Decrypting the functional design of unmodified translation elongation factor P.

Bacteria overcome ribosome stalling by employing translation elongation factor P (EF-P), which requires post-translational modification (PTM) for its full activity. However, EF-Ps of the PGKGP subfamily are unmodified. The mechanism behind the ability to avoid PTM while retaining active EF-P requires further examination. Here, we investigate the design principles governing the functionality of unmodified EF-Ps in Escherichia coli. We screen for naturally unmodified EF-Ps with activity in E. coli and discover that the EF-P from Rhodomicrobium vannielii rescues growth defects of a mutant lacking the modification enzyme EF-P-(R)-ß-lysine ligase. We identify amino acids in unmodified EF-P that modulate its activity. Ultimately, we find that substitution of these amino acids in other marginally active EF-Ps of the PGKGP subfamily leads to fully functional variants in E. coli. These results provide strategies to improve heterologous expression of proteins with polyproline motifs in E. coli and give insights into cellular adaptations to optimize protein synthesis.

Pronucleotide Probes Reveal a Diverging Specificity for AMPylation vs UMPylation of Human and Bacterial Nucleotide Transferases.

AMPylation is a post-translational modification utilized by human and bacterial cells to modulate the activity and function of specific proteins. Major AMPylators such as human FICD and bacterial VopS have been studied extensively for their substrate and target scope in vitro. Recently, an AMP pronucleotide probe also facilitated the in situ analysis of AMPylation in living cells. Based on this technology, we here introduce a novel UMP pronucleotide probe and utilize it to profile uninfected and Vibrio parahaemolyticus infected human cells. Mass spectrometric analysis of labeled protein targets reveals an unexpected promiscuity of human nucleotide transferases with an almost identical target set of AMP- and UMPylated proteins. Vice versa, studies in cells infected by V. parahaemolyticus and its effector VopS revealed solely AMPylation of host enzymes, highlighting a so far unknown specificity of this transferase for ATP. Taken together, pronucleotide probes provide an unprecedented insight into the in situ activity profile of crucial nucleotide transferases, which can largely differ from their in vitro activity.

Cu-Catalyzed Azide-Alkyne-Thiol Reaction Forms Ubiquitous Background in Chemical Proteomic Studies.

We report here a Cu-catalyzed azide-alkyne-thiol reaction forming thiotriazoles as the major byproduct under widely used bio-orthogonal protein labeling "click" conditions. The development of Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) had a tremendous impact on many biological discoveries. However, the considered chemoselectivity of CuAAC is hampered by the high reactivity of cysteine free thiols, yielding thiotriazole protein conjugates. The reaction byproducts generate false-positive protein hits in functional proteomic studies. The reported detail investigation of conjugates between chemical probes containing terminal alkynes, azide tags, and cell lysates reveals the formation of thiotriazoles, which can be readily detected by in-gel fluorescence scanning or after peptide and protein enrichment by mass spectrometry-based proteomics. In protein level identification and quantification experiments, the produced fluorescent bands or enriched proteins may not result from the important enzymatically driven reaction and can be falsely assigned as hits. This study provides a complete list of the most common background proteins. The knowledge of this previously overlooked reactivity now leads to the introduction of modified CuAAC conditions, which avoids the undesired product formation, diminishes the background, and hence improves the signal-to-noise ratio.

Chemical Proteomics Reveals Protein Tyrosination Extends Beyond the Alpha-Tubulins in Human Cells.

Tubulin detyrosination-tyrosination cycle regulates the stability of microtubules. With respect to α-tubulins, the tyrosination level is maintained by a single tubulin-tyrosine ligase (TTL). However, the precise dynamics and tubulin isoforms which undergo (de)tyrosination in neurons are unknown. Here, we exploit the substrate promiscuity of the TTL to introduce an O-propargyl-l-tyrosine to neuroblastoma cells and neurons. Mass spectrometry-based chemical proteomics in neuroblastoma cells using the O-propargyl-l-tyrosine probe revealed previously discussed tyrosination of TUBA4A, MAPRE1, and other non-tubulin proteins. This finding was further corroborated in differentiating neurons. Together we present the method for tubulin tyrosination profiling in living cells. Our results show that detyrosination-tyrosination is not restricted to α-tubulins with coded C-terminal tyrosine and is thus involved in fine-tuning of the tubulin and non-tubulin proteins during neuronal differentiation.

Transforming Chemical Proteomics Enrichment into a High-Throughput Method Using an SP2E Workflow.

Protein post-translational modifications (PTMs) play a critical role in the regulation of protein catalytic activity, localization, and protein-protein interactions. Attachment of PTMs onto proteins significantly diversifies their structure and function, resulting in proteoforms. However, the sole identification of post-translationally modified proteins, which are often cell type and disease-specific, is still a highly challenging task. Substoichiometric amounts and modifications of low abundant proteins necessitate the purification or enrichment of the modified proteins. Although the introduction of mass spectrometry-based chemical proteomic strategies has enabled the screening of protein PTMs with increased throughput, sample preparation remains highly time-consuming and tedious. Here, we report an optimized workflow for the enrichment of PTM proteins in a 96-well plate format, which could be extended to robotic automation. This platform allows us to significantly lower the input of total protein, which opens up the opportunity to screen specialized and difficult-to-culture cell lines in a high-throughput manner. The presented SP2E protocol is robust and time- and cost-effective, as well as suitable for large-scale screening of proteoforms. The application of the SP2E protocol will thus enable the characterization of proteoforms in various processes such as neurodevelopment, neurodegeneration, and cancer. This may contribute to an overall acceleration of the recently launched Human Proteoform Project.

Clickable report tags for identification of modified peptides by mass spectrometry.

The identification and quantification of modified peptides are critical for the functional characterization of post-translational protein modifications (PTMs) to elucidate their biological function. Nowadays, quantitative mass spectrometry coupled with various bioinformatic pipelines has been successfully used for the determination of a wide range of PTMs. However, direct characterization of low abundant protein PTMs in bottom-up proteomic workflow remains challenging. Here, we present the synthesis and evaluation of tandem mass spectrometry tags (TMT) which are introduced via click-chemistry into peptides bearing alkyne handles. The fragmentation properties of the two mass tags were validated and used for screening in a model system and analysis of AMPylated proteins. The presented tags provide a valuable tool for diagnostic peak generation to increase confidence in the identification of modified peptides and potentially for direct peptide-PTM quantification from various experimental conditions.

AMPylation profiling during neuronal differentiation reveals extensive variation on lysosomal proteins.

Protein AMPylation is a posttranslational modification with an emerging role in neurodevelopment. In metazoans two highly conserved protein AMP-transferases together with a diverse group of AMPylated proteins have been identified using chemical proteomics and biochemical techniques. However, the function of AMPylation remains largely unknown. Particularly problematic is the localization of thus far identified AMPylated proteins and putative AMP-transferases. We show that protein AMPylation is likely a posttranslational modification of luminal lysosomal proteins characteristic in differentiating neurons. Through a combination of chemical proteomics, gel-based separation of modified and unmodified proteins, and an activity assay, we determine that the modified, lysosomal soluble form of exonuclease PLD3 increases dramatically during neuronal maturation and that AMPylation correlates with its catalytic activity. Together, our findings indicate that AMPylation is a so far unknown lysosomal posttranslational modification connected to neuronal differentiation and it may provide a molecular rationale behind lysosomal storage diseases and neurodegeneration.

Extracellular LGALS3BP regulates neural progenitor position and relates to human cortical complexity.

Basal progenitors (BPs), including intermediate progenitors and basal radial glia, are generated from apical radial glia and are enriched in gyrencephalic species like humans, contributing to neuronal expansion. Shortly after generation, BPs delaminate towards the subventricular zone, where they further proliferate before differentiation. Gene expression alterations involved in BP delamination and function in humans are poorly understood. Here, we study the role of LGALS3BP, so far known as a cancer biomarker, which is a secreted protein enriched in human neural progenitors (NPCs). We show that individuals with LGALS3BP de novo variants exhibit altered local gyrification, sulcal depth, surface area and thickness in their cortex. Additionally, using cerebral organoids, human fetal tissues and mice, we show that LGALS3BP regulates the position of NPCs. Single-cell RNA-sequencing and proteomics reveal that LGALS3BP-mediated mechanisms involve the extracellular matrix in NPCs' anchoring and migration within the human brain. We propose that its temporal expression influences NPCs' delamination, corticogenesis and gyrification extrinsically.

Synthetic post-translational modifications of elongation factor P using the ligase EpmA.

Canonically, tRNA synthetases charge tRNA. However, the lysyl-tRNA synthetase paralog EpmA catalyzes the attachment of (R)-ß-lysine to the ε-amino group of lysine 34 of the translation elongation factor P (EF-P) in Escherichia coli. This modification is essential for EF-P-mediated translational rescue of ribosomes stalled at consecutive prolines. In this study, we determined the kinetics of EpmA and its variant EpmA_A298G to catalyze the post-translational modification of K34 in EF-P with eight noncanonical substrates. In addition, acetylated EF-P was generated using an amber suppression system. The impact of these synthetically modified EF-P variants on in vitro translation of a polyproline-containing NanoLuc luciferase reporter was analyzed. Our results show that natural (R)-ß-lysylation was more effective in rescuing stalled ribosomes than any other synthetic modification tested. Thus, our work not only provides new biochemical insights into the function of EF-P, but also opens a new route to post-translationally modify proteins using EpmA.

MS-Based in Situ Proteomics Reveals AMPylation of Host Proteins during Bacterial Infection.

Bacteria utilize versatile strategies to propagate infections within human cells, e.g., by the injection of effector proteins, which alter crucial signaling pathways. One class of such virulence-associated proteins is involved in the AMPylation of eukaryotic Rho GTPases with devastating effects on viability. In order to get an inventory of AMPylated proteins, several technologies have been developed. However, as they were designed for the analysis of cell lysates, knowledge about AMPylation targets in living cells is largely lacking. Here, we implement a chemical-proteomic method for deciphering AMPylated host proteins in situ during bacterial infection. HeLa cells treated with a previously established cell permeable pronucleotide probe (pro-N6pA) were infected with Vibrio parahaemolyticus, and modified host proteins were identified upon probe enrichment and LC-MS/MS analysis. Three already known targets of the AMPylator VopS-Rac1, RhoA, and Cdc42-could be confirmed, and several other Rho GTPases were additionally identified. These hits were validated in comparative studies with V. parahaemolyticus wild type and a mutant producing an inactive VopS (H348A). The method further allowed to decipher the sites of modification and facilitated a time-dependent analysis of AMPylation during infection. Overall, the methodology provides a reliable detection of host AMPylation in situ and thus a versatile tool in monitoring infection processes.

Protein-AMPylierungs-Identifikation in lebenden Zellen.

Protein AMPylation is a prevalent protein post-translational modification in human cells involved in endoplasmic reticulum stress regulation and neural development. In this article we describe the design, synthesis and application of a pronucleotide probe suitable for in situ fluorescence imaging and chemical protemics profiling of AMPylated proteins. Our probe utilizes straightforward strain-promoted azidealkyne click reaction for fluorescence labeling in living cells.

From Young to Old: AMPylation Hits the Brain.

Protein post-translational modifications (PTMs) are implicated in numerous physiological processes and significantly contribute to complex regulatory networks of protein functions. Recently, a protein PTM called AMPylation was found to play a role in modulation of neurodevelopment and neurodegeneration. Combination of biochemical and chemical proteomic studies has uncovered the prevalence of this PTM in regulation of diverse metabolic pathways. In metazoans, thus far two protein AMP transferases have been identified to introduce AMPylation: FICD and SELO. These two proteins were found to be involved in unfolded protein response and redox homeostasis on the cellular level and in the case of FICD to adjust the development of glial cells and neurons in Drosophila and cerebral organoids, respectively. Together with findings on AMPylation and its association with toxic protein aggregation, we summarize in this Perspective the knowledge and putative future directions of protein AMPylation research.

Structure and Function of an Elongation Factor P Subfamily in Actinobacteria.

Translation of consecutive proline motifs causes ribosome stalling and requires rescue via the action of a specific translation elongation factor, EF-P in bacteria and archaeal/eukaryotic a/eIF5A. In Eukarya, Archaea, and all bacteria investigated so far, the functionality of this translation elongation factor depends on specific and rather unusual post-translational modifications. The phylum Actinobacteria, which includes the genera Corynebacterium, Mycobacterium, and Streptomyces, is of both medical and economic significance. Here, we report that EF-P is required in these bacteria in particular for the translation of proteins involved in amino acid and secondary metabolite production. Notably, EF-P of Actinobacteria species does not need any post-translational modification for activation. While the function and overall 3D structure of this EF-P type is conserved, the loop containing the conserved lysine is flanked by two essential prolines that rigidify it. Actinobacteria's EF-P represents a unique subfamily that works without any modification.

ECE2 regulates neurogenesis and neuronal migration during human cortical development.

During embryonic development, excitatory projection neurons migrate in the cerebral cortex giving rise to organised layers. Periventricular heterotopia (PH) is a group of aetiologically heterogeneous disorders in which a subpopulation of newborn projection neurons fails to initiate their radial migration to the cortex, ultimately resulting in bands or nodules of grey matter lining the lateral ventricles. Although a number of genes have been implicated in its cause, currently they only satisfactorily explain the pathogenesis of the condition for 50% of patients. Novel gene discovery is complicated by the extreme genetic heterogeneity recently described to underlie its cause. Here, we study the neurodevelopmental role of endothelin-converting enzyme-2 (ECE2) for which two biallelic variants have been identified in two separate patients with PH. Our results show that manipulation of ECE2 levels in human cerebral organoids and in the developing mouse cortex leads to ectopic localisation of neural progenitors and neurons. We uncover the role of ECE2 in neurogenesis, and mechanistically, we identify its involvement in the generation and secretion of extracellular matrix proteins in addition to cytoskeleton and adhesion.

A Pronucleotide Probe for Live-Cell Imaging of Protein AMPylation.

Conjugation of proteins to AMP (AMPylation) is a prevalent post-translational modification (PTM) in human cells, involved in the regulation of unfolded protein response and neural development. Here we present a tailored pronucleotide probe suitable for in situ imaging and chemical proteomics profiling of AMPylated proteins. Using straightforward strain-promoted azide-alkyne click chemistry, the probe provides stable fluorescence labelling in living cells.

FICD activity and AMPylation remodelling modulate human neurogenesis.

Posttranslational modification (PTM) of proteins represents an important cellular mechanism for controlling diverse functions such as signalling, localisation or protein-protein interactions. AMPylation (also termed adenylylation) has recently been discovered as a prevalent PTM for regulating protein activity. In human cells AMPylation has been exclusively studied with the FICD protein. Here we investigate the role of AMPylation in human neurogenesis by introducing a cell-permeable propargyl adenosine pronucleotide probe to infiltrate cellular AMPylation pathways and report distinct modifications in intact cancer cell lines, human-derived stem cells, neural progenitor cells (NPCs), neurons and cerebral organoids (COs) via LC-MS/MS as well as imaging methods. A total of 162 AMP modified proteins were identified. FICD-dependent AMPylation remodelling accelerates differentiation of neural progenitor cells into mature neurons in COs, demonstrating a so far unknown trigger of human neurogenesis.

Selective Activation of Human Caseinolytic Protease†P (ClpP).

Caseinolytic protease P (ClpP) is the proteolytic component of the ClpXP protein degradation complex. Eukaryotic ClpP was recently found to act within the mitochondria-specific unfolded protein response (UPRmt ). However, its detailed function and dedicated regulation remain largely unexplored. A small molecule (D9) acts as a potent and species-selective activator of human ClpP (hClpP) by mimicking the natural chaperone ClpX. Structure-activity relationship studies highlight the importance of a halogenated benzyl motif within D9 that interacts with a unique aromatic amino acid network in hClpP. Mutational and structural studies suggest that this YYW motif tightly controls hClpP activity and regulates substrate turnover by interaction with cognate ligands. This signature motif is unique to ClpP from higher organisms and does not exist in tested bacterial homologues, allowing a species-selective analysis. Thus, D9 is a versatile tool to analyze mechanistic features of hClpP.

5-Substituted Pyrimidine and 7-Substituted 7-Deazapurine dNTPs as Substrates for DNA Polymerases in Competitive Primer Extension in the Presence of Natural dNTPs.

A complete series of 5-substituted uracil or cytosine, as well as 7-substituted 7-deazaadenine and 7-deazaguanine 2'-deoxyribonucleoside triphosphates (dNTPs) bearing substituents of increasing bulkiness (H, Me, vinyl, ethynyl, and phenyl) were systematically studied in competitive primer extension in the presence of their natural counterparts (nonmodified dNTPs), and their kinetic data were determined. The results show that modified dNTPs bearing π-electron-containing substituents (vinyl, ethynyl, Ph) are typically excellent substrates for DNA polymerases comparable to or better than natural dNTPs. The kinetic studies revealed that these modified dNTPs have higher affinity to the active site of the enzyme-primer-template complex, and the calculations (semiempirical quantum mechanical scoring function) suggest that it is due to the cation-π interaction of the modified dNTP with Arg629 in the active site of Bst DNA polymerase.

Flexible double-headed cytosine-linked 2'-deoxycytidine nucleotides. Synthesis, polymerase incorporation to DNA and interaction with DNA methyltransferases.

New types of double-headed 2'-deoxycytidine 5'-O-triphosphates (dC(XC)TPs) bearing another cytosine or 5-fluorocytosine linked through a flexible propargyl, homopropargyl or pent-1-ynyl linker to position 5 were prepared by the aqueous Sonogashira cross-coupling reactions of 5-iodo-dCTP with the corresponding (fluoro)cytosine-alkynes. The modified dC(XC)TPs were good substrates for DNA polymerases and were used for enzymatic synthesis of cytosine-functionalized DNA by primer extension or PCR. The cytosine- or fluorocytosine-linked DNA probes did not significantly inhibit DNA methyltransferases and did not cross-link to these proteins.

7-Aryl-7-deazaadenine 2'-deoxyribonucleoside triphosphates (dNTPs): better substrates for DNA polymerases than dATP in competitive incorporations.

A series of 7-substituted 7-deazaadenine and 5-substituted cytosine 2'-deoxyribonucleoside triphosphates (dNTPs) were tested for their competitive incorporations (in the presence of dATP and dCTP) into DNA by several DNA polymerases by using analysis based on cleavage by restriction endonucleases. 7-Aryl-7-deazaadenine dNTPs were more efficient substrates than dATP because of their higher affinity for the active site of the enzyme, as proved by kinetic measurements and calculations.