SelT, SelW, SelH, and Rdx12: genomics and molecular insights into the functions of selenoproteins of a novel thioredoxin-like family

Selenium is an essential trace element in many life forms due to its occurrence as a selenocysteine (Sec) residue in selenoproteins. The majority of mammalian selenoproteins, however, have no known function. Herein, we performed extensive sequence similarity searches to define and characterize a new protein family, designated Rdx, that includes mammalian selenoproteins SelW, SelV, SelT and SelH, bacterial SelW-like proteins and cysteine-containing proteins of unknown function in all three domains of life. An additional member of this family is a mammalian cysteine-containing protein, designated Rdx12, and its fish selenoprotein orthologue. Rdx proteins are proposed to possess a thioredoxin-like fold and a conserved CxxC or CxxU (U is Sec) motif, suggesting a redox function. We cloned and characterized three mammalian members of this family, which showed distinct expression patterns in mouse tissues and different localization patterns in cells transfected with the corresponding GFP fusion proteins. By analogy to thioredoxin, Rdx proteins can use catalytic cysteine (or Sec) to form transient mixed disulfides with substrate proteins. We employed this property to identify cellular targets of Rdx proteins using affinity columns containing mutant versions of these proteins. Rdx12 was found to interact with glutathione peroxidase 1, whereas 14-3-3 protein was identified as one of the targets of mammalian SelW, suggesting a mechanism for redox regulation of the 14-3-3 family of proteins.     

CA-MMP: a matrix metalloproteinase with a novel cysteine array, but without the classic cysteine switch.

A matrix metalloproteinase (MMP)-like gene was identified in mouse to contain a conserved MMP catalytic domain and an RRRR motif. It lacks a classic cysteine switch, but it possesses two novel motifs: a cysteine array (Cys-X(6)-Cys-X(8)-Cys-X(10)-Cys-X(3)-Cys-X(2)-Cys), and a novel Ig-fold. It is named CA-MMP after the distinct cysteine array motif, and little is known about its biochemical function. In an attempt to characterize CA-MMP activity, the full-length sequence was expressed in mammalian cells and its product found to be cell-associated without detectable secretion. In light of this unusual finding, a chimera combining the catalytic domain of CA-MMP with the prodomain of stromelysin-3 was constructed to express a fully active enzyme in mammalian cells. Purified CA-MMP catalytic domain expresses proteolytic activity against protein substrates in an MMP inhibitor sensitive fashion. Taken together, it is concluded that CA-MMP is an MMP with distinct structure, biochemical properties and evolutionary history that may define a new subclass of the MMP superfamily.     

Cytoplasmic, nuclear, and golgi localization of RGS proteins. Evidence for N-terminal and RGS domain sequences as intracellular targeting motifs

RGS proteins comprise a family of proteins named for their ability to negatively regulate heterotrimeric G protein signaling. Biochemical studies suggest that members of this protein family act as GTPase-activating proteins for certain Galpha subunits, thereby accelerating the turn-off mechanism of Galpha and terminating signaling by both Galpha and Gbetagamma subunits. In the present study, we used confocal microscopy to examine the intracellular distribution of several RGS proteins in COS-7 cells expressing RGS-green fluorescent protein (GFP) fusion proteins and in cells expressing RGS proteins endogenously. RGS2 and RGS10 accumulated in the nucleus of COS-7 cells transfected with GFP constructs of these proteins. In contrast, RGS4 and RGS16 accumulated in the cytoplasm of COS-7 transfectants. As observed in COS-7 cells, RGS4 exhibited cytoplasmic localization in mouse neuroblastoma cells, and RGS10 exhibited nuclear localization in human glioma cells. Deletion or alanine substitution of an N-terminal leucine repeat motif present in both RGS4 and RGS16, a domain identified as a nuclear export sequence in HIV Rev and other proteins, promoted nuclear localization of these proteins in COS-7 cells. In agreement with this observation, treatment of mouse neuroblastoma cells with leptomycin B to inhibit nuclear protein export by exportin1 resulted in accumulation of RGS4 in the nucleus of these cells. GFP fusions of RGS domains of RGS proteins localized in the nucleus, suggesting that nuclear localization of RGS proteins results from nuclear targeting via RGS domain sequences. RGSZ, which shares with RGS-GAIP a cysteine-rich string in its N-terminal region, localized to the Golgi complex in COS-7 cells. Deletion of the N-terminal domain of RGSZ that includes the cysteine motif promoted nuclear localization of RGSZ. None of the RGS proteins examined were localized at the plasma membrane. These results demonstrate that RGS proteins localize in the nucleus, the cytoplasm, or shuttle between the nucleus and cytoplasm as nucleo-cytoplasmic shuttle proteins. RGS proteins localize differentially within cells as a result of structural differences among these proteins that do not appear to be important determinants for their G protein-regulating activities. These findings suggest involvement of RGS proteins in more complex cellular functions than currently envisioned.     

Identification of a novel mouse membrane-bound family 1 glycosidase-like protein,which carries an atypical active site structure

We have identified a novel mouse gene klph (Klotho-LPH related protein; where LPH stands for lactase-phlorizin hydrolase) that encodes a novel mammalian family 1 glycosidase-like protein. KLPH was a putative type I membrane protein that consists of N-terminal signal sequence, glycosidase domain, transmembrane region and short cytoplasmic tail. Despite its overall structural similarity to other family 1 glycosidases, the glutamic acid for the acid-base catalyst was not conserved in this protein. klph mRNA was predominantly expressed in the kidney and skin. Epitope-tagged KLPH was localized to the perinuclear tubular network structure of the endoplasmic reticulum in cultured cells.     

Functional analysis of the fission yeast Prp4 protein kinase involved in pre-mRNA splicing and isolation of a putative mammalian homologue.

The prp4 gene of Schizosaccharomyces pombe encodes a protein kinase. A physiological substrate is not yet known. A mutational analysis of prp4 revealed that the protein consists of a short N-terminal domain, containing several essential motifs, which is followed by the kinase catalytic domain comprising the C-terminus of the protein. Overexpression of N-terminal mutations disturbs mitosis and produces elongated cells, Using a PCR approach, we isolated a putative homologue of Prp4 from human and mouse cells. The mammalian kinase domain is 53% identical to the kinase domain of Prp4. The short N-terminal domains share <20% identical amino acids, but contain conserved motifs. A fusion protein consisting of the N-terminal region from S. pombe followed by the mammalian kinase domain complements a temperature-sensitive prp4 mutation of S. pombe. Prp4 and the recombinant yeast/mouse protein kinase phosphorylate the human SR splicing factor ASF/SF2 in vitro in its RS domain.     

Sequence requirement for the nucleolar localization of human I-mfa domain-containing protein (HIC p40)

The human I-mfa domain-containing protein (HIC) mRNA produces two protein isoforms, HIC p32 and p40, synthesized from alternative translational initiations. p32 translation is initiated from a standard AUG codon and p40 is an N-terminal extension of p32 generated from an upstream GUG codon. The two isoforms show different subcellular localization: p32 is distributed throughout the cytoplasm whereas p40 can be found both in the cytoplasm and the nucleolus. To investigate the possibility that p40 contains a nucleolus targeting sequence in its N-terminal region, COS cells were transfected with an eukaryotic expression vector coding for green fluorescent protein (GFP) fused to the p40 N terminus. The localization of this fusion protein in the nucleolus indicated that the N-terminal amino acids of p40 probably contain a nucleolar localization signal (NoLS). To find the structural motifs required for nucleolar localization of p40, deletion mutants were expressed in COS cells as fusion polypeptides with GFP. We defined a domain of 19 amino acids near the N terminus that contains an arginine-rich subdomain that conforms to other known NoLS. To demonstrate that this sequence is an authentic NoLS, the sequence was fused to GFP. This fusion protein was observed to migrate into the nucleolus. Taken together, our studies demonstrate that p40 contains a NoLS.     

Molecular function of WhiB4/Rv3681c of Mycobacterium tuberculosis H37Rv: a [4Fe-4S] cluster co-ordinating protein disulphide reductase

The genome sequence of Mycobacterium tuberculosis H37Rv revealed the presence of seven whiB-like open reading frames. In spite of several genetic studies on whiB genes, the biochemical properties of WhiB proteins are poorly understood. All WhiB-like proteins have four conserved cysteine residues, out of which two are present in a CXXC motif. We report for the first time the detailed biochemical and biophysical properties of M. tuberculosis WhiB4/Rv3681c and demonstrate the functional relevance of four conserved cysteines and the CXXC motif. UV-visible absorption spectra of freshly purified mWhiB4 showed the presence of a [2Fe-2S] cluster, whereas the electron paramagnetic resonance (EPR) spectra of reconstituted protein showed the presence of a [4Fe-4S] cluster. The iron-sulphur cluster was redox sensitive but stably co-ordinated to the protein even in the presence of high concentration of chaotropic agents. Despite primary sequence divergence from thioredoxin family proteins, the apo mWhiB4 has properties similar to thioredoxins and functions as a protein disulphide reductase, whereas holo mWhiB4 is enzymatically inactive. Apart from the cysteine thiol of CXXC motif the distantly placed thiol pair also contributes equally to the enzymatic activity of mWhiB4. A functional model of mWhiB4 in redox signaling during oxidative stress in M. tuberculosis has been presented.     

The search of partners of a new mammalian selenium-containing protein V (SelV) and expression it's mRNA during ontogenesis and spermatogenesis

Selenium is a biologically active trace elements, which is part of several proteins, and thus linked with the activity of many organs, tissues and systems of organism. There are 25 mammalian selenoproteins at present, one of which is SelV (Selenoprotein V). Since this protein has thioredoxin-like folding and a conserved motif (CXXU, where C is cysteine, U-selenocysteine) in its catalytic center, it belongs to the family of redox proteins, whose members are involved in redox reactions. In this paper, we show that the redox protein SelV can interact with O-linked N-acetylglucosamine transferase (OGT) and proteins belonging to the family of ASB: Asb-17, and Asb-9. The specificity of interactions SelV with OGT and Asb-9, but not with Asb-17 is confirmed by coimmunopretsipitation. In addition, expression of SelV mRNA in the later stages spermatogenesis, as well as in puberty and reproductive periods of rats is shown.     

Nuclear localization of enzymatically active green fluorescent protein-CTP:phosphocholine cytidylyltransferase alpha fusion protein is independent of cell cycle conditions and cell types

To address the recent controversy about the subcellular localization of CTP:phosphocholine cytidylyltransferase alpha (CTalpha), this study was designed to visualize green fluorescent protein (GFP). CTalpha fusion proteins directly and continuously under different conditions of cell cycling and in various cell lines. The GFP. CTalpha fusion proteins were enzymatically active and capable of rescuing mutant cells with a temperature-sensitive CT. The expressed GFP.CTalpha fusion protein was localized to the nucleus in all cell lines and required the N-terminal nuclear targeting sequence. Serum depletion/replenishment did not cause shuttling of CTalpha between the nucleus and cytoplasm. Moreover, the subcellular localization of CTalpha was examined continuously through all stages of the cell cycle in synchronized cells. No shuttling of CTalpha between the nucleus and cytoplasm was observed at any stage of the cell cycle. Stimulation of cells with oleate had no effect on the localization of CTalpha. The GFP.CTalpha lacking the nuclear targeting sequence stayed exclusively in the cytoplasm. Regardless of their localization, the GFP.CTalpha fusion proteins were equally active for phosphatidylcholine synthesis and mutant rescue. We conclude that the nuclear localization of CTalpha is a biological event independent of cell cycle in most mammalian cells and is unrelated to activation of phosphatidylcholine synthesis.     

Attenuation of green fluorescent protein half-life in mammalian cells

The half-life of the green fluorescent protein (GFP) was determined biochemically in cultured mouse LA-9 cells. The wild-type protein was found to be stable with a half-life of approximately 26 h, but could be destabilized by the addition of putative proteolytic signal sequences derived from proteins with shorter half-lives. A C-terminal fusion of a PEST sequence from the mouse ornithine decarboxylase gene reduced the half-life to 9.8 h, resulting in a GFP variant suitable for the study of dynamic cellular processes. In an N-terminal fusion containing the mouse cyclin B1 destruction box, it was reduced to 5.8 h, with most degradation taking place at metaphase. The combination of both sequences produced a similar GFP half-life of 5.5 h. Thus, the stability of this marker protein can be controlled in predetermined ways by addition of the appropriate proteolytic signals.     

Lamina-associated polypeptide 2beta (LAP2beta) is contained in a protein complex together with A- and B-type lamins

Lamina-associated polypeptide 2beta (LAP2beta) of vertebrates is an integral membrane protein of the inner nuclear membrane that is generated by alternative splicing from the LAP2 gene. In the majority of Xenopus somatic cells including cultured kidney epithelial cells (A6 cells) there is only one major LAP2 isoform expressed that has the highest similarities with the mammalian LAP2beta whereas isoforms corresponding in size to the mammalian LAP2gamma and alpha are not detectable. We selected A6 cells and A6 cells stably expressing GFP fusion proteins of Xenopus LAP2beta (XLAP2Pbeta) as a model system to study interactions between LAP2beta and lamins. In vitro binding experiments with GST-XLAP2beta fusion proteins and immunoprecipitations with antibodies to GFP revealed that XLAP2beta is part of a complex that contains A- and B-type lamins. For the targeting to the nuclear envelope and the in vivo formation of this complex, GFP fusion proteins were sufficient comprising only the carboxyterminal 135 amino acids of XLAP2beta or the comparable region of zebrafish LAP2beta. A highly conserved 36 amino acids long sequence is located in this region of LAP2beta that is part of the lamina-binding domain previously identified in rat LAP2beta. GFP-LAP2beta fusion proteins of Xenopus, zebrafish, and rat that contained this sequence do compete with endogenous LAP2 in transfected cells for the same binding sites in the lamina. Our data indicate that the lamina-binding site of LAP2beta has been highly conserved during vertebrate evolution and suggests that this region of LAP2beta mediates the interactions between polymers of A- and B-type lamins.     

Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana

Ara6 of Arabidopsis thaliana is a novel member of the Rab/Ypt GTPase family with unique structural features. It resembles Rab5 GTPases best, but lacks a large part of the C-terminal hypervariable region and the cysteine motif, and instead harbors an extra stretch of amino acid residues containing myristoylation and palmitoylation sites at the N-terminus. Ara6 is tightly associated with membranes and is expressed constitutively. In contrast, the conventional Rab5 ortholog, Ara7, is highly expressed only in actively dividing cells. Examination of green fluorescent protein (GFP)-tagged proteins indicates that both Ara6 and Ara7 are distributed on a subpopulation of endosomes and suggests their roles in endosomal fusion. The endosomal localization of Ara6 requires N-terminal fatty acylation, nucleotide binding and the C-terminal amino acid sequence coordinately. Proteins similar to Ara6 are found only in higher plants and thus represent a novel class of Rab GTPases regulating endocytic function in a plant- specific manner.     

Functional and mutational characterization of human MIA40 acting during import into the mitochondrial intermembrane space

A first component involved in import into the mitochondrial intermembrane space, named Mia40, has been described recently in yeast. Here, we identified the human MIA40 as a novel and ubiquitously expressed component of human mitochondria. It belongs to a novel protein family whose members share six highly conserved cysteine residues constituting a -CXC-CX9C-CX9C- motif. Human MIA40 is significantly smaller than the fungal protein and lacks the N-terminal extension including a transmembrane region and mitochondrial targeting signal. It forms soluble complexes within the intermembrane space of human mitochondria. Depletion of MIA40 in human cells by RNA interference specifically affected steady-state levels of small and cysteine-containing intermembrane space proteins like DDP1 and TIM10A, suggesting that MIA40 acts along the import pathway into the intermembrane space. Studies on the in vivo redox state of human MIA40 demonstrated that it contains intramolecular disulfide bonds. Thiol-trapping assays revealed the co-existence of different oxidation states of human MIA40 within the cell. Furthermore, we show that the twin -CX9C- motif is specifically required for import and stability of MIA40 in mitochondria. Partial mutation of this motif affects stable accumulation of MIA40 in the intermembrane space, whereas mutation of all cysteine residues in this motif inhibits import in mitochondria. Taken together, we conclude that the biogenesis and function of MIA40 in the mitochondrial intermembrane space is dependent on redox processes involving conserved cysteine residues.     

小鼠脂联素与绿色荧光蛋白融合基因表达载体的构建及在COS-7细胞中的表达

以绿色荧光蛋白(GFP)基因(gfp)为报告基因,构建小鼠脂联素(mADPN)基因(mAd)与gfp的融合基因mAd/gfp表达载体pCI-neo-apoEHCR-hAATp-mAd-gfp,脂质体法转染体外培养的COS-7细胞,荧光显微镜观察GFP在细胞中的表达可间接反映mADPN的表达,并通过RT-PCR在核酸水平进一步确证mAd的表达.荧光显微镜观察及RT-PCR结果均证明mADPN在COS-7细胞中获得了高效表达,表明mADPN重组表达载体pCI-neo-apoEHCR-hAATp-mAd可以在真核细胞COS-7中高效表达mADPN,为进一步探讨mAd在小鼠体内的表达提供了可行性依据.     

Cloning and characterization of a mouse cysteine proteinase

cDNA clones encoding a mouse cysteine proteinase were isolated from a cDNA library constructed from mRNA derived from the macrophage-like cell line J774. The DNA sequence predicts a protein that is closely related to, but distinct from, the lysosomal enzyme cathepsin H. Alignment of the predicted amino acid sequence with the known protein sequences for seven other cysteine proteinases suggests that the cloned DNA encodes a 334-residue protein containing both a 17-amino acid pre-region and a 96-amino acid pro-region. Consistent with this prediction, antiserum raised to a recombinant fusion protein expressed in Escherichia coli immunoprecipitated multiple forms of the cysteine proteinase in mouse peritoneal macrophages and fibroblasts. In pulse-chase experiments, a 36-kDa precursor, presumedly the pro-form, was converted intracellularly into a 28-kDa protein and subsequently into a 21-kDa protein. Indirect immunofluorescence microscopy results suggested that the cysteine proteinase was localized to lysosomes. Western blot analysis detected significantly more of the proteinase in thioglycolate-elicited peritoneal macrophages than in resident peritoneal macrophages. Northern blot analysis revealed that several cell lines failed to express mouse cysteine proteinase mRNA.     

The Golgi-localized Arabidopsis endomembrane protein12 contains both endoplasmic reticulum export and Golgi retention signals at its C terminus

Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12 in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.e., an endoplasmic reticulum export motif and a Golgi retention signal that interacted with COPII and COPI subunits, respectively); and (4) the Golgi retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in plant cells.     

Identification of a novel male germ cell-specific gene TESF-1 in mice

Mammalian spermatogenesis is precisely regulated by many germ cell-specific factors. In search for such a germ cell-specific factor, we have identified a novel mouse gene testis-specific factor 1 (TESF-1). Messenger RNA of TESF-1 was found only in the testis and its expression appeared to be regulated in a developmental manner. Further analysis demonstrated that the expression of TESF-1 was specifically in male germ cells, supported by the observation that we were not able to detect the TESF-1 mRNA from at/at homozygous mutant testes, which lack germ cells. The deduced amino acid sequence of TESF-1 contains a leucine-zipper motif, a potential nuclear localization signal, and two cAMP- and cGMP-dependent protein kinase phosphorylation sites. The green fluorescent protein (GFP)-tagged TESF-1 fusion protein was expressed in COS-7 cells and localized primarily in the nucleus. Taken together, these results indicate that TESF-1 is a novel male germ cell-specific gene, and its protein product may function as a nuclear factor involved in the regulation of spermatogenesis.     

Mouse Nudt13 is a Mitochondrial Nudix Hydrolase with NAD(P)H Pyrophosphohydrolase Activity

The mammalian NUDT13 protein possesses a sequence motif characteristic of the NADH pyrophosphohydrolase subfamily of Nudix hydrolases. Due to the persistent insolubility of the recombinant product expressed in Escherichia coli, active mouse Nudt13 was expressed in insect cells from a baculovirus vector as a histidine-tagged recombinant protein. In vitro, it efficiently hydrolysed NADH to NMNH and AMP and NADPH to NMNH and 2′,5′-ADP and had a marked preference for the reduced pyridine nucleotides. Much lower activity was obtained with other nucleotide substrates tested. K _m and k _cat values for NADH were 0.34 mM and 7 s^?1 respectively. Expression of Nudt13 as an N-terminal fusion to green fluorescent protein revealed that it was targeted exclusively to mitochondria by the N-terminal targeting peptide, suggesting that Nudt13 may act to regulate the concentration of mitochondrial reduced pyridine nucleotide cofactors and the NAD(P)⁺/NAD(P)H ratio in this organelle and elsewhere. Future studies of the enzymology of pyridine nucleotide metabolism in relation to energy homeostasis, redox control, free radical production and cellular integrity should consider the possible regulatory role of Nudt13.