Pulmonary maternal immune activation does not cross the placenta but leads to fetal metabolic adaptation.

The fetal development of organs and functions is vulnerable to perturbation by maternal inflammation which may increase susceptibility to disorders after birth. Because it is not well understood how the placenta and fetus respond to acute lung- inflammation, we characterize the response to maternal pulmonary lipopolysaccharide exposure across 24 h in maternal and fetal organs using multi-omics, imaging and integrative analyses. Unlike maternal organs, which mount strong inflammatory immune responses, the placenta upregulates immuno-modulatory genes, in particular the IL-6 signaling suppressor Socs3. Similarly, we observe no immune response in the fetal liver, which instead displays metabolic changes, including increases in lipids containing docosahexaenoic acid, crucial for fetal brain development. The maternal liver and plasma display similar metabolic alterations, potentially increasing bioavailability of docosahexaenoic acid for the mother and fetus. Thus, our integrated temporal analysis shows that systemic inflammation in the mother leads to a metabolic perturbation in the fetus.

TBK1 phosphorylation activates LIR-dependent degradation of the inflammation repressor TNIP1.

Limitation of excessive inflammation due to selective degradation of pro-inflammatory proteins is one of the cytoprotective functions attributed to autophagy. In the current study, we highlight that selective autophagy also plays a vital role in promoting the establishment of a robust inflammatory response. Under inflammatory conditions, here TLR3-activation by poly(I:C) treatment, the inflammation repressor TNIP1 (TNFAIP3 interacting protein 1) is phosphorylated by Tank-binding kinase 1 (TBK1) activating an LIR motif that leads to the selective autophagy-dependent degradation of TNIP1, supporting the expression of pro-inflammatory genes and proteins. This selective autophagy efficiently reduces TNIP1 protein levels early (0-4 h) upon poly(I:C) treatment to allow efficient initiation of the inflammatory response. At 6 h, TNIP1 levels are restored due to increased transcription avoiding sustained inflammation. Thus, similarly as in cancer, autophagy may play a dual role in controlling inflammation depending on the exact state and timing of the inflammatory response.

Identification of ester-linked ubiquitylation sites during TLR7 signalling increases the number of inter-ubiquitin linkages from 8 to 12.

The E3 ligase HOIL-1 forms ester bonds in vitro between ubiquitin and serine/threonine residues in proteins. Here, we exploit UbiSite technology to identify serine and threonine residues undergoing HOIL-1 catalysed ubiquitylation in macrophages stimulated with R848, an activator of the TLR7/8 heterodimer. We identify Thr12, Thr14, Ser20 and Thr22 of ubiquitin as amino acid residues forming ester bonds with the C-terminal carboxylate of another ubiquitin molecule. This increases from 8 to 12 the number of ubiquitin linkage types that are formed in cells. We also identify Ser175 of IRAK4, Ser136, Thr163 and Ser168 of IRAK2 and Thr141 of MyD88 as further sites of HOIL-1-catalysed ubiquitylation together with lysine residues in these proteins that also undergo R848-dependent ubiquitylation. These findings establish that the ubiquitin chains attached to components of myddosomes are initiated by both ester and isopeptide bonds. Ester bond formation takes place within the proline, serine, threonine-rich (PST) domains of IRAK2 and IRAK4 and the intermediate domain of MyD88. The ubiquitin molecules attached to Lys162, Thr163 and Ser168 of IRAK2 are attached to different IRAK2 molecules.

Deubiquitinating enzymes and the proteasome regulate preferential sets of ubiquitin substrates.

The ubiquitin-proteasome axis has been extensively explored at a system-wide level, but the impact of deubiquitinating enzymes (DUBs) on the ubiquitinome remains largely unknown. Here, we compare the contributions of the proteasome and DUBs on the global ubiquitinome, using UbiSite technology, inhibitors and mass spectrometry. We uncover large dynamic ubiquitin signalling networks with substrates and sites preferentially regulated by DUBs or by the proteasome, highlighting the role of DUBs in degradation-independent ubiquitination. DUBs regulate substrates via at least 40,000 unique sites. Regulated networks of ubiquitin substrates are involved in autophagy, apoptosis, genome integrity, telomere integrity, cell cycle progression, mitochondrial function, vesicle transport, signal transduction, transcription, pre-mRNA splicing and many other cellular processes. Moreover, we show that ubiquitin conjugated to SUMO2/3 forms a strong proteasomal degradation signal. Interestingly, PARP1 is hyper-ubiquitinated in response to DUB inhibition, which increases its enzymatic activity. Our study uncovers key regulatory roles of DUBs and provides a resource of endogenous ubiquitination sites to aid the analysis of substrate specific ubiquitin signalling.

Detailed dynamics of direct three-body recombination of singly charged ions.

We consider the main aspects of detailed dynamics of the reactions of direct three-body ion-ion recombination Cs+ + X- + R → CsX + R (X- = F-, I- and R = Ar, Xe) for non-central encounters of the ions. The reactions are simulated by the quasiclassical trajectory method using diabatic semiempirical potential energy surfaces proposed previously. The recombination mechanisms are studied via visualization of randomly selected trajectories for each of the four systems. Comparison of trajectories for different systems with identical sets of initial conditions is carried out. For most of the presented trajectories, the ion encounter energy and the third body energy are equal to 1 eV. The characteristic function of recombination is defined, this function depends on 13 arguments including eight kinematic parameters. It is shown that the transfer of excess energy from the ion pair to the neutral atom can occur, in particular, via an encounter of the R atom with the Cs+ ion, via an encounter of the R atom with the X- ion, or via successive encounters of the R atom with both the ions, as well as via an "insertion" of the R atom between the ions.

Magnitude of Ubiquitination Determines the Fate of Epidermal Growth Factor Receptor Upon Ligand Stimulation.

Receptor tyrosine kinases (RTK) bind growth factors and are critical for cell proliferation and differentiation. Their dysregulation leads to a loss of growth control, often resulting in cancer. Epidermal growth factor receptor (EGFR) is the prototypic RTK and can bind several ligands exhibiting distinct mitogenic potentials. Whereas the phosphorylation on individual EGFR sites and their roles for downstream signaling have been extensively studied, less is known about ligand-specific ubiquitination events on EGFR, which are crucial for signal attenuation and termination. We used a proteomics-based workflow for absolute quantitation combined with mathematical modeling to unveil potentially decisive ubiquitination events on EGFR from the first 30 seconds to 15 minutes of stimulation. Four ligands were used for stimulation: epidermal growth factor (EGF), heparin-binding-EGF like growth factor, transforming growth factor-α and epiregulin. Whereas only little differences in the order of individual ubiquitination sites were observed, the overall amount of modified receptor differed depending on the used ligand, indicating that absolute magnitude of EGFR ubiquitination, and not distinctly regulated ubiquitination sites, is a major determinant for signal attenuation and the subsequent cellular outcomes.

Proteomic investigation of Cbl and Cbl-b in neuroblastoma cell differentiation highlights roles for SHP-2 and CDK16.

Neuroblastoma is a highly heterogeneous embryonal solid tumor of the sympathetic nervous system. As some tumors can be treated to undergo differentiation, investigating this process can guide differentiation-based therapies of neuroblastoma. Here, we studied the role of E3 ubiquitin ligases Cbl and Cbl-b in regulation of long-term signaling responses associated with extracellular signal-regulated kinase phosphorylation and neurite outgrowth, a morphological marker of neuroblastoma cell differentiation. Using quantitative mass spectrometry (MS)-based proteomics, we analyzed how the neuroblastoma cell line proteome, phosphoproteome, and ubiquitylome were affected by Cbl and Cbl-b depletion. To quantitatively assess neurite outgrowth, we developed a high-throughput microscopy assay that was applied in combination with inhibitor studies to pinpoint signaling underlying neurite outgrowth and to functionally validate proteins identified in the MS data sets. Using this combined approach, we identified a role for SHP-2 and CDK16 in Cbl/Cbl-b-dependent regulation of extracellular signal-regulated kinase phosphorylation and neurite outgrowth, highlighting their involvement in neuroblastoma cell differentiation.

MaxQuant.Live Enables Enhanced Selectivity and Identification of Peptides Modified by Endogenous SUMO and Ubiquitin.

Small ubiquitin-like modifiers (SUMO) and ubiquitin are frequent post-translational modifications of proteins that play pivotal roles in all cellular processes. We previously reported mass spectrometry-based proteomics methods that enable profiling of lysines modified by endogenous SUMO or ubiquitin in an unbiased manner, without the need for genetic engineering. Here we investigated the applicability of precursor mass filtering enabled by MaxQuant.Live to our SUMO and ubiquitin proteomics workflows, which efficiently avoided sequencing of precursors too small to be modified but otherwise indistinguishable by mass-to-charge ratio. Using precursor mass filtering, we achieved a much higher selectivity of modified peptides, ultimately resulting in up to 30% more SUMO and ubiquitin sites identified from replicate samples. Real-time exclusion of unmodified peptides by MQL resulted in 90% SUMO-modified precursor selectivity from a 25% pure sample, demonstrating great applicability for digging deeper into ubiquitin-like modificomes. We adapted the precursor mass filtering strategy to the new Exploris 480 mass spectrometer, achieving comparable gains in SUMO precursor selectivity and identification rates. Collectively, precursor mass filtering via MQL significantly increased identification rates of SUMO- and ubiquitin-modified peptides from the exact same samples, without the requirement for prior knowledge or spectral libraries.

The multifunctional role of SPANX-A/D protein subfamily in the promotion of pro-tumoural processes in human melanoma.

Human sperm protein associated with the nucleus on the X chromosome (SPANX) genes encode a protein family (SPANX-A, -B, -C and -D), whose expression is limited to the testis and spermatozoa in normal tissues and various tumour cells. SPANX-A/D proteins have been detected in metastatic melanoma cells, but their contribution to cancer development and the underlying molecular mechanisms of skin tumourigenesis remain unknown. Combining functional and proteomic approaches, the present work describes the presence of SPANX-A/D in primary and metastatic human melanoma cells and how it promotes pro-tumoural processes such as cell proliferation, motility and migration. We provide insights into the molecular features of skin tumourigenesis, describing for the first time a multifunctional role of the SPANX-A/D protein family in nuclear function, energy metabolism and cell survival, considered key hallmarks of cancer. A better comprehension of the SPANX-A/D protein subfamily and its molecular mechanisms will help to describe new aspects of tumour cell biology and develop new therapeutic targets and tumour-directed pharmacological drugs for skin tumours.

Dynamics of third order direct three-body recombination of heavy ions.

The direct three-body recombination reactions Cs+ + X- + R → CsX + R (X = F, I and R = Ar, Xe) are studied within the quasiclassical trajectory method using diabatic semiempirical potential energy surfaces, the encounters of the ions being non-central. The collision energies range between 1 and 10 eV (values typical for low temperature plasma), while the so-called delay parameter, which characterizes the delay in the arrival of the neutral atom R in relation to the time instant when the distance between the ions attains its minimum, is equal to 0 or 20%. The calculation results include the recombination excitation functions, the opacity functions, and the vibrational and rotational energy distributions of the recombination products. All the four reactions considered exhibit similar overall statistical dynamics, but each process has its own features. On the whole, for both the recombining pairs Cs+ + F- and Cs+ + I-, xenon is more effective than argon as an acceptor of excess energy from the ion pair. The rotational energy distributions of the salt molecules CsF and CsI are almost equilibrium, whereas the vibrational energy distributions are strongly non-equilibrium.

Kappa- opioid receptor regulates human sperm functions via SPANX-A/D protein family.

The kappa-opioid receptor (KOR) is involved in the regulation of the fertilizing capacity of human sperm. Recently, a testicular-specific protein family, SPANX-A/D, has also been found to be involved in regulating this process. In order to determine if KOR has a role in the regulation of sperm fertility through the SPANX-A/D protein family, we activated the kappa opioid receptor adding its selective agonist, U50488H to normozoospermic human spermatozoa. Then, we performed immunofluorescence assays and immunoprecipitation experiments followed by LC-MS/MS. According to our results, KOR activation may cause the translocation of SPANX-A/D into the nucleus of human spermatozoa. Phosphoproteomic studies show that KOR does not cause phosphorylation changes in SPANX-A/D residues. However, interactome assays demonstrate that KOR activation provokes changes in SPANX-A/D potential interactors involved in sperm motility, energy metabolism and nuclear processes. Taking these results into account, KOR may regulate human sperm fertility through SPANX-A/D protein family, modifying its subcellular location and interactions. Although further studies are needed, this finding could help us describing the molecular mechanisms underlying sperm fertility as well as developing new strategies for treating infertility.

DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1.

The Ddi1/DDI2 proteins are ubiquitin shuttling factors, implicated in a variety of cellular functions. In addition to ubiquitin-binding and ubiquitin-like domains, they contain a conserved region with similarity to retroviral proteases, but whether and how DDI2 functions as a protease has remained unknown. Here, we show that DDI2 knockout cells are sensitive to proteasome inhibition and accumulate high-molecular weight, ubiquitylated proteins that are poorly degraded by the proteasome. These proteins are targets for the protease activity of purified DDI2. No evidence for DDI2 acting as a de-ubiquitylating enzyme was uncovered, which could suggest that it cleaves the ubiquitylated protein itself. In support of this idea, cleavage of transcription factor NRF1 is known to require DDI2 activity in vivo. We show that DDI2 is indeed capable of cleaving NRF1 in vitro but only when NRF1 protein is highly poly-ubiquitylated. Together, these data suggest that DDI2 is a ubiquitin-directed endoprotease.

SPANX-A/D protein subfamily plays a key role in nuclear organisation, metabolism and flagellar motility of human spermatozoa.

Human sperm protein associated with the nucleus on the X chromosome (SPANX) genes encode a protein family (SPANX-A, -B, -C and -D), whose expression is limited to the testis and spermatozoa in normal tissues and to a wide variety of tumour cells. Present only in hominids, SPANX-A/D is exclusively expressed in post-meiotic spermatids and mature spermatozoa. However, the biological role of the protein family in human spermatozoa is largely unknown. Combining proteomics and molecular approaches, the present work describes the presence of all isoforms of SPANX-A/D in human spermatozoa and novel phosphorylation sites of this protein family. In addition, we identify 307 potential SPANX-A/D interactors related to nuclear envelop, chromatin organisation, metabolism and cilia movement. Specifically, SPANX-A/D interacts with fumarate hydratase and colocalises with both fumarate hydratase and Tektin 1 proteins, involved in meeting energy demands for sperm motility, and with nuclear pore complex nucleoporins. We provide insights into the molecular features of sperm physiology describing for the first time a multifunctional role of SPANX-A/D protein family in nuclear envelope, sperm movement and metabolism, considered key functions for human spermatozoa. SPANX-A/D family members, therefore, might be promising targets for sperm fertility management.

Phosphoproteomic profiling reveals a defined genetic program for osteoblastic lineage commitment of human bone marrow-derived stromal stem cells.

Bone marrow-derived mesenchymal stem cells (MSCs) differentiate into osteoblasts upon stimulation by signals present in their niche. Because the global signaling cascades involved in the early phases of MSCs osteoblast (OB) differentiation are not well-defined, we used quantitative mass spectrometry to delineate changes in human MSCs proteome and phosphoproteome during the first 24 h of their OB lineage commitment. The temporal profiles of 6252 proteins and 15,059 phosphorylation sites suggested at least two distinct signaling waves: one peaking within 30 to 60 min after stimulation and a second upsurge after 24 h. In addition to providing a comprehensive view of the proteome and phosphoproteome dynamics during early MSCs differentiation, our analyses identified a key role of serine/threonine protein kinase D1 (PRKD1) in OB commitment. At the onset of OB differentiation, PRKD1 initiates activation of the pro-osteogenic transcription factor RUNX2 by triggering phosphorylation and nuclear exclusion of the histone deacetylase HDAC7.

Phosphoproteomic and Functional Analyses Reveal Sperm-specific Protein Changes Downstream of Kappa Opioid Receptor in Human Spermatozoa.

G-protein coupled receptors (GPCRs) belong to the seven transmembrane receptor superfamily that transduce signals via G proteins in response to external stimuli to initiate different intracellular signaling pathways which culminate in specific cellular responses. The expression of diverse GPCRs at the plasma membrane of human spermatozoa suggests their involvement in the regulation of sperm fertility. However, the signaling events downstream of many GPCRs in spermatozoa remain uncharacterized. Here, we selected the kappa-opioid receptor (KOR) as a study model and applied phosphoproteomic approach based on TMT labeling and LC-MS/MS analyses. Quantitative coverage of more than 5000 proteins with over 3500 phosphorylation sites revealed changes in the phosphorylation levels of sperm-specific proteins involved in the regulation of the sperm fertility in response to a specific agonist of KOR, U50488H. Further functional studies indicate that KOR could be involved in the regulation of sperm fertile capacity by modulation of calcium channels. Our findings suggest that human spermatozoa possess unique features in the molecular mechanisms downstream of GPCRs which could be key regulators of sperm fertility and improved knowledge of these specific processes may contribute to the development of useful biochemical tools for diagnosis and treatment of male infertility.

NADH dehydrogenase complex I is overexpressed in incipient metastatic murine colon cancer cells.

Colon cancer is one of the most frequently occurring types of cancers in the world. Primary tumours are treated very efficiently, but the metastatic cases are known to have severe outcomes. Therefore, the aim of the present study was to obtain a greater understanding of the transformation of primary colon cancer cells into metastatic phenotypes. Small changes in protein expression provoke the metastatic phenotype transformation. More sensitive methods to detect small variations are required. A murine colon cancer cell line with metastatic characteristics in a very early phase was created in order to investigate the first steps of transformation using a murine liver metastasis model. The protein expression patterns of metastatic and non­metastatic cells were compared using the stable isotope labelling by amino acids in cell culture method in combination with mass spectrometry. Quantitative proteomics data indicated that nicotinamide adenine dinucleotide hydride (NADH) dehydrogenase complex I was overexpressed in metastatic cells with respect to non­metastatic cells. Since the NADH dehydrogenase complex catalyses the oxidation of NADH to NAD+, the functionality of the complex was studied by measuring the amount of NADH. The results revealed that metastatic cells accumulate more NADH and reactive oxygen species. In addition, the mitochondrial membrane potential of metastatic cells was lower than that of non­metastatic cells, indicating that the activity of NADH dehydrogenase and the mitochondrial oxidative chain were decreased in metastatic cells. During the incipient transformation of primary cancer cells, NADH dehydrogenase complex I was overexpressed but then became inactive due to the Warburg effect, which inhibits mitochondrial activity. In the first step of transformation, the high energy demand required in an adverse environment is fulfilled by overexpressing components of the respiratory chain, a fact that should be considered for future anti­metastatic therapies.

UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites.

Ubiquitination is a post-translational modification (PTM) that is essential for balancing numerous physiological processes. To enable delineation of protein ubiquitination at a site-specific level, we generated an antibody, denoted UbiSite, recognizing the C-terminal 13 amino acids of ubiquitin, which remain attached to modified peptides after proteolytic digestion with the endoproteinase LysC. Notably, UbiSite is specific to ubiquitin. Furthermore, besides ubiquitination on lysine residues, protein N-terminal ubiquitination is readily detected as well. By combining UbiSite enrichment with sequential LysC and trypsin digestion and high-accuracy MS, we identified over 63,000 unique ubiquitination sites on 9,200 proteins in two human cell lines. In addition to uncovering widespread involvement of this PTM in all cellular aspects, the analyses reveal an inverse association between protein N-terminal ubiquitination and acetylation, as well as a complete lack of correlation between changes in protein abundance and alterations in ubiquitination sites upon proteasome inhibition.

Data on mass spectrometry-based proteomics for studying the involvement of CYLD in the ubiquitination events downstream of EGFR activation.

The present data article corresponds to the proteomic data of the involvement of Cylindromatosis protein (CYLD) in the ubiquitination signaling initiated by EGF stimulation. CYLD tumor suppressor protein has Lys63-chain deubiquitinase activity that has been proved essential for the negative regulation of crucial signaling mechanisms, namely the NFkB pathway. Previous results have suggested the involvement of CYLD in the EGF-dependent signal transduction as well, showing its engagement within the tyrosine-phosphorylated complexes formed following the addition of the growth factor. EGFR signaling participates in central cellular processes and its tight regulation, partly through ubiquitination cascades, is decisive for a balanced cellular homeostasis. We carried out the substitution of the endogenous pool of ubiquitin for a His-FLAG-tagged ubiquitin (Stable Ubiquitin Exchange, StUbEx), in combination with the shRNA silencing of CYLD and SILAC-labeling on HeLa cells. The subsequent tandem affinity purification of ubiquitinated proteins in control and CYLD-depleted cells was followed by mass spectrometric analysis. Therefore, we present an unbiased study investigating the impact of CYLD in the EGF-dependent ubiquitination. The data supplied herein is related to the research article entitled "Cylindromatosis tumor suppressor protein (CYLD) deubiquitinase is necessary for proper ubiquitination and degradation of the epidermal growth factor receptor" (Sanchez-Quiles et al., 2017) [1]. We provide the associated mass spectrometry raw files, excel tables and gene ontology enrichments. The data have been deposited in the ProteomeXchange with the identifier PXD003423.

StUbEx PLUS-A Modified Stable Tagged Ubiquitin Exchange System for Peptide Level Purification and In-Depth Mapping of Ubiquitination Sites.

Modulation of protein activities by reversible post-translational modifications (PTMs) is a major molecular mechanism involved in the control of virtually all cellular processes. One of these PTMs is ubiquitination, which regulates key processes including protein degradation, cell cycle, DNA damage repair, and signal transduction. Because of its importance for numerous cellular functions, ubiquitination has become an intense topic of research in recent years, and proteomics tools have greatly facilitated the identification of many ubiquitination targets. Taking advantage of the StUbEx strategy for exchanging the endogenous ubiquitin with an epitope-tagged version, we created a modified system, StUbEx PLUS, which allows precise mapping of ubiquitination sites by mass spectrometry. Application of StUbEx PLUS to U2OS cells treated with proteasomal inhibitors resulted in the identification of 41 589 sites on 7762 proteins, which thereby revealed the ubiquitous nature of this PTM and demonstrated the utility of the approach for comprehensive ubiquitination studies at site-specific resolution.

Cylindromatosis Tumor Suppressor Protein (CYLD) Deubiquitinase is Necessary for Proper Ubiquitination and Degradation of the Epidermal Growth Factor Receptor.

Cylindromatosis tumor suppressor protein (CYLD) is a deubiquitinase, best known as an essential negative regulator of the NFkB pathway. Previous studies have suggested an involvement of CYLD in epidermal growth factor (EGF)-dependent signal transduction as well, as it was found enriched within the tyrosine-phosphorylated complexes in cells stimulated with the growth factor. EGF receptor (EGFR) signaling participates in central cellular processes and its tight regulation, partly through ubiquitination cascades, is decisive for a balanced cellular homeostasis. Here, using a combination of mass spectrometry-based quantitative proteomic approaches with biochemical and immunofluorescence strategies, we demonstrate the involvement of CYLD in the regulation of the ubiquitination events triggered by EGF. Our data show that CYLD regulates the magnitude of ubiquitination of several major effectors of the EGFR pathway by assisting the recruitment of the ubiquitin ligase Cbl-b to the activated EGFR complex. Notably, CYLD facilitates the interaction of EGFR with Cbl-b through its Tyr15 phosphorylation in response to EGF, which leads to fine-tuning of the receptor's ubiquitination and subsequent degradation. This represents a previously uncharacterized strategy exerted by this deubiquitinase and tumors suppressor for the negative regulation of a tumorigenic signaling pathway.