Stress-induced Phosphoprotein Research Articles

Acute exposure to offshore produced water has an effect on stress- and secondary stress responses in three-spined stickleback Gasterosteus aculeatus

Pollution is one of today's greatest problems, and the release of contaminants into the environment can cause adverse changes in vitally important biological pathways. In this study, we exposed three-spined stickleback Gasterosteus aculeatus to produced water (PW), i.e. wastewater from offshore petroleum production. PW contains substances such as alkylphenols (APs) and aromatic hydrocarbons (PAHs) known to induce toxicant stress and endocrine disruption in a variety of organisms. Following exposure to PW, a standardized confinement treatment was applied as a second stressor (PW-stress), testing how fish already under stress from the pollutant would respond to an additional stressor. The endpoint for analysis was a combination of blood levels of cortisol and glucose, in addition to transcribed levels of a set of genes related to toxicant stress, endocrine disruption and general stress. The findings of this study indicate that low doses of PW do not induce vitellogenin in immature female stickleback, but do cause an upregulation of cytochrome (CYP1A) and UDP-glucuronsyltransferase (UDP-GT), two biomarkers related to toxicant stress. However, when the second stressor was applied, both genes were downregulated, indicating that the confinement exposure had a suppressive effect on the expression of toxicant biomarkers (CYP1A and UDP-GT). Further, two of the stress related genes, heat shock protein 90 (HSP90) and stress-induced phosphoprotein (STIP), were upregulated in both PW- and PW-stress-treatment, but not in the water control confinement treatment, indicating that PW posed as a larger stress-factor than confinement for these genes. The confinement stressor caused an increased level of glucose in both control and PW-treated fish, indicating hyperglycemia, a commonly reported stress response in fish.

Autism-specific maternal autoantibodies recognize critical proteins in developing brain

Autism spectrum disorders (ASDs) are neurodevelopmental in origin, affecting an estimated 1 in 88 children in the United States. We previously described ASD-specific maternal autoantibodies that recognize fetal brain antigens. Herein, we demonstrate that lactate dehydrogenase A and B (LDH), cypin, stress-induced phosphoprotein 1 (STIP1), collapsin response mediator proteins 1 and 2 (CRMP1, CRMP2) and Y-box-binding protein to comprise the seven primary antigens of maternal autoantibody-related (MAR) autism. Exclusive reactivity to specific antigen combinations was noted in 23% of mothers of ASD children and only 1% of controls. ASD children from mothers with specific reactivity to LDH, STIP1 and CRMP1 and/or cypin (7% vs 0% in controls; P<0.0002; odds ratios of 24.2 (95% confidence interval: 1.45–405)) had elevated stereotypical behaviors compared with ASD children from mothers lacking these antibodies. We describe the first panel of clinically significant biomarkers with over 99% specificity for autism risk thereby advancing our understanding of the etiologic mechanisms and therapeutic possibilities for MAR autism.

Expression of stress-induced phosphoprotein-1 (STIP-1) was associated with tumor progression and poor prognosis in epithelial ovarian cancer

Objective: Our previous study has suggested that stress-induced phosphoprotein-1 (STIP-1) is a putative biomarker candidate in epithelial ovarian cancer (EOC). In this study, we investigated in detail the expression of STIP-1 as well as its functions in EOC.

Immunohistological analysis of stress-induced phosphoprotein 1 in ovarian cancer patients with low serum cancer antigen 125 levels

ObjectiveStress-induced phosphoprotein 1 (STIP1) was recently identified as a potential tumor marker for human ovarian cancer. This study further evaluates the usefulness of STIP1 in ovarian tumor patients with normal CA125 serum levels. Materials and MethodsSTIP1 and CA125 were immunohistochemically analyzed in 84 primary ovarian cancer and 30 benign ovarian tumors in patients with serum CA125 levels < 35 U/mL before surgery. Histoscores (0–300) were calculated as staining intensities (0–3) multiplied by percentage of tumor tissue (0–100%). ResultsThe cell types of the 84 cancers included 11 serous, 10 clear-cell, 51 mucinous, and 12 endometrioid carcinomas. There were 55 patients with invasive cancer and 29 with borderline ovarian tumors. The histoscores of STIP1, but not of CA125, in invasive cancer (mean ± SD, 186.3 ± 82.5) were significantly (p < 0.0001) higher than those seen in borderline ovarian tumors (86.2 ± 85.5). When the STIP1 histoscore was set at 183.8, invasive cancers (n = 55) were identified from benign tumors (n = 30) with a sensitivity of 56.4%, a specificity of 93.3%, a positive predictive value of 93.9%, and a negative predictive value of 53.8%. Results of receiver operating characteristics analysis showed that the area under curve of the STIP1 histoscore was 0.755, which was superior to that of CA125 (0.599). ConclusionSTIP1 histoscores may be useful in detecting invasive human ovarian cancer in patients with low serum CA125 levels.

Tumor Stress-Induced Phosphoprotein1 (STIP1) as a Prognostic Biomarker in Ovarian Cancer

Stress-induced phosphoprotein 1 (STIP1) has been recently identified as a released biomarker in human ovarian cancer. In addition, STIP1 secreted by human ovarian cancer cells has been shown to promote tumor cell proliferation by binding to ALK2 (activin A receptor, type II-like kinase 2) and activating the SMAD-ID3 signaling pathways. In this study, a total of 330 ovarian cancer tumor samples were evaluated for STIP1 expression by immunohistochemistry and analyzed for a possible correlation with patient characteristics and survival. The quantification of immunoreactivity was accomplished by applying an immunohistochemical scoring system (histoscore). Patients with high-level STIP1 expression (histoscore ≥169) had a significantly worse survival (high STIP1, mean survival time = 76 months; low STIP1, mean survival time = 112 months; P<0.0001). Moreover, STIP1 histoscores were significantly higher in high-grade tumors (grade 3) than in low-grade (grade 1–2) malignancies (P<0.0001), suggesting that STIP1 may be a proxy for tumor aggressiveness. The results of multivariable analysis revealed that high STIP1 histoscores, advanced stages, histologic types, and the presence of residual disease (≥2 cm) were independent predictors of poor prognosis. The addition of STIP1 histoscores improved the prediction of overall and progression-free survival rates in the multivariable Cox proportional hazard model. The treatment of ovarian cancer cells with recombinant STIP1 stimulated cell proliferation and migration, but co-treatment with anti-STIP1 antibodies abrogated this effect. Our findings suggest that STIP1 expression may be related to prognosis and that the STIP1 pathway may represent a novel therapeutic target for human ovarian cancer.

Unique and differential protein signatures within the mononuclear cells of HIV-1 and HCV mono-infected and co-infected patients

BackgroundPathogenesis of liver damage in patients with HIV and HCV co-infection is complex and multifactorial. Although global awareness regarding HIV-1/HCV co-infection is increasing little is known about the pathophysiology that mediates the rapid progression to hepatic disease in the co-infected individuals.ResultsIn this study, we investigated the proteome profiles of peripheral blood mononuclear cells from HIV-1 mono-, HCV mono-, and HIV-1/HCV co-infected patients. The results of high-resolution 2D gel electrophoresis and PD quest software quantitative analysis revealed that several proteins were differentially expressed in HIV-1, HCV, and HIV-1/HCV co-infection. Liquid chromatography-mass spectrometry and Mascot database matching (LC-MS/MS analysis) successfully identified 29 unique and differentially expressed proteins. These included cytoskeletal proteins (tropomyosin, gelsolin, DYPLSL3, DYPLSL4 and profilin-1), chaperones and co-chaperones (HSP90-beta and stress-induced phosphoprotein), metabolic and pre-apoptotic proteins (guanosine triphosphate [GTP]-binding nuclear protein Ran, the detoxifying enzyme glutathione S-transferase (GST) and Rho GDP-dissociation inhibitor (Rho-GDI), proteins involved in cell prosurvival mechanism, and those involved in matrix synthesis (collagen binding protein 2 [CBP2]). The six most significant and relevant proteins were further validated in a group of mono- and co-infected patients (n = 20) at the transcriptional levels.ConclusionsThe specific pro- and anti- apoptotic protein signatures revealed in this study could facilitate the understanding of apoptotic and protective immune-mediated mechanisms underlying HIV-1 and HCV co-infection and their implications on liver disease progression in co-infected patients.

Secreted Stress-Induced Phosphoprotein 1 Activates the ALK2-SMAD Signaling Pathways and Promotes Cell Proliferation of Ovarian Cancer Cells

Stress-induced phosphoprotein 1 (STIP1), a cochaperone that organizes other chaperones, heat shock proteins (HSPs), was recently shown to be secreted by human ovarian cancer cells. In neuronal tissues, binding to prion protein was required for STIP1 to activate the ERK (extracellular-regulated MAP kinase) signaling pathways. However, we report that STIP1 binding to a bone morphogenetic protein (BMP) receptor, ALK2 (activin A receptor, type II-like kinase 2), was necessary and sufficient to stimulate proliferation of ovarian cancer cells. The binding of STIP1 to ALK2 activated the SMAD signaling pathway, leading to transcriptional activation of ID3 (inhibitor of DNA binding 3), promoting cell proliferation. In conclusion, ovarian-cancer-tissue-secreted STIP1 stimulates cancer cell proliferation by binding to ALK2 and activating the SMAD-ID3 signaling pathways. Although animal studies are needed to confirm these mechanisms in vivo, our results may pave the way for developing novel therapeutic strategies for ovarian cancer.

Phosphoproteomic analysis of electroacupuncture analgesia in an inflammatory pain rat model

The phosphorylation changes of nociceptive signaling proteins in the spinal cord dorsal horn (SCDH) are important in creating exaggerated pain following peripheral inflammation. Electroacupuncture (EA) has been widely used to relieve acute and chronic inflammatory pain in human and experimental pain models. In the present study, we performed a phosphoproteomic analysis to investigate whether EA alters protein phosphorylation in SCDH to attenuate pain development. Inflammatory hyperalgesia was induced by intraplantar injection of complete Freund's adjuvant (CFA) into the rat hind paw. EA treatment at ST36 and SP6 acupoints alleviated thermal hyperalgesia of the CFA-induced inflammatory pain model rats. The SCDH proteins from the control, inflammatory pain model and EA treatment rats were separated by 2-dimensional gel electrophoresis and the alterations in phosphoproteins were detected by Pro-Q Diamond staining. Eight proteins were differentially phosphorylated following EA treatment in the inflammatory pain model. AldolaseC, nascent polypeptide-associated complex α, stress-induced phosphoprotein 1 and heat shock protein 90 were identified as phosphoproteins whose expression was increased, whereas GDP dissociation inhibitor 1, thiamine triphosphatase, phosphoglycerate kinase 1 and 14-3-3 γ were phosphoproteins whose expression was decreased. This is the first phosphoproteomic screening study to elucidate the working mechanisms of EA analgesia. The results suggest that the regulation of cellular pathways in which the identified proteins are involved may be associated with an EA analgesic mechanism.

Association Between The STIP1 Polymorphism And The Response To Inhaled Steroid In Children With Asthma

Bronchial hyperresponsiveness is usually measured by bronchial challenges using direct stimuli by methacholine or indirect stimuli by adenosine 5’-monophosphate (AMP). Airway inflammation is more closely reflected by AMP challenge test. The stress-induced-phosphoprotein 1 (STIP1) is one of the genes in steroid pathway. And polymorphisms in STIP1 are correlated with lung function after corticosteroid treatment in white subjects. We investigated whether polymorphism (rs4980524) of the STIP1 was associated with clinical phenotypes and responsiveness to inhaled steroid in Korean asthmatic children.

IgG from patients with pulmonary arterial hypertension and/or systemic sclerosis binds to vascular smooth muscle cells and induces cell contraction

ObjectivesPulmonary arterial hypertension (PAH) is characterised by remodelling of pulmonary arteries with enhanced vascular smooth muscle cell (VSMC) contraction, migration and proliferation. The authors investigated the presence of antibodies to...

Anti-neuronal and stress-induced-phosphoprotein 1 antibodies in neuro-Behçet's disease

No disease-specific neuronal antibodies have so far been defined in neuro-Behçet's disease (NBD). Immunohistochemistry and immunocytochemistry studies showed antibodies to hippocampal and cerebellar molecular layers and the surface antigens of cultured hippocampal neurons in sera and/or cerebrospinal fluids (CSF) of 13 of 20 NBD and 6 of 20 BD patients but not in multiple sclerosis or headache controls. Screening with a protein macroarray led to identification of stress-induced-phosphoprotein-1 (STIP-1) as an antigenic target. High-titer STIP-1-antibodies were detected in 6 NBD patients' sera but not in controls. These results suggest that neuronal antibodies could be useful as diagnostic biomarkers in NBD.

Inhibition of stress-induced phosphoprotein 1 decreases proliferation of ovarian cancer cell lines

Objective: Stress-induced phosphoprotein 1 (STIP-1) was previously identified by comparing different tumor-associated antigen levels between pretreatment and posttreatment sera from patients with ovarian cancer. STIP-1 acts as an adapter to assemble HSP 90 to HSP 70–client protein complexes, which are associated with antiapoptosis in various cancers. We attempted to determine whether the inhibition of STIP-1 influences proliferation in ovarian cancer cell lines.

Label‐free quantitative proteomics and SAINT analysis enable interactome mapping for the human Ser/Thr protein phosphatase 5

Affinity purification coupled to mass spectrometry (AP-MS) represents a powerful and proven approach for the analysis of protein-protein interactions. However, the detection of true interactions for proteins that are commonly considered background contaminants is currently a limitation of AP-MS. Here using spectral counts and the new statistical tool, Significance Analysis of INTeractome (SAINT), true interaction between the serine/threonine protein phosphatase 5 (PP5) and a chaperonin, heat shock protein 90 (Hsp90), is discerned. Furthermore, we report and validate a new interaction between PP5 and an Hsp90 adaptor protein, stress-induced phosphoprotein 1 (STIP1; HOP). Mutation of PP5, replacing key basic amino acids (K97A and R101A) in the tetratricopeptide repeat (TPR) region known to be necessary for the interactions with Hsp90, abolished both the known interaction of PP5 with cell division cycle 37 homolog and the novel interaction of PP5 with stress-induced phosphoprotein 1. Taken together, the results presented demonstrate the usefulness of label-free quantitative proteomics and statistical tools to discriminate between noise and true interactions, even for proteins normally considered as background contaminants.

Proteomics Profiling and Cytotoxic Effect of Curcuma longa on Prostate Cancer

Turmeric (Curcuma longa) has been shown to possess anti-inflammatory, antioxidant and antitumor properties. Extraction, partition and column chromatography of the dry powder of C. longa rhizomes showed presence of biological activity only in ethyl acetate eluted fraction in clonogenic assays using highly metastatic PC-3M prostate cancer cell line. HPLC, UV-Vis and Mass spectra studies showed presence of three curcuminoids in this fraction. Accordingly, we have made an attempt to identify the proteins modulated by purified turmeric fraction in PC-3M human prostate cancer cell line using high-resolution two-dimensional gel electrophoresis (2-DE). Isoelectric focusing and 2-DE analysis showed a total of 29 proteins altered by treatment with ethyl acetate fraction (EAF) of C. longa. Out of 29 differentially expressed pro- teins, 15 were identified by peptide mass fingerprinting through LC/MS/MS sequencing. From these, a total of 7 were down-regulated and 8 were up-regulated spots. The down-regulated proteins identified by Peptide Mass Fingerprinting are Elongation Factor 2 (eEF2), Stress-induced phosphoprotein 1, Glutathione S-transferase (GST) Omega-1, Parvalbumin alpha, Succinyl-CoA: ketoacid, Lamin-A/C and Annexin A2. The up-regulated proteins identified by Peptide Mass Fin- gerprinting include 78 kDa Glucose-regulated protein precursor (GRP78), Protein disulfide isomerase (PDI) precursor, Actin cytoplasmic 2, protein SET, Calreticulin precursor, Nucleophosmin, Vimentin, and Aortic alpha-actin (ACTA2). The identified proteins have diverse cellular functions such as ER stress, Unfolded Protein Response (UPR), cytoskeletal, structural, regulatory, and apoptotic proteins that will require further in-depth studies to understand the biological signifi- cance.

Stress-induced Phosphoprotein 1 as a Secreted Biomarker for Human Ovarian Cancer Promotes Cancer Cell Proliferation

Ovarian cancers are frequently not diagnosed until advanced stages, resulting in a high case fatality rate. Because of this, more tumor markers, in addition to CA125, for detecting and monitoring ovarian cancer are needed. During a systematic search for potential biomarkers of ovarian cancer, we compared the protein profiles between tumor interstitial fluid and normal interstitial fluid of ovaries, rationalizing that abnormal levels of proteins in tumor interstitial fluid may be detected in peripheral blood and thus serve as easily accessible tumor markers. Here, we show that stress-induced phosphoprotein 1 (STIP1) was secreted by ovarian cancer tissues into the peripheral blood of patients, resulting in a significant increase of serum levels of STIP1 in cancer patients compared with those in age-matched normal controls. Our results further indicated that combined use of CA125 and STIP1 may increase early detection of ovarian cancer. Functionally, recombinant STIP1 significantly induced ERK phosphorylation, promoted DNA synthesis, and increased Ki-67 immunoreactivity in ovarian cancer cells, suggesting that STIP1 in vitro promotes cell proliferation. Colocalization of STIP1 and phospho-ERK in human ovarian cancer tissues also supports an in vivo activation of ERK by STIP1. Further understanding of molecular roles of STIP1 in human ovarian cancer may shed light on its pathophysiology and development of novel therapeutic strategies.

Association of the Influenza Virus RNA Polymerase Subunit PB2 with the Host Chaperonin CCT

The RNA polymerase of influenza A virus is a host range determinant and virulence factor. In particular, the PB2 subunit of the RNA polymerase has been implicated as a crucial factor that affects cell tropism as well as virulence in animal models. These findings suggest that host factors associating with the PB2 protein may play an important role during viral replication. In order to identify host factors that associate with the PB2 protein, we purified recombinant PB2 from transiently transfected mammalian cells and identified copurifying host proteins by mass spectrometry. We found that the PB2 protein associates with the cytosolic chaperonin containing TCP-1 (CCT), stress-induced phosphoprotein 1 (STIP1), FK506 binding protein 5 (FKBP5), alpha- and beta-tubulin, Hsp60, and mitochondrial protein p32. Some of these binding partners associate with each other, suggesting that PB2 might interact with these proteins in multimeric complexes. More detailed analysis of the interaction of the PB2 protein with CCT revealed that PB2 associates with CCT as a monomer and that the CCT binding site is located in a central region of the PB2 protein. PB2 proteins from various influenza virus subtypes and origins can associate with CCT. Silencing of CCT resulted in reduced viral replication and reduced PB2 protein and viral RNA accumulation in a ribonucleoprotein reconstitution assay, suggesting an important function for CCT during the influenza virus life cycle. We propose that CCT might be acting as a chaperone for PB2 to aid its folding and possibly its incorporation into the trimeric RNA polymerase complex.

Temporal proteome profiling of taxol‐induced mitotic arrest and apoptosis

Taxol (Paclitaxel) is a mitotic inhibitor widely used in cancer therapy. Temporal proteome profiling was performed to study changes of proteins during the different cellular states of HeLa cells caused by exposure to taxol. The changes of proteins over time could be associated with various cellular processes such as mitotic arrest, an intermediate between mitotic arrest and apoptosis, apoptosis, and late apoptosis. Calumenin, stress-induced phosphoprotein 1 (STIP1), and translationally controlled tumor protein (TCTP) were assigned to mitotic arrest and selected for further experiments using immunoblotting and subcellular fractionation. Calumenin translocated from membranes to the cytosol during mitotic arrest and late apoptosis, but was significantly reduced in the cytosol during apoptosis. Translocation of STIP1 to the nucleus was observed at apoptosis and to the cytoskeleton at late apoptosis. TCTP increased in the cytosol at mitotic arrest and in membranes at apoptosis. In addition, the quantitative time courses of Bim isoforms revealed differences between BimL and BimS in comparison with BimEL. In summary, temporal proteome profiling of HeLa cells incubated with taxol allowed the assignment of proteins to certain processes and additional experiments with complementary approaches enabled a more comprehensive understanding of spatial changes of selected proteins during mitotic arrest and apoptosis.

Immunohistochemical analysis of stress-induced phosphoprotein-1 (STIP1) in ovarian cancer tissues with low serum levels of CA125.

e15530 Background: Through a systemic search for biomarkers of human ovarian cancer by comparing proteomes between tumor interstitial fluid (TIF) and normal interstitial fluid (NIF), we have identi...

Decreased GRP78 Protein Expression is a Potential Prognostic Marker of Oral Squamous Cell Carcinoma in Taiwan

Oral squamous cell carcinoma (OSCC) is an aggressive tumor and its occurrence in Taiwan is closely related to chronic smoking, alcohol consumption, and especially to betel quid chewing. It became the fourth most common malignant tumor of Taiwanese men in 2006. Unfortunately, there are few biomarkers for diagnosis and treatment of this disease. To find potential markers, two domestic cell lines (OC2 and OCSL) derived from different grades of OSCC were established and their proteins were compared by global proteomic analysis. The expression differences of GRP78 protein in these two cell lines and clinical samples from OSCC patients were verified. Of the 11 candidate proteins expressed differentially in both cell lines, six [heat shock protein 90 kDa beta member 1 (94 kDa glucose-regulated protein; GRP94), protein disulfide-isomerase precursor, vimentin, tubulin beta-2C chain, 78 kDa glucose-regulated protein precursor (GRP78), and annexin A2] were increased in OC2 cells (low-grade OSCC), and five (heat shock protein 90-beta, annexin A1, stress-induced phosphoprotein 1, elongation factor-2, and integrin alpha-3 precursor) were increased in OCSL cells (high-grade OSCC). Some of these proteins have been previously associated with malignant tumors, but no previous association of GRP78 with OSCC has been reported. GRP78 protein expression in these two OSCC cell lines was confirmed by Western blotting. Immunohistochemical staining of clinical samples from OSCC patients revealed that decreased GRP78 protein expression was significantly correlated with advance tumor stage (p < 0.001) and neck lymph node metastasis (p = 0.001). GRP78 protein is a possible biomarker of oral cancer in Taiwan.

Autoantibodies against stress‐induced phosphoprotein‐1 as a novel biomarker candidate for ovarian cancer

Detection of autoantibodies against tumor-associated antigens (TAA) has recently been shown to be a powerful tool for early detection of various cancers. The aim of this study was to investigate the possibility of using autoantibodies against TAA as novel biomarkers by a proteomics-based approach in patients with ovarian cancer. We used two-dimensional differential gel electrophoresis analysis of immuno-precipitated tumor antigens (2D-DITA) to compare the levels of autoantibodies in pretreatment and posttreatment sera of patients with ovarian cancers. The identified autoantibodies were validated by SYBR Green real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC). We further evaluated the level of autoantibody in sera of 68 ovarian cancer patients by an enzyme-linked immunosorbent assay (ELISA). The autoantibody directed against stress-induced phosphoprotein-1 (STIP-1) emerged as a novel biomarker candidate for ovarian cancer. SYBR Green PCR and IHC confirmed that the STIP-1 mRNA and protein expression levels were significantly up-regulated in ovarian cancers compared with normal and benign tumors (P = 0.003 and P < 0.001, respectively). A preliminary ELISA study showed that the serum levels of anti-STIP-1 autoantibodies were significantly elevated in ovarian cancer patients compared with healthy controls (P = 0.03). The results suggest that 2D-DITA is a useful tool to detect autoantibodies and that STIP-1 is a potential biomarker candidate for ovarian cancers.