Proteomic analysis of acidic chaperones, and stress proteins in extreme halophile Halobacterium NRC-1: a comparative proteomic approach to study heat shock response

BACKGROUND: Halobacterium sp. NRC-1 is an extremely halophilic archaeon and has adapted to optimal growth under conditions of extremely high salinity. Its proteome is highly acidic with a median pI of 4.9, a unique characteristic which helps the organism to adapt high saline environment. In the natural growth environment, Halobacterium NRC-1 encounters a number of stressful conditions including high temperature and intense solar radiation, oxidative and cold stress. Heat shock proteins and chaperones play indispensable roles in an organism's survival under many stress conditions. The aim of this study was to develop an improved method of 2-D gel electrophoresis with enhanced resolution of the acidic proteome, and to identify proteins with diverse cellular functions using in-gel digestion and LC-MS/MS and MALDI-TOF approach. RESULTS: A modified 2-D gel electrophoretic procedure, employing IPG strips in the range of pH 3–6, enabled improved separation of acidic proteins relative to previous techniques. Combining experimental data from 2-D gel electrophoresis with available genomic information, allowed the identification of at least 30 cellular proteins involved in many cellular functions: stress response and protein folding (CctB, PpiA, DpsA, and MsrA), DNA replication and repair (DNA polymerase A α subunit, Orc4/CDC6, and UvrC), transcriptional regulation (Trh5 and ElfA), translation (ribosomal proteins Rps27ae and Rphs6 of the 30 S ribosomal subunit; Rpl31eand Rpl18e of the 50 S ribosomal subunit), transport (YufN), chemotaxis (CheC2), and housekeeping (ThiC, ThiD, FumC, ImD2, GapB, TpiA, and PurE). In addition, four gene products with undetermined function were also identified: Vng1807H, Vng0683C, Vng1300H, and Vng6254. To study the heat shock response of Halobacterium NRC-1, growth conditions for heat shock were determined and the proteomic profiles under normal (42°C), and heat shock (49°C) conditions, were compared. Using a differential proteomic approach in combination with available genomic information, bioinformatic analysis revealed five putative heat shock proteins that were upregulated in cells subjected to heat stress at 49°C, namely DnaJ, GrpE, sHsp-1, Hsp-5 and sHsp-2. CONCLUSION: The modified 2-D gel electrophoresis markedly enhanced the resolution of the extremely acidic proteome of Halobacterium NRC-1. Constitutive expression of stress proteins and chaperones help the organism to adapt and survive under extreme salinity and other stress conditions. The upregulated expression pattern of putative chaperones DnaJ, GrpE, sHsp-1, Hsp-5 and sHsp-2 under elevated temperature clearly suggests that Halobacterium NRC-1 has a sophisticated defense mechanism to survive in extreme environments

Role of Hsp90 in Systemic Lupus Erythematosus and Its Clinical Relevance

Heat shock proteins (HSP) are a family of ubiquitous and phylogenically highly conserved proteins which play an essential role as molecular chaperones in protein folding and transport. Heat Shock Protein 90 (Hsp90) is not mandatory for the biogenesis of most proteins, rather it participate in structural maturation and conformational regulation of a number of signaling molecules and transcription factors. Hsp90 has been shown to play an important role in antigen presentation, activation of lymphocytes, macrophages, maturation of dendritic cells, and in the enhanceosome mediated induction of inflammation. Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disease with complex immunological and clinical manifestations. Dysregulated expression of Type I interferon α, activation of B cells and production of autoantibodies are hallmarks of SLE. The enhanced levels of Hsp90 were detected in the serum of SLE patients. The elevated level of Hsp90 in SLE has also been correlated with increased levels of IL-6 and presence of autoantibodies to Hsp90. This suggests that Hsp90 may contribute to the inflammation and disease progression and that targeting of Hsp 90 expression may be a potential treatment of SLE. The pharmacologic inhibition of Hsp90 was successfully applied in mouse models of autoimmune encephalomyelitis and SLE—like autoimmune diseases. Thus targeting Hsp90 may be an effective treatment for SLE, especially if combined with other targeted therapeutic approaches

Comprehensive Analysis of Cancer-Proteogenome to Identify Biomarkers for the Early Diagnosis and Prognosis of Cancer

During the past century, our understanding of cancer diagnosis and treatment has been based on a monogenic approach, and as a consequence our knowledge of the clinical genetic underpinnings of cancer is incomplete. Since the completion of the human genome in 2003, it has steered us into therapeutic target discovery, enabling us to mine the genome using cutting edge proteogenomics tools. A number of novel and promising cancer targets have emerged from the genome project for diagnostics, therapeutics, and prognostic markers, which are being used to monitor response to cancer treatment. The heterogeneous nature of cancer has hindered progress in understanding the underlying mechanisms that lead to abnormal cellular growth. Since, the start of The Cancer Genome Atlas (TCGA), and the International Genome consortium projects, there has been tremendous progress in genome sequencing and immense numbers of cancer genomes have been completed, and this approach has transformed our understanding of the diagnosis and treatment of different types of cancers. By employing Genomics and proteomics technologies, an immense amount of genomic data is being generated on clinical tumors, which has transformed the cancer landscape and has the potential to transform cancer diagnosis and prognosis. A complete molecular view of the cancer landscape is necessary for understanding the underlying mechanisms of cancer initiation to improve diagnosis and prognosis, which ultimately will lead to personalized treatment. Interestingly, cancer proteome analysis has also allowed us to identify biomarkers to monitor drug and radiation resistance in patients undergoing cancer treatment. Further, TCGA-funded studies have allowed for the genomic and transcriptomic characterization of targeted cancers, this analysis aiding the development of targeted therapies for highly lethal malignancy. High-throughput technologies, such as complete proteome, epigenome, protein–protein interaction, and pharmacogenomics data, are indispensable to glean into the cancer genome and proteome and these approaches have generated multidimensional universal studies of genes and proteins (OMICS) data which has the potential to facilitate precision medicine. However, due to slow progress in computational technologies, the translation of big omics data into their clinical aspects have been slow. In this review, attempts have been made to describe the role of high-throughput genomic and proteomic technologies in identifying a panel of biomarkers which could be used for the early diagnosis and prognosis of cancer

Protein Domain Analysis of C. botulinum Type A Neurotoxin and Its Relationship with Other Botulinum Serotypes

Botulinum neurotoxins (BoNTs) are highly potent poisons produced by seven serotypes of Clostridium botulinum. The mechanism of neurotoxin action is a multistep process which leads to the cleavage of one of three different SNARE proteins essential for synaptic vesicle fusion and transmission of the nerve signals to muscles: synaptobrevin, syntaxin, or SNAP-25. In order to understand the precise mechanism of neurotoxin in a host, the domain structure of the neurotoxin was analyzed among different serotypes of C. botulinum. The results indicate that neurotoxins type A, C, D, E and F contain a coiled-coil domain while types B and type G neurotoxin do not. Interestingly, phylogenetic analysis based on neurotoxin sequences has further confirmed that serotypes B and G are closely related. These results suggest that neurotoxin has multi-domain structure, and coiled-coil domain plays an important role in oligomerisation of the neurotoxin. Domain analysis may help to identify effective antibodies to treat Botulinum toxin intoxication

Alterations in the red blood cell membrane proteome in alzheimer's subjects reflect disease-related changes and provide insight into altered cell morphology

Abstract Background Our earlier studies have shown that red blood cell (RBC) morphology in Alzheimer's disease (AD) subjects was altered (> 15% of the RBCs were elongated as compared to 5.9% in normal controls (p Results The data suggest, (1) RBC shape/morphology changes in AD subjects are possibly attributed primarily to the changes (elevation or decrease) in the level of a series of membrane/cytoskeleton proteins involved in regulating the stability and elasticity of the RBC membrane, and (2) changes (elevation or decrease) in the level of a second series of proteins in the RBC membrane proteome reflect similar changes reported earlier by various investigators in AD or animal model of AD. Of particular interest, elevation of oxidative stress response proteins such as heat shock 90 kDa protein 1 alpha in AD subjects has been confirmed by western blot analysis in the RBC membrane proteome. Conclusions The results suggest that this study provides a potential link between the alterations in RBC membrane proteome in AD subjects and AD pathology.</p

Immunotherapy, Radiotherapy, and Hyperthermia: A Combined Therapeutic Approach in Pancreatic Cancer Treatment

Pancreatic cancer (PC) has the highest mortality rate amongst all other cancers in both men and women, with a one-year relative survival rate of 20%, and a five-year relative survival rate of 8% for all stages of PC combined. The Whipple procedure, or pancreaticoduodenectomy, can increase survival for patients with resectable PC, however, less than 20% of patients are candidates for surgery at time of presentation. Most of the patients are diagnosed with advanced PC, often with regional and distant metastasis. In these advanced cases, chemotherapy and radiation have shown limited tumor control, and PC continues to be refractory to treatment and results in a poor survival outcome. In recent years, there has been intensive research on checkpoint inhibitor immunotherapy for PC, however, PC is characterized with dense stromal tissue and a tumor microenvironment (TME) that is highly immunosuppressive, which makes immunotherapy less effective. Interestingly, when immunotherapy is combined with radiation therapy (RT) and loco-regional hyperthermia (HT), it has demonstrated enhanced tumor responses. HT improves tumor killing via a variety of mechanisms, targeting both the tumor and the TME. Targeted HT raises the temperature of the tumor and surrounding tissues to 42&#8315;43 &#176;C and makes the tumor more immunoresponsive. HT can also modulate the immune system of the TME by inducing and synthesizing heat shock proteins (HSP), which also activate an anti-tumor response. It is well known that HT can enhance RT-induced DNA damage in cancer cells and simultaneously help to oxygenate hypoxic regions. Thus, it is envisaged that combined HT and RT might have immunomodulatory effects in the PC-TME, making PC more responsive to immunotherapies. Moreover, the combined tripartite approach of immunotherapy, RT, and HT could reduce the overall toxicity associated with each individual therapy, while concomitantly enhancing the immunotherapeutic effect of overall individual therapies to treat local and metastatic PC. Thus, the use of a tripartite combinatorial approach could be promising and more efficacious than monotherapy or dual therapy to treat and increase the survival of the PC patients

SOX2 and OCT4 mediate radiation and drug resistance in pancreatic tumor organoids

Abstract Pancreatic cancer has a five-year survival rate of only 10%, mostly due to late diagnosis and limited treatment options. In patients with unresectable disease, either FOLFIRINOX, a combination of 5-fluorouracil (5-FU), oxaliplatin and irinotecan, or gemcitabine plus nab-paclitaxel combined with radiation are frontline standard regimens. However, chemo-radiation therapy has shown limited success because patients develop resistance to chemotherapy and/or radiation. In this study, we evaluated the role of pancreatic cancer stem cells (CSC) using OCT4 and SOX2, CSC markers in mouse pancreatic tumor organoids. We treated pancreatic tumor organoids with 4 or 8 Gy of radiation, 10 μM of 5-FU (5-Fluorouracil), and 100 μM 3-Bromopyruvate (3BP), a promising anti-cancer drug, as a single treatment modalities, and in combination with RT. Our results showed significant upregulation of, OCT4, and SOX2 expression in pancreatic tumor organoids treated with 4 and 8 Gy of radiation, and downregulation following 5-FU treatment. The expression of CSC markers with increasing treatment dose exhibited elevated upregulation levels to radiation and downregulation to 5-FU chemotherapy drug. Conversely, when tumor organoids were treated with a combination of 5-FU and radiation, there was a significant inhibition in SOX2 and OCT4 expression, indicating CSC self-renewal inhibition. Noticeably, we also observed that human pancreatic tumor tissues exhibited heterogeneous and aberrant OCT4 and SOX2 expression as compared to normal pancreas, indicating their potential role in pancreatic cancer growth and therapy resistance. In addition, the combination of 5-FU and radiation treatment exhibited significant inhibition of the β-catenin pathway in pancreatic tumor organoids, resulting in sensitization to treatment and organoid death. In conclusion, our study emphasizes the crucial role of CSCs in therapeutic resistance in PC treatment. We recommend using tumor organoids as a model system to explore the impact of CSCs in PC and identify new therapeutic targets

Docetaxel radiosensitizes castration resistant prostate cancer by downregulating CAV-1

Purpose: Docetaxel (dox), a noted radiosensitizer, is one of the few chemotherapy drugs approved for castration-resistant prostate cancer (CRPC), though only a fraction of CRPCs respond to it. CAV-1, a critical regulator of radioresistance, has been known to modulate dox and radiation effects. Combining dox with radiotherapy may create a synergistic anticancer effect through CAV-1 and improve CRPC patients' response to therapy. Here, we investigate the effectiveness and molecular characteristics of dox and radiation combination therapy in vitro. Materials and Methods: We used live/dead assays to determine the IC50 of dox for PC3, DU-145, and TRAMP-C1 cells. Colony formation assay was used to determine the radioresponse of the same cells treated with radiation with/without IC50 dox (4, 8, 12 Gy). We performed gene expression analysis on public transcriptomic data collected from human-derived prostate cancer cell lines (C4-2, PC3, DU-145, LNCaP) treated with dox for 8, 16, and 72 hours. Cell cycle arrest and protein expression were assessed using flow cytometry and western blot, respectively. Results: Compared to radiation alone, combination therapy with dox significantly increased CRPC death in PC3 (1.48-fold, p  Conclusions: Our results suggest that dox sensitizes CRPC cells to radiation by downregulating CAV-1. Dox + radiation combination therapy may be effective at treating CRPC, especially subtypes associated with high CAV-1 expression, and should be studied further. </p