Substitution Of Asparagine Research Articles

Hb G-Waimanalo [A1] or α64(E13)Asp→Asn (α1) (HBA1: c.193G>A) Observed in a Bulgarian Family

The abnormal hemoglobin (Hb) with an aspartic acid to asparagine substitution at α64 has been found on both the α2- and α1-globin genes. It has been described in many different populations under different names, but never in Bulgaria. Using the recently proposed nomenclature, Hb G-Waimanalo [A1] refers to the HBA1: c.193G > A, while Hb G-Waimanalo [A2] refers to the HBA2: c.193G > A mutation. Here, we present the first family from Bulgaria with Hb G-Waimanalo [A1].

Rescue of Na+ Affinity in Aspartate 928 Mutants of Na+,K+-ATPase by Secondary Mutation of Glutamate 314

The Na(+),K(+)-ATPase binds Na(+) at three transport sites denoted I, II, and III, of which site III is Na(+)-specific and suggested to be the first occupied in the cooperative binding process activating phosphorylation from ATP. Here we demonstrate that the asparagine substitution of the aspartate associated with site III found in patients with rapid-onset dystonia parkinsonism or alternating hemiplegia of childhood causes a dramatic reduction of Na(+) affinity in the α1-, α2-, and α3-isoforms of Na(+),K(+)-ATPase, whereas other substitutions of this aspartate are much less disruptive. This is likely due to interference by the amide function of the asparagine side chain with Na(+)-coordinating residues in site III. Remarkably, the Na(+) affinity of site III aspartate to asparagine and alanine mutants is rescued by second-site mutation of a glutamate in the extracellular part of the fourth transmembrane helix, distant to site III. This gain-of-function mutation works without recovery of the lost cooperativity and selectivity of Na(+) binding and does not affect the E1-E2 conformational equilibrium or the maximum phosphorylation rate. Hence, the rescue of Na(+) affinity is likely intrinsic to the Na(+) binding pocket, and the underlying mechanism could be a tightening of Na(+) binding at Na(+) site II, possibly via movement of transmembrane helix four. The second-site mutation also improves Na(+),K(+) pump function in intact cells. Rescue of Na(+) affinity and Na(+) and K(+) transport by second-site mutation is unique in the history of Na(+),K(+)-ATPase and points to new possibilities for treatment of neurological patients carrying Na(+),K(+)-ATPase mutations.

Effects of N-glycosylation site removal in archaellins on the assembly and function of archaella in Methanococcus maripaludis.

In Methanococcus maripaludis S2, the swimming organelle, the archaellum, is composed of three archaellins, FlaB1S2, FlaB2S2 and FlaB3S2. All three are modified with an N-linked tetrasaccharide at multiple sites. Disruption of the N-linked glycosylation pathway is known to cause defects in archaella assembly or function. Here, we explored the potential requirement of N-glycosylation of archaellins on archaellation by investigating the effects of eliminating the 4 N-glycosylation sites in the wildtype FlaB2S2 protein in all possible combinations either by Asn to Glu (N to Q) substitution or Asn to Asp (N to D) substitutions of the N-glycosylation sequon asparagine. The ability of these mutant derivatives to complement a non-archaellated ΔflaB2S2 strain was examined by electron microscopy (for archaella assembly) and swarm plates (for analysis of swimming). Western blot results showed that all mutated FlaB2S2 proteins were expressed and of smaller apparent molecular mass compared to wildtype FlaB2S2, consistent with the loss of glycosylation sites. In the 8 single-site mutant complements, archaella were observed on the surface of Q2, D2 and D4 (numbers after N or Q refer to the 1st to 4th glycosylation site). Of the 6 double-site mutation complementations all were archaellated except D1,3. Of the 4 triple-site mutation complements, only D2,3,4 was archaellated. Elimination of all 4 N-glycosylation sites resulted in non-archaellated cells, indicating some minimum amount of archaellin glycosylation was necessary for their incorporation into stable archaella. All complementations that led to a return of archaella also resulted in motile cells with the exception of the D4 version. In addition, a series of FlaB2S2 scanning deletions each missing 10 amino acids was also generated and tested for their ability to complement the ΔflaB2S2 strain. While most variants were expressed, none of them restored archaellation, although FlaB2S2 harbouring a smaller 3-amino acid deletion was able to partially restore archaellation.

Basic concepts and potential applications of genetics and genomics for cardiovascular and stroke clinicians: a scientific statement from the American Heart Association.

Although genetics and genomics play an increasingly large role in the practice of medicine, the clinical care of patients suffering from cardiovascular disease or stroke has not been significantly affected. This is despite the tremendous strides being made to understand the genetic basis of both rare and common cardiovascular and stroke disorders through techniques such as genome-wide association studies (GWASs) and next-generation sequencing studies. Much of this knowledge remains to be translated to the clinic and must be subjected to clinical trials to ensure patient safety and a meaningful impact on clinical outcomes. However, even if this knowledge were to be successfully implemented into clinical practice, a potential barrier to widespread adoption is a lack of familiarity with basic concepts of genetics and genomics. Another concern is the possibility of the emergence of a significant gap in clinical care provided by practitioners who are informed about the clinical use of genetics and genomics knowledge and those who are not. Thus, there is a critical need to foster genetics/genomics literacy among all involved in the care of cardiovascular and stroke patients because it can be expected that these topics will transform the way medicine is practiced. The purpose of this document is to serve as a resource for practitioners in cardiovascular and stroke medicine on the application of genetics and genomics to patient care. Although not exhaustive, it contains an overview of the field written specifically to be accessible and relevant to practitioners. It also refers to additional educational materials available in the literature, in textbooks, and on the Internet. (Because this article is intended to be primarily educational in nature, rather than providing a review of the literature, citations are limited to a small number of research articles and reviews of exceptional interest.) It recommends a core knowledge base with …

Effect of asparagine substitutions in the YXN loop of a class C β-lactamase of Acinetobacter baumannii on substrate and inhibitor kinetics.

Class C cephalosporinases are a growing threat, and inhibitors of these enzymes are currently unavailable. Studies exploring the YXN loop asparagine in the Escherichia coli AmpC, P99, and CMY-2 enzymes have suggested that interactions between C6' or C7' substituents on penicillins or cephalosporins and this Asn are important in determining substrate specificity and enzymatic stability. We sought to characterize the YXN loop asparagine in the clinically important ADC-7 class C β-lactamase of Acinetobacter baumannii. Mutagenesis at the N148 position in ADC-7 yields functional mutants (N152G, -S, -T, -Q, -A, and -C) that retain cephalosporinase activity. Using standard assays, we show that N148G, -S, and -T variants possess good catalytic activity toward cefoxitin and ceftaroline but that cefepime is a poor substrate. Because N152 variants of CMY-2, another class C β-lactamase, are more readily inhibited by tazobactam due to higher rates of inactivation, we also tested if the N148 substitutions in ADC-7 would affect inactivation by sulfone inhibitors, sulbactam and tazobactam, class A β-lactamase, and A. baumannii penicillin-binding protein (PBP) inhibitors with in vitro activity against ADC-7. The 50% inhibitory concentrations (IC50s) for tazobactam and sulbactam were improved, with 7-fold and 2-fold reductions, respectively, for the N148S variant. A homology model of the N148S ADC-7 enzyme in a Michaelis-Menten complex with tazobactam showed a loss of interaction between N148 and the sulfone moiety of the inhibitor. We postulate that this may result in more-rapid secondary ring opening of the inhibitor, as the unbound sulfone is an excellent leaving group, leading to more-rapid formation of the stable linearized inhibitor.

Highly reduced binding to high and low affinity mouse Fc gamma receptors by L234A/L235A and N297A Fc mutations engineered into mouse IgG2a

The effects of the Fc silencing mutations such as leucine (L) to alanine (A) substitution at the position 234 and 235 (LALA) and the alanine (A) to asparagine (N) substitution at position 297 (N297A) are well investigated for human IgG. However, the effects of the same two silencing Fc mutations in a mouse IgG backbone are not yet well investigated in respect to binding to mouse Fc gamma receptors (FcγRs), complement and subsequent effector functions. By using a mouse IgG2a tool antibody directed against mouse OX40L, we demonstrate a strongly reduced binding of the two Fc mutants to high and low affinity recombinant and cell expressed mouse FcγRs, when compared to the mouse IgG2a with the wild type (wt) backbone. Reduced FcγR binding by the two investigated Fc mutants could further be confirmed on primary mouse macrophages expressing their native FcγRs. In addition, we reveal that the LALA and N297A mutations in the mIgG2a also slightly reduced binding to C1q of human origin. Thus, here we provide experimental evidence that the two investigated Fc mutations in the mouse IgG backbone lead to similar “silencing” properties as previously demonstrated for the human IgG and thus represent a useful method to alter effector functions in tool antibodies to be used in mouse models.

Mutations in the 50S ribosomal subunit of Brachyspira hyodysenteriae associated with altered minimum inhibitory concentrations of pleuromutilins

Brachyspira hyodysenteriae, the causative agent of swine dysentery, is responsible for severe mucohaemorrhagic colitis with considerable financial loss to worldwide swine production. Antimicrobial resistance against macrolides and lincosamides is widespread and the mechanisms are well known. Currently, the most common treatment for swine dysentery is the use of pleuromutilins and resistance to these drugs also is increasingly being reported. Although resistance mechanisms against pleuromutilins are less clear than for other drugs, they seem to involve alterations of the peptidyl transferase centre (PTC), including ribosomal RNA and the ribosomal protein L3. The present study was conducted to examine molecular mechanisms of resistance on a representative set of B. hyodysenteriae field strains with different resistance patterns. In total, we identified 24 single nucleotide polymorphisms (SNPs) in the 23S rRNA gene and genes of the ribosomal proteins L3, L4, L2 and L22. The SNP in the ribosomal protein gene L3 at position 443 led to an amino acid substitution of asparagine (Asn) by serine (Ser) at position 148, significantly associated with MICs for pleuromutilins. Based on this SNP a correct assignment of 71% of the strains with respect to a threshold of >0.625μgtiamulin/ml was reached. Unexpectedly low MICs in some of the Asn-strains were explained by a second SNP at position 2535 of the 23S rRNA.Our results clearly show the associations between MICs for pleuromutilins and mutations in their binding site. A complete list of SNPs that influence MICs of B. hyodysenteriae strains is needed to enable the interpretation of future molecular susceptibility testing.

Functional disruption of yeast metacaspase, Mca1, leads to miltefosine resistance and inability to mediate miltefosine-induced apoptotic effects

Miltefosine (MI) is a novel, potential antifungal agent with activity against some yeast and filamentous fungal pathogens. We previously demonstrated in the model yeast, Saccharomyces cerevisiae, that MI causes disruption of mitochondrial membrane potential and apoptosis-like cell death via interaction with the Cox9p sub-unit of cytochrome c oxidase (COX). To identify additional mechanisms of antifungal action, MI resistance was induced in S. cerevisiae by exposure to the mutagen, ethyl methanesulfonate, and gene mutation(s) responsible for resistance were investigated. An MI-resistant haploid strain (H-C101) was created. Resistance was retained in the diploid strain (D-C101) following mating, confirming dominant inheritance. Phenotypic assessment of individual D-C101 tetrads revealed that only one mutant gene contributed to the MI-resistance phenotype. To identify this gene, the genome of H-C101 was sequenced and 17 mutated genes, including metacaspase-encoding MCA1, were identified. The MCA1 mutation resulted in substitution of asparagine (N) with aspartic acid (D) at position 164 (MCA1N164D). MI resistance was found to be primarily due to MCA1N164D, as single-copy episomal expression of MCA1N164D, but not two other mutated genes (FAS1T1417I and BCK2T104A), resulted in MI resistance in the wild-type strain. Furthermore, an MCA1 deletion mutant (mca1Δ) was MI-resistant. MI treatment led to accumulation of reactive oxygen species (ROS) in MI-resistant (MCA1N164D-expressing and mca1Δ) strains and MI-susceptible (MCA1-expressing) strains, but failed to activate Mca1 in the MI-resistant strains, demonstrating that ROS accumulation does not contribute to the fungicidal effect of MI. In conclusion, functional disruption of Mca1, leads to MI resistance and inability to mediate MI-induced apoptotic effects. Mca1-mediated apoptosis is therefore a major mechanism of MI-induced antifungal action.

RNA editing in RHOQ promotes invasion potential in colorectal cancer

RNA editing can increase RNA sequence variation without altering the DNA sequence. By comparing whole-genome and transcriptome sequence data of a rectal cancer, we found novel tumor-associated increase of RNA editing in ras homologue family member Q (RHOQ) transcripts. The adenosine-to-inosine (A-to-I) editing results in substitution of asparagine with serine at residue 136. We observed a higher level of the RHOQ RNA editing in tumor compared with normal tissue in colorectal cancer (CRC). The degree of RNA editing was associated with RhoQ protein activity in CRC cancer cell lines. RhoQ N136S amino acid substitution increased RhoQ activity, actin cytoskeletal reorganization, and invasion potential. KRAS mutation further increased the invasion potential of RhoQ N136S in vitro. Among CRC patients, recurrence was more frequently observed in patients with tumors having edited RHOQ transcripts and mutations in the KRAS gene. In summary, we show that RNA editing is another mechanism of sequence alteration that contributes to CRC progression.

Functional characterization of naturally occurring melittin peptide isoforms in two honey bee species, Apis mellifera and Apis cerana

Insect-derived antimicrobial peptides (AMPs) have diverse effects on antimicrobial properties and pharmacological activities such as anti-inflammation and anticancer properties. Naturally occurring genetic polymorphism have a direct and/or indirect influence on pharmacological effect of AMPs, therefore information on single nucleotide polymorphism (SNP) occurring in natural AMPs provides an important clue to therapeutic applications. Here we identified nucleotide polymorphisms in melittin gene of honey bee populations, which is one of the potent AMP in bee venoms. We found that the novel SNP of melittin gene exists in these two honey bee species, Apis mellifera and Apis cerana. Nine polymorphisms were identified within the coding region of the melittin gene, of which one polymorphism that resulted in serine (Ser) to asparagine (Asp) substitution that can potentially effect on biological activities of melittin peptide. Serine-substituted melittin (Mel-S) showed more cytotoxic effect than asparagine-substituted melittin (Mel-N) against E. coli. Also, Mel-N and Mel-S had different inhibitory effects on the production of inflammatory factors such as IL-6 and TNF-α in BV-2 cells. Moreover, Mel-S showed stronger cytotoxic activities than Mel-N peptide against two human ovarian cancer cell lines. Using carbon nanotube-based transistor, we here characterized that Mel-S interacted with small unilamellar liposomes more strongly than Mel-N. Taken together, our present study demonstrates that there exist different characteristics of the gene frequency and the biological activities of the melittin peptide in two honey bee species, Apis mellifera and A. cerana.

Disruption of N-linked glycosylation promotes proteasomal degradation of the human ATP-binding cassette transporter ABCA3

The lipid transport protein, ABCA3, expressed in alveolar type 2 (AT2) cells, is critical for surfactant homeostasis. The first luminal loop of ABCA3 contains three putative N-linked glycosylation sites at residues 53, 124, and 140. A common cotranslational modification, N-linked glycosylation, is critical for the proper expression of glycoproteins by enhancing folding, trafficking, and stability through augmentation of the endoplasmic reticulum (ER) folding cycle. To understand its role in ABCA3 biosynthesis, we utilized EGFP-tagged fusion constructs with either wild-type or mutant ABCA3 cDNAs that contained glutamine for asparagine substitutions at the putative glycosylation motifs. In A549 cells, inhibition of glycosylation by tunicamycin increased the electrophoretic mobility (Mr) and reduced the expression level of wild-type ABCA3 in a dose-dependent manner. Fluorescence imaging of transiently transfected A549 or primary human AT2 cells showed that although single motif mutants exhibited a vesicular distribution pattern similar to wild-type ABCA3, mutation of N124 and N140 residues resulted in a shift toward an ER-predominant distribution. By immunoblotting, the N53 mutation exhibited no effect on either the Mr or ABCA3 expression level. In contrast, substitutions at N124 or N140, as well a N124/N140 double mutation, resulted in increased electrophoretic mobility indicative of a glycosylation deficiency accompanied by reduced overall expression levels. Diminished steady-state levels of glycan-deficient ABCA3 isoforms were rescued by treatment with the proteasome inhibitor MG132. These results suggest that cotranslational N-linked glycosylation at N124 and N140 is critical for ABCA3 stability, and its disruption results in protein destabilization and proteasomal degradation.

Perturbing the Water Cavity Surrounding the Manganese Cluster by Mutating the Residue D1-Valine 185 Has a Strong Effect on the Water Oxidation Mechanism of Photosystem II

The active site of water oxidation in Photosystem II (PSII) is a Mn4CaO5 cluster that is located in a cavity between the D1 and CP43 protein subunits by which it is coordinated. The remainder of this cavity is filled with water molecules, which serve as a source of substrate and participate in poorly understood hydrogen bond networks that may modulate the function of the Mn4CaO5 cluster. These water molecules interact with the first and second sphere amino acid ligands to the Mn4CaO5 cluster and some water interacts directly with the Mn4CaO5 cluster. Here, the results of mutations to the amino acids that line the walls of several predicted cavities in the immediate vicinity of the Mn4CaO5 cluster were examined in Synechocystis sp. PCC 6803. Of these, mutations of Val185 in the D1 subunit resulted in the most interesting functional alterations. The hydrophobic D1-Val185 occupies a location contacting water molecules that are positioned between the redox active tyrosine (YZ) and the putative proton gate residue, D1-Asp61, and at a position opposite the oxo bridge atom, O5, of the cluster. Mutations of the residue D1-Val185 were produced, with the intention that the substitute residue would extend into the water cavity that includes H2O molecules that interact with the Mn4CaO5 cluster, amino acid ligands of the Mn4CaO5 cluster, YZ and the chloride co-factor of PSII. Three of these mutants, D1-Val185Asn, D1-Val185Thr, and D1-Val185Phe, were able to accumulate significant levels of charge separating PSII and were characterized using polarographic and fluorescent techniques. Of the three substitutions, the phenylalanine substitution was the most severe with a complete inability to evolve oxygen, despite being able to accumulate PSII and to undergo stable charge separation. The threonine substitution had no apparent effect on oxygen evolution other than a 40% reduction in the steady state rate of O2 production compared to the case of wild-type Synechocystis , due to a reduced ability to accumulate PSII centers. The asparagine substitution produced the most complex phenotype with respect to O2 evolution. Although still able to evolve oxygen, D1-Val185Asn does so less efficiently than wild-type PSII, with a higher miss factor than that for the wild type. Most significantly, asparagine substitution dramatically retards the rate of O2 release and results in an extension of the kinetic lag phase prior to O2 release that is highly reminiscent of the effects of mutations produced at D1-Asp61. The observed effects of the D1-Val185Phe and D1-Val185Asn mutations may be due to alterations in the environment of nearby chloride co-factor of PSII and/or alterations in the hydrogen bond network, perhaps impeding the movement of water to a binding site on the metal cluster.

Asparagine Substitution at PB2 Residue 701 Enhances the Replication, Pathogenicity, and Transmission of the 2009 Pandemic H1N1 Influenza A Virus

The 2009/2010 pandemic influenza virus (H1N1pdm) contains an avian-lineage PB2 gene that lacks E627K and D701N substitutions important in the pathogenesis and transmission of avian-origin viruses in humans or other mammals. Previous studies have shown that PB2-627K is not necessary because of a compensatory Q591R substitution. The role that PB2-701N plays in the H1N1pdm phenotype is not well understood. Therefore, PB2-D701N was introduced into an H1N1pdm virus (A/New York/1682/2009 (NY1682)) and analyzed in vitro and in vivo. Mini-genome replication assay, in vitro replication characteristics in cell lines, and analysis in the mouse and ferret models demonstrated that PB2-D701N increased virus replication rates and resulted in more severe pathogenicity in mice and more efficient transmission in ferrets. In addition, compared to the NY1682-WT virus, the NY1682-D701N mutant virus induced less IFN-λ and replicated to a higher titer in primary human alveolar epithelial cells. These findings suggest that the acquisition of the PB2-701N substitution by H1N1pdm viruses may result in more severe disease or increase transmission in humans.

A genetic variant of 5-hydroxytryptamine receptor 3C (HTR3C): A novel link to chemotherapy-induced side effects.

9630 Background: Symptom clusters are defined as three or more concurrent symptoms that are related to each other. Clusters may stem from common physiological mechanisms and may better represent adverse effects to chemotherapy compared to individual symptoms. We aimed to identify association between the experience of symptom clusters and specific genetic alterations. Methods: Study population consisted of 108 breast cancer patients who received over two cycles of adjuvant doxorubicin and cyclophosphamide (AC) treatment at the oncology institute of the Sheba Medical Center. Participants completed the Memorial Symptom Assessment Scale, the Lee Fatigue Scale and the Center for Epidemiological Studies Depression Scale. Hierarchical cluster analysis was used to identify patients' subgroups based on their symptom experience. For the genetic analyses, DNA was extracted from peripheral blood and single nucleotide polymorphisms (SNPs) of candidate genes were tested using restriction endonuclease assays. Results: Two distinct subgroups were identified based on severity of fatigue, depression, nausea, and change in food tastes: "all high" (n=79) and "all low" (n=29) level of all symptoms. As patients did not have active cancer, symptoms were attributed solely to chemotherapy. A genetic variant of HTR3C (rs6766410) results in a substitution of asparagine to lysine (N163K) may be associated with nausea and vomiting. We tested the association between this variant and symptom score. 51 of 75 (68%) patients with high symptom score harbored the variant allele, compared to 13 of 28 (46%) of those with low symptom score (p=0.038). Conclusions: Analysis of genetic background of clusters, rather than for individual symptoms, represents a novel approach for the study of chemotherapy-induced side effects. This approach enabled the identification of HTR3C variant as a possible mediator of side effects following treatment with AC. Discovering the genetic basis of symptom clusters may lead to the development of novel diagnostic and therapeutic modalities able to improve symptom management. This may translate to improved outcome among chemotherapy-treated cancer patients.

Abstract 2564: Identification of a novel SPOP missense mutation from targeted next-generation sequencing of men with chromosome 17-q linkage.

Abstract Speckle-type POZ protein (SPOP), located on chromosome 17q21, encodes the substrate-binding subunit of a Cullin-based E3 ubiquitin ligase gene. The SPOP gene has been shown to be one of the most commonly mutated genes in studies focused on characterization of somatic mutations in prostate tumors. In light of the evidence for prostate cancer linkage to chromosome 17q21-22 markers, we hypothesized that germline variation in SPOP may increase the risk for prostate cancer. In this study, 94 unrelated familial prostate cancer cases from the University of Michigan Prostate Cancer Genetics Project (n= 54) and Johns Hopkins University (n=40) were selected from multiplex prostate cancer families based on evidence of linkage to chromosome 17q21-22. Germline DNA samples were subjected to targeted next-generation sequencing of more than 200 genes in the candidate region which included the exons and UTRs of SPOP. One individual, diagnosed with prostate cancer at age 43, was identified to have a novel missense mutation in exon 10 of the gene which results in an amino acid substitution of asparagine to isoleucine at codon 296 (N296I). Tumor DNA from this individual was positive for LOH at neighboring marker D17S943 as well as an ERG fusion documented by immunohistochemistry. Sanger sequencing was performed using genomic DNA from five additional male relatives and two female relatives. Notably, the N296I mutation completely segregates with prostate cancer affection status among the men in this family; the father and a brother with prostate cancer also carry the mutation, as does another brother affected with both prostate and kidney cancer. The mother and two unaffected brothers do not carry the N296I allele, however the sister of the proband was positive for the variant. To our knowledge, neither of the female relatives has been diagnosed with cancer. The SPOP N296I mutation was not detected in genomic DNA samples from a set of 1411 siblings with and without prostate cancer (775 cases and 636 controls) from 573 families. The N296I mutation is present in the evolutionarily conserved Bric-a-brac, Tramtrack, Broad-complex (BTB) domain. Changes to BTB may affect the ability of SPOP to recruit targets to Cul3 for degradation, thus suggesting a role for SPOP mutations in prostate cancer susceptibility. In conclusion, analysis of a set of chromosome 17q21-22 linked families has led to the identification of a novel rare missense mutation in the SPOP gene and further studies are ongoing to determine the potential role of this variant in prostate cancer susceptibility. Citation Format: Kimberly A. Zuhlke, Anna M. Johnson, Scott A. Tomlins, Nallasivam Palanisamy, Christiane M. Robbins, Waibhav A. Tembe, John D. Carpten, Ethan M. Lange, William B. Isaacs, Kathleen A. Cooney. Identification of a novel SPOP missense mutation from targeted next-generation sequencing of men with chromosome 17-q linkage. [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 2564. doi:10.1158/1538-7445.AM2013-2564

Enhanced secretion of biologically active, non-glycosylated VEGF from Saccharomyces cerevisiae

Vascular endothelial growth factor (VEGF) mediates angiogenesis, which plays a critical role in the development and differentiation of the vascular system. VEGF is a homodimeric glycoprotein that contains one N-glycosylation site. In this study, we evaluated Saccharomyces cerevisiae expression systems producing glycosylated and non-glycosylated splice variants of human VEGF, VEGF121, and VEGF165. The pre region of the mating factor α1 (MFα1) signal sequence was found to perform better than the entire MFα1 prepro signal sequence in secreting glycosylated VEGF. Secretion of non-glycosylated VEGF165 was completely blocked, indicating the importance of glycosylation in VEGF165 secretion. Interestingly, non-glycosylated VEGF165 was secreted when guided by the MFα1 prepro signal sequence, albeit to a lesser degree, compared to glycosylated VEGF165. N-glycosylation in the pro region was required for the prepro sequence to promote VEGF secretion. Furthermore, substitution of asparagine at the VEGF glycosylation site with lysine or glutamic acid increased secretion of non-glycosylated VEGF, a finding not previously reported. Our findings suggest that S. cerevisiae could be a suitable host for secreting biologically active, non-glycosylated VEGF for clinical use.

Interplay between drying and stability of a TIM barrel protein: a combined simulation-experimental study.

Recent molecular dynamics simulations have suggested important roles for nanoscale dewetting in the stability, function, and folding dynamics of proteins. Using a synergistic simulation-experimental approach on the αTS TIM barrel protein, we validated this hypothesis by revealing the occurrence of drying inside hydrophobic amino acid clusters and its manifestation in experimental measures of protein stability and structure. Cavities created within three clusters of branched aliphatic amino acids [isoleucine, leucine, and valine (ILV) clusters] were found to experience strong water density fluctuations or intermittent dewetting transitions in simulations. Individually substituting 10 residues in the large ILV cluster at the N-terminus with less hydrophobic alanines showed a weakening or diminishing effect on dewetting that depended on the site of the mutation. Our simulations also demonstrated that replacement of buried leucines with isosteric, polar asparagines enhanced the wetting of the N- and C-terminal clusters. The experimental results on the stability, secondary structure, and compactness of the native and intermediate states for the asparagine variants are consistent with the preferential drying of the large N-terminal cluster in the intermediate. By contrast, the region encompassing the small C-terminal cluster experiences only partial drying in the intermediate, and its structure and stability are unaffected by the asparagine substitution. Surprisingly, the structural distortions required to accommodate the replacement of leucine by asparagine in the N-terminal cluster revealed the existence of alternative stable folds in the native basin. This combined simulation-experimental study demonstrates the critical role of drying within hydrophobic ILV clusters in the folding and stability of the αTS TIM barrel.

Role of DNA Polymerases in Repeat-Mediated Genome Instability

Expansions of simple DNA repeats cause numerous hereditary diseases in humans. We analyzed the role of DNA polymerases in the instability of Friedreich's ataxia (GAA)(n) repeats in a yeast experimental system. The elementary step of expansion corresponded to ~160 bp in the wild-type strain, matching the size of Okazaki fragments in yeast. This step increased when DNA polymerase α was mutated, suggesting a link between the scale of expansions and Okazaki fragment size. Expandable repeats strongly elevated the rate of mutations at substantial distances around them, a phenomenon we call repeat-induced mutagenesis (RIM). Notably, defects in the replicative DNA polymerases δ and ε strongly increased rates for both repeat expansions and RIM. The increases in repeat-mediated instability observed in DNA polymerase δ mutants depended on translesion DNA polymerases. We conclude that repeat expansions and RIM are two sides of the same replicative mechanism.

Negative regulation of CCaMK is essential for symbiotic infection

One of the earliest responses of legumes to symbiotic signalling is oscillation of the calcium concentration in the nucleoplasm of root epidermal cells. Integration and decoding of the calcium-spiking signal involve a calcium- and calmodulin-dependent protein kinase (CCaMK) and its phosphorylation substrates, such as CYCLOPS. Here we describe the Lotus japonicus ccamk-14 mutant that originated from a har1-1 suppressor screen. The ccamk-14 mutation causes a serine to asparagine substitution at position 337 located within the calmodulin binding site, which we determined to be an in vitro phosphorylation site in CCaMK. We show that ccamk-14 exerts cell-specific effects on symbiosis. The mutant is characterized by an increased frequency of epidermal infections and significantly compromised cortical infections by Mesorhizobium loti and also the arbuscular mycorrhiza fungus Rhizophagus irregularis. The S337 residue is conserved across angiosperm CCaMKs, and testing discrete substitutions at this site showed that it participates in a negative regulation of CCaMK activity, which is required for the cell-type-specific integration of symbiotic signalling.

Study of the association between alleles of the growth hormone receptor and prolactin receptor genes of bulls and the milk productivity of their daughters

A substitution of thyrosine for phenylalanine (F297Y) in the transmembrane domain of the growth hormone receptor (GHR) was tested for significance for breeding evaluation of bulls of the holstenized Black-and-White breed. The breeding value was estimated by the method of daughter yield deviation to contemporaries with modification. The frequency of genotype FF in the bulls examined was 0.37, lower than in Holstein bulls (0.67). The F297Y substitution exerted the greatest effect on the milk fat content (1.5 sigma) and milk yield (0.8 sigma) and a lower effect on the milk fat yield (0.6 sigma), milk protein yield (0.5 sigma), and milk protein content (0.6 sigma). The GHR4.2 single nucleotide substitution (SNP) in the promoter of the GHR gene did not affect the milk production traits. A substitution of asparagine for serine (S18N) in the transmembrane domain of the prolactin receptor (PRLR) was also examined, but it did not significantly affect the milk production parameters. The results are discussed in the context of the hypothesis that multiplicity of causal mutations of a particular gene is common and should be taken into account in the genetics of quantitative traits.