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N-terminome analysis of tomato P69D knock-out mutant total leaf proteomes treated with and without recominant P69D

ABSTRACT: The intercellular space or apoplast constitutes the main interface in plant-pathogen interactions. Apoplastic subtilisin-like proteases -subtilases- may play an important role in defence and they have been identified as targets of pathogen-secreted effector proteins. However, most evidence is limited to the interaction of plants with non-vascular foliar pathogens. Here, we characterise the role of the Solanaceae-specific P69 subtilase family in the interaction between tomato and the vascular bacterial wilt pathogen Ralstonia solanacearum.

INSTRUMENT(S): impact II

ORGANISM(S): Solanum Lycopersicum

TISSUE(S): Leaf

SUBMITTER: Pitter Huesgen

LAB HEAD: Pitter Huesgen

PROVIDER: PXD044637 | Pride | 2023-12-27

REPOSITORIES: Pride

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Comparison of tomato wild type and P69D knock-out mutant apoplast proteome and N-terminomes

Project description:The intercellular space or apoplast constitutes the main interface in plant-pathogen interactions. Apoplastic subtilisin-like proteases -subtilases- may play an important role in defence and they have been identified as targets of pathogen-secreted effector proteins. However, most evidence is limited to the interaction of plants with non-vascular foliar pathogens. Here, we characterise the role of the Solanaceae-specific P69 subtilase family in the interaction between tomato and the vascular bacterial wilt pathogen Ralstonia solanacearum.

2023-12-27 | PXD044635 | Pride

Calpain Cleavage of Junctophilin-2 generates a Spectrum of Calcium-dependent Cleavage Products and DNA-rich NT1-Fragment Domains in Cardiomyocytes - PRM Dataset

Project description:Calpains are calcium (Ca2+) activated neutral proteases that are involved in several essential signaling pathways. One Calpain-specific proteolytic target is Junctophilin-2 (JP2), an important structural protein of Ca2+ release units required for the excitation-contraction coupling in cardiomyocytes. While downregulation of JP2 by Calpain cleavage in mouse models of heart failure has been reported previously, the precise molecular identity of the Calpain cleavage sites and the (patho-) physiological roles of the JP2 proteolytic products remain controversial. We systematically analyzed the JP2 cleavage fragments as function of Calpain-1 versus Calpain-2 proteolytic activities, revealing that both Calpain isoforms preferentially cleave mouse JP2 at R565, but subsequently also at three additional secondary Calpain cleavage sites. We identified the Calpain-specific primary cleavage products not only in mouse but also in human iPSC-derived cardiomyocytes (hiPSC-CMs). Knockout of RyR2 in hiPSC-CMs destabilized JP2 resulting in an increase of the Calpain-specifc cleavage fragments. The primary N-terminal cleavage product (NT1) accumulated in the nucleus of mouse and human cardiomyocytes in a Ca2+ dependent manner, closely associated with DNA-rich chromosomal regions, where NT1 is proposed to function as a cardioprotective transcriptional regulator in heart failure. Taken together, our data suggest that stabilizing NT1 by preventing secondary cleavage events by Calpain and other proteases could be an important therapeutic target for future studies.

2022-08-12 | PXD031424 | Pride

Proteome analysis of pathogen-induced regulated cell death in maize

Project description:Regulated cell death (RCD) is crucial for plant development, as well as in decision-making in plant-pathogen interactions. Previous studies revealed components of the molecular network controlling RCD, including different proteases. However, the identity, the proteolytic network as well as molecular components involved in the initiation and execution of distinct plant RCD processes, still remain largely elusive. We analyzed the proteome of Z. mays leaves treated with the effector avrRxo1, the mycotoxin Fumonisin B1 (FB1), or the phytohormone salicylic acid (SA) to dissect plant cellular processes related to cell death and plant immunity. We found highly distinct and time-dependent biological processes being activated on proteome level in response to avrRxo1, FB1 and SA. This study characterizes distinct RCD responses in Z. mays and provides a framework for the mechanistic exploration of components involved in the initiation and execution of cell death.

2023-07-13 | PXD041377 | Pride

N-terminome analysis of pathogen-induced cell death in maize

Project description:Regulated cell death (RCD) is crucial for plant development, as well as in decision-making in plant-pathogen interactions. Previous studies revealed components of the molecular network controlling RCD, including different proteases. However, the identity, the proteolytic network as well as molecular components involved in the initiation and execution of distinct plant RCD processes, still remain largely elusive. We analyzed the N-terminome of Z. mays leaves treated with the Xanthomonas effector avrRxo1, the mycotoxin Fumonisin B1 (FB1), or the phytohormone salicylic acid (SA) to dissect plant cellular processes related to cell death and plant immunity. We found highly distinct and time-dependent biological processes being activated on N-terminome level in response to avrRxo1, FB1 and SA. This study characterizes distinct RCD responses in Z. mays and provides a framework for the mechanistic exploration of components involved in the initiation and execution of cell death.

2023-07-13 | PXD041656 | Pride

A role for the Trim33 ubiquitin ligase in cellular response to replicative stress

Project description:Deregulation of RNA polymerase II (RNAPII) by oncoproteins, such as transcription factor Myc, interferes with DNA replication and is a major source of DNA damage and genomic instability. Ubiquitination is a key pathway controlling RNAPII activity via modification of RNAPII subunits or associated regulatory proteins. We uncover a mechanism for genome maintenance by ubiquitin ligase Trim33 and transcription factor E2f4. We show that Trim33 promotes E2f4 protein turnover, restricting interactions of E2f4 with chromatin and with the Recql DNA helicase. Replicative stress blunts Trim33-dependent regulation, which stimulates E2f4 and Recql recruitment to chromatin and facilitates recovery of DNA replication. Deletion of Trim33 triggers chronic recruitment of Recql to chromatin and accelerates DNA replication under stress, accompanied by compromised DDR signaling and DNA repair. Depletion of Trim33 in Myc-overexpressing cells leads to accumulation of replication-associated DNA damage and delays Myc-driven tumorigenesis. We propose that the Trim33-E2f4-Recql axis provides a mechanism to control DNA replication at transcriptionally active chromatin to maintain genome integrity.

2023-07-24 | PXD037854 | Pride

Impact of ultrastructural left-ventricular Ca2+ release unit remodeling in hemodynamic subtypes of severe aortic stenosis

Project description:Aortic valve stenosis (AS) frequently causes heart disease in elderly patients and is characterized by a broad spectrum of hemodynamic phenotypes, some with life-threatening arrhythmias, and increased risk of death. The guideline for management of severe AS in patients is to improve quality of life by transaortic catheter-based valve replacement (TAVR). However, the mechanisms underlying different AS-associated hemodynamic phenotypes remain poorly understood. Proteome profiling by data-independent acquisition mass spectrometry in individual left-ventricular biopsies quantified 2.273 proteins. 160 showed statistically significant abundance changes in AS hearts compared to non-failing samples. Unbiased hierchical clustering identified four different proteotypes which broadly corresponded to hemodynamic phenotypes. As adult cardiomyocytes undergo distinct stages of subcellular remodeling, we used quantitative superresolution microscopy of candidate proteins in left-ventricular biopsies to determine the consequences of dyadic RyR2 protein changes on AS dysfunction in vivo.

2022-10-17 | PXD019594 | Pride

Rhodnius prolixus hemolymph proteome

Project description:This project aims to investigate the immuno-physiology of the Chagas disease vector Rhodnius prolixus, by analysing the effect of blood ingestion and Trypanosoma cruzi colonisation of its digestive tract on the modulation of protein expression in the hemolymph.

2022-04-27 | PXD032787 | Pride

Benchmarking of 5.5 cm, 50 cm and 110 cm micropillar array columns as compared to packed bed columns

Project description:A comprehensive proteome map is essential to elucidate molecular pathways and protein functions. Although great improvements in sample preparation, instrumentation and data analysis already yield-ed impressive results, current studies suffer from a limited proteomic depth and dynamic range there-fore lacking low abundant or highly hydrophobic proteins. Here, we combine and benchmark advanced micro pillar array columns (µPAC) operated at nanoflow with Wide Window Acquisition (WWA) and the AI-based CHIMERYS search engine for data analysis to maximize chromatographic separation power, sensitivity and proteome coverage. Our data shows that µPAC columns clearly outperform classical packed bed columns boosting peptide IDs by up to 50% and protein IDs by up to 24%. Using the above-mentioned analysis platform, more than 10,000 proteins could be identified from a single 2 h gradient shotgun analysis for a triple proteome mix of human, yeast and E. coli digests. At high sample loads of 400 ng all three uPAC types yielded comparable number of protein identifications, whereas the 50cm neo column performed best when lower inputs of less than 200 ng were injected. Due to its unique architecture, the 5.5 cm brick column facilitates a highly meandering flow along the brick-shaped micropillars leading to an effective flow path length similar to the 50 cm neo column, while at the same time allowing high flow rates up to 2.5 µL/min. This enables to reduce overhead time by applying high flow rates during sample loading and column equilibration improving sample throughput to ~100 samples per day, while maintaining high protein ID numbers. Particularly for the single cell field, for which throughput is currently one of the most limiting factors, this column could present a valuable asset.

2024-02-13 | PXD039576 | Pride

A Multiplexed Parallel Reaction Monitoring Assay to monitor bovine Pregnancy-Associated Glycoproteins throughout pregnancy and after gestation - PRM Data

Project description:Bovine pregnancy-associated glycoproteins (boPAGs) are extensively glycosylated secretory proteins of trophoblast cells. Roughly 20 different boPAG members are known but their distribution patterns and degree of glycosylation during pregnancy are not well characterized. The objective of the present study was the development of a parallel reaction monitoring-based assay for the profiling of different boPAGs during pregnancy and after gestation. Furthermore, we investigated the effects of N-glycosylation on our analytical results. BoPAGs were purified from cotyledons of four different pregnancy stages. The assay detects 25 proteotypic peptides from 18 boPAGs in a single run. The highest abundances were found for boPAG 1 in both, glycosylated and deglycosylated samples. Strongest effects of glycosylation were detected during mid and late pregnancy as well as in afterbirth samples. Furthermore, we identified different boPAG-clusters based on the observed relative protein abundances between glycosylated and deglycosylated samples. A linkage between the impact of glycosylation and potential N-glycosylation sites or phylogenetic relation was not detected. In conclusion, the newly developed parallel reaction monitoring-based assay enables for the first time a comprehensive semi-quantitative profiling of 18 different boPAGs during pregnancy and post-partum on protein level, thereby investigating the influence of glycosylation. The results of this study provide new and important starting points to address further research on boPAGs to better understand their physiological role during pregnancy and for the development of new pregnancy detection tests.

2022-07-05 | PXD034108 | Pride

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