Thr Mutation Research Articles

The functional change of the P2X7R containing the Ala348 to Thr polymorphism is associated with the pathogenesis of gout

Our previous study has shown that ATP action on P2X7R could be the second signal to induce the onset of gouty arthritis. However, the functional changes of P2X7R single nucleotide polymorphisms (SNPs) on the effects of ATP-P2X7R-IL-1β signaling pathway and uric acid remained unknown. We aimed to investigate the association between the functional change of P2X7R containing the Ala348 to Thr polymorphisms (rs1718119) and the pathogenesis of gout. First, 270 gout patients and 70 hyperuricemic patients (without gout attack history in recent 5 years) were recruited for genotyping. In addition, the changes of ATP-induced pore formation were assessed in HEK-293T cells overexpressing different mutants in P2RX7, and the effects on P2X7R-NLRP3-IL-1β pathway activation were explored in P2RX7 overexpression THP-1 cells. The risk allele for gout was A at rs1718119, and the AA and AG genotypes exhibited a higher risk of gout. Furthermore, Ala348 to Thr mutants increased P2X7-dependent ethidium+ bromide uptake, upregulated IL-1β and NLRP3 levels as compared to the wild-type. We suggest that genetic polymorphisms of P2X7R containing the Ala348 to Thr are associated with the increased risk of gout, showing an enhanced gain-of-function effect on the development of this disease.

A novel steroid hydroxylase from Nigrospora sphaerica with various hydroxylation capabilities to different steroid substrates

Fungal hydroxylation of steroids is a key step in the industrial production of various steroid drugs. The main enzymes that enable these reactions are Cytochrome P450s (CYP), though very few industrially important CYPs have been identified and characterized. In this study, we identified a CYP enzyme (CYP-N2) and a cytochrome P450 reductase (CPRns) from Nigrospora sphaerica 722 by a combination of transcriptome sequencing and heterologous expression in Pichia pastoris. Gene CYP-N2 co-expressed with CPRns in Pichia pastoris GS115 showed 6β- and 15α-hydroxylation activities on progesterone. Different hydroxylation specificity of CYP-N2 was observed on different steroid substrates. CYP-N2 showed 1α-hydroxylation on cortisone and 1α-hydroxylation and 6β-hydroxylation activities on androstenedione (AD). With dehydroepiandrosterone (DHEA) as a substrate, the hydroxylated products of CYP-N2 included 7α-hydroxy-DHEA and 7α,15α-dihydroxy-DHEA. In order to precisely elucidate CYP-N2 biological function and find out the key amino acids influencing its hydroxylation capabilities in the binding pocket, new generation artificial intelligence technology AlphaFold 2 was used to predict the function-structure of CYP-N2 with high reliability. Through molecular docking, it was concluded that the residues almost binding all substrates were located in the same substrate binding pocket and the various hydroxylation abilities might be due to the different binding conformations of different substrates in the binding pocket. Alanine scanning mutagenesis was used to verify key amino acids identified by the molecular docking with steroid substrates. The 128 THR mutation resulted in conversion rate increase for substrates AD and cortisone by 2.6-fold and 2.1-fold respectively. The information obtained in this study is beneficial to facilitating the engineering of more efficient steroid hydroxylases for industrial applications.

Suppression of Linear Ubiquitination Ameliorates Cytoplasmic Aggregation of Truncated TDP-43.

TAR DNA-binding protein 43 (TDP-43) is a predominant component of inclusions in the brains and spines of patients with amyotrophic lateral sclerosis (ALS). The progressive accumulation of inclusions leads to proteinopathy in neurons. We have previously shown that Met1(M1)-linked linear ubiquitin, which is specifically generated by the linear ubiquitin chain assembly complex (LUBAC), is colocalized with TDP-43 inclusions in neurons from optineurin-associated familial and sporadic ALS patients, and affects NF-κB activation and apoptosis. To examine the effects of LUBAC-mediated linear ubiquitination on TDP-43 proteinopathies, we performed cell biological analyses using full-length and truncated forms of the ALS-associated Ala315→Thr (A315T) mutant of TDP-43 in Neuro2a cells. The truncated A315T mutants of TDP-43, which lack a nuclear localization signal, efficiently generated cytoplasmic aggregates that were colocalized with multiple ubiquitin chains such as M1-, Lys(K)48-, and K63-chains. Genetic ablation of HOIP or treatment with a LUBAC inhibitor, HOIPIN-8, suppressed the cytoplasmic aggregation of A315T mutants of TDP-43. Moreover, the enhanced TNF-α-mediated NF-κB activity by truncated TDP-43 mutants was eliminated in the presence of HOIPIN-8. These results suggest that multiple ubiquitinations of TDP-43 including M1-ubiquitin affect protein aggregation and inflammatory responses in vitro, and therefore, LUBAC inhibition ameliorates TDP-43 proteinopathy.

MET235THR POLYMORPHISM IN THE AGT GENE AS THE DEVELOPMENT FACTOR OF SEVERE PREECLAMPSIA

Objective: Every year, as a prediction risk of developing preeclampsia, experts are attracted by polymorphisms of some genes involved in the regulation of vascular tone and endothelial function. The purpose - to establish the features of Met235Thr polymorphism in the AGT gene in the development of severe preeclampsia. Design and method: We examined 139 pregnant women in the third trimester of gestation which were divided into 2 groups: The main group - 56 pregnant women with severe preeclampsia. The control group is 83 conditionally healthy women. The age of the pregnant women was 19–37 years. The material for the study was DNA samples from pregnant women. Isolation of DNA from blood and PCR analysis were performed with kits of reagents and test systems from Ampli Prime Ribot-prep. Results: The shares of the Thr allele, being at very close levels in values, were insignificantly less in the control group, where they were 36.9%, versus 38.4% among patients with severe preeclampsia. The detection rate of the homozygous wild Met / Met genotype of the Met235Thr polymorphism of the AGT gene was also insignificantly higher in the control group, being at the level of 43.0% (x2 = 0.15; p = 0.70; RR = 0.92; 95% CI: 0.33–2.54; OR = 0.87; 95% CI: 0.42–1.79), which was insignificantly higher than among patients with severe preeclampsia 39.5%. The heterozygous Met / Thr genotype, also relatively evenly distributed between the groups, was insignificantly less frequently detected in the control group, amounting to 40.2% (x2 = 0.20; p = 0.67; RR = 1.1; 95% CI: 0.41–2.97; OR = 1.18; 95 % CI: 0.58–2.41), versus 44.2% among patients with severe preeclampsia. The homozygous mutant Thr / Thr genotype, also being at a comparable level, prevailed statistically insignificantly in the control group (16.8%), relative to the group of patients (16.3%) (x2 = 0.01; p = 0.94; RR = 0.97; 95% CI: 0.25–3.72; OR = 0.96; 95% CI: 0.38–2.44) Conclusions: We can conclude that the Met235Thr polymorphism in the AGT gene in women with severe preeclampsia has clinical prognostic significance.

Entropy in Biochemical Failure

The ab-initio determination of the thermodynamic properties of the hydrolysis of the GTP gamma-phosphate in normal and abnormal cell functions of the RAS protein mutant Thr (Q61) leads to a description of energy cycle deviations in the abnormal mitogen-activated protein kinase cascade. 1 A predictive non-equilibrium probability statement describing the nonlinear changes for these open and finite-lifetime systems follows from reasonable enthalpy and entropy values between the normal and mutated forms based on structures of the GTPase states at the allosteric site. Recent advances in understanding entropy in terms of asymmetric and highly entropic catalysis lead to an investigation of the GTPase entropy, specifically with regard to a failure in catalysis of the phosphate fragment by a water hydrogen positioned by the enzyme. 2 Utilizing a simple atomic metal catalyst surface displacement model, a paradigm that reduces noise from the quantum entanglement plus atomic displacement terms results in the process entropy. The evaluation of entropies within the mixed ionic, covalent and entangled system requires a nonlinear Markovian approach utilizing von Neumann entropies achieved by a systematic accumulation of entangled potentials in a step-wise method. 3 , 4 Determination of the Hamiltonian for the entangled atomic state includes pure and mixed quantum states solved within the Araki–Leib triangle boundary resulting in only hard-entangled states, and the entanglement of Coulombic and Laughlin-like states can be evaluated by slicing the Hilbert spaces and solving the pure states, or mixed states separately, and then summing them. 5 Incorporating the resulting entanglement potentials as well as the Coulombic atomic displacement states into a derivative of the Fokker–Planck equation results in generated and produced entropy. 6

Genotypic Characterization of katG, inhA, and ahpC in Isoniazid-Resistant Mycobacterium tuberculosis Clinical Isolates in Shanghai, China

BackgroundMycobacterium tuberculosis is a pathogen that causes Tuberculosis and can invade various organs in infected patients. Its high morbidity and high mortality seriously threaten human health. In recent years, the continuous emergence of drug-resistant Tuberculosis bacteria has brought severe challenges to the prevention and control of Tuberculosis.ObjectivesThis study aimed to characterize the most frequent mutations of the katG, inhA, and ahpC genes in isoniazid (INH)-resistant M. tuberculosis clinical isolates in Shanghai Pulmonary Hospital, China, and investigate the relationship between gene mutations and the minimum inhibitory concentrations (MICs) of INH against M. tuberculosis.MethodsWe collected 92 INH-resistant and 30 INH-susceptible clinical isolates of M. tuberculosis. The drug resistance profiles of M. tuberculosis clinical isolates against common anti-tuberculosis drugs were determined and sequencing analysis was performed.ResultsOf 92 INH-resistant strains, mutations in the katG and inhA genes were observed in 64 (69.6%) isolates and five (5.4%) isolates, respectively, and only had one (1.1%) strain both katG and inhA mutations. Among them, 62 (67.4%) strains carried a single mutation at codon 315 of the katG gene and a new mutation site was found in the katG gene of two strains. We detected a single mutation site at codon 271 and three simultaneous mutation sites at codons 315, 431, and 439. Only one (3.3%) of the 30 isoniazid-sensitive strains had the katG mutation. The AhpC mutation was detected in no experimental strains. The KatG Ser 315 Thr (AGC315ACC) mutation occurred in 53 (68.8%) out of 77 strains with high MICs (≥ 1 g/mL) of isoniazid-resistant M. tuberculosis while five (33.3%) out of 15 strains with low MICs (less than 1 g/mL) had katG Ser 315 Thr (AGC315ACC) mutation.ConclusionsIsoniazid-resistant strains were dominated by Ser315 → Thr (AGC → ACC) substitution, which seems to be associated with multidrug resistance and high-level resistance to INH. Multisite mutations are related to multidrug-resistant M. tuberculosis and the discovery of new mutation sites provides a new basis for the detection of drug-resistant M. tuberculosis.

A phosphorylation-deficient mutant of retinoid X receptor α at Thr 167 alters fasting response and energy metabolism in mice

Retinoid X receptor α (RXRα) has a conserved phosphorylation motif at threonine 162 (humans) and threonine 167 (mice) within the DNA-binding domain. Here we have generated RXRα knock-in mice (RxrαT167A) bearing a single mutation of Thr 167 to alanine and examined the roles of Thr 167 in the regulation of energy metabolism within adipose, muscle, and liver tissues. RxrαT167A mice exhibited down-regulation of metabolic pathways converting glucose to fatty acids, such as acetyl-CoA carboxylase in the white adipose tissue (WAT) and ATP citrate lyase in the muscle. They also reduced gene expression for genes related to fatty acid catabolism and triglyceride synthesis in WAT and controlled heat factors such as adrenergic receptor β1 in muscles. In contrast, hepatic gluconeogenic pathways and synthetic pathways related to fatty acids remained unaffected by this mutation. Expression of multiple genes that were affected by the Thr 167 mutation in adipose tissue exhibited clear response to LG100268, a synthetic RXR agonist. Thus, the altered gene expression in mutant mice adipose appeared to be a direct effect of RXRα Thr 167 mutation and by some secondary effect of the mutation. Blood glucose levels remained normal in RxrαT167A during feeding, as observed with RXRα wild-type mice. However, RxrαT167A mice exhibited an attenuated decrease of blood glucose levels that occurred after fasting. This attenuation correlated with a concomitant down-regulation of lipid metabolism in WAT and was associated with RXRα phosphorylation at Thr 167. Thus, Thr 167 enabled RXRα to coordinate these three organs for regulation of energy metabolism and maintenance of glucose homeostasis.

Tryptophan hydroxylase (TRH) loss of function mutations in Daphnia deregulated growth, energetic, serotoninergic and arachidonic acid metabolic signalling pathways

Serotonin has a pivotal function regulating development, growth, reproduction and behavior in animals. In this paper, we studied the deregulatory effects of the deprivation of serotonin in Daphnia magna TRH CRISPR-Cas9 mutants. Bi-allelic in-del THR mutants and, to a lesser extent, mono-allelic ones grew less, reproduced later, and produced smaller clutches than wild type clones. Transcriptomic and functional gene analyses showed a down-regulation of growth/molting and energy metabolism signaling pathways in TRH mutants, while revealing marked differences between mono- and bi-allelic clones. Bi-allelic mutants, lacking serotonin, presented the serotonergic synapse and arachidonic acid metabolic pathways down-regulated while the tryptophan to kynurenine was upregulated, thus indicating a cross-talk between the serotonergic and arachidonic acid metabolic pathways. Finally, the effects on the insulin growth factor-mediated signaling pathway were marginal. These changes in functional and metabolic pathways are consistent with previously reported effects in D. magna exposed to pharmaceuticals that inhibited arachidonic metabolism or enhanced the levels of serotonin.

Statistical analysis of disease-causing and neutral mutations in human membrane proteins.

Mutations in transmembrane proteins (TMPs) have diverse effects on their structure and functions, which may lead to various diseases. In this present study, we have investigated variations in human membrane proteins and found that negatively charged to positively charged/polar and nonpolar to nonpolar changes are dominant in disease-causing and neutral mutations, respectively. Further, we analyzed the top 10 preferred mutations in 14 different disease classes and found that each class has at least two Arg mutations. Moreover, in cardiovascular diseases and congenital disorders of metabolism, Cys mutations occur more frequently in single-pass proteins, whereas Arg and nonpolar residues are more frequently substituted in multi-pass membrane proteins. The immune system diseases are enriched in C → R and C → Y mutations in inside and outside regions. On the other hand, in the membrane region, E → K and R → Q mutations are prevalent. The comparison of mutations in topologically similar regions of globular and membrane proteins showed that Ser and Thr mutations cause deleterious effects in membrane regions, whereas Cys and charged residues, Asp and Arg are prevalent in the buried regions of globular proteins. Our comprehensive analysis of disease-associated mutations in transmembrane proteins will be useful for developing prediction tools.

РАСПРОСТРАНЕННОСТЬ МУТАЦИЙ В ГЕНАХ МИКОБАКТЕРИЙ ТУБЕРКУЛЕЗА, КОДИРУЮЩИХ ЛЕКАРСТВЕННУЮ УСТОЙЧИВОСТЬ К ИЗОНИАЗИДУ И РИФАМПИЦИНУ, У БОЛЬНЫХ ТУБЕРКУЛЕЗОМ В РАЗНЫХ ВОЗРАСТНЫХ ГРУППАХ

The objective : to study the prevalence and ranges of mutations in katG, inhA, and ahpC genes coding resistance to isoniazid, and in rpoB gene coding resistance to rifampicin in patients with pulmonary tuberculosis from different age groups. Subjects and methods . 253 patients were examined; based on their age they were divided into 3 groups (Group 1 – from 18 to 30 years, Group 2 – from 31 to 60 years, group 3 – from 61 to 80 years). Biological microchips with TB-Biochip reagent kit (Russia) were used. Results . In Groups 1 and 2, compared to Group 3, mutations were recorded more often simultaneously in katG, inhA, ahpC genes and rpoB genes which meant that multiple drug resistance (MDR) prevailed (32.4 and 36.1% against 13.2%, respectively). In general, mutations in katG gene (including MDR + isolated isoniazid resistance) were also confidently more frequent in Groups 1 and 2 (30.9 and 32.7%) versus Group 3 (10.5%). Similar changes were found in the prevalence of the adverse type of ser 315-> Thr mutation of katG gene, which was registered in 23.5% of cases in Group 1, in 21.8% of cases in Group 2, and in 7.9% – in Group 3. In Group 3, mutations in rpoB gene (including MDR) were less frequently recorded and made 26.5% versus 45.5% in Group 1 and 46.9% in Group 2. There were no confident differences in ranges of mutations in rpoB gene between the examined groups of patients. Thus, a significantly lower level of resistance to isoniazid and rifampicin in elderly and senile tuberculosis patients was found compared to young and middle-aged patients, which was registered at the level of genetic mutations.

The phosphorylation of Tudor-SN mediated by JNK is involved in the regulation of milk protein synthesis induced by prolactin in BMECs.

Tudor staphylococcal nuclease (Tudor-SN) is a multifunctional protein involved in a variety of cellular processes and plays a critical role in the regulation of gene expression. Recently, Tudor-SN was found to be upregulated in mammary epithelial cells during lactation in response to prolactin, which further to regulate milk protein synthesis. However, the detailed regulatory mechanism of Tudor-SN to milk protein still remains to be elucidated. In our study, we observed that the levels of Tudor-SN and phosphor-Tudor-SN (Thr103) were both enhanced upon prolactin stimulation. Immunofluorescence assays demonstrated that prolactin treatment facilitated the nuclear transport of Tudor-SN. Further study revealed that the phosphorylation of Tudor-SN was depended on activated JNK. Coimmunoprecipitation assays disclosed that Tudor-SN might be phosphorylated directly by JNK. Using gene mutation assays, we further discovered that mutation of Thr to Ala at site of 103 prevented the nuclear transport of Tudor-SN. Thus, these results suggested the essential mechanism of the activated Tudor-SN in milk protein regulation in response to prolactin, which may provide some new sights into improve milk protein production.

Alicyclobacillus acidocaldarius Squalene-Hopene Cyclase: The Critical Role of Steric Bulk at Ala306 and the First Enzymatic Synthesis of Epoxydammarane from 2,3-Oxidosqualene.

The acyclic molecule squalene (1) is cyclized into 6,6,6,6,5-fused pentacyclic hopene (2) and hopanol (3; ca. 5:1) through the action of Alicyclobacillus acidocaldarius squalene-hopene cyclase (AaSHC). The polycyclization reaction proceeds with regio- and stereochemical specificity under precise enzymatic control. This pentacyclic hopane skeleton is generated by folding 1 into an all-chair conformation. The Ala306 residue in AaSHC is conserved in known squalene-hopene cyclases (SHCs); however, increasing the steric bulk (A306T and A306V) led to the accumulation of 6,6,6,5-fused tetracyclic scaffolds possessing 20R stereochemistry in high yield (94 % for A306V). The production of the 20R configuration indicated that 1 had been folded in a chair-chair-chair-boat conformation; in contrast, the normal chair-chair-chair-chair conformation affords the tetracycle with 20S stereochemistry, but the yield produced by the A306V mutant was very low (6 %). Consequently, bulk at position 306 significantly affects the stereochemical fate during the polycyclization reaction. The SHC also accepts (3R) and (3S)-2,3-oxidosqualenes (OXSQs) to generate 3α,β-hydroxyhopenes and 3α,β-hydroxyhopanols through polycyclization initiated at the epoxide ring. However, the Val and Thr mutants generated epoxydammarane scaffolds from (3R)-OXSQ; this indicated that the polycyclization cascade started in these instances at the terminal double bond position. This work is the first to report the polycyclization of oxidosqualene starting at the terminal double bond.

Nonstructural protein 9 residues 586 and 592 are critical sites in determining the replication efficiency and fatal virulence of the Chinese highly pathogenic porcine reproductive and respiratory syndrome virus

The highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has caused huge economic losses to the swine industry in China. Understanding the molecular basis in relation to the virulence of HP-PRRSV is essential for effectively controlling clinical infection and disease. In the current study, we constructed and rescued a serial of mutant viruses in nsp9 and nsp10 based on the differential amino acid sites between HP-PRRSV JXwn06 and LP-PRRSV HB-1/3.9. The replication efficiency in pulmonary alveolar macrophages (PAMs) and the pathogenicity of the mutant viruses for piglets were analyzed. Our results showed that the mutation of Thr to Ala in 586 and Ser to Thr in 592 of nsp9 decreased the replication efficiency of HP-PRRSV in PAMs, and could attenuate its virulence for piglets, suggesting that the residues 586 and 592 of nsp9 are critical sites natively in determining the fatal virulence of the Chinese HP-PRRSV for piglets.

Recognition of corn defense chitinases by fungal polyglycine hydrolases.

Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn (Zea mays ssp. mays). These target-specific endoproteases are unusual because they do not cut a specific peptide bond but select one of many Gly-Gly bonds within the polyglycine region. Some Gly-Gly bonds are cleaved frequently while others are never cleaved. Moreover, we have previously shown that PGHs from different fungal pathogens prefer to cleave different Gly-Gly peptide bonds. It is not understood how PGHs selectively cleave the ChitA linker, especially because its polyglycine structure lacks peptide sidechains. To gain insights into this process we synthesized several peptide analogs of ChitA to evaluate them as potential substrates and inhibitors of Es-cmp, a PGH from the plant pathogenic fungus Epicoccum sorghi. Our results showed that part of the PGH recognition site for substrate chitinases is adjacent to the polyglycine linker on the carboxy side. More specifically, four amino acid residues were implicated, each spaced four residues apart on an alpha helix. Moreover, analogous peptides with selective Gly->sarcosine (N-methylglycine) mutations or a specific Ser->Thr mutation retained inhibitor activity but were no longer cleaved by PGH. Additonally, our findings suggest that peptide analogs of ChitA that inhibit PGH activity could be used to strengthen plant defenses.

FIRST REPORT OF A NEW PEPPER MILD MOTTLE VIRUS PATHOTYPE 1,2 ON PEPPER IN SPAIN

During the summer of 2015, leaf deformations and chlorotic mosaic symptoms on leaves and unusual necrotic lesions on leaves, stems and fruits were observed on pepper (Capsicum annuum) plants grown in Almeria (Spain). Symptomatic leaf and fruit samples from eight collected plants, carrying L3 and L4 resistance against tobamovirus, were analyzed by ELISA specific for potyviruses, Cucumber mosaic virus and each of the following possible pepper-infecting viruses: Paprika mild mottle virus, Tobacco mosaic virus, Tomato mosaic virus, Tobacco mild green mosaic virus, and Pepper mild mottle virus (PMMoV). The eight plants tested negative for all viruses except for PMMoV. Mechanically inoculated extracts from two PMMoV-positive, singly infected symptomatic pepper plants systemically infected C. annuum cv Mazo (Unigenia), but not C. chinense PI159236, C. chacoense PI260429, four commercial L4, and two L3 resistant pepper plants, which exhibited local necrosis symptoms. Total RNAs from two plants inoculated with single infections were extracted, analyzed by reverse transcription PCR using primers designed to specifically amplify the coat protein gene (CP) of PMMoV (Antignus et al., 2008) and directly sequenced. Two independent samples yielded identical nt sequences (GenBank accession No. KT962848) and BLAST analysis showed the highest deduced aa identities of 99.4% with P1,2 isolates (M81413, AB000709, EF434393) of PMMoV. However, the predicted aa sequence possessed an Ala→Thr mutation at residue 66 of the CP. To our knowledge, although PMMoV has been shown to have variants that fail to infect L3 and L4 resistant pepper plants, based on unique CP coding sequences, this is the first report of a new P1,2 pathotype in Spain.

Amyloid precursor protein modulates Nav1.6 sodium channel currents through a Go-coupled JNK pathway.

Amyloid precursor protein (APP), commonly associated with Alzheimer’s disease, also marks axonal degeneration. In the recent studies, we demonstrated that APP aggregated at nodes of Ranvier (NORs) in myelinated central nervous system (CNS) axons and interacted with Nav1.6. However, the physiological function of APP remains unknown. In this study, we described reduced sodium current densities in APP knockout hippocampal neurons. Coexpression of APP or its intracellular domains containing a VTPEER motif with Nav1.6 sodium channels in Xenopus oocytes resulted in an increase in peak sodium currents, which was enhanced by constitutively active Go mutant and blocked by a dominant negative mutant. JNK and CDK5 inhibitor attenuated increases in Nav1.6 sodium currents induced by overexpression of APP. Nav1.6 sodium currents were increased by APPT668E (mutant Thr to Glu) and decreased by T668A (mutant Thr to ALa) mutant, respectively. The cell surface expression of Nav1.6 sodium channels in the white matter of spinal cord and the spinal conduction velocity is decreased in APP, p35 and JNK3 knockout mice. Therefore, APP modulates Nav1.6 sodium channels through a Go-coupled JNK pathway, which is dependent on phosphorylation of APP at Thr668.

Threonine532 phosphorylation in ClC‐3 channels is required for angiotensin II‐induced Cl− current and migration in cultured vascular smooth muscle cells

Angiotensin II (AngII) induces migration and growth of vascular smooth muscle cell (VSMC), which is responsible for vascular remodelling in some cardiovascular diseases. Ang II also activates a Cl(-) current, but the underlying mechanism is not clear. The A10 cell line and primary cultures of VSMC from control, ClC-3 channel null mice and WT mice made hypertensive with AngII infusions were used. Techniques employed included whole-cell patch clamp, co-immunoprecipitation, site-specific mutagenesis and Western blotting, In VSMC, AngII induced Cl(-) currents was carried by the chloride ion channel ClC-3. This current was absent in VSMC from ClC-3 channel null mice. The AngII-induced Cl(-) current involved interactions between ClC-3 channels and Rho-kinase 2 (ROCK2), shown by N- or C-terminal truncation of ClC-3 protein, ROCK2 siRNA and co-immunoprecipitation assays. Phosphorylation of ClC-3 channels at Thr(532) by ROCK2 was critical for AngII-induced Cl(-) current and VSMC migration. The ClC-3 T532D mutant (mutation of Thr(532) to aspartate), mimicking phosphorylated ClC-3 protein, significantly potentiated AngII-induced Cl(-) current and VSMC migration, while ClC-3 T532A (mutation of Thr(532) to alanine) had the opposite effects. AngII-induced cell migration was markedly decreased in VSMC from ClC-3 channel null mice that was insensitive to Y27632, an inhibitor of ROCK2. In addition, AngII-induced cerebrovascular remodelling was decreased in ClC-3 null mice, possibly by the ROCK2 pathway. ClC-3 protein phosphorylation at Thr(532) by ROCK2 is required for AngII-induced Cl(-) current and VSMC migration that are involved in AngII-induced vascular remodelling in hypertension.

LB03.08

Objective: hSLC39A8 (human solute carrier family 39 member 8) encodes a transmembrane protein that co-transports divalent heavy metal cations, such as Cd2+, with elusive physiological role. Recent genome-wide association studies have identified a non-synonymous single nucleotide polymorphism rs13107325 to be associated with hypertension. To investigate the functional impact of rs13107325 resulting in an amino acid substitution from Ala to Thr (A391T) in SLC39A8 on Cd2+ transport and the downstream signalling pathways. Design and method: Intracellular Cd2+ uptake was measured in HEK293 cells overexpressing SLC39A8 (Measure-iTTM Pb and Cd assay kit), and in human umbilical vascular endothelial cells (HUVECs) of different genotypes. Cd2+- and genotype-dependence of ERK1/2 and NF-kB pathway's activation were investigated by immunoblotting and dual-luciferase reporter assay. Cytotoxicity was measured by the lactate dehydrogenase assay and MTS assay. Molecular dynamics simulations were performed to predict in silico the effect of A391T on the structure and dynamics of SLC39A8 by using Robetta, TMHMM and etc. Results: Overexpression of Ala variant in HEK293 resulted in higher Cd2+ uptake and higher cytotoxicity as compared with the Thr variant. This is associated with increased phosphorylation of ERK1 and NF-kB activation. Similar trends were observed in HUVECs with endogenous SLC39A8. Bioinformatics tools also suggested a conformational change of the α-helical structural transition (residual 390–392) in the Thr mutant, which potentially attenuates the protein function. Conclusions: Increased Cd2+ uptake by SLC39A8 Ala variant (blood pressure raising allele) is associated with higher cell death in human kidney and endothelial cells. Therefore its altered function due to rs13107325 may indicate a potential therapeutic target in hypertension.

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.

Mutation Choice to Eliminate Buried Free Cysteines in Protein Therapeutics

Buried free-cysteine (Cys) residues can contribute to an irreversible unfolding pathway that promotes protein aggregation, increases immunogenic potential, and significantly reduces protein functional half-life. Consequently, mutation of buried free-Cys residues can result in significant improvement in the storage, reconstitution, and pharmacokinetic properties of protein-based therapeutics. Mutational design to eliminate buried free-Cys residues typically follows one of two common heuristics: either substitution by Ser (polar and isosteric), or substitution by Ala or Val (hydrophobic); however, a detailed structural and thermodynamic understanding of Cys mutations is lacking. We report a comprehensive structure and stability study of Ala, Ser, Thr, and Val mutations at each of the three buried free-Cys positions (Cys16, Cys83, and Cys117) in fibroblast growth factor-1. Mutation was almost universally destabilizing, indicating a general optimization for the wild-type Cys, including van der Waals and H-bond interactions. Structural response to Cys mutation characteristically involved changes to maintain, or effectively substitute, local H-bond interactions-by either structural collapse to accommodate the smaller oxygen radius of Ser/Thr, or conversely, expansion to enable inclusion of novel H-bonding solvent. Despite the diverse structural effects, the least destabilizing average substitution at each position was Ala, and not isosteric Ser.