Development of a fluorescently labeled thermostable DHFR for studying conformational changes associated with inhibitor binding

The tissue kallikrein (KLK) family contains 15 genes (KLK1–KLK15) tandemly arranged on chromosome 19q13.4 that forms the largest cluster of contiguous protease genes in the human genome. Here, we provide mechanistic evidence showing that the expression of KLK13, one of the most recently identified family members, is significantly up-regulated in metastatic lung adenocarcinoma. Whilst overexpression of KLK13 resulted in an increase in malignant cell behavior, knockdown of its endogenous gene expression caused a significant decrease in cell migratory and invasive properties. Functional studies further demonstrated that KLK13 is activated via demethylation of its upstream region. The elevated KLK13 protein then enhances the ability of tumor cells to degrade extracellular laminin that, subsequently, facilitates cell metastatic potential in the in vivo SCID mouse xenograft model. KLK13 was also found to induce the expression of N-cadherin to help promote tumor cell motility. Together, these results reveal the enhancing effects of KLK13 on tumor cell invasion and migration, and that it may serve as a diagnostic/prognostic marker and a potential therapeutic target for lung cancer.     

Increasing methotrexate resistance by combination of active-site mutations in human dihydrofolate reductase

Methotrexate-resistant forms of human dihydrofolate reductase have the potential to protect healthy cells from the toxicity of methotrexate (MTX), to improve prognosis during cancer therapy. It has been shown that synergistic MTX-resistance can be obtained by combining two active-site mutations that independently confer weak MTX-resistance. In order to obtain more highly MTX-resistant human dihydrofolate reductase (hDHFR) variants for this application, we used a semi-rational approach to obtain combinatorial active-site mutants of hDHFR that are highly resistant towards MTX. We created a combinatorial mutant library encoding various amino acids at residues Phe31, Phe34 and Gln35. In vivo library selection was achieved in a bacterial system on medium containing high concentrations of MTX. We characterized ten novel MTX-resistant mutants with different amino acid combinations at residues 31, 34 and 35. Kinetic and inhibition parameters of the purified mutants revealed that higher MTX-resistance roughly correlated with a greater number of mutations, the most highly-resistant mutants containing three active site mutations (Ki(MTX)=59-180 nM; wild-type Ki(MTX)<0.03 nM). An inverse correlation was observed between resistance and catalytic efficiency, which decreased mostly as a result of increased KM toward the substrate dihydrofolate. We verified that the MTX-resistant hDHFRs can protect eukaryotic cells from MTX toxicity by transfecting the most resistant mutants into DHFR-knock-out CHO cells. The transfected variants conferred survival at concentrations of MTX between 100-fold and >4000-fold higher than the wild-type enzyme, the most resistant triple mutant offering protection beyond the maximal concentration of MTX that could be included in the medium. These highly resistant variants of hDHFR offer potential for myeloprotection during administration of MTX in cancer treatment.     

Biochemical characterization of a protein tyrosine phosphatase from Trypanosoma cruzi involved in metacyclogenesis and cell invasion

Protein tyrosine phosphatases (PTPs) form a large family of enzymes involved in the regulation of numerous cellular functions in eukaryotes. Several protein tyrosine phosphatases have been recently identified in trypanosomatides. Here we report the purification and biochemical characterization of TcPTP1, a protein tyrosine phosphatase from Trypanosoma cruzi, the causing agent of Chagas’ disease. The enzyme was cloned and expressed recombinantly in Escherichia coli and purified by Ni-affinity chromatography. Biochemical characterization of recombinant TcPTP1 with the PTP pseudo-substrate pNPP allowed the estimation of a Michaelis–Menten constant K_m of 4.5 mM and a k_cat of 2.8 s⁻¹. We were able to demonstrate inhibition of the enzyme by the PTP1b inhibitor BZ3, which on its turn was able to accelerate the differentiation of epimastigotes into metacyclic forms of T. cruzi induced by nutritional stress. Additionally, this compound was able to inhibit by 50% the infectivity of T. cruzi trypomastigotes in a separate cellular assay. In conclusion our results indicate that TcPTP1 is of importance for cellular differentiation and invasivity of this parasite and thus is a valid target for the rational drug design of potential antibiotics directed against T. cruzi.     

Development of a sensitive method for quantitation of ABT-089 in plasma using fluorescence labeling with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole

A high-performance liquid chromatography method for the quantitation of ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine] (I), a new structural type of cholinergic channel modulators (ChCM), is described in this paper using 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescent-labeling reagent. The method combined an optimized liquid–liquid extraction from plasma followed by pre-column derivatization to yield a fluorescence product. The selectivity, sensitivity, and reproducibility of this method were found to be excellent. This method was applied to the determination of ng/ml plasma and tissue levels of ABT-089 and similar compounds in biological samples.     

Binding of ATP to TK1-like enzymes is associated with a conformational change in the quaternary structure

Human thymidine kinase 1 (hTK1) and structurally related TKs from other organisms catalyze the initial phosphorylation step in the thymidine salvage pathway. Though ATP is known to be the preferred phosphoryl donor for TK1-like enzymes, its exact binding mode and effect on the oligomeric state has not been analyzed. Here we report the structures of hTK1 and of the Thermotoga maritima thymidine kinase (TmTK) in complex with the bisubstrate inhibitor TP4A. The TmTK-TP4A structure reveals that the adenosine moiety of ATP binds at the subunit interface of the homotetrameric enzyme and that the majority of the ATP-enzyme interactions occur between the phosphate groups and the P-loop. In the hTK1 structure the adenosine group of TP4A exhibited no electron density. This difference between hTK1 and TmTK is rationalized by a difference in the conformation of their quaternary structure. A more open conformation, as seen in the TmTK-TP4A complex structure, is required to provide space for the adenosine moiety. Our analysis supports the formation of an analogous open conformation in hTK1 upon ATP binding.     

Electron-donating para-methoxy converts a benzamide-isoquinoline derivative into a highly Sigma-2 receptor selective ligand

The sigma-2 (σ2) receptor has been suggested to be a promising target for pharmacological interventions to curb tumor progression. Development of σ2-specific ligands, however, has been hindered by lack of understanding of molecular determinants that underlie selective ligand-σ2 interactions. Here we have explored effects of electron donating and withdrawing groups on ligand selectivity for the σ2 versus σ1 receptor using new benzamide-isoquinoline derivatives. The electron-donating methoxy group increased but the electron-withdrawing nitro group decreased σ2 affinity. In particular, an extra methoxy added to the para-position (5e) of the benzamide phenyl ring of 5f dramatically improved (631 fold) the σ2 selectivity relative to the σ1 receptor. This para-position provided a sensitive site for effective manipulation of the sigma receptor subtype selectivity using either the methoxy or nitro substituent. Our study provides a useful guide for further improving the σ2-over-σ1 selectivity of new ligands.     

Plasmodium falciparum: DNA sequence specificity of cisplatin and cisplatin analogues

In this paper, we provided evidence that cisplatin is able to form adducts with cellular DNA in Plasmodium falciparum. The DNA sequence specificity of cisplatin adduct formation was determined in trophozoite-enriched P. falciparum cells and this paper represents the first occasion that the sequence specificity of cisplatin DNA damage has been observed in malaria cells. Utilising a sub-telomeric, 692 bp repeat sequence in the P. falciparum genome, we were able to investigate the DNA adducts formed by cisplatin and five analogues. A run of eight consecutive guanines was the most prominent site of DNA damage in the malarial cells. This study suggests that the mechanism of P. falciparum cell death caused by cisplatin involves damage to DNA and hence inhibition of DNA replication and cell division.     

Structural changes in bacteriorhodopsin during in vitro refolding from a partially denatured state

We report on the formation of the secondary and tertiary structure of bacteriorhodopsin during its in vitro refolding from an SDS-denatured state. We used the mobility of single spin labels in seven samples, attached at various locations to six of the seven helical segments to engineered cysteine residues, to follow coil-to-helix formation. Distance measurements obtained by spin dipolar quenching in six samples labeled at either the cytoplasmic or extracellular ends of pairs of helices revealed the time dependence of the recovery of the transmembrane helical bundle. The secondary structure in the majority of the helical segments refolds with a time constant of <100–140 ms. Recovery of the tertiary structure is achieved by sequential association of the helices and occurs in at least three distinct steps with time constants of 1), well below 1 s; 2), 3–4 s; and 3), 60–130 s (the latter depending on the helical pair). The slowest of these processes occurs in concert with recovery of the retinal chromophore.     

Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors

Identification of all residues involved in the recognition and binding of cholinergic ligands (e.g. agonists, competitive antagonists, and noncompetitive agonists) is a primary objective to understand which structural components are related to the physiological function of the nicotinic acetylcholine receptor (AChR). The picture for the localization of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are located mainly on both alpha subunits in a pocket approximately 30-35 A above the surface membrane. Since both alpha subunits are identical, the observed high and low affinity for different ligands on the receptor is conditioned by the interaction of the alpha subunit with other non-alpha subunits. This molecular interaction takes place at the interface formed by the different subunits. For example, the high-affinity acetylcholine (ACh) binding site of the muscle-type AChR is located on the alphadelta subunit interface, whereas the low-affinity ACh binding site is located on the alphagamma subunit interface. Regarding homomeric AChRs (e.g. alpha7, alpha8, and alpha9), up to five binding sites may be located on the alphaalpha subunit interfaces. From the point of view of subunit arrangement, the gamma subunit is in between both alpha subunits and the delta subunit follows the alpha aligned in a clockwise manner from the gamma. Although some competitive antagonists such as lophotoxin and alpha-bungarotoxin bind to the same high- and low-affinity sites as ACh, other cholinergic drugs may bind with opposite specificity. For instance, the location of the high- and the low-affinity binding site for curare-related drugs as well as for agonists such as the alkaloid nicotine and the potent analgesic epibatidine (only when the AChR is in the desensitized state) is determined by the alphagamma and the alphadelta subunit interface, respectively. The case of alpha-conotoxins (alpha-CoTxs) is unique since each alpha-CoTx from different species is recognized by a specific AChR type. In addition, the specificity of alpha-CoTxs for each subunit interface is species-dependent.In general terms we may state that both alpha subunits carry the principal component for the agonist/competitive antagonist binding sites, whereas the non-alpha subunits bear the complementary component. Concerning homomeric AChRs, both the principal and the complementary component exist on the alpha subunit. The principal component on the muscle-type AChR involves three loops-forming binding domains (loops A-C). Loop A (from mouse sequence) is mainly formed by residue Y(93), loop B is molded by amino acids W(149), Y(152), and probably G(153), while loop C is shaped by residues Y(190), C(192), C(193), and Y(198). The complementary component corresponding to each non-alpha subunit probably contributes with at least four loops. More specifically, the loops at the gamma subunit are: loop D which is formed by residue K(34), loop E that is designed by W(55) and E(57), loop F which is built by a stretch of amino acids comprising L(109), S(111), C(115), I(116), and Y(117), and finally loop G that is shaped by F(172) and by the negatively-charged amino acids D(174) and E(183). The complementary component on the delta subunit, which corresponds to the high-affinity ACh binding site, is formed by homologous loops. Regarding alpha-neurotoxins, several snake and alpha-CoTxs bear specific residues that are energetically coupled with their corresponding pairs on the AChR binding site. The principal component for snake alpha-neurotoxins is located on the residue sequence alpha1W(184)-D(200), which includes loop C. In addition, amino acid sequence 55-74 from the alpha1 subunit (which includes loop E), and residues gammaL(119) (close to loop F) and gammaE(176) (close to loop G) at the low-affinity binding site, or deltaL(121) (close to the homologous region of loop G) at the high-affinity binding site, are i     

Probing the structural determinants of yellow fluorescence of a protein from Phialidium sp

Fluorescent proteins homologous to green fluorescent protein (avGFP) display pronounced spectral variability due to different chromophore structures and variable chromophore interactions with the surrounding amino acids. To gain insight into the structural basis for yellow emission, the 3D structure of phiYFP (λ_em = 537 nm), a protein from the sea medusa Phialidium sp., was built by a combined homology modeling – mass spectrometry approach. Mass spectrometry of the isolated chromophore-bearing peptide reveals that the chromophore of phiYFP is chemically identical to that of avGFP (λ_em = 508 nm). The experimentally acquired chromophore structure was combined with the homology-based model of phiYFP, and the proposed 3D structure was used as a starting point for identification of the structural features responsible for yellow fluorescence. Mutagenesis of residues in the local chromophore environment of phiYFP suggests that multiple factors cooperate to establish the longest-wavelength emission maximum among fluorescent proteins with an unmodified GFP-like chromophore.     

Binding site and interlobe interactions of the ionotropic glutamate receptor GluK3 ligand binding domain revealed by high resolution crystal structure in complex with (S)-glutamate

Ionotropic glutamate receptors (iGluRs) are involved in excitatory signal transmission throughout the central nervous system and their malfunction is associated with various health disorders. GluK3 is a subunit of iGluRs, belonging to the subfamily of kainate receptors (GluK1–5). Several crystal structures of GluK1 and GluK2 ligand binding domains have been determined in complex with agonists and antagonists. However, little is known about the molecular mechanisms underlying GluK3 ligand binding properties and no compounds displaying reasonable selectivity towards GluK3 are available today. Here, we present the first X-ray crystal structure of the ligand binding domain of GluK3 in complex with glutamate, determined to 1.6 Å resolution. The structure reveals a conserved glutamate binding mode, characteristic for iGluRs, and a water molecule network in the glutamate binding site similar to that seen in GluK1. In GluK3, a slightly lower degree of domain closure around glutamate is observed compared to most other kainate receptor structures with glutamate. The volume of the GluK3 glutamate binding cavity was found to be of intermediate size between those of GluK1 and GluK2. The residues in GluK3 contributing to the subfamily differences in the binding sites are primarily: Thr520, Ala691, Asn722, Leu736 and Thr742. The GluK3 ligand binding domain seems to be less stabilized through interlobe interactions than GluK1 and this may contribute to the faster desensitization kinetics of GluK3.     

Recombinant Gaussia luciferase with a reactive cysteine residue for chemical conjugation: Expression, purification and its application for bioluminescent immunoassays

The mutated recombinant Gaussia luciferase (hgGLase) having the hinge sequence with a reactive cysteine residue at the carboxyl terminal region was purified from Escherichia coli cells by nickel-chelate affinity chromatography and hydrophobic chromatography. The biotinylated hgGLase (Biotin-hgGLase) was prepared by chemical conjugation with a maleimide activated biotin and apply to bioluminescent immunoassay. In the streptavidin and biotin complex system using Biotin-hgGLase, the measurable range of α-fetoprotein as a model analyte was 0.02–100 ng/ml with the coefficient of variation between 2.5% and 5.2%. The sensitivity of Biotin-hgGLase was similar to that by using the detection system of aequorin, alkaline phosophatase and horseradish peroxidase as a label enzyme.     

T-817MA, a neuroprotective agent, attenuates the motor and cognitive impairments associated with neuronal degeneration in P301L tau transgenic mice

Tau pathology is implicated in mechanisms of neurodegenerative tauopathies, including Alzheimer’s disease (AD) and hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). It has been reported that transgenic mice expressing FTDP-17 mutation P301L of human tau (P301L mice) display extensive tau pathology and exhibit behavioral deficits with aging. In this study, we investigated the effects of T-817MA, a neuroprotective agent, on the motor and cognitive impairments associated with neuronal degeneration in P301L mice. T-817MA prevented the progression of motor deficit and the loss of spinal cord motor neurons in P301L mice. Furthermore, T-817MA significantly attenuated the spatial memory impairment and the reduction in synaptic terminal density in the hippocampal dentate gyrus of P301L mice. These results indicate that T-817MA improved the motor and cognitive impairments as a result of inhibiting neuronal degeneration derived from tau pathology in the P301L mice. Therefore, it is expected that T-817MA has a therapeutic potential for tau-related neurodegenerative diseases such as AD.     

Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR) = 50 more than 10⁵ FomA proteins could be incorporated in a bilayer array with a total membrane area of 2 mm² within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.     

GPS autoproteolysis is required for CD97 to up-regulate the expression of N-cadherin that promotes homotypic cell-cell aggregation

Most adhesion-class G protein-coupled receptors (adhesion-GPCRs) undergo a novel self-catalytic cleavage at the GPCR proteolysis site (GPS) to form a hetero-dimeric complex containing the extracellular and seven-span transmembrane subunits. However, little is known about the role of GPS auto-proteolysis in the function of adhesion-GPCRs. Here we show that GPS cleavage is essential for the homotypic cell aggregation promoted by CD97 receptor, a leukocyte-restricted adhesion-GPCR often aberrantly expressed in carcinomas. We find that CD97 does not mediate cell aggregation directly. Instead, expression of the wild type – but not the GPS cleavage-deficient CD97 up-regulates the expression of N-cadherin, leading to Ca⁺⁺-dependent cell–cell aggregation. Our results provide a clear evidence for the role of GPS proteolytic modification in the cellular function of adhesion-GPCRs.     

Identification of oxidized phospholipids by electrospray ionization mass spectrometry and LC-MS using a QQLIT instrument

Phospholipids are complex and varied biomolecules that are susceptible to lipid peroxidation after attack by free radicals or electrophilic oxidants and can yield a large number of different oxidation products. There are many available methods for detecting phospholipid oxidation products, but also various limitations and problems. Electrospray ionization mass spectrometry allows the simultaneous but specific analysis of multiple species with good sensitivity and has a further advantage that it can be coupled to liquid chromatography for separation of oxidation products. Here, we explain the principles of oxidized phospholipid analysis by electrospray mass spectrometry and describe fragmentation routines for surveying the structural properties of the analytes, in particular precursor ion and neutral loss scanning. These allow targeted detection of phospholipid headgroups and identification of phospholipids containing hydroperoxides and chlorine, as well as the detection of some individual oxidation products by their specific fragmentation patterns. We describe instrument protocols for carrying out these survey routines on a QTrap5500 mass spectrometer and also for interfacing with reverse-phase liquid chromatography. The article highlights critical aspects of the analysis as well as some limitations of the methodology.     

Structural characterization of inhibitor complexes with checkpoint kinase 2 (Chk2), a drug target for cancer therapy

Chk2 (checkpoint kinase 2) is a serine/threonine kinase that participates in a series of signaling networks responsible for maintaining genomic integrity and responding to DNA damage. The development of selective Chk2 inhibitors has recently attracted much interest as a means of sensitizing cancer cells to current DNA-damaging agents used in the treatment of cancer. Additionally, selective Chk2 inhibitors may reduce p53-mediated apoptosis in normal tissues, thereby helping to mitigate adverse side effects from chemotherapy and radiation. Thus far, relatively few selective inhibitors of Chk2 have been described and none have yet progressed into clinical trials. Here, we report crystal structures of the catalytic domain of Chk2 in complex with a novel series of potent and selective small molecule inhibitors. These compounds exhibit nanomolar potencies and are selective for Chk2 over Chk1. The structures reported here elucidate the binding modes of these inhibitors to Chk2 and provide information that can be exploited for the structure-assisted design of novel chemotherapeutics.     

Functional and physical competition between phospholamban and its mutants provides insight into the molecular mechanism of gene therapy for heart failure

We have used functional co-reconstitution of purified sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA) with phospholamban (PLB), its inhibitor in the heart, to test the hypothesis that loss-of-function (LOF) PLB mutants (PLB_M) can compete with wild-type PLB (PLB_W) to relieve SERCA inhibition. Co-reconstitution at varying PLB-to-SERCA ratios was conducted using synthetic PLB_W, gain-of-function mutant I40A, or LOF mutants S16E (phosphorylation mimic) or L31A. Inhibitory potency was defined as the fractional increase in K_Ca, measured from the Ca²⁺-dependence of ATPase activity. At saturating PLB, the inhibitory potency of I40A was about three times that of PLB_W, while the potency of each of the LOF PLB_M was about one third that of PLB_W. However, there was no significant variation in the apparent SERCA affinity for these four PLB variants. When SERCA was co-reconstituted with mixtures of PLB_W and LOF PLB_M, inhibitory potency was reduced relative to that of PLB_W alone. Furthermore, FRET between donor-labeled SERCA and acceptor-labeled PLB_W was decreased by both (unlabeled) LOF PLB_M. These results show that LOF PLB_M can compete both physically and functionally with PLB_W, provide a rational explanation for the partial success of S16E-based gene therapy in animal models of heart failure, and establish a powerful platform for designing and testing more effective PLB_M targeted for gene therapy of heart failure in humans.