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 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 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 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.     

Proteomic analysis of the proteins expressed by hydrogen peroxide treated cultured human dermal microvascular endothelial cells

Reactive oxygen species (ROS) have been traditionally regarded as toxic by-products of aerobic metabolism. However, ROS also act as intracellular signaling molecules and can mediate phenotypes in vascular endothelial cells, which may be physiological or pathological in nature. To clarify the molecular mechanisms of ROS signaling, we examined hydrogen peroxide (H(2)O(2))-responsive proteins in cultured human dermal microvascular endothelial cells (HMVEC) using proteomic tools. Protein expression in HMVEC was studied after they had been exposed to low- and high-levels of H(2)O(2) for various times, and intracellular ROS production was examined by flow cytometer and UV spectrophotometer. Proteins obtained from dose- and time-dependent series were separated by two-dimensional gel electrophoresis and tentatively identified by matrix-assisted laser desorption-time of flight mass spectrometry, by matching the tryptic mass maps obtained with entries in the NCBI and Swiss-Prot protein sequence database. At least 163 proteins were changed by H(2)O(2), and 60 proteins were identified. Oxidative stress triggered dramatic change in the expression of proteins in primary microvessel endothelial cells, and their mapping to cellular process provided a view of the ubiquitous cellular changes elicited by H(2)O(2). These results could provide a framework for the understanding of the mechanisms of cellular redox homeostasis and H(2)O(2) metabolism in microendothelium environment in various biological processes as well as pathological conditions.     

Proteomic analysis reveals an aflatoxin-triggered immune response in cotyledons of Arachis hypogaea infected with Aspergillus flavus

An immune response is triggered in host cells when host receptors recognize conserved molecular motifs, pathogen-associated molecular patterns (PAMPs), such as β-glucans, and chitin at the cell surface of a pathogen. Effector-triggered immunity occurs when pathogens deliver effectors into the host cell to suppress the first immune signaling. Using a differential proteomic approach, we identified an array of proteins responding to aflatoxins in cotyledons of peanut (Arachis hypogaea) infected with aflatoxin-producing (toxigenic) but not nonaflatoxin-producing (atoxigenic) strains of Aspergillus flavus. These proteins are involved in immune signaling and PAMP perception, DNA and RNA stabilization, induction of defense, innate immunity, hypersensitive response, biosynthesis of phytoalexins, cell wall responses, peptidoglycan assembly, penetration resistance, condensed tannin synthesis, detoxification, and metabolic regulation. Gene expression analysis confirmed the differential abundance of proteins in peanut cotyledons supplemented with aflatoxins, with or without infection with the atoxigenic strain. Similarly, peanut germination and A. flavus growth were altered in response to aflatoxin B1. These findings show an additional immunity initiated by aflatoxins. With the PAMP- and effector-triggered immune responses, this immunity constitutes the third immune response of the immune system in peanut cotyledon cells. The system is also a three-grade coevolution of plant-pathogen interaction.     

Proteomic analysis of human plasma in chronic rheumatic mitral stenosis reveals proteins involved in the complement and coagulation cascade

Background

Rheumatic fever in childhood is the most common cause of Mitral Stenosis in developing countries. The disease is characterized by damaged and deformed mitral valves predisposing them to scarring and narrowing (stenosis) that results in left atrial hypertrophy followed by heart failure. Presently, echocardiography is the main imaging technique used to diagnose Mitral Stenosis. Despite the high prevalence and increased morbidity, no biochemical indicators are available for prediction, diagnosis and management of the disease. Adopting a proteomic approach to study Rheumatic Mitral Stenosis may therefore throw some light in this direction. In our study, we undertook plasma proteomics of human subjects suffering from Rheumatic Mitral Stenosis (n = 6) and Control subjects (n = 6). Six plasma samples, three each from the control and patient groups were pooled and subjected to low abundance protein enrichment. Pooled plasma samples (crude and equalized) were then subjected to in-solution trypsin digestion separately. Digests were analyzed using nano LC-MS^E. Data was acquired with the Protein Lynx Global Server v2.5.2 software and searches made against reviewed Homo sapiens database (UniProtKB) for protein identification. Label-free protein quantification was performed in crude plasma only.

Results

A total of 130 proteins spanning 9–192 kDa were identified. Of these 83 proteins were common to both groups and 34 were differentially regulated. Functional annotation of overlapping and differential proteins revealed that more than 50% proteins are involved in inflammation and immune response. This was corroborated by findings from pathway analysis and histopathological studies on excised tissue sections of stenotic mitral valves. Verification of selected protein candidates by immunotechniques in crude plasma corroborated our findings from label-free protein quantification.

Conclusions

We propose that this protein profile of blood plasma, or any of the individual proteins, could serve as a focal point for future mechanistic studies on Mitral Stenosis. In addition, some of the proteins associated with this disorder may be candidate biomarkers for disease diagnosis and prognosis. Our findings might help to enrich existing knowledge on the molecular mechanisms involved in Mitral Stenosis and improve the current diagnostic tools in the long run.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-35) contains supplementary material, which is available to authorized users.     

Proteomic analysis of the hydrophobic fraction of mesenchymal stem cells derived from human umbilical cord blood

Mesenchymal stem cells (MSCs) are promising candidates for cell therapy and tissue engineering, but their application has been impeded by lack of knowledge of their core biological properties. In order to identify MSC-specific proteins, the hydrophobic protein fraction was individually prepared from two different umbilical cord blood (UCB)-derived MSC populations; these were then subjected to two-dimensional (2D) gel electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). Although the 2D gel patterns differed somewhat between the two samples, computer-assisted image analysis identified shared protein spots. 35 spots were reliably identified corresponding to 32 different proteins, many of which were chaperones. Based on their primary sub-cellular locations the proteins could be grouped into 6 categories: extracellular, cell surface, endoplasmic reticular, mitochondrial, cytoplasmic and cytoskeletal proteins. This map of the water-insoluble proteome may provide valuable insights into the biology of the cell surface and other compartments of human MSCs.     

Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH2-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking.     

Proteomic analysis of ERK1/2-mediated human sickle red blood cell membrane protein phosphorylation

Background

In sickle cell disease (SCD), the mitogen-activated protein kinase (MAPK) ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs). ERK1/2 is involved in activation of ICAM-4-mediated sickle RBC adhesion to the endothelium. However, other effects of the ERK1/2 activation in sickle RBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are unknown.

Results

To further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Sickle RBC treatment with U0126 decreased thirty-six phosphopeptides from twenty-one phosphoproteins involved in regulation of not only RBC shape, flexibility, cell morphology maintenance and adhesion, but also glucose and glutamate transport, cAMP production, degradation of misfolded proteins and receptor ubiquitination. Glycophorin A was the most affected protein in sickle RBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreases in both anion transport by band 3 and band 3 trafficking. The abundance of twelve of the thirty-six phosphopeptides were subsequently increased in normal RBCs co-incubated with recombinant ERK2 and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2.

Conclusions

These findings expand upon the current model for the involvement of ERK1/2 signaling in RBCs. These findings also identify additional protein targets of this pathway other than the RBC adhesion molecule ICAM-4 and enhance the understanding of the mechanism of small molecule inhibitors of MEK/1/2/ERK1/2, which could be effective in ameliorating RBC hemorheology and adhesion, the hallmarks of SCD.     

Genomic analysis of the TRIM family reveals two groups of genes with distinct evolutionary properties

Background  

The TRIM family is composed of multi-domain proteins that display the Tripartite Motif (RING, B-box and Coiled-coil) that can be associated with a C-terminal domain. TRIM genes are involved in ubiquitylation and are implicated in a variety of human pathologies, from Mendelian inherited disorders to cancer, and are also involved in cellular response to viral infection.     

Proteomic analysis of S-acylated proteins in human B cells reveals palmitoylation of the immune regulators CD20 and CD23

S-palmitoylation is a reversible post-translational modification important for controlling the membrane targeting and function of numerous membrane proteins with diverse roles in signalling, scaffolding, and trafficking. We sought to identify novel palmitoylated proteins in B lymphocytes using acyl-biotin exchange chemistry, coupled with differential analysis by liquid-chromatography tandem mass spectrometry. In total, we identified 57 novel palmitoylated protein candidates from human EBV-transformed lymphoid cells. Two of them, namely CD20 and CD23 (low affinity immunoglobulin epsilon Fc receptor), are immune regulators that are effective/potential therapeutic targets for haematological malignancies, autoimmune diseases and allergic disorders. Palmitoylation of CD20 and CD23 was confirmed by heterologous expression of alanine mutants coupled with bioorthogonal metabolic labeling. This study demonstrates a new subset of palmitoylated proteins in B cells, illustrating the ubiquitous role of protein palmitoylation in immune regulation.     

Proteomic analysis of early response lymph node proteins in mice treated with titanium dioxide nanoparticles

Human exposure to nanoparticles is inevitable from natural and anthropogenic sources. Titanium dioxide (TiO2) nanoparticles are increasingly being used in pharmaceutical and cosmetic products. Previous studies revealed that TiO2 levels were significantly increased in tissues (e.g., lymph nodes) after mice were injected with nanosized TiO2. To identify early response lymph node proteins to TiO2 nanoparticles, groups of mice were intradermally injected with a low dose of DeGussa P25 TiO2 nanoparticles or vehicle alone. The proteomes of lymph nodes at 24 h were quantitatively analyzed using trypsin-catalyzed 16O/18O labeling in conjunction with two-dimensional liquid chromatography separation and tandem mass spectrometry (2DLC-MS/MS). A total of 33 proteins were significantly changed (over 1.3-fold, p<0.05) in the mice treated with TiO2 nanoparticles, which accounted for approximately 1% of the total proteins identified. The differentially expressed proteins mainly involve the immune response (e.g., inflammation), lipid and fatty acid metabolism, mRNA processing, and nucleosome assembly. Regulation of functionally distinct classes of proteins could be mediated by estrogen receptor (ESR1), PPARγ, and c-Myc signalings, etc. The differentially expressed proteins identified in this experiment could represent early response proteins to TiO2 nanoparticle treatment in mouse lymph nodes.