Labeled Amino Acids Research Articles

Organocatalysis Enables the Isotopic Labelling of Amino Acids

Organocatalysis Enables the Isotopic Labelling of Amino Acids

Francisella tularensis Glyceraldehyde-3-Phosphate Dehydrogenase Is Relocalized during Intracellular Infection and Reveals Effect on Cytokine Gene Expression and Signaling.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known for its multifunctionality in several pathogenic bacteria. Our previously reported data suggest that the GAPDH homologue of Francisella tularensis, GapA, might also be involved in other processes beyond metabolism. In the present study, we explored GapA's potential implication in pathogenic processes at the host cell level. Using immunoelectron microscopy, we demonstrated the localization of this bacterial protein inside infected macrophages and its peripheral distribution in bacterial cells increasing with infection time. A quantitative proteomic approach based on stable isotope labeling of amino acids in cell culture (SILAC) combined with pull-down assay enabled the identification of several of GapA's potential interacting partners within the host cell proteome. Two of these partners were further confirmed by alternative methods. We also investigated the impact of gapA deletion on the transcription of selected cytokine genes and the activation of the main signaling pathways. Our results show that ∆gapA-induced transcription of genes encoding several cytokines whose expressions were not affected in cells infected with a fully virulent wild-type strain. That might be caused, at least in part, by the detected differences in ERK/MAPK signaling activation. The experimental observations together demonstrate that the F. tularensis GAPDH homologue is directly implicated in multiple host cellular processes and, thereby, that it participates in several molecular mechanisms of pathogenesis.

Development of PET Tracers of Glutamine Metabolism

The labeling of amino acids with positron-emitting radionuclides (such as fluorine-18) has been a widely used approach for the imaging of tumors as it often provides higher diagnostic accuracy than what is observed with [18F]FDG. In particular, PET tracers of glutamine metabolism have garnered significant attention in recent years. O-(2-[18F]fluoroethyl-L-tyrosine (18F-FET) is a promising PET tracer in this regard and is currently under investigation at Indiana University (IU) through an expanded access IND for patients with brain malignancies. Clinical production of 18F-FET at IU previously required the use of HPLC for purification, following the reaction of fluorine-18 with the precursor molecule for FET. While this method has been successful in removing undesirable impurities and byproducts, HPLC significantly increases synthesis time and is a common failure point in the synthesis of FET on our current radiochemistry module. To address this issue, we aimed to deploy a solid-phase-extraction (SPE) method for the purification of FET, thereby eliminating the need for HPLC purification. Several methods for the SPE purification of FET have been previously reported; however, none of these strategies afforded pure [18F]FET on our synthesis module, thus development of new methods was required.
 While several tracers capable of measuring different aspects of glutamine metabolism have been evaluated in both preclinical and clinical studies, there are metabolic liabilities that limit their utility and complicate data analysis. [18F]-4F-glutamine is one such tracer that has shown promise but has limitations due to undesirable metabolism in vivo. Herein we report our progress towards an improved synthesis of [18F]FET for ongoing clinical studies as well as our progress towards the development of a novel tracer that would address metabolic liabilities associated with currently available PET tracers of glutamine metabolism.

Probing structural topology and dynamics of human KCNE3 membrane protein in lipid bilayers using site-directed spin labeling and electron paramagnetic resonance spectroscopy.

KCNE3 is a single pass membrane spanning protein that modulates the function of the voltage gated potassium ion channel KCNQ1. Genetic mutations in the KCNE3 protein can result in multiple diseases such as long QT syndrome, atrial fibrillation, Mènière's disease, etc. Despite the biological significance, its structural and dynamic properties in a lipid bilayer are not yet fully understood. In this study, the structural topology and dynamic properties of KCNE3 were explored in lipid bilayers utilizing site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. Continuous wave EPR data indicated that the nitroxide spin labeled sidechains of KCNE3 in the transmembrane domain (TMD) are less mobile when compared to that of N and C-termini. Multiple nitroxide spin labeled amino acid sites have been studied using power saturation EPR to determine the membrane depth parameter (φ) of KCNE3 with respect to the POPC/POPG lipid bilayers. These results will be helpful for developing the topology model of KCNE3 in lipid bilayers.

Three-in-One Detector by 3D Printing: Simultaneous Contactless Conductivity, Ultraviolet Absorbance, and Laser-Induced Fluorescence Measurements for Capillary Electrophoresis.

We describe a 3-in-1 detector for simultaneous contactless conductivity (C4D), ultraviolet absorbance (UV-AD), and laser-induced fluorescence (LIF) measurements on a single detection point for capillary electrophoresis (CE). A key component of the detector was a rectangular detector head that was assembled with four 3D-printed parts. Two parts covering the detector head to function as a Faraday cage were fused deposition modeling printed using an electrically conductive material. The other two parts in between the conductive parts were stereolithography (SLA) printed with high-resolution (50 μm) constructions on the surface. After assembling the two SLA printed parts, several cavities were built with the surface constructions. Two electrodes and a Faraday shield for C4D were cast by injecting molten Wood's metal into the cavities. For UV-AD, a slit (100 μm width) was created by putting together two grooves (50 μm depth) on the surface of the SLA printed parts. A 255 nm UV-LED was used as the light source. The effective path length and stray light for a 50 μm id capillary were 39 μm and 13%, which were superior to those of other reported 3D-printed AD detectors. Confocal LIF detection was conducted by using an objective lens to focus the laser on the capillary via a through-hole. The detector was used to detect model analytes, including inorganic and organic ions, and fluorescein isothiocyanate labeled amino acids in a signal-run CE separation. In detecting fluorescein, LODs were 1.3 μM (C4D), 2.0 μM (UV-AD), and 1 nM (LIF). The calibration ranges covered from 0.01 μM to 500 μM.

Quantitative proteomic analysis reveals the effects of mu opioid agonists on HT22 cells.

Introduction: At present, the mu opioid receptor is the most important neuroaesthetics receptor in anesthesiology research, and the damage that it does to the nervous system is unknown. Methods: We investigated the effects of loperamide, an agonist of the mu opioid receptor, on protein expression in HT22 cells using stable isotope labeling of amino acids in cell culture (SILAC), immobilized metal affinity chromatography (IMAC) enrichment, and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 7,823 proteins were identified. Results and Discussion: Bioinformatic analysis revealed that mu opioid receptor agonism can induce distinct changes in the proteome of HT22 cells. These findings improve our understanding of narcotic drugs.

Water-soluble NHC Pd/Ni bimetallic nanoparticles for H/D exchange in aromatic amino-acids.

Labelling of amino-acids is important for the production of deuterated proteins. However, aromatic amino-acid reduction is a common undesired process with noble-metal nanocatalysts. In this work, we describe a new NHC-stabilized water-soluble Pd/Ni system able to perform H/D exchange reactions in an enantiospecific fashion without reducing the aromatic ring of phenylalanine and tyrosine thanks to a synergetic Pd-Ni effect.

Genetic Encoding of a Fluorescent Noncanonical Amino Acid as a FRET Donor for theAnalysis of Deubiquitinase Activities.

The genetic code expansion technology enables the genetic encoding of fluorescent noncanonical amino acids (ncAAs) for site-specific fluorescent labeling of proteins. These co-translational and internal fluorescent tags have been harnessed to establish genetically encoded Förster resonance energy transfer (FRET) probes for studying protein structural changes and interactions. Here, we describe the protocols for site-specific incorporation of an aminocoumarin-derived fluorescent ncAA into proteins in E. coli and preparation of a fluorescent ncAA-based FRET probe for assaying theactivities of deubiquitinases, a key class of enzymes involved in ubiquitination. We also describe the deployment of an in vitro fluorescence assay to screen and analyze small-molecule inhibitors against deubiquitinases.

Synthesis of 13C-methyl-labeled amino acids and their incorporation into proteins in mammalian cells.

Isotopic labeling of methyl-substituted proteinogenic amino acids with 13C has transformed applications of solution-based NMR spectroscopy and allowed the study of much larger and more complex proteins than previously possible with 15N labeling. Procedures are well-established for producing methyl-labeled proteins expressed in bacteria, with efficient incorporation of 13C-methyl labeled metabolic precursors to enable the isotopic labeling of Ile, Val, and Leu methyl groups. Recently, similar methodology has been applied to enable 13C-methyl labeling of Ile, Val, and Leu in yeast, extending the approach to proteins that do not readily fold when produced in bacteria. Mammalian or insect cells are nonetheless preferable for production of many human proteins, yet 13C-methyl labeling using similar metabolic precursors is not feasible as these cells lack the requisite biosynthetic machinery. Herein, we report versatile and high-yielding synthetic routes to 13C methyl-labeled amino acids based on palladium-catalyzed C(sp3)-H functionalization. We demonstrate the efficient incorporation of two of the synthesized amino acids, 13C-γ2-Ile and 13C-γ1,γ2-Val, into human receptor extracellular domains with multiple disulfides using suspension-cultured HEK293 cells. Production costs are reasonable, even at moderate expression levels of 2-3 mg purified protein per liter of medium, and the method can be extended to label other methyl groups, such as 13C-δ1-Ile and 13C-δ1,δ2-Leu. In summary, we demonstrate the cost-effective production of methyl-labeled proteins in mammalian cells by incorporation of 13C methyl-labeled amino acids generated de novo by a versatile synthetic route.

Computational assessment of amino acid-coupled benzanthrone 2-aminoacetamides as molecular probes for insulin amyloid fibril visualization

Fluorescent proteins are currently the most extensively studied fluorescent markers used in bio-imaging, and many such fluorescent markers are used in research to label amino acids and proteins.

Biocatalytic reductive amination as a route to isotopically labelled amino acids suitable for analysis of large proteins by NMR

We demonstrate an atom-efficient and easy to use H2-driven biocatalytic platform for the enantioselective synthesis of isotopically labelled amino acids for protein NMR studies.

Protein-Peptide Turnover Profiling reveals the order of PTM addition and removal during protein maturation

Post-translational modifications (PTMs) regulate various aspects of protein function, including degradation. Mass spectrometric methods relying on pulsed metabolic labeling are popular to quantify turnover rates on a proteome-wide scale. Such data have traditionally been interpreted in the context of protein proteolytic stability. Here, we combine theoretical kinetic modeling with experimental pulsed stable isotope labeling of amino acids in cell culture (pSILAC) for the study of protein phosphorylation. We demonstrate that metabolic labeling combined with PTM-specific enrichment does not measure effects of PTMs on protein stability. Rather, it reveals the relative order of PTM addition and removal along a protein’s lifetime—a fundamentally different metric. This is due to interconversion of the measured proteoform species. Using this framework, we identify temporal phosphorylation sites on cell cycle-specific factors and protein complex assembly intermediates. Our results thus allow tying PTMs to the age of the modified proteins.

Atypical E3 ligase ZFP91 promotes small-molecule-induced E2F2 transcription factor degradation for cancer therapy.

The E2F family of transcription factors play a crucial role in the development of various cancers. However, E2F members lack targetable binding pockets and are typically considered "undruggable". Unlike canonical small-molecule therapeutics, molecular glues mediate new E3 ligase-protein interactions to induce selective proteasomal degradation, which represents an attractive option to overcome these limitations. Human proteome microarray was utilized to identify a natural product-derived molecular glue for targeting E2F2 degradation. Co-IP analysis with stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics was carried out to further explore the E3 ligase for E2F2 degradation. In this study, we identified a molecular glue bufalin, which significantly promoted E2F2 degradation. Unexpectedly, E2F2 underwent ubiquitination and proteasomal degradation via a previously undisclosed atypical E3 ligase, zinc finger protein 91 (ZFP91). In particular, we observed that bufalin markedly promoted E2F2-ZFP91 complex formation, thereby leading to E2F2 polyubiquitination via K48-linked ubiquitin chains for degradation. E2F2 degradation subsequently caused transcriptional suppression of multiple oncogenes including c-Myc, CCNE1, CCNE2, MCM5 and CDK1, and inhibited hepatocellular carcinoma growth invitro and invivo. Collectively, our findings open up a new direction for transcription factors degradation by targeting atypical E3 ligase ZFP91. Meanwhile, the chemical knockdown strategy with molecular glue may promote innovative transcription factor degrader development in cancer therapy. This work was financially supported by the NationalKeyResearchandDevelopment Projectof China (2022YFC3501601), National Natural Sciences Foundation of China (81973505, 82174008, 82030114), and China Postdoctoral Science Foundation (2019M650396), the Fundamental Research Funds for the Central Universities.

Fluorescently Labeling Amino Acids in a Deep Eutectic Solvent.

The increased use of deep eutectic solvents (DESs) in recent years has been significant and provides new approaches to sample collection and preparation. At the same time, the use of these new solvents to prepare samples can present challenges for subsequent analyses. Common analytical approaches, such as fluorescent labeling, may not be compatible with the solvents. In this work, we explore how effective three traditional fluorescent labels can be at derivatizing amino acids in the most common DESs, formed from choline chloride and ethylene glycol. We demonstrate that the unique solvent characteristics of the DESs still allow for two of the fluorophores, fluorescein isothiocyanate and 5-carboxyfluorescein succinimidyl ester, to effectively label amino acids. Initial optimizations of the reaction conditions demonstrate that we can effectively label both d- and l-amino acids, in solution with concentrations of amino acids down to 4 μM. Capillary electrophoretic separations following this preparation can detect as little as 50 nM. This is possible without removal of any DES from the sample matrix. These results represent the first complete fluorescent labeling reaction in a DES and subsequent capillary electrophoretic separation of the analytes.

Using SILAC to Develop Quantitative Data-Independent Acquisition (DIA) Proteomic Methods.

Proteins are integral to biological systems and functions. Identifying and quantifying proteins can therefore offer systems-wide insights into protein-protein interactions, cellular signaling, and biological pathway activity. The use of quantitative proteomics has become a method of choice for identifying and quantifying proteins in complex matrices. Proteomics allows researchers to survey hundreds to thousands of proteins in a less biased manner than classical antibody-based protein capture strategies. Typically,discovery approacheshave used data-dependent acquisition (DDA) methods, but this approach suffers from stochasticity that compromises quantitation. Recent developments in data-independent acquisition (DIA) proteomics workflows enable proteomic profiling of thousands of samples with increased peak picking consistency making it an excellent candidate for discovering and assessing biomarkers in clinical samples. However, quantitation of peptides from DIA datasets is computationally intensive, and guidelines on how to establish DIA methods are lacking. Method development and optimization require novel tools to visualize and filter DIA datasets appropriately. Here, a protocol and novel script workflow for the optimization of quantitative DIA methods using stable isotope labeling of amino acids in culture (SILAC) are presented. This protocol includes steps for cell growth and labeling, peptide digestion and preparation, and optimization of quantitative DIA methods. In addition, important steps for (1) computational analysis to identify and quantify peptides, (2) data visualizations to identify the linear abundance ranges for all peptides in the sample, and (3) descriptions of how to find high confidence quantitation abundance thresholds are described herein.

LOXL2-dependent deacetylation of aldolase A induces metabolic reprogramming and tumor progression

Lysyl-oxidase like-2 (LOXL2) regulates extracellular matrix remodeling and promotes tumor invasion and metastasis. Altered metabolism is a core hallmark of cancer, however, it remains unclear whether and how LOXL2 contributes to tumor metabolism. Here, we found that LOXL2 and its catalytically inactive L2Δ13 splice variant boost glucose metabolism of esophageal tumor cells, facilitate tumor cell proliferation and promote tumor development in vivo. Consistently, integrated transcriptomic and metabolomic analysis of a knock-in mouse model expressing L2Δ13 gene revealed that LOXL2/L2Δ13 overexpression perturbs glucose and lipid metabolism. Mechanistically, we identified aldolase A, glyceraldehyde-3-phosphate dehydrogenase and enolase as glycolytic proteins that interact physically with LOXL2 and L2Δ13. In the case of aldolase A, LOXL2/L2Δ13 stimulated its mobilization from the actin cytoskeleton to enhance aldolase activity during malignant transformation. Using stable isotope labeling of amino acids in cell culture (SILAC) followed by proteomic analysis, we identified LOXL2 and L2Δ13 as novel deacetylases that trigger metabolic reprogramming. Both LOXL2 and L2Δ13 directly catalyzed the deacetylation of aldolase A at K13, resulting in enhanced glycolysis which subsequently reprogramed tumor metabolism and promoted tumor progression. High level expression of LOXL2/L2Δ13 combined with decreased acetylation of aldolase-K13 predicted poor clinical outcome in patients with esophageal cancer. In summary, we have characterized a novel molecular mechanism that mediates the pro-tumorigenic activity of LOXL2 independently of its classical amine oxidase activity. These findings may enable the future development of therapeutic agents targeting the metabolic machinery via LOXL2 or L2Δ13. Highlight of the studyLOXL2 and its catalytically inactive isoform L2Δ13 function as new deacetylases to promote metabolic reprogramming and tumor progression in esophageal cancer by directly activating glycolytic enzymes such as aldolase A.

Characterization of RARRES1 Expression on Circulating Tumor Cells as Unfavorable Prognostic Marker in Resected Pancreatic Ductal Adenocarcinoma Patients.

Simple SummaryOur explorative study used a microfluidic-based approach for circulating tumor cell (CTC) detection in 55 pancreatic ductal adenocarcinoma (PDAC) patients before treatment initiation (baseline) and during follow-up (FUP). For the first time, we assessed the expression of retinoic acid receptor responder 1 (RARRES1) on CTCs. CTCs were detected in 25.5% of patients at baseline, while the detection rate during FUP was higher (45.5%). Especially high CTC counts during FUP in resected patients were associated with early tumor relapse (p = 0.02). Combining CTC detection and RARRES1 protein expression showed that RARRES1-positive patients with high CTCs counts after curative operation during FUP had a worse prognosis (p = 0.001). In conclusion, RARRES1 is a new marker of interest for further research investigations on subtypes of CTCs in PDAC.Background: In pancreatic ductal adenocarcinoma (PDAC), the characterization of circulating tumor cells (CTCs) opens new insights into cancer metastasis as the leading cause of cancer-related death. Here, we focused on the expression of retinoic acid receptor responder 1 (RARRES1) on CTCs as a novel marker for treatment failure and early relapse. Methods: The stable isotope labeling of amino acids in cell culture (SILAC)—approach was applied for identifying and quantifying new biomarker proteins in PDAC cell lines HPDE and its chemoresistant counterpart, L3.6pl-Res. Fifty-five baseline and 36 follow-up (FUP) peripheral blood samples were processed via a marker-independent microfluidic-based CTC detection approach using RARRES1 as an additional marker. Results: SILAC-based proteomics identified RARRES1 as an abundantly expressed protein in more aggressive chemoresistant PDAC cells. At baseline, CTCs were detected in 25.5% of all PDAC patients, while FUP analysis (median: 11 months FUP) showed CTC detection in 45.5% of the resected patients. CTC positivity (≥3 CTC) at FUP was significantly associated with short recurrence-free survival (p = 0.002). Furthermore, detection of RARRES1 positive CTCs was indicative of an even earlier relapse after surgery (p = 0.001). Conclusions: CTC detection in resected PDAC patients during FUP is associated with a worse prognosis, and RARRES1 expression might identify an aggressive subtype of CTCs that deserves further investigation.

Amino acid oxidation methods to determine amino acid requirements: do we require lengthy adaptation periods?

Determination of indispensable amino acid (IAA) requirements necessitates a range of intakes of the test IAA and monitoring of the physiological response. Short-term methods are the most feasible for studying multiple intake levels in the same individual. Carbon oxidation methods measure the excretion of 13CO2 in breath from a labelled amino acid (AA) in response to varying intakes of the test AA following a period of adaptation. However, the length of adaptation to each AA intake level has been a source of debate and disagreement among researchers. The assertion of the minimally invasive indicator amino acid oxidation (IAAO) technique is that IAA requirements can be estimated after only a few hours (8 h) of adaptation to each test AA intake, suggesting that adaptation occurs rapidly in response to dietary adjustments. On the contrary, the assertion of most other techniques is that 6-7 d of adaptation is required when determining IAA needs. It has even been argued that a minimum of two weeks is needed to achieve complete adaptation. This review explores evidence regarding AA oxidation methods and whether long periods of adaptation to test IAA levels are necessary when estimating IAA requirements. It was found that the consumption of experimental diets containing lower test IAA intake for greater than 7 d violates the terms of a successful adaptive response. While there is some evidence that short-term 8 h IAAO is not different among different test amino acid intakes up to 7 d, it is unclear whether it impacts assessment of IAA requirements.

Automated Specific Amino Acid Footprinting Mass Spectrometry: Repurposing an HDX Platform for Determining Reagent Feasibility.

Protein footprinting is a mass spectrometry (MS)-based approach to measure protein conformational changes. One approach, specific amino acid labeling, imparts often an irreversible modification to protein side chains but requires careful selection of the reactive reagent and often time-consuming optimization of experimental parameters prior to submission to bottom-up MS analysis. In this work, we repurpose a hydrogen-deuterium exchange MS (HDX-MS) LEAP HDX system for automated specific amino acid footprinting MS, demonstrating its efficacy in reaction optimization and monitoring applicability to specific ligand binding systems. We screened reagent conditions for two model ligand-binding systems and demonstrate the method's efficacy for measuring differences induced by ligand binding. Our proof-of-concept experiments provide a platform for rapidly screening specific amino acid reagents and reaction conditions for protein systems to be studied by footprinting.

Dendritic cell-mediated cross presentation of tumor-derived peptides is biased against plasma membrane proteins

BackgroundFor effective tumor elimination, cytotoxic CD8+ T cells must recognize tumor-derived antigens presented on class I major histocompatibility complex (MHC-I). Despite a general association between the expression of immunogenic antigens,...