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Publikation

Frolov, A., Didio, A., Ihling, C., Chantzeva, V., Grishina, T., Hoehenwarter, W., Sinz, A., Smolikova, G., Bilova, T. & Medvedev, S. The effect of simulated microgravity on the Brassica napus seedling proteome. Funct Plant Biol 45, 440-452, (2018) DOI: 10.1071/FP16378

The magnitude and the direction of the gravitational field represent an important environmental factor affecting plant development. In this context, the absence or frequent alterations of the gravity field (i.e. microgravity conditions) might compromise extraterrestrial agriculture and hence space inhabitation by humans. To overcome the deleterious effects of microgravity, a complete understanding of the underlying changes on the macromolecular level is necessary. However, although microgravity-related changes in gene expression are well characterised on the transcriptome level, proteomic data are limited. Moreover, information about the microgravity-induced changes in the seedling proteome during seed germination and the first steps of seedling development is completely missing. One of the valuable tools to assess gravity-related issues is 3D clinorotation (i.e. rotation in two axes). Therefore, here we address the effects of microgravity, simulated by a two-axial clinostat, on the proteome of 24- and 48-h-old seedlings of oilseed rape (Brassica napus L.). The liquid chromatography-MS-based proteomic analysis and database search revealed 95 up- and 38 downregulated proteins in the tryptic digests obtained from the seedlings subjected to simulated microgravity, with 42 and 52 annotations detected as being unique for 24- and 48-h treatment times, respectively. The polypeptides involved in protein metabolism, transport and signalling were annotated as the functional groups most strongly affected by 3-D clinorotation. 
Publikation

Wessjohann, L. A., Kreye, O. & Rivera, D. G. One-Pot assembly of amino acid bridged hybrid macromulticyclic cages through multiple multicomponent macrocyclizations. Angew Chem Int Ed. = Angew Chem. 56 = 129 , 3501-3505 = 3555-3559., (2017) DOI: 10.1002/anie.201610801

An important development in the field of macrocyclization strategies towards molecular cages is described. The approach comprises the utilization of a double Ugi four-component macrocyclization for the assembly of macromulticycles with up to four different tethers, that is, hybrid cages. The innovation of this method rests on setting up the macromulticycle connectivities not through the tethers but through the bridgeheads, which in this case involve N-substituted amino acids. Both dilution and metal-template-driven macrocyclization conditions were implemented with success, enabling the one-pot formation of cryptands and cages including steroidal, polyether, heterocyclic, peptidic, and aryl tethers. This method demonstrates substantial complexity-generating character and is suitable for applications in molecular recognition and catalysis.
Publikation

Soboleva, A., Vikhnina, M., Grishina, T. & Frolov, A. Probing protein glycation by chromatography and mass spectrometry: analysis of glycation adducts Int J Mol Sci 18(12), 2557, (2017) DOI: 10.3390/ijms18122557

Glycation is a non-enzymatic post-translational modification of proteins, formed by the reaction of reducing sugars and α-dicarbonyl products of their degradation with amino and guanidino groups of proteins. Resulted early glycation products are readily involved in further transformation, yielding a heterogeneous group of advanced glycation end products (AGEs). Their formation is associated with ageing, metabolic diseases, and thermal processing of foods. Therefore, individual glycation adducts are often considered as the markers of related pathologies and food quality. In this context, their quantification in biological and food matrices is required for diagnostics and establishment of food preparation technologies. For this, exhaustive protein hydrolysis with subsequent amino acid analysis is the strategy of choice. Thereby, multi-step enzymatic digestion procedures ensure good recoveries for the most of AGEs, whereas tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) mode with stable isotope dilution or standard addition represents “a gold standard” for their quantification. Although the spectrum of quantitatively assessed AGE structures is continuously increases, application of untargeted profiling techniques for identification of new products is desired, especially for in vivo characterization of anti-glycative systems. Thereby, due to a high glycative potential of plant metabolites, more attention needs to be paid on plant-derived AGEs.
Publikation

Smolikova, G., Kreslavski, V., Shiroglazova, O., Bilova, T., Sharova, E., Frolov, A. & Medvedev, S. Photochemical activity changes accompanying the embryogenesis of pea (Pisum sativum) with yellow and green cotyledon. Functional Plant Biol. 45, 228-235, (2017) DOI: 10.1071/FP16379

The pea seeds are photosynthetically active until the end of the maturation phase, when the embryonic chlorophylls degrade. However, in some cultivars, the underlying mechanisms are compromised, and the mature seeds preserve green colour. The residual chlorophylls can enhance oxidative degradation of reserve biomolecules, and affect thereby the quality, shelf life and nutritive value of seeds. Despite this, the formation, degradation, and physical properties of the seed chlorophylls are still not completely characterised. So here we address the dynamics of seed photochemical activity in the yellow- and green-seeded pea cultivars by the pulse amplitude modulation (PAM) fluorometric analysis. The experiments revealed the maximal photochemical activity at the early- and mid-cotyledon stages. Thereby, the active centres of PSII were saturated at the light intensity of 15–20 µmol photons m–2 s–1. Despite of their shielding from the light by the pod wall and seed coat, photochemical reactions can be registered in the seeds with green embryo. Importantly, even at the low light intensities, the photochemical activity in the coats and cotyledons could be detected. The fast transients of the chlorophyll a fluorescence revealed a higher photochemical activity in the coat of yellow-seeded cultivars in comparison to those with the green-seeded ones. However, it declined rapidly in all seeds at the late cotyledon stage, and was accompanied with the decrease of the seed water content. Thus, the termination of photosynthetic activity in seeds is triggered by their dehydration.
Publikation

Soboleva, A., Modzel, M., Didio, A., Plociennik, H., Kijewska, M., Grischina, T., Karonova, T., Bilova, T., Stefanov, V., Stefanowicz, P. & Frolov, A. Quantification of prospective type 2 diabetes mellitus biomarkers by stable isotope dilution with bi-labeled standard glycated peptides. Anal Methods 9 , 409-418, (2017) DOI: 10.1039/C6AY02483A

Type 2 diabetes mellitus (T2DM) is a complex group of disorders, characterized by hyperglycemia, insulin resistance and insulin deficiency. In human blood, hyperglycemia ultimately results in the enhancement of glycation – a posttranslational modification formed by the interaction of protein amino groups with glucose. The resulting fructosamines (Amadori compounds) readily undergo further degradation resulting in advanced glycation end products (AGEs), known to be pro-inflammatory in humans. These compounds are highly heterogeneous and characteristic of advanced stages of the disease, whereas fructosamines are recognized markers of early diabetes stages (HbA1C, glycated albumin). Recently, individual plasma protein glycation sites were proposed as promising T2DM biomarkers sensitive to short-term fluctuations of plasma glucose. However, corresponding absolute quantification strategies, applicable in regular clinical practice, are still not established. Therefore, here we propose a new analytical approach aiming at reproducible and precise quantification of multiple glycated peptides in human plasma tryptic digests. Thereby, the standard peptides comprised a 13C,15N-labeled lysyl residue, a dabsyl moiety for determination of standard amounts, and a cleavable linker. Known amounts of these peptides were spiked to plasma samples prior to tryptic digestion, quantification relying on stable isotope dilution. The method was demonstrated to be applicable for quantification of individual glycated sites in T2DM patients and non-diabetic controls.

Publikation

Soboleva, A., Schmidt, R., Vikhnina, M., Grishina, T. & Frolov, A. Maillard Proteomics: opening new pages. Int J Mol Sci. 18(12), 2677, (2017) DOI: 10.3390/ijms18122677

Protein glycation is a ubiquitous non-enzymatic post-translational modification, formed by reaction of protein amino and guanidino groups with carbonyl compounds, presumably reducing sugars and α-dicarbonyls. Resulting advanced glycation end products (AGEs) represent a highly heterogeneous group of compounds, deleterious in mammals due to their pro-inflammatory effect, and impact in pathogenesis of diabetes mellitus, Alzheimer’s disease and ageing. The body of information on the mechanisms and pathways of AGE formation, acquired during the last decades, clearly indicates a certain site-specificity of glycation. It makes characterization of individual glycation sites a critical pre-requisite for understanding in vivo mechanisms of AGE formation and developing adequate nutritional and therapeutic approaches to reduce it in humans. In this context, proteomics is the methodology of choice to address site-specific molecular changes related to protein glycation. Therefore, here we summarize the methods of Maillard proteomics, specifically focusing on the techniques providing comprehensive structural and quantitative characterization of glycated proteome. Further, we address the novel break-through areas, recently established in the field of Maillard research, i.e., in vitro models based on synthetic peptides, site-based diagnostics of metabolism-related diseases (e.g., diabetes mellitus), proteomics of anti-glycative defense, and dynamics of plant glycated proteome during ageing and response to environmental stress. View Full-Text.
Publikation

Cotrim, C. A., Weidner, A., Strehmel, N., Bisol, T. B., Meyer, D., Brandt, W., Wessjohann, L. A. & Stubbs, M. T. A distinct aromatic prenyltransferase associated with the futalosine pathway. ChemistrySelect 2, 9319-9325, (2017) DOI: 10.1002/slct.201702151

Menaquinone (MK) is an electron carrier molecule essential for respiration in most Gram positive bacteria. A crucial step in MK biosynthesis involves the prenylation of an aromatic molecule, catalyzed by integral membrane prenyltransferases of the UbiA (4-hydroxybenzoate oligoprenyltransferase) superfamily. In the classical MK biosynthetic pathway, the prenyltransferase responsible is MenA (1,4-dihydroxy-2-naphthoate octaprenyltransferase). Recently, an alternative pathway for formation of MK, the so-called futalosine pathway, has been described in certain micro-organisms. Until now, five soluble enzymes (MqnA-MqnE) have been identified in the first steps. In this study, the genes annotated as ubiA from T. thermophilus and S. lividans were cloned, expressed and investigated for prenylation activity. The integral membrane proteins possess neither UbiA nor MenA activity and represent a distinct class of prenyltransferases associated with the futalosine pathway that we term MqnP. We identify a critical residue within a highly conserved Asp-rich motif that serves to distinguish between members of the UbiA superfamily.
Publikation

Frolov, A., Bilova, T., Paudel, G., Berger, R., Balcke, G. U., Birkemeyer, C. & Wessjohann, L. A. Early responses of mature Arabidopsis thaliana plants to reduced water potential in the agar-based polyethylene glycol infusion drought model. J Plant Physiol. 208, 70-83, (2017) DOI: 10.1016/j.jplph.2016.09.013

Drought is one of the most important environmental stressors resulting in increasing losses of crop plant productivity all over the world. Therefore, development of new approaches to increase the stress tolerance of crop plants is strongly desired. This requires precise and adequate modeling of drought stress. As this type of stress manifests itself as a steady decrease in the substrate water potential (ψw), agar plates infused with polyethylene glycol (PEG) are the perfect experimental tool: they are easy in preparation and provide a constantly reduced ψw, which is not possible in soil models. However, currently, this model is applicable only to seedlings and cannot be used for evaluation of stress responses in mature plants, which are obviously the most appropriate objects for drought tolerance research. To overcome this limitation, here we introduce a PEG-based agar infusion model suitable for 6–8-week-old A. thaliana plants, and characterize, to the best of our knowledge for the first time, the early drought stress responses of adult plants grown on PEG-infused agar. We describe essential alterations in the primary metabolome (sugars and related compounds, amino acids and polyamines) accompanied by qualitative and quantitative changes in protein patterns: up to 87 unique stress-related proteins were annotated under drought stress conditions, whereas further 84 proteins showed a change in abundance. The obtained proteome patterns differed slightly from those reported for seedlings and soil-based models.

Publikation

Bilova, T., Lukasheva, E., Brauch, D., Greifenhagen, U., Paudel, G., Tarakhovskaya, E., Frolova, N., Mittasch, J., Balcke, G. U., Tissier, A., Osmolovskaya, N., Vogt, T., Wessjohann, L. A., Birkemeyer, C., Milkowski, C. & Frolov A Snapshot of the Plant Glycated Proteome: structural, functional, and mechanistic aspects J. Biol. Chem. 291, 7621-7636, (2016) DOI: 10.1074/jbc.M115.678581

Glycation is the reaction of carbonyl compounds (reducing sugars and α-dicarbonyls) with amino acids, lipids, and proteins, yielding early and advanced glycation end products (AGEs). The AGEs can be formed via degradation of early glycation intermediates (glycoxidation) and by interaction with the products of monosaccharide autoxidation (autoxidative glycosylation). Although formation of these potentially deleterious compounds is well characterized in animal systems and thermally treated foods, only a little information about advanced glycation in plants is available. Thus, the knowledge of the plant AGE patterns and the underlying pathways of their formation are completely missing. To fill this gap, we describe the AGE-modified proteome of Brassica napus and characterize individual sites of advanced glycation by the methods of liquid chromatography-based bottom-up proteomics. The modification patterns were complex but reproducible: 789 AGE-modified peptides in 772 proteins were detected in two independent experiments. In contrast, only 168 polypeptides contained early glycated lysines, which did not resemble the sites of advanced glycation. Similar observations were made with Arabidopsis thaliana. The absence of the early glycated precursors of the AGE-modified protein residues indicated autoxidative glycosylation, but not glycoxidation, as the major pathway of AGE formation. To prove this assumption and to identify the potential modifying agents, we estimated the reactivity and glycative potential of plant-derived sugars using a model peptide approach and liquid chromatography-mass spectrometry-based techniques. Evaluation of these data sets together with the assessed tissue carbohydrate contents revealed dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, ribulose, erythrose, and sucrose as potential precursors of plant AGEs.

Bücher und Buchkapitel

Bilova, T., Greifenhagen, U., Paudel, G., Lukasheva, E., Brauch, D., Osmolovskaya, N., Tarakhovskaya, E., Balcke, G. U., Tissier, A., Vogt, T., Milkowski, C., Birkemeyer, C., Wessjohann, L. & Frolov, A. Glycation of Plant Proteins under Environmental Stress — Methodological Approaches, Potential Mechanisms and Biological Role. In: Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives (Shanker, A. K.; Shanker, C.). (2016) ISBN: 978-953-51-2250-0 DOI: 10.5772/61860

 Environmental stress is one of the major factors reducing crop productivity. Due to the oncoming climate changes, the effects of drought and high light on plants play an increasing role in modern agriculture. These changes are accompanied with a progressing contamination of soils with heavy metals. Independent of their nature, environmental alterations result in development of oxidative stress, i.e. increase of reactive oxygen species (ROS) contents, and metabolic adjustment, i.e. accumulation of soluble primary metabolites (amino acids and sugars). However, a simultaneous increase of ROS and sugar concentrations ultimately results in protein glycation, i.e. non-enzymatic interaction of reducing sugars or their degradation products (α-dicarbonyls) with proteins. The eventually resulting advanced glycation end-products (AGEs) are known to be toxic and pro-inflammatory in mammals. Recently, their presence was unambiguously demonstrated in vivo in stressed Arabidopsis thaliana plants. Currently, information on protein targets, modification sites therein, mediators and mechanisms of plant glycation are being intensively studied. In this chapter, we comprehensively review the methodological approaches for plant glycation research and discuss potential mechanisms of AGE formation under stress conditions. On the basis of these patterns and additional in vitro experiments, the pathways and mechanisms of plant glycation can be proposed.

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