Johan R. Lillehaug

Proteomics analyses reveal the evolutionary conservation and divergence of N-terminal acetyltransferases from yeast and humans

Nα-terminal acetylation is one of the most common protein modifications in eukaryotes. The COmbined FRActional DIagonal Chromatography (COFRADIC) proteomics technology that can be specifically used to isolate N-terminal peptides was used to determine the N-terminal acetylation status of 742 human and 379 yeast protein N termini, representing the largest eukaryotic dataset of N-terminal acetylation. The major N-terminal acetyltransferase (NAT), NatA, acts on subclasses of proteins with Ser-, Ala-, Thr-, Gly-, Cys- and Val- N termini. NatA is composed of subunits encoded by yARD1 and yNAT1 in yeast and hARD1 and hNAT1 in humans. A yeast ard1-Δ nat1-Δ strain was phenotypically complemented by hARD1 hNAT1, suggesting that yNatA and hNatA are similar. However, heterologous combinations, hARD1 yNAT1 and yARD1 hNAT1, were not functional in yeast, suggesting significant structural subunit differences between the species. Proteomics of a yeast ard1-Δ nat1-Δ strain expressing hNatA demonstrated that hNatA acts on nearly the same set of yeast proteins as yNatA, further revealing that NatA from humans and yeast have identical or nearly identical specificities. Nevertheless, all NatA substrates in yeast were only partially N-acetylated, whereas the corresponding NatA substrates in HeLa cells were mainly completely N-acetylated. Overall, we observed a higher proportion of N-terminally acetylated proteins in humans (84%) as compared with yeast (57%). N-acetylation occurred on approximately one-half of the human proteins with Met-Lys- termini, but did not occur on yeast proteins with such termini. Thus, although we revealed different N-acetylation patterns in yeast and humans, the major NAT, NatA, acetylates the same substrates in both species.

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.

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.

Using VAAST to identify an X-linked disorder resulting in lethality in male infants due to N-terminal acetyltransferase deficiency.

We have identified two families with a previously undescribed lethal X-linked disorder of infancy; the disorder comprises a distinct combination of an aged appearance, craniofacial anomalies, hypotonia, global developmental delays, cryptorchidism, and cardiac arrhythmias. Using X chromosome exon sequencing and a recently developed probabilistic algorithm aimed at discovering disease-causing variants, we identified in one family a c.109T>C (p.Ser37Pro) variant in NAA10, a gene encoding the catalytic subunit of the major human N-terminal acetyltransferase (NAT). A parallel effort on a second unrelated family converged on the same variant. The absence of this variant in controls, the amino acid conservation of this region of the protein, the predicted disruptive change, and the co-occurrence in two unrelated families with the same rare disorder suggest that this is the pathogenic mutation. We confirmed this by demonstrating a significantly impaired biochemical activity of the mutant hNaa10p, and from this we conclude that a reduction in acetylation by hNaa10p causes this disease. Here we provide evidence of a human genetic disorder resulting from direct impairment of N-terminal acetylation, one of the most common protein modifications in humans.

Gene Expression Profiling-Based Identification of Molecular Subtypes in Stage IV Melanomas with Different Clinical Outcome

Purpose: The incidence of malignant melanoma is increasing worldwide in fair-skinned populations. Melanomas respond poorly to systemic therapy, and metastatic melanomas inevitably become fatal. Although spontaneous regression, likely due to immune defense activation, rarely occurs, we lack a biological rationale and predictive markers in selecting patients for immune therapy. Experimental Design: We performed unsupervised hierarchical clustering of global gene expression data from stage IV melanomas in 57 patients. For further characterization, we used immunohistochemistry of selected markers, genome-wide DNA copy number analysis, genetic and epigenetic analysis of the CDKN2A locus, and NRAS/BRAF mutation screening. Results: The analysis revealed four distinct subtypes with gene signatures characterized by expression of immune response, pigmentation differentiation, proliferation, or stromal composition genes. Although all subtypes harbored NRAS and BRAF mutations, there was a significant difference between subtypes (P < 0.01), with no BRAF/NRAS wild-type samples in the proliferative subtype. Additionally, the proliferative subtype was characterized by a high frequency of CDKN2A homozygous deletions (P < 0.01). We observed a different prognosis between the subtypes (P = 0.01), with a particularly poor survival for patients harboring tumors of the proliferative subtype compared with the others (P = 0.003). Importantly, the clinical relevance of the subtypes was validated in an independent cohort of 44 stage III and IV melanomas. Moreover, low expression of an a priori defined gene set associated with immune response signaling was significantly associated with poor outcome (P = 0.001). Conclusions: Our data reveal a biologically based taxonomy of malignant melanomas with prognostic effect and support an influence of the antitumoral immune response on outcome. Clin Cancer Res; 16(13); 3356–67. ©2010 AACR.

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.

Expression of oncogenes in thyroid tumours: coexpression of c-erbB2/neu and c-erbB

The receptor-type oncogenes c-erbB2/neu and c-erbB have been found amplified and/or overexpressed in a number of tumours of epithelial origin. We have studied the expression of oncogenes in biopsies from human thyroid tumours. The c-erbB2/neu and c-erbB oncogenes showed two- to three-fold higher levels of RNA in papillary carcinomas and lymph node metastases as well as in one adenoma when compared to non-tumour tissue. The nuclear oncogenes c-myc and c-fos were found to be expressed at varying levels in both non-tumour and tumour tissue. RNA transcripts specific for the platelet-derived growth factor A and B chains and the N-ras oncogene were detected in one anaplastic carcinoma. Neither rearrangements nor amplifications of oncogenes were observed in the thyroid tumours. These data are particularly interesting in light of the recent findings that epidermal growth factor induces proliferation and dedifferentiation of normal thyroid epithelial cells in vitro. We suggest that the epidermal growth factor or other ligands for the c-erbB and c-erbB2/neu receptors may contribute to the development and/or maintenance of the malignant phenotype of papillary carcinomas of the thyroid.

RAR‐independent RXR signaling induces t(15;17) leukemia cell maturation

Although retinoic acid receptor alpha (RARα) agonists induce the maturation of t(15;17) acute promyelocytic leukemia (APL) cells, drug treatment also selects leukemic blasts expressing PML–RARα fusion proteins with mutated ligand‐binding domains that no longer respond to all‐trans retinoic acid (ATRA). Here we report a novel RARα‐independent signaling pathway that induces maturation of both ATRA‐sensitive and ATRA‐resistant APL NB4 cells, and does not invoke the ligand‐induced alteration of PML–RARα signaling, stability or compartmentalization. This response involves a cross‐talk between RXR agonists and protein kinase A signaling. Our results indicate the existence of a separate RXR‐dependent maturation pathway that can be activated in the absence of known ligands for RXR heterodimerization partners.

Induction of apoptosis in human cells by RNAi-mediated knockdown of hARD1 and NATH, components of the protein N - α -acetyltransferase complex

Protein N-ɛ-acetylation is recognized as an important modification influencing many biological processes, and protein deacetylase inhibitors leading to N-ɛ-hyperacetylation of histones are being clinically tested for their potential as anticancer drugs. In contrast to N-ɛ-acetyltransferases, the N-α-acetyltransferases transferring acetyl groups to the α-amino groups of protein N-termini have only been briefly described in mammalians. Human arrest defective 1 (hARD1), the only described human enzyme in this class, complexes with N-acetyltransferase human (NATH) and cotranslationally transfers acetyl groups to the N-termini of nascent polypeptides. Here, we demonstrate that knockdown of NATH and/or hARD1 triggers apoptosis in human cell lines. Knockdown of hARD1 also sensitized cells to daunorubicin-induced apoptosis, potentially pointing at the NATH–hARD1 acetyltransferase complex as a novel target for chemotherapy. Our results argue for an essential role of the NATH–hARD1 complex in cell survival and underscore the importance of protein N-α-acetylation in mammalian cells.

Proteome-derived Peptide Libraries Allow Detailed Analysis of the Substrate Specificities of Nα-acetyltransferases and Point to hNaa10p as the Post-translational Actin Nα-acetyltransferase

The impact of Nα-terminal acetylation on protein stability and protein function in general recently acquired renewed and increasing attention. Although the substrate specificity profile of the conserved enzymes responsible for Nα-terminal acetylation in yeast has been well documented, the lack of higher eukaryotic models has hampered the specificity profile determination of Nα-acetyltransferases (NATs) of higher eukaryotes. The fact that several types of protein N termini are acetylated by so far unknown NATs stresses the importance of developing tools for analyzing NAT specificities. Here, we report on a method that implies the use of natural, proteome-derived modified peptide libraries, which, when used in combination with two strong cation exchange separation steps, allows for the delineation of the in vitro specificity profiles of NATs. The human NatA complex, composed of the auxiliary hNaa15p (NATH/hNat1) subunit and the catalytic hNaa10p (hArd1) and hNaa50p (hNat5) subunits, cotranslationally acetylates protein N termini initiating with Ser, Ala, Thr, Val, and Gly following the removal of the initial Met. In our studies, purified hNaa50p preferred Met-Xaa starting N termini (Xaa mainly being a hydrophobic amino acid) in agreement with previous data. Surprisingly, purified hNaa10p preferred acidic N termini, representing a group of in vivo acetylated proteins for which there are currently no NAT(s) identified. The most prominent representatives of the group of acidic N termini are γ- and β-actin. Indeed, by using an independent quantitative assay, hNaa10p strongly acetylated peptides representing the N termini of both γ- and β-actin, and only to a lesser extent, its previously characterized substrate motifs. The immunoprecipitated NatA complex also acetylated the actin N termini efficiently, though displaying a strong shift in specificity toward its known Ser-starting type of substrates. Thus, complex formation of NatA might alter the substrate specificity profile as compared with its isolated catalytic subunits, and, furthermore, NatA or hNaa10p may function as a post-translational actin Nα-acetyltransferase.