Phosphorylated Amino Acid Research Articles

Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein-Protein Interactions.

The postsynaptic density protein of 95 kDa (PSD-95) is a key scaffolding protein that controls signaling at synapses in the brain through interactions of its PDZ domains with the C-termini of receptors, ion channels, and enzymes. PSD-95 is highly regulated by phosphorylation. To explore the effect of phosphorylation on PSD-95, we used semisynthetic strategies to introduce phosphorylated amino acids at four positions within the PDZ domains and examined the effects on interactions with a large set of binding partners. We observed complex effects on affinity. Most notably, phosphorylation at Y397 induced a significant increase in affinity for stargazin, as confirmed by NMR and single molecule FRET. Additionally, we compared the effects of phosphorylation to phosphomimetic mutations, which revealed that phosphomimetics are ineffective substitutes for tyrosine phosphorylation. Our strategy to generate site-specifically phosphorylated PDZ domains provides a detailed understanding of the role of phosphorylation in the regulation of PSD-95 interactions.

Chemical Approaches to Investigate Labile Peptide and Protein Phosphorylation.

Protein phosphorylation is by far the most abundant and most studied post-translational modification (PTM). For a long time, phosphate monoesters of serine (pSer), threonine (pThr), and tyrosine (pTyr) have been considered as the only relevant forms of phosphorylation in organisms. Recently, several research groups have dedicated their efforts to the investigation of other, less characterized phosphoamino acids as naturally occurring PTMs. Such apparent peculiar phosphorylations include the phosphoramidates of histidine (pHis), arginine (pArg), and lysine (pLys), the phosphorothioate of cysteine (pCys), and the anhydrides of pyrophosphorylated serine (ppSer) and threonine (ppThr). Almost all of these phosphorylated amino acids show higher lability under physiological conditions than those of phosphate monoesters. Furthermore, they are prone to hydrolysis under acidic and sometimes basic conditions as well as at elevated temperatures, which renders their synthetic accessibility and proteomic analysis particularly challenging. In this Account, we illustrate recent chemical approaches to probe the occurrence and function of these labile phosphorylation events. Within these endeavors, the synthesis of site-selectively phosphorylated peptides, in particular in combination with chemoselective phosphorylation strategies, was crucial. With these well-defined standards in hand, the appropriate proteomic mass spectrometry-based analysis protocols for the characterization of labile phosphosites in biological samples could be developed. Another successful approach in this research field includes the design and synthesis of stable analogues of these labile PTMs, which were used for the generation of pHis- and pArg-specific antibodies for the detection and enrichment of endogenous phosphorylated samples. Finally, other selective enrichment techniques are described, which rely for instance on the unique chemical environment of a pyrophosphate or the selective interaction between a phosphoamino acid and its phosphatase. It is worth noting that many of those studies are still in their early stages, which is also reflected in the small number of identified phosphosites compared to that of phosphate monoesters. Thus, many challenges need to be mastered to fully understand the biological role of these poorly characterized and rather uncommon phosphorylations. Taken together, this overview exemplifies recent efforts in a flourishing field of functional proteomic analysis and furthermore manifests the power of modern peptide synthesis to address unmet questions in the life sciences.

Bisdemethoxycurcumin sensitizes cisplatin-resistant lung cancer cells to chemotherapy by inhibition of CA916798 and PI3K/AKT signaling.

Curcumin, a dietary supplement or herbal medicine from Curcuma longa, has shown antitumor activity in different cancer cell lines and clinical trials. CA916798, a novel protein, is overexpressed in multidrug-resistant tumor cells. This study aimed to assess the effects of curcumin on regulating chemosensitivity in cisplatin-resistant non-small cell lung cancer (NSCLC) cells in vitro and to explore the underlying molecular mechanisms. Human cisplatin-sensitive A549 and cisplatin-resistant A549/CDDP lung adenocarcinoma cells were treated with curcumin to assess cell viability and gene modulations using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting. CA916798 shRNA and point mutations were used to assess the CA916798 functions and phosphorylation sites. Bisdemethoxycurcumin sensitized cisplatin-resistant lung cancer cells to various chemotherapeutic agents, including cisplatin. Bisdemethoxycurcumin reduced the levels of CA916798 mRNA and protein in A549 and A549/CDDP cells, while it also suppressed phosphatidylinositol-3-kinase (PI3K)/AKT signaling. CA916798, as a downstream gene, interacted with AKT after bisdemethoxycurcumin treatment in A549 and A549/CDDP cells. Moreover, A549/CDDP cells expressing the point-mutated CA916798-S20D protein were more resistant to cisplatin and bisdemethoxycurcumin, whereas tumor cells expressing CA916798-S20A, CA916798-S31A, CA916798-S60A, CA916798-S93A, or CA916798-T97A (different sites of amino acid phosphorylation) showed similar sensitivity or resistance to cisplatin and bisdemethoxycurcumin, compared with the control cells. Bisdemethoxycurcumin is able to sensitize cisplatin-resistant NSCLC cells to chemotherapeutic agents by inhibition of CA916798 and PI3K/AKT activities. Moreover, phosphorylation of CA916798 at the S20 residue plays a critical role in mediating bisdemethoxycurcumin antitumor activity.

Muscarinic receptor regulates extracellular signal regulated kinase by two modes of arrestin binding

Binding of agonists to G-protein-coupled receptors (GPCRs) activates heterotrimeric G proteins and downstream signaling. Agonist-bound GPCRs are then phosphorylated by protein kinases and bound by arrestin to trigger desensitization and endocytosis. Arrestin plays another important signaling function. It recruits and regulates activity of an extracellular signal-regulated kinase (ERK) cascade. However, molecular details and timing of ERK activation remain fundamental unanswered questions that limit understanding of how arrestin-dependent GPCR signaling controls cell functions. Here we validate and model a system that tracks the dynamics of interactions of arrestin with receptors and of ERK activation using optical reporters. Our intermolecular FRET measurements in living cells are consistent with β-arrestin binding to M1 muscarinic acetylcholine receptors (M1Rs) in two different binding modes, transient and stable. The stable mode persists for minutes after agonist removal. The choice of mode is governed by phosphorylation on key residues in the third intracellular loop of the receptor. We detect a similar intramolecular conformational change in arrestin in either binding mode. It develops within seconds of arrestin binding to the M1 receptor, and it reverses within seconds of arrestin unbinding from the transient binding mode. Furthermore, we observed that, when stably bound to phosphorylated M1R, β-arrestin scaffolds and activates MEK-dependent ERK. In contrast, when transiently bound, β-arrestin reduces ERK activity via recruitment of a protein phosphatase. All this ERK signaling develops at the plasma membrane. In this scaffolding hypothesis, a shifting balance between the two arrestin binding modes determines the degree of ERK activation at the membrane.

Synthesis and molecular docking study of novel alizarin derivatives containing phosphoryl amino acid moiety as potential antitumor agents

Series of novel alizarin and phosphoryl amino acid scaffold (4a–4d, 8a–8d) were synthesized and evaluated for the suppression of cancer cell proliferation in vitro against MGC-803, HepG2, T24, NCI-H460, and SK-OV-3 cell lines by standard 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay compared with commercial anticancer drug doxorubicin. Interestingly, all newly synthesized compounds exhibited relatively high cytotoxicity compared with alizarin and low cytotoxicity against human normal liver cell line HL-7702 cells. Especially, compound 8d showed the best cytotoxicity against SK-OV-3 cells with IC50 7.09 µM, which was slightly worse than that of drug doxorubicin. The cell apoptosis-inducing activity of representative compound 8d in SK-OV-3 cells revealed that the anticancer activity of this compound depended on apoptosis of cancer cells via regulation of Bcl-2 family members, activation of caspase-9 and caspase-3. Cell cycle analysis confirmed that compound 8d mainly arrested SK-OV-3 cells in G2 stage. In addition, molecular docking studies were performed to position compound 8d into the telomerase (5CQG) active site to determine the probable binding model.

Kinetic-based trapping by intervening sequence variants of the active sites of protein-disulfide isomerase identifies platelet protein substrates

Thiol isomerases such as protein-disulfide isomerase (PDI) direct disulfide rearrangements required for proper folding of nascent proteins synthesized in the endoplasmic reticulum. Identifying PDI substrates is challenging because PDI catalyzes conformational changes that cannot be easily monitored (e.g. compared with proteolytic cleavage or amino acid phosphorylation); PDI has multiple substrates; and it can catalyze either oxidation, reduction, or isomerization of substrates. Kinetic-based substrate trapping wherein the active site motif CGHC is modified to CGHA to stabilize a PDI-substrate intermediate is effective in identifying some substrates. A limitation of this approach, however, is that it captures only substrates that are reduced by PDI, whereas many substrates are oxidized by PDI. By manipulating the highly conserved -GH- residues in the CGHC active site of PDI, we created PDI variants with a slowed reaction rate toward substrates. The prolonged intermediate state allowed us to identify protein substrates that have biased affinities for either oxidation or reduction by PDI. Because extracellular PDI is critical for thrombus formation but its extracellular substrates are not known, we evaluated the ability of these bidirectional trapping PDI variants to trap proteins released from platelets and on the platelet surface. Trapped proteins were identified by mass spectroscopy. Of the trapped substrate proteins identified by mass spectroscopy, five proteins, cathepsin G, glutaredoxin-1, thioredoxin, GP1b, and fibrinogen, showed a bias for oxidation, whereas annexin V, heparanase, ERp57, kallekrein-14, serpin B6, tetranectin, and collagen VI showed a bias for reduction. These bidirectional trapping variants will enable more comprehensive identification of thiol isomerase substrates and better elucidation of their cellular functions.

Site-specific His/Asp phosphoproteomic analysis of prokaryotes reveals putative targets for drug resistance

BackgroundPhosphorylation of amino acid residues on proteins is an important and common post-translational modification in both eukaryotes and prokaryotes. Most research work has been focused on phosphorylation of serine, threonine or tyrosine residues, whereas phosphorylation of other amino acids are significantly less clear due to the controversy on their stability under standard bioanalytical conditions.ResultsHere we applied a shotgun strategy to analyze the histidine and aspartate phosphorylations in different microbes. Our results collectively indicate that histidine and aspartate phosphorylations frequently occur also in proteins that are not part of the two-component systems. Noticeably, a number of the modified proteins are pathogenesis-related or essential for survival in host. These include the zinc ion periplasmic transporter ZnuA in Acinetobacter baumannii SK17, the multidrug and toxic compound extrusion (MATE) channel YeeO in Klebsiella pneumoniae NTUH-K2044, branched amino acid transporter AzlC in Vibrio vulnificus and the RNA-modifying pseudouridine synthase in Helicobacter pylori.ConclusionsIn summary, histidine and aspartate phosphorylation is likely to be ubiquitous and to take place in proteins of various functions. This work also sheds light into how these functionally important proteins and potential drug targets might be regulated at a post-translational level.

Bioactive Peptides in Animal Food Products.

Proteins of animal origin represent physiologically active components in the human diet; they exert a direct action or constitute a substrate for enzymatic hydrolysis upon food processing and consumption. Bioactive peptides may descend from the hydrolysis by digestive enzymes, enzymes endogenous to raw food materials, and enzymes from microorganisms added during food processing. Milk proteins have different polymorphisms for each dairy species that influence the amount and the biochemical characteristics (e.g., amino acid chain, phosphorylation, and glycosylation) of the protein. Milk from other species alternative to cow has been exploited for their role in children with cow milk allergy and in some infant pathologies, such as epilepsy, by monitoring the immune status. Different mechanisms concur for bioactive peptides generation from meat and meat products, and their functionality and application as functional ingredients have proven effects on consumer health. Animal food proteins are currently the main source of a range of biologically-active peptides which have gained special interest because they may also influence numerous physiological responses in the organism. The addition of probiotics to animal food products represent a strategy for the increase of molecules with health and functional properties.

Riboflavin Responsive Mitochondrial Dysfunction in Neurodegenerative Diseases

Mitochondria are the repository for various metabolites involved in diverse energy-generating processes, like the TCA cycle, oxidative phosphorylation, and metabolism of amino acids, fatty acids, and nucleotides, which rely significantly on flavoenzymes, such as oxidases, reductases, and dehydrogenases. Flavoenzymes are functionally dependent on biologically active flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN), which are derived from the dietary component riboflavin, a water soluble vitamin. Riboflavin regulates the structure and function of flavoenzymes through its cofactors FMN and FAD and, thus, protects the cells from oxidative stress and apoptosis. Hence, it is not surprising that any disturbance in riboflavin metabolism and absorption of this vitamin may have consequences on cellular FAD and FMN levels, resulting in mitochondrial dysfunction by reduced energy levels, leading to riboflavin associated disorders, like cataracts, neurodegenerative and cardiovascular diseases, etc. Furthermore, mutations in either nuclear or mitochondrial DNA encoding for flavoenzymes and flavin transporters significantly contribute to the development of various neurological disorders. Moreover, recent studies have evidenced that riboflavin supplementation remarkably improved the clinical symptoms, as well as the biochemical abnormalities, in patients with neuronopathies, like Brown-Vialetto-Van-Laere syndrome (BVVLS) and Fazio-Londe disease. This review presents an updated outlook on the cellular and molecular mechanisms of neurodegenerative disorders in which riboflavin deficiency leads to dysfunction in mitochondrial energy metabolism, and also highlights the significance of riboflavin supplementation in aforementioned disease conditions. Thus, the outcome of this critical assessment may exemplify a new avenue to enhance the understanding of possible mechanisms in the progression of neurodegenerative diseases and may provide new rational approaches of disease surveillance and treatment.

Origin of Life and the Phosphate Transfer Catalyst.

In this paper, we revisit several issues relevant to origin-of-life research and propose a Phosphate Transfer Catalyst hypothesis that furthers our understanding of some of the key events in prebiotic chemical evolution. In the Phosphate Transfer Catalyst hypothesis, we assume the existence of hypothetical metallopeptides with phosphate transfer activity that use abundant polyphosphates as both substrates and energy sources. Nonspecific catalysis by this phosphate transfer catalyst would provide a variety of different products such as phosphoryl amino acids, nucleosides, polyphosphate nucleotides, nucleic acids, and aminoacylated nucleic acids. Moreover, being an autocatalytic set and metabolic driver at the same time, it could possibly replicate itself and produce a collective system of two polymerases; a nucleic acid able to catalyze peptide bond formation and a peptide able to polymerize nucleic acids. The genetic code starts at first as a system that reduces the energy barrier by bringing substrates (2'/3' aminoacyl-nucleotides) together, an ancestral form of the catalysis performed by modern ribosomes. Key Words: Origin of life-Prebiotic chemistry-Catalysis-Nucleic acids. Astrobiology 17, 277-285.

Anti‑lung cancer effect of glucosamine by suppressing the phosphorylation of FOXO.

Lung cancer is the most common cause of cancer‑associated mortality worldwide, and glucosamine has the potential to exhibit antitumor activity. To reveal its anti‑lung cancer mechanism, the present study investigated the effect of glucosamine on the transcriptional activity of forkhead box O (FOXO)1 and FOXO3, and associated signal transduction pathways in A549 cells. An MTT assay was performed to investigate cell viability and immunoblotting was performed to detect protein levels of FOXO1/3, phosphorylated (p)‑FOXO1/3, AKT, p‑AKT, extracellular signal‑regulated kinase (ERK) and p‑ERK, and the levels of β‑O‑linked N‑acetylglucosamine (O‑GlcNAc)‑modified FOXO1 protein. Immunoprecipitation was performed to purify O‑GlcNAc‑modified protein prior to immunoblotting. Glucosamine inhibited FOXO1‑ and FOXO3‑specific amino acid phosphorylation, which was correlated with its translocation from the nucleus to cytoplasm, indicating a possible anti‑lung cancer mechanism of glucosamine. The present study also examined the phosphoinositide 3‑kinase (PI3K)/AKT and mitogen‑activated protein kinase (MAPK)/ERK pathways, which induce FOXO1‑ and FOXO3‑specific site phosphorylation. The data showed that glucosamine suppressed the translocation of FOXO from the cytoplasm to the nucleus via glucosamine‑induced O‑GlcNAc modification. These observations suggested that glucosamine modulated A549 cell proliferation, possibly via O‑GlcNAc modification‑induced downregulation of the PI3K/AKT and MAPK/ERK pathways and their downstream signaling molecules, FOXO1 and FOXO3.

Chemical Approaches to Studying Labile Amino Acid Phosphorylation.

Phosphorylation of serine, threonine, and tyrosine residues is the archetypal posttranslational modification of proteins. While phosphorylation of these residues has become standard textbook knowledge, phosphorylation of other amino acid side chains is underappreciated and minimally characterized by comparison. This disparity is rooted in the relative instability of these chemically distinct amino acid side chain moieties, namely phosphoramidates, acyl phosphates, thiophosphates, and phosphoanhydrides. In the case of the O-phosphorylated amino acids, synthetic constructs were critical to assessing their stability and developing tools for their study. As the chemical biology community has become more aware of these alternative phosphorylation sites, methodology has been developed for the synthesis of well-characterized standards and close mimics of these phosphorylated amino acids as well. In this article, we review the synthetic chemistry that is a prerequisite to progress in this field.

Dynamic Lipid-dependent Modulation of Protein Topology by Post-translational Phosphorylation

Membrane protein topology and folding are governed by structural principles and topogenic signals that are recognized and decoded by the protein insertion and translocation machineries at the time of initial membrane insertion and folding. We previously demonstrated that the lipid environment is also a determinant of initial protein topology, which is dynamically responsive to post-assembly changes in membrane lipid composition. However, the effect on protein topology of post-assembly phosphorylation of amino acids localized within initially cytoplasmically oriented extramembrane domains has never been investigated. Here, we show in a controlled in vitro system that phosphorylation of a membrane protein can trigger a change in topological arrangement. The rate of change occurred on a scale of seconds, comparable with the rates observed upon changes in the protein lipid environment. The rate and extent of topological rearrangement were dependent on the charges of extramembrane domains and the lipid bilayer surface. Using model membranes mimicking the lipid compositions of eukaryotic organelles, we determined that anionic lipids, cholesterol, sphingomyelin, and membrane fluidity play critical roles in these processes. Our results demonstrate how post-translational modifications may influence membrane protein topology in a lipid-dependent manner, both along the organelle trafficking pathway and at their final destination. The results provide further evidence that membrane protein topology is dynamic, integrating for the first time the effect of changes in lipid composition and regulators of cellular processes. The discovery of a new topology regulatory mechanism opens additional avenues for understanding unexplored structure-function relationships and the development of optimized topology prediction tools.

Study of Peroxisomal Protein Phosphorylation by Functional Proteomics.

Reversible protein phosphorylation is a frequently occurring posttranslational modification mediated by protein kinases and phosphatases that plays an essential role in the regulation of a large number of cellular processes. Evidence is accumulating that protein phosphorylation is also an important mechanism governing processes associated with peroxisome biology. For an improved and detailed understanding of these processes and their regulation it is therefore crucial to study phosphorylation of peroxisome-associated proteins and to determine the phosphorylated amino acid(s). To place peroxisome-related processes into a larger, cellular context, it is further required to identify the kinases and phosphatases catalyzing phosphorylation and dephosphorylation events in peroxisomal proteins. We here provide a strategy for the targeted analysis of peroxisomal phosphoproteins of Saccharomyces cerevisiae combining affinity purification of epitope-tagged peroxisomal proteins with Phos-tag SDS-PAGE and high-resolution mass spectrometry (MS) for the identification and precise localization of in vivo phosphosites. Furthermore, we describe a protocol for an MS-based in vitro kinase assay using recombinant peroxisomal proteins and a selected kinase facilitating the site-resolved analysis of kinase-substrate relationships.

High-Throughput Analysis of Mammalian Receptor Tyrosine Kinase Activation in Yeast Cells.

Tyrosine phosphorylation is an essential posttranslational modification in intracellular signaling molecules. Since tyrosine phosphorylation occurs in less than 0.1 % of all phosphorylated amino acids in mammalian cells, it is difficult to detect the nascent phosphotyrosine at a high signal-to-noise ratio due to high intracellular backgrounds (i.e., unexpected crosstalks among endogenous signaling molecules). In order to address this issue, we reconstituted the mammalian signaling pathway involving an extracellular ligand and a receptor tyrosine kinase (RTK) in Saccharomyces cerevisiae, a lower eukaryote that lacks endogenous tyrosine kinases. In this chapter, we describe a method for high-throughput analysis of ligand-receptor interaction by combining the yeast cell-surface display technique with an automated single-cell analysis and isolation system. Yeast cells coexpressing the cell-wall-anchored form of the human epidermal growth factor (EGF) and the human EGF receptor (EGFR) fused with a signal peptide at the N terminus facilitated the interaction of EGF with EGFR in an autocrine manner, followed by EGFR oligomerization and subsequent autophosphorylation. Furthermore, yeast cells expressing cell-wall-anchored forms of a conformationally constrained random peptide library instead of EGF are treated with a fluorophore-labeled anti-phosphorylated EGFR antibody and then subjected to the automated single-cell analysis and isolation system. The yeast cells with the highest level of fluorescence were shown to display novel and efficient EGFR agonistic peptides. Thus, our yeast display technique serves as a quantitative measurement for RTK activation, which is applicable to high-throughput de novo screening of RTK agonistic peptides.

RNA-seq-based digital gene expression analysis reveals modification of host defense responses by rice stripe virus during disease symptom development in Arabidopsis.

BackgroundVirus infection induces and suppresses host gene expression on a global level. Rice stripe virus (RSV) is the type species of the genus Tenuivirus and infects rice and Arabidopsis plants. Microarray-based and next generation sequencing-based transcriptomic approaches have been used to study rice-RSV interactions. However, our knowledge of the response of Arabidopsis plants to RSV infection is limited, and it requires further investigation to determine the similarities (or differences) in virus-host interactions between monocot and dicot hosts infected with RSV.MethodsWe characterized transcriptome changes in Arabidopsis thaliana infected with rice stripe virus (RSV) with RNA-seq based digital gene expression (DGE) analysis. The transcriptomes of RSV-infected samples were compared to those of mock-treated samples at 14 and 21 days post-infection (dpi) during different stages of symptom development.ResultsWe identified 624 differentially expressed genes (DEGs) in Arabidopsis influenced by RSV at 14 dpi and 21 dpi, among which at 14 dpi, 255 transcripts were induced, and 38 were repressed; at 21 dpi, 146 were induced, and 237 were repressed. Functional annotation indicated that these DEGs were related to multiple biological functions, including defense response, secondary metabolism, protein amino acid phosphorylation and response to abiotic stress.ConclusionsImportantly, the transcription of genes related to host defense systems was activated by RSV infection at an early stage of symptom development (14 dpi), whereas over the infection period (21 dpi), the host defense response systems were suppressed. A total of 52 genes were continuously differentially expressed between the two time points, indicating that the majority of DEGs were transient and unique to a particular time point during symptom development. The DEGs, particularly the defense response genes, identified in this study are candidates suitable for further functional analysis during the RSV-Arabidopsis interaction.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0663-7) contains supplementary material, which is available to authorized users.

Strong correlation between ASPM gene expression and HCV cirrhosis progression identified by co-expression analysis

Hepatitis C virus (HCV) cirrhosis is at a high risk of hepatocellular carcinoma (HCC), and its progression is influenced by a complex network of gene interactions. A weighted gene co-expression network was constructed to identify gene modules associated with the seven-stage disease progression from HCV cirrhosis to HCV-related HCC (n=65). In the significant module (R2=0.86), a total of 25 network hub genes were identified, half of which were also hub nodes in the protein-protein interaction network of the module genes. In validation, most hub genes showed a moderate correlation with the disease progression, and only ASPM was highly correlated (R2=0.801). In the test set (n=63), ASPM was also more highly expressed in HCV cirrhosis with concomitant HCC than in those without HCC (P=0.0054). Gene set enrichment analysis (GSEA) demonstrated that the gene set of “regulation of protein amino acid phosphorylation” (n=20) was enriched in HCV cirrhosis samples with ASPM highly expressed (false discovery rate (FDR)=0.049). In gene ontology (GO) analysis, genes in the enriched set were associated with liver neoplasms and other neoplastic diseases. In conclusion, through co-expression analysis, ASPM was identified and validated in association with the progression of HCV cirrhosis probably by regulating tumor-related phosphorylation.

In Silico Methods for Analyzing Mutagenesis Targets.

Molecular dynamics of complex biological and chemical systems is possible using personal computers due to increased computer performance and improved software design. Here we describe molecular dynamics methods using Not Another Molecular Dynamics (NAMD) and Visual Molecular Dynamics (VMD) programs that aid in understanding the structural effects a mutation has on a protein. We describe in silico methods for site-specific mutation to standard and phosphorylated amino acids. Molecular dynamics equilibrations are used to provide a means for measuring structural fluctuations. These fluctuations assist in defining a distance coordinate, or reaction coordinate, that is relevant to the function of the protein. Adaptive biasing force molecular dynamics are then demonstrated to evaluate the energy landscape, or potential of mean force, along the chosen reaction coordinate. The potential of mean force identifies variations of the predominant structures among mutants that may affect function.

RNA-seq analysis of Brachypodium distachyon responses to Barley stripe mosaic virus infection

Barley stripe mosaic virus (BSMV) is the type member of the genus Hordeivirus. Brachypodium distachyon line Bd3-1 shows resistance to the BSMV ND18 strain, but is susceptible to an ND18 double mutant (β NDTGB1R390K, T392K) in which lysine is substituted for an arginine at position 390 and for threonine at position 392 of the triple gene block 1 (TGB1) protein. In order to understand differences in gene expression following infection with ND18 and double mutant ND18, Bd3-1 seedlings were subjected to RNA-seq analyses at 1, 6, and 14days post inoculation (dpi). The results revealed that basal immunity genes involved in cellulose synthesis and pathogenesis-related protein biosynthesis were enhanced in incompatible interactions between Bd3-1 and ND18. Most of the differentially expressed transcripts are related to trehalose biosynthesis, ethylene, jasmonic acid metabolism, protein phosphorylation, protein ubiquitination, transcriptional regulation, and transport process, as well as pathogenesis-related protein biosynthesis. In compatible interactions between Bd3-1 and ND18 mutant, Bd3-1 developed weak basal resistance responses to the virus. Many genes involved in cellulose biosynthesis, protein amino acid phosphorylation, protein biosynthesis, protein glycosylation, glycolysis and cellular macromolecular complex assembly that may be related to virus replication, assembly and movement were up-regulated. Some genes involved in oxidative stress responses were also up-regulated at 14dpi. BSMV ND18 mutant infection suppressed expression of genes functioning in regulation of transcription, protein kinase, cellular nitrogen compound biosynthetic process and photosynthesis. Differential expression patterns between compatible and incompatible interactions in Bd3-1 to the two BSMV strains provide important clues for understanding mechanism of resistance to BMSV in the model plant Brachypodium.

Positive regulation of the Candida albicans multidrug efflux pump Cdr1p function by phosphorylation of its N-terminal extension.

Overexpression of ATP-binding cassette (ABC) transporters is a frequent cause of multidrug resistance in cancer cells and pathogenic microorganisms. One example is the Cdr1p transporter from the human fungal pathogen Candida albicans that belongs to the pleiotropic drug resistance (PDR) subfamily of ABC transporters found in fungi and plants. Cdr1p is overexpressed in several azole-resistant clinical isolates, causing azole efflux and treatment failure. Cdr1p appears as a doublet band in western blot analyses, suggesting that the protein is post-translationally modified. We investigated whether Cdr1p is phosphorylated and the function of this modification. Phosphorylated residues were identified by MS. Their function was investigated by site-directed mutagenesis and expression of the mutants in a C. albicans endogenous system that exploits a hyperactive allele of the Tac1p transcription factor to drive high levels of Cdr1p expression. Fluconazole resistance was measured by microtitre plate and spot assays and transport activity by Nile red accumulation. We identified a cluster of seven phosphorylated amino acids in the N-terminal extension (NTE) of Cdr1p. Mutating all seven sites to alanine dramatically diminished the ability of Cdr1p to confer fluconazole resistance and transport Nile red, without affecting Cdr1p localization. Conversely, a Cdr1p mutant in which the seven amino acids were replaced by glutamate was able to confer high levels of fluconazole resistance and to export Nile red. Our results demonstrate that the NTE of Cdr1p is phosphorylated and that NTE phosphorylation plays a major role in regulating Cdr1p and possibly other PDR transporter function.