c-Myc| gene and protein structures| function| mutation

Table of content

• What is c-myc Gene Family 
• c-myc
• Structure of c-myc gene and protein  
• Mechanism of c-MYC Ativation
• Function of c-myc Gene

What is c-myc Gene Family 

MYC is a family of regulator gene and proto-oncogene that encode a protein which regulate the expression of other gene in an estimation it regulate nearly 15 % the expression of other gene and basically involved in cell   proliferation, cell growth, apoptosis and cell transformation. Infect they determine the fate of a cell. MYC gene family include other member like c-Myc (c-MYC), l-Myc (I-MYC), and n-Myc  (n-MYC). C-Myc is firstly discovered and referred as MYC.

c-myc

c-myc was discovered in human Burkitt's lymphoma, which show homologue to viral oncogene v-Myc which was isolated from an avian retrovirus. c-myc gene is located at chromosome no 8 at position 8q24.21, which encode the c-myc protein with in the nucleus which regulate the expression of other gene, in two ways.

• Firstly, these proteins act as transcription factors for other gene.
• Secondly these proteins act as histone modifier which help in transcription of a gene.

The encoded protein implicated in various cellular process like cell growth, proliferation, loss of differentiation and apoptosis.

Structure of c-myc gene and protein 

The structure of c-myc gene consist of three exons and two introns, more over the c-Myc transcription is regulated by multiple promoters. 

Figure 1: represents the structure and location of c-myc gene.

c-Myc protein is a 65 kDa nuclear phosphoro-protein, the basic structure of the protein consists of basic helix-loop-helix domain and leucine zipper (bHLH/LZ), bHLH is a DNA binding domain while LZ is bind with other protein (MAX).

Figure 3: represents secondary c-Myc protein structure.

Mechanism of c-MYC Ativation

The function of c-MYC is mediated by another protein that is Max, c-MYC when binds to the Max and generated a complex called Myc-Max complexes. This Myc-Max complexes releases the different type of transcription factors which regulate the expression of other gene that involve in cell proliferation, growth, apoptosis etc. Similarly, this Myc-Max complexes mechanism is controlled and inhibit by other factors to regulate the process.

Figure 3: represents Myc-Max complexe and their binding to DNA.

Function of c-myc Gene

c- myc gene involve in many function

• The primary functions of c-Myc are to promote cell proliferation and to arrest cell differentiation
• Have role in the regulation of stem cell function. With the cooperation of n-Myc, c-Myc inhibits the differentiation of stem cells, such as embryonic stem cells 
• c-Myc is not only an inducer of cell proliferation, but also has the ability to regulate cell apoptosis via various signaling pathways.
• Many observations suggested that protein synthesis is regulated by c-Myc in multiple ways, in which the transcription of various RNA, iRNA, and ribosomes is controlled by the oncogene product.
• Recently, evidence has been accumulating that c-Myc also regulates the expression of miRNAs, which are a set of small, non-protein-coding RNAs and regulate gene expression at the post-transcriptional level.

Gene/Protein Structure of C-Repeat binding Transcription Factor

Table of content 

• Gene/Protein Structure of C- Repeat Binding Transcription Factor
• CBF Gene family
• Function of DREBs/CBF (C-repeat binding factor)

Gene/Protein Structure of C- Repeat Binding Transcription Factor

CBF Gene family

CBF (C-repeat bindingfactor) also known  as DREB (dehydration-responsive element-binding protein). DREB is the class of transcription factors in plants that induces under stress stimuli, and involve in the regulation of series of cascade for the activation of stress responsive gene to bring tolerance in plants.

The CBF (CBF1, CBF2, and CBF3) genes in Arabidopsis organize in tandem on chromosome 4. Both CBF1 and CBF2 induced by cold, while CBF4 involved in drought resistance. It is difficult to determine the chromosomal location for a gene in plant of complex genome and where there high variation in chromosomal ploidy and aneuploidy level like sugarcane. However in a recent study about 110 SsDREB subfamily proteins identified in S. spontaneum. Which were classified into six groups, the number of identified protein is also greater than other species. The SsDREB genes variably distributed across all 29 chromosomes of S. spontaneum, while chromosome 2B contain the greater number of DREB genes.

The first cDNA of CBF1 (C-repeat/DREB Factor 1) was isolated from Arabidopsis thaliana in 1997 by Stockinger and colleague. Molecular mass of the protein is about 24 KDa, comprises of nuclear localization domain and acidic activation domain.  All AP2/ERF family members have conserved AP2 DNA-binding motif that is compose of ~ 60 amino acids residue.  AP2 DNA-binding domain recognize and bind to specific sequence of cis-regulatory elements present in promoters of many stress responsive gene  The AP2 domain further possess two regions: 1)-YRG region/element that consists 20 amino acids, it is present in N-terminal stretch and is rich with hydrophobic and basic amino acids, responsible for DNA-binding activities. 2)-The RAYD region/element contains 40 amino acids. At RAYD region about 18-amino-acid stretch is present at C-terminal and is responsible for amphipathic alpha helix formation, that involve in protein–protein interaction. Three-dimensional analysis by using nuclear magnetic resonance (NMR) of AP2/ERF motif/domain revealed a β-sheets having three strands that connected anti-parallel way by α-helix and loops. Similarly, at AP2/ERF domain's end the DSAW motif is present and at the C-terminal region end LWSY motif is present. However nuclear localization signal (NLS) involved in entrance of DREB proteins in nucleus, it is a stretch rich of basic-amino acid residues. Those TF proteins without the presence of NLSs enter the nucleus by protein–protein interaction with the help of TFs that have NLSs. In AP2/ERF protein domain at position 19 the glutamic acid and at position 14 amino acid Valine are quite conserved but, some species amino acid at 19 is replace by valine .

Representation of typical structure of CBF/DREB protein domain.

Function of DREBs/CBF (C-repeat binding factor)

The DREBs/CBF (C-repeat binding factor), belongs to ERF subfamily and play a key role in plants in response to stress stimuli. The DREB/CBF gene are mainly involved in expression of cold stress-responsive genes. Along with cold stress DREB greatly express under other abiotic stress like drought, salinity in ABA dependently and ABA independently. CBF family further includes three members namely DREB1a/CBF3, DREB1b/CBF1, and DREB1c/CBF2 that induce gene transcription of Cold-regulated genes (COR) under cold stress. The CBF TFs recognized and bind to the conserved CRT/DRE motif CCGAC present in the promoters of many cold-regulated (COR) genes which mediate plant freezing tolerance. In sugarcane CBF/DREB greatly expresses under drought and cold stresses and mediate wide range of COR gene that confer tolerance in many plant. Currently several inducer of CBF have been identified which includes MYB, Inducer of CBF expression (ICE1 and ICE2), Ethylene insensitive (EIN3), Calmodulin-binding transcription activator (CAMTA1, CAMTA2, CAMTA3), Phyto-chrome-interacting (PIF3/4/7) factors, Suppressor of overexpression of co1 (SOC1) and others. Which regulate the expression of CBF and continue the cold signaling pathway. Nevertheless ICE1-CBF-COR best known signaling cascade involve plants in cold tolerance responses.

Low temperature CBFs activation, CBFs bind to CRT/DRE cis-acting element, present in promoter of COR gene, activation of whole signaling cascade results in an increase freezing tolerance.

Beta-catenin (CTNNB1)| Gene Protein structure| Functions and Mutation.

Beta-catenin (CTNNB1) location and structure

Beta-catenin (CTNNB1) also known as catenin beta-1, is located on the short arm of chromosome 3 at position 3p22.1. The size of the complete gene was determined to be 23.2 kb. CTNNB1 has 16 exons according sequence analysis, the exon size ranged from 61 to 790 bp, half of the introns were smaller than 550 bp, with the smallest being 84 bp and the longest being 6700 bp.  Alternative splicing within exon 16 produced a splice variant that is 159-bp shorter in the 3-prime untranslated region. The promoter region was shown to be GC-rich and to contain a TATA box.

Protein structure

The primary structure of β-catenin consists of three domains: central domain known as armadillo repeat domain, an N-terminal domain, and a C-terminal.

• Armadillo repeat domain constitute of 550-amino-acid repeats occupy between N- terminus and C-terminus. The central armadillo domain composed 12 repeats of three helices, which is known as armadillo repeats, each of which contains approximately 42 amino acid residues. This structure forms a super helix for the binding of many factors, including the Tcf transcription factor, the cell adhesion protein cadherin, APC, Axin and other.
• N-terminal domain is composed of 150-amino-acid, the N-terminal domain is the phosphorylation site for GSK-3β and casein kinase-1. The N-terminus of b-catenin is phosphorylated when b-catenin is bound to the destruction complex.
• C-terminal domain is composed of approximately 100-amino-acid residues. The C-terminal segment of β-catenin can mimic the effects of the entire Wnt pathway.

       The N- and C-terminal regions are much smaller, and form flexible regions that interact with transcriptional activating factors. Both of terminal domain combine with the armadillo repeat domain and may regulate the partner-binding properties of the armadillo repeats.

Figure 1: represet the gene protein structure of catenin beta_1 along with its protein interacting domain.

The basic biological function of β-catenin

β-catenin is crucial for two important developmental processes:

  1: Establishment and maintenance of cell-type-specific through cell-to-cell adhesion and

  2: Regulation of target gene expression via the Wnt signaling pathway.

1: Role in cell-to-cell adhesion   

In most cases, β-catenin perform its functions in combination with several other proteins to. β-catenin is normally present in the cell membrane and can play a role in cell to cell adhesion by forming a complex, β-catenin associates with the cytoplasmic domain of E-cadherin (also known as uvomorulin that expressed by epithelial cells) and also with α-catenin, which in turn binds to F-actin protein. β-Catenin, therefore, provides the physical linkage between transmembrane adhesion proteins and the cytoskeleton proteins in the cells. This linkage to the cytoskeleton play an important role to the cell adhesion function. In summary, β-catenin provides obvious connections among extracellular signals, cell-cycle management, and gene transcription.

2: Role in Wnt signaling pathway

β-catenin-dependent Wnt signaling path way is also called the ‘canonical’ Wnt signaling pathway. The canonical Wnt/β-catenin pathway is more complex because high number of ligands and receptors involved in signaling that involve in variety of intracellular responses. Activity of β-catenin is mediated by the destruction complex, consisting of APC, AXIN-1, AXIN-2, casein kinase-1α (CK-1), protein phosphatase 2A (PP2A), and glycogen synthase kinase (GSK)-3β in the cytoplasm.

This pathway has two states dependent upon the presence or absence of Wnt ligands

1: In the absence of Wnt ligands

In normal inactivated cells GSK3-β kinases bind to and phosphorylates β-catenin in the APC/Axin destruction complex, leading to subsequent degradation of β-catenin in the proteasome.

 2: In the presence of Wnt ligands

However, upon Wnt stimulation, the GSK3-β kinase activity is inhibited, so the stable and nonphosphorylated β-catenin accumulates in the cytoplasm and then translocates into the nucleus. Nuclear localized β-catenin binds to the T Cell-Factor/Lymphoid-Enhancer Factor (TCF/LEF) DNA-binding proteins and regulates the transcription of many target genes, depending on its developmental stages and context.

      

Figure 2: represents β-catenin Wnt signaling pathway, in A the absence of wnt ligant and B presence of wnt ligant.

Mutation in β-catenin

Exon 3 of CTNNB1 is a key exon encoding serine-threonine phosphorylation sites for GSK-3β (Glycogen synthase kinase 3 beta) that activates degradation of β-catenin. The CTNNB1 mutations are frequently missense mutations, mostly localized in the hot-spot exon 3, and most of them have affect the phosphorylation sites for GSK-3β; S45 is the phosphorylation site for casein kinase-1; and D32 and G34 are essential for the interaction of β-catenin with Fbw1.

Gene mutations lead to activate the Wnt/β-catenin signaling pathway, which play a role in the development of some cancer cases. Moreover, a high level of β-catenin activity is required for cancer initiation. Initial characterization of mutations of CTNNB1 and deregulation of the canonical Wnt pathway were in colorectal cancer cases.  Similarly, another mutation of β-catenin (S37F) activates Wnt signaling in several melanoma cell lines. Such mutations have been shown to result in the accumulation of nuclear β-catenin and stabilization of the protein and tumorigenesis. These mutations stabilize β-catenin, as they cease the phosphorylation-dependent interaction of β-catenin with Fbw 1 (is a type of F-box protein), involve in degradation of β-catenin in association with casein kinase-1α and GSK-3β. In addition, other relatively benign tumors, such as desmoid-type fibromatosis, also have CTNNB1 mutations and abnormal nuclear β-catenin expression.

 

Gap Junction Protein Beta-2 (Gjb2)| Gene, Protein Structure of (Gjb2) and mutation.

Gap Junction Protein Beta-2 (Gjb2)

Gj2 gene is located on DFNB1 loci and is mapped to 13q12 of the chromosome, encode a gap junction beta-2 protein known as connexins 26 which express in cochlea and epidermis of the skin. Some of the alternative names for Gjb2, are DFNB1, DFNA3, DFNB1A, DFNA3A, CX26, NSRD1, KID, HID, PPK, (gap junction protein, beta 2, 26kDa), (gap junction protein, beta 2, 26kD (connexin 26)) [http://atlasgeneticsoncology.org/gene/40716/gjb2-(gap-junction-protein-beta-2)].

Gjb2 gene structure

Gjb2 comprising the simple structure, it consists of about 26kDa molecular weight, the size of the gene comprising 5500bp or 5.5 kb, that located on chromosome no 13 at short arm of locus 13q12.11. The size of the translated m RNA consists of 4.2Kb, which encode the protein having 226 amino acid residue. Flanked between GJA3 which encode connexin-46 present at centromeric side and GJB6 which encode connexibbbn-30 present at telomeric side. The gene comprising on two exons, the untranslated exon 1 is separated from exon 2 by an intron which is 3179 bp in length, exon 2 is only the coding region consisting the uninterrupted coding region and 3’ UTR (untranslated region).  

Figure 1: Present the location of Gjb2 on the 13 chromosome and typical structure of Gjb2 gene consist of exon 1 exon 2 and an intron 3’UTR, stop codon.

Connexin-26 

Connexin-26 is transmembrane protein which is encoded by gap junction beta-2 gene, mutation in which causes most common congenital, sensorineural deafness, it is member of connexin family. The naming is based upon the molecular weight of the particular connexin protein (CX-26, CX-43, CX-30, etc. To date up to 21 connexin gene have been identified for human.  That are classified into five group (alpha, beta, gamma, delta and epsilon) connexin-26 belongs to beta groups.  Connexin family consist of hemi-channel known as connexon, which contain the six subunit of connexin protein that arrange in such a way to form water filled channel in it, two connexon in the plasma membrane of adjacent cells adjoins by head to head docking to form gap junction channel in it. A typical CX-26 consists of four transmembrane domain segment (TM1_TM4) which are consider to be anti-parallel in conformation T1 and T2 predicated to be interior hydrophilic, whereas T3 and T4 predicated to be exterior hydrophobic environment. Two extra cellular loop EL1 between T1 and T2, EL2 which is present between T3 and T4. N terminus, the interior cytosolic C terminus   and the cytoplasmic loop between T2 and T3, shown in fig: 2. The CX-26 expresses both in the skin epidermis and inner cochlea cells, mostly with co-expression of other connexin, it expresses in hair follicles, sweat gland, also play an important role in keratinocyte proliferation, inter follicular cells. Cx-26 co-localized with CX-30 in the inner ear and perform important function in maintenance of homeostasis of inner ear. It is essential for cycling of K⁺ ion, which is required for the balance of ionic composition of endolymph and endo-cochlear potential, also play an important role in intercellular signaling, by the release of Ca⁺ oscillation and waves which are mediated by the diffusion of inositol 1,4,5-trisphosphate (IP₃) in corti of the inner ear. 

                 

Figure 2: Structure of cx-26, consist of CT, CL, NT four domain segment, and loop EL1, EL2.

Gjb2 mutation

Mutation in the Gjb2 gene encode defective protein which is the major cause of non-syndromic hearing loss, to date more than 100 mutations have been identified for Gjb2 which inherited either in recessive or dominant mode to cause deafness, syndromic disorder with hearing loss, and many skin diseases. These mutations may be appearing in the form of missense, nonsense, small deletion or insertion.  That leads to truncated protein, which bring defect in Gjb2 protein structure, like 35delG effect extracellular loop EL1, 235delC effect T2, V95M effect CL domain of Cx-26 all of these appear in autosomal recessive manner. AD inheritance includes like W44C, R75Q mutations which effect EL1 of CX-26. Their epidemiology varies in different ethnic group, for example 167delG mutation is more prevails among Jews, W24X and W77X appear to be most common in south Asian countries like India, Bangladesh and Pakistan, similarly high frequency of 235delC mutation prevails in East Asian country.