An increase in phosphorylation and truncation of crystallin with the progression of cataracts.

BACKGROUND: Cataracts are the leading cause of blindness worldwide; however, there is no evidence regarding the direct formation of cataracts. At present, there is no treatment method other than surgery to prevent the formation or progression of cataracts. OBJECTIVE: Understanding the protein changes during various stages of cataracts might help realize the mechanism of the formation and progression of cataracts. METHODS: Lens materials were collected from cataract surgery. Cataracts were classified according to lens opacity using the gradation of the Lens Opacities Classification System. Lens proteins were separated by 2-dimensional polyacrylamide gel electrophoresis. Protein spots were visualized by Coomassie blue staining, and expression patterns were analyzed. Protein spots of interest were excised from 2-dimensional polyacrylamide gel electrophoresis gels, digested in situ with trypsin, and analyzed by mass spectrometry and liquid chromatographic tandem mass spectrometry. RESULTS: Crystallin was the major protein in the cataract lens, and αA, ßB1, αB, and ßA4 were the dominant types. Crystallin αB and ßA4 increased with the formation of lens opacity. Moreover, phosphorylation and truncation of these proteins increased with the progression of cataracts. CONCLUSION: Crystallin αB and ßA4 and phosphorylation and truncation of crystallin in the lens might contribute to the formation of cataracts. In contrast, acetylation was not dominant in the progression of cataracts and did not play major role in the formation of cataracts.

Aloe-emodin suppressed NMDA-induced apoptosis of retinal ganglion cells through regulation of ERK phosphorylation.

A high concentration of glutamate in the vitreous body and optic nerves of the eyes activates N-methyl-D-aspartate (NMDA) receptors and is toxic to retina ganglion cells (RGCs) in glaucomatous patients. Aloe-emodin sulfates/glucuronides (s/g), the major metabolites of aloe-emodin, was found to be effective in decreasing NMDA-induced apoptosis in RGCs. In order to elucidate the mechanisms, an in vitro optic neuropathy model adding NMDA to N18 RGCs was used in this study. The phosphorylation level of extra-cellular signal-regulated kinase1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 kinase (cytokines-suppressive antiinflammatory drug binding protein kinase) were measured by western blotting and luciferase reporter assay. The results showed that aloe-emodin metabolites significantly decreased the activation of three major mitogen-activated protein (MAP) kinase pathways and the activation of downstream genes in nucleus induced by NMDA, which were verified by the addition of the respective inhibitors. Comparing the effect of the inhibitors of the three MAP kinase pathways, the ERK pathway was found to be the major route of aloe-emodin metabolites in decreasing the apoptosis of NMDA-treated RGCs. Besides, cfos rather then cjun was the target downstream gene. Aloe-emodin emodin metabolites could regulate the phosphorylation of ERK kinases and it was a promising candidate for NMDA-induced apoptosis of RGCs.

Proteomic analysis of up-regulated proteins in human promonocyte cells expressing severe acute respiratory syndrome coronavirus 3C-like protease.

The pathogenesis of severe acute respiratory syndrome coronavirus (SARS CoV) is an important issue for treatment and prevention of SARS. Previously, SARS CoV 3C-like protease (3CLpro) has been demonstrated to induce apoptosis via the activation of caspase-3 and caspase-9 (Lin, C. W., Lin, K. H., Hsieh, T. H., Shiu, S. Y. et al., FEMS Immunol. Med. Microbiol. 2006, 46, 375-380). In this study, proteome analysis of the human promonocyte HL-CZ cells expressing SARS CoV 3CLpro was performed using 2-DE and nanoscale capillary LC/ESI quadrupole-TOF MS. Functional classification of identified up-regulated proteins indicated that protein metabolism and modification, particularly in the ubiquitin proteasome pathway, was the main biological process occurring in SARS CoV 3CLpro-expressing cells. Thirty-six percent of identified up-regulated proteins were located in the mitochondria, including apoptosis-inducing factor, ATP synthase beta chain and cytochrome c oxidase. Interestingly, heat shock cognate 71-kDa protein (HSP70), which antagonizes apoptosis-inducing factor was shown to down-regulate and had a 5.29-fold decrease. In addition, confocal image analysis has shown release of mitochondrial apoptogenic apoptosis-inducing factor and cytochrome c into the cytosol. Our results revealed that SARS CoV 3CLpro could be considered to induce mitochondrial-mediated apoptosis. The study provides system-level insights into the interaction of SARS CoV 3CLpro with host cells, which will be helpful in elucidating the molecular basis of SARS CoV pathogenesis.

Aloe-emodin metabolites protected N-methyl-d-aspartate-treated retinal ganglion cells by Cu-Zn superoxide dismutase.

A high concentration of glutamate in the eyes not only activates N-methyl-D-aspartate (NMDA) receptors, but also is toxic to the retina ganglion cells (RGCs) in glaucomatous patients. Our previous study had found that aloe-emodin sulfates/glucuronides metabolites, an anthraquinone polyphenol, exerted a neuroprotective activity upon RGCs. In order to understand the mechanisms involved in this neuroprotective effect, this study aimed to determine the expressions of RNAs and proteins in various treatments. The proteins expressed in the control group, NMDA-treated group, and aloe-emodin metabolites-cotreated group were separated by two-dimensional gel electrophoresis (2-DE). Protein spots were excised from 2-DE and analyzed by nano-LC-MS/MS (nano-liquid chromatography with mass spectrometry; tandem MS). Quantitative polymerase chain reaction (Q-PCR) was used to investigate the RNA related to these proteins. There were 84 spots with significant differences in various treatments. Among the 84 spots, we identified 9 spots whose functions were closely related to regulate the apoptosis of cells. The results of Q-PCR were not completely unanimous with those of 2-DE. Our results suggested that aloe-emodin metabolites decreased NMDA-induced apoptosis of RGCs by preserving, and inducing, some proteins related to the antioxidation and regulation of cells' energy. Both the level of RNA and protein of superoxide dismutase (Cu-Zn) were significantly elevated after aloe-emodin metabolites were added. The mechanisms of neuroprotection are complicated, and involve not only the transcription and stability of mRNA, but also post-translation protein modifications, degradation, and protein-protein interaction.

S100A8 is identified as a biomarker of HPV18-infected oral squamous cell carcinomas by suppression subtraction hybridization, clinical proteomics analysis, and immunohistochemistry staining.

The purpose of this work is to differentiate between the Human papillomaviruses 18 positive (HPV18+) and negative (HPV18-) oral squamous cell carcinomas (OSCC) in oral cancer patients with cancer-associated oral habits (betel quid chewing, cigarette smoking, and alcohol drinking). Both gene and protein expression profiles of HPV18+ and HPV18- OSCC were compared: we then further explored the biological effect of HPV in oral cancer. Suppression subtraction hybridization (SSH), clinical proteomics analysis, and immunohistochemistry (IHC) staining were carried out in the HPV18+ and HPV18- OSCC groups. HPV typing detection revealed that 11 OSCC tissues from 82 patients were positive for HPV18. The SSH experiment showed that 4 cancer-associated genes were highly transcribed within 11 cDNA libraries of HPV18+ OSCC, including poly(ADP-ribose)polymerase I (PARP1), replication protein A2 (RPA2), S100A8, and S100A2. Clinical proteomics analysis indicated that there was over 10-fold overexpression of Stratifin, F-actin capping protein alpha-1 subunit (CapZ alpha-1), Apolipoprotein A-1 (ApoA-1), Heat-shock protein 27 (HSP27), Arginase-1, p16INK4A, and S100 calcium-binding protein A8 (S100A8) in HPV18+ OSCC. Interestingly, the results from SSH and protemics analysis showed that S100A8 was overexpressed in HPV18+ OSCC. Moreover, IHC staining demonstrated that S100A8 was up-regulated in HPV18+ OSCC tissues. Our results suggest that S100A8 plays an important role in oral carcinogenesis following HPV18 infection; therefore, S100A8 may be a powerful biomarker of HPV18 as well as a potential therapeutic target for HPV18+ OSCC patients. The study is the first to identify S100A8 as a biomarker in HPV-associated cancer. Furthermore, this is also the first study to discover a biomarker by combining SSH, clinical proteomics, and IHC stain analysis in oral cancer-associated research.

Identification of over-expressed proteins in oral squamous cell carcinoma (OSCC) patients by clinical proteomic analysis.

BACKGROUND: Oral cancer is a worldwide problem. It is a universal aggressive disease in the population of smoking and drinking. The oral cancer mortality has been ranked 5th place in Taiwan in male cancer patients. A number of protein markers for oral cancer are still not applicable in large populations. Proteomic technologies provide excellent tools for rapid screening of a large number of potential biomarkers in malignant cells. METHOD: Proteomics and real-time quantitative RT-PCR were used to analyze over-expressed proteins in 10 OSCC patients. RESULT: Forty-one proteins were identified as commonly over-expressed in OSCC tissues. In OSCC tissues, alphaB-crystallin, tropomyosin 2, myosin light chain 1, heat shock protein 27 (HSP27), stratifin, thioredoxin-dependent peroxide reductase, flavin reductase, vimentin, rho GDP-dissociation inhibitor 2 (rho GDI-2), glutathione S-transferase Pi (GST-pi) and superoxide dismutase [Mn] (MnSOD) were significantly over-expressed (an average of 7.2, 6.0, 5.7, 4.3, 3.6, 3.4, 3.0, 3.0, 2.6, 2.5, 2.1-fold, respectively). In real-time quantitative RT-PCR analysis, the gene expressions of alphaB-crystallin, HSP27 and MnSOD were also increased in the cancer tissues, consistent with proteomic results. CONCLUSION: The identified proteins in this experiment may be used in future studies of carcinogenesis or as diagnostic markers and therapeutic targets for OSCC.

Strategic shotgun proteomics approach for efficient construction of an expression map of targeted protein families in hepatoma cell lines.

An expression map of the most abundant proteins in human hepatoma HepG2 cells was established by a combination of complementary shotgun proteomics approaches. Two-dimensional liquid chromatography (LC)-nano electrospray ionization (ESI) tandem mass spectrometry (MS/MS) as well as one-dimensional LC-matrix-assisted laser desorption/ionization MS/MS were evaluated and shown that additional separation introduced at the peptide level was not as efficient as simple prefractionation of protein extracts in extending the range and total number of proteins identified. Direct LC-nanoESI MS/MS analyses of peptides from total solubilized fraction and the excised gel bands from one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionated insolubilized fraction afforded the best combination in efficient construction of a nonredundant cell map. Compiling data from multiple variations of rapid shotgun proteomics analyses is nonetheless useful to increase sequence coverage and confidence of hits especially for those proteins identified primarily by a single or two peptide matches. While the returned hit score in general reflects the abundance of the respective proteins, it is not a reliable index for differential expression. Using another closely related hepatoma Hep3B as a comparative basis, 16 proteins with more than two-fold difference in expression level as defined by spot intensity in two-dimensional gel electrophoresis analysis were identified which notably include members of the heat shock protein (Hsp) and heterogeneous nuclear ribonucleoprotein (hnRPN) families. The observed higher expression level of hnRNP A2/B1 and Hsp90 in Hep3B led to a search for reported functional roles mediated in concert by both these multifunctional cellular chaperones. In agreement with the proposed model for telomerase and telomere bound proteins in promoting their interactions, data was obtained which demonstrated that the expression proteomics data could be correlated with longer telomeric length in tumorigenic Hep3B. This biological significance constitutes the basis for further delineation of the dynamic interactions and modifications of the two protein families and demonstrated how proteomic and biological investigation could be mutually substantiated in a productive cycle of hypothesis and pattern driven research.