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The tumor suppressor protein fragile histidine triad (Fhit) is known to be associated with genomic instability and apoptosis. The tumor-suppressive function of Fhit depends on the interaction with the alarmone diadenosine triphosphate (Ap3A), a noncanonical nucleotide whose concentration increases upon cellular stress. How the Fhit–Ap3A complex exerts its signaling function is unknown. Here, guided by a chemical proteomics approach employing a synthetic stable Fhit–Ap3A complex, we found that the Fhit–Ap3A complex, but not Fhit or Ap3A alone, impedes translation. Our findings provide a mechanistic model in which Fhit translocates from the nucleolus into the cytosol upon stress to form an Fhit–Ap3A complex. The Fhit–Ap3A complex impedes translation both in vitro and in vivo, resulting in reduced cell viability. Overall, our findings provide a mechanistic model by which the tumor suppressor Fhit collaborates with the alarmone Ap3A to regulate cellular proliferation. published

Cross-linking mass spectrometry (XL-MS) has become an indispensable tool for the emerging field of systems structural biology over the recent years. However, the confidence in individual protein–protein interactions (PPIs) depends on the correct assessment of individual inter-protein cross-links. In this article, we describe a mono- and intralink filter (mi-filter) that is applicable to any kind of cross-linking data and workflow. It stipulates that only proteins for which at least one monolink or intra-protein cross-link has been identified within a given data set are considered for an inter-protein cross-link and therefore participate in a PPI. We show that this simple and intuitive filter has a dramatic effect on different types of cross-linking data ranging from individual protein complexes over medium-complexity affinity enrichments to proteome-wide cell lysates and significantly reduces the number of false-positive identifications for inter-protein links in all these types of XL-MS data. published

Intrinsically disordered regions (IDRs) are essential for membrane receptor regulation but often remain unresolved in structural studies. TRPV4, a member of the TRP vanilloid channel family involved in thermo- and osmosensation, has a large N-terminal IDR of approximately 150 amino acids. With an integrated structural biology approach, we analyze the structural ensemble of the TRPV4 IDR and identify a network of regulatory elements that modulate channel activity in a hierarchical lipid-dependent manner through transient long-range interactions. A highly conserved autoinhibitory patch acts as a master regulator by competing with PIP2binding to attenuate channel activity. Molecular dynamics simulations show that loss of the interaction between the PIP2-binding site and the membrane reduces the force exerted by the IDR on the structured core of TRPV4. This work demonstrates that IDR structural dynamics are coupled to TRPV4 activity and highlights the importance of IDRs for TRP channel function and regulation.

Intracellular dinucleoside polyphosphates (Npn Ns) have been known for decades but the functional role remains enigmatic. Diadenosine triphosphate (Ap3 A) is one of the most prominent examples, and its intercellular concentration was shown to increase upon cellular stress. By employment of previously reported Ap3 A-based photoaffinity-labeling probes (PALPs) in chemical proteomics, we investigated the Ap3 A interactome in the human lung carcinoma cell line H1299. The cell line is deficient of the fragile histidine triade (Fhit) protein, a hydrolase of Ap3 A and tumor suppressor. Overall, the number of identified potential interaction partners was significantly lower than in the previously investigated HEK293T cell line. Gene ontology term analysis revealed that the identified proteins participate in similar pathways as for HEK293T, but the percentage of proteins involved in RNA-related processes is higher for H1299. The obtained results highlight similarities and differences of the Ap3 A interaction network in different cell lines and give further indications regarding the importance of the presence of Fhit. published

The AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis that initiates cytoplasmic maturation of the large ribosomal subunit. Drg1 releases the shuttling maturation factor Rlp24 from pre-60S particles shortly after nuclear export, a strict requirement for downstream maturation. The molecular mechanism of release remained elusive. Here, we report a series of cryo-EM structures that captured the extraction of Rlp24 from pre-60S particles by Saccharomyces cerevisiae Drg1. These structures reveal that Arx1 and the eukaryote-specific rRNA expansion segment ES27 form a joint docking platform that positions Drg1 for efficient extraction of Rlp24 from the pre-ribosome. The tips of the Drg1 N domains thereby guide the Rlp24 C terminus into the central pore of the Drg1 hexamer, enabling extraction by a hand-over-hand translocation mechanism. Our results uncover substrate recognition and processing by Drg1 step by step and provide a comprehensive mechanistic picture of the conserved modus operandi of AAA-ATPases.

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.

Different forms of life are known to express RNA ligases that catalyse the condensation of a 3’-hydroxy group and a 5’-terminal phosphate of RNA. No such RNA ligases have yet been identified in vertebrates. Here, we report that the hitherto uncharacterised human protein chromosome 12 open reading frame 29 (C12orf29), which we identified by a chemical proteomics approach, is a 5’-3’ RNA ligase. C12orf29 catalyses RNA ligation via auto-AMPylation of a critical lysine residue by using ATP as a cosubstrate and subsequent AMP transfer to the 5’-phosphate of an RNA substrate followed by phosphodiester bond formation. Studies at the cellular level reveal the involvement of C12orf29 in maintaining RNA integrity upon cellular stress induced by reactive oxygen species. These findings highlight the importance of RNA ligation for cellular fitness.

Oocyte maturation and early embryo development occur in vertebrates in the near absence of transcription. Thus, sexual reproduction of vertebrates critically depends on the timely translation of mRNAs already stockpiled in the oocyte. Yet how translational activation of specific mRNAs is temporally coordinated is still incompletely understood. Here, we elucidate the function of Zar1l, a yet uncharacterized member of the Zar RNA-binding protein family, in Xenopus oocytes. Employing TRIM-Away, we demonstrate that loss of Zar1l accelerates hormone-induced meiotic resumption of Xenopus oocytes due to premature accumulation of the M-phase-promoting kinase cMos. We show that Zar1l is a constituent of a large ribonucleoparticle containing the translation repressor 4E-T and the central polyadenylation regulator CPEB1, and that it binds directly to the cMos mRNA. Partial, hormone-induced degradation of Zar1l liberates 4E-T from CPEB1, which weakens translational repression of mRNAs encoding cMos and likely additional M-phase-promoting factors. Thus, our study provides fundamental insights into the mechanisms that ensure temporally regulated translation of key cell cycle regulators during oocyte maturation, which is essential for sexual reproductivity.

Post-translational modification (PTM) with ADP-ribose and poly(ADP-ribose) using nicotinamide adenine dinucleotide (NAD+ ) as substrate is involved in the regulation of numerous cellular pathways in eukaryotes, notably the response to DNA damage caused by cellular stress. Nevertheless, due to intrinsic properties of NAD+ e.g., high polarity and associated poor cell passage, these PTMs are difficult to characterize in cells. Here, two new NAD+ derivatives are presented, which carry either a fluorophore or an affinity tag and, in combination with developed methods for mild cell delivery, allow studies in living human cells. We show that this approach allows not only the imaging of ADP-ribosylation in living cells but also the proteome-wide analysis of cellular adaptation by protein ADP-ribosylation as a consequence of environmental changes such as H2 O2 -induced oxidative stress or the effect of the approved anti-cancer drug olaparib. Our results therefore pave the way for further functional and clinical studies of the ADP-ribosylated proteome in living cells in health and disease. published

DEAD-box ATPases are ubiquitous enzymes essential in all aspects of RNA biology. However, the limited in vitro catalytic activities described for these enzymes is at odds with their complex cellular roles, most notably in driving large-scale RNA remodeling steps during the assembly of ribonucleoproteins (RNPs). We describe cryo-EM structures of 60S ribosomal biogenesis intermediates that reveal how context-specific RNA unwinding by the DEAD-box ATPase Spb4 results in extensive, sequence-directed remodeling of rRNA secondary structure. Multiple cis and trans interactions stabilize a post-catalytic, high-energy intermediate that drives the organization of the root helix structure within rRNA domain IV. This mechanism explains how limited strand separation by DEAD-box ATPases is leveraged to provide non-equilibrium directionality and ensure efficient and accurate RNP assembly.

Cross-Linking Mass Spectrometry (XL-MS) has become an indispensable tool for the emerging field of systems structural biology over the recent years. However, the confidence in individual protein-protein interactions (PPIs) depends on the correct assessment of individual inter protein cross-links. This can be challenging, in particularly in samples where relatively few PPIs are detected, as is often the case in complex samples containing low abundant proteins or in in-vivo settings. In this manuscript we are describing a novel mono- and intralink filter (mi-filter) that is applicable to any kind of crosslinking data and workflow. It stipulates that only proteins for which at least one monolink or intra-protein crosslink has been identified within a given dataset are considered for an inter-protein cross-link and therefore participate in a PPI. We show that this simple and intuitive filter has a dramatic effect on different types of crosslinking-data ranging from single protein complexes, over medium-complexity affinity enrichments to proteome-wide cell lysates and significantly lowers the number of false-positive identifications resulting in improved false-discovery rates for inter-protein links in all these types of XL-MS data.

SummaryAberrant liquid-to-solid phase transitions of biomolecular condensates have been linked to various neurodegenerative diseases. However, the underlying molecular interactions that drive aging remain enigmatic. Here, we develop quantitative time-resolved crosslinking mass spectrometry to monitor protein interactions and dynamics inside condensates formed by the protein fused in sarcoma (FUS). We identify misfolding of the RNA recognition motif (RRM) of FUS as a key driver of condensate ageing. We demonstrate that the small heat shock protein HspB8 partitions into FUS condensates via its intrinsically disordered domain and prevents condensate hardening via condensate-specific interactions that are mediated by its α-crystallin domain (αCD). These αCD-mediated interactions are altered in a disease-associated mutant of HspB8, which abrogates the ability of HspB8 to prevent condensate hardening. We propose that stabilizing aggregation-prone folded RNA-binding domains inside condensates by molecular chaperones may be a general mechanism to prevent aberrant phase transitions.

Proteome-wide cross-linking studies have spurred great interest as they facilitate structural probing of protein interactions in living cells and organisms. However, current studies have a bias for high-abundant proteins. In this study we demonstrate both experimentally and by a kinetic model that this bias is also caused by the propensity of cross-links to preferentially form on high abundant proteins and not by the inability to detect cross-links due to limitations in current technology. We further show, by using both an in vitro mimic of a crowded cellular environment and eukaryotic cell lysates, that parameters optimized toward a pseudo first order kinetics model result in a significant increase in the detection of lower-abundant proteins on a proteome-wide scale. Our study therefore explains the cause of a major limitation in current proteome-wide cross-linking studies and demonstrates how to address a larger part of the proteome by cross-linking.

The covalent attachment of the cytokine-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) to hundreds of substrate proteins leads to their rapid degradation by the 26 S proteasome independently of ubiquitylation. Here, we identify another function of FAT10, showing that it interferes with the activation of SUMO1/2/3 in vitro and down-regulates SUMO conjugation and the SUMO-dependent formation of promyelocytic leukemia protein (PML) bodies in cells. Mechanistically, we show that FAT10 directly binds to and impedes the activity of the heterodimeric SUMO E1 activating enzyme AOS1/UBA2 by competing very efficiently with SUMO for activation and thioester formation. Nevertheless, activation of FAT10 by AOS1/UBA2 does not lead to covalent conjugation of FAT10 with substrate proteins which relies on its cognate E1 enzyme UBA6. Hence, we report that one ubiquitin-like modifier (FAT10) inhibits the conjugation and function of another ubiquitin-like modifier (SUMO) by impairing its activation., FAT10 is an ubiquitin-like modifier that targets proteins to proteasomal degradation. Here, the authors show that FAT10 also regulates SUMO activation in vitro and in cells, providing evidence for functional crosstalk between two ubiquitin-like modifiers.

Small heat-shock proteins (sHSP) are important members of the cellular stress response in all species. Their best described function is the binding of early unfolding states and the resulting prevention of protein aggregation. Most sHSPs exist as oligomers but vary in size and subunit organization. Many sHSPs exist as a polydisperse composition of oligomers which undergoes changes in subunit composition, folding status and relative distribution upon heat activation. To date only an incomplete picture of the mechanism of sHSP activation exists and in particularly the molecular basis of how sHSPs bind client proteins and mediate client specificity is not fully understood. In this study we have applied cross-linking mass spectrometry (XL-MS) to obtain detailed structural information on sHSP activation and client binding for yeast Hsp26. Our cross-linking data reveals the middle domain of Hsp26 as client-independent interface in multiple Hsp26::client complexes and indicates that client-specificity is likely mediated via additional binding sites within its αCD and CTE. Our quantitative XL-MS data underpins the middle domain as the main driver of heat-induced activation and client binding but shows that global rearrangements spanning all domains of Hsp26 are taking place simultaneously. We also investigated a Hsp26::client complex in the presence of Ssa1 (Hsp70) and Ydj1(Hsp40) at the initial stage of refolding and observe that the interaction between refolding chaperones is altered by the presence of a client protein, pointing to a mechanism where interaction of Ydj1 with the HSP::client complex initiates assembly of the active refolding machinery.

SummaryEukaryotic ribosome biogenesis is facilitated and regulated by numerous ribosome biogenesis factors (RBFs). High-resolution cryo-EM maps have defined the molecular interactions of RBFs during maturation, but many transient and dynamic interactions, particularly during early assembly, remain uncharacterized. Using quantitative proteomics and crosslinking coupled to mass spectrometry (XL-MS) data from a extensive set of pre-ribosomal particles, we derived a comprehensive and time-resolved interaction map of RBF engagement during 60S maturation. A novel filter that efficiently eliminates false positive interactions and integration of our MS data with known structural information allowed us to localize 22 unmapped RBFs to specific biogenesis intermediates and to identify 9 proteins that represent potentially new RBFs. Our analysis reveals an extensive interaction network for the casein kinase complex in 60S maturation and elucidates the timing and molecular function of 60S engagement by DEAD-box ATPases. Our data provide a powerful resource for future studies of 60S ribosome biogenesis.

The abnormal amplification of a CAG repeat in the gene coding for huntingtin (HTT) leads to Huntington's disease (HD). At the protein level, this translates into the expansion of a polyglutamine (polyQ) stretch located at the HTT N terminus, which renders HTT aggregation prone by unknown mechanisms. Here we investigated the effects of polyQ expansion on HTT in a complex with its stabilizing interaction partner huntingtin-associated protein 40 (HAP40). Surprisingly, our comprehensive biophysical, crosslinking mass spectrometry and cryo-EM experiments revealed no major differences in the conformation of HTT-HAP40 complexes of various polyQ length, including 17QHTT-HAP40 (wild type), 46QHTT-HAP40 (typical polyQ length in HD patients), and 128QHTT-HAP40 (extreme polyQ length). Thus, HTT polyQ expansion does not alter the global conformation of HTT when associated with HAP40. published

SummaryThe abnormal amplification of a CAG repeat in the gene coding for huntingtin (HTT) leads to Huntington disease (HD). At the protein level, this translates into the expansion of a poly-glutamine (polyQ) stretch located at the HTT N-terminus, which renders it aggregation-prone by unknown mechanisms. Here we investigated the effects of polyQ expansion on HTT in a complex with its stabilizing interaction partner huntingtin-associated protein 40 (HAP40). Surprisingly, our comprehensive biophysical, crosslinking mass spectrometry and cryo-EM experiments revealed no major differences in the conformation of HTT-HAP40 complexes of various polyQ length, including 17QHTT-HAP40 (wild type), 46QHTT-HAP40 (typical polyQ length in HD patients) and 128QHTT-HAP40 (extreme polyQ length). Thus, HTT polyQ expansion does not alter the global structure of HTT when associated with HAP40.

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 E6AP. This indicates that both HPV types employ E6AP to organize the cellular proteome to viral needs. However, while 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 mass spectrometry. 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 approximately 190 potential substrate proteins for low risk HPV-11 E6 as well as 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. published

The attachment of differently linked ubiquitin (Ub) chains of varying length to proteins is a prevalent posttranslational modification in eukaryotic cells. We established an affinity enrichment approach for the identification of linkage- and length-selective ubiquitin binding proteins and could show that in addition to the linkage, the length of a Ub chain is an important determinant for Ub chain recognition.

Parkin is an E3 ubiquitin ligase belonging to the RING-between-RING family. Mutations in the Parkin-encoding gene PARK2 are associated with familial Parkinson's disease. Here, we investigate the interplay between Parkin and the inflammatory cytokine-induced ubiquitin-like modifier FAT10. FAT10 targets hundreds of proteins for degradation by the 26S proteasome. We show that FAT10 gets conjugated to Parkin and mediates its degradation in a proteasome-dependent manner. Parkin binds to the E2 enzyme of FAT10 (USE1), auto-FAT10ylates itself, and facilitates FAT10ylation of the Parkin substrate Mitofusin2 in vitro and in cells, thus identifying Parkin as a FAT10 E3 ligase. On mitochondrial depolarization, FAT10ylation of Parkin inhibits its activation and ubiquitin-ligase activity causing impairment of mitophagy progression and aggravation of rotenone-mediated death of dopaminergic neuronal cells. In conclusion, FAT10ylation inhibits Parkin and mitophagy rendering FAT10 a likely inflammation-induced exacerbating factor and potential drug target for Parkinson's disease. published

Genetic aberrations of the UBE3A gene encoding the E3 ubiquitin ligase E6AP underlie the development of Angelman syndrome (AS). Approximately 10% of AS individuals harbor UBE3A genes with point mutations, frequently resulting in the expression of full-length E6AP variants with defective E3 activity. Since E6AP exists in two states, an inactive and an active one, we hypothesized that distinct small molecules can stabilize the active state and that such molecules may rescue the E3 activity of AS-derived E6AP variants. Therefore, we established an assay that allows identifying modulators of E6AP in a high-throughput format. We identified several compounds that not only stimulate wild-type E6AP but also rescue the E3 activity of certain E6AP variants. Moreover, by chemical cross-linking coupled to mass spectrometry we provide evidence that the compounds stabilize an active conformation of E6AP. Thus, these compounds represent potential lead structures for the design of drugs for AS treatment. published

Proteome-wide crosslinking studies have spurred great interest as they facilitate structural probing of protein interactions in living cells and organisms. However, current studies have a bias for high-abundant proteins. In a paradigm shift, we demonstrate both experimentally and by a kinetic model that this bias is caused by the propensity of crosslinks to preferentially form on high abundant proteins and not by the inability to detect crosslinks due to limitations in current technology. We further show, by using both an in-vitro mimic of a crowded cellular environment and eukaryotic cell lysates, that parameters optimized towards a pseudo 1st order kinetics model result in a significant 3 to 10-fold overall increase in the detection of lower-abundant proteins on a proteome-wide scale. Our study therefore explains the cause of a major limitation in current proteome-wide crosslinking studies and demonstrates how to address a larger part of the proteome by crosslinking.

During their final maturation in the cytoplasm, pre-60S ribosomal particles are converted to translation-competent large ribosomal subunits. Here, we present the mechanism of peptidyltransferase centre (PTC) completion that explains how integration of the last ribosomal proteins is coupled to release of the nuclear export adaptor Nmd3. Single-particle cryo-EM reveals that eL40 recruitment stabilises helix 89 to form the uL16 binding site. The loading of uL16 unhooks helix 38 from Nmd3 to adopt its mature conformation. In turn, partial retraction of the L1 stalk is coupled to a conformational switch in Nmd3 that allows the uL16 P-site loop to fully accommodate into the PTC where it competes with Nmd3 for an overlapping binding site (base A2971). Our data reveal how the central functional site of the ribosome is sculpted and suggest how the formation of translation-competent 60S subunits is disrupted in leukaemia-associated ribosomopathies., eLife digest Biological machines called ribosomes make proteins in the cells of our body. Mammalian cells build roughly 7,500 new ribosomes every minute, each one containing 80 proteins and four RNA molecules. Problems that prevent ribosomes from assembling correctly have been linked to cancers such as leukemia, and a class of disorders called ribosomopathies that increase the likelihood of someone developing cancer. Understanding how ribosomes assemble could therefore help to develop new treatments for these diseases. Ribosomes are mostly constructed in the cell nucleus, but the final stages of assembly occur in the cytoplasm of the cell. A protein called Nmd3 binds to the partly constructed ribosome to export it out of the nucleus. Then, the final ribosomal proteins integrate into the structure to form a key site called the peptidyltransferase centre (PTC), which is where the ribosome joins together amino acids when making new proteins for the cell. Questions remained about how these final assembly steps occur, and how Nmd3 is removed from the ribosome. Kargas et al. have now examined how the PTC forms by using a method known as cryo-electron microscopy to determine the structures that the ribosome forms at different stages of assembly. This revealed that when the last two ribosomal proteins integrate into the ribosome, the ribosomal RNA goes through large shape changes that evict Nmd3 from the PTC. Quality control factors then check the structure of the newly formed ribosome and, if it passes their checks that it works correctly, license it to start making cell proteins. This stage of ribosome assembly is likely to occur in the same way in all plant, animal and other eukaryotic species. The results presented by Kargas et al. will also help researchers to better understand the consequences of the mutations that affect ribosomal proteins in cancer cells. Ultimately, this knowledge may help to uncover new ways to treat cancer and ribosomopathies.