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TES (protein)
Encyclopedia
Testin also known as TESS is a protein
that in humans is encoded by the TES gene
located on chromosome
7. TES is a 47 kDa
protein composed of 421 amino acids found at focal adhesions and is thought to have a role in regulation of cell motility. In addition to this, TES functions as a tumour suppressor. The TES gene is located within a fragile region of chromosome 7, and the promoter elements of the TES gene have been shown to be susceptible to methylation
- this prevents the expression
of the TES protein. TES came to greater prominence towards the end of 2007 as a potential mechanism for its tumour suppressor function was published.
The structures of the Cysteine rich domain and the PET domain are not known. LIM domains, however, are known as modulators of protein interactions. LIM domain consist of 2 zinc fingers separated by 2 hydrophobic amino acids (generally a phenylalanine
and then a leucine
).
; rather it is a protein that mediates/regulates cellular functions via Protein:protein interactions. Pull down
experiments reveal that TES has putative interactions mediated by the indicated domain:
Garvalov et al. showed that the interaction between TES & zyxin were direct, using recombinant
proteins expressed in E. coli
.
Some of the potential binding partners (Zyxin, mENA) can be found in focal adhesion
complexes; the range of binding partners indicates a potential role for TES in-between 'privileged' Actin
polymerisation and focal adhesion contacts to the extracellular matrix
. This tallies with the observation that GFP
-tagged TES can be seen at focal adhesions.
motif consisting of a Phenylalanine
residue, followed by four Proline
residues - known as a FPPPP motif. It is the EVH1 domain
s of VASP/EVL proteins that directly contact the FPPPP motif. The precise architecture of the TES:MENA binding was revealed by X-ray crystallography
, and showed that the 3rd LIM domain of TES covered up the FPPPP binding site within Menas EVH1 domain. Isothermal Titration Calorimetry
showed that TES has a greater affinity for Mena than its canonical FPPPP ligand, as presented in the focal adhesion protein Zyxin. Using microscopy it was shown that either over-expression of GFP
-tagged TES, or just the tagged third LIM domain displaced Mena from focal adhesions and reduced mean cell velocity.
These finding were significant given that Mena is often over expressed in cancer
cells, and is thought to be partly responsible for cancer cell motility, and therefore a factor is cancer Metastasis
. TES is conversely, often not produced in cancer cells. It is possible that a drug designed to mimic TESs interaction with Mena could be used to prevent Metastasis and thus development of secondary tumours in cancer patients.
Given this, the work was widely reported in the British press (the work was carried out by Cancer Research UK
), and also in the international press.
Garvalov et al. propose that TES exists in two conformational states: A 'closed' state where the N & C halves of TES interact, obscuring the Zyxin binding site in LIM1, and an 'open' state where the Zyxin binding site is accessible and the two halves no-longer interact in the same fashion, if at all. The regulatory mechanism switching between the two states is not presently fully understood.
experiments, cells that had impaired TES expression showed an inability to correctly organise their focal adhesions and actin
stress fibres.
In gene knockout experiments, transgenic mice lacking both copies of the TES gene displayed an increased susceptibility to tumour formation when challenged with a carcinogen. Mice retaining the TES gene were less susceptible: thus, TES is a tumour suppressor gene.
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
that in humans is encoded by the TES gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
located on chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
7. TES is a 47 kDa
KDA
KDA may refer to:* Karachi Development Authority* Kongsberg Defence & Aerospace* Kotelawala Defence Academy* Kramer Design Associates* Lithium diisopropylamide, KDA is the potassium analogue of lithium diisopropylamideOr kDa may refer to:...
protein composed of 421 amino acids found at focal adhesions and is thought to have a role in regulation of cell motility. In addition to this, TES functions as a tumour suppressor. The TES gene is located within a fragile region of chromosome 7, and the promoter elements of the TES gene have been shown to be susceptible to methylation
Methylation
In the chemical sciences, methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with, to be specific, a methyl group, rather than a larger carbon chain, replacing a hydrogen atom...
- this prevents the expression
Protein expression
Protein expression is a subcomponent of gene expression. It consists of the stages after DNA has been translated into polypeptide chains, which are ultimately folded into proteins...
of the TES protein. TES came to greater prominence towards the end of 2007 as a potential mechanism for its tumour suppressor function was published.
Domain organisation
Tes is composed of the following domains:| Domain Name | Boundaries | Domain type |
|---|---|---|
| Cysteine Cysteine Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid... rich domain |
1 - 90 | No Homology |
| PET domain | 90 - 200 | PET domain - no structure |
| Linker . | 201 - 233 | no domain |
| LIM1 | 234 - 300 | LIM domain LIM domain LIM domains are protein structural domains, composed of two contiguous zinc finger domains, separated by a two-amino acid residue hydrophobic linker. They are named after their initial discovery in the proteins Lin11, Isl-1 & Mec-3. LIM-domain containing proteins have been shown to play roles in... |
| LIM2 | 300 - 365 | LIM domain LIM domain LIM domains are protein structural domains, composed of two contiguous zinc finger domains, separated by a two-amino acid residue hydrophobic linker. They are named after their initial discovery in the proteins Lin11, Isl-1 & Mec-3. LIM-domain containing proteins have been shown to play roles in... |
| LIM3 | 366 - 421 | LIM domain LIM domain LIM domains are protein structural domains, composed of two contiguous zinc finger domains, separated by a two-amino acid residue hydrophobic linker. They are named after their initial discovery in the proteins Lin11, Isl-1 & Mec-3. LIM-domain containing proteins have been shown to play roles in... |
The structures of the Cysteine rich domain and the PET domain are not known. LIM domains, however, are known as modulators of protein interactions. LIM domain consist of 2 zinc fingers separated by 2 hydrophobic amino acids (generally a phenylalanine
Phenylalanine
Phenylalanine is an α-amino acid with the formula C6H5CH2CHCOOH. This essential amino acid is classified as nonpolar because of the hydrophobic nature of the benzyl side chain. L-Phenylalanine is an electrically neutral amino acid, one of the twenty common amino acids used to biochemically form...
and then a leucine
Leucine
Leucine is a branched-chain α-amino acid with the chemical formula HO2CCHCH2CH2. Leucine is classified as a hydrophobic amino acid due to its aliphatic isobutyl side chain. It is encoded by six codons and is a major component of the subunits in ferritin, astacin and other 'buffer' proteins...
).
Binding partners
TES does not appear to be an enzymeEnzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
; rather it is a protein that mediates/regulates cellular functions via Protein:protein interactions. Pull down
Immunoprecipitation
Immunoprecipitation is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins...
experiments reveal that TES has putative interactions mediated by the indicated domain:
| Partner | Domain | ref | Method |
|---|---|---|---|
| mENA/VASP | LIM3 | Yeast two Hybrid Two-hybrid screening Two-hybrid screening is a molecular biology technique used to discover protein–protein interactions and protein–DNA interactions by testing for physical interactions between two proteins or a single protein and a DNA molecule, respectively.The premise behind the test is the activation of... , Pull-down assay Immunoprecipitation Immunoprecipitation is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins... , Structure X-ray crystallography X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a... , ITC Isothermal Titration Calorimetry Isothermal titration calorimetry is a physical technique used to determine the thermodynamic parameters of interactions in solution. It is most often used to study the binding of small molecules to larger macromolecules .-Thermodynamic measurements:ITC is a quantitative technique that can... |
|
| Arp7a | ??? | Yeast two Hybrid Two-hybrid screening Two-hybrid screening is a molecular biology technique used to discover protein–protein interactions and protein–DNA interactions by testing for physical interactions between two proteins or a single protein and a DNA molecule, respectively.The premise behind the test is the activation of... |
|
| Zyxin | LIM1 | Yeast two Hybrid Two-hybrid screening Two-hybrid screening is a molecular biology technique used to discover protein–protein interactions and protein–DNA interactions by testing for physical interactions between two proteins or a single protein and a DNA molecule, respectively.The premise behind the test is the activation of... , Pull-down assay Immunoprecipitation Immunoprecipitation is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins... |
|
| Actin | PET? | Pull-down assay Immunoprecipitation Immunoprecipitation is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins... |
|
| α-Actinin | PET? | Pull-down assay Immunoprecipitation Immunoprecipitation is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins... |
|
| Paxillin | PET? | Pull-down assay Immunoprecipitation Immunoprecipitation is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins... |
Garvalov et al. showed that the interaction between TES & zyxin were direct, using recombinant
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
proteins expressed in E. coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
.
Some of the potential binding partners (Zyxin, mENA) can be found in focal adhesion
Focal adhesion
In cell biology, focal adhesions are specific types of large macromolecular assemblies through which both mechanical force and regulatory signals are transmitted. More precisely, they can be considered as sub-cellular macromolecules that mediate the regulatory effects In cell biology, focal...
complexes; the range of binding partners indicates a potential role for TES in-between 'privileged' Actin
Actin
Actin is a globular, roughly 42-kDa moonlighting protein found in all eukaryotic cells where it may be present at concentrations of over 100 μM. It is also one of the most highly-conserved proteins, differing by no more than 20% in species as diverse as algae and humans...
polymerisation and focal adhesion contacts to the extracellular matrix
Extracellular matrix
In biology, the extracellular matrix is the extracellular part of animal tissue that usually provides structural support to the animal cells in addition to performing various other important functions. The extracellular matrix is the defining feature of connective tissue in animals.Extracellular...
. This tallies with the observation that GFP
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
-tagged TES can be seen at focal adhesions.
TES as a tumour suppressor
In December 2007, Boeda, Briggs et al. showed that the third LIM domain of TES displaces Mena from its usual subcellular positions (focal adhesions or the cell leading edge). The ENA/VASP protein family (of which Mena is a member) are anchored to specific proteins within the cell by a peptidePeptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
motif consisting of a Phenylalanine
Phenylalanine
Phenylalanine is an α-amino acid with the formula C6H5CH2CHCOOH. This essential amino acid is classified as nonpolar because of the hydrophobic nature of the benzyl side chain. L-Phenylalanine is an electrically neutral amino acid, one of the twenty common amino acids used to biochemically form...
residue, followed by four Proline
Proline
Proline is an α-amino acid, one of the twenty DNA-encoded amino acids. Its codons are CCU, CCC, CCA, and CCG. It is not an essential amino acid, which means that the human body can synthesize it. It is unique among the 20 protein-forming amino acids in that the α-amino group is secondary...
residues - known as a FPPPP motif. It is the EVH1 domain
EVH1 domain
The EVH1 domain is an evolutionary conserved protein domain.The EVH1 domain is found in multi-domain proteins implicated in a diverse range of signalling, nuclear transport and cytoskeletal events. This domain of around 115 amino acids is present in species ranging from yeast to mammals...
s of VASP/EVL proteins that directly contact the FPPPP motif. The precise architecture of the TES:MENA binding was revealed by X-ray crystallography
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
, and showed that the 3rd LIM domain of TES covered up the FPPPP binding site within Menas EVH1 domain. Isothermal Titration Calorimetry
Isothermal Titration Calorimetry
Isothermal titration calorimetry is a physical technique used to determine the thermodynamic parameters of interactions in solution. It is most often used to study the binding of small molecules to larger macromolecules .-Thermodynamic measurements:ITC is a quantitative technique that can...
showed that TES has a greater affinity for Mena than its canonical FPPPP ligand, as presented in the focal adhesion protein Zyxin. Using microscopy it was shown that either over-expression of GFP
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
-tagged TES, or just the tagged third LIM domain displaced Mena from focal adhesions and reduced mean cell velocity.
These finding were significant given that Mena is often over expressed in cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
cells, and is thought to be partly responsible for cancer cell motility, and therefore a factor is cancer Metastasis
Metastasis
Metastasis, or metastatic disease , is the spread of a disease from one organ or part to another non-adjacent organ or part. It was previously thought that only malignant tumor cells and infections have the capacity to metastasize; however, this is being reconsidered due to new research...
. TES is conversely, often not produced in cancer cells. It is possible that a drug designed to mimic TESs interaction with Mena could be used to prevent Metastasis and thus development of secondary tumours in cancer patients.
Given this, the work was widely reported in the British press (the work was carried out by Cancer Research UK
Cancer Research UK
Cancer Research UK is a cancer research and awareness charity in the United Kingdom, formed on 4 February 2002 by the merger of The Cancer Research Campaign and the Imperial Cancer Research Fund. Its aim is to reduce the number of deaths from cancer. As the world's largest independent cancer...
), and also in the international press.
Conformational Change
Based on the observations that:- Mammalian cell derived TES binding Zyxin
- E. coliEscherichia coliEscherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
produced recombinantRecombinant DNARecombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
TES (rTES) does not bind Zyxin - An rTES construct composed of residues 201-421 (i.e, the linker and all 3 LIM domains) does bind Zyxin
- The above rTES construct binds an N-terminal rTES construct, consisting of the cysteine rich and PET domains - IE, the two halves of TES interact with each other.
Garvalov et al. propose that TES exists in two conformational states: A 'closed' state where the N & C halves of TES interact, obscuring the Zyxin binding site in LIM1, and an 'open' state where the Zyxin binding site is accessible and the two halves no-longer interact in the same fashion, if at all. The regulatory mechanism switching between the two states is not presently fully understood.
Phenotype
In RNAiRNAI
RNAI is a non-coding RNA that is an antisense repressor of the replication of some E. coli plasmids, including ColE1. Plasmid replication is usually initiated by RNAII, which acts as a primer by binding to its template DNA. The complementary RNAI binds RNAII prohibiting it from its initiation role...
experiments, cells that had impaired TES expression showed an inability to correctly organise their focal adhesions and actin
Actin
Actin is a globular, roughly 42-kDa moonlighting protein found in all eukaryotic cells where it may be present at concentrations of over 100 μM. It is also one of the most highly-conserved proteins, differing by no more than 20% in species as diverse as algae and humans...
stress fibres.
In gene knockout experiments, transgenic mice lacking both copies of the TES gene displayed an increased susceptibility to tumour formation when challenged with a carcinogen. Mice retaining the TES gene were less susceptible: thus, TES is a tumour suppressor gene.