Mechanistic analysis of A46V,H57Y,and D129N in pyrazinamidase associated with pyrazinamide resistance

Pyrazinamide (PZA) is a component of first-line drugs, active against latent Mycobacterium tuberculosis (MTB) isolates. The prodrug is activated into the active form, pyrazinoic acid (POA) via pncA gene-encoded pyrazinamidase (PZase). Mutations in pncA have been reported, most commonly responsible for PZA-resistance in more than 70% of the resistant cases. In our previous study, we detected many mutations in PZase among PZA-resistance MTB isolates including A46V, H71Y, and D129N. The current study was aimed to investigate the molecular mechanism of PZA-resistance behind mutants (MTs) A46V, H71Y, and D129N in comparison with the wild type (WT) through molecular dynamic (MD) simulation. MTB positive samples were subjected to PZA drug susceptibility testing (DST) against critical concentration (100ug/ml). The resistant samples were subjected to pncA sequencing. Thirty-six various mutations have been observed in the coding region of pncA of PZA-resistant isolates (GenBank accession No. MH461111) including A46V, H71Y, and D129N. The post-simulation analysis revealed a significant variation in MTs structural dynamics as compared to the WT. Root means square deviations (RMSD) and Root means square fluctuation (RMSF) has been found in variation between WT and MTs. Folding effect and pocket volume were altered in MTs when compared with WT. Geometric matching supports the effect of mutation A46V, H71Y, and D129N on PZase structure that may have an insight effect on PZase dynamics, making them vulnerable to convert pro-PZA into active form, POA. In conclusion, the current analyses will provide useful information behind PZA-resistance for better management of drug-resistant TB.     

Structural dynamics behind variants in pyrazinamidase and pyrazinamide resistance

Abstract

Pyrazinamide (PZA) is an important component of first-line anti-tuberculosis (anti-TB) drugs. The anti-TB agent is activated into an active form, pyrazinoic acid (POA), by Mycobacterium tuberculosis (MTB) pncA gene encoding pyrazinamidase (PZase). The major cause of PZA-resistance has been associated with mutations in the pncA gene. We have detected several novel mutations including V131F, Q141P, R154T, A170P, and V180F (GeneBank Accession No. MH461111) in the pncA gene of PZA-resistant isolates during PZA drug susceptibility testing followed by pncA gene sequencing. Here, we investigated molecular mechanism of PZA-resistance by comparing the results of experimental and molecular dynamics. The mutants (MTs) and wild type (WT) PZase structures in apo and complex with PZA were subjected to molecular dynamic simulations (MD) at the 40?ns. Multiple factors, including root mean square deviations (RMSD), binding pocket, total energy, dynamic cross correlation, and root mean square fluctuations (RMSF) of MTs and WT were compared. The MTs attained a high deviation and fluctuation compared to WT. Binding pocket volumes of the MTs, were found, lower than the WT, and the docking scores were high than WT while shape complementarity scores were lower than that of the WT. Residual motion in MTs are seemed to be dominant in anti-correlated motion. Mutations at locations, V131F, Q141P, R154T, A170P, and V180F, might be involved in the structural changes, possibly affecting the catalytic property of PZase to convert PZA into POA. Our study provides useful information that will enhance the understanding for better management of TB. Abbreviations DST drug susceptibility testing

Δelec electrostatic energy

LJ Lowenstein–Jensen medium

MGIT mycobacterium growth indicator tubes

MTs mutants

MD molecular dynamic simulations

MTB Mycobacterium tuberculosis

NALC–NaOH N-acetyl-l-cysteine–sodium hydroxide

NIH National Institutes of Health

NPT amount of substance (N), pressure (P) temperature (T)

NVT moles (N), volume (V) temperature (T)

PZase pyrazinamidase

Δps polar solvation energy

PTRL Provincial Tuberculosis Reference Laboratory

RMSD root mean square deviations

RMSF root mean square fluctuations

ΔSASA solvent accessible surface area energy

TB tuberculosis

GTotal total binding free energy

ΔvdW Van der Waals energy

WT wild type

Communicated by Ramaswamy H. Sarma     

Insight to pyrazinamide resistance inMycobacterium tuberculosisby molecular docking

Pyrazinamide (PZA) - an important drug in the anti-tuberculosis therapy, activated by an enzyme Pyrazinamidase (PZase). The basis of PZA resistance in Mycobacterium tuberculosis was owing to mutation in pncA gene coding for PZase. Homology modeling of PZase was performed using software Discovery Studio (DS) 2.0 based on the crystal structure of the PZase from Pyrococcus horikoshii (PDB code 1im5), in this study. The model comprises of one sheet with six parallel strands and seven helices with the amino acids Asp8, Asp49, Trp68, Lys96, Ala134, Thr135 and Cys138 at the active site. Five mutants were generated with Gly at position 8, Thr at position 96, Arg at position 104, Tyr and Ser at position 138. The Wild-type (WT) and five mutant models were docked with PZA. The results indicate that the mutants Lys96Thr, Ser104Arg Asp8Gly and Cys138Tyr may contribute to higher level drug resistance than Cys138Ser. These models provide the first in-silico evidence for the binding interaction of PZA with PZase and form the basis for rationalization of PZA resistance in naturally occurring pncA mutant strains of M. tuberculosis.     

Surveillance of pyrazinamide susceptibility among multidrug-resistant <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> isolates from Siriraj Hospital,Thailand

Background  

Susceptibility testing of pyrazinamide (PZA) against Mycobacterium tuberculosis is difficult to perform because the acidity of culture medium that is required for drug activity also inhibits the growth of bacteria. In Thailand, very limited information has been generated on PZA resistance, particularly among multidrug-resistant tuberculosis (MDR-TB) isolated from Thailand. Only two studies on PZA susceptibility among Thai M. tuberculosis strains have been reported; one used a pyrazinamidase assay, and the other used the BACTEC 460 TB for PZA susceptibility testing. In this study, we determined the percentage of strains possessing pyrazinamide resistance among pan-susceptible M. tuberculosis and MDR-TB isolates by using the pyrazinamidase assay, BACTEC MGIT 960 PZA method and pncA sequencing, and assessed the correlation in the data generated using these methods. The type and frequency of mutations in pncA were also determined.     

Exploring the cause of drug resistance by the detrimental missense mutations in KIT receptor: computational approach

In this work, we computationally identified the most detrimental missense mutations of KIT receptor causing gastrointestinal stromal tumors and analyzed the drug resistance of these missense mutations. Out of 31 missense mutations, 19 variants were commonly found less stable, deleterious and damaging by I-Mutant 2.0, SIFT and PolyPhen programs, respectively. Subsequently, we performed modeling of these 19 variants to understand their change in conformations with respect to native KIT receptor by computing their RMSD. Further, the native and 19 mutants were docked with the drug ‘Imatinib’ to explain the drug resistance of these detrimental missense mutations. Among the 19 mutants, we found by docking studies that 12 mutants, namely, F584C, F584L, V654A, L656P, T670I, R804W, D816F, D816V, D816Y, N822K, Y823D and E839K had less binding affinity with Imatinib than the native type. Finally, we analyzed that the loss of binding affinity of these 12 mutants, was due to altered flexibility in their binding amino acids with Imatinib as compared with native type by normal mode analysis. In our work, we found the novel data that the majority of the drug-binding amino acids in those 12 mutants had encountered loss of flexibility, which could be the theoretical basis for the cause of drug insensitivity.     

Site-directed mutagenesis studies on the L-arginine-binding sites of feedback inhibition in N-acetyl-L-glutamate kinase (NAGK) from Corynebacterium glutamicum

Arginine biosynthesis in Corynebacterium glutamicum proceeds via a pathway that is controlled by arginine through feedback inhibition of NAGK, the enzyme that converts N-acetyl-l-glutamate (NAG) to N-acety-l-glutamy-l-phosphate. In this study, the gene argB encoding NAGK from C. glutamicum ATCC 13032 was site-directed, and the l-arginine-binding sites of feedback inhibition in Cglu_NAGK are described. The N-helix and C-terminal residues were first deleted, and the results indicated that they are both necessary for Cglu_NAGK, whereas, the complete N-helix deletion (the front 28 residues) abolished the l-arginine inhibition. Further, we study here the impact on these functions of 12 site-directed mutations affecting seven residues of Cglu_NAGK, chosen on the basis of homology structural alignment. The E19R, H26E, and H268N variants could increase the I_0.5 ^R 50–60 fold, and the G287D and R209A mutants could increase the I_0.5 ^R 30–40 fold. The E281A mutagenesis resulted in the substrate kinetics being greatly influenced. The W23A variant had a lower specific enzyme activity. These results explained that the five amino acid residues (E19, H26, R209, H268, and G287) located in or near N-helix are all essential for the formation of arginine inhibition.     

Familial hemiplegic migraine mutations affect Na,K-ATPase domain interactions

Familial hemiplegic migraine (FHM) is a monogenic variant of migraine with aura. One of the three known causative genes, ATP1A2, which encodes the α2 isoform of Na,K-ATPase, causes FHM type 2 (FHM2). Over 50 FHM2 mutations have been reported, but most have not been characterized functionally. Here we study the molecular mechanism of Na,K-ATPase α2 missense mutations. Mutants E700K and P786L inactivate or strongly reduce enzyme activity. Glutamic acid 700 is located in the phosphorylation (P) domain and the mutation most likely disrupts the salt bridge with Lysine 35, thereby destabilizing the interaction with the actuator (A) domain. Mutants G900R and E902K are present in the extracellular loop at the interface of the α and β subunit. Both mutants likely hamper the interaction between these subunits and thereby decrease enzyme activity. Mutants E174K, R548C and R548H reduce the Na⁺ and increase the K⁺ affinity. Glutamic acid 174 is present in the A domain and might form a salt bridge with Lysine 432 in the nucleotide binding (N) domain, whereas Arginine 548, which is located in the N domain, forms a salt bridge with Glutamine 219 in the A domain. In the catalytic cycle, the interactions of the A and N domains affect the K⁺ and Na⁺ affinities, as observed with these mutants. Functional consequences were not observed for ATP1A2 mutations found in two sporadic hemiplegic migraine cases (Y9N and R879Q) and in migraine without aura (R51H and C702Y).     

Roles in inhibitor recognition and quinol oxidation of the amino acid side chains at positions of cytb providing resistance to Qo-inhibitors of the bc1 complex from Rhodobacter capsulatus

The substitutions M140I, F144S and L, G152S, T163A and V333A in cytochrome b of the ubiquinol-cytochrome c oxidoreductase (bc₁ complex) from Rhodobacter capsulatus provide resistance to the quinol oxidation (Q_o inhibitors myxothiazol, mucidin and stigmatellin. Site-directed mutagenesis with degenerate primers was used to define the role of these positions in inhibitor recognition and quinol oxidation, and a collection of various substitutions at each of these positions was obtained. The effects of these mutations on quinol oxidation, nature and level of inhibitor resistance, prosthetic group incorporation and assembly of the complex were analysed. Most of these mutations, unlike those at position 158 reported earlier, yielded functional bc₁, complexes able to support the photosynthetic growth of R. capsulatus. However, they perturbed steady-state quinol oxidation and inhibitor recognition indicating that they are important for the function of the Q_o site. In particular, the presence of a methyl group on the β-carbon (He and Val residues) at position 140, the absence of an aromatic ring (Phe, Tyr and Trp residues) at position 144 and the loss of residues with small side chains (Gly and Ala) at position 152 correlated with resistance to myxothiazol. On the other hand, no myxothiazol resistance was observed with the substitutions at positions 163 and 333 suggesting that they affected solely the recognition of stigmatellin. Five substitutions, M140R, F144H and R, G152P and T163R, yielded photosynthesis-deficient mutants with assembled but impaired bc₁ complexes. Unexpectedly, two substitutions at position 163 (T to F or P) yielded mutants lacking the three subunits of     

Molecular dynamics study of the dominant-negative E219K polymorphism in human prion protein

Human prion diseases are associated with misfolding or aggregation of the Human Prion Protein (HuPrP). Missense mutations in the HuPrP gene, contribute to conversion of HuPrP^C to HuPrP^Sc and amyloid formation. Based on our previous comprehensive study, three missense mutations, from two different functional groups, i.e. disease-related mutations, and protective mutations, were selected and extensive molecular dynamics simulations were performed on these three mutants to compare their dynamics and conformations with those of the wildtype HuPrP. In addition to simulations of monomeric forms of mutants, in order to study the dominant-negative effect of protective mutation (E219K), 30-ns simulations were performed on E219K-wildtype and wildtype-wildtype dimeric forms. Our results indicate that, although after 30-ns simulations the global three-dimensional structure of models remain fairly intact, the disease-related mutations (V210I and Q212P) introduce local structural changes, i.e. close contact changes and secondary structure changes, in addition to global flexibility changes. Furthermore, our results support the loss of hydrophobic interaction due to the mutations in hydrophobic core that has been reported by previous NMR and computational studies. On the other hand, this protective mutation (E219K) results in helix elongation, and significant increases of overall flexibility of E219K mutant during 30-ns simulation. In conclusion, the simulations of dimeric forms suggest that the dominant-negative effect of this protective mutation (E219K) is due to the incompatible structures and dynamics of allelic variants during conversion process.     

Mutations in human cardiac troponin I that are associated with restrictive cardiomyopathy affect basal ATPase activity and the calcium sensitivity of force development

Human cardiac Troponin I (cTnI) is the first sarcomeric protein for which mutations have been associated with restrictive cardiomyopathy. To determine whether five mutations in cTnI (L144Q, R145W, A171T, K178E, and R192H) associated with restrictive cardiomyopathy were distinguishable from hypertrophic cardiomyopathy-causing mutations in cTnI, actomyosin ATPase activity and skinned fiber studies were carried out. All five mutations investigated showed an increase in the Ca2+ sensitivity of force development compared with wild-type cTnI. The two mutations with the worst clinical phenotype (K178E and R192H) both showed large increases in Ca2+ sensitivity (deltapCa50 = 0.47 and 0.36, respectively). Although at least one of these mutations is not in the known inhibitory regions of cTnI, all of the mutations investigated caused a decrease in the ability of cTnI to inhibit actomyosin ATPase activity. Mixtures of wild-type and mutant cTnI showed that cTnI mutants could be classified into three different groups: dominant (L144Q, A171T and R192H), equivalent (K178E), or weaker (R145W) than wild-type cTnI in actomyosin ATPase assays in the absence of Ca2+. Although most of the mutants were able to activate actomyosin ATPase similarly to wild-type cTnI, L144Q had significantly lower maximal ATPase activities than any of the other mutants or wild-type cTnI. Three mutants (L144Q, R145W, and K178E) were unable to fully relax contraction in the absence of Ca2+. The inability of the five cTnI mutations investigated to fully inhibit ATPase activity/force development and the generally larger increases in Ca2+ sensitivity than observed for most hypertrophic cardiomyopathy mutations would likely lead to severe diastolic dysfunction and may be the major physiological factors responsible for causing the restrictive cardiomyopathy phenotype in some of the genetically affected individuals.     

Regulation of NADPH Oxidase Activity in Phagocytes: RELATIONSHIP BETWEEN FAD/NADPH BINDING AND OXIDASE COMPLEX ASSEMBLY*

The X⁺-linked chronic granulomatous disease (X⁺-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X⁺-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X⁺-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, Δ507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However, according to their position in the three-dimensional model of the cytosolic domain of Nox2, only Cys-369 could be in direct contact with cytosolic factors during oxidase assembly. In addition, the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus, the NADPH oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2.     

Structural basis for targeting the ribosomal protein S1 of Mycobacterium tuberculosis by pyrazinamide

Pyrazinamide (PZA) is a first‐line drug for tuberculosis (TB) treatment and is responsible for shortening the duration of TB therapy. The mode of action of PZA remains elusive. RpsA, the ribosomal protein S1 of Mycobacterium tuberculosis (Mtb), was recently identified as a target of PZA based on its binding activity to pyrazinoic acid (POA), the active form of PZA. POA binding to RpsA led to the inhibition of trans‐translation. However, the nature of the RpsA–POA interaction remains unknown. Key questions include why POA exhibits an exquisite specificity to RpsA of Mtb and how RpsA mutations confer PZA resistance. Here, we report the crystal structures of the C‐terminal domain of RpsA of Mtb and its complex with POA, as well as the corresponding domains of two RpsA variants that are associated with PZA resistance. Structural analysis reveals that POA binds to RpsA through hydrogen bonds and hydrophobic interactions, mediated mainly by residues (Lys303, Phe307, Phe310 and Arg357) that are essential for tmRNA binding. Conformational changes induced by mutation or sequence variation at the C‐terminus of RpsA abolish the POA binding activity. Our findings provide insights into the mode of action of PZA and molecular basis of PZA resistance associated with RpsA mutations.     

Unstable Mutants of R Factor

Thirty mutants sensitive to tetracycline were obtained from an R100 factor capable of conferring resistance to tetracycline (TC), chloramphenicol (CM), streptomycin (SM) and sulfanilamide (SA). Among the TC sensitive mutants, three showed a high frequency of spontaneous loss from host strains. The genetic loci governing the stability of R factor in host bacteria were denoted as stb. The stb^– R factors have lost many of the properties of a wild type R factor, such as, the capability to sexually transfer drug resistance and host chromosome, to confer superinfection immunity and to inhibit F function. All of these properties did not revert to a wild type phenotype, suggesting that these mutations are deletions including genetic determinants governing both TC resistance and stability of R factor. Recombinational analysis between stb^– and stb⁺ R factors indicated that crossovers between the stb loci and those governing CM (or SM.SA) resistance took place at high frequency. No crossovers were detected between stb loci and those governing TC resistance, indicating that the stb loci are linked closely to the loci governing TC resistance.     

Kinase domain mutants of Bcr-Abl exhibit altered transformation potency, kinase activity, and substrate utilization, irrespective of sensitivity to imatinib

Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays. Relative to unmutated (native) Bcr-Abl, the ATP binding loop mutants Y253F and E255K exhibited increased transformation potency, M351T and H396P were less potent, and the performance of T315I was assay dependent. The transformation potency of Y253F and M351T correlated with intrinsic Bcr-Abl kinase activity, whereas the kinase activity of E255K, H396P, and T315I did not correlate with transforming capabilities, suggesting that additional factors influence transformation potency. Analysis of the phosphotyrosine proteome by mass spectroscopy showed differential phosphorylation among the mutants, a finding consistent with altered substrate specificity and pathway activation. Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion, leading to gain- or loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors.     

Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis isolates from Korea and analysis of the correlation between the mutations and pyrazinamidase activity

To investigate the effect of natural pyrazinamidase (PncA) mutations on protein function, we analyzed expression and PncA activity of eight pncA point mutants identified in nineteen pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates. Among them, two mutants (Y99D and T135P) showed high expression level and solubility comparable to those of the wild-type PncA protein, two (K48E and G97D) displayed low expression level and solubility, and four (C14R, H51P, W68S, and A146V) were insoluble. Interestingly, when possible structural effects of these mutations were predicted by the CUPSAT program based on the proposed three-dimensional structure of M. tuberculosis PncA, only two highly soluble mutant proteins (Y99D and T135P) were predicted to be stabilizing and have favorable torsion angles. However, the others exhibiting either low solubility or precipitation were foreseen to be destabilizing and/or have unfavorable torsion angles, suggesting that the alterations could interfere with proper protein folding, thereby decreasing or depleting protein solubility. A PncA activity assay demonstrated that two mutants (G97D and T135P) showed virtually no activity, but two other mutants (K48E and Y99D) exhibited wild-type activity, indicating that the PncA residues (Cys14, His51, Trp68, Gly97, Thr135, and Ala146) may be important for PncA activity and/or proper protein folding.     

Dihydropyrimidine dehydrogenase (DPD) deficiency: identification and expression of missense mutations C29R, R886H and R235W

Dihydropyrimidine dehydrogenase (DPD) deficiency (McKusick 274270) is an autosomal recessive disease characterized by thymine-uraciluria in homozygous-deficient patients and associated with a variable clinical phenotype. Cancer patients with this defect should not be treated with the usual dose of 5-fluorouracil because of the expected lethal toxicity. In addition, heterozygosity for mutations in the DPD gene increases the risk of toxicity in cancer patients treated with this drug. Sequence analysis in a patient with complete DPD deficiency, previously shown to be heterozygous for the ΔC1897 frameshift mutation, revealed the presence of a novel missense mutation, R235W. Expression of this novel mutation and previously identified missense mutations C29R and R886H in Escherichia coli showed that both C29R and R235W lead to a mutant DPD protein without significant residual enzymatic activity. The R886H mutation, however, resulted in about 25% residual enzymatic activity and is unlikely to be responsible for the DPD-deficient phenotype. We show that the E. coli expression system is a valuable tool for examining DPD enzymatic variants. In addition, two new patients who were both heterozygous for the C29R mutation and the common splice donor site mutation were identified. Only one of these patients showed convulsive disorders during childhood, whereas the other showed no clinical phenotype, further illustrating the lack of correlation between genotype and phenotype in DPD deficiency. Received: 20 June 1997 / Accepted: 26 August 1997     

In vitro characterization of the intramelanosomal domain of human recombinant TYRP1 and its oculocutaneous albinism type 3-related mutant variants

Tyrosinase related protein 1 (TYRP1) is the most abundant melanosomal protein of the melanocyte, where plays an important role in the synthesis of eumelanin, possibly catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid to 5,6-quinone-2-carboxylic acid. Mutations to the TYRP1 gene can result in oculocutaneous albinism type 3 (OCA3), a rare disease characterized by reduced synthesis of melanin in skin, hair, and eyes. To investigate the effect of genetic mutations on the TYRP1 structure, function, and stability, we engineered the intramelanosomal domain of TYRP1 and its mutant variants mimicking either OCA3-related changes, C30R, H215Y, D308N, and R326H or R87G mutant variant, analogous to OCA1-related pathogenic effect in tyrosinase. Proteins were produced in Trichoplusia Ni larvae, then purified, and analyzed by biochemical methods. Data shows that D308N and R326H mutants keep the native conformations and demonstrate no change in their stability and enzymatic activity. In contrast, mutations C30R and R87G localized in the Cys-rich domain show the variants misfolding during the purification process. The H215Y variant disrupts the binding of Zn²⁺ in the active site and thus reduces the strength of the enzyme/substrate interactions. Our results, consistent with the clinical and in silico studies, show that mutations at the protein surface are expected to have a negligible phenotype change compared to that of TYRP1. For the mutations with severe phenotype changes, which were localized in the Cys-rich domain or the active site, we confirmed a complete or partial protein misfolding as the possible mechanism of protein malfunction caused by OCA3 inherited mutations.     

Cover Image,Volume 88, Issue 6

Hsp16.3, a molecular chaperone, plays a vital role in the growth and survival of Mycobacterium tuberculosis inside the host. We previously reported that deletion of three amino acid residues (¹⁴²STN¹⁴⁴) from C-terminal extension (CTE) of Hsp16.3 triggers its structural perturbation and increases its chaperone activity, which reaches its apex upon the deletion of its entire CTE (¹⁴¹RSTN¹⁴⁴). Thus, we hypothesized that Arg141 (R141) and Ser142 (S142) in the CTE of Hsp16.3 possibly hold the key in maintaining its native-like structure and chaperone activity. To test this hypothesis, we generated two deletion mutants in which R141 and S142 were deleted individually (Hsp16.3ΔR141 and Hsp16.3ΔS142) and three substitution mutants in which R141 was replaced by lysine (Hsp16.3R141K), alanine (Hsp16.3R141A), and glutamic acid (Hsp16.3R141E), respectively. Hsp16.3ΔS142 or Hsp16.3R141K mutant has native-like structure and chaperone activity. Deletion of R141 from the CTE (Hsp16.3ΔR141) perturbs the secondary and tertiary structure, lowers the subunit exchange dynamics and decreases the chaperone activity of Hsp16.3. But, the substitution of R141 with alanine (Hsp16.3R141A) or glutamic acid (Hsp16.3R141E) perturbs its secondary and tertiary structure. Surprisingly, such charge tampering of R141 enhances the subunit exchange dynamics and chaperone activity of Hsp16.3. Interestingly, neither the deletion of R141/S142 nor the substitution of R141 with lysine, alanine and glutamic acid affects the oligomeric mass/size of Hsp16.3. Overall, our study suggests that R141 (especially the positive charge on R141) plays a crucial role in maintaining the native-like structure as well as in regulating subunit exchange dynamics and chaperone activity of Hsp16.3.     

p110α Hot Spot Mutations E545K and H1047R Exert Metabolic Reprogramming Independently of p110α Kinase Activity

The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity.     

Studies on the Establishment of Multi-Drug-Resistant Strain BIO-4R of Streptococcus faecalis in the Intestinal Tract of Germ-Free Mice

Germ-free ICR mice were mono- or dicontaminated with a multi-drug-resistant strain BIO-4R of Streptococcus faecalis (BIO-4R) and Escherichia coli 026 : K60 (E. coli) and administered aminobenzyl penicillin (ABPC). BIO-4R was established in the intestinal tract at a level of 10⁸ viable cells per gram of stool on the fourth day following oral inoculation and the BIO-4R population was stably maintained thereafter. The drug resistance of BIO-4R remained unchanged in the intestinal tract of gnotobiotes throughout the experiment. Highly resistant cells of E. coli were isolated from the feces of some dicontaminated mice after ABPC administration. However, it seems that the high resistance of these E. coli is not due to the transfer of resistance of BIO-4R to E. coli. All animals given a large amount of BIO-4R (10⁸ cells) per os survived throughout the study period of two weeks without symptoms.