Showing posts with label Genetics. Show all posts
Showing posts with label Genetics. Show all posts

Protein Synthesis | Transcription phase | Translation phase |

Protein Synthesis

The cell preserves hereditary information in genes, which work as instructions for building proteins. The gene instructions from the DNA transfer are stored in the molecules of ribonucleic acid, the "mRNA," and then the mRNA transcript translates the instructions for the synthesis of protein. This one flow of hereditary information from DNA to mRNA and from mRNA to protein is known as the "center dogma of life."

There are three different forms of RNA in cells, each of which has a different function: messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). All three types of RNAs are essential for protein synthesis. The process of protein synthesis involves two stages known as transcription and translation. The process of transcription and translation is discussed below.

mRNA Transcription 

Transcription involves the transfer of information from a gene in DNA into an mRNA molecule. In eukaryotic organisms, transcription occurs inside the nucleus, while in prokaryotic organisms, it takes place in the cytoplasm. It is defined as

"The formation of RNA on a template of a DNA strand is called transcription."

Transcription includes the following steps:

Template binding

Transcription begins when an enzyme called RNA polymerase binds to the transcription start site of a gene on a region of DNA called a promoter. A promoter is a specific sequence of DNA that is important for the recognition of RNA polymerase, known as the transcription start site. It is known as the "TATA box" in eukaryotes because it is rich in thymine and adenine nitrogenous bases. RNA polymerase cannot initiate transcription on its own until the transcription factors recognize the sequence and help the RNA polymerase in their search for promoters to start the transcription. After RNA polymerase binds to a promoter, the enzyme starts to unwind and separate the two strands of the double helix, exposing the DNA's nitrogen-containing bases. The enzyme recognizes the nitrogen base and adds the complementary base on the growing chain, the two bases join each other by a phosphodiester bond and initiate the formation of the mRNA chain. DNA bases act as a template for the formation of the mRNA molecule. In any particular transcription, however, only one of the two strands of DNA is transcribed; this strand is called the template strand or antisense strand, while the opposite strand is called the coding sense or sense strand. Thus, the resulting mRNA is complementary to the DNA template from which it is formed.

Chain elongation

RNA polymerase separates apart the double helix and moves downstream along the DNA template strand from the initiation site, always in the 3’ to 5’ direction. While the growing mRNA always synthesize from 5’ to 3’ direction. The enzyme reads each nucleotide and adds it on complementary mRNA. When the RNA nucleotides are added, they are linked together with sugar-to-phosphate covalent bonds. In eukaryotic cells, the RNA nucleotides are found in the nucleus; in prokaryotic cells, they are in the cytoplasm. As the transcription grows downstream, the two strands of DNA close up by forming hydrogen bonds between complementary bases and reform the double helix.

Chain termination

RNA polymerase continues to elongate the mRNA until it reaches the termination site. It is the specific sequence of nucleotides that signals the termination or end of the mRNA transcription. The mRNA, the transcript of a gene, is released, and polymerase subsequently dissociates from the DNA.

In prokaryotes, the mRNA is directly released into the cytoplasm, where it is translated into protein. In eukaryotes, mRNA has to travel a long distance from the nucleus to the cytoplasm; therefore, mRNA must be processed before it is released.

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Figure 1: Labeled diagram of transcription. 

mRNA processing in eukaryotes

Before being released into the cytoplasm for protein synthesis, eukaryotic mRNA undergoes post-transcriptional modification, which includes 5' capping, mRNA splicing, and polyadenylation.

Addition  of 5 ’capping on mRNA:  

Before the release enzyme called guanyl transferase adds the cap, it is in the form of 7-methyl GTP (7-methyle-guanosine-5'-triphosphate) on the first nucleotide of mRNA at 5’. It is a highly methylated modified guanine base. The 5’ cap protects the mRNA from degradation, prevents it from being attacked by exonucleases, and even assists other post transcriptional events, including splicing, polyadenylation, and recruitment of translational machinery.

mRNA splicing

The growing mRNA stretch contain both intron (a noncoding region) and exon (a coding region), and the pre-mRNA undergoes alternative splicing, which leads to different combinations of mRNA before release. The different mRNAs produce different proteins.

Polyadenylation

The tail is in the form of poly A linked to 3’ end of mRNA by the help of an enzyme called poly-A polymerases. Poly-A tail is in fact a stretch of adenosine monophosphate, which contains almost 100 to 250 nucleotide residues. The newly synthesized mRNA contains a 3’ UTR region that is enriched with AU nucleotides; the 3’ UTR contains a sequence AAUAAA that assists the addition of a tail. The end 3’ region is cleaved by a set of proteins to cleave the hydroxyl group, and the enzyme poly-A polymerases assist in the addition of the poly-A tail. The tail protects the mRNA from a variety of cytoplasmic enzymes, stabilises the mRNA, prevents it from degradation, and allows the export of mature mRNA from the nucleus to the cytoplasm.

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Figure 2: Structure of mature mRNA.


Translation

The equipment for translation is located in the cytoplasm, where a cell keeps its protein supply. This translation machinery includes: transfer RNA (tRNA), which is a single strand of RNA folded into a compact shape with three loops, one of the loop has a three-nucleotide sequence called an anticodon. A codon can be complementary to one of the 64 codons of the genetic code. Opposite the anticodon on a tRNA molecule is a site at which the molecule carries an amino acid. The ribosome is composed of protein and rRNA; each ribosome has two subunits. The smaller subunit binds to the mRNA, and the larger subunit has three functional sites. Two sites called P and A bind to the tRNA, while the third site catalyzes the formation of peptide bonds between amino acids in growing protein chain.

tRNA and genetic code

In the presence of an enzyme called aminoacyl-tRNA synthetases, each tRNA is chemically linked with a specific amino acid. The tRNA is covalently linked with the specific amino acid, and ATP provides the energy for the binding reaction. There are different types of tRNA, ranging from 40 to 60, depending on the type of cell and species, each bearing the specific anticodon for the 20 different amino acids floating in the cytoplasm. There are 64 different codons; multiple codons can be encoded for the same amino acid. AUG on an mRNA read is a start codon and always encodes for methionine; similarly, UAA, UAG, and UGA are stop codons and do not encode for any amino acid.

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Figure 3: Genetic codes for particular amino acids. 
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Figure 4: Labeled diagram of structure of tRNA.


Translation definition

"Translation is the process by which a protein is synthesized; the liner amino acid sequence is directed by mRNA sequences that are specified by a gene."

The mechanism of translation The process consists of the following stages.

Translation Initiation

Translation begins when mRNA binds to the smaller ribosomal subunit in the cytoplasm. The ribosomal S1 protein plays an important role in the recognition of mRNA and its binding. Similarly, the ribosome also recognizes the 7-methyle-guanosine cap and scans the 5’ UTR (untranslated region) for binding and initiation of translation. The mRNA is oriented in such a way that the start codon (a codon that signals the beginning of a protein chain) is sitting in the P site, and at the same time, the initiator tRNA with the anticodon UAC base pairs with the initiation codon AUG bearing the amino acid methionine (met) and initiates the complex. The larger ribosomal subunit joins the initiation complex, and a protein called initiator factor brings these translation components together. In a bacterial cell, almost 3 initiator factors (IF1, IF2, and IF3) are required to initiate the translation, while in eukaryotes, which are complex organisms, there are a number of initiator factors, but at least 11 are required to initiate the translation (eIF2, eIF3, eIF1, eIF1A, eIF4A, eIF4F, eIF4G, eIF4B, eIF5 and eIF5B, eIF5 and eIF2B, respectively). GTP, which is closely related to ATP, provides energy for the initiation process. The larger subunit has three sites: the P (peptidyl) site, the A (aminoacyl) site, and the E (exit) site. The initiator tRNA first binds to the P site, which later holds the nascent peptide chain, and the A site is available for the tRNA (aminoacylated) bearing the next amino acid. The E site holds the deacylated tRNA before the tRNA leaves the complex after the transfer of amino acid.

Translation Initiation Mechanism:

  • The initiation phase of translation begins when initiation factors (eIF1, eIF3, and eIF1A) bind to the smaller ribosomal units, while initiation factor eIF2 along with GTP complexes with initiator tRNA brings it to the ribosome, and a group of eIF4 initiation factors brings mRNA to the ribosome.
  • As the translation machinery was brought to the ribosome, the ribosome then scanned the mRNA downstream to identify the initiation codon AUG. This process required energy, which was provided by ATP hydrolysis.
  • As the start codon was identified, another initiation factor known as eIF5 triggered the GTP hydrolysis, as a result, the eIF2_GTP complex dissociates and tRNA is released and binds to the A site.
  • The larger subunit then binds to the complex and continues the process.
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Figure 5: Mechanism of translation initiation.

The elongation cycle of translation

After the formation of the initiation complex, the very next step is elongation of the peptide chain. As the incoming aminoacylated tRNA binds to the codon at the P site, the elongation factor and GTP are required for the process. The amino acid at P site forms a covalent link with the amino acid at A site, and a peptide is formed between the new amino acid and the growing polypeptide chain. The reaction of peptide bond formation is catalysed by peptidyl transferase (a catalytic RNA). The growing peptide chain first transferred to the tRNA at A site. The ribosome moves along the mRNA downstream from 5’ to 3’. As the ribosome translocates downstream, there is a shift of one codon forward. The initiator tRNA or tRNA at P site shifted to the E site and the tRNA bearing the polypeptide chain translocated at P site, A site is again available for the next coming tRNA.

Mechanism of chain elongation

The following are the steps involved in chain elongation:

  • The elongation phase of translation involves the elongation of polypeptide chains, as the initiator methyonyl tRNA sits at the P site and the second aminoacyl tRNA is brought to the A site by the elongation factor EF-Tu complex with GTP, having an anticodon matched exactly to the code at the A site.
  • GTP hydrolysis the bond, and GTP_EF-Tu complex leaves the ribosome and newly coming aminoacylated tRNA sits at A site.
  • A peptide bond is formed between two amino acids, which results in the transfer of methionine (an amino acid at the P site) to the amino acid at the A site.
  • After the transfer, the ribosome is translocated three nucleotides forward; this process is conducted by the elongation factor EF-Gu.
  • As the ribosome translocate the deaminoacylated tRNA shifts at E site, the tRNA at A site bearing the peptide chain shifts at P site and A site sets free for incoming new aminoacylated tRNA.
  • The process is to continue until the whole mRNA is fully decoded and reaches to the end.
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Figure 6: Mechanism of translation elogation.

Termination of translation

When a ribosome reaches a termination codon on a strand of mRNA, a protein called release factor recognizes the stop codon and binds to the A site instead of tRNA. In eukaryotes, a single release factor, eRF1, recognizes all three stop codons, whereas prokaryotes have two release factors.RF1: It can recognize UGA and UUA, and RF2: It can recognize UUA and UGA. The release factor hydrolyzes the bond between the tRNA in the P-site and the last amino acid of the polypeptide chain. Both polypeptide and tRNA are then free to depart from the ribosome, and the two ribosomal units dissociate from the mRNA, and that’s how the translation ends and the process of protein synthesis is completed. However, one mRNA can be translated by several ribosomes at the same time.

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Figure 7: Mechanism of translation termination.


Post translational modification

Protein could not be synthesize or fully functional soon after the translation. As the translation ends, the nascent polypeptide chain undergoes posttranslational modification. These modifications include the removal of Met from certain proteins and the addition of functional groups that include methyl, phosphate, acetate, and amide. Lipids and sugar are also added to certain proteins. Other proteins undergo conformational changes, breakage of some bonds, formation of new covalent linkages, excision of some peptide chains, and reorientation of peptide chains. All these posttranslational changes lead to the final protein product, which is then available to perform its function.

Sources

Clark, D. P., & Pazdernik, N. (2012). Molecular biology. Elsevier
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c-Myc| gene and protein structures| function| mutation

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. 

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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).

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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.

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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.


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Gene/Protein Structure of C-Repeat binding Transcription Factor

Table of content 

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 .

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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.

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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.




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What is Molecular Chaperon or Ubiquitious Protein?

Table of contents 

Chaperon and Ubiquitination's Role in Protein Conformation or Function 

What is Molecular Chaperone?

Molecular chaperones are the class of proteins that assist in stabilising non-native protein (the biologically relevant components of a cell but are misfolded proteins) conformations and promote folding into the native state whenever possible. Alternatively, misfolded proteins are eliminated by a cell via a degradation mechanism called the ubiquitin–proteasome system.
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What is Protein folding?

Protein synthesis is carried out by ribosomes. In the beginning, ribosomes synthesise a linear chain of amino acids called the polypeptide chain. The stretch of mRNA specifies the amino acid sequence, and each amino acid in this polypeptide chain has its own unique property. For example, glycine is very hydrophobic, while arginine is very hydrophilic. These properties of amino acids depict the three-dimensional structure of the protein. The outside of the protein possesses the hydrophilic amino acids, while the inside of the protein must conserve the hydrophobic amino acids. The secondary structure of proteins (alpha helices and beta sheets) is acquired by hydrogen bonds between the polypeptide chains. After the primary and secondary structures, the helices constitute the tertiary structure. Protein folding must be precise in its three-dimensional form and must not be aggregated or degraded. The unfolded and misfolded proteins result in non-native protein aggregation, which is an inactive form but, in some cases, may lead to diseases. 

The Role of Molecular Chaperones and Ubiquitination in Protein Conformation 

In molecular biology, the chaperones are defined as "the molecular chaperones are the proteins that have the ability to assist and stabilise the conformation of non-native folding. The ubiquitous proteins, which are an amazingly diverse family of proteins, are also among the most abundant intracellular proteins. The basic function of chaperones is posttranslational modification of proteins, so ubiquitous proteins mediate correct protein folding, inhibit misfolding, and prevent other proteins from degradation and non-native aggregation during their tertiary structure formation. Chaperones sometimes interact with other packaging called ancillary proteins. Both proteins together regulate the folding of other proteins. Chaperones are ubiquitously expressed and are found in all cellular compartments of the eukaryotic cell (except for peroxisomes). In general, the concentration of chaperone increases in response to diverse stresses in order to prevent the proteins' folding and for the stabilisation of the protein. An example of chaperon proteins are the "heat shock proteins" (Hsps), which are discovered in bacteria under stress conditions. The bacteria produce a number of such proteins under stressful conditions, such as higher temperatures, pH variation, and hypoxic conditions. 

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Fig 2: Role of Chaperon in quality control of protein.

Two examples of Hsps are Hsp70 and Hsp60. 

Heat shock protein (Hsp70)

The Hsp70 chaperone proteins are folding catalysts that help in the refolding or misfolding of aggregated proteins and in the folding and assembling of new proteins. The Hsps 70 contains two different domains, namely the N terminal and the C terminal. The N terminal contains the ATPase site while the C terminal domain binds to the substrate. The N terminal ATPase hydrolysis site helps the C terminal to open and bind to the substrate. 
Hsp70 recognises the "extended region" of an unfolded polypeptide chain. This extended region contains many hydrophobic residues. HSP70-binding prevents the aggregation of these proteins. 
Fig 3: Domain organization and 3D structure of Hsp70

Heat shock protein (Hsp60)

Hsp60 chaperone proteins bind to exposed hydrophobic residues of unfolded proteins and help to form aggregates that are stable but inactive. Hsp60 proteins could not prevent a protein's aggregation, but rather they functioned to isolate and quarantine an unfolded protein. This isolation prevents a polypeptide chain from aggregating into clumps with other chains within the cytoplasm.
Hsp 60 contains 14 different protein components. These proteins are made of 7 proteins which are placed on top of each other and form two rings. Unfolded proteins can then fold safely within these rings because the Hsp60 protein does not interfere with the unfolded protein (newly formed proteins) or aggregate with other unfolded proteins. Hsp60 also has two different domains: the protein binding motif, where ATP binds, and the unfolded proteins can enter the hole between the two rings. The enclosed state, called the folding-active state, is then activated by ATP hydrolysis. This conformational change prevents the unfolded protein from leaving the ring and assists the protein folding. The state lasts for around 15 seconds and the folded protein  after proper folding, is released into the cytoplasm. The enclosed state then deactivates and the protein changes back to its conformation.

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Fig 4: Domain organization and 3D structure of Hsp60.
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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.

 

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