Protein biosynthesis ,Peptides ,Protein Processing, Post-Translational - Abstract
Methods to prepare proteins that include a specific modification at a desired position are essential for understanding their cellular functions and physical properties in living systems. Chemical protein synthesis, which relies on the chemoselective ligation of unprotected peptides, enables the preparation of modified proteins that are not easily fabricated by other methods. In contrast to recombinant approaches, chemical synthesis can be used to prepare protein analogues such as D-proteins, which are useful in protein structure determination and the discovery of novel therapeutics. Post-translationally modifying proteins is another example where chemical protein synthesis proved itself as a powerful approach for preparing samples with high homogeneity and in workable quantities. In this Review, we discuss the basic principles of the field, focusing on novel chemoselective peptide ligation approaches such as native chemical ligation and the recent advances based on this method with a proven record of success in the synthesis of highly important protein targets.
- Published
- 2016
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- View/download PDF
38. Protein ubiquitination via dehydroalanine: development and insights into the diastereoselective 1,4-addition step
- Author
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Sumeet K. Singh, Roman Meledin, Ashraf Brik, and Sachitanand M. Mali
- Subjects
Models, Molecular ,Protein Conformation ,Stereochemistry ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Protein structure ,Ubiquitin ,Dehydroalanine ,Amino Acid Sequence ,Physical and Theoretical Chemistry ,chemistry.chemical_classification ,Isopeptide bond ,Alanine ,Binding Sites ,biology ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,Ubiquitination ,Stereoisomerism ,Native chemical ligation ,Protein ubiquitination ,0104 chemical sciences ,Michael reaction ,biology.protein ,Stereoselectivity - Abstract
We report a strategy for site-specific protein ubiquitination using dehydroalanine (Dha) chemistry for the preparation of ubiquitin conjugates bearing a very close mimic of the native isopeptide bond. Our approach relies on the selective formation of Dha followed by conjugation with hexapeptide bearing a thiol handle derived from the C-terminal of ubiquitin. Subsequently, the resulting synthetic intermediate undergoes native chemical ligation with the complementary part of the ubiquitin polypeptide. It has been proposed that the Michael addition step could result in the formation of a diastereomeric mixture as a result of unselective protonation of the enolate intermediate. It has also been proposed that the chiral protein environment may influence such an addition step. In the protein context these questions remain open and no experimental evidence was provided as to how such a protein environment affects the diastereoselectivity of the addition step. As was previously proposed for the conjugation step on protein bearing Dha, the isopeptide bond formation step in our study resulted in the construction of two protein diastereomers. To assign the ratio of these diastereomers, trypsinization coupled with high-pressure liquid chromatography analysis were performed. Moreover, the obtained peptide diastereomers were compared with identical synthetic peptides having defined stereogenic centers, which enabled the determination of the configuration of the isopeptide mimic in each diastereomer. Our study, which offers a new method for isopeptide bond formation and protein ubiquitination, gives insights into the parameters that affect the stereoselectivity of the addition step to Dha for chemical protein modifications.
- Published
- 2016
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- View/download PDF
39. Halogen Substituents in the Isoquinoline Scaffold Switches the Selectivity of Inhibition between USP2 and USP7
- Author
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Roman Meledin, Pushparathinam Gopinath, Ganga B. Vamisetti, and Ashraf Brik
- Subjects
0301 basic medicine ,Scaffold ,Biochemistry ,Deubiquitinating enzyme ,Ubiquitin-Specific Peptidase 7 ,03 medical and health sciences ,chemistry.chemical_compound ,Structure-Activity Relationship ,Halogens ,In vivo ,Endopeptidases ,Carbohydrate Conformation ,Humans ,Isoquinoline ,Molecular Biology ,IC50 ,chemistry.chemical_classification ,Reactive oxygen species ,biology ,Dose-Response Relationship, Drug ,Molecular Structure ,Hydrocarbons, Halogenated ,Organic Chemistry ,Isoquinolines ,Combinatorial chemistry ,High-Throughput Screening Assays ,030104 developmental biology ,chemistry ,Halogen ,biology.protein ,Molecular Medicine ,Selectivity ,Ubiquitin Thiolesterase ,HeLa Cells - Abstract
Deubiquitinases are important components of the protein regulatory network and, hence, constitute a tempting drug target. We report herein structure-activity relationship studies to develop halogen-substituted isoquionoline-1,3-dione-based inhibitors of the deubiquitinase USP2. In contrast to our previous reports, the best compound discovered was found to act through a reactive oxygen species independent, uncompetitive mechanism with an IC50 of 250 nm. We show the crucial role of halogens in the common scaffold to provide potency and selectivity of our compound, where the introduction of the fluorine atom completely switches the selectivity of the inhibitor between USP2 and USP7. Our cellular studies highlight the potential applicability of the reported compound for in vivo experiments. The discovery of the isoquinoline-1,3-dione core and the knowledge obtained with regard to halogen substituents provide a platform towards understanding USP2 inhibition and the development of highly selective next-generation deubiquitinase inhibitors.
- Published
- 2018
40. FACT and Ubp10 collaborate to modulate H2B deubiquitination and nucleosome dynamics
- Author
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Tim Formosa, Laura McCullough, Zaily Connell, Cynthia Wolberger, Ashraf Brik, Michael T. Morgan, Melesse Nune, Muhammad Jbara, and Hao Sun
- Subjects
0301 basic medicine ,Transcription, Genetic ,Nucleosome assembly ,S. cerevisiae ,Histones ,0302 clinical medicine ,Transcription (biology) ,Gene Expression Regulation, Fungal ,Hydroxyurea ,Monoubiquitination ,Biology (General) ,Promoter Regions, Genetic ,deubiquitinating enzyme ,0303 health sciences ,biology ,Chemistry ,General Neuroscience ,High Mobility Group Proteins ,General Medicine ,Chromosomes and Gene Expression ,Nucleosomes ,Cell biology ,DNA-Binding Proteins ,Phenotype ,Histone ,embryonic structures ,Medicine ,Transcriptional Elongation Factors ,transcription ,Ubiquitin Thiolesterase ,Research Article ,Protein Binding ,Deubiquitination ,Saccharomyces cerevisiae Proteins ,QH301-705.5 ,Science ,Saccharomyces cerevisiae ,DNA replication ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Biochemistry and Chemical Biology ,ubiquitin ,Histone H2B ,Nucleosome ,Alleles ,030304 developmental biology ,General Immunology and Microbiology ,Ubiquitination ,030104 developmental biology ,histone chaperone ,nucleosome dynamics ,Mutation ,biology.protein ,030217 neurology & neurosurgery - Abstract
Monoubiquitination of histone H2B (H2B-Ub) plays a role in transcription and DNA replication, and is required for normal localization of the histone chaperone, FACT. In yeast, H2B-Ub is deubiquitinated by Ubp8, a subunit of SAGA, and Ubp10. Although they target the same substrate, loss of Ubp8 and Ubp10 causes different phenotypes and alters the transcription of different genes. We show that Ubp10 has poor activity on yeast nucleosomes, but that addition of FACT stimulates Ubp10 activity on nucleosomes and not on other substrates. Consistent with a role for FACT in deubiquitinating H2Bin vivo, a FACT mutant strain shows elevated levels of H2B-Ub. Combination of FACT mutants with deletion of Ubp10, but not Ubp8, confers increased sensitivity to hydroxyurea and activates a cryptic transcription reporter, suggesting that FACT and Ubp10 may coordinate nucleosome assembly during DNA replication and transcription. Our findings reveal unexpected interplay between H2B deubiquitination and nucleosome dynamics.
- Published
- 2018
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41. Chemical Synthesis of Ubiquitin Chains
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Ashraf Brik, Sumeet K. Singh, Somasekhar Bondalapati, and Hosahalli P. Hemantha
- Subjects
chemistry.chemical_classification ,biology ,Ubiquitin ,Chemistry ,Biomolecule ,Molecular Sequence Data ,General Medicine ,Chemical synthesis ,Combinatorial chemistry ,chemistry.chemical_compound ,Biochemistry ,Complex protein ,biology.protein ,Peptide synthesis ,Solid-Phase Synthesis Techniques ,Amino Acid Sequence ,Peptide sequence - Abstract
Chemical synthesis of complex biomolecules such as proteins is a challenging adventure, yet rewarding in driving various biochemical and biophysical research activities. Over the years, the refinement of peptide synthesis and invention of ligation methodologies have led to the successful synthesis of several complex protein targets. Ubiquitin bioconjugates, which are being studied intensively by many groups due to their involvement in numerous biological processes, represent a fine example where chemistry is greatly aiding these studies. In this article, we describe the synthetic routes and strategies to prepare different ubiquitin analogs with desired modifications, as well as di-ubiquitin chains.
- Published
- 2015
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42. Chemical Synthesis of Phosphorylated Ubiquitin and Diubiquitin Exposes Positional Sensitivities of E1-E2 Enzymes and Deubiquitinases
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Mark A. Nakasone, Wissam Mansour, Ashraf Brik, Suman Kumar Maity, Somasekhar Bondalapati, and Michael H. Glickman
- Subjects
inorganic chemicals ,chemistry.chemical_classification ,Ubiquitin-Specific Proteases ,biology ,Ubiquitin ,Chemistry ,Organic Chemistry ,macromolecular substances ,General Chemistry ,Ubiquitin-conjugating enzyme ,Chemical synthesis ,Catalysis ,Ubiquitin ligase ,Ligases ,enzymes and coenzymes (carbohydrates) ,Ubiquitins ,Enzyme ,Biochemistry ,biology.protein ,bacteria ,Phosphorylation - Abstract
Modification of ubiquitin by phosphorylation extends the signaling possibilities of this dynamic signal, as it could affect the activity of ligases and the processing of ubiquitin chains by deubiquitinases. The first chemical synthesis of phosphorylated ubiquitin and of Lys63-linked diubiquitin at the proximal, distal or both ubiquitins is reported. This enabled the examination of how such a modification alters E1-E2 activities of the ubiquitination machinery. It is found that E1 charging was not affected, while the assembly of phosphorylated ubiquitin chains was differentially inhibited with E2 enzymes tested. Moreover, this study shows that phosphorylation interferes with the recognition of linkage specific antibodies and the activities of several deubiquitinases. Notably, phosphorylation in the proximal or distal ubiquitin unit has differential effects on specific deubiquitinases. These results support a unique role of phosphorylation in the dynamics of the ubiquitin signal.
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- 2015
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43. Ubiquitin Signaling: Chemistry Comes to the Rescue
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Ashraf Brik, Sumeet K. Singh, Emad Eid, and Sachitanand M. Mali
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0301 basic medicine ,biology ,DNA repair ,Chemistry ,General Chemistry ,Cell cycle ,Protein Homeostasis ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Semisynthesis ,Catalysis ,0104 chemical sciences ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,Colloid and Surface Chemistry ,Ubiquitin ,Molecular mechanism ,biology.protein ,Posttranslational modification - Abstract
Posttranslational modification of proteins by ubiquitin (Ub), i.e., ubiquitination, mediates a variety of cellular processes, including protein homeostasis, cell cycle, DNA repair, and viral infections. Understanding the molecular mechanism of ubiquitination in these events is the basis for unraveling its precise role in health and disease. However, the inherent complexity of Ub signaling due to the high atomic complexity of Ub conjugates, where Ub is attached to other Ub molecules and to protein substrates in various forms, imposes a major challenge for these studies. In this regard, the enzymatic approaches employed for the preparation of important Ub conjugates have severe limitations to deliver them in high homogeneity and in adequate amounts for the desired study. Recent developments in the area of chemical synthesis and semisynthesis of proteins offer great solutions to the enzymatic limitations and enabling the preparation of various Ub conjugates with precise control over the atomic structure. These conjugates significantly contribute to deciphering Ub signaling at the molecular level, and with the synthetic tools in hand, chemical biologists have become key players in efforts toward understanding the complexity of the Ub code. In this Perspective, we highlight the key contributions of these synthetic approaches and how the development of novel Ub-based reagents is greatly assisting in uncovering unknown aspects of Ub signaling. We also discuss future aspirations to address unresolved questions in this exciting area of research.
- Published
- 2017
44. Total chemical synthesis of methylated analogues of histone 3 revealed KDM4D as a potential regulator of H3K79me3
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Nabieh Ayoub, Muhammad Jbara, Noga Guttmann-Raviv, Ashraf Brik, and Suman Kumar Maity
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0301 basic medicine ,Jumonji Domain-Containing Histone Demethylases ,Clinical Biochemistry ,Pharmaceutical Science ,Biochemistry ,Methylation ,Catalysis ,Histones ,03 medical and health sciences ,Histone H3 ,Coordination Complexes ,Drug Discovery ,Histone methylation ,Histone H2A ,Histone H2B ,Histone code ,Amino Acid Sequence ,Molecular Biology ,Chromatography, High Pressure Liquid ,biology ,Chemistry ,Lysine ,Organic Chemistry ,030104 developmental biology ,Histone ,Histone methyltransferase ,biology.protein ,Molecular Medicine ,Demethylase - Abstract
Histone H3 methylation plays an important role in regulating gene expression. In histones in general, this mark is dynamically regulated via various demethylases, which found to control cell fate decisions as well as linked to several diseases, including neurological and cancer. Despite major progress in studying methylation mark at various positions in H3 histone proteins, less is known about the regulation of methylated H3 at Lys79. Methylation at this site is known to have direct cross-talk with monoubiquitination of histone H2B at positions Lys120 and 34, as well as with acetylated H3 at Lys9. Herein we applied convergent total chemical protein synthesis to prepare trimethylated H3 at Lys79 to perform initial studies related to the regulation of this mark. Our study enabled us to identify KDM4D lysine demethylase as a potential regulator for trimethylated H3 at Lys79.
- Published
- 2017
45. Total chemical synthesis of SUMO-2-Lys63-linked diubiquitin hybrid chains assisted by removable solubilizing tags
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Somasekhar Bondalapati, Emad Eid, Cynthia Wolberger, Sachitanand M. Mali, and Ashraf Brik
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chemistry.chemical_classification ,biology ,010405 organic chemistry ,DNA repair ,genetic processes ,Peptide ,General Chemistry ,Computational biology ,macromolecular substances ,010402 general chemistry ,01 natural sciences ,Chemical synthesis ,environment and public health ,0104 chemical sciences ,enzymes and coenzymes (carbohydrates) ,Chemistry ,Ubiquitin ,chemistry ,Biochemistry ,Transcription (biology) ,biology.protein ,Protein biosynthesis ,health occupations ,Cleavable linker ,Linker - Abstract
We report the first total chemical synthesis of four different SUMO-2-Lys63-linked di-ubiquitin hybrid chains, in which the di-ubiquitin is linked to different lysines in SUMO., Small ubiquitin like modifier (SUMO) proteins are known to regulate many important cellular processes such as transcription and apoptosis. Recently, hybrid SUMO-ubiquitin chains containing SUMO-2 linked to Lys63-di-ubiquitin were found to play a major role in DNA repair. Despite some progress in understanding the role of these hybrid chains in DNA repair, there are various fundamental questions remaining to be answered. To further investigate the importance of hybrid SUMO-ubiquitin chains in DNA repair, the homogenous material of these chains, and their unique analogues, are needed in workable quantities. By applying advanced chemical strategies for protein synthesis, we report the first total chemical synthesis of four different SUMO-2-Lys63-linked di-ubiquitin hybrid chains, in which the di-ubiquitin is linked to different lysines in SUMO. In these syntheses, the usefulness of removable solubilizing tags is demonstrated, and two different approaches were examined in terms of reliability and efficiency. In the first approach, a poly-Arg tag was attached to the C-terminus of SUMO via a 3,4-diaminobenzoic acid cleavable linker, whereas in the second we attached the tag via a phenylacetamidomethyl linker, which can be cleaved by PdCl2. The comparison between these different strategies offers guidelines for future scale-up preparation of these analogues and other proteins, which currently use synthetic peptide intermediates that are difficult to handle and purify. The availability of the SUMO-ubiquitin hybrid chains opens up new opportunities for studying the role of these chains in DNA repair and other cellular processes.
- Published
- 2017
46. Harnessing the Oxidation Susceptibility of Deubiquitinases for Inhibition with Small Molecules
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Amir Aharoni, Maya Refua, Ashraf Brik, Shimrit Ohayon, and Adi Hendler
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chemistry.chemical_classification ,Reactive oxygen species ,biology ,Cancer therapy ,General Chemistry ,General Medicine ,medicine.disease_cause ,Small molecule ,Catalysis ,Residue (chemistry) ,Enzyme ,chemistry ,Ubiquitin ,Biochemistry ,medicine ,biology.protein ,Ubiquitin-Specific Proteases ,Reactive Oxygen Species ,Oxidation-Reduction ,Oxidative stress - Abstract
Deubiquitinases (DUBs) counteract ubiquitination by removing or trimming ubiquitin chains to alter the signal. Their diverse role in biological processes and involvement in diseases have recently attracted great interest with regard to their mechanism and inhibition. It has been shown that some DUBs are regulated by reactive oxygen species (ROS) in which the catalytic Cys residue undergoes reversible oxidation, hence modulating DUBs activity under oxidative stress. Reported herein for the first time, the observation that small molecules, which are capable of generating ROS efficiently, inhibit DUBs by selective and nonreversible oxidation of the catalytic Cys residue. Interestingly, the small molecule beta-lapachone, which is currently in clinical trials for cancer, is among the potent inhibitors, thus suggesting possible new cellular targets for its therapeutic effects. Our study describes a novel mechanism of DUBs inhibition and opens new opportunities in exploiting them for cancer therapy.
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- 2014
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47. Extended ubiquitin species are protein-based DUB inhibitors
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David Fushman, Mark A. Nakasone, Ashraf Brik, Donald S. Kirkpatrick, Steven P. Gygi, Noa Reis, Michael H. Glickman, Peter Siman, Daoning Zhang, and Daria Krutauz
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Proteasome Endopeptidase Complex ,Saccharomyces cerevisiae Proteins ,Leupeptins ,Saccharomyces cerevisiae ,Molecular Sequence Data ,Biology ,Ubiquitin-conjugating enzyme ,Article ,Deubiquitinating enzyme ,03 medical and health sciences ,0302 clinical medicine ,Ubiquitin ,Endopeptidases ,Amino Acid Sequence ,Polyubiquitin ,Molecular Biology ,Peptide sequence ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Ubiquitination ,Cell Biology ,biology.organism_classification ,Ubiquitin ligase ,Enzyme ,Biochemistry ,chemistry ,Proteasome ,biology.protein ,Proteasome Inhibitors ,030217 neurology & neurosurgery - Abstract
A frame-shift mutation in the transcript of the ubiquitin-B gene leads to a C-terminally extended ubiquitin, UBB+1. UBB+1 has been considered to inhibit proteasomes, and as such to be the underlying cause for toxic protein buildup correlated with certain neuropathological conditions. We demonstrated that expression of extended ubiquitin variants led to accumulation of heterogeneously-linked polyubiquitin conjugates indicating a pervasive effect on ubiquitin-dependent turnover. 20S proteasomes selectively proteolysed ubiquitin extensions, yet no evidence for inhibition of 26S holoenzymes was found. However, among susceptible targets for inhibition was Ubp6, the primary enzyme responsible for disassembly of lysine-48 linkages at 26S proteasomes. Processing of lysine-48 and lysine-63 linkages by other deubiquitinating enzymes (DUBs) was also inhibited. Disruption of ubiquitin-dependent degradation by extended ubiquitin variants may therefore be attributed to their inhibitory effect on select DUBs, thus shifting research efforts related to protein accumulation in neurodegenerative processes from proteasomes to DUBs.
- Published
- 2014
48. Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology
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Subramanian Vedhanarayanan Karthikeyan, Mahmood Haj-Yahya, Yifat Herman-Bachinsky, Sudhir N. Bavikar, Ashraf Brik, Bruno Fauvet, Aaron Ciechanover, Mirva Hejjaoui, and Hilal A. Lashuel
- Subjects
Alpha-synuclein ,0303 health sciences ,Multidisciplinary ,Amyloid ,biology ,Protein engineering ,010402 general chemistry ,01 natural sciences ,Pathophysiology ,3. Good health ,0104 chemical sciences ,Cell biology ,Pathogenesis ,03 medical and health sciences ,chemistry.chemical_compound ,chemistry ,Ubiquitin ,Proteasome ,biology.protein ,Phosphorylation ,030304 developmental biology - Abstract
Ubiquitination regulates, via different modes of modifications, a variety of biological processes, and aberrations in the process have been implicated in the pathogenesis of several neurodegenerative diseases. However, our ability to dissect the pathophysiological relevance of the ubiquitination code has been hampered due to the lack of methods that allow site-specific introduction of ubiquitin (Ub) chains to a specific substrate. Here, we describe chemical and semisynthetic strategies for site-specific incorporation of K48-linked di- or tetra-Ub chains onto the side chain of Lys12 of α-Synuclein (α-Syn). These advances provided unique opportunities to elucidate the role of ubiquitination and Ub chain length in regulating α-Syn stability, aggregation, phosphorylation, and clearance. In addition, we investigated the cross-talk between phosphorylation and ubiquitination, the two most common α-Syn pathological modifications identified within Lewy bodies and Parkinson disease. Our results suggest that α-Syn functions under complex regulatory mechanisms involving cross-talk among different posttranslational modifications.
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- 2013
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49. Modifying the Vicinity of the Isopeptide Bond To Reveal Differential Behavior of Ubiquitin Chains with Interacting Proteins: Organic Chemistry Applied to Synthetic Proteins
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Carlos A. Castañeda, Muhammad Jbara, Marlin Penner, Hosahalli P. Hemantha, Aaron Ciechanover, Ashraf Brik, David Fushman, Najat Haj-Yahya, Mahmood Haj-Yahya, and Liat Spasser
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chemistry.chemical_classification ,Isopeptide bond ,biology ,Ubiquitin binding ,Chemistry ,Peptide ,General Chemistry ,Ubiquitin-conjugating enzyme ,Combinatorial chemistry ,Chemical synthesis ,Catalysis ,Ubiquitin ligase ,Deubiquitinating enzyme ,Ubiquitin ,Biophysics ,biology.protein - Abstract
In Every Direction Chemical synthesis of proteins allowed the synthesis of ubiquitin chains modified in the vicinity of the isopeptide peptide to examine their behavior with deubiquitinases and ubiquitin binding domains. Our results set the ground for the generation of unique probes for studying the interactions of these chains with various ubiquitin-interacting proteins.
- Published
- 2013
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50. ChemInform Abstract: Chemical and Semisynthetic Approaches to Study and Target Deubiquitinases
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Pushparathinam Gopinath, Mickal Nawatha, Shimrit Ohayon, and Ashraf Brik
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Ubiquitin ,biology ,Chemistry ,Posttranslational modification ,biology.protein ,General Medicine ,Computational biology ,Target protein ,Selective inhibition ,Semisynthesis - Abstract
Ubiquitination is a key posttranslational modification, which affects numerous biological processes and is reversed by a class of enzymes known as deubiquitinases (DUBs). This family of enzymes cleaves mono-ubiquitin or poly-ubiquitin chains from a target protein through different mechanisms and mode of interactions with their substrates. Studying the role of DUBs in health and diseases has been a major goal for many laboratories both in academia and in industry. However, the field has been challenged by the difficulties in obtaining native substrates and novel reagents using traditional enzymatic and molecular biology approaches. Recent advancements in the synthesis and semisynthesis of proteins made it possible to prepare several unique ubiquitin conjugates to study various aspects of DUBs such as their specificities and structures. Moreover, these approaches enable the preparation of novel activity based probes and assays to monitor DUB activities in vitro and in cellular contexts. Efforts made to bring new chemical entities for the selective inhibition of DUBs based on these tools are also highlighted with selected examples.
- Published
- 2016
- Full Text
- View/download PDF
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