Outer membrane protein 25-a mitochondrial anchor and inhibitor of stress-activated protein kinase-3

Stress-activated protein kinase-3 (SAPK3) is unique amongst the mitogen-activated protein kinase (MAPK) family with its C-terminal 5 amino acids directing interaction with the PDZ domain-containing substrates alpha1-Syntrophin and SAP90/PSD95. Here, we identify three additional PDZ domain-containing binding partners, Lin-7C, Scribble, and outer membrane protein 25 (OMP25). This latter protein is localised together with SAPK3 at the mitochondria but it is not a SAPK3 substrate. Instead, OMP25 inhibits SAPK3 activity towards PDZ domain-containing substrates such as alpha1-Syntrophin and substrates without PDZ domains such as the mitochondrial protein Sab. This is a new mechanism for the regulation of SAPK3 and suggests that its intracellular activity should not be solely assessed by its phosphorylation status.     

Global Conservation of Protein Status between Cell Lines and Xenografts

Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery.     

大肠杆菌中高表达重组Era可溶性蛋白的纯化及其生化特性

质粒pCE31含有在P_L起动子控制下的重组era基因,在大肠杆菌中、42℃诱导高表达出Era蛋白,经溶菌酶处理裂菌、沉淀离心洗涤后所得的纯Era是不溶的,生物活性也不高。改在40℃诱导2h,在Era底物GDP的存在下裂菌,60％以上的Era处于可溶状态。用蛋白酶抑制剂防止Era蛋白降解,经盐析、Q-SepharoseFF柱层析分离,获得可溶性纯Era蛋白。该蛋白能特异地与鸟苷酸结合、并具有GTP酶活性,表明Era是一种G蛋白。动力学定量分析结果:在4℃时,每分子Era肽链能结合一分子GTP或GDP,表明所纯化的Era蛋白几乎都具有活性;4℃下,Era蛋白与GTP或GDP结合的解离常数,分别为5.49和1.01μmol/L;37℃时,Era蛋白GTP酶的Km值为9.0μmol/L,催化GTP水解的最大速度为9.8mmolCTP/mol Era/min。     

Leaf alkaloids of Rauwolfia volkensii

Thirteen alkaloids were isolated and identified from the leaves of Rauwolfia volkensii. The alkaloids included E-seco heteroyohimbine, heteroyohimbine, sarpagan, dihydroindole, pleiocarpamine, picrinine and akuammicine types together with peraksine.     

Identification of Cellular Targets in Human Intrahepatic Cholangiocarcinoma Using Laser Microdissection and Accurate Mass and Time Tag Proteomics

Obtaining accurate protein profiles from homogeneous cell populations in heterogeneous tissues can enhance the capability to discover protein biomarkers. In this context, methodologies to access specific cellular populations and analyze their proteome with exquisite sensitivity have to be selected. We report here the results of an investigation using a combination of laser microdissection and accurate mass and time tag proteomics. The study was aimed at the precise determination of proteome alterations in intrahepatic cholangiocarcinoma ICC, a markedly heterogeneous tumor. This cancer, which is difficult to diagnose and carries a very poor prognosis, has shown an unexplained increase in incidence over the last few years. Among a pool of 574 identified proteins, we were able to report on altered abundance patterns affecting 39 proteins conforming to a variety of potential tumorigenic pathways. The reliability of the proteomics results was confirmed by Western blot and immunohistochemistry on matched samples. Most of the proteins displaying perturbed abundances had not yet been described in the setting of ICC. These include proteins involved in cell mobility and actin cytoskeleton remodeling, which may participate in the epithelial to mesenchymal transition, a process invoked in migration and invasion of cancer cells. The biological relevance of these findings was explored using a tissue microarray. An increased abundance of vimentin was thus detected in 70% of ICC and none of the controls. These results suggest that vimentin could play a role in the aggressiveness of ICC and provide a basis for the serious outcome of this cancer.Cholangiocarcinoma (CCA),¹ which arises from the hepatic bile ducts, is the primary cancer accounting for ∼10–15% of all hepatobiliary malignancies. CCA is categorized by the International Classification of Diseases for Oncology into intrahepatic and extrahepatic forms, the latter including perihilar, hilar, and distal bile duct CCA. The global incidence rate of intrahepatic cholangiocarcinoma (ICC) has increased by 2–6% during recent decades, whereas the annual incidence rate of the more common, extrahepatic form has remained relatively stable (1, 2). The diagnosis of ICC remains particularly challenging because the disease can mimic metastasis to the liver or hepatocellular carcinoma (HCC). The only potentially curative treatment options available at present are surgical. Unfortunately, the majority of patients are diagnosed at an advanced, unresectable stage because of the initially silent clinical characteristics of this malignancy. The prognosis of ICC is therefore devastating with survival of less than 24 months following diagnosis. Although several risk factors have been reported as contributing to the development of the disease, most cases of ICC occur in the absence of known etiological factors (3–5). As a consequence, considerable efforts have been made to identify reliable markers to enable the early detection of biliary cancers and provide new insights into the pathogenesis of this deadly disease (6). Recent studies have focused on the cytokines and growth factors (7, 8) produced by CCA cells as well as on the proteomics analysis of serum and bile (9, 10). Follow-up studies are ongoing to determine the sensitivity and specificity of the markers that have emerged from these investigations.Tumor tissue is undeniably the most appropriate resource to investigate tumor-specific signals. Indeed, the determination of alterations to the protein profiles of ICC may offer opportunities to identify tumor-specific markers. To date, proteomics studies performed using tumor tissues have mainly tried to identify proteins with a differential expression between HCC and ICC to prevent the misclassification of these pathologies (11, 12). Furthermore, these studies, performed on total liver homogenates, were not appropriate to detect proteins with altered expression in tumorous cholangiocytes. Indeed, ICC tumor cells are essentially embedded in an abundant stroma containing inflammatory cells and fibroblasts, which may impair the detection of proteins displaying tumor-specific expression patterns (13). In summary, because no proteomics studies have so far been performed on isolated cholangiocytes, any proteome alterations in the setting of ICC remain a matter of speculation.Laser microdissection (LM) has emerged as a suitable tool to selectively extract cells of interest from their natural environment. This technology has been used extensively to profile global gene expression in purified tumor cells (14–16). It has also been used successfully in experiments designed to discover protein biomarkers through mass spectrometry-based proteomics studies (17–19). However, working with such small samples challenges conventional proteomics techniques in terms of sensitivity and the precision of quantitation.One particularly efficient method for proteome analysis is the so-called accurate mass and time (AMT) tag approach where high performance liquid chromatography (HPLC) and high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS or FTMS) work in synergy to achieve a broad proteome coverage even for small sample amounts (20, 21). A recent report demonstrated the significant advantage of the AMT tag method over more conventional proteomics technologies in enabling significantly broader proteome coverage using the minute protein quantities available from tissue microdissection (22).In the present study, we used an AMT tag strategy to determine the differential protein profile between tumorous and non-tumorous microdissected cholangiocytes to define specific proteome alterations of human ICC. Among proteins with altered expression in ICC, we detected a panel of proteins conforming to potential tumorigenic pathways that could be candidates as therapeutic targets and tumor markers for this lethal disease.     

Telomere architecture

Telomeres are protein–DNA complexes that cap chromosome ends and protect them from being recognized and processed as DNA breaks. Loss of capping function results in genetic instability and loss of cellular viability. The emerging view is that maintenance of an appropriate telomere structure is essential for function. Structural information on telomeric proteins that bind to double and single-stranded telomeric DNA shows that, despite a lack of extensive amino-acid sequence conservation, telomeric DNA recognition occurs via conserved DNA-binding domains. Furthermore, telomeric proteins have multidomain structures and hence are conformationally flexible. A possibility is that telomeric proteins take up different conformations when bound to different partners, providing a simple mechanism for modulating telomere architecture.