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Thousands of long noncoding RNAs are encoded in mammalian genomes, yet most remain uncharacterized. In this study, we functionally characterized a mouse long noncoding RNA named U90926. Analysis of U90926 RNA levels revealed minimal expression across multiple tissues at steady state. However, the expression of this gene was highly induced in macrophages and dendritic cells by TLR activation, in a p38 MAPK- and MyD88-dependent manner. To study the function of U90926, we generated U90926-deficient (U9-KO) mice. Surprisingly, we found minimal effects of U90926 deficiency in cultured macrophages. Given the lack of macrophage-intrinsic effect, we investigated the subcellular localization of U90926 transcript and its protein-coding potential. We found that U90926 RNA localizes to the cytosol, associates with ribosomes, and contains an open reading frame that encodes a novel glycosylated protein (termed U9-ORF), which is secreted from the cell. An in vivo model of endotoxic shock revealed that, in comparison with wild type mice, U9-KO mice exhibited increased sickness responses and mortality. Mechanistically, serum levels of IL-6 were elevated in U9-KO mice, and IL-6 neutralization improved endotoxemia outcomes in U9-KO mice. Taken together, these results suggest that U90926 expression is protective during endotoxic shock, potentially mediated by the paracrine and/or endocrine actions of the novel U9-ORF protein secreted by activated myeloid cells.

Raich-Rac1 Imaging, video 1

Raich-Rac1 Imaging, video 2

Glioblastoma is a primary brain cancer that is resistant to all treatment modalities. This resistance is due, in large part, to invasive cancer cells that disperse from the main tumor site, escape surgical resection, and contribute to recurrent secondary lesions. The adhesion and signaling mechanisms that drive glioblastoma cell invasion remain enigmatic, and as a result there are no effective anti-invasive clinical therapies. Here we have characterized a novel adhesion and signaling pathway comprised of the integrin αvβ8 and its intracellular binding partner, Spinophilin (Spn), which regulates glioblastoma cell invasion in the brain microenvironment. We show for the first time that Spn binds directly to the cytoplasmic domain of β8 integrin in glioblastoma cells. Genetically targeting Spn leads to enhanced invasive cell growth in preclinical models of glioblastoma. Spn regulates glioblastoma cell invasion by modulating the formation and dissolution of invadopodia. Spn-regulated invadopodia dynamics are dependent, in part, on proper spatiotemporal activation of the Rac1 GTPase. Glioblastoma cells that lack Spn showed diminished Rac1 activities, increased numbers of invadopodia, and enhanced extracellular matrix degradation. Collectively, these data identify Spn as a critical adhesion and signaling protein that is essential for modulating glioblastoma cell invasion in the brain microenvironment.Implications: Tumor cell invasion is a major clinical obstacle in glioblastoma and this study identifies a new signaling pathway regulated by Spinophilin in invasive glioblastoma. Mol Cancer Res; 14(12); 1277–87. ©2016 AACR.

Supplementary Table 1. Peptides identified by LC-MS/MS and SEQUEST searching for integrin αv, integrin β8, and Spinophilin. Supplemental Figure 1. Analysis of β8 integrin-Spn binding and Spn protein localization in vitro and in cultured cells. Supplemental Figure 2. Spn protein is expressed in recurrent human GBM samples. Supplemental Figure 3. Spn mRNA is differentially expressed in GBM regions, but expression levels do not correlate with patient survival. Supplemental Figure 4. Experimental strategy to generate mosaic mouse models of the brain cancer astrocytoma. Supplemental Figure 5. Genetically targeting Spn in human GBM cells using RNAi leads to enhanced tumor cell invasion. Supplemental Figure 6. Microscopic analysis of Spn-dependent invasive growth in LN229 tumors. Supplemental Figure 7. Immunohistochemical analysis of xenograft tumors derived from LN229 cells expressing control or Spn shRNAs. Supplemental Figure 8. Analysis of invadopodia formation in GBM cells. Supplemental Videos 1 and 2. Imaging Rac1 GTPase activities in live cells by FRET.

Basement membranes (BM) play fundamental roles in morphogenesis and tissue maintenance in multicellular organisms. Glutactin is a BM protein that belongs to the Cholinesterase-Like Adhesion Molecules (CLAMs) protein family. InDrosophilaembryos, Glutactin has been shown to outline internal organs and to play a role in synapse formation. Here, we report that Glutactin is broadly expressed in BM surrounding most vital tissues of the larva and the adult, and within the larval muscle sarcomere. Ubiquitous RNAi driven down-regulation of Glutactin expression (Tub>Glt-RNAi) resulted in pronounced impairments in larval and adult locomotor behavior, reduced oviposition, and shortened lifespan. Muscle-specific down-regulation of Glutactin resulted in reduced larval crawling speed indicating a secondary function for Glutactin independent of BM expression.Tub>Glt-RNAipupa showed abdominal scars, suggestive of defects in histoblast nest expansion and replacement of larval epidermal cells, and a high mortality rate at eclosion. Surviving adults showed a range of morphological and physiological defects including excess melanization and pigmentation, incomplete rotation and duplication of the genitalia, and abnormal heart morphology and contraction. Insofar excess melanization is symptomatic of internal tissue damage, we propose that Glutactin is essential for the mechanical stabilization of the BM and for its ability to withstand internal stresses.

Axon guidance is a critical process in forming the connections between a neuron and its target. The growth cone steers the growing axon toward the appropriate direction by integrating extracellular guidance cues and initiating intracellular signal transduction pathways downstream of these cues. The growth cone generates these responses by remodeling its cytoskeletal components. Regulation of microtubule dynamics within the growth cone is important for making guidance decisions. TACC3, as a microtubule plus-end binding (EB) protein, modulates microtubule dynamics during axon outgrowth and guidance. We have previously shown that Xenopus laevis embryos depleted of TACC3 displayed spinal cord axon guidance defects, while TACC3-overexpressing spinal neurons showed increased resistance to Slit2-induced growth cone collapse. Tyrosine kinases play an important role in relaying guidance signals to downstream targets during pathfinding events via inducing tyrosine phosphorylation. Here, in order to investigate the mechanism behind TACC3-mediated axon guidance, we examined whether tyrosine residues that are present in TACC3 have any role in regulating TACC3's interaction with microtubules or during axon outgrowth and guidance behaviors. We find that the phosphorylatable tyrosines within the TACC domain are important for the microtubule plus-end tracking behavior of TACC3. Moreover, TACC domain phosphorylation impacts axon outgrowth dynamics such as growth length and growth persistency. Together, our results suggest that tyrosine phosphorylation of TACC3 affects TACC3's microtubule plus-end tracking behavior as well as its ability to mediate axon growth dynamics in cultured embryonic neural tube explants.

The Vel blood group antigen is carried on the short extracellular segment of the 78-amino-acid-long, type II transmembrane protein SMIM1 of unknown function. Here, using biochemical analysis and flow cytometry of cells expressing wild-type and mutant alleles of SMIM1, we demonstrate that dimerization of SMIM1 promotes cell surface display of the Vel epitope. We show that SMIM1 dimerization is mediated both by an extracellular Cys77-dependent, homomeric disulfide linkage and via a GxxxG helix-helix interaction motif in the transmembrane domain. These results provide important context for the observed variability in reactivity patterns of clinically important anti-Vel identified in patient sera.

Flaviviruses are enveloped, positive-strand RNA viruses that cause millions of infections in the human population annually. Although Zika virus (ZIKV) had been detected in humans as early as the 1950s, its reemergence in South America in 2015 resulted in a global health crisis. While flaviviruses encode 10 proteins that can be post-translationally modified by host enzymes, little is known regarding post-translational modifications (PTMs) of the flavivirus proteome. We used mass spectrometry to comprehensively identify host-driven PTMs on the ZIKV proteome. This approach allowed us to identify 43 PTMs across 8 ZIKV proteins, including several that are highly conserved within theFlavivirusgenus. Notably, we found two phosphosites on the ZIKV envelope protein that are functionally important for viral propagation and appear to regulate viral budding. Additionally, we discovered 115 host kinases that interacted with ZIKV proteins and determined that Bosutinib, an FDA-approved tyrosine kinase inhibitor that targets ZIKV interacting host kinases, impairs ZIKV growth. Thus, we have defined a high-resolution map of host-driven PTMs on ZIKV proteins as well as cellular interacting kinases, uncovered a novel mechanism of host driven-regulation of ZIKV budding, and identified an FDA-approved inhibitor of ZIKV growth.

Semaphorins (Semas) are a family of secreted and transmembrane proteins that play critical roles in development. Interestingly, several vertebrate transmembrane Sema classes are capable of producing functional soluble ectodomains. However, little is known of soluble Sema6 ectodomains in the nervous system. Herein, we show that the soluble Sema6A ectodomain, sSema6A, exhibits natural and protein kinase C (PKC)-induced release. We show that PKC mediates Sema6A phosphorylation at specific sites and while this phosphorylation is not the primary mechanism regulating sSema6A production, we found that the intracellular domain confers resistance to ectodomain release. Finally, sSema6A is functional as it promotes the cohesion of zebrafish early eye field explants. This suggests that in addition to its canonical contact-mediated functions, Sema6A may have regulated, long-range, forward-signaling capacity.

The discoidin, CUB, and LCCL domain-containing (DCBLD) receptor family are composed of the type-I transmembrane proteins DCBLD1 and DCBLD2 (also ESDN and CLCP1). These proteins are highly conserved across vertebrates and possess similar domain structure to that of neuropilins, which act as critical co-receptors in developmental processes. Although DCBLD1 remains largely uncharacterized, the functional and mechanistic roles of DCBLD2 are emerging. This review provides a comprehensive discussion of this presumed receptor family, ranging from structural and signaling aspects to their associations with cancer, physiology, and development.

Background The development of the central nervous system (CNS) requires critical cell signaling molecules to coordinate cell proliferation and migration in order to structure the adult tissue. Chicken tumor virus #10 Regulator of Kinase (CRK) and CRK-like (CRKL) are adaptor proteins with pre-metazoan ancestry and are known to be required for patterning laminated structures downstream of Reelin (RELN), such as the cerebral cortex, cerebellum, and hippocampus. CRK and CRKL also play crucial roles in a variety of other growth factor and extracellular matrix signaling cascades. The neuronal retina is another highly laminated structure within the CNS that is dependent on migration for proper development, but the cell signaling mechanisms behind neuronal positioning in the retina are only partly understood. Results We find that crk and crkl have largely overlapping expression within the developing zebrafish nervous system. We find that their disruption results in smaller eye size and loss of retinal lamination. Conclusions Our data indicate that Crk adaptors are critical for proper development of the zebrafish neural retina in a crk/crkl dose-dependent manner.

Key Points TCR-γδ tetramer identified ligand expression by flow cytometry. TCR-γδ ligands were induced on activated monocytes or T cells. Bioinformatics combined with mass spectrometry produced an overlapping list of 16 candidate ligands., Visual Abstract, Lack of understanding of the nature and physiological regulation of γδ T cell ligands has considerably hampered full understanding of the function of these cells. We developed an unbiased approach to identify human γδ T cells ligands by the production of a soluble TCR-γδ (sTCR-γδ) tetramer from a synovial Vδ1 γδ T cell clone from a Lyme arthritis patient. The sTCR-γδ was used in flow cytometry to initially define the spectrum of ligand expression by both human tumor cell lines and certain human primary cells. Analysis of diverse tumor cell lines revealed high ligand expression on several of epithelial or fibroblast origin, whereas those of hematopoietic origin were largely devoid of ligand. This allowed a bioinformatics-based identification of candidate ligands using RNAseq data from each tumor line. We further observed that whereas fresh monocytes and T cells expressed low to negligible levels of TCR-γδ ligands, activation of these cells resulted in upregulation of surface ligand expression. Ligand upregulation on monocytes was partly dependent upon IL-1β. The sTCR-γδ tetramer was then used to bind candidate ligands from lysates of activated monocytes and analyzed by mass spectrometry. Surface TCR-γδ ligand was eliminated by treatment with trypsin or removal of glycosaminoglycans, and also suppressed by inhibition of endoplasmic reticulum–Golgi transport. Of particular interest was that inhibition of glycolysis also blocked TCR-γδ ligand expression. These findings demonstrate the spectrum of ligand(s) expression for human synovial Vδ1 γδ T cells as well as the physiology that regulates their expression.

The FYN- and ABL-regulated interactomes of DCBLD proteins have been determined using quantitative mass spectrometry-based proteomics. FYN and ABL drove the binding of several signaling molecules to DCBLD1 and DCBLD2, including members of the 14-3-3 family of adaptors. Biochemical investigation of the DCBLD2/14-3-3 interaction revealed ABL-induced binding of 14-3-3 family members directly to DCBLD2.The interactions identified in this screen present important steps in elucidating signaling events involving DCBLD proteins in developmental and oncogenic signaling., Graphical Abstract Highlights • FYN and ABL differentially regulate DCBLD Ser/Thr/Tyr phosphorylation. • DCBLD1 and DCBLD2 interactomes are modulated by FYN and ABL. • ABL drives a direct DCBLD2/14-3-3 interaction., The Discoidin, CUB, and LCCL domain-containing protein (DCBLD) family consists of two type-I transmembrane scaffolding receptors, DCBLD1 and DCBLD2, which play important roles in development and cancer. The nonreceptor tyrosine kinases FYN and ABL are known to drive phosphorylation of tyrosine residues in YXXP motifs within the intracellular domains of DCBLD family members, which leads to the recruitment of the Src homology 2 (SH2) domain of the adaptors CT10 regulator of kinase (CRK) and CRK-like (CRKL). We previously characterized the FYN- and ABL-driven phosphorylation of DCBLD family YXXP motifs. However, we have identified additional FYN- and ABL-dependent phosphorylation sites on DCBLD1 and DCBLD2. This suggests that beyond CRK and CRKL, additional DCBLD interactors may be regulated by FYN and ABL activity. Here, we report a quantitative proteomics approach in which we map the FYN- and ABL-regulated interactomes of DCBLD family members. We found FYN and ABL regulated the binding of several signaling molecules to DCBLD1 and DCBLD2, including members of the 14-3-3 family of adaptors. Biochemical investigation of the DCBLD2/14-3-3 interaction revealed ABL-induced binding of 14-3-3 family members directly to DCBLD2.

Incremental increases in a driver variable, such as nutrients or detritus, can trigger abrupt shifts in aquatic ecosys-tems. Once these ecosystems change state, a simple reduction in the driver variable may not return them to their original state. Because of the long time scales involved, we still have a poor understanding of the dynamics of ecosys-tem recovery after a state change. A model system for understanding ecosystem recovery is the aquatic microecosystem that inhabits the cup-shaped leaves of the pitcher plant Sarracenia purpurea. With enrichment of organic matter, this system flips within 1 to 3 days from an oxygen-rich state to an oxygen-poor (hypoxic) state. In a replicated green-house experiment, we enriched pitcher plant leaves at different rates with bovine serum albumin (BSA), a molecular substitute for detritus. Changes in dissolved oxygen ([O2]) and undigested BSA concentration were monitored during enrichment and recovery phases. At low enrichment rates, ecosystems showed a substantial lag in the recovery of [O2] (clockwise hysteresis). At intermediate enrichment rates, [O2] tracked the levels of undigested BSA with the same profile during the enrichment and recovery phases (no hysteresis). At high enrichment rates, we observed a novel response: changes in [O2] were proportionally larger during the recovery phase than during the enrichment phase (counter-clockwise hysteresis). These experiments demonstrate that detrital enrichment rate can modulate a diversity of hysteretic responses in a single aquatic ecosystem. With counter-clockwise hysteresis, rapid reduction of a driver variable following high enrichment rates may be a viable restoration strategy.

Axon guidance is a critical process in forming the connections between a neuron and its target. The growth cone steers the growing axon towards the appropriate direction by integrating extracellular guidance cues and initiating intracellular signal transduction pathways downstream of these cues. The growth cone generates these responses by remodeling its cytoskeletal components. Regulation of microtubule dynamics within the growth cone is important for making guidance decisions. TACC3, as a microtubule plus-end binding protein, modulates microtubule dynamics during axon outgrowth and guidance. We have previously shown that embryos depleted of TACC3 displayed spinal cord axon guidance defects, while TACC3-overexpressing spinal neurons showed increased resistance to Slit2-induced growth cone collapse. Here, in order to investigate the mechanism behind TACC3-mediated axon guidance, we studied the importance of tyrosine phosphorylation induced by Abelson tyrosine kinase. We find that the phosphorylatable tyrosines within the TACC domain are important for the microtubule plus-end tracking behavior of TACC3. Moreover, TACC domain phosphorylation impacts axon outgrowth and guidance, and it also regulates microtubule extension into the growth cone periphery. Together, our results suggest that phosphorylation of TACC3 is a key regulatory mechanism by which TACC3 controls axon outgrowth and pathfinding decisions of neurons during embryonic development.

Studies comparing endogenous and recombinant serum amyloid A (SAA) have generated conflicting data on the proinflammatory function of these proteins. In exploring this discrepancy, we found that in contrast to commercially sourced recombinant human SAA1 (hSAA1) proteins produced in Escherichia coli, hSAA1 produced from eukaryotic cells did not promote proinflammatory cytokine production from human or mouse cells, induce Th17 differentiation, or stimulate TLR2. Proteomic analysis of E. coli–derived hSAA1 revealed the presence of numerous bacterial proteins, with several being reported or probable lipoproteins. Treatment of hSAA1 with lipoprotein lipase or addition of a lipopeptide to eukaryotic cell–derived hSAA1 inhibited or induced the production of TNF-α from macrophages, respectively. Our results suggest that a function of SAA is in the binding of TLR2-stimulating bacterial proteins, including lipoproteins, and demand that future studies of SAA employ a recombinant protein derived from eukaryotic cells.

The type I cGMP-dependent protein kinase (PKG I) is an essential regulator of vascular tone. It has been demonstrated that the type Iα isoform can be constitutively activated by oxidizing conditions. However, the amino acid residues implicated in this phenomenon are not fully elucidated. To investigate the molecular basis for this mechanism, we studied the effects of oxidation using recombinant WT, truncated, and mutant constructs of PKG I. Using an in vitro assay, we observed that oxidation with hydrogen peroxide (H(2)O(2)) resulted in constitutive, cGMP-independent activation of PKG Iα. PKG Iα C42S and a truncation construct that does not contain Cys-42 (Δ53) were both constitutively activated by H(2)O(2). In contrast, oxidation of PKG Iα C117S maintained its cGMP-dependent activation characteristics, although oxidized PKG Iα C195S did not. To corroborate these results, we also tested the effects of our constructs on the PKG Iα–specific substrate, the large conductance potassium channel (K(Ca) 1.1). Application of WT PKG Iα activated by either cGMP or H(2)O(2) increased the open probabilities of the channel. Neither cGMP nor H(2)O(2) activation of PKG Iα C42S significantly increased channel open probabilities. Moreover, cGMP-stimulated PKG Iα C117S increased K(Ca) 1.1 activity, but this effect was not observed under oxidizing conditions. Finally, we observed that PKG Iα C42S caused channel flickers, indicating dramatically altered K(Ca) 1.1 channel characteristics compared with channels exposed to WT PKG Iα. Cumulatively, these results indicate that constitutive activation of PKG Iα proceeds through oxidation of Cys-117 and further suggest that the formation of a sulfur acid is necessary for this phenotype.

Plexins (Plxns) are semaphorin (Sema) receptors that play important signaling roles, particularly in the developing nervous system and vasculature. Sema-Plxn signaling regulates cellular processes such as cytoskeletal dynamics, proliferation, and differentiation. However, the receptor-proximal signaling mechanisms driving Sema-Plxn signal transduction are only partially understood. Plxn tyrosine phosphorylation is thought to play an important role in these signaling events as receptor and nonreceptor tyrosine kinases have been shown to interact with Plxn receptors. The Src family kinase Fyn can induce the tyrosine phosphorylation of PlxnA1 and PlxnA2. However, the Fyn-dependent phosphorylation sites on these receptors have not been identified. Here, using mass spectrometry-based approaches, we have identified highly conserved, Fyn-induced PlexinA (PlxnA) tyrosine phosphorylation sites. Mutation of these sites to phenylalanine results in significantly decreased Fyn-dependent PlxnA tyrosine phosphorylation. Furthermore, in contrast to wild-type human PLXNA2 mRNA, mRNA harboring these point mutations cannot rescue eye developmental defects when coinjected with a plxnA2 morpholino in zebrafish embryos. Together these data suggest that Fyn-dependent phosphorylation at two critical tyrosines is a key feature of vertebrate PlxnA1 and PlxnA2 signal transduction.

Background Semaphorin (Sema)/Plexin (Plxn) signaling is important for many aspects of neuronal development, however, the transcriptional regulation imposed by this signaling pathway is unknown. Previously, we identified an essential role for Sema6A/PlxnA2 signaling in regulating proliferation and cohesion of retinal precursor cells (RPCs) during early eye development. This study used RNA isolated from control, Sema6A-deficient and PlxnA2-deficient zebrafish embryos in a microarray analysis to identify genes that were differentially expressed when this signaling pathway was disrupted. Results We uncovered a set of 58 transcripts, and all but 1 were up-regulated in both sema6A and plxnA2 morphants. We validated gene expression changes in subset of candidates that are suggested to be involved in proliferation, migration or neuronal positioning. We further functionally evaluated one gene, rasl11b, as contributing to disrupted proliferation in sema6A and plxna2 morphants. Our results suggest rasl11b negatively regulates proliferation of RPCs in the developing zebrafish eye. Conclusions Microarray analysis has generated a resource of target genes downstream of Sema6A/PlxnA2 signaling, which can be further investigated to elucidate the downstream effects of this well-studied neuronal and vascular guidance signaling pathway. Developmental Dynamics 246:539-549, 2017. © 2017 Wiley Periodicals, Inc.

The phylogenetic relationships among genera of the subfamily Vespinae (yellowjackets and hornets) remain unclear. Yellowjackets and hornets constitute one of the only two lineages of highly eusocial wasps, and the distribution of key behavioral traits correlates closely with the current classification of the group. The potential of the Vespinae to elucidate the evolution of social life, however, remains limited due to ambiguous genus-level relationships. Here, we address the relationships among genera within the Vespinae using transcriptomic (RNA-seq) data. We sequenced the transcriptomes of six vespid wasps, including three of the four genera recognized in the Vespinae, combined our data with publicly available transcriptomes, and assembled two matrices comprising 1,507 and 3,356 putative single-copy genes. The results of our phylogenomic analyses recover Dolichovespula as more closely related to Vespa than to Vespula, therefore challenging the prevailing hypothesis of yellowjacket (Vespula+Dolichovespula) monophyly. This suggests that traits such as large colony size and high paternity arose in the genus Vespula following its early divergence from the remaining vespine genera.

Viral late domains are used by many viruses to recruit the cellular endosomal sorting complex required for transport (ESCRT) to mediate membrane scission during viral budding. Unlike the P(S/T)AP and YPX(1–3)L late domains, which interact directly with the ESCRT proteins Tsg101 and ALIX, the molecular linkage connecting the PPXY late domain to ESCRT proteins is unclear. The mammarenavirus lymphocytic choriomeningitis virus (LCMV) matrix protein, Z, contains only one late domain, PPXY. We previously found that this domain in LCMV Z, as well as the ESCRT pathway, are required for the release of defective interfering (DI) particles but not infectious virus. To better understand the molecular mechanism of ESCRT recruitment by the PPXY late domain, affinity purification-mass spectrometry was used to identify host proteins that interact with the Z proteins of the Old World mammarenaviruses LCMV and Lassa virus. Several Nedd4 family E3 ubiquitin ligases interact with these matrix proteins and in the case of LCMV Z, the interaction was PPXY-dependent. We demonstrated that these ligases directly ubiquitinate LCMV Z and mapped the specific lysine residues modified. A recombinant LCMV containing a Z that cannot be ubiquitinated maintained its ability to produce both infectious virus and DI particles, suggesting that direct ubiquitination of LCMV Z alone is insufficient for recruiting ESCRT proteins to mediate virus release. However, Nedd4 ligases appear to be important for DI particle release suggesting that ubiquitination of targets other than the Z protein itself is required for efficient viral ESCRT recruitment., Author summary Enveloped viruses derive their lipid bilayer from either the cellular plasma membrane or an intracellular organelle during the process of viral budding in which a virus particle is formed at a membrane. Many enveloped viruses recruit the cellular endosomal sorting complex required for transport (ESCRT) in order to efficiently cut the membrane that connects a newly budded, but not released, virus particle from its parent membrane. Late domains, which are short protein motifs found in numerous enveloped viruses, specifically recruit ESCRT for this process. Two types of late domains accomplish this by binding directly to ESCRT proteins. A third late domain, PPXY, recruits ESCRT proteins through an unknown, indirect linkage. In this study, we sought to identify proteins that may bridge the PPXY late domain and ESCRT proteins. We found that Nedd4 family ubiquitin ligases interact with the PPXY domain in the mammarenavirus Z protein resulting in ubiquitination of Z at two lysine residues. However, Z ubiquitination was largely dispensable for the virus. Conversely, Nedd4 ubiquitin ligases were critical during infection suggesting that the most important contribution made to virus release by Nedd4 ligases is not direct ubiquitination of the viral matrix protein, but possibly the ubiquitination of cellular proteins or other viral proteins.

Chagas disease, a neglected tropical disease endemic in Latin America, is caused by the protozoan parasite Trypanosoma cruzi and is responsible for significant health impacts, especially in rural communities. The parasite is transmitted by insect vectors in the Triatominae subfamily and due to lack of vaccines and limited treatment options, vector control is the main way of controlling the disease. Knowing what vectors are feeding on directly enhances our understanding of the ecology and biology of the different vector species and can potentially aid in engaging communities in active disease control, a concept known as Ecohealth management. We evaluated bloodmeals in rural community, house-caught insect vectors previously evaluated for bloodmeals via DNA analysis as part of a larger collaborative project from three countries in Central America, including Guatemala. In addition to identifying bloodmeals in 100% of all samples using liquid chromatography tandem mass spectrometry (LC-MS/MS) (n = 50), strikingly for 53% of these samples there was no evidence of a recent bloodmeal by DNA-PCR. As individual vectors often feed on multiple sources, we developed an enhanced detection pipeline, and showed the ability to quantify a bloodmeal using stable-isotope-containing synthetic references peptides, a first step in further exploration of species-specific bloodmeal composition. Furthermore, we show that a lower resolution mass spectrometer is sufficient to correctly identify taxa from bloodmeals, an important and strong attribute of our LC-MS/MS-based method, opening the door to using proteomics in countries where Chagas disease is endemic.

Glioblastoma is a primary brain cancer that is resistant to all treatment modalities. This resistance is due, in large part, to invasive cancer cells that disperse from the main tumor site, escape surgical resection, and contribute to recurrent secondary lesions. The adhesion and signaling mechanisms that drive glioblastoma cell invasion remain enigmatic, and as a result there are no effective anti-invasive clinical therapies. Here we have characterized a novel adhesion and signaling pathway comprised of the integrin αvβ8 and its intracellular binding partner, Spinophilin (Spn), which regulates glioblastoma cell invasion in the brain microenvironment. We show for the first time that Spn binds directly to the cytoplasmic domain of β8 integrin in glioblastoma cells. Genetically targeting Spn leads to enhanced invasive cell growth in preclinical models of glioblastoma. Spn regulates glioblastoma cell invasion by modulating the formation and dissolution of invadopodia. Spn-regulated invadopodia dynamics are dependent, in part, on proper spatiotemporal activation of the Rac1 GTPase. Glioblastoma cells that lack Spn showed diminished Rac1 activities, increased numbers of invadopodia, and enhanced extracellular matrix degradation. Collectively, these data identify Spn as a critical adhesion and signaling protein that is essential for modulating glioblastoma cell invasion in the brain microenvironment. Implications: Tumor cell invasion is a major clinical obstacle in glioblastoma and this study identifies a new signaling pathway regulated by Spinophilin in invasive glioblastoma. Mol Cancer Res; 14(12); 1277–87. ©2016 AACR.

We report that the lymphocytic choriomeningitis virus (LCMV) matrix protein, which drives viral budding, is phosphorylated at serine 41 (S41). A recombinant (r)LCMV bearing a phosphomimetic mutation (S41D) was impaired in infectious and defective interfering (DI) particle release, while a non-phosphorylatable mutant (S41A) was not. The S41D mutant was disproportionately impaired in its ability to release DI particles relative to infectious particles. Thus, DI particle production by LCMV may be dynamically regulated via phosphorylation of S41.

Flightin is a myosin binding phosphoprotein that originated in the ancestor to Pancrustacea ~500 MYA. In Drosophila melanogaster, flightin is essential for length determination and flexural rigidity of thick filaments. Here, we show that among 12 Drosophila species, the N-terminal region is characterized by low sequence conservation, low pI, a cluster of phosphorylation sites, and a high propensity to intrinsic disorder (ID) that is augmented by phosphorylation. Using mass spectrometry, we identified eight phosphorylation sites within a 29 amino acid segment in the N-terminal region of D. melanogaster flightin. We show that phosphorylation of D. melanogaster flightin is modulated during flight and, through a comparative analysis to orthologs from other Drosophila species, we found phosphorylation sites that remain invariant, sites that retain the charge character, and sites that are clade-specific. While the number of predicted phosphorylation sites differs across species, we uncovered a conserved pattern that relates the number of phosphorylation sites to pI and ID. Extending the analysis to orthologs of other insects, we found additional conserved features in flightin despite the near absence of sequence identity. Collectively, our results demonstrate that structural constraints demarcate the evolution of the highly variable N-terminal region.

The triggering receptor expressed on myeloid cells (TREM) protein family forms a class of type I transmembrane proteins expressed in immune cells that play important roles in innate and adaptive immune responses. The TREM family member TREM-like transcript 1 (TLT-1, also TREML1) is expressed in megakaryocytes and packaged into platelet granules. TLT-1 binds fibrinogen and plays a role in bleeding initiated by inflammatory insults. Here, we describe a proteomics screen that maps the TLT-1 interactome in resting and activated human platelets. Several identified TLT-1 interactors are involved in cell adhesion and migration, as well as platelet activation. Select interactors, including β3-integrin, RACK1, GRB2, and Rabs 5A, 7, and 11A, were additionally characterized in co-immunoprecipitation/immunoblotting experiments. Finally, several phosphorylation sites were found on immunoprecipitated TLT-1, including Thr280, a novel, regulated site on a conserved residue near the TLT-1 ITIM regulatory sequence. SIGNIFICANCE: Platelet function relies on the secretion of active molecules from intracellular vesicles, or granules, which contain soluble and membrane-bound proteins that are essential for platelet aggregation, coagulation reactions, and pathogen defense mechanisms. TLT-1 is sequestered in α-granules and transported to the plasma membrane, where it plays a unique role in hemostasis after inflammatory insults. Despite the known importance of TLT-1 in platelet biology, our knowledge of TLT-1 mechanistic signaling is limited. This study defines the TLT-1 interactome in resting and active human platelets, identifying several novel TLT-1 interactors, as well as TLT-1 phosphorylation sites, all with likely signaling implications in platelet aggregation dynamics.

Lassa mammarenavirus (LASV) is an enveloped RNA virus that can cause Lassa fever, an acute hemorrhagic fever syndrome associated with significant morbidity and high rates of fatality in endemic regions of western Africa. The arenavirus matrix protein Z has several functions during the virus life cycle, including coordinating viral assembly, driving the release of new virus particles, regulating viral polymerase activity, and antagonizing the host antiviral response. There is limited knowledge regarding how the various functions of Z are regulated. To investigate possible means of regulation, mass spectrometry was used to identify potential sites of phosphorylation in the LASV Z protein. This analysis revealed that two serines (S18, S98) and one tyrosine (Y97) are phosphorylated in the flexible N- and C-terminal regions of the protein. Notably, two of these sites, Y97 and S98, are located in (Y97) or directly adjacent to (S98) the PPXY late domain, an important motif for virus release. Studies with non-phosphorylatable and phosphomimetic Z proteins revealed that these sites are important regulators of the release of LASV particles and that host-driven, reversible phosphorylation may play an important role in the regulation of LASV Z protein function.

Background Chagas disease is highly prevalent in Latin America, and vector control is the most effective control strategy to date. We have previously shown that liquid chromatography tandem mass spectrometry (LC-MS/MS) is a valuable tool for identifying triatomine vector blood meals. Objectives The purpose of this study was to determine blood meal detection ability as a function of method [polymerase chain reaction (PCR) vs. LC-MS/MS], time since feeding, and the effect of molting in mouse-fed triatomine insect vectors targeting hemoglobin and albumin proteins with LC-MS/MS and short interspersed nuclear elements (SINE)-based PCR. Methods We experimentally fed Triatoma protracta on mice and used LC-MS/MS to detect hemoglobin and albumin peptides over time post-feeding and post-molting (≤ 12 weeks). We compared LC-MS/MS results with those of a standard PCR method based on SINEs. Findings Hemoglobin-based LC-MS/MS detected blood meals most robustly at all time points post-feeding. Post-molting, no blood meals were detected with PCR, whereas LC-MS/MS detected mouse hemoglobin and albumin up to 12 weeks. Main conclusions In our study, the hemoglobin signature in the insect abdomen lasted longer than that of albumin and DNA. LC-MS/MS using hemoglobin shows promise for identifying triatomine blood meals over long temporal scales and even post-molting. Clarifying the frequency of blood-feeding on different hosts can foster our understanding of vector behavior and may help devise sounder disease-control strategies, including Ecohealth (community based ecosystem management) approaches.

Motivation The development of proteomic methods for the characterization of domain/motif interactions has greatly expanded our understanding of signal transduction. However, proteomics-based binding screens have limitations including that the queried tissue or cell type may not harbor all potential interacting partners or post-translational modifications (PTMs) required for the interaction. Therefore, we sought a generalizable, complementary in silico approach to identify potentially novel motif and PTM-dependent binding partners of high priority. Results We used as an initial example the interaction between the Src homology 2 (SH2) domains of the adaptor proteins CT10 regulator of kinase (CRK) and CRK-like (CRKL) and phosphorylated-YXXP motifs. Employing well-curated, publicly-available resources, we scored and prioritized potential CRK/CRKL–SH2 interactors possessing signature characteristics of known interacting partners. Our approach gave high priority scores to 102 of the >9000 YXXP motif-containing proteins. Within this 102 were 21 of the 25 curated CRK/CRKL–SH2-binding partners showing a more than 80-fold enrichment. Several predicted interactors were validated biochemically. To demonstrate generalized applicability, we used our workflow to predict protein–protein interactions dependent upon motif-specific arginine methylation. Our data demonstrate the applicability of our approach to, conceivably, any modular binding domain that recognizes a specific post-translationally modified motif. Supplementary information Supplementary data are available at Bioinformatics online.

Discoidin, CUB, and LCCL domain containing 2 (DCBLD2) is a neuropilin-like transmembrane scaffolding receptor with known and anticipated roles in vascular remodeling and neuronal positioning. DCBLD2 is also up-regulated in several cancers and can drive glioblastomas downstream of activated epidermal growth factor receptor. While a few studies have shown either a positive or negative role for DCBLD2 in regulating growth factor receptor signaling, little is known about the conserved signaling features of DCBLD family members that drive their molecular activities. We previously identified DCBLD2 tyrosine phosphorylation sites in intracellular YxxP motifs that are required for the phosphorylation-dependent binding of the signaling adaptors CRK and CRKL (CT10 regulator of kinase and CRK-like). These intracellular YxxP motifs are highly conserved across vertebrates and between DCBLD family members. Here, we demonstrate that, as for DCBLD2, DCBLD1 YxxP motifs are required for CRKL–SH2 (Src homology 2) binding. We report that Src family kinases (SFKs) and Abl differentially promote the interaction between the CRKL–SH2 domain and DCBLD1 and DCBLD2, and while SFKs and Abl each promote DCBLD1 and DCBLD2 binding to the CRKL–SH2 domain, the effect of Abl is more pronounced for DCBLD1. Using high-performance liquid chromatography coupled with tandem mass spectrometry, we quantified phosphorylation at several YxxP sites in DCBLD1 and DCBLD2, mapping site-specific preferences for SFKs and Abl. Together, these data provide a platform to decipher the signaling mechanisms by which these novel receptors drive their biological activities.

Aquatic ecosystem enrichment can lead to distinct and irreversible changes to undesirable states. Understanding changes in active microbial community function and composition following organic-matter loading in enriched ecosystems can help identify biomarkers of such state changes. In a field experiment, we enriched replicate aquatic ecosystems in the pitchers of the northern pitcher plant, Sarracenia purpurea. Shotgun metaproteomics using a custom metagenomic database identified proteins, molecular pathways, and contributing microbial taxa that differentiated control ecosystems from those that were enriched. The number of microbial taxa contributing to protein expression was comparable between treatments; however, taxonomic evenness was higher in controls. Functionally active bacterial composition differed significantly among treatments and was more divergent in control pitchers than enriched pitchers. Aerobic and facultative anaerobic bacteria contributed most to identified proteins in control and enriched ecosystems, respectively. The molecular pathways and contributing taxa in enriched pitcher ecosystems were similar to those found in larger enriched aquatic ecosystems and are consistent with microbial processes occurring at the base of detrital food webs. Detectable differences between protein profiles of enriched and control ecosystems suggest that a time series of environmental proteomics data may identify protein biomarkers of impending state changes to enriched states.

Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. These viruses encode only four proteins to accomplish the viral life cycle, so each arenavirus protein likely plays unappreciated accessory roles during infection. Here we used immunoprecipitation and mass spectrometry to identify human proteins that interact with the nucleoproteins (NPs) of the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) and the New World arenavirus Junín virus (JUNV) strain Candid #1. Bioinformatic analysis of the identified protein partners of NP revealed that host translation appears to be a key biological process engaged during infection. In particular, NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2α. JUNV infection leads to increased expression of PKR as well as its redistribution to viral replication and transcription factories. Further, phosphorylation of PKR, which is a prerequisite for its ability to phosphorylate eIF2α, is readily induced by JUNV. However, JUNV prevents this pool of activated PKR from phosphorylating eIF2α, even following exposure to the synthetic dsRNA poly(I·C), a potent PKR agonist. This blockade of PKR function is highly specific, as LCMV is unable to similarly inhibit eIF2α phosphorylation. JUNV's ability to antagonize the antiviral activity of PKR appears to be complete, as silencing of PKR expression has no impact on viral propagation. In summary, we provide a detailed map of the host machinery engaged by arenavirus NPs and identify an antiviral pathway that is subverted by JUNV. IMPORTANCE Arenaviruses are important human pathogens for which FDA-approved vaccines do not exist and effective antiviral therapeutics are needed. Design of antiviral treatment options and elucidation of the mechanistic basis of disease pathogenesis will depend on an increased basic understanding of these viruses and, in particular, their interactions with the host cell machinery. Identifying host proteins critical for the viral life cycle and/or pathogenesis represents a useful strategy to uncover new drug targets. This study reveals, for the first time, the extensive human protein interactome of arenavirus nucleoproteins and uncovers a potent antiviral host protein that is neutralized during Junín virus infection. In so doing, it shows further insight into the interplay between the virus and the host innate immune response and provides an important data set for the field.

Resveratrol, a plant-derived polyphenol, regulates many cellular processes, including cell proliferation, aging and autophagy. However, the molecular mechanisms of resveratrol action in cells are not completely understood. Intriguingly, resveratrol treatment of cells growing in nutrient-rich conditions induces autophagy, while acute resveratrol treatment of cells in a serum-deprived state inhibits autophagy. In this study, we performed a phosphoproteomic analysis after applying resveratrol to serum-starved cells with the goal of identifying the acute signaling events initiated by resveratrol in a serum-deprived state. We determined that resveratrol in serum-starved conditions reduces the phosphorylation of several proteins belonging to the mTORC1 signaling pathway, most significantly, PRAS40 at T246 and S183. Under these same conditions, we also found that resveratrol altered the phosphorylation of several proteins involved in various biological processes, most notably transcriptional modulators, represented by p53, FOXA1, and AATF. Together these data provide a more comprehensive view of both the spectrum of phosphoproteins upon which resveratrol acts as well as the potential mechanisms by which it inhibits autophagy in serum-deprived cells.

The Abelson (Abl) non-receptor tyrosine kinase regulates the cytoskeleton during multiple stages of neural development, from neurulation, to the articulation of axons and dendrites, to synapse formation and maintenance. We previously showed that Abl is genetically linked to the microtubule (MT) plus end tracking protein (+TIP) CLASP in Drosophila. Here we show in vertebrate cells that Abl binds to CLASP and phosphorylates it in response to serum or PDGF stimulation. In vitro, Abl phosphorylates CLASP with a Km of 1.89 µM, indicating that CLASP is a bona fide substrate. Abl-phosphorylated tyrosine residues that we detect in CLASP by mass spectrometry lie within previously mapped F-actin and MT plus end interaction domains. Using purified proteins, we find that Abl phosphorylation modulates direct binding between purified CLASP2 with both MTs and actin. Consistent with these observations, Abl-induced phosphorylation of CLASP2 modulates its localization as well as the distribution of F-actin structures in spinal cord growth cones. Our data suggest that the functional relationship between Abl and CLASP2 is conserved and provides a means to control the CLASP2 association with the cytoskeleton. © 2014 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc.