Aberrant reactive oxygen and nitrogen species generation in rheumatoid arthritis (RA):causes and consequences for immune function, cell survival, and therapeutic intervention

The infiltration and persistence of hematopoietic immune cells within the rheumatoid arthritis (RA) joint results in elevated levels of pro-inflammatory cytokines, increased reactive oxygen (ROS) and -nitrogen (RNS) species generation, that feeds a continuous self-perpetuating cycle of inflammation and destruction. Meanwhile, the controlled production of ROS is required for signaling within the normal physiological reaction to perceived "foreign matter" and for effective apoptosis. This review focuses on the signaling pathways responsible for the induction of the normal immune response and the contribution of ROS to this process. Evidence for defects in the ability of immune cells in RA to regulate the generation of ROS and the consequence for their immune function and for RA progression is considered. As the hypercellularity of the rheumatoid joint and the associated persistence of hematopoietic cells within the rheumatoid joint are symptomatic of unresponsiveness to apoptotic stimuli, the role of apoptotic signaling proteins (specifically Bcl-2 family members and the tumor suppressor p53) as regulators of ROS generation and apoptosis are considered, evaluating evidence for their aberrant expression and function in RA. We postulate that ROS generation is required for effective therapeutic intervention.

Efficient purification of chromatin architectural proteins:histones, HMGB proteins and FKBP3 (FKBP25) immunophilin

A two-step process of high ionic strength lysis of chicken erythrocyte cell nuclei followed by cation-exchange chromatography has separated at very high yield all the histone and HMGB (high-mobility group B) nuclear proteins, except the less-soluble histone tetramers. Surprisingly high yields of the nuclear immunophilin FKBP3 (FKBP25) and Hsp70 (heat-shock protein 70) co-fractionate with HMGB1 and HMGB3. Furthermore, these proteins can be separated by anion-exchange chromatography. The purified nuclear proteins retain their native, post-translational modification (PTM) marks, including those associated with chromatin-fibre remodelling. These marks are intimately associated with the control of the cell cycle. The methods herein are therefore of value for targeting these and other nuclear proteins for future proteomic studies in healthy and diseased cells. This journal is © 2012 The Royal Society of Chemistry.

Identification of DNA-binding proteins by incorporating evolutionary information into pseudo amino acid composition via the top-n-gram approach

DNA-binding proteins are crucial for various cellular processes and hence have become an important target for both basic research and drug development. With the avalanche of protein sequences generated in the postgenomic age, it is highly desired to establish an automated method for rapidly and accurately identifying DNA-binding proteins based on their sequence information alone. Owing to the fact that all biological species have developed beginning from a very limited number of ancestral species, it is important to take into account the evolutionary information in developing such a high-throughput tool. In view of this, a new predictor was proposed by incorporating the evolutionary information into the general form of pseudo amino acid composition via the top-n-gram approach. It was observed by comparing the new predictor with the existing methods via both jackknife test and independent data-set test that the new predictor outperformed its counterparts. It is anticipated that the new predictor may become a useful vehicle for identifying DNA-binding proteins. It has not escaped our notice that the novel approach to extract evolutionary information into the formulation of statistical samples can be used to identify many other protein attributes as well.

EnDNA-Prot:identification of DNA-binding proteins by applying ensemble learning

DNA-binding proteins are crucial for various cellular processes, such as recognition of specific nucleotide, regulation of transcription, and regulation of gene expression. Developing an effective model for identifying DNA-binding proteins is an urgent research problem. Up to now, many methods have been proposed, but most of them focus on only one classifier and cannot make full use of the large number of negative samples to improve predicting performance. This study proposed a predictor called enDNA-Prot for DNA-binding protein identification by employing the ensemble learning technique. Experiential results showed that enDNA-Prot was comparable with DNA-Prot and outperformed DNAbinder and iDNA-Prot with performance improvement in the range of 3.97-9.52% in ACC and 0.08-0.19 in MCC. Furthermore, when the benchmark dataset was expanded with negative samples, the performance of enDNA-Prot outperformed the three existing methods by 2.83-16.63% in terms of ACC and 0.02-0.16 in terms of MCC. It indicated that enDNA-Prot is an effective method for DNA-binding protein identification and expanding training dataset with negative samples can improve its performance. For the convenience of the vast majority of experimental scientists, we developed a user-friendly web-server for enDNA-Prot which is freely accessible to the public. © 2014 Ruifeng Xu et al.

The synthesis of recombinant membrane proteins in yeast for structural studies

Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies.

The use of sodium carboxymethylcellulose in the preparation of spray-dried proteins for pulmonary drug delivery

The use of sodium carboxymethylcellulose (NaCMC) as a spray-drying excipient in the preparation of inhalable formulations of proteins was investigated, using alkaline phosphatase as a model functional protein. Two spray-dried powders were investigated: a control powder comprising 100% (w/w) alkaline phosphatase and a test powder comprising 67% (w/w) NaCMC and 33% (w/w) alkaline phosphatase. Following physicochemical characterisation, the powders were prepared as both dry powder inhaler (DPI) and pressurised metered dose inhaler (pMDI) formulations. The aerosolisation performance of the formulations was assessed using a Multi-Stage Liquid Impinger, both immediately after preparation and over a 16-week storage period. Formulating the control powder as a DPI resulted in a poor fine particle fraction (FPF: 10%), whereas the FPF of the NaCMC-modified DPI formulation was significantly greater (47%). When the powders were formulated as pMDI systems, the control and NaCMC-modified powders demonstrated FPFs of 52% and 55%, respectively. Following storage, reduced FPF was observed for all formulations except the NaCMC-modified pMDI system; the performance of this formulation following storage was statistically equivalent to that immediately following preparation. Co-spray-drying proteins and peptides with NaCMC may therefore offer an alternative method for the preparation of stable and respirable pMDI formulations for pulmonary delivery. © 2010 Elsevier B.V.

Synthesis of aromatic monothiols and aromatic dithiols to increase the folding rate and yield of disulfide containing proteins

Most pharmaceutically relevant proteins and many extracellular proteins contain disulfide bonds. Formation of the correct disulfide bonds is essential for stability in almost all cases. Disulfide containing proteins can be rapidly and inexpensively overexpressed in bacteria. However, the overexpressed proteins usually form aggregates inside the bacteria, called inclusion bodies, which contains inactive and non-native protein. To obtain native protein, inclusion bodies need to be isolated and resolubilized, and then the resulting protein refolded in vitro. In vitro protein folding is aided by the addition of a redox buffer, which is composed of a small molecule disulfide and/or a small molecule thiol. The most commonly used redox buffer contains reduced and oxidized glutathione. Recently, aliphatic dithiols and aromatic monothiols have been employed as redox buffers. Aliphatic dithiols improved the yield of native protein as compared to the aliphatic thiol, glutathione. Dithiols mimic the in vivo protein folding catalyst, protein disulfide isomerase, which has two thiols per active site. Furthermore, aromatic monothiols increased the folding rate and yield of lysozyme and RNase A relative to glutathione. By combining the beneficial properties of aliphatic dithiols and aromatic monothiols, aromatic dithiols were designed and were expected to increase in vitro protein folding rates and yields. Aromatic monothiols (1-4) and their corresponding disulfides (5-8), two series of ortho- and para-substituted ethylene glycol dithiols (9-15), and a series of aromatic quaternary ammonium salt dithiols (16-17) were synthesized on a multigram scale. Monothiols and disulfides (1-8) were utilized to fold lysozyme and bovine pancreatic trypsin inhibitor. Dithiols (11-17) were tested for their ability to fold lysozyme. At pH 7.0 and pH 8.0, and high protein concentration (1 mg/mL), aromatic dithiols (16, 17) and a monothiol (3) significantly enhanced the in vitro folding rate and yield of lysozyme relative to the aliphatic thiol, glutathione. Additionally, aromatic dithiols (16, 17) significantly enhance the folding yield as compared to the corresponding aromatic monothiol (3). Thus, the folding rate and yield enhancements achieved in in vitro protein folding at high protein concentration will decrease the volume of renaturation solution required for large scale processes and consequently reduce processing time and cost.

The role of redox signaling in the molecular mechanism of tamoxifen resistance in breast cancer.

The emergence of tamoxifen or aromatase inhibitor resistance is a major problem in the treatment of breast cancer. The molecular signaling mechanism of antiestrogen resistance is not clear. Understanding the mechanisms by which resistance to these agents arise could have major clinical implications for preventing or circumventing it. Therefore, in this dissertation we have investigated the molecular mechanisms underlying antiestrogen resistance by studying the contributions of reactive oxygen species (ROS)-induced redox signaling pathways in antiestrogen resistant breast cancer cells. Our hypothesis is that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with a progressive shift towards a pro-oxidant environment of cells as a result of oxidative stress. The hypothesis of this dissertation was tested in an in vitro 2-D cell culture model employing state of the art biochemical and molecular techniques, including gene overexpression, immunoprecipitation, Western blotting, confocal imaging, ChIP, Real-Time RT-PCR, and anchorage-independent cell growth assays. We observed that tamoxifen (TAM) acts like both an oxidant and an antioxidant. Exposure of tamoxifen resistant LCC2 cell to TAM or 17 beta-estradiol (E2) induced the formation of reactive oxidant species (ROS). The formation of E2-induced ROS was inhibited by co-treatment with TAM, similar to cells pretreated with antioxidants. In LCC2 cells, treatments with either E2 or TAM were capable of inducing cell proliferation which was then inhibited by biological and chemical antioxidants. Exposure of LCC2 cells to tamoxifen resulted in a decrease in p27 expression. The LCC2 cells exposed to TAM showed an increase in p27 phosphorylation on T157 and T187. Conversely, antioxidant treatment showed an increase in p27 expression and a decrease in p27 phosphorylation on T157 and T187 in TAM exposed cells which were similar to the effects of Fulvestrant. In line with previous studies, we showed an increase in the binding of cyclin E-Cdk2 and in the level of p27 in TAM exposed cells that overexpressed biological antioxidants. Together these findings highly suggest that lowering the oxidant state of antiestrogen resistant LCC2 cells, increases LCC2 susceptibility to tamoxifen via the cyclin dependent kinase inhibitor p27.

HIV infection and drugs of abuse: role of acute phase proteins

Background HIV infection and drugs of abuse such as methamphetamine (METH), cocaine, and alcohol use have been identified as risk factors for triggering inflammation. Acute phase proteins such as C-reactive protein (CRP) and serum amyloid A (SAA) are the biomarkers of inflammation. Hence, the interactive effect of drugs of abuse with acute phase proteins in HIV-positive subjects was investigated. Methods Plasma samples were utilized from 75 subjects with METH use, cocaine use, alcohol use, and HIV-positive alone and HIV-positive METH, cocaine, and alcohol users, and age-matched control subjects. The plasma CRP and SAA levels were measured by ELISA and western blot respectively and the CD4 counts were also measured. Results Observed results indicated that the CRP and SAA levels in HIV-positive subjects who are METH, cocaine and alcohol users were significantly higher when compared with either drugs of abuse or HIV-positive alone. The CD4 counts were also dramatically reduced in HIV-positive with drugs of abuse subjects compared with only HIV-positive subjects. Conclusions These results suggest that, in HIV-positive subjects, drugs of abuse increase the levels of CRP and SAA, which may impact on the HIV infection and disease progression.