Purification and characterisation of Azospirillum brasilense N-truncated NtrX protein.

The NtrX protein has been identified as a transcriptional activator of genes involved in the metabolic control of alternative nitrogen sources, acting as a member of a two-component regulatory system. The in silico analysis of the NtrX amino acid sequence shows that this protein contains an N-terminal receiver domain, a central AAA+ superfamily domain and a C-terminal DNA binding domain. To over-express and purify this protein, the ntrX gene of Azospirillum brasilense lacking the first eight codons was cloned into the vector pET29a+. The NtrX protein was over-expressed as an S.Tag fusion protein induced by l-arabinose in the Escherichia coli strain BL21AI and purified by ion exchange and affinity chromatography. The ATPase activity of NtrX was measured by coupling the ATP conversion to ADP with NADH oxidation. The ATPase activity of NtrX was stimulated in the presence of A. brasilense sigma(54)/NtrC-dependent promoter of the glnBA gene. Phosphorylation by carbamyl-phosphate also stimulated ATPase, in a manner similar to the NtrC protein. Together our results suggest that NtrX is active in the phosphorylated form and that there may be a cross-talk between the NtrYX and NtrBC regulatory systems in A. brasilense.

Expression and spectroscopic analysis of a mutant hepatitis B virus onco-protein HBx without cysteine residues.

Chronic infection of the hepatitis B virus (HBV) is one of the causes leading to liver cancer. The 3.2kb genome of HBV encodes four proteins: core antigen, surface antigen, a DNA polymerase and the X protein (HBx). The biological functions of HBx are not fully understood. It has been shown that HBx is a potent trans-activator, which activates transcription of many cellular and viral promoters indirectly via protein-protein interactions. These transactivating activities of HBx may contribute to the development of hepatocellular carcinoma. In this paper a truncated mini-HBx(-Cys) (18-142) protein, where the cysteines had been either deleted or substituted by serines, was constructed by site-directed mutagenesis and overexpressed as a 6xHis fusion protein in Escherichia coli. The 6xHis-mini-HBx(-Cys) protein was isolated from inclusion bodies, purified by Ni-affinity chromatography under denaturing conditions and refolded by sequential dialysis. The structure of the 6xHis-mini-HBx(-Cys) protein was analyzed by circular dichroism, fluorescence and one-dimensional NMR spectroscopic assays. The data presented here suggest that HBx is unstructured but has a propensity to gain secondary structure under specific experimental conditions. Its conformational flexibility might partially explain its functional complexity, namely its capacity to interact with a wide array of signaling proteins, transcriptional regulators and nucleic acids.

Expression of deletion mutants of the hepatitis B virus protein HBx in E. coli and characterization of their RNA binding activities.

The hepatitis B virus protein HBx has been implicated in the development of liver cancer. It has been shown that the HBx protein is able to bind to single-stranded DNA in a specific manner. This DNA binding activity might be relevant for HBx oncogene character. To study the HBx interaction with nucleic acids in more detail we expressed full-length HBx as well as several N- and C-terminally truncated HBx proteins as 6xHis and GST-fusions in E. coli. Using a gel shift assay, we were able to demonstrate that all of the truncated HBx proteins have the ability to bind to an AU-rich RNA. The affinity of GST-HBx #3 (residues 80-142) was an order of magnitude higher than that of GST-HBx #2 (residues 5-79), indicating that a high affinity RNA binding site is located in HBx C-terminal half. AUF1 is the protein ligand that binds to AU-rich RNA regions present in certain proto-oncogene mRNAs and causes their rapid degradation. By a competitive binding experiment of AUF1 and HBx to the AU-rich RNA oligonucleotide, we show that HBx is able to displace AUF1 from its binding site on the RNA oligonucleotide. This new aspect of HBx function is discussed in the context of cellular transformation.

In vitro interaction between STAT 5 and JAK 2; dependence upon phosphorylation status of STAT 5 and JAK 2.

A working model for haematopoietic cytokine signal transduction has been hypothesised as follows. Binding of cytokines to specific receptor molecules leads to phosphorylation and activation of receptor associated members of the Janus kinase family. This is followed by tyrosine phosphorylation of the associated receptor and members of the STAT (signal transducer and activator of transcription) family of DNA-binding transcription factors. Phosphorylation is accompanied by STAT dimerisation, nuclear transport and activation of gene transcription. Activation of gene transcription is mediated by the binding of STAT dimers to palindromic STAT response elements. A number of areas of confusion remain; not least the mechanism by which multiple cytokines signal via a limited number of STATs. A role has been suggested for phosphorylated receptor tyrosine residues as STAT docking sites on activated receptor-JAK complexes. According to this model the amino acid sequence context of key tyrosine residues confers receptor specificity upon STAT activation. There is some controversy as to whether this model applies to STAT 5. The heterologous expression of STAT 5 in Sf 9 insect cells using the baculovirus expression system is described here. Protein of the correct molecular weight was expressed and found to be phosphorylated on tyrosine residues and to bind to a STAT response DNA element. This binding was dependent upon the phosphorylation status of the STAT protein. DNA binding could be abolished in vitro by treatment with a phosphotyrosine phosphatase and restored in vitro by treatment with activated recombinant JAK 2. The protein was purified to near homogeneity using a simple ion exchange/gel filtration chromatography procedure. The interaction between purified recombinant STAT 5 and JAK 2, either expressed by baculovirus or endogenously expressed in Buffalo rat liver cells, was studied. In both cases STAT 5 in its non-phosphorylated form was found to form a stable complex with activated JAK 2. Non-activated JAK 2 and phosphorylated STAT 5 were unable to participate in complex formation. The results presented provide a mechanistic basis for the activation of STAT 5 by a wide range of cytokines capable of activating JAK 2.