Proteomic analysis of skin invasion by blood fluke larvae

Background

During invasion of human skin by schistosome blood fluke larvae (cercariae), a multicellular organism breaches the epidermis, basement membrane, and dermal barriers of skin. To better understand the pathobiology of this initial event in schistosome infection, a proteome analysis of human skin was carried out following invasion by cercariae of Schistosoma mansoni.

Methodology and Results

Human skin samples were exposed to cercariae for one-half hour to two hours. Controls were exposed to water used to collect cercariae in an identical manner, and punctured to simulate cercarial tunnels. Fluid from both control and experimental samples was analyzed by LC/MS/MS using a linear ion trap in “triple play” mode. The coexistence of proteins released by cercariae and host skin proteins from epidermis and basement membrane confirmed that cercarial tunnels in skin were sampled. Among the abundant proteins secreted by cercariae was the cercarial protease that has been implicated in degradation of host proteins, secreted proteins proposed to mediate immune invasion by larvae, and proteins implicated in protection of parasites against oxidative stress. Components of the schistosome surface tegument, previously identified with immune serum, were also released. Both lysis and apoptosis of epidermal cells took place during cercarial invasion of the epidermis. Components of lysed epidermal cells, including desmosome proteins which link cells in the stratum granulosum and stratum spinosum, were identified. While macrophage-derived proteins were present, no mast cell or lymphocyte cytokines were identified. There were, however, abundant immunoglobulins, complement factors, and serine protease inhibitors in skin. Control skin samples incubated with water for the same period as experimental samples ensured that invasion-related proteins and host protein fragments were not due to nonspecific degeneration of the skin samples.

Conclusions

This analysis identified secreted proteins from invasive larvae that are released during invasion of human skin. Analysis of specific host proteins in skin invaded by cercariae served to highlight both the histolytic events facilitating cercarial invasion, and the host defenses that attempt to arrest or retard invasion. Proteins abundant in psoriatic skin or UV and heat-stressed skin were not abundant in skin invaded by cercariae, suggesting that results did not reflect general stress in the surgically removed skin specimen. Abundant immunoglobulins, complement factors, and serine protease inhibitors in skin form a biochemical barrier that complements the structural barrier of the epidermis, basement membrane, and dermis. The fragmentation of some of these host proteins suggests that breaching of host defenses by cercariae includes specific degradation of immunoglobulins and complement, and either degradation of, or overwhelming the host protease inhibitor repertoire.     

Comparison of skin invasion among three major species of Schistosoma

A comparison of the host-finding behavior, mode of skin invasion and skin-migratory patterns of the three major schistosomes of humans reveals major differences. Among the three species, Schistosoma japonicum is remarkable at conserving energy during the host-finding process, and exhibiting swift migration through the skin to reach the predilection site sooner and mature earlier compared with Schistosoma mansoni and Schistosoma haematobium. In this article, we summarize and compare the penetration and migratory behavior of schistosomula of the three major human schistosomes through mouse and human skin.     

Proteomic analysis of human blood serum using peptide library beads

Human serum is thought to contain key information for diagnostics of human disease. However, no single technology is currently nor might ever be available to cope with the complexity and dynamic range of the serum proteome. We here report a large-scale proteomic study of human blood serum using peptide library beads and mass spectrometry. Serum proteins are adsorbed onto polymeric beads coated with a combinatorial library composed of millions of hexameric peptide baits. Analysis of the eluates from this combinatorial library (as obtained with 3 eluants of different strength, able to release 99% of the retentate) via liquid chromatography coupled to high-resolution mass spectrometry resulted in the identification of 1559 proteins or 3869 proteins, respectively, depending on how 95% confidence was estimated. In either case, the analysis showed that ligand beads are able to capture a large number of proteins in a single operation. The ligand bead bound fraction appeared to have a lower dynamic range when compared to the starting material, due to a "normalization" of the protein concentrations in the original mixture. We find that extensive information on the protein composition of complex samples such as serum can be obtained using ligand beads and that these beads enrich the proteomic tool box.     

Proteomic analysis of human osteoarthritic chondrocytes reveals protein changes in stress and glycolysis

Osteoarthritis (OA) is characterized by cartilage degradation. The chondrocyte is the only cell type present in mature cartilage, and it is important in the control of cartilage integrity. The aim of this study was to analyze, by a proteomic approach, the changes that are characteristic of OA chondrocytes, and to identify new OA-related proteins. Chondrocytes were isolated from the cartilage of ten OA patients undergoing joint replacement and ten donors with no history of joint disease. Whole-cell proteins were resolved by 2-DE and stained with SYPRO Ruby. Protein expression patterns of 2-DE gels from OA and normal chondrocyte proteins were analyzed with PDQuest 7.3.1 software. OA-related proteins were identified by MALDI-TOF or MALDI-TOF/TOF MS. The results were validated for ANXA1, GSTO1, GRP78, and HSP90beta in cells by Western blotting and in tissue cartilage by immunohistochemistry. Results showed an average of 700 protein spots that were present in the 2-DE gels. Compared to normal chondrocytes, 19 protein spots were found to be significantly increased in OA cells (ratio OA:N> or =2.0, p<0.05), whereas nine were decreased in OA chondrocytes (ratio OA:N< or =0.5, p<0.05). Three stress response proteins were increased (HSP90beta, GRP78, and GRP94) and three proteins involved in glycolysis were decreased (enolase, glyceraldehyde 3-phosphate dehydrogenase, and fructose biphosphate aldolase). Functionally, almost all proteins could be classified as proteins involved in cellular metabolism (33%), structure (21%), or protein targeting (21%).     

Proteomic analysis reveals the strategies of Bacillus thuringiensis YBT-1520 for survival under long-term heat stress

Bacillus thuringiensis (Bt) has been widely used for 50 years as a safe biopesticide for controlling agricultural and sanitary insect pests because of its insecticidal crystal proteins. In this study a proteomic approach was used to investigate the responses and survival strategies of Bt YBT-1520 under a long-term heat stress condition (42°C). Heat stress mainly influenced the characteristics of YBT-1520 on four aspects: (i) the abilities to synthesise insecticidal crystal proteins and other potential pathogenic factors were almost lost, (ii) cell adhesion and motility were also lost, (iii) cell did not sporulate, (iv) cell kept accumulating poly(3-hydroxybutyrate) (PHB). Proteomic analyses to the physiological changes of the strain revealed three strategies of YBT-1520 for survival under long-term heat stress. The first strategy is to up-regulate enzymes (BDH1, GuaB and PepA) for long-term heat stress tolerance. The second one is to down-regulate metabolic enzymes to reduce metabolic burden. The third strategy is to increase the synthesis and accumulation of PHB. Under heat stress condition, the bacterium adjusted its metabolism by up-/down-regulation and continuous accumulation of PHB. These strategies would help cells to gain more tolerance to heat stress.     

Proteomic analysis of testis biopsies in men treated with transient scrotal hyperthermia reveals the potential targets for contraceptive development

Mild testicular heating safely and reversibly suppresses spermatogenesis. In this study, we attempted to clarify the underlying molecular mechanism(s) involved in heat‐induced spermatogenesis suppression in human testis. We conducted global proteomic analyses of human testicular biopsies before, and at 2 and 9 wk after heat treatment. Thirty‐one and Twenty‐six known proteins were identified with significant differential expression at 2 and 9 wk after heat treatment, respectively. These were used to characterize the cellular and molecular events in the testes when seminiferous epithelia became damaged (2 wk) and recovered (9 wk). At 2 wk post‐treatment, the changed expression of a series of proteins could promote apoptosis or suppress proliferation and cell survival. At 9 wk post‐treatment, the changed expression of proteins mainly promoted cell proliferation, differentiation and survival, but resisted cell apoptosis. Among those heat‐regulated proteins, HNRNPH1 was selected for the further functional study. We found that HNRNPH1 was an anti‐apoptosis protein that could regulate the expression of other heat‐induced proteins. In conclusion, heat‐induced reversible suppression of spermatogenesis occurred by modulating the expression of proteins related to proliferation, differentiation, apoptosis and cell survival pathways. These differentially expressed proteins were found to be key molecular targets affecting spermatogenesis after heat treatment.     

Proteomic analysis of calcium/calmodulin-dependent protein kinase I and IV in vitro substrates reveals distinct catalytic preferences

The multifunctional calcium/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV) are closely related by primary sequence and predicted to have similar substrate specificities based on peptide studies. We identified a fragment of p300-(1-117) that is a substrate of both kinases, and through both mutagenesis and Edman phosphate ((32)P) release sequencing, established that CaMKI and CaMKIV phosphorylate completely different sites. The CaMKI site, Ser(89) ((84)LLRSGSSPNL(93)), fits the expected consensus whereas the CaMKIV site, Ser(24) ((19)SSPALSASAS(28)), is novel. To compare kinase substrate preferences more generally, we employed a proteomic display technique that allowed comparison of complex cell extracts phosphorylated by each kinase in a rapid in vitro assay, thereby demonstrating substrate preferences that overlapped but were clearly distinct. To validate this approach, one of the proteins labeled in this assay was identified by microsequencing as HSP25, purified as a recombinant protein, and examined as a substrate for both CaMKI and CaMKIV. Again, CaMKI and CaMKIV were different, this time in kinetics and stoichiometry of the phosphorylation sites, with CaMKI preferring Ser(15) ((10)LLRTPSWGPF(19)) to Ser(85) ((80)LNRQLSSGVS(89)) 3:1, but CaMKIV phosphorylating the two sites equally. These differences in substrate specificities emphasize the need to consider these protein kinases independently despite their close homology.     

Proteomic analysis reveals novel binding partners of MIP‐T3 in human cells

MIP‐T3 (microtubule‐interacting protein associated with TRAF3) is a microtubule‐interacting protein that evolutionarily conserved from worms to humans, but whose cellular functions remains unknown. To get insight into the functions of MIP‐T3, we set out to identify MIP‐T3 interacting proteins by immunoprecipitation in human embryonic kidney 293 cells and MS analysis. As the results, a total of 34 proteins were identified and most of them were novel MIP‐T3 putative partners. The MIP‐T3‐associated proteins could be grouped into nine clusters based on their molecule functions, including cytoskeleton, chaperone, nucleic acid binding, kinase and so on. Three MIP‐T3‐interacted proteins – actin, HSPA8 and tubulin – were further confirmed by reciprocal coimmunoprecipitations and colocalization analysis. The interaction of MIP‐T3 with both actin filaments and microtubule suggested that MIP‐T3 may play an important role in regulation of cytoskeleton dynamics in cells. Our results therefore not only uncover a large number of MIP‐T3‐associated proteins that possess a variety of cellular functions, but also provide new research directions for the study of the functions of MIP‐T3.     

Proteomic analysis of human metaphase chromosomes reveals topoisomerase II alpha as an Aurora B substrate

The essential Aurora B kinase is a chromosomal passenger protein that is required for mitotic chromosome alignment and segregation. Aurora B function is dependent on the chromosome passenger, INCENP. INCENP, in turn, requires sister chromatid cohesion for its appropriate behaviour. Relatively few substrates have been identified for Aurora B, so that the precise role it plays in controlling mitosis remains to be elucidated. To identify potential novel mitotic substrates of Aurora B, extracted chromosomes were prepared from mitotically-arrested HeLa S3 cells and incubated with recombinant human Aurora B in the presence of radioactive ATP. Immunoblot analysis confirmed the HeLa scaffold fraction to be enriched for known chromosomal proteins including CENP-A, CENP-B, CENP-C, ScII and INCENP. Mass spectrometry of bands excised from one-dimensional polyacrylamide gels further defined the protein composition of the extracted chromosome fraction. Cloning, fluorescent tagging and expression in HeLa cells of the putative GTP-binding protein NGB/CRFG demonstrated it to be a novel mitotic chromosome protein, with a perichromosomal localisation. Identi fication of the protein bands corresponding to those phosphorylated by Aurora B revealed topoisomerase II alpha (topo IIα) as a potential Aurora B substrate. Purified recombinant human topo IIα was phosphorylated by Aurora B in vitro, confirming this proteomic approach as a valid method for the initial definition of candidate substrates of key mitotic kinases.     

Proteomic analysis of cervical cancer cells treated with suberonylanilide hydroxamic acid

Suberonylanilide hydroxamic acid (SAHA) is an orally administered histone deacetylase inhibitor (HDACI) that has shown significant antitumour activity in a variety of tumour cells. To identify proteins involved in its antitumour activity, we utilized a proteomic approach to reveal protein expression changes in the human cervical cancer cell line HeLa following SAHA treatment. Protein expression profiles were analysed by 2-dimensional polyacrylamide gel electrophoresis (2-DE) and protein identification was performed on a MALDI-Q-TOF MS/MS instrument. As a result, a total of nine differentially expressed proteins were visualized by 2-DE and Coomassie brilliant blue (CBB) staining. Further, all the changed proteins were positively identified via mass spectrometry (MS)/MS analysis. Of these, PGAM1 was significantly downregulated in HeLa cells after treatment with SAHA. Moreover, PGAM1 has been proven to be downregulated in another cervical cancer cell line (CaSki) by western blot analysis. Together, using proteomic tools, we identified several differentially expressed proteins that underwent SAHA-induced apoptosis. These changed proteins may provide some clues to a better understanding of the molecular mechanisms underlying SAHA-induced apoptosis in cervical cancer.     

Proteomic analysis of SRm160-containing complexes reveals a conserved association with cohesin

In this study, we describe a rapid immunoaffinity purification procedure for gel-free tandem mass spectrometry-based analysis of endogenous protein complexes and apply it to the characterization of complexes containing the SRm160 (serine/arginine repeat-related nuclear matrix protein of 160 kDa) splicing coactivator. In addition to promoting splicing, SRm160 stimulates 3'-end processing via its N-terminal PWI nucleic acid-binding domain and is found in a post-splicing exon junction complex that has been implicated in coupling splicing with mRNA turnover, export, and translation. Consistent with these known functional associations, we found that the majority of proteins identified in SRm160-containing complexes are associated with pre-mRNA processing. Interestingly, SRm160 is also associated with factors involved in chromatin regulation and sister chromatid cohesion, specifically the cohesin subunits SMC1alpha, SMC3, RAD21, and SA2. Gradient fractionation suggested that there are two predominant SRm160-containing complexes, one enriched in splicing components and the other enriched in cohesin subunits. Co-immunoprecipitation and co-localization experiments, as well as combinatorial RNA interference in Caenorhabditis elegans, support the existence of conserved and functional interactions between SRm160 and cohesin.     

Proteomic analysis of secreted membrane vesicles of archaeal Sulfolobus species reveals the presence of endosome sorting complex components

The crenarchaea Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii, release membrane vesicles into the medium. These membrane vesicles consist of tetraether lipids and are coated with an S-layer. A proteomic analysis reveals the presence of proteins homologous to subunits of the eukaryotic endosomal sorting complex required for transport (ESCRT). Immunodetection of one of these homologs suggest a cell surface localization in intact cells. These data suggest that the membrane vesicles in Sulfolobus sp. emerge from a specific budding process with similarity to the endosomal sorting pathway.     

Phylogenetic reassessment of gorals with new evidence from northern Myanmar reveals five distinct species

Researchers have recognised one to seven species of goral (Naemorhedus spp., Caprinae, Bovidae). We compared all available whole mitogenomic sequences, including eight new ones from northern Myanmar, and now recognise five distinct species. Naemorhedus caudatus remains a valid species. Naemorhedus griseus is a synonym of Naemorhedus goral. Naemorhedus evansi is a valid species, with a geographic range that extends northwards into southwestern China. Naemorhedus cranbrooki and Naemorhedus baileyi are distinct species, not synonyms. Molecular divergence analyses suggest that goral speciation was closely related to the rapid uplift of the Qinghai-Tibetan Plateau and Hengduan Mountains from the early Pliocene to the Pleistocene.     

Proteomic analysis of ischemia-reperfusion injury upon human liver transplantation reveals the protective role of IQGAP1

Ischemia-reperfusion injury (IRI) represents a major determinant of liver transplantation. IRI-induced graft dysfunction is related to biliary damage, partly due to a loss of bile canaliculi (BC) integrity associated with a dramatic remodeling of actin cytoskeleton. However, the molecular mechanisms associated with these events remain poorly characterized. Using liver biopsies collected during the early phases of organ procurement (ischemia) and transplantation (reperfusion), we characterized the global patterns of expression and phosphorylation of cytoskeleton-related proteins during hepatic IRI. This targeted functional proteomic approach, which combined protein expression pattern profiling and phosphoprotein enrichment followed by mass spectrometry analysis, allowed us to identify IQGAP1, a Cdc42/Rac1 effector, as a potential regulator of actin cytoskeleton remodeling and maintenance of BC integrity. Cell fractionation and immunohistochemistry revealed that IQGAP1 expression and localization were affected upon IRI and related to actin reorganization. Furthermore using an IRI model in human hepatoma cells, we demonstrated that IQGAP1 silencing decreased the basal level of actin polymerization at BC periphery, reflecting a defect in BC structure coincident with reduced cellular resistance to IRI. In summary, this study uncovered new mechanistic insights into the global regulation of IRI-induced cytoskeleton remodeling and led to the identification of IQGAP1 as a regulator of BC structure. IQGAP1 therefore represents a potential target for the design of new organ preservation strategies to improve transplantation outcome.     

Translational analysis of mouse and human placental protein and mRNA reveals distinct molecular pathologies in human preeclampsia

Preeclampsia (PE) adversely impacts ~5% of pregnancies. Despite extensive research, no consistent biomarkers or cures have emerged, suggesting that different molecular mechanisms may cause clinically similar disease. To address this, we undertook a proteomics study with three main goals: (1) to identify a panel of cell surface markers that distinguish the trophoblast and endothelial cells of the placenta in the mouse; (2) to translate this marker set to human via the Human Protein Atlas database; and (3) to utilize the validated human trophoblast markers to identify subgroups of human preeclampsia. To achieve these goals, plasma membrane proteins at the blood tissue interfaces were extracted from placentas using intravascular silica-bead perfusion, and then identified using shotgun proteomics. We identified 1181 plasma membrane proteins, of which 171 were enriched at the maternal blood-trophoblast interface and 192 at the fetal endothelial interface with a 70% conservation of expression in humans. Three distinct molecular subgroups of human preeclampsia were identified in existing human microarray data by using expression patterns of trophoblast-enriched proteins. Analysis of all misexpressed genes revealed divergent dysfunctions including angiogenesis (subgroup 1), MAPK signaling (subgroup 2), and hormone biosynthesis and metabolism (subgroup 3). Subgroup 2 lacked expected changes in known preeclampsia markers (sFLT1, sENG) and uniquely overexpressed GNA12. In an independent set of 40 banked placental specimens, GNA12 was overexpressed during preeclampsia when co-incident with chronic hypertension. In the current study we used a novel translational analysis to integrate mouse and human trophoblast protein expression with human microarray data. This strategy identified distinct molecular pathologies in human preeclampsia. We conclude that clinically similar preeclampsia patients exhibit divergent placental gene expression profiles thus implicating divergent molecular mechanisms in the origins of this disease.     

Proteomic comparison of human and great ape blood plasma reveals conserved glycosylation and differences in thyroid hormone metabolism

Most blood plasma proteins are glycosylated. These glycoproteins typically carry sialic acid-bearing sugar chains, which can modify the observed molecular weights and isoelectric points of those proteins during electrophoretic analyses. To explore changes in protein expression and glycosylation that occurred during great ape and human evolution, we subjected multiple blood plasma samples from all these species to high-resolution proteomic analysis. We found very few species-specific differences, indicating a remarkable degree of conservation of plasma protein expression and glycosylation during approximately 12 million years of evolution. A few lineage-specific differences in protein migration were noted among the great apes. The only obvious differences between humans and all great apes were an apparent decrease in transthyretin (prealbumin) and a change in haptoglobin isoforms (the latter was predictable from prior genetic studies). Quantitative studies of transthyretin in samples of blood plasma (synthesized primarily by the liver) and of cerebrospinal fluid (synthesized locally by the choroid plexus of the brain) confirmed approximately 2-fold higher levels in chimpanzees compared to humans. Since transthyretin binds thyroid hormones, we next compared plasma thyroid hormone parameters between humans and chimpanzees. The results indicate significant differences in the status of thyroid hormone metabolism, which represent the first known endocrine difference between these species. Notably, thyroid hormones are known to play major roles in the development, differentiation, and metabolism of many organs and tissues, including the brain and the cranium. Also, transthyretin is known to be the major carrier of thyroid hormone in the cerebrospinal fluid, likely regulating delivery of this hormone to the brain. A potential secondary difference in retinoid (vitamin A) metabolism is also noted. The implications of these findings for explaining unique features of human evolution are discussed.     

Proteomic analysis of differentially expressed proteins in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory–secretory products

Severe Clonorchis sinensis infection is a significant risk factor for malignant changes in bile ducts and surrounding liver tissues occurring as a result of direct contact with C. sinensis worms and their excretory–secretory products (ESP). However, the intrinsic molecular mechanisms involved in these processes remain obscure. To determine the effects of C. sinensis infection on protein expression in host bile duct epithelium, we examined proteomic profile changes in the human cholangiocarcinoma cell line (HuCCT1) treated with ESP at 24 h. Using a combination of 2‐DE, quantitative image and MALDI‐TOF MS analysis, we identified 83 proteins that were translationally modulated in response to ESP, among which 49 were up‐regulated and 34 down‐regulated. These proteins were classified under various biological categories, including metabolism, cell structure and architecture, proteolysis, protein modification, transport, signal transduction, and reactive oxygen species (ROS) detoxification. In particular, ESP induced the expression of redox‐regulating proteins, including peroxiredoxins (Prdx 2, 3, and 6) and thioredoxin 1 (Trx 1), possibly via intracellular ROS generation. Application of the proteomic approach to identify ESP response proteins should be a prerequisite before further investigation to clarify the molecular pathways and mechanisms involved in C. sinensis infection of host cells. J. Cell. Biochem. 108: 1376–1388, 2009. © 2009 Wiley‐Liss, Inc.