Summary N 6 -methyladenosine (m 6 A), the most prevalent internal RNA modification on mammalian messenger RNAs (mRNAs), regulates fates and functions of modified transcripts through m 6 A-specific binding proteins 1 – 5 . m 6 A is abundant in the nervous system and modulates various neural functions 6 – 11 . While m 6 A marks groups of mRNAs for coordinated degradation in various physiological processes 12 – 15 , the relevance of m 6 A in mRNA translation remains largely unknown in vivo . Here we show that, through its binding protein Ythdf1, m 6 A promotes protein synthesis of target transcripts in response to neuronal stimuli in the adult mouse hippocampus, thereby facilitating learning and memory. Mice with genetic deletion of Ythdf1 ( Ythdf1 -KO) exhibit learning and memory defects as well as impaired hippocampal synaptic transmission and long-term potentiation. Ythdf1 re-expression in the hippocampus of adult Ythdf1 -KO mice rescues behavioral and synaptic defects, while hippocampus-specific acute knockdown of Ythdf1 or Mettl3 , the catalytic component of m 6 A methyltransferase complex, recapitulates the hippocampal deficiency. Transcriptome-wide mapping of Ythdf1 binding sites and m 6 A sites on hippocampal mRNAs uncovered key neuronal genes. Nascent protein labeling and tether reporter assays in hippocampal neurons revealed that Ythdf1 enhances protein synthesis in a neuronal-stimulus-dependent manner. Collectively, our results uncover a pathway of mRNA m 6 A methylation in learning and memory, which is mediated through Ythdf1 in response to stimuli.
Identification of deamidated sites in proteins is commonly used for assignment of N-glycosylation sites. It is also important for assessing the role of deamidation in vivo. However, nonenzymatic deamidation occurs easily in peptides under conditions commonly used in treatment with trypsin and PNGase F. The impact on proteomic sample preparation has not yet been evaluated systematically. In addition, the 13 C peaks of amidated peptides can be misassigned as monoisotopic peaks of the corresponding deamidated ones in database searches. The 19.34 mDa mass difference between them is proposed as a means for eliminating the resulting false positive identifications in large-scale proteomic analysis. We evaluated five groups of proteomic data, obtained mainly through an electrostatic repulsion-hydrophilic interaction chromatography (ERLIC)-reverse phase (RP) chromatography sequence, and ascertained that nonenzymatic asparagine deamidation occurred to some extent on 4 -9% of the peptides, resulting in the false positive identification of many N-glycosylation sites. A comprehensive investigation indicated that the chief causative factors were the mildly alkaline pH and prolonged incubations at 37°C during proteomic sample preparation. An improved protocol is proposed featuring tryptic digestion at pH 6 and deglycosylation at pH 5, resulting in a significant decrease in nonenzymatic deamidation while conserving adequate digestion efficiency. The number of identified deamidation sites was improved significantly by increasing the sample loading amount in liquid chromatography-tandem MS. This permitted the identification of a significant number of glutamine deamidation sites, which featured sequence motifs largely different from those for asparagine deami- Accurate identification of protein deamidation sites is important to understanding the role of deamidation in human diseases and aging and in the correct location of N-glycosylation using the PNGase F deglycosylation method. However, determination of protein deamidation with liquid chromatography-tandem MS (LC-MS/MS) 1 is compromised by current methods of proteomics sample preparation and database searching. Nonenzymatic deamidation of Asn and Gln occurs spontaneously on proteins and peptides both in vivo and in vitro. In vivo, deamidation is reported to be related to Alzheimer's disease and cataracts (1-3). It has also been proposed as a molecular clock in studies of aging (4, 5). In vitro, deamidation leads to the degradation of therapeutic proteins during production and storage (6, 7). It takes place relatively slowly in intact proteins with a half-life of 1-500 days for Asn and 100 -500 days for Gln (8). However, the deamidation rate of peptides increases significantly under conditions typical of tryptic digestion, i.e. incubation in digestion buffers at pH 8 for 8 -16 h at 37°C (9). Under mildly alkaline conditions, Asn deamidation happens mainly through the formation of a succinimide ring intermediate that is quickly hydrolyzed to D,LAsp and D,L-isoAsp with isoAsp pred...
The communication between cells and between cellular organelles is often controlled by the interaction of membrane proteins. Although many methods for the detection of protein-protein interactions (PPIs) exist, membrane PPIs remain difficult to detect. Here we developed a proximity-based tagging system, PUP-IT (pupylation-based interaction tagging), to identify membrane protein interactions. In this approach, a small protein tag, Pup, is applied to proteins that interact with a PafA-fused bait, enabling transient and weak interactions to be enriched and detected by mass spectrometry. Pup does not diffuse from the enzyme, which allows high-specificity labeling. We applied this approach to CD28, a critical costimulatory receptor for T lymphocyte activation, and identified known CD28 binding partners and multiple potential interacting proteins. In addition, we demonstrated that this method can identify the interaction between a cell surface receptor and its ligand.
Drug resistance and tolerance greatly diminish the therapeutic potential of antibiotics against pathogens. Antibiotic tolerance by bacterial biofilms often leads to persistent infections, but its mechanisms are unclear. Here we use a proteomics approach, pulsed stable isotope labelling with amino acids (pulsed-SILAC), to quantify newly expressed proteins in colistin-tolerant subpopulations of Pseudomonas aeruginosa biofilms (colistin is a ‘last-resort' antibiotic against multidrug-resistant Gram-negative pathogens). Migration is essential for the formation of colistin-tolerant biofilm subpopulations, with colistin-tolerant cells using type IV pili to migrate onto the top of the colistin-killed biofilm. The colistin-tolerant cells employ quorum sensing (QS) to initiate the formation of new colistin-tolerant subpopulations, highlighting multicellular behaviour in antibiotic tolerance development. The macrolide erythromycin, which has been previously shown to inhibit the motility and QS of P. aeruginosa, boosts biofilm eradication by colistin. Our work provides insights on the mechanisms underlying the formation of antibiotic-tolerant populations in bacterial biofilms and indicates research avenues for designing more efficient treatments against biofilm-associated infections.
Klein und zackig: Nach der Einführung eines minimalen Photovernetzers mit Eignung für Klick‐Reaktionen (siehe Schema) in zahlreiche niedermolekulare Kinase‐Inhibitoren wurden die resultierenden Sonden zum Proteom‐Profiling in vitro (in Zelllysaten) und in situ (in lebenden Zellen) eingesetzt. Auch bei der Identifizierung ihrer potenziellen Zielkinasen im großen Maßstab waren sie bekannten Sonden überlegen.
In shotgun proteomics, multidimensional liquid chromatography (MDLC) is commonly used to reduce sample complexity and increase dynamic range of protein identification. Since reversed-phase chromatography is mostly used as the second-dimensional separation before mass spectrometric analysis, the improvement of MDLC primarily depends on the first dimension of separation. Here, we present a novel whole proteome analysis method that separates peptides based on ERLIC. Tryptic peptides were retained on a weak anion exchange column through ERLIC with a high organic mobile phase. They were then distributed into multiple fractions based on both pI and polarity through the simultaneous effect of electrostatic repulsion and hydrophilic interaction when eluted using a salt-free pH gradient of increasing water content. Applying this to rat kidney tissue, we identified 4821 proteins and 30 659 unique peptides with high confidence from two replicates using LTQ-FT. This was 36.2% and 64.3% higher, respectively, than was obtained with the widely used SCX separation mode. Notably, the identification of both highly hydrophobic and basic peptides increased over 120% using the ERLIC method. The results indicate that ERLIC is a promising alternative to SCX as the first dimension of MDLC. In total, 5499 proteins and 35 847 unique peptides of rat kidney tissue are characterized.
Protein post-translational modifications (PTMs) are regulated separately from protein expression levels. Thus, simultaneous characterization of the proteome and its PTMs is pivotal to an understanding of protein regulation, function and activity. However, concurrent analysis of the proteome and its PTMs by mass spectrometry is a challenging task because the peptides bearing PTMs are present in sub-stoichiometric amounts and their ionization is often suppressed by unmodified peptides of high abundance. We describe here a method for concurrent analysis of phosphopeptides, glycopeptides and unmodified peptides in a tryptic digest of rat kidney tissue with a sequence of ERLIC and RP-LC-MS/MS in a single experimental run, thereby avoiding inter-experimental variation. Optimization of loading solvents and elution gradients permitted ERLIC to be performed with totally volatile solvents. Two SCX and four ERLIC gradients were compared in details, and one ERLIC gradient was found to perform the best, which identified 2929 proteins, 583 phosphorylation sites in 338 phosphoproteins and 722 N-glycosylation sites in 387 glycoproteins from rat kidney tissue. Two hundred low-abundance proteins with important functions were identified only from the glyco- or phospho-subproteomes, reflecting the importance of the enrichment and separation of modified peptides by ERLIC. In addition, this strategy enables identification of unmodified and corresponding modified peptides (partial phosphorylation and N-glycosylation) from the same protein. Interestingly, partially modified proteins tend to occur on proteins involved in transport. Moreover, some membrane or extracellular proteins, such as versican core protein and fibronectin, were found to have both phosphorylation and N-glycosylation, which may permit an assessment of the potential for cross talk between these two vital PTMs and their roles in regulation.
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