Chemical approaches to study the biology of ubiquitin-like proteins
The post-translational modification (PTM) of proteins gives rise to functional complexity in living organisms far beyond that encoded by their genomes. Among the myriad of PTMs found in Nature, one of the most dramatic chemical changes to a protein is its modification by conjugation with the small protein ubiquitin (Ub) and with ubiquitin-like proteins (Ubls). The enzymatic condensation of specific Lysine e-amines in proteins with the C-terminal carboxylate of Ub, or Ubls, leads to drastic changes in their function, localization, and/or proteasomal degradation.
The Small Ubiquitin-like Modifier (SUMO) is a highly studied, yet enigmatic, Ubl found in eukaryotes. Although the modification of proteins by SUMO (termed SUMOylation) is often a signal for their nuclear localization, several distinct functions for SUMOylation have also been proposed. These include the regulation of enzymatic activity, initiation of the DNA damage response, regulation of potassium channel function, modulation of Ab aggregate formation (Alzheimer’s disease), and chromatin regulation. The SUMOylation of chromatin is a reversible modification that is enhanced near telomeric regions, and has been associated with reduced histone acetylation and the repression of gene transcription through genetic studies in both yeast and humans.However, the molecular mechanism by which histone SUMOylation directs gene repression awaits investigation. Furthermore, the histone deSUMOylating proteases (SENPs) that confer reversibility to SUMOylation, and hence gene repression, remain unknown. We believe that a molecular understanding of how chromatin SUMOylation regulates gene transcription is not only important for understanding the mechanistic underpinnings of the histone code hypothesis of gene regulation, but can also lead to the identification of small-molecule modulators of chromatin function. Such an approach has already borne fruit in the instance of histone deacetylase (HDAC) inhibitors such as Vorinostat (Merck and Co.) and Romidepsin (Celgene) that are currently in clinical use against cutaneous T-cell lymphomas.
With these aims in mind, we are developing new chemical tools to probe the biochemical and biophysical consequences of histone SUMOylation and deSUMOylation.
The Prokaryotic ubiquitin-like protein (Pup) is a very recent addition to the Ubl protein family. Pupylation of proteins in Mycobacterium tuberculosis (Mtb) leads to their degradation by the 20S proteasome, which is necessary for Mtb infection and resistance to host macrophages. However, little is known regarding the non-covalent binding partners of Pup, and its turnover by the proteasome. Moreover, an enzyme with dePupylating activity that would be a therapeutic target specific to Mtb is yet to be discovered.
The increasing emergence of drug-resistant Mtb infections drives our interest in the Pup-proteasomal system, which is absent in humans. We believe that understanding the normal functions of Pup and its associated enzymes would set the stage for chemically inhibiting Mtb growth- an attractive prospect for tuberculosis therapy.
The general approach of our laboratory combines synthetic protein chemistry, protein engineering, molecular and cell biology to interrogate the mechanisms underlying the regulation of protein function by Ubiquitin-like proteins.