Suppression of centrosome duplication and amplification by deacetylases

Centrosome duplication is controlled both negatively and positively by a number of proteins. The activities and stabilities of those regulatory proteins are in many cases controlled by posttranslational modifications. Although acetylation and deacetylation are highly common posttranslational modifications, their roles in the regulation of centrosome duplication had not been closely examined. Here, through focusing on the deacetylases, we investigated the role of acetylation/deacetylation in the regulation of centrosome duplication and induction of abnormal amplification of centrosomes. We found that the deacetylation event negatively controls centrosome duplication and amplification. Of the 18 total known deacetylases (HDAC1–11, SIRT1–7), ten deacetylases possess the activity to suppress centrosome amplification, and their centrosome amplification suppressing activities are strongly associated with their abilities to localize to centrosomes. Among them, HDAC1, HDAC5 and SIRT1 show the highest suppressing activities, but each of them suppresses centrosome duplication and/or amplification with its unique mechanism.     

Centrosome Function: Sometimes Less Is More

Tight regulation of centrosome duplication is critical to ensure that centrosome number doubles once and only once per cell cycle. Superimposed onto this centrosome duplication cycle is a functional centrosome cycle in which they alternate between phases of quiescence and robust microtubule (MT) nucleation and MT-anchoring activities. In vertebrate cycling cells, interphase centrioles accumulate less pericentriolar material (PCM), reducing their MT nucleation capacity. In mitosis, centrosomes mature, accumulating more PCM to increase their nucleation and anchoring capacities to form robust MT asters. Interestingly, functional cycles of centrosomes can be altered to suit the cell's needs. Some interphase centrosomes function as a microtubule-organizing center by increasing their ability to anchor MTs to form centrosomal radial arrays. Other interphase centrosomes maintain their MT nucleation capacity but reduce/eliminate their MT-anchoring capacity. Recent work demonstrates that Drosophila cells take this to the extreme, whereby centrioles lose all detectable PCM during interphase, offering an explanation as to how centrosome-deficient flies develop to adulthood. Drosophila stem cells further modify the functional cycle by differentially regulating their two centrioles – a situation that seems important for stem cell asymmetric divisions, as misregulation of centrosome duplication in stem/progenitor cells can promote tumor formation. Here, we review recent findings that describe variations in the functional cycle of centrosomes.     

组蛋白去乙酰化酶4与人类疾病

组蛋白去乙酰化酶4（histone deacetylase 4,HDAC4）是一类依赖锌的去乙酰化酶,属于Ⅱ类组蛋白去乙酰化酶（histone deacetylases,HDACs）,主要具有去乙酰化酶的活性。HDAC4由去乙酰化酶结构域发挥去乙酰化酶的作用,还具有核定位序列和核输出序列,通过转录后与翻译后水平的修饰可在细胞核和细胞质之间穿梭,进而参与多种调节过程。近年来的研究发现,HDAC4可参与基因的转录调控、细胞凋亡、代谢等诸多生物进程,在多种疾病的发生发展中发挥重要作用。本文主要从HDAC4的结构、去乙酰作用、自身的修饰及其在核浆中的穿梭作用对其进行概述,同时对其在骨关节炎、心血管疾病、肌萎缩性侧索硬化症等不同疾病中的作用、相关的分子机制及组蛋白抑制剂在肿瘤中的应用等方面的研究进展进行综述。     

Regulation of ERα Signaling Pathway in Neuronal HN10 Cells: Role of Protein Acetylation and Hsp90

Estrogen has a variety of neuroprotective effects but the molecular basis of its function is still mainly unclear. Estrogen receptor (ER) signaling is highly dependent on posttranslational modifications and the assembly of coactivator and corepressor complexes. Several proteins involved in ERα signaling have recently been found to be acetylated, including ERα itself and Hsp90, a key chaperone in the functional regulation of ERα. ERα complexes also contain histone deacetylases (HDAC) which repress transactivation. Our purpose was to clarify the role of protein acetylation and Hsp90 function in the ERE-mediated ERα signaling in neuronal HN10 cells. We observed that increasing protein/histone acetylation status with trichostatin A, a potent HDAC inhibitor, increased the 17β-estradiol (E2)-induced transactivation of ERE-driven luciferase in non-transfected cells, and even more extensively in pERα-transfected cells. E2-induced ERE-driven transactivation was blocked by ICI 182.780. Several ER antagonists, such as raloxifene and tamoxifen, were unresponsive. Valproate, an antiepileptic drug which is recently characterized as a HDAC inhibitor, was also able to potentiate the E2-induced ERE-transactivation. Inhibition of the function of Hsp90 chaperone with geldanamycin strongly inhibited the E2-induced ERE-transactivation. Overexpression of SIRT2 protein deacetylase did not inhibit the acetylation-potentiated ERE-driven transactivation indicating that SIRT2 deacetylase is not involved in ERα signaling. Our results reveal that ERα signaling is dependent on protein acetylation and epigenetic regulation.     

Multiple histone deacetylases and the CREB-binding protein regulate pre-mRNA 3'-end processing

Trichostatin A (TSA), a specific inhibitor of histone deacetylases (HDACs), induces acetylation of various non-histone proteins such as p53 and alpha-tubulin. We purified several acetylated proteins by the affinity to an anti-acetylated lysine (AcLys) antibody from cells treated with TSA and identified them by mass spectrometry. Here we report on acetylation of CFIm25, a component of mammalian cleavage factor Im (CF Im), and poly(A) polymerase (PAP), a polyadenylating enzyme for the pre-mRNA 3'-end. The residues acetylated in these proteins were mapped onto the regions required for interaction with each other. Whereas CBP acetylated these proteins, HDAC1, HDAC3, HDAC10, SIRT1, and SIRT2 were involved in in vivo deacetylation. Acetylation of the CFIm25 occurred depending on the cleavage factor complex formation. Importantly, the interaction between PAP and CF Im complex was decreased by acetylation. We also demonstrated that acetylation of PAP inhibited the nuclear localization of PAP by inhibiting the binding to the importin alpha/beta complex. These results suggest that CBP and HDACs regulate the 3'-end processing machinery and modulate the localization of PAP through the acetylation and deacetylation cycle.     

综述: 组蛋白去乙酰化酶的结构及应用

组蛋白赖氨酸乙酰化是目前研究最为广泛和深入的组蛋白翻译后修饰之一,在染色质重塑和基因表达调控等方面发挥重要作用,这种修饰在体内受到组蛋白乙酰化酶和去乙酰化酶的高度动态调控．除了以组蛋白为底物外,组蛋白去乙酰化酶还可以催化多种非组蛋白的去乙酰化,参与多种生命过程的调节．本文围绕四类人源组蛋白去乙酰化酶,综述了其分类依据、结构与功能特点、催化反应的分子机制,以及针对这些组蛋白去乙酰化酶的抑制剂和激动剂的开发和应用等方面的研究进展．     

An enzyme-coupled assay measuring acetate production for profiling histone deacetylase specificity

Histone deacetylases catalyze the hydrolysis of an acetyl group from post-translationally modified acetyl-lysine residues in a wide variety of essential cellular proteins, including histones. Because these lysine modifications can alter the activity and properties of affected proteins, aberrant acetylation/deacetylation may contribute to disease states. Many fundamental questions regarding the substrate specificity and regulation of these enzymes have yet to be answered. Here, we optimize an enzyme-coupled assay to measure low micromolar concentrations of acetate, coupling acetate production to the formation of NADH (nicotinamide adenine dinucleotide, reduced form) that is measured by changes in either absorbance or fluorescence. Using this assay, we measured the steady-state kinetics of peptides representing the H4 histone tail and demonstrate that a C-terminally conjugated methylcoumarin enhances the catalytic efficiency of deacetylation catalyzed by cobalt(II)-bound histone deacetylase 8 [Co(II)–HDAC8] compared with peptide substrates containing a C-terminal carboxylate, amide, and tryptophan by 50-, 2.8-, and 2.3-fold, respectively. This assay can be adapted for a high-throughput screening format to identify HDAC substrates and inhibitors.     

Involvement of poly(ADP-Ribose) polymerase 1 and poly(ADP-Ribosyl)ation in regulation of centrosome function

The regulatory mechanism of centrosome function is crucial to the accurate transmission of chromosomes to the daughter cells in mitosis. Recent findings on the posttranslational modifications of many centrosomal proteins led us to speculate that these modifications might be involved in centrosome behavior. Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes poly(ADP-ribosyl)ation to various proteins. We show here that PARP-1 localizes to centrosomes and catalyzes poly(ADP-ribosyl)ation of centrosomal proteins. Moreover, centrosome hyperamplification is frequently observed with PARP inhibitor, as well as in PARP-1-null cells. Thus, it is possible that chromosomal instability known in PARP-1-null cells can be attributed to the centrosomal dysfunction. P53 tumor suppressor protein has been also shown to be localized at centrosomes and to be involved in the regulation of centrosome duplication and monitoring of the chromosomal stability. We found that centrosomal p53 is poly(ADP-ribosyl)ated in vivo and centrosomal PARP-1 directly catalyzes poly(ADP-ribosyl)ation of p53 in vitro. These results indicate that PARP-1 and PARP-1-mediated poly(ADP-ribosyl)ation of centrosomal proteins are involved in the regulation of centrosome function.     

Nepsilon-thioacetyl-lysine: a multi-facet functional probe for enzymatic protein lysine Nepsilon-deacetylation

Peptides containing Nepsilon-thioacetyl-lysine and Nepsilon-acetyl-lysine were evaluated for their de(thio)acetylation catalyzed by human HDAC8 and SIRT1, two distinct protein deacetylases. Nepsilon-Thioacetyl-lysine was found to be a mimic of Nepsilon-acetyl-lysine for HDAC8-catalyzed reaction, but to confer inhibition against SIRT1. These results point to further diverse applications based on Nepsilon-thioacetyl-lysine.     

Daughter centrioles assemble preferentially towards the nuclear envelope in Drosophila syncytial embryos

Centrosomes are important organizers of microtubules within animal cells. They comprise a pair of centrioles surrounded by the pericentriolar material, which nucleates and organizes the microtubules. To maintain centrosome numbers, centrioles must duplicate once and only once per cell cycle. During S-phase, a single new ‘daughter’ centriole is built orthogonally on one side of each radially symmetric ‘mother’ centriole. Mis-regulation of duplication can result in the simultaneous formation of multiple daughter centrioles around a single mother centriole, leading to centrosome amplification, a hallmark of cancer. It remains unclear how a single duplication site is established. It also remains unknown whether this site is pre-defined or randomly positioned around the mother centriole. Here, we show that within Drosophila syncytial embryos daughter centrioles preferentially assemble on the side of the mother facing the nuclear envelope, to which the centrosomes are closely attached. This positional preference is established early during duplication and remains stable throughout daughter centriole assembly, but is lost in centrosomes forced to lose their connection to the nuclear envelope. This shows that non-centrosomal cues influence centriole duplication and raises the possibility that these external cues could help establish a single duplication site.     

HDACs, histone deacetylation and gene transcription： from molecular biology to cancer therapeutics

Histone deacetylases （HDACs） and histone acetyl transferases （HATs） are two counteracting enzyme families whose enzymatic activity controls the acetylation state of protein lysine residues, notably those contained in the N-terminal extensions of the core histones. Acetylation of histones affects gene expression through its influence on chromatin conformation. In addition, several non-histone proteins are regulated in their stability or biological function by the acetylation state of specific lysine residues. HDACs intervene in a multitude of biological processes and are part of a multiprotein family in which each member has its specialized functions. In addition, HDAC activity is tightly controlled through targeted recruitment, protein-protein interactions and post-translational modifications. Control of cell cycle progression, cell survival and differentiation are among the most important roles of these enzymes. Since these processes are affected by malignant transformation, HDAC inhibitors were developed as antineoplastic drugs and are showing encouraging efficacy in cancer patients.     

Centrosome-associated NDR kinase regulates centrosome duplication

Human NDR kinases are upregulated in some cancer types, yet their functions still remain undefined. Here, we report the first known function of a mammalian NDR kinase by demonstrating that human NDR directly contributes to centrosome duplication. A subpopulation of endogenous NDR localizes to centrosomes in a cell-cycle-dependent manner. Overexpression of NDR resulted in centrosome overduplication in a kinase-activity-dependent manner, while expression of kinase-dead NDR or depletion of NDR by small interfering RNA (siRNA) negatively affected centrosome duplication. By targeting NDR to the centrosome, we show that the centrosomal pool of NDR is sufficient to generate supernumerary centrosomes. Furthermore, our data indicate that NDR-driven centrosome duplication requires Cdk2 activity and that Cdk2-induced centrosome amplification is affected upon reduction of NDR activity. Overall, considering that centrosome overduplication is linked to cellular transformation, our observations may also provide a molecular link between mammalian NDR kinases and cancer.     

Role of histone deacetylase 2 and its posttranslational modifications in cardiac hypertrophy

Cardiac hypertrophy is a form of global remodeling, although the initial step seems to be an adaptation to increased hemodynamic demands. The characteristics of cardiac hypertrophy include the functional reactivation of the arrested fetal gene program, where histone deacetylases (HDACs) are closely linked in the development of the process. To date, mammalian HDACs are divided into four classes: I, II, III, and IV. By structural similarities, class II HDACs are then subdivided into IIa and IIb. Among class I and II HDACs, HDAC2, 4, 5, and 9 have been reported to be involved in hypertrophic responses; HDAC4, 5, and 9 are negative regulators, whereas HDAC2 is a pro-hypertrophic mediator. The molecular function and regulation of class IIa HDACs depend largely on the phosphorylation-mediated cytosolic redistribution, whereas those of HDAC2 take place primarily in the nucleus. In response to stresses, posttranslational modification (PTM) processes, dynamic modifications after the translation of proteins, are involved in the regulation of the activities of those hypertrophy-related HDACs. In this article, we briefly review 1) the activation of HDAC2 in the development of cardiac hypertrophy and 2) the PTM of HDAC2 and its implications in the regulation of HDAC2 activity. [BMB Reports 2015; 48(3): 131-138]     

Centrosome abnormalities during a Chlamydia trachomatis infection are caused by dysregulation of the normal duplication pathway

The presence of supernumerary centrosomes in cells infected with Chlamydia trachomatis may provide a mechanism to explain the association of C. trachomatis genital infection with cervical cancer. We show that the amplified centrosomal foci induced during a chlamydial infection contain both centriolar and pericentriolar matrix markers, demonstrating that they are bona fide centrosomes. As there were multiple immature centrioles but approximately one mature centriole per cell, aborted cytokinesis alone cannot account for centrosome amplification during a chlamydial infection. Production of supernumerary centrosomes required the kinase activities of Cdk2 and Plk4, which are known regulators of centrosome duplication, and progression through S-phase, which is the stage in the cell cycle when duplication of the centrosome occurs. These requirements indicate that centrosome amplification during a chlamydial infection depends on the host centrosome duplication pathway, which normally produces a single procentriole from each template centriole. However, C. trachomatis induces a loss of numerical control so that multiple procentrioles are formed per template.