Dihydrofolate Reductase Domain Research Articles

Lanthanide-based resonance energy transfer biosensors for live-cell applications.

Lanthanide-based, Förster resonance energy transfer (LRET) biosensors enable sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living mammalian cells. LRET biosensors are polypeptides that consist of an alpha-helical linker sequence sandwiched between a lanthanide complex-binding domain and a fluorescent protein (FP) with two interacting domains residing at each terminus. Interaction between the terminal affinity domains brings the lanthanide complex and FP in close proximity such that lanthanide-to-FP, LRET-sensitized emission is increased. A recent proof-of-concept study examined model biosensors that incorporated the affinity partners FKBP12 and the rapamycin-binding domain of m-Tor (FRB) as well as p53 (1-92) and HDM2 (1-128). The sensors contained an Escherichia coli dihydrofolate reductase (eDHFR) domain that binds with high selectivity and affinity to Tb(III) complexes coupled to the ligand trimethoprim (TMP). When cell lines that stably expressed the sensors were treated with TMP-Tb(III), TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. Much larger signal changes (>2500%) and Z'-factors of 0.5 or more were observed when cells were grown in 96-well or 384-well plates and analyzed using a TGL plate reader. In this chapter, we elaborate on the design and performance of LRET biosensors and provide detailed protocols to guide their use for live-cell microscopic imaging studies and high-throughput library screening.

A Destabilizing Domain Allows for Fast, Noninvasive, Conditional Control of Protein Abundance in the Mouse Eye - Implications for Ocular Gene Therapy.

PurposeTemporal and reversible control of protein expression in vivo is a central goal for many gene therapies, especially for strategies involving proteins that are detrimental to physiology if constitutively expressed. Accordingly, we explored whether protein abundance in the mouse retina could be effectively controlled using a destabilizing Escherichia coli dihydrofolate reductase (DHFR) domain whose stability is dependent on the small molecule, trimethoprim (TMP).MethodsWe intravitreally injected wild-type C57BL6/J mice with an adeno-associated vector (rAAV2/2[MAX]) constitutively expressing separate fluorescent reporters: DHFR fused to yellow fluorescent protein (DHFR.YFP) and mCherry. TMP or vehicle was administered to mice via oral gavage, drinking water, or eye drops. Ocular TMP levels post treatment were quantified by LC-MS/MS. Protein abundance was measured by fundus fluorescence imaging and western blotting. Visual acuity, response to light stimulus, retinal structure, and gene expression were evaluated after long-term (3 months) TMP treatment.ResultsWithout TMP, DHFR.YFP was efficiently degraded in the retina. TMP achieved ocular concentrations of ∼13.6 μM (oral gavage), ∼331 nM (drinking water), and ∼636 nM (eye drops). Oral gavage and TMP eye drops stabilized DHFR.YFP as quickly as 6 hours, whereas continuous TMP drinking water could stabilize DHFR.YFP for ≥3 months. Stabilization was completely and repeatedly reversible following removal/addition of TMP in all regimens. Long-term TMP treatment had no impact on retina function/structure and had no effect on >99.9% of tested genes.ConclusionsThis DHFR-based conditional system is a rapid, efficient, and reversible tool to effectively control protein expression in the retina.

A barcode of multilocus nuclear DNA identifies genetic relatedness in pre- and post-Artemether/Lumefantrine treated Plasmodium falciparum in Nigeria

BackgroundThe decline in the efficacy of artemisinin-based combination treatment (ACT) in some endemic regions threatens the progress towards global elimination of malaria. Molecular surveillance of drug resistance in malaria-endemic regions is vital to detect the emergence and spread of mutant strains.MethodsWe observed 89 malaria patients for the efficacy of artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum infections in Lagos, Nigeria and determined the prevalence of drug resistant strains in the population. Parasite clearance rates were determined by microscopy and the highly sensitive var gene acidic terminal sequence (varATS) polymerase chain reaction for 65 patients with samples on days 0, 1, 3, 7, 14, 21 and 28 after commencement of treatment. The genomic finger print of parasite DNA from pre- and post-treatment samples were determined using 24 nuclear single nucleotide polymorphisms (SNP) barcode for P. falciparum. Drug resistance associated alleles in chloroquine resistance transporter gene (crt-76), multidrug resistance genes (mdr1–86 and mdr1–184), dihydropteroate synthase (dhps-540), dihydrofolate reductase (dhfr-108) and kelch domain (K-13580) were genotyped by high resolution melt analysis of polymerase chain reaction (PCR) fragments.ResultsBy varATS qPCR, 12 (18.5%) of the participants had detectable parasite DNA in their blood three days after treatment, while eight (12.3%) individuals presented with genotypable day 28 parasitaemia. Complexity of infection (CoI) was 1.30 on day 0 and 1.34 on day 28, the mean expected heterozygosity (HE) values across all barcodes were 0.50 ± 0.05 and 0.56 ± 0.05 on days 0 and 28 respectively. Barcode (π) pairwise comparisons showed high genetic relatedness of day 0 and day 28 parasite isolates in three (37.5%) of the eight individuals who presented with re-appearing infections. Crt-76 mutant allele was present in 38 (58.5%) isolates. The mdr1–86 mutant allele was found in 56 (86.2%) isolates. No mutation in the K-13580 was observed.ConclusionsPersistence of DNA-detectable parasitaemia in more than 18% of cases after treatment and indications of genetic relatedness between pre- and post-treatment infections warrants further investigation of a larger population for signs of reduced ACT efficacy in Nigeria.

Targeting plant DIHYDROFOLATE REDUCTASE with antifolates and mechanisms for genetic resistance.

The folate biosynthetic pathway and its key enzyme dihydrofolate reductase (DHFR) is a popular target for drug development due to its essential role in the synthesis of DNA precursors and some amino acids. Despite its importance, little is known about plant DHFRs, which, like the enzymes from the malarial parasite Plasmodium, are bifunctional, possessing DHFR and thymidylate synthase (TS) domains. Here using genetic knockout lines we confirmed that either DHFR-TS1 or DHFR-TS2 (but not DHFR-TS3) was essential for seed development. Screening mutated Arabidopsis thaliana seeds for resistance to antimalarial DHFR-inhibitor drugs pyrimethamine and cycloguanil identified causal lesions in DHFR-TS1 and DHFR-TS2, respectively, near the predicted substrate-binding site. The different drug resistance profiles for the plants, enabled by the G137D mutation in DHFR-TS1 and the A71V mutation in DHFR-TS2, were consistent with biochemical studies using recombinant proteins and could be explained by structural models. These findings provide a great improvement in our understanding of plant DHFR-TS and suggest how plant-specific inhibitors might be developed, as DHFR is not currently targeted by commercial herbicides.

Spatial organization of protein export in malaria parasite blood stages.

Plasmodium falciparum, which causes malaria, extensively remodels its human host cells, particularly erythrocytes. Remodelling is essential for parasite survival by helping to avoid host immunity and assisting in the uptake of plasma nutrients to fuel rapid growth. Host cell renovation is carried out by hundreds of parasite effector proteins that are exported into the erythrocyte across an enveloping parasitophorous vacuole membrane (PVM). The Plasmodium translocon for exported (PTEX) proteins is thought to span the PVM and provide a channel that unfolds and extrudes proteins across the PVM into the erythrocyte. We show that exported reporter proteins containing mouse dihydrofolate reductase domains that inducibly resist unfolding become trapped at the parasite surface partly colocalizing with PTEX. When cargo is trapped, loop-like extensions appear at the PVM containing both trapped cargo and PTEX protein EXP2, but not additional components HSP101 and PTEX150. Following removal of the block-inducing compound, export of reporter proteins only partly recovers possibly because much of the trapped cargo is spatially segregated in the loop regions away from PTEX. This suggests that parasites have the means to isolate unfoldable cargo proteins from PTEX-containing export zones to avert disruption of protein export that would reduce parasite growth.

NOVEL SCHIFF'S BASES OF SUBSTITUTED 2-AMINO BENZOTHIAZOLES: DESIGN, SYNTHESIS AND ANTIMICROBIAL ACTIVITY

Novel Schiff’s bases bearing substituted 2-amino benzothiazole were synthesized by single step process through simple condensation of 2-amino benzothiazole and substituted benzaldehydes and further characterized by FTIR, 1HNMR, and Mass spectrometry data. Antimicrobial activity of compounds was performed by agar diffusion method against a panel of bacterial strains such as S. aureus, B. subtilis (Gram-positive bacteria), E. coli, P. aeruginosa (Gram-negative bacteria) and fungal strains such as C. albicans and A. niger. Compound S13 and S17 had shown potent antifungal activity against C. albicans and A. niger respectively among the novel Schiff’s base compounds when compared to standard, and S13 compound had only shown moderate antibacterial activity against S. aureus amongst all. Molecular docking study was carried out against C. albicans DHFR (Dihydrofolate Reductase) domain to confirm their activity.

Conditional, Genetically Encoded, Small Molecule-Regulated Inhibition of NFκB Signaling in RPE Cells.

PurposeNuclear factor κB (NFκB) is a ubiquitously expressed, proinflammatory transcription factor that controls the expression of genes involved in cell survival, angiogenesis, complement activation, and inflammation. Studies have implicated NFκB-dependent cytokines or complement-related factors as being detrimentally involved in retinal diseases, thus making inhibition of NFκB signaling a potential therapeutic target. We sought to develop a conditional and reversible method that could regulate pathogenic NFκB signaling by the addition of a small molecule.MethodsWe developed a genetically based, trimethoprim (TMP)-regulated approach that conditionally inhibits NFκB signaling by fusing a destabilized dihydrofolate reductase (DHFR) domain to an inhibitor of NFκB, IκBα, in ARPE-19 cells. We then challenged ARPE-19 cells with a number of stimuli that have been demonstrated to trigger NFκB signaling, including LPS, TNFα, IL-1α, and A2E. Western blotting, electrophoretic mobility shift assay, quantitative PCR, ELISA, and NFκB reporter assays were used to evaluate the effectiveness of this DHFR-IκBα approach.ResultsThis destabilized domain approach, coupled with doxycycline-inducibility, allowed for accurate control over the abundance of DHFR-IκBα. Stabilization of DHFR-IκBα with TMP prevented IL-1α-, A2E-, LPS-, and TNFα-induced NFκB-mediated upregulation and release of the proinflammatory cytokines IL-1β and IL-6 from ARPE-19 cells (by as much as 93%). This strategy is dosable, completely reversible, and can be cycled “on” or “off” within the same cell population repeatedly to confer protection at desired time points.ConclusionsThese studies lay the groundwork for the use of destabilized domains in retinal pigment epithelium (RPE) cells in vivo and in this context, demonstrate their utility for preventing inflammatory signaling.

Understanding the molecular mechanism of substrate channeling and domain communication in protozoal bifunctional TS-DHFR.

Most species, such as humans, have monofunctional forms of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) that are key folate metabolism enzymes making critical folate components required for DNA synthesis. In contrast, several parasitic protozoa, including Leishmania major (Lm), Plasmodium falciparum (Pf), Toxoplasma gondii (Tg) and Cryptosporidium hominis (Ch), contain a unique bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) having the two sequential catalytic activities contained on a single polypeptide chain. It has been suggested that the bifunctional nature of the two catalytic activities may enable substrate channeling. The 3D structures for each of these enzymes reveals distinct features for each species. While three of the four species (Pf, Tg and Ch) contain a junctional region linking the two domains, this is lacking in Lm. The Lm and Pf contain N-terminal amino acid extensions. A multidisciplinary approach using structural studies and transient kinetic analyses combined with mutational analysis has investigated the roles of these unique structural features for each enzyme. Additionally, the possibility of substrate channeling behavior was explored. These studies have identified unique, functional regions in both the TS and DHFR domains that govern efficient catalysis for each species. Surprisingly, even though there are structural similarities among the species, each is regulated in a distinct manner. This structural and mechanistic information was also used to exploit species-specific inhibitor design.

Substrate Polyubiquitination Increases Proteasomal Processivity

The 26S proteasome in eukaryotes is the macromolecular machine responsible for the degradation of misfolded and damaged polypeptides, as well as signaling and regulatory proteins that accumulate intracellularly. Most proteins are marked for degradation and targeted to the proteasome by the covalent attachment of polyubiquitin chains, which are recognized by receptors on the 19S regulatory particle of the proteasome. After initiating degradation, the polyubiquitin tag is removed and the proteasome moves processively along the protein substrate, unfolding domains as they are encountered and translocating the unfolded polypeptide to the protease active sites in the 20S core for degradation into small peptides. However, the proteasome can stall during this process and rather than successfully unfolding the encountered domain, the proteasome will release the substrate as a partially degraded protein, which can assume a new biological function or form a potentially toxic byproduct. While our understanding of proteasomal processivity remains limited, it has been shown that ubiquitinated substrates can activate the proteasome's ATPase and peptidase activities. We set out to determine if substrate ubiquitination affected the proteasome's ability to unfold its substrates, since the ubiquitin chain is removed early on in the degradation process. To accomplish this, a previously established processivity assay was used, in which the unfolding and subsequent degradation versus the release of a stabilized dihydrofolate reductase (DHFR) domain are quantified. The ratio of degradation to release rates is defined as the unfolding ability U, a measure of the proteasome's processivity. We compared the unfolding abilities of ubiquitin‐independent substrates, as well as substrates ubiquitinated with the E3 ligase Rsp5 (reported to give K63‐linked chains) or the E3 ligase complex Cul3/Rbx1/Keap1 (reported to give primarily K48‐linked chains). We found that significantly less folded DHFR fragment was produced during degradation of the Cul3/Rbx1/Keap1‐ubiquitinated substrate, resulting in a higher unfolding ability, compared to the Rsp5‐ubiquitinated substrate. The unfolding ability for ubiquitin‐independent substrates was much lower than that of either Rsp5‐ or Keap1 ubiquitinated substrates. These results suggest that the presence and chain architecture of polyubiquitin on a substrate can shift the proteasome towards a more active state during engagement, which results in a greater unfolding ability.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under Grant No. 1515229.

Substrate Polyubiquitination Increases the Unfolding Ability of the 26S Proteasome

The 26S proteasome is responsible for the bulk of intracellular protein degradation in eukaryotes. Most proteins are targeted to the proteasome by the addition of one or more polyubiquitin tags. After binding to the proteasome via the ubiquitin tag and engaging proteasomal ATPases via an unstructured region, substrates are deubiquitinated, processively unfolded, translocated into the core of the proteasome, and degraded into small peptides. The factors that allow the proteasome to successfully unfold a folded domain (or prevent it from doing so) are still poorly understood. We sought to determine whether the polyubiquitin chain that brings a substrate to the proteasome affected the proteasome's ability to unfold the substrate. Using a previously established processivity assay, in which the unfolding and degradation versus irreversible release of a stabilized dihydrofolate reductase (DHFR) domain are quantified, we compared the unfolding abilities of substrates bearing either K48-linked or K63-linked polyubiquitin chains. We found that substrates targeted to the proteasome via K48-linked ubiquitin chains were degraded more processively than those bearing K63-linked chains, even though the ubiquitin chain was presumably removed earlier in the degradation process, before the DHFR domain began to be unfolded. A substrate targeted to the proteasome via a ubiquitin-independent degron showed an even lower unfolding ability in the same assay. These results suggest that polyubiquitin chains activate the proteasome's unfolding ability even after removal from the substrate, and that different polyubiquitin chain linkages activate the proteasome to different extents.

First Three-Dimensional Structure of Toxoplasma gondii Thymidylate Synthase–Dihydrofolate Reductase: Insights for Catalysis, Interdomain Interactions, and Substrate Channeling

Most species, such as humans, have monofunctional forms of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) that are key folate metabolism enzymes making critical folate components required for DNA synthesis. In contrast, several parasitic protozoa, including Toxoplasma gondii , contain a unique bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) having the catalytic activities contained on a single polypeptide chain. The prevalence of T. gondii infections across the world, especially for those immunocompromised, underscores the need to understand TS-DHFR enzyme function and to find new avenues to exploit for the design of novel antiparasitic drugs. As a first step, we have solved the first three-dimensional structures of T. gondii TS-DHFR at 3.7 Å and of a loop truncated TS-DHFR, removing several flexible surface loops in the DHFR domain, improving resolution to 2.2 Å. Distinct structural features of the TS-DHFR homodimer include a junctional region containing a kinked crossover helix between the DHFR domains of the two adjacent monomers, a long linker connecting the TS and DHFR domains, and a DHFR domain that is positively charged. The roles of these unique structural features were probed by site-directed mutagenesis coupled with presteady state and steady state kinetics. Mutational analysis of the crossover helix region combined with kinetic characterization established the importance of this region not only in DHFR catalysis but also in modulating the distal TS activity, suggesting a role for TS-DHFR interdomain interactions. Additional kinetic studies revealed that substrate channeling occurs in which dihydrofolate is directly transferred from the TS to DHFR active site without entering bulk solution. The crystal structure suggests that the positively charged DHFR domain governs this electrostatically mediated movement of dihydrofolate, preventing release from the enzyme. Taken together, these structural and kinetic studies reveal unique, functional regions on the T. gondii TS-DHFR enzyme that may be targeted for inhibition, thus paving the way for designing species specific inhibitors.

Caracterización bioquímica de la enzima bifuncional dihidrofolato reductasa-timidilato sintasa de Leishmania (Viannia) y su evaluación como blanco molecular

Dihydrofolate reductase (DHFR) has been used successfully as a drug target in the area of anti-bacterial, anti-cancer and anti-malarial therapy. Although this bifunctional enzyme is also a potential drug target for treatment of leishmaniasis, there have been no reports on its efficacy against Leishmania (Viannia) species. The gene encoding the bifunctional DHFR and thymidylate synthase (TS) of Le. (V.) braziliensis was isolated and expressed in E. coli. The enzyme was purified and characterized. The inhibitory effects of antifolates and four aporphine alkaloids on its activity were evaluated. The full-length gene consists of a 1560-bp open reading frame encoding a 58 kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. The recombinant DHFR-TS enzyme revealed Km and Vmax values of 55.35 ± 4.02 µ M (mean ± SE) and 0.02 ± 5.34 x 10 -4 µ M/min respectively for dihydrofolic acid (H₂F). The Le. braziliensis rDHFR-TS have Ki values for antimicrobial antifolates in the µM range. Methotrexate (MTX) was a more-potent inhibitor of enzymatic activity (Ki = 22.0 µM) than trimethoprim (Ki = 33 µM) and pyrimethamine (Ki = 68 µM). These Ki values are significantly lower than those obtained for the aporphine alkaloids. The results of the study show the inhibitory effect of antifolate drugs on enzymatic activity, indicating that Le. braziliensis rDHFR-TS could be a model to studying antifolate compounds as potential antiprotozoal drugs.

A single copy of SecYEG is sufficient for preprotein translocation

The heterotrimeric SecYEG complex comprises a protein-conducting channel in the bacterial cytoplasmic membrane. SecYEG functions together with the motor protein SecA in preprotein translocation. Here, we have addressed the functional oligomeric state of SecYEG when actively engaged in preprotein translocation. We reconstituted functional SecYEG complexes labelled with fluorescent markers into giant unilamellar vesicles at a natively low density. Förster's resonance energy transfer and fluorescence (cross-) correlation spectroscopy with single-molecule sensitivity allowed for independent observations of the SecYEG and preprotein dynamics, as well as complex formation. In the presence of ATP and SecA up to 80% of the SecYEG complexes were loaded with a preprotein translocation intermediate. Neither the interaction with SecA nor preprotein translocation resulted in the formation of SecYEG oligomers, whereas such oligomers can be detected when enforced by crosslinking. These data imply that the SecYEG monomer is sufficient to form a functional translocon in the lipid membrane.

Protein-Ligand interaction studies on 2, 4, 6- trisubstituted triazine derivatives as anti-malarial DHFR agents using AutoDock.

The dihydrofolate reductase (DHFR) domain of P. falciparum is one of the few well defined targets in malarial chemotherapy. The enzyme catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH) dependent reduction of dihydrofolate to tetrahydrofolate. Protein-ligand interactions were studied using DHFR protein 2BL9, extracted from PDB to evaluate the strength of affinity of various molecules towards ligand binding site and to study the extent of correlation between experimental values and computational dock scores. AutoDock runs resulted in binding energy scores from -7.14 to -10.72 kcal/mol. Among the five inhibitors (Bioorganic and Medicinal Chemistry Letters 15 2005 531-533) selected for docking studies, an excellent correlation was observed in all cases, for instance, experimentally reported most active molecule 2a (MIC: 1µg/ml) showed a high dock score (-10.72 kcal/mol) than the remaining inhibitors. Therefore, molecular docking using AutoDock suggests the importance of evaluating the prediction accuracy of various molecules as evidenced by a correlation coefficient of 0.961 between experimental activities and AutoDock binding energies.

Disruption of the crossover helix impairs dihydrofolate reductase activity in the bifunctional enzyme TS–DHFR from Cryptosporidium hominis

In contrast with most species, including humans, which have monofunctional forms of the folate biosynthetic enzymes TS (thymidylate synthase) and DHFR (dihydrofolate reductase), several pathogenic protozoal parasites, including Cryptosporidium hominis, contain a bifunctional form of the enzymes on a single polypeptide chain having both catalytic activities. The crystal structure of the bifunctional enzyme TS-DHFR C. hominis reveals a dimer with a 'crossover helix', a swap domain between DHFR domains, unique in that this helical region from one monomer makes extensive contacts with the DHFR active site of the other monomer. In the present study, we used site-directed mutagenesis to probe the role of this crossover helix in DHFR catalysis. Mutations were made to the crossover helix: an 'alanine-face' enzyme in which the residues on the face of the helix close to the DHFR active site of the other subunit were mutated to alanine, a 'glycine-face' enzyme in which the same residues were mutated to glycine, and an 'all-alanine' helix in which all residues of the helix were mutated to alanine. These mutant enzymes were studied using a rapid transient kinetic approach. The mutations caused a dramatic decrease in the DHFR activity. The DHFR catalytic activity of the alanine-face mutant enzyme was 30 s(-1), the glycine-face mutant enzyme was 17 s(-1), and the all-alanine helix enzyme was 16 s(-1), all substantially impaired from the wild-type DHFR activity of 152 s(-1). It is clear that loss of helix interactions results in a marked decrease in DHFR activity, supporting a role for this swap domain in DHFR catalysis. The crossover helix provides a unique structural feature of C. hominis bifunctional TS-DHFR that could be exploited as a target for species-specific non-active site inhibitors.

Step-size Analyses of the Mitochondrial Hsp70 Import Motor Reveal the Brownian Ratchet in Operation

Newly synthesized mitochondrial precursor proteins have to become unfolded by the mitochondrial Hsp70 (mtHsp70) import motor to cross the mitochondrial membranes. To assess the mechanism of unfolding of precursor proteins by mtHsp70, we designed a system to measure step sizes of the mtHsp70 import motor, which are distances at which the motor system moves along polypeptide chains during a single turnover of ATP. We made a series of fusion proteins consisting of a mitochondrial presequence containing the first mtHsp70 binding site, a spacer sequence containing an Hsp70 avoidance segment followed by the second mtHsp70 binding site, and different folded mature domains. Analyses of the dependence of the import rates of those fusion proteins on the lengths of Hsp70 avoidance segments allowed us to estimate the step sizes, which differ for different mature domains and different lengths of the spacers. These results suggest that the mtHsp70 import motor functions at least as a molecular Brownian ratchet to unfold mitochondrial precursor proteins.

Dissecting virulence pathways of Mycobacterium tuberculosis through protein–protein association

The sudden increase in information derived from the completed Mycobacterium tuberculosis (Mtb) genome sequences has revealed the need for approaches capable of converting raw genome sequence data into functional information. To date, an experimental system for studying protein-protein association in mycobacteria is not available. We have developed a simple system, termed mycobacterial protein fragment complementation (M-PFC), that is based upon the functional reconstitution of two small murine dihydrofolate reductase domains independently fused to two interacting proteins. Using M-PFC, we have successfully demonstrated dimerization of yeast GCN4, interaction between Mtb KdpD and KdpE, and association between Esat-6 and Cfp-10. We established the association between the sensor kinase, DevS, and response regulator, DevR, thereby demonstrating the potential of M-PFC to study protein associations in the mycobacterial membrane. To validate our system, we screened an Mtb library for proteins that associate with the secreted antigen Cfp-10 and consistently identified Esat-6 in our screens. Additional proteins that specifically associate with Cfp-10 include Rv0686 and Rv2151c (FtsQ), a component and substrate, respectively, of the evolutionary conserved signal recognition pathway; and Rv3596c (ClpC1), an AAA-ATPase chaperone involved in protein translocation and quality control. Our results provide empirical evidence that directly links the Mtb specialized secretion pathway with the evolutionary conserved signal recognition and SecA/SecYEG pathways, suggesting they share secretory components. We anticipate that M-PFC will be a major contributor to the systematic assembly of mycobacterial protein interaction maps that will lead to the development of better strategies for the control of tuberculosis.

Nuclear transcription is essential for specification of mammalian replication origins

I have demonstrated that nuclear transcription modulates the distribution of replication origins along mammalian chromosomes. Chinese Hamster Ovary (CHO) cells were exposed to transcription inhibitors in early G1 phase and replication origin sites in the dihydrofolate reductase (DHFR) gene locus were mapped several hours later. DNA within nuclei prepared from control and transcription-deficient G1-phase cells was replicated with similar efficiencies when introduced into Xenopus egg extracts. Replication initiated in the intergenic region within control late-G1 nuclei, but randomly within transcriptionally repressed nuclei. Random initiation was not a consequence of inability to produce an essential protein(s), since initiation was site-specific within cells exposed to the translation inhibitor cycloheximide during the same interval of G1 phase. Furthermore, in vivo inhibition of transcription within late-G1-phase cells reduced the frequency of usage of pre-established DHFR replication origin sites. Transcription rates in the DHFR domain were very low and did not change throughout G1 phase. This implies that, although ongoing nuclear transcription is required, local expression of the genes in the DHFR locus alone is not sufficient to create a site-specific replication initiation pattern. I conclude that epigenetic factors, including general nuclear transcription, play a role in replication origin selection in mammalian nuclei.

Manipulation of Membrane Protein Topology on the Endoplasmic Reticulum by a Specific Ligand in Living Cells

Almost all integral membrane proteins in the secretory pathway are cotranslationally inserted into the endoplasmic reticulum membrane. Their membrane topology is determined by their amino acid sequences. Here we show that the topology can be manipulated by a factor other than the amino acid sequence. A dihydrofolate reductase (DHFR) domain was fused to the N-terminus of the type I signal-anchor sequence of synaptotagmin II, which mediates translocation of the preceding portion. The DHFR domain was translocated through the membrane in COS7 cells and a transmembrane (TM) topology was achieved. When a DHFR ligand, methotrexate, was added to the culture medium, translocation of the DHFR domain was suppressed and both ends of the signal-anchor sequence remained on the cytoplasmic side. In contrast, translocation of the DHFR domain fused after the signal peptide, which translocates the following region, was not affected by the ligand. The topology-altered fusion protein was anchored to the membrane in a high salt-resistant state, and partially extracted from the membrane under alkali conditions. We concluded that the topology of membrane proteins can be manipulated by a trans-acting factor, even in living cells.

Translocation of a long amino-terminal domain through ER membrane by following signal-anchor sequence

Type I signal-anchor sequences mediate translocation of the N-terminal domain (N-domain) across the endoplasmic reticulum (ER) membrane. To examine the translocation in detail, dihydrofolate reductase (DHFR) was fused to the N-terminus of synaptotagmin II as a long N-domain. Translocation was arrested by the DHFR ligand methotrexate, which stabilizes the folding of the DHFR domain, and resumed after depletion of methotrexate. The targeting of the ribosome-nascent chain complex to the ER requires GTP, whereas N-domain translocation does not require any nucleotide triphosphates. Significant translocation was observed even in the absence of a lumenal hsp70 (BiP). When the nascent polypeptide was released from the ribosomes after the membrane targeting, the N-domain translocation was suppressed and the nascent chain was released from the translocon. Ribosomes have a crucial role in maintaining the translocation-intermediate state. The translocation of the DHFR domain was greatly impaired when it was separated from the signal-anchor sequence. Unfolding and translocation of the DHFR domain must be driven by the stroke of the signal-anchor sequence into translocon.