Intracellular fraction of zona pellucida protein 3 is required for the oocyte-to-embryo transition in mice.

In oocyte biology, the zona pellucida has long been known to operate three extracellular functions downstream of the secretory pathway, namely, encasing the oocytes in ovarian follicles, mediating sperm-oocyte interaction, and preventing premature embryo contact with oviductal epithelium. The present study uncovers a fourth function that is fundamentally distinct from the other three, being critical for embryonic cell survival in mice. Intriguingly, the three proteins of the mouse zona pellucida (ZP1, ZP2, ZP3) were found abundantly present also inside the embryo 4 days after fertilization, as shown by mass spectrometry, immunoblotting, and immunofluorescence. Contrary to current understanding of the roles of ZP proteins, ZP3 was associated more with the cytoskeleton than with secretory vesicles in the subcortical region of metaphase II oocytes and zygotes, and was excluded from regions of cell-cell contact in cleavage-stage embryos. Trim-away-mediated knockdown of ZP3 in fertilized oocytes hampered the first zygotic cleavage, while ZP3 overexpression supported blastocyst formation. Transcriptome analysis of ZP3-knockdown embryos pointed at defects of cytoplasmic translation in the context of embryonic genome activation. This conclusion was supported by reduced protein synthesis in the ZP3-knockdown and by the lack of cleavage arrest when Trim-away was postponed from the one-cell to the late two-cell stage. These data place constraints on the notion that zona proteins only operate in the extracellular space, revealing also a role during the oocyte-to-embryo transition. Ultimately, these data recruit ZP3 into the family of maternal factors that contribute to developmental competence of mouse oocytes.

A Ran-binding protein facilitates nuclear import of human papillomavirus type 16.

Human papillomaviruses (HPVs) utilize an atypical mode of nuclear import during cell entry. Residing in the Golgi apparatus until mitosis onset, a subviral complex composed of the minor capsid protein L2 and viral DNA (L2/vDNA) is imported into the nucleus after nuclear envelope breakdown by associating with mitotic chromatin. In this complex, L2 plays a crucial role in the interactions with cellular factors that enable delivery and ultimately tethering of the viral genome to mitotic chromatin. To date, the cellular proteins facilitating these steps remain unknown. Here, we addressed which cellular proteins may be required for this process. Using label-free mass spectrometry, biochemical assays, microscopy, and functional virological assays, we discovered that L2 engages a hitherto unknown protein complex of Ran-binding protein 10 (RanBP10), karyopherin alpha2 (KPNA2), and dynein light chain DYNLT3 to facilitate transport towards mitotic chromatin. Thus, our study not only identifies novel cellular interactors and mechanism that facilitate a poorly understood step in HPV entry, but also a novel cellular transport complex.

Dopamine signaling regulates hematopoietic stem and progenitor cell function.

Hematopoietic stem and progenitor cell (HSPC) function in bone marrow (BM) is controlled by stroma-derived signals, but the identity and interplay of these signals remain incompletely understood. Here, we show that sympathetic nerve-derived dopamine directly controls HSPC behavior through D2 subfamily dopamine receptors. Blockade of dopamine synthesis, as well as pharmacological or genetic inactivation of D2 subfamily dopamine receptors, leads to reduced HSPC frequency, inhibition of proliferation, and low BM transplantation efficiency. Conversely, treatment with a D2-type receptor agonist increases BM regeneration and transplantation efficiency. Mechanistically, dopamine controls expression of the lymphocyte-specific protein tyrosine kinase (Lck), which, in turn, regulates MAPK-mediated signaling triggered by stem cell factor in HSPCs. Our work reveals critical functional roles of dopamine in HSPCs, which may open up new therapeutic options for improved BM transplantation and other conditions requiring the rapid expansion of HSPCs.

Dissecting the mechanism of signaling-triggered nuclear export of newly synthesized influenza virus ribonucleoprotein complexes.

Influenza viruses (IV) exploit a variety of signaling pathways. Previous studies showed that the rapidly accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway is functionally linked to nuclear export of viral ribonucleoprotein (vRNP) complexes, suggesting that vRNP export is a signaling-induced event. However, the underlying mechanism remained completely enigmatic. Here we have dissected the unknown molecular steps of signaling-driven vRNP export. We identified kinases RSK1/2 as downstream targets of virus-activated ERK signaling. While RSK2 displays an antiviral role, we demonstrate a virus-supportive function of RSK1, migrating to the nucleus to phosphorylate nucleoprotein (NP), the major constituent of vRNPs. This drives association with viral matrix protein 1 (M1) at the chromatin, important for vRNP export. Inhibition or knockdown of MEK, ERK or RSK1 caused impaired vRNP export and reduced progeny virus titers. This work not only expedites the development of anti-influenza strategies, but in addition demonstrates converse actions of different RSK isoforms.

The integrin-linked kinase is required for chemokine-triggered high-affinity conformation of the neutrophil ß2-integrin LFA-1.

Neutrophil adhesion and extravasation into tissue at sites of injury or infection depend on binding of the integrin lymphocyte function-associated antigen 1 (LFA-1) to ICAM-1 expressed on activated endothelial cells. The activation-dependent conformational change of LFA-1 to the high-affinity conformation (H+) requires kindlin-3 binding to the ß2-integrin cytoplasmic domain. Here we show that genetic deletion of the known kindlin interactor integrin-linked kinase (ILK) impaired neutrophil adhesion and extravasation in the cremaster muscle and in a clinically relevant model of renal ischemia reperfusion injury. Using in vitro microfluidic adhesion chambers and conformation-specific antibodies, we show that knockdown of ILK in HL-60 cells reduced the conformational change of ß2-integrins to the H+ conformation. Mechanistically, we found that ILK was required for protein kinase C (PKC) membrane targeting and chemokine-induced upregulation of its kinase activity. Moreover, PKC-α deficiency also resulted in impaired leukocyte adhesion in bone marrow chimeric mice. Mass spectrometric and western blot analyses revealed stimulation- and ILK-dependent phosphorylation of kindlin-3 upon activation. In summary, our data indicate an important role of ILK in kindlin-3-dependent conformational activation of LFA-1.

TrkC Is Essential for Nephron Function and Trans-Activates Igf1R Signaling.

BACKGROUND: Injury to kidney podocytes often results in chronic glomerular disease and consecutive nephron malfunction. For most glomerular diseases, targeted therapies are lacking. Thus, it is important to identify novel signaling pathways contributing to glomerular disease. Neurotrophic tyrosine kinase receptor 3 (TrkC) is expressed in podocytes and the protein transmits signals to the podocyte actin cytoskeleton. METHODS: Nephron-specific TrkC knockout (TrkC-KO) and nephron-specific TrkC-overexpressing (TrkC-OE) mice were generated to dissect the role of TrkC in nephron development and maintenance. RESULTS: Both TrkC-KO and TrkC-OE mice exhibited enlarged glomeruli, mesangial proliferation, basement membrane thickening, albuminuria, podocyte loss, and aspects of FSGS during aging. Igf1 receptor (Igf1R)-associated gene expression was dysregulated in TrkC-KO mouse glomeruli. Phosphoproteins associated with insulin, erb-b2 receptor tyrosine kinase (Erbb), and Toll-like receptor signaling were enriched in lysates of podocytes treated with the TrkC ligand neurotrophin-3 (Nt-3). Activation of TrkC by Nt-3 resulted in phosphorylation of the Igf1R on activating tyrosine residues in podocytes. Igf1R phosphorylation was increased in TrkC-OE mouse kidneys while it was decreased in TrkC-KO kidneys. Furthermore, TrkC expression was elevated in glomerular tissue of patients with diabetic kidney disease compared with control glomerular tissue. CONCLUSIONS: Our results show that TrkC is essential for maintaining glomerular integrity. Furthermore, TrkC modulates Igf-related signaling in podocytes.

The COP9 signalosome subunit 3 is necessary for early embryo survival by way of a stable protein deposit in mouse oocytes.

Investigations of genes required in early mammalian development are complicated by protein deposits of maternal products, which continue to operate after the gene locus has been disrupted. This leads to delayed phenotypic manifestations and underestimation of the number of genes known to be needed during the embryonic phase of cellular totipotency. Here we expose a critical role of the gene Cops3 by showing that it protects genome integrity during the 2-cell stage of mouse development, in contrast to the previous functional assignment at postimplantation. This new role is mediated by a substantial deposit of protein (94th percentile of the proteome), divided between an exceptionally stable cortical rim, which is prevalent in oocytes, and an ancillary deposit in the embryonic nuclei. Since protein abundance and stability defeat prospects of DNA- or RNA-based gene inactivation in oocytes, we harnessed a classical method next to an emerging method for protein inactivation: antigen masking (for functional inhibition) versus TRIM21-mediated proteasomal degradation, also known as 'Trim away' (for physical removal). Both resulted in 2-cell embryo lethality, unlike the embryos receiving anti-green fluorescent protein. Comparisons between COPS3 protein-targeted and non-targeted embryos revealed large-scale transcriptome differences, which were most evident for genes associated with biological functions critical for RNA metabolism and for the preservation of genome integrity. The gene expression abnormalities associated with COPS3 inactivation were confirmed in situ by the occurrence of DNA endoreduplication and DNA strand breaks in 2-cell embryos. These results recruit Cops3 to the small family of genes that are necessary for early embryo survival. Overall, assigning genes with roles in embryogenesis may be less safe than assumed, if the protein products of these genes accumulate in oocytes: the inactivation of a gene at the protein level can expose an earlier phenotype than that identified by genetic techniques such as conventional gene silencing.

Proximity proteomics of endothelial Weibel-Palade bodies identifies novel regulator of von Willebrand factor secretion.

Weibel-Palade bodies (WPB) are unique secretory organelles of endothelial cells that store factors regulating vascular hemostasis and local inflammation. Endothelial activation triggers rapid exocytosis of WPB, leading to the surface presentation of adhesion molecules relevant for leukocyte rolling (P-selectin) and platelet capture (von Willebrand factor [VWF]). Despite its role as an important secretory organelle, a comprehensive compilation of factors associated with WPB has not been carried out. We addressed this via a proximity proteomics approach employing the peroxidase APEX2 coupled with 2 known WPB-associated proteins: the Rab GTPases Rab3b and Rab27a. We show that APEX2-Rab3b/27a fusion constructs are correctly targeted to WPB of primary endothelial cells, and that proteins in their close proximity can be biotinylated through the WPB-recruited APEX2. Mass spectrometry analysis of the biotinylated proteins identified 183 WPB-associated proteins. Whereas these include factors reported before to localize to WPB, the majority comprises proteins not previously associated with WPB biology. Among them, the SNARE-interacting protein Munc13-2 was shown here to specifically localize to WPB and to serve as a novel factor promoting histamine-evoked WPB exocytosis and VWF secretion. Thus, APEX2-based proximity proteomics can be used to specifically identify novel organelle-associated factors in primary endothelial cells.

VE-PTP inhibition stabilizes endothelial junctions by activating FGD5.

Inhibition of VE-PTP, an endothelial receptor-type tyrosine phosphatase, triggers phosphorylation of the tyrosine kinase receptor Tie-2, which leads to the suppression of inflammation-induced vascular permeability. Analyzing the underlying mechanism, we show here that inhibition of VE-PTP and activation of Tie-2 induce tyrosine phosphorylation of FGD5, a GTPase exchange factor (GEF) for Cdc42, and stimulate its translocation to cell contacts. Interfering with the expression of FGD5 blocks the junction-stabilizing effect of VE-PTP inhibition in vitro and in vivo. Likewise, FGD5 is required for strengthening cortical actin bundles and inhibiting radial stress fiber formation, which are each stimulated by VE-PTP inhibition. We identify Y820 of FGD5 as the direct substrate for VE-PTP. The phosphorylation of FGD5-Y820 is required for the stabilization of endothelial junctions and for the activation of Cdc42 by VE-PTP inhibition but is dispensable for the recruitment of FGD5 to endothelial cell contacts. Thus, activation of FGD5 is a two-step process that comprises membrane recruitment and phosphorylation of Y820. These steps are necessary for the junction-stabilizing effect stimulated by VE-PTP inhibition and Tie-2 activation.

Vascular Endothelial Receptor Tyrosine Phosphatase: Identification of Novel Substrates Related to Junctions and a Ternary Complex with EPHB4 and TIE2.

Vascular endothelial protein tyrosine phosphatase (VE-PTP, PTPRB) is a receptor type phosphatase that is crucial for the regulation of endothelial junctions and blood vessel development. We and others have shown recently that VE-PTP regulates vascular integrity by dephosphorylating substrates that are key players in endothelial junction stability, such as the angiopoietin receptor TIE2, the endothelial adherens junction protein VE-cadherin and the vascular endothelial growth factor receptor VEGFR2. Here, we have systematically searched for novel substrates of VE-PTP in endothelial cells by utilizing two approaches. First, we studied changes in the endothelial phosphoproteome on exposing cells to a highly VE-PTP-specific phosphatase inhibitor followed by affinity isolation and mass-spectrometric analysis of phosphorylated proteins by phosphotyrosine-specific antibodies. Second, we used a substrate trapping mutant of VE-PTP to pull down phosphorylated substrates in combination with SILAC-based quantitative mass spectrometry measurements. We identified a set of substrate candidates of VE-PTP, of which a remarkably large fraction (29%) is related to cell junctions. Several of those were found in both screens and displayed very high connectivity in predicted functional interaction networks. The receptor protein tyrosine kinase EPHB4 was the most prominently phosphorylated protein on VE-PTP inhibition among those VE-PTP targets that were identified by both proteomic approaches. Further analysis revealed that EPHB4 forms a ternary complex with VE-PTP and TIE2 in endothelial cells. VE-PTP controls the phosphorylation of each of these two tyrosine kinase receptors. Despite their simultaneous presence in a ternary complex, stimulating each of the receptors with their own specific ligand did not cross-activate the respective partner receptor. Our systematic approach has led to the identification of novel substrates of VE-PTP, of which many are relevant for the control of cellular junctions further promoting the importance of VE-PTP as a key player of junctional signaling.

Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels.

Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate quantification. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials and one end-capped C18 reference material as stationary phases for reversed-phase separation. We found that these matrices have complementing retention and separation characteristics, including the capability to separate structural isomers. PGC and C18 matrices yielded excellent signal-to-noise ratios in nLC-MS while differing in the separation capability and sensitivity for various nucleosides. This emphasizes the need for tailored LC-MS setups for optimally detecting as many nucleoside modifications as possible. Detection ranges spanning up to six orders of magnitude enable the analysis of individual ribonucleosides down to femtomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications.

Extrinsic immune cell-derived, but not intrinsic oligodendroglial factors contribute to oligodendroglial differentiation block in multiple sclerosis.

Multiple sclerosis (MS) is the most frequent demyelinating disease in young adults and despite significant advances in immunotherapy, disease progression still cannot be prevented. Promotion of remyelination, an endogenous repair mechanism resulting in the formation of new myelin sheaths around demyelinated axons, represents a promising new treatment approach. However, remyelination frequently fails in MS lesions, which can in part be attributed to impaired differentiation of oligodendroglial progenitor cells into mature, myelinating oligodendrocytes. The reasons for impaired oligodendroglial differentiation and defective remyelination in MS are currently unknown. To determine whether intrinsic oligodendroglial factors contribute to impaired remyelination in relapsing-remitting MS (RRMS), we compared induced pluripotent stem cell-derived oligodendrocytes (hiOL) from RRMS patients and controls, among them two monozygous twin pairs discordant for MS. We found that hiOL from RRMS patients and controls were virtually indistinguishable with respect to remyelination-associated functions and proteomic composition. However, while analyzing the effect of extrinsic factors we discovered that supernatants of activated peripheral blood mononuclear cells (PBMCs) significantly inhibit oligodendroglial differentiation. In particular, we identified CD4+ T cells as mediators of impaired oligodendroglial differentiation; at least partly due to interferon-gamma secretion. Additionally, we observed that blocked oligodendroglial differentiation induced by PBMC supernatants could not be restored by application of oligodendroglial differentiation promoting drugs, whereas treatment of PBMCs with the immunomodulatory drug teriflunomide prior to supernatant collection partly rescued oligodendroglial differentiation. In summary, these data indicate that the oligodendroglial differentiation block is not due to intrinsic oligodendroglial factors but rather caused by the inflammatory environment in RRMS lesions which underlines the need for drug screening approaches taking the inflammatory environment into account. Combined, these findings may contribute to the development of new remyelination promoting strategies.

A framework for TRIM21-mediated protein depletion in early mouse embryos: recapitulation of Tead4 null phenotype over three days.

BACKGROUND: While DNA and RNA methods are routine to disrupt the expression of specific genes, complete understanding of developmental processes requires also protein methods, because: oocytes and early embryos accumulate proteins and these are not directly affected by DNA and RNA methods. When proteins in the oocyte encounter a specific antibody and the TRIpartite Motiv-containing 21 (TRIM21) ubiquitin-protein ligase, they can be committed to degradation in the proteasome, producing a transient functional knock-out that reveals the role of the protein. However, there are doubts about whether this targeted proteolysis could be successfully used to study mammalian development, because duration of the transient effect is unknown, and also because amounts of reagents delivered must be adequate in relation to the amount of target protein, which is unknown, too. RESULTS: We show that the mouse egg contains up to 1E-02 picomoles/protein, as estimated by mass spectrometry using the intensity-based absolute quantification (iBAQ) algorithm. However, the egg can only accommodate ≈1E-04 picomoles of antibody or TRIM21 without incurring toxic effects. Within this framework, we demonstrate that TRIM21-mediated protein depletion efficiently disrupts the embryonic process of trophectoderm formation, which critically depends on the TEA domain family member 4 (Tead4) gene. TEAD4 depletion starting at the 1-cell stage lasts for 3 days prior to a return of gene and protein expression to baseline. This time period is long enough to result in a phenotype entirely consistent with that of the published null mutation and RNA interference studies: significant underexpression of trophectodermal genes Cdx2 and Gata3 and strongly impaired ability of embryos to cavitate and implant in the uterus. Omics data are available via ProteomeXchange (PXD012613) and GEO (GSE124844). CONCLUSIONS: TRIM21-mediated protein depletion can be an effective means to disrupt gene function in mouse development, provided the target gene is chosen carefully and the method is tuned accurately. The knowledge gathered in this study provides the basic know-how (prerequisites, requirements, limitations) to expedite the protein depletion of other genes besides Tead4.

FUS pathology in ALS is linked to alterations in multiple ALS-associated proteins and rescued by drugs stimulating autophagy.

Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by motor neuron degeneration and associated with aggregation of nuclear RNA-binding proteins (RBPs), including FUS. How FUS aggregation and neurodegeneration are prevented in healthy motor neurons remain critically unanswered questions. Here, we use a combination of ALS patient autopsy tissue and induced pluripotent stem cell-derived neurons to study the effects of FUS mutations on RBP homeostasis. We show that FUS' tendency to aggregate is normally buffered by interacting RBPs, but this buffering is lost when FUS mislocalizes to the cytoplasm due to ALS mutations. The presence of aggregation-prone FUS in the cytoplasm causes imbalances in RBP homeostasis that exacerbate neurodegeneration. However, enhancing autophagy using small molecules reduces cytoplasmic FUS, restores RBP homeostasis and rescues motor function in vivo. We conclude that disruption of RBP homeostasis plays a critical role in FUS-ALS and can be treated by stimulating autophagy.

An evolutionary approach uncovers a diverse response of tRNA 2-thiolation to elevated temperatures in yeast.

Chemical modifications of transfer RNA (tRNA) molecules are evolutionarily well conserved and critical for translation and tRNA structure. Little is known how these nucleoside modifications respond to physiological stress. Using mass spectrometry and complementary methods, we defined tRNA modification levels in six yeast species in response to elevated temperatures. We show that 2-thiolation of uridine at position 34 (s(2)U34) is impaired at temperatures exceeding 30°C in the commonly used Saccharomyces cerevisiae laboratory strains S288C and W303, and in Saccharomyces bayanus. Upon stress relief, thiolation levels recover and we find no evidence that modified tRNA or s(2)U34 nucleosides are actively removed. Our results suggest that loss of 2-thiolation follows accumulation of newly synthesized tRNA that lack s(2)U34 modification due to temperature sensitivity of the URM1 pathway in S. cerevisiae and S. bayanus. Furthermore, our analysis of the tRNA modification pattern in selected yeast species revealed two alternative phenotypes. Most strains moderately increase their tRNA modification levels in response to heat, possibly constituting a common adaptation to high temperatures. However, an overall reduction of nucleoside modifications was observed exclusively in S288C. This surprising finding emphasizes the importance of studies that utilize the power of evolutionary biology, and highlights the need for future systematic studies on tRNA modifications in additional model organisms.

Proteomic Analysis of Mouse Oocytes Identifies PRMT7 as a Reprogramming Factor that Replaces SOX2 in the Induction of Pluripotent Stem Cells.

The reprogramming process that leads to induced pluripotent stem cells (iPSCs) may benefit from adding oocyte factors to Yamanaka's reprogramming cocktail (OCT4, SOX2, KLF4, with or without MYC; OSK(M)). We previously searched for such facilitators of reprogramming (the reprogrammome) by applying label-free LC-MS/MS analysis to mouse oocytes, producing a catalog of 28 candidates that are (i) able to robustly access the cell nucleus and (ii) shared between mature mouse oocytes and pluripotent embryonic stem cells. In the present study, we hypothesized that our 28 reprogrammome candidates would also be (iii) abundant in mature oocytes, (iv) depleted after the oocyte-to-embryo transition, and (v) able to potentiate or replace the OSKM factors. Using LC-MS/MS and isotopic labeling methods, we found that the abundance profiles of the 28 proteins were below those of known oocyte-specific and housekeeping proteins. Of the 28 proteins, only arginine methyltransferase 7 (PRMT7) changed substantially during mouse embryogenesis and promoted the conversion of mouse fibroblasts into iPSCs. Specifically, PRMT7 replaced SOX2 in a factor-substitution assay, yielding iPSCs. These findings exemplify how proteomics can be used to prioritize the functional analysis of reprogrammome candidates. The LC-MS/MS data are available via ProteomeXchange with identifier PXD003093.

Esm1 modulates endothelial tip cell behavior and vascular permeability by enhancing VEGF bioavailability.

RATIONALE: Endothelial cell-specific molecule 1 (Esm1) is a secreted protein thought to play a role in angiogenesis and inflammation. However, there is currently no direct in vivo evidence supporting a function of Esm1 in either of these processes. OBJECTIVE: To determine the role of Esm1 in vivo and the underlying molecular mechanisms. METHODS AND RESULTS: We generated and analyzed Esm1 knockout (Esm1(KO)) mice to study its role in angiogenesis and inflammation. Esm1 expression is induced by the vascular endothelial growth factor A (VEGF-A) in endothelial tip cells of the mouse retina. Esm1(KO) mice showed delayed vascular outgrowth and reduced filopodia extension, which are both VEGF-A-dependent processes. Impairment of Esm1 function led to a decrease in phosphorylated Erk1/2 (extracellular-signal regulated kinases 1/2) in sprouting vessels. We also found that Esm1(KO) mice displayed a 40% decrease in leukocyte transmigration. Moreover, VEGF-induced vascular permeability was decreased by 30% in Esm1(KO) mice and specifically on stimulation with VEGF-A165 but not VEGF-A121. Accordingly, cerebral edema attributable to ischemic stroke-induced vascular permeability was reduced by 50% in the absence of Esm1. Mechanistically, we show that Esm1 binds directly to fibronectin and thereby displaces fibronectin-bound VEGF-A165 leading to increased bioavailability of VEGF-A165 and subsequently enhanced levels of VEGF-A signaling. CONCLUSIONS: Esm1 is simultaneously a target and modulator of VEGF signaling in endothelial cells, playing a role in angiogenesis, inflammation, and vascular permeability, which might be of potential interest for therapeutic applications.

Toll-like receptor 9 protects non-immune cells from stress by modulating mitochondrial ATP synthesis through the inhibition of SERCA2.

Toll-like receptor 9 (TLR9) has a key role in the recognition of pathogen DNA in the context of infection and cellular DNA that is released from damaged cells. Pro-inflammatory TLR9 signalling pathways in immune cells have been well investigated, but we have recently discovered an alternative pathway in which TLR9 temporarily reduces energy substrates to induce cellular protection from stress in cardiomyocytes and neurons. However, the mechanism by which TLR9 stimulation reduces energy substrates remained unknown. Here, we identify the calcium-transporting ATPase, SERCA2 (also known as Atp2a2), as a key molecule for the alternative TLR9 signalling pathway. TLR9 stimulation reduces SERCA2 activity, modulating Ca(2+) handling between the SR/ER and mitochondria, which leads to a decrease in mitochondrial ATP levels and the activation of cellular protective machinery. These findings reveal how distinct innate responses can be elicited in immune and non-immune cells--including cardiomyocytes--using the same ligand-receptor system.

Differences in embryo quality are associated with differences in oocyte composition: a proteomic study in inbred mice.

Current models of early mouse development assign roles to stochastic processes and epigenetic regulation, which are considered to be as influential as the genetic differences that exist between strains of the species Mus musculus. The aim of this study was to test whether mouse oocytes vary from each other in the abundance of gene products that could influence, prime, or even predetermine developmental trajectories and features of derivative embryos. Using the paradigm of inbred mouse strains, we quantified 2010 protein groups (SILAC LC-MS/MS) and 15205 transcripts (RNA deep sequencing) present simultaneously in oocytes of four strains tested (129/Sv, C57Bl/6J, C3H/HeN, DBA/2J). Oocytes differed according to donor strain in the abundance of catalytic and regulatory proteins, as confirmed for a subset (bromodomain adjacent to zinc finger domain, 1B [BAZ1B], heme oxygenase 1 [HMOX1], estrogen related receptor, beta [ESRRB]) via immunofluorescence in situ. Given a Pearson's r correlation coefficient of 0.18-0.20, the abundance of oocytic proteins could not be predicted from that of cognate mRNAs. Our results document that a prerequisite to generate embryo diversity, namely the different abundances of maternal proteins in oocytes, can be studied in the model of inbred mouse strains. Thus, we highlight the importance of proteomic quantifications in modern embryology. All MS data have been deposited in the ProteomeXchange with identifier PXD001059 (http://proteomecentral.proteomexchange.org/dataset/PXD001059).