Search

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.

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.

Preparation of proteins composed entirely of D-amino acids is an elegant approach to assemble completely orthogonal living systems. In this issue of Cell Chemical Biology, Weidmann et al. (2019) demonstrate the chemical synthesis and characterization of mirror image DNA-ligase, which represents an important step for the creation of artificial life.

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.

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.

Chemical synthesis offers unique opportunities to prepare proteins with precise control of the atomic composition. Thanks to recent breakthroughs in synthetic methods, the preparation of large and complex proteins composed of 200-300 residues has now become possible. With these advances, a unique toolbox has been created to enable chemical biologists to investigate proteins that are difficult or even impossible to achieve otherwise, such as posttranslationally modified proteins and proteins composed of d-amino acids. In this review we describe the latest achievements in constructing protein conjugates of record sizes, such as those that are involved in the ubiquitin system.

A strategy for the synthesis of dehydroalanine based diubiquitin activity probes is described. The site-specific introduction of dehydroalanine was achieved from diubiquitin bearing Cys residue near the scissile bond between two ubiquitins linked through Lys48, Lys63 or in a head to tail fashion. The probes were characterized for their activities with various deubiquitinases, which open new opportunities in studying deubiquitinases in various settings.

SIRT1 specificity: The multispecific SIRT1 enzyme catalyzes the deacetylation of acetyllysine residues within protein targets. However, little is known regarding the molecular basis for SIRT1 substrate recognition. Kinetic analysis of SIRT1 with a panel of peptide substrates shows the high importance of the region N-flanking the target acetyllysine and its high conservation through evolution.