Predicted Phosphorylation Sites Research Articles

Identification of phosphorylated small ORF-encoded peptides in Hep3B cells by LC/MS/MS

Small ORF-encoded peptides (SEPs) are a class of low molecular weight proteins and peptides comprising <100 amino acids with important functions in various life activities. Although the sequence length is short, SEPs might also have post-translational modification (PTM). Phosphorylation is one of the most essential PTMs of proteins. In this work, we enriched phosphopeptides with IMAC and TiO2 materials and analyzed the phosphorylated SEPs in Hep3B cells. A total of 24 phosphorylated SEPs were identified, and 11 SEPs were coded by ncRNA. For the sequence analysis, we found that the general characteristics of phosphorylated SEPs are roughly the same as canonical proteins. Besides, two phosphorylation SEPs have the Stathmin family signature 2 motif, which can regulate the microtubule cytoskeleton. Some SEPs have domains or signal peptides, indicating their specific functions and subcellular locations. Kinase network analysis found a small number of kinases that may be a clue to the specific functions of some SEPs. However, only one-fifth of the predicted phosphorylation sites were identified by LC/MS/MS, indicating that many SEP PTMs are hidden in the dark, waiting to be uncovered and verified. This study helps expand our understanding of SEP and provides information for further SEP function investigation. SignificanceSmall ORF-encoded peptides (SEPs) are important in various life activities. Although the sequence length is short (<100AA), SEPs might also have post-translational modification (PTM). Phosphorylation is one of the most essential PTMs of proteins. We enriched phosphopeptides and analyzed the phosphorylated SEPs in Hep3B cells. That is the first time to explore the PTM of SPEs systematically. Kinase network analysis found a small number of kinases that may be a clue to the specific functions of SEPs. More SEP PTMs are hidden in the dark and waiting to be uncovered and verified. This study helps expand our understanding of SEP and provides information for further SEP function investigation.

Transcriptional Regulation of the Human 5-HT1A Receptor Gene by Lithium: Role of Deaf1 and GSK3β.

Serotonin 1A (5-HT1A) autoreceptors located on serotonin neurons inhibit their activity, and their upregulation has been implicated in depression, suicide and resistance to antidepressant treatment. Conversely, post-synaptic 5-HT1A heteroreceptors are important for antidepressant response. The transcription factor deformed epidermal autoregulatory factor 1 (Deaf1) acts as a presynaptic repressor and postsynaptic enhancer of 5-HT1A transcription, but the mechanism is unclear. Because Deaf1 interacts with and is phosphorylated by glycogen synthase kinase 3β (GSK3β)-a constitutively active protein kinase that is inhibited by the mood stabilizer lithium at therapeutic concentrations-we investigated the role of GSK3β in Deaf1 regulation of human 5-HT1A transcription. In 5-HT1A promoter-reporter assays, human HEK293 kidney and 5-HT1A-expressing SKN-SH neuroblastoma cells, transfection of Deaf1 reduced 5-HT1A promoter activity by ~45%. To identify potential GSK3β site(s) on Deaf1, point mutations of known and predicted phosphorylation sites on Deaf1 were tested. Deaf1 repressor function was not affected by any of the mutants tested except the Y300F mutant, which augmented Deaf1 repression. Both lithium and the selective GSK3 inhibitors CHIR-99021 and AR-014418 attenuated and reversed Deaf1 repression compared to vector. This inhibition was at concentrations that maximally inhibit GSK3β activity as detected by the GSK3β-sensitive TCF/LEF reporter construct. Our results support the hypothesis that GSK3β regulates the activity of Deaf1 to repress 5-HT1A transcription and provide a potential mechanism for actions of GSK3 inhibitors on behavior.

Fatty acid binding protein 1 and fatty acid synthetase over-expression have differential effects on collagen III synthesis and cross-linking in Zongdihua pig primary adipocytes.

The purpose of this study was to determine whether FABP1 and FAS regulate expression of collagen and its crosslinking via lysyl oxidase in isolated adipocytes from Zongdihua pigs. We aimed to identify biochemical processes affecting meat quality using molecular tools to provide a basis for breeding improvement of these animals. We measured expression levels of FABP1 and related genes using qRT-PCR in longissimus dorsi muscle and subcutaneous adipose tissues. Primary adipocytes from fat tissues were isolated and FABP1 and FAS were over-expressed from recombinant plasmids. Sequence analysis of the cloned genes indicated that FABP1 encodes a hydrophobic protein of 128 amino acids and contained 12 predicted phosphorylation sites and no transmembrane regions. The basal levels of FABP1 and FAS expression in pig tissues were 3-3.5-fold higher in subcutaneous fat compared with muscle (P < 0.01). Recombinant expression plasmids were successfully transfected into the cloned preadipocytes and (a) over-expression of FAS resulted in significantly increased expression of COL3A1 (P < 0.05) and significantly inhibited lysyl oxidase LOX expression (P < 0.01); (b) over-expression of FABP1 significantly increased COL3A1 expression (P < 0.01) and significantly inhibited LOX expression (P< 0.05) and significantly reduced lysyl oxidase activity (P < 0.01). Therefore, FAS enhanced FABP1 expression resulting in increased collagen accumulation and this preliminarily suggested that FAS and FABP1 can serve as fat-related candidate genes and provide a theoretical basis for the study of fat deposition in Zongdihua pigs.

Specific Residues in the C-Terminal Domain of the Human Metapneumovirus Phosphoprotein Are Indispensable for Formation of Viral Replication Centers and Regulation of the Function of the Viral Polymerase Complex.

Human metapneumovirus (HMPV) is a negative-strand RNA virus that frequently causes respiratory tract infections in infants, the elderly, and the immunocompromised. A hallmark of HMPV infection is the formation of membraneless, liquid-like replication and transcription centers in the cytosol termed inclusion bodies (IBs). The HMPV phosphoprotein (P) and nucleoprotein (N) are the minimal viral proteins necessary to form IB-like structures, and both proteins are required for the viral polymerase to synthesize RNA during infection. HMPV P is a homotetramer with regions of intrinsic disorder and has several known and predicted phosphorylation sites of unknown function. In this study, we found that the P C-terminal intrinsically disordered domain (CTD) must be present to facilitate IB formation with HMPV N, while either the N-terminal intrinsically disordered domain or the central oligomerization domain was dispensable. Alanine substitution at a single tyrosine residue within the CTD abrogated IB formation and reduced coimmunoprecipitation with HMPV N. Mutations to C-terminal phosphorylation sites revealed a potential role for phosphorylation in regulating RNA synthesis and P binding partners within IBs. Phosphorylation mutations which reduced RNA synthesis in a reporter assay produced comparable results in a recombinant viral rescue system, measured as an inability to produce infectious viral particles with genomes containing these single P mutations. This work highlights the critical role HMPV P plays in facilitating a key step of the viral life cycle and reveals the potential role for phosphorylation in regulating the function of this significant viral protein. IMPORTANCE Human metapneumovirus (HMPV) infects global populations, with severe respiratory tract infections occurring in infants, the elderly, and the immunocompromised. There are currently no FDA-approved therapeutics available to prevent or treat HMPV infection. Therefore, understanding how HMPV replicates is vital for the identification of novel targets for therapeutic development. During HMPV infection, viral RNA synthesis proteins localize to membraneless structures called inclusion bodies (IBs), which are sites of genome replication and transcription. The HMPV phosphoprotein (P) is necessary for IBs to form and for the virus to synthesize RNA, but it is not known how this protein contributes to IB formation or if it is capable of regulating viral replication. We show that the C-terminal domain of P is the location of a molecular interaction driving IB formation and contains potential phosphorylation sites where amino acid charge regulates the function of the viral polymerase complex.

Unravel the regulatory mechanism of Yrr1p phosphorylation in response to vanillin stress in Saccharomyces cerevisiae

Improving the resistance of Saccharomyces cerevisiae to vanillin, derived from lignin, will benefit the design of robust cell factories for lignocellulosic biorefining. The transcription factor Yrr1p mediates S. cerevisiae resistance to various compounds. In this study, eleven predicted phosphorylation sites were mutated, among which 4 mutants of Yrr1p, Y134A/E and T185A/E could improve vanillin resistance. Both dephosphorylated and phosphorylated mutations at Yrr1p 134 and 185 gathered in the nucleus regardless of the presence or absence of vanillin. However, the phosphorylated mutant Yrr1p inhibited target gene expression, while dephosphorylated mutants promoted expression. Transcriptomic analysis showed that the dephosphorylated Yrr1p T185 mutant, under vanillin stress, upregulated ribosome biogenesis and rRNA processing. These results demonstrate the mechanism by which Yrr1p phosphorylation regulates the expression of target genes. The identification of key phosphorylation sites in Yrr1p offers novel targets for the rational construction of Yrr1p mutants to improve resistance to other compounds.

Role of DevR phosphorylation in nitric oxide homeostasis and signaling of Streptomyces coelicolor A3(2) M145.

Our previous studies revealed that a two-component system (TCS), DevS, and DevR, regulate both nitric oxide (NO) signaling and NO homeostasis in the actinobacterium Streptomyces coelicolor A3(2) M145, suggesting a reasonable system for NO-dependent metabolism. In this study, sequence alignment of DevR and DevR homologs found Asp66 (D66) and Thr196 (T196) as predicted phosphorylation sites of DevR. Phos-tag gel electrophoretic mobility shift assay suggested that D66 and T196 are involved in the phosphorylation of DevR. The respective point mutations of D66 and T196 significantly decreased the transcriptional activity of DevR, which affected nitrite production and aerial mycelium formation. These results suggested that both D66 and T196 of DevR are important for the regulation of NO homeostasis and signaling in S. coelicolor A3(2) M145.

Bioinformatic analysis of the S protein of human respiratory coronavirus

The present study aimed to apply bioinformatic methods to analyze the structure of the S protein of human respiratory coronaviruses, including severe respiratory disease syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus HKU1 (HCoV-HKU1), and severe respiratory disease syndrome coronavirus type 2 (SARS-CoV-2). We predicted and analyzed the physicochemical properties, hydrophilicity and hydrophobicity, transmembrane regions, signal peptides, phosphorylation and glycosylation sites, epitopes, functional domains, and motifs of the S proteins of human respiratory coronaviruses. All four S proteins contain a transmembrane region, which enables them to bind to host cell surface receptors. All four S proteins contain a signal peptide, phosphorylation sites, glycosylation sites, and epitopes. The predicted phosphorylation sites might mediate S protein activation, the glycosylation sites might affect the cellular orientation of the virus, and the predicted epitopes might have implications for the design of antiviral inhibitors. The S proteins of all four viruses have two structural domains, S1 (C-terminal and N-terminal domains) and S2 (homology region 1 and 2). Our bioinformatic analysis of the structural and functional domains of human respiratory coronavirus S proteins provides a basis for future research to develop broad-spectrum antiviral drugs, vaccines, and antibodies.

Pancancer Analyses Reveal Genomics and Clinical Characteristics of the SETDB1 in Human Tumors.

Background Malignant tumor is one of the most common diseases that seriously affect human health. The prior literature has reported the biological function and potential therapeutic targets of SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) as an oncogene. However, SETDB1 has rarely been analyzed from a pan-cancer perspective. Methods Bioinformatics analysis tools and databases, including GeneCards, National Center for Biotechnology Information (NCBI), UniProt, Illustrator for Biological Sequences (IBS), Human Protein Atlas (HPA), GEPIA, TIMER2, Sangerbox 3.0, UALCAN, Kaplan-Meier (K-M) plotter, cBioPortal, Catalogue Of Somatic Mutations In Cancer (COSMIC), PhosphoSitePlus, TISIDB, STRING, and GeneMANIA, were utilized to clarify the biological functions and clinical significance of SETDB1 from a pan-cancer perspective. Results In this study, the pan-cancer analysis demonstrated that SETDB1 showed significantly differential expression in most tumor tissues and paracancerous tissues, and SETDB1 expression was associated with clinicopathological features and clinical prognosis. We also found that SETDB1 mutations occurred in most tumors and were related to tumorigenesis. In addition, DNA methylation of SETDB1 primarily occurred at the cg10444928 site and was associated with prognosis in several human tumors. The predicted phosphorylation site of SETDB1 was Ser1006. We found that SETDB1 was significantly related to the specific tumor-infiltrating immune cell populations and expression of clinically targetable immune checkpoints and may be a promising immunotherapy target. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses also indicated that SETDB1 may function as crucial regulator in carcinogenesis of human cancers. Conclusions SETDB1 is an important oncogene involved in tumorigenesis and tumor progression through different biological mechanisms. Furthermore, SETDB1 may be a potential therapeutic target for cancer treatment.

Molecular cloning, characterization, and functional analysis of the uncharacterized C11orf96 gene

BackgroundThe mammalian genome encodes millions of proteins. Although many proteins have been discovered and identified, a large part of proteins encoded by genes are yet to be discovered or fully characterized. In the present study, we successfully identified a host protein C11orf96 that was significantly upregulated after viral infection.ResultsFirst, we successfully cloned the coding sequence (CDS) region of the cat, human, and mouse C11orf96 gene. The CDS region of the C11orf96 gene is 372 bp long, encodes 124 amino acids, and is relatively conserved in different mammals. From bioinformatics analysis, we found that C11orf96 is rich in Ser and has multiple predicted phosphorylation sites. Moreover, protein interaction prediction analysis revealed that the protein is associated with several transmembrane family proteins and zinc finger proteins. Subsequently, we found that C11orf96 is strictly distributed in the cytoplasm. According to the tissue distribution characteristics, C11orf96 is distributed in all tissues and organs, with the highest expression levels in the kidney. These results indicate that C11orf96 may play a specific biological role in the kidney.ConclusionsSummarizing, these data lay the foundation for studying the biological functions of C11orf96 and for exploring its role in viral replication.

Mutations in FgPrp6 suppressive to the Fgprp4 mutant in Fusarium graminearum

The pre-mRNA processing factor Prp6 is an essential component of the U4/U6.U5 tri-small nuclear ribonucleoprotein (snRNP). In a previous study, mutations were identified in the PRP6 ortholog in four suppressors of Fgprp4 that was deleted of the only kinase FgPrp4 among the spliceosome components in the plant pathogenic fungus Fusarium graminearum. In this study, we identified additional suppressor mutations in FgPrp6 and determined the suppressive effects of selected mutations. In total, 12 mutations of FgPRP6 were identified in 20 suppressors of Fgprp4 by sequencing analysis. Whereas three mutation sites are in the linker region of FgPrp6, seven are in the first two HAT repeats. RNA-seq analysis showed that suppressor mutations on different sites caused different splicing efficiency recovery. The suppressive effects of E308K and R230H were verified. Similar to human and fission yeast, the FgPrp6 was phosphorylated by the FgPrp4 kinase. Interestingly, the conserved Prp4-phosphorylation sites T261, T219&T221, and predicted phosphorylation sites T199&T200 on FgPrp6 were dispensable for the function of FgPrp6 in hyphal growth and sexual reproduction but important in plant infection. They are required for the infectious growth of F. graminearum in wheat lemma. RNA-seq analysis of the wheat lemma infected with Fgprp6/FgPRP6Δ199–221-GFP or Fgprp6/FgPRP6Δ250–262-GFP showed that 28 and 35% introns had splicing defects, respectively, which may be responsible for their defects in plant infection.

Mutation of MtrA at the Predicted Phosphorylation Site Abrogates Its Role as a Global Regulator in Streptomyces venezuelae.

ABSTRACTThe global regulator MtrA controls development and primary and secondary metabolism in Streptomyces species. However, residues critical for its function have not yet been characterized. In this study, we identified residue D53 as the potential phosphorylation site of MtrA from Streptomyces venezuelae, a model Streptomyces strain. MtrA variants with amino acid substitutions at the D53 site were generated, and the effects of these substitutions were evaluated in vitro and in vivo. We showed that, although substitutions at D53 did not alter MtrA’s secondary structure, the MtrA D53 protein variants lost the ability to bind known MtrA recognition sequences (MtrA sites) in electrophoretic mobility shift assays. Complementation of the ΔmtrA strain with MtrA D53 protein variants did not affect overall strain growth. However, in comparison to the wild-type strain, chloramphenicol and jadomycin production were aberrant in the D53 variant strains, with levels similar to the levels in the ΔmtrA strain. Transcriptional analysis showed that the expression patterns of genes were also similar in the ΔmtrA strain and the D53 variant strains. Although the D53 protein variants and wild-type MtrA were produced at similar levels in S. venezuelae, chromatin immunoprecipitation-quantitative PCR results indicated that replacing the D53 residue rendered the altered proteins unable to bind MtrA sites in vivo, including MtrA sites that regulate genes involved in nitrogen metabolism and in chloramphenicol and jadomycin biosynthesis. In conclusion, our study demonstrates that the predicted phosphorylation site D53 is critical for the role of MtrA in regulation and suggests that MtrA functions in a phosphorylated form in the genus Streptomyces.IMPORTANCE Although phosphorylation has been shown to be essential for the activation of many response regulator proteins of two-component systems, the role of the phosphorylation site in the function of the global regulator MtrA in the genus Streptomyces has not been reported. In this study, we generated Streptomyces mutants that had amino acid substitutions at the predicted phosphorylation site of MtrA, and the effects of the substitutions were investigated by comparing the phenotypes of the resulting strains and their gene expression patterns with those of the wild-type strain and an MtrA deletion mutant. The ability of the altered proteins to bind known promoter targets in vitro was also evaluated. Our analyses showed that the predicted phosphorylation site D53 is critical for MtrA binding in vitro and for the normal functioning of MtrA in vivo. These studies further demonstrate the importance of MtrA as a global regulator in the genus Streptomyces.

FgSfl1 and Its Conserved PKA Phosphorylation Sites Are Important for Conidiation, Sexual Reproduction, and Pathogenesis in Fusarium graminearum

The fungal plant pathogen, Fusarium graminearum, contains two genes, FgCPK1 and FgCPK2, encoding the catalytic subunits of cAMP-dependent protein kinase A. FgCPK1 and FgCPK2 are responsible for most of the PKA activities and have overlapping functions in various cellular processes in F. graminearum. The cpk1 cpk2 double mutant was significantly reduced in growth, rarely produced conidia, and was non-pathogenic. In this study, we found that the cpk1 cpk2 double mutant was unstable and produced fast-growing spontaneous sectors that were defective in plant infection. All spontaneous suppressor strains had mutations in FgSFL1, a transcription factor gene orthologous to SFL1 in yeast. Thirteen suppressor strains had non-sense mutations at Q501, three suppressor strains had frameshift mutations at W198, and five suppressor strains had mutations in the HSF binding domain of FgSfl1. Only one suppressor strain had both a non-synonymous mutation at H225 and a non-sense mutation at R490. We generated the SFL1 deletion mutant and found that it produced less than 2% of conidia than that of the wild-type strain PH-1. The sfl1 mutant was significantly reduced in the number of perithecia on carrot agar plates at 7 days post-fertilization (dpf). When incubated for more than 12 days, ascospore cirrhi were observed on the sfl1 mutant perithecia. The infection ability of the sfl1 deletion mutant was also obviously defective. Furthermore, we found that in addition to the S223 and S559 phosphorylation sites, FgSFL1 had another predicted phosphorylation site: T452. Interestingly, the S223 phosphorylation site was responsible for sexual reproduction, and the T452 phosphorylation site was responsible for growth and sexual reproduction. Only the S559 phosphorylation site was found to play an important role in conidiation, sexual reproduction, and infection. Overall, our results indicate that FgSFL1 and its conserved PKA phosphorylation sites are important for vegetative growth, conidiation, sexual reproduction, and pathogenesis in F. graminearum.

Prp19-associated splicing factor Cwf15 regulates fungal virulence and development in the rice blast fungus.

The splicing factor Cwf15 is an essential component of the Prp19-associated component of the spliceosome and regulates intron splicing in several model species, including yeasts and human cells. However, the roles of Cwf15 remain unexplored in plant pathogenic fungi. Here, we report that MoCWF15 in the rice blast fungus Magnaporthe oryzae is non-essential to viability and important to fungal virulence, growth and conidiation. MoCwf15 contains a putative nuclear localization signal (NLS) and is localized into the nucleus. The NLS sequence but not the predicted phosphorylation site or two sumoylation sites was essential for the biological functions of MoCwf15. Importantly, MoCwf15 physically interacted with the Prp19-associated splicing factors MoCwf4, MoSsa1 and MoCyp1, and negatively regulated protein accumulations of MoCyp1 and MoCwf4. Furthermore, with the deletion of MoCWF15, aberrant intron splicing occurred in near 400 genes, 20 of which were important to the fungal development and virulence. Taken together, MoCWF15 regulates fungal growth and infection-related development by modulating the intron splicing efficiency of a subset of genes in the rice blast fungus.

Hybrid Histidine Kinase BinK Represses Vibrio fischeri Biofilm Signaling at Multiple Developmental Stages.

The symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and its exclusive light organ symbiont, Vibrio fischeri, provides a natural system in which to study host-microbe specificity and gene regulation during the establishment of a mutually beneficial symbiosis. Colonization of the host relies on bacterial biofilm-like aggregation in the squid mucus field. Symbiotic biofilm formation is controlled by a two-component signaling (TCS) system consisting of regulators RscS-SypF-SypG, which together direct transcription of the symbiosis polysaccharide Syp. TCS systems are broadly important for bacteria to sense environmental cues and then direct changes in behavior. Previously, we identified the hybrid histidine kinase BinK as a strong negative regulator of V. fischeri biofilm regulation, and here we further explore the function of BinK. To inhibit biofilm formation, BinK requires the predicted phosphorylation sites in both the histidine kinase (H362) and receiver (D794) domains. Furthermore, we show that RscS is not essential for host colonization when binK is deleted from strain ES114, and imaging of aggregate size revealed no benefit to the presence of RscS in a background lacking BinK. Strains lacking RscS still suffered in competition. Finally, we show that BinK functions to inhibit biofilm gene expression in the light organ crypts, providing evidence for biofilm gene regulation at later stages of host colonization. Overall, this study provides direct evidence for opposing activities of RscS and BinK and yields novel insights into biofilm regulation during the maturation of a beneficial symbiosis. IMPORTANCE Bacteria are often in a biofilm state, and transitions between planktonic and biofilm lifestyles are important for pathogenic, beneficial, and environmental microbes. The critical nature of biofilm formation during Vibrio fischeri colonization of the Hawaiian bobtail squid light organ provides an opportunity to study development of this process in vivo using a combination of genetic and imaging approaches. The current work refines the signaling circuitry of the biofilm pathway in V. fischeri, provides evidence that biofilm regulatory changes occur in the host, and identifies BinK as one of the regulators of that process. This study provides information about how bacteria regulate biofilm gene expression in an intact animal host.

The Lipid Transfer Protein 1 from Nicotiana benthamiana Assists Bamboo mosaic virus Accumulation.

Host factors play a pivotal role in regulating virus infection. Uncovering the mechanism of how host factors are involved in virus infection could pave the way to defeat viral disease. In this study, we characterized a lipid transfer protein, designated NbLTP1 in Nicotiana benthamiana, which was downregulated after Bamboo mosaic virus (BaMV) inoculation. BaMV accumulation significantly decreased in NbLTP1-knockdown leaves and protoplasts compared with the controls. The subcellular localization of the NbLTP1-orange fluorescent protein (OFP) was mainly the extracellular matrix. However, when we removed the signal peptide (NbLTP1/ΔSP-OFP), most of the expressed protein targeted chloroplasts. Both NbLTP1-OFP and NbLTP1/ΔSP-OFP were localized in chloroplasts when we removed the cell wall. These results suggest that NbLTP1 may have a secondary targeting signal. Transient overexpression of NbLTP1 had no effect on BaMV accumulation, but that of NbLTP1/ΔSP significantly increased BaMV expression. NbLTP1 may be a positive regulator of BaMV accumulation especially when its expression is associated with chloroplasts, where BaMV replicates. The mutation was introduced to the predicted phosphorylation site to simulate the phosphorylated status, NbLTP/ΔSP/P(+), which could still assist BaMV accumulation. By contrast, a mutant lacking calmodulin-binding or simulates the phosphorylation-negative status could not support BaMV accumulation. The lipid-binding activity of LTP1 was reported to be associated with calmodulin-binding and phosphorylation, by which the C-terminus functional domain of NbLTP1 may play a critical role in BaMV accumulation.

Genome-wide characterization and expression analysis of fatty acid desaturase gene family in Camelina sativa

Camelina is a flexible crop with several positive properties such as resistant to adverse environmental conditions, high potential for biodiesel production that make it distinctive among oilseed crops. Fatty acid desaturases (FADs) play a crucial role in regulating FA compositions, but there are no well characteristics and comprehensive genome-wide study of the FAD gene family in Camelina sativa. An exhaustive analysis was fulfilled to identify the FAD genes from Camelina sativa genome and described their phylogenetic relationship, the conserved motifs and histidine boxes, expression profile, co-expression analysis, physical and chemical properties, prediction of post-translation modifications and promoter analysis. In the present study, 24 desaturase genes were identified from Camelina sativa genome. The conserved histidine boxes were observed at deduced FAD proteins with various sequences, the four histidine boxes were figured out at omega desaturase genes, and it seems that the conserved histidine box, HHHGH, was detected for the first time. Also, three SAD genes were identified in camelina genome. Moreover, the result of the expression profile revealed that FAD genes have the tissue-specific expression pattern and diverse function in response to adverse conditions. In addition, 55 genes with high expression correlation were identified as co-expression genes with FAD genes in camelina. Besides, the studied FAD proteins varied based on predicted phosphorylation and N-glycosylation site. For instance, LOC104699734 protein showed the highest N-glycosylation site, while the LOC104765502, LOC104776214, LOC104734563, and LOC104745425 had the highest predicted phosphorylation sites. This study was the first report that widely analyzes the FAD gene family in the genome of Camelina sativa using available bioinformatics tools that will be used in future functional analyses related to molecular mechanisms of FAD genes in camelina.

Label-Free Quantitative Phosphoproteomics Reveals Signaling Dynamics Involved in Embryogenic Competence Acquisition in Sugarcane.

In this study, a label-free quantitative phosphoproteomic analysis was performed to identify and quantify signaling events related to the acquisition of embryogenic competence in sugarcane. Embryogenic and nonembryogenic calli were compared at the multiplication phase, resulting in the identification of 163 phosphoproteins unique to embryogenic calli, 9 unique to nonembryogenic calli, and 51 upregulated and 40 downregulated in embryogenic calli compared to nonembryogenic calli. Data are available via ProteomeXchange with identifier PXD018054. Motif-x analysis revealed the enrichment of [xxxpSPxxx], [RxxpSxxx], and [xxxpSDxxx] motifs, which are predicted phosphorylation sites for several kinases related to stress responses. The embryogenic-related phosphoproteins (those unique and upregulated in embryogenic calli) identified in the present study are related to abscisic acid-induced signaling and abiotic stress response; they include OSK3, ABF1, LEAs, and RD29Bs. On the other hand, the nonembryogenic-related phosphoproteins EDR1 and PP2Ac-2 are negative regulators of abscisic acid signaling, suggesting a relationship between phosphoproteins involved in the abscisic acid and stress responses in the acquisition of embryogenic competence. Moreover, embryogenic-related phosphoproteins associated with epigenetic modifications, such as HDA6, HDA19, and TOPLESS, and with RNA metabolism, including AGO1, DEAH5, SCL30, UB2C, and SR45, were identified to play potential roles in embryogenic competence. These results reveal novel phosphorylation sites for several proteins and identify potential candidate biomarkers for the acquisition of embryogenic competence in sugarcane.

Analysis of Adaptive Evolution and Coevolution of rbcL Gene in the Genus Hildenbrandia (Rhodophyta).

The adaptive evolution and coevolution of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene in the genus Hildenbrandia were studied based on phylogenetic tree construction and the physicochemical properties and the secondary structures of protein encoded by rbcL (Rubisco large subunit) were analyzed. The amino acids compositions and grand average of hydropathicity of freshwater H. rivularis and marine H. rubra were similar. Rubisco large subunit of Hildenbrandia was hydrophilic and the secondary structure was primarily composed of α-helixes and β-sheets, revealing the relatively stable structure of this protein. The predicted phosphorylation sites in H. rivularis and H. rubra were 33 and 36, respectively. No positive selection sites were detected in the genus Hildenbrandia, implying that rbcL gene evolved either neutrally or under purifying selection. A total of 41 coevolutionary groups were detected in the Rubisco large subunit of Hildenbrandia and the coevolving sites are in closer proximity in 3-dimensional structure of the protein. Despite the long evolutionary history, rbcL gene in genus Hildenbrandia under different environments is rather conservative.

Phospho-mutant activity assays provide evidence for alternative phospho-regulation pathways of the transcription factor FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR.

Summary The key basic helix–loop–helix (bHLH) transcription factor in iron (Fe) uptake, FER‐LIKE IRON DEFICIENCY‐INDUCED TRANSCRIPTION FACTOR (FIT), is controlled by multiple signaling pathways, important to adjust Fe acquisition to growth and environmental constraints. FIT protein exists in active and inactive protein pools, and phosphorylation of serine Ser272 in the C‐terminus, a regulatory domain of FIT, provides a trigger for FIT activation.Here, we use phospho‐mutant activity assays and study phospho‐mimicking and phospho‐dead mutations of three additional predicted phosphorylation sites, namely at Ser221 and at tyrosines Tyr238 and Tyr278, besides Ser 272.Phospho‐mutations at these sites affect FIT activities in yeast, plant, and mammalian cells. The diverse array of cellular phenotypes is seen at the level of cellular localization, nuclear mobility, homodimerization, and dimerization with the FIT‐activating partner bHLH039, promoter transactivation, and protein stability. Phospho‐mimicking Tyr mutations of FIT disturb fit mutant plant complementation.Taken together, we provide evidence that FIT is activated through Ser and deactivated through Tyr site phosphorylation. We therefore propose that FIT activity is regulated by alternative phosphorylation pathways.

The Evolution of Tau Phosphorylation and Interactions.

Tau is a neuronal microtubule-associated protein (MAP) that is involved in the regulation of axonal microtubule assembly. However, as a protein with intrinsically disordered regions (IDRs), tau also interacts with many other partners in addition to microtubules. Phosphorylation at selected sites modulates tau’s various intracellular interactions and regulates the properties of IDRs. In Alzheimer’s disease (AD) and other tauopathies, tau exhibits pathologically increased phosphorylation (hyperphosphorylation) at selected sites and aggregates into neurofibrillary tangles (NFTs). By bioinformatics means, we tested the hypothesis that the sequence of tau has changed during the vertebrate evolution in a way that novel interactions developed and also the phosphorylation pattern was affected, which made tau prone to the development of tauopathies. We report that distinct regions of tau show functional specialization in their molecular interactions. We found that tau’s amino-terminal region, which is involved in biological processes related to “membrane organization” and “regulation of apoptosis,” exhibited a strong evolutionary increase in protein disorder providing the basis for the development of novel interactions. We observed that the predicted phosphorylation sites have changed during evolution in a region-specific manner, and in some cases the overall number of phosphorylation sites increased owing to the formation of clusters of phosphorylatable residues. In contrast, disease-specific hyperphosphorylated sites remained highly conserved. The data indicate that novel, non-microtubule related tau interactions developed during evolution and suggest that the biological processes, which are mediated by these interactions, are of pathological relevance. Furthermore, the data indicate that predicted phosphorylation sites in some regions of tau, including a cluster of phosphorylatable residues in the alternatively spliced exon 2, have changed during evolution. In view of the “antagonistic pleiotropy hypothesis” it may be worth to take disease-associated phosphosites with low evolutionary conservation as relevant biomarkers into consideration.