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1.
Gut Microbes ; 13(1): 1994836, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34763597

RESUMO

Intestinal microbiota plays a key role in shaping host homeostasis by regulating metabolism, immune responses and behavior. Its dysregulation has been associated with metabolic, immune and neuropsychiatric disorders and is accompanied by changes in bacterial metabolic regulation. Although proteomics is well suited for analysis of individual microbes, metaproteomics of fecal samples is challenging due to the physical structure of the sample, presence of contaminating host proteins and coexistence of hundreds of taxa. Furthermore, there is a lack of consensus regarding preparation of fecal samples, as well as downstream bioinformatic analyses following metaproteomics data acquisition. Here we assess sample preparation and data analysis strategies applied to mouse feces in a typical mass spectrometry-based metaproteomic experiment. We show that subtle changes in sample preparation protocols may influence interpretation of biological findings. Two-step database search strategies led to significant underestimation of false positive protein identifications. Unipept software provided the highest sensitivity and specificity in taxonomic annotation of the identified peptides of unknown origin. Comparison of matching metaproteome and metagenome data revealed a positive correlation between protein and gene abundances. Notably, nearly all functional categories of detected protein groups were differentially abundant in the metaproteome compared to what would be expected from the metagenome, highlighting the need to perform metaproteomics when studying complex microbiome samples.

2.
Cancers (Basel) ; 13(21)2021 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-34771574

RESUMO

Immune checkpoint inhibitors are used to restore or augment antitumor immune responses and show great promise in the treatment of melanoma and other types of cancers. However, only a small percentage of patients are fully responsive to immune checkpoint inhibition, mostly due to tumor heterogeneity and primary resistance to therapy. Both of these features are largely driven by the accumulation of patient-specific mutations, pointing to the need for personalized approaches in diagnostics and immunotherapy. Proteogenomics integrates patient-specific genomic and proteomic data to study cancer development, tumor heterogeneity and resistance mechanisms. Using this approach, we characterized the mutational landscape of four clinical melanoma patients. This enabled the quantification of hundreds of sample-specific amino acid variants, among them many that were previously not reported in melanoma. Changes in abundance at the protein and phosphorylation site levels revealed patient-specific over-represented pathways, notably linked to melanoma development (MAPK1 activation) or immunotherapy (NLRP1 inflammasome). Personalized data integration resulted in the prediction of protein drug targets, such as the drugs vandetanib and bosutinib, which were experimentally validated and led to a reduction in the viability of tumor cells. Our study emphasizes the potential of proteogenomic approaches to study personalized mutational landscapes, signaling networks and therapy options.

3.
Mol Cell Proteomics ; 20: 100163, 2021 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-34673281

RESUMO

Analysis of nucleotide variants is a cornerstone of cancer medicine. Although only 2% of the genomic sequence is protein coding, mutations occurring in these regions have the potential to influence protein structure or modification status and may have severe impact on disease aetiology. Proteogenomics enables the analysis of sample-specific nonsynonymous nucleotide variants with regard to their effect at the proteome and phosphoproteome levels. Here, we developed a proof-of-concept proteogenomics workflow and applied it to the malignant melanoma cell line A375. Initially, we studied the resistance to serine/threonine-protein kinase B-raf (BRAF) inhibitor (BRAFi) vemurafenib in A375 cells. This allowed identification of several oncogenic nonsynonymous nucleotide variants, including a gain-of-function variant on aurora kinase A (AURKA) at F31I. We also detected significant changes in abundance among (phospho)proteins, which led to reactivation of the MAPK signaling pathway in BRAFi-resistant A375 cells. Upon reconstruction of the multiomic integrated signaling networks, we predicted drug therapies with the potential to disrupt BRAFi resistance mechanism in A375 cells. Notably, we showed that AURKA inhibition is effective and specific against BRAFi-resistant A375 cells. Subsequently, we investigated amino acid variants that interfere with protein posttranslational modification (PTM) status and potentially influence A375 cell signaling irrespective of BRAFi resistance. Mass spectrometry (MS) measurements confirmed variant-driven PTM changes in 12 proteins. Among them was the runt-related transcription factor 1 (RUNX1) displaying a variant on a known phosphorylation site S(Ph)276L. We confirmed the loss of phosphorylation site by MS and demonstrated the impact of this variant on RUNX1 interactome.

4.
J Biol Chem ; 297(5): 101339, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34688664

RESUMO

Mitochondria are important organelles in eukaryotes. Turnover and quality control of mitochondria are regulated at the transcriptional and posttranslational level by several cellular mechanisms. Removal of defective mitochondrial proteins is mediated by mitochondria resident proteases or by proteasomal degradation of individual proteins. Clearance of bulk mitochondria occurs via a selective form of autophagy termed mitophagy. In yeast and some developing metazoan cells (e.g., oocytes and reticulocytes), mitochondria are largely removed by ubiquitin-independent mechanisms. In such cases, the regulation of mitophagy is mediated via phosphorylation of mitochondria-anchored autophagy receptors. On the other hand, ubiquitin-dependent recruitment of cytosolic autophagy receptors occurs in situations of cellular stress or disease, where dysfunctional mitochondria would cause oxidative damage. In mammalian cells, a well-studied ubiquitin-dependent mitophagy pathway induced by mitochondrial depolarization is regulated by the mitochondrial protein kinase PINK1, which upon activation recruits the ubiquitin ligase parkin. Here, we review mechanisms of mitophagy with an emphasis on posttranslational modifications that regulate various mitophagy pathways. We describe the autophagy components involved with particular emphasis on posttranslational modifications. We detail the phosphorylations mediated by PINK1 and parkin-mediated ubiquitylations of mitochondrial proteins that can be modulated by deubiquitylating enzymes. We also discuss the role of accessory factors regulating mitochondrial fission/fusion and the interplay with pro- and antiapoptotic Bcl-2 family members. Comprehensive knowledge of the processes of mitophagy is essential for the understanding of vital mitochondrial turnover in health and disease.

5.
mSystems ; 6(4): e0054921, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34427514

RESUMO

During antibiotic persistence, bacterial cells become transiently tolerant to antibiotics by restraining their growth and metabolic activity. Detailed molecular characterization of antibiotic persistence is hindered by low count of persisting cells and the need for their isolation. Here, we used sustained addition of stable isotope-labeled lysine to selectively label the proteome during hipA-induced persistence and hipB-induced resuscitation of Escherichia coli cells in minimal medium after antibiotic treatment. Time-resolved, 24-h measurement of label incorporation allowed detection of over 500 newly synthesized proteins in viable cells, demonstrating low but widespread protein synthesis during persistence. Many essential proteins were newly synthesized, and several ribosome-associated proteins such as RaiA and Sra showed high synthesis levels, pointing to their roles in maintenance of persistence. At the onset of resuscitation, cells synthesized the ribosome-splitting GTPase HflX and various ABC transporters, restored translation machinery, and resumed metabolism by inducing glycolysis and biosynthesis of amino acids. IMPORTANCE While bactericidal antibiotics typically require actively growing cells to exploit their function, persister cells are slowly replicating which makes them tolerant to the lethal action of antimicrobials. Here, we used an established in vitro model of bacterial persistence based on overexpression of the paradigm toxin-antitoxin (TA) system hipA/hipB to devise a generic method for temporal analysis of protein synthesis during toxin-induced persistence and antitoxin-mediated resuscitation. Our time-resolved, 24-h measurement of label incorporation demonstrated low but widespread protein synthesis during persistence. At the onset of resuscitation, cells restored translation machinery and resumed metabolism by inducing glycolysis and biosynthesis of amino acids. Our study provides the first global analysis of protein synthesis in persisting and resuscitating bacterial cells, and as such, presents an unprecedented resource to study the processes governing antibiotic persistence.

6.
J Mol Biol ; 433(19): 167175, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34303721

RESUMO

Virulence-associated type III secretion systems (T3SS) are utilized by Gram negative bacterial pathogens for injection of effector proteins into eukaryotic host cells. The transmembrane export apparatus at the core of T3SS is composed of a unique helical complex of the hydrophobic proteins SctR, SctS, SctT, and SctU. These components comprise a number of highly conserved charged residues within their hydrophobic domains. The structure of the closed state of the core complex SctR5S4T1 revealed that several of these residues form inter- and intramolecular salt bridges, some of which have to be broken for pore opening. Mutagenesis of individual residues was shown to compromise assembly or secretion of both, the virulence-associated and the related flagellar T3SS. However, the exact role of these conserved charged residues in the assembly and function of T3SS remains elusive. Here we performed an in-depth mutagenesis analysis of these residues in the T3SS of Salmonella Typhimurium, coupled to blue native PAGE, in vivo photocrosslinking and luciferase-based secretion assays. Our data show that these conserved salt bridges are not critical for assembly of the respective protein but rather facilitate the incorporation of the following subunit into the assembling complex. Our data also indicate that these conserved charged residues are critical for type III-dependent secretion and reveal a functional link between SctSE44 and SctTR204 and the cytoplasmic domain of SctU in gating the T3SS injectisome. Overall, our analysis provides an unprecedented insight into the delicate requirements for the assembly and function of the machinery at the core of T3SS.


Assuntos
Salmonella enterica/metabolismo , Sistemas de Secreção Tipo III/química , Sistemas de Secreção Tipo III/metabolismo , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Mutação , Conformação Proteica , Domínios Proteicos , Salmonella enterica/genética , Salmonella enterica/patogenicidade , Sistemas de Secreção Tipo III/genética , Virulência
7.
New Phytol ; 231(3): 1123-1137, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34058021

RESUMO

Cyanobacteria are the only prokaryotes that perform plant-like oxygenic photosynthesis. They evolved an inorganic carbon-concentrating mechanism to adapt to low CO2 conditions. Quantitative phosphoproteomics was applied to analyze regulatory features during the acclimation to low CO2 conditions in the model cyanobacterium Synechocystis sp. PCC 6803. Overall, more than 2500 proteins were quantified, equivalent to c. 70% of the Synechocystis theoretical proteome. Proteins with changing abundances correlated largely with mRNA expression levels. Functional annotation of the noncorrelating proteins revealed an enrichment of key metabolic processes fundamental for maintaining cellular homeostasis. Furthermore, 105 phosphoproteins harboring over 200 site-specific phosphorylation events were identified. Subunits of the bicarbonate transporter BCT1 and the redox switch protein CP12 were among phosphoproteins with reduced phosphorylation levels at lower CO2 , whereas the serine/threonine protein kinase SpkC revealed increased phosphorylation levels. The corresponding ΔspkC mutant was characterized and showed decreased ability to acclimate to low CO2 conditions. Possible phosphorylation targets of SpkC including a BCT1 subunit were identified by phosphoproteomics. Collectively, our study highlights the importance of posttranscriptional regulation of protein abundances as well as posttranslational regulation by protein phosphorylation for the successful acclimation towards low CO2 conditions in Synechocystis and possibly among cyanobacteria.


Assuntos
Synechocystis , Aclimatação , Proteínas de Bactérias/metabolismo , Dióxido de Carbono , Fotossíntese , Proteoma , Synechocystis/genética , Synechocystis/metabolismo
8.
Am J Hum Genet ; 108(6): 1069-1082, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34022130

RESUMO

BCAS3 microtubule-associated cell migration factor (BCAS3) is a large, highly conserved cytoskeletal protein previously proposed to be critical in angiogenesis and implicated in human embryogenesis and tumorigenesis. Here, we established BCAS3 loss-of-function variants as causative for a neurodevelopmental disorder. We report 15 individuals from eight unrelated families with germline bi-allelic loss-of-function variants in BCAS3. All probands share a global developmental delay accompanied by pyramidal tract involvement, microcephaly, short stature, strabismus, dysmorphic facial features, and seizures. The human phenotype is less severe compared with the Bcas3 knockout mouse model and cannot be explained by angiogenic defects alone. Consistent with being loss-of-function alleles, we observed absence of BCAS3 in probands' primary fibroblasts. By comparing the transcriptomic and proteomic data based on probands' fibroblasts with those of the knockout mouse model, we identified similar dysregulated pathways resulting from over-representation analysis, while the dysregulation of some proposed key interactors could not be confirmed. Together with the results from a tissue-specific Drosophila loss-of-function model, we demonstrate a vital role for BCAS3 in neural tissue development.


Assuntos
Mutação com Perda de Função , Perda de Heterozigosidade , Proteínas de Neoplasias/genética , Transtornos do Neurodesenvolvimento/etiologia , Adolescente , Adulto , Animais , Movimento Celular , Criança , Pré-Escolar , Drosophila , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Lactente , Masculino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/metabolismo , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/patologia , Linhagem , Proteoma/análise , Adulto Jovem
9.
Open Biol ; 11(4): 200405, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33823663

RESUMO

Fluctuations in TOR, AMPK and MAP-kinase signalling maintain cellular homeostasis and coordinate growth and division with environmental context. We have applied quantitative, SILAC mass spectrometry to map TOR and nutrient-controlled signalling in the fission yeast Schizosaccharomyces pombe. Phosphorylation levels at more than 1000 sites were altered following nitrogen stress or Torin1 inhibition of the TORC1 and TORC2 networks that comprise TOR signalling. One hundred and thirty of these sites were regulated by both perturbations, and the majority of these (119) new targets have not previously been linked to either nutritional or TOR control in either yeasts or humans. Elimination of AMPK inhibition of TORC1, by removal of AMPKα (ssp2::ura4+), identified phosphosites where nitrogen stress-induced changes were independent of TOR control. Using a yeast strain with an ATP analogue-sensitized Cdc2 kinase, we excluded sites that were changed as an indirect consequence of mitotic control modulation by nitrogen stress or TOR signalling. Nutritional control of gene expression was reflected in multiple targets in RNA metabolism, while significant modulation of actin cytoskeletal components points to adaptations in morphogenesis and cell integrity networks. Reduced phosphorylation of the MAPKK Byr1, at a site whose human equivalent controls docking between MEK and ERK, prevented sexual differentiation when resources were sparse but not eliminated.

10.
Front Microbiol ; 12: 657562, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33889145

RESUMO

Understanding phosphorylation-mediated regulation of metabolic enzymes, pathways, and cell phenotypes under metabolic shifts represents a major challenge. The kinases associated with most phosphorylation sites and the link between phosphorylation and enzyme activity remain unknown. In this study, we performed stable isotope labeling by amino acids in cell culture (SILAC)-based proteome and phosphoproteome analysis of Escherichia coli ΔyeaG, a strain lacking a poorly characterized serine/threonine kinase YeaG, to decipher kinase-substrate interactions and the effects on metabolic phenotype during shifts from glucose to malate. The starting point of our analysis was the identification of physiological conditions under which ΔyeaG exhibits a clear phenotype. By metabolic profiling, we discovered that ΔyeaG strain has a significantly shorter lag phase than the wild type during metabolic shift from glucose to malate. Under those conditions, our SILAC analysis revealed several proteins that were differentially phosphorylated in the ΔyeaG strain. By focusing on metabolic enzymes potentially involved in central carbon metabolism, we narrowed down our search for putative YeaG substrates and identified isocitrate lyase AceA as the direct substrate of YeaG. YeaG was capable of phosphorylating AceA in vitro only in the presence of malate, suggesting that this phosphorylation event is indeed relevant for glucose to malate shift. There is currently not enough evidence to firmly establish the exact mechanism of this newly observed regulatory phenomenon. However, our study clearly exemplifies the usefulness of SILAC-based approaches in identifying proteins kinase substrates, when applied in physiological conditions relevant for the activity of the protein kinase in question.

11.
Plant Cell ; 33(2): 248-269, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33793824

RESUMO

Although regulatory small RNAs have been reported in photosynthetic cyanobacteria, the lack of clear RNA chaperones involved in their regulation poses a conundrum. Here, we analyzed the full complement of cellular RNAs and proteins using gradient profiling by sequencing (Grad-seq) in Synechocystis 6803. Complexes with overlapping subunits such as the CpcG1-type versus the CpcL-type phycobilisomes or the PsaK1 versus PsaK2 photosystem I pre(complexes) could be distinguished, supporting the high quality of this approach. Clustering of the in-gradient distribution profiles followed by several additional criteria yielded a short list of potential RNA chaperones that include an YlxR homolog and a cyanobacterial homolog of the KhpA/B complex. The data suggest previously undetected complexes between accessory proteins and CRISPR-Cas systems, such as a Csx1-Csm6 ribonucleolytic defense complex. Moreover, the exclusive association of either RpoZ or 6S RNA with the core RNA polymerase complex and the existence of a reservoir of inactive sigma-antisigma complexes is suggested. The Synechocystis Grad-seq resource is available online at https://sunshine.biologie.uni-freiburg.de/GradSeqExplorer/ providing a comprehensive resource for the functional assignment of RNA-protein complexes and multisubunit protein complexes in a photosynthetic organism.


Assuntos
Membranas Intracelulares/metabolismo , Fotossíntese , Análise de Sequência de RNA , Synechocystis/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Complexos Multiproteicos/metabolismo , Fotossíntese/genética , Filogenia , Ligação Proteica , Biossíntese de Proteínas , Proteoma/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Tilacoides/metabolismo , Sistemas Toxina-Antitoxina , Transcriptoma/genética
12.
Proc Natl Acad Sci U S A ; 118(6)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33526690

RESUMO

Nitrogen limitation imposes a major transition in the lifestyle of nondiazotrophic cyanobacteria that is controlled by a complex interplay of regulatory factors involving the pervasive signal processor PII Immediately upon nitrogen limitation, newly fixed carbon is redirected toward glycogen synthesis. How the metabolic switch for diverting fixed carbon toward the synthesis of glycogen or of cellular building blocks is operated was so far poorly understood. Here, using the nondiazotrophic cyanobacterium Synechocystis sp. PCC 6803 as model system, we identified a novel PII interactor, the product of the sll0944 gene, which we named PirC. We show that PirC binds to and inhibits the activity of 2,3-phosphoglycerate-independent phosphoglycerate mutase (PGAM), the enzyme that deviates newly fixed CO2 toward lower glycolysis. The binding of PirC to either PII or PGAM is tuned by the metabolite 2-oxoglutarate (2-OG), which accumulates upon nitrogen starvation. In these conditions, the high levels of 2-OG dissociate the PirC-PII complex to promote PirC binding to and inhibition of PGAM. Accordingly, a PirC-deficient mutant showed strongly reduced glycogen levels upon nitrogen deprivation, whereas polyhydroxybutyrate granules were overaccumulated compared to wild-type. Metabolome analysis revealed an imbalance in 3-phosphoglycerate to pyruvate levels in the pirC mutant, confirming that PirC controls the carbon flux in cyanobacteria via mutually exclusive interaction with either PII or PGAM.


Assuntos
Proteínas de Bactérias/genética , Cianobactérias/genética , Proteínas PII Reguladoras de Nitrogênio/genética , Fosfoglicerato Mutase/genética , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Cianobactérias/metabolismo , Nitrogênio/metabolismo , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Fosfoglicerato Mutase/metabolismo , Synechocystis/genética , Synechocystis/metabolismo
13.
Proc Natl Acad Sci U S A ; 118(5)2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33509926

RESUMO

Phycobilisomes are the major pigment-protein antenna complexes that perform photosynthetic light harvesting in cyanobacteria, rhodophyte, and glaucophyte algae. Up to 50% of the cellular nitrogen can be stored in their giant structures. Accordingly, upon nitrogen depletion, phycobilisomes are rapidly degraded following an intricate genetic program. Here, we describe the role of NblD, a cysteine-rich, small protein in this process in cyanobacteria. Deletion of the nblD gene in the cyanobacterium Synechocystis sp. PCC 6803 prevented the degradation of phycobilisomes, leading to a nonbleaching (nbl) phenotype, which could be complemented by a plasmid-localized gene copy. Competitive growth experiments between the ΔnblD and the wild-type strain provided direct evidence for the physiological importance of NblD under nitrogen-limited conditions. Ectopic expression of NblD under nitrogen-replete conditions showed no effect, in contrast to the unrelated proteolysis adaptors NblA1 and NblA2, which can trigger phycobilisome degradation. Transcriptome analysis indicated increased nblA1/2 transcript levels in the ΔnblD strain during nitrogen starvation, implying that NblD does not act as a transcriptional (co)regulator. However, immunoprecipitation and far-western experiments identified the chromophorylated (holo form) of the phycocyanin ß-subunit (CpcB) as its target, while apo-CpcB was not bound. The addition of recombinant NblD to isolated phycobilisomes caused a reduction in phycocyanin absorbance and a broadening and shifting of the peak to lower wavelengths, indicating the occurrence of structural changes. These data demonstrate that NblD plays a crucial role in the coordinated dismantling of phycobilisomes and add it as a factor to the genetically programmed response to nitrogen starvation.


Assuntos
Proteínas de Bactérias/metabolismo , Ficobilissomas/metabolismo , Synechocystis/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência Conservada , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Modelos Biológicos , Mutação/genética , Nitrogênio/deficiência , Nitrogênio/farmacologia , Fenótipo , Fotossíntese , Filogenia , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Synechocystis/efeitos dos fármacos , Synechocystis/genética , Transcriptoma/genética
14.
Nat Commun ; 11(1): 5299, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33082345

RESUMO

Parasitic plants of the genus Cuscuta penetrate shoots of host plants with haustoria and build a connection to the host vasculature to exhaust water, solutes and carbohydrates. Such infections usually stay unrecognized by the host and lead to harmful host plant damage. Here, we show a molecular mechanism of how plants can sense parasitic Cuscuta. We isolated an 11 kDa protein of the parasite cell wall and identified it as a glycine-rich protein (GRP). This GRP, as well as its minimal peptide epitope Crip21, serve as a pathogen-associated molecular pattern and specifically bind and activate a membrane-bound immune receptor of tomato, the Cuscuta Receptor 1 (CuRe1), leading to defense responses in resistant hosts. These findings provide the initial steps to understand the resistance mechanisms against parasitic plants and further offer great potential for protecting crops by engineering resistance against parasitic plants.


Assuntos
Parede Celular/metabolismo , Cuscuta/metabolismo , Lycopersicon esculentum/metabolismo , Lycopersicon esculentum/parasitologia , Doenças das Plantas/parasitologia , Proteínas de Plantas/metabolismo , Parede Celular/genética , Cuscuta/genética , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Parasita , Lycopersicon esculentum/genética , Doenças das Plantas/genética , Proteínas de Plantas/genética
15.
Front Cell Dev Biol ; 8: 544043, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33072739

RESUMO

Disease modeling requires appropriate cellular models that best mimic the underlying pathophysiology. Human origin and an adequate expression of the disease protein are pre-requisites that support information from a model to be meaningful. In this study we investigated expression profiles of (i) PBMCs and (ii) fibroblasts as patient derived cells as well as (iii) lymphoblasts and (iv) induced pluripotent stem cells (iPSC) as immortalized sources, and (v) iPSC-derived cortical neurons to assess their aptitude to model motor neuron diseases (MNDs) including hereditary spastic paraplegia (HSP), amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). We generated all five different cell types from two healthy donors and performed RNA sequencing to display expression patterns in MND-related genes. For the ten most common HSP genotypes we validated gene expression by qPCR. To verify the results on protein level, proteome analysis of fibroblasts, iPSCs and cortical neurons was performed. Depending on the specific MND gene we found largely different expression patterns. Out of 168 MND-related genes, 50 had their highest expression in iPSC-derived cortical neurons, 41 were most strongly expressed in fibroblasts, 26 in lymphoblasts, 22 in iPSCs, and 14 in PBMCs. Pathophysiologically related MNDs like HSPs associated with axonal transport deficits shared highest expression in cortical neurons. 15 MND-related genes were not detectable in any of the analyzed cell types. This may reflect the critical dependency of motor neurons on support of other cell types like oligodendrocytes which express myelin proteins like L1CAM (SPG1), PLP1 (SPG2) and MAG (SPG75) which are lacking in neurons but cause MNDs if mutated. This study provides comprehensive information on expression of genes associated with a large spectrum of MNDs. Expression profiles can be used to inform on appropriate cell models for genotype specific motor neuron research.

16.
Sci Rep ; 10(1): 13336, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770129

RESUMO

Efficient mRNA splicing is a prerequisite for protein biosynthesis and the eukaryotic splicing machinery is evolutionarily conserved among species of various phyla. At its catalytic core resides the activated splicing complex Bact consisting of the three small nuclear ribonucleoprotein complexes (snRNPs) U2, U5 and U6 and the so-called NineTeen complex (NTC) which is important for spliceosomal activation. CWC15 is an integral part of the NTC in humans and it is associated with the NTC in other species. Here we show the ubiquitous expression and developmental importance of the Arabidopsis ortholog of yeast CWC15. CWC15 associates with core components of the Arabidopsis NTC and its loss leads to inefficient splicing. Consistent with the central role of CWC15 in RNA splicing, cwc15 mutants are embryo lethal and additionally display strong defects in the female haploid phase. Interestingly, the haploid male gametophyte or pollen in Arabidopsis, on the other hand, can cope without functional CWC15, suggesting that developing pollen might be more tolerant to CWC15-mediated defects in splicing than either embryo or female gametophyte.


Assuntos
Arabidopsis/genética , Spliceossomos/genética , Pólen/genética , Splicing de RNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
17.
Mol Cell Proteomics ; 19(9): 1485-1502, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32554711

RESUMO

During Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, we examined protein-protein associations of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells allowed us to identify 26 previously unknown associations, besides recovering several well characterized interactions. We identified interactions between RBPs and several splicing factors, providing links between nuclear and cytoplasmic events of mRNA regulation. Additionally, components of the translational and RNA decay machineries were selectively co-purified with some baits, suggesting a mechanism for how RBPs may regulate maternal transcripts. Given the evolutionary conservation of the studied RBPs, the interaction network presented here provides the foundation for future functional and structural studies of mRNA localization across metazoans.


Assuntos
Proteínas de Drosophila/metabolismo , Oogênese , Ovário/metabolismo , Mapas de Interação de Proteínas , Proteínas de Ligação a RNA/metabolismo , Animais , Animais Geneticamente Modificados , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Drosophila , Proteínas de Drosophila/genética , Feminino , Ontologia Genética , Células HEK293 , Humanos , Imunoprecipitação , Espectrometria de Massas , Metaboloma , MicroRNAs/genética , MicroRNAs/metabolismo , Oogênese/genética , Biossíntese de Proteínas , Mapas de Interação de Proteínas/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Estabilidade de RNA , RNA Mensageiro Estocado/genética , RNA Mensageiro Estocado/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes
18.
Plant Physiol ; 183(1): 371-384, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32152212

RESUMO

Plants have evolved effective strategies to defend themselves against pathogen invasion. Starting from the plasma membrane with the recognition of microbe-associated molecular patterns (MAMPs) via pattern recognition receptors, internal cellular signaling pathways are induced to ultimately fend off the attack. Phospholipase D (PLD) hydrolyzes membrane phospholipids to produce phosphatidic acid (PA), which has been proposed to play a second messenger role in immunity. The Arabidopsis (Arabidopsis thaliana) PLD family consists of 12 members, and for some of these, a specific function in resistance toward a subset of pathogens has been shown. We demonstrate here that Arabidopsis PLDγ1, but not its close homologs PLDγ2 and PLDγ3, is specifically involved in plant immunity. Genetic inactivation of PLDγ1 resulted in increased resistance toward the virulent bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Botrytis cinerea As pldγ1 mutant plants responded with elevated levels of reactive oxygen species to MAMP treatment, a negative regulatory function for this PLD isoform is proposed. Importantly, PA levels in pldγ1 mutants were not affected compared to stressed wild-type plants, suggesting that alterations in PA levels are not likely the cause for the enhanced immunity in the pldγ1 line. Instead, the plasma-membrane-attached PLDγ1 protein colocalized and associated with the BAK1-INTERACTING RECEPTOR-LIKE KINASES BIR2 and BIR3, which are known negative regulators of pattern-triggered immunity. Moreover, complex formation of PLDγ1 and BIR2 was further promoted upon MAMP treatment. Hence, we propose that PLDγ1 acts as a negative regulator of plant immune responses in complex with immunity-related proteins BIR2 and BIR3.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipases/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Botrytis/patogenicidade , Proteínas de Membrana/genética , Fosfolipase D/metabolismo , Fosfolipases/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Pseudomonas syringae/patogenicidade , Espécies Reativas de Oxigênio/metabolismo
19.
Cell Microbiol ; 22(1): e13111, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31515903

RESUMO

Staphylococcus aureus is a facultative intracellular pathogen. Recently, it has been shown that the protein part of the lipoprotein-like lipoproteins (Lpls), encoded by the lpl cluster comprising of 10 lpls paralogue genes, increases pathogenicity, delays the G2/M phase transition, and also triggers host cell invasion. Here, we show that a recombinant Lpl1 protein without the lipid moiety binds directly to the isoforms of the human heat shock proteins Hsp90α and Hsp90ß. Synthetic peptides covering the Lpl1 sequence caused a twofold to fivefold increase of S. aureus invasion in HaCaT cells. Antibodies against Hsp90 decrease S. aureus invasion in HaCaT cells and in primary human keratinocytes. Additionally, inhibition of ATPase function of Hsp90 or silencing Hsp90α expression by siRNA also decreased the S. aureus invasion in HaCaT cells. Although the Hsp90ß is constitutively expressed, the Hsp90α isoform is heat-inducible and appears to play a major role in Lpl1 interaction. Pre-incubation of HaCaT cells at 39°C increased both the Hsp90α expression and S. aureus invasion. Lpl1-Hsp90 interaction induces F-actin formation, thus, triggering an endocytosis-like internalisation. Here, we uncovered a new host cell invasion principle on the basis of Lpl-Hsp90 interaction.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Lipoproteínas/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Actinas/metabolismo , Proteínas de Bactérias/genética , Células Cultivadas , Endocitose , Prepúcio do Pênis/citologia , Proteínas de Choque Térmico HSP90/genética , Células HaCaT , Interações Hospedeiro-Patógeno , Humanos , Queratinócitos/microbiologia , Lipoproteínas/genética , Masculino , Proteínas Recombinantes
20.
FEBS J ; 287(2): 325-344, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31323700

RESUMO

Enzyme-catalyzed proximity labeling (PL) with the engineered ascorbate peroxidase APEX2 is a novel approach to map organelle compartmentalization and protein networks in living cells. Current procedures developed for mammalian cells do not allow delivery of the cosubstrate, biotin-phenol, into living yeast cells. Here, we present a new method based on semipermeabilized yeast cells. Combined with stable isotope labeling by amino acids in cell culture (SILAC), we demonstrate proteomic mapping of a membrane-enclosed and a semiopen compartment, the mitochondrial matrix and the nucleus. APEX2 PL revealed nuclear proteins that were previously not identified by conventional techniques. One of these, the Yer156C protein, is highly conserved but of unknown function. Its human ortholog, melanocyte proliferating gene 1, is linked to developmental processes and dermatological diseases. A first characterization of the Yer156C neighborhood reveals an array of proteins linked to proteostasis and RNA binding. Thus, our approach establishes APEX2 PL as another powerful tool that complements the methods palette for the model system yeast.


Assuntos
Ascorbato Peroxidases/metabolismo , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Ascorbato Peroxidases/química , Núcleo Celular/metabolismo , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Proteínas Mitocondriais/metabolismo , Mapeamento de Interação de Proteínas/normas , Proteômica/normas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química
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