Chronic Myeloid Leukaemia (CML) is caused by the BCR/ABL1 fusion gene. Both the presence and the levels of BCR/ABL1 expression seem to be critical for CML progression from chronic phase (CP) to blast crisis (BC). After the oncogenic translocation, the BCR/ABL1 gene is under the transcriptional control of BCR promoter but the molecular mechanisms involved in the regulation of oncogene expression are mostly unknown.A region of 1443bp of the functional BCR promoter was studied for transcription factor binding sites through in-silico analysis and Chromatin Immunoprecipitation experiments. BCR and BCR/ABL1 expression levels were analysed in CML cell lines after over-expression or silencing of MYC transcription factor. A luciferase reporter assay was used to confirm its activity on BCR promoter.In the present study we demonstrate that MYC and its partner MAX bind to the BCR promoter, leading to up-regulation of BCR and BCR/ABL1 at both transcriptional and protein levels. Accordingly, silencing of MYC expression in various BCR/ABL1 positive cell lines causes significant downregulation of BCR and BCR/ABL1, which consequently leads to decreased proliferation and induction of cell death.Here we describe a regulatory pathway modulating BCR and BCR/ABL1 expression, showing that the BCR promoter is under the transcriptional control of the MYC/MAX heterodimer. Since MYC is frequently over-expressed in BC, this phenomenon could play a critical role in BCR/ABL1 up-regulation and blast aggressiveness acquired during CML evolution. Chronic Myeloid Leukaemia (CML) is a clonal myeloproliferative disorder caused by the constitutive tyrosine kinase activity of the BCR/ABL1 fusion protein, the product of the Philadelphia (Ph) chromosome, generated from the t translocation  (sc-197X)(Santa Cruz Biotechnology), mouse anti c-MYC[9E10] (sc-40)(Santa Cruz Biotechnology), rabbit anti-c-ABL[K-12] (sc-131)(Santa Cruz Biotechnology), rabbit anti PARP-1[E102] (ab32138) and anti-ACTIN (A2066).Western Blot was performed as previously described  using th5′GAGGTAGGTGGTGGGGCTTGGCTGTTCCAGGACTGCAGGACTG3′; MycMax-34-Rw 5′CAGTCCTGCAGTCCTGGAACAGCCAAGCCCCACCACCTACCTC3′.pGL3_BCR promoter constructs were obtained as in . Briefly, FUGW-H1-scrambled control shRNA (shNC) was a gift from Sally Temple [MYC silencing was generated infecting K562, LAMA-84 and KCL-22 cells with lentiviral particles obtained from modified FUGW lentiviral vectors: FUGW-MYC-shRNA(shMYC) kindly provided from Dr. RN Eisenman , FUGW-H1y Temple   and introducing artificial HindIII and EcoRI sites at the 5′ and 3′ ends of the coding region were used for amplification and the PCR product was cloned into the pCDNA3 vector. Sequences of the primers were: MAX_Fw iam Hahn   or the scrambled-shRNA(shNC). RT-qPCR and Western Blot were used to confirm MYC down-regulation. Infected 293 cells were then co-transfected with pRL and pGL3/BCR promoter constructs prepared as described in Marega et al.  and luciCell viability was monitored by CellTiter 96 AQ One Solution Cell Proliferation Assay (Promega). The MTS tetrazolium reagent was added to the cells after 24h, 48h, 72h and 96h from seeding. Absorbance was assayed with a Wallac 1450 MicroBeta Trilux (PerkinElmer).Cells were fixed in ethanol and stained with propidium-iodide (Sigma-Aldrich). Flow cytometry was performed on a Becton Dickinson FACSCalibur  and data were analysed by FCS Express 4 Flow Research Edition software.T-test) were performed by the GraphPad Prism  statistical package.All the statistical analyses (unpaired two-tailed


Background: Naturally occurring mutations in solute carrier 6 (SLC6) family members impair folding of these transporters.Results: Folding-deficient SERT mutants bound heat shock protein (HSP)70-1A, HSP90β, and co-chaperones. Noribogaine synergized with HSP inhibitors in rescuing these SERT mutants.Conclusion: Folding of SERT is assisted by a cytosolic HSP relay.Significance: The folding trajectory of SERT is relevant to folding diseases arising from mutated SLC6 transporters. e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90β by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90β. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay.Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90β. Consistent with an HSP relay, co-chaperones ( Point mutations within the coding sequence of human membrane proteins can result in their retention in the endoplasmic reticulum and thus give rise to clinically relevant phenotypes . These a5–9–i.e. its ability to rescue a folding defect resulting from mutations in the C terminus, suggests that the hydrophobic core and the C terminus cooperate during the folding reaction. This conjecture is also supported by the fact that C-terminal truncations beyond the last 16 amino acids result in inactive SERT variants  and [3H]imipramine (47.5 Ci/mmol) were purchased from PerkinElmer Life Sciences. Cell culture media, supplements, and antibiotics were from Invitrogen. The Malachite Green Phosphate Assay kit (POMG-25H) was from BioAssay Systems . The HSP inhibitors pifithrin-μ (2-phenylethynesulfonamide) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) were purchased from Sigma-Aldrich. Noribogaine was donated by Sacrament of Transition . Bovine serum albumin (BSA) and CompleteTM protease inhibitor mixture were from Roche Applied Science, SDS was from BioMol GmbH , and Tris and scintillation mixture (Rotiszint®-eco plus) were from Carl Roth GmbH . Anti-GFP antibody (ab290), anti-HSP70-1A antibody (ab47455), anti-HSC70 antibody (ab2788), anti-HSP90α antibody (ab79849), anti-HSP90β antibody (ab53497), anti-HSC70-HSP90-organizing protein (HOP) antibody (ab56873), anti-C terminus of HSP70-interacting protein (CHIP) antibody (ab109103), and anti-p23 antibody (ab2814) were all from Abcam Plc . Protein A-Sepharose and anti-rabbit IgG1 antibody linked to horseradish peroxidase were from Amersham Biosciences. The recombinant purified protein HSP70-1A (ADI-NSP55-D) was purchased from Enzo Life Sciences . All other chemicals were of analytical grade.imipramine and [3H]5-HT uptake assays were carried out as described previously  and the cDNA of human SERT cloned into the pEYFP vector as template. Oligonucleotides were from Operon Biotechnologies . HEK293 cells were grown at 37 °C in a 5% COEscherichia coli. Bacteria (2 liters of liquid culture) were grown at 37 °C to an absorbance at 600 nm (A600) of 0.6, and then bacteria were transferred to 22 °C and grown to an A600 of 0.8–0.9. Protein expression was induced at 22 °C by addition of isopropyl 1-thio-β-d-galactopyranoside (0.7 mm) for 3 h. Thereafter, the cells were harvested by centrifugation at 5,000 × g for 15 min at 4 °C. Bacterial pellets were resuspended in 25 ml of buffer containing Tris-HCl (pH 8.0), 150 mm NaCl, 0.1% Tween 20 (TBST), and Complete protease inhibitor mixture. Bacteria were incubated with lysozyme (1 mg/ml) for 30 min on ice. Lysates were supplemented with Triton X-100 to a final concentration of 1% and sonicated thrice on ice for 60 s with a Branson Sonifier® cell disruptor B15. The homogenate was cleared from cell debris by centrifugation at 48,000 × g for 25 min at 4 °C and incubated for 2 h at 4 °C with 5 ml of glutathione-SepharoseTM 4 Fast Flow , which had been pre-equilibrated in TBST, under mild stirring in a glass beaker. The suspension was transferred into chromatography columns, and the Sepharose was washed three times in 50 ml of TBST. Proteins were eluted as 1-ml fractions in 100 mm reduced l-glutathione (Sigma-Aldrich) in TBST (pH 8.0). The protein concentration of each fraction was measured using Bradford reagent. The four fractions with the highest protein concentration were combined and dialyzed against TBST using Spectra/Por® dialysis membranes . Aliquots were frozen in liquid nitrogen and stored at −80 °C.The sequence corresponding to the C terminus of SERT was inserted into the pGEX5 vector. Mutations  were introduced as described above. Plasmids were used to transform XL10-Gold g at 4 °C), the pellets were lysed in 0.5 ml of lysis buffer (20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, Complete protease inhibitor mixture) by gentle rotation at 4 °C for 30 min, and the lysates were centrifuged at 16,000 × g for 15 min at 4 °C. Supernatants containing cytosolic components were frozen in liquid nitrogen and stored at −80 °C. For experiments involving recombinant HSP70, 0.15 ml of TBST containing 2 μg of recombinant HSP70 was combined with Sepharose-bound GST proteins. The reactions were carried out by gentle rotation for 60 min at room temperature. The Sepharose beads were recovered by centrifugation and washed thrice with buffer . Bound proteins were released by heat denaturation in 50 μl of sample buffer containing β-mercaptoethanol. Aliquots of the samples (10 μl) were separated by denaturing gel electrophoresis, the resolved proteins were transferred to nitrocellulose membranes, and HSP70 was visualized by immunoblotting.For pulldown experiments, GST fusion proteins (10 μg) were immobilized on glutathione-Sepharose (25 μl of packed resin) in 0.15 ml of TBST by gentle rotation for 45 min at room temperature. The Sepharose was recovered by centrifugation and washed by resuspension in 1 ml of TBST. For experiments involving endogenous HSP70, HEK293 cell lysates (0.15 ml) were applied to the Sepharose-bound GST proteins. These cell lysates were prepared as follows. Confluent 10-cm dishes of HEK293 cells were washed twice with ice-cold PBS, the cells were harvested in 1 ml of PBS and centrifuged (5 min at 800 × m Tris-HCl (pH 8), 150 mm NaCl, 1% dodecyl maltoside, 1 mm EDTA, protease mixture inhibitors. Alternatively, at the end of the incubation period, HEK293 cells were washed with PBS, and intracellular proteins were cross-linked with 1 mm dithiobis(succinimidyl propionate) in PBS. After 45 min at room temperature, the reaction was quenched by addition of buffer ), and the cells were scraped off the dishes, harvested by centrifugation , and lysed in 0.1 ml of lysis buffer , 5 mm EDTA, 1% Nonidet P-40). The two approaches (i.e. immunoprecipitation with or without prior cross-linking) gave equivalent results. The insoluble material was removed by centrifugation at 50,000 × g for 30 min at 4 °C. Equal amounts of protein (2 mg/sample) were incubated overnight with 4 μl of anti-GFP antibody (20 μg of IgG). Subsequently, pre-equilibrated protein A-Sepharose (20 mg of protein A/sample) was added and incubated at 4 °C for 5 h with gentle rotation. The beads were collected by centrifugation and then washed three times with lysis buffer. Bound proteins were eluted by denaturation in 0.1 ml of sample buffer containing 40 mm dithiothreitol and 1% mercaptoethanol at 45 °C for 30 min. Aliquots were loaded onto SDS-polyacrylamide gels. After the proteins had been resolved by denaturing electrophoresis, they were transferred to nitrocellulose membranes. Immunoreactive bands were detected by appropriate antibodies  and a horseradish peroxidase-conjugated second antibody using enhanced chemiluminescence.Co-immunoprecipitation experiments were performed as described previously  with minm) for 2 h prior to measuring FRET. The images were acquired using a 6× oil immersion objective and an automated filter wheel  to allow for rapid switching between the fluorescence excitation and emission filters for CFP , YFP , and FRET . The images were captured by a charge-coupled device camera and analyzed using the ImageJ PixFRET plug-in  domain that is reminiscent of the canonical sequence NRLLLTG, which is recognized by the bacterial HSP70 homologue DnaK (595YRLIITP601 is the only candidate binding site within the intracellular portions of SERT. We verified this prediction in pulldown assays with GST fusion proteins comprising the SERT C terminus (35 amino acids) and mutants thereof. These were bound to glutathione-Sepharose and incubated with cell lysates of HEK293 cells  and purified recombinant HSP70-1A . Immunoblotting against bound HSP70 showed robust binding between HSP70 and the wild type C terminus of SERT; however, no binding was observed between HSP70 and the GST protein alone . Mutation of the hydrophobic core region (LII) of the putative binding motif to three serine residues (RSSSTP) or two serines (RSSITP) substantially reduced this interaction . We ruled out that introducing serine residues suppressed a nonspecific interaction between HSP70 and the C terminus of SERT by deleting the hydrophobic amino acids LII from the sequence. The resulting GST protein (with the sequence RTPGT at the fusion site) did not bind either cytosolic or purified HSP70-1A . We also examined whether the mere presence of the 596RLIIT600 motif sufficed to promote HSP70 binding by creating a GST protein that carried only these five amino acid residues. In fact, GST-RLIIT bound more HSP70-1A than any other mutant . Taken together, these data indicate that HSP70 interacted directly with the SERT C terminus and that this binding was supported by the RLIIT motif.Inspection of the sequence of SERT reveals the stretch of residues gue DnaK , 27. In C) or recombinant HSC70 . The 24-mer peptide, which covered residues 596–620  stimulated ATPase activity of HSP70 by ∼4-fold . In contrast, the 20-mer peptide, which lacked the first four residues, was substantially less effective, resulting in ∼2-fold stimulation . Similar results were obtained for the constitutively expressed HSP70 isoform HSC70 .HSP70 has a very low intrinsic ATPase activity, which is increased by binding of substrate and of HSP40/J domain proteins , 29. AccFig. 1top and bottom rows, left-hand images) with cells in which CFP-tagged SERT had already reached the cell surface (middle row). FRET was not detectable if SERT had reached the plasma membrane (middle row). In contrast, FRET was recorded between ER-resident CFP-tagged SERT and YFP-tagged HSP70-1A . Because HSP70-1A is a soluble cytosolic protein, we used YFP, which accumulates in the cytosol as a negative control. When expressed to levels comparable with YFP-tagged HSP70-1A, YFP did not give rise to FRET with ER-resident CFP-SERT (bottom right panel).Our hypothesis posits that HSP70 assists folding of SERT in the endoplasmic reticulum. Accordingly, we visualized the interaction of YFP-tagged HSP70-1A and CFP-tagged SERT by FRET microscopy in live cells. We specifically compared those transiently transfected HEK293 cells in which copious amounts of CFP-tagged SERT still resided in the endoplasmic reticulum (lane 2). In addition, these immunoprecipitates also contained the highest levels of HOP, p23/HSP90 co-chaperone, and the E3-ligase CHIP. These observations are consistent with the fact that SERT-P601A/G602A is a stalled folding mutant . Accordingly, we reasoned that the association of SERT-R607A/I608A with HSP70-1A may be enhanced by trapping the complex. We first verified that SERT-R607A/I608A was confined to the endoplasmic reticulum by examining its co-localization with calnexin. It is evident from the confocal imaging in Fig. 4A that YFP-tagged SERT-R607A/I608A and CFP-tagged calnexin were strictly co-localized. We then made use of VER-155008. This adenosine analog competitively inhibits HSP70 with an IC50 value of 500 nm, disrupts the cycle of HSP70 association and dissociation, and blocks it in a state that is halfway between the closed (ADP-liganded) and the open state . A statistical comparison showed that there was a statistically significant increase in resonance energy transfer in cells that had been treated with VER-155008 . We ruled out that the increased association of SERT-R607A/I608A and YFP-tagged HSP70-1A was due to a change in the expression levels of endogenous HSP70-1A: if HEK293 cells were pretreated with VER-155008 for 2 h (i.e. the conditions used for FRET), then the compound also promoted the association of endogenous HSP70-1A with SERT-R607A/I608A and SERT-P601A/G602A . However, the levels of endogenous HSP70-1A were comparable in control cells and VER-155008-treated cells regardless of whether the HEK293 cells expressed the mutants SERT-R607A/I608A and SERT-P601A/G602A or not .The folding deficiency of SERT-R607A/I608A is less pronounced than that of SERT-P601A/G602A. However, appreciable levels of HSP70-1A and of HOP were still co-immunoprecipitated with SERT-R607A/I608A (en state . VER-155C), and SERT and mutants thereof were subsequently transiently expressed in these cells. Specific [3H]5-HT uptake was used to quantify cell surface levels of SERT because only transporters located at the plasma membrane can take up the substrate: depletion of HSP90β, but not of HSP70-1A, resulted in a statistically significant increase in substrate uptake by SERT . This is consistent with the chaperone-COPII exchange model: the HSP relay terminates in HSP90, the release of which is (at least in part) rate-limiting for recruitment of SEC24. Likewise, depletion of HSP90β also enhanced the ability of SERT-F604Q to transport [3H]5-HT . In contrast, neither transport by SERT-R607A/I608A  nor by SERT-P601A/G602A was affected by depletion of HSP90β in a statistically significant manner . This may reflect quantitative rather than qualitative differences because depletion of HSP90β also resulted in a numerical increase in transport by these two mutants.The recently proposed chaperone-COPII exchange model posits that the SEC24 binding site becomes accessible upon release of HSP90 C, and SEcf. Figs. 5B and 6C). We combined siRNA-mediated depletion of HSP90β with noribogaine based on the assumption that pharmacochaperoning by noribogaine resulted in advancing SERT-P601A/G602A along its folding trajectory. Thus, noribogaine was predicted to render SERT-P601A/G602A susceptible to knockdown of HSP90β. This was the case .ER-trapped mutants can be rescued by various chemical and pharmacological chaperones . In our m) in the presence or absence of the HSP70 inhibitor pifithrin-μ  with a comparable EC50 . Accordingly, in the presence of pifithrin-μ, higher concentrations of noribogaine were required to elicit a half-maximum effect : the lower (labeled C) and the upper bands (labeled M) were shown previously to correspond to the ER-resident, core glycosylated transporter and the mature glycosylated transporter, respectively . Noribogaine increased the mature glycosylated form (M) ; the HSP inhibitors per se did not have any appreciable effect but augmented the action of 10 μm noribogaine with 17-DMAG being more effective than pifithrin-μ . We also blotted for the levels of G protein β-subunits  using an antiserum that recognizes all Gβ isoforms . Analogous observations were made with SERT-F604Q and SERT-R607A/I608A (data not shown).As a complementary approach, we also explored the pharmacochaperoning action of noribogaine in the absence and presence of HSP inhibitors: HEK293 cells stably expressing the mutated versions of SERT were treated with increasing concentrations of the pharmacochaperone noribogaine , SERT-R607A/I608A , and SERT-P601A/G602A  from cells that had been treated with noribogaine and HSP inhibitors and examined the association with HSP70-1A and HSP90β. Inhibition of HSP90 by 17-DMAG uniformly decreased the association of SERT mutants with HSP90β (A–C) but increased the association with HSP70-1A. This was to be expected if these mutants were stalled within the relay. By contrast with SERT-F604Q and SERT-R607A/I608A, blockage of HSP90 by 17-DMAG did not promote the association SERT-P601A/G602A with HSP70-1A . Similarly, inhibition by pifithrin-μ caused a decline in SERT-P601A/G602A, but not of SERT-F604Q and of SERT-R607A/I608A, complexed to HSP90β. This is consistent with the conclusion that SERT-P601A/G602A is trapped at an early stage, i.e. prior to the HSP70-HSP90 relay. Accordingly and contrary to SERT-F604Q  and SERT-R607A/I608A , noribogaine did not reduce complex formation of SERT-P601A/G602A with HSP70-1A and HSP90β  presumably because it increased the flux of folding intermediates through the folding trajectory and thus promoted the association of SERT-P601A/G602A with HSPs.In the HSP relay, folding intermediates advance from an HSP70-bound state to conformations engaging HSP90 . PharmacFig. 7A), and siRNA-mediated depletion of HSP90β resulted in increased cell surface levels as assessed by substrate uptake (see A). Accordingly, we also verified that inhibition of HSP90 increased the surface levels of the transporter. In HEK293 cells, which stably expressed wild type SERT, pretreatment with 17-DMAG enhanced substrate uptake  and raised the level of the mature form of the transporter . It was of obvious interest to recapitulate these findings with endogenously expressed SERT. Hence, we incubated the choriocarcinoma cell line JAR with 17-DMAG. These cells endogenously express SERT. Pretreatment of JAR cells with 17-DMAG substantially increased both maximal uptake velocity (Vmax) of 5-HT  and binding of the SERT-specific radioligand [3H]imipramine  without affecting the affinity for substrate or radioligand  be recruited to the C terminus of the receptor. Conversely, if the transporter is stalled in a misfolded state, the chaperones reroute it to ER-associated degradation. Our experiments were designed to explore the involvement of cytosolic chaperones in the folding trajectory of SERT. They demonstrate that HSP70-1A and HSP90β associate with SERT, assist in its folding in a relay, and thus determine its availability for ER export. This conclusion is based on the following findings. (i) SERT has a canonical DnaK/HSP70 binding motif in the proximal portion of its C terminus. (ii) In live cells, the interaction between HSP70-1A and SERT was confined to ER-resident rather than to surface-expressed transporters. (iii) Complex formation of SERT and in particular folding-deficient mutants of SERT with HSP70-1A was confirmed by co-immunoprecipitation. (iv) The association with HSP70-1A was per se not limiting for ER export of SERT: SERT and SERT mutants were relayed to HSP90β. Accordingly, co-chaperones  were recovered in the immunoprecipitates of stalled SERT mutants. Depletion of HSP90β by siRNA or its inhibition by 17-DMAG increased the cell surface expression of wild type SERT but was devoid of any action on the more severely affected folding-deficient mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) These mutants were rendered susceptible to the inhibition of HSP70 and HSP90 by pharmacochaperoning with noribogaine: this not only increased surface levels and the mature form of the transporter, but it also altered the level of HSP70-1A and/or HSP90β in a manner consistent with a shift in steady-state distribution of folding intermediates. (vi) Finally, in JAR cells, inhibition of HSP90 also increased the levels of the transporter SERT, indicating that endogenously expressed SERT was also susceptible to control by HSP90β. Taken together, these findings support the concept that the folded state of SERT is sampled by a cytoplasmic chaperone relay prior to recruitment of the COPII coat to the C terminus of SERT.When compared with their bacterial orthologs, eukaryotic SLC6 transporters have extended N and C termini. These extensions reflect the complexity of their regulation because they allow for the recruitment of interacting proteins and thus for modulatory input. This is presumably more important in a compartmentalized cell (in particular if it resides in a multicellular organism) than in bacterial cells. In fact, many regulatory proteins are known to bind to the N or C terminus of mammalian SERT , specifically its most prevalent representative CFTR-ΔF508. The folding state of CFTR-ΔF508 is monitored by a cytoplasmic heat shock protein relay  is thought to result from changes in the expression of HSPs (Our observations are of relevance for diseases that arise from misfolded SLC6 transporters . The mos5–9–45–5– of HSPs . We reca of HSPs . Given t


The membrane proximal external region (MPER) is a highly conserved membrane-active region located at the juxtamembrane positions within class I viral fusion glycoproteins and essential for membrane fusion events during viral entry. The MPER in the human immunodeficiency virus type I (HIV-1) envelope protein (Env) interacts with the lipid bilayers through a cluster of tryptophan (Trp) residues and a C-terminal cholesterol-interacting motif. The inclusion of the MPER N-terminal sequence contributes to the membrane reactivity and anti-viral efficacy of the first two anti-HIV peptidyl fusion inhibitors T20 and T1249. As a type I transmembrane protein, Env also interacts with the cellular membranes during its biosynthesis and trafficking. Here we investigated the roles of MPER membrane-active sequences during both viral entry and assembly, specifically, their roles in the design of peptidyl fusion inhibitors and the biosynthesis of viral structural proteins. We found that elimination of the membrane-active elements in MPER peptides, namely, penta Trp→alanine (Ala) substitutions and the disruption of the C-terminal cholesterol-interacting motif through deletion inhibited the anti-viral effect against the pseudotyped HIV-1. Furthermore, as compared to C-terminal dimerization, N-terminal dimerization of MPER peptides and N-terminal extension with five helix-forming residues enhanced their anti-viral efficacy substantially. The secondary structure study revealed that the penta-Trp→Ala substitutions also increased the helical content in the MPER sequence, which prompted us to study the biological relevance of such mutations in pre-fusion Env. We observed that Ala mutations of Trp664, Trp668 and Trp670 in MPER moderately lowered the intracellular and intraviral contents of Env while significantly elevating the content of another viral structural protein, p55/Gag and its derivative p24/capsid. The data suggest a role of the gp41 MPER in the membrane-reactive events during both viral entry and budding, and provide insights into the future development of anti-viral therapeutics. The envelope protein (Env) of human immunodeficiency virus type I HIV-1) is a class I fusion glycoprotein  is a cla–4. RecogThe post-6-HB-formation lipid mixing and subsequent membrane fusion is mediated by the membrane proximal external region (MPER) in gp41 , a hydroPrevious studies have shown that Ala substitutions of the five Trp residues abrogated the ability of MPER-containing peptides to partition into and destabilize liposomal membranes , 19. In The membrane destabilizing ability of MPER sequences has implication in designing viral fusion inhibitors. T20 and T1249, two of the first fusion inhibitors, are synthetic peptides containing sequences from both the HR2 and the N-terminus of MPER  27, 28], 2827, 2Apart from its role in viral entry, Env also interacts with host cellular membranes during its biosynthesis and trafficking to the viral budding site. As a type I transmembrane protein, Env is co-translationally translocated into the rough endoplasmic reticulum (ER) and further transported to the Golgi complex for maturation into gp160 and subsequent proteolytic processing , 35. TheHere we describe the roles of the Trp residues in the membrane-active MPER sequence in anti-HIV fusion inhibitor design and a surprising role in the biosynthesis of viral structural proteins. Six peptides ranging from 19 to 37 amino acids (a.a.) were designed to contain the MPER sequence in its full length, with C-terminal truncation, or with penta Trp→Ala substitution . Their aHIV-1 gp41-derived monomeric peptides were custom synthesized by Synpeptide Co Ltd  and the dimeric peptides by Pepscan Presto BV . The antibody to gp160/41 (Chessie 8) and Vif (#319) were obtained through the NIH AIDS Research and Reference Reagent Program . The secHLtat (Cat. #1293) and TZM-bl (Cat. #8129) cell lines were obtained through NIH AIDS Research and Reference Reagent Program –47 and cvpu-deficient HIV-1 genome with the signal sequence of env truncated and was constructed from pNL4-3 with Quick Change II XL (Stratagene) [CCGGGATACAACCTATAATAGTAGCAATAG-3’, (reverse) 5’-CTATTGCTACTATTATAGGTTGTATCCCGGTACTACTTACTGC-3’. The same mutation was introduced into ΔnSS-gp160 [CCGGGATACAACCTATAATAGTAGC-3’, (reverse) 5’-GCTACTATTATAGGTTGTATCCCGGGTACTCCGTGCGCGCTTC-3’. The XmaI-NheI fragment of the constructed mutant ΔnSS-gp160 was then directionally inserted into pUCEnvxΔu to create pUCEnvxΔuss11, in which site-directed mutagenesis was carried out with primer pair(Δss): (forward) 5’-GGATATTGATAATCTGTAGTGCTATGGAAAAATTGTGGGTC-3’, (reverse) 5’-GACCCACAATTTTTCCATAGCACTACAGATTATCAATATCC-3’ to introduce gp160 signal sequence mutation. The EcoRI-NheI fragment of the resulted mutant construct was then subcloned into p83-10 [pNLHIVxΔuΔss contains a atagene) . SpecifiSS-gp160 , encodino p83-10 , of whicGCGCTGGCGTATATAAAATTATTCATAATGATAGTAGGAGG-3’, (reverse) 5’- CCTCCTACTATCATTATGAATAATTTTATATACGCCAGCGCATTTGTTATGTTAAACCAATTCCACAAACTTGC -3’. pNL1.5EU+W3A was constructed using the primer pair B(W664.668.670): (forward) 5’- GAATGAACAAGAATTATTGGAATTAGATAAAGCGGCAAGTTTGGCGAATGCGTTTAACATAACAAATTGGCTGTGGTA -3’, (reverse) 5’- TACCACACCACATTTGTTATGTTAAACGCATTCGCCAAACTTGCCGCTTTATCTAATTCCAATAATTCTTGTTCATTC -3’. pNL1.5EU+W5A was constructed from pNL1.5EU+W3A using primer pair A (W676.678). The mutated pNL1.5EU+ plasmids were sequenced between nucleotide 7988 to 8533 with the sequencing primer (7988F-CTCCTGGGGATTTGGGGTTG and 8533R-GTCTCTCAAGCGGTGGTAGC).pNL1.5EU+W5A, pNL1.5EU+W3A and pNL1.5+W2A were constructed from pNL1.5EU+, using Quick Change II (Agilent Technologies). pNL1.5EU+W2A was constructed using the primer pair A(W676.678): (forward) 5’-GCAAGTTTGTGGAATTGGTTTAACATAACAAAT5) were co-transfected with the HIV-1 proviral clone pNLHIVxΔuΔss, and with the Env- and Vpu-expressing plasmid pNL1.5EU+, using FugeneHD (Roche). For the production of mutant pseudotyped HIV-1, we used pNL1.5EU+W5A, pNL1.5EU+W3A or pNL1.5EU+W2A during transfection, instead of pNL1.5EU+. The cell culture supernatant was collected 48 hours (h) post-transfection, clarified by centrifugation and 0.45-μm filtration, used for virion precipitation or stored at -80°C until further use.HLtat cells  polyethylene glycol 6000 containing 0.667M NaCl. The precipitated particles were further concentrated by centrifugation at 16,000 times g for 20 min at 4°C. The virus pellets were dissolved in radio-immunoprecipitation assay (RIPA) buffer containing 50 mM Tris-HCL (pH = 7.4), 1% Triton X-100, 1% deoxycholate, 150 mM NaCl, 1 mM EDTA, and 0.1% sodium dodecyl sulfate (SDS) and supplemented with Complete protease inhibitor cocktail (Roche).4 CD4+, CCR5+ TZM-bl cells. TZM-bl cells contain integrated copies of an LTR -driven luciferase reporter gene. Seventy-two h post-infection, the infectivity of pseudovirus was assessed as luciferase activity, using the One-Glo Luciferase assay system (Promega). The TCID50 of pseudotyped HIV-1 was calculated based on the luciferase activity of the infected TZM-bl cells, using the Reed–Muench method and the cut-off value set at 3 times of the background signal [Cell-free pseudotyped HIV-1 virus of 100TCID50 was either pre-incubated with peptides of various concentrations at 37°C for 1 h, or applied directly to infect 10d signal .50) and 80% (IC80) of viral infectivity were estimated with GraphPad Prism. The IC50 and IC80 values were estimated from the dose-response curves that were curve-fitted with the sigmoidal dose-response non-linear regression model on Prism GraphPad software, using the percentage of inhibition data and the log values of peptide concentrations.Effective concentrations of peptides inhibiting 50% , according to the manufacturer’s protocol. Briefly, peptides of different concentrations were added to 10,000 TZM-bl cells seeded in 96-well plates. Upon 24 h of incubation, Prestoblue cell viability reagent was added to the cells and incubated for 30 min at 37°C. Resulting absorbance values were recorded at 570nm and 600nm (baseline). Final spectrum was obtained by normalizing the 570 nm values to the 600 nm values.Circular dichroism spectroscopy analysis was performed to study the secondary structure of the monomeric peptides in trifluoroethanol (TFE). TFE was used to mimic the hydrophobic environment at the membrane fusion junction. The measurements were made on Chirascan circular dichroism spectrometer (Applied Photophysics). Fifty μM peptide was dissolved in 10%, 20%, or 40% TFE and subjected to the measurement with three repeats in a cell of 0.1mm pathlength (Hellma Uk Ltd.) at 25°C. Samples were measured between 190nm and 260nm, with a 0.5nm step resolution, a measurement speed of 60nm/min and a 1nm bandwidth. The baseline was measured with 10% TFE, with three repeats. The final spectrum was generated by subtracting the averaged sample spectrum with the baseline spectrum, followed by smoothing with a Savitsky-Golay filter. The secondary structures of the peptides were estimated from deconvoluting the respective circular dichroism spectra using the CDSSTR deconvolution algorithm on Dichroweb, with a cut-off NRMSD value set at 0.15 –55.5) expressing WT or mutant pseudotyped HIV-1 were lysed in RIPA buffer on ice, centrifuged and mixed with Laemmli reducing buffer. Precipitated pseudotyped HIV-1 viral particles dissolved in RIPA buffer were also prepared in Laemmli reducing buffer. Cell lysates or virus lysates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to nitro-cellulous membranes and immunoblotted with anti-gp41 antisera and followed by HRP-conjugated anti-rabbit secondary antibody for Env expression, or with anti-p24 antisera and followed by HRP-conjugated anti-rabbit secondary antibody for p24 and p55/Gag expression, or with anti-Nef antibody and followed by HRP-conjugated anti-rabbit secondary antibody for Nef expression, or with anti-Vif and followed by HRP-conjugated anti-mouse secondary antibody for Vif expression, or with anti-β-actin and followed by HRP-conjugated anti-mouse secondary antibody for β-actin expression. The membranes were either exposed to film or analyzed with G:BOX Chemi XX6 (Syngene). The band intensities were quantified by ImageJ software.HLtat cells (5 X 10p24 levels in the cell-free lysate of the virus-producing HLtat cells or the cell culture supernatants were quantified by the automated system ARCHITECT (Abbott). A standard curve was generated using p24 of known concentration and curve-fitted with the linear non-regression model on Prism Graphpad software. Prior to each measurement, the samples were diluted to the concentrations within the linear range of the standard curve.50 and IC80 values to 3.9 μM and 8.8 μM, respectively. However, further N-terminal extension with the addition of nine and sixteen HR2 residues to LK21, resulting in peptides EK30 and EK37, did not lead to any substantial increase in their anti-viral potency. However, adding the HR2 hydrophilic residues greatly enhanced the solubility and structural stability of the MPER-containing peptides QK26, EK30 and EK37, allowing further application and design, such as peptide dimerization that will be elaborated on in section 2.Six MPER-containing peptides  ranging To determine the necessity of the conserved MPER C-terminal cholesterol-interacting motif (LWYIK) for the antiviral effect of the peptides, the MPER C-terminal sequence, NWLWYIK, was deleted from the most active peptide, QK26, creating the peptide QT19. This deletion abrogated the antiviral activity, as QT19 failed to inhibit viral infection at concentration up to 33.3μM . In addiTo investigate the structure-function relationship of MPER-containing peptides in inhibiting the entry of pseudo-HIV-1, the secondary structures of the peptides were determined by circular dichroism . The HR2We next investigated if the antiviral effect of our peptides could be enhanced by dimerization at either the N- or C-terminus. We hypothesized that N-terminally dimerized MPER-containing peptides would mimic the fusion-active oligomerization state of gp41 MPER, thus having an enhanced binding affinity with their interaction partners (e.g. FP), and thereby possess an improved viral inhibitory effect.via a thiazolidine linkage to a linker molecule consisting of two Ser branching from Lys  and with the WT or mutant pNL1.5EU+ to produce pseudo-typed HIV-1 (NL4-3) viral particles containing WT, W5A, W3A or W2A Env. Accordingly, the expressed pseudo-HIV-1 particles were termed HIV(WT), HIV(W5A), HIV(W3A) or HIV(W2A). At 48 h post-transfection, it was found that the Env levels in cell lysates containing HIV(W5A), HIV(W3A) or HIV(W2A) were lower than that in lysate containing the HIV(WT) , with thvia the secretory pathway, the p55/Gag and Gag-Pol are expressed in the cytosol. The two viral cytosolic precursor structural proteins meet up with Env at the plasma membrane where p55/Gag directly or indirectly interacts with cytosolic tail of Env to recruit it into the assembling viral particles [More striking effects of Trp mutations were observed in the other viral structural proteins, specifically p55/Gag and its derivative proteins. The HIV-1 genome encodes three structural proteins, which are the precursor protein Env, p55/Gag, and Gag-Pol. While Env is translated into the ER and is transported to the site of viral assembly at the plasma membrane articles . During articles , 59.Analysis of the p24 levels in cells producing WT or mutant pseudo-HIV-1 revealed that at 48 h post-transfection p24 in cells producing HIV(W3A) was approximately 45%  higher as compared to p24 in cells producing HIV(WT) . HoweverThe extracellular p24 levels in the HIV(W3A)-expressing cultures, analyzed by the automated system ARCHIECT (Abbot), were further found to be 67% higher than those of the HIV(WT) , consistent with the increased intracellular p24 expression levels from this mutant . A smallThe influence of the Trp substitutions to Ala in the MPER on viral entry and infectivity was also tested by adding equal volumes of the respective cell free culture supernatant to the TZM-bl cells. Forty-eight hours post-infection, the Tat-activated expression of luciferase in the TZM-bl cells were measured and showed that in contrast to the HIV(WT), all the mutant particles HIV(W5A), HIV(W3A) and HIV(W2A) lost their ability to infect the TZM-bl cells .These data collectively suggest that, while substitutions of the Trp to Ala in the MPER sequence of gp160 decreased its intracellular expression levels and consequently moderately reduced the Env incorporation into the viral particles, the mutations significantly influenced the intracellular expression levels of p55/Gag. In particular, the substitutions of the Trp664, Trp668 and Trp670 in the MPER significantly elevated the intracellular p55/Gag expression and subsequently the viral particle production. Furthermore, the substitutions of the Trp to Ala rendered the viral particles non-infectious, in accordance with the previous literature .NL4-3: LDKWASLWNWFNITNWLWYIK) induces membrane-fusion-required membrane perturbation in the viral envelope and cellular membranes, through its two conserved membrane-active sequence elements; the enrichment of aromatic residues (e.g. Trp) and a C-terminal cholesterol interacting motif (LWYIK) [The gp41 MPER (HIV-1 (LWYIK) . Here we50 values at 8.0 μM. N-terminal extension of LK21 with five amphipathic residues derived from HR1-biding region of HR2 (628 a.a.–666 a.a.) enhanced the anti-viral effect and reduced the IC50 by half. Further N-terminal extension of LK21 did not enhance its anti-viral effect substantially, probably because this sequence does not include enough residues to mediate a stable interaction between the peptide and the viral HR1 as 6-HB formation. Instead, the enhancement of the anti-viral efficacy through addition of five amphipathic residues correlated with the subtle increase in the helicity of the resulting peptides while maintaining the general structural profiles, as estimated from the respective circular dichroism spectrum. In the context of gp41, this amphipathic sequence upstream to the MPER serves as an extension into HR2 and induces the N-terminus of MPER in fusogenic gp41 to transform from an extended conformation to a helix upon increases in local lipidity [In this study, peptide LK21, containing the entire MPER sequence, inhibited pseudotyped HIV-1(NL4-3) entry and infection with the IClipidity . This suet.al also observed that the C-terminal trimerization of T20 failed to enhance its anti-viral efficacy significantly, presumably because the trimerization constrains its membrane-active MPER sequence, offsetting the activity enhancement due to the potential cooperative interaction between its HR2 sequences and the viral gp41 HR1 [The formation of 6-HB has been suggested to induce not only a secondary structural transformation in the MPER N-terminus, but also to result in its quaternary structural rearrangement and oligomerization , 61. Meagp41 HR1 , 62. Thegp41 HR1 . Trp congp41 HR1 –66. In tgp41 HR1 . The datgp41 HR1 –32. Of nAside from its involvement in membrane fusion events, Trp residues have also been shown to modulate the interaction between MPER and other viral domain(s) during viral entry. Specifically, gp41 MPER interacts with FP to form a continuous hydrophobic track along with the 6-HB and promote membrane juxta-positioning , 69. Sho50 and IC80 values up to 5-fold. Our data would suggest that N-terminally dimerized MPER-containing peptides mimic the fusion-active oligomerization state of gp41 MPER, thus enhancing binding affinity with their interaction partners (e.g. FP and membrane), thereby having an improved anti-viral effect. The optimal length of the linker between the monomers could be explored to enhance the flexibility of the unit peptide, which may further enhance the cooperative interactions between the peptide multimer and gp41. The differential effects of N- and C-dimerization on the anti-viral effects of MPER-containing peptides have been previously observed in our group, with SARS-CoV as the model virus  in events that require protein-membrane interactions during both viral entry and assembly. These results indicate the importance of the five Trp residues and C-terminal sequence (NWLWYIK) in MPER for the design of future MPER-based fusion inhibitors and offer further insights into the understanding of viral structural proteins biosynthesis. The role of gp41 MPER Trp residues in modulating the viral contents of Gag proteins might guide the discovery of potential therapeutic targets against HIV-1 infection.S1 Fig293T cells were transiently transfected to express wild type spike protein (Swt). Twenty-four h post-transfection, cells were harvested and lysed on ice in 1% Triton X-100 TNE lysis buffer, and the cell postnuclear extracts were fractionated by 5%-30% sucrose gradient ultracentrifugation. Eleven fractions were collected from top to bottom after centrifugation. Samples were resolved by SDS-PAGE and western blot, with or without PNGase F treatment. Caveolin-1 serves as a positive marker for lipid raft.(TIF)Click here for additional data file.S2 Figvia PNGase F confirmed the gp180 and gp170 species originated from a common precursor but differed in glycosylation stage. The majority of Swt gp180 was directed to lipid-raft containing fractions, while Swt gp170 was predominantly retained in the bottom fractions. Triple Trp→Ala substitutions resulted in an altered trafficking pattern of the mature form of the S protein. In S3w3a, both S3w3a gp180 and S3w3a gp170 were found in the upper and bottom fractions at equal amounts, suggesting that a lower percentage of mature S3w3a was recruited to the lipid raft. The data suggest that the Trp residues function to fine-tune the clustering of fully mature S protein into lipid rafts during budding.The triple Trp→Ala substituted mutant of Swt was expressed in 293T cells and the lipid raft of the transfected cells were extracted, as described in (TIF)Click here for additional data file.S3 FigSARS, a peptide containing the SARS-CoV S protein MPER sequence (KYEQYIKWPWYVWLGF) and its N- and C-terminal dimers, N-MSARS and C-MSARS, were tested as fusion inhibitors against pseudotyped SARS-CoV. Pseudotyped SARS-CoV was prepared by co-transfecting 293T cells using calcium phosphate transfection method with pNL4-3Luc+Env-Vpr- and pcDNA3.1-OPT9-S mutant plasmids. pNL4-3Luc+Env-Vpr- was kindly provided by Prof. Zhang Linqi . Peptides were incubated with the virus for 1 h under 5% CO2 at 37°C, prior to being added to Vero E6 cells and incubated for another 72 h. Inhibitory activities of the peptides were calculated from the luciferase activities of the Vero E6 cells, determined by a TD-20/20 Luminometer (Tuner Designs).Peptide M(TIF)Click here for additional data file.


The ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) in the human adrenal gland causes significant hypercortisolemia after ingestion of each meal and leads to Cushing’s syndrome, implying that human GIPR activation is capable of robustly activating adrenal glucocorticoid secretion. In this study, we transiently transfected the human GIPR expression vector into cultured human adrenocortical carcinoma cells (H295R) and treated them with GIP to examine the direct link between GIPR activation and steroidogenesis. Using quantitative RT-PCR assay, we examined gene expression of steroidogenic related proteins, and carried out immunofluorescence analysis to prove that forced GIPR overexpression directly promotes production of steroidogenic enzymes CYP17A1 and CYP21A2 at the single cell level. Immunofluorescence showed that the transfection efficiency of the GIPR gene in H295R cells was approximately 5%, and GIP stimulation enhanced CYP21A2 and CYP17A1 expression in GIPR-introduced H295R cells (H295R-GIPR). Interestingly, these steroidogenic enzymes were also expressed in the GIPR (–) cells adjacent to the GIPR (+) cells. The mRNA levels of a cholesterol transport protein required for all steroidogenesis, StAR, and steroidogenic enzymes, HSD3β2, CYP11A1, CYP21A2, and CYP17A1 increased 1.2-2.1-fold in GIP-stimulated H295R-GIPR cells. These changes were reflected in the culture medium in which 1.5-fold increase in the cortisol concentration was confirmed. Furthermore, the levels of adenocorticotropic hormone (ACTH) receptor and ACTH precursor proopiomelanocortin (POMC) mRNA were upregulated 2- and 1.5-fold, respectively. Immunofluorescence showed that ACTH expression was detected in GIP-stimulated H295R-GIPR cells. An ACTH-receptor antagonist significantly inhibited steroidogenic gene expression and cortisol production. Immunostaining for both CYP17A1 and CYP21A2 was attenuated in cells treated with ACTH receptor antagonists as well as with POMC siRNA. These results demonstrated that GIPR activation promoted production and release of ACTH, and that steroidogenesis is activated by endogenously secreted ACTH following GIP administration, at least in part, in H295R cells. Glucose-dependent insulinotropic polypeptide (GIP) is a 42 amino acid peptide hormone released from intestinal K cells upon nutrient ingestion. GIP exerts multiple biological effects via GIP receptor (GIPR), which is a G-protein-coupled receptor (GPCR), through cAMP production, resulting in glucose-stimulated insulin production and secretion, cell proliferation, and anti-apoptosis in pancreatic beta-cells 7, thyrotropin, luteinizing hormone, V2-vasopressin etc). The underlying pathophysiology has been thought to be independent of ACTH et al. recently reported that cortisol secretion from the adrenal glands of patients with macronodular hyperplasia of Cushing’s syndrome appears to be regulated by ACTH, which is produced by a subpopulation of steroidogenic cells in the hyperplastic adrenal glands, but not by pituitary adrenocorticotroph cells. Tissues containing aberrant GPCRs release ACTH and cortisol during perifusion with GIP, serotonin, or human chorionic gonadotropin. The ACTH-receptor antagonist ACTH (7–38) inhibits cortisol secretion by 40% in these tissues. Thus, they showed that cortisol production is apparently controlled dually by aberrant GPCRs and by ACTH produced within the adrenocortical tissue, amplifying the effect of the aberrant receptors Adenocorticotropic hormone (ACTH) is a physiological modulator of steroidogenesis in the adrenal cortex. Binding to its receptor, melanocortin 2 receptor (MC2R), activates adenylyl cyclase and leads to cAMP production with cAMP-dependent protein kinase A (PKA) activation and phosphorylation of specific transcriptional factors, which regulate free cholesterol availability and activate steroidogenic enzyme expression et al. reported that bovine adrenal cells transfected with the GIPR and injected under the renal capsule of mice lead to the development of hyperplastic adrenal glands and hypercortisolism The ectopic expression of GIPR in the human adrenal gland causes significant hypercortisolemia after ingestion of a meal and leads to food-dependent Cushing’s syndrome (FD-CS), demonstrating that activation of human GIPR is capable of robustly activating adrenal glucocorticoid secretion The aim of our study was to investigate whether activated GIPR mediates ACTH secretion and steroidogenesis, and whether GIPR-induced steroidogenesis is operated through secreted ACTH in adrenocortical cells. H295R cells are transformed human adrenal cells that secrete all of the steroid intermediates of the steroidogenesis pathway, and have been found useful for studying cAMP/PKA signaling and steroidogenesis in adrenocortical cells The protocol for the collection of adrenal samples and the experimental procedures were approved by Ethical Review Committee of National Defense Medical College (No. 773). Written informed consent was obtained from all patients.5′-ATACTCGAGGCCACCATGACTACCTCTCCGATCCTGCAGCTGC-3′ (forward) and 5′-ATACTCGAGCTACTTGTCATCGTCGTCCTTGTAGTCGCAGTAACTTTCCAACTCCCGGCT-3′ (reverse) that encoded FLAG-tag sequence. The PCR product was subcloned into pcDNA3.1Zeo (+) at EcoRV site. Eight-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) and forskolin were purchased from Sigma-Aldrich . GIP was purchased from Peptide Institute Inc . ACTH (7–38) was purchased from Wako Pure Chemical Industries, Ltd (Osaka Japan). H-89 dihydrochloride was purchased from Calbiochem . The following antibodies were used in this study: primary antibodies: anti-mouse FLAG M2 monoclonal , anti-rabbit GIPR monoclonal , anti-goat CYP21A2 polyclonal (C-17) , anti-rabbit CYP17A1 polyclonal  antibodies, and secondary antibodies: Alexa Fluor 647 goat a anti-mouse/rabbit IgG (H+L) , Alexa Fluor 647 donkey anti-goat IgG (H+L) , Alexa Fluor 546 goat anti-mouse IgG (H+L) , Alexa Fluor 546 donkey anti-mouse/rabbit IgG . Anti-rabbit ACTH (1–39) polyclonal antibody was given by S. Tanaka C-terminally FLAG-tagged GIPR was PCR-amplified using human GIPR cDNA . The primer set was as follows, 2. The serum-free starvation medium consisted of DMEM/Ham’s F-12 medium, and penicillin/streptomycin. The cells for experiments were plated in 6-well plates at a density of 6.0×105 cells per well and cultured in the growth medium for 24 h.Human adrenal cortical cells  were purchased from American Type Culture Collection . The cells were cultured in DMEM/F-12K (1∶1) (ATCC) supplemented with 1% ITS+Premix  , 2.5% NuSerum  (BD Biosciences), and Penicillin/Streptomycin (100 U/ml penicillin and 0.1 mg/ml streptomycin) , at 37°C in a humidified atmosphere containing 5% COFor the RT-PCR experiments, the cells were cultured in the starvation medium for 24 h and then treated with 8-Br-cAMP (500 µM) and forskolin (10 µM) for 6 h. For the immunofluorescent experiments and cortisol measurements, the cells were cultured in the growth medium for 24 h, and then treated with 8-Br-cAMP and forskolin for 48 h.−7 M human GIP for 6 or 24 h. For the immunofluorescent experiments and cortisol assays, at 24 h after transfection, the medium was changed to the growth medium, and on the next day, the cells were treated with 10−7 M human GIP for 48 h.One day after plating, pcDNA3.1Zeo (+) (empty vector) or pcDNA3.1Zeo (+)-FLAG-tagged human GIPR vector (the hGIPR-c’FLAG) was introduced into H295R cells using Lipofectamine 2000 Reagent . For the RT-PCR experiments, at 24 h after transfection, the medium was changed to the starvation medium. The cells were then incubated for another 24 h, and followed by the treatment with 10Control siRNA and mixtures of siRNAs for the ACTH precursor proopiomelanocortin (POMC) were obtained from Dharmacon/ThermoFisher Scientific, Inc. . siRNAs were co-transfected with the hGIPR-c’FLAG vector using Lipofectamine 2000 according to the manufacturer’s instructions.Cortisol production from H295R cells and adrenal cells from patients was analyzed by measuring its concentration in culture medium using a Chemiluminescent Enzyme Immunoassay (LSI Medience corporation). Three independent experiments were performed in triplicate, and followed by statistical analyses.GAPDH was used as an internal control because this gene, along with the cyclophilin gene, was widely employed as an internal control for the changes of mRNA levels of the steroidogenic enzyme genes −ΔΔCt method Total RNA was extracted with RNA iso-Plus  according to the manufacturer’s instructions. Total RNA (1 µg) was treated with 122.5 U DNase I (Invitrogen) in a 100 µl reaction for 1 hour at 37°C. The enzyme was denatured at 90°C for 10 minutes, and then 2.5 µl of the solution was added to each reaction tube. Quantitative RT-PCR (qRT-PCR) was performed using a One Step SYBR PrimeScript RT-PCR kit Ver. 1  and a Thermal Cycler Dice Real –Time System  according to the manufacturer’s instructions. The thermal cycling conditions consisted of an initial denaturation step at 95°C for 30 seconds followed by 40 cycles of PCR under the following conditions: 95°C for 5 s and 60°C for 60 s. nd antibodies were used at a concentration of 1∶500.For immunofluorescence analysis, the cells were cultured on coverslips in 6-well plates. The cells were fixed with 4% paraformaldehyde and 4% sucrose in phosphate-buffered saline (PBS) for 20 min at room temperature. Permeabilization was carried out with 0.25% Triton X-100 in PBS for 5 min at room temperature. Nonspecific binding was blocked by incubation in 10% bovine serum albumin and 0.1% Triton X-100 in PBS for 30 min at 37°C. The antibodies were diluted in the above blocking solution at the indicated concentrations and incubated for 2 h at 37°C. Secondary antibodies were also diluted in the blocking solution and incubated for 30 min at 37°C. Nuclei were stained with 2 µg/ml 4′6-diamidine-2′-phenylindole dihydrochloride (DAPI)  in PBS for 15 min at room temperature. Images were acquired using a laser-scanning confocal image system . The primary antibodies, anti-flag, anti-GIPR, anti-CYP17A1, anti-CYP21A2, and anti-ACTH were used at concentrations of 1∶1000, 1∶50, 1∶250, 1∶50, 1∶100 respectively, and 2t test, or one way ANOVA followed by post-hoc Tukey using JMP9 software.Quantitative data are expressed as the mean ± standard error (SE). Statistical analysis was performed by Student’s To determine whether cAMP activation induces steroidogenic gene expression, we treated H295R cells with 8-Br-cAMP, a cAMP analog and PKA activator, and forskolin, a direct activator of adenylyl cyclase. Quantitative RT-PCR (qRT-PCR) experiments were performed using H295R with or without 8-Br-cAMP or forskolin for 6 h in the starved condition. The levels of StAR, HSD3β2, CYP11A1, CYP17A1, and CYP21A2 mRNA transcripts were significantly increased by 8-Br-cAMP to 2.6-, 1.6-, 1.7-, 1.7-, and 1.4-fold, respectively , p<0.05.Having obtained evidence that cAMP activation induced steroidogenesis in H295R cells, we asked whether steroidogenesis is induced by GIP in GIPR-expressing H295R. We transiently introduced the human GIPR gene (hGIPR-c’FLAG vector) into H295R cells and treated them with GIP. The existence of both GIPR- and FLAG-positive cells in cells transfected with the hGIPR-c’FLAG vector was confirmed by immunofluorescence analysis with double staining of anti-FLAG and anti-GIPR antibodies . FLAG-poThen, the cells were double-stained with antibodies against FLAG and CYP17A1 or CYP21A2. In the cells transfected with the empty vector treated with or without GIP, there were no FLAG-, CYP17A1-, or CYP21A2-positive cells. In the cells transfected with GIPR, but not with GIP, there were some FLAG-positive [FLAG (+)] cells, but they expressed neither CYP17A1  nor CYP2Having obtained evidence for the presence of a steroidogenesis-inducing factor secreted from GIP-stimulated and GIPR-expressing cells, we investigated whether ACTH, the most important steroidogenesis-inducing factor in adrenocortical cells, is produced and secreted by GIP-GIPR H295R cells. The prohormone POMC undergoes post-translational processing to ACTH before secretion into the systematic circulation. As shown in To investigate the role of secreted ACTH in steroidogenesis, human corticotropin inhibiting peptide  was used as a substance with respect to its antagonistic action toward the MC2R. GIP-stimulated StAR, HSD3β2, CYP11A1, CYP17A1, and CYP21A2 mRNA transcripts were partially inhibited by ACTH (7–38), whereas H89, a PKA inhibitor, completely inhibited their expression . Immunof; Wako] wFinally, we performed quantitative analysis of steroidogenic enzyme-positive cells in FLAG-tagged GIPR-transfected cells. The rate of steroidogenic enzyme-positive cells was increased both in FLAG (+) and FLAG (–) cells . Of noteIn this study, we elucidated the mechanisms regulating steroidogenesis promoted by GIP-GIPR in human adrenal H295R cells, and conclude that GIPR activation provoked steroidogenesis via the secretion of ACTH in autocrine and paracrine manners.et al. recently reported that 8-Br-cAMP promoted HSD3β2 expression and the synthesis of steroid hormones in H295R cells et al. reported that GIP activates steroidogenic gene expression in Y1 mouse adrenocortical cells stably expressing GIPR Cyclic AMP leads to the activation of kinases that phosphorylate steroidogenic transcription factors, the induction of steroidogenic enzyme expression, and subsequently steroidogenesis. ACTH, the key hormone regulating glucocorticoid and androgen biosynthesis in the adrenal cortex, exerts its effects via the GPCR, MC2R which predominantly activates the second messenger cAMP What is the secondary factor? The first candidate factor to mediate steroidogenesis is ACTH. ACTH is a 39 amino acid polypeptide that is synthesized predominantly in and secreted from the anterior lobe of the pituitary gland. The synthesis and secretion of ACTH are tightly controlled by the hypothalamic-pituitary-adrenal axis. Under stress conditions, the paraventricular nucleus of the hypothalamus secretes corticotropin-releasing hormone (CRH). CRH regulates the anterior lobe of the pituitary gland and stimulates the secretion of ACTH. Additionally, various factors such as IL-6, LIF, arginine-vasopressin, and oncostatin M induce ACTH synthesis and secretion et al. recently reported that corticotropin (ACTH) is produced by subpopulation of steroidogenic cells in the hyperplastic adrenal glands of patients with macronodular hyperplasia, and the secreted ACTH controls subsequent steroidogenesis Of particular interest, Louiset We investigated whether secreted ACTH is involved in steroidogenesis by activated GIPR. The GIP-induced secretion of cortisol was partially reduced by the MC2R antagonist ACTH (7–38), which is a fragment of corticotropin corresponding to amino acids 7 to 38 of the peptide. Further, siRNA against POMC inhibited steroidogenesis in a manner similar to ACTH (7–38). These results indicate that the activated GIPR-mediated steroidogenesis occurs, at least in part, through an interaction between ACTH and MC2R. It is noted that H89, an inhibitor of PKA, completely inhibited the mRNA expression of steroidogenic factors, whereas its inhibition by ACTH (7–38) was partial. Immunofluorescence analysis revealed that after treatment with ACTH (7–38), CYP17A1 and CYP21A2 disappeared largely in FLAG (–) cells, while they remained partially in FLAG (+), GIPR-expressing cells . These iThese observations are consistent with the notion that a process mediated via GIPR, and activated by a factor other than ACTH, is surely present. Thus another receptor, for example, the receptor of corticotropin releasing hormone (CRH), the luteinizing hormone receptor (LHR) and other G protein coupled receptors should be considered. In this paper, we did not check expression of other receptors in H295R cells, and are not able to deny the possibility that another receptor located on H295R cells that could be stimulated by GIP, is able to regulate CYP17A1 and CYP21A2 expression. The involvement of other receptors would be investigated in future study.In this study, we used serum-free medium in qRT-PCR experiments, and serum-containing medium in immunofluorescence and cortisol assay. First, we performed all experiments under starved condition, because serum includes many factors, which may affect cortisol synthesis and secretion. However, in cortisol assay, the concentration of cortisol in the medium of control, and GIP-GIPR-, forskolin- or 8-bromo cAMP-stimulated cells was below the measurable limit in starved condition. In growth condition, secretion of cortisol was clearly stimulated by GIP-GIPR, forskolin or 8-bromo cAMP, but not in the control medium. In immunofluorescence, CYP17A1 and CYP21A2 were expressed in stimulated H295R cells in both medium conditions, but the level of expression (but not rate of these enzyme-positive cells) was slightly higher in growth condition rather in starved condition, indicating that serum may affect CYP17A1 and CYP21A2 protein stability. Overall, these suggest that downstream pathway of CYP17A1 and CYP21A2 protein synthesis in cortisol production may need synergistically some factor(s) in the serum to stimulate cortisol secretion.in vitro study provides direct evidence that steroidogenesis mediated by activated GIPR is controlled by GIP-GIPR via MC2R-ACTH neo-axis- dependent or independent mechanism using immunofluorescent microscopic experiments at the single cell level. It is reported that ectopic expression of GIPR in adrenal gland induces steroidogenesis, resulting in FD-CS in vitro experiments might well reflect the pathological condition of the patient’s adrenal gland.Our Here, we have focused on the relationship between GIPR and steroidogenesis, but we believe that the principles of our assay are generalizable to other factors including GPCRs. In recent years, genomic research on adrenocortical tumors has yielded a number of new findings; comprehensive data on gene and miRNA expression, the genome, and methylation alterations in adrenocortical carcinomas are now available In summary, we showed that GIP activates steroidogenesis in GIPR-expressing H295R adrenocortical cells through the expression of genes for steroidogenic factors and enzymes and the accumulation of their respective proteins. Steroidogenesis proceeds via two pathways, the ACTH-MC2R and GIP-GIPR systems. These results were confirmed by immunofluorescent microscopic experiments at the single cell level. This study provides information that may be helpful in the elucidation of the pathology, subsequent development of therapy, and maintenance of adrenocortical tumors.Figure S1Expression of FLAG/GIPR in H295R cells transfected with human FLAG-tagged GIPR gene. H295R cells were transiently transfected with the empty vector or human GIPR expression vector. Immunostaining for FLAG and GIPR. Green staining shows the anti-FLAG antibody, red staining shows the anti-GIPR antibody, and blue staining shows DAPI (cell nuclei). Scale bars represent 100 µm.(TIF)Click here for additional data file.Figure S2Inhibitory effect of POMC siRNA on the expression of POMC. Relative mRNA expression of POMC gene was analyzed by qRT-PCR. H295R cells were transfected with the indicated siRNA. At 24 h after transfection, the culture medium was changed to the starvation medium. After 24 h, the cells were treated with GIP (10−7 M) for 24 h, and following this, RNA was extracted. Data are presented as mean ± SE of three independent experiments. *P<0.05 vs. control siRNA.(TIF)Click here for additional data file.Figure S3Analysis of adrenal gland tumor samples from a patient with FD-CS. (A) GIPR expression in samples of adrenal gland tumor from a patient with FD-CS. Normal portion in adrenal gland from a patient with aldosterone-producing adrenal tumor was used as a control. Immunostaining for GIPR and CYP21A2. Green staining shows the anti-GIPR antibody, red staining shows the anti-CYP21A2 antibody, and blue staining shows DAPI (cell nuclei). Scale bars represent 100 µm. (B) GIP stimulated cortisol production in cultured cells derived from an adrenal tumor specimen of a patient with FD-CS. The cells were treated with GIP  for 24 h. Cortisol concentration of the culture medium was measured using ELISA. *P<0.05 vs. GIP 0.0 (nM).(TIF)Click here for additional data file.Table S1Primer sequences for quantitative RT-PCR.(DOCX)Click here for additional data file.File S1Methods for experiments of patients’ samples .(DOCX)Click here for additional data file.


Background: Inhibiting PfDHODH kills malaria parasites, but the potential for drug resistance is unknown.Results: Selections gave several categories of resistance mutations. Several mutants were hypersensitive to other drugs.Conclusion: Resistance to PfDHODH inhibitors is largely though mutations in or amplification of the target gene, PfDHODH.Significance: Resistance to PfDHODH inhibitors is possible but often increases sensitivity to other compounds. Plasmodium falciparum (Pf) DHODH inhibitors via in vitro resistance selections. We found 11 point mutations in the PfDHODH target. Target gene amplification and unknown mechanisms also contributed to resistance, albeit to a lesser extent. These mutant parasites were often hypersensitive to other PfDHODH inhibitors, which immediately suggested a novel combination therapy approach to preventing resistance. Indeed, a combination of wild-type and mutant-type selective inhibitors led to resistance far less often than either drug alone. The effects of point mutations in PfDHODH were corroborated with purified recombinant wild-type and mutant-type PfDHODH proteins, which showed the same trends in drug response as the cognate cell lines. Comparative growth assays demonstrated that two mutant parasites grew less robustly than their wild-type parent, and the purified protein of those mutants showed a decrease in catalytic efficiency, thereby suggesting a reason for the diminished growth rate. Co-crystallography of PfDHODH with three inhibitors suggested that hydrophobic interactions are important for drug binding and selectivity.Malaria is a preventable and treatable disease; yet half of the world's population lives at risk of infection, and an estimated 660,000 people die of malaria-related causes every year. Rising drug resistance threatens to make malaria untreatable, necessitating both the discovery of new antimalarial agents and the development of strategies to identify and suppress the emergence and spread of drug resistance. We focused on in-development dihydroorotate dehydrogenase (DHODH) inhibitors. Characterizing resistance pathways for antimalarial agents not yet in clinical use will increase our understanding of the potential for resistance. We identified resistance mechanisms of  Plasmodium parasites. In 2010, there were an estimated 219 million cases, resulting in 660,000 malaria-related deaths  and the nucleobases thymine, cytosine, and uracil, are ubiquitous and essential in cells. There are two ways to obtain pyrimidines: salvage and de novo synthesis. Malaria parasites lack pyrimidine salvage pathways and are completely reliant upon de novo synthesis , albeit with an alternate codon. Comparative growth assays demonstrated that the E182D mutant and the E182D and D182E wild-type revertant both grew poorly when compared with the wild-type 3D7 parent, but they had similar growth rates to each other. Simultaneously selecting parasites with a combination of wild-type and mutant-type selective PfDHODH inhibitors, a test of targeting resistance, maintained a wild-type population and prevented the emergence of resistance far more often than selection with either drug alone. In contrast, simultaneously selecting parasites with compounds that did not exhibit mutually incompatible resistance did not protect from the development of resistance.pfdhodh are located near the 5′ end and are thus difficult to genetically alter via traditional allelic exchange strategies. In lieu of genetic evidence, we pursued biochemical approaches. Recombinant PfDHODH protein recapitulated the drug responses seen in mutant parasites, suggesting that point mutations in pfdhodh are the main factor in drug resistance. PfDHODH E182D protein had lower catalytic efficiency than the wild-type protein, which may provide an explanation for the mutant parasite's altered growth. Crystallography of wild-type PfDHODH protein with three inhibitors implied that selectivity is largely due to hydrophobic interactions rather than hydrogen bonding or steric clashes.Many of the resistance mutations in P. falciparum were grown at 37 °C in solutions of 4% O+ hematocrit in RPMI 1640 medium supplemented with 28 mm NaHCO3, 25 mm HEPES, and 5% albumax II (w/v). Human blood was supplied from Research Blood Components or Interstate Blood Bank. Cultures were grown in a gas mixture of 5% O2, 5% CO2, and 90% N2. Cultures were maintained with media changes every other day and were subcultured to maintain parasitemia below 4%. At least 25% of the hematocrit was replaced weekly. Parasite growth was synchronized by treatment with sorbitol . A 2.2-kb fragment encompassing the complete pfdhodh ORF was PCR-amplified from drug-resistant clones and parental lines (primer sequences below). PCR-amplified fragments were fully sequenced using pfdhodh-specific primers as follows: PfDHODH forward 5′-GATCCCTAGGATGATCTCTAAATTGAAACCTCAATTTATG-3′, PfDHODH reverse 5′-GATACTCGAGTTAACTTTTGCTATGCTTTCGGCCAATG-3′, and PfDHODH internal 5′-CATTATTTGGATTATATGGTTTTTTTGAATCTTATAATCCTG-3′.Genomic DNA was extracted from parasites using the QIAamp blood kit (Qiagen) for sequence analysis of the pfdhodh gene was amplified with Pfu Ultra II Fusion DNA polymerase (Agilent) with the following conditions: step 1, 95 °C for 120 s; step 2, 95 °C for 30 s; step 3, 55 °C for 30 s, and step 4, 60 °C for 140 s, go to step two and repeat 30 times. PCR amplicons were purified with a PCR clean-up kit (Qiagen) and sequenced. Cytochrome b  and cytochrome c oxidase  were amplified and sequenced with the following primers: Mal_mito_1 forward 5′-GTAATTTATCAAATATAAAAGCACATCTAGTTTC-3′ and Mal_mito_1 reverse 5′-CTGAATAGAATAAGAACTCTATAAATAACCAG-3′; Mal_mito_3 forward 5′-GCTCATGAACTTTTACTCTATTAATTTAGTTAAAGC-3′ and Mal_mito_3 reverse 5′-GATCTTATATGTTTGCTTGGGAGCTG-3′.The Genomic DNA was sheared and made into a 200-bp fragment Illumina sequencing library and sequenced with paired-end reads on an Illumina GAIIx machine at the Broad Institute .One microgram of genomic DNA in 50 μl of water was sheared with a Covaris sonicator. 300 ng of sheared DNA in 15 μl of water was used to create libraries in an Apollo324 machine (IntegenX) using adaptors from BioO NEXTFlex. Library quality was assessed with a bioanalyzer DNA chip and quantitative PCR . These Illumina sequencing libraries were prepared at the Harvard Bauer Core facility and sequenced with paired-end 2 × 250-bp MiSeq at the Broad Institute.P. falciparum 3D7 reference from PlasmoDB (version 7.1*)   using BWon 7.1*) . Duplicaon 7.1*)  and the on 7.1*)  mpileup Drug susceptibility was measured using the SYBR Green method , and com50 values were calculated using the log(inhibitor) versus response-variable slope equation in GraphPad Prism version 5.0d  was cloned into a pET28b plasmid with an amino-terminal His6 tag followed by a recombinant tobacco etch virus cleavage site as described by Deng et al. (6-rTEV-PfDHODH del(1–158). The listed mutations were created with the following primers and cloned into the tagged pET28b construct described above. The N61S/G254A double mutant was not made because residue 61 is not part of the heterologous expression plasmid due to solubility issues, and there is thus no complete model for this mutant protein as follows: L172F forward 5′-CCTGTACGATATTTTCTTCAAATTTTGTTTGAAGTA-3′ and L172F reverse 5′-GTACTTCAAACAAAATTTGAAGAAAATATCGTACA-3′; E182D forward 5′-GTACATCGATGGTGACATTTGCCATGACCTG-3′ and E182D reverse 5′-CAGGTCATGGCAAATGTCACCATCGATGTAC-3′; F188I forward 5′-AGCCATGACCTGATTTTGCTGCTTGG-3′ and F188I reverse 5′-CAAGCAGCAAAATCAGGTCATGGCT-3′; and F188L forward 5′-ATTTGCCATGACCTGCTGTTGCTGCTTGG-3′ and F188L reverse 5′-CCAAGCAGCAACAGCAGGTCATGGCAAAT-3′; F227I forward 5′-GTTGCAGCTGGAATCGATAAAAACGGTG-3′ and F227I reverse 5′-CACCGTTTTTATCGATTCCAGCTGCAAC-3′; I263F forward 5′-GAAACCGCGGTTCTTTCGTGACGTC-3′ and I263F reverse 5′-GACGTCACGAAAGAACCGCGGTTTC-3′; L527I forward 5′-CTTCCGTGTGTCAGATCTATTCGTGCTT-3′ and L527I reverse 5′-AAGCACGAATAGATCTGACACACGGAAG-3′; and L531F forward 5′-GCTCTATTCGTGCTTCGTTTTCAACGGTATG-3′ and L531F reverse 5′-CATACCGTTGAAAACGAAGCACGAATAGAGC-3′.A synthetic codon-optimized gene encoding residues 159–565 of PfDHODH  were transformed with pET28b-PfDHODH expression constructs. Transformed cells were grown in Terrific Broth with 100 μg/ml kanamycin at 37 °C and 180 rpm and then dropped to 20 °C and 150 rpm for induction. Cultures were induced with 200 μm isopropyl d-thiogalactoside at A600 = 0.6–0.8. Cultures were grown 12–16 h post-induction, then pelleted by centrifugation at 10,000 × g, and frozen at −80 °C for later use. For purification, thawed pellets were resuspended in buffer A . The cells were lysed by sonication on ice . The cell lysate was clarified by centrifugation at 40,000 × g for 45 min at 4 °C and then filtered through a 0.45-μm filter. The clarified and filtered lysate was applied to a HisPrep FF 16/10 column  pre-equilibrated with buffer A on an AKTA Purifier FPLC. The column was washed with 30 column volumes of buffer A and then eluted with stepwise increments of buffer B . The eluted protein was concentrated by centrifugation in concentrator spin columns (Pierce) and then injected into a HiLoad 16/60 Superdex 200 size exclusion column pre-equilibrated with size exclusion buffer . Fractions containing PfDHODH were pooled and concentrated. Protein concentration was assessed by Bradford assay (Pierce). Protein aliquots were flash-frozen in liquid nitrogen and stored at −80 °C.BL21(DE3)Star in vitro assay in 384-well clear plates (Corning 3702) as described by Malmquist et al. (m protein with 500 μml-dihydroorotate substrate (excess), 18 mm dodecylubiquinone electron acceptor (∼mK), and 100 μm 2,6-dichloroindophenol indicator dye in assay buffer . Assays were incubated at 25 °C for 20 min and then assessed via A600. Atmospheric oxygen, which can act as an electron acceptor, was depleted through the addition of 0.1 mg/ml glucose oxidase, 0.02 mg/ml catalase, and 50 mm glucose, followed by incubation at 25 °C for 5 min prior to assay assembly. Data were normalized to 1% DMSO and excess inhibitor (500 nm Genz-669178 unless another compound or concentration was appropriate). Steady-state kinetic parameters were determined as described by Malmquist et al. (296 = 4.3 mm−1 cm−1) in UV-transparent 384-well plates (Corning 3672). For enzyme oxidase activity, 100 nm enzyme was assayed against 5–500 μml-DHO. For oxidant affinity, 5–50 nm enzyme was assayed against a range of CoQd (1–150 μm) with excess substrate (500 μml-DHO). Data were fitted to the Michaelis-Menten equation in GraphPad Prism 5.0d  and Diamond (I03). Molecular models of the data are presented with CCP4mg  was produced as described  by Glaxop < 0.05.Data are shown as mean ± S.D. and were analyzed by one-way analysis of variance with Dunnett's multiple comparison post hoc test via GraphPad Prism 5.0d software. Differences were considered significant for Plasmodium-selective DHODH inhibitors  as follows: mutation in the target, copy number variation of the target, or a broad “other” category that could include adopting a quiescent state, increasing the quantity or activity of multidrug efflux pumps, altered drug metabolism, or altering pyrimidine biosynthetic metabolism or use through some other means.There are three theoretically possible mechanisms of resistance , A and BIn six sets of primary selections, we identified five instances of wild-type target gene amplification and six instances of single nucleotide polymorphisms (SNPs) in PfDHODH as follows: E182D (twice), F188I, F188L, F227I, and L531F . NotablyFour sets of sequential selections gave target gene amplification twice and SNPs in PfDHODH nine times . The L17Four sets of simultaneous selections gave one instance of resistance, and this was associated with a SNP in PfDHODH, I263F . Combinipfdhodh gene. The other five lines were resistant through unknown mechanisms.All PfDHODH inhibitors, whether selective for wild-type or mutant-type, are highly synergistic with other PfDHODH inhibitors (data not shown). As a control, we identified compound pairs with similar levels of synergy as determined by a modified fixed-ratio isobologram  that didB).Primary selection gave resistance in 81.25% of attempts, sequential selection, 86%, and simultaneous selection, only 6.67%. Importantly, simultaneous selection with synergistic compounds that do not exhibit mutually incompatible resistance led to resistance in 100% of attempts B.C) (The resistance-associated SNPs lined the “species-selective inhibitor site” in PfDHODH C 23). T. TC) , and E182D and F227I were more sensitive to IDI-6273 . I263F gave resistance to both selecting agents as follows: 6.7-fold resistance to Genz-669178 and 66.7-fold resistance to GSK3 . The mutations F188I and F188L increased sensitivity to the wild-type-selective inhibitor DSM74 about 25-fold . The addition of an L172F mutation to the F227I mutant line increased the sensitivity to DSM74 15.8-fold, from 490 to 31 nm. The L531F mutant gave only slight, nonstatistically significant resistance to the corresponding selection agent DSM74 (490 to 600 nm). Perhaps PfDHODH L531F has a survival advantage that does not directly relate to changes in drug IC50, such as increased catalytic efficiency.The different pathways to resistance gave markedly different responses to drug pressure. SNPs in PfDHODH often led to large changes in drug sensitivity . We obse50, and this reached significance. Copy number of the multidrug-resistant transporter gene pfmdr1 did not correlate with dihydroartemisinin resistance, and there is no known link yet between pyrimidine pools or PfDHODH-influenced mitochondrial physiology and dihydroartemisinin resistance.The unrelated antimalarial dihydroartemisinin was used as a control, although most resistant parasite lines showed no significant changes in sensitivity, F227I and F227I and L527I (but not F227I and L172F) both gave an ∼1.8-fold increase in ICpfdhodh generally gave lower levels of resistance  to 600–610 nm .Parasite lines with target gene amplification and no mutation in sistance . We obse50 of 490 nm dropping to 74 or 110 nm). F227I-amplified lines were also more sensitive to the mutant-selective inhibitors IDI-6253 (6600 to 630 or 570 nm) and IDI-6273 (1400 to 540 or 520 nm). Two copies of wild-type pfdhodh in a Dd2 background increased sensitivity to the wild-type-selective inhibitor DSM74, with IC50 values dropping from 130 to 59 nm, a 2.2-fold decrease. It is unclear how increasing gene dose increases sensitivity to certain compounds, although these parasite lines may certainly have unknown differences outside of pfdhodh copy number and sequence. Producing transgenic parasite lines in isogenic backgrounds could help dissect this point.Parasites with multiple copies of PfDHODH: F227I were more sensitive to the wild-type-selective inhibitor DSM74 , FMN, and pfdhodh gene , another parasite with seven copies of pfmdr1 had no change in sensitivity. The parasites selected with atovaquone, a cytochrome bc1 inhibitor, had no mutations in cytochrome b or cytochrome c oxidase (data not shown). PfDHODH and cytochrome bc1 are both embedded in the inner mitochondrial membrane. PfDHODH requires functional cytochrome bc1 for generation and maintenance of a mitochondrial proton gradient, and thereby a supply of respiratory chain ubiquinone.Finally, we identified five parasite lines that were resistant to PfDHODH inhibitors without mutation in or copy number variation of the odh gene . All of P. falciparum, it is possible that rare codon effects such as reduced mRNA stability and translation elongation pausing affect this “wild-type revertant” parasite  identified an I239V mutation in cytochrome c oxidase  relative to the 3D7 reference, but this mutation was also present in the 3D7: E182D line. Cytochrome c oxidase I239V may be important as PfDHODH activity relies on electron flow to and from the mitochondrial electron transport chain.Indeed, the 3D7 E182D/D182E revertant was not perfectly wild-type, although the sensitivity to some drugs was more similar to wild-type than the E182D mutant , some were not. IDI-6273 changed from an ICrent see . Whole gA). However, when the two mutants were grown together, no change was seen in relative allele frequency, indicating roughly equal fitness in the conditions tested. It is possible that the E182D mutation and the effects of a rare codon in the E182D/D182E revertant line counterbalance each other. Organismal fitness could be further assessed by determining cell cycle time and burst size by flow sorting. Protein stability could be assessed via ribosomal profiling and/or Western blot, although this task is hampered by the lack of commercially available PfDHODH antibodies.We measured comparative growth rates by growing all pairwise combinations of these three cell lines. Over six generations, both the E182D mutant and the E182D/D182E revertant declined to about 40% prevalence when grown with 3D7 and then held steady, indicating altered growth rates in both mutants relative to wild type A. HoweveB and C). The E182D mutation decreased kcat and increased mK values for both the l-dihydroorotate (l-DHO) substrate and the CoQd co-substrate, although it should be noted that the E182D mutant protein appears to degrade more easily than the wild-type protein and is thus not a homogeneous species. We do not know if differences observed between wild-type and E182D proteins are due to differences in enzyme activity and/or stability. Additionally, expression of full-length protein that may better reflect physiology is not yet possible due to an amino-terminal membrane anchor and mitochondrial targeting sequence that are deleted for all protein work shown. Enzyme kinetic changes were all less than 6-fold and are clearly compatible with viability in culture.We determined the steady-state parameters of recombinant wild-type and E182D PfDHODH protein by directly measuring the production of orotate product , B and Cpfdhodh gene are the driving factor in resistance and not mere coincidence. For example, inhibition of E182D protein activity required more Genz-669178 compound than wild-type protein, which is also true in cells (B). The fit between protein and cell data was substantially closer to the wild-type than many of the mutants, indicating that the in vitro protein activity assays may be missing components that affect mutant PfDHODH in cells, such as protein-folding chaperones or biological membranes to cross. Interestingly, many of the inhibitors were more potent in cellular assays than against purified protein. This indicates that these inhibitors may have multiple targets, active metabolites in cells, and/or a cellular accumulation effect. Several mutant proteins were not inhibited by the maximum amount of drug permissible in the assay (2 mm).We cloned and expressed codon-optimized PfDHODH proteins with engineered resistance mutations . We usedin cells B. The fiD), and subtle changes in helix packing slightly shifted other residues. His-185 is one of only two polar residues to contact the ligands (the other is Arg-265). Mutation of Phe-188 to leucine or isoleucine gave high level resistance to wild-type PfDHODH inhibitors, so it is interesting that Phe-188 is so flexible. Two main possibilities could explain this: one, the large aromatic ring of phenylalanine has more favorable interactions with the inhibitors than the small aliphatic chains of leucine and isoleucine, perhaps pi stacking or edge-to-face interactions. Two, Phe-188 acts as a gatekeeper to the inhibitor-binding site tunnel, and the small residues do not hold the inhibitors in place.In an attempt to better understand selectivity between and resistance to PfDHODH inhibitors, we performed co-crystallography experiments. We crystallized wild-type PfDHODH with the following three inhibitors: Genz-669178, IDI-6253, and IDI-6273 . The inhFig. 7ribbon diagram in Fig. 1C), we can speculate that the bulky phenylalanine substitution in I263F may prevent multiple structural classes of PfDHODH inhibitors from binding, whether through direct steric clashes, altered helix packing, or an altered path of electron flow.I263F and the combination G254A and N61S both arose after simultaneous selection with two compounds. I263F came from the combination of Genz-669178, a wild-type-selective inhibitor, and GSK3, a mutant-selective inhibitor. From the Genz-669178 structure  tended to give high level resistance with little apparent consequence. Mutations lining the presumed electron tunnel  tended to give low level resistance, increased susceptibility to other compounds, and an anecdotal reduced protein stability. Tunnel mutations appear more liable to compromise enzyme function than lid mutations, and thus have graver consequences.In general, mutations near the top or bottom of the amino-terminal helix-turn-helix lid  as the main resistance locus for mefloquine . Surprisingly, chloroquine-resistant parasites were hypersensitive to lumefantrine. This induced sensitivity was traced to mutually incompatible resistance mechanisms ; pfcrt afantrine , 35. As fantrine .P. falciparum dihydrofolate reductase (An area for improvement in the targeting resistance strategy would be to identify mutant-specific (rather than mutant-selective) compounds. The mutant-selective compounds have relatively little activity against the wild type, but it is not negligible. This selective force can lead to resistance developing in the wild-type population, which could compromise the effectiveness of the mutant-selective compound when needed. The wild-type population is large and thus has a large potential for diversity. The emerging resistant population is small and thus has relatively little diversity. The less time the nascent resistant population is allowed to exist, the less time it has to accumulate diversity and thus develop competitively viable resistance. Therefore, the goal is to neutralize emerging threats as soon as possible, before they have a chance to spread or become more fit through compensatory mutations. Identifying mutant-specific inhibitors would eliminate selective pressure for resistance in the wild-type population. Another strategy is to identify inhibitors that are highly active against both wild-type and mutant-type parasites, as was neatly demonstrated for eductase .By targeting both the wild-type population and the most fit resistance pathways, we can accomplish two goals, kill parasites and help prevent the emergence of resistance. Selecting partner drugs or creating double-warhead molecules that block resistance increases the useful lifetime of an antimalarial agent, and it should be considered before putting therapeutics in clinical use.


We found that the methyl 13C chemical shifts of the M230 resonance of HIV-1 RT bound to these drugs exhibited a high correlation with their anti-HIV-1 RT activities. This methionine residue is located in proximity to the NNRTI-binding pocket but not directly involved in drug interactions and serves as a conformational probe, indicating that the open conformation of HIV-1 RT was more populated with NNRTIs with higher inhibitory activities. Thus, the NMR approach offers a useful tool to screen for novel NNRTIs in developing anti-HIV drugs.Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is an important target for antiviral therapy against acquired immunodeficiency syndrome. However, the efficiency of available drugs is impaired most typically by drug-resistance mutations in this enzyme. In this study, we applied a nuclear magnetic resonance (NMR) spectroscopic technique to the characterization of the binding of HIV-1 RT to various non-nucleoside reverse transcriptase inhibitors (NNRTIs) with different activities, i.e., nevirapine, delavirdine, efavirenz, dapivirine, etravirine, and rilpivirine.  Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) plays an important role in HIV-1 replication by catalyzing the conversion of single-stranded RNA into double-stranded DNA. This enzyme is one of the most promising targets for anti-HIV drug development to suppress the production of new viral particles. The structure of HIV-1 RT consists of an asymmetric heterodimer of two subunits, a 66 kDa subunit (p66) containing both polymerase and RNase H domains, and a 51 kDa subunit (p51) containing only a polymerase domain121HIV-1 RT inhibitors can be divided into two classes, nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). NRTIs are nucleoside analogs lacking the 3′-OH group and acts as a chain terminator of DNA synthesis. NNRTIs are small molecules that bind to a hydrophobic pocket located in proximity to the polymerase active site on the p66 subunit513C at the methyl side chain of methionine offers useful spectroscopic probes for investigating the structures and dynamics of larger proteins14151617et al. previously reported heteronuclear single-quantum coherence (HSQC) spectra for observing signals from the methionine methyl groups of the HIV-1 RT p66 subunit in the absence and presence of nevirapine, with assignments based on the site-directed mutagenesis method1613C resonances.Biophysical and structural approaches are useful for rapid, efficient development of small molecule inhibitors targeting HIV-1 RT. X-ray crystallography offers atomic images of the different binding modes of HIV-1 RT between NRTIs and NNRTIs56810111213C chemical shift of M230 in the p66 subunit, which is located in close proximity to the inhibitor binding pocket, serves as a useful indicator of the efficacy of these NNRTIs.In the present study, we have applied the NMR technique to characterize the interactions of HIV-1 RT with various NNRTIs with different inhibitory activities, nevirapine, delavirdine, efavirenz, dapivirine, etravirine, and rilpivirine . We foun13C-labeled p66 and unlabeled p51 was prepared by bacterial expression using [methyl-13C]methionine. The recombinant p66 subunit possesses six intrinsic methionine residues and an extra methionine residue at its N-terminus. The 1H-13C HSQC spectrum of the apo form of the 13C-labeled HIV-1 RT protein gave four peaks , as shown in Spectral comparison among the six NNRTI-bound forms of HIV-1 RT indicated significant chemical variation of the M230 peak . The met13C chemical shift of the methionine methyl group can refer to side chain conformations, associated with χ3 values: the 13C chemical shifts of 16 ppm and 19 ppm correspond to gauche and trans conformations, respectively, whereas 17 ppm indicates flexibly exchanging trans and gauche conformationsgauche conformation, while it occasionally adapted a trans conformation in the rilpivirine-bound open form (PDB code 3MEE), presumably with increased mobility. Under this circumstance, 13C chemical shift of M230 reflects drug-induced population shifts of the open and closed conformations. The intensity of the M230 resonance in the rilpivirine- and etravirine-bound forms of HIV-1 RT was higher than that in the nevirapine-, delavirdine- and efavirenz-bound forms. This indicates that diarylpyrimidine-based inhibitors (DAPYs) such as rilpivirine and etravirine fix the open conformation most tightly, rendering M230 exposed with its highest mobility  in comparison with nevirapine, delavirdine, and efavirenz. In other words, fixation in open conformation by nevirapine, delavirdine, and efavirenz are not complete. Consistent with this interpretation, the enzyme bound to efavirenz remains under dynamic equilibrium between the open and closed formsThe 13C resonances from the p66 subunit. We confirmed that the compounds examined in this study share the same binding pocket. Moreover, our NMR data revealed that the methyl 13C chemical shift of M230 can be used as an indicator of the efficacy of NNRTI, offering a useful tool in screening for novel inhibitors in developing anti-HIV drugs.In summary, we applied NMR spectroscopy to probe NNRTI binding to HIV-1 RT using the methionine methyl Powdered rilpivirine, delavirdine, dapivirine, and etravirine were purchased from MedChem Express. Nevirapine and efavirenz were synthesized by Dr. Supanna Techasakul .DpnI (TOYOBO) and transformed into the Escherichia coli strain DH5α. The mutations were confirmed by DNA sequencing using an ABI 3130xl genetic analyzer (Applied Biosystems).Site-directed mutagenesis was performed to prepare six variants of the p66 subunit of HIV-1 RT, M16L, M41L, M164L, M184L, M230L, and M357L. The recombinant plasmids containing HIV-1 RT genes, pGEX3X E. coli BL21(DE3)-RIL strain (Agilent Technologies) in M9 medium containing 50 μg/ml of ampicillin with L-[methyl-13C]methionine (Cambridge Isotope Laboratories) using previously described protocols27D-thiogalactopyranoside at 16 °C for 16–18 h. The harvested cells of p51 and p66 HIV-1 RT were suspended and mixed into 50 mM Tris-HCl (pH 7.5) containing 0.5 mM EDTA, 50 mM NaCl, 1 mM DTT, 5% (w/v) glycerol, 0.5% (w/v) Triton X-100, and protease inhibitor cocktail (Sigma-Aldrich) and then disrupted by sonication. The HIV-1 RT protein was sequentially purified from cell lysates with a DEAE cellulose column (Whatman), a phosphocellulose P11 column (Whatman), a Chelating Sepharose Fast Flow  charged with nickel sulfate, and a RESOURCE S column . Finally, the HIV-1 RT protein was purified by Superdex-200 (HiLoad 16/60) gel filtration using a FPLC column . All purification steps of HIV-1 RT were performed at 4 °C with buffer containing protease inhibitors.Wild-type and mutated p66 subunit proteins were individually expressed in the To confirm the existence of the extra N-terminal methionine, HIV-1 RT protein was incubated with methionine aminopeptidase (Clontech) at 37 °C for 12 h, and checked by NMR.11 (pD 7.6) containing 200 mM KCl, 1.5 mM sodium azide, and 4 mM MgCl2. A 20 mM stock solution of each inhibitor was prepared in dimethylsulfoxide-d6 and was added in five-fold molar excess to the protein. Spectra were processed and analyzed with the programs Topspin and SPARKYAll NMR measurements were made using an AVANCE800 (Bruker BioSpin) spectrometer equipped with a cryogenic probe. The probe temperature was set at 25 °C. The wild-type or mutated HIV-1 RT was dissolved at a concentration of 27 μM in 10 mM Tris-HCl-dHow to cite this article: Thammaporn, R. et al. NMR characterization of HIV-1 reverse transcriptase binding to various non-nucleoside reverse transcriptase inhibitors with different activities. Sci. Rep.5, 15806; doi: 10.1038/srep15806 (2015).