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.
Subject(s)
Parkinson Disease/physiopathology , Polyubiquitin/chemistry , Protein Engineering/methods , alpha-Synuclein/chemistry , Humans , Parkinson Disease/metabolism , Phosphorylation , Polyubiquitin/chemical synthesis , Protein Stability , Ubiquitination , alpha-Synuclein/chemical synthesisABSTRACT
Biopolymers with repeating modules composed of either folded peptides or tertiary protein domains are considered some of the basic biomaterials that nature has evolved to optimize for energy efficient synthesis and unique functions. Such biomaterials continue to inspire scientists to mimic their exceptional properties and the ways that nature adopts to prepare them. Ubiquitin chains represent another example of nature's approach to use a protein-repeating module to prepare functionally important biopolymers. In the current work, we utilize a novel synthetic strategy to prepare bifunctional ubiquitin monomers having a C-terminal thioester and a nucleophilic 1,2-aminothiol at a desired position to examine their polymerization products under different conditions. Our study reveals that such analogues, when subjected to polymerization conditions under different folding states, afford distinct patterns of polymerization products where both the dynamic and the tertiary structures of the chains play important roles in such processes. Moreover, we also show that the presence of a specific ubiquitin-binding domain, which binds specifically to some of these chains, could interfere selectively with the polymerization outcome. Our study represents the first example of examining the polymerization of designed and synthetic repeating modules based on tertiary protein domains and affords early lessons in the design and synthesis of biomaterial. In regards to the ubiquitin system, our study may have implications on the ease of synthesis of ubiquitin chains with varying lengths and types for structural and functional analyses. Importantly, such an approach could also assist in understanding the enzymatic machinery and the factors controlling the assembly of these chains with a desired length.
Subject(s)
Biopolymers/chemistry , Ubiquitin/chemistry , Binding Sites , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Models, MolecularABSTRACT
The 1,3-dipolar cycloaddition of azomethine ylides derived from substituted isatins and 1,3-thiazolane-4-carboxylic acid to a series of 1-methyl-3,5-bis[(E)-arylmethylidene]-tetrahydro-4(1H)-pyridinones afforded novel spiro-pyrrolothiazoles chemo-, regio- and stereoselectively in quantitative yields. These compounds were screened for their in vitro activity against Mycobacterium tuberculosis H37Rv (MTB) and multi-drug resistant M. tuberculosis (MDR-TB) using agar dilution method. Among the synthesized compounds, spiro[5.3'']-5''-nitrooxindole-spiro-[6.3']-1'-methyl-5'-(2,4-di-chlorophenylmethylidene)tetrahydro-4'(1H)-pyridinone-7-(2,4-dichlorophenyl)tetra-hydro-1H-pyrrolo[1,2-c][1,3]thiazole (9k) was found to be the most active with a minimum inhibitory concentration (MIC) of 0.6microM against MTB and MDR-TB.
Subject(s)
Antitubercular Agents/chemical synthesis , Pyrroles/chemistry , Spiro Compounds/chemistry , Thiazoles/chemical synthesis , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Crystallography, X-Ray , Drug Resistance, Multiple, Bacterial/drug effects , Molecular Conformation , Mycobacterium tuberculosis/drug effects , Stereoisomerism , Thiazoles/chemistry , Thiazoles/pharmacologyABSTRACT
A series of novel 2-aryl-3,4-dihydro-2H-thieno[3,2-b]indoles has been synthesised regioselectively in good yields from the reaction of 5-aryldihydro-3(2H)-thiophenones and arylhydrazine hydrochloride. This reaction is found to be assisted by microwaves. The thieno[3,2-b]indoles were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv (MTB) and multi-drug resistant M. tuberculosis (MDR-TB). Among 22 compounds screened, [2-(2,4-dichlorophenyl)-7-fluoro-3,4-dihydro-2H-thieno[3,2-b]indole] (6t) was found to the most active compound with MIC of 0.4 microg/mL against MTB and MDR-TB.
Subject(s)
Antitubercular Agents/chemical synthesis , Indoles/chemical synthesis , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Indoles/chemistry , Indoles/pharmacology , Microwaves , Mycobacterium tuberculosis/drug effects , Stereoisomerism , Structure-Activity RelationshipSubject(s)
Histones/chemistry , Lysine/chemistry , Amino Acid Sequence , Chromatin/metabolism , Histones/chemical synthesis , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/chemistry , Protein Processing, Post-Translational , Ubiquitin/chemistry , Ubiquitin/metabolism , UbiquitinationABSTRACT
The desulfurization reaction introduced by Yan and Dawson as a postnative chemical ligation step greatly expanded the scope of ligation chemistry beyond Xaa-Cys (Xaa is any amino acid) by making ligation at Xaa-Phe, Xaa-Val, Xaa-Lys, Xaa-Leu, Xaa-Thr, and Xaa-Pro junctions accessible in the synthesis of functional proteins. A new ligation site based on Xaa-Gln utilizing γ-mercaptoglutamine is reported, and several examples on the efficiency of ligation coupled with desulfurization are provided.