Jeffrey H. Ringrose

Proteomic Studies Reveal Coordinated Changes in T-Cell Expression Patterns upon Infection with Human Immunodeficiency Virus Type 1

ABSTRACT We performed an extensive two-dimensional differential in-gel electrophoresis proteomic analysis of the cellular changes in human T cells upon human immunodeficiency virus type 1 (HIV-1) infection. We detected 2,000 protein spots, 15% of which were differentially expressed at peak infection. A total of 93 proteins that changed in relative abundance were identified. Of these, 27 were found to be significantly downregulated and 66 were upregulated at peak HIV infection. Early in infection, only a small group of proteins was changed. A clear and consistent program of metabolic rerouting could be seen, in which glycolysis was downregulated and mitochondrial oxidation enhanced. Proteins that participate in apoptotic signaling were also significantly influenced. Apart from these changes, the virus also strongly influenced levels of proteins involved in intracellular transport. These and other results are discussed in light of previous microarray and proteomic studies regarding the impact of HIV-1 infection on cellular mRNA and protein content.

Highly efficient depletion strategy for the two most abundant erythrocyte soluble proteins improves proteome coverage dramatically.

In-depth human erythrocyte proteome studies are severely hampered by the presence of hemoglobin and carbonic anhydrase-1, which account for more than 98% of the total erythrocyte soluble protein content. We developed a specific depletion approach that resulted in a drastic increase in the number of identified proteins. This depletion technique is valuable for proteome studies of human erythrocyte disorders with unknown etiology and of tissue samples that contain blood.

Acute anterior uveitis and spondyloarthropathies.

Acute anterior uveitis (AAU) is characterized by sudden-onset, mostly unilateral exacerbations of an inflammation of the iris and ciliary body. The duration of illness is short if the patient is treated with corticosteroids. Half of all patients with any type of anterior uveitis are HLA-B27-positive, and more than half of the B27-positive patients have spondyloarthropathy. Ophthalmologists should therefore refer all patients with AAU who are HLA-B27-positive to a rheumatologist. Because attacks of AAU are extremely painful and frightening, most spondyloarthropathy patients with AAU will seek out an ophthalmologist on their own. The anterior chamber of the eye and the joints are mesenchymal cavities that are cleaned by macrophages. Anterior chamber-associated immune deviation is the mechanism by which specific regulatory T cells normally produce sufficient transforming growth factor-beta to impair inflammatory reactions that might hamper vision. Another mechanism of immune privilege is Fas-ligand induced apoptosis. Because the cells of the anterior eye express Fas-ligand, infiltrating cells are apoptotically killed. Comparable mechanisms may occur at a lower level in joints. The cause of AAU and spondyloarthropathy is unknown. B27 is probably only responsible for one quarter of the pathogenesis, other non-B27 genetic factors for another quarter, and unknown exogenous factors for the remaining half. It is possible that Gram-negative bacteria such as Klebsiella or Yersinia are involved in the pathogenesis in a yet unknown way.

Deep proteome profiling of Trichoplax adhaerens reveals remarkable features at the origin of metazoan multicellularity

Genome sequencing of arguably the simplest known animal, Trichoplax adhaerens, uncovered a rich array of transcription factor and signalling pathway genes. Although the existence of such genes allows speculation about the presence of complex regulatory events, it does not reveal the level of actual protein expression and functionalization through posttranslational modifications. Using high-resolution mass spectrometry, we here semi-quantify 6,516 predicted proteins, revealing evidence of horizontal gene transfer and the presence at the protein level of nodes important in animal signalling pathways. Moreover, our data demonstrate a remarkably high activity of tyrosine phosphorylation, in line with the hypothesized burst of tyrosine-regulated signalling at the instance of animal multicellularity. Together, this Trichoplax proteomics data set offers significant new insight into the mechanisms underlying the emergence of metazoan multicellularity and provides a resource for interested researchers.

Spatially resolving the secretome within the mycelium of the cell factory Aspergillus niger.

Aspergillus niger is an important cell factory for the industrial production of enzymes. These enzymes are released into the culture medium, from which they can be easily isolated. Here, we determined with stable isotope dimethyl labeling the secretome of five concentric zones of 7-day-old xylose-grown colonies of A. niger that had either or not been treated with cycloheximide. As expected, cycloheximide blocked secretion of proteins at the periphery of the colony. Unexpectedly, protein release was increased by cycloheximide in the intermediate and central zones of the mycelium when compared to nontreated colonies. Electron microscopy indicated that this is due to partial degradation of the cell wall. In total, 124 proteins were identified in cycloheximide-treated colonies, of which 19 secreted proteins had not been identified before. Within the pool of 124 proteins, 53 secreted proteins were absent in nontreated colonies, and additionally, 35 proteins were released ≥4-fold in the central and subperipheral zones of cycloheximide-treated colonies when compared to nontreated colonies. The composition of the secretome in each of the five concentric zones differed. This study thus describes spatial release of proteins in A. niger, which is instrumental in understanding how fungi degrade complex substrates in nature.

Comparison of Peptides Eluted from the Groove of HLA-B27 from Salmonella Infected and Non-Infected Cells

SummaryReactive arthritis (ReA) is associated with the MHC class- I molecule HLA- B27 and caused by certain Gram- negative bacteria. The mechanism by which HLA- B27 confers a higher susceptibility for this disease compared to other MHC Class-I alleles is still not known. We investigated whether infection of human HLA- B27+ cells is able to change the peptide repertoire presented by these HLA- B27 molecules. To this end large quantities of a B- cell line (C1R- B27) transfected with HLA- B2705 were infected with S.typhimurium. Peptides were eluted from the B27 molecules and separated by Reversed Phase Chromatography (RPC). We then compared the peptide profiles obtained from S.typhimurium infected C1R B- cells with that obtained from non infected cells. p]Apart from a few additional peaks present in the profile derived from the infected batch the peptide profiles were almost identical. A few fractions were subjected to sequencing by Edman degradation. All peptides found were nonameres with arginine (Arg) at position 2 which is in agreement with the previously described HLA- B27 peptide binding motif. The majority of peaks expressed a mixture of at least four different peptides. The analysis of differences between HLA- B27 bound peptides from Salmonella infected and non infected cells might lead to the identification of T- cell epitopes shared by Salmonella and autoantigens.

Carbohydrate Moieties of Microsporidian Polar Tube Proteins Are Targeted by Immunoglobulin G in Immunocompetent Individuals

ABSTRACT Microsporidia of the Encephalitozoon species are frequently found as opportunistic pathogens of immunocompromised patients, but very little is known about the prevalence and significance of Encephalitozoon infection in immunocompetent individuals. It was reported previously that 8% of Dutch blood donors and 5% of pregnant French women had an immunoglobulin G (IgG) immune response against specific organelles of Encephalitozoon intestinalis. These organelles, the so-called polar tube and anchoring disk, are used to penetrate membranes of host cells during infection. The unexpectedly high percentage of immunocompetent individuals with IgG against these organelles suggested that infection of humans with microsporidia might be more common than previously recognized. In the present study, we analyzed this anti-Encephalitozoon IgG response by using indirect immunofluorescence, Western blotting, two-dimensional gel electrophoresis, and chemical deglycosylation. Our results show that the antibody response is directed against the posttranslational carbohydrate modification of the major polar tube protein (polar tube protein 1) and carbohydrate moieties of proteins in the anchoring region of the polar tube of Encephalitozoon. In addition, the antibodies were found to decrease the infectivity of E. intestinalis in vitro. The significance and possible origin of these prevalent antibodies are discussed.

Influence of infection of cells with bacteria associated with reactive arthritis on the peptide repertoire presented by HLA-B27

Reactive arthritis (ReA) after infections with various gram-negative bacteria is strongly associated with the MHC class I molecule HLA-B27. It is supposed that the B27 molecule itself plays a role in the pathogenesis of ReA by presenting antigenic peptides to cytotoxic T lymphocytes. The peptide repertoires presented by Salmonella-, Shigella- and non-infected cells were compared to identify such peptides. From the peptides isolated from the B27 molecules of these cells, profiles were generated by reversed-phase chromatography and peaks present in the profiles from infected cells but not in profiles from non-infected cells were studied for their peptide compositions. Some sequences with identity to those in human histone H3, human ribosomal protein S17 and the heavy chain of HLA-B27 itself were detected only in profiles from infected cells. All peptides identified from infected cells contained the B*2705 peptide-binding motif. The data suggest that HLA-B27-positive cells infected with ReA-inducing bacteria show an increased presentation of certain self-peptides. There was no evidence for altered peptide-binding specificity of B27 after infection. However, the interpretations were hampered by the variation in peptide presentation between different experiments.

Major histocompatibility complex class I peptide presentation after Salmonella enterica serovar typhimurium infection assessed via stable isotope tagging of the B27-presented peptide repertoire

ABSTRACT Reactive arthritis (ReA) induced by infection with several gram-negative bacteria is strongly associated with expression of the major histocompatibility complex class I molecule HLA-B27. It is thought that due to the intracellular lifestyle of ReA-inducing bacteria, bacterial fragments can be presented by HLA-B27. Cytotoxic T cells recognizing such bacterial peptides or other induced host peptides could cross-react with self peptides presented in the joints, giving rise to disease. Studies to analyze the B27 peptide repertoire in relation to infection were severely hampered, as complex peptide profiles obtained from separate infected and noninfected cell preparations had to be compared. For this study, we applied a new approach to examine the effect of Salmonella enterica serovar Typhimurium infection on the B27 peptide repertoire presented by the HLA-B*2704 subtype associated with disease. Firstly, we showed that both host cell and S. enterica serovar Typhimurium proteins can be tagged metabolically with stable-isotope-labeled arginine. We then designed experiments so that either the tagged endogenous or tagged bacterial B*2704-presented peptide repertoires from infected cells could be analyzed by mass spectrometry from single peptide preparations that included uninfected controls. Using this new approach, we found no evidence for significant changes in endogenous B*2704 peptide presentation after infection or for any S. enterica serovar Typhimurium-derived B27-bound peptide. In conclusion, the hypothesis that S. enterica serovar Typhimurium induces changes in B27 peptide presentation could not be supported.

Proteomic analysis of HIV–T cell interaction: an update

This mini-review summarizes techniques applied in, and results obtained with, proteomic studies of human immunodeficiency virus type 1 (HIV-1)–T cell interaction. Our group previously reported on the use of two-dimensional differential gel electrophoresis (2D-DIGE) coupled to matrix assisted laser-desorption time of flight peptide mass fingerprint analysis, to study T cell responses upon HIV-1 infection. Only one in three differentially expressed proteins could be identified using this experimental setup. Here we report on our latest efforts to test models generated by this data set and extend its analysis by using novel bioinformatic algorithms. The 2D-DIGE results are compared with other studies including a pilot study using one-dimensional peptide separation coupled to MSE, a novel mass spectrometric approach. It can be concluded that although the latter method detects fewer proteins, it is much faster and less labor intensive. Last but not least, recent developments and remaining challenges in the field of proteomic studies of HIV-1 infection and proteomics in general are discussed.