Substitution Of Lysine Research Articles

The role of MHC class II polymorphisms for DM-mediated peptide editing and autoimmunity

Abstract Polymorphisms in MHC class II (MHCII) genes are the strongest risk factors for the development of autoimmune diseases, such as type I diabetes (T1D). T1D is associated with allelic variants of the MHCII molecule HLA-DQ. In the nonobese diabetic (NOD) mouse, the orthologous molecule, I-Ag7, also predisposes mice to spontaneous diabetes development. To investigate the functional impact of natural MHCII polymorphisms on DM binding and T1D we introduced selected mutations in the genome of the NOD mouse by CRISPR/Cas9 genomic editing. Our approach was to first identify unique polymorphisms in the putative interface of I-Ag7, and then assess the impact of these polymorphisms on DM-mediated peptide editing in vitro. Several polymorphisms increased the interaction between I-Ag7 and DM and augmented peptide binding. The substitution of lysine (K) 40 in I-Aα with glutamic acid (E) had the strongest effect on DM editing and accelerated peptide binding more than 9-fold. Lysine at position α40 is unique to the diabetes associated I-Ag7 molecule and was therefore selected for in vivo evaluation. Homozygous and heterozygous K40E mutant mice showed increased H2-DM protein expression compared to WT littermate controls. Increased H2-DM levels were accompanied by an increase in intra- and extracellular I-Ag7 expression. This suggests that the K40E mutation promotes the interaction between H2-DM and I-Ag7, and likely, represents increased intracellular dimerization and more stable I-Ag7/peptide complexes on the cell surface. Currently, we are evaluating whether increased editing of the MHCII peptide repertoire mediated by the K40E mutation influences spontaneous diabetes development.

Keap1/Cullin3 Modulates p62/SQSTM1 Activity via UBA Domain Ubiquitination

p62/SQSTM1 (p62) is a scaffolding protein that facilitates the formation and degradation of ubiquitinated aggregates via its self-interaction and ubiquitin binding domains. The regulation of this process is unclear but may relate to the post-translational modification of p62. In the present study, we find that Keap1/Cullin3 ubiquitinates p62 at lysine 420 within its UBA domain. Substitution of lysine 420 with an arginine diminishes p62 sequestration and degradation activity similar what is seen when the UBA domain is deleted. Overexpression of Keap1/Cullin3 in p62-WT-expressing cells increases ubiquitinated inclusion formation and p62's association with LC3 and rescues proteotoxicity. This effect is not seen in cells expressing a mutant p62 that fails to interact with Keap1. Interestingly, p62 disease mutants have diminished or absent UBA domain ubiquitination. These data suggest that the ubiquitination of p62's UBA domain at lysine 420 may regulate p62's function and be disrupted in p62-associated disease.

Mechanism of plasmin generation by S100A10.

Plasminogen (Pg) is cleaved to form plasmin by the action of specific plasminogen activators such as the tissue plasminogen activator (tPA). Although the interaction of tPA and Pg with the surface of the fibrin clot has been well characterised, their interaction with cell surface Pg receptors is poorly understood. S100A10 is a cell surface Pg receptor that plays a key role in cellular plasmin generation. In the present report, we have utilised domain-switched/deleted variants of tPA, truncated plasminogen variants and S100A10 site-directed mutant proteins to define the regions responsible for S100A10-dependent plasmin generation. In contrast to the established role of the finger domain of tPA in fibrin-stimulated plasmin generation, we show that the kringle-2 domain of tPA plays a key role in S100A10-dependent plasmin generation. The kringle-1 domain of plasminogen, indispensable for fibrin-binding, is also critical for S100A10-dependent plasmin generation. S100A10 retains activity after substitution or deletion of the carboxyl-terminal lysine suggesting that internal lysine residues contribute to its plasmin generating activity. These studies define a new paradigm for plasminogen activation by the plasminogen receptor, S100A10.

PP14. NETWORK ANALYSIS REVEALS INSIGHTS INTO H3F3A BIOLOGY IN PEDIATRIC GLIOBLASTOMA

AbstractBACKGROUND: H3 histone, family 3A (H3F3A) encodes H3.3 histone protein monomers, which plays a central role in chromatin structure and gene regulation. Recent studies have reported highly recurrent mutations affecting the N-terminal tail of H3.3 and causing amino acid substitution of lysine 27 to methionine (K27M) and of glycine 34 to arginine or valine (G34A or G34V) in pediatric glioblastoma (GBM), which in turns linked to two subgroups with distinct clinical and pathological features. However, effect of H3F3A mutations on other closely related genes is not yet fully explored. Thus, we performed a network analysis to gain insights into the biology of H3F3A in pediatric GBM. METHODS: We constructed H3F3A network by extracting gene products that directly interact with H3F3A from protein-protein interaction (PPI) databases. To examine the network wiring around H3F3A, we combined pediatric GBM gene expression data from three independent studies and computed the correlation between H3F3A and all interacting genes for each mutation status (K27M or G34) and H3F3A wild type (WT). We then identified network of differentially co-expressed gene pairs that either gained or lost correlation in mutation relative to the WT tumours. RESULTS: Through mining of PPI networks, 106 gene products were identified to interact with H3F3A. Many of these genes were known to be involved in chromatin modification (47 genes; FDR = 2.17E-38), DNA metabolic process (34 genes; FDR = 3.23E-16), DNA repair (22 genes; FDR = 8.18E-12), and histone methylation (13 genes; FDR = 2.75E-11). H3F3A network was largely perturbed by K27M mutation than G34 (44 interactions changed in K27M and 24 in G34 when compared with WT, FDR < 0.05). Of these interactions, only four were perturbed by both mutations: H3F3A - ASB9, H3F3A - CBX3, H3F3A - HIST2H2BE, and H3F3A - PDGFRA, suggesting distinct mechanisms by which H3F3A mutations might be inducing transcriptional programming in pediatric GBM. A relatively small number of H3F3A interactions were identified between IDH1 mutation and WT tumours from adult GBM (7 interactions from K27M and 1 from G34) and in pediatric medulloblastomas (2 interactions from K27M and 1 from G34), demonstrating the robustness and specificity of identified interactions in pediatric GBM. CONCLUSION: In conclusion, the network analysis represents a new approach for the prioritization of H3F3A interacting partners in pediatric GBM, providing new insights into network dependencies in pediatric GBM subgroups and potentially enabling the identification of new diagnostic and therapeutic targets.

Rapid capillary mixing experiments for the analysis of hydrophobic membrane complexes directly from aqueous lipid bilayer solutions.

In this study the gas-phase conformer preferences of Gramicidin A (GA), a linear antimicrobial pentadecapeptide, were investigated directly from aqueous solutions of lipid vesicle bilayers using a mixing tee-electrospray ionization (MT-ESI) setup coupled with ion mobility mass spectrometry (IM-MS). The required time for GA sample preparation was decreased by approximately 50% using MT-ESI when compared to previously reported methods which required freeze-drying of samples. Using an MT-ESI approach to analyze samples of GA associated with POPC (16:0, 18:1 PC) and DEPC (22:1 PC) lipid bilayers yielded dimer conformer preferences comparable to results obtained using more lengthy protocols. GA analogues that contain leucine to lysine substitutions were analyzed; these analogues yielded more hydrophilic GA dimers owing to the hydrophilicity of lysine head groups. The conformer preferences of lipid bilayer associated hydrophilic GA analogues can be obtained owing to disassociation of lipids during the fast mixing time MT-ESI process. The data for both GA analogues associated with negatively charged POPC/POPG (16:0, 18:1 PC/PG) lipid bilayers reveal a preference for antiparallel double helix (ADH) formation. The adoption of nascent conformers for both GA analogues was observed using MT-ESI for samples associated with DMPC/DMPG (12:0 PC/PG) bilayers.

Methylarginines within the RGG-Motif Region of hnRNP A1 Affect Its IRES Trans-Acting Factor Activity and Are Required for hnRNP A1 Stress Granule Localization and Formation

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a stress granule-associated RNA-binding protein that plays a role in apoptosis and cellular stress recovery. HnRNP A1 is a major non-histone target of protein arginine methyltransferase 1, which asymmetrically dimethylates hnRNP A1 at several key arginine residues within its arginine–glycine–glycine (RGG)-motif region. Although arginine methylation is known to regulate general RNA binding of hnRNP A1 in vitro, the functional role of arginine methylation in hnRNP A1 cytoplasmic activity is unknown. To test the impact of key methylarginine residues on hnRNP A1 cytoplasmic activity and stress granule association, cytoplasmically restricted Flag-tagged mutants of hnRNP A1 were generated in which key methylarginine residues within the RGG-motif region were changed to either lysine or alanine. Lysine substitution, which mimics unmethylated arginine, resulted in a 40% increase in internal ribosome entry site trans-acting factor (ITAF) activity and the protein readily associates with stress granules. Alanine substitution resulted in a loss of ITAF activity and reduced mRNA binding. The alanine mutant also displays reduced stress granule association and suppresses stress granule formation. Our data suggest that arginine residues within the RGG-motif region are critical for hnRNP A1 cytoplasmic activities and that endogenous asymmetric dimethylation of the RGG-motif region suppresses hnRNP A1 ITAF activity in cells. Our findings indicate that methylarginine residues within the RGG-motif region of hnRNP A1 are important for its cytoplasmic activities and that hypomethylation and/or mutation of the RGG-motif region may contribute to the role of hnRNP A1 in diseases such as cancer.

Host-derived extracellular RNA promotes adhesion of Streptococcus pneumoniae to endothelial and epithelial cells.

Streptococcus pneumoniae is the most frequent cause of community-acquired pneumonia. The infection process involves bacterial cell surface receptors, which interact with host extracellular matrix components to facilitate colonization and dissemination of bacteria. Here, we investigated the role of host-derived extracellular RNA (eRNA) in the process of pneumococcal alveolar epithelial cell infection. Our study demonstrates that eRNA dose-dependently increased S. pneumoniae invasion of alveolar epithelial cells. Extracellular enolase (Eno), a plasminogen (Plg) receptor, was identified as a novel eRNA-binding protein on S. pneumoniae surface, and six Eno eRNA-binding sites including a C-terminal 15 amino acid motif containing lysine residue 434 were characterized. Although the substitution of lysine 434 for glycine (K434G) markedly diminished the binding of eRNA to Eno, the adherence to and internalization into alveolar epithelial cells of S. pneumoniae strain carrying the C-terminal lysine deletion and the mutation of internal Plg-binding motif were only marginally impaired. Accordingly, using a mass spectrometric approach, we identified seven novel eRNA-binding proteins in pneumococcal cell wall. Given the high number of eRNA-interacting proteins on pneumococci, treatment with RNase1 completely inhibited eRNA-mediated pneumococcal alveolar epithelial cell infection. Our data support further efforts to employ RNAse1 as an antimicrobial agent to combat pneumococcal infectious diseases.

Effects of lactoferrin derived peptides on simulants of biological warfare agents

Lactoferrin (LF) is an important immune protein in neutrophils and secretory fluids of mammals. Bovine LF (bLF) harbours two antimicrobial stretches, lactoferricin and lactoferampin, situated in close proximity in the N1 domain. To mimic these antimicrobial domain parts a chimeric peptide (LFchimera) has been constructed comprising parts of both stretches (LFcin17–30 and LFampin265–284). To investigate the potency of this construct to combat a set of Gram positive and Gram negative bacteria which are regarded as simulants for biological warfare agents, the effect on bacterial killing, membrane permeability and membrane polarity were determined in comparison to the constituent peptides and the native bLF. Furthermore we aimed to increase the antimicrobial potency of the bLF derived peptides by cationic amino acid substitutions. Overall, the bactericidal activity of the peptides could be related to membrane disturbing effects, i.e. membrane permeabilization and depolarization. Those effects were most prominent for the LFchimera. Arginine residues were found to be crucial for displaying antimicrobial activity, as lysine to arginine substitutions resulted in an increased antimicrobial activity, affecting mostly LFampin265–284 whereas arginine to lysine substitutions resulted in a decreased bactericidal activity, predominantly in case of LFcin17–30.

Role of E542 and E545 missense mutations of PIK3CA in breast cancer: a comparative computational approach

Recent statistics describe breast cancer as the leading cause of death among women across the world with varied causes and reasons. Lifestyle, diet, genetic and environmental factors introduce their generous contributions towards breast cancer, among which genetic factors have lately become one of the most important aspects in understanding the mechanism. Although various genes have already been reported in causing breast cancer, PIK3CA stands second on the list. Mutations observed in this gene have the ability to trigger the different activities of the cell, thereby bypassing the regular cellular cycle. Among the mutations in PIK3CA, three hotspot mutations were commonly reported, one in the catalytic domain (position HIS1047) and other two in the helical domain (position GLU542 and GLU545). In the helical domain of PIK3CA, the lysine substitution at 542–545 positions was significantly studied in causing breast cancer. To compare the deleterious effect of these mutations, in silico prediction tools along with molecular dynamics simulations and molecular docking approach was initiated to analyse the change in binding landscape upon mutation. In this comparative analysis, we report that the mere existence of mutant E545K can trigger the function of the protein but may not be as harmful as H1047R. Among the two mutations E542K and E545K, the latter shows the most deleterious effect that correlates with the previous reported experimental studies. We assume the results observed in this combinatorial computational study might further pave a better way for providing better treatment procedures.

Functional analysis of iodotyrosine deiodinase from drosophila melanogaster.

The flavoprotein iodotyrosine deiodinase (IYD) was first discovered in mammals through its ability to salvage iodide from mono- and diiodotyrosine, the by-products of thyroid hormone synthesis. Genomic information indicates that invertebrates contain homologous enzymes although their iodide requirements are unknown. The catalytic domain of IYD from Drosophila melanogaster has now been cloned, expressed and characterized to determine the scope of its potential catalytic function as a model for organisms that are not associated with thyroid hormone production. Little discrimination between iodo-, bromo-, and chlorotyrosine was detected. Their affinity for IYD ranges from 0.46 to 0.62 μM (Kd ) and their efficiency of dehalogenation ranges from 2.4 - 9 x 103 M-1 s-1 (kcat /Km ). These values fall within the variations described for IYDs from other organisms for which a physiological function has been confirmed. The relative contribution of three active site residues that coordinate to the amino acid substrates was subsequently determined by mutagenesis of IYD from Drosophila to refine future annotations of genomic and meta-genomic data for dehalogenation of halotyrosines. Substitution of the active site glutamate to glutamine was most detrimental to catalysis. Alternative substitution of an active site lysine to glutamine affected substrate affinity to the greatest extent but only moderately affected catalytic turnover. Substitution of phenylalanine for an active site tyrosine was least perturbing for binding and catalysis.

Acetylation of androgen receptor by ARD1 promotes dissociation from HSP90 complex and prostate tumorigenesis.

Prostate cancer is an androgen receptor (AR)-driven disease and post-translational modification of AR is critical for AR activation. We previously reported that Arrest-defective protein 1 (ARD1) is an oncoprotein in prostate cancer. It acetylates and activates AR to promote prostate tumorigenesis. However, the ARD1-targeted residue within AR and the mechanisms of the acetylation event in prostate tumorigenesis remained unknown. In this study, we show that ARD1 acetylates AR at lysine 618 (K618) in vitro and in vivo. An AR construct with the charged lysine substitution by arginine (AR-618R) reduces RNA Pol II binding, AR transcriptional activity, prostate cancer cell growth, and xenograft tumor formation due to attenuation of AR nuclear translocation, whereas, construct mimicking neutral polar substitution acetylation at K618 by glutamine (AR-618Q) enhanced these effects beyond that of the wild-type AR. Mechanistically, ARD1 forms a ternary complex with AR and HSP90 in vitro and in vivo. Expression of ARD1 increases levels of AR acetylation and AR-HSP90 dissociation in a dose dependent manner. Moreover, the AR acetylation defective K618R mutant is unable to dissociate from HSP90 while the HSP90-dissociated AR is acetylated following ligand exposure. This work identifies a new mechanism for ligand-induced AR-HSP90 dissociation and AR activation. Targeting ARD1-mediated AR acetylation may be a potent intervention for AR-dependent prostate cancer therapy.

Initial response of renal cell carcinoma to vemurafenib in a patient treated for metastatic melanoma.

Vemurafenib is a selective inhibitor of overactive BRAF oncogene with a substitution of lysine for glutamic acid at residue 600 (BRAFV600E), a mutation expressed in approximately 50% of all melanomas. We report a case of a patient with metastatic melanoma treated with vemurafenib, who subsequently presented with a biopsy-proven conventional renal cell carcinoma (RCC). We observed an initial complete regression of the mass while on vemurafenib. This was unexpected, given that vemurafenib is a specific inhibitor of BRAFV600E and most RCCs do not harbour this mutation.

SMYD3-mediated lysine methylation in the PH domain is critical for activation of AKT1.

AKT1 is a cytosolic serine/threonine kinase that is overexpressed in various types of cancer and has a central role in human tumorigenesis. Although it is known that AKT1 is post-translationally modified in various ways including phosphorylation and ubiquitination, methylation has not been reported so far. Here we demonstrate that the protein lysine methyltransferase SMYD3 methylates lysine 14 in the PH domain of AKT1 both in vitro and in vivo. Lysine 14-substituted AKT1 shows significantly lower levels of phosphorylation at threonine 308 than wild-type AKT1, and knockdown of SMYD3 as well as treatment with a SMYD3 inhibitor significantly attenuates this phosphorylation in cancer cells. Furthermore, substitution of lysine 14 diminishes the plasma membrane accumulation of AKT1, and cancer cells overexpressing lysine 14-substiuted AKT1 shows lower growth rate than those overexpressing wild-type AKT1. These results imply that SMYD3-mediated methylation of AKT1 at lysine 14 is essential for AKT1 activation and that SMYD3-mediated AKT1 methylation appears to be a good target for development of anti-cancer therapy.

Enolase of Mycobacterium tuberculosis is a surface exposed plasminogen binding protein

BackgroundEnolase, a glycolytic enzyme, has long been studied as an anchorless protein present on the surface of many pathogenic bacteria that aids in tissue remodeling and invasion by binding to host plasminogen. MethodsAnti-Mtb enolase antibodies in human sera were detected using ELISA. Immunoelectron microscopy, immunofluorescence microscopy and flow cytometry were used to show surface localization of Mtb enolase. SPR was used to determine the affinity of enolase-plasminogen interaction. Plasmin formation upon plasminogen binding to enolase and Mtb surface was measured by ELISA. Mice challenge and histopathological studies were undertaken to determine the protective efficacy of enolase immunization. ResultsEnolase of Mtb is present on its surface and binds human plasminogen with high affinity. There was an average of 2-fold increase in antibody mediated recognition of Mtb enolase in human sera from TB patients with an active disease over control individuals. Substitution of C-terminal lysine to alanine in rEno decreased its binding affinity with human plasminogen by >2-folds. Enolase bound plasminogen showed urokinase mediated conversion into plasmin. Binding of plasminogen to the surface of Mtb and its conversion into fibrinolytic plasmin was significantly reduced in the presence of anti-rEno antibodies. Immunization with rEno also led to a significant decrease in lung CFU counts of mice upon infection with Mtb H37Rv. ConclusionsMtb enolase is a surface exposed plasminogen binding protein which upon immunization confers significant protection against Mtb challenge. General significancePlasminogen binding has been recognized for Mtb, however, proteins involved have not been characterized. We show here that Mtb enolase is a moonlighting plasminogen binding protein.

Abstract 4613: SMYD3-mediated lysine methylation is critical for the activation of AKT1 in human cancer

Abstract v-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT1), a serine/threonine-protein kinase also known as protein kinase B Alpha (PKB Alpha), is a key mediator of a signaling pathway that governs various cellular processes regulating cell growth, survival, glucose metabolism, genome stability and neovascularization. It is known that dysregulation of AKT1 activity is one of the critical factors for the development and/or progression of a variety of human cancers. Posttranslational modifications of AKT1, including phosphorylation, ubiquitination and glycosylation, are well-analyzed, and it is known that the phosphorylation status of AKT1 is strongly correlated with its oncogenic activity. However, biological functions of methylation on AKT1 have never been analyzed so far. In the present study, we demonstrated that the protein lysine methyltrasnferase SMYD3 methylates lysine 14 in the PH domain of AKT1, which is known to play a critical role in the regulation of AKT1 activity through binding to phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) or phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2), in vitro and in vivo. Lysine 14-substituted AKT1 shows significantly lower levels of phosphorylation at threonine 308, which is the major indicator of AKT1 activity, than wild-type AKT1, and knockdown of SMYD3 as well as treatment with a SMYD3 inhibitor significantly attenuates this phosphorylation in cancer cells. Furthermore, substitution of lysine 14 diminishes the plasma membrane accumulation of AKT1, and cancer cells overexpressing lysine 14-substiuted AKT1 shows lower growth rate than those overexpressing wild-type AKT1. These results imply that SMYD3-mediated methylation of AKT1 at lysine 14 plays a central role in the oncogenic activity of AKT1. Although Phosphatidylinositide 3-kinase (PI3K)-dependent AKT1 phosphorylation by Pyruvate Dehydrogenase Kinase, Isozyme 1 (PDK1) and mTOR Complex 2 (mTORC2) has long been considered to be the primary mechanism accounting for AKT1 activation, these members are not sufficient enough to explain how AKT1 hyperactivation can occur in tumors with normal levels of PI3K/Phosphatase And Tensin Homolog (PTEN) activity. Our findings unveiled the novel mechanism of constitutive hyperphosphorylation on AKT1 in cancer cells, mediated by SMYD3-mediated methylation. The inhibition of this methylation pathway appears to be a promising strategy to develop anti-cancer drugs. Citation Format: Yuichiro Yoshioka, Yusuke Nakamura, Ryuji Hamamoto. SMYD3-mediated lysine methylation is critical for the activation of AKT1 in human cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4613.

Importance of a Potential Protein Kinase A Phosphorylation Site of Na+,K+-ATPase and Its Interaction Network for Na+ Binding

The molecular mechanism underlying PKA-mediated regulation of Na(+),K(+)-ATPase was explored in mutagenesis studies of the potential PKA site at Ser-938 and surrounding charged residues. The phosphomimetic mutations S938D/E interfered with Na(+) binding from the intracellular side of the membrane, whereas Na(+) binding from the extracellular side was unaffected. The reduction of Na(+) affinity is within the range expected for physiological regulation of the intracellular Na(+) concentration, thus supporting the hypothesis that PKA-mediated phosphorylation of Ser-938 regulates Na(+),K(+)-ATPase activity in vivo Ser-938 is located in the intracellular loop between transmembrane segments M8 and M9. An extended bonding network connects this loop with M10, the C terminus, and the Na(+) binding region. Charged residues Asp-997, Glu-998, Arg-1000, and Lys-1001 in M10, participating in this bonding network, are crucial to Na(+) interaction. Replacement of Arg-1005, also located in the vicinity of Ser-938, with alanine, lysine, methionine, or serine resulted in wild type-like Na(+) and K(+) affinities and catalytic turnover rate. However, when combined with the phosphomimetic mutation S938E only lysine substitution of Arg-1005 was compatible with Na(+),K(+)-ATPase function, and the Na(+) affinity of this double mutant was reduced even more than in single mutant S938E. This result indicates that the positive side chain of Arg-1005 or the lysine substituent plays a mechanistic role as interaction partner of phosphorylated Ser-938, transducing the phosphorylation signal into a reduced affinity of Na(+) site III. Electrostatic interaction of Glu-998 is of minor importance for the reduction of Na(+) affinity by phosphomimetic S938E as revealed by combining S938E with E998A.

Pepino mosaic virus RNA-Dependent RNA Polymerase POL Domain Is a Hypersensitive Response-Like Elicitor Shared by Necrotic and Mild Isolates.

Pepino mosaic virus (PepMV) is an emerging pathogen that represents a serious threat to tomato production worldwide. PepMV-induced diseases manifest with a wide range of symptoms, including systemic necrosis. Our results showed that PepMV accumulation depends on the virus isolate, tomato cultivar, and environmental conditions, and associates with the development of necrosis. Substitution of lysine for glutamic acid at position 67 in the triple gene block 3 (TGB3) protein, previously described as a necrosis determinant, led to increased virus accumulation and was necessary but not sufficient to induce systemic necrosis. Systemic necrosis both in tomato and Nicotiana benthamiana shared hypersensitive response (HR) features, allowing the assessment of the role of different genomic regions on necrosis induction. Overexpression of both TGB3 and the polymerase domain (POL) of the RNA-dependent RNA polymerase (RdRp) resulted in necrosis, although only local expression of POL triggered HR-like symptoms. Our results also indicated that the necrosis-eliciting activity of POL resides in its highly conserved "palm" domain, and that necrosis was jasmonic acid-dependent but not salicylic acid-dependent. Altogether, our data suggest that the RdRp-POL domain plays an important role in PepMV necrosis induction, with necrosis development depending on the virus accumulation level, which can be modulated by the nature of TGB3, host genotype and environmental conditions.

Ionic Interactions of the C-Terminal Domain of Apolipoprotein A-I are Responsible for Oligomerization

Human apoA-I is an anti-atherogenic protein circulating in plasma. The protein is a single polypeptide of 243 amino acid residues, and is the major protein component of high density lipoprotein. The three-dimensional structure of the protein remains unknown, however, secondary structure analysis revealed that apoA-I is predominately α-helical in nature. The N-terminal (NT) domain is made of a bundle of amphipathic α-helices, while the structure of the C-terminal (CT) domain is less defined. Lipid-free apoA-I exists as a heterogeneous population of oligomers. The CT domain is responsible for oligomerization, and several critical lysine residues that may be responsible for oligomerization have been identified. Site-directed mutagenesis was used to design a series of mutant proteins in which lysine residues of the CT domain were changed into glutamine (K195, K206, K208, K226, K238, and K239). The proteins were over-expressed in Escherichia coli and purified with Ni-affinity and gel filtration chromatography. Circular dichroism analysis showed that the helical content and midpoint of guanidine denaturation was not affected by the amino acid changes. Crosslinking with dimethylsuberimidate and size-exclusion FPLC showed that the extent of oligomerization of apoA-I decreased incrementally with each lysine substitution. The lysines of CT-apoA-I may form ionic bonds with oppositely charged residues in neighboring apoA-I. E234 and E235 are in close proximity to K238 and K239, and may form intermolecular ionic bonds upon self-association. To test this, E234 and E235 were mutated to glutamine. These mutant proteins retained their secondary structure and stability properties were not changed. However, the amount of cross-linking was significantly reduced. This result indicates that both lysine and glutamate residues in CT-apoA-I are critical residues responsible for self-association. Therefore, the glutamine variants will be excellent candidates to obtain the high-resolution structure of the protein.

Molecular Mechanism by which Two Lysine Substitutions Alter Na/K-Pump Stoichiometry to Confer High-Salinity Adaptation in Brine Shrimp

In most animal cells, the Na/K pump maintains a large electrochemical gradient for Na+ across the plasma membrane by extruding 3 Na+ and importing 2 K+ per ATP molecule hydrolyzed. When adapted to high salinity, brine shrimp (Artemia franciscana) express a pump that has two asparagine-to-lysine substitutions within the normally conserved ion-binding region. To address the molecular mechanisms by which these substitutions may be advantageous, we used two-electrode voltage clamp to evaluate the functional effects of the equivalent substitutions (N333K and N785K) in the Xenopus Na/K pump α subunit. Compared to wild-type, the apparent affinity for K+-activation of pump current without Na+o was reduced 10-fold by N785K, but nearly unaffected by N333K or N333K/N785K. In contrast, both individual mutants displaced the center of the charge-voltage curve measured with Na+o, without K+o, by −80 mV, reflecting a 16-fold decrease in Na+o affinity (20 mV shift per 2-fold change), whereas N333K/N785K induced only a ∼ -40 mV shift. Molecular dynamic simulations (3.3 μs) of these mutants based on the available crystal structures provide an explanation of the effects of individual and concurrent mutations and how these residues are coupled. Simulations of N333K/N785K with a single Na+ or K+ removed from the structures restored ion-binding site stability, consistent with a previous proposal of altered stoichiometry (Jorgensen and Amat, 2008, J. Memb. Biol. 221: 39-49, 2008). To directly assess stoichiometry, we measured 86Rb+ (a K+ congener) uptake in voltage-clamped oocytes and found that, for every charge extruded, wild-type pumps imported 2.11 ± 0.07 (n=40) Rb+, as expected, while N333K/N785K imported 1.01 ± 0.05 (n=13) Rb+. This reduced 2 Na:1 K stoichiometry is essential for the brine shrimp to maintain a larger Na+ gradient than other animals. NSF MCB-1515434.

An in silico approach to investigate the source of the controversial interpretations about the phenotypic results of the human AhR-gene G1661A polymorphism

Aryl hydrocarbon receptor (AhR) acts as an enhancer binding ligand-activated intracellular receptor. Chromatin remodeling components and general transcription factors such as TATA-binding protein (TBP) are evoked on AhR-target genes by interaction with its flexible transactivation domain (TAD). AhR-G1661A single nucleotide polymorphism (SNP: rs2066853) causes an arginine to lysine substitution in the acidic sub-domain of TAD at position 554 (R554K). Although, numerous studies associate the SNP with some abnormalities such as cancer, other reliable investigations refuse the associations. Consequently, the interpretation of the phenotypic results of G1661A-transition has been controversial. In this study, an in silico analysis were performed to investigate the possible effects of the transition on AhR-mRNA, protein structure, interaction properties and modifications. The analysis revealed that the R554K substitution affects secondary structure and solvent accessibility of adjacent residues. Also, it causes to decreasing of the AhR stability; altering the hydropathy features of the local sequence and changing the pattern of the residues at the binding site of the TAD-acidic sub-domain. Generating of new sites for ubiquitination and acetylation for AhR-K554 variant respectively at positions 544 and 560 was predicted. Our findings intensify the idea that the AhR-G1661A transition may affects AhR-TAD interactions, especially with the TBP, which influence AhR-target genes expression. However, the previously reported flexibility of the modular TAD could act as an intervening factor, moderate the SNP effects and causes distinct outcomes in different individuals and tissues.