Arg Mutation Research Articles

Subtle Changes in the Combining Site of the Chlamydiaceae-Specific mAb S25-23 Increase the Antibody-Carbohydrate Binding Affinity by an Order of Magnitude.

Murine antibodies S25-23, S25-26, and S25-5 derive from a common germ-line origin, and all bind the Chlamydiaceae family-specific epitope αKdo(2→8)αKdo(2→4)αKdo (where Kdo is 3-deoxy-α-d- manno-oct-2-ulosonic acid) with high affinity and specificity. These antibodies recognize the entire trisaccharide antigen in a linkage-dependent manner via a groove composed largely of germ-line residues. Despite sharing identical heavy and light chain genes, S25-23 binds the family-specific epitope with nanomolar affinity, which is an order of magnitude higher than that of S25-26, while S25-5 displays an affinity between those of S25-23 and S25-26. We determined the high-resolution crystal structures of S25-23 and S25-5 antigen binding fragments in complex with a pentasaccharide derived from the LPS of Chlamydia and measured the affinity of S25-5 for chlamydial LPS antigens using isothermal titration microcalorimetry. The 1.75 Å resolution structure of S25-23 shows how subtle conservative mutations Arg(L)-27E to lysine and Ser(H)-56 to threonine lead to an order of magnitude increase in affinity. Importantly, comparison between previous S25-26 structures and the 1.99 and 2.05 Å resolution liganded and unliganded structures of S25-5, respectively, shows how a Ser(L)-27E mutation results in an intermediate affinity due to the reduced enthalpic penalty associated with complex formation that would otherwise be required for arginine in this position. This strategy allows for subtle adjustments in the combining site via affinity maturation that have dramatic consequences for the affinity of an antibody for its antigen.

Glu2.53(90) of the GnRH receptor is part of the conserved G protein-coupled receptor structure and does not form a salt-bridge with Lys3.32(121)

GnRH receptor mutations, Glu2.53(90)Lys and Glu2.53(90)Asp, cause congenital hypogonadotropic hypogonadism. The Glu2.53(90) side-chain has been proposed to form an intramolecular salt-bridge with Lys3.32(121), but conserved intramolecular interaction networks in G protein-coupled receptor crystal structures predict that it interacts with Ser3.35(124) and Trp6.48(280). We investigated interhelical interactions of Glu2.53(90) that stabilise GnRH receptor folding using functional analyses and computational modelling of mutant receptors. The Glu2.53(90)Asp mutant was non-functional, but mutants with hydrophobic amino acids or Arg substituted for Glu2.53(90) were functional, excluding a salt-bridge interaction. The Glu2.53(90)Arg and Trp6.48(280)Arg mutants had decreased affinity for GnRH. Models showed that congenital Glu2.53(90)Lys and Glu2.53(90)Asp mutations disrupt interactions with Ser3.35(124) and Trp6.48(280) respectively, whereas the Glu2.53(90)Arg and Trp6.48(280)Arg mutations preserve intramolecular contacts, but increase distance between the transmembrane helices. Our results show that disruption of interhelical contacts that are conserved in G protein-coupled receptors accounts for the effects of some disease-associated GnRH receptor mutations.

Improving the thermostability by introduction of arginines on the surface of α-L-rhamnosidase (r-Rha1) from Aspergillus niger

To improve the thermostability of α-L-rhamnosidase (r-Rha1), an enzyme previously identified from Aspergillus niger JMU-TS528, multiple arginine (Arg) residues were introduced into the r-Rha1 sequence to replace several lysine (Lys) residues that located on the surface of the folded r-Rha1. Hinted by in silico analysis, five surface Lys residues (K134, K228, K406, K440, K573) were targeted to produce a list of 5 single-residue mutants and 4 multiple-residue mutants using site-directed mutagenesis. Among these mutants, a double Lys to Arg mutant, i.e. K406R/K573R, showed the best thermostability improvement. The half-life of this mutant's enzyme activity increased 3 h at 60 °C, 23 min at 65 °C, and 3.5 min at 70 °C, when compared with the wild type. The simulated protein structure based interaction analysis and molecular dynamics calculation indicate that the thermostability improvement of the mutant K406R-K573R was possibly due to the extra hydrogen bonds, the additional cation-π interactions, and the relatively compact conformation. With the enhanced thermostability, the α-L-rhamnosidase mutant, K406R-K573R, has potentially broadened the r-Rha1 applications in food processing industry.

A novel factor X mutation Cys81 by Arg and a reported factor VII polymorphism Arg353 replaced by Gln co-occured in a patient

: Coagulation factor X and factor VII (FVII) are both very important components in blood coagulation. To study the molecular pathogenic mechanism of the inherited factor X and FVII deficiency, the factor X activity (FX:C) and FVII activity were tested with one-stage clotting methods. The factor X antigen and factor FVII antigen were tested with ELISA. All the exons, intron-exon boundaries and 5',3'-flanking regions of F10 and F7 genes were amplified by PCR with direct sequencing. The ClustalX software was used to analyze the conservative property of the mutation sites. The PolyPhen-2 and Sorting Intolerant From Tolerant (SIFT) online bioinformatics softwares were taken to predict whether the point mutation could affect protein function. The software Swiss-pdb Viewer was brought to analyze the impact of mutations on the structure and function of the protein. The thrombin generation tests were applied to evaluate whether there were obstacles in producing thrombin about the mutant protein. The FX:C and FVII activity of the proband were reduced to 35 and 42%, and the factor X antigen and factor FVII antigen were decreased to 43 and 55%, simultaneously. Correspondingly, a FX:Cys81Arg (Cys81 by Arg) mutation and a FVII:Arg353 replaced by Gln polymorphism were detected in the proband. The Cys81 of factor X was conserved among homologous species, but the Arg353 of FVII was not. All softwares analysis results indicated protein features and structures might be affected by the mutation and the polymorphism. And the thrombin generation tests showed that the mutant protein had obstacles in thrombin generation. We identified a FX:Cys81Arg mutation and a FVII:Arg353 replaced by Gln polymorphism in the proband. And they accounted for the decrease of the activity and antigen of factor X and FVII. Of note, the Cys81Arg of factor X was first reported in the world.

Dysregulated human Tyrosyl-DNA phosphodiesterase I acts as cellular toxin.

Tyrosyl-DNA phosphodiesterase I (TDP1) hydrolyzes the drug-stabilized 3’phospho-tyrosyl bond formed between DNA topoisomerase I (TOPO1) and DNA. TDP1-mediated hydrolysis uses a nucleophilic histidine (Hisnuc) and a general acid/base histidine (Hisgab). A Tdp1Hisgab to Arg mutant identified in patients with the autosomal recessive neurodegenerative disease SCAN1 causes stabilization of the TDP1-DNA intermediate. Based on our previously reported Hisgab-substitutions inducing yeast toxicity (Gajewski et al. J. Mol. Biol. 415, 741-758, 2012), we propose that converting TDP1 into a cellular poison by stabilizing the covalent enzyme-DNA intermediate is a novel therapeutic strategy for cancer treatment. Here, we analyzed the toxic effects of two TDP1 catalytic mutants in HEK293 cells. Expression of human Tdp1HisnucAla and Tdp1HisgabAsn mutants results in stabilization of the covalent TDP1-DNA intermediate and induces cytotoxicity. Moreover, these mutants display reduced in vitro catalytic activity compared to wild type. Co-treatment of Tdp1mutant with topotecan shows more than additive cytotoxicity. Overall, these results support the hypothesis that stabilization of the TDP1-DNA covalent intermediate is a potential anti-cancer therapeutic strategy.

Acquisition of pro-oxidant activity of fALS-linked SOD1 mutants as revealed using circular dichroism and UV-resonance Raman spectroscopy

The acquisition of pro-oxidant activity of the mutated form of human Cu, Zn-superoxide dismutase (SOD1) has been investigated to clarify the relationship between mutations in SOD1 and the pathogenesis of amyotrophic lateral sclerosis (ALS). Ala4→Val (A4V) and Gly93→Ala (G93A) mutants, which are representative ALS-linked SOD1 mutants, have been found to exhibit both the denaturation and the gain of pro-oxidant activity after incubation in the apo-form at a physiological condition of 37°C and pH 7.4 and the rebinding of Cu2+. These characteristics are similar to those previously reported for the His43→Arg (H43R) mutant. UV-resonance Raman spectra indicated that the coordination structure of the Cu-binding site catalyzing the oxidation reaction is the same among the denatured A4V, G93A, and H43R. Since wild-type SOD1 does not exhibit the denaturation in its apo-form at 37°C and pH 7.4, the instability of the protein structure due to mutation can be considered as a significant factor that induces the denaturation and the subsequent pro-oxidant activity.

Abstract 2761: Cellular consequences of human tyrosyl-DNA phosphodiesterase I dysregulation

Abstract Tyrosyl-DNA phosphodiesterase I (Tdp1) is a highly conserved eukaryotic DNA repair enzyme that catalyzes the resolution of 3’ and 5’ phospho-DNA adducts. Tdp1 has been implicated in the repair of DNA topoisomerase I (Topo1)-DNA covalent complexes reversibly stabilized by camptothecins (CPTs) such as the FDA approved CPT derivatives topotecan and irinotecan. Tdp1 utilizes a two-step catalytic cycle that centers on the formation of an obligatory Tdp1-DNA covalent complex (Tdp1-cc) through its nucleophilic histidine (Hisnuc), resulting in dissociation of the adduct, while its general acid/base histidine (Hisgab) mediates Tdp1 dissociation. A Tdp1Hisgab to Arg (H493R) mutant stabilizes the Tdp1-cc and is associated with autosomal recessive ataxia SCAN1. Alternative substitutions of Hisgab or substitutions of the Hisnuc transforms yeast Tdp1 into a potent toxin via stabilization of Tdp1-cc. We propose that stabilization of this Tdp1-DNA covalent complex is a potential novel therapeutic anti-cancer strategy. As proof-of-concept, we analyzed two catalytic Hisgab (H493R or H493N) mutants and one Hisnuc (H263A) mutant of hTdp1 in HEK293 cells. Doxycycline-induced expression of Tdp1H263A, Tdp1H493R, and Tdp1H493N mutant enzymes induced Tdp1-dependent cytotoxicity without additional genotoxic stress. Utilizing two different immuno-assays, we validated that the observed Tdp1-dependent toxicity correlates with stabilization of their enzyme-DNA covalent complex. Moreover, all of these Tdp1 catalytic mutants show reduced catalytic activity compared to wild type hTdp1, but they do not all show a stabilized Tdp1-cc in this in vitro assay. This indicates a significant difference between in vitro Tdp1 activity and cellular Tdp1 activity, which is most likely due to the difference in substrate; a small oligonucleotide with a 3’phospho-tyrosyl modification versus covalent complex of full length Topo1 with genomic DNA. However, these results confirm our previous yeast studies: Stabilization of the Tdp1-cc converts a DNA repair enzyme into a cellular toxin, which constitutes a potential novel therapeutic strategy to treat cancer. In addition, we are comparing schedule dependent ‘drug’-combinations of our toxic Tdp1 mutant expression with topotecan, etoposide (targets Topo2-cc) and cisplatin to evaluate the potential therapeutic value of this novel Tdp1 targeted strategy. This work is in part supported by the ADDA, UAB ACS-IRG, and DOD OCRP WX81WH-15-1-0198. Citation Format: Selma M. Cuya, Kellie M. Regal, Robert C.A.M. Van Waardenburg. Cellular consequences of human tyrosyl-DNA phosphodiesterase I dysregulation. [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 2761.

Point mutation (R153H or R153C) in Escherichia coli isocitrate dehydrogenase: Biochemical characterization and functional implication.

Arginine 132 (R132) mutations to histidine or cysteine frequently occur to cytosolic NADP+ -isocitrate dehydrogenase (IDH1) in secondary glioblastoma multiforme (GBM) patients, in which GBM develops from a lower grade astroctyoma. Mutant enzymes lose the normal IDH activity, but acquire a neomorphic ability of producing 2-hydroxyglutarate (2-HG) from α-ketoglutarate (α-KG). In the present study, the analogous mutations, Arg to His or Cys, were employed to homologous Arg153 of the NADP+ -IDH from Escherichia coli (EcIDH), generating two mutants: EcIDH R153 H and EcIDH R153C. The mutations dramatically reduced the catalytic efficiencies (kcat /Km ) of EcIDH R153H and EcIDH R153C for isocitrate oxidation, which dropped to only 0.6 and 1.5% of the wild-type enzyme, respectively. Neoenzymatic activities of catalyzing α-KG to 2-HG by EcIDH R153H and EcIDH R153C were confirmed by GC/TOF-MS analysis. The Km values of EcIDH R153H and EcIDH R153C displayed for α-KG were 3.3 ± 0.12 and 2.2 ± 0.13 mM, respectively, and the catalytic efficiencies (kcat /Km ) of the two mutants for α-KG were 300 and 450 M-1 s-1 , respectively. As human IDH1 Arg132 mutation is cancer-associated, the present study provides new information for the in-depth investigation of the metabolic influence of EcIDH Arg mutation in vivo.

Conformational Change of Human Checkpoint Kinase 1 (Chk1) Induced by DNA Damage

Phosphorylation of Chk1 by ataxia telangiectasia-mutated and Rad3-related (ATR) is critical for checkpoint activation upon DNA damage. However, how phosphorylation activates Chk1 remains unclear. Many studies suggest a conformational change model of Chk1 activation in which phosphorylation shifts Chk1 from a closed inactive conformation to an open active conformation during the DNA damage response. However, no structural study has been reported to support this Chk1 activation model. Here we used FRET and bimolecular fluorescence complementary techniques to show that Chk1 indeed maintains a closed conformation in the absence of DNA damage through an intramolecular interaction between a region (residues 31-87) at the N-terminal kinase domain and the distal C terminus. A highly conserved Leu-449 at the C terminus is important for this intramolecular interaction. We further showed that abolishing the intramolecular interaction by a Leu-449 to Arg mutation or inducing ATR-dependent Chk1 phosphorylation by DNA damage disrupts the closed conformation, leading to an open and activated conformation of Chk1. These data provide significant insight into the mechanisms of Chk1 activation during the DNA damage response.

Cholesterol Influence on Arginine-Containing Transmembrane Peptides

An essential component of animal cells, cholesterol exerts significant influence on the physical properties of the membrane and in turn, its constituents. One such constituent, the membrane protein, often contains polar amino acids. Although sparse, polar residues are highly conserved and play pivotal roles in determining specific structural and functional properties. To gain greater understanding of the membrane, and more broadly, cellular function, a model peptide framework termed “GWALP23” (acetyl-GGALWLALALAL12AL14ALALWLAGA-amide) is useful. The limited dynamic averaging of NMR observables such as the deuterium quadrupolar splittings of labeled alanine residues makes GWALP23 favorable for single residue replacements. Previously, GWALP23 family peptides were characterized with single Leu to Arg mutations at positions 12 and 14 in single-lipid membranes [J. Am. Chem. Soc., 132, 5803-5811, 2010]. GWALP23-R14 adopts a defined tilted orientation in DOPC bilayers, whereas GWALP23-R12 displays multi-state behavior. The goal of this research is to further characterize these peptides in cholesterol-containing bilayers. Specific deuterium-labeled alanine residues were incorporated into the R12 and R14 sequences to identify transmembrane peptide orientation by means of solid-state deuterium NMR. Both peptides were incorporated into phospholipid bilayers with varying cholesterol content (0%, 10%, or 20%). Our findings suggest that 10% or 20% cholesterol content has minimal impact on the orientation of GWALP23-R14 peptide. (Although the NMR signals are broader and weaker in the presence of 20% cholesterol, the deuterium quadrupolar splittings for 2H-Ala residues in GWALP23-R14 change little.) Conversely, cholesterol appears to reduce the multi-state behavior of GWALP23-R12, favoring a single transmembrane state for the helix. With 10% or 20% cholesterol content, the spectra exhibit defined quadrupolar splittings, suggesting that GWALP23-R12 adopts a predominant, tilted orientation in the presence of cholesterol. These results convey a conditional sensitivity of a complex multi-state peptide helix to the presence of cholesterol.

Bisalbuminaemia due to novel mutation at a critical residue involved in recycling; Albumin Lyon (510His → Arg)

ObjectivesTo define the underlying cause of bisalbuminaemia in an individual presenting with spontaneous venous thrombosis. MethodPlasma was examined by electrospray time-of-flight mass spectrometry (TOF MS) to assess albumin mutations and to quantify variant expression level. Tryptic peptide mapping and DNA sequencing were used to precisely define the mutation. ResultsWhole protein MS indicated a 19Da increase in the mass of 50% of the albumin molecules suggesting a His→Arg substitution. A novel heterozygous 510His→Arg mutation was identified by peptide mass mapping and confirmed by DNA sequencing of exon 12 of the albumin gene. ConclusionThe nature and location of the mutation suggest it would have no direct influence on haemostasis through altered warfarin binding or increased fibrinogen attachment and it appears to be incidental to the thrombotic phenotype. However the highly conserved His510 residue is recognised as being of critical importance in albumin recycling through interaction with its savaging neonatal Fc receptor. The normal albumin level of 41.1g/l and the coequal expression of albumin Lyon demonstrate that the conservative 510His→Arg substitution does not interfere with the pH dependant capture and release of albumin by the receptor.

Processivity, Substrate Positioning, and Binding: The Role of Polar Residues in a Family 18 Glycoside Hydrolase

The enzymatic degradation of recalcitrant polysaccharides such as cellulose (β-1,4-linked glucose) and chitin (β-1,4-linked N-acetylglucosamine) by glycoside hydrolases (GHs) is of significant biological and economical importance. In nature, depolymerization is primarily accomplished by processive GHs, which remain attached to the substrate between subsequent hydrolytic reactions. Recent computational efforts have suggested that the processive ability of a GH is directly linked to the ligand binding free energy. The contribution of individual aromatic residues in the active site of these enzymes has been extensively studied. In this study, we offer the first experimental evidence confirming correlation of binding free energy and degree of processivity and evidence that polar residues are essential for maintaining processive ability. Exchanging Thr(276) with Ala in substrate binding subsite -2 in the processive ChiA of Serratia marcescens results in a decrease in both the enthalpy (2.6 and 3.8 kcal/mol) and free energy (0.5 and 2.2 kcal/mol) for the binding to the substrate (GlcNAc)6 and the inhibitor allosamidin, respectively, compared to that of the wild type. Moreover, the initial apparent processivity as measured by [(GlcNAc)2]/[GlcNAc] ratios (17.1 ± 0.4) and chitin degradation efficiency (20%) are greatly reduced for ChiA-T276A versus those of the wild type (30.1 ± 1.5 and 75%, respectively). Mutation of Arg(172) to Ala reduces the level of recognition and positioning of the substrate into the active site. Molecular dynamics simulations indicate ChiA-R172A behaves like the wild type, but the dynamics of ChiA-T276A are greatly influenced by mutation, which is reflective of their influence on processivity.

Abstract 3628: Tyrosyl-DNA phosphodiesterase I as a therapeutic target: lessons from yeast functional studies

Abstract Tyrosyl-DNA phosphodiesterase I (Tdp1) resolves various 3′phospho-adducts within DNA breaks induced by numerous chemotherapeutics. This includes Tdp1 ability to repair DNA topoisomerase I (Topo1)-DNA covalent complexes reversibly stabilized by camptothecins (CPTs) such as the FDA approved CPT derivatives topotecan and irinotecan. Tdp1 uses a two-step catalytic cycle that requires the formation of a Tdp1-DNA covalent intermediate through its nucleophilic histidine (Hisnuc) resulting in dissociation of the adduct, while the general acid/base histidine (Hisgab) mediates Tdp1 dissociation. A Tdp1Hisgab to Arg mutant stabilizes the Tdp1-DNA intermediate and is associated with autosomal recessive ataxia SCAN1. Alternative substitutions of Hisgab transforms yeast Tdp1 into a potent Topo1-depended toxin via stabilization of enzyme-DNA intermediates. We propose that stabilization of the Tdp1-DNA complex is a potential novel therapeutic anti-cancer strategy. As proof-of-concept, we analyzed two different catalytic human Tdp1 mutants in HEK293 cells without additional stress. Expression of hTdp1HisnucAla and hTdp1HisgabAsn mutants induced cytotoxicity, which correlates with stabilization of their enzyme-DNA intermediates. Serendipitously, we discovered that Tdp1's N-terminal residues are critical for Tdp1HisnucAla catalytic activity but not for wild type Tdp1. In addition, analyzing the effect of the N-terminal domain of other Tdp1 catalytic mutants revealed that these residues influence the formation of covalent Tdp1-DNA intermediates in vitro, which is conserved from yeast to human. To better understand the interaction between Tdp1 and its substrates, we examined the cellular role of the poorly conserved N-terminal domain (∼80aa yTdp1 and 140aa hTdp1). This domain is not essential for catalytic activity per se, however, we observed that it is critical for Tdp1 cellular function in yeast and human cells. Comparison of the full-length and N-terminal truncated Tdp1 mutants showed similar cellular distribution, but a converse toxicity. This suggests that the N-terminal domain is a critical determinant of Tdp1 cellular function and this function is conserved from yeast to human. Thus, understanding the mechanism of interaction between Tdp1 and Topo1-DNA intermediate is important for the development of Tdp1 as a therapeutic target. Overall, these results support our concept that stabilization of Tdp1-DNA covalent intermediates converting this DNA repair enzyme into a cellular toxin is a potential novel anti-cancer therapeutic strategy, which is different from the intuitive strategy of inhibiting (preventing) Tdp1 catalytic activity. This work is in part supported by the ADDA. Citation Format: Selma M. Cuya, Ashley C. Conoway, Robert C.A.M. van Waardenburg. Tyrosyl-DNA phosphodiesterase I as a therapeutic target: lessons from yeast functional studies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3628. doi:10.1158/1538-7445.AM2015-3628

Bone metastasis in prostate cancer: Recurring mitochondrial DNA mutation reveals selective pressure exerted by the bone microenvironment

BackgroundCancer progression and metastasis occur such that cells with acquired mutations enhancing growth and survival (or inhibiting cell death) increase in number, a concept that has been recognized as analogous to Darwinian evolution of species since Peter C. Nowell's description in 1976. Selective forces include those intrinsic to the host (including metastatic site) as well as those resulting from anti-cancer therapies. By examining the mutational status of multiple tumor sites within an individual patient some insight may be gained into those genetic variants that enhance site-specific metastasis. By comparing these data across multiple individuals, recurrent patterns may identify alterations that are fundamental to successful site-specific metastasis. MethodsWe sequenced the mitochondrial genome in 10 prostate cancer patients with bone metastases enrolled in a rapid autopsy program. Patients had late stage disease and received androgen ablation and frequently other systemic therapies. For each of 9 patients, 4 separate tissues were sequenced: the primary prostate cancer, a soft tissue metastasis, a bone metastasis and an uninvolved normal tissue that served as the non-cancerous control. An additional (10th) patient had no primary prostate available for sequencing but had both metastatic sites (and control DNA) sequenced. We then examined the number and location of somatically acquired mitochondrial DNA (mtDNA) mutations in the primary tumor and two metastatic sites in each individual patient. Finally, we compared patients with each other to determine any common patterns of somatic mutation. ResultsSomatic mutations were significantly more numerous in the bone compared to either the primary tumor or soft tissue metastases. A missense mutation at nucleotide position (n.p.) 10398 (A10398G; Thr114Ala) in the respiratory complex I gene ND3 was the most common (7 of 10 patients) and was detected only in the bone. Other notable somatic mutations that occurred in more than one patient include a tRNA Arg mutation at n.p. 10436 and a tRNA Thr mutation at n.p. 15928. The tRNA Arg mutation was restricted to bone metastases and occurred in three of 10 patients (30%). Somatic mutation at 15928 was not restricted to the bone and also occurred in three patients. ConclusionsMitochondrial genomic variation was greater in metastatic sites than in the primary tumor and bone metastases had statistically significantly greater numbers of somatic mutations than either the primary or the soft tissue metastases. The genome was not mutated randomly. At least one mutational “hot-spot” was identified at the individual base level (nucleotide position 10398 in bone metastases) indicating a pervasive selective pressure for bone metastatic cells that had acquired the 10398 mtDNA mutation. Two additional recurrent mutations (tRNA Arg and tRNA Thr) support the concept of bone site-specific “survival of the fittest” as revealed by variation in the mitochondrial genome and selective pressure exerted by the metastatic site.

Dissociation of a BRICHOS trimer into monomers leads to increased inhibitory effect on Aβ42 fibril formation

The BRICHOS domain is associated with human amyloid disease, and it efficiently prevents amyloid fibril formation of the amyloid β-peptide (Aβ) in vitro and in vivo. Recombinant human prosurfactant protein C (proSP-C) BRICHOS domain forms a homotrimer as observed by x-ray crystallography, analytical ultracentrifugation, native polyacrylamide gel electrophoresis, analytical size exclusion chromatography and electrospray mass spectrometry. It was hypothesized that the trimer is an inactive storage form, as a putative substrate-binding site identified in the monomer, is buried in the subunit interface of the trimer. We show here increased dissociation of the BRICHOS trimer into monomers, by addition of detergents or of bis-ANS, known to bind to the putative substrate-binding site, or by introducing a Ser to Arg mutation expected to interfere with trimer formation. This leads to increased capacity to delay Aβ42 fibril formation. Cross-linking of the BRICHOS trimer with glutaraldehyde, in contrast, renders it unable to affect Aβ42 fibril formation. Moreover, proSP-C BRICHOS expressed in HEK293 cells is mainly monomeric, as detected by proximity ligation assay. Finally, proteolytic cleavage of BRICHOS in a loop region that is cleaved during proSP-C biosynthesis results in increased capacity to delay Aβ42 fibril formation. These results indicate that modulation of the accessibility of the substrate-binding site is a means to regulate BRICHOS activity.

Abl2/Abl-related Gene Stabilizes Actin Filaments, Stimulates Actin Branching by Actin-related Protein 2/3 Complex, and Promotes Actin Filament Severing by Cofilin

Both Arp2/3 complex and the Abl2/Arg nonreceptor tyrosine kinase are essential to form and maintain diverse actin-based structures in cells, including cell edge protrusions in fibroblasts and cancer cells and dendritic spines in neurons. The ability of Arg to promote cell edge protrusions in fibroblasts does not absolutely require kinase activity, raising the question of how Arg might modulate actin assembly and turnover in the absence of kinase function. Arg has two distinct actin-binding domains and interacts physically and functionally with cortactin, an activator of the Arp2/3 complex. However, it was not known whether and how Arg influences actin filament stability, actin branch formation, or cofilin-mediated actin severing or how cortactin influences these reactions of Arg with actin. Arg or cortactin bound to actin filaments stabilizes them from depolymerization. Low concentrations of Arg and cortactin cooperate to stabilize filaments by slowing depolymerization. Arg stimulates formation of actin filament branches by Arp2/3 complex and cortactin. An Arg mutant lacking the C-terminal calponin homology actin-binding domain stimulates actin branch formation by the Arp2/3 complex, indicative of autoinhibition. ArgΔCH can stimulate the Arp2/3 complex even in the absence of cortactin. Arg greatly potentiates cofilin severing of actin filaments, and cortactin attenuates this enhanced severing. The ability of Arg to stabilize filaments, promote branching, and increase severing requires the internal (I/L)WEQ actin-binding domain. These activities likely underlie important roles that Arg plays in the formation, dynamics, and stability of actin-based cellular structures.

Modifications of human growth differentiation factor 9 to improve the generation of embryos from low competence oocytes.

Growth differentiation factor 9 (GDF9) is an oocyte-derived growth factor that plays a critical role in ovarian folliculogenesis and oocyte developmental competence and belongs to the TGF-β family of proteins. Recombinant human GDF9 (hGDF9) is secreted in a latent form, which in the case of the fully processed protein, has the proregion noncovalently associated with the mature region. In this study, we investigated a number of amino acid residues in the mature region of hGDF9 that are different from the corresponding residues in the mouse protein, which is not latent. We designed, expressed, and purified 4 forms of chimeric hGDF9 (M1-M4) that we found to be active in a granulosa cell bioassay. Using a porcine in vitro maturation model with inherent low developmental competence (yielding 10%-20% blastocysts), we tested the ability of the chimeric hGDF9 proteins to improve oocyte maturation and developmental competence. Interestingly, one of the chimeric proteins, M3, was able to significantly increase the level of embryo production using such low competence oocytes. Our molecular modeling studies suggest that in the case of hGDF9 the Gly(391)Arg mutation probably increases receptor binding affinity, thereby creating an active protein for granulosa cells in vitro. However, for an improvement in oocyte developmental competence, a second mutation (Ser(412)Pro), which potentially decreases the affinity of the mature region for the proregion, is also required.

Mechanisms of Activation of Slo2.1 Channels by Intracellular Na+

The open probability of Slo2 K+ channels is low under physiological conditions, but is increased dramatically by elevation of [Na+]i. Mutation of the Asp in the Na+ coordination motif (DXRXXH) of Kir3 or Slo2.2 channels was previously reported to greatly reduce channel sensitivity to [Na+]i. The intracellular C-terminus of Slo2.1 subunits contains five potential Na+ binding motifs. The Asp residue in each of these five sequences was mutated to Arg. Surprisingly, unlike Slo2.2, Slo2.1 channel currents showed little or no activity with high [Na+]i (100 or 300 mM) in excised inside-out macro-patches from Xenopus oocytes or HEK cells. Addition of PIP2, Ca2+, cAMP, NAD+, GTP and ATP to the high Na+ solution also failed to activate Slo2.1. The presence of functional Slo2.1 channels in the patch was confirmed by activation with niflumic acid (NFA). To circumvent this problem, activation of channels by elevated [NaCl]i was studied using whole cell currents. Channels containing a single Asp to Arg mutation were expressed in Xenopus oocytes and currents were recorded by using two-microelectrode voltage clamp. [NaCl]i was elevated by using microelectrodes filled with 2M NaCl. All mutant channels other than D757R responded to intracellular NaCl loading similar to WT channels. D757R channels were activated by 1 mM NFA, confirming their functional expression. Similar results were obtained for whole cell currents recorded in HEK cells expressing WT or D757R Slo2.1 channels. With 70 mM NaCl in the pipette solution, WT Slo2.1 current increased 30-fold, whereas D757R currents were not activated. Both WT and D757R channel currents were increased >150-fold by 0.5-1 mM NFA. Together these findings indicate that a crucial soluble cofactor required for activation of channels is lost upon patch excision and that Asp757 is the primary determinant of Na+ activation of Slo2.1.

Conformational Activation of Antithrombin by Heparin Involves an Altered Exosite Interaction with Protease

Heparin allosterically activates antithrombin as an inhibitor of factors Xa and IXa by enhancing the initial Michaelis complex interaction of inhibitor with protease through exosites. Here, we investigate the mechanism of this enhancement by analyzing the effects of alanine mutations of six putative antithrombin exosite residues and three complementary protease exosite residues on antithrombin reactivity with these proteases in unactivated and heparin-activated states. Mutations of antithrombin Tyr(253) and His(319) exosite residues produced massive 10-200-fold losses in reactivity with factors Xa and IXa in both unactivated and heparin-activated states, indicating that these residues made critical attractive interactions with protease independent of heparin activation. By contrast, mutations of Asn(233), Arg(235), Glu(237), and Glu(255) exosite residues showed that these residues made both repulsive and attractive interactions with protease that depended on the activation state and whether the critical Tyr(253)/His(319) residues were mutated. Mutation of factor Xa Arg(143), Lys(148), and Arg(150) residues that interact with the exosite in the x-ray structure of the Michaelis complex confirmed the importance of all residues for heparin-activated antithrombin reactivity and Arg(150) for native serpin reactivity. These results demonstrate that the exosite is a key determinant of antithrombin reactivity with factors Xa and IXa in the native as well as the heparin-activated state and support a new model of allosteric activation we recently proposed in which a balance between attractive and repulsive exosite interactions in the native state is shifted to favor the attractive interactions in the activated state through core conformational changes induced by heparin binding.

Solution NMR structures of immunoglobulin-like domains 7 and 12 from obscurin-like protein 1 contribute to the structural coverage of the human cancer protein interaction network

High-quality solution NMR structures of immunoglobulin-like domains 7 and 12 from human obscurin-like protein 1 were solved. The two domains share 30% sequence identity and their structures are, as expected, rather similar. The new structures contribute to structural coverage of human cancer associated proteins. Mutations of Arg 812 in domain 7 cause the rare 3-M syndrome, and this site is located in a surface area predicted to be involved in protein-protein interactions.