Isobaric labeling-based quantitative proteomics of FACS-purified immune cells and epithelial cells from the intestine of Crohn's disease patients reveals proteome changes of potential importance in disease pathogenesis.

Crohn's disease (CD) is a chronic condition characterized by recurrent flares of inflammation in the gastrointestinal tract. Disease etiology is poorly understood and is characterized by dysregulated immune activation that progressively destroys intestinal tissue. Key cellular compartments in disease pathogenesis are the intestinal epithelial layer and its underlying lamina propria. While the epithelium contains predominantly epithelial cells, the lamina propria is enriched in immune cells. Deciphering proteome changes in different cell populations is important to understand CD pathogenesis. Here, using isobaric labeling-based quantitative proteomics, we perform an exploratory study to analyze in-depth proteome changes in epithelial cells, immune cells and stromal cells in CD patients compared to controls using cells purified by FACS. Our study revealed increased proteins associated with neutrophil degranulation and mitochondrial metabolism in immune cells of CD intestinal mucosa. We also found upregulation of proteins involved in glycosylation and secretory pathways in epithelial cells of CD patients, while proteins involved in mitochondrial metabolism were reduced. The distinct alterations in protein levels in immune- versus epithelial cells underscores the utility of proteome analysis of defined cell types. Moreover, our workflow allowing concomitant assessment of cell-type specific changes on an individual basis enables deeper insight into disease pathogenesis.

A stationary phase with a positively charged surface allows for minimizing formic acid concentration in the mobile phase, enhancing electrospray ionization in LC-MS proteomic experiments.

The default choice of mobile phase acidifier for bottom-up LC-MS proteomic analyses is 0.10% formic acid because of its decent acidity, decent ion pairing ability, and low suppression of electrospray ionization. In recent years, state-of-the-art columns have been designed specifically to provide efficient separation even when using an MS-friendly mobile phase of low ionic strength. Despite this, no attempts have been made to improve the sensitivity of the MS-based analytical methods by reducing the amount of formic acid in the mobile phase. In this study, we evaluated the effect of reduced formic acid concentration in the mobile phase on the chromatographic behavior and MS response of peptides when separated using columns packed with a C18 stationary phase with a positively charged surface. Using 0.01% formic acid in the mobile phase maintained excellent chromatographic performance and increased MS signal response compared to the standard of 0.10%. The enhanced MS response translated to about 50% improved peptide identifications depending on the complexity and amount of sample injected. The increased retention of peptides at a reduced formic acid concentration was directly proportional to the number of acidic residues in the peptide sequence. The study was carried out by covering a spectrum of protein samples with varied complexity using analytical flow, micro-, and nanoflow regimes to expand the applicability in routine practice.

Sense and Nonsense of Elevated Column Temperature in Proteomic Bottom-up LC-MS Analyses.

Elevated column temperature represents a simple means for improving chromatographic separation of peptides. Here, we demonstrated the advantages of the column temperature in peptide separation using state-of-the-art columns. More importantly, we also determined how temperature can impair proteomic bottom-up analyses. We found that an elevated temperature in combination with the acidic pH of the mobile phase induced in-column peptide hydrolysis with high specificity to Asp and accelerated five modification reactions of amino acids. The positive effects of temperature dominated in the 30 min long gradients since the column operated at 90 °C provided the largest number of identified peptides and proteins. However, the adverse effects of temperature on peptide integrity in longer liquid chromatography-mass spectrometry (LC-MS) analyses required its reduction to obtain optimum results. The largest number of peptides was identified using the column maintained at 75 °C in 60 min long gradients, at 60 °C in 120 min long gradients, and at 45 °C in 240 min long gradients. Our results indicate that no universal column temperature exists for bottom-up LC-MS analyses. Quite the contrary, the temperature setting must be selected rationally to exploit the full capabilities of the state-of-the-art mass spectrometers in proteomic LC-MS analyses, with the gradient time being a critical factor.

Altered ceramide metabolism is a feature in the extracellular vesicle-mediated spread of alpha-synuclein in Lewy body disorders.

Mutations in glucocerebrosidase (GBA) are the most prevalent genetic risk factor for Lewy body disorders (LBD)-collectively Parkinson's disease, Parkinson's disease dementia and dementia with Lewy bodies. Despite this genetic association, it remains unclear how GBA mutations increase susceptibility to develop LBD. We investigated relationships between LBD-specific glucocerebrosidase deficits, GBA-related pathways, and α-synuclein levels in brain tissue from LBD and controls, with and without GBA mutations. We show that LBD is characterised by altered sphingolipid metabolism with prominent elevation of ceramide species, regardless of GBA mutations. Since extracellular vesicles (EV) could be involved in LBD pathogenesis by spreading disease-linked lipids and proteins, we investigated EV derived from post-mortem cerebrospinal fluid (CSF) and brain tissue from GBA mutation carriers and non-carriers. EV purified from LBD CSF and frontal cortex were heavily loaded with ceramides and neurodegeneration-linked proteins including alpha-synuclein and tau. Our in vitro studies demonstrate that LBD EV constitute a "pathological package" capable of inducing aggregation of wild-type alpha-synuclein, mediated through a combination of alpha-synuclein-ceramide interaction and the presence of pathological forms of alpha-synuclein. Together, our findings indicate that abnormalities in ceramide metabolism are a feature of LBD, constituting a promising source of biomarkers, and that GBA mutations likely accelerate the pathological process occurring in sporadic LBD through endolysosomal deficiency.

The role of innate immunity and inflammation in Parkinson´s disease.

For many years, it was postulated that the brain is the organ behind the barrier with an autonomous need for its maintenance. This view has been changed by the concept that the central nervous system is sensitive to the immune processes occurring in the periphery as well as to the infiltration of peripheral immune cells. However, how the immune system might contribute to the development of neurodegenerative diseases, such as Parkinson's disease (PD), remains unclear. PD is a chronic neurodegenerative disorder that affects motor and cognitive functions. Although the precise cause of PD is unknown, studies in both mice and human suggest that alterations in the innate immunity may play a critical role in modulating PD progression. Here, we review recent advancements in our understanding of inflammation and the innate immune mechanisms in PD pathology.

Bordetellapertussis Acetylome is Shaped by Lysine Deacetylase Bkd1.

Post-translational modifications of proteins enable swift physiological adaptation of cells to altered growth conditions and stress. Aside from protein phosphorylation, acetylation on ε-amino groups of lysine residues (N-ε-lysine acetylation) represents another important post-translational modification of proteins. For many bacterial pathogens, including the whooping cough agent Bordetella pertussis, the role and extent of protein acetylation remain to be defined. We expressed in Escherichia coli the BP0960 and BP3063 genes encoding two putative deacetylases of B. pertussis and show that BP0960 encodes a lysine deacetylase enzyme, named Bkd1, that regulates acetylation of a range of B. pertussis proteins. Comparison of the proteome and acetylome of a Δbkd1 mutant with the proteome and acetylome of wild-type B. pertussis (PRIDE ID. PXD016384) revealed that acetylation on lysine residues may modulate activities or stabilities of proteins involved in bacterial metabolism and histone-like proteins. However, increased acetylation of the BvgA response regulator protein of the B. pertussis master virulence-regulating BvgAS two-component system affected neither the total levels of produced BvgA nor its phosphorylation status. Indeed, the Δbkd1 mutant was not impaired in the production of key virulence factors and its survival within human macrophages in vitro was not affected. The Δbkd1 mutant exhibited an increased growth rate under carbon source-limiting conditions and its virulence in the in vivo mouse lung infection model was somewhat affected. These results indicate that the lysine deacetylase Bkd1 and N-ε-lysine acetylation primarily modulate the general metabolism rather than the virulence of B. pertussis.

The Early Dendritic Cell Signaling Induced by Virulent Francisella tularensis Strain Occurs in Phases and Involves the Activation of Extracellular Signal-Regulated Kinases (ERKs) and p38 In the Later Stage.

Dendritic cells (DCs) infected by Francisella tularensis are poorly activated and do not undergo classical maturation process. Although reasons of such unresponsiveness are not fully understood, their impact on the priming of immunity is well appreciated. Previous attempts to explain the behavior of Francisella-infected DCs were hypothesis-driven and focused on events at later stages of infection. Here, we took an alternative unbiased approach by applying methods of global phosphoproteomics to analyze the dynamics of cell signaling in primary DCs during the first hour of infection by Francisella tularensis Presented results show that the early response of DCs to Francisella occurs in phases and that ERK and p38 signaling modules induced at the later stage are differentially regulated by virulent and attenuated ΔdsbA strain. These findings imply that the temporal orchestration of host proinflammatory pathways represents the integral part of Francisella life-cycle inside hijacked DCs.

Application of SILAC labeling to primary bone marrow-derived dendritic cells reveals extensive GM-CSF-dependent arginine metabolism.

Although dendritic cells (DCs) control the priming of the adaptive immunity response, a comprehensive description of their behavior at the protein level is missing. The introduction of the quantitative proteomic technique of metabolic labeling (SILAC) into the field of DC research would therefore be highly beneficial. To achieve this, we applied SILAC labeling to primary bone marow-derived DCs (BMDCs). These cells combine both biological relevance and experimental feasibility, as their in vitro generation permits the use of (13)C/(15)N-labeled amino acids. Interestingly, BMDCs appear to exhibit a very active arginine metabolism. Using standard cultivation conditions, ∼20% of all protein-incorporated proline was a byproduct of heavy arginine degradation. In addition, the dissipation of (15)N from labeled arginine to the whole proteome was observed. The latter decreased the mass accuracy in MS and affected the natural isotopic distribution of peptides. SILAC-connected metabolic issues were shown to be enhanced by GM-CSF, which is used for the differentiation of DC progenitors. Modifications of the cultivation procedure suppressed the arginine-related effects, yielding cells with a proteome labeling efficiency of ≥90%. Importantly, BMDCs generated according to the new cultivation protocol preserved their resemblance to inflammatory DCs in vivo, as evidenced by their response to LPS treatment.

Serving the new masters - dendritic cells as hosts for stealth intracellular bacteria.

Dendritic cells (DCs) serve as the primers of adaptive immunity, which is indispensable for the control of the majority of infections. Interestingly, some pathogenic intracellular bacteria can subvert DC function and gain the advantage of an ineffective host immune reaction. This scenario appears to be the case particularly with so-called stealth pathogens, which are the causative agents of several under-diagnosed chronic diseases. However, there is no consensus how less explored stealth bacteria like Coxiella, Brucella and Francisella cross-talk with DCs. Therefore, the aim of this review was to explore the issue and to summarize the current knowledge regarding the interaction of above mentioned pathogens with DCs as crucial hosts from an infection strategy view. Evidence indicates that infected DCs are not sufficiently activated, do not undergo maturation and do not produce expected proinflammatory cytokines. In some cases, the infected DCs even display immunosuppressive behaviour that may be directly linked to the induction of tolerogenicity favouring pathogen survival and persistence.

Radiosensitization of human leukemic HL-60 cells by ATR kinase inhibitor (VE-821): phosphoproteomic analysis.

DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)-triggered by radiation-induced double strand breaks-is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.

Impact of anesthetics on rat hippocampus and neocortex: A comprehensive proteomic study based on label-free mass spectrometry.

Anesthesia is regarded as an important milestone in medicine. However, the negative effect on memory and learning has been observed. In addition, the impact of anesthetics on postoperative cognitive functions is still discussed. In this work, in vivo experiment simulating a general anesthesia and ICU sedation was designed to assess the impact of two intravenous (midazolam, dexmedetomidine) and two inhalational (isoflurane, desflurane) agents on neuronal centers for cognition (neocortex), learning, and memory (hippocampus). More than 3600 proteins were quantified across both neocortex and hippocampus. Proteomic study revealed relatively mild effects of anesthetics, nevertheless, protein dysregulation uncovered possible different effect of isoflurane (and midazolam) compared to desflurane (and dexmedetomidine) to neocortical and hippocampal proteins. Isoflurane induced the upregulation of hippocampal NMDAR and other proteins of postsynaptic density and downregulation of GABA signaling, whereas desflurane and dexmedetomidine rather targeted mitochondrial VDAC isoforms and protein regulating apoptotic activity.

SILAC-IodoTMT for Assessment of the Cellular Proteome and Its Redox Status.

Stable isotope labeling by amino acids in cell culture (SILAC) and iodoacetyl tandem mass tag (iodoTMT) are well-implemented mass spectrometry-based approaches for quantification of proteins and for site-mapping of cysteine modification. We describe here a combination of SILAC and iodoTMT to assess ongoing changes in the global proteome and cysteine modification levels using liquid chromatography separation coupled with high-resolution mass spectrometry (LC-MS/MS).

Lung macrophages utilize unique cathepsin K-dependent phagosomal machinery to degrade intracellular collagen.

Resident tissue macrophages are organ-specialized phagocytes responsible for the maintenance and protection of tissue homeostasis. It is well established that tissue diversity is reflected by the heterogeneity of resident tissue macrophage origin and phenotype. However, much less is known about tissue-specific phagocytic and proteolytic macrophage functions. Here, using a quantitative proteomics approach, we identify cathepsins as key determinants of phagosome maturation in primary peritoneum-, lung-, and brain-resident macrophages. The data further uncover cathepsin K (CtsK) as a molecular marker for lung phagosomes required for intracellular protein and collagen degradation. Pharmacological blockade of CtsK activity diminished phagosomal proteolysis and collagenolysis in lung-resident macrophages. Furthermore, profibrotic TGF-ß negatively regulated CtsK-mediated phagosomal collagen degradation independently from classical endocytic-proteolytic pathways. In humans, phagosomal CtsK activity was reduced in COPD lung macrophages and non-COPD lung macrophages exposed to cigarette smoke extract. Taken together, this study provides a comprehensive map of how peritoneal, lung, and brain tissue environment shapes phagosomal composition, revealing CtsK as a key molecular determinant of lung phagosomes contributing to phagocytic collagen clearance in lungs.

Peroxiredoxin 6 protects irradiated cells from oxidative stress and shapes their senescence-associated cytokine landscape.

Cellular senescence is a complex stress response defined as an essentially irreversible cell cycle arrest mediated by the inhibition of cell cycle-specific cyclin dependent kinases. The imbalance in redox homeostasis and oxidative stress have been repeatedly observed as one of the hallmarks of the senescent phenotype. However, a large-scale study investigating protein oxidation and redox signaling in senescent cells in vitro has been lacking. Here we applied a proteome-wide analysis using SILAC-iodoTMT workflow to quantitatively estimate the level of protein sulfhydryl oxidation and proteome level changes in ionizing radiation-induced senescence (IRIS) in hTERT-RPE-1 cells. We observed that senescent cells mobilized the antioxidant system to buffer the increased oxidation stress. Among the antioxidant proteins with increased relative abundance in IRIS, a unique 1-Cys peroxiredoxin family member, peroxiredoxin 6 (PRDX6), was identified as an important contributor to protection against oxidative stress. PRDX6 silencing increased ROS production in senescent cells, decreased their resistance to oxidative stress-induced cell death, and impaired their viability. Subsequent SILAC-iodoTMT and secretome analysis after PRDX6 silencing showed the downregulation of PRDX6 in IRIS affected protein secretory pathways, decreased expression of extracellular matrix proteins, and led to unexpected attenuation of senescence-associated secretory phenotype (SASP). The latter was exemplified by decreased secretion of pro-inflammatory cytokine IL-6 which was also confirmed after treatment with an inhibitor of PRDX6 iPLA2 activity, MJ33. In conclusion, by combining different methodological approaches we discovered a novel role of PRDX6 in senescent cell viability and SASP development. Our results suggest PRDX6 could have a potential as a drug target for senolytic or senomodulatory therapy.

Paramagnetic antibody-modified microparticles coupled with voltammetry as a tool for isolation and detection of metallothionen as a bioindicator of metal pollution.

Low-molecular mass proteins rich in cysteines called metallothioneins (MT) can be considered as markers for the pollution of the environment by metals. Here, we report on suggestion for an automated procedure for the isolation of MT followed by voltammetric analysis. Primarily, we optimized the automated detection of MT using an electrochemical analyser. It was found that the most sensitive and repeatable analyses are obtained at a temperature of 4 °C for the supporting electrolyte. Further, we optimized experimental conditions for the isolation of MT by using antibody-linked paramagnetic microparticles. Under the optimal conditions (4 h long interaction between the microparticles and MT), the microparticles were tested on isolation of various amounts of MT. The lowest isolated amount of MT by antibody-linked paramagnetic microparticles was 5 µg ml(-1) of MT (50 ng). The automated procedure of MT isolation was further tested on isolation of MT from guppy fish (Poecilia reticulata) treated with silver(i) ions (50 µM AgNO(3)). The whole process lasted less than five hours and was fully automated. We attempted to correlate these results with the standard method for MT isolation. The correlation coefficient is 0.9901, which confirms that results are in good agreement. Moreover, the concentration of silver ions in tissues of fish treated with Ag(i) ions was determined by high performance liquid chromatography with electrochemical detection.

Cell Wall Stress Stimulates the Activity of the Protein Kinase StkP of Streptococcus pneumoniae, Leading to Multiple Phosphorylation.

Streptococcus pneumoniae is an opportunistic human pathogen that encodes a single eukaryotic-type Ser/Thr protein kinase StkP and its functional counterpart, the protein phosphatase PhpP. These signaling enzymes play critical roles in coordinating cell division and growth in pneumococci. In this study, we determined the proteome and phosphoproteome profiles of relevant mutants. Comparison of those with the wild-type provided a representative dataset of novel phosphoacceptor sites and StkP-dependent substrates. StkP phosphorylates key proteins involved in cell division and cell wall biosynthesis in both the unencapsulated laboratory strain Rx1 and the encapsulated virulent strain D39. Furthermore, we show that StkP plays an important role in triggering an adaptive response induced by a cell wall-directed antibiotic. Phosphorylation of the sensor histidine kinase WalK and downregulation of proteins of the WalRK core regulon suggest crosstalk between StkP and the WalRK two-component system. Analysis of proteomic profiles led to the identification of gene clusters regulated by catabolite control mechanisms, indicating a tight coupling of carbon metabolism and cell wall homeostasis. The imbalance of steady-state protein phosphorylation in the mutants as well as after antibiotic treatment is accompanied by an accumulation of the global Spx regulator, indicating a Spx-mediated envelope stress response. In summary, StkP relays the perceived signal of cell wall status to key cell division and regulatory proteins, controlling the cell cycle and cell wall homeostasis.

An adsorptive transfer technique coupled with brdicka reaction to reveal the importance of metallothionein in chemotherapy with platinum based cytostatics.

The drugs based on platinum metals represent one of the oldest, but also one of the most effective groups of chemotherapeutic agents. Thanks to many clinical studies it is known that resistance of tumor cells to drugs is a frequent cause of chemotherapy failure. With regard to platinum based drugs, multidrug resistance can also be connected with increased expression of low-molecular weight protein metallothionein (MT). This study aimed at investigating the interactions of MT with cisplatin or carboplatin, using the adsorptive transfer technique coupled with differential pulse voltammetry Brdicka reaction (AdTS DPV Brdicka reaction), and a comparison of in vitro results with results obtained in vivo. The results obtained from the in vitro study show a strong affinity between platinum based drugs and MT. Further, we analyzed extracts of neuroblastoma cell lines treated with cisplatin or carboplatin. It is clear that neuroblastoma UKF-NB-4 cisplatin-resistant and cisplatin-sensitive cell lines unlikely respond to the presence of the platinum-based cytostatics cisplatin and carboplatin. Finally, we determined the level of MT in samples from rabbits treated with carboplatin and patients with retinoblastoma treated with the same drug.

Lys05 - A Promising Autophagy Inhibitor in the Radiosensitization Battle: Phosphoproteomic Perspective.

BACKGROUND: Autophagy is a crucial factor contributing to radioresistance during radiotherapy. Although Lys05 has proven its ability to improve the results of radiotherapy through the inhibition of autophagy, molecular mechanisms of this inhibition remain elusive. We aimed to describe the molecular mechanisms involved in Lys05-induced inhibition of autophagy. MATERIALS AND METHODS: Radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative) and methods of quantitative phosphoproteomics were employed to define the molecular mechanisms involved in Lys05-induced inhibition of autophagy. RESULTS: We confirmed that at an early stage after irradiation, autophagy was induced, whereas at a later stage after irradiation, it was inhibited. The early-stage induction of autophagy was characterized mainly by the activation of biosynthetic and metabolic processes through up- or down-regulation of the critical autophagic regulatory proteins Sequestosome-1 (SQSTM1) and proline-rich AKT1 substrate 1 (AKT1S1). The late-stage inhibition of autophagy was attributed mainly to down-regulation of Unc-51 like autophagy-activating kinase 1 (ULK1) through phosphorylation at Ser638. CONCLUSION: This work contributes to emerging phosphoproteomic insights into autophagy-mediated global signaling in lung cancer cells, which might consequently facilitate the development of precision medicine therapeutics.

Study of Interactions between Metallothionein and Cisplatin by using Differential Pulse Voltammetry Brdickás reaction and Quartz Crystal Microbalance.

Treatment strategies for tumour diseases are progressively focusing on personalization of medicine. However, this focus requires methods revealing the early general biological mechanisms, including the formation anti-cancer drugs' resistance. The low molecular mass protein metallothionein is thought to be the crucial for the formation of resistance in tumour treatment based on the platinum-cytostatics. The interactions between metallothionein (MT) and cisplatin were determined by the adsorptive transfer stripping technique coupled with the differential pulse votlammetry Brdickás reaction. The signals related to the MT-cisplatin complex appeared at -0.9 V. The formation of this complex depended on the time of interaction between cisplatin and MT. The complex formation was consequently confirmed by quartz crystal microbalance analyses. The formation of this complex was detectable even after a 20 s long interaction. Moreover, we detected presence of MT-cisplatin complex in the blood of male rats treated with this drug.

Quantification of cellular protein and redox imbalance using SILAC-iodoTMT methodology.

Under normal conditions, the cellular redox status is maintained in a steady state by reduction and oxidation processes. These redox alterations in the cell are mainly sensed by protein thiol residues of cysteines thus regulating protein function. The imbalance in redox homeostasis may therefore regulate protein turnover either directly by redox modulating of transcription factors or indirectly by the degradation of damaged proteins due to oxidation. A new analytical method capable of simultaneously assessing cellular protein expression and cysteine oxidation would provide a valuable tool for the field of cysteine-targeted biology. Here, we show a workflow based on protein quantification using metabolic labeling and determination of cysteine oxidation using reporter ion quantification. We applied this approach to determine protein and redox changes in cells after 5-min, 60-min and 32-h exposure to H2O2, respectively. Based on the functional analysis of our data, we confirmed a biological relevance of this approach and its applicability for parallel mapping of cellular proteomes and redoxomes under diverse conditions. In addition, we revealed a specific pattern of redox changes in peroxiredoxins in a short time-interval cell exposure to H2O2. Overall, our present study offers an innovative, versatile experimental approach to the multifaceted assessment of cellular proteome and its redox status, with broad implications for biomedical research towards a better understanding of organismal physiology and diverse disease conditions.