- Chemical and Semi-synthesis of Posttranslationally Modified Proteins:Posttranslational modifications of proteins play crucial roles in health and disease by affecting numerous aspects of protein structure, function, stability and sub-cellular localization. Yet understanding the effects of these modifications at the molecular level has been hindered by the lack of homogeneously modified proteins obtained via traditional biochemical and molecular biology approaches. Moreover, the preparation of such complex bioconjugates at a workable level is highly demanding. Recent advances in protein chemistry applying chemical and semisynthetic approaches are becoming increasingly beneficial to overcome these challenges. Our lab is interested in developing novel synthetic methods as well as utilizing the available state of art approaches to prepare site specifically modified proteins for biochemical, biophysical and functional analyses.
- Studying the Ubiquitin and Ubiquitin Like Modifiers Signals Using Chemical Biology Approaches:
Ubiquitination is the cellular process where a ubiquitin monomer, composed of 76 amino acids, or of a polyubiquitin chain are linked to a protein target, affecting a variety of biological processes such as protein degradation, trafficking, transcription and the DNA damage response. Not surprisingly, ubiquitination plays a key role in various diseases, such as neurological disorders, infectious diseases and cancer. Hence, understanding this signal at the molecular level is crucial for understanding and combating various diseases. In ubiquitination, three enzymes, E1, E2, and E3, collaborate to link the C-terminal Gly of ubiquitin to the Lys side chain of the protein target through an isopeptide bond. Conjugation of the ubiquitin molecule to a protein target may involve an ubiquitin monomer or a chain of ubiquitins of various lengths and linkage types. The evolving complexity of the ubiquitin signal and the recent discoveries of its involvement in a wide range of biological functions continue to dazzle scientists in many ways and hence engage several research groups aiming to decipher the molecular bases of this signal and its importance in health and diseases. Yet, research in this field, including structural and functional analyses, the development of reagents and understanding the enzymatic machineries and the factors involved in this signal, has been challenged by the inability to obtain homogenous ubiquitin bioconjugates at a workable level from cells, and even from cell free reconstituted enzymatic systems where conjugation often proceeds in an uncontrolled manner. We have recently reported a number of novel chemical methods that offer solutions to these challenges and prepare any ubiquitin conjugate in high homogeneity, purity and large quantities to shed light on various processes related to the ubiquitin signal. Specifically, our group has pioneered the development of delta mercaptolysine (thiolysine) to mediate the transthioesterification step with ubiquitin thioester, followed by an S-N acyl transfer to form the isopeptide bond between ubiquitin and a specific substrate. The thiol handle can then be removed by applying a desulfurization reaction to furnish the native isopeptide linkage. The above-described tools allowed us, for the first time, to synthesize all Lys linked di-ubiquitin chains in which thiolysine was introduced at the desired position (i.e., K63, K48, K33, K29, K27, K11, or K6), thus facilitating the site-specific attachment of the sequential ubiquitin molecule. The establishment of synthetic/semisynthetic routes for di-ubiquitin chains and analogs by us paved the way for many rigorous studies in order to shed light on the various aspects of ubiquitin systems. It has been shown for example that some small proteins undergo proteasomal degradation with monoubiquitination, however, for other proteins it is known that a chain of four ubiquitin units is necessary to execute the degradation signal. There are numerous cellular activities for which the preferred chain’s length is also unknown. Such questions can be attempted by establishing preparative methods for longer ubiquitin chains anchored to the specific substrate. Our group was the first to execute the chemical synthesis of K48-linked tetra-ubiquitin chain, which is the largest protein to be assembled chemically to date. In addition, we have also reported the synthesis of peptides linked to mono, di-, tri- and tetra-ubiquitin chains linked via K48 or K63 to examine their behaviors with specific deubiquitinases. our group has also accomplished the semi-synthesis of alpha-syn linked to chains of various lengths and investigated the role of the chain on alpha-synuclein aggregation and phosphorylation by various kinases. More recently, we have also extended these methods for polyubiquitination of expressed proteins and expand the use of these synthetic tools to more complex protein targets.The recent breakthrough in chemical and semisynthetic approaches reported by our group makes it possible now to assemble ubiquitin chains and ubiquitinated peptides and proteins in high purity and large quantity. As a result, great opportunities have become now available for chemical biologists and biologists to study the ubiquitin signal at the molecular level to reveal unknown aspects of this amazing and diverse signal. Similarly, these tools could be extended to the family of ubiquitin like modifiers, e.g. SOMU, which uses similar enzymatic machinery to link these proteins to their substrates. Along these adventures, chemistry will continue to play a vital role in creating new opportunities in studying these signals and their importance in health and disease.
- Chemical Synthesis of Modified Histones:
Packing the massive DNA polymer into a small and compact size structure within the nucleus is facilitated by nucleosomal proteins, H2A, H2B, H3 and H4. In a typical nucleosome, the DNA is wrapped into two turns around the histone octamer core of four histone partners; H3-H4 tetramer and two H2A-H2B dimers. These proteins are known to undergo a variety of modifications such as phosphorylation, methylation, acetylation and ubiquitination, which play crucial roles in regulating chromatin dynamics, gene expression and DNA repair. We are interested in understanding how these modifications achieve their function by chemically preparing homogeneously modified histones to support biochemical, biophysical and proteomic studies. For example, we have reported the efficient synthesis of monoubiquitinated H2B at Lys120 and Lys34 and studied its role in stem cell differentiation as well used these conjugates to find new protein that interact with the modified chromatin.
- Modulating Enzymes Activities using Small Molecules, Peptides and Peptidomimetis:
We are also interested in developing novel strategies to modulate various enzymes using different type of chemical entities. For example, our group is interested in studying and inhibiting deubiquitinases (DUBs). These enzymes have been implicated in various diseases, including neurological disorders, infectious diseases and cancer. Hence, it is not surprising that they are emerging as potential therapeutic targets and have encouraged the development of assays for the discovery of inhibitors for pharmaceutical needs and for studying the roles of DUBs in various biological functions. However none of these strategies have identified inhibitors against any DUB that has reached clinical stages. This is due to several challenges that are encountered in the discovery process, such as the absence of a highly sensitive and practically available high-throughput screening (HTS) assay to identify lead compounds against a DUB of interest. One of the reasons for the lack of such an assay is related to synthetic difficulties in preparing an efficient assay that takes into consideration the relatively complex structure of the natural substrate. We designed and generate an HTS assay based on ubiquitinated peptides to target UCH-L3/L1 that are known to cleave small adducts attached to ubiquitin. These types of assays are now being extended to other important DUBs involved in cancer. For example, we reported that small molecules that are capable of generating ROS efficiently inhibit DUBs by selective and non-reversible oxidation of the catalytic Cys residue. Interestingly, molecule beta-lapachone, which is currently in clinical trials for cancer, is among our list of the potent inhibitors suggesting possible new cellular targets for its therapeutic effects.
Professor Ashraf Brik completed his undergraduate studies in Chemistry in 1996 at the Ben-Gurion University of the Negev. Brik attended the Technion-Israel Institute of Technology for his M.Sc. degree and became interested in organic synthesis of natural products. Under the guidance of Dr. Nizar Haddad, Brik worked on the total chemical synthesis of Borrelidin, a natural antibiotic macrolide. In 1998, he moved to The Scripps Research Institute (TSRI) where he worked under the guidance of Professor Ehud Keinan and Professor Philip E. Dawson on a joint program between the Technion and TSRI, in the area of chemical synthesis of proteins. He used the developed chemical tools to study the mechanism of 4-Oxalocrotonate tautomerase and alter its activity from tautomerase to decarboxylase through chemical point mutations. He completed his Ph.D. in 2001, thereafter started his postdoctoral position with Professor Chi-Huey Wong (TSRI). During this time, Brik developed methods to facilitate the discovery of inhibitors against various enzymes. Specifically, Brik developed an approach named microtiter plate based chemistry and in situ screening for the discovery of potent inhibitors against HIV protease, beta-aryl sulfotransferase, and SARS human corona virus protease. In 2004, he was promoted to a Sr. Research Associate in the Wong Laboratory and became involved in the synthesis of glycopeptides and glycoproteins applying his knowledge and skills in protein chemistry. In 2007, Brik returned to his Alma Mater as an Assistant Professor at the Chemistry Department in BGU and was promoted to an Associate Professor in 2011 and to a Full Professor in 2012. In 2015, Brik moved to the Technion where he is today a Neubauer Professor in the Schulich Faculty of Chemistry. Professor Brik is the recipient of the Bessel Award of the Humboldt Foundation for 2015, the 11th Hirata Award, Teva Award for Excellence in memory of Eli Hurvitz for 2013, the Tetrahedron Young Investigator Award in Bioorganic and Medicinal Chemistry for 2013 and the 2011 Israel Chemical Society prize for Outstanding Young Chemist.
- Suman Kumar Maity#, Guy Mann#, Muhammad Jbara, Shay Laps, Guy Kamnesky, and Ashraf Brik; Palladium-Assisted Removal of a Solubilizing Tag from a Cys Side Chain To Facilitate Peptide and Protein Synthesis (# these authors contributed equally), Organic Letters, 2016, 18, 3026–3029, ACS Editor Choise.
- Roman Meledin, Sachitanand M. Mali, Sumeet Singh and Ashraf Brik; Protein ubiquitination via Dehydroalanine: Development and Insights into the Diastereoselective 1,4-Addition Step, Organic & Biomolecular Chemistry, 2016, 14, 4817 – 4823.
- Suman Kumar Maity#, Muhammad Jbara#, Shay Laps and Ashraf Brik; Efficient Palladium Assisted One-pot and Rapid Deprotection of Cysteine(Acetamidomethyl) Following Native Chemical Ligation and/or Desulfurization to Expedite Chemical Protein Synthesis (# these authors contributed equally), AngewandteChemie ,2016, 55, 1 – 6.
- Muhammad Jbara#, Suman Kumar Maity#, MallikantiSeenaiah, and Ashraf Brik, Palladium Mediated Rapid Deprotection of N-terminal Cysteine Under Native Chemical Ligation Conditions for the Efficient Preparation of Synthetically Challenging Proteins (# these authors contributed equally), Journal of American Chemical Society, 2016, 138 ,5069–5075.
- PushparathinamGopinath, ShimritOhayon, MickalNawatha and Ashraf Brik, Chemical and Semisynthetic Approaches to Study and Target Deubiquitinases, Chemical Soiciety Reviews, Advanced Article.
- Michael Morgan, Mahmood Haj-Yahya, Alison E. Ringel, PrasanthiBandi, Ashraf Brik and Cynthia Wolberger*, Structural Basis for Histone H2B deubiquitination by SAGA DUB Module,Science, 2016, 351 725-728.
- SomasekharBondalapati#, Muhammad Jbara# and Ashraf Brik*; Expanding the Chemical Toolbox for the Synthesis of Large and Uniquely Modified Proteins (# these authors contributed equally), Nature Chemistry, 2016, 8, 407–418.
- Muhammad Jbara, Suman Kumar Maity, Michael Morgan, Cynthia Wolberger* and Ashraf Brik*: Total Chemical Synthesis of Phosphorylated Histone H2A at Tyr57 Reveals Insight into the Inhibition Mode of the SAGA Deubiquitinating Module, AngewandteChemie ,2016,55, 4972-6.
- PushparathinamGopinath and Ashraf Brik*, 2,2′-Azobis[2-(2-imidazolin-2-yl)propane] Dihydrochloride, e-EROS, In press.
- Suman Kumar Maity, Muhammad Jbara and Ashraf Brik*: Chemical and Semisynthesis of Modified Histones, Journal of Peptide Science, first published online: 18 JAN 2016 (Festschrift for the journal on occasion of the 70th birthday of Stephen BH Kent).
- Roman Meledin, Sachitanand M. Mali and Ashraf Brik; Pushing the Boundaries of Chemical Protein Synthesis: The Case of Ubiquitin Chains, Polyubiquitinated Peptides and Proteins; The Chemical Record, 2016, 16, 509–519.
|Dr. Guy Kamnesky, Lab firstname.lastname@example.org|
|Dr.Hao Sun (Postdoc)||email@example.com|
|Dr. Babu Ganga (Postdoc)||firstname.lastname@example.org|
|Dr. Sathish Gandhesiri (Postdoc)||email@example.com|
|Roman Meledin (Postdoc)||firstname.lastname@example.org|
|Sumeet Singh (Ph.D.)||email@example.com|
|Muhammad Jbara (Ph.D.)||Jbara@technion.ac.il|
|Mickal Abd Alhadifirstname.lastname@example.org|