ABSTRACT
Cannabinoid receptor 1 (CB1) is the principal target of Δ9-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 Å crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals.
Subject(s)
Cannabinoid Receptor Antagonists/chemistry , Morpholines/chemistry , Pyrazoles/chemistry , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptor, Cannabinoid, CB1/chemistry , Binding Sites , Cannabinoids/pharmacology , Cannabis/chemistry , Crystallography, X-Ray , Dronabinol/pharmacology , Endocannabinoids/pharmacology , Humans , Ligands , Morpholines/chemical synthesis , Protein Binding , Protein Conformation, alpha-Helical , Pyrazoles/chemical synthesisABSTRACT
Adhesion G protein-coupled receptors (aGPCRs) are essential for a variety of physiological processes such as immune responses, organ development, cellular communication, proliferation and homeostasis1-7. An intrinsic manner of activation that involves a tethered agonist in the N-terminal region of the receptor has been proposed for the aGPCRs8,9, but its molecular mechanism remains elusive. Here we report the G protein-bound structures of ADGRD1 and ADGRF1, which exhibit many unique features with regard to the tethered agonism. The stalk region that proceeds the first transmembrane helix acts as the tethered agonist by forming extensive interactions with the transmembrane domain; these interactions are mostly conserved in ADGRD1 and ADGRF1, suggesting that a common stalk-transmembrane domain interaction pattern is shared by members of the aGPCR family. A similar stalk binding mode is observed in the structure of autoproteolysis-deficient ADGRF1, supporting a cleavage-independent manner of receptor activation. The stalk-induced activation is facilitated by a cascade of inter-helix interaction cores that are conserved in positions but show sequence variability in these two aGPCRs. Furthermore, the intracellular region of ADGRF1 contains a specific lipid-binding site, which proves to be functionally important and may serve as the recognition site for the previously discovered endogenous ADGRF1 ligand synaptamide. These findings highlight the diversity and complexity of the signal transduction mechanisms of the aGPCRs.
Subject(s)
Receptors, G-Protein-Coupled , Signal Transduction , Humans , Ligands , Oncogene Proteins/agonists , Oncogene Proteins/metabolism , Protein Binding , Protein Domains , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/metabolismABSTRACT
Data-independent acquisition (DIA) mass spectrometry (MS) has emerged as a powerful technology for high-throughput, accurate, and reproducible quantitative proteomics. This review provides a comprehensive overview of recent advances in both the experimental and computational methods for DIA proteomics, from data acquisition schemes to analysis strategies and software tools. DIA acquisition schemes are categorized based on the design of precursor isolation windows, highlighting wide-window, overlapping-window, narrow-window, scanning quadrupole-based, and parallel accumulation-serial fragmentation-enhanced DIA methods. For DIA data analysis, major strategies are classified into spectrum reconstruction, sequence-based search, library-based search, de novo sequencing, and sequencing-independent approaches. A wide array of software tools implementing these strategies are reviewed, with details on their overall workflows and scoring approaches at different steps. The generation and optimization of spectral libraries, which are critical resources for DIA analysis, are also discussed. Publicly available benchmark datasets covering global proteomics and phosphoproteomics are summarized to facilitate performance evaluation of various software tools and analysis workflows. Continued advances and synergistic developments of versatile components in DIA workflows are expected to further enhance the power of DIA-based proteomics.
Subject(s)
Proteomics , Software , Proteomics/methods , Mass Spectrometry/methods , Gene Library , Proteome/analysisABSTRACT
Transmembrane (TM) proteins constitute over 30% of the mammalian proteome and play essential roles in mediating cell-cell communication, synaptic transmission, and plasticity in the central nervous system. Many of these proteins, especially the G protein-coupled receptors (GPCRs), are validated or candidate drug targets for therapeutic development for mental diseases, yet their expression profiles are underrepresented in most global proteomic studies. Herein, we establish a brain TM protein-enriched spectral library based on 136 data-dependent acquisition runs acquired from various brain regions of both naïve mice and mental disease models. This spectral library comprises 3043 TM proteins including 171 GPCRs, 231 ion channels, and 598 transporters. Leveraging this library, we analyzed the data-independent acquisition data from different brain regions of two mouse models exhibiting depression- or anxiety-like behaviors. By integrating multiple informatics workflows and library sources, our study significantly expanded the mental stress-perturbed TM proteome landscape, from which a new GPCR regulator of depression was verified by in vivo pharmacological testing. In summary, we provide a high-quality mouse brain TM protein spectral library to largely increase the TM proteome coverage in specific brain regions, which would catalyze the discovery of new potential drug targets for the treatment of mental disorders.
Subject(s)
Brain , Disease Models, Animal , Mental Disorders , Mice, Inbred C57BL , Proteome , Proteomics , Animals , Proteome/metabolism , Brain/metabolism , Proteomics/methods , Mice , Mental Disorders/metabolism , Membrane Proteins/metabolism , Male , Receptors, G-Protein-Coupled/metabolismABSTRACT
The recently developed double-click reaction sequence [G. Meng et al., Nature 574, 86-89 (2019)] is expected to vastly expand the number and diversity of synthetically accessible 1,2,3-triazole derivatives. However, it remains elusive how to rapidly navigate the extensive chemical space created by double-click chemistry for bioactive compound discovery. In this study, we selected a particularly challenging drug target, the glucagon-like-peptide-1 receptor (GLP-1R), to benchmark our new platform for the design, synthesis, and screening of double-click triazole libraries. First, we achieved a streamlined synthesis of customized triazole libraries on an unprecedented scale (composed of 38,400 new compounds). By interfacing affinity-selection mass spectrometry and functional assays, we identified a series of positive allosteric modulators (PAMs) with unreported scaffolds that can selectively and robustly enhance the signaling activity of the endogenous GLP-1(9-36) peptide. Intriguingly, we further revealed an unexpected binding mode of new PAMs which likely act as a molecular glue between the receptor and the peptide agonist. We anticipate the merger of double-click library synthesis with the hybrid screening platform allows for efficient and economic discovery of drug candidates or chemical probes for various therapeutic targets.
Subject(s)
Glucagon-Like Peptide-1 Receptor , Peptides , Allosteric Regulation , Glucagon-Like Peptide 1/metabolism , Glucagon-Like Peptide-1 Receptor/agonists , Peptides/chemistry , Triazoles/chemistryABSTRACT
Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kα. The application of chemical cross-linking mass spectrometry (CXMS) facilitated the identification of the p85 domains BH, cSH2, and SH3 as well as their docking positions on the PI3Kα catalytic core. Binding of individual nanobodies to PI3Kα induced activation or inhibition of enzyme activity and caused conformational changes that could be correlated with enzyme function. Binding of nanobody Nb3-126 to the BH domain of p85α substantially improved resolution for parts of the PI3Kα complex, and binding of nanobody Nb3-159 induced a conformation of PI3Kα that is distinct from known PI3Kα structures. The analysis of CXMS data also provided mechanistic insights into the molecular underpinning of the flexibility of PI3Kα.
Subject(s)
Catalytic Domain , Class I Phosphatidylinositol 3-Kinases , Class Ia Phosphatidylinositol 3-Kinase , Class I Phosphatidylinositol 3-Kinases/chemistry , Class Ia Phosphatidylinositol 3-Kinase/chemistry , Humans , Mass Spectrometry/methods , Single-Domain AntibodiesABSTRACT
G protein-coupled receptor (GPCR) structural studies with in-solution spectroscopic approaches have offered distinctive insights into GPCR activation and signaling that highly complement those yielded from structural snapshots by crystallography or cryo-EM. While most current spectroscopic approaches allow for probing structural changes at selected residues or loop regions, they are not suitable for capturing a holistic view of GPCR conformational rearrangements across multiple domains. Herein, we develop an approach based on limited proteolysis mass spectrometry (LiP-MS) to simultaneously monitor conformational alterations of a large number of residues spanning both flexible loops and structured transmembrane domains for a given GPCR. To benchmark LiP-MS for GPCR conformational profiling, we studied the adenosine 2A receptor (A2AR) in response to different ligand binding (agonist/antagonist/allosteric modulators) and G protein coupling. Systematic and residue-resolved profiling of A2AR conformational rearrangements by LiP-MS precisely captures structural mechanisms in multiple domains underlying ligand engagement, receptor activation, and allostery, and may also reflect local conformational flexibility. Furthermore, these residue-resolution structural fingerprints of the A2AR protein allow us to readily classify ligands of different pharmacology and distinguish the G protein-coupled state. Thus, our study provides a new structural MS approach that would be generalizable to characterizing conformational transition and plasticity for challenging integral membrane proteins.
Subject(s)
Mass Spectrometry , Protein Conformation , Receptor, Adenosine A2A , Receptor, Adenosine A2A/chemistry , Receptor, Adenosine A2A/metabolism , Humans , Ligands , Models, Molecular , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolismABSTRACT
Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms1,2. As central mediators in this complex signalling pathway, FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research. Here we present an atomic-resolution structure of the human Frizzled 4 receptor (FZD4) transmembrane domain in the absence of a bound ligand. The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed, and is distinct from all other GPCR structures reported so far. Within this unique transmembrane fold is an extremely narrow and highly hydrophilic pocket that is not amenable to the binding of traditional GPCR ligands. We show that such a pocket is conserved across all FZDs, which may explain the long-standing difficulties in the development of ligands for these receptors. Molecular dynamics simulations on the microsecond timescale and mutational analysis uncovered two coupled, dynamic kinks located at helix VII that are involved in FZD4 activation. The stability of the structure in its ligand-free form, an unfavourable pocket for ligand binding and the two unusual kinks on helix VII suggest that FZDs may have evolved a novel ligand-recognition and activation mechanism that is distinct from that of other GPCRs.
Subject(s)
Frizzled Receptors/chemistry , Binding Sites , Crystallography, X-Ray , Cysteine/metabolism , Dishevelled Proteins/metabolism , Frizzled Receptors/genetics , Humans , Ligands , Models, Molecular , Molecular Dynamics Simulation , Protein Domains , Wnt Signaling PathwayABSTRACT
The concept of subtype selectivity and functional bias has recently reshaped current GPCR drug discovery for G protein-coupled receptors. A series of new N-H aporphines with A-ring modifications have been synthesized, and their efficacy on 5-HT2 receptor activation was evaluated. SAR analysis led to the discovery of several more potent and selective 5-HT2C receptor agonists (e.g., 11b and 11f) with low nanomolar activity. Molecular docking analysis of this series of aporphines was in accordance with our SAR results. The functional selectivity of specific compounds was tested via both Gq-mediated calcium flux and ß-arrestin recruitment assays, which revealed that these compounds exhibited no ß-arrestin recruitment activity. Further ADMET study combined with behavioral assessment using a methamphetamine-induced hyperactivity model identified compound 11b and 11f possessing promising drug-like profiles and having antipsychotic potential. These agonists with an exclusive bias toward Gq signaling may serve as valuable pharmacological probes to facilitate the elucidation of therapeutically relevant 5-HT2C signaling pathways and the development of alternative antipsychotic medications.
Subject(s)
Antipsychotic Agents , Aporphines , Antipsychotic Agents/chemistry , Antipsychotic Agents/pharmacology , Aporphines/pharmacology , Molecular Docking Simulation , Receptor, Serotonin, 5-HT2C , SerotoninABSTRACT
Misoprostol is a life-saving drug in many developing countries for women at risk of post-partum hemorrhaging owing to its affordability, stability, ease of administration and clinical efficacy. However, misoprostol lacks receptor and tissue selectivities, and thus its use is accompanied by a number of serious side effects. The development of pharmacological agents combining the advantages of misoprostol with improved selectivity is hindered by the absence of atomic details of misoprostol action in labor induction. Here, we present the 2.5 Å resolution crystal structure of misoprostol free-acid form bound to the myometrium labor-inducing prostaglandin E2 receptor 3 (EP3). The active state structure reveals a completely enclosed binding pocket containing a structured water molecule that coordinates misoprostol's ring structure. Modeling of selective agonists in the EP3 structure reveals rationales for selectivity. These findings will provide the basis for the next generation of uterotonic drugs that will be suitable for administration in low resource settings.
Subject(s)
Misoprostol/chemistry , Receptors, Prostaglandin E, EP3 Subtype/chemistry , Receptors, Prostaglandin E, EP3 Subtype/metabolism , Binding Sites , Crystallography, X-Ray , Dinoprostone/analogs & derivatives , Dinoprostone/chemistry , Dinoprostone/metabolism , Humans , Misoprostol/metabolism , Molecular Docking Simulation , Mutagenesis, Site-Directed , Protein Conformation , Receptors, Prostaglandin E, EP3 Subtype/agonists , Receptors, Prostaglandin E, EP3 Subtype/genetics , Signal Transduction , Water/chemistryABSTRACT
In the version of this article originally published, the present address for Petr Popov was incorrectly listed as 'Koltech Institute of Science & Technology, Moscow, Russia'. The correct present address is 'Skolkovo Institute of Science and Technology, Moscow, Russia'. The error has been corrected in the HTML and PDF versions of the paper.
ABSTRACT
Affinity mass spectrometry (MS) enables rapid screening of compound mixtures for ligands bound to a specific protein target, yet its current throughput is limited to individually assay pools of 400-2000 compounds. Typical affinity MS screens implemented in pharmaceutical industry laboratories identify putative ligands based on qualitative analysis of compound binding to the target whereas no quantitative information is acquired to discriminate high- and low-affinity ligands in the screening phase. Furthermore, these screens require purification of a stabilized form of the protein target, which poses a great challenge for membrane receptor targets. Here, we describe a new, potentially general affinity MS strategy that allows screening of 20,000 compounds in one pool for highly efficient ligand discovery toward a G protein-coupled receptor (GPCR) target. Quantitative measurement of compound binding to the receptor enables high-affinity ligand selection using both the purified receptor and receptor-embedded cell membranes. This high-throughput, label-free and quantitative affinity MS screen resulted in discovery of three new antagonists of the A2A adenosine receptor.
Subject(s)
High-Throughput Screening Assays/methods , Mass Spectrometry/methods , Receptors, G-Protein-Coupled/metabolism , Adenosine A2 Receptor Antagonists/analysis , Cell Membrane/metabolism , Humans , Ligands , Protein Binding , Receptors, G-Protein-Coupled/antagonists & inhibitorsABSTRACT
Although natural herbs have been a rich source of compounds for drug discovery, identification of bioactive components from natural herbs suffers from low efficiency and prohibitive cost of the conventional bioassay-based screening platforms. Here we develop a new strategy that integrates virtual screening, affinity mass spectrometry (MS) and targeted metabolomics for efficient discovery of herb-derived ligands towards a specific protein target site. Herb-based virtual screening conveniently selects herbs of potential bioactivity whereas affinity MS combined with targeted metabolomics readily screens candidate compounds in a high-throughput manner. This new integrated approach was benchmarked on screening chemical ligands that target the hydrophobic pocket of the nucleoprotein (NP) of Ebola viruses for which no small molecule ligands have been reported. Seven compounds identified by this approach from the crude extracts of three natural herbs were all validated to bind to the NP target in pure ligand binding assays. Among them, three compounds isolated from Piper nigrum (HJ-1, HJ-4 and HJ-6) strongly promoted the formation of large NP oligomers and reduced the protein thermal stability. In addition, cooperative binding between these chemical ligands and an endogenous peptide ligand was observed, and molecular docking was employed to propose a possible mechanism. Taken together, we established a platform integrating in silico and experimental screening approaches for efficient discovery of herb-derived bioactive ligands especially towards non-enzyme protein targets.
Subject(s)
Biological Products/metabolism , Mass Spectrometry/methods , Metabolomics/methods , Nucleoproteins/metabolism , Plant Extracts/metabolism , Viral Core Proteins/metabolism , Binding Sites , Biological Products/chemistry , Biological Products/isolation & purification , Drug Discovery/methods , Ebolavirus/chemistry , Ligands , Molecular Docking Simulation , Nucleocapsid Proteins , Nucleoproteins/chemistry , Ophiopogon/chemistry , Piper nigrum/chemistry , Plant Components, Aerial/chemistry , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Protein Binding , Salvia miltiorrhiza/chemistry , Seeds/chemistry , Viral Core Proteins/chemistryABSTRACT
Posttranslational modifications of certain stress granule (SG) proteins are closely related to the assembly of SGs, a type of cytoplasmic foci structure. Our previous studies revealed that the Tudor staphylococcal nuclease (Tudor-SN) protein participates in the formation of SGs. However, the functional significance of potential Tudor-SN modifications during stress has not been reported. In this study, we demonstrated that the Tudor-SN protein was phosphorylated at threonine 103 (T103) upon stimulation with arsenite. In addition, c-Jun N-terminal kinase (JNK) was found to be responsible for Tudor-SN phosphorylation at the T103 site. We further illustrated that either a T103A mutation or the suppression of phosphorylation of T103 by the JNK inhibitor SP600125 inhibited the efficient recruitment of Tudor-SN into SGs. In addition, the T103A mutation could affect the physical binding of Tudor-SN with the G3BP (Ras-GAP SH3 domain-binding protein) protein but not with the HuR (Hu antigen R) protein and AGTR1-3'UTR (3'-untranslated region of angiotensin II receptor, type 1) mRNA cargo. These data suggested that JNK-enhanced Tudor-SN phosphorylation promotes the interaction between Tudor-SN and G3BP and facilitates the efficient recruitment of Tudor-SN into SGs under conditions of sodium arsenite-induced oxidative stress. This finding provides novel insights into the physiological function of Tudor-SN modification.
Subject(s)
Carrier Proteins/genetics , Cytoplasmic Granules/metabolism , JNK Mitogen-Activated Protein Kinases/genetics , Nuclear Proteins/genetics , Protein Processing, Post-Translational , Anthracenes/pharmacology , Arsenites/pharmacology , Carrier Proteins/metabolism , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/ultrastructure , DNA Helicases , ELAV-Like Protein 1/genetics , ELAV-Like Protein 1/metabolism , Endonucleases , HeLa Cells , Humans , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/metabolism , Mutation , Nuclear Proteins/metabolism , Oxidative Stress , Phosphorylation/drug effects , Poly-ADP-Ribose Binding Proteins , Protein Binding , Protein Kinase Inhibitors/pharmacology , RNA Helicases , RNA Recognition Motif Proteins , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, Angiotensin, Type 1/genetics , Receptor, Angiotensin, Type 1/metabolism , Sodium Compounds/pharmacology , Threonine/metabolismABSTRACT
To gain a deep understanding of yeast-cell response to heat stress, multiple laboratory strains have been intensively studied via genome-wide expression analysis for the mechanistic dissection of classical heat-shock response (HSR). However, robust industrial strains of Saccharomyces cerevisiae have hardly been explored in global analysis for elucidation of the mechanism of thermotolerant response (TR) during fermentation. Herein, we employed data-independent acquisition and sequential window acquisition of all theoretical mass spectra based proteomic workflows to characterize proteome remodeling of an industrial strain, ScY01, responding to prolonged thermal stress or transient heat shock. By comparing the proteomic signatures of ScY01 in TR versus HSR as well as the HSR of the industrial strain versus a laboratory strain, our study revealed disparate response mechanisms of ScY01 during thermotolerant growth or under heat shock. In addition, through proteomics data-mining for decoding transcription factor interaction networks followed by validation experiments, we uncovered the functions of two novel transcription factors, Mig1 and Srb2, in enhancing the thermotolerance of the industrial strain. This study has demonstrated that accurate and high-throughput quantitative proteomics not only provides new insights into the molecular basis for complex microbial phenotypes but also pinpoints upstream regulators that can be targeted for improving the desired traits of industrial microorganisms.
Subject(s)
Gene Regulatory Networks , Heat-Shock Response , Proteome/analysis , Saccharomyces cerevisiae/physiology , Thermotolerance/genetics , Fermentation , Mediator Complex/physiology , Repressor Proteins/physiology , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae Proteins/physiology , Species Specificity , Time Factors , Transcription FactorsABSTRACT
Proteomic analysis with data-independent acquisition (DIA) approaches represented by the sequential window acquisition of all theoretical fragment ion spectra (SWATH) technique has gained intense interest in recent years because DIA is able to overcome the intrinsic weakness of conventional data-dependent acquisition (DDA) methods and afford higher throughout and reproducibility for proteome-wide quantification. Although the raw mass spectrometry (MS) data quality and the data-mining workflow conceivably influence the throughput, accuracy and consistency of SWATH-based proteomic quantification, there lacks a systematic evaluation and optimization of the acquisition and data-processing parameters for SWATH MS analysis. Herein, we evaluated the impact of major acquisition parameters such as the precursor mass range, isolation window width and accumulation time as well as the data-processing variables including peak extraction criteria and spectra library selection on SWATH performance. Fine tuning these interdependent parameters can further improve the throughput and accuracy of SWATH quantification compared to the original setting adopted in most SWATH proteomic studies. Furthermore, we compared the effectiveness of two widely used peak extraction software PeakView and Spectronaut in discovery of differentially expressed proteins in a biological context. Our work is believed to contribute to a deeper understanding of the critical factors in SWATH MS experiments and help researchers optimize their SWATH parameters and workflows depending on the sample type, available instrument and software.
Subject(s)
Protein Processing, Post-Translational/genetics , Proteome/genetics , Proteomics/statistics & numerical data , Software , Chromatography, Liquid/statistics & numerical data , Data Mining , Tandem Mass Spectrometry/methodsABSTRACT
Quantification of targeted metabolites, especially trace metabolites and structural isomers, in complex biological materials is an ongoing challenge for metabolomics. Initially developed for proteomic analysis, the parallel reaction monitoring (PRM) technique exploiting high-resolution MS2 fragment ion data has shown high promise for targeted metabolite quantification. Notably, MS1 ion intensity data acquired independently as part of each PRM scan cycle are often underutilized in the PRM assay. In this study, we developed an MS1/MS2-combined PRM workflow for quantification of central carbon metabolism intermediates, amino acids and shikimate pathway-related metabolites on an orthogonal QqTOF system. Concentration curve assessment revealed that exploiting both MS1 and MS2 scans in PRM analysis afforded higher sensitivity, wider dynamic range and better reproducibility than relying on either scan mode for quantification. Furthermore, Skyline was incorporated into our workflow to process the MS1/MS2 ion intensity data, and eliminate noisy signals and transitions with interferences. This integrated MS1/MS2 PRM approach was applied to targeted metabolite quantification in engineered E. coli strains for understanding of metabolic pathway modulation. In addition, this new approach, when first implemented in a dynamic 13C-labeling experiment, showed its unique advantage in capturing and correcting isotopomer labeling curves to facilitate nonstationary 13C-labeling metabolism analysis.
Subject(s)
Escherichia coli/metabolism , Metabolomics , Carbon Isotopes , Escherichia coli/cytology , Mass SpectrometryABSTRACT
Saccharomyces cerevisiae has been intensively studied in responses to different environmental stresses such as heat shock through global omic analysis. However, the S. cerevisiae industrial strains with superior thermotolerance have not been explored in any proteomic studies for elucidating the tolerance mechanism. Recently a new diploid strain was obtained through evolutionary engineering of a parental industrial strain, and it exhibited even higher resistance to prolonged thermal stress. Herein, we performed iTRAQ-based quantitative proteomic analysis on both the parental and evolved industrial strains to further understand the mechanism of thermotolerant adaptation. Out of â¼ 2600 quantifiable proteins from biological quadruplicates, 193 and 204 proteins were differentially regulated in the parental and evolved strains respectively during heat-stressed growth. The proteomic response of the industrial strains cultivated under prolonged thermal stress turned out to be substantially different from that of the laboratory strain exposed to sudden heat shock. Further analysis of transcription factors underlying the proteomic perturbation also indicated the distinct regulatory mechanism of thermotolerance. Finally, a cochaperone Mdj1 and a metabolic enzyme Adh1 were selected to investigate their roles in mediating heat-stressed growth and ethanol production of yeasts. Our proteomic characterization of the industrial strain led to comprehensive understanding of the molecular basis of thermotolerance, which would facilitate future improvement in the industrially important trait of S. cerevisiae by rational engineering.
Subject(s)
Adaptation, Physiological , Heat-Shock Response , Industrial Microbiology , Proteomics/methods , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Temperature , Carbon/metabolism , Ethanol/metabolism , Saccharomyces cerevisiae/growth & development , Transcription Factors/metabolismABSTRACT
Detection of proteins containing single amino acid polymorphisms (SAPs) encoded by nonsynonymous SNPs (nsSNPs) can aid researchers in studying the functional significance of protein variants. Most proteogenomic approaches for large-scale SAPs mapping require construction of a sample-specific database containing protein variants predicted from the next-generation sequencing (NGS) data. Searching shotgun proteomic data sets against these NGS-derived databases allowed for identification of SAP peptides, thus validating the proteome-level sequence variation. Contrary to the conventional approaches, our study presents a novel strategy for proteome-wide SAP detection without relying on sample-specific NGS data. By searching a deep-coverage proteomic data set from an industrial thermotolerant yeast strain using our strategy, we identified 337 putative SAPs compared to the reference genome. Among the SAP peptides identified with stringent criteria, 85.2% of SAP sites were validated using whole-genome sequencing data obtained for this organism, which indicates high accuracy of SAP identification with our strategy. More interestingly, for certain SAP peptides that cannot be predicted by genomic sequencing, we used synthetic peptide standards to verify expression of peptide variants in the proteome. Our study has provided a unique tool for proteogenomics to enable proteome-wide direct SAP identification and capture nongenetic protein variants not linked to nsSNPs.
Subject(s)
Amino Acids/chemistry , Proteome , Tandem Mass Spectrometry/methodsABSTRACT
Tubulin is known to undergo unique post-translational modifications (PTMs), such as detyrosination and polyglutamylation, particularly in the unstructured carboxy-terminal tails (CTTs). However, more conventional PTMs of tubulin and their roles in the regulation of microtubule properties and functions remain poorly defined. Here, we report the comprehensive profiling of tubulin phosphorylation, acetylation, ubiquitylation, and O-GlcNAcylation in HeLa cells with a proteomic approach. Our tubulin-targeted analysis has identified 80 residues bearing single or multiple conventional PTMs including 24 novel PTM sites not covered in previous global proteomic surveys. By using a series of PTM-deficient or PTM-mimicking mutants, we further find that tubulin phosphorylation and acetylation play important roles in the control of microtubule assembly and stability. In addition, these tubulin PTMs have distinct effects on the retrograde transport of adenoviruses along microtubules. These findings thus enlarge the repertoire of tubulin PTMs and foster our understanding of their versatile roles in the regulation of microtubule dynamics and cellular functions.