Fluorescence Resonance Energy Transfer Data Research Articles

Abstract 3718: FOXM1 switches protein conformations upon C-terminal domain-mediated-autoregulation dictating its transcriptional activity in cancer cell proliferation

Abstract Transcription factor Forkhead box M1 (FOXM1) participates in tumor initiation, tumor development and tumor metastasis through regulating transcriptional network of its target genes. Activated FOXM1 promotes G1/S and G2/M transition via manipulating Cdc25B, Cyclin B1, TOPOIIa, Aurora and Polo-like kinases, as well as cancer cell survival and invasion through upregulating Survivin, SNAIL and MMP-9. The depletion of Foxm1 repressed the oncogenic Kras (G12D) driven mice lung cancer, Rb/Trp53/Myc triple knockout mice small cell lung cancer and carcinogen induced mice hepatocellular carcinoma. Given the roles of FOXM1 in tumorigenesis and its relevance to clinical outcomes, deciphering the mechanism underlying the regulation of FOXM1 activity might provide new therapeutic target for cancer study. It is known that FOXM1 activity drops when the C-terminal transcription activation domain (TAD) is blocked by the N-terminal repression domain (NRD), which is called autorepression. Furthermore, the NRD-deleted FOXM1 displays extraordinarily high transactivity. Compared to the NRD, the TAD is relatively longer and consists of around 420 amino acid with most of them being intrinsically disordered region (IDR). Previous studies showed that the NRD binds to a relatively ordered small region on the TAD. However, it is poorly understood about whether the IDR of TAD partakes in the FOXM1 self-regulated activity and how FOXM1 protein structure switches in living cells. Our co-immunoprecipitation (co-IP) and dual-luciferase reporter assay (DLR) suggested that FOXM1 TAD self-interacts via its IDR and the C-terminal αβα motif, which promotes FOXM1 transactivation. The IDR-driven FOXM1 autoactivation was mimicked by the chemically-induced dimerization system (CID) that forces IDR-αβα proximity intermolecularly. Using fluorescence resonance energy transfer (FRET) microscopy, we observed that the IDR-αβα interaction was elevated when cell cycle progression from G1/S to G2/M in living cells. The IDR-αβα interaction and FOXM1 activity were both suppressed by PLK1 inhibition. Furthermore, the characteristics of IDR-αβα interaction did not resemble NRD-TAD interaction as revealed by FRET data where we showed the NRD-TAD interaction dropped after G1/S. The NRD-TAD interaction underlying FOXM1 autorepression was recapitulated by CID. Through co-IP and yeast-two hybrid assays (Y2H), we further identified the minimal yet biological functional unit ββαβ on NRD that suppressed FOXM1 activity and lung adenocarcinoma A549 cells in vitro and in vivo. Collectively, our work suggested that FOXM1 might be switched from the NRD-TAD mediated autorepression toward the IDR-αβα mediated activation by Plk1 during S-G2 phase. And ββαβ peptide treatment might be applied to lung cancer as a potential therapeutic strategy. Citation Format: Chia-Chan Hsu, Xiang Yao, I-Ching Wang. FOXM1 switches protein conformations upon C-terminal domain-mediated-autoregulation dictating its transcriptional activity in cancer cell proliferation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3718.

Fluorescence lifetime analysis of smFRET with contribution of PIFE on donor and acceptor.

Single-molecule fluorescence resonance energy transfer (FRET) is a powerful technique based on dipole-dipole interaction between donor and acceptor fluorophores to observe inter- and intra-molecular dynamics in real time with sensitivity to macro-molecular distances (∼2.5-10 nm). That said, some fluorophores have an inherent characteristic known as protein induced fluorescence enhancement (PIFE). PIFE is a photo-physical feature of dyes undergoing cis-trans transitions and occurs for protein-dye interactions closer than 3 nm where FRET is less sensitive. Here, the challenge is uncoupling the PIFE effect in the FRET data. Ignoring the PIFE effect in the analysis of the FRET data may lead to misinterpretation of the system under investigation. As a solution to this problem, we develop a computational framework based on Bayesian statistics to analyze the fluorescence lifetime signals of the donor and acceptor channels which allows us to uncouple the PIFE effects from the FRET. Our framework can extract any changes in the FRET efficiency simultaneously with any changes in the fluorescence lifetimes of the donor and acceptor due to the PIFE effect. In addition, our framework can provide other parameters, such as the donor and acceptor excitation rates, background photon rates, and detectors’ cross-talk ratios. Our framework extracts all these parameters by analyzing a single photon arrival time trace with only a few thousand photons.

Chirality-Dependent Dynamic Evolution of the Protein Corona on the Surface of Quantum Dots.

Elucidating the biological behavior of engineered nanoparticles, for example, the protein corona, is important for the development of safe and efficient nanomedicine, but our current understanding is still limited due to its highly dynamic nature and lack of adequate analytical tools. In the present work, we demonstrate the establishment of a fluorescence resonance energy transfer (FRET)-based platform for monitoring the dynamic evolution behavior of the protein corona in complex biological media. With human serum albumin and lysozyme as the model serum proteins, the protein exchange process of the preformed corona on the surface of chiral quantum dots (QDs) upon feeding either individual protein or human serum was monitored in situ by FRET. Important parameters characterizing the evolution process of protein corona could be obtained upon quantitative analysis of FRET data. Further combining real-time FRET monitoring with gel electrophoresis experiments revealed that the nature of the protein initially adsorbed on the surface of QDs significantly affects the subsequent dynamic exchange behavior of the protein corona. Furthermore, our results also revealed that only a limited proportion of proteins are involved in the protein exchange, and the exchange process exhibits a significant dependence on the surface chirality of QDs. This work demonstrates the feasibility of FRET as a powerful tool to exploit the dynamic evolution process of the protein corona, which can provide theoretical guidance for further design of advanced nanomaterials for biomedical applications.

Synergism in the Molecular Crowding of Ligand-Induced Riboswitch Folding: Kinetic/Thermodynamic Insights from Single-Molecule Spectroscopy.

Conformational dynamics in riboswitches involves ligand binding and folding of RNA, each of which can be influenced by excluded volume effects under "crowded" in vivo cellular conditions and thus incompletely characterized by in vitro studies under dilute buffer conditions. In this work, temperature-dependent single-molecule fluorescence resonance energy transfer (FRET) spectroscopy is used to characterize the thermodynamics of (i) cognate ligand and (ii) molecular crowders (PEG, polyethylene glycol) on folding of the B. subtilis LysC lysine riboswitch. With the help of detailed kinetic analysis, we isolate and study the effects of PEG on lysine binding and riboswitch folding steps individually, from which we find that PEG crowding facilitates riboswitch folding primarily via a surprising increase in affinity for the cognate ligand. This is furthermore confirmed by temperature-dependent studies, which reveal that PEG crowding is not purely entropic and instead significantly impacts both enthalpic and entropic contributions to the free energy landscape for folding. The results indicate that PEG molecular crowding/stabilization of the lysine riboswitch is more mechanistically complex and requires extension beyond the conventional picture of purely repulsive solvent-solute steric interactions arising from excluded volume and entropy. Instead, the current experimental FRET data support an alternative multistep mechanism, whereby PEG first entropically crowds the unfolded riboswitch into a "pre-folded" conformation, which in turn greatly increases the ligand binding affinity and thereby enhances the overall equilibrium for riboswitch folding.

MASH-FRET: A Simplified Approach for Single-Molecule Multiplexing Using FRET.

Multiplexed detection has been a big motivation in biomarker analysis as it not only saves cost and labor but also improves the reliability of diagnosis. Among the many approaches for multiplexed detection, fluorescence resonance energy transfer (FRET)-based multiplexing is gaining popularity particularly due to its low background and quantitative nature. Although several FRET-based approaches have been developed for multiplexing, they require either multiple FRET pairs in combination with multiple excitation sources or complicated algorithms to accurately assign signals for individual FRET pairs. Therefore, the need for multiple FRET pairs and multiple excitation sources not only complicates the experimental design but also increases the cost and labor. In this regard, multiplexed sensing by tuning the interdye distance of a single FRET pair could be an ideal solution if identification of multiple FRET efficiencies in a single imaging is possible. Here, implementing a program called MASH-FRET, we evaluated the rigor and capability of this program in identifying seemingly overlapped FRET populations obtained from a multiplexed detection experiment using a single FRET pair. Through MASH-FRET-enabled bootstrap-based analysis of FRET data (also called BOBA-FRET), we demonstrated that the resolution and statistical confidence of the poorly resolved or even unresolved FRET populations can be readily determined. Using simulated FRET data, we further demonstrated that the program can easily identify FRET populations separated by ∼0.1 in mean FRET values, indicating an upper limit of ∼9-fold multiplexing without the need for complicated labeling schemes and multiexcitation sources. Therefore, this paper presents a data analysis approach on an existing platform that has a great potential to simplify the technological needs for multiplexing and to broaden the scope of FRET-based single-molecule analyses.

High-resolution crystal structure of the Borreliella burgdorferi PlzA protein in complex with c-di-GMP: new insights into the interaction of c-di-GMP with the novel xPilZ domain.

ABSTRACTIn the tick-borne pathogens, Borreliella burgdorferi and Borrelia hermsii, c-di-GMP is produced by a single diguanylate cyclase (Rrp1). In these pathogens, the Plz proteins (PlzA, B and C) are the only c-di-GMP receptors identified to date and PlzA is the sole c-di-GMP receptor found in all Borreliella isolates. Bioinformatic analyses suggest that PlzA has a unique PilZN3-PilZ architecture with the relatively uncommon xPilZ domain. Here, we present the crystal structure of PlzA in complex with c-di-GMP (1.6 Å resolution). This is the first structure of a xPilz domain in complex with c-di-GMP to be determined. PlzA has a two-domain structure, where each domain comprises topologically equivalent PilZ domains with minimal sequence identity but remarkable structural similarity. The c-di-GMP binding site is formed by the linker connecting the two domains. While the structure of apo PlzA could not be determined, previous fluorescence resonance energy transfer data suggest that apo and holo forms of the protein are structurally distinct. The information obtained from this study will facilitate ongoing efforts to identify the molecular mechanisms of PlzA-mediated regulation in ticks and mammals.

Probing the effects of synthesized silver nanowire/reduced graphene oxide composites on the structure and esterase-like activity of human serum albumin and its impacts on human endometrial stem cells: A new platform in nanomedicine

Objective(s): Nowadays, the unique and fascinating properties of graphene‐based nanocomposites make them one of the most promising materials for therapeutics, delivery carriers as well as tissue engineering. On the other hand, silver nanowire has been attracting more attention in nanomedicine applications, too. In this study, the effects of synthesized silver nanowire/reduced graphene oxide (AgNWs/rGO) composites on the structure and esterase-like activity of Human Serum Albumin (HSA), as well as its impacts on Human Endometrial Stem Cells (hEnSCs), were evaluatedMaterials and Methods: AgNWs/rGO composite was first synthesized and fabricated. Subsequently, its effects on the structure and esterase-like activity of HSA were evaluated by UV-Visible spectroscopy, circular dichroism spectroscopy, and fluorescence spectroscopy. Afterward, its impacts on the viability and growth of hEnSCs were studied by MTT assay, DAPI staining, and flow cytometry analysis.Results: The spectroscopic results showed that AgNWs/rGO composite could form a complex with HSA, however, did not affect the secondary structure of HSA and the binding constant for this complex was found to be 5.4×104 mL.mg-1. Furthermore, HSA maintained most of its activity in the presence of the AgNWs/rGO composite. Based on FRET (fluorescence resonance energy transfer) data the value of r0 was less than 7 nm signifying that the energy transfer from HSA to AgNWs/rGO composite occurs with a high level of possibility. The MTT assay, DAPI staining, and flow cytometry analysis indicated that the AgNWs/rGO composite was non-toxic towards hEnSCs. Conclusion: Our results suggest that the prepared AgNWs/rGO composite, potentially, is suitable in nanomedicine applications such as tissue engineering and drug delivery.

In Situ Investigation on the Protein Corona Formation of Quantum Dots by Using Fluorescence Resonance Energy Transfer.

A fundamental understanding of nanoparticle-protein corona and its interactions with biological systems is essential for future application of engineered nanomaterials. In this work, fluorescence resonance energy transfer (FRET) is employed for studying the protein adsorption behavior of nanoparticles. The adsorption of human serum albumin (HSA) onto the surface of InP@ZnS quantum dots (QDs) with different chirality (d- and l-penicillamine) shows strong discernible differences in the binding behaviors including affinity and adsorption orientation that are obtained upon quantitative analysis of FRET data. Circular dichroism spectroscopy further confirms the differences in the conformational changes of HSA upon interaction with d- and l-chiral QD surfaces. Consequently, the formed protein corona on chiral surfaces may affect their following biological interactions, such as possible protein exchange with serum proteins plasma as well as cellular interactions. These results vividly illustrate the potential of the FRET method as a simple yet versatile platform for quantitatively investigating biological interactions of nanoparticles.

Constitutive inhibitory G protein activity upon adenylyl cyclase-dependent cardiac contractility is limited to adenylyl cyclase type 6.

PurposeWe previously reported that inhibitory G protein (Gi) exerts intrinsic receptor-independent inhibitory activity upon adenylyl cyclase (AC) that regulates contractile force in rat ventricle. The two major subtypes of AC in the heart are AC5 and AC6. The aim of this study was to determine if this intrinsic Gi inhibition regulating contractile force is AC subtype selective.MethodsWild-type (WT), AC5 knockout (AC5KO) and AC6 knockout (AC6KO) mice were injected with pertussis toxin (PTX) to inactivate Gi or saline (control).Three days after injection, we evaluated the effect of simultaneous inhibition of phosphodiesterases (PDE) 3 and 4 with cilostamide and rolipram respectively upon in vivo and ex vivo left ventricular (LV) contractile function. Also, changes in the level of cAMP were measured in left ventricular homogenates and at the membrane surface in cardiomyocytes obtained from the same mouse strains expressing the cAMP sensor pmEPAC1 using fluorescence resonance energy transfer (FRET).ResultsSimultaneous PDE3 and PDE4 inhibition increased in vivo and ex vivo rate of LV contractility only in PTX-treated WT and AC5KO mice but not in saline-treated controls. Likewise, Simultaneous PDE3 and PDE4 inhibition elevated total cAMP levels in PTX-treated WT and AC5KO mice compared to saline-treated controls. In contrast, simultaneous PDE3 and PDE4 inhibition did not increase in vivo or ex vivo rate of LV contractility or cAMP levels in PTX-treated AC6KO mice compared to saline-treated controls. Using FRET analysis, an increase of cAMP level was detected at the membrane of cardiomyocytes after simultaneous PDE3 and PDE4 inhibition in WT and AC5KO but not AC6KO. These FRET data are consistent with the functional data indicating that AC6 activity and PTX inhibition of Gi is necessary for simultaneous inhibition of PDE3 and PDE4 to elicit an increase in contractility.ConclusionsTogether, these data suggest that AC6 is tightly regulated by intrinsic receptor-independent Gi activity, thus providing a mechanism for maintaining low basal cAMP levels in the functional compartment that regulates contractility.

Elucidation of the FKBP25-60S Ribosomal Protein L7a Stress Response Signaling During Ischemic Injury

Background/Aims: Peptidyl-prolyl cis-trans isomerase FKBP25 is a member of the FK506-binding proteins family which has peptidyl-prolyl cis/trans isomerase domain. The biological function and pathophysiologic role of FKBP25 remain elusive. Methods: The spatio-temporal changes in expression of endothelial FKBP25 upon oxygen-glucose deprivation (OGD) treatment were examined by Western blot and immunofluorescence. The immunoprecipitation and fluorescence resonance energy transfer (FRET) were used to address the interacting proteins with FKBP25. Results: In the present study, nuclear translocation of FKBP25 was observed following OGD in cultured endothelial cells. Intriguingly, FKBP25 nuclear translocation was further validated in peroxynitrite (ONOO^-)-treated endothelial cells. Coimmunoprecipitation and FRET data indicated that FKBP25 translocated into the nucleus, in which it interacted with 60S ribosomal protein L7a, while overexpression FKBP25 protect endothelial cells against OGD injury. Conclusion: Our findings reveal that the nuclear import of FKBP25 and binding with 60S ribosomal protein L7a are protective stress responses to ischemia/nitrosaive stress injury.

Crystal Structures of Cyanine Fluorophores Stacked onto the End of Double-Stranded RNA

The indodicarbocyanine fluorophores Cy3 and Cy5 are extensively used as donor-acceptor pairs in fluorescence resonance energy transfer experiments, especially those involving single molecules. When terminally attached to double-stranded nucleic acids via the 5′ phosphate group these fluorophores stack onto the ends of the molecule. Knowledge of the positions of the fluorophores is critical to the interpretation of fluorescence resonance energy transfer data. The positions have been demonstrated for double-stranded (ds) DNA using NMR spectroscopy. Here, we have used x-ray crystallography to analyze the location of Cy3 and Cy5 on dsRNA, using complexes of an RNA stem-loop bound to L5 protein determined at 2.4 Å resolution. This confirms the tendency of both fluorophores to stack on the free end of RNA, with the long axis of the fluorophores approximately parallel to that of the terminal basepair. However, the manner of interaction of both Cy3 and Cy5 with the terminus of the dsRNA is significantly different from that deduced for dsDNA using NMR. The fluorophores are stacked on the terminal basepair such that their indole nitrogen atoms lie on the major groove side, and thus their pendant methyl groups are on the minor groove side.

Experimental and computational characterization of photosensitized conformational effects mediated by protoporphyrin ligands on human serum albumin.

When investigating the interaction between proteins and protoporphyrins in aqueous solution, one typically has to contend with the tendency of the latter to form polydispersed aggregates. The interference of aggregated protoporphyrins manifests, at least, at two levels: aggregates sequester the majority of the protoporphyrin molecules in solution and prevent their interaction with the proteins, but also their presence interferes with optical experiments such as absorption and fluorescence spectroscopy. In this study we present a protocol which uses dialysis and centrifugation to eliminate the aggregates and yield solutions dominated by non-covalent complexes of albumin (HSA) and protoporphyrins. The elimination of the aggregates enabled us to observe effects which had not been previously observed such as eliminating the discrepancy between the binding constants obtained through the quenching of HSA fluorescence and the one obtained through the emission of the protoporphyrins. Moreover the elimination of the aggregated protoporphyrins enabled us to reveal the occurrence of fluorescence resonance energy transfer (FRET) between the Trp214 residue of HSA and the porphyrin ligands. FRET data were then used to estimate the location of metal free as well as Zn-protoporphyrin IX relative to the well-known location of Trp214. This information was used to refine docking simulations to find the best binding site for the two protoporphyrins. In addition we observed that the irradiation of the protoporphyrins in the visible region prompts small conformational changes in HSA that appear to be largely due to tertiary modifications.

The Arginine Pairs and C-Termini of the Sso7c4 from Sulfolobus solfataricus Participate in Binding and Bending DNA.

The Sso7c4 from Sulfolobus solfataricus forms a dimer, which is believed to function as a chromosomal protein involved in genomic DNA compaction and gene regulation. Here, we present the crystal structure of wild-type Sso7c4 at a high resolution of 1.63 Å, showing that the two basic C-termini are disordered. Based on the fluorescence polarization (FP) binding assay, two arginine pairs, R11/R22′ and R11′/R22, on the top surface participate in binding DNA. As shown in electron microscopy (EM) images, wild-type Sso7c4 compacts DNA through bridging and bending interactions, whereas the binding of C-terminally truncated proteins rigidifies and opens DNA molecules, and no compaction of the DNA occurs. Moreover, the FP, EM and fluorescence resonance energy transfer (FRET) data indicated that the two basic and flexible C-terminal arms of the Sso7c4 dimer play a crucial role in binding and bending DNA. Sso7c4 has been classified as a repressor-like protein because of its similarity to Escherichia coli Ecrep 6.8 and Ecrep 7.3 as well as Agrobacterium tumefaciens ACCR in amino acid sequence. Based on these data, we proposed a model of the Sso7c4-DNA complex using a curved DNA molecule in the catabolite activator protein-DNA complex. The DNA end-to-end distance measured with FRET upon wild-type Sso7c4 binding is almost equal to the distance measured in the model, which supports the fidelity of the proposed model. The FRET data also confirm the EM observation showing that the binding of wild-type Sso7c4 reduces the DNA length while the C-terminal truncation does not. A functional role for Sso7c4 in the organization of chromosomal DNA and/or the regulation of gene expression through bridging and bending interactions is suggested.

Photon-by-Photon Hidden Markov Model Analysis for Microsecond Single-Molecule FRET Kinetics.

The function of biological macromolecules involves large-scale conformational dynamics spanning multiple time scales, from microseconds to seconds. Such conformational motions, which may involve whole domains or subunits of a protein, play a key role in allosteric regulation. There is an urgent need for experimental methods to probe the fastest of these motions. Single-molecule fluorescence experiments can in principle be used for observing such dynamics, but there is a lack of analysis methods that can extract the maximum amount of information from the data, down to the microsecond time scale. To address this issue, we introduce H2MM, a maximum likelihood estimation algorithm for photon-by-photon analysis of single-molecule fluorescence resonance energy transfer (FRET) experiments. H2MM is based on analytical estimators for model parameters, derived using the Baum-Welch algorithm. An efficient and effective method for the calculation of these estimators is introduced. H2MM is shown to accurately retrieve the reaction times from ∼1 s to ∼10 μs and even faster when applied to simulations of freely diffusing molecules. We further apply this algorithm to single-molecule FRET data collected from Holliday junction molecules and show that at low magnesium concentrations their kinetics are as fast as ∼104 s-1. The new algorithm is particularly suitable for experiments on freely diffusing individual molecules and is readily incorporated into existing analysis packages. It paves the way for the broad application of single-molecule fluorescence to study ultrafast functional dynamics of biomolecules.

Crystal structures of the bifunctional tRNA methyltransferase Trm5a.

tRNA methyltransferase Trm5 catalyses the transfer of a methyl group from S-adenosyl-L-methionine to G37 in eukaryotes and archaea. The N1-methylated guanosine is the product of the initial step of the wyosine hypermodification, which is essential for the maintenance of the reading frame during translation. As a unique member of this enzyme family, Trm5a from Pyrococcus abyssi (PaTrm5a) catalyses not only the methylation of N1, but also the further methylation of C7 on 4-demethylwyosine at position 37 to produce isowyosine, but the mechanism for the double methylation is poorly understood. Here we report four crystal structures of PaTrm5a ranging from 1.7- to 2.3-Å, in the apo form or in complex with various SAM analogues. These structures reveal that Asp243 specifically recognises the base moiety of SAM at the active site. Interestingly, the protein in our structures all displays an extended conformation, quite different from the well-folded conformation of Trm5b from Methanocaldococcus jannaschii reported previously, despite their similar overall architectures. To rule out the possibilities of crystallisation artefacts, we conducted the fluorescence resonance energy transfer (FRET) experiments. The FRET data suggested that PaTrm5a adopts a naturally extended conformation in solution, and therefore the open conformation is a genuine state of PaTrm5a.

Binding of carvedilol to serum albumins investigated by multi-spectroscopic and molecular modeling methods

Carvedilol (CAR) binding to human and bovine serum albumins (HSA and BSA) was studied using fluorescence, UV–vis absorption and Fourier transform infrared spectroscopy (FTIR) and molecular docking techniques at different temperatures (288, 298 and 308K) under physiologic pH. Results obtained from fluorescence data indicated that values of binding sites (n), effective quenching constants (Ka) and binding constants (Kb) decreased under higher temperature and that the quenching mechanism was static. The thermodynamic parameters including enthalpy (ΔH), entropy (ΔS) and Gibb׳s free energy (ΔG) changes were calculated by the van׳t Hoff equation and these data showed that hydrogen bonds and Van der Waals contacts were the main binding force in HSA–CAR and BSA–CAR systems. Binding distance (r) between HSA–CAR and BSA–CAR were calculated by the Fӧrster (fluorescence resonance energy transfer (FRET)) method. FTIR absorption studies showed that the secondary structure was changed according to the interaction of HSA/BSA and CAR. Results determined by molecular docking were in agreement with thermodynamic and FRET data and confirmed that the binding mechanism of Carvedilol to HSA and BSA is different.

Single Molecule Cluster Analysis dissects splicing pathway conformational dynamics.

We report Single Molecule Cluster Analysis (SiMCAn), which utilizes hierarchical clustering of hidden Markov modeling-fitted single-molecule fluorescence resonance energy transfer (smFRET) trajectories to dissect the complex conformational dynamics of biomolecular machines. We used this method to study the conformational dynamics of a precursor mRNA during the splicing cycle as carried out by the spliceosome. By clustering common dynamic behaviors derived from selectively blocked splicing reactions, SiMCAn was able to identify the signature conformations and dynamic behaviors of multiple ATP-dependent intermediates. In addition, it identified an open conformation adopted late in splicing by a 3' splice-site mutant, invoking a mechanism for substrate proofreading. SiMCAn enables rapid interpretation of complex single-molecule behaviors and should prove useful for the comprehensive analysis of a plethora of dynamic cellular machines.

Abstract 2899: Mitophagy imparts enzalutamide resistance in prostate cancer

Abstract Purpose: Anti-androgens are widely used in androgen deprivation therapy (ADT), a standard-of-care for patients with recurrent prostate cancer (PCa). Unfortunately, most patients ultimately develop resistance to ADT and progress to castrate-resistant prostate cancer (CRPC). Recently, two agents that block androgen receptor activation [abiraterone acetate (Zytiga, J&J) and enzalutamide (Xtandi, Medivation/Astellas)] have been approved for CRPC therapy. While some patients respond to these therapies, many fail. We investigated if sequestration of metabolically aberrant mitochondria in the autophagosomes (mitophagy) imparts anti-androgen resistance. Method: We studied the effects of anti-androgen enzalutamide on the autophagy of androgen-dependent LNCaP and -independent C4-2 cells. Autophagy was monitored by cellular fluorescence in cells treated with monodansylcadavarine (MDC). Cellular fluorescence due to Mitosox dye oxidation was used to identify mitochondria producing high superoxide (O2-). Mitophagy of O2- producing mitochondria was monitored using fluorescence resonance energy transfer (FRET) between MDC and Mitosox in a 96-well plate based high throughput (HTS) assay and by visualization of FRET images and quantitation of FRET image intensities using a Nikon A1 fluorescence confocal microscope and associated software. Results: Our data show that in low androgen media, the degree of autophagy is less in C4-2 cells than in LNCaP cells. Enzalutamide treatment induces autophagy in both cell lines, but the increase in autophagy is more pronounced in the androgen-independent C4-2 than in the -dependent LNCaP cells. FRET data from both HTS and fluorescence microscopy show that while enzalutamide increases mitophagy of O2- producing mitochondria in C4-2 cells in a dose dependent manner, it decreases after an initial increase in LNCaP cells. Mitophagy of such mitochondria has also been observed by FRET based fluorescence microscopy in live circulating tumor cells (CTCs) isolated from blood sample of a patient undergoing enzalutamide therapy. Conclusion: Our data demonstrate that PCa cells resistant to enzalutamide show high degree of mitophagy of O2- mitochondria in autophagosomes. If this effect correlates with CTCs from blood collected from CRPC patients undergoing enzalutamide therapy, it can become a clinically useful method of identifying patients who are most likely to benefit from enzalutamide treatment. Citation Format: Hirak S. Basu, Cynthia L. Schrieber, Jamie M. Sperger, Maryanne Naundorf, Ashley M. Weichman, Farideh Mehraein-Ghomi, Dawn R. Church, Joshua M. Lang, George Wilding. Mitophagy imparts enzalutamide resistance in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2899. doi:10.1158/1538-7445.AM2015-2899

Three-color confocal Förster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells.

Experiments using live cell 3-color Förster (or fluorescence) resonance energy transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation. A novel application of 3-color FRET data is demonstrated, extending the analysis beyond the customary energy-transfer efficiency (E%) calculations. MDCK cells were transfected for coexpression of Teal-N-WASP/Venus-IQGAP1/mRFP1-Rac1, Teal-N-WASP/Venus-IQGAP1/mRFP1-Cdc42, CFP-Rac1/Venus-IQGAP1/mCherry-actin, or CFP-Cdc42/Venus-IQGAP1/mCherry-actin, and with single-label equivalents for spectral bleedthrough correction. Using confirmed E% as an entry point, fluorescence levels and related ratios were correlated at discrete accumulating levels at cell peripheries. Rising ratios of CFP-Rac1:Venus-IQGAP1 were correlated with lower overall actin fluorescence, whereas the CFP-Cdc42:Venus-IQGAP1 ratio correlated with increased actin fluorescence at low ratios, but was neutral at higher ratios. The new FRET analyses also indicated that rising levels of mRFP1-Cdc42 or mRFP1-Rac1, respectively, promoted or suppressed the association of Teal-N-WASP with Venus-IQGAP1. These 3-color FRET assays further support our in vitro results about the role of IQGAP1, Rac1, and Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N-WASP with IQGAP1. In addition, this study emphasizes the power of 3-color FRET as a systems biology strategy for simultaneous evaluation of multiple interacting proteins in individual live cells.

Structural Dynamics of the Glycine-binding Domain of the N-Methyl-d-Aspartate Receptor

N-Methyl-D-aspartate receptors mediate the slow component of excitatory neurotransmission in the central nervous system. These receptors are obligate heteromers containing glycine- and glutamate-binding subunits. The ligands bind to a bilobed agonist-binding domain of the receptor. Previous x-ray structures of the glycine-binding domain of NMDA receptors showed no significant changes between the partial and full agonist-bound structures. Here we have used single molecule fluorescence resonance energy transfer (smFRET) to investigate the cleft closure conformational states that the glycine-binding domain of the receptor adopts in the presence of the antagonist 5,7-dichlorokynurenic acid (DCKA), the partial agonists 1-amino-1-cyclobutanecarboxylic acid (ACBC) and L-alanine, and full agonists glycine and D-serine. For these studies, we have incorporated the unnatural amino acid p-acetyl-L-phenylalanine for specific labeling of the protein with hydrazide derivatives of fluorophores. The single molecule fluorescence resonance energy transfer data show that the agonist-binding domain can adopt a wide range of cleft closure states with significant overlap in the states occupied by ligands of varying efficacy. The difference lies in the fraction of the protein in a more closed-cleft form, with full agonists having a larger fraction in the closed-cleft form, suggesting that the ability of ligands to select for these states could dictate the extent of activation.