6 research outputs found
Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology
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
Polymerization Behavior of a Bifunctional Ubiquitin Monomer as a Function of the Nucleophile Site and Folding Conditions
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
Nonenzymatic Polyubiquitination of Expressed Proteins
Ubiquitination
is one of the most ubiquitous posttranslational
modifications in eukaryotes and is involved in various cellular events
such as proteasomal degradation and DNA repair. The overwhelming majority
of studies aiming to understand ubiquitination and deubiquitination
have employed unanchored ubiquitin chains and mono-ubiquitinated proteins.
To shed light on these processes at the molecular level, it is crucial
to have facile access to ubiquitin chains linked to protein substrates.
Such conjugates are highly difficult to prepare homogenously and in
workable quantities using the enzymatic machinery. To address this
formidable challenge we developed new chemical approaches to covalently
attach ubiquitin chains to a protein substrate through its Cys residue.
A key aspect of this approach is the installation of acyl hydrazide
functionality at the C-terminus of the proximal Ub, which allows,
after ubiquitin chain assembly, the introduction of various reactive
electrophiles for protein conjugation. Employing α-globin as
a model substrate, we demonstrate the facile conjugation to K48-linked
ubiquitin chains, bearing up to four ubiquitins, through disulfide
and thioether linkages. These bioconjugates were examined for their
behavior with the USP2 enzyme, which was found to cleave the ubiquitin
chain in a similar manner to unanchored ones. Furthermore, proteasomal
degradation study showed that di-ubiquitinated α-globin is rapidly
degraded in contrast to the mono-ubiquitinated counterpart, highlighting
the importance of the chain lengths on proteasomal degradation. The
present work opens unprecedented opportunities in studying the ubiquitin
signal by enabling access to site-specifically polyubiquitinated proteins
with an increased size and complexity
Synthesis of chimeric tetrapeptide-linked cholic acid derivatives: impending synergistic agents
Tetrapeptides derived from glycine and β-alanine were hooked at the C-3β position of the modified cholic acid to realize novel linear tetrapeptide-linked cholic acid derivatives. All the synthesized compounds were tested against a wide variety of microorganisms (Gram-negative bacteria, Gram-positive bacteria and fungi) and their cytotoxicity was evaluated against human embryonic kidney (HEK293) and human mammary adenocarcinoma (MCF-7) cell lines. While relatively inactive by themselves, these compounds interact synergistically with antibiotics such as fluconazole and erythromycin to inhibit growth of fungi and bacteria, respectively, at 1-24 μg/mL. The synergistic effect shown by our novel compounds is due to their inherent amphiphilicity. The fractional inhibitory concentrations reported are comparable to those reported for Polymyxin B derivatives