Protein Phosphorylation Mechanisms Research Articles

The phosphorylation of a WD40-repeat protein negatively regulates flavonoid biosynthesis in Camellia sinensis under drought stress.

Flavonoids constitute the main nutraceuticals in the leaves of tea plants (Camellia sinensis). To date, although it is known that drought stress can negatively impact the biosynthesis of flavonoids in tea leaves, the mechanism behind this phenomenon is unclear. Herein, we report a protein phosphorylation mechanism that negatively regulates the biosynthesis of flavonoids in tea leaves in drought conditions. Transcriptional analysis revealed the downregulation of gene expression of flavonoid biosynthesis and the upregulation of CsMPK4a encoding a mitogen-activated protein kinase in leaves. Luciferase complementation and yeast two-hybrid assays disclosed that CsMPK4a interacted with CsWD40. Phosphorylation assay in vitro, specific protein immunity, and analysis of protein mass spectrometry indicated that Ser-216, Thr-221, and Ser-253 of CsWD40 were potential phosphorylation sites of CsMPK4a. Besides, the protein immunity analysis uncovered an increased phosphorylation level of CsWD40 in tea leaves under drought conditions. Mutation of the three phosphorylation sites generated dephosphorylated CsWD403A and phosphorylated CsWD403D variants, which were introduced into the Arabidopsis ttg1 mutant. Metabolic analysis showed that the anthocyanin and proanthocyanidin content was lower in ttg1:CsWD403D transgenic plants than ttg1::CsWD403A transgenic and wild type plants. The transient overexpression of CsWD403D downregulated the anthocyanidin biosynthesis in tea leaves. The dual-fluorescein protein complementation experiment showed that CsWD403D did not interact with CsMYB5a and CsAN2, two key transcription factors of procyanidins and anthocyanidins biosynthesis in tea plant. These findings indicate that the phosphorylation of CsWD40 by CsMPK4a downregulates the flavonoid biosynthesis in tea plants in drought stresses.

Protein Phosphorylation Mechanism of Mesenchymal Stem Cells in the Treatment of Sepsis: A Systematic Review and Meta-analysis.

The severity and mortality of sepsis are related to excessive inflammation and cytokine storm. Nevertheless, little is known about why sepsis has a significant increase in proinflammatory cytokine production, which leads to more severe inflammatory damage. Mesenchymal stem cells have achieved certain results in the treatment of sepsis, but the specific mechanism remains to be further clarified. Therefore, this paper will elaborate on the currently recognized mechanism of mesenchymal stem cells in the treatment of sepsis, the protein phosphorylation mechanism of sepsis inflammatory response, and the possibility that mesenchymal stem cells may block the occurrence and development of sepsis by regulating relevant pathways or protein phosphorylation. It provides a novel target for mesenchymal stem cells to prevent intervention or therapeutically block the development of sepsis.

The carbon and nitrogen metabolisms of Ardisia quinquegona were altered in different degrees by canopy and understory nitrogen addition in a subtropical forest

Although effects of atmospheric nitrogen (N) deposition on forest plants have been widely investigated, N interception and absorption effects by forest canopy should not be neglected. Moreover, how N deposition change the molecular biological process of understory dominant plants, which was easily influenced by canopy interception so as to further change physiological performance, remains poorly understood. To assess the effects of N deposition on forest plants, we investigated the effects of understory (UAN) and canopy N addition (CAN) on the transcriptome and physiological properties of Ardisia quinquegona, a dominant subtropical understory plant species in an evergreen broad-leaved forest in China. We identified a total of 7394 differentially expressed genes (DEGs). Three of these genes were found to be co-upregulated in CAN as compared to control (CK) after 3 and 6h of N addition treatment, while 133 and 3 genes were respectively found to be co-upregulated and co-downregulated in UAN as compared to CK. In addition, highly expressed genes including GP1 (a gene involved in cell wall biosynthesis) and STP9 (sugar transport protein 9) were detected in CAN, which led to elevated photosynthetic capacity and accumulation of protein and amino acid as well as decrease in glucose, sucrose, and starch contents. On the other hand, genes associated with transport, carbon and N metabolism, redox response, protein phosphorylation, cell integrity, and epigenetic regulation mechanism were affected by UAN, resulting in enhanced photosynthetic capacity and carbohydrates and accumulation of protein and amino acid. In conclusion, our results showed that the CAN compared to UAN treatment had less effects on gene regulation and carbon and N metabolism. Canopy interception of N should be considered through CAN treatment to simulate N deposition in nature.

Modulation of potassium conductances optimizes fidelity of auditory information.

Potassium channels in auditory neurons are rapidly modified by changes in the auditory environment. In response to elevated auditory stimulation, short-term mechanisms such as protein phosphorylation and longer-term mechanisms such as accelerated channel synthesis increase the amplitude of currents that promote high-frequency firing. It has been suggested that this allows neurons to fire at high rates in response to high sound levels. We have carried out simple simulations of the response to postsynaptic neurons to patterns of neurotransmitter release triggered by auditory stimuli. These demonstrate that the amplitudes of potassium currents required for optimal encoding of a low-amplitude auditory signal differ from those for louder sounds. Specifically, the cross-correlation of the output of a neuron with an auditory stimulus is improved by increasing potassium currents as sound amplitude increases. Temporal fidelity for low-frequency stimuli is improved by increasing potassium currents that activate at negative potentials, while that for high-frequency stimuli requires increases in currents that activate at positive membrane potentials. These effects are independent of the firing rate. Moreover, levels of potassium currents that maximize the fidelity of the output of an ensemble of neurons differ from those that maximize fidelity for a single neuron. This suggests that the modulatory mechanisms must coordinate channel activity in groups of neurons or an entire nucleus. The simulations provide an explanation for the modulation of the intrinsic excitability of auditory brainstem neurons by changes in environmental sound levels, and the results may extend to information processing in other neural systems.

Modulation of potassium channels preserves temporal fidelity in auditory network models.

Potassium channels in auditory neurons are rapidly modified by changes in the auditory environment. In response to elevated auditory stimulation, short-term mechanisms such as protein phosphorylation and long-term mechanisms linked to channel synthesis increase the activity of channels that promote high frequency firing. We have now used simple simulations of cochlear hair cells and postsynaptic neurons to demonstrate that the amplitudes of potassium currents in neurons required for optimal encoding of a low-level auditory signal differs substantially from that for louder sounds. Specifically, the cross correlation of the output of a neuron with an auditory stimulus is increased by increasing potassium currents as sound amplitude increases. Although it has been suggested that channel modulation allows neurons to fire at high rates in response to high sound levels, we found that the cross correlation is entirely independent of firing rate and that combinations of currents that maximize firing to a stimulus provide very poor temporal fidelity. We also found that levels of potassium currents that maximize the temporal fidelity of the output of an ensemble of thirty neurons differ from those that maximize temporal fidelity for a single neuron. This suggests that, to maximize preservation of temporal information, modulatory mechanisms must coordinate channel activity in groups of neurons receiving similar synaptic inputs. The simulations provide an explanation for the modulation of the intrinsic excitability of auditory brainstem neurons by changes in environmental sound levels, and the results may extend to information processing in other neural systems.

Customized synthesis of phosphoprotein bearing phosphoserine or its nonhydrolyzable analog

Studies on the mechanism of protein phosphorylation and therapeutic interventions of its related molecular processes are limited by the difficulty in the production of purpose-built phosphoproteins harboring site-specific phosphorylated amino acids or their nonhydrolyzable analogs. Here we address this limitation by customizing the cell-free protein synthesis (CFPS) machinery via chassis strain selection and orthogonal translation system (OTS) reconfiguration screening. The suited chassis strains and reconfigured OTS combinations with high orthogonality were consequently picked out for individualized phosphoprotein synthesis. Specifically, we synthesized the sfGFP protein and MEK1 protein with site-specific phosphoserine (O-pSer) or its nonhydrolyzable analog, 2-amino-4-phosphonobutyric acid (C-pSer). This study successfully realized building cell-free systems for site-specific incorporation of phosphonate mimics into the target protein. Our work lays the foundation for developing a highly expansible CFPS platform and the streamlined production of user-defined phosphoproteins, which can facilitate research on the physiological mechanism and potential interference tools toward protein phosphorylation.

Quantitative phosphoproteomics analyses reveal the regulatory mechanisms related to frozen-thawed sperm capacitation and acrosome reaction in yak (Bos grunniens).

Mammalian spermatozoa are not mature after ejaculation and must undergo additional functional and structural changes within female reproductive tracts to achieve subsequent fertilization, including both capacitation and acrosome reaction (AR), which are dominated by post-translational modifications (PTMs), especially phosphorylation. However, the mechanism of protein phosphorylation during frozen-thawed sperm capacitation and AR has not been well studied. In this study, the phosphoproteomics approach was employed based on tandem mass tag (TMT) labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy to analyze frozen-thawed sperm in Ashidan yak under three sequential conditions (density gradient centrifugation-based purification, incubation in the capacitation medium and induction of AR processes by the calcium ionophore A23187 treatment). The identification of 1,377 proteins with 5,509 phosphorylation sites revealed changes in phosphorylation levels of sperm-specific proteins involved in regulation of spermatogenesis, sperm motility, energy metabolism, cilium movement, capacitation and AR. Some phosphorylated proteins, such as AKAP3, AKAP4, SPA17, PDMD11, CABYR, PRKAR1A, and PRKAR2A were found to regulate yak sperm capacitation and AR though the cAMP/PKA signaling pathway cascades. Notably, the phosphorylation level of SPA17 at Y156 increased in capacitated sperm, suggesting that it is also a novel functional protein besides AKAPs during sperm capacitation. Furthermore, the results of this study suggested that the phosphorylation of PRKAR1A and PRKAR2A, and the dephosphorylation of CABYR both play key regulatory role in yak sperm AR process. Protein-protein interaction analysis revealed that differentially phosphorylated proteins (AKAP3, AKAP4, FSIP2, PSMD11, CABYR, and TPPP2) related to capacitation and AR process played a key role in protein kinase A binding, sperm motility, reproductive process, cytoskeleton and sperm flagella function. Taken together, these data provide not only a solid foundation for further exploring phosphoproteome of sperm in yak, but an efficient way to identify sperm fertility-related marker phosphorylated proteins.

Global proteome analyses of phosphorylation and succinylation of barley root proteins in response to phosphate starvation and recovery.

Phosphate (Pi) stress is an important environmental factor that limits plant growth and development. Of various posttranslational modifications (PTMs), protein phosphorylation and succinylation are the two most important PTMs that regulate multiple biological processes in response to Pi stress. However, these PTMs have been investigated individually but their interactions with proteins in response to Pi stress remain poorly understood. In this study, to elucidate the underlying mechanisms of protein phosphorylation and succinylation in response to Pi stress, we performed a global analysis of the barley root phosphorylome and succinylome in Pi starvation and recovery stages, respectively. A total of 3,634 and 884 unique phosphorylated and succinylated proteins, respectively, corresponding to 11,538 and 2,840 phospho- and succinyl-sites, were identified; of these, 275 proteins were found to be simultaneously phosphorylated and succinylated. Gene Set Enrichment Analysis was performed with a Kyoto Encyclopedia of Genes and Genomes pathway database revealing pathways that significantly enriched in the phosphorylome and succinylome. Such pathways, were dynamically regulated by Pi starvation and recovery treatments, and could be partitioned into distinct metabolic processes. In particular, phosphorylated proteins related to purine, the mitogen-activated protein kinase (MAPK) signaling pathway, pyrimidine, and ATP-binding cassette (ABC) transporters were upregulated in both Pi deprivation and recovery stages. Succinylated proteins, significantly upregulated by both Pi starvation and recovery, were enriched in nitrogen metabolism and phenylpropanoid biosynthesis. Meanwhile, succinylated proteins that were significantly downregulated by both Pi starvation and recovery were enriched in lysine degradation and tryptophan metabolism. This highlighted the importance of these metabolic pathways in regulating Pi homeostasis. Furthermore, protein-protein interaction network analyses showed that the response of central metabolic pathways to Pi starvation and recovery was significantly modulated by phosphorylation or succinylation, both individually and together. In addition, we discovered relevant proteins involved in MAPK signaling and phenylpropanoid biosynthetic pathways existing in interactions between phosphorylated and succinylated proteins in response to Pi recovery. The current study not only provides a comprehensive analysis of phosphorylated and succinylated proteins in plant responses to Pi starvation and recovery, but also reveals detailed interactions between phosphorylated and succinylated proteins in barley roots.

Preparation of Core‐Shell Structured Magnetic Superhydrophilic Extractant for Enrichment of Phosphopeptides

A core-shell structured magnetic superhydrophilic Fe3O4@C@TiO2 microspheres are successfully prepared. The as-prepared Fe3O4@C@TiO2 magnetic microspheres possess fast magnetic response, superhydrophilic and high affinity. The magnetic superhydrophilic Fe3O4@C@TiO2 microspheres are employed for selective enrichment phosphopeptides and exhibit excellent enrichment performance such as high selectivity, low detection and excellent reusability. Sixteen phosphopeptides and four endogenous phosphopeptides can be identified from non-fat milk and human serum by Fe3O4@C@TiO2 magnetic microspheres, indicating Fe3O4@C@TiO2 will be a powerful and helpful IMAC material in understanding and analyzing essential biological phosphorylation processes in phosphorylated proteomics research.

The abnormal phosphorylation of the Rac1, Lim-kinase 1, and Cofilin proteins in the pathogenesis of Hirschsprung’s disease

ABSTRACT Rac1 can affect the migration of neural crest cells by regulating the polymerization of actin and the membrane formation process. But the role of the Rac1 signaling pathway in the pathogenesis of Hirschsprung’s disease (HSCR) remains unclear. In order to investigate the mechanism of the abnormal protein phosphorylation of Rac1, Lim-kinase 1 (Limk1) and Cofilin involved in the pathogenesis of HSCR. The protein phosphorylation levels of these proteins were detected by Western blot in 30 samples of HSCR narrow segment, 30 samples of transitional segment tissues, and 14 samples of normal intestinal tissues. Subsequently, in the SH-SY5Y human neuroblastoma cell line, a Rac1, Limk1, and Cofilin inhibitor group, a Rac1 overexpression group (PDGF-BB group), a Rac1 overexpression group + a Limk1 inhibitor group (P-B group), a Rac1 overexpression group + a Cofilin inhibitor group (P-C group) were established. The results showed that the expressions of p-Rac1, p-Limk1, and p-Cofilin in HSCR narrow segment and transitional segment were lower than those in normal intestine (p < 0.05). The expression levels of p-Rac1, p-Limk1, and p-Cofilin in the relative inhibitor group were significantly lower than those in the control group (p < 0.05), and the proliferation and migration levels in the control group and Rac1 overexpression group were significantly higher than those in the Rac1, Limk1, and Cofilin inhibitor group (p < 0.05). In conclusion, the decreased phosphorylation of the Rac1/Limk1/Cofilin signaling pathway in HSCR could inhibit the proliferation and migration of SH-SY5Y cells, and this might be associated with the pathogenesis of HSCR.

Text Mining and Machine Learning Protocol for Extracting Human-Related Protein Phosphorylation Information from PubMed.

In the modern health care research, protein phosphorylation has gained an enormous attention from the researchers across the globe and requires automated approaches to process a huge volume of data on proteins and their modifications at the cellular level. The data generated at the cellular level is unique as well as arbitrary, and an accumulation of massive volume of information is inevitable. Biological research has revealed that a huge array of cellular communication aided by protein phosphorylation and other similar mechanisms imply different and diverse meanings. This led to a collection of huge volume of data to understand the biological functions of human evolution, especially for combating diseases in a better way. Text mining, an automated approach to mine the information from an unstructured data, finds its application in extracting protein phosphorylation information from the biomedical literature databases such as PubMed. This chapter outlines a recent text mining protocol that applies natural language parsing (NLP) for named entity recognition and text processing, and support vector machines (SVM), a machine learning algorithm for classifying the processed text related human protein phosphorylation. We discuss on evaluating the text mining system which is the outcome of the protocol on three corpora, namely, human Protein Phosphorylation (hPP) corpus, Integrated Protein Literature Information and Knowledge corpus (iProLink), and Phosphorylation Literature corpus (PLC). We also present a basic understanding on the chemistry and biology that drive the protein phosphorylation process in a human body. We believe that this basic understanding will be useful to advance the existing text mining systems for extracting protein phosphorylation information from PubMed.

How the structural properties of the indole derivatives are important in kinase targeted drug design?: A case study on tyrosine kinase inhibitors

Kinases are considered as important signalling enzymes that illustrate 20% of the druggable genome. Human kinase family comprises >500 protein kinases and about 20 lipid kinases. Protein kinases are responsible for the mechanism of protein phosphorylation. These are necessary for regulation of various cellular activities including proliferation, cell cycle, apoptosis, motility, growth, differentiation, etc. Their deregulation leads to disruption of many cellular processes leading to different diseases most importantly cancer. Thus, kinases are considered as valuable targets in different types of cancer as well as other diseases. Researchers around the world are actively engaged in developing inhibitors based on distinct chemical scaffolds. Indole represents as a versatile scaffold in the naturally occurring and bioactive molecules. It is also used as a privileged scaffold for the target-based drug design against different diseases. This present article aim to review the applications of indole scaffold in the design of inhibitors against different tyrosine kinases such as epidermal growth factor receptors (EGFRs), vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptors (PDGFRs), etc. Important structure activity relationships (SARs) of indole derivatives were discussed. The present work is an attempt to summarize all the crucial structural information which is essential for the development of indole based tyrosine kinase inhibitors with improved potency.

Structural Insights into Protein Regulation by Phosphorylation and Substrate Recognition of Protein Kinases/Phosphatases

Protein phosphorylation is one of the most widely observed and important post-translational modification (PTM) processes. Protein phosphorylation is regulated by protein kinases, each of which covalently attaches a phosphate group to an amino acid side chain on a serine (Ser), threonine (Thr), or tyrosine (Tyr) residue of a protein, and by protein phosphatases, each of which, conversely, removes a phosphate group from a phosphoprotein. These reversible enzyme activities provide a regulatory mechanism by activating or deactivating many diverse functions of proteins in various cellular processes. In this review, their structures and substrate recognition are described and summarized, focusing on Ser/Thr protein kinases and protein Ser/Thr phosphatases, and the regulation of protein structures by phosphorylation. The studies reviewed here and the resulting information could contribute to further structural, biochemical, and combined studies on the mechanisms of protein phosphorylation and to drug discovery approaches targeting protein kinases or protein phosphatases.

The Journey from Two-Step to Multi-Step Phosphorelay Signaling Systems.

BackgroundThe two-component signaling (TCS) system is an important signal transduction machinery in prokaryotes and eukaryotes, excluding animals, that uses a protein phosphorylation mechanism for signal transmission.ConclusionProkaryotes have a primitive type of TCS machinery, which mainly comprises a membrane-bound sensory histidine kinase (HK) and its cognate cytoplasmic response regulator (RR). Hence, it is sometimes referred to as two-step phosphorelay (TSP). Eukaryotes have more sophisticated signaling machinery, with an extra component - a histidine-containing phosphotransfer (HPT) protein that shuttles between HK and RR to communicate signal baggage. As a result, the TSP has evolved from a two-step phosphorelay (His–Asp) in simple prokaryotes to a multi-step phosphorelay (MSP) cascade (His–Asp–His–Asp) in complex eukaryotic organisms, such as plants, to mediate the signaling network. This molecular evolution is also reflected in the form of considerable structural modifications in the domain architecture of the individual components of the TCS system. In this review, we present TCS system's evolutionary journey from the primitive TSP to advanced MSP type across the genera. This information will be highly useful in designing the future strategies of crop improvement based on the individual members of the TCS machinery.

Effects of Single-Dose and Long-Term Ketamine Administration on Tau Phosphorylation-Related Enzymes GSK-3β, CDK5, PP2A, and PP2B in the Mouse Hippocampus.

Ketamine is a recreational drug that causes emotional and cognitive impairments, but its specific mechanisms of action are still unclear. Recent evidence suggests that Tau protein phosphorylation and targeted delivery to the postsynaptic area are closely related to its neurotoxicity, and our recent studies have shown that long-term ketamine administration causes excessive Tau protein phosphorylation. However, the regulatory mechanism of Tau protein phosphorylation induced by ketamine has not been clarified. In the present study, we administered a single ketamine injection and long-term (6months) ketamine injections in C57BL/6 mice, to investigate the effects of different doses of ketamine on the expression levels of Tau protein and its phosphorylation, the expression levels and activities of the related protein phosphokinases GSK-3β and CDK5, and the expression levels and activities of the related protein phosphatases PP2A and PP2B in the mouse hippocampus. Our results showed that both single-dose and long-term ketamine administration induced excessive phosphorylation of the Tau protein at ser202/thr205 and ser396. A single ketamine administration caused an increase in the activity of GSK-3β (at high doses) and a decrease in the activity of PP2A. On the other hand, long-term ketamine administration resulted in an increase in the activities of GSK-3β (at high doses) and CDK5, and a decrease in the activity of PP2A. Our results indicate that GSK-3β, CDK5, and PP2A may be involved in ketamine-induced Tau protein phosphorylation.

Quantitative Phosphoproteomic Analysis in Alpha-Synuclein Transgenic Mice Reveals the Involvement of Aberrant p25/Cdk5 Signaling in Early-stage Parkinson's Disease.

A30P and A53T mutations in the gene encoding alpha-synuclein-a presynaptic protein-are the most frequently identified genetic causes of Parkinson's disease (PD). Aberrant alpha-synuclein likely plays central roles in dopaminergic neuronal death and motor symptoms in PD. This study investigated the protein phosphorylation profile in early-stage PD through phosphoproteomic analyses of tissue samples from the substantia nigra pars compacta (SNpc) of 6-month-old alpha-synuclein transgenic mice (A30P/A53T double-mutant human alpha-synuclein; hm2α-SYN-39 strain). We identified 5351 phosphorylation sites in 2136 phosphoproteins. Of these, 357 upregulated sites in 245 proteins and 50 downregulated sites in 46 proteins were differentially phosphorylated between alpha-synuclein transgenic and wildtype mice. Bioinformatic analyses, including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway enrichment, and motif analyses, were used to elucidate the molecular and cellular mechanisms underlying double-mutant human alpha-synuclein overexpression. Scansite-based computational analysis and prediction of differentially quantitated phosphoproteins identified the neuronal protein cyclin-dependent kinase 5 (Cdk5) as the most significantly enriched kinase. Biochemical experiments suggested that the p25/Cdk5 pathway was activated in an MPP+-induced cell culture model and MPTP-induced mouse model. Moreover, Cdk5 could directly phosphorylate the Ank2 protein at Ser1889 in vitro. Therefore, quantitative phosphoproteomic using an alpha-synuclein transgenic mouse model offers a powerful approach for elucidating the protein phosphorylation mechanism underlying SNpc dopaminergic neuronal death in an animal model of PD.

Detection of Phosphorylation on Immunoprecipitates from Total Protein Extracts of Arabidopsis thaliana Seedlings.

Phosphorylation is a versatile posttranslational modification that can regulate the localization, stability, and conformation of proteins; protein-protein interactions; and enzyme activities. Phosphorylation of plasma membrane proteins, for example, can serve as recognition signals for ubiquitin ligases and hence can trigger its endocytic degradation. Key determinants of protein phosphorylation are kinases and phosphatases that are spatiotemporally regulated to phosphorylate or dephosphorylate specific target proteins. To understand the dynamics and regulatory mechanisms of protein phosphorylation, it is essential to analyze the phosphorylation status of the proteins and identify phosphorylation sites as well as the modifying enzymes. In this chapter, we describe methods that can be used for the detection of phosphoproteins that are immunoprecipitated from Arabidopsis total extracts.

Identification and Characterization of Phosphoproteins in Somatic Embryogenesis Acquisition during Oil Palm Tissue Culture.

Somatic embryogenesis during oil palm tissue culture is a long process. The identification of the proteins that control this process may help to shorten the time of oil palm tissue culture. We collected embryogenic callus and somatic embryos at the globular, torpedo, and cotyledon maturation stages, as well as from plantlets, for total protein extraction. An enrichment column was used to enrich the phosphoproteins, which were subjected to tryptic enzyme digestion. Each sample was analyzed with nano-liquid chromatography–tandem mass spectrometry (nano LC-MS/MS). A total of 460 phosphoproteins were identified and analyzed. The functional characterization of phosphoproteins were observed as highest in the metabolic process, protein/nucleotide/ion binding, and membrane component. The different phosphoproteins are involved in the control of vegetative growth, cellular differentiation, cell morphogenesis, and signaling roles in plants. The Quantitative Real-Time Reverse Transcription-PCR technique (qPCR) was successfully used to verify the expression of genes, and the results were consistent with the level of protein expression from nano-LC-MS/MS. The E3 ubiquitin-protein ligase and sister chromatid cohesion PDS5 were specifically expressed only in the somatic embryo and plantlet, and these could be used as protein biomarkers to determine the oil palm somatic embryo maturation stage. This study sheds light on the protein phosphorylation mechanism that regulates somatic embryogenesis transition during oil palm tissue culture.

The Major Storage Protein in Potato Tuber Is Mobilized by a Mechanism Dependent on Its Phosphorylation Status

The role of the protein phosphorylation mechanism in the mobilization of vegetative storage proteins (VSPs) is totally unknown. Patatin is the major VSP of the potato (Solanum tuberosum L.) tuber that encompasses multiple differentially phosphorylated isoforms. In this study, temporal changes in the phosphorylation status of patatin isoforms and their involvement in patatin mobilization are investigated using phosphoproteomic methods based on targeted two-dimensional electrophoresis (2-DE). High-resolution 2-DE profiles of patatin isoforms were obtained in four sequential tuber life cycle stages of Kennebec cultivar: endodormancy, bud break, sprouting and plant growth. In-gel multiplex identification of phosphorylated isoforms with Pro-Q Diamond phosphoprotein-specific stain revealed an increase in the number of phosphorylated isoforms after the tuber endodormancy stage. In addition, we found that the phosphorylation status of patatin isoforms significantly changed throughout the tuber life cycle (P < 0.05) using the chemical method of protein dephosphorylation with hydrogen fluoride-pyridine (HF-P) coupled to 2-DE. More specifically, patatin phosphorylation increased by 32% from endodormancy to the tuber sprouting stage and subsequently decreased together with patatin degradation. Patatin isoforms were not randomly mobilized because highly phosphorylated Kuras-isoforms were preferably degraded in comparison to less phosphorylated non-Kuras isoforms. These results lead us to conclude that patatin is mobilized by a mechanism dependent on the phosphorylation status of specific isoforms.

Protein Phosphorylation Mechanisms Underlying Chronic Pain in Sickle Cell Disease

Pain is one of the most dreadful symptoms in sickle cell disease (SCD) and is often refractory to currently available analgesics. Besides acute painful vaso-occlusive crises, SCD is also accompanied by intractable chronic pain. This persistent, and often unrelieved, pain starts early in childhood and continues throughout life. The neurobiological mechanisms of chronic pain in SCD remain unclear, which markedly limits effective pain management and the quality of life in patients with SCD. Taking advantage of two humanized mouse models of SCD, this study aimed to investigate protein phosphorylation mechanisms for chronic pain in SCD.We characterized pain in two transgenic mice models of SCD that exclusively express human alleles encoding normal α- and sickle β-globin. Berkeley SCD mice (BERK mice) and Townes' SCD mice (TOW mice) exhibited ongoing spontaneous pain behavior and increased sensitivity to evoked pain stimuli compared with littermate control mice expressing normal human hemoglobins. To investigate the underlying protein phosphorylation mechanisms of chronic pain in SCD, we examined PKC isoform mediated nociceptive signaling. Prominent activation of multiple PKC isoforms were observed in the superficial laminae of the spinal cord dorsal horn in BERK and TOW mice. Functional inhibition and silencing of specific PKC isoforms attenuated spontaneous pain, mechanical allodynia, and heat hyperalgesia in both transgenic SCD mice. Furthermore, employing hematopoietic stem cell transplantation approach, we were able to generate a sickle cell anemia model in PKC-null mice, allowing us to specifically target neuronal PKC in SCD. Neither spontaneous pain nor evoked pain was detected in the mice lacking specific PKC isoform despite the full establishment of SCD phenotypes.In summary, this study is the first to identify the presence of ongoing spontaneous pain in preclinical sickle cell models, which closely mimic the most prevalent manifestation in patients with SCD. Moreover, we found that spinal PKC is a critical mechanism for the generation and maintenance of ongoing and evoked pain in SCD. These findings offer insights into sickle cell pain mechanisms, which may become a potential target for pharmacological interventions. DisclosuresNo relevant conflicts of interest to declare.