Search

Given the plasticity of hematopoietic stem and progenitor cells, multiple routes of differentiation must be blocked in the the pathogenesis of acute myeloid leukemia, the molecular basis of which is incompletely understood. We report that posttranscriptional repression of the transcription factor ARID3A by miR-125b is a key event in the pathogenesis of acute megakaryoblastic leukemia (AMKL). AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndrome are at a high risk of developing the disease. The results of our study showed that chromosome 21–encoded miR-125b synergizes with Gata1s to drive leukemogenesis in this context. Leveraging forward and reverse genetics, we uncovered Arid3a as the main miR-125b target behind this synergy. We demonstrated that, during normal hematopoiesis, this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFβ-induced apoptosis and cell cycle arrest in complex with SMAD2/3. Although Gata1s mutations perturb erythroid differentiation and induce hyperproliferation of megakaryocytic progenitors, intact ARID3A expression assures their megakaryocytic differentiation and growth restriction. Upon knockdown, these tumor suppressive functions are revoked, causing a blockade of dual megakaryocytic/erythroid differentiation and subsequently of AMKL. Inversely, restoring ARID3A expression relieves the arrest of megakaryocytic differentiation in AMKL patient-derived xenografts. This work illustrates how mutations in lineage-determining transcription factors and perturbation of posttranscriptional gene regulation can interact to block multiple routes of hematopoietic differentiation and cause leukemia. In AMKL, surmounting this differentiation blockade through restoration of the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease.

In order to reduce nitrate in vivo, the spore-specific respiratory nitrate reductase, Nar1, of Streptomyces coelicolor relies on an active cytochrome bcc-aa3 oxidase supercomplex (bcc-aa3 supercomplex). This suggests that membrane-associated Nar1, comprising NarG1, NarH1, and NarI1 subunits, might not act as a classical menaquinol oxidase but could either receive electrons from the bcc-aa3 supercomplex, or require the supercomplex to stabilize the reductase in the membrane to allow it to function. To address the biochemical basis for this dependence on the bcc-aa3 supercomplex, we purified two different Strep-tagged variants of Nar1 and enriched the native enzyme complex from spore extracts using different chromatographic and electrophoretic procedures. Polypeptides associated with the isolated Nar1 complexes were identified using mass spectrometry and included components of the bcc-aa3 supercomplex, along with an alternative, spore-specific cytochrome b component, QcrB3. Surprisingly, we also co-enriched the Nar3 enzyme with Nar1 from the wild-type strain of S. coelicolor. Two differentially migrating active Nar1 complexes could be identified after clear native polyacrylamide gel electrophoresis; these had masses of approximately 450 and 250 kDa. The distribution of active Nar1 in these complexes was influenced by the presence of cytochrome bd oxidase and by QcrB3; the presence of the latter shifted Nar1 into the larger complex. Together, these data suggest that several respiratory complexes can associate in the spore membrane, including Nar1, Nar3, and the bcc-aa3 supercomplex. Moreover, these findings provide initial support for the hypothesis that Nar1 and the bcc-aa3 supercomplex physically associate.

The combination of cross-linking/mass spectrometry (XL-MS) and ion mobility is still underexplored for conducting protein conformational and protein-protein interaction studies. We present a method for analyzing cross-linking mixtures on a timsTOF Pro mass spectrometer that allows separating ions based on their gas phase mobilities. Cross-linking was performed with three urea-based MS-cleavable cross-linkers that deliver distinct fragmentation patterns for cross-linked species upon collisional activation. The discrimination of cross-linked species from non-cross-linked peptides was readily performed based on their collisional cross sections. We demonstrate the general feasibility of our combined XL-MS/ion mobility approach for three protein systems of increasing complexity: (i) Bovine serum albumin, (ii) E. coli ribosome, and (iii) HEK293T cell nuclear lysates. We identified a total of 508 unique cross-linking sites for BSA, 868 for the E. coli ribosome, and 1,623 unique cross-links for nuclear lysates, corresponding to 1,088 intra- and 535 interprotein interactions and yielding 564 distinct protein-protein interactions. Our results underline the strength of combining XL-MS with ion mobility not only for deriving 3D-structures of single proteins, but also for performing system-wide protein interaction studies.TOC Graphic

Given the plasticity of hematopoietic stem/progenitor cells, multiple routes of differentiation must be blocked during acute myeloid leukemia pathogenesis – the molecular basis of which is incompletely understood. Here we report that post-transcriptional repression of transcription factor ARID3A by miR-125b is a key event in megakaryoblastic leukemia (AMKL) pathogenesis. AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndrome are at a high risk of developing this disease. We show that chromosome 21-encoded miR-125b synergizes with Gata1s to drive leukemogenesis in this context. Leveraging forward and reverse genetics, we uncover Arid3a as the main miR-125b target underlying this synergy. We demonstrate that during normal hematopoiesis this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFβ-induced apoptosis and cell cycle arrest in complex with SMAD2/3. While Gata1s mutations perturb erythroid differentiation and induce hyperproliferation of megakaryocytic progenitors, intact ARID3A expression assures their megakaryocytic differentiation and growth restriction. Upon knockdown, these tumor suppressive functions are revoked, causing a dual megakaryocytic/erythroid differentiation blockade and subsequently AMKL. Inversely, restoring ARID3A expression relieves the megakaryocytic differentiation arrest in AMKL patient-derived xenografts. This work illustrates how mutations in lineage-determining transcription factors and perturbation of post-transcriptional gene regulation interplay to block multiple routes of hematopoietic differentiation and cause leukemia. Surmounting this differentiation blockade in megakaryoblastic leukemia by restoring the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease.Key pointsRepression of megakaryocytic transcription factor ARID3A by miR-125b synergizes with GATA1s to induce leukemiaRestoring ARID3A expression relieves megakaryocytic differentiation arrest in megakaryoblastic leukemiaGraphical abstract

Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing human acid-sensing ion channel 1a (hASIC1a) variants in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay, and live-cell crosslinking provides insight into the hASIC1a–psalmotoxin 1 (PcTx1) interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells., This study describes a method to rapidly screen hundreds of ion channel variants containing non-canonical amino acids. A proof-of-principle introducing photocrosslinking non-canonical amino acids into the human ion channel hASIC1a shows how this approach can provide insights into function and pharmacology.

Epithelial-to-mesenchymal transition (EMT) is a hallmark of aggressive, mesenchymal-like high-grade serous ovarian carcinoma (HG-SOC). The SRC kinase is a key driver of cancer-associated EMT promoting adherens junction (AJ) disassembly by phosphorylation-driven internalization and degradation of AJ proteins. Here we show, that the IGF2 mRNA binding protein 1 (IGF2BP1) is up-regulated in mesenchymal-like HG-SOC and promotes SRC activation by a previously unknown protein-ligand-induced, but RNA-independent mechanism. IGF2BP1-driven invasive growth of ovarian cancer cells essentially relies on the SRC-dependent disassembly of AJs. Concomitantly, IGF2BP1 enhances ERK2 expression in a RNA-binding dependent manner. Together this reveals a post-transcriptional mechanism of interconnected stimulation of SRC/ERK signaling in ovarian cancer cells. The IGF2BP1-SRC/ERK2 axis is targetable by the SRC-inhibitor saracatinib and MEK-inhibitor selumetinib. However, due to IGF2BP1-directed stimulation only combinatorial treatment effectively overcomes the IGF2BP1-promoted invasive growth in 3D culture conditions as well as intraperitoneal mouse models. In conclusion, we reveal an unexpected role of IGF2BP1 in enhancing SRC/MAPK-driven invasive growth of ovarian cancer cells. This provides a rational for the therapeutic benefit of combinatorial SRC/MEK inhibition in mesenchymal-like HG-SOC.Graphical Abstract

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their AUX/IAA targets perceive the phytohormone auxin. The F-box protein TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, by adopting biochemical, structural proteomics and in vivo approaches we unveil how flexibility in AUX/IAAs and regions in TIR1 affect their conformational ensemble allowing surface accessibility of degrons. We resolve TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron’s vicinity, cooperatively position AUX/IAAs on TIR1. We identify essential residues at the TIR1 N- and C-termini, which provide non-native interaction interfaces with IDRs and the folded PB1 domain of AUX/IAAs. We thereby establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for a multiplicity of functional states., Auxin-mediated recruitment of AUX/IAAs by the F-box protein TIR1 prompts rapid AUX/IAA ubiquitylation and degradation. By resolving auxin receptor topology, the authors show that intrinsically disordered regions near the degrons of two Aux/IAA proteins reinforce complex assembly and position Aux/IAAs for ubiquitylation.

In the modern world, crop plants represent a major source of daily consumed foods. Among them, cereals and legumes — i.e. the crops accumulating oils, carbohydrates and proteins in their seeds — dominate in European agriculture, tremendously impacting global protein consumption and biodiesel production. Therefore, the seeds of crop plants attract the special attention of biologists, biochemists, nutritional physiologists and food chemists. Seed development and germination, as well as age- and stress-related changes in their viability and nutritional properties, can be addressed by a variety of physiological and biochemical methods. In this context, the methods of functional genomics can be applied to address characteristic changes in seed metabolism, which can give access to stress-resistant genotypes. Among these methods, proteomics is one of the most effective tools, allowing mining metabolism changes on the protein level. Here we discuss the main methodological approaches of seed proteomics in the context of physiological changes related to environmental stress and ageing. We provide a comprehensive comparison of gel- and chromatographybased approaches with a special emphasis on advantages and disadvantages of both strategies in characterization of the seed proteome.

Streptomyces coelicolor A3(2), an obligately aerobic, oxidase-positive, and filamentous soil bacterium, lacks a soluble cytochrome c in its respiratory chain, having instead a membrane-associated diheme c-type cytochrome, QcrC. This necessitates complex formation to allow electron transfer between the cytochrome bcc and aa3 oxidase respiratory complexes. Combining genetic complementation studies with in-gel cytochrome oxidase activity staining, we demonstrate that the complete qcrCAB-ctaCDFE gene locus on the chromosome, encoding, respectively, the bcc and aa3 complexes, is required to manifest a cytochrome oxidase enzyme activity in both spores and mycelium of a qcr-cta deletion mutant. Blue-native-PAGE identified a cytochrome aa3 oxidase complex of approximately 270 kDa, which catalyzed oxygen-dependent diaminobenzidine oxidation without the requirement for exogenously supplied cytochrome c, indicating association with QcrC. Furthermore, higher molecular mass complexes were identified upon addition of soluble cytochrome c, suggesting the supercomplex is unstable and readily dissociates into subcomplexes lacking QcrC. Immunological and mass spectrometric analyses of active, high-molecular mass oxidase-containing complexes separated by clear-native PAGE identified key subunits of both the bcc complex and the aa3 oxidase, supporting supercomplex formation. Our data also indicate that the cytochrome b QcrB of the bcc complex is less abundant in spores compared with mycelium.

Glycation is a post-translational modification resulting from the interaction of protein amino and guanidino groups with carbonyl compounds. Initially, amino groups react with reducing carbohydrates, yielding Amadori and Heyns compounds. Their further degradation results in formation of advanced glycation end products (AGEs), also originating from α-dicarbonyl products of monosaccharide autoxidation and primary metabolism. In mammals, AGEs are continuously formed during the life of the organism, accumulate in tissues, are well-known markers of aging, and impact age-related tissue stiffening and atherosclerotic changes. However, the role of AGEs in age-related molecular alterations in plants is still unknown. To fill this gap, we present here a comprehensive study of the age-related changes in the Arabidopsis thaliana glycated proteome, including the proteins affected and specific glycation sites therein. We also consider the qualitative and quantitative changes in glycation patterns in terms of the general metabolic background, pathways of AGE formation, and the status of plant anti-oxidative/anti-glycative defense. Although the patterns of glycated proteins were only minimally influenced by plant age, the abundance of 96 AGE sites in 71 proteins was significantly affected in an age-dependent manner and clearly indicated the existence of age-related glycation hot spots in the plant proteome. Homology modeling revealed glutamyl and aspartyl residues in close proximity (less than 5 A) to these sites in three aging-specific and eight differentially glycated proteins, four of which were modified in catalytic domains. Thus, the sites of glycation hot spots might be defined by protein structure that indicates, at least partly, site-specific character of glycation.

Translational repression of maternal mRNAs is an essential regulatory mechanism during early embryonic development. Repression of theDrosophila nanosmRNA, required for the formation of the anterior-posterior body axis, depends on the protein Smaug binding to two Smaug recognition elements (SREs) in thenanos3’ UTR. In a comprehensive mass-spectrometric analysis of the SRE-dependent repressor complex, we identified Smaug, Cup, Me31B, Trailer hitch, eIF4E and PABPC, in agreement with earlier data. As a novel component, the RNA-dependent ATPase Belle (DDX3) was found, and its involvement in deadenylation and repression ofnanoswas confirmedin vivo. Smaug, Cup and Belle bound stoichiometrically to the SREs, independently of RNA length. Binding of Me31B and Tral was also SRE-dependent, but their amounts were proportional to the length of the RNA and equimolar to each other. We suggest that ‘coating’ of the RNA by a Me31B•Tral complex may be at the core of repression.

Previous studies have shown the benefits of the amine-reactive, CID-MS/MS-cleavable cross-linker disuccinimidyl dibutyric urea (DSBU) for structural proteomics studies via cross-linking/MS (XL-MS). To further facilitate the automation of XL-MS experiments, we synthesized a deuterated (D12) version of the DSBU cross-linker combining the advantages of MS-cleavable linkers and isotope labeling. The rationale of conducting XL-MS with a mixture of unlabeled and stable isotope-labeled DSBU is to obtain characteristic mass differences at the MS level indicating cross-linked species. These cross-linked species can then be selected for fragmentation by collisional activation. At the MS/MS level, the characteristic 26-u doublets arising from cleavage of the central urea group in DSBU confirm the amino acid sequences of cross-linked peptides as well as the exact cross-linking sites. D12-labeled DSBU was tested on three systems with increasing complexity: (i) bovine serum albumin as purified protein, (ii) Escherichia coli ribosome as large, multimeric protein assembly, and (iii) Drosophila embryo extract as complete proteome. We demonstrate the benefits arising from the use of isotope-labeled DSBU for an automated assignment of cross-linked products. Combining isotope labeling and MS cleavability in one cross-linker resulted in higher cross-link identification numbers especially for highly complex protein mixtures.

Exploring the interactions between the Ca2+ binding protein calmodulin (CaM) and its target proteins remains a challenging task. Members of the Munc13 protein family play an essential role in short-term synaptic plasticity, modulated via the interaction with CaM at the presynaptic compartment. In this study, we focus on the bMunc13-2 isoform expressed in the brain, as strong changes in synaptic transmission were observed upon its mutagenesis or deletion. The CaM−bMunc13-2 interaction was previously characterized at the molecular level using short bMunc13-2-derived peptides only, revealing a classical 1−5−10 CaM binding motif. Using larger protein constructs, we have now identified for the first time a novel and unique CaM binding site in bMunc13-2 that contains an N-terminal extension of a classical 1−5−10 CaM binding motif. We characterize this motif using a range of biochemical and biophysical methods and highlight its importance for the CaM−bMunc13-2 interaction.

Rapid and comprehensive genetic sequencing has shed light on the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and allowed timely implementation of PCR tests to determine the presence of viral RNA. PCR tests for SARS-CoV-2 are some way from being reliably qualitative and will never indicate how the disease might progress in an individual. As COVID-19 becomes endemic, there is a concomitant need for accurate serological assays to detect antibodies to SARS-CoV-2 antigens and ultimately tests for prognostic markers to target treatment options.1,2 With this considerable genetic insight, and the emerging structural information, comes associated questions regarding the molecular descriptors that contribute to disease progression, especially when we consider spread across different populations. The power of mass spectrometry to generate rapid, precise, and reproducible diagnostic information that complements genomic information and accelerates our understanding of the disease, is now becoming a reality.

Introduction: Calmodulin (CaM) is a highly conserved Ca2+-binding protein that is exceptionally abundant in the brain. In the presynaptic compartment of neurons, CaM transduces changes in Ca2+ concentration into the regulation of synaptic transmission dynamics.Areas covered: We review selected literature including published CaM interactor screens and outline established and candidate presynaptic CaM targets. We present a workflow of biochemical and structural proteomic methods that were used to identify and characterize the interactions between CaM and Munc13 proteins. Finally, we outline the potential of ion mobility-mass spectrometry (IM-MS) for conformational screening and of protein-protein cross-linking for the structural characterization of CaM complexes.Expert commentary: Cross-linking/MS and native MS can be applied with considerable throughput to protein mixtures under near-physiological conditions, and thus effectively complement high-resolution structural biology techniques. Experimenta...

Chemical cross-linking/mass spectrometry (MS) is gradually developing into a routine method to investigate protein conformation and to decipher protein interaction networks. To increase identification rates of the frequently low abundant cross-linked products in LC/MS/MS experiments, fast and reliable sample preparation protocols are indispensable. We present simplified solid phase extraction methods using C18/SCX StageTips and mixed-mode OASIS MCX cartridges for a single-step enrichment of cross-linked products prior to LC/MS/MS analysis. Our improved protocols result in 3.5 to 4.6 times higher numbers of cross-link identifications for the model protein bovine serum albumin compared to non-processed samples.

Highlight Osmotic stress enhances the rate of protein glycation and monosaccharide autoxidation is the main pathway., Among the environmental alterations accompanying oncoming climate changes, drought is the most important factor influencing crop plant productivity. In plants, water deficit ultimately results in the development of oxidative stress and accumulation of osmolytes (e.g. amino acids and carbohydrates) in all tissues. Up-regulation of sugar biosynthesis in parallel to the increasing overproduction of reactive oxygen species (ROS) might enhance protein glycation, i.e. interaction of carbonyl compounds, reducing sugars and α-dicarbonyls with lysyl and arginyl side-chains yielding early (Amadori and Heyns compounds) and advanced glycation end-products (AGEs). Although the constitutive plant protein glycation patterns were characterized recently, the effects of environmental stress on AGE formation are unknown so far. To fill this gap, we present here a comprehensive in-depth study of the changes in Arabidopsis thaliana advanced glycated proteome related to osmotic stress. A 3 d application of osmotic stress revealed 31 stress-specifically and 12 differentially AGE-modified proteins, representing altogether 56 advanced glycation sites. Based on proteomic and metabolomic results, in combination with biochemical, enzymatic and gene expression analysis, we propose monosaccharide autoxidation as the main stress-related glycation mechanism, and glyoxal as the major glycation agent in plants subjected to drought.

We investigated the interaction network of human PKD2 in the cytosol and in Golgi-enriched subcellular protein fractions by an affinity enrichment strategy combined with chemical cross-linking/mass spectrometry (MS). Analysis of the subproteomes revealed the presence of distinct proteins in the cytosolic and Golgi fractions. The covalent fixation of transient or weak interactors by chemical cross-linking allowed capturing interaction partners that might otherwise disappear during conventional pull-down experiments. In total, 31 interaction partners were identified for PKD2, including glycogen synthase kinase-3 beta (GSK3B), 14-3-3 protein gamma (YWHAG), and the alpha isoform of 55 kDa regulatory subunit B of protein phosphatase 2A (PPP2R2A). Remarkably, the entire seven-subunit Arp2/3 complex (ARPC1B, ARPC2, ARPC3, ARPC4, ARPC5, ACTR3, ACTR2) as well as ARPC1A and ARPC5L, which are putative substitutes of ARPC1B and ARPC5, were identified. We provide evidence of a direct protein-protein interaction between PKD2 and Arp2/3. Our findings will pave the way for further structural and functional studies of PKD2 complexes, especially the PKD2/Arp2/3 interaction, to elucidate the role of PKD2 for transport processes at the trans-Golgi network. Data are available via ProteomeXchange with identifiers PXD003909 (enrichment from cytosolic fractions), PXD003913 (enrichment from Golgi fractions), and PXD003917 (subcellular fractionation).

Cleavage factor II (CF II) is a poorly characterized component of the multi-protein complex catalyzing 3’ cleavage and polyadenylation of mammalian mRNA precursors. We have reconstituted CF II as a heterodimer of hPcf11 and hClp1. The heterodimer is active in partially reconstituted cleavage reactions, whereas hClp1 by itself is not. Pcf11 moderately stimulates the RNA 5’ kinase activity of hClp1; the kinase activity is dispensable for RNA cleavage. CF II binds RNA with nanomolar affinity. Binding is mediated mostly by the two zinc fingers in the C-terminal region of hPcf11. RNA is bound without pronounced sequence-specificity, but extended G-rich sequences appear to be preferred. We discuss the possibility that CF II contributes to the recognition of cleavage/polyadenylation substrates through interaction with G-rich far-downstream sequence elements.

Pore-forming toxins (PFT) are virulence factors that transform from soluble to membrane-bound states. The Yersinia YaxAB system represents a family of binary α-PFTs with orthologues in human, insect, and plant pathogens, with unknown structures. YaxAB was shown to be cytotoxic and likely involved in pathogenesis, though the molecular basis for its two-component lytic mechanism remains elusive. Here, we present crystal structures of YaxA and YaxB, together with a cryo-electron microscopy map of the YaxAB complex. Our structures reveal a pore predominantly composed of decamers of YaxA–YaxB heterodimers. Both subunits bear membrane-active moieties, but only YaxA is capable of binding to membranes by itself. YaxB can subsequently be recruited to membrane-associated YaxA and induced to present its lytic transmembrane helices. Pore formation can progress by further oligomerization of YaxA–YaxB dimers. Our results allow for a comparison between pore assemblies belonging to the wider ClyA-like family of α-PFTs, highlighting diverse pore architectures., The Yersinia YaxAB system is a pore-forming toxin of so far unknown structure. Here authors present X-ray and cryo-EM to structures of individual subunits and of the YaxAB pore complex, and find that YaxA binds to membranes first and recruits YaxB for subsequent oligomerization.

Munc13 proteins are essential presynaptic regulators that mediate synaptic vesicle priming and play a role in the regulation of neuronal short-term synaptic plasticity. All four Munc13 isoforms share a common domain structure, including a calmodulin (CaM) binding site in their otherwise divergent N-termini. Here, we summarize recent results on the investigation of the CaM/Munc13 interaction. By combining chemical cross-linking, photoaffinity labeling, and mass spectrometry, we showed that all neuronal Munc13 isoforms exhibit similar CaM binding modes. Moreover, we demonstrated that the 1-5-8-26 CaM binding motif discovered in Munc13-1 cannot be induced in the classical CaM target skMLCK, indicating unique features of the Munc13 CaM binding motif.

Legume crops represent the major source of food protein and contribute to human nutrition and animal feeding. An essential improvement of their productivity can be achieved by symbiosis with beneficial soil microorganisms&mdash, rhizobia (Rh) and arbuscular mycorrhizal (AM) fungi. The efficiency of these interactions depends on plant genotype. Recently, we have shown that, after simultaneous inoculation with Rh and AM, the productivity gain of pea (Pisum sativum L) line K-8274, characterized by high efficiency of interaction with soil microorganisms (EIBSM), was higher in comparison to a low-EIBSM line K-3358. However, the molecular mechanisms behind this effect are still uncharacterized. Therefore, here, we address the alterations in pea seed proteome, underlying the symbiosis-related productivity gain, and identify 111 differentially expressed proteins in the two lines. The high-EIBSM line K-8274 responded to inoculation by prolongation of seed maturation, manifested by up-regulation of proteins involved in cellular respiration, protein biosynthesis, and down-regulation of late-embryogenesis abundant (LEA) proteins. In contrast, the low-EIBSM line K-3358 demonstrated lower levels of the proteins, related to cell metabolism. Thus, we propose that the EIBSM trait is linked to prolongation of seed filling that needs to be taken into account in pulse crop breeding programs. The raw data have been deposited to the ProteomeXchange with identifier PXD013479.

Calmodulin (CaM) is a highly conserved intracellular Ca(2+)-binding protein that exerts important functions in many cellular processes. Prominent examples of CaM-regulated proteins are adenylyl cyclases (ACs), which synthesize cAMP as a central second messenger. The interaction of ACs with CaM represents the link between Ca(2+)-signaling and cAMP-signaling pathways. Thereby, different AC isoforms stimulated by CaM, comprise diverse mechanisms of regulation by the Ca(2+) sensor. To extend the structural information about the detailed mechanisms underlying the regulation of AC8 by CaM, we employed an integrated approach combining chemical cross-linking and mass spectrometry with two peptides representing the CaM-binding regions of AC8. These experiments reveal that the structures of CaM/AC8 peptide complexes are similar to that of the CaM/skeletal muscle myosin light chain kinase peptide complex where CaM is collapsed around the target peptide that binds to CaM in an antiparallel orientation. Cross-linking experiments were complemented by investigating the binding of AC8 peptides to CaM thermodynamically with isothermal titration calorimetry. There were no hints on a complex, in which both AC8 peptides bind simultaneously to CaM, refining our current understanding of the interaction between CaM and AC8.

In vitro amyloid formation has been suggested to be a common property of any polypeptide chain depending on particular environmental conditions although in vivo amyloid fibril formation can be promoted by point mutations or triplet expansions. Here, we explored the influence of agitation on fibril formation of amyloidogenic alanine segments fused to Cold Shock Protein B (CspB) of Bacillus subtilis. While without agitation fibril formation was clearly dependent on the presence of an amyloidogenic alanine segment, fibril formation was independent of the amyloidogenic segment under agitation. Agitation even led to fibrillation of native CspB lacking the amyloidogenic segment. Furthermore, agitation not only influenced the kinetics of fibril formation, but also resulted in completely different fibril morphologies. These results indicate that experimental conditions can alter the region that undergoes a conformational change during in vitro fibrillation. Moreover, the data show that deductions from in vitro assays on in vivo fibril formation mechanisms are afflicted with a certain degree of uncertainty and therefore need to be cautiously discussed.

Combining the properties of a zero-length cross-linker with cleavability by tandem mass spectrometry (MS/MS) poses great advantages for protein structure analysis using the cross-linking/MS approach. These include a reliable, automated data analysis and the possibility to obtain short-distance information of protein 3D-structures. We introduce 1,1'-carbonyldiimidazole (CDI) as an easy-to-use and commercially available, low-cost reagent that ideally fulfils these features. CDI bridges primary amines and hydroxy groups in proteins with the lowest possible spacer length of one carbonyl unit (ca. 2.6 Å). The cross-linking reaction can be conducted under physiological conditions in the pH range between 7.2 and 8. Urea and carbamate cross-linked products are cleaved upon collisional activation during MS/MS experiments generating characteristic product ions, greatly improving the unambiguous identification of cross-links. Our innovative analytical concept is exemplified and applied for bovine serum albumin (BSA), wild-type tumor suppressor p53, an intrinsically disordered protein, and retinal guanylyl cyclase activating protein-2 (GCAP-2).

Tyrosine kinase inhibitors, such as erlotinib, display reliable responses and survival benefits for the treatment of human non-small cell lung cancer (NSCLC) patients. However, primary or acquired resistance limits their therapeutic success. In this study, we conducted in-depth mass spectrometric analyses of NSCLC cell secretomes. To identify secreted proteins that are differentially regulated in erlotinib-sensitive (PC-9) and -resistant (PC-9ER) NSCLC cell lines, SILAC experiments were performed. On average, 900 proteins were identified in each sample with low variations in the numbers of identified proteins. Fourteen proteins were found to be differently regulated among erlotinib-sensitive and -resistant NSCLC cell lines, with five proteins (tissue-type plasminogen activator, epidermal growth factor receptor, urokinase-type plasminogen activator, platelet-derived growth factor D, and myeloid-derived growth factor) showing the most prominent regulation. Tissue-type plasminogen activator (t-PA) was up to 10-times upregulated in erlotinib-resistant NSCLC cells compared with erlotinib-sensitive cells. T-PA is an established tumor marker for various cancer types and seems to be a promising prognostic marker to differentiate erlotinib-sensitive from erlotinib-resistant NSCLC cells. To gain further insights into t-PA-regulated pathways, a t-PA variant was expressed in E. coli cells and its interactions with proteins secreted from erlotinib-sensitive and -resistant NCSLC cells were studied by a combined affinity enrichment chemical cross-linking/mass spectrometry (MS) approach. Fourteen proteins were identified as potential t-PA interaction partners, deserving a closer inspection to unravel the mechanisms underlying erlotinib resistance in NSCLC cells.

Munc13s are presynaptic proteins that mediate synaptic vesicle priming and thereby control the size of the readily releasable pool of vesicles. During high synaptic activity, Munc13-1 and its closely related homolog, ubMunc13-2, bind Ca(2+)/calmodulin, resulting in enhanced priming activity and in changes of short-term synaptic plasticity characteristics. Here, we studied whether bMunc13-2 and Munc13-3, two remote isoforms of Munc13-1 with a neuronal subtype-specific expression pattern, mediate synaptic vesicle priming and regulate short-term synaptic plasticity in a Ca(2+)/calmodulin-dependent manner. We identified a single functional Ca(2+)/calmodulin binding site in these isoforms and provide structural evidence that all Munc13s employ a common mode of interaction with calmodulin despite the lack of sequence homology between their Ca(2+)/calmodulin binding sites. Electrophysiological analysis showed that, during high-frequency activity, Ca(2+)/calmodulin binding positively regulates the priming activity of bMunc13-2 and Munc13-3, resulting in an increase in the size of the readily releasable pool of vesicles and subsequently in strong short-term synaptic enhancement of neurotransmission. We conclude that Ca(2+)/calmodulin-dependent regulation of priming activity is structurally and functionally conserved in all Munc13 proteins, and that the composition of Munc13 isoforms in a neuron differentially controls its short-term synaptic plasticity characteristics.

Asymmetric dimethylation of arginine side chains is a common post-translational modification of eukaryotic proteins, which serves mostly to regulate protein-protein interactions. The modification is catalyzed by type I protein arginine methyltransferases, PRMT1 being the predominant member of the family. Determinants of substrate specificity of these enzymes are poorly understood. The Nuclear poly(A) binding protein 1 (PABPN1) is methylated by PRMT1 at 13 arginine residues located in RXR sequences in the protein's C-terminal domain. We have identified a preferred site for PRMT1-catalyzed methylation in PABPN1 and in a corresponding synthetic peptide. Variants of these substrates were analyzed by steady-state kinetic analysis and mass spectrometry. The data indicate that initial methylation is directed toward the preferred arginine residue by an N-terminally adjacent proline. Enhanced methylation upon peptide cyclization suggests that induction of a reverse turn structure is the basis for the ability of the respective proline residue to enable preferred methylation of the neighboring arginine residue, and this notion is supported by far-UV circular dichroism spectroscopy. We suggest that the formation of a reverse turn facilitates the access of arginine side chains to the active sites of PRMT1, which are located in the central cavity of a doughnut-shaped PRMT1 homodimer.

Four enzymes with phospholipase A1 (PLA1) activity were purified from the fruiting bodies of the basidiomycete Armillaria ostoyae. The enzymes (PLA1-1, -2, -3 and -4) showed similar isoelectric points (4.3, 3.9, 4.0 and 4.0) and apparent molecular masses in the range of 35–47 kDa. Mass spectrometric analyses of proteolytic fragments revealed sequences homologous to α/β-hydrolase fold enzymes. The enzymes share one conserved region with fungal phospholipases B and the active site sequence with bacterial esterases and PLA1s. PLA1-1 cleaves phospholipids and lysophospholipids with an optimum activity at pH 5.3. In contrast, PLA1-2, -3 and -4 are characterized by broad pH optima in the slightly acidic to neutral range and are additionally capable of hydrolyzing mono- and diglycerides as well as fatty acid methyl esters. All enzymes favor glycerol-based lipids with a single medium-sized fatty acid moiety in the sn-1 position but show reduced activity towards the corresponding 1,2-diacyl derivatives with bulky long-chain or inflexible saturated fatty acid moieties in the sn-2 position. The enzymes prefer zwitterionic phospholipid substrates and are unable to hydrolyze triglycerides. From the selectivity of these broad-spectrum α/β-hydrolase fold enzymes towards the different classes of their substrates a regiospecific steric hindrance and a head group recognition are concluded.

Rationale Chemical cross-linking in combination with a mass spectrometric analysis of the created cross-linked products is an area of growing interest for deriving low-resolution structural information of proteins and protein complexes. One of the greatest challenges is the complexity of the created cross-linking mixtures, which can be met by a charge-based enrichment of cross-linked peptides after proteolytic digestion using strong cation-exchange (SCX) chromatography. Methods SCX chromatography was used for the enrichment of cross-linked peptides with the N-hydroxysuccinimide ester bis(sulfosuccinimidyl)succinate (BS(3)) prior to a mass spectrometric analysis by nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS. Bovine serum albumin (BSA) and glutathione S-transferase (GST) were employed as model proteins. Results Conditions for SCX enrichment were optimized for obtaining as many interpeptide cross-linked peptides as possible in order to maximize the amount of structural information from a single experiment. With an SCX-based enrichment step of cross-linked products within BSA using the cross-linker BS(3), 154 interpeptidal cross-linking products were identified during nano-HPLC/nano-ESI-MS/MS analyses, whereas analyses without a prior SCX enrichment allowed the identification of merely 20 cross-linked products. The application of the SCX enrichment strategy for the analysis of cross-linked products of GST with BS(3) allowed the identification of 26 interpeptidal cross-linked products compared with 16 without SCX enrichment. Conclusions For both proteins investigated herein, BSA and GST, the introduction of an SCX-based enrichment step prior to nano-HPLC/nano-ESI-MS/MS of cross-linked products led to a considerable gain in structural information.

In the thyroid, cAMP controls both thyroid growth and function. Gain-of-function mutations in the thyroid-stimulating hormone receptor (TSHR) lead to constitutive cAMP formation and are a major cause of autonomous thyroid adenomas. The impact of activating TSHR mutations on the signal transduction network of the thyrocyte is not fully understood. To gain more insights into constitutive TSHR signaling, rat thyrocytes (FRTL-5 cells) with stable expression of three activating TSHR mutants (mutTSHR: A623I, L629F and Del613-621), which differ in their functional characteristics in vitro, were analyzed by a quantitative proteomic approach and compared to the wild-type TSHR (WT-TSHR). This study revealed (1) differences in the expression of Rab proteins suggesting an increased TSHR internalization in mutTSHR but not in the WT-TSHR; (2) differential stimulation of PI3K/Akt signaling in mutTSHR vs. WT-TSHR cells, (3) activation of Epac, impairing short-time Akt phosphorylation in both, mutTSHR and WT-TSHR cells. Based on the analysis of global changes in protein expression patterns, our findings underline the complexity of gain-of-function TSHR signaling in thyrocytes, which extends beyond pure cAMP and/or IP formation. Moreover, evidence for augmented endocytosis in the mutTSHR, adds to a new concept of TSHR signaling in thyroid autonomy. Further studies are required to clarify whether the observed differences in Rab, PI3K and Epac signaling may contribute to differences in the phenotypic presentation, i.e. stimulation of function and growth of thyroid autonomy in vivo.

A poly(glycidyl methacrylate-co-acrylamide-co-ethylene dimethacrylate) monolith and a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were prepared in fused silica capillaries (100 μm ID) and modified with monomeric avidin using the glutaraldehyde technique. The biotin binding capacity of monolithic affinity columns with immobilized monomeric avidin (MACMAs) was determined by fluorescence spectroscopy using biotin (5-fluorescein) conjugate, as well as biotin- and fluorescein-labeled bovine serum albumin (BSA). The affinity columns were able to bind 16.4 and 3.7 μmol biotin/mL, respectively. Columns prepared using the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith retained 7.1 mg BSA/mL, almost six times more than commercially available monomeric avidin beads. Protocols based on MALDI-TOF mass spectrometry monitoring were optimized for the enrichment of biotinylated proteins and peptides. A comparison of enrichment efficiencies between MACMAs and commercially available monomeric avidin beads yielded superior results for our novel monolithic affinity columns. However, the affinity medium presented in this work suffers from a significant degree of nonspecific binding, which might hamper the analysis of more complex mixtures. Further modifications of the monolith's surface are envisaged for the future development of monoliths with improved enrichment characteristics.

Chemical crosslinking in combination with mass spectrometry has matured into an alternative approach to derive low-resolution structural information of proteins and protein complexes. Yet, one of the major drawbacks of this strategy remains the lack of software that is able to handle the large MS datasets that are created after chemical crosslinking and enzymatic digestion of the crosslinking reaction mixtures. Here, we describe a software, termed StavroX, which has been specifically designed for analyzing highly complex crosslinking datasets. The StavroX software was evaluated for three diverse biological systems: (1) the complex between calmodulin and a peptide derived from Munc13, (2) an N-terminal ß-laminin fragment, and (3) the complex between guanylyl cyclase activating protein-2 and a peptide derived from retinal guanylyl cyclase. We show that the StavroX software is advantageous for analyzing crosslinked products due to its easy-to-use graphical user interface and the highly automated analysis of mass spectrometry (MS) and tandem mass spectrometry (MS/MS) data resulting in short times for analysis. StavroX is expected to give a further push to the chemical crosslinking approach as a routine technique for protein interaction studies.

Basement membranes are thin extracellular protein layers, which separate endothelial and epithelial cells from the underlying connecting tissue. The main noncollagenous components of basement membranes are laminins, trimeric glycoproteins, which form polymeric networks by interactions of their N-terminal (LN) domains; however, no high-resolution structure of laminin LN domains exists so far. To construct models for laminin β(1) and γ(1) LN domains, 14 potentially suited template structures were determined using fold recognition methods. For each target/template-combination comparative models were created with Rosetta. Final models were selected based on their agreement with experimentally obtained distance constraints from natural cross-links, that is, disulfide bonds as well as chemical cross-links obtained from reactions with two amine-reactive cross-linkers. We predict that laminin β(1) and γ(1) LN domains share the galactose-binding domain-like fold.

Activation of the glucagon-like peptide-1 receptor (GLP-1R) upon ligand binding leads to the release of insulin from pancreatic cells. This strictly glucose-dependent process renders the receptor and its ligands useful in the treatment of type II diabetes mellitus. To enable a biophysical characterization in vitro, we expressed the human full-length GLP-1R in the cytosol of Escherichia coli as inclusion bodies. After purification, refolding of the SDS-solubilized receptor was achieved by the exchange of SDS against the detergent Brij78 using an artificial chaperone system. Far-UV circular dichroism spectroscopic studies revealed that the receptor adopts a characteristic alpha-helical structure in Brij78 micelles. Ligand binding of the renatured protein was quantified by fluorescence quenching and surface plasmon resonance spectroscopy. In the presence of Brij micelles, the refolded receptor binds the agonist exendin-4 with an apparent dissociation constant of approximately 100 nM in a reversible one-step mechanism. To demonstrate that the detected ligand binding activity is not only due to an autonomously functional N-terminal domain (nGLP-1R) but also due to additional contacts with the juxtamembrane part, we separately expressed and refolded the extracellular domain relying on identical protocols established for the full-length GLP-1R. In support of the suggested multidomain binding mode, the nGLP-1R binds exendin-4 with a lower affinity (K(app) in the micromolar range) and a different kinetic mechanism. The lower ligand affinity of the nGLP-1R results entirely from a decreased kinetic stability of the receptor-ligand complex, dissociation of which is approximately 40-fold faster in the case of the nGLP-1R compared to the full-length GLP-1R. In summary, a framework was developed to produce functional human full-length GLP-1R by recombinant expression in E. coli as a prerequisite for eventual structure determination and a rigorous biophysical characterization including protein variants.

The CCR4-NOT complex is the main enzyme catalyzing the deadenylation of mRNA. We have investigated the composition of this complex in Drosophila melanogaster by immunoprecipitation with a monoclonal antibody directed against NOT1. The CCR4, CAF1 (=POP2), NOT1, NOT2, NOT3, and CAF40 subunits were associated in a stable complex, but NOT4 was not. Factors known to be involved in mRNA regulation were prominent among the other proteins coprecipitated with the CCR4-NOT complex, as analyzed by mass spectrometry. The complex was localized mostly in the cytoplasm but did not appear to be a major component of P bodies. Of the known CCR4 paralogs, Nocturnin was found associated with the subunits of the CCR4-NOT complex, whereas Angel and 3635 were not. RNAi experiments in Schneider cells showed that CAF1, NOT1, NOT2, and NOT3 are required for bulk poly(A) shortening and hsp70 mRNA deadenylation, but knock-down of CCR4, CAF40, and NOT4 did not affect these processes. Overexpression of catalytically dead CAF1 had a dominant-negative effect on mRNA decay. In contrast, overexpression of inactive CCR4 had no effect. We conclude that CAF1 is the major catalytically important subunit of the CCR4-NOT complex in Drosophila Schneider cells. Nocturnin may also be involved in mRNA deadenylation, whereas there is no evidence for a similar role of Angel and 3635.

Signal transducer and activator of transcription 3 (Stat3) is the major mediator of interleukin-6 (IL-6) family cytokines. In addition, Stat3 is known to be involved in the pathophysiology of many malignancies. Here, we show that the cis-trans peptidyl-prolyl isomerase cyclophilin (Cyp) B specifically interacts with Stat3, whereas the highly related CypA does not. CypB knockdown inhibited the IL-6-induced transactivation potential but not the tyrosine phosphorylation of Stat3. Binding of CypB to Stat3 target promoters and alteration of the intranuclear localization of Stat3 on CypB depletion suggested a nuclear function of Stat3/CypB interaction. By contrast, CypA knockdown inhibited Stat3 IL-6-induced tyrosine phosphorylation and nuclear translocation. The Cyp inhibitor cyclosporine A (CsA) caused similar effects. However, Stat1 activation in response to IL-6 or interferon-gamma was not affected by Cyp silencing or CsA treatment. As a result, Cyp knockdown shifted IL-6 signaling to a Stat1-dominated pathway. Furthermore, Cyp depletion or treatment with CsA induced apoptosis in IL-6-dependent multiple myeloma cells, whereas an IL-6-independent line was not affected. Thus, Cyps support the anti-apoptotic action of Stat3. Taken together, CypA and CypB both play pivotal roles, yet at different signaling levels, for Stat3 activation and function. These data also suggest a novel mechanism of CsA action.

Asymmetric dimethylation of arginine residues is a common posttranslational modification of proteins carried out by type I protein arginine methyltransferases, including PRMT1 and -3. We report that the consecutive transfer of two methyl groups to a single arginine side chain by PRMT1 and -3 occurs in a distributive manner, i.e. with intermittent release of the monomethylated intermediate. The oligomeric state of PRMTs together with the clustering of methylated arginine residues in most proteins carrying this type of modification suggests that multiple methyl transfers to a single polypeptide chain might proceed in a processive manner by cooperation of multiple active sites. However, three different types of experiments provide evidence that the reaction is distributive even with substrates containing multiple methyl-accepting arginines, including one with 13 such residues. PRMT1 also does not prefer substrates already containing one or more singly or doubly methylated arginine residues. Even though the reaction is distributive, the efficiency of methylation of one particular protein strongly depends on the number of methyl-accepting arginine residues it contains.

Calcitonin gene-related peptide is a 37 amino acidneuropeptide. Although calcitonin gene-relatedpeptide activates a G-protein-coupled receptor,recent evidence suggests that calcitonin gene-rela-ted peptide induces more complex signaling cas-cades including the activation of MAP kinases [EurJ Pharmacol; 389:125–130 (2000), Neuropeptides;34:229–233 (2000)]. However, the molecular mech-anisms of this activation still remain to be elucida-ted. For the first time we applied a proteomicsapproach in order to identify molecular targets ofcalcitonin gene-related peptide downstream signa-ling in the neuroblastoma cell line SK-N-MC andidentified proteins that changed either theirexpression, location, or their post-translationalmodifications in a time-dependent manner aftercalcitonin gene-related peptide stimulation.Key words: calcitonin gene-related peptide, G-protein-coupledreceptor, peptide mass fingerprinting, proteomics, signal transduction,two-dimensional gel electrophoresisReceived 10 December 2006, revised 22 December 2006 and acceptedfor publication 25 December 2006

We demonstrate that the Y3/Y3** noncoding RNAs (ncRNAs) bind to the CPSF (cleavage and polyadenylation specificity factor) and that Y3** associates with the 3′ untranslated region (UTR) of histone pre-mRNAs. The depletion of Y3** impairs the 3′ end processing of histone pre-mRNAs as well as the formation and protein dynamics of histone locus bodies (HLBs), the site of histone mRNA synthesis and processing. HLB morphology is also disturbed by knockdown of the CPSF but not the U7-snRNP components. In conclusion, we propose that the Y3** ncRNA promotes the 3′ end processing of histone pre-mRNAs by enhancing the recruitment of the CPSF to histone pre-mRNAs at HLBs.

Under various pathological conditions, such as inflammation, ischemia and in solid tumors, physiological parameters (local oxygen tension or extracellular pH) show distinct tissue abnormalities (hypoxia and acidosis). For tumors, the prevailing microenvironment exerts a strong influence on the phenotype with respect to proliferation, invasion, and metastasis formation and therefore influences prognosis. In this study, we investigate the impact of extracellular metabolic acidosis (pH 7.4 versus 6.6) on the proteome patterns of a prostate cancer-derived tumor cell type (AT-1) using isobaric labeling and LC-MS/MS analysis. In total, 2710 proteins were identified and quantified across four biological replicates, of which seven were significantly affected with changes50% and used for validation. Glucose transporter 1 and farnesyl pyrophosphatase were found to be down-regulated after 48 h of acidic treatment, and metallothionein 2A was reduced after 24 h and returned to control values after 48 h. After 24 and 48 h at pH 6.6, glutathione S transferase A3 and NAD(P)H dehydrogenase 1, cellular retinoic acid-binding protein 2, and Na-bicarbonate transporter 3 levels were found to be increased. The changes in protein levels were confirmed by transcriptome and functional analyses. In addition to the experimental in-depth investigation of proteins with changes50%, functional profiling (statistical enrichment analysis) including proteins with changes20% revealed that acidosis upregulates GSH metabolic processes, citric acid cycle, and respiratory electron transport. Metabolism of lipids and cholesterol biosynthesis were downregulated. Our data comprise the first comprehensive report on acidosis-induced changes in proteome patterns of a tumor cell line.

Calmodulin serves as a calcium-dependent regulator in many metabolic pathways and is known to bind with high affinity to various target proteins and peptides. One such target is the small peptide melittin, the principal component of honeybee venom. The calmodulin-melittin system was used as a model system to gain further insight into target recognition of calmodulin. Using chemical cross-linking in combination with high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS), we have determined the interacting regions within the calcium-dependent calmodulin-melittin complex and thus the orientation of bound melittin. Using ambiguous distance restraints derived from the chemical cross-linking data in combination with recently developed computational methods of conjoined rigid body/torsion angle simulated annealing, we were able to generate low-resolution three-dimensional structure models of the calmodulin-melittin complex, for which no high-resolution structure exists to date. Our data provide evidence for the first time that calmodulin can recognize target peptides in two opposing orientations simultaneously. The general procedure for mapping interacting regions within the complex involves conjugation of calmodulin and melittin with several cross-linking reagents possessing different specificities and spacer lengths, followed by enzymatic proteolysis of the cross-linked complex. The highly complex peptide mixtures were subsequently analyzed by nano-HPLC, which was online coupled to a FTICR mass spectrometer equipped with a nano-electrospray ionization source. The mass spectra obtained in this manner were screened for possible cross-linking products using customized software programs. This integrated approach, exemplified for mapping the topology of the calmodulin-melittin complex, is likely to have wide-ranging implications for structural studies on protein-protein interactions.

Visible symptoms of tepal senescence in cut Iris x hollandica (cv. Blue Magic) flowers were delayed by placing one cut daffodil flower (Narcissus pseudonarcissus, cv. Carlton) in the same vase. Addition of mucilage, exuded by daffodil stems, to the vase water had the same effect as the flowering daffodil stem. The active compound in the mucilage was identified as narciclasine (using LC/MS, GC/MS, 1H and 13C-NMR, and comparison with an authentic sample of narciclasine). The delay of senescence, either by mucilage or purified narciclasine, was correlated with a delayed increase in protease activity, and with a considerable reduction of maximum protease activity. Narciclasine did not affect in vitro protease activity, but is known to inhibit protein synthesis at the ribosomal level. Its effects on senescence and protease activity were similar to those of cycloheximide (CHX), another inhibitor of protein synthesis, but the effective narciclasine concentration was about 100 times lower than that of CHX. It is concluded that the delay of Iris tepal senescence by daffodil stems is due to narciclasine in daffodil mucilage, which apparently inhibits the synthesis of proteins involved in senescence.

The magnitude and the direction of the gravitational field represent an important environmental factor affecting plant development. In this context, the absence or frequent alterations of the gravity field (i.e. microgravity conditions) might compromise extraterrestrial agriculture and hence space inhabitation by humans. To overcome the deleterious effects of microgravity, a complete understanding of the underlying changes on the macromolecular level is necessary. However, although microgravity-related changes in gene expression are well characterised on the transcriptome level, proteomic data are limited. Moreover, information about the microgravity-induced changes in the seedling proteome during seed germination and the first steps of seedling development is completely missing. One of the valuable tools to assess gravity-related issues is 3D clinorotation (i.e. rotation in two axes). Therefore, here we address the effects of microgravity, simulated by a two-axial clinostat, on the proteome of 24- and 48-h-old seedlings of oilseed rape (Brassica napus L.). The liquid chromatography-MS-based proteomic analysis and database search revealed 95 up- and 38 downregulated proteins in the tryptic digests obtained from the seedlings subjected to simulated microgravity, with 42 and 52 annotations detected as being unique for 24- and 48-h treatment times, respectively. The polypeptides involved in protein metabolism, transport and signalling were annotated as the functional groups most strongly affected by 3-D clinorotation.

Differential proteome analysis is used to study body fluids from patients suffering from rheumatoid arthritis (RA), reactive arthritis (reaA) or osteoarthritis (OA). Mass spectrometric structure characterization of gel-separated proteins provided a detailed view of the protein-processing events that lead to distinct protein species present in the respective body fluids. (i) Fibrin(ogen) beta-chain degradation products, presumably plasmin-derived, appeared solely in synovial fluids (SF) from both patient collectives, (ii) calgranulin B (MRP14) was exclusively identified in SF samples derived from 5 out of 6 patients suffering from RA. Calgranulin B was not observed in synovial fluids from OA patients, nor in plasmas from either patient group. In all cases where calgranulin B was detected, calgranulin C was identified as well. (iii) Serum amyloid A protein spots were determined in plasmas and synovial fluids from patients with RA, but not in patients with OA. In addition to disease-relevant differences, interindividual differences in haptoglobin patterns of the patients under investigation were observed. Hence, in-depth proteome analysis of body fluids has proven effective for identification of multiple molecular markers and determination of associated protein structure modifications, that are thought to play a role for specifically determining a defined pathological state of diseased joints.

We present a novel approach that relies on the affinity capture of protein interaction partners from a complex mixture, followed by their covalent fixation via UV-induced activation of incorporated diazirine photoreactive amino acids (photo-methionine and photo-leucine). The captured protein complexes are enzymatically digested and interacting proteins are identified and quantified by label-free LC/MS analysis. Using HeLa cell lysates with photo-methionine and photo-leucine-labeled proteins, we were able to capture and preserve protein interactions that are otherwise elusive in conventional pull-down experiments. Our approach is exemplified for mapping the protein interaction network of protein kinase D2, but has the potential to be applied to any protein system. Data are available via ProteomeXchange with identifiers PXD005346 (photo amino acid incorporation) and PXD005349 (enrichment experiments).

Photo-induced cross-linking is a highly promising technique to investigate protein conformations and protein-protein interactions in their natural cellular environment. One strategy relies on the non-directed incorporation of diazirine-containing photo-activatable amino acids into proteins and a subsequent cross-link formation induced by UV-A irradiation. The advantage of this photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can also be targeted, which is advantageous for investigating membrane proteins. Here, we present a simplified protocol that relies on the use of mineral salts medium without any special requirements for the incorporation of photo-methionines into proteins in Escherichia coli cells. The possibility to perform these experiments in E. coli is especially valuable as it is the major system for recombinant protein production. The method is exemplified for the Ca(2+) regulating protein calmodulin containing nine methionines, which were found to be replaced by their photo-activatable analogues. Our protocol allows the facile and stochastic incorporation of photo-methionines as the basis for conducting photo-cross-linking experiments in E. coli in an efficient manner.

During anaerobic growth Escherichia coli synthesizes two large, highly homologous respiratory formate dehydrogenases (Fdh's), Fdh-N and Fdh-O, which are associated with the inner membrane but have their respective active site located within the periplasm. The Fdh-N enzyme extends 90 A into the periplasmic compartment, which in E. coli ranges between 100 and 150 A from the inner to the outer membrane leaflet. To date, little is known about the interaction partners of Fdh-N and Fdh-O in the periplasmic space that might be involved in stabilizing these enzymes after maturation and translocation across the cytoplasmic membrane has occurred. To address this question, we performed chemical cross-linking in combination with mass spectrometry. We present for the first time the identification of cell envelope interaction partners of Fdh-N and -O from anaerobically grown E. coli using a heterobifunctional amine/photo-reactive cross-linker followed by mass spectrometric analysis of the cross-linked products. We additionally mapped the interface regions within the Fdh/protein complexes for four selected Fdh-binding partners, the chaperone Skp, the l,d-transpeptidase ErfK, OppA, and TolB. Our work yields first structural and functional insights into the mechanisms that support the postmaturation of the multisubunit enzymes Fdh-N and Fdh-O in the periplasm of E. coli.

We describe the use of the (13)C-labeled artificial amino acid p-benzoyl-L-phenylalanine (Bpa) to improve the reliability of cross-linked product identification. Our strategy is exemplified for two protein-peptide complexes. These studies indicate that in many cases the identification of a cross-link without additional stable isotope labeling would result in an ambiguous assignment of cross-linked products. The use of a (13)C-labeled photoreactive amino acid is considered to be preferred over the use of deuterated cross-linkers as retention time shifts in reversed phase chromatography can be ruled out. The observation of characteristic fragment ions additionally increases the reliability of cross-linked product assignment. Bpa possesses a broad reactivity towards different amino acids and the derived distance information allows mapping of spatially close amino acids and thus provides more solid structural information of proteins and protein complexes compared to the longer deuterated amine-reactive cross-linkers, which are commonly used for protein 3D-structure analysis and protein-protein interaction studies.

VP1 is the major coat protein of murine polyomavirus and forms virus-like particles (VLPs) in vitro. VLPs consist of 72 pentameric VP1 subunits held together by a terminal clamp structure that is further stabilized by disulfide bonds and chelation of calcium ions. Yeast-derived VLPs (yVLPs) assemble intracellularly in vivo during recombinant protein production. These in vivo assembled yVLPs differ in several properties from VLPs assembled in vitro from bacterially produced pentamers. We found several intermolecular disulfide linkages in yVLPs involving 5 of the 6 cysteines of VP1 (Cys(115)-Cys(20), Cys(12)-Cys(20), Cys(16)-Cys(16), Cys(12)/ Cys(16)-Cys(115), and Cys(274)-Cys(274)), indicating a highly coordinated disulfide network within the in vivo assembled particles involving the N-terminal region of VP1. Cryoelectron microscopy revealed structured termini not resolved in the published crystal structure of the bacterially expressed VLP that appear to clamp the pentameric subunits together. These structural features are probably the reason for the observed higher stability of in vivo assembled yVLPs compared with in vitro assembled bacterially expressed VLPs as monitored by increased thermal stability, higher resistance to trypsin cleavage, and a higher activation enthalpy of the disassembly reaction. This high stability is decreased following disassembly of yVLPs and subsequent in vitro reassembly, suggesting a role for cellular components in optimal assembly.