Hereditary hemochromatosis protein, HFE, interaction with transferrin receptor 2 suggests a molecular mechanism for mammalian iron sensing

HFE and transferrin receptor 2 (TFR2) are membrane proteins integral to mammalian iron homeostasis and associated with human hereditary hemochromatosis. Here we demonstrate that HFE and TFR2 interact in cells, that this interaction is not abrogated by disease-associated mutations of HFE and TFR2, and that TFR2 competes with TFR1 for binding to HFE. We propose a new model for the mechanism of iron status sensing that results in the regulation of iron homeostasis.     

Molecular and functional interactions between Escherichia coli nucleoside-diphosphate kinase and the uracil-DNA glycosylase Ung

Escherichia coli nucleoside-diphosphate kinase (Ndk) catalyzes nucleoside triphosphate synthesis and maintains intracellular triphosphate pools. Mutants of E. coli lacking Ndk exhibit normal growth rates but show a mutator phenotype that cannot be entirely attributed to the absence of Ndk catalytic activity or to an imbalance in cellular triphosphates. It has been suggested previously that Ndk, similar to its human counterparts, possesses nuclease and DNA repair activities, including the excision of uracil from DNA, an activity normally associated with the Ung and Mug uracil-DNA glycosylases (UDGs) in E. coli. Here we have demonstrated that recombinant Ndk purified from wild-type E. coli contains significant UDG activity that is not intrinsic, but rather, is a consequence of a direct physical and functional interaction between Ung and Ndk, although a residual amount of intrinsic UDG activity exists as well. Co-purification of Ung and Ndk through multicolumn low pressure and nickel-nitrilotriacetic acid affinity chromatography suggests that the interaction occurs in a cellular context, as was also suggested by co-immunoprecipitation of endogenous Ung and Ndk from cellular extracts. Glutathione S-transferase pulldown and far Western analyses demonstrate that the interaction also occurs at the level of purified protein, suggesting that it is specific and direct. Moreover, significant augmentation of Ung catalytic activity by Ndk was observed, suggesting that the interaction between the two enzymes is functionally relevant. These findings represent the first example of Ung interacting with another E. coli protein and also lend support to the recently discovered role of nucleoside-diphosphate kinases as regulatory components of multiprotein complexes.     

Infrared spectroscopic study of the structural and functional properties of the Na/H antiporter MjNhaP1 from Methanococcus jannaschii

In this study, structural, functional, and mechanistic properties of the Na⁺/H⁺ antiporter MjNhaP1 from Methanococcus jannaschii were analyzed by infrared spectroscopic techniques. Na⁺/H⁺ antiporters are generally responsible for the regulation of cytoplasmic pH and Na⁺ concentration. MjNhaP1 is active in the pH range between pH 6 and pH 6.5; below and above it is inactive.The secondary structure analysis on the basis of ATR-IR spectra provides the first insights into the structural changes between inactive (pH 8) and active (pH 6) state of MjNhaP1. It results in decreased ordered structural elements with increasing the pH-value i.e. with inactivation of the protein. Analysis of temperature-dependent FTIR spectra indicates that MjNhaP1 in the active state exhibits a much higher unfolding temperature in the spectral region assigned to α-helical segments. In contrast, the temperature-induced structural changes for β-sheet structure are similar for inactive and active state. Consequently, this structure element is not the part of the activation region of the protein. The surface accessibility of the protein was analyzed by following the extent of H/D exchange. Due to higher content of unordered structural elements a higher accessibility for amide protons is observed for the inactive as compared to the active state of MjNhaP1. Altogether, the results present the active state of MjNhaP1 as the state with ordered structural elements which exhibit high thermal stability and increased hydrophobicity.     

Inheritance and variation of erucic acid content in a transgenic rapeseed (Brassica napus L.) doubled haploid population

Erucic acid (22:1) is a valuable renewable resource for the oleochemical industry. Currently available high erucic acid rapeseed cultivars contain only about 50% erucic acid in the seed oil. A substantial increase of the erucic acid content of the rapeseed oil could increase market prospects. The transgenic line TNKAT, over expressing the rapeseed fatty acid elongase gene (fae1) and expressing the Ld-LPAAT gene from Limnanthes douglasii was crossed with the line 6575-1 HELP (high erucic and low polyunsaturated fatty acid). A from the F₁ plants produced population of 90 doubled haploid (DH) lines was tested in a greenhouse with three replicates. Parental lines TNKAT and 6575-1 HELP contained 46 and 50% erucic acid in the seed oil, respectively. In the DH population the erucic acid content ranged between 35 and 59%. The Ld-LPAAT + Bn-fae1.1 transgene showed a 1:1 segregation. The transgenic DH lines contained up to 8% trierucolyglycerol, but surprisingly had a by 2.3% lower erucic acid content compared to the non-transgenic segregants. Results indicated that the ectopically expressed fae1.1 gene may not be functional. The DH population also showed a large quantitative variation for PUFA content ranging from 6 to 28% (TNKAT: 21%, 6575-1 HELP: 8%). Regression analysis showed that in the DH population a 10% reduction in PUFA content led to a 4.2% increase in erucic acid content. Development of locus specific PCR primers for the two resident erucic acid genes fae1.1 (A-genome) and fae1.2 genes (C-genome) of rapeseed allowed sequencing of the respective alleles from TNKAT and 6575-1 HELP. Single nucleotide polymorphisms were only found for the fae1.1 gene. Use of allele specific fae1.1 PCR primers, however, did not reveal a significant effect of the fae1.1 allele from either parent on erucic acid content. The high erucic acid low polyunsaturated fatty acid DH lines and the fae1 locus specific primers developed in the present study should be useful in future studies aimed at increasing erucic acid content in rapeseed.     

Large catalase based bioelectrode for biosensor application

A large catalase (CAT) (Mr ~ 90 kDa), immobilized on multiwalled carbon nanotubes—Nafion^® (MWCNT-NF) matrix and encapsulated with polyethylenimine (PEI) on glassy carbon electrode (GCE), showed a pair of nearly reversible cyclic voltammetric peaks for Fe^(III)/Fe^(II) couple with formal potential of about −0.45 V (vs. Ag/AgCl electrode at pH 7.5). PEI significantly reduced the charge transfer resistance and stabilized the bioelectrode through electrostatic interaction. The electron transfer rate constant and surface coverage of the immobilized CAT were 1.05 ± 0.2 s⁻¹ and 2.1 × 10⁻¹⁰ mol cm⁻², respectively. Studies on electrocatalytic activity and kinetics of GCE/MWCNT-NF/CAT/PEI for hydrogen peroxide (H₂O₂) showed the apparent Michaelis-Menten constant of 3 mM, linear response in the range of 10 μM to 5 mM, response time of ~ 2 s for steady state current, and detection limit of ~ 1 μM. A high operational and storage stability was also demonstrated for the bioelectrode. Hence, the direct electrochemistry of the large catalase and its potential biosensor application have been established through this investigation.     

Development and characterization of two new cell lines from common carp, Cyprinus carpio (Linn)

Two new cell lines (CCF and CCH) were established from fin and heart tissues of common carp, Cyprinus carpio. The cells were optimally maintained in Leibovitz-15 medium supplemented with 10% fetal bovine serum (FBS) and 10 ng/ml of basic fibroblastic growth factor (bFGF). The effects of temperature, concentration of FBS and bFGF on the growth of CCF and CCH cells were examined. The temperature ranged from 24 to 32°C for good growth of the cells. The growth rate of cells was higher in medium containing 10% FBS and the addition of bFGF to the medium significantly increased the growth rate. The CCF cells were found to be epithelial, while the CCH cells were fibroblastic in nature. The cytogenetic analysis of the cell lines revealed a diploid number of 100 chromosomes in C. carpio. The viability of CCF and CCH cell lines were 70 and 72%, respectively, after six months of storage in liquid nitrogen (-196° C). Molecular characterization of the cell lines using 16S rRNA and Cytochrome Oxidase Subunit I (COI) revealed the origin of the cell lines. These new cell lines will be useful for isolation of fish viruses and other in vitro biotechnological studies.     

Co-chaperone CHIP associates with expanded polyglutamine protein and promotes their degradation by proteasomes

A major hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various chaperones and proteasome components. But, how the polyglutamine proteins are ubiquitinated and degraded by the proteasomes are not known. Here, we demonstrate that CHIP (C terminus of Hsp70-interacting protein) co-immunoprecipitates with the polyglutamine-expanded huntingtin or ataxin-3 and associates with their aggregates. Transient overexpression of CHIP increases the ubiquitination and the rate of degradation of polyglutamine-expanded huntingtin or ataxin-3. Finally, we show that overexpression of CHIP suppresses the aggregation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when CHIP is overexpressed along with Hsc70.     

Evidence for antagonism of BMP-4 signals by MAP kinase during Xenopus axis determination and neural specification

We have previously shown that mitogen-activated protein (MAP) kinase activity is required for neural specification in Xenopus. In mammalian cells, the BMP-4 effector Smad1 is inhibited by phosphorylation at MAP kinase sites (Kretzschmar et al., 1997). To test the hypothesis that MAP kinase inhibits the BMP-4/Smad1 pathway during early Xenopus development, we have generated a Smad1 mutant lacking the MAP kinase phosphorylation sites (M4A-Smad1) and compared the effects of wild-type (WT)- and M4A-Smad1 on axial pattern and neural specification in Xenopus embryos. Although overexpression of either WT- or M4A-Smad1 produced ventralized embryos, at each mRNA concentration, M4A-Smad1 had a greater ventralizing effect than WT-Smad1. Interestingly, overexpression of either form of Smad1 in ventral blastomeres disrupted posterior pattern and morphogenesis; again, more severe defects were produced by expression of M4A-Smad1 than by equal amounts of WT-Smad1. Ectodermal expression of M4A-Smad1 disrupted expression of the anterior neural gene otx2 in vivo and inhibited neural specification in response to endogenous signals in mesoderm-ectoderm recombinates. In contrast, overexpression of WT-Smad1 at identical levels had little effect on either neural specification or otx2 expression. Comparisons of protein levels following overexpression of either WT- or M4A-Smad1 indicate that WT-Smad1 may be slightly more stable than M4A-Smad1; thus, differences in stability cannot account for the increased effectiveness of M4A-Smad1. Our results demonstrate that mutations disrupting the MAPK phosphorylation sites act collectively as a gain-of-function mutation in Smad1 and that inhibitory phosphorylation of Smad1 may be a significant mechanism for the regulation of BMP-4/Smad1 signals during Xenopus development.