The atypical histone macroH2A1.2 interacts with HER-2 protein in cancer cells.

Because HER-2 has been demonstrated in the nuclei of cancer cells, we hypothesized that it might interact with transcription factors that activate ERBB2 transcription. Macrohistone 2A1 (H2AFY; mH2A1) was found to interact with HER-2 in cancer cells that overexpress HER-2. Of the two human mH2A1 isoforms, mH2A1.2, but not mH2A1.1, interacted with HER-2 in human cancer cell lines. Overexpression of mH2A1.2, but not mH2A1.1, in cancer cells significantly increased HER-2 expression and tumorigenicity. Inhibition of HER-2 kinase activity diminished mH2A1 expression and mH2A1.2-induced ERBB2 transcription in cancer cells. Chromatin immunoprecipitation of mH2A1.2 in cancer cells stably transfected with mH2A1.2 showed enrichment of mH2A1.2 at the HER-2 promoter, suggesting a role for mH2A1.2 in driving HER-2 overexpression. The evolutionarily conserved macro domain of mH2A1.2 was sufficient for the interaction between HER-2 and mH2A1.2 and for mH2A1.2-induced ERBB2 transcription. Within the macro domain of mH2A1.2, a trinucleotide insertion (-EIS-) sequence not found in mH2A1.1 was essential for the interaction between HER-2 and mH2A1.2 as well as mH2A1.2-induced HER-2 expression and cell proliferation.

Ex vivo identification of protein-protein interactions involving the dopamine transporter.

The dopamine (DA) transporter (DAT) is a key regulator of dopaminergic signaling as it mediates the reuptake of extrasynaptic DA and thereby terminates dopaminergic signaling. Emerging evidence indicates that DAT function is influenced through interactions with other proteins. The current report describes a method to identify such interactions following DAT immunoprecipitation from a rat striatal synaptosomal preparation. This subcellular fraction was selected since DAT function is often determined ex vivo by measuring DA uptake in this preparation and few reports investigating DAT-protein interactions have utilized this preparation. Following SDS-PAGE and colloidal Coomassie staining, selected protein bands from a DAT-immunoprecipitate were excised, digested with trypsin, extracted, and analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). From the analysis of the tryptic peptides, several proteins were identified including DAT, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) ß, CaMKII δ, protein kinase C (PKC) ß, and PKC γ. Co-immunoprecipitation of PKC, CaMKII, and protein interacting with C kinase-1 with DAT was confirmed by Western blotting. Thus, the present study highlights a method to immunoprecipitate DAT and to identify co-immunoprecipitating proteins using LC/MS/MS and Western blotting. This method can be utilized to evaluate DAT protein-protein interactions but also to assess interactions involving other synaptic proteins. Ex vivo identification of protein-protein interactions will provide new insight into the function and regulation of a variety of synaptic, membrane-associated proteins, including DAT.

Translational bypassing without peptidyl-tRNA anticodon scanning of coding gap mRNA.

Half the ribosomes translating the mRNA for phage T4 gene 60 topoisomerase subunit bypass a 50 nucleotide coding gap between codons 46 and 47. The pairing of codon 46 with its cognate peptidyl-tRNA anticodon dissociates, and following mRNA slippage, peptidyl-tRNA re-pairs to mRNA at a matched triplet 5' adjacent to codon 47, where translation resumes. Here, in studies with gene 60 cassettes, it is shown that the peptidyl-tRNA anticodon does not scan the intervening sequence for potential complementarity. However, certain coding gap mutants allow peptidyl-tRNA to scan sequences in the bypassed segment. A model is proposed in which the coding gap mRNA enters the ribosomal A-site and forms a structure that precludes peptidyl-tRNA scanning of its sequence. Dissipation of this RNA structure, together with the contribution of 16S rRNA anti-Shine-Dalgarno sequence pairing with GAG, facilitates peptidyl-tRNA re-pairing to mRNA.

A case for "StopGo": reprogramming translation to augment codon meaning of GGN by promoting unconventional termination (Stop) after addition of glycine and then allowing continued translation (Go).

When a eukaryotic mRNA sequence specifying an amino acid motif known as 2A is directly followed by a proline codon, two nonoverlapping proteins are synthesized. From earlier work, the second protein is known to start with this proline codon and is not created by proteolysis. Here we identify the C-terminal amino acid of an upstream 2A-encoded product from Perina nuda picorna-like virus that is glycine specified by the last codon of the 2A-encoding sequence. This is an example of recoding where 2A promotes unconventional termination after decoding of the glycine codon and continued translation beginning with the 3' adjacent proline codon.

Human eosinophil major basic protein 2: location of disulfide bonds and free sulfhydryl groups.

Eosinophil granule major basic protein 2 (MBP2 or major basic protein homolog) is a paralog of major basic protein (MBP1) and, similar to MBP1, is cytotoxic and cytostimulatory in vitro. MBP2, a small protein of 13,433 Da molecular weight, contains 10 cysteine residues. Mass spectrometry shows two cystine disulfide linkages (Cys20-Cys115 and Cys92-Cys107) and 6 cysteine residues with free sulfhydryl groups (Cys2, Cys23, Cys42, Cys43, Cys68, and Cys96). MBP2, similar to MBP1, has conserved motifs in common with C-type lectins. The disulfide bond locations are conserved among human MBP1, MBP2 and C-type lectins.

Thioredoxin reductase is required for the inactivation of tumor suppressor p53 and for apoptosis induced by endogenous electrophiles.

Previous studies demonstrate that the covalent modification of thioredoxin reductase (TrxR) by both endogenous and exogenous electrophiles results in disruption of the conformation of the tumor suppressor protein p53. Here we report that the loss of normal cellular TrxR enzymatic activity by electrophilic modification or deletion of the C-terminal catalytic selenocysteine residue has functional consequences that are distinct from those resulting from depletion of TrxR protein in human RKO colon cancer cells. A thorough kinetic analysis was performed on purified TrxR in order to characterize the mechanism of its inhibition by electrophiles. Furthermore, electrospray mass spectrometry confirmed the alkylation of TrxR by lipid electrophiles and liquid chromatography-mass spectrometry/mass spectrometry identified the C-terminus as one target for alkylation. Then the consequences of TrxR modification by electrophiles on p53 conformation, transactivation and apoptosis were compared and contrasted with the effects of depletion of TrxR protein by treatment of cells with small interfering RNA directed against TrxR1. We found that cells depleted of TrxR were actually less sensitive to electrophile-induced disruption of p53 conformation and apoptosis than were cells expressing normal levels of TrxR. When RKO cells depleted of wild-type TrxR were transfected with C-terminal mutants of TrxR lacking the catalytic selenocysteine, p53 was found to be conformationally deranged, similar to cells treated with electrophiles. These results lead us to conclude that C-terminal modification of TrxR is both necessary and sufficient for the disruption of p53 and for the induction of apoptosis. Endogenous lipid electrophiles have been our primary focus; however, metabolic activation of hormones can generate endogenous mutagens, and we demonstrate that estrone-quinone attenuates p53 function in human MCF7 cells.

Factors that influence selection of coding resumption sites in translational bypassing: minimal conventional peptidyl-tRNA:mRNA pairing can suffice.

This study investigates bypassing initiated from codons immediately 5' of a stop codon. The mRNA slips and is scanned by the peptidyl-tRNA for a suitable landing site, and standard decoding resumes at the next 3' codon. This work shows that landing sites with potentially strong base pairing between the peptidyl-tRNA anticodon and mRNA are preferred, but sites with little or no potential for Watson-Crick or wobble base pairing can also be utilized. These results have implications for re-pairing in ribosomal frameshifting. Shine-Dalgarno sequences in the mRNA can alter the distribution of landing sites observed. The bacteriophage T4 gene 60 nascent peptide, known to influence take-off in its native context, imposes stringent P-site pairing requirements, thereby limiting the number of suitable landing sites.