Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome

Comparative characteristics of inducible NO synthase inhibitor and nitric oxide donor in endothelial dysfunction correction caused by osteoarthrosis under experimental conditions

Study have been carried out on white Wistar line rats (age – 3 months, weight – 180-220 g). According to the tasks the animals were divided into 7 groups. 1st group is intact (n = 20). 2nd group is rats, which were modeled osteoarthritis without further correction and were withdrawn from the experiment in the first stage (7th day) (n=40). 3rd group is rats, which were modeled osteoarthritis without further correction and removed from the experiment in the second stage (21st day) (n=40). 4th group is rats, in which experimental osteoarthritis was corrected with nonsteroidal anti-inflammatory drugs (NSAIDs) (Diclofenac) and aminoguanidine and removed from the experiment in the first stage (7th day) (n=20). 5th group is rats, in which experimental osteoarthritis was corrected with NSAIDs (Diclofenac) and aminoguanidine and withdrawn from the experiment in the second stage (21st day) (n=20). 6th group is rats, where experimental osteoarthritis was corrected using NSAIDs and a 7% L-arginine solution and withdrawn from the experiment in the first stage (7th day) (n=20). 7th group is rats, in which experimental osteoarthritis was corrected with NSAIDs and 7% L-arginine solution and withdrawn from the experiment in the second stage (21st day) (n=20) Animals were withdrawn from the experiment for the 7th day and the 21st day after the simulation of the pathological condition. NSAIDs (Diclofenac), aminoguanidine and L-arginine were administered from the beginning of the study. We have obtained the following results: The increase in the content of von Willebrand factor (VWF) in the animals blood proves that endothelial dysfunction is an important part of experimental osteoarthritis pathogenesis. It’s revealed the tendency which directed on normalization of the endothelial dysfunction investigated marker at correction by aminoguadine as a part of complex therapy. L-arginine involvement in the complex correction in experimental osteoarthritis more pronouncedly normalized the VWF level, which indicates the endothelial function normalization. The use of nitric oxide donor is more effective in comparison with the inhibition of inducible NO synthase also in the endothelial nitric oxide synthase activity analysis

Magnetoresistive study of antiferromagnetic--weak ferromagnetic transition in single-crystal La2_{2}CuO4+δ_{4+\delta}

The resistive measurements were made to study the magnetic field-induced antiferromagnetic (AF) - weak ferromagnetic (WF) transition in La$_2$CuO$_4$ single-crystal. The magnetic field (DC or pulsed) was applied normally to the CuO$_2$ layers. The transition manifested itself in a drastic decrease of the resistance in critical fields of ~5-7 T. The study is the first to display the effect of the AF -WF transition on the conductivity of the La$_2$CuO$_4$ single-crystal in the parallel - to - CuO$_2$ layers direction. The results provide support for the 3-dimensional nature of the hopping conduction of this layered oxide.Comment: 8 pages, 7 figures, RevTe

Structural characterization of human Vaccinia-Related Kinases (VRK) bound to small-molecule inhibitors identifies different P-loop conformations

The human genome encodes two active Vaccinia-related protein kinases (VRK), VRK1 and VRK2. These proteins have been implicated in a number of cellular processes and linked to a variety of tumors. However, understanding the cellular role of VRKs and establishing their potential use as targets for therapeutic intervention has been limited by the lack of tool compounds that can specifically modulate the activity of these kinases in cells. Here we identified BI-D1870, a dihydropteridine inhibitor of RSK kinases, as a promising starting point for the development of chemical probes targeting the active VRKs. We solved co-crystal structures of both VRK1 and VRK2 bound to BI-D1870 and of VRK1 bound to two broad-spectrum inhibitors. These structures revealed that both VRKs can adopt a P-loop folded conformation, which is stabilized by different mechanisms on each protein. Based on these structures, we suggest modifications to the dihydropteridine scaffold that can be explored to produce potent and specific inhibitors towards VRK1 and VRK2

Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev: Determinants of DNA Binding and Redox Regulation by Disulfide Bond Formation.

Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40-200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors

Risk of breast cancer and other cancers in heterozygotes for ataxia-telangiectasia

Mortality from cancer among 178 parents and 236 grandparents of 95 British patients with ataxia-telangiectasia was examined. For neither parents nor grandparents was mortality from all causes or from cancer appreciably elevated over that of the national population. Among mothers, three deaths from breast cancer gave rise to a standardized mortality ratio of 3.37 (95% confidence interval (CI): 0.69–9.84). In contrast, there was no excess of breast cancer in grandmothers, the standardized mortality ratio being 0.89 (95% CI: 0.18–2.59), based on three deaths. This is the largest study of families of ataxia-telangiectasia patients conducted in Britain but, nonetheless, the study is small and CIs are wide. However, taken together with data from other countries, an increased risk of breast cancer among female heterozygotes is still apparent, though lower than previously thought. © 1999 Cancer Research Campaig

The Association between ATM IVS 22-77 T>C and Cancer Risk: A Meta-Analysis

BACKGROUND AND OBJECTIVES: It has become increasingly clear that ATM (ataxia-telangiectasia-mutated) safeguards genome stability, which is a cornerstone of cellular homeostasis, and ATM IVS 22-77 T>C affects the normal activity of ATM proteins. However, the association between the ATM IVS 22-77 T>C genetic variant and cancer risk is controversial. Therefore, we conducted a systematic meta-analysis to estimate the overall cancer risk associated with the polymorphism and to quantify any potential between-study heterogeneity. METHODS: A total of nine studies including 4,470 cases and 4,862 controls were analyzed for ATM IVS 22-77 T>C association with cancer risk in this meta-analysis. Heterogeneity among articles and their publication bias were also tested. RESULTS: Our results showed that no association reached the level of statistical significance in the overall risk. Interestingly, in the stratified analyses, we observed an inverse relationship in lung and breast cancer. CONCLUSION: Further functional research on the ATM mechanism should be performed to explain the inconsistent results in different cancer types