The serine threonine kinase Akt is a core survival factor that underlies a variety of human diseases. Although regulatory phosphorylation and dephosphorylation have been well documented, the other posttranslational mechanisms that modulate Akt activity remain unclear. We show here that tetratricopeptide repeat domain 3 (TTC3) is an E3 ligase that interacts with Akt. TTC3 contains a canonical RING finger motif, a pair of tetratricopeptide motifs, a putative Akt phosphorylation site, and nuclear localization signals, and is encoded by a gene within the Down syndrome (DS) critical region on chromosome 21. TTC3 is an Akt-specific E3 ligase that binds to phosphorylated Akt and facilitates its ubiquitination and degradation within the nucleus. Moreover, DS cells exhibit elevated TTC3 expression, reduced phosphorylated Akt, and accumulation in the G(2)M phase, which can be reversed by TTC3 siRNA or Myr-Akt. Thus, interaction between TTC3 and Akt may contribute to the clinical symptoms of DS.

Fusion protein AML1-ETO, resulting from t(8;21) translocation, is highly related to leukemia development. It has been reported that full-length AML1-ETO blocks AML1 function and requires additional mutagenic events to promote leukemia. We have previously shown that the expression of AE9a, a splice isoform of AML1-ETO, can rapidly cause leukemia in mice. To understand how AML1-ETO is involved in leukemia development, we took advantage of our AE9a leukemia model and sought to identify its interacting proteins from primary leukemic cells. Here, we report the discovery of a novel AE9a binding partner PRMT1 (protein arginine methyltransferase 1). PRMT1 not only interacts with but also weakly methylates arginine 142 of AE9a. Knockdown of PRMT1 affects expression of a specific group of AE9a-activated genes. We also show that AE9a recruits PRMT1 to promoters of AE9a-activated genes, resulting in enrichment of H4 arginine 3 methylation, H3 Lys9/14 acetylation, and transcription activation. More importantly, knockdown of PRMT1 suppresses the self-renewal capability of AE9a, suggesting a potential role of PRMT1 in regulating leukemia development. (Blood. 2012;119(21):4953-4962) IntroductionAcute myeloid leukemia (AML) is closely associated with chromosomal abnormalities. 1 The AML1 gene (also known as CBFA2, PEBP2␣B, and RUNX1) was initially identified as a target of chromosomal translocation in t(8;21), which is associated with 15% of de novo AML cases and Յ 40% in the AML subtype M2 of the French-American-British classification. 2,3 This specific translocation at t(8;21) involves the AML1 gene on chromosome 21 and the ETO (also known as MTG8) gene on chromosome 8 and generates an AML1-ETO fusion transcription factor. 4 AML1-ETO inherits the DNA binding RUNT domain from AML1 and is functionally characterized as a transcription factor for both gene repression and activation. 3,[5][6][7] It has been shown that AML1-ETO negatively regulates AML1 target genes, possibly through interaction with corepressor proteins such as mSin3A, N-CoR/SMRT (nuclear receptor corepressor/silencing mediator for retinoic acid receptor and thyroid hormone receptor), and HDACs (histone deacetylases). [8][9][10] AML1-ETO could also act as a transactivator on certain genes. One of the possible mechanisms is by recruiting histone modifiers to make chromatin structure more accessible to the transcription activation machinery, resulting in gene activation. A recent finding shows that p300 binds to NHR1 domain of AML1-ETO to facilitate transcription. 11 A variety of posttranslational modifications, including acetylation, methylation, and phosphorylation, on specific residues of histones and their corresponding enzymes has been discovered. 12 It is well documented that a specific histone modification on a promoter could determine the state of transcription. Specifically, methylation on histone H4 arginine 3 (Arg3) by PRMT1 (protein arginine methyltransferase 1) generally correlates with transcription activation. 13 PRMT1 is the most predominant arginine ...

L-Carnosine is a bioactive dipeptide (␤-alanyl-L-histidine) present in mammalian tissues, including the central nervous system, and has potential neuroprotective and neurotransmitter functions. In mammals, two types of L-carnosine-hydrolyzing enzymes (CN1 and CN2) have been cloned thus far, and they have been classified as metallopeptidases of the M20 family. The enzymatic activity of CN2 requires Mn 2؉ , and CN2 is inhibited by a nonhydrolyzable substrate analog, bestatin. Here, we present the crystal structures of mouse CN2 complexed with bestatin together with Zn 2؉ at a resolution of 1.7 Å and that with Mn 2؉ at 2.3 Å . CN2 is a homodimer in a noncrystallographic asymmetric unit, and the Mn 2؉ and Zn 2؉ complexes closely resemble each other in the overall structure. Each subunit is composed of two domains: domain A, which is complexed with bestatin and two metal ions, and domain B, which provides the major interface for dimer formation. The bestatin molecule bound to domain A interacts with several residues of domain B of the other subunit, and these interactions are likely to be essential for enzyme activity. Since the bestatin molecule is not accessible to the bulk water, substrate binding would require conformational flexibility between domains A and B. The active site structure and substrate-binding model provide a structural basis for the enzymatic activity and substrate specificity of CN2 and related enzymes.L-Carnosine (␤-alanyl-L-histidine) and structurally related dipeptides, such as homocarnosine (␥-aminobutyryl-L-histidine) and anserine (␤-alanyl-L-1-methylhistidine) are distributed in a wide variety of vertebrate tissues (1). L-Carnosine is present at particularly high concentrations in mammalian skeletal muscles and the brain, and it has been implicated in neuroprotection (2), the olfactory system (1), and hypothalamic neuronal networks (3). Our recent observations suggest that central and peripheral administration of L-carnosine at low doses attenuates 2-deoxyglucose-induced hyperglycemia (4) and suppresses peripheral sympathetic nerve activity (5, 6). These effects of L-carnosine are suppressed by central administration of thioperamide, a histamine H3 blocker. This suggests that L-carnosine regulates the autonomic nervous system via the hypothalamic histaminergic neurons (4 -6). In addition, the dipeptide exhibits antioxidant and free radical scavenger properties via complexation of transition metals, such as zinc and copper, suggesting that it is also involved in neuroprotection from oxidative stress (2, 8, 9). L-Carnosine is synthesized from ␤-alanine and L-histidine by carnosine synthetase and is degraded by intra-and extracellular dipeptidases known as carnosinases. Their enzymatic activities are regulated under various physiological conditions (10). Carnosinase was first isolated (11) from the porcine kidney in 1949 and was subsequently found to be widely distributed in tissues of rodents and higher mammals (12-15). Recently, two types of carnosinases were identified in humans and mice: h...

Background: Double-stranded RNA-activated protein kinase (PKR) is activated by virus-derived RNA and inhibits protein translation. Results: PKR is covalently modified by interferon-stimulated gene 15 (ISG15), and ISG15-PKR fusion protein is active without virus RNA. Conclusion: PKR is able to be activated by ISG15 modification. Significance: PKR might be an anti-tumor molecule by inhibiting protein translation in ISG15-positive cancer cells.

IntroductionAcute myeloid leukemia (AML) is a heterogeneous disease that is classified based on the presence of specific cytogenetic abnormalities as well as the French-American-British (FAB) classification of the leukemic cells and immunophenotype. One of the common translocations identified in leukemia is between chromosome 8q22 and chromosome 21q22 ( Figure 1a). 1 It is associated with nearly 40% of cases of FAB-M2 AML and 8% to 20% of all cases of AML depending on the genetic background and geographic location of the population. The (8;21) translocation is also observed in approximately 6% of AML M1 and, more rarely, in AML M0, M4, M5, and other myeloproliferative syndromes. 2,3 The involved genes are, on chromosome 8, MTG8 or ETO, meaning myeloid translocation gene or eight twenty-one, respectively, 4,5 and AML1 (acute myeloid leukemia factor 1) on chromosome 21. 4 The commonly used name for the t(8;21) fusion protein is AML1-MTG8 or AML1-ETO, and we refer to it as AML1-ETO in this review. AML1 was also discovered from other studies that are not related to t(8;21) and has several different names. 6 Its HUGO (Nomenclature Committee of the Human Genome Organization) name is RUNX1. In correlation, MTG8/ETO is named RUNX1T1 for RUNX1 translocation 1.The t(8;21) generates the 2 fusion genes AML1-ETO and ETO-AML1 ( Figure 1B). AML1-ETO mRNA is easily detectable using polymerase chain reaction (PCR) primers on 2 sides of the fusion point. However, ETO-AML1 mRNA was not identified using a similar approach (E. Kanbe, D.-E.Z., unpublished data, February 2003). This result indicates that the ETO-AML1 transcript is not expressed, is expressed at an extremely low level, or is highly unstable due to degradation. All of the studies on t(8;21) have therefore focused on AML1-ETO.Most of the coding region of the ETO gene is fused to the AML1 amino terminus containing the DNA-binding runt homology domain (RHD) to generate an AML1-ETO fusion protein ( Figure 1C). 4,5,7 The ETO gene has 14 exons. The original cloned AML1-ETO cDNA contained ETO exons 2 through 11; the fusion transcript produces an AML1-ETO protein of 752 amino acids ( Figure 1C). 8 The ETO portion of the full-length AML1-ETO protein contains 3 proline-serine-threonine (PST)-rich regions and 4 Nervy homology regions (NHR1-4) ( Figure 1C). 9 The PST-rich regions have multiple potential kinase phosphorylation sites (SP [Serine-Proline] and TP [Threonine-Proline]). Phosphorylation of ETO has been reported although no kinase involved in its phosphorylation has been identified. 10 NHR1, also called the TAF (TATA box binding protein associated factor) homology domain, shares a sequence similarity with TAF110 and other related TAFs. NHR2 has a hydrophobic amino acid heptad repeat, which is critical for ETO oligomerization. 11 NHR3 contains a predicted coiled-coil structure. NHR4 is a myeloid-Nervy-DEAF1 (MYND) homology domain with 2 predicted zinc finger motifs.Expression of the AML1-ETO fusion gene is under the control of the AML1 promoter. The AML1 gene has 2 promot...

A reciprocal translocation involving chromosomes 8 and 21 generates the AML1/ETO oncogenic transcription factor that initiates acute myeloid leukemia by recruiting co-repressor complexes to DNA. AML1/ETO interferes with the function of its wild-type counterpart, AML1, by directly targeting AML1 binding sites. However, transcriptional regulation determined by AML1/ETO probably relies on a more complex network, since the fusion protein has been shown to interact with a number of other transcription factors, in particular E-proteins, and may therefore target other sites on DNA. Genome-wide chromatin immunoprecipitation and expression profiling were exploited to identify AML1/ETO-dependent transcriptional regulation. AML1/ETO was found to co-localize with AML1, demonstrating that the fusion protein follows the binding pattern of the wild-type protein but does not function primarily by displacing it. The DNA binding profile of the E-protein HEB was grossly rearranged upon expression of AML1/ETO, and the fusion protein was found to co-localize with both AML1 and HEB on many of its regulated targets. Furthermore, the level of HEB protein was increased in both primary cells and cell lines expressing AML1/ETO. Our results suggest a major role for the functional interaction of AML1/ETO with AML1 and HEB in transcriptional regulation determined by the fusion protein.

The cellular endomembrane system requires the proper kinetic balance of synthesis and degradation of its individual components, which is maintained in part by a specific membrane fusion apparatus. In this study, we describe the molecular properties of D12, which was identified from a mouse expression library. This C-terminal anchored membrane protein has sequence similarity to both a yeast soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE), Use1p/Slt1p, and a recently identified human syntaxin 18-binding protein, p31. D12 formed a tight complex with syntaxin 18 as well as Sec22b and bound to ␣-SNAP, indicating that D12 is a SNARE protein. Although the majority of D12 is located in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartments at steady state, overexpression or knockdown of D12 had no obvious effects on membrane trafficking in the early secretory pathway. However, suppression of D12 expression caused rapid appearance of lipofuscin granules, accompanied by apoptotic cell death without the apparent activation of the unfolded protein response. The typical cause of lipofuscin formation is the impaired degradation of mitochondria by lysosomal degradative enzymes, and, consistent with this, we found that proper post-Golgi maturation of cathepsin D was impaired in D12-deficient cells. This unexpected observation was supported by evidence that D12 associates with VAMP7, a SNARE in the endosomallysosomal pathway. Hence, we suggest that D12 participates in the degradative function of lysosomes.

The distribution of palladium nanoparticles and their influence on the phase separation on a poly(2-vinylpyridine)-block-polyisoprene diblock copolymer were visualized by element spectroscopic imaging in the transmission electron microscope. High palladium nanoparticle concentration suppresses the microphase separation of the diblock copolymer, while a comparatively low palladium nanoparticle concentration still induces the microphase separation where the nanoparticles are exclusively located in the poly(2-vinylpyridine) microphase. A careful analysis of the palladium particle distribution revealed that the palladium particles tend to be located close to the interface of the two coexisting microdomains.