Acetylation of proteins on lysine residues is a dynamic posttranslational modification that is known to play a key role in regulating transcription and other DNA-dependent nuclear processes. However, the extent of this modification in diverse cellular proteins remains largely unknown, presenting a major bottleneck for lysine-acetylation biology. Here we report the first proteomic survey of this modification, identifying 388 acetylation sites in 195 proteins among proteins derived from HeLa cells and mouse liver mitochondria. In addition to regulators of chromatin-based cellular processes, nonnuclear localized proteins with diverse functions were identified. Most strikingly, acetyllysine was found in more than 20% of mitochondrial proteins, including many longevity regulators and metabolism enzymes. Our study reveals previously unappreciated roles for lysine acetylation in the regulation of diverse cellular pathways outside of the nucleus. The combined data sets offer a rich source for further characterization of the contribution of this modification to cellular physiology and human diseases.

The monocytic leukemia zinc finger protein MOZ and the related factor MORF form tetrameric complexes with ING5 (inhibitor of growth 5), EAF6 (Esa1-associated factor 6 ortholog), and the bromodomain-PHD finger protein BRPF1, -2, or -3. To gain new insights into the structure, function, and regulation of these complexes, we reconstituted them and performed various molecular analyses. We found that BRPF proteins bridge the association of MOZ and MORF with ING5 and EAF6. An N-terminal region of BRPF1 interacts with the acetyltransferases; the enhancer of polycomb (EPc) homology domain in the middle part binds to ING5 and EAF6. The association of BRPF1 with EAF6 is weak, but ING5 increases the affinity. These three proteins form a trimeric core that is conserved from Drosophila melanogaster to humans, although authentic orthologs of The gene of MOZ (monocytic leukemia zinc finger protein, also referred to as MYST3 and KAT6A), located on chromosome 8p11, was first identified as a fusion partner in chromosome translocation t(8;16)(p11;p13) (2, 52). This recurrent translocation is associated with a monocytic subtype of acute myeloid leukemia and results in the fusion of the MOZ Nterminal domain to the C-terminal part of the transcription coactivator CBP. Two other leukemia-associated chromosomal rearrangements lead to the expression of proteins fusing MOZ fragments to the CBP paralog p300 and the p300/CBP-interacting nuclear receptor coactivator TIF2 (transcription intermediary factor 2, also known as steroid receptor coactivator 2 [SRC-2] and nuclear receptor coactivator 2 [NCOA2]) (6,8,29,34). One of the resulting fusion proteins, MOZ-TIF2, is known to promote self-renewal of leukemic stem cells (17,25), suggesting that the chromosome abnormalities play a causal role in leukemogenesis. In addition, it was recently reported that MOZ is fused to NCOA3 (22), a TIF2 paralog synonymous with SRC-3 and AIB1 (amplified in breast cancer 1). MOZ is highly homologous to MORF (MOZ-related factors, also named Querkopf, MYST4, and KAT6B) (11,64). The MORF gene is rearranged in leukemia patients with t(10; 16)(q22;p13) (46) and in leiomyoma cases with t(10;17)(p11; q21) (40). The CBP gene is the fusion partner in the former translocation, while the GCN5 gene is a potential candidate in the latter translocation. All of these findings suggest that deregulated acetylation has an important role in oncogenesis. In addition, recent studies indicate that MOZ and MORF play key roles in hematopoiesis, skeletogenesis, neurogenesis, and other developmental processes (16,26,38,39,62,64). Therefore, MOZ and MORF are intimately linked to both normal development and cancer development (63,69).At the molecular level, available data suggest that this pair of paralogs functions as transcriptional coactivators with intrinsic histone acetyltransferase (HAT) activity (3,11,12,27,28,48). Both possess the MYST domain, a catalytic core conserved among members of the MYST family of acetyltransferases (2, 52). Within this family, there are five members in hu...

By fusing the extracellular domain of the natural killer (NK) cell receptor NKG2D to DAP12, we constructed a chimeric antigen receptor (CAR) to improve NK cell tumor responses. An RNA electroporation approach that provides transient expression of the CAR was adopted as a risk mitigation strategy. Expression of the NKG2D RNA CAR significantly augmented the cytolytic activity of NK cells against several solid tumor cell lines in vitro and provided a clear therapeutic benefit to mice with established solid tumors. Three patients with metastatic colorectal cancer were then treated with local infusion of the CAR-NK cells. Reduction of ascites generation and a marked decrease in number of tumor cells in ascites samples were observed in the first two patients treated with intraperitoneal infusion of low doses of the CAR-NK cells. The third patient with metastatic tumor sites in the liver was treated with ultrasound-guided percutaneous injection, followed by intraperitoneal infusion of the CAR-NK cells. Rapid tumor regression in the liver region was observed with Doppler ultrasound imaging and complete metabolic response in the treated liver lesions was confirmed by positron emission tomography (PET)-computed tomographic (CT) scanning. Our results highlight a promising therapeutic potential of using RNA CAR-modified NK cells to treat metastatic colorectal cancer.

A eukaryotic protein is often subject to regulation by multiple modifications like phosphorylation, acetylation, ubiquitination, and sumoylation. How these modifications are coordinated in vivo is an important issue that is poorly understood but is relevant to many biological processes. We recently showed that human MEF2D (myocyte enhancer factor 2D) is sumoylated on Lys-439. Adjacent to the sumoylation motif is Ser-444, which like Lys-439 is highly conserved among MEF2 proteins from diverse species. Here we presented several lines of evidence to demonstrate that Ser-444 of MEF2D is required for sumoylation of Lys-439. Histone deacetylase 4 (HDAC4) stimulated this modification by acting through Ser-444. In addition, phosphorylation of Ser-444 by Cdk5, a cyclin-dependent kinase known to inhibit MEF2 transcriptional activity, stimulated sumoylation. Opposing the actions of HDAC4 and Cdk5, calcineurin (also known as protein phosphatase 2B) dephosphorylated Ser-444 and inhibited sumoylation of Lys-439. This phosphatase, however, exerted minimal effects on the phosphorylation catalyzed by ERK5, an extracellular signal-regulated kinase known to activate MEF2D. These results identified an essential role for Ser-444 in MEF2D sumoylation and revealed a novel mechanism by which calcineurin selectively "edits" phosphorylation at different sites, thereby reiterating that interplay between different modifications represents a general mechanism for coordinated regulation of eukaryotic protein functions in vivo.In higher eukaryotes, each cell type has a unique gene expression pattern that is ultimately determined by a specific network of transcription factors. The question how cell signaling regulates activities of transcription factors is thus of central importance to many biological processes. The MEF2 3 family of transcription factors comprises four members in mammals, MEF2A, -B, -C, and -D. They were originally identified as major transcriptional activators for muscle differentiation (1, 2). Indeed, some of them were subsequently shown to be important for cardiac myogenesis; null mutation of the murine MEF2A or MEF2C gene led to cardiac death (3-5), and a mutation on the human MEF2A gene was recently suggested to play a role in coronary artery disease (6). MEF2 proteins also have important roles in non-muscle cells by regulating growth factor response, viral gene expression, neuronal survival, T-cell apoptosis, and tumorigenesis (7-12). Consistent with this, the human MEF2D gene is rearranged in pre-B acute lymphoblastic leukemia (13,14), and large scale retrovirus-mediated insertion mutagenesis identified the mouse MEF2D gene as a potential oncogene (15, 16). Therefore, MEF2 transcription factors are key players in diverse cellular programs.At the molecular level, MEF2 is composed of a highly conserved N-terminal domain responsible for DNA recognition and a C-terminal domain with trans-acting function. Regulation of MEF2 function is complex and occurs at multiple levels, including tissue-specific expression (1, 5), altern...

The myocyte enhancer factor 2 (MEF2) family of transcription factors is not only important for controlling gene expression in normal cellular programs, like muscle differentiation, T-cell apoptosis, neuronal survival, and synaptic differentiation, but has also been linked to cardiac hypertrophy and other pathological conditions. Lysine acetylation has been shown to modulate MEF2 function, but it is not so clear which deacetylase(s) is involved. We report here that treatment of HEK293 cells with trichostatin A or nicotinamide upregulated MEF2D acetylation, suggesting that different deacetylases catalyze the deacetylation. Related to the trichostatin A sensitivity, histone deacetylase 4 (HDAC4) and HDAC5, two known partners of MEF2, exhibited little deacetylase activity towards MEF2D. In contrast, HDAC3 efficiently deacetylated MEF2D in vitro and in vivo. This was specific, since HDAC1, HDAC2, and HDAC8 failed to do so. While HDAC4, HDAC5, HDAC7, and HDAC9 are known to recognize primarily the MEF2-specific domain, we found that HDAC3 interacts directly with the MADS box. In addition, HDAC3 associated with the acetyltransferases p300 and p300/CBP-associated factor (PCAF) to reverse autoacetylation. Furthermore, the nuclear receptor corepressor SMRT (silencing mediator of retinoid acid and thyroid hormone receptor) stimulated the deacetylase activity of HDAC3 towards MEF2 and PCAF. Supporting the physical interaction and deacetylase activity, HDAC3 repressed MEF2-dependent transcription and inhibited myogenesis. These results reveal an unexpected role for HDAC3 and suggest a novel pathway through which MEF2 activity is controlled in vivo.Protein lysine acetylation refers to transfer of the acetyl moiety from acetyl coenzyme A (acetyl-CoA) to the ε-amino group of a lysine residue and is an important posttranslational modification that has recently emerged and rivals phosphorylation (41, 61). Proteins known to be subject to lysine acetylation include histones, over 50 transcription factors, and various other proteins (10,40,41,61,77). This dynamic modification is controlled by the opposing actions of acetyltransferases and deacetylases in vivo. Histones were the first substrates identified, so these two families of enzymes are known as histone acetyltransferases (HATs) and histone deacetylases (HDACs), although most of them also act on nonhistone proteins. In the past decade, many proteins have been shown to possess HDAC activity (4,22,39,66,78). On the basis of homology to budding yeast counterparts, human HDACs are grouped into four classes, with HDAC1, -2, -3, and -8, homologs of yeast Rpd3, forming class I. Class II comprises HDAC4, -5, -6, -7, -9, and -10, which possess deacetylase domains highly related to that of yeast Hda1. HDAC4, -5, -7, and -9 have similar domain organization and thus belong to a subgroup known as class IIa.Class III consists of SIRT1 and other Sir2-related proteins. A recent phylogenetic analysis revealed that HDAC11 represents class IV (21). Members of classes I, II, and IV are zinc-depend...