Preventing Inappropriate Signals Pre- and Post-Ligand Perception by a Toggle-Switch Mechanism of ERECTA.

Dynamic control of signaling events requires swift regulation of receptors at an active state. By focusing on Arabidopsis ERECTA (ER) receptor kinase, which perceives peptide ligands to control multiple developmental processes, we report a mechanism preventing inappropriate receptor activity. The ER C-terminal tail (ER_CT) functions as an autoinhibitory domain: its removal confers higher kinase activity and hyperactivity during inflorescence and stomatal development. ER_CT is required for the binding of a receptor kinase inhibitor, BKI1, and two U-box E3 ligases PUB30 and PUB31 that inactivate activated ER. We further identify ER_CT as a phosphodomain trans-phosphorylated by the co-receptor BAK1. The phosphorylation impacts the tail structure, likely releasing from autoinhibition. The phosphonull version enhances BKI1 association, whereas the phosphomimetic version promotes PUB30/31 association. Thus, ER_CT acts as an off-on-off toggle switch, facilitating the release of BKI1 inhibition, enabling signal activation, and swiftly turning over the receptors afterwards. Our results elucidate a mechanism fine-tuning receptor signaling via a phosphoswitch module, keeping the receptor at a low basal state and ensuring the robust yet transient activation upon ligand perception. Significance: Cells perceive and process external signals through their cell-surface receptors, whose activity must be tightly maintained to prevent the spread of misinformation. How do plant cells prevent the inappropriate receptor activity? We identify a structural module within the C-terminal tail of the ERECTA (ER_CT), that inhibits the receptor pre- and post-signal activation. The ER_CT comprises of a linker and an α-helix. Before activation, ER_CT is autoinhibitory and associates with an inhibitory protein. Ligand perception triggers the transphosphorylation of ER_CT by the co-receptor, which then recruits a degradation machinery to swiftly turn over the activated receptor. Thus, we reveal an off-on-off toggle switch mechanism that finely adjusts the activity of the plant receptor, enabling the precise control over cell signaling.

The fitness cost of spurious phosphorylation.

The fidelity of signal transduction requires the binding of regulatory molecules to their cognate targets. However, the crowded cell interior risks off-target interactions between proteins that are functionally unrelated. How such off-target interactions impact fitness is not generally known. Here, we use Saccharomyces cerevisiae to inducibly express tyrosine kinases. Because yeast lacks bona fide tyrosine kinases, the resulting tyrosine phosphorylation is biologically spurious. We engineered 44 yeast strains each expressing a tyrosine kinase, and quantitatively analysed their phosphoproteomes. This analysis resulted in ~30,000 phosphosites mapping to ~3500 proteins. The number of spurious pY sites generated correlates strongly with decreased growth, and we predict over 1000 pY events to be deleterious. However, we also find that many of the spurious pY sites have a negligible effect on fitness, possibly because of their low stoichiometry. This result is consistent with our evolutionary analyses demonstrating a lack of phosphotyrosine counter-selection in species with tyrosine kinases. Our results suggest that, alongside the risk for toxicity, the cell can tolerate a large degree of non-functional crosstalk as interaction networks evolve.

Mistranslating tRNA variants have anticodon- and sex-specific impacts on Drosophila melanogaster.

Transfer RNAs (tRNAs) are vital in determining the specificity of translation. Mutations in tRNA genes can result in the misincorporation of amino acids into nascent polypeptides in a process known as mistranslation. Since mistranslation has different impacts, depending on the type of amino acid substitution, our goal here was to compare the impact of different mistranslating tRNASer variants on fly development, lifespan, and behaviour. We established two mistranslating fly lines, one with a tRNASer variant that misincorporates serine at valine codons (V→S) and the other that misincorporates serine at threonine codons (T→S). While both mistranslating tRNAs increased development time and developmental lethality, the severity of the impacts differed depending on amino acid substitution and sex. The V→S variant extended embryonic, larval, and pupal development whereas the T→S only extended larval and pupal development. Females, but not males, containing either mistranslating tRNA presented with significantly more anatomical deformities than controls. Since mistranslation disrupts cellular translation and proteostasis, we also tested the hypothesis that tRNA variants impact fly lifespan. Interestingly, mistranslating females experienced extended lifespan whereas mistranslating male lifespan was unaffected. Consistent with delayed neurodegeneration and beneficial effects of mistranslation, mistranslating flies from both sexes showed improved locomotion as they aged. The ability of mistranslating tRNA variants to have both positive and negative effects on fly physiology and behaviour has important implications for human health given the prevalence of tRNA variants in humans.

Impact of tRNA-induced proline-to-serine mistranslation on the transcriptome of Drosophila melanogaster.

Mistranslation is the misincorporation of an amino acid into a polypeptide. Mistranslation has diverse effects on multicellular eukaryotes and is implicated in several human diseases. In Drosophila melanogaster, a serine transfer RNA (tRNA) that misincorporates serine at proline codons (P→S) affects male and female flies differently. The mechanisms behind this discrepancy are currently unknown. Here, we compare the transcriptional response of male and female flies to P→S mistranslation to identify genes and cellular processes that underlie sex-specific differences. Both males and females downregulate genes associated with various metabolic processes in response to P→S mistranslation. Males downregulate genes associated with extracellular matrix organization and response to negative stimuli such as wounding, whereas females downregulate aerobic respiration and ATP synthesis genes. Both sexes upregulate genes associated with gametogenesis, but females also upregulate cell cycle and DNA repair genes. These observed differences in the transcriptional response of male and female flies to P→S mistranslation have important implications for the sex-specific impact of mistranslation on disease and tRNA therapeutics.

Mistranslating tRNA variants have anticodon- and sex-specific impacts on Drosophila melanogaster.

Transfer RNAs (tRNAs) are vital in determining the specificity of translation. Mutations in tRNA genes can result in the misincorporation of amino acids into nascent polypeptides in a process known as mistranslation. Since mistranslation has different impacts, depending on the type of amino acid substitution, our goal here was to compare the impact of different mistranslating tRNASer variants on fly development, lifespan, and behaviour. We established two mistranslating fly lines, one with a tRNASer variant that misincorporates serine at valine codons (V→S) and the other that misincorporates serine at threonine codons (T→S). While both mistranslating tRNAs increased development time and developmental lethality, the severity of the impacts differed depending on amino acid substitution and sex. The V→S variant extended embryonic, larval, and pupal development whereas the T→S only extended larval and pupal development. Females, but not males, containing either mistranslating tRNA presented with significantly more anatomical deformities than controls. Mistranslating females also experienced extended lifespan whereas mistranslating male lifespan was unaffected. In addition, mistranslating flies from both sexes showed improved locomotion as they aged, suggesting delayed neurodegeneration. Therefore, although mistranslation causes detrimental effects, we demonstrate that mistranslation also has positive effects on complex traits such as lifespan and locomotion. This has important implications for human health given the prevalence of tRNA variants in humans.

Impact of tRNA-induced proline-to-serine mistranslation on the transcriptome of Drosophila melanogaster.

Mistranslation is the misincorporation of an amino acid into a polypeptide. Mistranslation has diverse effects on multicellular eukaryotes and is implicated in several human diseases. In Drosophila melanogaster, a serine transfer RNA (tRNA) that misincorporates serine at proline codons (P→S) affects male and female flies differently. The mechanisms behind this discrepancy are currently unknown. Here, we compare the transcriptional response of male and female flies to P→S mistranslation to identify genes and cellular processes that underlie sex-specific differences. Both males and females downregulate genes associated with various metabolic processes in response to P→S mistranslation. Males downregulate genes associated with extracellular matrix organization and response to negative stimuli such as wounding, whereas females downregulate aerobic respiration and ATP synthesis genes. Both sexes upregulate genes associated with gametogenesis, but females also upregulate cell cycle and DNA repair genes. These observed differences in the transcriptional response of male and female flies to P→S mistranslation have important implications for the sex-specific impact of mistranslation on disease and tRNA therapeutics.

A conserved function of corepressors is to nucleate assembly of the transcriptional preinitiation complex.

The plant corepressor TPL is recruited to diverse chromatin contexts, yet its mechanism of repression remains unclear. Previously, we have leveraged the fact that TPL retains its function in a synthetic transcriptional circuit in the yeast model Saccharomyces cerevisiae to localize repressive function to two distinct domains. Here, we employed two unbiased whole genome approaches to map the physical and genetic interactions of TPL at a repressed locus. We identified SPT4, SPT5 and SPT6 as necessary for repression with the SPT4 subunit acting as a bridge connecting TPL to SPT5 and SPT6. We also discovered the association of multiple additional constituents of the transcriptional preinitiation complex at TPL-repressed promoters, specifically those involved in early transcription initiation events. These findings were validated in yeast and plants through multiple assays, including a novel method to analyze conditional loss of function of essential genes in plants. Our findings support a model where TPL nucleates preassembly of the transcription activation machinery to facilitate rapid onset of transcription once repression is relieved.

Microdissected tumor cuboids: a microscale cancer model for large-scale testing that retains a complex tumor microenvironment.

Current cancer disease models fail to faithfully recapitulate key features of the human tumor microenvironment (TME), such as immune and vascular cells, while simultaneously enabling high-throughput drug tests. We have recently developed a precision slicing method that optimizes the yield of large numbers of cuboidal microtissues ("cuboids", ∼(400 µm) 3 ) from a single tumor biopsy. Here we demonstrate that cuboids from syngeneic mouse tumor models and human tumors retain a complex TME, making them amenable for drug and immunotherapy evaluation. We characterize relevant TME parameters, such as cellular architecture, cytokine secretion, proteomics profiles, and response to drug panels in multi-well arrays. Despite the cutting procedure and the time spent in culture (up to 7 days), the cuboids display strong cytokine and drug responses, including to immunotherapy. Overall, our results suggest that cuboids could provide invaluable therapeutic information for personalized oncology applications, and could help the development of TME-dependent therapeutics and cancer disease models, including for clinical trials.

Efficient and economic protein labeling for NMR in mammalian expression systems: Application to a preT-cell and T-cell receptor protein.

Protein nuclear magnetic resonance (NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with 15N and 13C, respectively. Moreover, E. coli can grow and express proteins in uniformly deuterium-substituted water (D2O), a strategy useful for experiments targeting high molecular weight proteins. Unfortunately, many proteins, particularly those requiring specific posttranslational modifications like disulfide bonding or glycosylation for proper folding and/or function, cannot be readily expressed in their functional forms using E. coli-based expression systems. One such class of proteins includes T-cell receptors and their related preT-cell receptors. In this study, we present an expression system for isotopic labeling of proteins using a nonadherent human embryonic kidney cell line, Expi293F, and a specially designed media. We demonstrate the application of this platform to the ß subunit common to both receptors. In addition, we show that this expression system and media can be used to specifically label amino acids Phe, Ile, Val, and Leu in this system, utilizing an amino acid-specific labeling protocol that allows targeted incorporation at high efficiency without significant isotopic scrambling. We demonstrate that this system can also be used to express proteins with fluorinated amino acids. We were routinely able to obtain an NMR sample with a concentration of 200 µM from 30 mL of culture media, utilizing less than 20 mg of the labeled amino acids.

High-Throughput Identification of Calcium Regulated Proteins Across Diverse Proteomes.

Calcium ions play important roles in nearly every biological process, yet whole-proteome analysis of calcium effectors has been hindered by lack of high-throughput, unbiased, and quantitative methods to identify proteins-calcium engagement. To address this, we adapted protein thermostability assays in the budding yeast, human cells, and mouse mitochondria. Based on calcium-dependent thermostability, we identified 2884 putative calcium-regulated proteins across human, mouse, and yeast proteomes. These data revealed calcium engagement of novel signaling hubs and cellular processes, including metabolic enzymes and the spliceosome. Cross-species comparison of calcium-protein engagement and mutagenesis experiments identified residue-specific cation engagement, even within well-known EF-hand domains. Additionally, we found that the dienoyl-CoA reductase DECR1 binds calcium at physiologically-relevant concentrations with substrate-specific affinity, suggesting direct calcium regulation of mitochondrial fatty acid oxidation. These unbiased, proteomic analyses of calcium effectors establish a key resource to dissect cation engagement and its mechanistic effects across multiple species and diverse biological processes.

The regulatory landscape of the yeast phosphoproteome.

The cellular ability to react to environmental fluctuations depends on signaling networks that are controlled by the dynamic activities of kinases and phosphatases. Here, to gain insight into these stress-responsive phosphorylation networks, we generated a quantitative mass spectrometry-based atlas of early phosphoproteomic responses in Saccharomyces cerevisiae exposed to 101 environmental and chemical perturbations. We report phosphosites on 59% of the yeast proteome, with 18% of the proteome harboring a phosphosite that is regulated within 5 min of stress exposure. We identify shared and perturbation-specific stress response programs, uncover loss of phosphorylation as an integral early event, and dissect the interconnected regulatory landscape of kinase-substrate networks, as we exemplify with target of rapamycin signaling. We further reveal functional organization principles of the stress-responsive phosphoproteome based on phosphorylation site motifs, kinase activities, subcellular localizations, shared functions and pathway intersections. This information-rich map of 25,000 regulated phosphosites advances our understanding of signaling networks.

The fitness cost of spurious phosphorylation.

The fidelity of signal transduction requires the binding of regulatory molecules to their cognate targets. However, the crowded cell interior risks off-target interactions between proteins that are functionally unrelated. How such off-target interactions impact fitness is not generally known, but quantifying this is required to understand the constraints faced by cell systems as they evolve. Here, we use the model organism S. cerevisiae to inducibly express tyrosine kinases. Because yeast lacks bona fide tyrosine kinases, most of the resulting tyrosine phosphorylation is spurious. This provides a suitable system to measure the impact of artificial protein interactions on fitness. We engineered 44 yeast strains each expressing a tyrosine kinase, and quantitatively analysed their phosphoproteomes. This analysis resulted in ~30,000 phosphosites mapping to ~3,500 proteins. Examination of the fitness costs in each strain revealed a strong correlation between the number of spurious pY sites and decreased growth. Moreover, the analysis of pY effects on protein structure and on protein function revealed over 1000 pY events that we predict to be deleterious. However, we also find that a large number of the spurious pY sites have a negligible effect on fitness, possibly because of their low stoichiometry. This result is consistent with our evolutionary analyses demonstrating a lack of phosphotyrosine counter-selection in species with bona fide tyrosine kinases. Taken together, our results suggest that, alongside the risk for toxicity, the cell can tolerate a large degree of non-functional crosstalk as interaction networks evolve.

Anticodon sequence determines the impact of mistranslating tRNAAla variants.

Transfer RNAs (tRNAs) maintain translation fidelity through accurate charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA at the ribosome. Mistranslation occurs when an amino acid not specified by the genetic message is incorporated into proteins and has applications in biotechnology, therapeutics and is relevant to disease. Since the alanyl-tRNA synthetase uniquely recognizes a G3:U70 base pair in tRNAAla and the anticodon plays no role in charging, tRNAAla variants with anticodon mutations have the potential to mis-incorporate alanine. Here, we characterize the impact of the 60 non-alanine tRNAAla anticodon variants on the growth of Saccharomyces cerevisiae. Overall, 36 tRNAAla anticodon variants decreased growth in single- or multi-copy. Mass spectrometry analysis of the cellular proteome revealed that 52 of 57 anticodon variants, not decoding alanine or stop codons, induced mistranslation when on single-copy plasmids. Variants with G/C-rich anticodons resulted in larger growth deficits than A/U-rich variants. In most instances, synonymous anticodon variants impact growth differently, with anticodons containing U at base 34 being the least impactful. For anticodons generating the same amino acid substitution, reduced growth generally correlated with the abundance of detected mistranslation events. Differences in decoding specificity, even between synonymous anticodons, resulted in each tRNAAla variant mistranslating unique sets of peptides and proteins. We suggest that these differences in decoding specificity are also important in determining the impact of tRNAAla anticodon variants.

The mitochondrially targeted peptide elamipretide (SS-31) improves ADP sensitivity in aged mitochondria by increasing uptake through the adenine nucleotide translocator (ANT).

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function. We measured the response to ADP stimulation in young and old muscle mitochondria with ELAM treatment, in vivo heart and muscle function, and compared protein abundance, phosphorylation, and S-glutathionylation of ADP/ATP pathway proteins. ELAM treatment increased ADP sensitivity in old muscle mitochondria by increasing uptake of ADP through the ANT and rescued muscle force and heart systolic function. Protein abundance in the ADP/ATP transport and synthesis pathway was unchanged, but ELAM treatment decreased protein s-glutathionylation incuding of ANT. Mitochondrial ADP sensitivity is rapidly modifiable. This research supports the hypothesis that ELAM improves ANT function in aging and links mitochondrial ADP sensitivity to physiological function. ELAM binds directly to ANT and ATP synthase and ELAM treatment improves ADP sensitivity, increases ATP production, and improves physiological function in old muscles. ADP (adenosine diphosphate), ATP (adenosine triphosphate), VDAC (voltage-dependent anion channel), ANT (adenine nucleotide translocator), H+ (proton), ROS (reactive oxygen species), NADH (nicotinamide adenine dinucleotide), FADH2 (flavin adenine dinucleotide), O2 (oxygen), ELAM (elamipretide), -SH (free thiol), -SSG (glutathionylated protein).

Automated Enrichment of Phosphotyrosine Peptides for High-Throughput Proteomics.

Phosphotyrosine (pY) enrichment is critical for expanding the fundamental and clinical understanding of cellular signaling by mass spectrometry-based proteomics. However, current pY enrichment methods exhibit a high cost per sample and limited reproducibility due to expensive affinity reagents and manual processing. We present rapid-robotic phosphotyrosine proteomics (R2-pY), which uses a magnetic particle processor and pY superbinders or antibodies. R2-pY can handle up to 96 samples in parallel, requires 2 days to go from cell lysate to mass spectrometry injections, and results in global proteomic, phosphoproteomic, and tyrosine-specific phosphoproteomic samples. We benchmark the method on HeLa cells stimulated with pervanadate and serum and report over 4000 unique pY sites from 1 mg of peptide input, strong reproducibility between replicates, and phosphopeptide enrichment efficiencies above 99%. R2-pY extends our previously reported R2-P2 proteomic and global phosphoproteomic sample preparation framework, opening the door to large-scale studies of pY signaling in concert with global proteome and phosphoproteome profiling.

Analysis of networks in the dorsolateral prefrontal cortex in chronic schizophrenia: Relevance of altered immune response.

The dorsolateral prefrontal cortex (DLPFC) has a crucial role in cognitive functioning and negative symptoms in schizophrenia. However, limited information of altered protein networks is available in this region in schizophrenia. We performed a proteomic analysis using single-shot liquid chromatography-tandem mass spectrometry of grey matter of postmortem DLPFC in chronic schizophrenia subjects (n = 20) and unaffected subjects (n = 20) followed by bioinformatic analysis to identify altered protein networks in schizophrenia (PXD024939 identifier in ProteomeXchange repository). Our results displayed a proteome profile in the DLPFC of 1989 proteins. 43 proteins were found significantly altered in schizophrenia. Analysis of this panel showed an enrichment of biological processes implicated in vesicle-mediated transport, processing and antigen presentation via MHC class II, intracellular transport and selenium metabolism. The enriched identified pathways were MHC class II antigen presentation, vesicle-mediated transport, Golgi ER retrograde transport, Nef mediated CD8 downregulation and the immune system. All these enriched categories were found to be downregulated. Furthermore, our network analyses showed crosstalk between proteins involved in MHC class II antigen presentation, membrane trafficking, Golgi-to-ER retrograde transport, Nef-mediated CD8 downregulation and the immune system with only one module built by 13 proteins. RAB7A showed eight interactions with proteins of all these pathways. Our results provide an altered molecular network involved in immune response in the DLPFC in schizophrenia with a central role of RAB7A. These results suggest that RAB7A or other proteins of this network could be potential targets for novel pharmacological strategies in schizophrenia for improving cognitive and negative symptoms.

Elamipretide Improves ADP Sensitivity in Aged Mitochondria by Increasing Uptake through the Adenine Nucleotide Translocator (ANT).

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function. We measured the response to ADP stimulation in young and old muscle mitochondria with ELAM treatment, in vivo heart and muscle function, and compared protein abundance, phosphorylation, and S-glutathionylation of ADP/ATP pathway proteins. ELAM treatment increased ADP sensitivity in old muscle mitochondria by increasing uptake of ADP through the ANT and rescued muscle force and heart systolic function. Protein abundance in the ADP/ATP transport and synthesis pathway was unchanged, but ELAM treatment decreased protein s-glutathionylation incuding of ANT. Mitochondrial ADP sensitivity is rapidly modifiable. This research supports the hypothesis that ELAM improves ANT function in aging and links mitochondrial ADP sensitivity to physiological function.

Automated Enrichment of Phosphotyrosine Peptides for High-Throughput Proteomics.

Phosphotyrosine (pY) enrichment is critical for expanding fundamental and clinical understanding of cellular signaling by mass spectrometry-based proteomics. However, current pY enrichment methods exhibit a high cost per sample and limited reproducibility due to expensive affinity reagents and manual processing. We present rapid-robotic phosphotyrosine proteomics (R2-pY), which uses a magnetic particle processor and pY superbinders or antibodies. R2-pY handles 96 samples in parallel, requires 2 days to go from cell lysate to mass spectrometry injections, and results in global proteomic, phosphoproteomic and tyrosine specific phosphoproteomic samples. We benchmark the method on HeLa cells stimulated with pervanadate and serum and report over 4000 unique pY sites from 1 mg of peptide input, strong reproducibility between replicates, and phosphopeptide enrichment efficiencies above 99%. R2-pY extends our previously reported R2-P2 proteomic and global phosphoproteomic sample preparation framework, opening the door to large-scale studies of pY signaling in concert with global proteome and phosphoproteome profiling.

A Python Package for the Localization of Protein Modifications in Mass Spectrometry Data.

Determining the correct localization of post-translational modifications (PTMs) on peptides aids in interpreting their effect on protein function. While most algorithms for this task are available as standalone applications or incorporated into software suites, improving their versatility through access from popular scripting languages facilitates experimentation and incorporation into novel workflows. Here we describe pyAscore, an efficient and versatile implementation of the Ascore algorithm in Python for scoring the localization of user defined PTMs in data dependent mass spectrometry. pyAscore can be used from the command line or imported into Python scripts and accepts standard file formats from popular software tools used in bottom-up proteomics. Access to internal objects for scoring and working with modified peptides adds to the toolbox for working with PTMs in Python. pyAscore is available as an open source package for Python 3.6+ on all major operating systems and can be found at pyascore.readthedocs.io.

Elamipretide effects on the skeletal muscle phosphoproteome in aged female mice.

The age-related decline in skeletal muscle mass and function is known as sarcopenia. Sarcopenia progresses based on complex processes involving protein dynamics, cell signaling, oxidative stress, and repair. We have previously found that 8-week treatment with elamipretide improves skeletal muscle function, reverses redox stress, and restores protein S-glutathionylation changes in aged female mice. This study tested whether 8-week treatment with elamipretide also affects global phosphorylation in skeletal muscle consistent with functional improvements and S-glutathionylation. Using female 6-7-month-old mice and 28-29-month-old mice, we found that phosphorylation changes did not relate to S-glutathionylation modifications, but that treatment with elamipretide did partially reverse age-related changes in protein phosphorylation in mouse skeletal muscle.