Signaling Pathways that Regulate Cell Division

Cell division requires careful orchestration of three major events: entry into mitosis, chromosomal segregation, and cytokinesis. Signaling within and between the molecules that control these events allows for their coordination via checkpoints, a specific class of signaling pathways that ensure the dependency of cell-cycle events on the successful completion of preceding events. Multiple positive- and negative-feedback loops ensure that a cell is fully committed to division and that the events occur in the proper order. Unlike other signaling pathways, which integrate external inputs to decide whether to execute a given process, signaling at cell division is largely dedicated to completing a decision made in G1 phase—to initiate and complete a round of mitotic cell division. Instead of deciding if the events of cell division will take place, these signaling pathways entrain these events to the activation of the cell-cycle kinase cyclin-dependent kinase 1 (CDK1) and provide the opportunity for checkpoint proteins to arrest cell division if things go wrong.     

Signaling Pathways in Murine Dendritic Cells That Regulate the Response to Vesicular Stomatitis Virus Vectors That Express Flagellin

Vesicular stomatitis virus (VSV) vectors that express heterologous antigens have shown promise as vaccines in preclinical studies. The efficacy of VSV-based vaccines can be improved by engineering vectors that enhance innate immune responses. We previously generated a VSV vaccine vector that incorporates two enhancing strategies: an M protein mutation (M51R) that prevents the virus from suppressing host antiviral responses and a gene encoding bacterial flagellin (M51R-F vector). The rationale was that intracellular expression of flagellin would activate innate immune pathways in addition to those activated by virus alone. This was tested with dendritic cells (DCs) from mice containing deletions in key signaling molecules. Infection of DC with either M51R or M51R-F vector induced the production of interleukin-12 (IL-12) and IL-6 and increased surface expression of T cell costimulatory molecules. These responses were dramatically reduced in DCs from IPS-1^−/− mice. Infection with M51R-F vector also induced the production of IL-1β. In addition, in approximately half of the DCs, M51R-F vector induced pyroptosis, a proinflammatory-type of cell death. These responses to flagellin were ablated in DCs from NLRC4^−/− mice but not Toll-like receptor 5-deficient (TLR5^−/−) mice, indicating that they resulted from inflammasome activation. These results demonstrate that flagellin induces additional innate immune mechanisms over those induced by VSV alone.     

mTORC2调节细胞骨架的信号通路

近几年来,关于哺乳动物雷帕霉素靶（mammalian target of rapamycin,mTOR）在各种哺乳动物细胞中调节肌动蛋白微丝极化及肌球蛋白微丝网形成的研究一直在不断地取得新的进展。尽管到目前为止,包括mTORC2上游和下游在内的相关的调控路径还未明确,但是因为mTORC6,的物学多样性,使其成为了当今生物学研究的焦点之一。基于长久以来特别是近五年对mTORC2的研究,在涉及细胞运动迁移、增殖分化、蛋白质合成、凋亡及自噬等生物学功能的研究中,一些重要的下游相关调控分子和蛋白相继被发现,比如P—Rexl／2、Rho家族GTPases、PKC、cAMP、p27kip1等。该综述着重总结了mTORC2实现这些生物学功能所可能通过的四条路径。当然,仍然需要大量的实验数据和研究证据进一步地证实和完善这些已经发现的可能存在的路径。     

Systematic Discovery of RNA Binding Proteins that Regulate MicroRNA Levels

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EP2 Receptor Signaling Pathways Regulate Classical Activation of Microglia

Activation of EP2 receptors by prostaglandin E2 (PGE₂) promotes brain inflammation in neurodegenerative diseases, but the pathways responsible are unclear. EP2 receptors couple to Gα_s and increase cAMP, which associates with protein kinase A (PKA) and cAMP-regulated guanine nucleotide exchange factors (Epacs). Here, we studied EP2 function and its signaling pathways in rat microglia in their resting state or undergoing classical activation in vitro following treatment with low concentrations of lipopolysaccharide and interferon-γ. Real time PCR showed that PGE₂ had no effect on expression of CXCL10, TGF-β1, and IL-11 and exacerbated the rapid up-regulation of mRNAs encoding cyclooxygenase-2, inducible NOS, IL-6, and IL-1β but blunted the production of mRNAs encoding TNF-α, IL-10, CCL3, and CCL4. These effects were mimicked fully by the EP2 agonist butaprost but only weakly by the EP1/EP3 agonist 17-phenyl trinor PGE₂ or the EP4 agonist CAY10598 and not at all by the EP3/EP1 agonist sulprostone and confirmed by protein measurements of cyclooxygenase-2, IL-6, IL-10, and TNF-α. In resting microglia, butaprost induced cAMP formation and altered the mRNA expression of inflammatory mediators, but protein expression was unchanged. The PKA inhibitor H89 had little or no effect on inflammatory mediators modulated by EP2, whereas the Epac activator 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate acetoxymethyl ester mimicked all butaprost effects. These results indicate that EP2 activation plays a complex immune regulatory role during classical activation of microglia and that Epac pathways are prominent in this role.     

The Role of MicroRNA in Regulation of Signaling Pathways in Gliomas

Gliomas are invasive brain tumors characterized by high rates of recurrence and mortality. Glioblastoma multiforme (GBM) is the most aggressive form of glioma with nearly 100% rate of recurrence and unfavorable prognosis in patients. MicroRNAs (miR) are a class of wide-spread short noncoding RNAs that inhibit translation via binding to the mRNA of target genes. The aim of this review is to analyze studies and experimental results on changes in the expression profiles of microRNA which are characteristic for gliomas/ glioblastomas and targeted to components of signaling pathways Hedgehog, Notch, Wnt, EGFR, TGFβ, and HIF1α, aberrantly regulated in the gliomas. Special attention has been paid to the links of microRNA to the targets of 2-hydroxyglutarate, the product of mutant isocitrate dehydrogenase (R132H IDH1), mutational changes of which are specific for the pathogenesis of gliomas. Detection of certain types of microRNA in tissues and blood serum can be used for diagnostics and prediction, including responsiveness of individual patients to therapy, and development of new therapeutic strategies.     

Genome-Wide CRISPR-Cas9 Screen Identifies MicroRNAs That Regulate Myeloid Leukemia Cell Growth

Mammalian microRNA expression is dysregulated in human cancer. However, the functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of a myeloid leukemia cell line. This approach identified evolutionarily conserved human microRNAs that suppress or promote cell growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. miR-155 was identified as a top microRNA candidate promoting cellular fitness, which we confirmed with two distinct miR-155-targeting CRISPR-Cas9 lentiviral constructs. Further, we performed anti-correlation functional profiling to predict relevant microRNA-tumor suppressor gene or microRNA-oncogene interactions in these cells. This analysis identified miR-150 targeting of p53, a connection that was experimentally validated. Taken together, our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how microRNAs contribute to human disease.     

Identification of Small Molecules That Suppress Ricin-Induced Stress-Activated Signaling Pathways

Ricin is a member of the ribosome-inactivating protein (RIP) family of plant and bacterial toxins. In this study we used a high-throughput, cell-based assay to screen more than 118,000 compounds from diverse chemical libraries for molecules that reduced ricin-induced cell death. We describe three compounds, PW66, PW69, and PW72 that at micromolar concentrations significantly delayed ricin-induced cell death. None of the compounds had any demonstrable effect on ricin''s ability to arrest protein synthesis in cells or on ricin''s enzymatic activity as assessed in vitro. Instead, all three compounds appear to function by blocking downstream stress-induced signaling pathways associated with the toxin-mediated apoptosis. PW66 virtually eliminated ricin-induced TNF-α secretion by J774A.1 macrophages and concomitantly blocked activation of the p38 MAPK and JNK signaling pathways. PW72 suppressed ricin-induced TNF-α secretion, but not p38 MAPK and JNK signaling. PW69 suppressed activity of the executioner caspases 3/7 in ricin toxin- and Shiga toxin 2-treated cells. While the actual molecular targets of the three compounds have yet to be identified, these data nevertheless underscore the potential of small molecules to down-regulate inflammatory signaling pathways associated with exposure to the RIP family of toxins.     

Proteome-wide Tyrosine Phosphorylation Analysis Reveals Dysregulated Signaling Pathways in Ovarian Tumors

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Highlights

• •Multiplexed PTM assays on HuProt array were developed using ovarian tumor lysates.
• •Proteome-wide Tyr phosphorylation with 102 ovarian tumors were performed and analyzed.
• •19 kinases were predicted to have elevated activities in ovarian tumor.
• •Elevated activities of PTK2 and PTK2B were confirmed in ovarian cancer cell lines.

     

Brassinosteroids Regulate Plant Growth through Distinct Signaling Pathways in Selaginella and Arabidopsis

Brassinosteroids (BRs) are growth-promoting steroid hormones that regulate diverse physiological processes in plants. Most BR biosynthetic enzymes belong to the cytochrome P450 (CYP) family. The gene encoding the ultimate step of BR biosynthesis in Arabidopsis likely evolved by gene duplication followed by functional specialization in a dicotyledonous plant-specific manner. To gain insight into the evolution of BRs, we performed a genomic reconstitution of Arabidopsis BR biosynthetic genes in an ancestral vascular plant, the lycophyte Selaginella moellendorffii. Selaginella contains four members of the CYP90 family that cluster together in the CYP85 clan. Similar to known BR biosynthetic genes, the Selaginella CYP90s exhibit eight or ten exons and Selaginella produces a putative BR biosynthetic intermediate. Therefore, we hypothesized that Selaginella CYP90 genes encode BR biosynthetic enzymes. In contrast to typical CYPs in Arabidopsis, Selaginella CYP90E2 and CYP90F1 do not possess amino-terminal signal peptides, suggesting that they do not localize to the endoplasmic reticulum. In addition, one of the three putative CYP reductases (CPRs) that is required for CYP enzyme function co-localized with CYP90E2 and CYP90F1. Treatments with a BR biosynthetic inhibitor, propiconazole, and epi-brassinolide resulted in greatly retarded and increased growth, respectively. This suggests that BRs promote growth in Selaginella, as they do in Arabidopsis. However, BR signaling occurs through different pathways than in Arabidopsis. A sequence homologous to the Arabidopsis BR receptor BRI1 was absent in Selaginella, but downstream components, including BIN2, BSU1, and BZR1, were present. Thus, the mechanism that initiates BR signaling in Selaginella seems to differ from that in Arabidopsis. Our findings suggest that the basic physiological roles of BRs as growth-promoting hormones are conserved in both lycophytes and Arabidopsis; however, different BR molecules and BRI1-based membrane receptor complexes evolved in these plants.     

Phosphoproteome Analysis Reveals Regulatory Sites in Major Pathways of Cardiac Mitochondria

Mitochondrial functions are dynamically regulated in the heart. In particular, protein phosphorylation has been shown to be a key mechanism modulating mitochondrial function in diverse cardiovascular phenotypes. However, site-specific phosphorylation information remains scarce for this organ. Accordingly, we performed a comprehensive characterization of murine cardiac mitochondrial phosphoproteome in the context of mitochondrial functional pathways. A platform using the complementary fragmentation technologies of collision-induced dissociation (CID) and electron transfer dissociation (ETD) demonstrated successful identification of a total of 236 phosphorylation sites in the murine heart; 210 of these sites were novel. These 236 sites were mapped to 181 phosphoproteins and 203 phosphopeptides. Among those identified, 45 phosphorylation sites were captured only by CID, whereas 185 phosphorylation sites, including a novel modification on ubiquinol-cytochrome c reductase protein 1 (Ser-212), were identified only by ETD, underscoring the advantage of a combined CID and ETD approach. The biological significance of the cardiac mitochondrial phosphoproteome was evaluated. Our investigations illustrated key regulatory sites in murine cardiac mitochondrial pathways as targets of phosphorylation regulation, including components of the electron transport chain (ETC) complexes and enzymes involved in metabolic pathways (e.g. tricarboxylic acid cycle). Furthermore, calcium overload injured cardiac mitochondrial ETC function, whereas enhanced phosphorylation of ETC via application of phosphatase inhibitors restored calcium-attenuated ETC complex I and complex III activities, demonstrating positive regulation of ETC function by phosphorylation. Moreover, in silico analyses of the identified phosphopeptide motifs illuminated the molecular nature of participating kinases, which included several known mitochondrial kinases (e.g. pyruvate dehydrogenase kinase) as well as kinases whose mitochondrial location was not previously appreciated (e.g. Src). In conclusion, the phosphorylation events defined herein advance our understanding of cardiac mitochondrial biology, facilitating the integration of the still fragmentary knowledge about mitochondrial signaling networks, metabolic pathways, and intrinsic mechanisms of functional regulation in the heart.Mitochondria are the source of energy to sustain life. In addition to their evolutionary origin as an energy-producing organelle, their functionality has integrated into every aspect of life, including the cell cycle, ROS¹ production, apoptosis, and ion balance (1, 2). Our understanding of mitochondrial biology is still growing. Several systems biology approaches have been dedicated to exploring the molecular infrastructure and dynamics of the functional versatility associated with this organelle (3–5).To meet tissue-specific functional demands, mitochondria acquire heterogeneous properties in individual organs, a first statement of their plasticity in function and proteome composition (1, 6). The heterogeneity is evident even in an individual cardiomyocyte (7). A catalogue of the cardiac mitochondrial proteome is emerging via a joint effort (3–5). The dynamics of the mitochondrial proteome manifest at multiple levels, including post-translational modifications, such as phosphorylation. Our investigative goal is to decode this organellar proteome and its post-translational modification in a biological and functional context. In cardiomyocytes, mitochondria are also constantly exposed to fluctuation in energy demands and in ionic conditions. The capacity of mitochondria to cope with such a dynamic environment is essential for the functional role of mitochondria in normal and disease phenotypes (8–10). Unique protein features enabling the mitochondrial proteome to adapt to these biological changes can be interrogated by proteomics tools (10–12). Protein phosphorylation as a rapid and reversible chemical event is an integral component of these protein features (12–14).It has been estimated that one-third of cellular proteins exist in a phosphorylated state at least one time in their lifetime (15). However, only a handful of phosphorylation events have been identified to tune mitochondrial functionality (13, 14, 16) despite the fact that the first demonstration of phosphorylation was reported on a mitochondrial protein more than 5 decades ago (17). Kinases and phosphatases comprise nearly 3% of the human genome (18, 19). In mitochondria, ∼30 kinases and phosphatases have been identified thus far within the expected organellar proteome of a few thousand (3–5, 16). The number of identified mitochondrial phosphoproteins is far below one-third of its proteome size (20). Thus, it appears that the current pool of reported phosphoproteins represents only a small fraction of the anticipated mitochondrial phosphoproteome. The seminal studies from several groups (12–14, 16) demonstrated the prevalence as well as the dynamic nature of phosphorylation in cardiac mitochondria, suggesting that obtaining a comprehensive map of the mitochondrial phosphoproteome is feasible.In this study, we took a systematic approach to tackle the phosphorylation of murine cardiac mitochondrial pathways. We applied the unique strengths of both electron transfer dissociation (ETD) and collision-induced dissociation (CID) LC-MS/MS to screen phosphorylation events in a site-specific fashion. A total of 236 phosphorylation sites in 203 unique phosphopeptides were identified and mapped to 181 phosphoproteins. Novel phosphorylation modifications were discovered in diverse pathways of mitochondrial biology, including ion balance, proteolysis, and apoptosis. Consistent with the role of mitochondria as the major source of energy production under delicate control, metabolic pathways claimed one-third of phosphorylation sites captured in this analysis. To study molecular players steering mitochondrial phosphorylation, we probed the effects of calcium loading on phosphorylation. In addition, a number of kinases with previously unappreciated mitochondrial residence are suggested as potential players modulating mitochondrial pathways. Taken together, the cohort of novel phosphorylation events discovered in this study constitutes an essential step toward the full delineation of the cardiac mitochondrial phosphoproteome.     

Genome-Wide Pathway Analysis Reveals Different Signaling Pathways between Secreted Lactoferrin and Intracellular Delta-Lactoferrin

Human lactoferrin (LF) is a multifunctional protein involved in immunomodulation, cellular growth, and differentiation. In addition to its secreted form (sLF), an alternative form (ΔLF) lacking the signal sequence has been found to be downregulated in cancer. Although the signaling pathways mediated by LF have been studied in a few cell models, there have been no relevant systemic approaches. Therefore, this study was carried out to identify and compare signaling networks provoked by the two LF isoforms. For this, the two forms were overexpressed in HEK293 cells using the Flp-In T-Rex system, after which genome-wide expression analysis of 18,367 genes was conducted. Pathway analysis of the genes showing altered expression identified pathways which are responsible for cell survival and apoptosis. In addition, the pathways mediated by the two LF forms were within distantly related networks. GPCR, PI3K complex, and POU5F1, which are involved in receptor-mediated pathways, were centered in the sLF network, whereas RIF1, NOS3, and RNPS1, which are involved in intracellular signaling, were centered in the ΔLF network. These results suggest that structural differences between the LF isoforms, mainly glycosylation, determine the fate of LF signaling. Furthermore, these findings provide information relating to the role of ΔLF which is downregulated during carcinogenesis.     

Whole Genome,Whole Population Sequencing Reveals That Loss of Signaling Networks Is the Major Adaptive Strategy in a Constant Environment

Molecular signaling networks are ubiquitous across life and likely evolved to allow organisms to sense and respond to environmental change in dynamic environments. Few examples exist regarding the dispensability of signaling networks, and it remains unclear whether they are an essential feature of a highly adapted biological system. Here, we show that signaling network function carries a fitness cost in yeast evolving in a constant environment. We performed whole-genome, whole-population Illumina sequencing on replicate evolution experiments and find the major theme of adaptive evolution in a constant environment is the disruption of signaling networks responsible for regulating the response to environmental perturbations. Over half of all identified mutations occurred in three major signaling networks that regulate growth control: glucose signaling, Ras/cAMP/PKA and HOG. This results in a loss of environmental sensitivity that is reproducible across experiments. However, adaptive clones show reduced viability under starvation conditions, demonstrating an evolutionary tradeoff. These mutations are beneficial in an environment with a constant and predictable nutrient supply, likely because they result in constitutive growth, but reduce fitness in an environment where nutrient supply is not constant. Our results are a clear example of the myopic nature of evolution: a loss of environmental sensitivity in a constant environment is adaptive in the short term, but maladaptive should the environment change.