Renjian Zheng

Mechanisms of Isoniazid Resistance in Mycobacterium tuberculosis: Enzymatic Characterization of Enoyl Reductase Mutants Identified in Isoniazid-Resistant Clinical Isolates

Mutants in the structural gene of the inhA-encoded NADH-dependent 2-trans enoyl-acyl carrier protein reductase were identified from isoniazid-resistant clinical isolates of Mycobacterium tuberculosis. Recombinant InhA proteins with defined single amino acid replacements were expressed in Escherichia coli and purified to homogeneity. Steady-state kinetic parameters for wild type (WT) and I16T, I21V, I47T, and I95P mutants of the enoyl reductase were measured spectrophotometrically. NADH binding to WT and I16T, I21V, I47T, S94A, and I95P mutant reductases were determined by fluorescence spectroscopy and demonstrated that all mutant enzymes had reduced NADH affinity and that NADH binding to all mutants was cooperative as compared with the hyperbolic binding of NADH to the WT enzyme. Since KatG-produced electrophilic derivatives of isoniazid have been suggested to inactivate the enoyl reductase-NADH complex, the kinetics of inactivation for the WT and I21V and I95P mutants was determined. Both mutations resulted in significantly increased values for the apparent first-order rate constant of inactivation.

The metabolism of nitrosothiols in the mycobacteria: identification and characterization of S-nitrosomycothiol reductase

When grown in culture Mycobacterium smegmatis metabolized S-nitrosoglutathione to oxidized glutathione and nitrate, which suggested a possible involvement of an S-nitrosothiol reductase and mycobacterial haemoglobin. The mycothiol-dependent formaldehyde dehydrogenase from M. smegmatis was purified by a combination of Ni2+-IMAC (immobilized metal ion affinity chromatography), hydrophobic interaction, anion-exchange and affinity chromatography. The enzyme had a subunit molecular mass of 38263 kDa. Steady-state kinetic studies indicated that the enzyme catalyses the NAD+-dependent conversion of S-hydroxymethylmycothiol into formic acid and mycothiol by a rapid-equilibrium ordered mechanism. The enzyme also catalysed an NADH-dependent decomposition of S-nitrosomycothiol (MSNO) by a sequential mechanism and with an equimolar stoichiometry of NADH:MSNO, which indicated that the enzyme reduces the nitroso group to the oxidation level of nitroxyl. Vmax for the MSNO reductase reaction indicated a turnover per subunit of approx. 116700 min(-1), which was 76-fold faster than the formaldehyde dehydrogenase activity. A gene, Rv2259, annotated as a class III alcohol dehydrogenase in the Mycobacterium tuberculosis genome was cloned and expressed in M. smegmatis as the C-terminally His6-tagged product. The purified recombinant enzyme from M. tuberculosis also catalysed both activities. M. smegmatis S-nitrosomycothiol reductase converted MSNO into the N -hydroxysulphenamide, which readily rearranged to mycothiolsulphinamide. In the presence of MSNO reductase, M. tuberculosis HbN (haemoglobin N) was converted with low efficiency into metHbN [HbN(Fe3+)] and this conversion was dependent on turnover of MSNO reductase. These observations suggest a possible route in vivo for the dissimilation of S-nitrosoglutathione.

Functional Interactions between KCNE1 C-Terminus and the KCNQ1 Channel

The KCNE1 gene product (minK protein) associates with the cardiac KvLQT1 potassium channel (encoded by KCNQ1) to create the cardiac slowly activating delayed rectifier, IKs. Mutations throughout both genes are linked to the hereditary cardiac arrhythmias in the Long QT Syndrome (LQTS). KCNE1 exerts its specific regulation of KCNQ1 activation via interactions between membrane-spanning segments of the two proteins. Less detailed attention has been focused on the role of the KCNE1 C-terminus in regulating channel behavior. We analyzed the effects of an LQT5 point mutation (D76N) and the truncation of the entire C-terminus (Δ70) on channel regulation, assembly and interaction. Both mutations significantly shifted voltage dependence of activation in the depolarizing direction and decreased IKs current density. They also accelerated rates of channel deactivation but notably, did not affect activation kinetics. Truncation of the C-terminus reduced the apparent affinity of KCNE1 for KCNQ1, resulting in impaired channel formation and presentation of KCNQ1/KCNE1 complexes to the surface. Complete saturation of KCNQ1 channels with KCNE1-Δ70 could be achieved by relative over-expression of the KCNE subunit. Rate-dependent facilitation of K+ conductance, a key property of IKs that enables action potential shortening at higher heart rates, was defective for both KCNE1 C-terminal mutations, and may contribute to the clinical phenotype of arrhythmias triggered by heart rate elevations during exercise in LQTS mutations. These results support several roles for KCNE1 C-terminus interaction with KCNQ1: regulation of channel assembly, open-state destabilization, and kinetics of channel deactivation.

Aminoglycoside Resistance Resulting from Tight Drug Binding to an Altered Aminoglycoside Acetyltransferase

ABSTRACT The aacA29b gene, which confers an atypical aminoglycoside resistance pattern to Escherichia coli, was identified on a class 1 integron from a multidrug-resistant isolate of Pseudomonas aeruginosa. On the basis of amino acid sequence homology, it was proposed that the gene encoded a 6′-N-acetyltransferase. The resistance gene was cloned into the pET23a(+) vector, and overexpression conferred high-level resistance to the usual substrates of the aminoglycoside N-acetyltransferase AAC(6′)-I, except netilmicin. The level of resistance conferred by aacA29b correlated perfectly with the level of expression of the gene. The corresponding C-terminal six-His-tagged AAC(6′)-29b protein was purified and found to exist as a dimer in solution. With a spectrophotometric assay, an extremely feeble AAC activity was detected with acetyl coenzyme A (acetyl-CoA) as an acetyl donor. Fluorescence titrations of the protein with aminoglycosides demonstrated the very tight binding of tobramycin, dibekacin, kanamycin A, sisomicin (Kd, ≤1 μM) and a weaker affinity for amikacin (Kd, ≈60 μM). The binding of netilmicin and acetyl-CoA could not be detected by either fluorescence spectroscopy or isothermal titration calorimetry. The inability of AAC(6′)-29b to efficiently bind acetyl-CoA is supported by an alignment analysis of its amino acid sequence compared with those of other AAC(6′)-I family members. AAC(6′)-29b lacks a number of residues involved in acetyl-CoA binding. These results lead to the conclusion that AAC(6′)-29b is able to confer aminoglycoside resistance by sequestering the drug as a result of tight binding.

Pore mutants of HERG and KvLQT1 downregulate the reciprocal currents in stable cell lines

We previously reported a transgenic rabbit model of long QT syndrome based on overexpression of pore mutants of repolarizing K(+) channels KvLQT1 (LQT1) and HERG (LQT2).The transgenes in these rabbits eliminated the slow and fast components of the delayed rectifier K(+) current (I(Ks) and I(Kr), respectively), as expected. Interestingly, the expressed pore mutants of HERG and KvLQT1 downregulated the remaining reciprocal repolarizing currents, I(Ks) and I(Kr), without affecting the steady-state levels of the native polypeptides. Here, we sought to further explore the functional interactions between HERG and KvLQT1 in heterologous expression systems. Stable Chinese hamster ovary (CHO) cell lines expressing KvLQT1-minK or HERG were transiently transfected with expression vectors coding for mutant or wild-type HERG or KvLQT1. Transiently expressed pore mutant or wild-type KvLQT1 downregulated I(Kr) in HERG stable CHO cell lines by 70% and 44%, respectively. Immunostaining revealed a severalfold lower surface expression of HERG, which could account for the reduction in I(Kr) upon KvLQT1 expression. Deletion of the KvLQT1 NH(2)-terminus did not abolish the downregulation, suggesting that the interactions between the two channels are mediated through their COOH-termini. Similarly, transiently expressed HERG reduced I(Ks) in KvLQT1-minK stable cells. Coimmunoprecipitations indicated a direct interaction between HERG and KvLQT1, and surface plasmon resonance analysis demonstrated a specific, physical association between the COOH-termini of KvLQT1 and HERG. Here, we present an in vitro model system consistent with the in vivo reciprocal downregulation of repolarizing currents seen in transgenic rabbit models, illustrating the importance of the transfection method when studying heterologous ion channel expression and trafficking. Moreover, our data suggest that interactions between KvLQT1 and HERG are mediated through COOH-termini.

Analysis of the interactions between the C-terminal cytoplasmic domains of KCNQ1 and KCNE1 channel subunits

Ion channel subunits encoded by KCNQ1 and KCNE1 produce the slowly activating K+ current (IKs) that plays a central role in myocardial repolarization. The KCNQ1 alpha-subunit and the KCNE1 beta-subunit assemble with their membrane-spanning segments interacting, resulting in transformation of channel activation kinetics. We recently reported a functional interaction involving C-terminal portions of the two subunits with ensuing regulation of channel deactivation. In the present study, we provide evidence characterizing a physical interaction between the KCNQ1-CT (KCNE1 C-terminus) and the KCNE1-CT (KCNE1 C-terminus). When expressed in cultured cells, the KCNE1-CT co-localized with KCNQ1, co-immunoprecipitated with KCNQ1 and perturbed deactivation kinetics of the KCNQ1 currents. Purified KCNQ1-CT and KCNE1-CT physically interacted in pull-down experiments, indicating a direct association. Deletion analysis of KCNQ1-CT indicated that the KCNE1-CT binds to a KCNQ1 region just after the last transmembrane segment, but N-terminal to the tetramerization domain. SPR (surface plasmon resonance) corroborated the pull-down results, showing that the most proximal region (KCNQ1 amino acids 349-438) contributed most to the bimolecular interaction with a dissociation constant of approximately 4 microM. LQT (long QT) mutants of the KCNE1-CT, D76N and W87F, retained binding to the KCNQ1-CT with comparable affinity, indicating that these disease-causing mutations do not alter channel behaviour by disruption of the association. Several LQT mutations involving the KCNQ1-CT, however, showed various effects on KCNQ1/KCNE1 association. Our results indicate that the KCNQ1-CT and the KCNE1-CT comprise an independent interaction domain that may play a role in IKs channel regulation that is potentially affected in some LQTS (LQT syndrome) mutations.

Molecular cloning and characterization of mitogen-activated protein kinase 2 in Trypanosoma cruzi

Mitogen-activated protein kinase (MAPK) pathways are major signal transduction systems by which eukaryotic cells convert environmental cues to intracellular events such as proliferation and differentiation. We have identified a Trypanosoma cruzi homologue of the MAPK family that we have called TcMAPK2. Sequence analyses demonstrates TcMAPK2 has high homology with lower eukaryotic ERK2 but has significant differences from mammalian ERK2. Enzymatic assays of both recombinant TcMAPK2 and native protein obtained by immunoprecipitation using anti-TcMAPK2 demonstrated that both preparations of TcMAPK2 were catalytically active. Immunofluorescence analysis of the subcellular localization of TcMAPK2 determined it is mainly cytoplasmic in epimastigotes, along the flagella in trypomastigotes and on the plasma membrane of intracellular amastigotes. Phosphorylated TcMAPK2 was highest in trypomastigotes and lowest in amastigotes. Recombinant TcMAPK2 was able to phosphorylate the recombinant protein of a cAMP specific phosphodiesterase. Over-expression of TcMAPK2 in epimastigotes inhibited growth and development leading to death. TcMAPK2 has an important role in the stress response of the parasite and may be important in regulating proliferation and differentiation.

Partially Dominant Mutant Channel Defect Corresponding with Intermediate LQT2 Phenotype

Background: The hereditary Long QT Syndrome is a common cardiac disorder where ventricular repolarization is delayed, abnormally prolonging the QTc interval on electrocardiograms. LQTS is linked to various genetic loci, including the KCNH2 (HERG) gene that encodes the α‐subunit of the cardiac potassium channel that carries IKr. Here, we report and characterize a novel pathologic missense mutation, G816V HERG, in a patient with sudden cardiac death.

Mechanisms underlying the protein-kinase mediated regulation of the HERG potassium channel synthesis.

The HERG (human ether-a-go-go related gene) potassium channel aids in the repolarization of the cardiomyocyte membrane at the end of each action potential. We have previously shown that sustained protein kinase A or C (PKA and PKC) activity specifically enhances channel synthesis over the course of hours to days in heterologous expression and cardiac myocytes. The kinase-mediated augmentation of the channel is post-transcriptional and occurs near or at the endoplasmic reticulum. Here we report our further investigations into the mechanisms of kinase-mediated augmentation of HERG channel protein. We show that HERG channel phosphorylation alone is not sufficient for the PKA-dependent increase to occur. In vitro translation studies indicate that an additional factor is required for the process. Pharmacologic inhibitors suggest that the channel augmentation is not due to kinase-mediated alteration in proteasome or lysosome activity. PKA activation had no effect on stability of HERG mRNA and polyribosomal profiling showed that kinase activity did not elevate translation from low to high rates. Transcriptional inhibition results suggest that the additional cellular factor is a PKA-regulated protein. Together, these findings suggest that PKA-mediated augmentation of HERG abundance is more complex than previously appreciated involving enhancement of already active translation rates, phosphorylation of the channel protein and at least one other cyclic-AMP/PKA-responsive protein. Further exploration of molecular components of this regulatory pathway will be necessary to determine exact mechanism and the biomedical impact of this process in vivo.

Structural determinants of the 5'-methylthioinosine specificity of Plasmodium purine nucleoside phosphorylase.

Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that leads to the phosphorylysis of both purines and 5′-methylthiopurines, byproducts of polyamine synthesis. We have explored structural features in Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We used site directed mutagenesis to identify residues critical for PfPNP catalytic activity as well as critical residues within a hydrophobic pocket required for accommodation of the 5′-methylthio group. Kinetic analysis data shows that several mutants had disrupted binding of the 5′-methylthio group while retaining activity for inosine. A triple PfPNP mutant that mimics Toxoplasma gondii PNP had significant loss of 5′-methylthio activity with retention of inosine activity. Crystallographic investigation of the triple mutant PfPNP with Tyr160Phe, Val66Ile, andVal73Ile in complex with the transition state inhibitor immucillin H reveals fewer hydrogen bond interactions for the inhibitor in the hydrophobic pocket.