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Bromodomain 4 protein 1

Information about the gene of the protein including mRNA isoforms generated and genes that are conserved in different species

Bromodomain(BRD4) is a pleiotropic protein that is a member of the bromodomain and extra terminal (BET) that preferentially binds to chromatin of an acetylated lysine residue which is one of the main modification that occurs at the histone tails and has three other isoforms in mammals namely BRD2, BRD3, BRDT proteins. Bromodomains of BRD4 are commonly found in chromatin binding proteins for example HATs, TAFI, CBP/p300 and remodelling factors such as SWI and SNF, located in tandem within the N-terminus and mediates binding of the BRD4 to chromatin via interaction with the acetylated lysine on histone H3 and H4. BET functions as cell cycle regulators where the BRD4 regulates the expression of genes required for M to early G1 phase transition. There is evidence of a functional relationship between the human Brd and Notch gene families when considering the positions of the human Brd genes i.e. Brd 2 and Notch 4 on chromosome 6, Brd 4 and Notch 3 on chromosome 19, etc. the domain organisation of the mammalian Brd 4 protein is conserved and extends to homologues in most other species. Majority of the detected biological activity accounted for in humans and mouse proteins isoforms is usually the long form of the Brdt4, whereas the short forms functionality is still undefined when expressed while a fusion protein of human BRD4 linked to the nuclear protein in testis (Nut) has been reportedly found in some epithelial carcinomas [2]. A crystal structure of human Brd2 BDI reveals a formation of a BDI homodimer which contains two acetyl-lysine-binding pockets and a negatively charged secondary binding pocket formed at the dimer interface which most likely helps in the determination of the binding specificity between bromodomains and different acetyl-lysine residues, the secondary pocket may also an independent surface for acetyl-lysine-independent bromodomain-histone interactions and show that of a bromodomain in a BET protein does not necessarily eliminate its chromatin binding and transcriptional activity. The amino acids involved in human Brd4 homodimeric interactions include, but not limited to Gln-62, Met-126, Gln-127, etc. While in mouse the Brd4 has been observed interacting with multiple cellular and viral proteins including the cyclin T1 component of the human positive transcription elongation factor b (P-TEFb) with CDK9 which promotes mRNA transcriptional elongation through phosphorylation of elongation repressors and RNA polymerase 2, and many other proteins [1].

Structure and post-translational modifications of the protein

Bromodomain 4 protein structure consist of 1362 amino acids with two bromodomain(BD1 and BD2), two conserved motifs(A and B), N-terminal cluster of phosphorylation site, a basic residue-enriched interaction domain, an ET domain, a SEED Domain enriched in serine residues mixed with glutamic and aspartic acid and CTM ( C-terminal interaction motif). The two domains consist of the Archetypal bromodomain folds comprised of two interconnected loops (ZA AND BC) from their N and C terminal regions[10]. Each consist of 4 alpha helices (Z, A, B AND C) which forms a pocket that encircle the recognition site for acetylated lysine’s. The 4 helices create a left handed helical hydrophobic core in each domain of which domain 1 (BD1) forms an intact homodimer and bromodomain-4 protein dimerizes through it[9].

Function of the protein and its role in cell signalling

Bromodomain 4 protein (BRD4) is essential for cellular growth and has been implicated in cell cycle control, transcription, differentiation and carcinogenesis [3,4]. In cell cycle control it is necessary for mitosis to proceed normally [ 4]. It plays a role in transcription as a scaffold for various transcription factors and chromatin, a nucleator of superenhancers, regulation of transcription elongation via the kinase activity and remodelling of chromatin via the HAT activity [4]. Lastly in carcinogenesis it is a therapeutic target because of its regulation of cell cycle genes, especially MYC [4]. BRD4 have a role in targeting chromatin-modifying enzymes to specific sites. These enzymes often act with other protein-interaction modules to guarantee a high level of targeting specificity for these essential enzymes [6]. BRD4 has a double bromodomains that fuse with the NUT protein and may change the gene expression profile to contribute to NMC (NUT midline carcinoma) tumorigenesis [5].

Alternative splicing of the BRD4 gene gives rise to a short 722- amino acid isoform C that retains the 2 bromodomains but lacts all the regions C- terminal to the SEED domain and this isoforms occurs in two forms, the long and the short isoforms of BRD4. The abundance of this isoforms vary within different cell types Isoform B and C.



Its role in disease

BET proteins, particularly BRD4, plays a critical role in many haematological and solid tumours, by acting as coactivators for the expression of proliferative genes. Proliferation is defined as growth or production of cells in an organism. Controlling proliferation, which is achieved by the genes of a cell, maintains the homeostasis of the body. Proliferative genes such as proto-oncogenes, promote cell division and facilitates growth. Antiproliferative genes, on the other hand, inhibit proliferation and are important in limiting positive cell growth. Notably, BRD4 is required for the expression of myelocytomatosis (myc); an oncogenic driver in many cancers. In a rare, but more aggressive form of squamous cell carcinoma known as Nuclear Protein in Testis (NUT) midline carcinoma, BRD4, or more occasionally BRD3, are fused in frame to the NUT gene of unknown function. This results in a fusion that is controlled by the BRD4 promoter and encodes the two bromodomains and ET domain of BRD4, and almost the entire coding region of NUT. BET proteins, particularly BRD4, plays a critical role in many haematological and solid tumours, by acting as coactivators for the expression of proliferative genes. Notably, BRD4 is required for the expression of myelocytomatosis (MYC); an oncogenic driver in many cancers [13].

P-TEF b (with CDK9) promotes mRNA transcriptional elongation through phosphorylation of elongation repressors and RNA polymerase 2. Structures of CDK9/Cyclin T1 and free Cyclin T2 have been determined. Upon determination, there are notably distinct differences between CDK9/CyclinT1 and CDK2/Cyclin A (the cell cycle CDK) manifested by a relative rotation of 26°C of CyclinT1 with respect to the CDK, showing for the first-time plasticity in CDK cyclin interactions. The CDK9/CyclinT1 interface is relatively sparse but retains some core CDK-cyclin interactions. The CyclinT1 C-terminal interaction motif shows flexibility that may be important for the interaction of this region with HIV TAT and HEXIM. Flavopiridol which is an anticancer drug in phase 2 clinical trials, binds to the ATP site of CDK9 inducing conformational changes. These structural changes bury inhibitors. BRD4 has emerged as an important transcriptional factor of NF-kB-dependent inflammatory gene expression. However, the in vivo physiological function of BRD4 in the inflammatory response remains poorly defined. Gene-expression microarray analysis of bone marrow-derived macrophages (BMDMs) reveal that deletion of BRD4 decreases the expression of a significant amount of LPS-induced inflammatory genes while reversing the expression of a small subset of LPS-suppressed genes, including MAP Kinase – interacting Serine/Threonine – Protein Kinase 2 (MKNK2). [11].

Liver fibrosis and cirrhosis are chronic liver diseases, resulting in life-threatening conditions with no FDA-approved therapy. Liver fibrosis is characterized by the persistent deposition of extracellular matrix components by Hepatic Stellate Cell-derived myofibroblasts. BRD4 has been identified as a critical regulator for enhancer-mediated profibrotic gene expression in hepatic stellate cells (previously known as lipocytes, or fat storing cells). These are responsible for liver fibrosis when activated into their fibrogenic myofibroblast-like cells. It has been found that loaded enhancers are associated with multiple profibrotic pathways in HSCs and that pharmacological inhibition of BRD4 blocks HSC activation. [14]

The effect of small-molecule inhibitor on cell cycle

Small molecules inhibitors of BRD4 are not only antagonists, they can further limit the fibrotic response in eukaryotic cells induced by CCl4. Thieno-1,4-diazepine(JQ1) is the first known inhibitor of BET bromodomain along with I-BET762 and others. Inhibition takes place at the acetylated lysine-binding pockets of the bromodomain which is optimal for charged-neutralised interaction displacing BRD4 fusion oncoprotein from chromatin. BRD4 inhibition cause a global downregulation of gene activity and P-TEF-b, blocking the transition of RNA POL II from initiation to elongation and alters transcriptional pathways involved in cell proliferation, tumour invasion and survival. JQ1 inhibits interaction between protein and transcription factors along with their targets(enhancers), affecting stem cells and suppresses signalling pathway. JQ1 downregulates MYC transcription and genomic-wide MYC-dependent target genes, promoting cell cycle arrest and cellular senescence. The inhibitor is induced in G1 cell cycle arrest, apoptosis and also express changes in key genes reducing cell viability and have an anti-growth effect. BET inhibitors jointly targets angiogenesis and the hypoxic response making it an effective anti-tumour combination.

The binding of BRD4 BD1 and BD2 to diferent patners were anylysed. These analysis showed that the strongest interaction took place with di- and tetra-acetylated peptides derived from histone 4 N-terminal tail. It was also found that 4 residues neighboring the acetylated lysines significantly influenced binding. 10 different BRD4 BD1 mutants and thier affinities were analysed to acetylated histone tailsand to the BET inhibitor JQ1 using a number of biochemical and biophysical methods which are complementary. Results showed that Trp-81, Tyr-97, Asn-140 and Met-149 play similarly important roles in the recognition of acetylated histones and JQ1. Pro-82, Leu-94, Asp-145 and IIe-146 have a more converted role. This suggest that different kinds of interactions takes place and that resistant mutations compatible with BRD4 function are possible. This study extends the knowledge on the contribution of individual BRD4 amino acids to histone and JQ1. binding and may help scientist design new BET antagonist with improved pharmacological properties [15].

Bibliography

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