Gene cloning, protein expression, and enzymatic characterization of a double-stranded RNA degrading enzyme in Apolygus lucorum.

RNA interference (RNAi) is a powerful tool that post-transcriptionally silences target genes in eukaryotic cells. However, silencing efficacy varies greatly among different insect species. Recently, we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA injection. The disappearance of double-stranded RNA (dsRNA) could be a potential factor that restricts RNAi efficiency. Here, we found that dsRNA can be degraded in midgut fluids, and a dsRNase of A. lucorum (AldsRNase) was identified and characterized. Sequence alignment indicated that its 6 key amino acid residues and the Mg2+ -binding site were similar to those of other insects' dsRNases. The signal peptide and endonuclease non-specific domain shared high sequence identity with the brown-winged green stinkbug Plautia stali dsRNase. AldsRNase showed high salivary gland and midgut expression and was continuously expressed through the whole life cycle, with peaks at the 4th instar ecdysis in the whole body. The purified AldsRNase protein obtained by heterologously expressed can rapidly degrade dsRNA. When comparing the substrate specificity of AldsRNase, 3 specific substrates (dsRNA, small interfering RNA, and dsDNA) were all degraded, and the most efficient degradation is dsRNA. Subsequently, immunofluorescence revealed that AldsRNase was expressed in the cytoplasm of midgut cells. Through cloning and functional study of AldsRNase, the enzyme activity and substrate specificity of the recombinant protein, as well as the subcellular localization of nuclease, the reason for the disappearance of dsRNA was explained, which was useful in improving RNAi efficiency in A. lucorum and related species.

Boosting CO2 Hydrogenation to Formate over Edge-Sulfur Vacancies of Molybdenum Disulfide.

Synthesis of formate from hydrogenation of carbon dioxide (CO2 ) is an atom-economic reaction but is confronted with challenges in developing high-performance non-precious metal catalysts for application of the process. Herein, we report a highly durable edge-rich molybdenum disulfide (MoS2 ) catalyst for CO2 hydrogenation to formate at 200 °C, which delivers a high selectivity of over 99 % with a superior turnover frequency of 780.7 h-1 surpassing those of previously reported non-precious metal catalysts. Multiple experimental characterization techniques combined with theoretical calculations reveal that sulfur vacancies at MoS2 edges are the active sites and the selective production of formate is enabled via a completely new water-mediated hydrogenation mechanism, in which surface OH* and H* species in dynamic equilibrium with water serve as moderate hydrogenating agents for CO2 with residual O* reduced by hydrogen. This study provides a new route for developing low-cost high-performance catalysts for CO2 hydrogenation to formate.

A highly sensitive and robust LC-MS platform for host cell protein characterization in biotherapeutics.

Host cell proteins (HCPs) are a major class of process-related impurities that need to be closely monitored during the production of biotherapeutics. Mass spectrometry (MS) has emerged as a promising tool for HCP analysis due to its specificity for individual HCP's identification and quantitation. However, utilization of MS as a routine characterization tool is still limited due to the time-consuming procedures, non-standardized instrumentation and methodologies, and the limited sensitivity compared to the enzyme-linked immunosorbent assays (ELISA). In this study, we introduced a sensitive (limit of detection (LOD) at 1-2 ppm) and robust HCP profiling platform method with suitable precision and accuracy that can be readily adopted to antibodies and other biotherapeutic modalities without the need for HCP enrichment. The NIST mAb and multiple in-house antibodies were analyzed, and results were benchmarked with other reported studies. In addition, a targeted analysis method with optimized sample preparation for absolute quantitation of lipases was developed and qualified with an LOD of 0.6 ppm and precision of <15%, which can be further improved to an LOD of 5 ppb by using the nano-flow LC.

iTRAQ Proteomic Analysis of Interactions Between 20E and Phospholipase C in Apolygus lucorum (Meyer-Dür).

Polyphagous Apolygus lucorum has become the dominant insect in Bacillus thuringiensis (Bt) cotton fields. Hormone 20-hydroxyecdysone (20E) regulates multiple insect development and physiology events. 20E responses are controlled by pathways triggered by phospholipase C (PLC)-associated proteins. However, 20E-modulated genes and related proteins that can be affected by PLC still remain unknown. Here, isobaric tag for relative and absolute quantitation (iTRAQ) and immunoblotting techniques were used to compare differentially expressed proteins (DEPs) in A. lucorum in response to the treatment of 20E and the PLC inhibitor U73122 as well as their combination. A total of 1,624 non-redundant proteins and 97, 248, 266 DEPs were identified in the 20E/control, U73122/control, and 20E + U73122/control groups, respectively. Only 8 DEPs, including pathogenesis-related protein 5-like, cuticle protein 19.8, trans-sialidase, larval cuticle protein A2B-like, cathepsin L1, hemolymph juvenile hormone-binding protein, ATP-dependent RNA helicase p62-like, and myosin-9 isoform X1, were detected in all three groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEPs were involved in diverse signaling pathways. The results were validated by immunoblotting, which highlighted the reliability of proteomics analysis. These findings provided novel insights into the function of PLC in 20E signaling pathway in A. lucorum.

Regulation of Respiration and Apoptosis by Cytochrome c Threonine 58 Phosphorylation.

Cytochrome c (Cytc) is a multifunctional protein, acting as an electron carrier in the electron transport chain (ETC), where it shuttles electrons from bc1 complex to cytochrome c oxidase (COX), and as a trigger of type II apoptosis when released from the mitochondria. We previously showed that Cytc is regulated in a highly tissue-specific manner: Cytc isolated from heart, liver, and kidney is phosphorylated on Y97, Y48, and T28, respectively. Here, we have analyzed the effect of a new Cytc phosphorylation site, threonine 58, which we mapped in rat kidney Cytc by mass spectrometry. We generated and overexpressed wild-type, phosphomimetic T58E, and two controls, T58A and T58I Cytc; the latter replacement is found in human and testis-specific Cytc. In vitro, COX activity, caspase-3 activity, and heme degradation in the presence of H2O2 were decreased with phosphomimetic Cytc compared to wild-type. Cytc-knockout cells expressing T58E or T58I Cytc showed a reduction in intact cell respiration, mitochondrial membrane potential (∆Ψm), ROS production, and apoptotic activity compared to wild-type. We propose that, under physiological conditions, Cytc is phosphorylated, which controls mitochondrial respiration and apoptosis. Under conditions of stress Cytc phosphorylations are lost leading to maximal respiration rates, ∆Ψm hyperpolarization, ROS production, and apoptosis.

Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity.

Cytochrome c (Cytc) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain and plays a key role in apoptosis. We have previously shown that tissue-specific phosphorylations of Cytc in the heart, liver, and kidney play an important role in the regulation of cellular respiration and cell death. Here, we report that Cytc purified from mammalian brain is phosphorylated on S47 and that this phosphorylation is lost during ischemia. We have characterized the functional effects in vitro using phosphorylated Cytc purified from pig brain tissue and a recombinant phosphomimetic mutant (S47E). We crystallized S47E phosphomimetic Cytc at 1.55 Å and suggest that it spatially matches S47-phosphorylated Cytc, making it a good model system. Both S47-phosphorylated and phosphomimetic Cytc showed a lower oxygen consumption rate in reaction with isolated Cytc oxidase, which we propose maintains intermediate mitochondrial membrane potentials under physiologic conditions, thus minimizing production of reactive oxygen species. S47-phosphorylated and phosphomimetic Cytc showed lower caspase-3 activity. Furthermore, phosphomimetic Cytc had decreased cardiolipin peroxidase activity and is more stable in the presence of H2O2. Our data suggest that S47 phosphorylation of Cytc is tissue protective and promotes cell survival in the brain.-Kalpage, H. A., Vaishnav, A., Liu, J., Varughese, A., Wan, J., Turner, A. A., Ji, Q., Zurek, M. P., Kapralov, A. A., Kagan, V. E., Brunzelle, J. S., Recanati, M.-A., Grossman, L. I., Sanderson, T. H., Lee, I., Salomon, A. R., Edwards, B. F. P, Hüttemann, M. Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity.

High-throughput LC-MS quantitation of cell culture metabolites.

Monitoring cell culture metabolites, including media components and cellular byproducts, during bio manufacturing is critical for gaining insights into cell growth, productivity and product quality. Historically, cell culture metabolite analysis was a complicated process requiring several orthogonal methods to cover the large number of metabolites with diverse properties over wide concentration ranges. These off-line analyses are time consuming and not suitable for real time bioreactor monitoring. In this study, we present a high-throughput LC-MS method with a 17-min cycle time that is capable of simultaneously monitoring 93 cell culture metabolites, including amino acids, nucleic acids, vitamins, sugars and others. This method has high precision and accuracy and has been successfully applied to the daily profiling of bioreactors and raw material qualification. Information obtained in these studies has been used to identify limiting amino acids during production, which guided adjustments to the feed strategy that prevented the potential misincorporation of amino acids. This type of metabolite profiling can be further utilized to build predictive process models for adaptive feedback control and pave the road for continuous manufacturing and real-time release testing.

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE.

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

The catalytic activity of the kinase ZAP-70 mediates basal signaling and negative feedback of the T cell receptor pathway.

T cell activation by antigens binding to the T cell receptor (TCR) must be properly regulated to ensure normal T cell development and effective immune responses to pathogens and transformed cells while avoiding autoimmunity. The Src family kinase Lck and the Syk family kinase ZAP-70 (ζ chain-associated protein kinase of 70 kD) are sequentially activated in response to TCR engagement and serve as critical components of the TCR signaling machinery that leads to T cell activation. We performed a mass spectrometry-based phosphoproteomic study comparing the quantitative differences in the temporal dynamics of phosphorylation in stimulated and unstimulated T cells with or without inhibition of ZAP-70 catalytic activity. The data indicated that the kinase activity of ZAP-70 stimulates negative feedback pathways that target Lck and thereby modulate the phosphorylation patterns of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 and ζ chain components of the TCR and of signaling molecules downstream of Lck, including ZAP-70. We developed a computational model that provides a mechanistic explanation for the experimental findings on ITAM phosphorylation in wild-type cells, ZAP-70-deficient cells, and cells with inhibited ZAP-70 catalytic activity. This model incorporated negative feedback regulation of Lck activity by the kinase activity of ZAP-70 and predicted the order in which tyrosines in the ITAMs of TCR ζ chains must be phosphorylated to be consistent with the experimental data.

Wide-scale quantitative phosphoproteomic analysis reveals that cold treatment of T cells closely mimics soluble antibody stimulation.

The activation of T lymphocytes through antigen-mediated T cell receptor (TCR) clustering is vital in regulating the adaptive immune response. Although T cell receptor signaling has been extensively studied, the fundamental mechanisms for signal initiation are not fully understood. Reduced temperatures have initiated some of the hallmarks of TCR signaling, such as increased phosphorylation and activation on ERK and calcium release from the endoplasmic reticulum, as well as coalesced the T cell membrane microdomains. The precise mechanism of the TCR signaling initiation due to temperature change remains obscure. One critical question is whether the signaling initiated by the cold treatment of T cells differs from the signaling initiated by the cross-linking of the T cell receptor. To address this uncertainty, we performed a wide-scale, quantitative mass-spectrometry-based phosphoproteomic analysis on T cells stimulated either by temperature shifts or through the cross-linking of the TCR. Careful statistical comparisons between the two stimulations revealed a striking level of identity among the subset of 339 sites that changed significantly with both stimulations. This study demonstrates for the first time, in unprecedented detail, that T cell cold treatment was sufficient to initiate signaling patterns that were nearly identical to those of soluble antibody stimulation, shedding new light on the mechanism of activation of these critically important immune cells.

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) N-terminal tyrosine residues regulate a dynamic signaling equilibrium involving feedback of proximal T-cell receptor (TCR) signaling.

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a cytosolic adaptor protein that plays an important role in the T-cell receptor-mediated T-cell signaling pathway. SLP-76 links proximal receptor stimulation to downstream effectors through interaction with many signaling proteins. Previous studies showed that mutation of three tyrosine residues, Tyr(112), Tyr(128), and Tyr(145), in the N terminus of SLP-76 results in severely impaired phosphorylation and activation of Itk and PLCγ1, which leads to defective calcium mobilization, Erk activation, and NFAT activation. To expand our knowledge of the role of N-terminal phosphorylation of SLP-76 from these three tyrosine sites, we characterized nearly 1000 tyrosine phosphorylation sites via mass spectrometry in SLP-76 reconstituted wild-type cells and SLP-76 mutant cells in which three tyrosine residues were replaced with phenylalanines (Y3F mutant). Mutation of the three N-terminal tyrosine residues of SLP-76 phenocopied SLP-76-deficient cells for the majority of tyrosine phosphorylation sites observed, including feedback on proximal T-cell receptor signaling proteins. Meanwhile, reversed phosphorylation changes were observed on Tyr(192) of Lck when we compared mutants to the complete removal of SLP-76. In addition, N-terminal tyrosine sites of SLP-76 also perturbed phosphorylation of Tyr(440) of Fyn, Tyr(702) of PLCγ1, Tyr(204), Tyr(397), and Tyr(69) of ZAP-70, revealing new modes of regulation on these sites. All these findings confirmed the central role of N-terminal tyrosine sites of SLP-76 in the pathway and also shed light on novel signaling events that are uniquely regulated by SLP-76 N-terminal tyrosine residues.

Quantitative phosphoproteomic analysis identifies activation of the RET and IGF-1R/IR signaling pathways in neuroblastoma.

Neuroblastoma is an embryonal tumor of childhood with a heterogenous clinical presentation that reflects differences in activation of complex biological signaling pathways. Protein phosphorylation is a key component of cellular signal transduction and plays a critical role in processes that control cancer cell growth and survival. We used shotgun LC/MS to compare phosphorylation between a human MYCN amplified neuroblastoma cell line (NB10), modeling a resistant tumor, and a human neural precursor cell line (NPC), modeling a normal baseline neural crest cell. 2181 unique phosphorylation sites representing 1171 proteins and 2598 phosphopeptides were found. Protein kinases accounted for 6% of the proteome, with a predominance of tyrosine kinases, supporting their prominent role in oncogenic signaling pathways. Highly abundant receptor tyrosine kinase (RTK) phosphopeptides in the NB10 cell line relative to the NPC cell line included RET, insulin-like growth factor 1 receptor/insulin receptor (IGF-1R/IR), and fibroblast growth factor receptor 1 (FGFR1). Multiple phosphorylated peptides from downstream mediators of the PI3K/AKT/mTOR and RAS pathways were also highly abundant in NB10 relative to NPC. Our analysis highlights the importance of RET, IGF-1R/IR and FGFR1 as RTKs in neuroblastoma and suggests a methodology that can be used to identify potential novel biological therapeutic targets. Furthermore, application of this previously unexploited technology in the clinic opens the possibility of providing a new wide-scale molecular signature to assess disease progression and prognosis.

Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

Microfluidic cytometer based on dual photodiode detection for cell size and deformability analysis.

Cellular mechanical properties play an important role in disease diagnosis. Distinguishing cells based on their mechanical properties provides a potential method for label-free diagnosis. In this work, a convenient and low-cost microfluidic cytometer was developed to study cell mechanical properties and cell size based on the change of transmission intensity, using a low-cost commercial laser as a light source and two photodiodes as detectors. The cells pass through a narrow microchannel with a width smaller than the cell dimension, integrated in a polydimethylsiloxane chip, below which the laser is focused. The transit time of individual cells is measured by the time difference detected by two photodiodes. This device was used to study the difference in cell mechanical properties between HL60 cells treated with and without Cytochalasin D. Furthermore, it was also applied to distinguish cells with different diameters, HL60 cells and red blood cells, by measuring the transmission intensity.

Benzyl N'-(2-chloro-benzyl-idene)hydrazinecarbodithio-ate.

The asymmetric unit of the title compound, C(15)H(13)ClN(2)S(2), contains two independent mol-ecules, which are linked into a pseudo-centrosymmetric dimer by inter-molecular N-H⋯S hydrogen bonds. The aromatic rings form dihedral angles of 67.06 (3) and 81.85 (2)° in the two independent mol-ecules.