Line M. Myklebust

The world of protein acetylation

Acetylation is one of the major post-translational protein modifications in the cell, with manifold effects on the protein level as well as on the metabolome level. The acetyl group, donated by the metabolite acetyl-coenzyme A, can be co- or post-translationally attached to either the α-amino group of the N-terminus of proteins or to the ε-amino group of lysine residues. These reactions are catalyzed by various N-terminal and lysine acetyltransferases. In case of lysine acetylation, the reaction is enzymatically reversible via tightly regulated and metabolism-dependent mechanisms. The interplay between acetylation and deacetylation is crucial for many important cellular processes. In recent years, our understanding of protein acetylation has increased significantly by global proteomics analyses and in depth functional studies. This review gives a general overview of protein acetylation and the respective acetyltransferases, and focuses on the regulation of metabolic processes and physiological consequences that come along with protein acetylation.

LEDGF/p75 has increased expression in blasts from chemotherapy-resistant human acute myelogenic leukemia patients and protects leukemia cells from apoptosis in vitro

BackgroundRelapse due to chemoresistant residual disease is a major cause of death in acute myelogenous leukemia (AML). The present study was undertaken to elucidate the molecular mechanisms of chemoresistance by comparing differential gene expression in blasts from patients with resistant relapsing AML and chemosensitive AML.ResultsAbout 20 genes were identified as preferentially expressed in blasts pooled from patients with resistant disease, as compared to chemosensitive AML blasts, based on differential gene expression screening. Half of these genes encoded proteins related to protein translation, of these a novel protein related to the ribosomal stalk protein P0. Other upregulated mRNAs coded for cytochrome C oxidase III, the transcription factors ERF-2/TIS11d, and the p75 and p52 splice variants of Lens Epithelial Derived Growth Factor (LEDGF). Analysis of blasts from single patients disclosed that LEDGF/p75 was the most consistently upregulated mRNA in resistant AML. Transfection experiments demonstrated that LEDGF/p75 and p52b antagonized daunorubicin-induced and cAMP-induced apoptosis in an AML cell line. Also HEK-293 cells were protected against daunorubicin by LEDGF/p75 and p52b, whereas LEDGF/p52 splice variants lacking exon 6 had proapoptotic effects. Interestingly, full length LEDGF/p75 protected against truncated pro-apoptotic LEDGF/p75.ConclusionOur results provide evidence for an association between the overexpression of genes encoding survival proteins like LEDGF/p75 and chemo-resistance in acute myelogenous leukemia. LEDGF/p75 has previously not been shown to protect against chemotherapy, and is a potential drug target in AML.

Downregulation of N-terminal acetylation triggers ABA-mediated drought responses in Arabidopsis.

N-terminal acetylation (NTA) catalysed by N-terminal acetyltransferases (Nats) is among the most common protein modifications in eukaryotes, but its significance is still enigmatic. Here we characterize the plant NatA complex and reveal evolutionary conservation of NatA biochemical properties in higher eukaryotes and uncover specific and essential functions of NatA for development, biosynthetic pathways and stress responses in plants. We show that NTA decreases significantly after drought stress, and NatA abundance is rapidly downregulated by the phytohormone abscisic acid. Accordingly, transgenic downregulation of NatA induces the drought stress response and results in strikingly drought resistant plants. Thus, we propose that NTA by the NatA complex acts as a cellular surveillance mechanism during stress and that imprinting of the proteome by NatA is an important switch for the control of metabolism, development and cellular stress responses downstream of abscisic acid.

Biochemical and cellular analysis of Ogden syndrome reveals downstream Nt-acetylation defects

The X-linked lethal Ogden syndrome was the first reported human genetic disorder associated with a mutation in an N-terminal acetyltransferase (NAT) gene. The affected males harbor an Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of NatA, the major human NAT involved in the co-translational acetylation of proteins. Structural models and molecular dynamics simulations of the human NatA and its S37P mutant highlight differences in regions involved in catalysis and at the interface between Naa10 and the auxiliary subunit hNaa15. Biochemical data further demonstrate a reduced catalytic capacity and an impaired interaction between hNaa10 S37P and Naa15 as well as Naa50 (NatE), another interactor of the NatA complex. N-Terminal acetylome analyses revealed a decreased acetylation of a subset of NatA and NatE substrates in Ogden syndrome cells, supporting the genetic findings and our hypothesis regarding reduced Nt-acetylation of a subset of NatA/NatE-type substrates as one etiology for Ogden syndrome. Furthermore, Ogden syndrome fibroblasts display abnormal cell migration and proliferation capacity, possibly linked to a perturbed retinoblastoma pathway. N-Terminal acetylation clearly plays a role in Ogden syndrome, thus revealing the in vivo importance of N-terminal acetylation in human physiology and disease.

De novo missense mutations in the NAA10 gene cause severe non-syndromic developmental delay in males and females

Recent studies revealed the power of whole-exome sequencing to identify mutations in sporadic cases with non-syndromic intellectual disability. We now identified de novo missense variants in NAA10 in two unrelated individuals, a boy and a girl, with severe global developmental delay but without any major dysmorphism by trio whole-exome sequencing. Both de novo variants were predicted to be deleterious, and we excluded other variants in this gene. This X-linked gene encodes N-alpha-acetyltransferase 10, the catalytic subunit of the NatA complex involved in multiple cellular processes. A single hypomorphic missense variant p.(Ser37Pro) was previously associated with Ogden syndrome in eight affected males from two different families. This rare disorder is characterized by a highly recognizable phenotype, global developmental delay and results in death during infancy. In an attempt to explain the discrepant phenotype, we used in vitro N-terminal acetylation assays which suggested that the severity of the phenotype correlates with the remaining catalytic activity. The variant in the Ogden syndrome patients exhibited a lower activity than the one seen in the boy with intellectual disability, while the variant in the girl was the most severe exhibiting only residual activity in the acetylation assays used. We propose that N-terminal acetyltransferase deficiency is clinically heterogeneous with the overall catalytic activity determining the phenotypic severity.

RINF (CXXC5) is overexpressed in solid tumors and is an unfavorable prognostic factor in breast cancer

BACKGROUND We have previously described the essential role of the retinoid-inducible nuclear factor (RINF) during differentiation of hematopoietic cells and suggested its putative involvement in myeloid leukemia and preleukemia. Here, we have investigated whether this gene could have a deregulated expression in malignant tissues compared with their normal tissues of origin and if this potential deregulation could be associated with important clinicopathological parameters. PATIENTS AND METHODS RINF messenger RNA expression was examined in biopsies from locally advanced breast tumors, metastatic malignant melanomas, and papillary thyroid carcinomas and compared with their paired or nonpaired normal reference samples. Further, the prognostic role of RINF expression was evaluated in locally advanced breast cancer. RESULTS RINF expression was significantly higher in all tumor forms (primary breast, and thyroid cancers and metastatic melanomas) as compared with normal control tissues (P < 0.001 for each comparison). Importantly, high levels of RINF expression correlated to a poor overall survival in breast cancer (P = 0.013). This finding was confirmed in three independent public microarray datasets (P = 0.043, n = 234; P = 0.016, n = 69; P = 0.001, n = 196) and was independent of tamoxifen therapy. Notably, high levels of RINF was strongly associated with TP53 wild-type status (P = 0.002) possibly indicating that high levels of RINF could substitute for TP53 mutations as an oncogenic mechanism during the malignant development of some cases of breast cancer. CONCLUSIONS Our data indicate that (i) RINF overexpression is associated with the malignant phenotype in solid tumors and (ii) RINF overexpression represents an independent molecular marker for poor prognosis in breast tumors.

The N-terminal acetyltransferase Naa10 is essential for zebrafish development

The Naa10 (Nα acetyltransferase 10) N-terminal acetyltransferase is implicated in cancer and developmental syndromes in humans. We show that its enzymatic activity is conserved in zebrafish, and that Naa10 depletion leads to developmental abnormalities.

Receptor for Activated Protein C Kinase 1 (RACK1) Is Overexpressed in Papillary Thyroid Carcinoma

BACKGROUND The receptor for activated C kinase 1 (RACK1) has been shown to be overexpressed in several types of cancers such as breast, colon, melanomas, and lung. RACK1 is linked to Ras-Raf-mediated signal transduction and transformed foci formation of 3T3 cells in vitro, and since this pathway is central in papillary thyroid carcinoma (PTC) oncogenesis, we hypothesized that RACK1 could play a role in the development or maintenance of PTC. No report on RACK1 expression in thyroid tissue is available; the present study was therefore aimed at identifying possible correlation of RACK1 expression at the mRNA or protein level in normal thyroid tissue compared to PTC. METHODS We used TaqMan quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry to study the RACK1 gene and protein expression in matched tumor and nontumor samples from 59 PTC patients. The tumor samples were divided into two main categories, low-risk (group 1-3) and high-risk (group 4-6), in accordance with both histological classification and clinical appearance. RESULTS RACK1 mRNA and protein levels were found highly overexpressed in tumor samples, whereas Ki-Ras mRNA was found to be relatively unchanged. B-Raf mRNA expression was low and detected only in tumor samples. Sequencing analysis detected no mutations in RACK1 or Ki-Ras, but 62.7% of the patients harbored the B-Raf single-nucleotide substitution T1799A (codon V600E). Phosphorylated extracellular signal-regulated kinase (pERK) immunohistochemistry analysis demonstrated activation of the mitogen-activated protein kinase (MAPK) pathway in tumor cells. Poorly differentiated and undifferentiated PTCs expressed significantly higher RACK1 mRNA levels than well-differentiated PTCs (p<0.017). CONCLUSIONS Taken together, our findings point to an important role of RACK1 protein in PTC development and progression. Our data also emphasize the importance of assessing protein expression and not only mRNA levels.

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 RACK1ΔWD1, lacked the N‐terminal 49 amino acids encoding the first of the 7 WD40 repeats in RACK1. RACK1ΔWD1 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 α, βI and δ, compromising the transduction of an activated Ras signal to the Raf‐MEK‐ERK pathway. The cellular localization of RACK1ΔWD1 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 α and δ was abrogated in RACK1ΔWD1‐expressing cells. Our data support a model where RACK1 acts as a key factor in Ki‐Ras‐mediated morphological transformation.

A Role for Human N-alpha Acetyltransferase 30 (Naa30) in Maintaining Mitochondrial Integrity

N-terminal acetylation (Nt-acetylation) by N-terminal acetyltransferases (NATs) is one of the most common protein modifications in eukaryotes. The NatC complex represents one of three major NATs of which the substrate profile remains largely unexplored. Here, we defined the in vivo human NatC Nt-acetylome on a proteome-wide scale by combining knockdown of its catalytic subunit Naa30 with positional proteomics. We identified 46 human NatC substrates, expanding our current knowledge on the substrate repertoire of NatC which now includes proteins harboring Met-Leu, Met-Ile, Met-Phe, Met-Trp, Met-Val, Met-Met, Met-His and Met-Lys N termini. Upon Naa30 depletion the expression levels of several organellar proteins were found reduced, in particular mitochondrial proteins, some of which were found to be NatC substrates. Interestingly, knockdown of Naa30 induced the loss of mitochondrial membrane potential and fragmentation of mitochondria. In conclusion, NatC Nt-acetylates a large variety of proteins and is essential for mitochondrial integrity and function.