Daniel Schneider

Dissecting ubiquitin signaling with linkage-defined and protease resistant ubiquitin chains.

Ubiquitylation is a complex posttranslational protein modification and deregulation of this pathway has been associated with different human disorders. Ubiquitylation comes in different flavors: Besides mono-ubiquitylation, ubiquitin chains of various topologies are formed on substrate proteins. The fate of ubiquitylated proteins is determined by the linkage-type of the attached ubiquitin chains, however, the underlying mechanism is poorly characterized. Herein, we describe a new method based on codon expansion and click-chemistry-based polymerization to generate linkage-defined ubiquitin chains that are resistant to ubiquitin-specific proteases and adopt native-like functions. The potential of these artificial chains for analyzing ubiquitin signaling is demonstrated by linkage-specific effects on cell-cycle progression.

Click chemistry for targeted protein ubiquitylation and ubiquitin chain formation

Herein we describe a simple protocol for the efficient generation of site-specific ubiquitin-protein conjugates using click chemistry. By using two different methods to expand the genetic code, the two bio-orthogonal functionalities that are necessary for CuI-catalyzed azide-alkyne cycloaddition (CuAAC), an alkyne and an azide, are co-translationally incorporated into the proteins of interest with unnatural amino acids. Protein ubiquitylation is subsequently carried out with the purified proteins in vitro by CuAAC. In addition, we provide a protocol for the incorporation of two unnatural amino acids into a single ubiquitin, resulting in a bifunctional protein that contains both an alkyne and an azide functionality, thereby enabling assembly of free ubiquitin chains as well as ubiquitin chains conjugated to a target protein. Our procedure enables the synthesis of nonhydrolyzable ubiquitin-protein conjugates within 1 week (given that the relevant cDNAs are at hand), and it yields conjugates in milligram quantities from 1-liter expression cultures. The approach described herein is faster and less laborious than other methods, and it requires only standard molecular biology equipment. Moreover, the protocol can be readily adapted to achieve conjugation at any site of any target protein, which facilitates the generation of custom-tailored ubiquitin-protein conjugates.

Fluorogenic ATP Analogues for Online Monitoring of ATP Consumption: Observing Ubiquitin Activation in Real Time†

The conjugation of ubiquitin to proteins plays an important role in the regulation of numerous cellular processes. Deregulation of this pathway has been associated with different human disorders including cancer and neurodege nerative diseases. For ubiquitylation, ubiquitin is initially activated by a ubiquitin activating enzyme (E1) at the expense of adenosine triphosphate (ATP; see Figure 1a) to form a thioester bond between the C terminal glycine of ubiquitin and the catalytic cysteine residue of E1. Subse quent transfer to a cysteine residue of a ubiquitin conjugating enzyme (E2) initiates the conjugation of ubiquitin to a target protein (mostly to a lysine residue by means of an isopeptide bond), a process which in many cases requires the help of a ubiquitin ligase (E3). As UBA1 is one of only two known human E1 enzymes for ubiquitin, modulation of its activity may prove beneficial in the treatment of certain disorders. Hence, assays for studying the activation of ubiquitin by UBA1 directly and without the interfering effects of the downstream enzymatic cascade are important tools to analyze UBA1 activity and identify modulators of this enzyme. So far, only a few assays that directly measure E1 activity have been described. However, these are laborious and not suitable for the continuous monitoring of E1 activity. In fact, until now no assay is available that allows the direct detection of ubiquitin activation in real time and that, for example, can be easily used to screen for E1 effectors. We, therefore, decided to elaborate on a conceptually novel assay: real time detection of ubiquitin activation by monitoring the cleavage of an ATP analogue (Figure 1b) that harbors two different fluorophores with the potential to undergo Fçrster resonance energy transfer (FRET). In the envisioned doubly labeled ATP analogue, excitation of the fluorescence donor (D) leads to transfer of the excitation energy to the fluorescence acceptor (A) whose fluorescence is monitored. Upon cleavage of the a/b anhydride bond of ATP, FRET is no longer possible and, thus, direct emission of the fluorescence donor can be detected. In this way, E1 activity results in a large change of fluorescence characteristics of the ATP analogue. Similar approaches based on the cleavage of FRET cassettes have been used for studying other hydro lyzing enzymes like proteases. For the envisaged time resolved ATPase sensor (TRASE) approach two fluorophores have to be attached to ATP. As the N6 position of ATP has been proven to be an attractive site for modifications without compromising UBA1 activity, the second modification has to be placed at the phosphoanhydride chain so that the two fluorophores are spatially separated upon cleavage. As earlier studies revealed that phosphate esters of ATP are stable, we tested g modified triphosphate II and d modified tetraphosphate III in promoting the autoubiquity lation of E6AP, a reaction that involves UBA1 and the E2 enzyme UbcH5b (Figure 2a,b). E6AP is an E3 ligase that has been causally associated with the development of three different human disorders: cervical cancer, Angelman syn drome, and autism spectrum disorders. As only the UBA1 reaction is ATP dependent, the appearance of polyubiquity lated E6AP (E6AP Ub), which does not significantly migrate in the applied gel electrophoresis, and consequently Figure 1. a) Mechanism of the activation of ubiquitin by UBA1. Ubiquitin is loaded onto UBA1 through the formation of a thioester bond with consumption of ATP. AMP: adenosine monophosphate, PP: pyrophosphate. b) Concept of signaling ATP consumption. The intact ATP analogue shows fluorescence of the acceptor (A) upon excitation of the donor (D) due to FRET. Upon cleavage by UBA1 direct donor fluorescence can be observed.

Improving bioorthogonal protein ubiquitylation by click reaction

Posttranslational modification of proteins with ubiquitin (ubiquitylation) regulates numerous cellular processes. Besides functioning as a signal for proteasomal degradation, ubiquitylation has also non-proteolytic functions by altering the biochemical properties of the modified protein. To investigate the effect(s) of ubiquitylation on the properties of a protein, sufficient amounts of homogenously and well-defined ubiquitylated proteins are required. Here, we report on the elaboration of a method for the generation of high amounts of site-specifically mono-ubiquitylated proteins. Firstly, a one-step affinity purification scheme was developed for ubiquitin containing the unnatural amino acid azidohomoalanine at the C-terminal position. This ubiquitin was conjugated in a click reaction to recombinant DNA polymerase β, equipped with an alkyne function at a distinct position. Secondly, addition of defined amounts of SDS to the reaction significantly improved product formation. With these two technical improvements, we have developed a straight forward procedure for the efficient generation of site-specifically ubiquitylated proteins that can be used to study the effect(s) of ubiquitylation on the activities/properties of a protein.

Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination

Significance Deregulation of components of the ubiquitin–proteasome system contributes to the development of various diseases. A prominent example is the ubiquitin ligase E6AP/UBE3A, which is associated with three disorders: in complex with the E6 oncoprotein of human papillomaviruses, it contributes to cervical carcinogenesis; loss of E6AP expression results in the development of Angelman syndrome; and increased E6AP expression has been associated with autism spectrum disorders. This indicates that E6AP has to be tightly controlled; however, only little is known about how this is achieved. By analyzing the role of ubiquitin and the E6 oncoprotein in E6AP-mediated ubiquitination, we provide evidence that E6AP exists in an active state and a latent state and that its activity is controlled by allosteric effectors. Deregulation of the ubiquitin ligase E6 associated protein (E6AP) encoded by the UBE3A gene has been associated with three different clinical pictures. Hijacking of E6AP by the E6 oncoprotein of distinct human papillomaviruses (HPV) contributes to the development of cervical cancer, whereas loss of E6AP expression or function is the cause of Angelman syndrome, a neurodevelopmental disorder, and increased expression of E6AP has been involved in autism spectrum disorders. Although these observations indicate that the activity of E6AP has to be tightly controlled, only little is known about how E6AP is regulated at the posttranslational level. Here, we provide evidence that the hydrophobic patch of ubiquitin comprising Leu-8 and Ile-44 is important for E6AP-mediated ubiquitination, whereas it does not affect the catalytic properties of the isolated catalytic HECT domain of E6AP. Furthermore, we show that the HPV E6 oncoprotein rescues the disability of full-length E6AP to use a respective hydrophobic patch mutant of ubiquitin for ubiquitination and that it stimulates E6AP-mediated ubiquitination of Ring1B, a known substrate of E6AP, in vitro and in cells. Based on these data, we propose that E6AP exists in at least two different states, an active and a less active or latent one, and that the activity of E6AP is controlled by noncovalent interactions with ubiquitin and allosteric activators such as the HPV E6 oncoprotein.

Anionic surfactants enhance click reaction-mediated protein conjugation with ubiquitin.

The Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) has become increasingly important in the conjugation chemistry of biomolecules. For example, it is an efficient and convenient method to generate defined ubiquitin-protein conjugates. Here, we investigate the effect of surfactants on the efficiency of CuAAC for chemical protein ubiquitylation. We found that anionic surfactants enhance conjugate formation by up to 10-fold resulting in high yields even at low (i.e., micromolar) concentrations of the reactants. Notably, the herein investigated conjugates are functional and thus properly folded.

Analyse des „Ubiquitin-Codes“ mithilfe Protease-resistenter Ubiquitinketten

Modification of proteins by ubiquitin plays a fundamental role in maintaining a functional proteome. Proteins can be modified by single ubiquitin moieties or by various types of ubiquitin chains differing in linkage type and topology. Different ubiquitin chains are assumed to signal target proteins for different fates, but how this is achieved is only partially understood. We developed an easy-to-use strategy to generate large amounts of linkage-defined, non-hydrolyzable ubiquitin chains and show their potential to dissect ubiquitin signalling.