Time-resolved in vivo ubiquitinome profiling by DIA-MS reveals USP7 targets on a proteome-wide scale.

Mass spectrometry (MS)-based ubiquitinomics provides system-level understanding of ubiquitin signaling. Here we present a scalable workflow for deep and precise in vivo ubiquitinome profiling, coupling an improved sample preparation protocol with data-independent acquisition (DIA)-MS and neural network-based data processing specifically optimized for ubiquitinomics. Compared to data-dependent acquisition (DDA), our method more than triples identification numbers to 70,000 ubiquitinated peptides in single MS runs, while significantly improving robustness and quantification precision. Upon inhibition of the oncology target USP7, we simultaneously record ubiquitination and consequent changes in abundance of more than 8,000 proteins at high temporal resolution. While ubiquitination of hundreds of proteins increases within minutes of USP7 inhibition, we find that only a small fraction of those are ever degraded, thereby dissecting the scope of USP7 action. Our method enables rapid mode-of-action profiling of candidate drugs targeting DUBs or ubiquitin ligases at high precision and throughput

Molecular classification of the placebo effect in nausea

In this proof-of-concept study, we tested whether placebo effects can be monitored and predicted by plasma proteins. In a randomized controlled design, 90 participants were exposed to a nauseating stimulus on two separate days and were randomly allocated to placebo treatment or no treatment on the second day. Significant placebo effects on nausea, motion sickness, and (in females) gastric activity could be verified. Using label-free tandem mass spectrometry, 74 differentially regulated proteins were identified as correlates of the placebo effect. Gene ontology (GO) enrichment analyses identified acute-phase proteins and microinflammatory proteins to be involved, and the identified GO signatures predicted day-adjusted scores of nausea indices in the placebo group. We also performed GO enrichment analyses of specific plasma proteins predictable by the experimental factors or their interactions and identified 'grooming behavior' as a prominent hit. Finally, Receiver Operator Characteristics (ROC) allowed to identify plasma proteins differentiating placebo responders from non-responders, comprising immunoglobulins and proteins involved in oxidation reduction processes and complement activation. Plasma proteomics is a promising tool to identify molecular correlates and predictors of the placebo effect in humans

Studies on the Coordination of Ribosomal Protein Assembly Events Involved in Prosessing and Stabiliz

Cellular production of ribosomes involves the formation of highly defined interactions between ribosomal proteins (r-proteins) and ribosomal RNAs (rRNAs). Moreover in eukaryotic cells, efficient ribosome maturation requires the transient association of a large number of ribosome biogenesis factors (RBFs) with newly forming ribosomal subunits. Here, we investigated how r-protein assembly events in the large ribosomal subunit (LSU) rRNA domain II are coordinated with each other and with the association of RBFs in early LSU precursors of the yeast Saccharomyces cerevisiae. Specific effects on the pre-ribosomal association of RBFs could be observed in yeast mutants blocked in LSU rRNA domain II assembly. Moreover, formation of a cluster of r-proteins was identified as a downstream event in LSU rRNA domain II assembly. We analyzed in more detail the functional relevance of eukaryote specific bridges established by this r-protein cluster between LSU rRNA domain II and VI and discuss how they can support the stabilization and efficient processing of yeast early LSU precursor RNAs

MaxDIA enables library-based and library-free data-independent acquisition proteomics

MaxDIA is a software platform for analyzing data-independent acquisition (DIA) proteomics data within the MaxQuant software environment. Using spectral libraries, MaxDIA achieves deep proteome coverage with substantially better coefficients of variation in protein quantification than other software. MaxDIA is equipped with accurate false discovery rate (FDR) estimates on both library-to-DIA match and protein levels, including when using whole-proteome predicted spectral libraries. This is the foundation of discovery DIA—hypothesis-free analysis of DIA samples without library and with reliable FDR control. MaxDIA performs three- or four-dimensional feature detection of fragment data, and scoring of matches is augmented by machine learning on the features of an identification. MaxDIA’s bootstrap DIA workflow performs multiple rounds of matching with increasing quality of recalibration and stringency of matching to the library. Combining MaxDIA with two new technologies—BoxCar acquisition and trapped ion mobility spectrometry—both lead to deep and accurate proteome quantification.publishedVersio

Studies on the assembly process of large subunit ribosomal proteins in S.cerevisiae

Ribosomes are ribonucleoprotein (RNP) particles that catalyze the translation of the genetic information of messenger RNA (mRNA) into proteins. The biogenesis of eukaryotic ribosomes is a highly complex process that starts in the nucleolus where the ribosomal RNA (rRNA) is synthesized by RNA Polymerases I and III. Three of the four rRNA molecules are produced as one precursor molecule (pre-rRNA) that contains spacer sequences that are not part of mature ribosomal subunits but are removed during ribosome biogenesis. Besides these processing events, rRNA is heavily modified. In the course of ribosome assembly, the numerous (around 80) ribosomal proteins (r-proteins) and the rRNA (precursors) join together in a highly dynamic and defined manner. Initial interactions of most of the r-proteins to preribosomes occur already in the nucleolus or in the nucleus; some seem to be incorporated in the cytoplasm where the some final maturation events happen. Most of the r-proteins are required for an efficient accumulation of functional ribosomal subunits in the cytoplasm. Interestingly, depletion of essential LSU r-proteins resulted not in one common but in several different pre-rRNA processing phenotypes indicating their requirement for specific maturation events. In vitro reconstitution experiments on prokaryotic ribosomal subunits (whose structures are highly similar to the ones of their eukaryotic counterparts) helped to analyze the hierarchical interrelationships between the individual r-protein assembly events. In eukaryotic cells, the production of ribosomal subunits does not occur in a “self assembly” mechanism but requires numerous transiently interacting proteins, the so called ribosome biogenesis factors, and many small nucleolar RNAs (snoRNAs). Most of our current knowledge on eukaryotic ribosome biogenesis comes from studies in the yeast S.cerevisiae. Highly resolved structural information of mature yeast ribosomes (which is available since a few years) contributed to a better understanding of ribosome function but also ribosome biogenesis. Although the knowledge of the binding sites of the structural components (r-proteins) in the mature ribosomal subunits might not fully reflect how these “endpoints” are established in vivo, the final structure is very helpful to better understand features like hierarchical interrelationship of r-protein assembly events and/or their requirement for specific maturation events. In this work, the assembly process of the 46 r-proteins of the yeast large ribosomal subunit was aimed to be characterized in terms of several previously fragmentary or unresolved aspects. By comparing the respective amounts of the individual LSU r-proteins in differently maturated LSU precursors to each other and/or to the ones in mature LSUs, the assembly characteristics of each of the 46 LSU r-proteins was addressed applying a combination of affinity purification (ex vivo) and quantitative mass spectrometry. In a complementary approach, the interaction of a number of essential LSU r-proteins with the nascent particles was investigated in a comparative way by affinity purification of epitope tagged LSU r-proteins and quantitative analysis of the co-purified (pre-) rRNA species. In order to better understand the requirement of individual LSU r-proteins for certain assembly or maturation events, various LSU r-protein expression mutants were then used to investigate changes in the composition of the mutant preribosomes. Both, changes in the association of other LSU r-proteins and LSU ribosome biogenesis factors were analyzed. The results were then compared to previously published data from in vivo and in vitro experiments. The comparative analyses on the composition of differently maturated LSU precursors indicated that most LSU r-proteins seem to already start interacting with preribosomes of early maturation stages. In average, the affinity of these initial interactions seems to be rather low, though. The binding strength of most LSU r-proteins is then stabilized in the course of ribosome maturation. Therefore, the stable incorporation of most LSU r-proteins seems to be a multistep, rather than a one step event. Some LSU r-proteins however showed a specific assembly behavior which was characterized by an underrepresentation in early (or early and intermediately) maturated LSU precursors. Their (stable) incorporation seems to occur at later stages; for one group of LSU r-proteins in the cytoplasm. Clear evidences for hierarchical interrelationships among LSU r-protein assembly events in vivo could be deduced from the analyses of the RPL mutants. They can be categorized into two kinds of effects. One effect observed after depletion of most LSU r-proteins was characterized by a partial destabilization of the mutant preribosomes in the direct neighborhood of the depleted LSU r-protein or in the same secondary structure domain. Second, more general effects which can be related to the depletion phenotype of the respective r-protein were observed in most cases. A comparison to the results of previously published in vitro reconstitution experiments of prokaryotic LSUs revealed clear differences between the observed hierarchical interrelationships in vivo (in yeast) and in vitro (in E.coli), which are discussed. In addition, specific changes in the association of several ribosome biogenesis factors to the mutant preribosomes were observed. While in some cases the recruitment of several biogenesis factors to the preribosomes was disturbed, evidences for an inhibited release of others were provided. Altogether these results can contribute to a better understanding of the interplay between r-protein assembly events and the transient association of ribosome biogenesis factors. In addition, information of the molecular prerequisites for several maturation events, which are disturbed in the individual mutants (as pre-rRNA processing or nuclear export), can be deduced from these results

Local Tertiary Structure Probing of Ribonucleoprotein Particles by Nuclease Fusion Proteins

Analyses of the conformational dynamics of the numerous cellular ribonucleoprotein particles (RNP) significantly contribute to the understanding of their modes of action. Here, we tested whether ribonuclease fusion proteins incorporated into RNPs can be used as molecular probes to characterize the local RNA environment of these proteins. Fusion proteins of micrococcal nuclease (MNase) with ribosomal proteins were expressed in S. cerevisae to produce in vivo recombinant ribosomes which have a ribonuclease tethered to specific sites. Activation of the MNase activity by addition of calcium led to specific rRNA cleavage events in proximity to the ribosomal binding sites of the fusion proteins. The dimensions of the RNP environment which could be probed by this approach varied with the size of the linker sequence between MNase and the fused protein. Advantages and disadvantages of the use of MNase fusion proteins for local tertiary structure probing of RNPs as well as alternative applications for this type of approach in RNP research are discussed

In vitro reconstitution of yeast tUTP/UTP A and UTP B subcomplexes provides new insights into their modular architecture.

Eukaryotic ribosome biogenesis is a multistep process involving more than 150 biogenesis factors, which interact transiently with pre-ribosomal particles to promote their maturation. Some of these auxiliary proteins have been isolated in complexes found separate from the ribosomal environment. Among them, are 3 large UTP subcomplexes containing 6 or 7 protein subunits which are involved in the early steps of ribosome biogenesis. The composition of the UTP subcomplexes and the network of binary interactions between protein subunits have been analyzed previously. To obtain further insights into the structural and biochemical properties of UTP subcomplexes, we established a heterologous expression system to allow reconstitution of the yeast tUTP/UTP A and UTP B subcomplexes from their candidate subunits. The results of a series of reconstitution experiments involving different combinations of protein subunits are in good agreement with most of the previously observed binary interactions. Moreover, in combination with additional biochemical analyses, several stable building blocks of the UTP subcomplexes were identified. Based on these findings, we present a refined model of the tUTP/UTP A and UTP B architecture

Structural Probing with MNase Tethered to Ribosome Assembly Factors Resolves Flexible RNA Regions within the Nascent Pre-Ribosomal RNA

The synthesis of ribosomes involves the correct folding of the pre-ribosomal RNA within pre-ribosomal particles. The first ribosomal precursor or small subunit processome assembles stepwise on the nascent transcript of the 35S gene. At the earlier stages, the pre-ribosomal particles undergo structural and compositional changes, resulting in heterogeneous populations of particles with highly flexible regions. Structural probing methods are suitable for resolving these structures and providing evidence about the architecture of ribonucleoprotein complexes. Our approach used MNase tethered to the assembly factors Nan1/Utp17, Utp10, Utp12, and Utp13, which among other factors, initiate the formation of the small subunit processome. Our results provide dynamic information about the folding of the pre-ribosomes by elucidating the relative organization of the 5′ETS and ITS1 regions within the 35S and U3 snoRNA around the C-terminal domains of Nan1/Utp17, Utp10, Utp12, and Utp13

Plasmids: Description of plasmids used in this work. Database Number, plasmid backbone used to clone the indicated genes is specified. Original References for previously used plasmids are indicated. When required, plasmids used during the recombination reaction are also indicated.

<p>Plasmids: Description of plasmids used in this work. Database Number, plasmid backbone used to clone the indicated genes is specified. Original References for previously used plasmids are indicated. When required, plasmids used during the recombination reaction are also indicated.</p

Identification of different UTP B building blocks.

<p>Tagged proteins were purified from cell extracts containing different UTP B components in one or two step affinity purifications. Correct identification by MS analysis of the corresponding protein is indicated as Pwp2, ▪; Utp6, •; Utp12, ♦; Utp13, ◊; Utp18, ○ and Utp21, ▴. Expression of the tagged proteins is indicated as +: untagged protein expressed; T:TAP-tagged; F: FLAG-tagged; *: bait protein. (<b>A</b>) Combinations of the indicated proteins were co-expressed in SF21 insect cells infected with baculoviruses containing the bacmids K2137, K1987, K2134, K2135, K2136, K1991 and K1978. The bait proteins were purified from lysates of 5×10⁷ infected insect cells with IgG-coupled beads and eluted with TEV protease (Lanes 1–6) or with anti-FLAG affinity beads and elution with FLAG peptide (Lane 7). Samples of the elution were analyzed with SDS-PAGE and MS analysis. (<b>B</b>) Combinations of the indicated proteins were co-expressed in SF21 insect cells infected with baculoviruses containing the bacmids K2137, K2134, K2136, K2138 and K2139. Expression of the tagged proteins is indicated. The bait proteins were purified from lysates of 5×10⁷ infected insect cells with anti-FLAG affinity matrix and eluted with the FLAG peptide. Samples of the elution were analyzed with SDS-PAGE and MS analysis. Note that a band compatible with the size of Utp4-TAP is observed in Lane 3 but was not possible to characterize by MS analysis. (<b>C</b>) Combinations of the indicated proteins were co-expressed in SF21 insect cells infected with baculoviruses containing the bacmids K1991, K2134, K2135, K2136, K2137 and K1987. The bait proteins were purified from lysates of 5×10⁷ infected insect cells with IgG-coupled beads and eluted with TEV protease. Aliquots of the elution (upper panel) or of the corresponding cell lysate (lower panel) were analyzed by WB with anti-HA antibody. The corresponding co-expressed proteins are indicated at the top of the figure.</p