Introduction Of Point Mutations Research Articles

Structure–activity relationship studies of Niemann-Pick type C1-like 1 (NPC1L1) ligands identified by screening assay monitoring pharmacological chaperone effect

A number of hereditary diseases are caused by defective protein trafficking due to a folding defect resulting from point mutations in proteins. Ligands that bind to the folding intermediates of such mutant proteins and rescue their trafficking defects, known as pharmacological chaperones, have promise for the treatment of certain genetic diseases, including Fabry disease, cystic fibrosis, and Niemann-Pick disease type C. Here we show that this pharmacological chaperone effect can be used for ligand screening, that is, binding of candidate ligands can be detected by monitoring the ligand-mediated correction of a localization defect caused by artificially introduced point mutations of the protein of interest. Using this method, we discovered novel steroidal ligands of Niemann-Pick type C1-like 1 (NPC1L1), an intestinal cholesterol transporter that is the target of the cholesterol absorption inhibitor ezetimibe, and conducted structure–activity relationship studies. We also present data indicating that the binding site of the new ligands is distinct from both the N-terminal sterol-binding domain and the ezetimibe-binding site.

Identification of amphiphysin 1 as an endogenous substrate for CDKL5, a protein kinase associated with X-linked neurodevelopmental disorder

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in brain and mutations of its gene are known to be associated with neurodevelopmental disorders such as X-linked West syndrome and Rett syndrome. However, the physiological substrates of CDKL5 that are directly linked to these neurodevelopmental disorders are currently unknown. In this study, we explored endogenous substrates for CDKL5 in mouse brain extracts fractionated by a liquid-phase isoelectric focusing. In conjunction with CDKL5 phosphorylation assay, this approach detected a protein band with an apparent molecular mass of 120kDa that is remarkably phosphorylated by CDKL5. This 120-kDa protein was identified as amphiphysin 1 (Amph1) by LC-MS/MS analysis, and the site of phosphorylation by CDKL5 was determined to be Ser-293. The phosphorylation mimic mutants, Amph1(S293E) and Amph1(S293D), showed significantly reduced affinity for endophilin, a protein involved in synaptic vesicle endocytosis. Introduction of point mutations in the catalytic domain of CDKL5, which are disease-causing missense mutations found in Rett patients, resulted in the impairment of kinase activity toward Amph1. These results suggest that Amph1 is the cytoplasmic substrate for CDKL5 and that its phosphorylation may play crucial roles in the neuronal development.

Quantitative and qualitative involvement of P3N-PIPO in overcoming recessive resistance against Clover yellow vein virus in pea carrying the cyv1 gene.

In pea carrying cyv1, a recessive gene for resistance to Clover yellow vein virus (ClYVV), ClYVV isolate Cl-no30 was restricted to the initially infected cells, whereas isolate 90-1 Br2 overcame this resistance. We mapped the region responsible for breaking of cyv1-mediated resistance by examining infection of cyv1 pea with chimeric viruses constructed from parts of Cl-no30 and 90-1 Br2. The breaking of resistance was attributed to the P3 cistron, which is known to produce two proteins: P3, from the main open reading frame (ORF), and P3N-PIPO, which has the N-terminal part of P3 fused to amino acids encoded by a small open reading frame (ORF) called PIPO in the +2 reading frame. We introduced point mutations that were synonymous with respect to the P3 protein but nonsynonymous with respect to the P3N-PIPO protein, and vice versa, into the chimeric viruses. Infection of plants with these mutant viruses revealed that both P3 and P3N-PIPO were involved in overcoming cyv1-mediated resistance. Moreover, P3N-PIPO quantitatively affected the virulence of Cl-no30 in cyv1 pea. Additional expression in trans of the P3N-PIPO derived from Cl-no30, using White clover mosaic virus as a vector, enabled Cl-no30 to move to systemic leaves in cyv1 pea. Susceptible pea plants infected with chimeric ClYVV possessing the P3 cistron of 90-1 Br2, and which were therefore virulent toward cyv1 pea, accumulated more P3N-PIPO than did those infected with Cl-no30, suggesting that the higher level of P3N-PIPO in infected cells contributed to the breaking of resistance by 90-1 Br2. This is the first report showing that P3N-PIPO is a virulence determinant in plants resistant to a potyvirus.

Abstract 1734: Drug- induced generation of Mcl-1128-350 fragment triggers apoptosis in multiple myeloma cells through upregulation of c-Jun.

Abstract Myeloid cell leukemia-1 (Mcl-1) gene, a pro-survival member of the Bcl-2 family, has been suggested to play a key role in Multiple Myeloma (MM) pathogenesis and drug resistance. Our present data demonstrate significant overexpression of Mcl-1 gene in a data set comparing samples from more than 500 MM patients with samples from healthy donors. These findings indicate Mcl-1 as a potential therapeutic target in MM. One strategy to inhibit Mcl-1 activity in MM and other malignancies is the induction of proteasome- independent Mcl-1 degradation. Indeed, our own and other previous studies demonstrate that a caspase- induced 28 kD Mcl-1 fragment, Mcl-1128-350 inhibits MM cell proliferation and survival. Importantly, our present results show that bortezomib, staurosporine and adaphostin, but not doxorubicin, thalidomide or enzastaurin induce generation of Mcl-1128-350 in MM cells. We next sought to identify molecular sequelae downstream of Mcl-1128-350, which mediate its pro-apoptotic effect. Surprisingly, we observed nuclear accumulation of drug- induced or transfected Mcl-1128-350, followed by elevated mRNA and protein levels of c-Jun as well as enhanced AP-1 reporter activity. Supporting a key role of Mcl-1128-350 in c-Jun transcription, drug- induced c-Jun upregulation was blocked after introduction of point mutations into the two highly conserved Mcl-1 caspase- cleavage sites Asp127 and Asp157. Consistent with these data, treatment with bortezomib triggered c-Jun upregulation followed by apoptosis in Mcl-1wt/wt, but not Mcl-1d/null murine embryonic fibroblasts (MEFs). Conversely, transfection of a plasmid carrying Mcl-1wt into Mcl-1d/null MEFs restored bortezomib- induced Mcl-1 fragementation, c-Jun upregulation and cell apoptosis. Importantly, Mcl-1 sequencing in several MM cell lines and primary cells didn't show the existence of point mutations in Mcl-1 caspase- cleavage sites, excluding a potential mechanism of drug resistance. In order to identify c-Jun downstream effectors of MM cell apoptosis, gene array profiling was finally performed on MM cells transfected with Mcl-1wt or Mcl-1128-350. Taken together, the present study demonstrates a promising therapeutic role of Mcl-1128-350 in MM. Moreover, our preliminary data indicate that a similar therapeutic role of Mcl-1128-350 may also exist in a variety of solid and other hematologic malignancies. Citation Format: Fengjuan Fan, Giovanni Tonon, Martin Sattler, Hartmut Goldschmidt, Joseph Opferman, Kenneth C. Anderson, Dirk Jäger, Klaus Podar. Drug- induced generation of Mcl-1128-350 fragment triggers apoptosis in multiple myeloma cells through upregulation of c-Jun. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1734. doi:10.1158/1538-7445.AM2013-1734

Rearrangements of Nascent Peptide Inside the Ribosomal Exit Tunnel

All proteins, from bacteria to man, are made in the ribosome and are elongated, one residue at a time, at the peptidyl transferase center (PTC). This growing peptide chain wends its way through the ribosomal tunnel to the exit port, ∼ 100 angstroms from the PTC. Regulation of the movement of the peptide within the tunnel and allosteric communication along the tunnel during translation are not well understood. Previously, we demonstrated that solvent-accessible volume surrounding a modifiable cysteine increases monotonically with increase in the van der Waal's volume of the adjacent side chain (Lu et al., J.Mol.Biol. 411: 499-510, 2011) and that the magnitude of this effect depends on location within the ribosomal tunnel. using a photocrosslinking approach, we confirm these results. We extend these studies to investigate whether mutations in the nascent peptide deep in the tunnel affect the accessibility of a modifiable reporter cysteine at the exit port and whether specific regions of the tunnel instigate these effects. Tryptophan vis-a-vis alanine, engineered into the nascent peptide at a distance of 17-19 residues from the PTC, alters the accessibility of residues at the exit port, a distance of 33 residues from the PTC, roughly 50 angstroms from the introduced point mutations. These findings are consistent with long-range rearrangements and may contribute to mechanisms governing sequence-specific signaling from different regions of the tunnel during translation. Supported by NIH grant R01GM52302.

Combinatorial Mutagenesis and Selection to Understand and Improve Yeast Promoters

Microbial promoters are important targets both for understanding the global gene expression and developing genetic tools for heterologous expression of proteins and complex biosynthetic pathways. Previously, we have developed and used combinatorial mutagenesis methods to analyse and improve bacterial expression systems. Here, we present for the first time an analogous strategy for yeast. Our model promoter is the strong and inducible P AOX1 promoter in methylotrophic Pichia pastoris. The Zeocin resistance gene was applied as a valuable reporter for mutant P AOX1 promoter activity, and we used an episomal plasmid vector to ensure a constant reporter gene dosage in the yeast host cells. This novel design enabled direct selection for colonies of recombinant cells with altered Zeocin tolerance levels originating solely from randomly introduced point mutations in the P AOX1 promoter DNA sequence. We demonstrate that this approach can be used to select for P AOX1 promoter variants with abolished glucose repression in large mutant libraries. We also selected P AOX1 promoter variants with elevated expression level under induced conditions. The properties of the selected P AOX1 promoter variants were confirmed by expressing luciferase as an alternative reporter gene. The tools developed here should be useful for effective screening, characterization, and improvement of any yeast promoters.

The pseudophosphatase MK‐STYX inhibits stress granule assembly independently of Ser149 phosphorylation of G3BP‐1

The pseudophosphatase MK-STYX (mitogen-activated protein kinase phosphoserine/threonine/tyrosine-binding protein) has been implicated in the stress response pathway. The expression of MK-STYX inhibits the assembly of stress granules, which are cytoplasmic storage sites for mRNA that form as a protective mechanism against stressors such as heat shock, UV irradiation and hypoxia. Furthermore, MK-STYX interacts with a key component of stress granules: G3BP-1 (Ras-GTPase activating protein SH3 domain binding protein-1). Because G3BP-1 dephosphorylation at Ser149 induces stress granule assembly, we initially hypothesized that the inhibition of stress granules by MK-STYX was G3BP-1 phosphorylation-dependent. However, in the present study, using MK-STYX constructs and G3BP-1 phosphomimetic or nonphosphorylatable mutants, we show that MK-STYX inhibits stress granule formation independently of G3BP-1 phosphorylation at Ser149. The introduction of point mutations at the ‘active site’ of MK-STYX that convert serine and phenylalanine to histidine and cysteine, respectively, is sufficient to generate an active enzyme. In separate experiments, we show that this active mutant, MK-STYXactive, has opposite effects to wild-type MK-STYK. Not only does MK-STYXactive induce stress granules, but also it has the capacity to dephosphorylate G3BP-1. Taken together, these results provide evidence that the pseudophosphatase MK-STYX plays a key role in the cellular response to stress.

Abstract 19603: Disruption of βPDGF Receptor-Dependent PI3k and Plcγ Signaling Protects from Hypoxia-Induced Pulmonary Hypertension

Background: Vascular remodeling is critical during the pathogenesis of pulmonary hypertension (PH). Growth factors, such as platelet-derived growth factor (PDGF), appear to be involved in these processes. With pharmacological inhibitors, we demonstrated previously that phosphatidyl inositol-3 kinase (PI3K) and phospholipase Cγ (PLCγ) are the critical mediators of βPDGF receptor (βPDGFR)-dependent proliferation and migration in pulmonary arterial smooth muscle cells (PASMC). Whereas expression data and data with the tyrosine kinase inhibitor imatinib suggest a role for PDGF in PH, data from genetic models is lacking. Methods and results: We utilized βPDGFR F3/F3 (“F3”) mice that were generated by introducing point mutations of the PI3K and PLCγ binding sites into the βPDGFR locus by gene targeting. We isolated PASMC from F3 and wild-type (WT) mice. Co-immunoprecipitation experiments after PDGF treatment showed that PI3K and PLCγ did not bind to the activated F3 receptor, whereas overall phosphorylation and binding of RasGap and SHP-2 were unaffected. In line with these observations, PDGF-mediated phosphorylation of Akt and Erk 1/2 was inhibited in F3-PASMC compared to WT. To address, if cellular responses were altered, we analyzed βPDGFR-dependent proliferation (as BrdU incorporation) and migration (modified Boyden chamber). These experiments revealed that these responses were essentially completely blocked in F3-PASMC compared to WT. To analyze the development and severity of PH in vivo, we exposed F3 and WT mice to hypoxia for 3 weeks. Following this treatment, right ventricular systolic pressure (RVSP) was measured by right heart catheterization, and right ventricular (RV) hypertrophy was assessed as RV weight/left ventricular + septal weight (RV/LV+S). These analyses revealed that RVSP and RV/LV+S were significantly reduced in F3 compared to WT mice. Conclusions: These data show that disruption of βPDGFR-dependent PI3K and PLCγ signaling blocks PDGF-dependent proliferation and migration of PASMC in vitro and protects from the development of hypoxia-induced PH in vivo. Consequently, these data support a critical role of PDGF during the pathogenesis of PH.

Targeting Mcl-1 for Multiple Myeloma (MM) Therapy: Drug-Induced Generation of Mcl-1 Fragment Mcl-1128–350 Triggers MM Cell Death Via c- Jun Upregulation

AbstractAbstract 3959Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein of the Bcl-2 family located on chromosome 1q21. Gains of 1q21 are unfavorable genetic prognostic factors for Multiple Myeloma (MM) patients. Our present data confirm significant overexpression of Mcl-1 gene in tumor versus plasma cells derived from healthy donors. Moreover, a significant link between Mcl-1 overexpression and poor prognosis was found in a data set of 590 MM patients, supporting a key role for Mcl-1 in MM survival and drug resistance. Mcl-1 is therefore a potential therapeutic target in MM. Strategies to inhibit Mcl-1 activity in MM and other malignancies may not only include induction of proteasome-dependent Mcl-1 degradation and modulation of Mcl-1 interaction with BH3-only proteins, but also induction of proteasome- independent Mcl-1 degradation. Indeed, our own and other previous data demonstrate that in contrast to full length Mcl-1 a caspase- induced 28kD Mcl-1 fragment, Mcl-1D128–350, inhibits MM cell proliferation and triggers apoptosis. We therefore performed a large-scale drug screen for agents which induce generation of Mcl-1D128–350 in MM cells. Our results show that bortezomib and staurosporine (STS) but not melphalan, doxorubicin or thalidomide induce generation of Mcl-1D128–350. We next sought to identify molecular sequelae downstream of Mcl-1D128–350 which mediate its anti-proliferative and pro-apoptotic effect in MM cells. Surprisingly, we observed nuclear accumulation of drug- induced Mcl-1D128–350, enhanced AP-1 reporter activity as well as elevated protein levels of c-Jun. Conversely, drug- induced c-Jun upregulation was blocked after introduction of point mutations (A127D, A157D) into the two highly conserved Mcl-1 caspase-cleavage sites Asp127 and Asp157. Gene profiles on MM cells transfected with either Mcl-1wt or Mcl-1D128–350 identified a group of differentially expressed genes associated with MM cell proliferation, survival and drug resistance. Consistent with these data, treatment with bortezomib or STS triggered c-Jun upregulation followed by apoptosis in Mcl-1wt/wt but not Mcl-1d/null murine embryonic fibroblasts (MEFs). Importantly, STS-induced c-Jun upregulation and cell death was restored in Mcl-1d/null MEFs transfected with a plasmid carrying Mcl-1wt. In ongoing studies we are analyzing genome sequencing data in MM cells for the existence of point mutations in the Mcl-1 caspase cleavage sites. The presence of such mutations could represent a potential new mechanism of drug resistance. Taken together, our data add another facet to the complex cellular functions of Mcl-1 in MM and derive a promising therapeutic and potential prognostic role for Mcl-1D128–350 in MM. Disclosures:No relevant conflicts of interest to declare.

Mitochondrial DNA Mutations Provoke Dominant Inhibition of Mitochondrial Inner Membrane Fusion

Mitochondria are highly dynamic organelles that continuously move, fuse and divide. Mitochondrial dynamics modulate overall mitochondrial morphology and are essential for the proper function, maintenance and transmission of mitochondria and mitochondrial DNA (mtDNA). We have investigated mitochondrial fusion in yeast cells with severe defects in oxidative phosphorylation (OXPHOS) due to removal or various specific mutations of mtDNA. We find that, under fermentative conditions, OXPHOS deficient cells maintain normal levels of cellular ATP and ADP but display a reduced mitochondrial inner membrane potential. We demonstrate that, despite metabolic compensation by glycolysis, OXPHOS defects are associated to a selective inhibition of inner but not outer membrane fusion. Fusion inhibition was dominant and hampered the fusion of mutant mitochondria with wild-type mitochondria. Inhibition of inner membrane fusion was not systematically associated to changes of mitochondrial distribution and morphology, nor to changes in the isoform pattern of Mgm1, the major fusion factor of the inner membrane. However, inhibition of inner membrane fusion correlated with specific alterations of mitochondrial ultrastructure, notably with the presence of aligned and unfused inner membranes that are connected to two mitochondrial boundaries. The fusion inhibition observed upon deletion of OXPHOS related genes or upon removal of the entire mtDNA was similar to that observed upon introduction of point mutations in the mitochondrial ATP6 gene that are associated to neurogenic ataxia and retinitis pigmentosa (NARP) or to maternally inherited Leigh Syndrome (MILS) in humans. Our findings indicate that the consequences of mtDNA mutations may not be limited to OXPHOS defects but may also include alterations in mitochondrial fusion. Our results further imply that, in healthy cells, the dominant inhibition of fusion could mediate the exclusion of OXPHOS-deficient mitochondria from the network of functional, fusogenic mitochondria.

Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression

HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5′ splice sites (5′ ss) and three 3′ splice sites (3′ ss) normally used in HPV16+ cervical cancer and its derived cell lines. The choice of two novel alternative 5′ ss (nt 221 5′ ss and nt 191 5′ ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5′ ss and nt 409 3′ ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3′ ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3′ ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of 91QYNK94 to 91PSFW94 displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression.

FRUIT, a Scar-Free System for Targeted Chromosomal Mutagenesis, Epitope Tagging, and Promoter Replacement in Escherichia coli and Salmonella enterica

Recombineering is a widely-used approach to delete genes, introduce insertions and point mutations, and introduce epitope tags into bacterial chromosomes. Many recombineering methods have been described, for a wide range of bacterial species. These methods are often limited by (i) low efficiency, and/or (ii) introduction of “scar” DNA into the chromosome. Here, we describe a rapid, efficient, PCR-based recombineering method, FRUIT, that can be used to introduce scar-free point mutations, deletions, epitope tags, and promoters into the genomes of enteric bacteria. The efficiency of FRUIT is far higher than that of the most widely-used recombineering method for Escherichia coli. We have used FRUIT to introduce point mutations and epitope tags into the chromosomes of E. coli K-12, Enterotoxigenic E. coli, and Salmonella enterica. We have also used FRUIT to introduce constitutive and inducible promoters into the chromosome of E. coli K-12. Thus, FRUIT is a versatile, efficient recombineering approach that can be applied in multiple species of enteric bacteria.

Co-Evolution of Mitochondrial tRNA Import and Codon Usage Determines Translational Efficiency in the Green Alga Chlamydomonas

Mitochondria from diverse phyla, including protozoa, fungi, higher plants, and humans, import tRNAs from the cytosol in order to ensure proper mitochondrial translation. Despite the broad occurrence of this process, our understanding of tRNA import mechanisms is fragmentary, and crucial questions about their regulation remain unanswered. In the unicellular green alga Chlamydomonas, a precise correlation was found between the mitochondrial codon usage and the nature and amount of imported tRNAs. This led to the hypothesis that tRNA import might be a dynamic process able to adapt to the mitochondrial genome content. By manipulating the Chlamydomonas mitochondrial genome, we introduced point mutations in order to modify its codon usage. We find that the codon usage modification results in reduced levels of mitochondrial translation as well as in subsequent decreased levels and activities of respiratory complexes. These effects are linked to the consequential limitations of the pool of tRNAs in mitochondria. This indicates that tRNA mitochondrial import cannot be rapidly regulated in response to a novel genetic context and thus does not appear to be a dynamic process. It rather suggests that the steady-state levels of imported tRNAs in mitochondria result from a co-evolutive adaptation between the tRNA import mechanism and the requirements of the mitochondrial translation machinery.

Mitochondrial ADP/ATP Carrier: Preventing Conformational Changes by Point Mutations Inactivates Nucleotide Transport Activity

The mitochondrial ADP/ATP carrier (Ancp) is a paradigm of the mitochondrial carrier family (MCF); its members allow metabolic fluxes between mitochondria and the cytosol. The members of the MCF share numerous structural and functional characteristics. Ancp is very specifically inhibited by two classes of compounds, which stabilize the carrier in two different conformations involved in nucleotide transport. Resolution of the atomic structure of the bovine Ancp, in complex with one of its specific inhibitors, is that of the carrier open toward the intermembrane space. To gain insights into the interconversion from one conformation to the other, we introduced point mutations in the yeast carrier at positions Cys73 in the first matrix loop and Tyr97 and Gly298 in transmembrane helices 2 and 6. We demonstrate in this paper that they impair stabilization of the carrier in one conformation or the other, resulting in an almost complete inactivation of nucleotide transport in both cases. The results are discussed on the basis of the atomic structure of the conformation open to the cytosol. These mutant proteins could afford convenient tools for undertaking structural studies of both conformations of the yeast carrier.

Frank-ter Haar Syndrome Protein Tks4 Regulates Epidermal Growth Factor-dependent Cell Migration

Mutations in the SH3PXD2B gene coding for the Tks4 protein are responsible for the autosomal recessive Frank-ter Haar syndrome. Tks4, a substrate of Src tyrosine kinase, is implicated in the regulation of podosome formation. Here, we report a novel role for Tks4 in the EGF signaling pathway. In EGF-treated cells, Tks4 is tyrosine-phosphorylated and associated with the activated EGF receptor. This association is not direct but requires the presence of Src tyrosine kinase. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutations of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks4. Furthermore, a PX domain mutant (R43W) Tks4 carrying a reported point mutation in a Frank-ter Haar syndrome patient showed aberrant intracellular expression and reduced phosphoinositide binding. Finally, silencing of Tks4 was shown to markedly inhibit HeLa cell migration in a Boyden chamber assay in response to EGF or serum. Our results therefore reveal a new function for Tks4 in the regulation of growth factor-dependent cell migration.

A catalytic and non-catalytic role for the Yen1 nuclease in maintaining genome integrity in Kluyveromyces lactis

Yen1 is a nuclease identified in Saccharomyces cerevisiae that cleaves the Holliday junction (HJ) intermediate formed during homologous recombination. Alternative routes to disjoin HJs are performed by the Mus81/Mms4- and Sgs1/Top3/Rmi1-complexes. Here, we investigate the role of the Yen1 protein in the yeast Kluyveromyces lactis. We demonstrate that both yen1 mus81 and yen1 sgs1 double mutants displayed negative genetic interactions in the presence of DNA-damaging chemicals. To test if these phenotypes required the catalytic activity of Yen1, we introduced point mutations targeting the catalytic site of Yen1, which abolished the nuclease activity in vitro. Remarkably, catalytically inactive Yen1 did not exacerbate the hydroxyurea sensitivity of the sgs1Δ strain, which the yen1Δ allele did. In addition, overexpression of catalytically inactive Yen1 partially rescued the DNA damage sensitivity of both mus81 and sgs1 mutant strains albeit less efficiently than WT Yen1. These results suggest that Yen1 serves both a catalytic and non-catalytic role in its redundant function with Mus81 and Sgs1. Diploids lacking Mus81 had a severe defect in sporulation efficiency and crossover frequency, but diploids lacking both Mus81 and Yen1 showed no further reduction in spore formation. Hence, Yen1 had no evident role in meiosis. However, overexpression of WT Yen1, but not catalytically inactive Yen1 partially rescued the crossover defect in mus81/mus81 mutant diploids. Yen1 is a member of the RAD2/XPG-family of nucleases, but genetic analyses revealed no genetic interaction between yen1 and other family members (rad2, exo1 and rad27). In addition, yen1 mutants had normal nonhomologous end-joining efficiency. We discuss the similarities and differences between K. lactis Yen1 and Yen1/GEN1 from other organisms.

Expression of recombinant Ara h 6 in Pichia pastoris but not in Escherichia coli preserves allergic effector function and allows assessment of specific mutations

Ara h 6 has recently been recognized as an important peanut allergen. Recombinant allergens have been used for analysis of IgE binding, but have not been used to analyze the allergic effector activity that is more relevant to allergic reactions. Ara h 6 was expressed as a recombinant protein in both Escherichia coli and Pichia pastoris (rAra h 6-E. coli and rAra h 6-Pichia, respectively). Effector activity was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from patients with severe peanut allergy. Compared to native Ara h 6 (nAra h 6), rAra h 6-Pichia had intact effector function whereas rAra h 6-E. coli had significantly reduced function. The lower effector activity in rAra h 6-E. coli compared to nAra h 6 and rAra h 6-Pichia did not appear to be due to differences in posttranslational modifications (analyzed by mass spectrometry and staining for carbohydrates) and may be due to subtle alteration(s) of folding seen on CD analysis and on nonreduced gels. Finally, we introduced point mutations in four important IgE-binding linear epitopes of Ara h 6 and found dramatically reduced allergic effector activity. Our studies demonstrate the utility of fully functional rAra h 6-Pichia as a starting point for analysis of specific mutations that adversely affect allergic effector function.

Crystallization and preliminary X-ray crystallographic analysis of bacterial tRNA(Sec) in complex with seryl-tRNA synthetase.

Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNA(Sec)) is first ligated with serine by seryl-tRNA synthetase (SerRS). To elucidate the tertiary structure of bacterial tRNA(Sec) and its specific interaction with SerRS, the bacterial tRNA(Sec) from Aquifex aeolicus was crystallized as the heterologous complex with the archaeal SerRS from Methanopyrus kandleri. Although X-ray diffraction by crystals of tRNA(Sec) in complex with wild-type SerRS was rather poor (to 5.7 Å resolution), the resolution was improved by introducing point mutations targeting the crystal-packing interface. Heavy-atom labelling also contributed to resolution improvement. A 3.2 Å resolution diffraction data set for phase determination was obtained from a K(2)Pt(CN)(4)-soaked crystal.

Abstract 56: Thrombin Induces Protease-Activated Receptor 1 and PAR4 Heterodimers

Thrombin is a potent platelet agonist. Thrombin activates platelets and other cells of the cardiovascular system by cleaving its receptors, protease activated receptor 1 (PAR1), PAR4 or both. PARs are G-protein coupled receptors that activate cellular signaling through Gq and G12/13. Our previous studies have determined that PAR4 forms homodimers and have mapped the homodimer interface to specific residues in transmembrane helix 4 (TM4). We have also shown that coexpression of PAR1 with PAR4 lowers the threshold for PAR4 activation by thrombin ∼10-fold. The purpose of the current study is to examine the physical interaction between PAR1 and PAR4 and how these interactions influence PAR1’s ability to enhance PAR4 activation. The PAR1-PAR4 heterodimers were examined by bioluminescence resonance energy transfer (BRET). PAR1-PAR4 heterodimers were not detected under basal conditions. However, when the cells were stimulated with 10 nM thrombin, we were able to detect a strong interaction between PAR1 and PAR4. In contrast, stimulating PAR1, PAR4, or both with agonist peptides TFLLRN (100 microM) or AYPGKF (500 microM), respectively did not induce heterodimers. Further, point mutations that prevent PAR1 or PAR4 cleavage did not from heterodimers when stimulated with thrombin. Other proteases that activate PAR1 or PAR4 such as activated protein C (APC) and plasmin do not induce PAR1-PAR4 heterodimers. Finally, we have introduced point mutations in transmembrane helix 4 of PAR1 and PAR4 that prevent heterodimer formation when stimulated with thrombin. These data demonstrate that PAR1 and PAR4 require allosteric changes induced by receptor cleavage by thrombin to mediate heterodimer formation and have determined the PAR1-PAR4 heterodimer interface. Taken together, these data suggest that PAR1 and PAR4 have a dynamic interaction depending on the context of their expression. Since PAR1 is an attractive antiplatelet target, the molecular interactions of this receptor on the cells surface must be taken into account when developing and characterizing these antagonists.

HDQ, a Potent Inhibitor of Plasmodium falciparum Proliferation, Binds to the Quinone Reduction Site of the Cytochrome bc 1 Complex

The mitochondrial bc(1) complex is a multisubunit enzyme that catalyzes the transfer of electrons from ubiquinol to cytochrome c coupled to the vectorial translocation of protons across the inner mitochondrial membrane. The complex contains two distinct quinone-binding sites, the quinol oxidation site of the bc(1) complex (Q(o)) and the quinone reduction site (Q(i)), located on opposite sides of the membrane within cytochrome b. Inhibitors of the Q(o) site such as atovaquone, active against the bc(1) complex of Plasmodium falciparum, have been developed and formulated as antimalarial drugs. Unfortunately, single point mutations in the Q(o) site can rapidly render atovaquone ineffective. The development of drugs that could circumvent cross-resistance with atovaquone is needed. Here, we report on the mode of action of a potent inhibitor of P. falciparum proliferation, 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ). We show that the parasite bc(1) complex--from both control and atovaquone-resistant strains--is inhibited by submicromolar concentrations of HDQ, indicating that the two drugs have different targets within the complex. The binding site of HDQ was then determined by using a yeast model. Introduction of point mutations into the Q(i) site, namely, G33A, H204Y, M221Q, and K228M, markedly decreased HDQ inhibition. In contrast, known inhibitor resistance mutations at the Q(o) site did not cause HDQ resistance. This study, using HDQ as a proof-of-principle inhibitor, indicates that the Q(i) site of the bc(1) complex is a viable target for antimalarial drug development.