[Show abstract][Hide abstract] ABSTRACT: NOA36/ZNF330 is an evolutionarily well-preserved protein present in the nucleolus and mitochondria of mammalian cells. We have previously reported that the pro-apoptotic activity of this protein is mediated by a characteristic cysteine-rich domain. We now demonstrate that the nucleolar localization of NOA36 is due to a highly-conserved nucleolar localization signal (NoLS) present in residues 1-33. This NoLS is a sequence containing three clusters of two or three basic amino acids. We fused the amino terminal of NOA36 to eGFP in order to characterize this putative NoLS. We show that a cluster of three lysine residues at positions 3 to 5 within this sequence is critical for the nucleolar localization. We also demonstrate that the sequence as found in human is capable of directing eGFP to the nucleolus in several mammal, fish and insect cells. Moreover, this NoLS is capable of specifically directing the cytosolic yeast enzyme polyphosphatase to the target of the nucleolus of HeLa cells, wherein its enzymatic activity was detected. This NoLS could therefore serve as a very useful tool as a nucleolar marker and for directing particular proteins to the nucleolus in distant animal species.
PLoS ONE 03/2013; 8(3):e59065. DOI:10.1371/journal.pone.0059065 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO) is a free radical gas involved in a variety of
physiological processes in invertebrates. Surprisingly, little is
known about the involvement of NO synthase (NOS) in the
immune system of crustaceans. This work is focused on the study
of the NOS gene of the spiny lobster Panulirus argus, and its
relationship with the immune response. A NOS full-length DNA
was isolated from hemocytes by reverse transcription-polymerase
chain reaction (RT-PCR). The open reading frame (ORF) encodes a protein of 1200 aa, with an estimated molecular mass of 135.9 kDa. NOS gene expression in lobster tissues was studied
by Real Time qPCR and was higher in hemocytes, heart and
gills. In addition, when lobster hemocytes and gills were exposed
to Escherichia coli O55:B5 lipopolysaccharide (LPS), an increase
in the NOS activity and also in the NOS gene expression was
observed. The 3D NOS structure was predicted by comparative
modeling, allowing the selection of a fragment of 666 bp that
was cloned and subsequently expressed in E. coli BL21, in which
a recombinant product of 31 kDa was obtained. Hyperimmune
serum obtained from immunized rabbits was tested and employed
to specifically detect the endogenous NOS from lobster hemocytes
by western blot and immunofluorescence. Additionally, the
antibacterial activity of the hemolymph of the shrimp Litopenaeus
vannamei decreased after the immunoneutralization of NOS.
These results demonstrate the presence of an inducible crustacean
NOS, that will be useful for evaluating the immunological
response to bacterial infections in these organisms.
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO) is a short-lived radical generated by nitric oxide synthases (NOS). NO is involved in a variety of functions in invertebrates, including host defense. In a previous study, we isolated and sequenced for the first time the NOS gene from hemocytes of Panulirus argus, demonstrating the inducibility of this enzyme by lipopolysaccharide (LPS) in vitro. In the present work, lobster hemocytes and gills exposed to Escherichia coli O55:B5 LPS showed an increase in both NOS activity and NOS gene expression in vivo. This response was dose and time dependent. The 3D NOS structure was predicted by comparative modeling showing the oxygenase and reductase domains. These domains contain the conserved binding motifs of NOS already found in a variety of organisms. The 3D structure prediction analysis allowed the selection of a fragment of 666bp that was cloned and subsequently expressed in E. coli BL21, in which a recombinant product of around 31KDa was obtained. Hyperimmune serum obtained from immunized rabbits was tested and employed to specifically detect the recombinant polypeptide or the endogenous NOS from lobster hemocytes by western blot and immunofluorescence. This study contributes to enlarge the existing knowledge related to NOS structure and NOS participation in the immune response in lobsters. The evaluation of an antibody capable to recognize NOS from lobsters constitutes a novel and interesting tool for the implementation of further studies on NOS functions in crustaceans.
[Show abstract][Hide abstract] ABSTRACT: Translocations of regulator proteins from or to the mitochondria are key events in apoptosis regulation. NOA36/ZNF330 is a highly evolutionary conserved protein with a characteristic cystein-rich domain. In this work we address its mitochondrial localization and we demonstrate that a blockage of endogenous NOA36/ZNF330 expression by small-interfering RNA (siRNA) reduced apoptotic response to etoposide (ETO), camptothecin (CPT) and staurosporine (STS) but not to CH11 anti-Fas antibody or tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) in HeLa cells. In contrast, when ectopically expressed in the cytoplasm, NOA36/ZNF330 induces apoptotic cell death. We also found that the domain responsible for this proapoptotic activity is located its cystein-rich region. We propose that NOA36/ZNF330 is translocated from the mitochondria to the cytoplasm when apoptosis is induced and that it contributes to cytochrome c release.
[Show abstract][Hide abstract] ABSTRACT: We have recently described a novel zinc finger cDNA, ZNF330, which was immunologically characterized as a new human autoantigen, highly conserved during evolution from nematodes to humans. The protein was found at the nucleolus and the cytoplasm in interphase and transiently associates with centromeres in mitosis as determined by immunofluorescence analysis. We now describe that the association of ZNF330 with the nucleolus but not with the cytoplasm is RNA-dependent as shown by RNAse treatment of fixed culture cells, since ZNF330 localization was unaffected by DNAse treatment. We also report the cloning, structural organization and chromosome location of the human ZNF330 gene. The gene is comprised of 10 exons and spans approximately 16 kb of genomic DNA. The conserved residues forming nine CXXC motifs are contained in exons 3 to 9. Several major transcription initiation sites were located 126, 124 and 121 bp upstream of the translation initiation codon ATG, as determined by primer extension analysis. The human ZNF330 gene was mapped by FISH to chromosome 4q31.1-->q31.2, the site of the FRA4C locus previously described as a common fragile site for acquired chromosome instability in humans.
Cytogenetics and cell genetics 02/2001; 93(3-4):234-8. DOI:10.1159/000056989
[Show abstract][Hide abstract] ABSTRACT: We have cloned a novel human autoimmune antigen in a patient suffering from rheumatoid arthritis with high levels of antibodies to the nucleolus organizer regions. Initially the human autoimmune serum was used to select a cDNA of 317 amino acids from a hamster expression library. Using the hamster DNA as a probe, we isolated the human homologous cDNA of 320 amino acids. Human and hamster polypeptides share a 95% amino acid homology. The deduced 36-kDa protein contains a putative amino-terminal NLS signal, nine cysteine-X-X-cysteine motifs highly conserved, and a carboxyl-terminal poly acidic region. Several homologous expressed sequence tags have been identified in data bases suggesting that orthologous proteins are present throughout evolution from worms to humans. A Drosophila expressed sequence tag was further completely sequenced for a full-length protein with 60% amino acid identity to the human homologue. Northern blot analysis revealed that this novel protein is widely distributed in human tissues with significantly higher expression levels in heart and skeletal muscle. Specific antibodies to the recombinant protein and transfection experiments demonstrated by immunofluorescence the localization of the protein predominantly but not exclusively to the nucleolus of interphase mammalian cells. In actinomycin D-treated cells the protein remains associated with the nucleolus but is not segregated, like other ribosomal factors such as upstream binding factor. In mitosis the protein was found to be associated with centromeres and concentrated at the midbody in cytokinesis. Transient distribution of this evolutionarily conserved zinc finger nucleolar autoantigen to the mitotic centromeres may provide the means for several aspects of cell cycle control and transcriptional regulation.
[Show abstract][Hide abstract] ABSTRACT: A human autoantigen (NOR-90) previously shown to be the upstream binding factor UBF, binds to the ribosomal RNA genes clustered at the nucleolus organizer regions (NORs). Truncated recombinant forms of hamster UBF were expressed in E. coli and it served to demonstrate that the DNA binding domains of UBF are major autoepitopes as shown by immunoblots analyses with a human autoimmune anti-NOR serum. Several monospecific antibodies to those recombinant truncated UBF polypeptides were generated as shown by immunofluorescence (IF) and Western blots. Immunoblots and IF studies using these anti-UBF sera in cell cultures of various vertebrates species from fish to mammals, indicate the conservation of the DNA binding domains of the ribosomal transcription factor during evolution.
Cellular and molecular biology 04/1999; 45(2):277-84. · 1.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We describe a new human autoimmune antigen in a patient suffering from scleroderma with high levels of antibodies to nucleolus and cytoplasmic antigens. Using a Chinese hamster ovary cell expression library, we have shown that this antigen corresponds to the autosomal Fragile-X-related gene FXR1. The deduced amino acid sequence from the hamster cDNA is 97, 98, and 58% homologous to the human, mouse, and Xenopus laevis FXR1 genes, respectively. Expression of the hamster cDNA clone in Escherichia coli and antibody production indicates unequivocally the location of the FXR1 protein in the cytoplasm of hamster cells. Affinity chromatography followed by immunofluorescence microscopy analysis and immunoblots demonstrated the presence of autoimmune IgGs to FXR1 in the scleroderma patient. Immunolabeling studies in Jurkat cells, induced to apoptosis by anti-Fas/APO1 serum, indicated that the FXR1 antigens were clearly displaced from their original cytoplasmic location to several punctuated foci, resembling the bleb-like membranous structures characteristic of cells at certain stages of apoptosis. This phenomenon could be part of a putative mechanism in which the FXR1 protein is presented as a target for the autoimmune response in humans.
[Show abstract][Hide abstract] ABSTRACT: Centromere autoantibodies are commonly found in the serum of patients with some systemic autoimmune diseases. Previous studies have shown that a major human centromere autoantigen is the histone H3-like protein CENP-A. Although the human cDNA has been cloned, native CENP-A has been neither isolated nor expressed in Escherichia coli, and specific antibodies to this chromatin-associated centromere protein are not available yet. In this report, a highly charged peptide on CENP-A (residues 3-17) was used to generate a monospecific antibody that reacts by immunoblots with the 17 kDa centromeric protein. Immunofluorescence analysis showed reactivity of this anti-CENP-A serum in several but not all mammalian culture cells analyzed, suggesting that the sequence of this histone-like centromere protein could be more variable throughout evolution than originally thought. Selective extractions of human placenta nuclear proteins and immunoblot analysis indicated that CENP-A behaves in a similar way to the core histone polypeptides after nuclease digestion of chromatin. Also, immunoblot analysis demonstrated that the CENP-A peptide used as immunogen is a target region on the CENP-A molecule in several but not all CREST patients analyzed with high titers of autoantibodies to the centromere. Lastly, we found that in Jurkat cells induced to apoptosis, CENP-A remains associated with the centromere, in contrast to other human autoantigens studied during apoptosis.
[Show abstract][Hide abstract] ABSTRACT: A mammalian autoantigen of the nucleolus organizing regions (NORs) was identified by a human autoantibody from the serum of a rheumatoid arthritis patient. The distribution and changes of NORs during the cell cycle of mammals were followed by using this autoantiserum in indirect-immunofluorescence microscopy. In interphase cells the staining pattern indicated that the autoantigen is restricted exclusively within the nucleolus. This fluorescence appeared punctuated rather than uniform, and it was reorganized during inhibition of transcription in cells treated with actinomycin D. During mitosis, the autoantigen was detected by light microscopy at the chromosomal nucleolus organizer regions, indicating that presumably the protein remains bound to the rRNA genes. Biochemical analysis by immunoblotting showed that the NOR autoantigen consists of two polypeptides with molecular masses apparent of 90-92 kDa in all of the mammalian cell lines tested. The identity of some epitopes, recognized by this autoantibody as the ribosomal transcription factor UBF, is discussed.
Cellular and molecular biology 01/1993; 38(8):841-51. · 1.23 Impact Factor