Glutamate 1-semialdehyde aminotransferase is connected to GluTR by GluTR-binding protein and contributes to the rate-limiting step of 5-aminolevulinic acid synthesis

Tetrapyrroles play fundamental roles in crucial processes including photosynthesis, respiration, and catalysis. In plants, 5-aminolevulinic acid (ALA) is the common precursor of tetrapyrroles. ALA is synthesized from activated glutamate by the enzymes glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde aminotransferase (GSAAT). ALA synthesis is recognized as the rate-limiting step in this pathway. We aimed to explore the contribution of GSAAT to the control of ALA synthesis and the formation of a protein complex with GluTR. In Arabidopsis thaliana, two genes encode GSAAT isoforms: GSA1 and GSA2. A comparison of two GSA knockout mutants with the wild-type revealed the correlation of reduced GSAAT activity and ALA-synthesizing capacity in leaves with lower chlorophyll content. Growth and green pigmentation were more severely impaired in gsa2 than in gsa1, indicating the predominant role of GSAAT2 in ALA synthesis. Interestingly, GluTR accumulated to higher levels in gsa2 than in the wild-type and was mainly associated with the plastid membrane. We propose that the GSAAT content modulates the amount of soluble GluTR available for ALA synthesis. Several different biochemical approaches revealed the GSAAT–GluTR interaction through the assistance of GluTR-binding protein (GBP). A modeled structure of the tripartite protein complex indicated that GBP mediates the stable association of GluTR and GSAAT for adequate ALA synthesis.Peer Reviewe

Pretubulysin derived probes as novel tools for monitoring the microtubule network via activity-based protein profiling and fluorescence microscopy

Microtubules (mt) are highly dynamic polymers composed of alpha- and beta-tubulin monomers that are present in all dividing and non-dividing cells. A broad variety of natural products exists that are known to interfere with the microtubule network, by either stabilizing or de-stabilizing these rope-like polymers. Among those tubulysins represent a new and potent class of cytostatic tetrapeptides originating from myxobacteria. Early studies suggested that tubulysins interact with the eukaryotic cytoskeleton by inhibition of tubulin polymerization with EC50 values in the picomolar range. Recently, pretubulysins have been described to retain the high tubulindegradation activity of their more complex tubulysin relatives and represent an easier synthetic target with an efficient synthesis already in place. Although tubulin has been suggested as the dedicated target of tubulysin a comprehensive molecular target analysis of pretubulysin in the context of the whole proteome has not been carried out so far. Here we utilize synthetic chemistry to develop two pretubulysin photoaffinity probes which were applied in cellular activity-based protein profiling and imaging studies in order to unravel and visualize dedicated targets. Our results clearly show a remarkable selectivity of pretubulysin for beta-tubulin which we independently confirmed by a mass-spectrometry based proteomic profiling platform as well as by tubulin antibody based co-staining on intact cells

Lysine acetylation regulates moonlighting activity of the E2 subunit of the chloroplast pyruvate dehydrogenase complex in Chlamydomonas

The dihydrolipoamide acetyltransferase subunit DLA2 of the chloroplast pyruvate dehydrogenase complex (cpPDC) in the green alga Chlamydomonas reinhardtii has previously been shown to possess moonlighting activity in chloroplast gene expression. Under mixotrophic growth conditions, DLA2 forms part of a ribonucleoprotein particle (RNP) with the psbA mRNA that encodes the D1 protein of the photosystem II (PSII) reaction center. Here, we report on the characterization of the molecular switch that regulates shuttling of DLA2 between its functions in carbon metabolism and D1 synthesis. Determination of RNA‐binding affinities by microscale thermophoresis demonstrated that the E3‐binding domain (E3BD) of DLA2 mediates psbA‐specific RNA recognition. Analyses of cpPDC formation and activity, as well as RNP complex formation, showed that acetylation of a single lysine residue (K197) in E3BD induces the release of DLA2 from the cpPDC, and its functional shift towards RNA binding. Moreover, Förster resonance energy transfer microscopy revealed that psbA mRNA/DLA2 complexes localize around the chloroplast's pyrenoid. Pulse labeling and D1 re‐accumulation after induced PSII degradation strongly suggest that DLA2 is important for D1 synthesis during de novo PSII biogenesis.Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Peer Reviewe

Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acetylation

Protein acetylation is a highly frequent protein modification. However, comparatively little is known about its enzymatic machinery. N-alpha-acetylation (NTA) and epsilon-lysine acetylation (KA) are known to be catalyzed by distinct families of enzymes (NATs andKATs, respectively), although the possibility that the sameGCN5-relatedN-acetyltransferase (GNAT) can perform both functions has been debated. Here, we discovered a new family of plastid-localizedGNATs, which possess a dual specificity. All characterizedGNATfamily members display a number of unique features. Quantitative mass spectrometry analyses revealed that these enzymes exhibit both distinctKAand relaxedNTAspecificities. Furthermore, inactivation ofGNAT2 leads to significantNTAorKAdecreases of several plastid proteins, while proteins of other compartments were unaffected. The data indicate that these enzymes have specific protein targets and likely display partly redundant selectivity, increasing the robustness of the acetylation processin vivo. In summary, this study revealed a new layer of complexity in the machinery controlling this prevalent modification and suggests that other eukaryoticGNATs may also possess these previously underappreciated broader enzymatic activities

ManuKnowVis: How to Support Different User Groups in Contextualizing and Leveraging Knowledge Repositories

We present ManuKnowVis, the result of a design study, in which we contextualize data from multiple knowledge repositories of a manufacturing process for battery modules used in electric vehicles. In data-driven analyses of manufacturing data, we observed a discrepancy between two stakeholder groups involved in serial manufacturing processes: Knowledge providers (e.g., engineers) have domain knowledge about the manufacturing process but have difficulties in implementing data-driven analyses. Knowledge consumers (e.g., data scientists) have no first-hand domain knowledge but are highly skilled in performing data-driven analyses. ManuKnowVis bridges the gap between providers and consumers and enables the creation and completion of manufacturing knowledge. We contribute a multi-stakeholder design study, where we developed ManuKnowVis in three main iterations with consumers and providers from an automotive company. The iterative development led us to a multiple linked view tool, in which, on the one hand, providers can describe and connect individual entities (e.g., stations or produced parts) of the manufacturing process based on their domain knowledge. On the other hand, consumers can leverage this enhanced data to better understand complex domain problems, thus, performing data analyses more efficiently. As such, our approach directly impacts the success of data-driven analyses from manufacturing data. To demonstrate the usefulness of our approach, we carried out a case study with seven domain experts, which demonstrates how providers can externalize their knowledge and consumers can implement data-driven analyses more efficiently

Thiol Redox Proteomics for Identifying Redox-Sensitive Cysteine Residues Within the Protein of Interest During Stress

Vogelsang L, Eirich J, Finkemeier I, Dietz K-J. Thiol Redox Proteomics for Identifying Redox-Sensitive Cysteine Residues Within the Protein of Interest During Stress. Methods in Molecular Biology . 2024;2832.Redox modulation is a common posttranslational modification to regulate protein activity. The targets of oxidizing agents are cysteine residues(Cys), which have to be exposed at the surface of the proteins and are characterized by an environment that favors redox modulation. This includes their protonation state and the neighboring amino acids. The Cys redox state can be assessed experimentally by redox titrations to determine the midpoint redox potential in the protein. Exposed cysteine residues and putative intramolecular disulfide bonds can be predicted by alignments with structural data using dedicated software tools and information on conserved cysteine residues. Labeling with light and heavy reagents, such as N-ethylmaleimide (NEM), followed by mass spectrometric analysis, allows for the experimental determination of redox-responsive cysteine residues. This type of thiol redox proteomics is a powerful approach to assessing the redox state of the cell, e.g., in dependence on environmental conditions and, in particular, under abiotic stress. © 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

Specificity and dynamics of H2O2 detoxification by the cytosolic redox regulatory network as revealed by in vitro reconstitution

Vogelsang L, Eirich J, Finkemeier I, Dietz K-J. Specificity and dynamics of H2O2 detoxification by the cytosolic redox regulatory network as revealed by in vitro reconstitution. Redox Biology. 2024;72: 103141.The thiol redox state is a decisive functional characteristic of proteins in cell biology. Plasmatic cell compartments maintain a thiol-based redox regulatory network linked to the glutathione/glutathione disulfide couple (GSH/GSSG) and the NAD(P)H system. The basic network constituents are known and in vivo cell imaging with gene-encoded probes have revealed insight into the dynamics of the [GSH]2/[GSSG] redox potential, cellular H2O2 and NAD(P)H+H+ amounts in dependence on metabolic and environmental cues. Less understood is the contribution and interaction of the network components, also because of compensatory reactions in genetic approaches. Reconstituting the cytosolic network of Arabidopsis thaliana in vitro from fifteen recombinant proteins at in vivo concentrations, namely glutathione peroxidase-like (GPXL), peroxiredoxins (PRX), glutaredoxins (GRX), thioredoxins, NADPH-dependent thioredoxin reductase A and glutathione reductase and applying Grx1-roGFP2 or roGFP2-Orp1 as dynamic sensors, allowed for monitoring the response to a single H2O2 pulse. The major change in thiol oxidation as quantified by mass spectrometry-based proteomics occurred in relevant peptides of GPXL, and to a lesser extent of PRX, while other Cys-containing peptides only showed small changes in their redox state and protection. Titration of ascorbate peroxidase (APX) into the system together with dehydroascorbate reductase lowered the oxidation of the fluorescent sensors in the network but was unable to suppress it. The results demonstrate the power of the network to detoxify H2O2, the partially independent branches of electron flow with significance for specific cell signaling and the importance of APX to modulate the signaling without suppressing it and shifting the burden to glutathione oxidation. Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved

Pretubulysin derived probes as novel tools for monitoring the microtubule network via activity-based protein profiling and fluorescence microscopy

Microtubules (mt) are highly dynamic polymers composed of alpha- and beta-tubulin monomers that are present in all dividing and non-dividing cells. A broad variety of natural products exists that are known to interfere with the microtubule network, by either stabilizing or de-stabilizing these rope-like polymers. Among those tubulysins represent a new and potent class of cytostatic tetrapeptides originating from myxobacteria. Early studies suggested that tubulysins interact with the eukaryotic cytoskeleton by inhibition of tubulin polymerization with EC50 values in the picomolar range. Recently, pretubulysins have been described to retain the high tubulindegradation activity of their more complex tubulysin relatives and represent an easier synthetic target with an efficient synthesis already in place. Although tubulin has been suggested as the dedicated target of tubulysin a comprehensive molecular target analysis of pretubulysin in the context of the whole proteome has not been carried out so far. Here we utilize synthetic chemistry to develop two pretubulysin photoaffinity probes which were applied in cellular activity-based protein profiling and imaging studies in order to unravel and visualize dedicated targets. Our results clearly show a remarkable selectivity of pretubulysin for beta-tubulin which we independently confirmed by a mass-spectrometry based proteomic profiling platform as well as by tubulin antibody based co-staining on intact cells

ManEx: The Visual Analysis of Measurements for the Assessment of Errors in Electrical Engines

Electrical engines are a key technology all automotive manufacturers must master to stay competitive. Engineers need to analyze an overwhelming number of engine measurements to improve the manufacturing for this technology. They are hindered in the task of analyzing large numbers of engines, however, by the following challenges: 1) Engines comprise a complex hierarchical structure of subcomponents. 2) Locating the cause of errors along manufacturing processes is a difficult procedure. 3) Large numbers of heterogeneous measurements impair the ability to explain errors in engines. We address these challenges in a design study with automotive engineers and by developing the visual analytics system Manufacturing Explorer (ManEx), which provides interactive interfaces to analyze measurements of engines across the manufacturing process. ManEx was validated by five experts. Our results suggest high usability and usefulness scores and the improvement of a real-world manufacturing process. Specifically, with ManEx, experts reduced scraped parts by over 3%

IRVINE: A Design Study on Analyzing Correlation Patterns of Electrical Engines

In this design study, we present IRVINE, a Visual Analytics (VA) system, which facilitates the analysis of acoustic data to detect and understand previously unknown errors in the manufacturing of electrical engines. In serial manufacturing processes, signatures from acoustic data provide valuable information on how the relationship between multiple produced engines serves to detect and understand previously unknown errors. To analyze such signatures, IRVINE leverages interactive clustering and data labeling techniques, allowing users to analyze clusters of engines with similar signatures, drill down to groups of engines, and select an engine of interest. Furthermore, IRVINE allows to assign labels to engines and clusters and annotate the cause of an error in the acoustic raw measurement of an engine. Since labels and annotations represent valuable knowledge, they are conserved in a knowledge database to be available for other stakeholders. We contribute a design study, where we developed IRVINE in four main iterations with engineers from a company in the automotive sector. To validate IRVINE, we conducted a field study with six domain experts. Our results suggest a high usability and usefulness of IRVINE as part of the improvement of a real-world manufacturing process. Specifically, with IRVINE domain experts were able to label and annotate produced electrical engines more than 30% faster