Maarten Dhaenens

Tryptic peptide analysis of protein binders in works of art by liquid chromatography-tandem mass spectrometry.

A proteomics approach was used for the identification of protein binders in historical paints: the proteins were digested enzymatically into peptides using trypsin before being separated and detected by high performance liquid chromatography-electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS). Mascot (Matrix Science) was used to analyse the resulting data and for protein identification. In contrast to amino acid analysis, amino acid sequences could be studied that retain much more information about the proteins. The best extraction strategy was selected based on the number of peptides that were identified in the protein content of paint replicas using different methods. The influence of pigments on the extraction method was studied and the analytical characteristics of the selected method were determined. Finally this method was applied to historical paint microsamples on the anonymous early 15th century panel painting Crucifixion with St Catherine and St Barbara (Calvary of the Tanners), the St Catherine Altarpiece by Joes Beyaert (c. 1479) and two paintings by Pieter Brueghel the Younger (1617-1628).

Mass spectrometry-based proteomics as a tool to identify biological matrices in forensic science

In forensic casework analysis, identification of the biological matrix and the species of a forensic trace, preferably without loss of DNA, is of major importance. The biological matrices that can be encountered in a forensic context are blood (human or non-human), saliva, semen, vaginal fluid, and to a lesser extent nasal secretions, feces, and urine. All these matrices were applied on swabs and digested with trypsin in order to obtain peptides. These peptides were injected on a mass spectrometer (ESI Q-TOF) resulting in the detection of several biomarkers that were used to build a decision tree for matrix identification. Saliva and blood were characterized by the presence of alpha-amylase 1 and hemoglobin, respectively. In vaginal fluid, cornulin, cornifin, and/or involucrin were found as biomarkers while semenogelin, prostate-specific antigen, and/or acid phosphatase were characteristic proteins for semen. Uromodulin or AMBP protein imply the presence of urine, while plunc protein is present in nasal secretions. Feces could be determined by the presence of immunoglobulins without hemoglobin. The biomarkers for the most frequently encountered biological matrices (saliva, blood, vaginal fluid, and semen) were validated in blind experiments and on real forensic samples. Additionally, by means of this proteomic approach, species identification was possible. This approach has the advantage that the analysis is performed on the first “washing” step of the chelex DNA extraction, a solution which is normally discarded, and that one single test is sufficient to determine the identity and the species of the biological matrix, while the conventional methods require cascade testing. This technique can be considered as a useful additional tool for biological matrix identification in forensic science and holds the promise of further automation.

Dendritic cells from spondylarthritis‐prone HLA–B27–transgenic rats display altered cytoskeletal dynamics, class II major histocompatibility complex expression, and viability

OBJECTIVE Spondylarthritis (SpA) is characterized by spinal and peripheral joint inflammation, frequently combined with extraarticular manifestations. Despite the well-established association of SpA with the class I major histocompatibility complex (MHC) allele HLA-B27, there are still different, parallel hypotheses on the relationship between HLA-B27 and disease mechanisms. The present study was undertaken to investigate several characteristics of mature dendritic cells (DCs), which are believed to be essential for triggering disease in a model of SpA in HLA-B27-transgenic rats. METHODS We combined different whole-proteome approaches (2-dimensional polyacrylamide gel electrophoresis and iTRAQ) to define the most aberrant molecular processes occurring in spleen DCs. Videomicroscopy and flow cytometry were used to confirm both cytoskeletal and class II MHC expression deficiencies. RESULTS Our proteome studies provided evidence of up-regulation of proteins involved in class I MHC loading, and unfolded protein response, along with a striking down-regulation of several cytoskeleton-reorganizing proteins. The latter result was corroborated by findings of deficient motility, altered morphology, and decreased immunologic synapse formation. Furthermore, class II MHC surface expression was reduced in DCs from B27-transgenic rats, and this could be linked to differences in class II MHC-induced apoptotic sensitivity. Finally, we found reduced viability of the CD103+CD4- DC subpopulation, which likely exerts tolerogenic function. CONCLUSION Taken together, our findings have different important implications regarding the physiology of B27-transgenic rat DCs, which have a putative role in spontaneous disease in these rats. In particular, the reduced motility and viability of putatively tolerogenic CD4+ DCs could play an important role in initiating the inflammatory process, resulting in SpA.

In vivo relevance of citrullinated proteins and the challenges in their detection

Citrullination is a posttranslational modification of arginine. It plays both a physiological role, for instance during apoptosis and epigenetics, and a pathological role in cancer or diseases of the central nervous system. Most research on citrullination to date focuses on its role in auto‐immune diseases such as multiple sclerosis and rheumatoid arthritis. In this context, the exact knowledge of citrullination sites in a protein can provide invaluable information about the etiological importance of these citrullinated proteins. However, few techniques exist that can accurately detect citrullination on the peptide level. This review aims to give an overview of the different methods available to date for the detection of citrullinated proteins and peptides. These include 2D‐SDS‐PAGE and immunodetection, as well as specific mass spectrometry (MS) approaches, both labeled and unlabeled. These MS approaches have been developed to pinpoint the exact location of citrullination on the peptide level. Improving the currently existing detection strategies while focusing on the role of citrullinated proteins will be invaluable to elucidate the importance of this posttranslational modification in vivo.

T-2 toxin induced Salmonella Typhimurium intoxication results in decreased Salmonella numbers in the cecum contents of pigs, despite marked effects on Salmonella-host cell interactions

The mycotoxin T-2 toxin and Salmonella Typhimurium infections pose a significant threat to human and animal health. Interactions between both agents may result in a different outcome of the infection. Therefore, the aim of the presented study was to investigate the effects of low and relevant concentrations of T-2 toxin on the course of a Salmonella Typhimurium infection in pigs. We showed that the presence of 15 and 83 μg T-2 toxin per kg feed significantly decreased the amount of Salmonella Typhimurium bacteria present in the cecum contents, and a tendency to a reduced colonization of the jejunum, ileum, cecum, colon and colon contents was noticed. In vitro, proteomic analysis of porcine enterocytes revealed that a very low concentration of T-2 toxin (5 ng/mL) affects the protein expression of mitochondrial, endoplasmatic reticulum and cytoskeleton associated proteins, proteins involved in protein synthesis and folding, RNA synthesis, mitogen-activated protein kinase signaling and regulatory processes. Similarly low concentrations (1-100 ng/mL) promoted the susceptibility of porcine macrophages and intestinal epithelial cells to Salmonella Typhimurium invasion, in a SPI-1 independent manner. Furthermore, T-2 toxin (1-5 ng/mL) promoted the translocation of Salmonella Typhimurium over an intestinal porcine epithelial cell monolayer. Although these findings may seem in favour of Salmonella Typhimurium, microarray analysis showed that T-2 toxin (5 ng/mL) causes an intoxication of Salmonella Typhimurium, represented by a reduced motility and a downregulation of metabolic and Salmonella Pathogenicity Island 1 genes. This study demonstrates marked interactions of T-2 toxin with Salmonella Typhimurium pathogenesis, resulting in bacterial intoxication.

Proteome characterization of human articular chondrocytes leads to novel insights in the function of small heat-shock proteins in chondrocyte homeostasis

OBJECTIVE In recent years, studies have been initiated to disclose the proteome of human chondrocytes and cartilage. Despite these studies, comprehensive information of the chondrocyte proteome remains limited. This study aimed to further explore the proteome expressed by human knee chondrocytes, and to study the functional aspects of heat-shock protein 27 (HSP27), a protein related to the previously described alphaBcrystallin, in chondrocyte biology. METHODS Chondrocytes isolated from human knee articular cartilage were cultured in a three-dimensional alginate culture system. To simplify the protein mixtures, proteins extracted from chondrocyte cell lysates were fractionated based on hydrophobicity and molecular weight. Proteins were digested and the resulting peptides were separated and identified by an on-line two-dimensional (2-D) nanoliquid chromatography (nanoLC)-system coupled to a quadrupole time-of-flight (Qq-TOF) mass spectrometer. Differential expression analysis of HSP27 was performed by Western Blotting and quantitative polymerase chain reaction (QPCR). The effects of HSP27 on chondrocyte biology were explored by suppression of HSP27 expression induced by RNA interference (RNAi). RESULTS In this study, we identified proteins with unknown functions together with membrane proteins, transcription factors and other low abundant proteins, which have not yet been described in chondrocytes. Based on previous knowledge on the related protein alphaBcrystallin, we selected HSP27 from the chondrocyte proteome database. Differential expression analysis revealed a decreased expression of HSP27 in Osteoarthritic (OA) chondrocytes. RNAi experiments revealed that HSP27 is involved in interleukin-1beta (IL-1beta) induced IL-6 secretion. CONCLUSION These findings highlight that small HSPs, especially HSP27, play a prominent role in the maintenance of human articular chondrocyte homeostasis.

Proteomic Analysis of the Excretory-Secretory Products from Larval Stages of Ascaris suum Reveals High Abundance of Glycosyl Hydrolases

Background Ascaris lumbricoides and Ascaris suum are socioeconomically important and widespread parasites of humans and pigs, respectively. The excretory-secretory (ES) molecules produced and presented at the parasite-host interface during the different phases of tissue invasion and migration are likely to play critical roles in the induction and development of protective immune and other host responses. Methodology/Principal Findings The aim of this study was to identify the ES proteins of the different larval stages (L3-egg, L3-lung and L4) by LC-MS/MS. In total, 106 different proteins were identified, 20 in L3-egg, 45 in L3-lung stage and 58 in L4. Although most of the proteins identified were stage-specific, 15 were identified in the ES products of at least two stages. Two proteins, i.e. a 14-3-3-like protein and a serpin-like protein, were present in the ES products from the three different larval stages investigated. Interestingly, a comparison of ES products from L4 with those of L3-egg and L3-lung showed an abundance of metabolic enzymes, particularly glycosyl hydrolases. Further study indicated that most of these glycolytic enzymes were transcriptionally upregulated from L4 onwards, with a peak in the adult stage, particularly in intestinal tissue. This was also confirmed by enzymatic assays, showing the highest glycosidase activity in protein extracts from adult worms gut. Conclusions/Significance The present proteomic analysis provides important information on the host-parasite interaction and the biology of the migratory stages of A. suum. In particular, the high transcriptional upregulation of glycosyl hydrolases from the L4 stage onwards reveals that the degradation of complex carbohydrates forms an essential part of the energy metabolism of this parasite once it establishes in the small intestine.

Batrachochytrium dendrobatidis zoospore secretions rapidly disturb intercellular junctions in frog skin

Global amphibian declines are in part driven by the chytrid fungus Batrachochytrium dendrobatidis, causing superficial dermatomycosis with epidermal hyperplasia and hyperkeratosis in infected amphibians. The susceptibility to chytridiomycosis and the severity of epidermal lesions in amphibians with chytridiomycosis are not consistent across species or even among individuals. Severe infections cause death of the animal most likely through disturbance of ion homeostasis. The mechanism by which this superficial skin infection results in epidermal lesions has so far eluded precise definition. It was the aim of this study to unravel how B. dendrobatidis causes alterations that affect skin integrity. Exposure of Xenopus laevis skin to B. dendrobatidis zoospore supernatant using skin explants and Ussing chambers caused rapid disruption of intercellular junctions, demonstrated using histology and transmission electron microscopy. The loss of intercellular junctions led to detachment-induced cell apoptosis, or anoikis. The zoospore supernatant induced neither apoptosis nor necrosis in isolated primary keratinocytes of X. laevis. This supports the idea that the loss of cell contacts triggered apoptosis in the skin explants. Mass spectrometric analysis of the protein composition of the supernatant revealed a complex mixture, including several new virulence associated proteins, such as proteases, biofilm-associated proteins and a carotenoid ester lipase. Protease and lipase activity of the supernatant was confirmed with a protease and lipase assay. In conclusion, B. dendrobatidis zoospores produce a complex mixture of proteins that quickly disturbs epidermal intercellular junctions leading to anoikis in the anuran skin. The role of the identified proteins in this process remains to be determined.

Identification of histone H3 clipping activity in human embryonic stem cells

Posttranslational histone modifications are essential features in epigenetic regulatory networks. One of these modifications has remained largely understudied: regulated histone proteolysis. In analogy to the histone H3 clipping during early mouse embryonic stem cell differentiation, we report for the first time that also in human embryonic stem cells this phenomenon takes place in the two different analyzed cell lines. Employing complementary techniques, different cleavage sites could be identified, namely A21, R26 and residue 31. The enzyme responsible for this cleavage is found to be a serine protease. The formation of cleaved H3 follows a considerably variable pattern, depending on the timeframe, culture conditions and culture media applied. Contrary to earlier findings on H3 clipping, our results disconnect the link between declining Oct4 expression and H3 cleavage.

Histone proteolysis: A proposal for categorization into ‘clipping’ and ‘degradation’

We propose for the first time to divide histone proteolysis into “histone degradation” and the epigenetically connoted “histone clipping”. Our initial observation is that these two different classes are very hard to distinguish both experimentally and biologically, because they can both be mediated by the same enzymes. Since the first report decades ago, proteolysis has been found in a broad spectrum of eukaryotic organisms. However, the authors often not clearly distinguish or determine whether degradation or clipping was studied. Given the importance of histone modifications in epigenetic regulation we further elaborate on the different ways in which histone proteolysis could play a role in epigenetics. Finally, unanticipated histone proteolysis has probably left a mark on many studies of histones in the past. In conclusion, we emphasize the significance of reviving the study of histone proteolysis both from a biological and an experimental perspective.