Serum Protein Signatures Differentiating Autoimmune Pancreatitis versus Pancreatic Cancer

Autoimmune pancreatitis (AIP) is defined by characteristic lymphoplasmacytic infiltrate, ductal strictures and a pancreatic enlargement or mass that can mimic pancreatic cancer (PaCa). The distinction between this benign disease and pancreatic cancer can be challenging. However, an accurate diagnosis may pre-empt the misdiagnosis of cancer, allowing the appropriate medical treatment of AIP and, consequently, decreasing the number of unnecessary pancreatic resections.Mass spectrometry (MS) and two-dimensional differential gel electrophoresis (2D-DIGE) have been applied to analyse serum protein alterations associated with AIP and PaCa, and to identify protein signatures indicative of the diseases. Patients'' sera were immunodepleted from the 20 most prominent serum proteins prior to further 2D-DIGE and image analysis. The identity of the most-discriminatory proteins detected, was performed by MS and ELISAs were applied to confirm their expression. Serum profiling data analysis with 2D-DIGE revealed 39 protein peaks able to discriminate between AIP and PaCa. Proteins were purified and further analysed by MALDI-TOF-MS. Peptide mass fingerprinting led to identification of eleven proteins. Among them apolipoprotein A-I, apolipoprotein A-II, transthyretin, and tetranectin were identified and found as 3.0-, 3.5-, 2-, and 1.6-fold decreased in PaCa sera, respectively, whereas haptoglobin and apolipoprotein E were found to be 3.8- and 1.6-fold elevated in PaCa sera. With the exception of haptoglobin the ELISA results of the identified proteins confirmed the 2D-DIGE image analysis characteristics. Integration of the identified serum proteins as AIP markers may have considerable potential to provide additional information for the diagnosis of AIP to choose the appropriate treatment.     

Effects of deoxynivalenol (DON), zearalenone (ZEN), and related metabolites on equine peripheral blood mononuclear cells (PBMC) in vitro and background occurrence of these toxins in horses

Both deoxynivalenol (DON), zearalenone (ZEN), and their metabolites are known to modulate immune cells in various species whereby viability and proliferation are influenced. Such effects were rarely examined in horses. Therefore, one aim of the present study was to titrate the inhibitory concentrations of DON, 3-acetyl-DON (3AcDON), de-epoxy-DON (DOM-1), ZEN, and α- and β-zearalenol (ZEL) at which viability and proliferation of equine PBMC were reduced by 50 % (IC₅₀) and 10 % (IC₁₀) in vitro. For evaluation of practical relevance of the in vitro findings, a further aim was to screen horses for the background occurrence of DON, ZEN, and their metabolites in systemic circulation and to relate toxin residues both to the inhibitory toxin concentrations and to hematological and clinical-chemical characteristics.The IC₅₀ (μM) for DON, 3AcDON, β-ZEL, α-ZEL, and ZEN were determined at 3.09, 25.90, 75.44, 97.44, and 98.15 in unstimulated cells, respectively, while in proliferating cells, the corresponding IC₅₀ values were 0.73, 6.89, 45.16, 75.96, and 82.51. Neither viability nor proliferation was influenced by DOM-1 up to a concentration of 100 μM.The in vivo screening (N?=?49) revealed the occurrence of ZEN (N?=?24), α-ZEL (N?=?3), β-ZEL (N?=?37), DON, and DOM-1 (N?=?2). The detected concentrations were much lower than the corresponding IC₅₀ while the IC₁₀ of DON and β-ZEL for proliferating PBMC corresponded to approximately 26 and 35 ng/mL which might be relevant when contaminated diets are fed.Clinical-chemical and hematological traits were not related to mycotoxin residue levels excepting blood urea nitrogen which was positively correlated to the sum of β-ZEL, α-ZEL, and ZEN concentration. Whether this reflects simply the feeding history of the horses or renal failures giving rise to a prolonged half-life of the toxins needs to be clarified further.     

Optimising Seagrass Conservation for Ecological Functions

Ecosystems - Animals are central to numerous ecological processes that shape the structure and function of ecosystems. It follows that species that are strongly linked to specific functions can...     

Preventing arginine-to-proline conversion in a cell-line-independent manner during cell cultivation under stable isotope labeling by amino acids in cell culture (SILAC) conditions

Quantitative proteomics has increasingly gained impact in life science research as a tool to describe changes in protein expression between different cellular states. Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful technique for relative quantification of proteins. However, the accuracy of quantification is impaired by the metabolic conversion of arginine to proline resulting in additional heavy labeled proline peptide satellites. Here we reinvestigated the addition of unlabeled proline during cell cultivation under SILAC conditions considering several thousand peptides and demonstrated that the arginine-to-proline conversion is prevented independent of the cell line used.     

Proteomic Analysis of Tardigrades: Towards a Better Understanding of Molecular Mechanisms by Anhydrobiotic Organisms

Background

Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered.

Principal Findings

Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions.

Conclusions

The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades.     

De Novo Proteome Analysis of Genetically Modified Tumor Cells By a Metabolic Labeling/Azide-alkyne Cycloaddition Approach

Activin receptor type II (ACVR2) is a member of the transforming growth factor type II receptor family and controls cell growth and differentiation, thereby acting as a tumor suppressor. ACVR2 inactivation is known to drive colorectal tumorigenesis. We used an ACVR2-deficient microsatellite unstable colon cancer cell line (HCT116) to set up a novel experimental design for comprehensive analysis of proteomic changes associated with such functional loss of a tumor suppressor. To this end we combined two existing technologies. First, the ACVR2 gene was reconstituted in an ACVR2-deficient colorectal cancer (CRC) cell line by means of recombinase-mediated cassette exchange, resulting in the generation of an inducible expression system that allowed the regulation of ACVR2 gene expression in a doxycycline-dependent manner. Functional expression in the induced cells was explicitly proven. Second, we used the methionine analog azidohomoalanine for metabolic labeling of newly synthesized proteins in our cell line model. Labeled proteins were tagged with biotin via a Click-iT chemistry approach enabling specific extraction of labeled proteins by streptavidin-coated beads. Tryptic on-bead digestion of captured proteins and subsequent ultra-high-performance LC coupled to LTQ Orbitrap XL mass spectrometry identified 513 proteins, with 25 of them differentially expressed between ACVR2-deficient and -proficient cells. Among these, several candidates that had already been linked to colorectal cancer or were known to play a key role in cell growth or apoptosis control were identified, proving the utility of the presented experimental approach. In principle, this strategy can be adapted to analyze any gene of interest and its effect on the cellular de novo proteome.Human tumors acquire a large number of genetic and epigenetic alterations that arise during progression from preneoplastic lesions to metastatic disease. However, the diversity of these alterations reflects the intratumoral heterogeneity and represents the genomic landscape of tumors. Among a high background number of irrelevant passenger alterations, only a limited number of genetic alterations are considered to be driving events that confer a selective advantage to tumor cells. Major signaling pathways affected by such driver mutations include the TGFβ, BMP, Activin, Wnt, and Notch pathways, abrogating normal regulation of key cellular processes such as cell fate, cell survival, and genome maintenance.Both tumor-relevant driver mutations in a major signaling receptor and tumor-irrelevant passenger mutations can cause changes at the proteomic level. Passenger-mutation-associated proteomic patterns are propagated randomly and do not represent generic tumor-associated changes (1). Therefore, a focus on proteome alterations associated with single driver mutations is necessary in order for specific changes that underlie tumor development to be identified. However, such analyses encounter two major limitations at different levels.At the molecular level, the genetic heterogeneity of tumors—especially those of the microsatellite unstable and mutator phenotype—poses a significant problem in determining mutation-specific effects. Two principal strategies for detecting cellular consequences of a single mutation have been applied. First, targeted gene knock-out in target-gene-proficient cell lines by means of homologous recombination, adeno-associated viral delivery, or zinc finger nucleases has been used successfully (2–4). However, these approaches are often limited by their low efficiency, are laborious and time-consuming, and bear the potential for confounding off-target effects. Second, transfer of the target gene into deficient cell lines via gene insertion or gene targeting methods has been extensively applied. Unfortunately, insertion methods are often affected by random insertion, a variable number of integrated gene copies per cell, and inconsistent integration sites, eventually resulting in unpredictable expression patterns (5). However, many non-integrating vectors, such as adenoviral DNA, are not often replicated during cell division, which limits their use in basic research.At the protein level, sample complexity is a major limiting factor. In addition to prefractionation methods, metabolic labeling is a versatile tool in work focusing on proteomic changes induced by gene activation. Because the activation of tumor suppressor pathways directly regulates target gene expression, analysis of tumor-suppressor-dependent alterations of newly synthesized proteins via metabolic labeling is a reasonable approach for restricting proteomic complexity. Conventional methods for metabolic labeling usually rely on amino acids containing either radioactive or stable isotopes. Although radioactive labeling enables extremely sensitive detection methods, its use for proteomic analysis is limited because of the need for special handling and precautions against contamination of the analytical instrumentation. Stable isotopic labeling, in particular the SILAC methodology, is currently the preferred method for most metabolic labeling approaches in proteomic analyses, and especially for cell lines (6). However, when applying the SILAC technology, mass spectrometric detection of labeled peptides has to be conducted in the presence of numerous irrelevant, unlabeled peptides, which hampers the detection of labeled low-abundance peptides. A relatively new method, termed Click-iT labeling, that enables labeling of nascent proteins comparable to that by a radioactive compound can overcome this problem, because upon incorporation of the labeled compound a handle for specific extraction of the labeled protein is worked in. The click reaction makes use of a methionine derivative that is functionalized with an azide (l-azidohomoalanine (AHA)).¹ During protein synthesis AHA is incorporated into proteins, which can then be tagged with a biotin alkyne (PEG4 carboxamide-propargyl biotin), resulting in the specific biotinylation of the metabolically labeled proteins (7). The final step is extraction of the labeled proteins by streptavidin beads.In the present study we pursued a completely new strategy to determine proteomic changes caused by a tumor-specific mutation in a major signaling pathway exemplified by the activin receptor type II (ACVR2) tumor suppressor. Disruption of activin signaling is a frequent event during colorectal tumorigenesis and can be caused by different molecular mechanisms. In colon tumors with chromosomal instability but microsatellite stability, ACVR2 inactivation mainly occurs via epigenetic mechanisms, whereas in colon cancers with a high level of microsatellite instability (MSI) loss of ACVR2 emerges from frameshift mutation (8). MSI has been defined as a change in length (insertions/deletions) of small repetitive DNA sequences (microsatellites) that arises specifically in tumor cells with impaired DNA mismatch repair functions (mutator phenotype) but not in corresponding normal tissue (9, 10). The A8 coding microsatellite in exon 10 of the ACVR2 gene has been identified as one of the most frequent mutation targets in high-MSI colorectal tumors that abrogates normal ACVR2 protein expression (11–13). As a consequence, phosphorylation of downstream signaling mediators such as Smad2/3, as well as regulation of several target genes such as SERPINE, SMAD7, and MYC, is significantly impaired, and this contributes to MSI colorectal tumorigenesis.Our strategy relied on a combination of two established technologies. First, we employed a recombination-mediated genomic targeting approach (14–16) to generate a genetically modified MSI colorectal tumor cell line that enabled doxycycline-inducible and reversible expression of an ACVR2 transgene and activation of ligand-dependent signaling in an isogenic background. Second, we performed metabolic labeling via a click-chemistry approach (7) in this MSI cell line to uncover alterations in the pattern of newly synthesized proteins (de novo proteome). In particular, we determined the constituents of the total cellular de novo proteome and subsets derived thereof that were regulated in an ACVR2-dependent manner. Our combinatorial strategy represents a versatile approach allowing one to overcome the limitations inherent in the genetic heterogeneity and proteomic complexity of MSI tumor cells. Moreover, this model system allows one to address any tumor target gene and thereby possesses broad applicability for studies of cancer proteome complexity, which is the logical starting point for identifying diagnostic biomarkers and therapeutic targets for cancer.     

Stoichiometry of the CD95 death-inducing signaling complex: experimental and modeling evidence for a death effector domain chain model

The CD95 (Fas/APO-1) death-inducing signaling complex (DISC) is essential for the initiation of CD95-mediated apoptotic and nonapoptotic responses. The CD95 DISC comprises CD95, FADD, procaspase-8, procaspase-10, and c-FLIP proteins. Procaspase-8 and procaspase-10 are activated at?the DISC, leading to the formation of active caspases and apoptosis initiation. In this study we analyzed the?stoichiometry of the CD95 DISC. Using quantitative western blots, mass spectrometry, and mathematical modeling, we reveal that the amount of DED proteins procaspase-8/procaspase-10 and c-FLIP at the DISC exceeds that of FADD by several-fold. Furthermore, our findings imply that procaspase-8, procaspase-10, and c-FLIP could form DED chains at the DISC, enabling the formation of dimers and efficient activation of caspase-8. Taken together, our findings provide an enhanced understanding of caspase-8 activation and initiation of apoptosis at the DISC.     

The proteome of rat olfactory sensory cilia

Olfactory sensory neurons expose to the inhaled air chemosensory cilia which bind odorants and operate as transduction organelles. Odorant receptors in the ciliary membrane activate a transduction cascade which uses cAMP and Ca²⁺ for sensory signaling in the ciliary lumen. Although the canonical transduction pathway is well established, molecular components for more complex aspects of sensory transduction, like adaptation, regulation, and termination of the receptor response have not been systematically identified. Moreover, open questions in olfactory physiology include how the cilia exchange solutes with the surrounding mucus, assemble their highly polarized set of proteins, and cope with noxious substances in the ambient air. A specific ciliary proteome would promote research efforts in all of these fields. We have improved a method to detach cilia from rat olfactory sensory neurons and have isolated a preparation specifically enriched in ciliary membrane proteins. Using LC‐ESI‐MS/MS analysis, we identified 377 proteins which constitute the olfactory cilia proteome. These proteins represent a comprehensive data set for olfactory research since more than 80% can be attributed to the characteristic functions of olfactory sensory neurons and their cilia: signal processing, protein targeting, neurogenesis, solute transport, and cytoprotection. Organellar proteomics thus yielded decisive information about the diverse physiological functions of a sensory organelle.