Phospholamban C-terminal Residues Are Critical Determinants of the Structure and Function of the Calcium ATPase Regulatory Complex

To determine the structural and regulatory role of the C-terminal residues of phospholamban (PLB) in the membranes of living cells, we fused fluorescent protein tags to PLB and sarco/endoplasmic reticulum calcium ATPase (SERCA). Alanine substitution of PLB C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB, suggesting a change in quaternary conformation of PLB pentamer and SERCA-PLB regulatory complex. Val to Ala substitution at position 49 (V49A) had particularly large effects on PLB pentamer structure and PLB-SERCA regulatory complex conformation, increasing and decreasing probe separation distance, respectively. We also quantified a decrease in oligomerization affinity, an increase in binding affinity of V49A-PLB for SERCA, and a gain of inhibitory function as quantified by calcium-dependent ATPase activity. Notably, deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. C-terminal truncations also resulted in progressive loss of PLB-PLB FRET due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the binding affinity of truncated PLB for SERCA and loss of inhibitory potency. However, despite decreased SERCA-PLB binding, intermolecular FRET for Val⁴⁹-stop (V49X) truncation mutant was paradoxically increased as a result of an 11.3-Å decrease in the distance between donor and acceptor fluorophores. We conclude that PLB C-terminal residues are critical for localization, oligomerization, and regulatory function. In particular, the PLB C terminus is an important determinant of the quaternary structure of the SERCA regulatory complex.     

Childhood thyroid cancer, radiation dose from Chernobyl, and dose uncertainties in Bryansk Oblast, Russia: a population-based case-control study

A population-based case-control study was conducted to estimate the radiation-related risk of thyroid cancer in persons who were exposed in childhood to (131)I from the Chernobyl accident of April 26, 1986 and to investigate the impact of uncertainties in individual dose estimates. Included were all 66 confirmed cases of primary thyroid cancer diagnosed from April 26, 1986 through September 1998 in residents of Bryansk Oblast, Russia, who were 0-19 years old at the time of the accident, along with two individually matched controls for each case. Thyroid radiation doses, estimated using a semi-empirical model based on environmental contamination data and individual characteristics, ranged from 0.00014 Gy to 2.73 Gy and had large uncertainties (median geometric standard deviation 2.2). The estimated excess relative risk (ERR) associated with radiation exposure, 48.7/Gy, was significantly greater than 0 (P = 0.00013) but had an extremely wide 95% confidence interval (4.8 to 1151/Gy). Adjusting for dose uncertainty nearly tripled the ERR to 138/Gy, although this was likely an overestimate due to limitations in the modeling of dose uncertainties. The radiation-related excess risk observed in this study is quite large, especially if the uncertainty of dose estimation is taken into account, but is not inconsistent with estimates previously reported for risk after (131)I exposure or acute irradiation from external sources.     

Electrophysical characteristics of Azospirillum brasilense Sp245 during interaction with antibodies to various cell surface epitopes

This work was undertaken to examine the electrooptical characteristics of cells of Azospirillum brasilense Sp245 during their interaction with antibodies developed to various cell surface epitopes. We used the dependences of the cell suspension optical density changes induced by electroorientation on the orienting field frequency (740, 1000, 1450, 2000, and 2800kHz). Cell interactions with homologous strain-specific antibodies to the A. brasilense Sp245 O antigen and with homologous antibodies to whole bacterial cells brought about considerable changes in the electrooptical properties of the bacterial suspension. When genus-specific antibodies to the flagellin of the Azospirillum sheathed flagellum and antibodies to the serologically distinct O antigen of A. brasilense Sp7 were included in the A. brasilense Sp245 suspension, the changes caused in the electrooptical signal were slight and had values close to those for the above changes. These findings agree well with the immunochemical characteristics of the Azospirillum O antigens and with the data on the topographical distribution of the Azospirillum major cell surface antigens. The obtained results can serve as a basis for the development of a rapid test for the intraspecies detection of microorganisms.     

Mapping of functional domains in F plasmid partition proteins reveals a bipartite SopB-recognition domain in SopA

Active partition of the F plasmid to dividing daughter cells is assured by interactions between proteins SopA and SopB, and a centromere, sopC. A close homologue of the sop operon is present in the linear prophage N15 and, together with sopC-like sequences, it ensures stability of this replicon. We have exploited this sequence similarity to construct hybrid sop operons with the aim of locating specific interaction determinants within the SopA and SopB proteins that are needed for partition function and for autoregulation of sopAB expression. Centromere binding was found to be specified entirely by a central 25 residue region of SopB strongly predicted to form a helix-turn-helix structure. SopB protein also carries a species-specific SopA-interaction determinant within its N-terminal 45 amino acids, and, as shown by Escherichia coli two-hybrid analysis, a dimerization domain within its C-terminal 75 (F) or 97 (N15) residues. Promoter-operator binding specificity was located within an N-terminal 66 residue region of SopA, which is predicted to contain a helix-turn-helix motif. Two other regions of SopA protein, one next to the ATPase Walker A-box, the other C-terminal, specify interaction with SopB. Yeast two-hybrid analysis indicated that these regions contact SopB directly. Evidence for the involvement of the SopA N terminus in autoinhibition of SopA function was obtained, revealing a possible new aspect of the role of SopB in SopA activation.     

Identification of new human cadherin genes using a combination of protein motif search and gene finding methods

We have combined protein motif search and gene finding methods to identify genes encoding proteins containing specific domains. Particularly, we have focused on finding new human genes of the cadherin superfamily proteins, which represent a major group of cell-cell adhesion receptors contributing to embryonic neuronal morphogenesis. Models for three cadherin protein motifs were generated from over 100 already annotated cadherin domains and used to search the complete translated human genome. The genomic sequence regions containing motif "hits" were analyzed by eukaryotic GeneMark.hmm to identify the exon-intron structure of new genes. Three new genes CDH-J, PCDH-J and FAT-J were found. The predicted proteins PCDH-J and FAT-J were classified into protocadherin and FAT-like subfamilies, respectively, based on the number and organization of cadherin domains and presence of subfamily-specific conserved amino acid residues. Expression of FAT-J was shown in almost all tested tissues. The exon-intron organization of CDH-J was experimentally verified by PCR with specifically designed primers and its tissue-specific expression was demonstrated. The described methodology can be applied to discover new genes encoding proteins from families with well-characterized structural and functional domains.     

NMR structures of loop B RNAs from the stem-loop IV domain of the enterovirus internal ribosome entry site: a single C to U substitution drastically changes the shape and flexibility of RNA

The 5'-untranslated region of positive-strand RNA viruses harbors many cis-acting RNA structural elements that are important for various viral processes such as replication, translation, and packaging of new virions. Among these is loop B RNA of the stem-loop IV domain within the internal ribosomal entry site (IRES) of enteroviruses, including Poliovirus type 1 (PV1). Studies on PV1 have shown that specific recognition of loop B by the first KH (hnRNP K homology) domain of cellular poly(rC)-binding protein 2 (PCBP2) is essential for efficient translation of the viral mRNA. Here we report the NMR solution structures of two representative sequence variants of enteroviral loop B RNA. The two RNA variants differ at only one position (C vs U) within a six-nucleotide asymmetric internal loop sequence that is the binding site for the PCBP2 KH1 domain. Surprisingly, the two RNAs are drastically different in the overall shape and local dynamics of the bulge region. The RNA with the 5'-AUCCCU bulge sequence adopts an overall L shape. Its bulge nucleotides, especially the last four, are highly flexible and not very well defined by NMR. The RNA with the 5'-AUUCCU bulge sequence adopts an overall U shape, and its bulge sequence exhibits only limited flexibility. A detailed analysis of the two RNA structures and their dynamic properties, as well as available sequence data and known KH domain-RNA complex structures, not only provides insights into how loop B RNA might be recognized by the PCBP2 KH1 domain but also suggests a possible correlation between structural flexibility and pre-existing structural features for protein recognition.     

Daily copper supplement effects on copper balance in trained subjects during prolonged restriction of muscular activity

The aim of this study was to assess the effect of a daily intake of copper supplements on negative copper balance during prolonged exposure to hypokinesia (decreased number of kilometers per day). During hypokinesia (HK), negative copper balance is shown by increased, not by decreased, serum copper concentration, as it happens in other situations. Studies were done during a 30-d prehypokinetic period and a 364-d hypokinetic period. Forty male trained volunteers aged 22–26 yr with a peak oxygen uptake of 66.4 mL/min/kg and with an average of 13.7 km/d running distance were chosen as subjects. They were equally divided into four groups: unsupplemented ambulatory control subjects (UACS), unsupplemented hypokinetic subjects (UHKS), supplemented hypokinetic subjects (SHKS), and supplemented ambulatory control subjects (SACS). The SACS and SHKS groups took 0.09 mg copper carbonate/kg body weight daily. The SHKS and UHKS groups were maintained under an average running distance of 1.7 km/d, whereas the SACS and UACS groups did not experience any modifications in their normal training routines. During the 30-d prehypokinetic period and the 346-d hypokinetic period, urinary excretion of copper, calcium, and magnesium and serum concentrations of copper, calcium, and magnesium were measured. Copper loss in feces and copper balance was also determined. In both UHKS and SHKS groups, urinary excretion of copper, calcium, and magnesium and concentrations of copper, magnesium, and calcium in serum increased significantly when compared with the SACS and UACS groups. Loss of copper in feces was also increased significantly in the SHKS and UHKS groups when compared with the UACS and SACS groups. Throughout the study, the copper balance was negative in the SHKS and UHKS groups, whereas in the SACS and UACS groups, the copper balance was positive. It was concluded that a daily intake of copper supplements cannot be used to prevent copper deficiency shown by increased copper concentration. Copper supplements also failed to prevent negative copper balance and copper losses in feces and urine in endurancetrained subjects during prolonged exposure to HK.