A synthetic protein as efficient multitarget regulator against complement over-activation
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Physcomitrella
TISSUE(S): Protonema
SUBMITTER: 
Ralf Reski
LAB HEAD: Ralf Reski
PROVIDER: PXD025471 | Pride | 2024-04-17
REPOSITORIES: Pride
Dataset's files
Items per page: 5 1 - 5 of 5 |
Similar Datasets
Project description:The hydroxylation of proline residues to 4-trans-hydroxyproline (Hyp) is a common post-translational protein modification in plants, mediated by prolyl 4-hydroxylases (P4Hs). Hyps predominantly occur in a group of cell wall proteins, the Hydroxyproline-rich glycoproteins (HRGPs), where they are frequently O-glycosylated. While prolyl-hydroxylation and O-glycosylation are important, e.g. for cell wall stability, they are not desirable in plant-made pharmaceuticals. Sequence motifs recognized for prolyl-hydroxylation derived from vascular plants were proposed but did not include data from mosses, such as Physcomitrella. Here, a phylogenetic reconstruction of plant P4Hs identified six P4Hs in four subfamilies in mosses. We analysed the amino acid sequences and structural environments around Hyps in Physcomitrella utilizing 73 Hyp sites in 24 secretory proteins from multiple MS/MS datasets, and found that prolines in close proximity to other prolines, alanine, serine, threonine and valine were preferentially hydroxylated. About 95 % of the Hyp sites were predictable with a combination of previously defined motifs and methods. In our data, AOV (Ala-Hyp-Val) was the most frequent prolyl-hydroxylation pattern. Additionally, short arabinose chains were attached to Hyps in two cell-wall pectinesterases. A combination of 443 AlphaFold structure models and our MS data of peptides with nearly 3000 proline sites found Hyps predominantly on protein surfaces in disordered regions. Moss-produced human erythropoietin (EPO) exhibited plant-specific O-glycosylation with arabinose chains on two Hyps. This modification was significantly reduced in a P4H1 single knock-out (KO) Physcomitrella mutant. Quantitative proteomics after isotope labelling with different P4H-KO moss mutants revealed specific changes in the amount of proteins, including HRGPs, and a modified prolyl-hydroxylation pattern from the mutants, suggesting a differential function of the six Physcomitrella P4Hs. Quantitative RT-PCR proved a differential effect of single P4H KOs on the expression of the other five p4h genes, suggesting a partial compensation of the mutation. AlphaFold-Multimer models for Physcomitrella P4H1 and its target EPO peptide superposed with the crystal structure of Chlamydomonas P4H1 and a peptide substrate suggested significant amino acids in the active centre of the enzyme that form H-bonds with the peptide substrate, and revealed differences between P4H1 and the other five Physcomitrella P4Hs.
2024-08-10 | PXD051497 | Pride
Project description:Protein oxidation results from the reaction of amino-acid side chains with reactive oxygen species (ROS) and is mostly irreversible. In non-photosynthetic tissues, mitochondria are a main source of ROS, whereas plastids are the major source in photosynthetic tissues. Oxidized proteins suffer from decreased structural integrity and even loss of function, and their accumulation ultimately leads to cytotoxic aggregates. In mammals, this correlates with aging and is linked to several age-related pathologies. Mammalian proteolytic pathways for clearance of oxidized proteins are under intensive research, while mechanistic insights into this process in plants is scarce. AARE enzymes are ATP-independent serine proteases presumably acting on oxidized proteins and operating in a dual exo-/endopeptidase mode. They are found in all domains of life. Here, we investigated AARE enzymes in the moss Physcomitrella and the angiosperm Arabidopsis and identified three homologous nuclear genes in Physcomitrella (PpAARE1-3) and a single nuclear gene in Arabidopsis (AtAARE). Surprisingly, we observed triple localization of the proteins AtAARE and PpAARE1 to plastids, mitochondria and the cytosol in vivo, likely conserved across the plant lineage. This represents an ATP-independent possibility for degradation of oxidized proteins in the major source organelles of ROS in plants, which is distinct to mammals. Combinatorial knockout plants and protein interaction analysis revealed specific interactions of the moss AARE isoforms and functions in progressive aging. Analysis of an AtAARE T-DNA mutant further suggests conservation of AARE function in age-related development.
2023-03-11 | PXD033854 | Pride
Project description:The red/far-red light photoreceptor phytochrome mediates photomorphological responses in plants. For light sensing and signaling, phytochromes need to associate with open-chain tetrapyrrole molecules as the chromophore. Biosynthesis of tetrapyrrole chromophores requires members of ferredoxin-dependent bilin reductases (FDBRs). There are two FDBRs in Physcomitrella patens, HY2 and PUBS. Knocking out both generates the phytochrome-deficient mutant. Datasets here provides the transcriptome profiling of Physcomitrella protonema grown in the dark and exposed to one hour red light. Wild type and the hy2 pubs double mutant were used to dissect the regulated genes of moss phytochromes. 4 samples, dark-grown wild-type and pubs hy2 protonema as time 0 control, followed by red light irradiation for one hour respectively
2012-05-08 | E-GEOD-36274 | biostudies-arrayexpress
Project description:This project aimed to discover genes that regulate the transition from 2D to 3D growth in the moss Physcomitrella patens. Mutants were generated that do not pattern 3D growth. Bulk segregant analysis was conducted to identify the causative genes. This experiment contains two samples - WT-pool, Mt-pool.
2020-12-11 | E-MTAB-7502 | biostudies-arrayexpress
Project description:Reactive oxygen species (ROS) are constant by-products of aerobic life. In excess, ROS lead to cytotoxic protein aggregates, which are a hallmark of ageing in animals and linked to age-related pathologies in humans. Acylamino acid-releasing enzymes (AARE) are bifunctional serine proteases, acting on oxidized proteins. AARE are found in all domains of life, albeit under different names, such as acylpeptide hydrolase (APEH/ACPH), acylaminoacyl peptidase (AAP), or oxidized protein hydrolase (OPH). In humans, AARE malfunction is associated with age-related pathologies, while their function in plants is less clear. Here, we provide a detailed analysis of AARE genes in the plant lineage and an in-depth analysis of AARE localization and function in the moss Physcomitrella and the angiosperm Arabidopsis. AARE loss-of-function mutants have not been described for any organism so far. We generated and analysed such mutants and describe a connection between AARE function, aggregation of oxidized proteins and plant ageing, including accelerated developmental progression and reduced life span. Our findings complement similar findings in animals and humans, and suggest a unified concept of ageing may exist in different life forms
2023-03-11 | PXD038742 | Pride
Project description:Protein arginylation is a post-translational modification at the N-terminus of proteins and is crucial for viability and physiology in higher eukaryotes. The lack of arginylation causes severe developmental defects in plants and embryo lethality in Drosophila and in mice. Although several studies investigated impact and function of the responsible enzyme, the arginyl-tRNA protein transferase (ATE) in plants, identification of arginylated proteins by mass spectrometry was not hitherto achieved. In the present study, we report the identification of targets and interaction partners of ATE in the model plant Physcomitrella patens by mass spectrometry employing two different immuno-affinity strategies and a recently established transgenic ATE:GUS reporter line (Schuessele et al., 2015 New Phytol., DOI: 10.1111/nph.13656). Here we use a commercially available antibody against the fused reporter protein (GUS) to pull down ATE and its interacting proteins and validate the in vivo interaction with a class I small heatshock protein via Förster resonance energy transfer (FRET). Additionally, we apply and modify a method that already successfully identified arginylated proteins from mouse proteomes by using custom-made antibodies specific for N-terminal arginine. As a result, we identify four arginylated proteins from Physcomitrella patens with high confidence.
2016-04-21 | PXD003232 | Pride
Project description:Endogenous 24nt short interfering RNAs (siRNAs) derived mostly from intergenic and repetitive genomic regions constitute a major class of endogenous small RNAs in Arabidopsis thaliana. Accumulation of A. thaliana 24nt siRNAs requires the Dicer family member DCL3, and clear homologs of DCL3 exist in both flowering and non-flowering plants. However, the absence of a conspicuous 24nt peak in the total RNA populations of several non-flowering plants has raised the question of whether this class of siRNAs might, in contrast to the ancient 21nt microRNAs (miRNAs) and 21-22nt trans-acting siRNAs (tasiRNAs), be an angiosperm-specific innovation. Analysis of non-miRNA, non-tasiRNA hotspots of small RNA production within the genome of the moss Physcomitrella patens revealed multiple loci which consistently produced a mixture of 21-24nt siRNAs with a peak at 23nts. These Pp23SR loci were significantly enriched in transposon content, depleted in overlap with annotated genes, and typified by dense concentrations of the 5-methyl cytosine (5mC) DNA modification. Deep sequencing of small RNAs from two independent Ppdcl3 mutants showed that the P. patens DCL3 homolog is required for the accumulation of 22-24nt siRNAs, but not 21nt siRNAs, at Pp23SR loci. The 21nt component of Pp23SR-derived siRNAs was also unaffected by a mutation in the RNA-dependent RNA polymerase mutant Pprdr6. Transcriptome-wide, Ppdcl3 mutants specifically failed to accumulate 23 and 24nt small RNAs from repetitive regions. We conclude that intergenic/repeat-derived siRNAs are indeed a broadly conserved, distinct class of small regulatory RNAs within land plants. Our results also suggest that Pp DCL3 produces siRNAs of heterogenous size, unlike its A. thaliana homolog which generates exclusively 24nt siRNAs. Small RNAs from Wild-type (two technical replicates), rdr6-19 (two technical replicates), dcl3-5 (one sample), and dcl3-10 (one sample) were sequenced using a Solexa GA. Three different linkers were used for different samples and mixed prior to sequencing. linker 1: CTGTAGGCACCATCAAT (GPL7174 AND GSM313212, GSM313213) linker 2: CACTCGGGCACCAAGGA (GPL7175 AND GSM313214, GSM313215) linker 3: TTTAACCGCGAATTCCAG (GPL7176 AND GSM313216, GSM313217) Raw data: Two FASTQ files represent the raw data underlying the processed sample data in GSE12468. 080616_s_7_seq_GAK-2.fastq corresponds to GSM313212, GSM313214, and GSM313216. 080616_s_8_seq_GAK-3.fastq corresponds to GSM313213, GSM313215, and GSM313217. Experimentally, samples GSM313212, GSM313214, and GSM313216 (i.e., all three genotypes) were sequenced in the same run (hence the same FASTQ file). The sequencing run was a mixture of three different libraries with different linker sequences (as indicated above). These 'barcoded' samples were computationally extracted based on the linker sequences. This is also true for samples GSM313213, GSM313215, and GSM313217. Raw data were filtered to identify small RNAs associated with each linker. Subsequently, small RNAs were filtered to remove rRNA fragments. The remainder were mapped to the Physcomitrella patens draft genome version 1.2 (using oligomap) and filtered to retain only those with one or more perfect match. See Cho et al. for details.
2008-12-19 | E-GEOD-12468 | biostudies-arrayexpress
Project description:Production of biopharmaceuticals relies on the expression of mammalian cDNAs in host organisms. Here we show that the expression of a human cDNA in the moss Physcomitrium patens generates the expected full-length and four additional transcripts due to unexpected splicing. This mRNA splicing results in non-functional protein isoforms, cellular misallocation of the proteins and low product yields. We integrated these results together with the results of our analysis of all 32,926 protein-encoding Physcomitrella genes and their 87,533 annotated transcripts in a web application, physCO, for automatized optimization. A thus optimized cDNA results in about twelve times more protein, which correctly localizes to the ER. An analysis of codon preferences of different production hosts suggests that similar effects occur also in non-plant hosts. We anticipate that the use of our methodology will prevent so far undetected mRNA heterosplicing resulting in maximized functional protein amounts for basic biology and biotechnology.
2022-01-10 | PXD026651 | Pride
Project description:To obtain insight into the molecular mechanism of FH intracellular function in hTERT-RPE1 cells, we analyzed the interactome of FH via its immunoprecipitation followed by MS-based analysis. We found that FH interacts with essential components of the ubiquitin-proteasomal pathway (UPS) as well as with RB1/E2F signalling in a complement-pathway independent manner. Moreover, we found, that FH silencing affects mRNA levels of the E3 Ubiquitin-Protein Ligase Parkin and PTEN induced putative kinase (Pink1), both of them are associated with UPS. As inhibition of mTORC1 has been previoulsy shown to result in increased overall protein degradation via UPS and as FH mRNA and protein levels were shown to be affected by inhibition of UPS, our data stress a potential regulatory link between endogenous FH activity and UPS.
2022-02-16 | PXD029408 | Pride
Project description:Purpose: The plant hormone abscisic acid (ABA) coordinates responses leading to desiccation tolerance in all land plants. RNA-seq was employed to obtain differential expression data in response to ABA, dehydration and mannitol treatments in wild-type tissue. This was compared against the targeted gene knockout line PpanrKO. The ANR gene encodes a novel ABA regulator identified from ABA non-responsive (anr) mutant screens. Methods: RNA-seq on the Illumina HiSeq platform and mapping to the V3.0 genome and transcriptome assemblies using tophat2 enabled gene abundance data to be generated for differential gene expression analysis with edgeR in the Trinity package. Results: We observe a coordinated up-regulation of ca. 900 genes, with extensive overlaps between hormone and stress treatments as well as ca. 900 down-regulated genes. Of the up-regulated genes, those encoding LEA proteins, membrane channel proteins and transporters and oxidative-stress-associated genes comprised a significant fraction while those encoding growth-associated cell wall enzymes (e.g. expansins) and components of secondary metabolism (notably enzymes in the phenylpropanoid pathway) were prominent in the down-regulated genes. The ANR gene, a modular MAP3 kinase comprising an N-terminal PAS domain, a central EDR domain and a C-terminal MAPKKK-like domain unique to basal plant lineages, is found to play a central role in these responses with mutants failing to accumulate the wide spectrum of dehydration tolerance-associated gene products characteristic of the wild-type response and do not acquire ABA-dependent desiccation tolerance. Conclusions: Our study adds considerable data to the stress responses in bryophytes helping understand the mechanisms behind vegetative dehydration tolerance; an important trait for crop species. We also confirmed the vital role that PpANR plays in these responses in the moss Physcomitrella patens likely representing an ancestral mechanism for ABA-mediated dehydration tolerance vital in the conquest of land. RNA from 3 biological replicas was bulked per library. 8 libraries were generated from two genotypes, wild-type and PpanrkO, which included 3 treatments and a control sample for each.
2016-07-25 | E-GEOD-72583 | biostudies-arrayexpress