英国医学类留学生作业指导：Histone deacetylases (HDACs) Progress in

Tumorigenesis is a complex pathological process , affected by multiple factors, including individual genetic factors, environmental factors, physical and chemical factors , molecular biological factors and so on. With the rapid development of life sciences and related pathogenic mechanisms and pathogenesis of tumor molecular biology research in depth, molecular biological factors in tumorigenesis prominent role is gradually revealed , affects cell growth, proliferation and gene involved in cell growth and proliferation of a variety of factors regulating the inactivation of a tumor of the key incentives. With the further development of the life sciences , the pathogenesis of tumor molecular biology and pathogenesis research for the development of low toxicity and high specificity of molecular targets for anticancer drugs provide a basis [ 1 ] . Histone deacetylase (histone deacetylases, HDACs) is to maintain the basic unit of chromosome nucleosome histone acetylation balance in key enzymes , one of its catalytic histone deacetylation role in transcriptional repression and gene closely related gene silencing involves promoting the many process design is a popular anticancer drug target . In this paper, the role of epigenetic HDACs , classification and structure , and the relationship between tumor development are reviewed in order to provide for the subsequent anti-tumor research new ideas.
肿瘤的发生是一个复杂的病理过程，受多重因素的影响，涵盖个体遗传因素、背景因素、物理化学因素、分子生物科学因素等等。随着性命科学的迅疾进展和相关肿瘤致病机制和发病机制的分子生物科学研讨的深化开展，分子生物科学因素在肿瘤发生中的冒尖效用被逐层揭示，影响细胞成长、增生的基因和参加细胞成长增生调节控制的各种因数的失活变成肿瘤发生的关键诱因。

1HDACs epigenetic effect

Epigenetics (epigenetics) does not involve DNA sequence changes heritable changes in gene expression studies , in its many forms , the covalent modification of histones plays an important role , with the closely related gene expression and regulation , including phosphorylation , acetylation, methylation , etc. . Histone acetylation and deacetylation modification is the most important way , regulation of gene expression is the main driving force , this reversible dynamic modification of the histone acetyltransferase (HAT) and HDACs co- catalyst , jointly controlled each chromatin regional core histone acetylation degree . Histone acetylation and transcriptional activity is closely related to the degree : transcriptional activity of regional core histone acetylation high density, without active area acetylated low density . HAT induce chromosome depolymerization activates transcription ; while HDACs are closed DNA, thereby inhibiting the transcription process . Under normal physiological conditions , HAT and HDACs of the role of histone acetylation in a balanced state regulation . The cells in the state of conversion to occur , HDACs activity was significantly enhanced , so that the original gene expression balance is broken , resulting in some affect cell proliferation and cell cycle regulation molecule expression imbalance , leading to malignant cells . Epigenetic HDACs become important in the design of anticancer drugs and potential targets.
表观遗传基因学(epigenetics)是不牵涉到DNA序列变更的可遗传的基因表现变动研讨，在其好些个方式中，组蛋白的共价修饰霸占幢幢地位，其与基因的表现调节控制关系近关涉，涵盖磷酸化、乙酰化、甲基化修饰等。

2 HDACs classification and structural characteristics

2.1 Classification
Yeast-based phylogenetic structure of the different HDACs homology analysis , eukaryotic HDACs are divided into four categories : Ⅰ class HDACs homologous to yeast Rpd3 , including HDAC1, HDAC2, HDAC3, HDAC8; Ⅱ class HDACs and yeast Hda1 homology , including HDAC4, HDAC5, HDAC6, HDAC7, HDAC9 and HDAC10, which differ according to the catalytic region can be divided into two categories : Ⅱ a class has a catalytic domain , including HDAC4, HDAC5, HDAC7 and HDAC9, Ⅱ b class with two catalytic domains , including HDAC6 and HDAC10; Ⅲ class HDACs that silent information regulator factor 2 (silent information regulator2, Sir2) - related enzymes (Sir2-related enzymes, sirtuins), which is nicotinamide adenine dinucleotide acid (nicotinamide adenine dinucleotide, NAD +) -dependent histone deacetylase class ; Ⅳ class HDACs mainly HDAC11, with Ⅰ, Ⅱ class was quite different HDACs placed under a separate category . HDACs family members mainly localized in the nucleus and the cytoplasm , and another small portion located in the cytoplasmic organelles such as mitochondria ( mostly Class Ⅲ HDACs in SIRT3, SIRT4 with SIRT5), HDACs play a catalytic role in a wide range of target proteins , common such as tumor suppressor protein p53, heat shock protein HSP70, Smads proteins , etc. , HDACs is through a variety of physiological processes and catalytic key proteins in the regulation of tumor processes play an important role .

2.2 Structural Features
2.2.1 Ⅰ class HDACs Ⅰ class of HDACs HDAC1 has been studied confirmed that its phosphorylation can promote the catalytic activity of the enzyme itself and the catalytic complex formation , phosphorylation occurs in HDAC1 in 421 serine sites Ser421 and Ser423 sites serine 423 . The use of K-trap (K- tartrate-resistant acid phosphatase ) affinity purified resin HDAC1 , SDS-PAGE ( polyacrylamide gel electrophoresis ) separated K-trap purified HDAC1, Coomassie blue staining proteins using mass spectrometry identification of phosphorylated group , showing the size of HDAC1 molecular weight markers were 75 000,66 000 , other kinds of class Ⅰ HDACs that HDAC2, HDAC3 HDAC8 protein identification and analysis applications Clustal method completed , HDAC1, HDAC2, HDAC8 respectively, by 482,488 , 377 kinds of amino acids .

2.2.2 Ⅱ class HDACs
2.2.2.1 Ⅱ a class of HDACs study confirmed [10], Ⅱ a class of HDACs a conserved zinc- binding domain - HDAC4 catalytic sequences distinguish class Ⅰ HDACs are characterized by a zinc ion binding domain , which accommodates two kinds of separation from the center of the molecule the protein fragment ( Figure 1A). First a fragment formed a 17 - amino acid ring (Lα1-α2), which contributed three zinc ions ligands : His665, Cys667, and His678 ( Figure 1A). The second fragment is one containing 35 -residue insert (α6-α7-β3-β4), the formation of a helical structure , including its tip followed by a fourth ligand Cys751 of zinc β- hairpin . It is worth noting that four zinc ligands in all Ⅱ a highly conserved class of HDACs in both , but in other types of HDACs missing . This zinc- binding domain and a ring structure is different compared to the point of causing HDAC4 HDAC8 HDAH have a more active and active site (Figure 1B, C). Meanwhile, in HDAC8, HDLP and HDAH acetate catalyst does not exist in the aqueous reaction product of the release passage ( Figure 1B, C).

Cell analysis results showed that the zinc- binding domain located Cys669 and Cys700 from between neighboring molecules , formation of disulfide bonds within a molecule . To exclude this disulfide inhibitors and active sites on the interatomic potential impact on wild- type (WT type ) HDAC4 and HDAC4 catalytic sequence of functional mutants (GOF-HDAC4cd) ( including two mutants containing C669A/C700A single mutant and C700A ) inhibitor structures were conducted demonstration shows the structure of the mutant without the corresponding wild-type and mutant disordered state excluding zinc binding domain mutants same . Reference HDAC4cd- inhibitor complexes in the spatial domain structure of bent and folded , and an intramolecular disulfide with some parts have been in its closed .

2.2.2.2 Ⅱ b class HDACs research has shown [11], Ⅱ b HDAC10 is a class of HDACs containing 669 amino acid residues of the polypeptide from the N-terminal sequence of deacetylation Hda1 associated with the C-terminal leucine rich sequence of modular structures. Shown in Figure 2 , HDAC10 sequence of deacetylation (DAC) indicated by white frames , leucine rich sequence (LRD) to clarify the shaded box . Another deacetylation HDAC10 HDAC6 sequence and a region corresponding to the same degree of similarity are also reflected in the figure , the two acetylated HDAC10 sequence corresponding to the sequence of HDAC6 same / similarity of 54 % / 62% and 53% / 60% .

2.2.3 Ⅲ class of HDACs mammals have seven kinds of Sir2-related enzymes, sirtuins, namely Class Ⅲ HDACs, including SIRT1-7. All members contain a NAD + -dependent catalytic core sequence , which plays a single -ADP- ribosyl transferase (mono-ADP-ribosyl transferase (ART)) , or NAD + -dependent deacetylase (DAC) of the role , this catalytic variable length sequence ends around the N-terminal and C-terminal sequences in the cell type of the positioning of different sirtuins . Figure 3 shows , SIRT1 activity with SIRT5 has only DAC , SIRT4 with SIRT6 has only ART activity, while SIRT2 with SIR3 DAC with ART has two active ; in cell positioning , SIRT1 mainly located in the euchromatin in , SIRT2 important positioning in the cytoplasm , SIRT3-SIRT5 are mainly located in the mitochondria , while SIRT7 is located in the nucleolus [ 12 ] .

2.2.4 Ⅳ class HDACsGao [13 ] the use of the human cDNA clone HDAC11 predicted amino acid sequence reveals HDAC11 has 347 amino acid open reading frame ( corresponding to a molecular weight of 39 000 ) . Experts predict HDAC11 gene database gene is located on human chromosome 3p25.2 , spanning the length of 25 kb, contains nine exons and eight introns at the amino acid sequence comparison shows the level : HDAC11 whole proteins with known family members of HDACs (HDAC1-HDAC10) only minor homology ( including yeast HOS3 protein ) , but HDAC11 contains functions for deacetylation potentially important role in all nine parts conserved sequence , HDAC11 and other HDACs family members together reveals a putative catalytic core sequence , this sequence may include almost all of HDACs in eukaryotes and prokaryotes, HDAC- like protein present in the catalytic activity of the same sequence.

3HDACs relationship with tumor development

HDACs different types of acetylated nuclear transcription factors and protein , inhibition of the expression of multiple tumor suppressor protein , and with a variety of closely related oncogene , resulting cell proliferation and tumorigenesis [ 14 ] .

3.1 -induced chromatin remodeling , inhibition of gene transcription
HDACs catalytic core histone N-terminal tail region lysine residues in the induction of deacetylation play an important role in gene silencing , which went through the histone acetylation modification with a positive charge , and thus with the negatively charged DNA closely combination of dense chromatin structure curly inhibition , inhibition of transcription [ 15 ] . Study confirmed , t (15; 17) (q22; q21) in acute promyelocytic leukemia (APL) of the most common chromosomal translocation encoding PML-RARα fusion protein can inhibit RA abnormal reaction proceeds HDACs gene transcription , causes myeloid cell maturation disorder. RAR is a nuclear hormone receptor superfamily , with the RA receptor RXR another form RAR / RXR heterodimers , heterodimers bind to DNA to be able to raise a transcriptional repressor complex N-CoR-mSin3-HDAC inhibition of RA response genes [ 16 ] , which led to RA response genes silenced . Class Ⅰ HDACs in HDAC1 and HDAC2 and RbAp48, Sin3A/Sin3B, SAP18, SAP30 together constitute Sin3 complexes play a role , TGF-β/BMP signaling pathway genes that this compound as a target . BMP signaling system can cause Smad1 protein phosphorylation in mouse chondrocytes which play a role in prompting and Smad4 protein , Sin3 complexes at this time that the role of Smad proteins and inhibit the signaling system TGF-β/BMP target genes [ 17 ] .

3.2 role in cell cycle -related factors affecting cell proliferation and differentiation
Tumor usually presents information on loss of normal cell cycle regulation and in the disordered state of disorder . In the cell cycle , cell G1 to S phase progression and G2 phase to M phase of the cell cycle, two restriction point (restrict point), abnormal proliferation of tumor cells smoothly requires the following three conditions: First p21WAFI/CIP1 inhibitory factor ( an important cyclin-dependent kinase inhibitor CDKI) low ; Second cyclins (cyclin) and cyclin-dependent kinases (cyclin-dependent kinase, CDK) and the kinase activity of the series , such as CDK2 ; three human retinoblastoma gene (RB) and phosphorylation levels sufficiently active because RB is the G1 phase cyclin / CDK substrate specificity [ 18 ] . HDACs can affect more abnormal proliferation of tumor cells acting factor of three kinds of tumor cell proliferation and differentiation of a significant impact.

3.2.1 inhibiting p21WAFI/CIP1 gene expression, the regulation of tumor cell proliferation and differentiation Wilson et al [ 19 ] illustrates an important member of Class Ⅱ HDACs HDAC4 hyperplasia site in the small intestine and cecum expression was significantly reduced during their differentiation in vitro cloning of cancer HCT116 SiRNA interference in the use of reduced HDAC4 expression was found to induce growth inhibition , delay the growth of xenograft tumors grafted increased P21 transcription. In addition , the use of co-immunoprecipitation and sequencing analysis to clarify the role of co-immunoprecipitation with P21 neighboring HDAC4 dependent Sp1 promoter binding , possibly directly through HDAC4-HDAC3-N-COR/SMRT of repressor complexes to complete, while the reduction in HDAC4 or SMRT improve the nearby loci P21 promoter histone H3 acetylation , HDAC4 confirmed by inhibiting the expression of P21 clone a new type of cell proliferation regulatory factors . In addition , depending on the P21 SP family of negative regulatory role has proven performance in class Ⅰ HDACs ( eg HDAC1, HDAC2 and HDAC3) and other molecular biological functions [ 20 ] .

3.2.2 The role of cell cycle proteins and protein kinases affect tumor development Rampalli , etc. [ 21 ] found that breast cancer cell lines SMAR1 ( one matrix- associated protein ) 160-350 loci by recruiting a complex role and HDAC1 physical structure -SIN3 and bags retinoblastoma protein binding , the new complexes formed by cyclin D1 promoter up to 5 kb upstream of loci exert deacetylation inhibition of cyclin D1 promoter , and found that this breast cancer cell lines induced expression of cyclin D1 high and decreased levels of SMAR1 were significantly associated ; while Iguchi , etc. [ 22 ] in insulinoma cells using chromatin immunoprecipitation assay showed : Growth of sex-determining region Y-box 6 ( i.e. SOX6) could induce the expression of cyclin D1 promoter H3 and H4 acetylation level , with HDACs inhibitors and immunoprecipitation analysis SOX6 with HDAC1 and β- Lianzhu action and inhibit cyclin D1 protein activity. Description Class Ⅰ HDACs important member of HDAC1 in influencing cell cycle has an important role.

3.2.3 Rb gene interactions affect tumor cell cycle Siddiqui , etc. [ 23 ] found that the activation of the Rb gene protein product of retinoblastoma gene suppressor protein (RB) transfer mediated cyclin A, cdc2, topoisomerase Ⅱ α , etc. promoter deacetylation state , and this deacetylation dependent HDACs play a role , not only confirmed that RB binding transcription factor E2F , and through LXCXE motifs and multiple co-repressor molecules ( such as HDACs) interaction [ 24 ] .

3.3 effect on apoptosis-related proteins , affecting the process of apoptosis
Escaffit , etc. [ 25 ] found that in Jurkat cells ( human T-cell lymphoma cells ) in , Ⅰ class of HDACs as a variety of pro-apoptotic genes HDAC3 nuclear repressor , its easy in a cell and species -independent proteolytic cleavage the way , this depends on the half- split caspases (caspase) and results in the C-terminal portion of HDAC3 deletion . HDAC3 activation of such a division of the anti-apoptotic target genes in HDAC3 -Fas gene encoding histone acetylation and transcriptional activity , and then by death receptor -induced apoptosis of Jurkat cells pathway . Another Zhu et al [ 26 ] found that , HDAC2 in most clones APC tumor suppressor genes with mice that lack normal mucosa and adenomas expressed at high levels , due to the absence of APC clone HT-29 tumor cells, small RNA (siRNA) for high expression of HDAC2 interference effect may be sufficient to induce apoptosis , indicating that this isoenzyme (HDAC2) in inhibiting tumor clone HT-29 cell apoptosis plays a special role .

3.4 Joint angiogenic factors affecting migration and the formation of tumor blood vessels
Ha , etc. [ 27 ] found that vascular endothelial growth factor (VEGF) in endothelial cells through one kind VEFG receptor 2 - phospholipase Cγ- protein kinase C (PKC) - protein kinase D (PKD) dependent pathway stimulation in Class Ⅱ HDACs the HDAC5 phosphorylation and nuclear output, PKD -dependent phosphorylation of HDAC5 in a specific deletion mutants inhibit VEGF -mediated NR4A1 ( an angiogenesis in oligonucleotide receptor ) expression of endothelial cell migration and in vitro angiogenesis . The use of siRNA technology to silence HDAC5, found fibroblast growth factor 2 (FGF2) and vascular growth , including Slit2 including guiding factor expression was inhibited , indicating HDAC5 also on endothelial cells by inhibiting capillary style " sprouting " play an important the role of angiogenic genes ( such as FGF2 and Slit2) exert anti-angiogenic effects [ 28 ] .

Among the many endogenous angiogenic substances , VEGF and tumor angiogenesis are closely related . Zhu Fang , etc. [ 29 ] found that : VEGF low expression cell lines do not appear vasculogenic mimicry phenomena , VEGF high expression cell lines may not appear vasculogenic mimicry phenomenon, and vascular mimicry phenomena cell line expression of VEGF high, indicating VEGF and angiogenesis, the formation of a certain relationship between mimicry , only cells with high expression of VEGF in the formation of vascular mimicry have potential. HDACs influence through interaction with VEGF vascular migration and formation .

Wang et al [ 30 ] also found that the HDAC7 Class Ⅱ HDACs via PKC / PKD dependent pathway completed by the VEGF -mediated phosphorylation and nuclear export , such HDAC7 mediated by VEGF molecule reactions for VEGF-induced signaling pathway and migration of endothelial cells, is required , by inhibiting muscle cell -dependent or not dependent promoting factor 2 (MEF2) gene expression and regulation of endothelial cell function [ 31 ] ; addition , Ⅱ class HDAC4, HDAC5 may also be mediated by VEGF complete phosphorylation, its nuclear output process may completely dependent on VEGF completed , suggesting that it may act on different targets in the VEGF-induced angiogenesis plays a role [ 28 ] .

Furthermore , Hait et al [ 32 ] found that HDACs are S1P ( sphingosine phosphonate ) direct intracellular target , which was originally present in the nucleus is a biologically active lipid messenger , the sphingosine kinase type 2 (sphK2) production. SphK2 selective gathered in encoding cyclin-dependent kinase inhibitor p21 or the transcription factor c-fos gene promoter . S1P through specific binding HDAC1 and HDAC2, inhibiting its activity , thereby protecting histidine, lysine is not the end of deacetylation , thereby increasing histone H3 acetylation levels , and promote transcription .

4 Prospects

HDACs as a catalytic role of histone deacetylation key enzymes involved in tumor cell growth and proliferation of its expression and regulation , and many other processes and in the development of epigenetic cancer research increasingly attracted academic attention and attention. Current studies focus on the existing HDACs inhibitory activity with chemical modification of anticancer drugs and structural reform in order to enhance its efficacy and reduce side effects , etc., and from the structural characteristics and biological function of HDACs starting design itself acting on specific target point with a specific pathway of anticancer drugs is still a minority. With the structural biology and computer-aided drug design and other disciplines, development, targeting HDACs develop novel anti-tumor activity of HDACs inhibitors bound to have broad prospects .