Piperlongumine and some of its analogs inhibit selectively the human immunoproteasome over the constitutive proteasome.

The natural small molecule piperlongumine A is toxic selectively to cancer cells in vitro and in vivo. This toxicity has been correlated with cancer cell ROS, DNA damage and apoptotic cell death increases. We demonstrate here a new mechanistic property of piperlongumine: it inhibits selectively human immunoproteasome with no noticeable inhibition of human constitutive proteasome. This result suggests that immunoproteasome inhibition, a mechanism independent of ROS elevation, may also partly play a role in the anticancer effects observed with piperlongumine. Structure-activity relationships of piperlongumine analogs suggest that the lactam (piperidonic) ring of piperlongumine A may be replaced by the linear olefin -NHCO-CH2=CH2 to improve both in vitro inhibitory efficiency against immunoproteasome and cellular toxicity.

Identification and characterization of a Streptococcus equi ssp. zooepidemicus immunogenic GroEL protein involved in biofilm formation.

Streptococcus equi ssp. zooepidemicus (S. equi spp. zooepidemicus) is an opportunistic pathogen that causes major economic losses in the swine industry in China and is also a threat for human health. Biofilm formation by this bacterium has been previously reported. In this study, we used an immunoproteomic approach to search for immunogenic proteins expressed by biofilm-grown S. equi spp. zooepidemicus. Seventeen immunoreactive proteins were found, of which nine common immunoreactive proteins were identified in planktonic and biofilm-grown bacteria. The immunogenicity and protective efficacy of the S. equi spp. zooepidemicus immunoreactive GroEL chaperone protein was further investigated in mice. The protein was expressed in vivo and elicited high antibody titers following S. equi spp. zooepidemicus infections of mice. An animal challenge experiment with S. equi spp. zooepidemicus showed that 75% of mice immunized with the GroEL protein were protected. Using in vitro biofilm inhibition assays, evidence was obtained that the chaperonin GroEL may represent a promising target for the prevention and treatment of persistent S. equi spp. zooepidemicus biofilm infections. In summary, our results suggest that the recombinant GroEL protein, which is involved in biofilm formation, may efficiently stimulate an immune response, which protects against S. equi spp. zooepidemicus infections. It may therefore be a candidate of interest to be included in vaccines against S. equi spp. zooepidemicus infections.

Identification of early diagnostic antigens from Spirometra erinaceieuropaei sparganum soluble proteins using immunoproteomics.

In order to identify early specific diagnostic antigens of Spirometra erinaceieuropaei (syn. S. erinacei or S. mansoni) sparganum, soluble proteins were analyzed by two-dimensional electrophoresis (2-DE) and western blotting probed with immune sera from infected mice at 14 days post-infection. From a total of approximately 462 proteins spots mainly distributed in pI range of 5-6.6 and with molecular mass of 25-48 kDa, 6 immuno-reactive protein spots with molecular mass of 31.8-38.3 kDa were characterized by MALDI-TOF/TOF-MS. Three proteins were identified as S. erinaceieuropaei cysteine protease, Toxoplasma gondii hypothetical protein and Pecten spp actin, while the remaining were unidentified. The cysteine protease from S. erinaceieuropaei soluble proteins recognized by early infection sera might be developed as diagnostic reagent for early detection of sparganosis.

The Immunoproteasome: An Emerging Target in Cancer and Autoimmune and Neurological Disorders.

The immunoproteasome (iCP) is an isoform of the 20S proteasome that is expressed when cells are stressed or receive an inflammatory signal. The primary role of the iCP is to hydrolyze proteins into peptides that are compatible with being loaded into a MHC-I complex. When the activity of the iCP is dysregulated or highly expressed, it can lead to unwanted cell death. Some cancer types express the iCP rather than the standard proteasome, and selective inhibitors have been developed to exploit this difference. Here, we describe diseases known to be influenced by iCP activity and the current status for targeting the iCP to elicit a therapeutic response. We also describe a variety of chemical tools that have been developed to monitor the activity of the iCP in cells. Finally, we present the future outlook for targeting the iCP in a variety of disease types and with mechanisms besides inhibition.

Harnessing Immunoproteostasis to Treat Neurodegenerative Disorders.

Immunoproteostasis is a term used to reflect interactions between the immune system and the proteinopathies that are presumptive "triggers" of many neurodegenerative disorders. The study of immunoproteostasis is bolstered by several observations. Mutations or rare variants in genes expressed in microglial cells, known to regulate immune functions, or both can cause, or alter risk for, various neurodegenerative disorders. Additionally, genetic association studies identify numerous loci harboring genes that encode proteins of known immune function that alter risk of developing Alzheimer's disease (AD) and other neurodegenerative proteinopathies. Further, preclinical studies reveal beneficial effects and liabilities of manipulating immune pathways in various neurodegenerative disease models. Although there are concerns that manipulation of the immune system may cause more harm than good, there is considerable interest in developing immune modulatory therapies for neurodegenerative disorders. Herein, I highlight the promise and challenges of harnessing immunoproteostasis to treat neurodegenerative proteinopathies.

Immunoproteasome in Macaca fascicularis: no age-dependent modification of abundance and activity in the brain and insight into an in silico structural model.

In this study, we investigated proteasome composition and activity in the brain of Macaca fascicularis, in order to test whether this nonhuman primate species might be a suitable animal model for anti-aging therapies in the central nervous system, addressed to the ubiquitin-proteasome system. We detected the catalytic beta subunits of constitutive proteasome, as well as the PA28 regulator and a subunit of immunoproteasome (i.e., beta1i [LMP2]), in seven adult, six old, and one young nonhuman primate brains. Subunit expression and proteasome activity were not influenced by the age of the animal in any of the brain regions (temporal and frontal cortex and cerebellum) we studied. However, an area-specific susceptibility to aged-related oxidative stress emerged. On the whole, the results suggest that, compared to humans, Macaca fascicularis primates may have a different age-dependent regulation of the ubiquitin-proteasome system and, possibly, of neuroinflammation in the brain. An in silico model of the 20S immunoproteasome containing the Macaca fascicularis alpha and beta subunits, present in database or identified by our group (i.e., LMP2), has been developed. Additional information was obtained by de novo sequencing of the beta1 (delta) subunit of Macaca fascicularis. A comparison with humans suggests that in multiprotein complexes some functional subunits, such as alpha subunits, appear to be preferentially conserved during evolution.

Identification of antibody reactive proteins in pancreatic cancer using 2D immunoblotting and mass spectrometry.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by early invasiveness and resistance to treatment. Surgery in early stages is the only effective treatment, thus finding new biomarkers for the early detection of PDAC remains a major challenge. The present study aimed to compare the immunoproteome between PDAC patients and healthy controls using serological proteome analysis method. Firstly, cell lysates from two different pancreatic cancer cell lines were separated by two dimensional (2D) gels, and then transferred onto membranes probed with sera from 20 PDAC patients and 10 healthy controls. Proteins differentially reacting with autoantibodies in PDAC patients and control groups and were identified using mass spectrometry. This process led to the identification of 18 pancreatic immunoreactive antigens such as laminin, superoxide dismutase, ATP synthase, Rho GDP-dissociation inhibitor II, septin, glyceraldehyde 3-phosphate-dehydrogenase, phosphoglycerate mutase B, tubulin ß8 channel and prohibit in. In the present study, we identified 18 immunoreactive proteins in PDAC. While the identified proteins were critically involved in PDAC pathogenesis, further investigation in a large scale population will determine the applicability of these potential biomarkers for the early diagnosis or treatment of the disease.

Regulation of T-cell immunity by T-cell immunoglobulin and mucin domain proteins.

The ability of T helper (TH) precursor cells to differentiate into T effector populations confers the adaptive immune system with a means to protect the host from microbes and react to "foreign" antigenic tissues. T-cell immunoglobulin and mucin domain (TIM) proteins have recently been shown to be novel and critical regulators of T cell subset-driven dependent immune responsiveness. A dichotomy is emerging as to how Tim-3- and Tim-2- related signals respectively impact TH1 and TH2 responses. By comparison, the influence of the Tim-1 pathway seems to be broader and is probably not restricted to a specific type of T helper response. Beyond the mere control of the TH1/TH2 balance, Tim proteins are likely to target other regulatory components of the T cell response. Likewise, it is tempting to speculate that Tim proteins might also modulate the function of other T helper cell subsets such as TH3, TR1 and TH17 cells, among others.

[The construction and expression of eukaryotic expression plasmid of Legionella pneumophila immunogenic protein].

OBJECTIVE: To construct the recombinant plasmid of Legionella pneumophila immunogenic protein gene (ip) and detect the immunoprotein expression in NIH3T3 cells. METHODS: The immunogenic protein gene was amplified from DNA of Legionella pneumophila by polymerase chain reaction (PCR), then inserted into pcDNA3. 1 (+) vector. The recombinant plasmid was named as pcDNA3. 1-ip and analyzed with restriction-endonuclease Hind III and BamH I digestion, PCR and DNA sequencing techniques. The NIH3T3 cell was transfected by recombinant plasmid pcDNA3. 1-ip with lipofection strategy. The transient and stable expression products of immunogenic protein gene were detected by immunofluorescence and Western blot. RESULTS: We have successfully constructed the recombinant plasmid of Legionella pneumophila immunogenic protein gene. It was found that there was dark green fluorescence on the cell membrane and inside the cell. Our results showed that NIH3T3 cell was transfected by pcDNA3. 1-ip successfully. The rabbit serum antibody of Legionella pneumophila detected the NIH3T3 cell transfected stably by pcDNA3. 1-ip to express the immunogenic protein with the relative molecular mass 29 X 1093). CONCLUSION: We have successfully expressed immunogenic protein of Legionella pneumophila. The expressed product showed the good immunogenicity and immunoreactivity.

Mouse Models for Assessing Protein Immunogenicity: Lessons and Challenges.

The success of clinical and commercial therapeutic proteins is rapidly increasing, but their potential immunogenicity is an ongoing concern. Most of the studies that have been conducted over the past few years to examine the importance of various product-related attributes (in particular several types of aggregates and particles) and treatment regimen (such as dose, dosing schedule, and route of administration) in the development of unwanted immune responses have utilized one of a variety of mouse models. In this review, we discuss the utility and drawbacks of different mouse models that have been used for this purpose. Moreover, we summarize the lessons these models have taught us and some of the challenges they present. Finally, we provide recommendations for future research utilizing mouse models to improve our understanding of critical factors that may contribute to protein immunogenicity.

Immunoproteomic analysis of Schistosoma japonicum schistosomulum proteins recognized by immunoglobulin G in the sera of susceptible and non-susceptible hosts.

The aim of this study was to search for immunogenic schistosomula proteins in the hope of identifying novel intervention targets. Schistosomula proteins were analyzed by immunoproteomic which the probes were sera derived from BALB/c mice (susceptible hosts) and Microtus fortis (resistant hosts). A total of 116 immunoreactive proteins recognized by 10 days post-infected BALB/c mice, M. fortis sera, and uninfected M. fortis sera were selected for further analysis. Finally, 95 protein spots were identified by mass spectrometry (MS) analysis. Bioinformatics analysis showed that the differentially identified immunogenic proteins participated mainly in cytoskeleton organization, cell motility, energy metabolism, responses to stimuli, and protein folding. Many of these proteins were the tegument or excretory-secretory products of schistosomes reported in previous studies. Among of them, Schistosoma japonicum DnaJ (Hsp40) homologue (SjDnaJ) was successfully expressed and the purified recombinant product was evaluated by immunoprotective experiment. After immunization of BALB/c mice with recombinant SjDnaJ, it could induce 34.5% and 48.9% reductions in the numbers of worms and eggs in the liver. These results contribute to a better understanding of the molecular mechanisms underlying the host-parasite relationship and provide a major dataset to facilitate the further development of new vaccine candidates and/or diagnostic markers for schistosomiasis. BIOLOGICAL SIGNIFICANCE: Schistosomiasis is caused by parasitic blood-dwelling flukes in tropical and subtropical areas, and it is one of the world's most prevalent tropical diseases. The lack of effective vaccine and reliable diagnostic methods make this disease difficult to control. In China, S. japonicum can infect more than 40 different susceptible mammals for this parasite. However, M. fortis is the only known mammal where the schistosome cannot develop and it exhibits no significant pathological effects. Many studies' results showed that native antibodies against S. japonicum are present in M. fortis that may have important anti-schistosomiasis roles during the infection process. The aim of this study was to search for immunogenic schistosomula proteins in the hope of identifying novel intervention targets. We present a comparative immunoproteomics analysis of the proteins recognized by susceptible and resistant host antibodies before and 10-days after infections. The results of this analysis will be helpful for identifying the key molecules required for the survival and development of schistosomes. At the same time, the study contributes to a better understanding of the molecular mechanisms underlying the host-parasite relationship associated with schistosomes and they also provide a major dataset to facilitate the further development of new diagnostic assays and/or vaccine candidates for schistosomiasis.

Generation and characterization of dimeric small immunoproteins specific for neuroblastoma associated antigen GD2.

GD2 is a disialoganglioside expressed at high density on the surface of malignant cells of neuroectodermal origin, especially in neuroblastoma (NB) and melanoma. Since its expression in normal tissues is very restricted, GD2 represents an excellent target for neuroectodermal tumor targeting. Mini-antibody technology allows the production of dimeric single-chain antibodies, also called small immunoproteins (SIPs), which are composed of a scFv fused to a dimerizing domain of immunoglobulin heavy chains. Dimerization results in an increase of the total apparent affinity and a slower clearance in vivo than scFvs. These properties make SIPs very attractive molecules for tumor targeting. We isolated the variable regions from an anti-GD2 monoclonal antibody and exploited the SIP technology to generate two novel anti-GD2 SIPs. The first anti-GD2 SIP is a fully murine molecule containing the CH3 domain of mouse IgG1, whereas the second construct is a hybrid mouse-human molecule containing the CH4 domain of human IgE. Both mini-antibodies were successfully produced and shown to retain binding specificity as well as an affinity similar to that of the original antibody.

Immunoproteomic approach for identification of Ascaris suum proteins recognized by pigs with porcine ascariasis.

Ascaris suum, the causative agent of porcine ascariasis, is responsible for marked economic losses in pig farms worldwide. Despite recent advances in research, including the characterization of the genome of A. suum, knowledge about the parasite/host relationship in porcine ascariasis at the molecular level is scarce and chemotherapy is the only effective option for parasite control. The aim of this study was to identify immunogenic proteins of A. suum somatic antigens associated with the pathogenicity/survival mechanisms of the parasite, by using two-dimensional (2-D) electrophoresis, 2-D Western blot and mass spectrometry (MS). A total of 24 parasite proteins recognized by serum samples from pigs naturally infected with A. suum were identified. Most of them (23/24) were identified as being involved in parasite survival mechanisms, including functions related to energy generation (12 proteins) and redox processes (5 proteins). These results may aid the search for effective chemo-therapeutic targets in porcine ascariasis. Further studies are needed, however, to illustrate the effect of the host immune response on the survival mechanisms of A. suum.

Chaperonin GroEL: a novel phylogenetically conserved protein with strong immunoreactivity of Avian Pathogenic Escherichia coli isolates from duck identified by immunoproteomics.

Avian Pathogenic Escherichia coli (APEC) is one of the most important bacterial pathogens of poultry. The lack of suitable vaccines and the emergence of multi-resistant strains have hampered the control of avian colibacillosis. To identify immunogenic proteins of APEC as vaccine candidates, immunoproteomics and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) were applied. Proteins from total cell lysates of APEC DE205B isolated from the brain of a duck with septicemia and neurological symptom in China were separated by two-dimensional electrophoresis (2-DE) and reacted with hyperimmune duck serum against DE205B. Fourteen immunoreactive spots were found, representing 11 distinct proteins. These included two predominant immunogenic components, outer membrane protein A (OmpA) and flagellin (FliC). GroEL, which is a member of the molecular chaperone family and identical structurally to eukaryotic heat shock protein 60 (Hsp60), and the other eight antigens are reported here as immunoreactive proteins of APEC for the first time. Subsequently, nine genes encoding the identified proteins were successfully cloned and expressed in E. coli BL21 (DE3). Seven of the recombinant proteins were able to react with hyperimmune duck serum and three of them, GroEL, OmpA and FliC, showed stronger immunoreactivity. Challenge studies revealed that, just like OmpA and FliC, recombinant GroEL stimulated a strong antibody response and supported protective efficacy against APEC infection in ducks. With high phylogenetic conservation, it is considered that GroEL would be an ideal immunogen of APEC for vaccine development.

Immunoproteasome-deficiency has no effects on NK cell education, but confers lymphocytes into targets for NK cells in infected wild-type mice.

Natural killer (NK) cells are part of the innate immune system and contribute to the eradication of virus infected cells and tumors. NK cells express inhibitory and activating receptors and their decision to kill a target cell is based on the balance of signals received through these receptors. MHC class I molecules are recognized by inhibitory receptors, and their presence during NK cell education influences the responsiveness of peripheral NK cells. We here demonstrate that mice with reduced MHC class I cell surface expression, due to deficiency of immunoproteasomes, have responsive NK cells in the periphery, indicating that the lower MHC class I levels do not alter NK cell education. Following adoptive transfer into wild-type (wt) recipients, immunoproteasome-deficient splenocytes are tolerated in naive but rejected in virus-infected recipients, in an NK cell dependent fashion. These results indicate that the relatively low MHC class I levels are sufficient to protect these cells from rejection by wt NK cells, but that this tolerance is broken in infection, inducing an NK cell-dependent rejection of immunoproteasome-deficient cells.

MAGE Xp-2: a member of the MAGE gene family isolated from an expression library using systemic lupus erythematosus sera.

Two regions of the genome contain members of the MAGE gene family; Xq27-qter and Xp21.3. We isolated a transcript, MAGE Xp-2, by screening a cDNA library from the human epithelial carcinoma cell line, HEp-2, using autoantibodies from patients with systemic lupus erythematosus (SLE). The open reading frame (ORF) of MAGE Xp-2 is entirely contained in exon 4, a signature feature of the MAGE gene family. While MAGE Xp-2 shares genomic homology with MAGE Xp-1, the predicted proteins are quite divergent. Specific primers were designed to reliably distinguish between MAGE Xp-1 and MAGE Xp-2 expression. MAGE Xp-2 is expressed in testis, but not in other normal tissues. It is also expressed strongly in two of seven melanoma cell lines and one of four breast carcinomas. MAGE gene expression may be important not only for tumor recognition and cancer therapy, but, because it is the apparent target of autoantibodies in SLE sera, it may also play a role in autoimmune diseases.