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.

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.z 1998 Federation of European Biochemical Societies.

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.

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.

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