Chemical biology tools to study Deubiquitinases and Ubl proteases

The reversible attachment of ubiquitin (Ub) and ubiquitin like modifiers (Ubls) to proteins are crucial post-translational modifications (PTMs)for many cellular processes. Not only do cells possess hundreds of ligases to mediate substrate specific modification with Ub and Ubls, but they also have a repertoire of more than 100 dedicated enzymes for the specific removal of ubiquitin (Deubiquitinases or DUBs) and Ubl modifications (Ubl-specific proteases or ULPs). Over the past two decades, there has been significant progress in our understanding of how DUBs and ULPs function ata molecular level and many novel DUBs and ULPs, including several new DUB classes, have been identified. Here, the development of chemical tools that can bind and trap active DUBs has played a key role. Since the introduction of the first activity-based probe for DUBs in 1986, several innovations have led to the development of more sophisticated tools to study DUBs and ULPs. In this review we discuss how chemical biology has led to the development of activity-based probes and substrates that have been invaluable to the study of DUBs and ULPs. We summarise our currently available toolbox, highlight the main achievements and give an outlook of how these tools may be applied to gain a better understanding of the regulatory mechanisms of DUBs and ULPs

Linkage between fitness of yeast cells and adenylate kinase catalysis

Enzymes have evolved with highly specific values of their catalytic parameters kcat and KM. This poses fundamental biological questions about the selection pressures responsible for evolutionary tuning of these parameters. Here we are address these questions for the enzyme adenylate kinase (Adk) in eukaryotic yeast cells. A plasmid shuffling system was developed to allow quantification of relative fitness (calculated from growth rates) of yeast in response to perturbations of Adk activity introduced through mutations. Biophysical characterization verified that all variants studied were properly folded and that the mutations did not cause any substantial differences to thermal stability. We found that cytosolic Adk is essential for yeast viability in our strain background and that viability could not be restored with a catalytically dead, although properly folded Adk variant. There exist a massive overcapacity of Adk catalytic activity and only 12% of the wild type kcat is required for optimal growth at the stress condition 20°C. In summary, the approach developed here has provided new insights into the evolutionary tuning of kcat for Adk in a eukaryotic organism. The developed methodology may also become useful for uncovering new aspects of active site dynamics and also in enzyme design since a large library of enzyme variants can be screened rapidly by identifying viable colonies

Structural characterisation of MDM2 RING domain: E2-ubiquitin binding and activation by phosphorylation

The RING E3 ligase MDM2 is a primary negative regulator of the tumour suppressor protein p53. It blocks transcriptional activity and ubiquitinates p53, resulting in proteasomal degradation. MDM2’s ligase activity depends on the dimerisation of its C-terminal RING domain with either itself or its homologue MDMX. The crystal structure of the MDM2-MDMX heterodimer RING domain in complex with E2-ubiquitin has recently been crystallised. In this complex, only the MDM2 RING domain binds an E2-ubiquitin complex whereas the MDMX RING domain does not. However, MDMX’s C-terminal tail supports ubiquitin binding. This complex assembly results in one MDM2-MDMX RING heterodimer bound to one E2-ubiquitin complex. Due to extensive aggregation of the MDM2 homodimer, no structural information of the homodimeric MDM2-E2-ubiquitin complex has been obtained to date. During the course of my studies, I developed a purification protocol to generate non-aggregated homodimeric MDM2 RING domain. Sufficient amounts of homogeneous protein could be isolated for crystallisation purposes and crystal structures of the MDM2 homodimer alone and in complex with E2-ubiquitin were obtained. The crystal structures show that the homodimer can simultaneously bind two molecules of E2ubiquitin. The E2-ubiquitin binding surface resembles the heterodimer but shows significant differences in the arrangement of helices adjacent to the RING domain. Upon DNA damage, p53 needs to be stabilised in order to trigger cell cycle arrest or apoptosis. This requires p53 to be uncoupled from MDM2-mediated downregulation and is achieved by a number of phosphorylation events on both proteins, which reduce the binding affinity between p53 and MDM2. However, mouse models suggest that additional mechanisms exist as p53 is stabilised even when the corresponding phosphorylation sites are mutated. In addition, p53 is reportedly stabilised by phosphorylation of MDM2 near the RING domain, a region that is sequentially far away from the p53-binding domain. So far, the molecular basis of this mechanism has been elusive. Here, I show that phosphorylation near the RING domain enhances MDM2’s catalytic activity. With my MDM2 purification protocol, homodimeric phospho-MDM2 was generated and the crystal structure in complex with E2-ubiquitin was obtained. The molecular basis and homodimer-specificity of this novel phosphoregulation will be discussed. The results presented here help to understand the molecular function of MDM2, particularly under DNA damage conditions, and might be beneficial in diagnostics. The purification protocol of homogeneous MDM2 RING domain will be helpful for further structure-based studies such as the design of an MDM2 RING domain inhibitor

Catalytic activity of Adk variants at 20°C.

<p>Adk variants analyzed were designed to have a broad coverage of <i>k</i>_cat which is illustrated by a display of <i>k</i>_cat from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163115#pone.0163115.t001" target="_blank">Table 1</a> vs the corresponding Adk variant.</p

Expression levels, growth rate constants, relative fitness and apparent <i>k</i>_cat values of Adk_eco variants at 20°C.

<p>Expression levels, growth rate constants, relative fitness and apparent <i>k</i>_cat values of Adk_eco variants at 20°C.</p

Thermal stability of Adk variants.

<p>The thermal stability of Adk1_yeast (black), (red), (green), (blue), (turquoise), (pink), (purple), (yellow) and (olive) was quantified by observing normalized circular dichroism signals at a wavelength of 220 nm as a function of temperature. The data are displayed assuming a two-state unfolding model. Associated melting temperatures (<i>T</i>_M) are displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163115#pone.0163115.t001" target="_blank">Table 1</a>.</p

Structures of yeast and <i>E</i>. <i>coli</i> adenylate kinase in closed and active states.

<p>The stereo-view was made by superposition of C^α atoms of Adk1_yeast [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163115#pone.0163115.ref026" target="_blank">26</a>] (2AKY) and Adk_eco [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163115#pone.0163115.ref017" target="_blank">17</a>] (1AKE.pdb). The inhibitor Ap5A [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163115#pone.0163115.ref029" target="_blank">29</a>] is displayed with a ball and stick representation. Adk1_yeast and the corresponding Ap5A molecule is colored blue while Adk_eco with its corresponding Ap5A molecule is colored red.</p

Serial dilution growth assays at 30°C and 20°C.

<p>Yeast <i>adk1Δ</i> cells expressing Adk1_yeast or indicated variants of Adk_eco proteins were serially diluted, spotted on SC-Leu plates, and incubated at 30°C and 20°C for 3–4 days.</p

Nomenclature, catalytic parameters and melting temperatures of Adk variants.

<p>Nomenclature, catalytic parameters and melting temperatures of Adk variants.</p

Yeast plasmid shuffling assay system.

<p>The yeast <i>ADK1</i> open reading frame was exchanged with the KanMX cassette. Viability of the resulting strain depends on the presence of a wild-type yeast <i>ADK1</i> gene in a low-copy number <i>URA3</i>-based vector, pRS316. A second low-copy number <i>LEU2</i>-based vector was used to introduce different alleles of the <i>E</i>. <i>coli adk</i> gene into this strain. Thus, a strain harboring both the <i>URA3</i> plasmid (wild-type yeast <i>ADK1</i> gene) and the <i>LEU2</i> plasmid (mutated <i>E</i>. <i>coli adk</i> gene) can be obtained. If such a strain is plated on medium containing 5-FOA, the <i>URA3</i> vector will be counter-selected as the <i>URA3</i> gene product converts 5-FOA to a toxic compound [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163115#pone.0163115.ref022" target="_blank">22</a>]. Thus, this plasmid shuffling procedure can reveal the phenotype conferred by a mutated <i>E</i>. <i>coli adk</i> gene located in the <i>LEU2</i> plasmid.</p