Biomedical subjects

Johan R Lillehaug

Publications and source records attributed to Johan R Lillehaug.

At least 19 recordsLinked to original sources

PubMed2007

RACK1 regulates Ki-Ras-mediated signaling and morphological transformation of NIH 3T3 cells.

Activating Ras mutations are involved in a significant fraction of human tumors. A suppressor screen using a retroviral mouse fibroblast cDNA library was performed to identify novel factors in Ras-mediated transformation. We identified a novel potent inhibitor of Ras-mediated morphological transformation encoded by a truncated version of the receptor for activated C-kinase (RACK1). The truncated protein, designated RACK1DeltaWD1, lacked the N-terminal 49 amino acids encoding the first of the 7 WD40 repeats in RACK1. RACK1DeltaWD1 expression restored contact inhibition, stress fiber formation and reduced ERK phosphorylation in Ki-Ras transformed NIH 3T3 cells. We demonstrate that truncated RACK1 is involved in complexes consisting of wild-type RACK1 and protein kinase C isoforms alpha, betaI and delta, compromising the transduction of an activated Ras signal to the Raf-MEK-ERK pathway. The cellular localization of RACK1DeltaWD1 differed from wtRACK1, indicating that signaling complexes containing the truncated version of RACK1 are incorrectly localized. Notably, 12-O-tetradecanoyl-13-phorbol acetate (TPA) mediated intracellular translocation of RACK1-interacting PKC alpha and delta was abrogated in RACK1DeltaWD1-expressing cells. Our data support a model where RACK1 acts as a key factor in Ki-Ras-mediated morphological transformation.

Actins↗

PubMed2006

Characterization of hARD2, a processed hARD1 gene duplicate, encoding a human protein N-alpha-acetyltransferase.

BACKGROUND: Protein acetylation is increasingly recognized as an important mechanism regulating a variety of cellular functions. Several human protein acetyltransferases have been characterized, most of them catalyzing epsilon-acetylation of histones and transcription factors. We recently described the human protein acetyltransferase hARD1 (human Arrest Defective 1). hARD1 interacts with NATH (N-Acetyl Transferase Human) forming a complex expressing protein N-terminal alpha-acetylation activity. RESULTS: We here describe a human protein, hARD2, with 81 % sequence identity to hARD1. The gene encoding hARD2 most likely originates from a eutherian mammal specific retrotransposition event. hARD2 mRNA and protein are expressed in several human cell lines. Immunoprecipitation experiments show that hARD2 protein potentially interacts with NATH, suggesting that hARD2-NATH complexes may be responsible for protein N-alpha-acetylation in human cells. In NB4 cells undergoing retinoic acid mediated differentiation, the level of endogenous hARD1 and NATH protein decreases while the level of hARD2 protein is stable. CONCLUSION: A human protein N-alpha-acetyltransferase is herein described. ARD2 potentially complements the functions of ARD1, adding more flexibility and complexity to protein N-alpha-acetylation in human cells as compared to lower organisms which only have one ARD.

Acetylation↗

PubMed2006

Cloning and characterization of hNAT5/hSAN: an evolutionarily conserved component of the NatA protein N-alpha-acetyltransferase complex.

The human hARD1-NATH complex, cotranslationally acetylating the alpha-amino groups of proteins, was recently described. In S. cerevisiae and D. melanogaster this NatA complex contains a third subunit, Nat5p or San, respectively. Based on phylogenetic analyses and database searches, we here describe the human homologue, hNAT5, of these proteins. RT-PCR experiments demonstrated that hNat5 mRNA was expressed in several human cell lines. The candidacy of hNAT5 as a third subunit of the hARD1-NATH complex was investigated using anti-NATH or anti-hARD1 in co-immunoprecipitation experiments followed by Mass Spectrometry analysis of tryptic peptides. Oligopeptides specific for hNAT5 were identified. This verified the expression of endogenous hNAT5 protein in human cells and also identified hNAT5 as a NATH and hARD1 interacting partner. hNAT5 localized to the cytoplasm in accordance with hNAT5-hARD1-NATH complexes playing a role in cotranslational N-alpha-acetylation. Sequence alignment revealed a high degree of similarity of the NAT5 protein between species supporting its conserved role as a part of the complex. The predicted acetyltransferase domain within hNAT5 suggested that this protein, like hARD1, is an enzymatically active component. In summary, we present the first description of the human homologue of Nat5p/San, hNAT5, the third component of the human NatA N-alpha-acetyltransferase complex.

Acetyltransferases↗

PubMed2006

Nuclear localisation of endogenous SUMO-1-modified PDGF-C in human thyroid tissue and cell lines.

We investigated post-translational modification and subcellular localisation of endogenous platelet-derived growth factor-C (PDGF-C) in human thyroid papillary carcinomas (PTC), non-neoplastic thyroid tissues, and a selection of cultured cell lines. PDGF-C expressed nuclear localisation in 95% of all tested cell types in culture and in 10% of the thyrocytes from both PTC and non-neoplastic tissue. The cell lines expressed two forms of full-length PDGF-C, approximately 39 and approximately 55 kDa, in cell membrane and cytosol, while the approximately 55 kDa form dominated in the nucleus where it was partly chromatin-associated. The approximately 55 kDa form was post-translationally modified by SUMO-1. The putative PDGF-C SUMOylation site is the surface exposed (314)lysine part of a positively charged loop ((312)RPKTGVRGLHK(322)) with characteristics of a nuclear localisation signal. The tissue thyrocytes expressed a non-SUMOylated approximately 43 kDa and the 55 kDa PDGF-C. The SUMO-1 modified approximately 55 kDa PDGF-C expression was low in PTC where the approximately 43 kDa PDGF-C dominated. This is in contrast to non-neoplastic tissue and cultured cells where the SUMOylated approximately 55 kDa PDGF-C was strongly expressed. Our data provide novel evidence for nuclear localisation of PDGF-C, post-translational modification by SUMOylation and the expression of a novel form of PDGF-C in human papillary thyroid carcinomas.

Amino Acid Sequence↗

PubMed2006

A novel type of deletion in the CDKN2A gene identified in a melanoma-prone family.

Linkage to the CDKN2A locus has been demonstrated in approximately 50% of families with hereditary malignant melanoma but only a subgroup of these harbor identified mutations. We here report a Norwegian melanoma-prone family with a novel large germline deletion removing 13707 bps of the CDKN2A gene, including exon 1alpha and approximately half of exon 2. Our finding is the first reported large CDKN2A germline deletion with a breakpoint located within an exon. This type of deletion is not detectable through the direct exon sequencing and may also escape identification by use of multiplex ligation-dependent probe amplification (MLPA) analysis. Here, the defect was identified through detection of a truncated p14(ARF) mRNA and loss of p16(INK4a) mRNA expression from the affected allele. Our finding suggests that atypical, large deletions in the CDKN2A gene may explain linkage to the 9p21 chromosome band without identified gene mutations among melanoma-prone families. Thus, it illustrates the need to include p14(ARF)- and p16(INK4a) transcript analysis when searching for unknown mutations within the CDKN2A locus in melanoma-prone families. Similar deletions with atypical breakpoints may affect other genes involved in cancer disposition, and the need to examine gene transcripts in high-risk families with no mutation identified through conventional testing should be considered.

Adult↗

PubMed2006

Gene expression in poorly differentiated papillary thyroid carcinomas.

We used cDNA microarrays to study gene expression in fresh frozen papillary thyroid carcinoma (PTC) specimens. Seven clinically aggressive carcinomas were included, comprising poorly differentiated PTC and tumors with extensive local invasion or synchronous distant metastases. Ten differentiated (classic) papillary thyroid carcinomas (PTC) and non-neoplastic thyroid tissues were also investigated. TaqMan quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry verified the differential gene expression. The B-Raf gene was mutated with a T-->A transversion at nucleotide 1799 (V600E) in 8 of 10 differentiated PTC, and in 4 of 7 aggressive carcinomas. Among genes markedly and equally over-expressed in carcinomas of both the aggressive and classic PtC groups, compared to normal thyroid tissue, were CBP/p300 transactivator (CItED1), fibronectin, growth/differentiation factor 15, potassium inwardly rectifying channel KCNJ2, glutaminyl peptide cyclotransferase, WNT7A, and dipeptidyl peptidase IV. A marked upregulation in carcinomas of P-cadherin mRNA and protein concomitant with E-cadherin downregulation, indicates a possible P-E cadherin "switch" in PTC. The growth factor homologue Nel-like 2, dual specificity phosphatase 5, the serine protease kallikrein 10, and also the tight junction genes claudin 1 and claudin 16, were upregulated in classic PTC but not in aggressive tumors, which may be consistent with altered cell polarity in the dedifferentiated PtC. The aggressive, poorly differentiated PtC group was specifically characterized by marked upregulation of several genes related to cell proliferation such as cell division cycle 2 (CDC2), CDC7, kinesin-like 5, ubiquitin conjugating enzyme E2C, and topoisomerase IIalpha, and by upregulation of genes encoding extracellular matrix proteins such as seprase, extracellular matrix protein 1, and several collagens. These aggressive tumors were also characterized by overexpression of the integrin ligand periostin, and in some biopsies also of osteopontin and of the upstream Rac-regulator dedicator of cytokinesis 10 (DOCK10). These data are interpreted to be consistent with altered cell motility, extracellular matrix remodeling and increased cell proliferation, as important processes in PTC tumor progression.

Adult↗

PubMed2005

Analysing the outer membrane subproteome of Methylococcus capsulatus (Bath) using proteomics and novel biocomputing tools.

High-resolution two-dimensional gel electrophoresis and mass spectrometry has been used to identify the outer membrane (OM) subproteome of the Gram-negative bacterium Methylococcus capsulatus (Bath). Twenty-eight unique polypeptide sequences were identified from protein samples enriched in OMs. Only six of these polypeptides had previously been identified. The predictions from novel bioinformatic methods predicting beta-barrel outer membrane proteins (OMPs) and OM lipoproteins were compared to proteins identified experimentally. BOMP ( http://www.bioinfo.no/tools/bomp ) predicted 43 beta-barrel OMPs (1.45%) from the 2,959 annotated open reading frames. This was a lower percentage than predicted from other Gram-negative proteomes (1.8-3%). More than half of the predicted BOMPs in M. capsulatus were annotated as (conserved) hypothetical proteins with significant similarity to very few sequences in Swiss-Prot or TrEMBL. The experimental data and the computer predictions indicated that the protein composition of the M. capsulatus OM subproteome was different from that of other Gram-negative bacteria studied in a similar manner. A new program, Lipo, was developed that can analyse entire predicted proteomes and give a list of recognised lipoproteins categorised according to their lipo-box similarity to known Gram-negative lipoproteins ( http://www.bioinfo.no/tools/lipo ). This report is the first using a proteomics and bioinformatics approach to identify the OM subproteome of an obligate methanotroph.

Bacterial Outer Membrane Proteins↗

PubMed2005

Interaction between HIF-1 alpha (ODD) and hARD1 does not induce acetylation and destabilization of HIF-1 alpha.

Hypoxia inducible factor-1 alpha (HIF-1 alpha) is a central component of the cellular responses to hypoxia. Hypoxic conditions result in stabilization of HIF-1 alpha and formation of the transcriptionally active HIF-1 complex. It was suggested that mammalian ARD1 acetylates HIF-1 alpha and thereby enhances HIF-1 alpha ubiquitination and degradation. Furthermore, ARD1 was proposed to be down-regulated in hypoxia thus facilitating the stabilization of HIF-1 alpha. Here we demonstrate that the level of human ARD1 (hARD1) protein is not decreased in hypoxia. Moreover, hARD1 does not acetylate and destabilize HIF-1 alpha. However, we find that hARD1 specifically binds HIF-1 alpha, suggesting a putative, still unclear, connection between these proteins.

Acetylation↗

PubMed2005

Identification and characterization of the human ARD1-NATH protein acetyltransferase complex.

Protein acetyltransferases and deacetylases have been implicated in oncogenesis, apoptosis and cell cycle regulation. Most of the protein acetyltransferases described acetylate epsilon-amino groups of lysine residues within proteins. Mouse ARD1 (homologue of yeast Ard1p, where Ard1p stands for arrest defective 1 protein) is the only known protein acetyltransferase catalysing acetylation of proteins at both alpha-(N-terminus) and epsilon-amino groups. Yeast Ard1p interacts with Nat1p (N-acetyltransferase 1 protein) to form a functional NAT (N-acetyltransferase). We now describe the human homologue of Nat1p, NATH (NAT human), as the partner of the hARD1 (human ARD1) protein. Included in the characterization of the NATH and hARD1 proteins is the following: (i) endogenous NATH and hARD1 proteins are expressed in human epithelial, glioma and promyelocytic cell lines; (ii) NATH and hARD1 form a stable complex, as investigated by reciprocal immunoprecipitations followed by MS analysis; (iii) NATH-hARD1 complex expresses N-terminal acetylation activity; (iv) NATH and hARD1 interact with ribosomal subunits, indicating a co-translational acetyltransferase function; (v) NATH is localized in the cytoplasm, whereas hARD1 localizes both to the cytoplasm and nucleus; (vi) hARD1 partially co-localizes in nuclear spots with the transcription factor HIF-1alpha (hypoxia-inducible factor 1alpha), a known epsilon-amino substrate of ARD1; (vii) NATH and hARD1 are cleaved during apoptosis, resulting in a decreased NAT activity. This study identifies the human homologues of the yeast Ard1p and Nat1p proteins and presents new aspects of the NATH and hARD1 proteins relative to their yeast homologues.

Acetylation↗

PubMed2005

Characterization of a prokaryotic haemerythrin from the methanotrophic bacterium Methylococcus capsulatus (Bath).

For a long time, the haemerythrin family of proteins was considered to be restricted to only a few phyla of marine invertebrates. When analysing differential protein expression in the methane-oxidizing bacterium, Methylococcus capsulatus (Bath), grown at a high and low copper-to-biomass ratio, respectively, we identified a putative prokaryotic haemerythrin expressed in high-copper cultures. Haemerythrins are recognized by a conserved sequence motif that provides five histidines and two carboxylate ligands which coordinate two iron atoms. The diiron site is located in a hydrophobic pocket and is capable of binding O(2). We cloned the M. capsulatus haemerythrin gene and expressed it in Escherichia coli as a fusion protein with NusA. The haemerythrin protein was purified to homogeneity cleaved from its fusion partner. Recombinant M. capsulatus haemerythrin (McHr) was found to fold into a stable protein. Sequence similarity analysis identified all the candidate residues involved in the binding of diiron (His22, His58, Glu62, His77, His81, His117, Asp122) and the amino acids forming the hydrophobic pocket in which O(2) may bind (Ile25, Phe59, Trp113, Leu114, Ile118). We were also able to model a three-dimensional structure of McHr maintaining the correct positioning of these residues. Furthermore, UV/vis spectrophotometric analysis demonstrated the presence of conjugated diiron atoms in McHr. A comprehensive genomic database search revealed 21 different prokaryotes containing the haemerythrin signature (PROSITE 00550), indicating that these putative haemerythrins may be a conserved prokaryotic subfamily.

Amino Acid Sequence↗

PubMed2005

Structural and functional specificities of PDGF-C and PDGF-D, the novel members of the platelet-derived growth factors family.

The platelet-derived growth factor (PDGF) family was for more than 25 years assumed to consist of only PDGF-A and -B. The discovery of the novel family members PDGF-C and PDGF-D triggered a search for novel activities and complementary fine tuning between the members of this family of growth factors. Since the expansion of the PDGF family, more than 60 publications on the novel PDGF-C and PDGF-D have been presented, highlighting similarities and differences to the classical PDGFs. In this paper we review the published data on the PDGF family covering structural (gene and protein) similarities and differences among all four family members, with special focus on PDGF-C and PDGF-D expression and functions. Little information on the protein structures of PDGF-C and -D is currently available, but the PDGF-C protein may be structurally more similar to VEGF-A than to PDGF-B. PDGF-C contributes to normal development of the heart, ear, central nervous system (CNS), and kidney, while PDGF-D is active in the development of the kidney, eye and brain. In adults, PDGF-C is active in the kidney and the central nervous system. PDGF-D also plays a role in the lung and in periodontal mineralization. PDGF-C is expressed in Ewing family sarcoma and PDGF-D is linked to lung, prostate and ovarian cancers. Both PDGF-C and -D play a role in progressive renal disease, glioblastoma/medulloblastoma and fibrosis in several organs.

Alternative Splicing↗

PubMed2005

Identification of a copper-repressible C-type heme protein of Methylococcus capsulatus (Bath). A member of a novel group of the bacterial di-heme cytochrome c peroxidase family of proteins.

Genomic sequencing of the methanotrophic bacterium, Methylococcus capsulatus (Bath), revealed an open reading frame (MCA2590) immediately upstream of the previously described mopE gene (MCA2589). Sequence analyses of the deduced amino acid sequence demonstrated that the MCA2590-encoded protein shared significant, but restricted, sequence similarity to the bacterial di-heme cytochrome c peroxidase (BCCP) family of proteins. Two putative C-type heme-binding motifs were predicted, and confirmed by positive heme staining. Immunospecific recognition and biotinylation of whole cells combined with MS analyses confirmed expression of MCA2590 in M. capsulatus as a protein noncovalently associated with the cellular surface of the bacterium exposed to the cell exterior. Similar to MopE, expression of MCA2590 is regulated by the bioavailability of copper and is most abundant in M. capsulatus cultures grown under low copper conditions, thus indicating an important physiological role under these growth conditions. MCA2590 is distinguished from previously characterized members of the BCCP family by containing a much longer primary sequence that generates an increased distance between the two heme-binding motifs in its primary sequence. Furthermore, the surface localization of MCA2590 is in contrast to the periplasmic location of the reported BCCP members. Based on our experimental and bioinformatical analyses, we suggest that MCA2590 is a member of a novel group of bacterial di-heme cytochrome c peroxidases not previously characterized.

Amino Acid Sequence↗

PubMed2004

Genomic insights into methanotrophy: the complete genome sequence of Methylococcus capsulatus (Bath).

Methanotrophs are ubiquitous bacteria that can use the greenhouse gas methane as a sole carbon and energy source for growth, thus playing major roles in global carbon cycles, and in particular, substantially reducing emissions of biologically generated methane to the atmosphere. Despite their importance, and in contrast to organisms that play roles in other major parts of the carbon cycle such as photosynthesis, no genome-level studies have been published on the biology of methanotrophs. We report the first complete genome sequence to our knowledge from an obligate methanotroph, Methylococcus capsulatus (Bath), obtained by the shotgun sequencing approach. Analysis revealed a 3.3-Mb genome highly specialized for a methanotrophic lifestyle, including redundant pathways predicted to be involved in methanotrophy and duplicated genes for essential enzymes such as the methane monooxygenases. We used phylogenomic analysis, gene order information, and comparative analysis with the partially sequenced methylotroph Methylobacterium extorquens to detect genes of unknown function likely to be involved in methanotrophy and methylotrophy. Genome analysis suggests the ability of M. capsulatus to scavenge copper (including a previously unreported nonribosomal peptide synthetase) and to use copper in regulation of methanotrophy, but the exact regulatory mechanisms remain unclear. One of the most surprising outcomes of the project is evidence suggesting the existence of previously unsuspected metabolic flexibility in M. capsulatus, including an ability to grow on sugars, oxidize chemolithotrophic hydrogen and sulfur, and live under reduced oxygen tension, all of which have implications for methanotroph ecology. The availability of the complete genome of M. capsulatus (Bath) deepens our understanding of methanotroph biology and its relationship to global carbon cycles. We have gained evidence for greater metabolic flexibility than was previously known, and for genetic components that may have biotechnological potential.

Bacterial Proteins↗

PubMed2003

Participation of phospholipase D and alpha/beta-protein kinase C in growth factor-induced signalling in C3H10T1/2 fibroblasts.

We have studied phospholipase D (PLD) activation in relation to protein kinase C (PKC) and the involvement of PLD in extracellularly regulated kinase 1 (MAPK) (ERK1) activation and c-fos mRNA expression in C3H/10T1/2 (Cl8) fibroblasts. In these cells, the PLD activity was significantly increased by porcine platelet-derived growth factor (PDGF-BB), phorbol 12-myristate 13-acetate (PMA), and epidermal growth factor (EGF). PLD activation by PDGF-BB and PMA, but not EGF, was inhibited in Cl8 cells expressing the HAbetaC2-1 peptide (Cl8 HAbetaC2-1 cells), with a sequence (betaC2-1) shown to bind receptor for activated C kinase 1 (RACK1) and inhibit c-PKC-mediated cell functions [Science 268 (1995) 247]. A role of alpha-PKC in PLD activation is further underscored by co-immunoprecipitation of alpha-PKC with PLD1 and PLD2 in non-stimulated as well as PMA- and PDGF-BB-stimulated Cl8 cells. However, only PKC in PLD1 precipitates was activated by these agonists, while the PKC in the PLD2 precipitates was constitutively activated. The c-fos mRNA levels in Cl8 cells increased more than 30-fold in response to either PDGF-BB, EGF, or PMA. Approximately 60% inhibition of this increase in c-fos mRNA levels was observed in Cl8 HAbetaC2-1 cells. Formation of phosphatidylbutanol (PtdBut) at the expense of phosphatidic acid (PtdH) in the presence of n-butanol inhibited ERK1 activation and c-fos mRNA expression in PDGF-BB-treated Cl8 cells. ERK activation by PMA was unaffected by n-butanol in Cl8 cells but almost abolished by n-butanol in Cl8 HAbetaC2-1 cells, showing that ERK activation by PMA is heavily dependent on PKC and PLD1. In contrast, ERK activation by EGF in both cell types was not sensitive to n-butanol. These results indicate (1) a role of a functional interaction between the RACK1 scaffolding protein and a alphaPKC-PLD complex for achieving full PLD activity in PDGF-BB- and PMA-stimulated Cl8 cells; (2) PLD-mediated PtdH formation is needed for optimal ERK1 activation by PDGF-BB and maximal increase in c-fos mRNA expression. These findings place PLD as an important component in PDGF-BB- and PMA-stimulated intracellular signalling leading to gene activation in Cl8 cells, while EGF does not require PLD.

1-Butanol↗

PubMed2003

Platelet-derived growth factor (PDGF)-C, a PDGF family member with a vascular endothelial growth factor-like structure.

Platelet-derived growth factor (PDGF)-C is a novel member of the PDGF family that binds to PDGF alphaalpha and alphabeta receptors. The growth factor domain of PDGF-C (GFD-PDGF-C) was expressed in high yields in Escherichia coli and was purified and refolded from inclusion bodies obtaining a biologically active growth factor with dimeric structure. The GFD-PDGF-C contains 12 cysteine residues, and Ellman assay analysis indicates that it contains three intramonomeric disulfide bonds, which is in accordance with GFD-PDGF-C being a member of the cystine knot superfamily of growth factors. The recombinant GFD-PDGF-C was characterized by CD, fluorescence, NMR, and infrared spectroscopy. Together, our data indicate that GFD-PDGF-C is a highly thermostable protein that contains mostly beta-sheet secondary structure and some (6%) alpha-helix structure. The structural model of PDGF-C, obtained by homology-based molecular modeling using the structural representatives of this family of growth factors, shows that GFD-PDGF-C has a higher structural homology to the vascular endothelial growth factor than to PDGF-B. The modeled structure can give further insights into the function and specificity of this molecule.

Amino Acid Sequence↗

PubMed2003

Expression and purification of receptor for activated C-kinase 1 (RACK1).

Receptor for activated C-kinase (RACK1) binds to protein kinase C and functions as an anchor for several other cellular components. Most in vitro studies of RACK1 have been carried out with RACK1 fused to a soluble fusion protein partner, such as GST or MBP. Here, we show that fusion complexes may exist as large soluble aggregates and thereby lead to false conclusions about the biological activity of RACK1. We developed a purification procedure that gave soluble monodisperse molecules of the protein. The RACK1 gene was cloned and expressed in a pMAL vector. After purification of the resulting MBP-RACK1 fusion protein, RACK1 was excised from MBP by thrombin, rendering RACK1 in a soluble monodisperse form as monitored by fluorimetric static light scattering, gel filtration, and ultracentrifugation. Circular dichroism analysis revealed that RACK1 was properly folded with a T(m) of approximately 62 degrees C and contained the predicted portions of secondary structures. The biological activity of the purified protein was verified by binding to activated protein kinase C. The production of soluble, high-purity RACK1 will allow structural studies and functional in vitro studies to identify interacting partners to this important scaffold protein.

Amylose↗

PubMed2003

Increased expression of genes encoding mitochondrial proteins in papillary thyroid carcinomas.

To investigate differences in gene expression between normal thyroid tissue and papillary thyroid carcinomas, we performed differential display (DD) polymerase chain reaction (PCR) using total RNA from fresh-frozen surgically removed thyroid specimens. Four DD fragments that were overexpressed in tumor tissue were identified as parts of genes from the mitochondrial genome: nicotinamide adenine dinucleotide (NADH) dehydrogenase 5, adenosine triphosphate (ATP) synthase 6, cytochrome b, and cytochrome c oxidase I. The expression profiles of these genes were confirmed by hybridization using a DNA dot-blot array and radioactively labeled complex cDNA probes generated from tumor (30 biopsies) and nontumor (15 biopsies) total RNA. Cytochrome c oxidase III was also found to be overexpressed in papillary carcinomas, while the nuclear-encoded mitochondrial transcription factor A showed similar mRNA expression levels in tumor and nontumor tissue. Electron microscopy showed increased number and size of mitochondria in papillary carcinomas. Immunohistochemistry using a monoclonal antibody recognizing a nuclear-encoded mitochondrial protein showed positivity in all cases of papillary carcinoma (44 samples), while normal thyroid tissue (34 samples) was negative in all cases except 3, in which there was a weak, focal cytoplasmic staining. We conclude that papillary thyroid carcinomas show increased expression of mitochondrial mRNA and proteins, encoded by nuclear as well as mitochondrial genes.

Carcinoma, Papillary↗

PubMed2002

NATH, a novel gene overexpressed in papillary thyroid carcinomas.

In this study a replica cDNA screening (RCS) approach to identify genes differentially expressed in papillary thyroid carcinomas (PTC) was used, as compared to non-neoplastic thyroid tissues. RCS is based on hybridization of radioactively labeled cDNA probes made from the biopsies to replica membranes with 15 000 clones from a PTC cDNA library. Among the genes overexpressed in PTC, and especially in clinically aggressive tumors with histologic evidence of poorly differentiated or undifferentiated areas, a novel gene named NATH was found. NATH has two mRNA species, 4.6 and 5.8 kb, both harboring the same open reading frame encoding a putative protein of 866 amino acids. The NATH protein is homologous to yeast N-acetyltransferase (NAT)1 and to mouse NARG1 (mNAT1) and contains four tetratricopeptide repeat (TPR) domains, suggesting that NATH may be part of a multiprotein complex. Overlapping RT-PCR fragments from several PTC biopsies confirmed the NATH mRNA sequence. Northern blots, semiquantitative RT-PCR experiments, TaqMan real-time RT-PCR experiments, and in situ hybridization verified the overexpression of NATH mRNA localized to tumor cells in PTC biopsies. NATH was expressed at a low level in most human adult tissues, including the normal thyroid gland. Increased NATH expression was seen especially in a Burkitt lymphoma cell line and in adult human testis. Recombinant in vitro expression showed that NATH protein was located mainly in the cytoplasm, and was present as a single protein band of the expected 105 kDa molecular weight. Heterologous expression of NATH in the papillary carcinoma cell line (NPA) and 293 cells did not alter the cellular proliferation rate. The biological function of NATH remains to be elucidated, but the overexpression in classic PTC and especially in poorly differentiated or undifferentiated components may indicate a function in the progression of papillary thyroid carcinomas.

Acetyltransferases↗