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SummaryThe B-cell receptor (BCR) is essential for B-cell development and a crucial clinical target in immuno-oncology. However, therapeutic success against the BCR and downstream signaling pathways is hampered by enhanced NF-κB activation as a resistance mechanism. Using a multiomic approach, we discover the c-REL proto-oncogenic subunit of the NF-κB family as a key transcription factor regulating BCR subunit levels in B-cell lymphoma. Subsequent ChIP- seq, cell biology experiments, and patient data analysis reveal that OTUD4 is a critical deubiquitinase for inhibiting proteasomal degradation of c-REL and for stabilizing a multi-loop positive feedback of NF-κB to the BCR pathway. Remarkably,OTUD4downregulation destabilizes c-REL and BCR levels and inhibits cell growth of B cell lymphoma. Thus, we shed light on the malignant potential of c-REL abundance, identify a positive feedback from c-REL to upstream BCR and present OTUD4 as a vulnerability to synergistically target NF-κB and BCR pathways in B-cell lymphoid malignancies.

Autocrine signaling pathways regulated by RAPID ALKALINIZATION FACTORs (RALFs) control cell wall integrity during pollen tube germination and growth in Arabidopsis. To investigate the role of pollen-specific RALFs in another plant species, we combined gene expression data with phylogenetic and biochemical studies to identify candidate orthologs in maize. We show that Clade IBZmRALF2/3mutants, but not that of Clade IIIZmRALF1/5caused cell wall instability in the sub-apical region of the growing pollen tube. ZmRALF2/3 are mainly located to the cell wall and are partially able to complement the pollen germination defect of their Arabidopsis orthologs AtRALF4/19. Mutations inZmRALF2/3compromise pectin distribution pattern leading to altered cell wall thickness, hyperphosphorylation of ZmPEX cell wall proteins and pollen tube burst. Clade IB, but not Clade III ZmRALFs are capable to interact with pollen-specific CrRLK1L receptor kinases ZmFERL4/7/9 and GPI-anchored co-receptors ZmLLG1/2 at similar binding affinities. In contrast, binding affinity to ZmPEX2/4 cell wall proteins is about five times higher. Based on these data, we now propose a dosage-dependent model showing how Clade IB RALFs act as extracellular sensors to regulate cell wall integrity and thickness during pollen tube growth in plants.One sentence summaryPollen-specific RALFs interact at different binding affinities with receptor kinases, GPI-anchored proteins and cell wall proteins to regulate cell wall integrity during pollen tube growth in maize.

Leseprobe Leseprobe 2 ----- Zusammenfassung Hochschulpolitische Forderungen nach hoheren Studienerfolgsquoten haben in den letzten Jahren zu vielfaltigen Masnahmen gefuhrt, die den Ubergang an die Hochschule unterstutzen sollen. Zu den Wirkungen dieser Angebote fur den Studieneinstieg liegen bislang nur punktuelle Befunde fur einzelne Masnahmen und Erfolgsindikatoren vor. Fur einen naheren Einblick nutzt der vorliegende Beitrag einen qualitativen Ansatz und geht dem komplexen Wirkungszusammenhang von Studieneinstiegsangeboten aus der Sicht von Studierenden nach. Im Zentrum der auf 19 Leitfadeninterviews basierenden Fallstudie steht dabei die Frage, wie die Teilnahme an Studieneinstiegsangeboten zur Bewaltigung von Studienanforderungen beitragt. Mittels qualitativer Inhaltsanalyse werden unterschiedliche Wirkungsweisen rekonstruiert, die sich sowohl aus den individuellen Nutzungsformen als auch aus den Gestaltungsmerkmalen von Studieneinstiegsangeboten ergeben. Die Ergebnisse liefern nicht nur Hinweise fur weiterfuhrende Mixed-Methods-Studien, sondern legen fur die Praxis nahe, den unterschiedlichen Nutzungsformen mit einem breiten Spektrum an Studieneinstiegsangeboten Rechnung zu tragen. Schlusselworter: Studieneingangsphase, Studienanforderungen, Studieneinstiegsangebote, Wirkungsweisen, Leitfadeninterviews, Qualitative Inhaltsanalyse ----- „And that has REALLY helped.“ – Effects of first-year support programmes in higher education Abstract Political demands to improve study success rates have lately led to a variety of programmes that seek to support the transition to higher education. So far though, findings on the effects of these programmes are limited to selected interventions and single success factors. To gain further insights, the present study adopts a qualitative approach and explores the complex effects of first year support programmes from the students’ perspective. Based on 19 semi-structured interviews, the casestudy examines how the participation in first-year support programmes helps students to handle the formal and informal requirements that are characteristic for the first year. Thematic analysis reveals that the students experience different types of effects that depend on both, the individual ways in which students use the programmes and the particular design of the programmes. The findings may not only contribute to future mixed-methods studies but also suggest that a broad range of support programmes is needed in order to meet the students’ individual ways of using first-year support. Keywords: Transition to higher education, first-year requirements, support programmes, effects, semistructured interviews, thematic analysis ----- Bibliographie: Bosse, Elke/Mergner, Julia: „Und das hat RICHTIG geholfen.“ − Wirkungsweisen von Angeboten fur den Studieneinstieg, ZeHf – Zeitschrift fur empirische Hochschulforschung, 2-2019, S. 105-122. https://doi.org/10.3224/zehf.v3i2.02

Proteins are intimately involved in executing and controlling virtually all cellular processes. To understand the molecular mechanisms that underlie plant phenotypes, it is essential to investigate protein expression, interactions, and modifications, to name a few. The proteome is highly dynamic in time and space, and a plethora of protein modifications, protein interactions, and network constellations are at play under specific conditions and developmental stages. Analysis of proteomes aims to characterize the entire protein complement of a particular cell type, tissue, or organism—a challenging task, given the dynamic nature of the proteome. Modern mass spectrometry–based proteomics technology can be used to address this complexity at a system-wide scale by the global identification and quantification of thousands of proteins. In this review, we present current methods and technologies employed in mass spectrometry–based proteomics and provide examples of dynamic changes in the plant proteome elucidated by proteomic approaches.

Plant growth and development are modulated by both biotic and abiotic stress. Increasing evidence suggests that cellular integration of the corresponding signals occurs within preformed hubs at the plasma membrane called nanodomains. These membrane sub-compartments are organized by multivalent molecular scaffold proteins, such as remorins. Here, we demonstrate that Group 1 remorins form a hetero-oligomeric complex at the plasma membrane. While these remorins are functionally redundant for some pathways their multivalency also allows the recruitment of specific interaction partners. One of them, the receptor-like kinase REMORIN-INTERACTING RECEPTOR 1 (RIR1), that acts redundantly with the closely related receptor NILR2, is specifically recruited by REM1.2 in a phosphorylation-dependent manner. Overlapping developmental phenotypes suggest that the REM/RIR complex regulates key developmental pathways.

Small RHO-type G-proteins act as signaling hubs and master regulators of polarity in eukaryotic cells. Their activity is tightly controlled, as defective RHO signaling leads to aberrant growth and developmental defects. Two major processes regulate G-protein activity: canonical shuttling between different nucleotide bound states and posttranslational modification (PTM), of which the latter can support or suppress RHO signaling, depending on the individual PTM. In plants, regulation of Rho of plants (ROPs) signaling activity has been shown to act through nucleotide exchange and GTP hydrolysis, as well as through lipid modification, but there is little data available on phosphorylation or ubiquitination of ROPs. Hence, we applied proteomic analyses to identify PTMs of the barley ROP RACB. We observed in vitro phosphorylation by barley ROP binding kinase 1 and in vivo ubiquitination of RACB. Comparative analyses of the newly identified RACB phosphosites and human RHO protein phosphosites revealed conservation of modified amino acid residues, but no overlap of actual phosphorylation patterns. However, the identified RACB ubiquitination site is conserved in all ROPs from Hordeum vulgare, Arabidopsis thaliana and Oryza sativa and in mammalian Rac1 and Rac3. Point mutation of this ubiquitination site leads to stabilization of RACB. Hence, this highly conserved lysine residue may regulate protein stability across different kingdoms.

Plants are essential for life and are extremely diverse organisms with unique molecular capabilities(1). Here we present a quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana. Our analysis provides initial answers to how many genes exist as proteins (more than 18,000), where they are expressed, in which approximate quantities (a dynamic range of more than six orders of magnitude) and to what extent they are phosphorylated (over 43,000 sites). We present examples of how the data may be used, such as to discover proteins that are translated from short open-reading frames, to uncover sequence motifs that are involved in the regulation of protein production, and to identify tissue-specific protein complexes or phosphorylation-mediated signalling events. Interactive access to this resource for the plant community is provided by the ProteomicsDB and ATHENA databases, which include powerful bioinformatics tools to explore and characterize Arabidopsis proteins, their modifications and interactions.A quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana provides a valuable resource for plant research.

The cullin-RING E3 ligases (CRLs) regulate diverse cellular processes in all eukaryotes. CRL activity is controlled by several proteins or protein complexes, including NEDD8, CAND1, and the CSN Recently, a mammalian protein called Glomulin (GLMN) was shown to inhibit CRLs by binding to the RING BOX (RBX1) subunit and preventing binding to the ubiquitin-conjugating enzyme. Here, we show that Arabidopsis ABERRANT LATERAL ROOT FORMATION4 (ALF4) is an ortholog of GLMN The alf4 mutant exhibits a phenotype that suggests defects in plant hormone response. We show that ALF4 binds to RBX1 and inhibits the activity of SCFTIR1, an E3 ligase responsible for degradation of the Aux/IAA transcriptional repressors. In vivo, the alf4 mutation destabilizes the CUL1 subunit of the SCF Reduced CUL1 levels are associated with increased levels of the Aux/IAA proteins as well as the DELLA repressors, substrate of SCFSLY1 We propose that the alf4 phenotype is partly due to increased levels of the Aux/IAA and DELLA proteins.

ProteomicsDB (https://www.ProteomicsDB.org) started as a protein-centric in-memory database for the exploration of large collections of quantitative mass spectrometry-based proteomics data. The data types and contents grew over time to include RNA-Seq expression data, drug-target interactions and cell line viability data. In this manuscript, we summarize new developments since the previous update that was published in Nucleic Acids Research in 2017. Over the past two years, we have enriched the data content by additional datasets and extended the platform to support protein turnover data. Another important new addition is that ProteomicsDB now supports the storage and visualization of data collected from other organisms, exemplified by Arabidopsis thaliana. Due to the generic design of ProteomicsDB, all analytical features available for the original human resource seamlessly transfer to other organisms. Furthermore, we introduce a new service in ProteomicsDB which allows users to upload their own expression datasets and analyze them alongside with data stored in ProteomicsDB. Initially, users will be able to make use of this feature in the interactive heat map functionality as well as the drug sensitivity prediction, but ultimately will be able to use all analytical features of ProteomicsDB in this way.

We have used a mass spectrometry-based proteomic approach to compile an atlas of the thermal stability of 48,000 proteins across 13 species ranging from archaea to humans and covering melting temperatures of 30–90 °C. Protein sequence, composition and size affect thermal stability in prokaryotes and eukaryotic proteins show a nonlinear relationship between the degree of disordered protein structure and thermal stability. The data indicate that evolutionary conservation of protein complexes is reflected by similar thermal stability of their proteins, and we show examples in which genomic alterations can affect thermal stability. Proteins of the respiratory chain were found to be very stable in many organisms, and human mitochondria showed close to normal respiration at 46 °C. We also noted cell-type-specific effects that can affect protein stability or the efficacy of drugs. This meltome atlas broadly defines the proteome amenable to thermal profiling in biology and drug discovery and can be explored online at http://meltomeatlas.proteomics.wzw.tum.de:5003/ and http://www.proteomicsdb.org. The meltome atlas compiles the thermal stability of 48,000 proteins across 13 species ranging from archaea to humans, providing a resource for analyzing protein stability in the context of function and interactions.

Plant growth and development are regulated by a tightly controlled interplay between cell division, cell expansion and cell differentiation during the entire plant life cycle from seed germination to maturity and seed propagation. To explore some of the underlying molecular mechanisms in more detail, we selected different aerial tissue types of the model plant Arabidopsis thaliana, namely rosette leaf, flower and silique/seed and performed proteomic, phosphoproteomic and transcriptomic analyses of sequential growth stages using tandem mass tag-based mass spectrometry and RNA sequencing. With this exploratory multi-omics dataset, development dynamics of photosynthetic tissues can be investigated from different angles. As expected, we found progressive global expression changes between growth stages for all three omics types and often but not always corresponding expression patterns for individual genes on transcript, protein and phosphorylation site level. The biggest difference between proteomic- and transcriptomic-based expression information could be observed for seed samples. Proteomic and transcriptomic data is available via ProteomeXchange and ArrayExpress with the respective identifiers PXD018814 and E-MTAB-7978., Measurement(s) transcriptome • Proteome • Phosphoproteome Technology Type(s) RNA sequencing • liquid chromatography-tandem mass spectrometry Factor Type(s) organism part • growth stage Sample Characteristic - Organism Arabidopsis thaliana Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12998993

The phytohormone abscisic acid (ABA) regulates plant responses to abiotic stress, such as drought and high osmotic conditions. The multitude of functionally redundant components involved in ABA signaling poses a major challenge for elucidating individual contributions to the response selectivity and sensitivity of the pathway. Here, we reconstructed single ABA signaling pathways in yeast for combinatorial analysis of ABA receptors and coreceptors, downstream-acting SnRK2 protein kinases, and transcription factors. The analysis shows that some ABA receptors stimulate the pathway even in the absence of ABA and that SnRK2s are major determinants of ABA responsiveness by differing in the ligand-dependent control. Five SnRK2s, including SnRK2.4 known to be active under osmotic stress in plants, activated ABA-responsive transcription factors and were regulated by ABA receptor complexes in yeast. In the plant tissue, SnRK2.4 and ABA receptors competed for coreceptor interaction in an ABA-dependent manner consistent with a tight integration of SnRK2.4 into the ABA signaling pathway. The study establishes the suitability of the yeast system for the dissection of core signaling cascades and opens up future avenues of research on ligand-receptor regulation.

The evolutionarily conserved 8-kD protein NEDD8 (NEURAL PRECURSOR CELL EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED8) belongs to the family of ubiquitin-like modifiers. Like ubiquitin, NEDD8 is conjugated to and deconjugated from target proteins. Many targets and functions of ubiquitylation have been described; by contrast, few targets of NEDD8 have been identified. In plants as well as in non-plant organisms, the cullin subunits of cullin-RING E3 ligases are NEDD8 conjugates with a demonstrated functional role for the NEDD8 modification. The existence of other non-cullin NEDD8 targets has generally been questioned. NEDD8 is translated as a precursor protein and proteolytic processing exposes a C-terminal glycine required for NEDD8 conjugation. In animals and yeast, DENEDDYLASE1 (DEN1) processes NEDD8. Here, we show that mutants of a DEN1 homolog from Arabidopsis thaliana have no detectable defects in NEDD8 processing but do accumulate a broad range of NEDD8 conjugates; this provides direct evidence for the existence of non-cullin NEDD8 conjugates. We further identify AUXIN RESISTANT1 (AXR1), a subunit of the heterodimeric NEDD8 E1 activating enzyme, as a NEDD8-modified protein in den1 mutants and wild type and provide evidence that AXR1 function may be compromised in the absence of DEN1 activity. Thus, in plants, neddylation may serve as a regulatory mechanism for cullin and non-cullin proteins.

The cullin‐RING E3 ligases (CRLs) regulate diverse cellular processes in all eukaryotes. CRL activity is controlled by several proteins or protein complexes, including NEDD8, CAND1, and the CSN. Recently, a mammalian protein called Glomulin (GLMN) was shown to inhibit CRLs by binding to the RING BOX (RBX1) subunit and preventing binding to the ubiquitin‐conjugating enzyme. Here, we show that Arabidopsis ABERRANT LATERAL ROOT FORMATION4 (ALF4) is an ortholog of GLMN. The alf4 mutant exhibits a phenotype that suggests defects in plant hormone response. We show that ALF4 binds to RBX1 and inhibits the activity of SCFTIR 1, an E3 ligase responsible for degradation of the Aux/IAA transcriptional repressors. In vivo, the alf4 mutation destabilizes the CUL1 subunit of the SCF. Reduced CUL1 levels are associated with increased levels of the Aux/IAA proteins as well as the DELLA repressors, substrate of SCFSLY 1. We propose that the alf4 phenotype is partly due to increased levels of the Aux/IAA and DELLA proteins.

In eukaryotes, the conjugation of the ubiquitin-like protein NEDD8 onto protein targets is an important post-translational modification. The best understood neddylation targets are the cullins, scaffold subunits of E3 ubiquitin ligases, where neddylation as well as deneddylation, facilitated by the protease activity of the CSN (COP9 signalosome), are required to control ubiquitin ligase assembly, function, and ultimately substrate degradation. Little is known about the role of other deneddylating enzymes besides CSN and the role of neddylation and deneddylation of their substrates. We previously characterized Arabidopsis thaliana mutants with defects in the conserved NEDD8-specific protease DEN1 (DENEDDYLASE1). These mutants display only subtle growth phenotypes despite the strong accumulation of a broad range of neddylated proteins. Specifically, we identified AXR1 (AUXIN-RESISTANT1), a subunit of the heterodimeric NAE (E1 NEDD8-ACTIVATING ENZYME), as highly neddylated in den1 mutants. Here, we examined the mechanism and consequences of AXR1 neddylation in more detail. We find that AXR1 as well as other neddylation enzymes are autoneddylated at multiple lysines. NAE autoneddylation can be linked to reduced NCE (E2 NEDD8-CONJUGATING ENZYME) NEDD8 thioester levels, either by critically reducing the pool of free NEDD8 or by reducing NAE activity. In planta, increasing NEDD8 gene dosage is sufficient to suppress den1 mutant phenotypes. We therefore suggest that DEN1 serves to recover diverted NEDD8 moieties from autoneddylated NAE subunits, and possibly also other neddylated proteins, to maintain NEDD8 pathway activity toward other NEDD8-dependent processes such as cullin E3 ligase regulation.

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Embryogenesis in flowering plants is initiated by an asymmetric zygote division, generating two daughter cells that are the precursors of different cell lineages. Little is known about the molecular players regulating activation and progression of zygote development, establishment of asymmetry, and the plant-specific process of cell-plate formation. Here, we report the function of the ubiquitin-like modifier DiSUMO-LIKE (DSUL) for early embryo development in maize. Introducing a DSUL-RNAi construct by sperm cells affects cytokinesis generating non-separated zygotic daughter nuclei or multinucleate embryonic cells lacking cell plates. DSUL accumulates in the cytoplasm partly in granules, in the nucleus, as well as in the cell division zone. The enzymatic DSULyation cascade involves maturation and the same enzymatic machinery for activation and conjugation as was previously shown for SUMO1. Identification of DSUL targets suggests predominant roles of DSULylation in regulation of cytoplasmic RNA metabolism as well as in cell-cycle progression and cell-plate formation. A comparison of DSUL and SUMO1 localization during the cell cycle and of their substrates indicates strong functional diversification between these two SUMO family modifiers.

Integrated analysis of genomes, transcriptomes, proteomes and drug responses of cancer cell lines (CCLs) is an emerging approach to uncover molecular mechanisms of drug action. We extend this paradigm to measuring proteome activity landscapes by acquiring and integrating quantitative data for 10,000 proteins and 55,000 phosphorylation sites (p-sites) from 125 CCLs. These data are used to contextualize proteins and p-sites and predict drug sensitivity. For example, we find that Progesterone Receptor (PGR) phosphorylation is associated with sensitivity to drugs modulating estrogen signaling such as Raloxifene. We also demonstrate that Adenylate kinase isoenzyme 1 (AK1) inactivates antimetabolites like Cytarabine. Consequently, high AK1 levels correlate with poor survival of Cytarabine-treated acute myeloid leukemia patients, qualifying AK1 as a patient stratification marker and possibly as a drug target. We provide an interactive web application termed ATLANTiC (http://atlantic.proteomics.wzw.tum.de), which enables the community to explore the thousands of novel functional associations generated by this work., Proteome activity has a major role in cancer progression and response to drugs. Here, the authors use comprehensive proteomic and phosphoproteomic data, in conjunction with drug-sensitivity screens, to generate a community resource consisting of landscapes of pathway and kinase activity across different cell lines