Outer membrane vesicles secreted by gram-negative bacteria play an important role in bacterial physiology as well as in virulence and host–pathogen interaction. Isolated vesicles of some bacteria ...have also been studied for their immunomodulatory potential in the vaccine development. However, the production of vesicles in sufficient amount, purity and reproducibility remains a critical challenge for subsequent analyses in most bacteria. In the present review methods of production, isolation, purification and quantification of outer membrane vesicles are summarized and discussed.
Although the role of high-risk human papillomaviruses (hrHPVs) as etiological agents in cancer development has been intensively studied during the last decades, there is still the necessity of ...understanding the impact of the HPV
and
oncogenes on host cells, ultimately leading to malignant transformation. Here, we used newly established immortalized human keratinocytes with a well-defined HPV16
expression cassette to get a more complete and less biased overview of global changes induced by HPV16 by employing transcriptome sequencing (RNA-Seq) and stable isotope labeling by amino acids in cell culture (SILAC). This is the first study combining transcriptome and proteome data to characterize the impact of HPV oncogenes in human keratinocytes in comparison with their virus-negative counterparts. To enhance the informative value and accuracy of the RNA-Seq data, four different bioinformatic workflows were used. We identified potential novel upstream regulators (e.g., CNOT7, SPDEF, MITF, and PAX5) controlling distinct clusters of genes within the HPV-host cell network as well as distinct factors (e.g., CPPED1, LCP1, and TAGLN) with essential functions in cancer. Validated results in this study were compared to data sets from The Cancer Genome Atlas (TCGA), demonstrating that several identified factors were also differentially expressed in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) and HPV-positive head and neck squamous cell carcinomas (HNSCs). This highly integrative approach allows the identification of novel HPV-induced cellular changes that are also reflected in cancer patients, providing a promising omics data set for future studies in both basic and translational research.
Human papillomavirus (HPV)-associated cancers still remain a big health problem, especially in developing countries, despite the availability of prophylactic vaccines. Although HPV oncogenes have been intensively investigated for decades, a study applying recent advances in RNA-Seq and quantitative proteomic approaches to a precancerous model system with well-defined HPV oncogene expression alongside HPV-negative parental cells has been missing until now. Here, combined omics analyses reveal global changes caused by the viral oncogenes in a less biased way and allow the identification of novel factors and key cellular networks potentially promoting malignant transformation. In addition, this system also provides a basis for mechanistic research on novel key factors regulated by HPV oncogenes, especially those that are confirmed
in cervical cancer as well as in head and neck cancer patient samples from TCGA data sets.
The adenylate cyclase toxin (CyaA) of the whooping cough agent Bordetella pertussis subverts immune functions of host myeloid cells expressing the α
β
integrin (CD11b/CD18, CR3 or Mac-1). CyaA ...delivers into cytosol of cells an extremely catalytically active adenylyl cyclase enzyme, which disrupts the innate and adaptive immune functions of phagocytes through unregulated production of the key signaling molecule cAMP. We have used phosphoproteomics to analyze cAMP signaling of CyaA in murine bone marrow-derived dendritic cells. CyaA action resulted in alterations of phosphorylation state of a number of proteins that regulate actin cytoskeleton homeostasis, including Mena, Talin-1 and VASP. CyaA action repressed mTOR signaling through activation of mTORC1 inhibitors TSC2 and PRAS40 and altered phosphorylation of multiple chromatin remodelers, including the class II histone deacetylase HDAC5. CyaA toxin action further elicited inhibitory phosphorylation of SIK family kinases involved in modulation of immune response and provoked dephosphorylation of the transcriptional coactivator CRTC3, indicating that CyaA-promoted nuclear translocation of CRTC3 may account for CyaA-induced IL-10 production. These findings document the complexity of subversive physiological manipulation of myeloid phagocytes by the CyaA toxin, serving in immune evasion of the pertussis agent.
The initiation of T-cell signaling is critically dependent on the function of the member of Src family tyrosine kinases, Lck. Upon T-cell antigen receptor (TCR) triggering, Lck kinase activity ...induces the nucleation of signal-transducing hubs that regulate the formation of complex signaling network and cytoskeletal rearrangement. In addition, the delivery of Lck function requires rapid and targeted membrane redistribution, but the mechanism underpinning this process is largely unknown. To gain insight into this process, we considered previously described proteins that could assist in this process
their capacity to interact with kinases and regulate their intracellular translocations. An adaptor protein, receptor for activated C kinase 1 (RACK1), was chosen as a viable option, and its capacity to bind Lck and aid the process of activation-induced redistribution of Lck was assessed. Our microscopic observation showed that T-cell activation induces a rapid, concomitant, and transient co-redistribution of Lck and RACK1 into the forming immunological synapse. Consistent with this observation, the formation of transient RACK1-Lck complexes were detectable in primary CD4
T-cells with their maximum levels peaking 10 s after TCR-CD4 co-aggregation. Moreover, RACK1 preferentially binds to a pool of kinase active pY394
, which co-purifies with high molecular weight cellular fractions. The formation of RACK1-Lck complexes depends on functional SH2 and SH3 domains of Lck and includes several other signaling and cytoskeletal elements that transiently bind the complex. Notably, the F-actin-crosslinking protein, α-actinin-1, binds to RACK1 only in the presence of kinase active Lck suggesting that the formation of RACK1-pY394
-α-actinin-1 complex serves as a signal module coupling actin cytoskeleton bundling with productive TCR/CD4 triggering. In addition, the treatment of CD4
T-cells with nocodazole, which disrupts the microtubular network, also blocked the formation of RACK1-Lck complexes. Importantly, activation-induced Lck redistribution was diminished in primary CD4
T-cells by an adenoviral-mediated knockdown of RACK1. These results demonstrate that in T cells, RACK1, as an essential component of the multiprotein complex which upon TCR engagement, links the binding of kinase active Lck to elements of the cytoskeletal network and affects the subcellular redistribution of Lck.
Wheat belongs to six major food allergens inducing IgE-mediated hypersensitivity reaction manifesting as cutaneous, gastrointestinal, and respiratory symptoms. Although cereals are a staple food item ...in most diets, only a few wheat proteins causing hypersensitivity have been identified. To characterize wheat allergens, salt-soluble wheat extracts were separated by 1-DE and 2-DE and IgE-binding proteins were detected by immunoblotting using sera of patients with allergy to ingested wheat. Proteins, frequently recognized by IgE on 2-DE were analyzed by MALDI-TOF and QTOF and their spectrum was completed by 1-DE and LCQDECA nLC-MS/MS IT technique. Using all three techniques we identified 19 potential wheat allergens such as α-amylase inhibitors, β-amylase, profilin, serpin, β-D-glucan exohydrolase, and 27K protein. Employing newly developed ELISA, levels of IgE Abs against Sulamit wheat extract and α-amylase inhibitors type 1 and 3 were quantified and shown to be significantly elevated in sera of allergic patients compared to those of healthy controls. The level of IgE Abs against α-amylase inhibitor type 3 was lower, slightly above the cut-off value in the majority of patients' sera. Our findings contribute to the identification of wheat allergens aimed to increase the specificity of serum IgE and cell activation diagnostic assays.
Due to their cardiac origin, H9c2 cells rank among the most popular cell lines in current cardiovascular research, yet molecular phenotype remains elusive. Hence, in this study we used proteomic ...approach to describe molecular phenotype of H9c2 cells in their undifferentiated (i.e., most frequently used) state, and its functional response to cardiotoxic drug doxorubicin. Of 1671 proteins identified by iTRAQ IEF/LC–MSMS analysis, only 12 proteins were characteristic for striated muscle cells and none was cardiac phenotype-specific. Targeted LC-SRM and western blot analyses confirmed that undifferentiated H9c2 cells are phenotypically considerably different to both primary neonatal cardiomyocytes and adult myocardium. These cells lack proteins essential for formation of striated muscle myofibrils or they express only minor amounts thereof. They also fail to express many proteins important for metabolism of muscle cells. The challenge with clinically relevant concentrations of doxorubicin did not induce a proteomic signature that has been previously noted in primary cardiomyocytes or adult hearts. Instead, several alterations previously described in other cells of mesodermal origin, such as fibroblasts, were observed (e.g., severe down-regulation of collagen synthesis pathway). In conclusion, the molecular phenotype of H9c2 cells resembles very immature myogenic cells with skeletal muscle commitment upon differentiation and thus, translatability of findings obtained in these cells deserves caution.
•Proteomics was used to characterize phenotype of undifferentiated H9c2 cells.•Heart-derived H9c2 cells were found considerably different from cardiac myocytes.•Doxorubicin does not induce typical cardiotoxic proteomic signature in H9c2 cells.•Interpretation of findings obtained on H9c2 cells deserves caution and verification.
The intracellular pathogens have the unique capacity to sense the host cell environment and to respond to it by alteration in gene expression and protein synthesis. Proteomic analysis of bacteria ...exposed directly to the host cell milieu might thus greatly contribute to the elucidation of processes leading to bacterial adaptation and proliferation inside the host cell. Here we have performed a global proteome analysis of a virulent Francisella tularensis subsp. holarctica strain during its intracellular cycle within the macrophage-like murine cell line J774.2 using the metabolic pulse-labeling of bacterial proteins with ³⁵S-methionine and ³⁵S-cysteine in various periods of infection. The two-dimensional gel analysis revealed macrophage-induced bacterial proteome changes in which 64 identified proteins were differentially expressed in comparison to controls grown in tissue culture medium. Nevertheless, activation of macrophages with interferon gamma before in vitro infection decreased the number of detected alterations in protein levels. Thus, these proteomic data indicate the F. tularensis ability to adapt to the intracellular hostile environment that is, however, diminished by prior interferon gamma treatment of host cells.
Celotno besedilo
Dostopno za:
CEKLJ, DOBA, EMUNI, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK
Determining disease-associated changes in protein glycosylation provides a better understanding of pathogenesis. This work focuses on human immunoglobulin A1 (IgA1), where aberrant O-glycosylation ...plays a key role in the pathogenesis of IgA nephropathy (IgAN). Normal IgA1 hinge region carries 3 to 6 O-glycans consisting of N-acetylgalactosamine (GalNAc) and galactose (Gal); both sugars may be sialylated. In IgAN patients, some O-glycans on a fraction of IgA1 molecules are Gal-deficient. Here we describe a sample preparation protocol with optimized cysteine alkylation of a Gal-deficient polymeric IgA1 myeloma protein prior to in-gel digestion and analysis of the digest by MALDI-TOF/TOF mass spectrometry (MS). Following a novel strategy, IgA1 hinge-region O-glycopeptides were fractionated by reversed-phase liquid chromatography using a microgradient device and identified by MALDI-TOF/TOF tandem MS (MS/MS). The acquired MS/MS spectra were interpreted manually and by means of our own software. This allowed assigning up to six O-glycosylation sites and demonstration, for the first time, of the distribution of isomeric O-glycoforms having the same molecular mass, but a different glycosylation pattern. The most abundant Gal-deficient O-glycoforms were GalNAc4Gal3 and GalNAc5Gal4 with one Gal-deficient site and GalNAc5Gal3 and GalNAc4Gal2 with two Gal-deficient sites. The most frequent Gal-deficient sites were at Ser230 and/or Thr236.
In this work, we studied the O-glycosylation in the hinge region of human immunoglobulin A1 (IgA1). Aberrant glycosylation of the protein plays a key role in the pathogenesis of IgA nephropathy. Thus identification of the O-glycan composition of IgA1 is important for a deeper understanding of the disease mechanism, biomarker discovery and validation, and implementation and monitoring of disease-specific therapies. We developed a new procedure for elucidating the heterogeneity of IgA1 O-glycosylation. After running a polyacrylamide gel electrophoresis under denaturing conditions, the heavy chain of IgA1 was subjected to in-gel digestion by trypsin. O-glycopeptides were separated from the digest on capillary columns using a microgradient chromatographic device (replacing commonly used liquid chromatographs) and subjected to MALDI-TOF/TOF mass spectrometry (MS) and tandem mass spectrometry (MS/MS) involving post-source decay fragmentation. We show that the complete modification of cysteines by iodoacetamide prior to electrophoresis is critical for successful MS/MS analyses on the way to deciphering the microheterogeneity of O-glycosylation in IgA1. Similarly, the removal of the excess of the reagent is equally important. The acquired MS/MS allowed assigning up to six O-glycosylation sites and identification of isomeric O-glycoforms. We show that our simplified approach is efficient and has a high potential to provide a method for the rapid assessment of IgA1 heterogeneity that is a less expensive and yet corroborating alternative to LC-(high-resolution)-MS protocols. The novelty and biological significance reside in the demonstration, for the first time, of the distribution of the most abundant isoforms of HR O-glycopeptides of IgA1. As another new feature, we introduce a software solution for the interpretation of MS/MS data of O-glycopeptide isoforms, which provides the possibility of fast and easier data processing.
This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.
Amino-acid sequence segments of selected heavy-chain O-glycopeptide isoforms originating from the hinge region of human myeloma pIgA1 (Ale) protein, which were detected in the present study by a software processing of MALDI-TOF/TOF MS/MS data. Frames indicate abundant isoforms; characteristic y-type fragment ions observed in the corresponding MS/MS spectra are labeled. For each isoform, a code with the number of attached GalNAc and Gal residues is provided. Display omitted
•This work focuses on the O-glycosylation of immunoglobulin A1 (IgA1).•IgA1 hinge-region glycopeptides were chromatographed using a microgradient device.•MALDI-TOF/TOF tandem mass spectrometry allowed assigning O-glycosylation sites.•The most frequent galactose-deficient sites were at Ser230 and/or Thr236.•The distribution of isomeric O-glycoforms is demonstrated using a new software.
Abstract Chronic anthracycline cardiotoxicity is a feared complication of cancer chemotherapy. However, despite several decades of primarily hypothesis-driven research, the molecular basis of this ...phenomenon remains poorly understood. The aim of this study was to obtain integrative molecular insights into chronic anthracycline cardiotoxicity and the resulting heart failure. Cardiotoxicity was induced in rabbits (daunorubicin 3 mg/kg, weekly, 10 weeks) and changes in the left ventricular proteome were analyzed by 2D-DIGE. The protein spots with significant changes (p < 0.01, > 1.5-fold) were identified using MALDI-TOF/TOF. Key data were corroborated by immunohistochemistry, qRT-PCR and enzyme activity determination and compared with functional, morphological and biochemical data. The most important alterations were found in mitochondria — especially in proteins crucial for oxidative phosphorylation, energy channeling, antioxidant defense and mitochondrial stress. Furthermore, the intermediate filament desmin, which interacts with mitochondria, was determined to be distinctly up-regulated and disorganized in its expression pattern. Interestingly, the latter changes reflected the intensity of toxic damage in whole hearts as well as in individual cells. In addition, a marked drop in myosin light chain isoforms, activation of proteolytic machinery (including the proteasome system), increased abundance of chaperones and proteins involved in chaperone-mediated autophagy, membrane repair as well as apoptosis were found. In addition, dramatic changes in proteins of basement membrane and extracellular matrix were documented. In conclusion, for the first time, the complex proteomic signature of chronic anthracycline cardiotoxicity was revealed which enhances our understanding of the basis for this phenomenon and it may enhance efforts in targeting its reduction.
The success of pathogens depends on their ability to circumvent immune defences. Francisella tularensis is one of the most infectious bacteria known. The remarkable virulence of Francisella is ...believed to be due to its capacity to evade or subvert the immune system, but how remains obscure. Here, we show that Francisella triggers but concomitantly inhibits the Toll‐like receptor, RIG‐I‐like receptor, and cytoplasmic DNA pathways. Francisella subverts these pathways at least in part by inhibiting K63‐linked polyubiquitination and assembly of TRAF6 and TRAF3 complexes that control the transcriptional responses of pattern recognition receptors. We show that this mode of inhibition requires a functional type VI secretion system and/or the presence of live bacteria in the cytoplasm. The ability of Francisella to enter the cytosol while simultaneously inhibiting multiple pattern recognition receptor pathways may account for the notable capacity of this bacterium to invade and proliferate in the host without evoking a self‐limiting innate immune response.
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