ABSTRACT
Organophosphoate (OP) chemicals are known to inhibit the enzyme acetylcholinesterase (AChE). Studying OP poisoning is difficult because common small animal research models have serum carboxylesterase, which contributes to animals' resistance to OP poisoning. Historically, guinea pigs have been used for this research; however, a novel genetically modified mouse strain (KIKO) was developed with nonfunctional serum carboxylase (Es1 KO) and an altered acetylcholinesterase (AChE) gene, which expresses the amino acid sequence of the human form of the same protein (AChE KI). KIKO mice were injected with 1xLD50 of an OP nerve agent or vehicle control with or without atropine. After one to three minutes, animals were injected with 35 mg/kg of the currently fielded Reactivator countermeasure for OP poisoning. Postmortem brains were imaged on a Bruker RapifleX ToF/ToF instrument. Data confirmed the presence of increased acetylcholine in OP-exposed animals, regardless of treatment or atropine status. More interestingly, we detected a small amount of Reactivator within the brain of both exposed and unexposed animals; it is currently debated if reactivators can cross the blood-brain barrier. Further, we were able to simultaneously image acetylcholine, the primary affected neurotransmitter, as well as determine the location of both Reactivator and acetylcholine in the brain. This study, which utilized sensitive MALDI-MSI methods, characterized KIKO mice as a functional model for OP countermeasure development.
Subject(s)
Acetylcholinesterase , Disease Models, Animal , Organophosphate Poisoning , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Animals , Mice , Humans , Acetylcholinesterase/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Atropine/pharmacology , Brain/metabolism , Brain/pathology , Brain/drug effects , Mice, Knockout , Cholinesterase Inhibitors , Acetylcholine/metabolismABSTRACT
Despite the importance of proline conformational equilibria (trans versus cis amide and exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19F NMR-based probe of proline conformational biases and cis-trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (ΔδFF = 0-3 ppm) when a trans X-Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (ΔδFF = 5-12 ppm) difference in chemical shift in a cis X-Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that ΔδFF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e., the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e., pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a proline is disordered (a mixture of exo and endo ring puckers, as at trans-Pro in peptides in water), it exhibits a small Δδ. In contrast, when the Pro is ordered (i.e., when one ring pucker is strongly preferred, as in cis-Pro amide bonds, where the endo ring pucker is strongly favored), a large Δδ is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline cis-trans isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the exo and endo proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n â π* interactions for the stabilization of PPII. Dfp was also employed to quantify proline cis-trans isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline cis-trans isomerization, protein folding, and local order in intrinsically disordered proteins.
Subject(s)
Fluorine , Proline , Proline/chemistry , Proline/analogs & derivatives , Fluorine/chemistry , Crystallography, X-Ray/methods , Protein Conformation , Magnetic Resonance Spectroscopy/methods , Peptides/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Molecular ConformationABSTRACT
Metabolic reprogramming is a hallmark of cancer, enabling cancer cells to rapidly proliferate, invade, and metastasize. We show that creatine levels in metastatic breast cancer cell lines and secondary metastatic tumors are driven by the ubiquitous mitochondrial creatine kinase (CKMT1). We discover that, while CKMT1 is highly expressed in primary tumors and promotes cell viability, it is downregulated in metastasis. We further show that CKMT1 downregulation, as seen in breast cancer metastasis, drives up mitochondrial reactive oxygen species (ROS) levels. CKMT1 downregulation contributes to the migratory and invasive potential of cells by ROS-induced upregulation of adhesion and degradative factors, which can be reversed by antioxidant treatment. Our study thus reconciles conflicting evidence about the roles of metabolites in the creatine metabolic pathway in breast cancer progression and reveals that tight, context-dependent regulation of CKMT1 expression facilitates cell viability, cell migration, and cell invasion, which are hallmarks of metastatic spread.
Subject(s)
Breast Neoplasms , Creatine Kinase, Mitochondrial Form , Reactive Oxygen Species , Animals , Female , Humans , Mice , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/genetics , Cell Line, Tumor , Cell Movement , Cell Survival , Creatine Kinase , Creatine Kinase, Mitochondrial Form/metabolism , Disease Progression , Gene Expression Regulation, Neoplastic , Mitochondria/metabolism , Neoplasm Invasiveness , Neoplasm Metastasis , Reactive Oxygen Species/metabolismABSTRACT
Breast cancer is the leading cancer-related cause of death in women. Here we show that solute carrier family 38-member 3 (SLC38A3) is overexpressed in breast cancer, particularly in triple-negative breast cancer (TNBC) cells and tissues. Our study reveals that SLC38A3 regulates cellular glutamine, glutamate, asparagine, aspartate, alanine, and glutathione (GSH) levels in breast cancer cells. Our data demonstrate that SLC38A3 enhances cell viability, cell migration and invasion in vitro, and promotes tumor growth and metastasis in vivo, while reducing apoptosis and oxidative stress. Mechanistically, we show that SLC38A3 suppresses the activity of glycogen synthase kinase 3-ß (Gsk3ß), a negative regulator of ß-catenin, and increases protein levels of ß-catenin, leading to the upregulation of epithelial-to-mesenchymal-transition (EMT)-inducing transcription factors and EMT markers in breast cancer. In summary, we show that SLC38A3 is overexpressed in breast cancer and promotes breast cancer metastasis via the GSK3ß/ß-catenin/EMT pathway, presenting a novel therapeutic target to explore for breast cancer.
Subject(s)
Triple Negative Breast Neoplasms , beta Catenin , Female , Humans , beta Catenin/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Epithelial-Mesenchymal Transition , Glutamine , Glycogen Synthase Kinase 3 beta/metabolism , Triple Negative Breast Neoplasms/pathology , Wnt Signaling PathwayABSTRACT
Here, the authors report that co-crystallization of fluorophores with matrix-assisted laser desorption/ionization (MALDI) imaging matrices significantly enhances fluorophore brightness up to 79-fold, enabling the amplification of innate tissue autofluorescence. This discovery facilitates FluoMALDI, the imaging of the same biological sample by both fluorescence microscopy and MALDI imaging. The approach combines the high spatial resolution and specific labeling capabilities of fluorescence microscopy with the inherently multiplexed, versatile imaging capabilities of MALDI imaging. This new paradigm simplifies registration by avoiding physical changes between fluorescence and MALDI imaging, allowing to image the exact same cells in tissues with both modalities. Matrix-fluorophore co-crystallization also facilitates applications with insufficient fluorescence brightness. The authors demonstrate feasibility of FluoMALDI imaging with endogenous and exogenous fluorophores and autofluorescence-based FluoMALDI of brain and kidney tissue sections. FluoMALDI will advance structural-functional microscopic imaging in cell biology, biomedicine, and pathology.
Subject(s)
Brain , Kidney , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Crystallization , Microscopy, Fluorescence , Kidney/diagnostic imagingABSTRACT
BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and leads to the poorest patient outcomes despite surgery and chemotherapy treatment. Exploring new molecular mechanisms of TNBC that could lead to the development of novel molecular targets are critically important for improving therapeutic options for treating TNBC. METHODS: We sought to identify novel therapeutic targets in TNBC by combining genomic and functional studies with lipidomic analysis, which included mechanistic studies to elucidate the pathways that tie lipid profile to critical cancer cell properties. Our studies were performed in a large panel of human breast cancer cell lines and patient samples. RESULTS: Comprehensive lipid profiling revealed that phospholipid metabolism is reprogrammed in TNBC cells. We discovered that patatin-like phospholipase domain-containing lipase 8 (PNPLA8) is overexpressed in TNBC cell lines and tissues from breast cancer patients. Silencing of PNPLA8 disrupted phospholipid metabolic reprogramming in TNBC, particularly affecting the levels of phosphatidylglycerol (PG), phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and glycerophosphocholine (GPC). We showed that PNPLA8 is essential in regulating cell viability, migration and antioxidation in TNBC cells and promoted arachidonic acid and eicosanoid production, which in turn activated PI3K/Akt/Gsk3ß and MAPK signaling. CONCLUSIONS: Our study highlights PNPLA8 as key regulator of phospholipid metabolic reprogramming and malignant phenotypes in TNBC, which could be further developed as a novel molecular treatment target.
Subject(s)
Triple Negative Breast Neoplasms , Humans , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Phenotype , Phosphatidylinositol 3-Kinases/genetics , Phospholipids/therapeutic use , Triple Negative Breast Neoplasms/pathologyABSTRACT
The premetastatic niche hypothesis proposes an active priming of the metastatic site by factors secreted from the primary tumor prior to the arrival of the first cancer cells. We investigated several extracellular matrix (ECM) structural proteins, ECM degrading enzymes, and ECM processing proteins involved in the ECM remodeling of the premetastatic niche. Our in vitro model consisted of lung fibroblasts, which were exposed to factors secreted by nonmalignant breast epithelial cells, nonmetastatic breast cancer cells, or metastatic breast cancer cells. We assessed ECM remodeling in vivo in premetastatic lungs of female mice growing orthotopic primary breast tumor xenografts, as compared to lungs of control mice without tumors. Premetastatic lungs contained significantly upregulated Collagen (Col) Col4A5, matrix metalloproteinases (MMPs) MMP9 and MMP14, and decreased levels of MMP13 and lysyl oxidase (LOX) as compared to control lungs. These in vivo findings were consistent with several of our in vitro cell culture findings, which showed elevated Col14A1, Col4A5, glypican-1 (GPC1) and decreased Col5A1 and Col15A1 for ECM structural proteins, increased MMP2, MMP3, and MMP14 for ECM degrading enzymes, and decreased LOX, LOXL2, and prolyl 4-hydroxylase alpha-1 (P4HA1) for ECM processing proteins in lung fibroblasts conditioned with metastatic breast cancer cell media as compared to control. Taken together, our data show that premetastatic priming of lungs by primary breast tumors resulted in significant ECM remodeling which could facilitate metastasis by increasing interstitial fibrillar collagens and ECM stiffness (Col14A1), disruptions of basement membranes (Col4A5), and formation of leaky blood vessels (MMP2, MMP3, MMP9, and MMP14) to promote metastasis.
Subject(s)
Breast Neoplasms , Mammary Neoplasms, Animal , Humans , Female , Mice , Animals , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinase 14/metabolism , Matrix Metalloproteinase 3/metabolism , Lung/pathology , Extracellular Matrix/metabolism , Mammary Neoplasms, Animal/metabolism , Extracellular Matrix Proteins/metabolism , Breast Neoplasms/pathologyABSTRACT
Multimodal tissue imaging techniques that integrate two complementary modalities are powerful discovery tools for unraveling biological processes and identifying biomarkers of disease. Combining Raman spectroscopic imaging (RSI) and matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry imaging (MSI) to obtain fused images with the advantages of both modalities has the potential of providing spatially resolved, sensitive, specific biomolecular information, but has so far involved two separate sample preparations, or even consecutive tissue sections for RSI and MALDI MSI, resulting in images with inherent disparities. We have developed RaMALDI, a streamlined, integrated, multimodal imaging workflow of RSI and MALDI MSI, performed on a single tissue section with one sample preparation protocol. We show that RaMALDI imaging of various tissues effectively integrates molecular information acquired from both RSI and MALDI MSI of the same sample, which will drive discoveries in cell biology, biomedicine, and pathology, and advance tissue diagnostics.
Subject(s)
Biosensing Techniques , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Multimodal Imaging , Serogroup , Specimen HandlingABSTRACT
NMR spectroscopy and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) are both commonly used to detect large numbers of metabolites and lipids in metabolomic and lipidomic studies. We have demonstrated a new workflow, highlighting the benefits of both techniques to obtain metabolomic and lipidomic data, which has realized for the first time the combination of these two complementary and powerful technologies. NMR spectroscopy is frequently used to obtain quantitative metabolite information from cells and tissues. Lipid detection is also possible with NMR spectroscopy, with changes being visible across entire classes of molecules. Meanwhile, MALDI MSI provides relative measures of metabolite and lipid concentrations, mapping spatial information of many specific metabolite and lipid molecules across cells or tissues. We have used these two complementary techniques in combination to obtain metabolomic and lipidomic measurements from triple-negative human breast cancer cells and tumor xenograft models. We have emphasized critical experimental procedures that ensured the success of achieving NMR spectroscopy and MALDI MSI in a combined workflow from the same sample. Our data show that several phospholipid metabolite species were differentially distributed in viable and necrotic regions of breast tumor xenografts. This study emphasizes the power of combined NMR spectroscopy-MALDI imaging to advance metabolomic and lipidomic studies.
Subject(s)
Lipidomics , Metabolomics , Humans , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , PhospholipidsABSTRACT
Rationale: Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is emerging as an alternative to gadolinium-based contrast MRI. We have evaluated the possibility of CEST MRI of orthotopic breast tumor xenografts with unlabeled aspirin's conversion to salicylic acid (SA) through various enzymatic activities, most notably inhibition of cyclooxygenase (COX)-1/-2 enzymes. Methods: We measured the COX-1/-2 expression in four breast cancer cell lines by Western Blot analysis and selected the highest and lowest expressing cell lines. We then performed CEST MRI following aspirin treatment to detect SA levels and ELISA to measure levels of downstream prostaglandin E2 (PGE2). We also injected aspirin into the tail vein of mice growing orthotopic tumor xenografts which expressed high and low COX-1/-2 and acquired SA CEST MR images of these tumor xenografts for up to 70 minutes. Tumors were then harvested to perform Western Blot and ELISA experiments to measure COX-1/-2 expression and PGE2 levels, respectively. Results: Western Blots determined that SUM159 cells contained significantly higher COX-1/-2 expression levels than MDA-MB-231 cells, in line with higher levels of downstream PGE2. SA CEST MRI yielded similar contrast at approximately 3% for both cell lines, independent of COX-1/-2 expression level. PGE2 levels decreased by about 50% following aspirin treatment. Results from our mouse study aligned with cultured cells, the overall SA CEST MRI contrast in both MDA-MB-231 and SUM159 tumor xenograft models was 5~8% at one hour post injection. PGE2 levels were ten times higher in SUM159 than MDA-MB-231 and decreased by 50%. The CEST contrast directly depended on the injected dose, with ~6%, ~3% and ~1.5% contrast observed following injection of 100 µL of 300 mM, 200 mM and 150 mM aspirin, respectively. Conclusions: Our data demonstrate the feasibility of using aspirin as a noninvasive activatable CEST MRI contrast agent for breast tumor detection.
Subject(s)
Breast Neoplasms , Animals , Aspirin , Breast Neoplasms/diagnostic imaging , Breast Neoplasms/drug therapy , Contrast Media , Dinoprostone , Female , Humans , Magnetic Resonance Imaging/methods , Mice , Theranostic NanomedicineABSTRACT
BACKGROUND: Although aberrant glycosylation is recognized as a hallmark of cancer, glycosylation in clinical breast cancer (BC) metastasis has not yet been studied. While preclinical studies show that the glycocalyx coating of cancer cells is involved in adhesion, migration, and metastasis, glycosylation changes from primary tumor (PT) to various metastatic sites remain unknown in patients. METHODS: We investigated N-glycosylation profiles in 17 metastatic BC patients from our rapid autopsy program. Primary breast tumor, lymph node metastases, multiple systemic metastases, and various normal tissue cores from each patient were arranged on unique single-patient tissue microarrays (TMAs). We performed mass spectrometry imaging (MSI) combined with extensive pathology annotation of these TMAs, and this process enabled spatially differentiated cell-based analysis of N-glycosylation patterns in metastatic BC. RESULTS: N-glycan abundance increased during metastatic progression independently of BC subtype and treatment regimen, with high-mannose glycans most frequently elevated in BC metastases, followed by fucosylated and complex glycans. Bone metastasis, however, displayed increased core-fucosylation and decreased high-mannose glycans. Consistently, N-glycosylated proteins and N-glycan biosynthesis genes were differentially expressed during metastatic BC progression, with reduced expression of mannose-trimming enzymes and with elevated EpCAM, N-glycan branching, and sialyation enzymes in BC metastases versus PT. CONCLUSION: We show in patients that N-glycosylation of breast cancer cells undergoing metastasis occurs in a metastatic site-specific manner, supporting the clinical importance of high-mannose, fucosylated, and complex N-glycans as future diagnostic markers and therapeutic targets in metastatic BC. FUNDING: NIH grants R01CA213428, R01CA213492, R01CA264901, T32CA193145, Dutch Province Limburg "LINK", European Union ERA-NET TRANSCAN2-643638.
Subject(s)
Breast Neoplasms/genetics , Mannose/metabolism , Polysaccharides/metabolism , Adult , Aged , Breast Neoplasms/pathology , Female , Glycosylation , Humans , Middle Aged , Neoplasm MetastasisABSTRACT
Using citrate-capped gold nanoparticles (AuNPs) for laser desorption ionization mass spectrometry (LDI-MS) is an approach that has demonstrated broad applicability to ionization of different classes of molecules. Here, we show a simple AuNP-based approach for the ionization of neurotransmitters. Specifically, the detection of acetylcholine, dopamine, epinephrine, glutamine, 4-aminobutyric acid, norepinephrine, octopamine, and serotonin was achieved at physiologically relevant concentrations in serum and homogenized tissue. Additionally, pneumatic spraying of AuNPs onto tissue sections facilitated mass spectrometry imaging (MSI) of rabbit brain tissue sections, zebrafish embryos, and neuroblastoma cells for several neurotransmitters simultaneously using this quick and simple sample preparation. AuNP LDI-MS achieved mapping of neurotransmitters in fine structures of zebrafish embryos and neuroblastoma cells at a lateral spatial resolution of 5 µm. The use of AuNPs to ionize small aminergic neurotransmitters in situ provides a fast, high-spatial resolution method for simultaneous detection of a class of molecules that typically evade comprehensive detection with traditional matrixes.
Subject(s)
Neurotransmitter Agents/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Animals , Brain Chemistry , Cell Line, Tumor , Gold/chemistry , Humans , Metal Nanoparticles/chemistry , Rabbits , Zebrafish/embryologyABSTRACT
19F NMR spectroscopy provides the ability to quantitatively analyze single species in complex solutions but is often limited by the modest sensitivity inherent to NMR. 4R- and 4S-Perfluoro-tert-buyl hydroxyproline contain 9 equivalent fluorines, in amino acids with strong conformational preferences. In order to test the ability to use these amino acids as sensitive probes of protein modifications, the perfluoro-tert-buyl hydroxyprolines were incorporated into substrate peptides of the protein kinases PKA and Akt. Peptides containing each diastereomeric proline were rapidly phosphorylated by each protein kinase and exhibited 19F chemical shift changes as a result of phosphorylation. The sensitivity of the perfluoro-tert-butyl group allowed quantitative analysis of the kinetics of phosphorylation over three half-lives at single-digit micromolar concentrations of each species. The distinct conformational preferences of these amino acids allowed the optimization of the substrate with a conformationally matched amino acid, in order to maximize the rate of phosphorylation. PKA preferred the 4R-amino acid at the -1 position, whereas the closely related AGC kinase Akt preferred the 4S-amino acid. These data, combined with analysis of structures of the Michaelis complexes of these kinases in the PDB, suggest that PKA recognizes the PPII conformation at the P-1 position relative to the phosphorylation site, while Akt/PKB recognizes an extended conformation at this position. These results suggest that conformational targeting may be employed to increase specificity in recognition by protein kinases. Perfluoro-tert-butyl hydroxyprolines were applied to the real-time detection and quantification of PKA activity and inhibition of PKA activity in HeLa cell extracts via 19F NMR spectroscopy. The coupling of proline ring pucker with main chain conformation suggests broad application of perfluoro-tert-butyl hydroxyprolines in molecular sensing and imaging.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/analysis , Hydrocarbons, Fluorinated/chemistry , Molecular Probes/chemistry , Peptides/chemistry , Proline/analogs & derivatives , Proto-Oncogene Proteins c-akt/analysis , Cyclic AMP-Dependent Protein Kinases/chemistry , Enzyme Assays/methods , Fluorine/chemistry , HeLa Cells , Humans , Molecular Conformation , Nuclear Magnetic Resonance, Biomolecular/methods , Phosphorylation , Protein Conformation , Proto-Oncogene Proteins c-akt/chemistry , StereoisomerismABSTRACT
Activated choline metabolism is a hallmark of carcinogenesis and tumor progression, which leads to elevated levels of phosphocholine and glycerophosphocholine in all types of cancer tested so far. Magnetic resonance spectroscopy applications have played a key role in detecting these elevated choline phospholipid metabolites. To date, the majority of cancer-related studies have focused on phosphocholine and the Kennedy pathway, which constitutes the biosynthesis pathway for membrane phosphatidylcholine. Fewer and more recent studies have reported on the importance of glycerophosphocholine in cancer. In this review article, we summarize the recent literature on glycerophosphocholine metabolism with respect to its cancer biology and its detection by magnetic resonance spectroscopy applications.
Subject(s)
Choline/metabolism , Glycerylphosphorylcholine/metabolism , Metabolic Networks and Pathways , Neoplasms/metabolism , Animals , Humans , Substrate Specificity , Transcription Factors/metabolismABSTRACT
Protein kinases and phosphatases modulate protein structure and function, which in turn regulate cellular activities. The development of novel proteins and protein motifs that are responsive to protein phosphorylation provides new ways to probe the functions of individual protein kinases and the intracellular effects of their activation and downregulation. Herein we develop a minimal motif that is responsive to protein phosphorylation, termed a minimal protein kinase-inducible domain. The encodable protein motif comprises a 7- or 8-residue sequence (DKDADXW or DKDADXXW), derived from EF-Hand calcium-binding domains, that is necessary but not sufficient for binding terbium, combined with a protein phosphorylation site (Ser or Thr at residue 9) that, upon phosphorylation, completes the metal-binding motif. Thus, the motif binds metal poorly and exhibits weak terbium luminescence when not phosphorylated. Upon phosphorylation, the peptide binds metal with significantly higher affinity and exhibits robust terbium luminescence. Phosphorylation results in up to a 23× increase in terbium luminescence. Minimal phosphorylation-dependent motifs as small as 9 residues (DKDADGWIS) were developed. NMR spectroscopy on this lanthanum(iii)·phosphopeptide complex confirmed that binding occurs in a manner similar to that in an EF-Hand, despite the absence of the conserved Glu12 typically present in an EF-Hand. By combining molecular design with known protein kinase recognition sequences, minimal protein kinase-inducible domains were developed that were responsive to phosphorylation by Protein Kinase A (PKA: DKDADRRW(S/pS)IIAK), Protein Kinase C (PKC: DKDADGWI(T/pT)FRRKA), and Casein Kinase 1 (CK1: DKDADDWA(S/pS)I). Phosphorylation by PKA was quantified in HeLa cell extracts, with a 4.4× increase in fluorescence (terbium luminescence) observed at 544 nm. The optimized minimal motif includes alternating aspartate residues at positions 1, 3, and 5, plus binding through the main-chain carbonyl at position 7; a lysine at position 2 to provide electrostatic balance and reduce binding in the absence of phosphorylation; an alanine at residue 4 to promote the αL conformation observed at that position of the EF Hand; a tryptophan at residue 7 or 8 to sensitize terbium luminescence; and a phosphorylation site with serine or threonine at residue 9. Residues at positions 6; 7 or 8; and 10 or later may be changed to provide kinase specificity. In the CK1-responsive peptide, the acidic residues in the proto-terbium-binding motif are employed as part of the kinase recognition sequence. This work thus presents fundamental rules for the design of compact phosphorylation-responsive terbium-binding motifs, with potential further application to motifs responsive to other protein post-translational modifications.
Subject(s)
Phosphoric Monoester Hydrolases/metabolism , Protein Kinases/metabolism , Humans , Phosphoric Monoester Hydrolases/chemistry , Phosphorylation , Protein Kinases/chemistryABSTRACT
The Kryptopterus bicirrhis (glass catfish) is known to respond to electromagnetic fields (EMF). Here we tested its avoidance behavior in response to static and alternating magnetic fields stimulation. Using expression cloning we identified an electromagnetic perceptive gene (EPG) from the K. bicirrhis encoding a protein that responds to EMF. This EPG gene was cloned and expressed in mammalian cells, neuronal cultures and in rat's brain. Immunohistochemistry showed that the expression of EPG is confined to the mammalian cell membrane. Calcium imaging in mammalian cells and cultured neurons expressing EPG demonstrated that remote activation by EMF significantly increases intracellular calcium concentrations, indicative of cellular excitability. Moreover, wireless magnetic activation of EPG in rat motor cortex induced motor evoked responses of the contralateral forelimb in vivo. Here we report on the development of a new technology for remote, non-invasive modulation of cell function.
Subject(s)
Avoidance Learning , Electromagnetic Fields , Fishes/physiology , Animals , Calcium/metabolism , Cells, Cultured , Fish Proteins/genetics , Fish Proteins/metabolism , Fishes/genetics , HEK293 Cells , Humans , Neurons/metabolism , Rats , Rats, Sprague-Dawley , Wireless TechnologyABSTRACT
Highly fluorinated amino acids can stabilize proteins and complexes with proteins, via enhanced hydrophobicity, and provide novel methods for identification of specific molecular events in complex solutions, via selective detection by 19F NMR and the absence of native 19F signals in biological contexts. However, the potential applications of 19F NMR in probing biological processes are limited both by the strong propensities of most highly fluorinated amino acids for the extended conformation and by the relatively modest sensitivity of NMR spectroscopy, which typically constrains measurements to mid-micromolar concentrations. Herein, we demonstrate that perfluoro-tert-butyl homoserine exhibits a propensity for compact conformations, including α-helix and polyproline helix (PPII), that is similar to that of methionine. Perfluoro-tert-butyl homoserine has nine equivalent fluorines that do not couple to any other nuclei, resulting in a sharp singlet that can be sensitively detected rapidly at low micromolar concentrations. Perfluoro-tert-butyl homoserine was incorporated at sites of leucine residues within the α-helical LXXLL short linear motif of estrogen receptor (ER) coactivator peptides. A peptide containing perfluoro-tert-butyl homoserine at position i + 3 of the ER coactivator LXXLL motif exhibited a Kd of 2.2 µM for the estradiol-bound estrogen receptor, similar to that of the native ligand. 19F NMR spectroscopy demonstrated the sensitive detection (5 µM concentration, 128 scans) of binding of the peptide to the ER and of inhibition of protein-protein interaction by the native ligand or by the ER antagonist tamoxifen. These results suggest diverse potential applications of perfluoro-tert-butyl homoserine in probing protein function and protein-protein interfaces in complex solutions.
Subject(s)
Estrogen Receptor alpha/chemistry , Estrogen Receptor alpha/metabolism , Halogenation , Homoserine/analogs & derivatives , Homoserine/chemistry , Homoserine/pharmacology , Amino Acid Sequence , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein DomainsABSTRACT
A practical synthesis of the novel highly fluorinated amino acid Fmoc-perfluoro-tert-butyl tyrosine was developed. The sequence proceeds in two steps from commercially available Fmoc-4-NH2-phenylalanine via diazotization followed by diazonium coupling reaction with perfluoro-tert-butanol. In peptides, perfluoro-tert-butyl tyrosine was detected in 30 s by NMR spectroscopy at 500 nM peptide concentration due to nine chemically equivalent fluorines that are a sharp singlet by 19F NMR. Perfluoro-tert-butyl ether has an estimated σp Hammett substituent constant of +0.30.
Subject(s)
Diazonium Compounds/chemistry , Tyrosine/chemical synthesis , Fluorine , Magnetic Resonance Spectroscopy , Molecular Conformation , Stereoisomerism , Tyrosine/analogs & derivatives , Tyrosine/chemistryABSTRACT
(2S,4R)- and (2S,4S)-perfluoro-tert-butyl 4-hydroxyproline were synthesized (as Fmoc-, Boc-, and free amino acids) in 2-5 steps. The key step of each synthesis was a Mitsunobu reaction with perfluoro-tert-butanol, which incorporated a perfluoro-tert-butyl group, with nine chemically equivalent fluorines. Both amino acids were incorporated in model α-helical and polyproline helix peptides. Each amino acid exhibited distinct conformational preferences, with (2S,4R)-perfluoro-tert-butyl 4-hydroxyproline promoting polyproline helix. Peptides containing these amino acids were sensitively detected by (19)F NMR, suggesting their use in probes and medicinal chemistry.