Biology subjects

Imami, K.

Publications and source records attributed to Imami, K..

31 records · Page 2Linked to original sources

bioRxiv2023

Liquid-liquid extraction of lipidated peptides for direct identification of lipidation sites

Proteins can be modified by lipids in various ways, for example by myristoylation, palmitoylation, farnesylation, and geranylgeranylation--these processes are collectively referred to as lipidation. Current chemical proteomics using alkyne lipids has enabled the identification of lipidated protein candidates but does not identify endogenous lipidation sites and is not readily applicable to in vivo systems. Here, we introduce a proteomic methodology for global analyses of endogenous lipidation sites that combines liquid-liquid extraction of hydrophobic lipidated peptides with liquid chromatography-tandem mass spectrometry using a gradient program of acetonitrile in the high concentration range. We applied this method to explore lipidation sites in HeLa cells, and identified a total of 90 lipidation sites, including 75 protein N-terminal myristoylation sites, which is more than the number of high-confidence lipidated proteins identified by myristic acid analog-based chemical proteomics. Isolation of lipidated peptides from digests prepared with different proteases enabled the identification of different lipidated sites, extending the coverage. Moreover, our peptide-centric approach successfully identified dually modified peptides having myristoylation and palmitoylation. Finally, we analyzed in vivo myristoylation sites in mouse tissues and found that the lipidation profile is tissue-specific. This simple method (not requiring chemical labeling or affinity purification) should be a promising tool for global profiling of various protein lipidations.

biochemistry↗

bioRxiv2023

Znf598-mediated Rps10/eS10 ubiquitination contributes to the ribosome ubiquitination dynamics during zebrafish development

Ribosome is a translational apparatus that comprises about 80 ribosomal proteins and four rRNAs. Recent studies reported that ribosome ubiquitination is crucial for translational regulation and ribosome-associated quality control (RQC). However, little is known about the dynamics of ribosome ubiquitination under complex biological processes of multicellular organisms. To explore ribosome ubiquitination during animal development, we generated a zebrafish strain that expresses a FLAG-tagged ribosomal protein Rpl36/eL36 from its endogenous locus. We examined ribosome ubiquitination during zebrafish development by combining affinity purification of ribosomes from rpl36-FLAG zebrafish embryos with immunoblotting analysis. Our findings showed that ubiquitination of ribosomal proteins dynamically changed as development proceeded. We also showed that during zebrafish development, the ribosome was ubiquitinated by Znf598, an E3 ubiquitin ligase that activates RQC. Ribosomal protein Rps10/eS10 was found to be a key ubiquitinated protein during development. Furthermore, we showed that Rps10/eS10 ubiquitination-site mutations reduced the overall ubiquitination pattern of ribosome. These results demonstrate the complexity and dynamics of ribosome ubiquitination during zebrafish development.

molecular biology↗

bioRxiv2022

Classification of Extracellular Vesicles based on Surface Glycan Structures by Spongy-like Separation Media

Extracellular vesicles (EVs) are lipid bilayer vesicles that enclose various biomolecules. EVs hold promise as sensitive biomarkers to detect and monitor various diseases. However, they have heterogenous molecular compositions. The compositions of EVs from identical donor cells obtained using the same purification methods may differ, which is a significant obstacle for elucidating objective biological functions. Herein the potential of a novel lectin-based affinity chromatography (LAC) method to classify EVs based on their glycan structures is demonstrated. The proposed method utilizes a spongy-like monolithic polymer (spongy monolith, SPM), which consists of poly(ethylene-co-glycidyl methacrylate) with continuous micropores and allows an efficient in-situ protein reaction with epoxy groups. Two distinct lectins with different specificities, Sambucus sieboldiana agglutinin and concanavalin A, are effectively immobilized on SPM without impacting the binding activity. Moreover, high recovery rates of liposomal nanoparticles as a model of EVs are achieved due to the large flow-through pores (>10 m) of SPM. Finally, lectin-immobilized SPMs are employed to classify EVs based on the surface glycan structures and demonstrate different subpopulations by proteome profiling.

biochemistry↗

bioRxiv2021

pSNAP: Proteome-wide analysis of elongating nascent polypeptide chains

Cellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the level of elongating nascent polypeptide chains (NPCs) and associated co-translational events. Here we describe pSNAP, a method for proteome-wide profiling of NPCs by affinity enrichment of puromycin- and stable isotope-labeled polypeptides. pSNAP does not require ribosome purification and/or chemical labeling, and captures bona fide NPCs that characteristically exhibit protein N-terminus-biased positions. We applied pSNAP to evaluate the effect of silmitasertib, a potential molecular therapy for cancer, and revealed acute translational repression through casein kinase II and mTOR pathways. We also characterized modifications on NPCs and demonstrated that the combination of different types of modifications, such as acetylation and phosphorylation in the N-terminal region of histone H1.5, can modulate interactions with ribosome-associated factors. Thus, pSNAP provides a framework for dissecting co-translational regulations on a proteome-wide scale.

biochemistry↗

bioRxiv2021

Novel roles of small extracellular vesicles in regulating the quiescence and proliferation of neural stem cells

Neural stem cells (NSCs) quiescence plays pivotal roles in securing sustainable neurogenesis and avoiding stemness exhaustion in the adult brain. The maintenance of quiescence and transition between proliferation and quiescence are complex processes associated with multiple niche signals, and environmental stimuli. Though the mechanisms of the transitions between NSC states have been extensively investigated, they remain to be fully elucidated. Exosomes are small extracellular vesicles (sEVs) containing functional units such as proteins, microRNAs, and mRNAs. It has already been demonstrated that sEVs actively participate in cancer cell proliferation and metastasis. However, the role of sEVs in NSC quiescence has not been investigated. Here, we applied proteomics to analyze the protein cargos of sEVs derived from proliferating, quiescent, and reactivating NSCs. Our findings revealed expression level fluctuations of NSCs sEV protein cargo at different proliferative conditions. We also identified functional clusters of gene ontology annotations from differentially expressed proteins in three sources of exosomes. Moreover, the use of exosome inhibitors revealed the contribution of exosomes to NSC quiescence at the entrance into quiescence, as well as in quiescence maintenance. Exosome inhibition delayed the entrance into quiescence by proliferating NSCs and allowed quiescent NSCs to exit from the G0 phase of the cell cycle. Protein translation was significantly upregulated in both quiescent NSCs and quiescent-induced NSCs via the exosome inhibition. Our results demonstrated that NSC exosomes are involved in regulating the quiescence of NSCs and provide a functional prediction of NSCs exosome protein cargos in terms of cell-cycle regulation and protein synthesis.

developmental biology↗

bioRxiv2021

Timed global reorganization of protein synthesis during neocortex neurogenesis at codon resolution

Translation modulates the timing and amplification of gene expression after transcription. Brain development requires uniquely complex gene expression patterns, but large-scale measurements of translation directly in the prenatal brain are lacking. We measure the reactants, synthesis, and products of translation spanning mouse neocortex neurogenesis, and discover a transient window of dynamic regulation at mid-gestation. Timed translation upregulation of chromatin binding proteins like Satb2, which is essential for neuronal subtype differentiation, restricts protein expression in neuronal lineages despite broad transcriptional priming in progenitors. In contrast, translation downregulation of ribosomal proteins sharply decreases ribosome number, coinciding with a major shift in protein synthesis dynamics at mid-gestation. Changing levels of eIF4EBP1, a direct inhibitor of ribosomal protein translation, are concurrent with ribosome downregulation and controls Satb2 fate acquisition during neuronal differentiation. Thus, the refinement of transcriptional programs by translation is central to the molecular logic of brain development. Modeling of the developmental neocortex translatome is provided as an open-source searchable resource: https://shiny.mdc-berlin.de/cortexomics/.

neuroscience↗

bioRxiv2021

Escherichia coli S2P family intramembrane protease RseP is engaged in the regulated sequential cleavages of FecR in the ferric citrate signaling

Escherichia coli RseP, a member of the S2P family of intramembrane proteases, is involved in the activation of the {sigma}E extracytoplasmic stress response and elimination of remnant signal peptides. However, whether RseP has additional cellular functions is unclear. In this study, we attempted to identify new RseP substrates to explore still unknown physiological roles of this protease. Our mass spectrometry-based quantitative proteomic analysis revealed that the levels of several Fec system proteins encoded by the fecABCDE operon (fec operon) were significantly decreased in an RseP-deficient strain. The Fec system is responsible for the uptake of ferric citrate, and the transcription of the fec operon is controlled by FecI, an alternative sigma factor, and its regulator FecR, a single-pass transmembrane protein. Assays with the fec operon expression reporter demonstrated that the proteolytic activity of RseP is essential for the ferric citrate-dependent upregulation of the fec operon. Analysis using the FecR protein and FecR-derived model proteins showed that FecR undergoes sequential processing at the membrane and that RseP participates in the last step of this sequential processing to generate the N-terminal cytoplasmic fragment of FecR that participates in the transcription of the fec operon with FecI. Ferric citrate signal-dependent generation of this cleavage product is the essential and sufficient role of RseP in the transcriptional activation of the fec operon. Our study unveiled that E. coli RseP performs the intramembrane proteolysis of FecR, a novel physiological role that is essential for regulating iron uptake by the ferric citrate transport system.

molecular biology↗

bioRxiv2021

Monitoring mitochondrial translation by pulse SILAC

Mitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, which shapes the oxidative phosphorylation (OXPHOS) complexes essential for cellular energy metabolism. Despite the importance of mitochondrial translation control, it is challenging to identify and quantify the mitochondrial-encoded proteins due to their hydrophobic nature and low abundance. Here, we introduce a mass spectrometry-based proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture (pSILAC). Our method provides the highest protein identification rate with the shortest measurement time among currently available methods, enabling us to quantify 12 out of the 13 mitochondrial-encoded proteins. We applied this method to uncover the global picture of (post-)translational regulation of both mitochondrial- and nuclear-encoded subunits of OXPHOS complexes. We found that inhibition of mitochondrial translation led to degradation of orphan nuclear-encoded subunits that are considered to form subcomplexes with the mitochondrial-encoded subunits. The results also allowed us to infer the subcomplex members of each OXPHOS complex. This method should be readily applicable to study mitochondrial translation programs in many contexts, including oxidative stress and mitochondrial disease.

biochemistry↗

bioRxiv2020

Exploiting a PAX3-FOXO1-induced synthetic lethal ATR dependency for rhabdomyosarcoma therapy

Pathognomonic PAX3-FOXO1 fusion oncogene expression is associated with poor outcome in rhabdomyosarcoma. Combining genome-wide CRISPR screening with cell- based functional genetic approaches, we here provide evidence that PAX3-FOXO1 induces replication stress, resulting in a synthetic lethal dependency to ATR-mediated DNA damage-response signaling in rhabdomyosarcoma. Expression of PAX3-FOXO1 in muscle progenitor cells was not only sufficient to induce hypersensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibited increased sensitivity to structurally diverse inhibitors of ATR, a dependency that could be validated genetically. Mechanistically, ATR inhibition led to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increased sensitivity of rhabdomyosarcoma cells to PARP inhibition in vitro, and combined ATR and PARP inhibition induced regression of primary patient-derived alveolar rhabdomyosarcoma xenografts in vivo. Moreover, a genome-wide CRISPR activation screen (CRISPRa) identified FOS gene family members as inducers of resistance against ATR inhibitors. Mechanistically, FOS gene family members reduced replication stress in rhabdomyosarcoma cells. Lastly, compassionate use of ATR inhibitors in two pediatric patients suffering from relapsed PAX3-FOXO1-expressing alveolar rhabdomyosarcoma showed signs of tolerability, paving the way to clinically exploit this novel synthetic lethal dependency in rhabdomyosarcoma.

cancer biology↗

bioRxiv2020

Proximity Labeling-assisted Identification of Endogenous Kinase Substrates

Mass spectrometry-based phosphoproteomics can identify more than 10,000 phosphorylated sites in a single experiment. But, despite the fact that enormous phosphosite information has been accumulated in public repositories, protein kinase-substrate relationships remain largely unknown. Here, we describe a method to identify endogenous substrates of kinases by means of proximity labeling. We used a proximity-dependent biotin identification approach, called BioID, in combination with kinase-perturbed phosphoproteomics profiling and phosphorylation sequence motifs derived from in vitro kinase assay to find molecules that interact with a target kinase, that show altered phosphorylation in response to kinase perturbation, and that are directly phosphorylated by the kinase in vitro; i.e., endogenous kinase substrates. Application of this methodology to casein kinase 2 (CK2) and protein kinase A (PKA) identified 33 and 52 putative substrates, respectively. We also show that known cancer-associated missense mutations near phosphosites of substrates affect phosphorylation by CK2 or PKA, and thus might alter downstream signaling in cancer cells bearing these mutations. This study extends our knowledge of kinase-substrate networks by proposing a new large-scale approach to identify endogenous substrates of kinases.

molecular biology↗

bioRxiv2020

Quantitative nascent proteome profiling by dual pulse labeling with O-propargyl-puromycin and stable isotope labeled amino acids

Monitoring translational regulation in response to environmental signals is crucial for understanding cellular proteostasis. However, only limited approaches are currently available for quantifying acute changes in protein synthesis induced by stimuli. Recently, a clickable puromycin analog, O-propargyl-puromycin (OPP), was developed and applied to label the C-termini of nascent polypeptide chains (NPCs). Following affinity purification via a click reaction, OPP allows for a proteomic analysis of NPCs. Despite its advantage, the affinity purification of NPCs using magnetic beads or resins inherently suffers from significant non-specific protein binding, which hinders accurate quantification of the nascent proteins. To address this issue, we employed dual pulse labeling of NPCs with both OPP and stable isotope labeled amino acids to distinguish bona fide NPCs from non-specific proteins, thereby enabling the accurate quantitative profiling of NPCs. We applied this method to dissecting the transcription-coupled translation responses and quantified ~3,000 nascent proteins. We found that the translation of a subset of ribosomal proteins (e.g., RPSA, RPLP0) as well as signaling proteins (e.g., BCAR3, EFNA1, DUSP1) was significantly repressed by transcription inhibition. Together, the present method provides an accurate and broadly applicable nascent proteome profiling for many biological applications at the level of translation.

biochemistry↗

bioRxiv2020

The architecture of protein synthesis in the developing neocortex at near-atomic resolution reveals Ebp1-mediated neuronal proteostasis at the 60S tunnel exit

Protein synthesis must be finely tuned in the nervous system, as it represents an essential feature of neurodevelopmental gene expression, and dominant pathology in neurological disease. However, the architecture of ribosomal complexes in the developing mammalian brain has not been analyzed at high resolution. This study investigates the architecture of ribosomes ex vivo from the embryonic and perinatal mouse neocortex, revealing Ebp1 as a 60S peptide tunnel exit binding factor at near-atomic resolution by multiparticle cryo-electron microscopy. The impact of Ebp1 on the neuronal proteome was analyzed by pSILAC and BONCAT coupled mass spectrometry, implicating Ebp1 in neurite outgrowth proteostasis, with in vivo embryonic Ebp1 knockdown resulting in dysregulation of neurite outgrowth. Our findings reveal Ebp1 as a central component of neocortical protein synthesis, and the 60S peptide tunnel exit as a focal point of gene expression control in the molecular specification of neuronal morphology. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/939488v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1231351org.highwire.dtl.DTLVardef@1c940f7org.highwire.dtl.DTLVardef@1c81e2corg.highwire.dtl.DTLVardef@135008b_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2019

Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis

Herpesviruses encode conserved protein kinases to stimulate phosphorylation-sensitive processes during infection. How these kinases bind to cellular factors and how this impacts their regulatory functions is poorly understood. Here, we use quantitative proteomics to determine cellular interaction partners of human herpesvirus (HHV) kinases. We find that these kinases can target key regulators of transcription and replication. The interaction with Cyclin A and associated factors is identified as a specific signature of {beta}-herpesvirus kinases. Cyclin A is recruited via RXL-motifs that overlap with nuclear localization signals (NLS) and locate in the non-catalytic N-terminal regions. This architecture is conserved for viral kinases of HHV6, HHV7 and rodent CMVs. Docking to Cyclin A competes with NLS function, enabling dynamic changes in kinase localization and substrate phosphorylation. The viral kinase redirects Cyclin A to the cytosol, which is essential for the inhibition of cellular DNA replication during infection. Our data highlight a fine-tuned and physiologically important interplay between a cellular cyclin and viral kinases.

microbiology↗