kD Protein Research Articles

Next-Generation Cancer Immunotherapy Targeting Glypican-3.

Glypican-3 (GPC3), a 65 kD protein consisting of 580 amino acids, is a heparan sulfate proteoglycan bound to the cell membrane by glycosylphosphatidylinositol. This protein is expressed in the liver and the kidney of healthy fetuses but is hardly expressed in adults, except in the placenta. Contrarily, GPC3 is specifically expressed in hepatocellular carcinoma (HCC), ovarian clear cell carcinoma, melanoma, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma (Wilms tumor), yolk sac tumor, and some pediatric cancers. Although the precise function of GPC3 remains unclear, it has been strongly suggested that it is related to the malignant transformation of HCC. We identified GPC3 as a promising target for cancer immunotherapy and have been working on the development of cancer immunotherapeutic agents targeting it through clinical trials. In some trials, it was revealed that the GPC3 peptide vaccines we developed using human leukocyte antigen-A24- and A2-restricted GPC3-derived peptides could induce GPC3-specific cytotoxic T cells in most vaccinated patients and thereby improve their prognosis. To further improve the clinical efficacy of cancer immunotherapy targeting GPC3, we are also developing next-generation therapeutic strategies using T cells engineered to express antigen-specific T-cell receptor or chimeric antigen receptor. In addition, we have successfully monitored the levels of serum full-length GPC3 protein, which is somehow secreted in the blood. The utility of GPC3 as a biomarker for predicting tumor recurrence and treatment efficacy is now being considered. In this review article, we summarize the results of clinical trials carried out by our team and describe the novel agent targeting the cancer-specific shared antigen, GPC3.

Identification and Interaction Between Centrosomal Protein Centriolin and the E3 Ubiquitin Ligase, HECTD1

Uncontrolled cellular division is a central hallmark of cancer. Thus, studying the molecular functions and interactions of the proteins that regulate the complex processes of cell division is crucial. Two proteins are at the focal point of this study. One is centriolin, a 270 kD protein that has been demonstrated as essential for cytokinesis. The second is a 289 kD E3 ubiquitin ligase called HECTD1. Prior experiments utilizing a yeast two hybrid screen concluded that there is an interaction between centriolin and HECTD1. Furthermore, results of immunoprecipation and western blot from synchronized cells showed that centriolin was diminished in G0, whereas HECTD1 levels were elevated compared to other phases of the cell cycle. In contrast, centriolin concentrations were much more robust in S phase, G1, and M phases, compared to that of G0. In separate experiments, we have used CRISPR to eliminate either centriolin or HECTD1 in HeLa cells to observe the repercussions of the loss of these proteins and their global effects on the cell cycle. Interestingly, we observed significant cell death when we eliminated the HECTD1 gene. Our further experiments revealed that the cell death is due to robust induction of apoptosis. Currently we are investigating whether or not the established interaction between centriolin and HECTD1 plays a role in the cell death that we observed.Support or Funding InformationThis research was supported by Lincoln Memorial University‐DeBusk College of Osteopathic Medicine Intramural Grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sacs R272C missense homozygous mice develop an ataxia phenotype

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS [MIM 270550]) is an early-onset neurodegenerative disorder caused by mutations in the SACS gene. Over 200 SACS mutations have been identified. Most mutations lead to a complete loss of a sacsin, a large 520 kD protein, although some missense mutations are associated with low levels of sacsin expression. We previously showed that Sacs knock-out mice demonstrate early-onset ataxic phenotype with neurofilament bundling in many neuronal populations. To determine if the preservation of some mutated sacsin protein resulted in the same cellular and behavioral alterations, we generated mice expressing an R272C missense mutation, a homozygote mutation found in some affected patients. Though SacsR272C mice express 21% of wild type brain sacsin and sacsin is found in many neurons, they display similar abnormalities to Sacs knock-out mice, including the development of an ataxic phenotype, reduced Purkinje cell firing rates, and somatodendritic neurofilament bundles in Purkinje cells and other neurons. Together our results support that Sacs missense mutation largely lead to loss of sacsin function.

The prognostic value of glycosylphosphatidylinositol-anchored 80 kD protein (GPI-80) positive myeloid derived suppressor cells (MDSCs) in peripheral blood of metastatic renal cell carcinoma (mRCC) patients.

601 Background: Myeloid derived suppressor cells (MDSCs) are found to play essential roles in various tumors and to associate with the prognosis of patients. However, the prognostic values of MDSCs in metastatic renal cell cancer (mRCC) are still undefined. Glycosylphosphatidylinositol-anchored 80 kD protein (GPI-80) is known as a regulator of Mac-1 (CD11b/CD18) and closely related with differentiation of myeloid cells. The object of our study was to interrogate the association of MDSCs in peripheral blood with clinical outcomes of mRCC patients by using GPI-80. Methods: Peripheral blood samples, collected from 23 mRCC patients before treatment and 16 healthy volunteers, were stained with several myeloid cell antigens such as CD16, CD15, CD33, and GPI-80 to mark neutrophils and monocytes. We measured the expression levels (mean of florescence intensity, MFI) and deviation (coefficient variation, CV) of myeloid antigens by flow cytometry. To investigate the immunosuppressive function of GPI-80 positive cells, reactive oxygen species (ROS) and LAP-1 (precursor of TGF-β1) were measured. The impact of the cell surface antigens on overall survival (OS) was evaluated using the univariate and multivariate Cox regression analysis. Results: The MFI or CV of GPI-80 on neutrophilic or monocytic cells were significantly difference in between patients and healthy volunteers. LAP-1 expression and ROS production in monocytic cells were increased in patients than in healthy volunteers, and correlated with expression of GPI-80 MFI (p = 0.0107 and p = 0.0381, respectively). Regarding the prognostic values, both GPI-80 CV of neutrophilic cells and GPI-80 MFI of monocytic cells were associated with poor OS of mRCC patients (p < 0.01). Multivariate analysis showed that GPI-80 MFI of monocytic cells were independent unfavorable prognosis marker (HR 2.427, p = 0.044). Conclusions: These results showed that change of GPI-80 expression on myeloid cells in peripheral blood representing appearance of MDSCs was likely to be negative predictors in patients with mRCC.

Towards the Inhibition of Calpain-Dependent Desmoplakin Cleavage in Arrhythmogenic Cardiomyopathies

Desmoplakin (DSP) is a large (260 kD) protein found in the desmosome, a subcellular structure that links the cytoskeleton of one myocyte to that of its neighbor. In cardiomyocytes, desmoplakin's main function is to maintain cell-to-cell adhesion and synchronization during heart contractions. A mutation hot spot, centered around the SH3 domain of DSP, is associated with arrhythmogenic cardiomyopathy, but the underlying mechanism(s) of this association isn’t well studied. Here, we show that many of these disease-causing mutations display increased calpain sensitivity. Additionally, structural and computational studies on DSP variants show that this cleavage event is driven not through a gross structural change, but instead through the discrete exposure of a normally occluded calpain cleavage site. Initial in silico and in vitro screens of small molecules suggest the feasibility of developing a pharmaceutical solution to prevent calpain-mediated DSP cleavage.

Targeting on glycosylation of mutant FLT3 in acute myeloid leukemia

ABSTRACTObjective: To summarize the abnormal location of FLT3 caused by different glycosylation status which further leads to the distinguishing signaling pathways and discuss targeting on FLT3 glycosylation by drugs reported in recent literatures.Methods: We review FLT3 glycosylation in endoplasmic reticulum. The abnormal signal of mutant FLT3 with different glycosylation status is discussed. We also address potential FLT3 glycosylation-targeting strategies for the treatment.Results: Inhibition of FLT3 mutant cells by drugs reported in recent literatures involves the influence of glycosylation of FLT3: 2-deoxy-D-glucose, Tunicamycin and Fluvastatin are reported to inhibit N-glycosylation of FLT3; Pim-1 inhibitors are proved to block the inhibition of Pim-1 on FLT3 Oglycosylation; HSP90 inhibitors and Tyrosine Kinase Inhibitors are shown to increase fully glycosylated form of FLT3.Discussion: The FMS-like tyrosine kinase 3 (FLT3) gene expressed only in CD34+ progenitor cells in bone marrow is located on chromosome 13q12 encoding FLT3 protein. FLT3 is initially synthesized as a 110 KD protein, which glycosylated in the endoplasmic reticulum to a 130 KD immature protein rich in mannose, and further processed into a mature 160 KD protein in the Golgi apparatus, which could be transferred to the cell surface. Therapy targeting on FLT3 glycosylation is a promising direction for AML treatment.Conclusions: The abnormal location of FLT3 caused by different glycosylation status leads to the distinguishing signaling pathways. Targeting on FLT3 glycosylation may provide a new perspective for therapeutic strategies.Abbreviations: ABCG2: ATP-binding cassette transporter breast cancer resistance protein; ATF: activating transcription factor; AML: acute myeloid leukemia; CHOP: CCAAT-enhancer-binding protein homologous protein; 2-DG: 2-deoxy-D-glucose; EFS: event free survival; EPO: erythropoietin; EPOR: erythropoietin receptor; ERS: endoplasmic reticulum stress; FLT3: FMS-like tyrosine kinase 3; GPI: glycosylphosphatidylinositol; HSP: heat shock protein; ITD: internal tandem duplication; IRE1a: inositol-requiring enzyme 1 alpha; JNK: c-Jun N-terminal kinase; JMD: juxtamembrane domain; JAK: janus kinase; MAPK/ERK: mitogen activated protein kinase/extracellular signal-regulated protein kinase; OS: overall survival; PI3K/AKT: phosphatidylinositide 3-kinases/protein kinase B; PERK: RNA-activated protein kinase-like endoplasmic reticulum kinase; Pgp: P-glycoprotein; PTX3: human pentraxin-3; STAT: signal transducer and activator of transcriptions; TKD: tyrosine-kinase domain; TKI: tyrosine kinase inhibitor; TM: Tunicamycin; UPR: unfolded protein reaction

GPI-80 as a Useful Index for Myeloid Cell Heterogeneity and a Potential Prognostic Biomarker for Metastatic Renal Cell Carcinoma.

Myeloid-derived suppressor cells (MDSCs), which include neutrophilic MDSCs and monocytic MDSCs, exhibit high immunosuppressive activity. Glycosylphosphatidylinositol-anchored 80 kD protein (GPI-80) is selectively expressed on mature neutrophils in healthy individuals. Increased GPI-80 expression on monocytes and variations in GPI-80 expression on neutrophils indicate the appearance of MDSCs in the peripheral blood of cancer patients. However, it is still unclear whether GPI-80 expression on myeloid cells, neutrophilic MDSCs and monocytic MDSCs, is correlated with the clinical outcomes of patients with cancer. In this study, we investigated the characteristics of myeloid cells expressing GPI-80 and the implication of GPI-80 expression in the clinical outcomes of patients with metastatic renal cell carcinoma (mRCC), in which primary renal cell carcinoma spreads from the kidney to other organs. The study included 20 patients with mRCC (a mean age of 66.0 years) and 16 healthy volunteers (a mean age of 47.8 years). To determine the heterogeneity of myeloid cells in peripheral blood samples, we performed the three-dimensional principal component analysis using the combination of GPI-80, CD16, and latency-associated peptide-1 (LAP), derived from the N-terminal region of transforming growth factor-β1 precursor. The results showed that myeloid cells in mRCC patients were widely distributed and clearly distinguishable from those in the healthy volunteers. The survival analysis revealed that GPI-80 expression on neutrophils and monocytes was correlated with poor prognostic outcomes of patients with mRCC. In conclusion, the expression of GPI-80 on myeloid cells, a useful index for the heterogeneity of MDSCs, serves as a potential prognostic biomarker for mRCC.

Allergenic extracts from natural and genetically modified soybean

Background. The wide spread of soybeans both natural and genetically modified (GM) in agriculture and food industry arises a question about the safety of its use, as soy is the most common food allergen among leguminous plants. Meanwhile, there are no registered domestic diagnostic allergens from soybeans in Russia. Objective. The aim of this study was to obtain allergenic extracts from natural and GM soybeans resistant to the herbicide «Roundup» and evaluate their biochemical and allergenic properties. Methods. Soybean extracts were obtained by the Evans-Kok method. The amount of protein nitrogen was determined by the Nessler method. The protein composition of the soybean was determined by the SDSPAGE. Specific activity was assessed in the reaction of NDTK. Allergenic activity was assessed in ELISA according to the sIgE levels to soy in the sera of patients with food allergy. Results. The protein fractions corresponding to known allergens weare revealed by SDS-PAGE in the samples of extracts: natural soybeans - Gly m 3, Gly m 5, and Gly m 6, while GM soybeans - Gly m Bd 30k. In addition to those proteins, in both extracts the 20 kD protein was clearly detected, which can correspond to the inhibitor of trypsin Kunitsa (Hor v 1, CMb, BDR). Allergenic soybean extracts bind sIgE and sIgG in the sera of patients with allergies. Conclusion. The obtained data confirm the high allergenic potential of extracts from natural soybeans, whereas the allergenic activity of GM soybeans extracts is reduced.

Active Calpain Promotes Fibrin Clot Contraction By Strengthening the Coupling of Fibrin-Bound αIIbβ3 to the Platelet Cytoskeleton

Platelet-driven blood clot contraction reduces and compacts clot volume, promoting hemostasis while restoring blood flow past otherwise obstructive thrombi. Clot contraction is driven by traction forces in the range of tens of pN per platelet that are generated by platelet non-muscle myosin IIa and actin on αIIbβ3 bound to extracellular fibrin fibers. In an analysis of the kinetics of thrombin-induced clot formation and contraction in platelet-rich plasma, we found that clot contraction has two discernable phases: an exponential initiation phase and a second exponential contraction phase. It is noteworthy that Azam et al (Mol Cell Biol21:2213-20, 2001) observed that deleting the µ-calpain gene in mice impairs platelet-mediated clot contraction. In this context, we found that inhibiting calpain activity with ALLM (N-Acetyl-Leu-Leu-Methional), a cell-permeable calpain inhibitor, significantly prolonged the duration of the initiation phase and decreased the kinetic rate constants for both the initiation and contraction phases of thrombin-stimulated platelet-mediated clot contraction, but without affecting the overall extent of contraction. The calpain inhibitor ALLN (N-acetyl-Leu-Leu-Norleu) had the same effect. Thus, these studies imply that activation of platelet calpain facilitates the transmission of traction forces from the platelet cytoskeleton to the αIIbβ3 bound to fibrin fibers. μ-Calpain is calcium-dependent cytosolic neutral cysteine protease that is activated 30-60 seconds after the onset of platelet aggregation stimulated by platelet agonists such as thrombin. To identify the calpain-cleaved protein or proteins involved in clot contraction, we incubated washed human platelets with the thrombin activation peptide TRAP in the presence or absence of calpain inhibitors and identified calpain-cleaved proteins using subtiligase-mediated biotin-labeling of nascent N-termini followed by mass spectrometry. We identified 32 proteins in the platelet cytosol that undergo calpain-mediated cleavage after TRAP stimulation. Many of these are cytoskeletal proteins, most prominently talin which connects integrins to the cytoskeleton and vinculin which is required for myosin-contractility-dependent effects on traction force and adhesion strength. Accordingly, we focused our attention on these two proteins. Talin, a 250 kD protein, is composed of a 45 kD N-terminal head domain attached via a flexible linker to a 200 kD C-terminal rod domain. The rod domain consists of 62 amphipathic α-helices organized into a series of four- and five-helix bundles. Vinculin is a 116 kD protein composed of 5 domains and has an autoinhibited structure in which domains 1-3 bind to domain 5. We detected 8 calpain-mediated cleavages in talin, 2 previously identified cleavage sites in unstructured regions and 6 others in α-helical regions known to interact with other proteins. Four of the latter are located within the first 12 talin helices, a region that contains four vinculin binding sites (VBSs). Likewise, the remaining two cleavage sites are in proximity to a VBS. A VBS is a vinculin-binding α-helix that is buried in a helical bundle due to extensive hydrophobic interactions with other amphipathic helices. Because VBS are packed into the interior of a helical bundle, a structural rearrangement is required to initiate vinculin binding. Using magnetic tweezers and atomic force microscopy, del Rio et al demonstrated that force-induced stretching of single talin rod molecules activates VBSs (Science323:638-41, 2009)), implying that stretching causes the conformational change required to expose buried VBSs. Thus, based on the location of the calpain-mediated cleavages in talin, we hypothesize that by cleaving talin in proximity to a VBS, calpain facilitates vinculin to binding talin, thereby strengthening the coupling of fibrin-bound αIIbβ3 to the platelet cytoskeleton to promote fibrin clot contraction. DisclosuresNo relevant conflicts of interest to declare.

Production and Function of Different Regions from Mytichitin-1 of Mytilus coruscus

Chitinase is an important enzyme for many physiological processes. Mytichitin-1 is a chitinase-like protein in Mytilus coruscus, and its C-terminal 55-AA fragment (mytichitin-CB) is a novel antimicrobial peptide, suggesting a new immune process in which chitinase is involved; mytichtin-1 may have various forms in the different biological processes of M. coruscus. Thus, the study of mytichitin-1 will be helpful for understanding the mechanism of mussel immune biology and the functional diversity of chitinase. In this study, mytichitin-1 was recombinantly expressed with different lengths, full-length mytichtin-1 (rMchi-F) and the N-terminal region (rMchi-N) in Escherichia coli BL21 with codon optimization. The results of SDS-PAGE, Western blotting, and mass spectrometry confirmed that the two forms of mytichitin-1 had been successfully recombinant expressed with a yield of 40 mg purified enzyme per L culture. In addition, the 55-AA fragment of mytichitin-CB was chemically synthesized (sMchi-CB). After purification and oxidation, the functions of the three protein products were analysed, including chitin degradation, chitin binding, and antimicrobial activities. Both rMchi-F and rMchi-N displayed enzymatic activity with the optimum pH of 4.0 and optimum temperature of 40 °C, and rMchi-N showed a stronger activity than rMchi-F. Enzymatic activities of rMchi-F and rMchi-N were stimulated by the metal ions Fe2+, Ba2+, and Na+ and partially inhibited by Cu2+, Ni2+ and Zn2+. rMchi-F, rMchi-N, and sMchi-CB had the ability to combine with colloid chitin. The antimicrobial activities of these proteins were tested against bacteria and fungi, and the results indicated the strongest activity for sMchi-CB and the weakest activity for rMchi-N. Using a prepared anti-rMchi-F polyclonal antibody, immunohistochemistry and immunoprecipitation were performed and the results revealed the location of mytichitin-1 in mantle, digestive gland and blood cells. In addition, two forms of mytichitin-1, mytichitin-CB (6 kD) and full-length mytichitin-1 (48 kD), were detected, and a 35 kD protein was identified as the third form of mytichitin-1, existing in various tissues of M. coruscus. These findings suggest that mytichitin-1 may play different roles, with at least three forms, in different M. coruscus tissues.

Differentially Expressed Proteins in Rat Hippocampus after Chronic Immobilization Stress and Intervention Using Xiao Yao San Decoction

ABSTRACT Objective To identify differentially expressed proteins in the hippocampus of rats after chronic immobilization stress (CIS) using a proteomics approach, and to study the effect of the Xiao Yao San (XYS) decoction on differentially expressed proteins. Methods Twenty-four Sprague Dawley rats were randomly assigned to one of four groups of equal body weight: control (non-stress), 7-day stress, 21-day stress and 21-day stress + XYS treatment groups. Two-dimensional gel electrophoresis (2-DE) was used to detect differences in protein expression in rat hippocampus. One differentially expressed protein was measured and verified by western blotting. Results Seventeen proteins showed differential expression. Among these, eight could be identified: glial fibrillary acidic protein-2 (GFAP-2), tubulin alpha-1c, cytoplasmic muscle actin 2, 14-3-3 protein, β-2a tubulin, phosphatidylethanolamine binding protein, synuclein α syn3, and a low molecular weight (18 kD) protein. Six of these proteins exhibited increased expression, one showed decreased expression, and the other protein, which comprised five subtypes, were either increased or decreased. These proteins are known to be involved in immunity, signal transduction, cell cycle control, apoptosis, regulation of enzyme activity, cytoskeleton structure, and synaptic plasticity. GFAP-2 was further analyzed, and its differential expression confirmed by western blotting. Conclusion Some proteins are differentially expressed in the hippocampus of rats under chronic stress. The biological functions of these differentially expressed proteins are varied. Finally, the XYS decoction can significantly up- or down-regulate these protein expression levels.

Secretion of heat shock ‐60, ‐70 kD protein, IL‐1β and TNFα levels in serum of a term normal pregnancy and patients with pre‐eclampsia development

The extracellular heat shock proteins (eHsp) family act as molecular chaperones regulating folding, transporting protein and are associated with immune modulation in different physiological and pathological processes. They have been localized in different gestational tissues and their concentration in amniotic fluid and serum has been determined. In the present study, we proposed to determine the concentration of eHsp‐60, ‐70, IL‐1β and TNFα in the serum of pregnant patients with 34 weeks of gestation with and without clinical evidences of preeclampsia (PE). Our results indicate significant increase of these markers in patients with PE with respect to healthy pregnant patients without active labor. Finally, the concentration of eHsp‐60 and ‐70 correlated positively with the hepatic dysfunction markers uric acid, lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT) glutamic pyruvic transaminase (GPT), and inflammatory IL‐1β and TNFα response. In conclusion, our results demonstrate a strong associated between Hsp and marker of hepatic dysfunction.

Sensitivity and clinical utility of the anti-cytosolic 5′-nucleotidase 1A (cN1A) antibody test in sporadic inclusion body myositis: Report of 40 patients from a single neuromuscular center

Sporadic inclusion body myositis (IBM) is the most common acquired myopathy affecting patients over age 50. The discovery of an autoantibody directed against a 43–44 kD protein (anti-cytosolic-5′-nucleotidase 1A or anti-cN1A) has provided support for the hypothesis of an immune-mediated pathogenesis. Previous studies have reported variable test sensitivity and specificity, and inconsistent results on the predictive value. In our cohort of 40 patients with clinico-pathologically or clinically defined IBM, we found the sensitivity of the anti-cN1A antibody test to be 50%. Comparing characteristics for test positive and test negative groups, we found that patients in our cohort testing positive for the anti-cN1A antibody were significantly more likely to be older than age 60 years at symptom onset. We found no positive association between anti-cN1A reactivity and other clinical, laboratory, and muscle histopathologic findings. Based on all clinical studies published to date including the present, the anti-cN1A antibody test shows high diagnostic specificity, moderate sensitivity, and a low predictive value in regards to age of onset, disease severity and other associated clinicopathological findings.

Rickettsiae reservoirs among small mammals (Rats, Mice And Shrews) and their Arthropod Vectors in Sri Lanka

Rickettsioses are a group of emerging diseases caused by closely related bacteria. In Sri Lanka, to date, studies have been focused mainly on human subjects. The present study aimed to identify small mammal reservoir hosts and vectors of Rickettsia spp. and Orientia tsutsugamushi in two districts of Sri Lanka. Quantitative-PCR was carried out to detect Rickettsia using citrate synthase gene and Orientia using 47-kD outer membrane protein antigen gene in blood of small rodents and their infested ectoparasites. In both districts ~7.5% blood samples were positive for Rickettsia . Rattus rattus, Bandicota indica and Mus fernandoni were carriers. Three individuals of Suncus murinus, B. indica and Golunda ellioti had only infected ectoparasites. Copies of gltA/100 μL ranged from 133-1.2 × 10 4 in blood and 197-1.9 × 10 7 in ectoparasites. Of small mammals with ectoparasites, 43% had Rickettsia positive ectoparasites. Rhipicephalus haemaphysaloides, Ixodes ceylonensis, Haemaphysalis spinigera, Haemaphysalis spp., Stivalius aporus and Xenopsylla cheopis were positive. All study sites except three had infected small mammals or ectoparasites. All samples were negative for O. tsutsugamushi . This is the first study to report Rickettsia spp. in small mammals and their ectoparasites in Sri Lanka. Haemaphysalis spinigera , I. ceylonensis and S. aporus are new records of vectors for Rickettsia . This is also the first report of endemic M. fernandoni as a carrier of Rickettsia and G. ellioti with Rickettsia infected ectoparasites. Though rickettsiosis is not life threatening in most cases, it can lead to severe or fatal disease in vertebrate animals and humans. Hence, the knowledge of the distribution of said pathogen in the reservoirs is essential to control the disease.

The Tomato Mitogen-Activated Protein Kinase SlMPK1 Is as a Negative Regulator of the High-Temperature Stress Response.

High-temperature (HT) stress is a major environmental stress that limits plant growth and development. MAPK cascades play key roles in plant growth and stress signaling, but their involvement in the HT stress response is poorly understood. Here, we describe a 47-kD MBP-phosphorylated protein (p47-MBPK) activated in tomato (Solanum lycopersicum) leaves under HT and identify it as SlMPK1 by tandem mass spectrometry analysis. Silencing of SlMPK1 in transgenic tomato plants resulted in enhanced tolerance to HT, while overexpression resulted in reduced tolerance. Proteomic analysis identified a set of proteins involved in antioxidant defense that are significantly more abundant in RNA interference-SlMPK1 plants than nontransgenic plants under HT stress. RNA interference-SlMPK1 plants also showed changes in membrane lipid peroxidation and antioxidant enzyme activities. Furthermore, using yeast two-hybrid screening, we identified a serine-proline-rich protein homolog, SlSPRH1, which interacts with SlMPK1 in yeast, in plant cells, and in vitro. We demonstrate that SlMPK1 can directly phosphorylate SlSPRH1. Furthermore, the serine residue serine-44 of SlSPRH1 is a crucial phosphorylation site in the SlMPK1-mediated antioxidant defense mechanism activated during HT stress. We also demonstrate that heterologous expression of SlSPRH1 in Arabidopsis (Arabidopsis thaliana) led to a decrease in thermotolerance and lower antioxidant capacity. Taken together, our results suggest that SlMPK1 is a negative regulator of thermotolerance in tomato plants. SlMPK1 acts by regulating antioxidant defense, and its substrate SlSPRH1 is involved in this pathway.

697 Epidermal tissue differentiation requires the uncharacterized protein RED1

The human genome encodes >4,000 uncharacterized open reading frames (ORFs) with undiscovered biological functions. To identify potentially novel mediators of epidermal differentiation from this candidate pool, RNA-Seq of primary human keratinocytes during calcium-induced differentiation was used to screen for uncharacterized ORFs whose transcript levels are increased during this process. The top up-regulated ORF encodes a 50 kD protein that we have termed Regulator of Epidermal Differentiation (RED) 1. An orthogonal screen also identified RED1 as highly down-regulated in primary human squamous cell carcinomas compared to patient-matched normal skin. As malignant neoplasms frequently demonstrate loss of differentiation, we hypothesized that RED1 is required for epidermal differentiation. To test this possibility, we undertook gain- and loss-of-function experiments in organotypic human epidermal tissue. Enforced expression of RED1 accelerated differentiation in regenerated human skin while CRISPR/Cas9-mediated depletion attenuated this process. In each case, whole transcriptome profiling to assess the global impact of altering RED1 expression identified gene groups that control skin differentiation, development, and function. Proximity-dependent biotin ligation assays in organotypic human epidermal tissue identified validated RED1 interactors including envoplakin (EVPL) and periplakin (PPL), genes known to be essential to skin differentiation, as well as genes not previously implicated in skin homeostasis, such as calcyclin binding protein (CACYBP). Gene set enrichment analysis suggests RED1 and its interacting proteins co-control distinct subsets of genes that induce epidermal differentiation and drive neoplastic transformation. Efforts are underway to study RED1s impacts on epidermal neoplasia. RED1 therefore represents a new regulator of epidermal differentiation with potential roles in skin carcinogenesis.

Interaction effect between the receptor for advanced glycation end products (RAGE) and high-mobility group box-1 (HMGB-1) for the migration of a squamous cell carcinoma cell line.

The receptor for advanced glycation end products (RAGE) is a multiligand cell surface receptor of the immunoglobulin superfamily and a newly recognized invasion-related gene. High mobility group box-1 (HMGB-1) is a 30-kD protein binding to RAGE and acting as a transcription-factor-like protein that regulates the expression of several genes. In this study, the interaction effect between RAGE and HMGB-1 on the migration of SCC7 cells was investigated along with the inhibitory effect of the drug nifedipine on this interaction effect. Ten surgical specimens from patients with squamous cell carcinoma (SCC) of the head and neck and a SCC7 cell line were stained using immunohistochemical and immunocytochemical methods. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect RAGE expression in SCC7 cells; Western blot analysis was used to detect HMGB-1 expression in SCC7 cells. An in vitro migration assay (Boyden chamber migration assay) was used for evaluating the interaction effect between RAGE and HMGB-1 on the migration of SCC7 cells. HMGB-1 and various concentrations of nifedipine were tested for their effect on SCC7 cell migration with in vitro migration assays. RAGE and HMGB-1 were expressed in almost all human head and neck SCC tissues and in SCC7 cells as detected by immunostaining. The migration assay showed that the interaction between RAGE and HMGB-1 increased SCC7 migration activity depending on the level of HMGB-1, and nifedipine inhibited the interaction effect between RAGE and HMGB-1 on SCC7 cells in a dose-dependent manner. The interaction between RAGE and HMGB-1 could be closely associated with metastasis of SCC of the head and neck. Nifedipine may have an inhibitory effect on tumor metastasis.

The development of methods to measure exposure to a major rabbit allergen (Ory c 1).

Rabbits are used as laboratory animal models and are also popular domestic pets. Allergic responses to rabbit allergens have been documented in both settings, and several rabbit allergens identified. We have purified an 18 kD protein extracted from rabbit fur that was shown by N-terminal sequencing and mass spectrometry (MS) to be a lipocalin, identical to that identified as an odorant binding protein and an allergen with the formal nomenclature of Ory c 1. De novo sequencing of the MS peptide fragments gave additional primary sequence data of this protein. Polyclonal antisera were raised against the purified protein and used to develop two types of immunoassay. Ory c 1 content was measured in used rabbit bedding and household dust samples from homes keeping rabbits as pets. Atmospheric sampling was also undertaken in an animal facility undertaking rabbit experimental work. Ory c 1 levels in house dust where rabbits were kept as pets were between undetectable–41,290 ng·g−1, and in used bedding between 370–26,740 ng·g−1. Significantly higher house dust levels were found where rabbits spent large amounts, or all of, their time indoors. Personal air sampler levels within the animal facility were between 65–216 ng·m−3. Low levels (0.8–2 ng·m−3) were found in the facility's changing rooms, but undetected in the entrance lobby, office and laundry. We believe that these immunochemical assays may be used to identify activities in the occupational and domestic setting which produce higher levels of exposure to rabbit allergens, and where measures to control exposure may be warranted to reduce potential risk of allergic outcomes.

Identification of antigens from nosocomial Acinetobacter baumannii clinical isolates in sera from ICU staff and infected patients using the antigenome technique

Nosocomial infections with a bacterial origin are considered one of the most dangerous threats to global health. Among the causes of these infections, Acinetobacter baumannii is playing a significant role, and the present study aimed‏ to determine the immunogenic proteins of this bacteria. Clinical isolates of A. baumannii were obtained from positive sputum cultures of intensive care unit (ICU) patients confirmed by Polymerase chain reaction (PCR) of the OXA-51 gene, and sera was obtained from 20 colonized patients. In addition, 20 and 30 serum samples were collected from ICU nurses and healthy controls, respectively. All the samples were screened in the presence of antibodies against A. baumannii by enzyme-linked immunosorbent assay (ELISA). IgG purified from the serum samples by affinity chromatography was used to isolate the bacteria by the Magnetic-activated cell sorting (MACS) procedure. After the bacteria were cultured, the identified antigen proteins were studied by western blotting and Mass spectrometry (MS). The MS results were analyzed with MASCOT software and revealed a 35 KD protein, which corresponds to outer membrane protein A (OmpA) of A. baumannii, a 25 KD band, which is a carbapenem-associated resistance protein precursor, and a 60 KD protein band, identified as a stress-induced bacterial acidophilic repeat motif protein. According to the properties of immunogen antigens and bio informatics tools, the outer membrane proteins (OMPs) can be used as a vaccine candidate in animal models.

Proteomic Profiling of Liver and Plasma in Chronic Ethanol Feeding Model of Hepatic Alcohol Dehydrogenase-Deficient Deer Mice.

Chronic alcohol abuse, a major risk factor for such diseases as hepatitis and cirrhosis, impairs hepatic alcohol dehydrogenase (ADH; key ethanol [EtOH]-metabolizing enzyme). Therefore, differentially altered hepatic and plasma proteomes were identified in chronic EtOH feeding model of hepatic ADH-deficient (ADH- ) deer mice to understand the metabolic basis of alcoholic liver disease (ALD). ADH- deer mice were fed 3.5g% EtOH via Lieber-DeCarli liquid diet daily for 3months and histology of the liver assessed. Liver and plasma proteins were separated by 2-dimensional gel electrophoresis. The proteins differentially expressed were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Histology of the liver showed panlobular steatosis and infiltration of T lymphocytes. Using the criteria of ≥1.5 for fold change (p-value ≤0.05) with expectation value (E ≤10-3 ) and protein score (≥64), 18 proteins in the livers and 5 in the plasma of EtOH-fed mice were differentially expressed and identified. Prolyl 4-hydroxylase, cytochrome b-5, endo A cytokeratin, ATP synthase, heat-shock 70kD proteins, enoyl CoA hydratase, stress-70 protein, peroxiredoxin 1, and ornithine carbamoyl transferase were up-regulated in the livers. However, carbonic anhydrase 3, mitochondrial ATP synthase, aldolase 2, actin γ, laminin receptor, and carbamoyl phosphate synthase were down-regulated. Contrary to the increased expression of creatine kinase M-type, a decreased expression of serine protease inhibitor A3A precursor, sulfated glycoprotein-2 (clusterin), and apolipoprotein E isoforms were found in the plasma of EtOH group. Chronic EtOH feeding in ADH- deer mice causes steatosis and infiltration of T lymphocytes in the livers along with increased expression of proteins involved in endoplasmic reticulum (ER) stress, fibrosis, fatty acid β oxidation and biogenesis, and decreased expression of proteins involved in ATP synthesis, carbohydrate metabolism, in cell regulation and architecture. Reduced expression of various carrier proteins as found in the plasma of EtOH group has a biomarker potential.