Dihydrofolate Reductase ( DHFR ) enzyme is responsible for the reaction in which dihydrofolate is reduced to tetrahydrofolate. Tetrahydrofolate is critical for synthesis of Purines and Thymidylate. For this ground the ordinance of DHFR look in the cell is imperative in order for cells to turn and proliferate.. DHFR is an extensively studied as a theoretical account cistron due to some of its typical belongingss. These include deficiency of a TATA booster, interactions with written text factors at different phases of the cell rhythm, autoregulation of interlingual rendition. This reappraisal summarizes the current apprehension of the ordinance of human dihydrofolate reductase look in the cell. Initially the construction and map of the enzyme are discussed in order to set up an understanding as to its importance in the cell. Next the reappraisal looks at ordinance of the cistron from many degrees ; Transcription, Post- Transcriptional alterations and Translation.
Dihydrofolate reductase is one of the cardinal enzymes in the vitamin Bc metamorphosis tract.
Figure 1 represents a simplified version of the folate metamorphosis tract. Genes and proteins can be seen in bluish while the substrates are the green rounded rectangles. The country of involvement to this reappraisal is denoted by an pointer. ( Leeder and Mitchel, 2007 ) .
Dihydrofolate reductase ( DHFR ) enzyme catalyses the decrease of 7,8-dihydrofolate ( H2F ) 5,6,7,8-tetrafolate ( H4F ) using NADPH as a cofactor ( Abali et al, 2008 ) . This reaction is depicted in figure 2 below.
Figure 2: adapted from hypertext transfer protocol: //research.chem.psu.edu/sjbgroup/projects/dihydro.htm
A DHFR is the lone enzyme in the tract capable of keeping the pool of tetrahydrofolate ( H4F ) . Tetrahydrofolate is indispensable in the synthesis of thymidylate, purines and some amino acids, including methionine, glycine and serine. ( Abali et al, 2008 ) . Thymidylate and purines are the structural constituents of the DNA molecule.
The suppression of DHFR consequences in DNA synthesis surcease and cell decease. The enzyme has been exploited as a mark in a figure of diseases such as malignant neoplastic disease and arthritis. Extensive research has been conducted since the 50s on the construction and map of DHFR. The drug Methotrexate is a structural parallel which inhibits DHFR by competitively adhering to the active site. It is used as an antineoplastic drug every bit good as an antinflammatory agent in the intervention of arthritis. ( B.Schweitzer et al, 1990 ) .
The focal point of this reappraisal is the ordinance of human DHFR at the transcriptional and translational degree. Presently a complete apprehension of the regulative control of the DHFR cistron has non been established nevertheless it is an country of increased research activity in recent old ages.
Structure and Mechanism of action of the enzyme
Figure 3: The 3D construction of the dihydrofolate reductase enzyme. Its substrate ( DHF ) and cofactor ( NADPH ) can be seen in ruddy. ( Wong et Al, 2005 ) .
The DHFR enzyme has been extensively studied, ab initio in E.coli and more late in mammals in order to find its construction and understand how it functions. The aminic acids involved in contact action and the secondary construction of the molecule have been conserved throughout development in bacteriums and mammalian species. The chief difference between Bacterial and human DHFR is the flexibleness of the molecules. Human DHFR is a more stiff molecule, and this stems from the DHFR lacking subdomain rotary motion constructions. ( Abali et al, 2008 and Sawaya and Kraut, 1997 ) .
The DHFR cistron is about 30 kilobits long and is located in chromosome part q11.2-q13.3 of chromosome 5. After written text the most abundant transcript is 3800 bases in length and the other 2 transcripts are 800 and 1,000 bases long. The ground for the different lengths of the transcripts lies on the fact that three different polyadenylation signals are found on exon 6. A polyadenylation signal is the sequence on the messenger RNA at which the post-transcriptional alteration procedure polyadenylation occurs ; this is the add-on of a polyA tail to the transcript. ( Abali et al, 2008, Chen et Al, 1984, Maurer et Al, 1984 ) .
Figure 4 depicts the location of human DHFR on chromosome 5. The country surrounded by the blue frame indicates the location of the DHFR cistron at place q11.2-q13.3. The ruddy line on the chromosome indicates the place of one of the DHFR pseudogenes. ( Adapted from image at: hypertext transfer protocol: //www.genecards.org/cgi-bin/carddisp.pl? gene=DHFR )
DHFR is a housekeeping cistron, a cistron that is invariably expressed because it codes for a protein that is ever required by the cell. As with many housekeeping cistrons DHFR lacks a TATA box booster, alternatively it possesses multiple GC boxes ( Abali et al, 2008 ) .TATA and GC boxes are types of boosters, a group of short sequence elements located upstream of the coding sequence of the cistron. Promoters act as acknowledgment sites for written text factors. The TATA box is merely a sequence of about 20 base brace of T and A bases with a nucleus sequence of: 5′-TATAAA-3 ‘ . Human cistrons normally have GC boxes as their nucleus boosters and the sequence is rich in G and C residues.
Pseudogenes can be defined as non-functional, intronless versions of a functional cistron. Anagnou and his squad discovered that there were at least 4 pseudogene versions of the DHFR cistron, largely located on different chromosomes. These were identified every bit pseudogenes as they are composed of sequences homologous to the functional DHFR coding DNAs but missing noncoding DNAs. ( Anagnou N.P. et Al, 1984 )
Regulation of Transcription and Translation
The cardinal tenet of molecular biological science is the written text of DNA to mRNA and eventually its interlingual rendition to protein. This tenet underlies the look of every cistron in the genome and is characterised in 4 major stairss ; written text, RNA processing, interlingual rendition and station translational alteration.
Figure 5: shows how eucaryotic Deoxyribonucleic acid is transcribed to RNA and so translated to a protein. ( Available at: hypertext transfer protocol: //www.accessexcellence.org/RC/VL/GG/central.php )
Merely a little part of the full Deoxyribonucleic acid contained in cells is of all time transcribed.
The synthesis of RNA is accomplished by an RNA polymerase, with DNA as a templet strand and ATP, CTP, GTP and UTP as RNA precursors. There are three eucaryotic RNA polymerases each transcribe a specific category of RNAs. RNA polymerase II is the polymerase that maps to transcribe all protein coding cistrons.
RNA polymerases can non originate written text independently, they require transcription factors ; proteins that bind to specific DNA sequences in order to steer the polymerases.
Short sequence elements located in the immediate locality of a cistron, frequently upstream of the cryptography sequence, jointly represent a booster. Promoters act as acknowledgment sites for written text factors.
Transcription is regulated by represser proteins which repress written text by barricading the fond regard of RNA polymerases to the booster.
The messenger RNA transcript undergoes a figure of alterations before it is translated to a protein. These alterations include splicing ; the remotion of coding DNAs and connection of the noncoding DNAs together, Capping ; add-on of 7-methyguanosine triphosphate to the 5 ‘ terminal of the messenger RNA and polyadenylation ; add-on of a poly A tail to the 3’end.
Once the messenger RNA transcript has been created and modified it moves out to the cytol where it interacts with ribosomes to bring forth a concatenation of polypeptides. The right folding of the protein into its 3D construction is straight related to the overall map of the molecule.
The proteins are eventually modified by the add-on of saccharide groups to the side ironss of certain aminic acids in a procedure termed glycosylation. Glycosylation helps the protein to turn up right and confers stableness to the molecule. ( Strachan and Read 2004 ) .
The look of a cistron is regulated at many degrees. The conformation and construction of the Deoxyribonucleic acid can impact the handiness of RNA polymerase, the rate at which written text occurs and the stableness of the RNA relate to the degrees of RNA available for interlingual rendition and the alterations the attendant protein undergoes. However, the principal agencies by which DHFR degrees are regulated in the cell is attributable to transcriptional control. ( Slansky and Farnham, 1996 ) .
Regulation of Transcription
As mentioned antecedently DHFR lacks a TATA box booster, alternatively it possesses 2 bidirectional boosters which consists of two instigator elements and multiple GC boxes. ( Fujii et al, 1992 ) .The major transcripts produced by the downstream booster are responsible for the written text of 99 % of the messenger RNA. ( Masters and Attardi, 1985 ) The upstream, minor transcript initiates 400 bases upriver from the major written text site. ( Blume et al. 2003 )
Regulation at chromosomal degree
As mentioned antecedently written text is regulated by written text factors but at another degree it is besides regulated by composites that re-model chromatin construction in order to modulate the look of single cistrons. The chromatin -remodelling complex is defined as “ A polypeptide composite that can pack or loosen up the secondary and third construction of chromatin ” . This re-modeling of chromatin is based on the acetylation, methylation and phosphorylation of histones, the structural constituents of chromatin. Once the written text factors have bound to the DNA histone ethanoyl group transferases ( HAT ) are attracted to the site heightening relaxation of the chromatin thereby increasing the degree of written text. Repression of written text can besides happen through interaction of the written text factors with histone deacetylases ( HDAC ) that remove acetyl groups and therefore stabilise the nucleosomal construction. ( Roberts and Orkin, 2004 ) . Hence transcriptional activity is modulated by the balance between histone acetylation and deacetylation. ( Park et Al, 2003 ) .
Regulation during cell rhythm by Sp1 and E2F
As discussed earlier Transcription factors are indispensable for the ordinance of written text at the cellular degree. There are two cardinal written text factor households that play a major function in the transition of DHFR degrees ; Sp and E2F. The most of import members of these households ; Sp1 and E2F1 are responsible for the ordinance of the DHFR booster. Sp1 and E2F are located in the locality of the DHFR written text induction part. ( Jensen et al, 1997 ) Sp1 recognizes the consensus GC box sequence ; 5’GGGGCGGGGC 3 ‘ in the DHFR booster. ( Abali et al, 2008 ) . The finding of the importance of SP1 to written text was discovered from many in vitro and in vivo surveies. The DHFR booster unambiguously contains four binding sites for Sp1. ( Park et al,2003 ) .
E2F1 is mediated by the sequence 5 ‘ TTTCGCGCCAAA 3 ‘ on DHFR, which is near to a written text start site. ( Abali et al, 2008 )
Figure 6: Conventional representation of the DHFR major booster. The consensus binding sites for Sp1 and E2F can be seen. The pointer indicates the way and location of the major written text start site. ( Wells et al, 1997 ) .
DHFR is continually transcribed at low degrees throughout the cell rhythm but the rate of written text addition dramatically during G1/S stage. ( Slansky and Farnham,1996 ) .
Figure 7: The above diagram represents the 4 stages of the cell rhythm. ( Sadava et al, 2008 ) .
Surveies in 1996 suggested 3 cardinal constituents involved in the ordinance of written text of DHFR ; CTD, SP1 and E2F. Slansky suggested a theoretical account for written text of DHFR during the cell rhythm. This theoretical account proposes that adhering of multiple written text factors to the booster is necessary for increased written text degrees at the G1/S stage of the rhythm. ( Slansky and Farnham, 1996 ) .
It is evident that Sp1 forms a basal composite and uninterrupted low degrees of DHFR are transcribed through all phases of the cell rhythms. ( Slansky and Farnham, 1996 ) .
The E2F household of written text factors nevertheless was discovered to hold more complex interactions with other proteins that can either repress or increase the degree of written text at different stages in the cycle.It has been determined that there are seven members of the E2F household ; 5 different E2Fs and 2 different DPs. In order for efficient adhering to happen heterodimers must organize between a member of the E2Fsub-group and the DP sub-group. ( Bandara et al, 1993, Helin et Al, 1993 and Krek et Al, 1993 ) . Following on from this find it was ascertained that degrees of certain E2F household members increase at the G1/S boundary, connoting that alterations in E2F binding to the DHFR booster could be responsible for ordinance of written text. ( Slansky and Farnham, 1996 ) .
At the G0/G1 boundary E2F interacts with negative regulators of cell growing ; retinoblastoma ( Rb ) , p107 and p130 doing the formation of a represser composite. This complex so binds to the booster guaranting a really low degree of DHFR written text. ( Cobrinik, 1995 ) . At the G1/S phase the negative regulators become phosphorylated and are released from the E2F composite, leting E2F to interact with other proteins every bit good as the basal written text composite. ( Slansky and Farnham, 1996 ) . After G1/S stage written text from the DHFR booster is merely activated by the SP1 sites. E2F is bound to an inhibitory protein and the degree of written text of DHFR returns to the same degree as during early G0/G1. ( Slansky and Farnham, 1996 and Cobrinik, 1995 ) .
RNA polymerase II contains a C-terminal sphere ( CTD ) which has been shown to be indispensable for in vitro written text of DHFR in mice ; Slansky suggests that this is besides the instance for human DHFR. A figure of observations indicate that the phosphorylation province of the CTD determines its construction and these observations were confirmed by in vitro and in vivo cross associating experiments. A hypothesis emerged that the CTD must be in an unphosphorylated province in order to organize a stable written text composite and so must go phosphoylated to let elongation of written text. ( Dahmus, 1994 ) . During the G0/G1 stage the phosphorylation of CTD by TFIIH ( present in all written text composites ) allows written text to happen at low degrees. At the G1/S boundary a sudden addition of written text of DHFR is observed, this may be explained by the interaction of E2F with a cyclin that caused an addition in the phosphorylation of CTD. ( Slansky and Farnham, 1996 ) .
Although Slansky ‘s survey was good researched and employed legion experimental methods to turn out its cogency the theoretical account proposed remains a hypothesis.
The exact functions of the written text factors SP1 and E2F in the ordinance of written text are non yet clear and have sparked considerable argument.
Following on from Slansky ‘s work, recent surveies by Sahin and Sladek have proposed a double function for E2F-1 depending on the cell rhythm. They suggest that in turning cells E2F-1 binding to retinoblastoma protein ( pRb ) causes repression of DHFR look while in cells re-entering the cell rhythm E2F-1 Acts of the Apostless as an activator of look of DHFR. ( Sahin and Sladek, 2010 ) .
Figure 8: theoretical accounts for the functions of E2F and pRb in cistron look of a figure of cistrons including our cistron of involvement human DHFR. ( Sahin and Sladek, 2010 ) .
In vitro surveies have demonstrated that mutants within the E2F-binding sites cause small consequence to the degrees of written text from the DHFR booster. However, point mutants in the E2F sites consequence in loss of the transcriptional addition at the G1/S boundary. Through experiments affecting serum stimulation it was determined that it is improbable that E2F could account for the addition in DHFR activity at the G1/S stage. ( Means et al, 1991 )
It has besides been showed that in the absence of functional Sp1 proteins or Sp1-binding sites in the booster, DHFR written text is about wholly prevented. ( ref transition ofaˆ¦ ) Equally good as farther foregrounding the importance of Sp1 in the written text of DHFR this besides indicates that E2F is a really weak activator of written text. ( ref transition ofaˆ¦ park ) V. Noe, C. Chen, C. Alemany, M. Nicolas, I. Caragol, L.A. Chasin and C.J. Ciudad, Cell-growth ordinance of the hamster dihydrofolate reductase cistron booster by written text factor Sp1. Eur. J. Biochem. 249 ( 1997 ) , pp. 13-20
Furthers surveies have shown it is necessary for a figure of elements to interact with each other in order to modulate written text. Sp1 and E2F interact with each other and with the maestro regulator, tumor suppresser proteins, Rb and two other Rb homologues, p107 and p130 in order to modulate the look of the DHFR. ( Nevins 2001 ) .
New Layer of Regulations-transcriptional repression
Martianov discovered a phenomenon by which the regulative transcript produced from the DHFR minor booster plays a critical function in the alteration of cistron look through repression of written text. As discussed antecedently the downstream major booster is responsible for written text of 99 % of DHFR RNA. By proving the consequence of the non-coding transcript in trans, a inhibitory consequence in the major booster was apparent, bespeaking the sequence of the major booster is the indispensable portion of the regulative RNA.The analysis of Martianov and his squad revealed an RNA-dependent mechanism of transcriptional repression reliant on the production of the regulative transcript from the minor booster. ( Martianov et al, 2007 )
Enhancement of Transcription
An upside-down repetition sequence has been shown to heighten written text of the DHFR cistron by adding stableness to E2F binding. This overlapping upside-down repetition sequence 5′-TTTCGCGCCAAA-3 ‘ is extremely conserved near the written text start site in the boosters of 3 mammalian cistrons that encode DHFR. The sequence is composed of 2 imbrication, oppositely orientated sites that match the E2F consensus sequence. It was demonstrated that mutants in the motive that eliminate the nature of the upside-down repetition significantly decrease the adhering stableness of E2F. ( Wade et Al, 1995 ) .
Do n’t cognize if this is of import.
Post transcriptional alterations are an built-in portion of the ordinance of eucaryotic cistrons. There are legion mechanisms that operate station transcriptionally to modulate the look of any cistron. The procedure of station transcriptional alteration chiefly relies on proteins interacting with the messenger RNA transcript produced at the written text phase. ( Tai et al, 2008 ) . These RNA-protein interactions result in either targeted debasement of the messenger RNA or forestall the ribosome from accessing the interlingual rendition start codon. Proper processing of the messenger RNA involves splicing, 5′- and 3 ‘ terminal alterations and export every bit good as their half life and interlingual rendition rate. . ( Tai et al, 2008 ) The bulk of the sequences responsible for post-transcriptional ordinance are localized in the untranslated parts ( UTRs ) .
MicroRNAs ( miRNAs ) are a comparatively new find in molecular biological science and are considered to play a critical function in cistron ordinance by a post-transcriptional mechanism. It is predicted that 30 % of all human cistrons are regulated by MicroRNAs by aiming sequences in their 3’UTR. ( Mishra et al, 2007 ) miRNAs have been shown to modulate cistron look through translational repression, mRNA cleavage, mRNA deadenylation, or transcriptional silencing. ( Abali et al,2008 ) . Mishra have by experimentation determined that a specific microRNA, miR-24 is partially responsible for the ordinance of DHFR messenger RNA and protein degrees. miR-24 is located next to a individual base polymorphism ( i.e a alteration in base brace at one specific place on the familial sequence which may or may non take to an consequence on cell map ) . The SNP is a alteration from C to thymine at place 829 on the sequence ( 829Ca†’T ) . In persons who have the normal allele 829C miR-24 downregulates DHFR protein, while people who carry the SNP 829T, over look of DHFR is caused by interfering with miR-24 map. Peoples carry SNP 829Ca†’T tend to hold increased opposition to methotrexate when treated with the drug than those who have the wild type DHFR ( i.e the typical signifier ) .This grounds could explicate the ground why certain patients do non react to intervention with MTX. Hence the SNP 829Ca†’T Acts of the Apostless as a loss-of -function mutant. ( Mishra et al, 2007 )
Regulation of cistron look in eucaryotes is controlled at many degrees. During the last decennary the importance of translational ordinance in cistron look has become progressively clear. Translational control is an energy efficient procedure that maps to regulation the look of a given protein and it helps keep the ordered nature of the cell rhythm. ( liu et Al, 2002 ) .
It was Bertino who foremost identified an increased degree of DHFR in patients being intervention with antifolates. He observed a six fold addition that he tested utilizing a partly purified enzyme from patients immune to the drug and comparing it to cells from the same patient before intervention, confiming his findings that the degrees of DHFR addition upon intervention with Methotrexate. ( Bertino et al, 1962 ) . Following on from this it was determined that a translational ordinance procedure underlies this addition in DHFR degrees. Hillcoat predicted this saying that an addition in DHFR activity during log stage of cell growing to be due to a stable species of messenger RNA. ( Hillcoat et al, 1967 ) .
DHFR regulates its ain interlingual rendition by adhering to its ain blood relation messenger RNA. This phenomenon of autoregulation of interlingual rendition is a well-established mechanism in procaryotes but had non been observed in eucaryotes. DHFR is one of the first eucaryotic cistrons whose look has been shown to follow translational autoregulation. ( Tai et al, 2004 ) . Translational autoregulation airss major advantages to the cell as it gives them a Swift and adept method of changing cistron look in response to external stimulations and cytotoxic emphasis. ( Tai et al, 2004 ) .
Through methods such as in vitro RNA-binding and filter-binding checks and in vivo experiments such as transfection, a cis-acting component in the protein coding part that interacts with high affinity to DHFR protein has been identified. This component has been found to be localized to a 164 nucleotide RNA sequence matching to nucleotides 401-564. This sequence binds to DHFR protein with a similar affinity to that of full-length DHFR messenger RNA. The 164-nt sequence was cloned with the newsman plasmid luciferase and was transfected into human cells to find its biological activity. The findings from this experiment illustrated that the 401-564 sequence was affected by changes in DHFR protein look and was hence a true DHFR-response component. ( Tai et al, 2004 ) . The component was farther localized to an 82 National Trusts sequence related to nucleotides 401-482. Further in vitro surveies identified 2 other sequences contained within the coding part that DHFR might besides interact with. These sequences correspond to DHFR:1-200 and DHFR:220-370. New surveies have even farther localized this Commonwealth of Independent States component to a 27nt sequence which binds with high affinity to human DHFR and forms a ribonucleoprotein composite. They confirmed that DHFR27 RNA sequence is a true DHFR response component and that an integral DHFR protein is necessary for its interlingual rendition regulative effects. ( Tai et al, 2008 ) .
Figure 9: Translational autoregulation ( Tai et al, 2004 ) .
The above theoretical account represents the autoregulation of interlingual rendition of DHFR. When cells are in the quiescent stage i.e. the G0 stage of the cell rhythm, DHFR binds to its blood relation messenger RNA and hence represses interlingual rendition. However when growing demands or substrate degrees addition or when cells are exposed to a cytotoxic emphasis as in the instance of intervention with amethopterin the DHFR protein is no longer able to adhere to its mark DHFR messenger RNA, thereby alleviating translational autoregulation and leting interlingual rendition and synthesis of extra DHFR protein to happen. ( Tai et al, 2004 and Emine et Al, 1997 ) . This procedure has immense biological significance given that it is an efficient mechanism by which DHFR degrees in a cell can be accurately controlled. Equally good as holding biological importance the procedure of translational autoregulation besides explains why some patients being treated with an repressive compound of DHFR, such as Methotrexate, develop opposition to the drug. ( Tai et al, 2004 ) .
The enzyme DHFR is extremely regulated at many degrees. One of the most of import of these is written text. There is still a immense sum of research to be carried out-exact functions of Sp1 and E2F
DHFR has a figure of intriguing and alone properties- this is the ground why it is studied as a theoretical account.
First mark for malignant neoplastic disease therapeutics.
Still provides a rich country for research.
Understanding the ordinance of written text and interlingual rendition of the dihydrofolate cistron may show fresh curative marks for human diseases. ( and better upon antecedently established 1s ) .