Refine
Document Type
- Article (16)
Language
- English (16)
Has Fulltext
- yes (16)
Is part of the Bibliography
- no (16)
Keywords
- - (10)
- proteomics (4)
- mass spectrometry (2)
- secretome (2)
- ADCC (1)
- AICC (1)
- Affymetrix (1)
- BCL11B (1)
- CNS (1)
- CRISPR/Cas (1)
Institute
- Interfakultäres Institut für Genetik und Funktionelle Genomforschung (MNF) (4)
- Interfakultäres Institut für Genetik und Funktionelle Genomforschung (UMG) (4)
- Kliniken und Polikliniken für Innere Medizin (2)
- Abteilung für Mikrobiologie und Molekularbiologie (1)
- Institut für Anatomie und Zellbiologie (1)
- Institut für Humangenetik (1)
- Institut für Immunologie u. Transfusionsmedizin - Abteilung Transfusionsmedizin (1)
- Institut für Med. Biochemie u. Molekularbiologie (1)
- Klinik und Poliklinik für Kinder- und Jugendmedizin (1)
- Mathematisch-Naturwissenschaftliche Fakultät (1)
Publisher
- MDPI (8)
- Frontiers Media S.A. (2)
- Wiley (2)
- Hindawi (1)
- S. Karger AG (1)
- Springer Nature (1)
- Taylor & Francis (1)
Microglia are the resident immune cells of the central nervous system (CNS) and play a major role in the regulation of brain homeostasis. To maintain their cellular protein homeostasis, microglia express standard proteasomes and immunoproteasomes (IP), a proteasome isoform that preserves protein homeostasis also in non-immune cells under challenging conditions. The impact of IP on microglia function in innate immunity of the CNS is however not well described. Here, we establish that IP impairment leads to proteotoxic stress and triggers the unfolded and integrated stress responses in mouse and human microglia models. Using proteomic analysis, we demonstrate that IP deficiency in microglia results in profound alterations of the ubiquitin-modified proteome among which proteins involved in the regulation of stress and immune responses. In line with this, molecular analysis revealed chronic activation of NF-κB signaling in IP-deficient microglia without further stimulus. In addition, we show that IP impairment alters microglial function based on markers for phagocytosis and motility. At the molecular level IP impairment activates interferon signaling promoted by the activation of the cytosolic stress response protein kinase R. The presented data highlight the importance of IP function for the proteostatic potential as well as for precision proteolysis to control stress and immune signaling in microglia function.
Tissue sections, which are widely used in research and diagnostic laboratories and have already been examined by immunohistochemistry (IHC), may subsequently provide a resource for proteomic studies, even though only small amount of protein is available. Therefore, we established a workflow for tandem mass spectrometry-based protein profiling of IHC specimens and characterized defined brain area sections. We investigated the CA1 region of the hippocampus dissected from brain slices of adult C57BL/6J mice. The workflow contains detailed information on sample preparation from brain slices, including removal of antibodies and cover matrices, dissection of region(s) of interest, protein extraction and digestion, mass spectrometry measurement, and data analysis. The Gene Ontology (GO) knowledge base was used for further annotation. Literature searches and Gene Ontology annotation of the detected proteins verify the applicability of this method for global protein profiling using formalin-fixed and embedded material and previously used IHC slides.
Background: Hyperthyroidism is known to induce a hypercoagulable state. It stimulates plasma levels of procoagulative factors and reduces fibrinolytic activity. So far most of the data have been derived from patients with endogenous hyperthyroidism with a wide variability in the underlying pathogenesis and severity of the disease. Objectives: In this study we experimentally induced thyrotoxicosis in healthy volunteers to explore the effects of thyroxine excess on the plasma proteome. Using a shotgun proteomics approach, the abundance of plasma proteins was monitored before, during and after thyrotoxicosis. Methods: Sixteen healthy male subjects were sampled at baseline, 4 and 8 weeks under 250 µg/day thyroxine p.o., as well as 4 and 8 weeks after stopping the application. Plasma proteins were analyzed after depletion of 6 high-abundance proteins (MARS6) by LC-ESI-MS/MS mass spectrometry. Mass spectrometric raw data were processed using a label-free, intensity-based workflow. Subsequently, the linear dependence between protein abundances and fT<sub>4</sub> levels were calculated using a Pearson correlation. Results: All subjects developed biochemical thyrotoxicosis, and this effect was reversed within the first 4 weeks of follow-up. None of the volunteers noticed any subjective symptoms. Levels of 10 proteins involved in the coagulation cascade specifically correlated with fT<sub>4</sub>, supporting an influence of thyroid hormone levels on blood coagulation even at nonpathological levels. Conclusions: The results suggest that experimental thyrotoxicosis exerts selective and specific thyroxine-induced effects on coagulation markers. Our study design allows assessment of thyroid hormone effects on plasma protein levels without secondary effects of other diseases or therapies.
Under hyperosmotic conditions, bacteria accumulate compatible solutes through synthesis or import. Bacillus subtilis imports a large set of osmostress protectants via five osmotically controlled transport systems (OpuA to OpuE). Biosynthesis of the particularly effective osmoprotectant glycine betaine requires the exogenous supply of choline. While OpuB is rather specific for choline, OpuC imports a broad spectrum of compatible solutes, including choline and glycine betaine. One previously mapped antisense RNA of B. subtilis, S1290, exhibits strong and transient expression in response to a suddenly imposed salt stress. It covers the coding region of the opuB operon and is expressed from a strictly SigB-dependent promoter. By inactivation of this promoter and analysis of opuB and opuC transcript levels, we discovered a time-delayed osmotic induction of opuB that crucially depends on the S1290 antisense RNA and on the degree of the imposed osmotic stress. Time-delayed osmotic induction of opuB is apparently caused by transcriptional interference of RNA-polymerase complexes driving synthesis of the converging opuB and S1290 mRNAs. When our data are viewed in an ecophysiological framework, it appears that during the early adjustment phase of B. subtilis to acute osmotic stress, the cell prefers to initially rely on the transport activity of the promiscuous OpuC system and only subsequently fully induces opuB. Our data also reveal an integration of osmostress-specific adjustment systems with the SigB-controlled general stress response at a deeper level than previously appreciated.
Human donor milk (HDM) provides appropriate nutrition and offers protective functionsin preterm infants. The aim of the study is to examine the impact of different storage conditions onthe stability of the human breast milk peptidome. HDM was directly frozen at−80◦C or stored at−20◦C (120 h), 4◦C (6 h), or room temperature (RT for 6 or 24 h). The milk peptidome was profiledby mass spectrometry after peptide collection by ultrafiltration. Profiling of the peptidome covered3587 peptides corresponding to 212 proteins. The variance of the peptidome increased with storagetemperature and time and varied for different peptides. The highest impact was observed whensamples were stored at RT. Smaller but significant effects were still observed in samples stored at4◦C, while samples showed highest similarity to those immediately frozen at−80◦C when storedat−20◦C. Peptide structures after storage at RT for 24 h point to the increased activity of thrombinand other proteases cleaving proteins at lysine/arginine. The results point to an ongoing proteindegradation/peptide production by milk-derived proteases. They underline the need for immediatefreezing of HDM at−20◦C or−80◦C to prevent degradation of peptides and enable reproducibleinvestigation of prospectively collected samples.
Fibroblasts contribute to approximately 20% of the non-cardiomyocytic cells in the heart. They play important roles in the myocardial adaption to stretch, inflammation, and other pathophysiological conditions. Fibroblasts are a major source of extracellular matrix (ECM) proteins whose production is regulated by cytokines, such as TNF-α or TGF-β. The resulting myocardial fibrosis is a hallmark of pathological remodeling in dilated cardiomyopathy (DCM). Therefore, in the present study, the secretome and corresponding transcriptome of human cardiac fibroblasts from patients with DCM was investigated under normal conditions and after TNF-α or TGF-β stimulation. Secreted proteins were quantified via mass spectrometry and expression of genes coding for secreted proteins was analyzed via Affymetrix Transcriptome Profiling. Thus, we provide comprehensive proteome and transcriptome data on the human cardiac fibroblast’s secretome. In the secretome of quiescent fibroblasts, 58% of the protein amount belonged to the ECM fraction. Interestingly, cytokines were responsible for 5% of the total protein amount in the secretome and up to 10% in the corresponding transcriptome. Furthermore, cytokine gene expression and secretion were upregulated upon TNF-α stimulation, while collagen secretion levels were elevated after TGF-β treatment. These results suggest that myocardial fibroblasts contribute to pro-fibrotic and to inflammatory processes in response to extracellular stimuli.
Simple Summary
Neuronal plasticity refers to the brain’s ability to adapt in response to activity-dependent changes. This process, among others, allows the brain to acquire memory or to compensate for a neurocognitive deficit. We analyzed adult FTSJ1-deficient mice in order to gain insight into the role of FTSJ1 in neuronal plasticity. These mice displayed alterations in the hippocampus (a brain structure that is involved in memory and learning, among other functions) e.g., in the form of changes in dendritic spines. Changes in dendritic spines are considered to represent a morphological hallmark of altered neuronal plasticity, and thus FTSJ1 deficiency might have a direct effect upon the capacity of the brain to adapt to plastic changes. Long-term potentiation (LTP) is an electrophysiological correlate of neuronal plasticity, and is related to learning and to processes attributed to memory. Here we show that LTP in FTSJ1-deficient mice is reduced, hinting at disturbed neuronal plasticity. These findings suggest that FTSJ1 deficiency has an impact on neuronal plasticity not only morphologically but also on the physiological level.
Abstract
The role of the tRNA methyltransferase FTSJ1 in the brain is largely unknown. We analyzed whether FTSJ1-deficient mice (KO) displayed altered neuronal plasticity. We explored open field behavior (10 KO mice (aged 22–25 weeks)) and 11 age-matched control littermates (WT) and examined mean layer thickness (7 KO; 6 WT) and dendritic spines (5 KO; 5 WT) in the hippocampal area CA1 and the dentate gyrus. Furthermore, long-term potentiation (LTP) within area CA1 was investigated (5 KO; 5 WT), and mass spectrometry (MS) using CA1 tissue (2 each) was performed. Compared to controls, KO mice showed a significant reduction in the mean thickness of apical CA1 layers. Dendritic spine densities were also altered in KO mice. Stable LTP could be induced in the CA1 area of KO mice and remained stable at for at least 1 h, although at a lower level as compared to WTs, while MS data indicated differential abundance of several proteins, which play a role in neuronal plasticity. FTSJ1 has an impact on neuronal plasticity in the murine hippocampal area CA1 at the morphological and physiological levels, which, in conjunction with comparable changes in other cortical areas, might accumulate in disturbed learning and memory functions.
Beta-2-glycoprotein I (β2GPI) is a blood protein and the major antigen in the autoimmune disorder
antiphospholipid syndrome (APS). β2GPI exists mainly in closed or open conformations and
comprises of 11 disulfides distributed across five domains. The terminal Cys288/Cys326 disulfide
bond at domain V has been associated with different cysteine redox states. The role of this disulfide
bond in conformational dynamics of this protein has not been investigated so far. Here, we report
on the enzymatic driven reduction by thioredoxin-1 (recycled by Tris(2-carboxyethyl)phosphine;
TCEP) of β2GPI. Specific reduction was demonstrated by Western blot and mass spectrometry
analyses confirming majority targeting to the fifth domain of β2GPI. Atomic force microscopy images
suggested that reduced β2GPI shows a slightly higher proportion of open conformation and is more
flexible compared to the untreated protein as confirmed by modelling studies. We have determined a
strong increase in the binding of pathogenic APS autoantibodies to reduced β2GPI as demonstrated
by ELISA. Our study is relevant for understanding the effect of β2GPI reduction on the protein
structure and its implications for antibody binding in APS patients.