Interactome of intact chromatosome variants with site-specifically ubiquitylated and acetylated linker histone H1.2.

Post-translational modifications (PTMs) of histones have fundamental effects on chromatin structure and function. While the impact of PTMs on the function of core histones are increasingly well understood, this is much less the case for modifications of linker histone H1, which is at least in part due to a lack of proper tools. In this work, we establish the assembly of intact chromatosomes containing site-specifically ubiquitylated and acetylated linker histone H1.2 variants obtained by a combination of chemical biology approaches. We then use these complexes in a tailored affinity enrichment mass spectrometry workflow to identify and comprehensively characterize chromatosome-specific cellular interactomes and the impact of site-specific linker histone modifications on a proteome-wide scale. We validate and benchmark our approach by western-blotting and by confirming the involvement of chromatin-bound H1.2 in the recruitment of proteins involved in DNA double-strand break repair using an in vitro ligation assay. We relate our data to previous work and in particular compare it to data on modification-specific interaction partners of free H1. Taken together, our data supports the role of chromatin-bound H1 as a regulatory protein with distinct functions beyond DNA compaction and constitutes an important resource for future investigations of histone epigenetic modifications.

Cobalamins Function as Allosteric Activators of an Angelman Syndrome-Associated UBE3A/E6AP Variant.

Genetic aberrations of the maternal UBE3A allele, which encodes the E3 ubiquitin ligase E6AP, are the cause of Angelman syndrome (AS), an imprinting disorder. In most cases, the maternal UBE3A allele is not expressed. Yet, approximately 10 percent of AS individuals harbor distinct point mutations in the maternal allele resulting in the expression of full-length E6AP variants that frequently display compromised ligase activity. In a high-throughput screen, we identified cyanocobalamin, a vitamin B12-derivative, and several alloxazine derivatives as activators of the AS-linked E6AP-F583S variant. Furthermore, we show by cross-linking coupled to mass spectrometry that cobalamins affect the structural dynamics of E6AP-F583S and apply limited proteolysis coupled to mass spectrometry to obtain information about the regions of E6AP that are involved in, or are affected by binding cobalamins and alloxazine derivatives. Our data suggest that dietary supplementation with vitamin B12 can be beneficial for AS individuals.

Biochemical and Structural Consequences of NEDD8 Acetylation.

Similar to ubiquitin, the ubiquitin-like protein NEDD8 is not only conjugated to other proteins but is itself subject to posttranslational modifications including lysine acetylation. Yet, compared to ubiquitin, only little is known about the biochemical and structural consequences of site-specific NEDD8 acetylation. Here, we generated site-specifically mono-acetylated NEDD8 variants for each known acetylation site by genetic code expansion. We show that, in particular, acetylation of K11 has a negative impact on the usage of NEDD8 by the NEDD8-conjugating enzymes UBE2M and UBE2F and that this is likely due to electrostatic and steric effects resulting in conformational changes of NEDD8. Finally, we provide evidence that p300 acts as a position-specific NEDD8 acetyltransferase.

Titin kinase ubiquitination aligns autophagy receptors with mechanical signals in the sarcomere.

Striated muscle undergoes remodelling in response to mechanical and physiological stress, but little is known about the integration of such varied signals in the myofibril. The interaction of the elastic kinase region from sarcomeric titin (A168-M1) with the autophagy receptors Nbr1/p62 and MuRF E3 ubiquitin ligases is well suited to link mechanosensing with the trophic response of the myofibril. To investigate the mechanisms of signal cross-talk at this titin node, we elucidated its 3D structure, analysed its response to stretch using steered molecular dynamics simulations and explored its functional relation to MuRF1 and Nbr1/p62 using cellular assays. We found that MuRF1-mediated ubiquitination of titin kinase promotes its scaffolding of Nbr1/p62 and that the process can be dynamically down-regulated by the mechanical unfolding of a linker sequence joining titin kinase with the MuRF1 receptor site in titin. We propose that titin ubiquitination is sensitive to the mechanical state of the sarcomere, the regulation of sarcomere targeting by Nbr1/p62 being a functional outcome. We conclude that MuRF1/Titin Kinase/Nbr1/p62 constitutes a distinct assembly that predictably promotes sarcomere breakdown in inactive muscle.

Generation and Characterization of Site-Specifically Mono-Ubiquitylated p53.

The tumor suppressor p53 is regulated by various posttranslational modifications including different types of ubiquitylation, which exert distinct effects on p53. While modification by ubiquitin chains targets p53 for degradation, attachment of single ubiquitin moieties (mono-ubiquitylation) affects the intracellular location of p53 and/or its interaction with chromatin. However, how this is achieved at the molecular level remains largely unknown. Similarly, since p53 can be ubiquitylated at different lysine residues, it remains unclear if the eventual effect depends on the position of the lysine modified. Here, we combined genetic code expansion with oxime ligation to generate p53 site-specifically mono-ubiquitylated at position 120. We found that mono-ubiquitylation at this position neither interferes with p53 ubiquitylation by the E3 ligases HDM2 and E6AP in complex with the viral E6 oncoprotein nor affects p53 binding to a cognate DNA sequence. Thus, ubiquitylation per se does not affect physiologically relevant properties of p53.

Interactome of Site-Specifically Acetylated Linker Histone H1.

Linker histone H1 plays a key role in chromatin organization and maintenance, yet our knowledge of the regulation of H1 functions by post-translational modifications is rather limited. In this study, we report on the generation of site-specifically mono- and di-acetylated linker histone H1.2 by genetic code expansion. We used these modified histones to identify and characterize the acetylation-dependent cellular interactome of H1.2 by affinity purification mass spectrometry and show that site-specific acetylation results in overlapping but distinct groups of interacting partners. Among these, we find multiple translational initiation factors and transcriptional regulators such as the NAD+-dependent deacetylase SIRT1, which we demonstrate to act on acetylated H1.2. Taken together, our data suggest that site-specific acetylation of H1.2 plays a role in modulating protein-protein interactions.

A ubiquitin variant-based affinity approach selectively identifies substrates of the ubiquitin ligase E6AP in complex with HPV-11 E6 or HPV-16 E6.

The E6 protein of both mucosal high-risk human papillomaviruses (HPVs) such as HPV-16, which have been causally associated with malignant tumors, and low-risk HPVs such as HPV-11, which cause the development of benign tumors, interacts with the cellular E3 ubiquitin ligase E6-associated protein (E6AP). This indicates that both HPV types employ E6AP to organize the cellular proteome to viral needs. However, whereas several substrate proteins of the high-risk E6-E6AP complex are known, e.g. the tumor suppressor p53, potential substrates of the low-risk E6-E6AP complex remain largely elusive. Here, we report on an affinity-based enrichment approach that enables the targeted identification of potential substrate proteins of the different E6-E6AP complexes by a combination of E3-selective ubiquitination in whole-cell extracts and high-resolution MS. The basis for the selectivity of this approach is the use of a ubiquitin variant that is efficiently used by the E6-E6AP complexes for ubiquitination but not by E6AP alone. By this approach, we identified ∼190 potential substrate proteins for low-risk HPV-11 E6 and high-risk HPV-16 E6. Moreover, subsequent validation experiments in vitro and within cells with selected substrate proteins demonstrate the potential of our approach. In conclusion, our data represent a reliable repository for potential substrates of the HPV-16 and HPV-11 E6 proteins in complex with E6AP.

Ubiquitin binding by a CUE domain regulates ubiquitin chain formation by ERAD E3 ligases.

Ubiquitin-binding domains (UBDs) differentially recognize ubiquitin (ub) modifications. Some of them specifically bind mono-ub, as has been shown for the CUE domain. Interestingly, so far no significant ubiquitin binding has been observed for the CUE domain of yeast Cue1p. Cue1p is receptor and activator of the ubiquitin-conjugating enzyme Ubc7p. It integrates Ubc7p into endoplasmic reticulum (ER) membrane-bound ubiquitin ligase complexes, and thus, it is crucial for ER-associated protein degradation (ERAD). Here we show that the CUE domain of Cue1p binds ubiquitin chains, which is pivotal for the efficient formation of K48-linked polyubiquitin chains in vitro. Mutations that abolish ubiquitin binding by Cue1p affect the turnover of ERAD substrates in vivo. Our data strongly imply that the CUE domain facilitates substrate ubiquitylation by stabilizing growing ubiquitin chains at the ERAD ubiquitin ligases. Hence, we demonstrate an unexpected function of a UBD in the regulation of ubiquitin chain synthesis.

Effects of the antifungal agent ciclopirox in HPV-positive cancer cells: Repression of viral E6/E7 oncogene expression and induction of senescence and apoptosis.

The malignant growth of human papillomavirus (HPV)-positive cancer cells is dependent on the continuous expression of the viral E6/E7 oncogenes. Here, we examined the effects of iron deprivation on the phenotype of HPV-positive cervical cancer cells. We found that iron chelators, such as the topical antifungal agent ciclopirox (CPX), strongly repress HPV E6/E7 oncogene expression, both at the transcript and protein level. CPX efficiently blocks the proliferation of HPV-positive cancer cells by inducing cellular senescence. Although active mTOR signaling is considered to be critical for the cellular senescence response towards a variety of prosenescent agents, CPX-induced senescence occurs under conditions of severely impaired mTOR signaling. Prolonged CPX treatment leads to p53-independent Caspase-3/7 activation and induction of apoptosis. CPX also eliminates HPV-positive cancer cells under hypoxic conditions through induction of apoptosis. Taken together, these results show that iron deprivation exerts profound antiviral and antiproliferative effects in HPV-positive cancer cells and suggest that iron chelators, such as CPX, possess therapeutic potential as HPV-inhibitory, prosenescent and proapoptotic agents in both normoxic and hypoxic environments.

Fluorescently Labelled ATP Analogues for Direct Monitoring of Ubiquitin Activation.

Simple and robust assays to monitor enzymatic ATP cleavage with high efficiency in real-time are scarce. To address this shortcoming, we developed fluorescently labelled adenosine tri-, tetra- and pentaphosphate analogues of ATP. The novel ATP analogues bear - in contrast to earlier reports - only a single acridone-based dye at the terminal phosphate group. The dye's fluorescence is quenched by the adenine component of the ATP analogue and is restored upon cleavage of the phosphate chain and dissociation of the dye from the adenosine moiety. Thereby the activity of ATP-cleaving enzymes can be followed in real-time. We demonstrate this proficiency for ubiquitin activation by the ubiquitin-activating enzymes UBA1 and UBA6 which represents the first step in an enzymatic cascade leading to the covalent attachment of ubiquitin to substrate proteins, a process that is highly conserved from yeast to humans. We found that the efficiency to serve as cofactor for UBA1/UBA6 very much depends on the length of the phosphate chain of the ATP analogue: triphosphates are used poorly while pentaphosphates are most efficiently processed. Notably, the novel pentaphosphate-harbouring ATP analogue supersedes the efficiency of recently reported dual-dye labelled analogues and thus, is a promising candidate for broad applications.

The Length of a Ubiquitin Chain: A General Factor for Selective Recognition by Ubiquitin-Binding Proteins.

The attachment of ubiquitin (Ub) chains of various length to proteins is a prevalent posttranslational modification in eukaryotes. The fate of a modified protein is determined by Ub-binding proteins (UBPs), which interact with Ub chains in a linkage-selective manner. However, the impact and functional consequences of chain length on the binding selectivity of UBPs remain mostly elusive. We have generated Ub chains of defined length and linkage by using click chemistry and GELFrEE fractionation. These defined polymers were used in affinity-based enrichment assays to identify length- and linkage-selective interaction partners on a proteome-wide scale. For the first time, it is revealed that the length of a Ub chain generally has a major impact on its ability to be selectively recognized by UBPs.

Artificially Linked Ubiquitin Dimers Characterised Structurally and Dynamically by NMR Spectroscopy.

As one of the most prevalent post-translational modifications in eukaryotic cells, ubiquitylation plays vital roles in many cellular processes, such as protein degradation, DNA metabolism, and cell differentiation. Substrate proteins can be tagged by distinct types of polymeric ubiquitin (Ub) chains, which determine the eventual fate of the modified protein. A facile, click chemistry based approach for the efficient generation of linkage-defined Ub chains, including Ub dimers, was recently established. Within these chains, individual Ub moieties are connected through a triazole linkage, rather than the natural isopeptide bond. Herein, it is reported that the conformation of an artificially K48-linked Ub dimer resembles that of the natively linked dimer, with respect to structural and dynamic characteristics, as demonstrated by means of high-resolution NMR spectroscopy. Thus, it is proposed that artificially linked Ub dimers, as generated by this approach, represent potent tools for studying the inherently different properties and functions of distinct Ub chains.

Towards a molecular basis of ubiquitin signaling: A dual-scale simulation study of ubiquitin dimers.

Covalent modification of proteins by ubiquitin or ubiquitin chains is one of the most prevalent post-translational modifications in eukaryotes. Different types of ubiquitin chains are assumed to selectively signal respectively modified proteins for different fates. In support of this hypothesis, structural studies have shown that the eight possible ubiquitin dimers adopt different conformations. However, at least in some cases, these structures cannot sufficiently explain the molecular basis of the selective signaling mechanisms. This indicates that the available structures represent only a few distinct conformations within the entire conformational space adopted by a ubiquitin dimer. Here, molecular simulations on different levels of resolution can complement the structural information. We have combined exhaustive coarse grained and atomistic simulations of all eight possible ubiquitin dimers with a suitable dimensionality reduction technique and a new method to characterize protein-protein interfaces and the conformational landscape of protein conjugates. We found that ubiquitin dimers exhibit characteristic linkage type-dependent properties in solution, such as interface stability and the character of contacts between the subunits, which can be directly correlated with experimentally observed linkage-specific properties.

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

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.

Small-Molecule Inhibitors of the Tumor Suppressor Fhit.

The tumor suppressor Fhit and its substrate diadenosine triphosphate (Ap3 A) are important factors in cancer development and progression. Fhit has Ap3 A hydrolase activity and cleaves Ap3 A into adenosine monophosphate (AMP) and adenosine diphosphate (ADP); this is believed to terminate Fhit-mediated signaling. How the catalytic activity of Fhit is regulated and how the Fhit⋅Ap3 A complex might exert its growth-suppressive function remain to be discovered. Small-molecule inhibitors of the enzymatic activity of Fhit would provide valuable tools for the elucidation of its tumor-suppressive functions. Here we describe the development of a high-throughput screen for the identification of such small-molecule inhibitors of Fhit. Two clusters of inhibitors that decreased the activity of Fhit by at least 90 % were identified. Several derivatives were synthesized and exhibited in vitro IC50 values in the nanomolar range.

Different Enzymatic Processing of γ-Phosphoramidate and γ-Phosphoester-Modified ATP Analogues.

Monitoring the activity of ATP-consuming enzymes provides the basis for elucidating their modes of action and regulation. Although a number of ATP analogues have been developed for this, their scope is restricted because of the limited acceptance by respective enzymes. In order to clarify which kind of phosphate-modified ATP analogues are accepted by the α-ß-phosphoanhydride-cleaving ubiquitin-activating enzyme 1 (UBA1) and the ß-γ-phosphoanhydride-cleaving focal adhesion kinase (FAK), we tested phosphoramidate- and phosphoester-modified ATP analogues. UBA1 and FAK were able to convert phosphoramidate-modified ATP analogues, even with a bulky modification like biotin. In contrast, a phosphoester-modified analogue was poorly accepted. These results demonstrate that minor variations in the design of ATP analogues for monitoring ATP utilization have a significant impact on enzymatic acceptance.

Dependence of intracellular and exosomal microRNAs on viral E6/E7 oncogene expression in HPV-positive tumor cells.

Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells.

Identification of Proteins Interacting with Ubiquitin Chains.

Ubiquitylation, the modification of proteins with ubiquitin (Ub), is one of the most versatile post-translational modifications in eukaryotic cells. Since Ub also serves as its own substrate, proteins can be modified by numerous different Ub chains, in which the individual moieties are linked via one or several of the seven lysines of Ub. Homogeneous Ub chains, in which the moieties are sequentially linked via the same residue, have been most extensively studied. However, due to their restricted availability, the functions of Ub chains linked via K27, K29, or K33 are poorly understood. We have developed an approach that, for the first time, allows the generation of all seven homogeneous Ub chains in large quantities. The potential of our approach is demonstrated by the identification of previously unknown interaction partners of K27-, K29-, and K33-linked Ub chains by affinity-based proteomics.

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.