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Role of E2 in Ubiquitination

Abstract:

Ubiquitin is ubiquitously present in every eukaryotic tissue and it appears to function through covalent attachment to other proteins, a novel posttranslational modification that is turning out to have a great biological importance. It was studied that some E3 enzymes help in activating the E2 enzymes. The study was designed mainly to see the role of E2 in the ubiquitination process and to screen the expression levels in the absence of E3. In this study the protein of interest was extracted from the recombinant bacterial clones then purified using the affinity, ion exchange and gel exclusion chromatography and confirmation was done using SDS PAGE and western blot. Furthermore direct UV absorption was done to determine protein concentration and Dynamic light scattering was used to measure the size of E2 protein. The protein was studied for the ubiquitination and deubiquitination assays.  E3 is normally required for the substrate complex formation, but in contrast we found that in the absence of E3, conjugating activity was found. Our results showed that E2 alone was responsible for the polyubiquitination, which was confirmed on the SDS PAGE in both the assays. This peculiar feature of E2 can unravel many questions of genetic disorders in humans.

Introduction:

Ubiquitin is a very small protein which exists in all the eukaryotic cells and functions by conjugating to large number of target or obsolete proteins. In simple words Ubiquitin helps to tag all those obsolete proteins for destruction. It consists of about 76 amino acids and is approximately 8.5kDa in molecular size. Ubiquitin is a cell used to post-translational modifications of other proteins to which it is bound by isopeptide linkage through a carboxyl group at its C-terminus. The target protein binding site is amino groups of lysine side chains. Ubiquitin itself also contains seven lysines  (K6, K11, K27, K29, K33, K48 and K63), enabling its chaining to form polyubiquitin chains. The possibility of chaining through different lysines, including the possibility of branching, means the large theoretical diversity of the resulting ubiquitin chains, and a different signaling function associated therewith. For the interaction of ubiquitin, a hydrophobic region is important, including the amino acids Ile44, Leu8, Val70 and His68. Different chaining of polyubiquitin also results in different diffusion of hydrophobicity. Interaction sites on the surface of the chain, creating specific binding surfaces recognized by the respective receptors.

Figure 1. Structure of ubiquitin from two different views. For ubiquitin and its similar structures, it is characteristic of the arrangement of the β-sheet and the α-helix shown in the figure. The figure on the left (a) contains the blue highlighted hydrophobic amino acids Ile44, Leu8, Val70 and His68, forming the major interaction surface. In the figure on the right (b), all amino acid residues that are covalently linked to other proteins: Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, Lys63, N-terminal Met1, and C-terminal Gly76 . Taken from doi: 10.1146/annurev-biochem-060310-170328.

The sequence of ubiquitin is evolutionarily highly conserved and differs only in very remote species of organisms. In humans and yeasts, four ubiquitin genes have been discovered – UB52, RPS27A, UBB, UBC. Two encode ubiquitin as a fusion protein with ribosomal proteins: the RPS27A gene produces ubiquitin fusion protein with the ribosomal protein S27a from the 40S subunit, the UB52 gene product with the 60S subunit of the ribosomal L40 protein. UBB and UBC produce polymeric ubiquitin precursors of three and nine subunits, respectively, which are subsequently cleaved into individual ubiquitins by specific DUBs. Prokaryotes do not pocesses these types of proteins which attach to another protein by covalent attachment. But instead they do contain proteins which seem to be the ancestors of ubiquitin. ThiS and MoaD are the bacterial proteins which aid in the insertion of sulfur into the thiamin and molybdopterin, respectively. The protein confirmations are very much similar to that of ubiquitin [A. P. VanDemark et al, 2002].

https://cdn.rcsb.org/pdb101/motm/images/ubiquitin.gif

 

Figure 1: Diagram showing the Ubiquitin (at the top) and group of four ubiquitin molecules bound to a protein (bottom). Image retrieved from https://pdb101.rcsb.org/motm/60

Cells build up the proteins (structural or physiological) in a continuous process. Most of these proteins are used for the process and discarded when not in use. This sort of homeostasis is maintained by the cell machinery. Signalling proteins like transcription regulators, and cyclins which control the process of cell division have very short half lives as such they need to be recycled back immediately [Glickman MH, 2002]. Even in the case of some specilaized enzymes, cells build them and discard them immediately after use. Such a system would be a mere waste but it is very much needed to keep in pace with the minute by minute activities of the cell.   But such destruction of the obsolete proteins will be taken care of by the cell machinery with the help of small proteins. Ubiquitin comes and attaches to all the proteins which need recycling.  For successful protein degradation, a string of at least four ubiquitins is required and the correct type of binding between them. The most common is the binding via lysine 48, which is considered to be a clear signal determining the protein to degrade. The linkage through lysine 63 commonly plays a role in DNA repair and endocytosis, although it has been shown that the K63 chain is a sufficient signal for in vitro degradation. For example, K11 chains play an important role in ERAD (ER-related degradation).

Three different enzymes allow for ubiquitination. The first one binds the ubiquitin molecule to the consumption of ATP and is the so-called ubiquitin-activating enzyme E1. In this step, glycine is activated ATP at the C-terminus of ubiquitin. Subsequently, a thioester linkage occurs between the cysteine enzyme E1 and the C-terminus of ubiquitin. Thus activated ubiquitin is subsequently recognized by ubiquitin-conjugating enzyme E2 and attached to it by thioester binding. As soon as such a complex approaches the protein of degradation, the final binding of ubiquitin to this protein occurs. This step mediates the enzyme ubiquitin-ligase E3. E3 can specifically recognize what protein is to be sent for disposal and thereby provide the ultimate ubiquitin. Since E3 ligases break down proteins based on specificity, the cell has to produce many different E3 enzymes. Using comparative genomics, it has been shown that there are only a few genes encoding E1 but about a dozen genes encoding E2 and about one hundred E3 ligase genes.

Ubiquitin ligases contains two domains namely RING (Really Interesting New Gene) and those bearing the HECT domain (Homologous to E6-Associated Protein C-Terminus). E3 with the HECT domain contain cysteines in the structure through which they directly bind ubiquitin before the target protein is tagged. In contrast, the E3 ligases with the RING domain do not bind themselves to the ubiquitin but catalyze its transfer from E2 directly to the target protein.

E2-E3 complex structure

Figure 5: E2 and E3 complex structure. E2 complex is coloured in sandy brown and E3 complex is cloured in light sea green. Image created using JSmol viewer. doi: 10.2210/pdb4ccg/pdb.

Ubiquitination usually affects the cellular process by controlling the breakdown of proteins, coordinating all the cellular localizations, protein activation and inactivation and protein-protein interactions modulation. These effects are usually regulated by different types like Monoubiquitination and Polyubiquitination.

Monoubiquitination is the addition of one ubiquitin protein to a substrate molecule. Multi-monoubiquitination refers to the addition of a single ubiquitin molecule to many of the substrate residues. This addition is required for the formation of polyubiquitin chains. This mechanism also effects the cellular processes like endocytosis and viral budding.

Polyubiquitylation is addition of chain of ubiquitin protein on lysine residues of a substrate.  These chains are made by establishing a link between glycine and lysine residues of ubiquitin proteins. N-terminus of lysine serve as a point of ubiquitylation. Among the seven lysine residues, 48 lysine linked polyubiquitylation are targeted for proteolysis. To recognized by 26S proteasome, at least four ubiquitin molecules should be attached to a lysine residue and this also require to form a barrel shape, in center of which is a proteolytic core. 63 lysine linked ubiquitin protein serve as target for inflammation, endocytic trafficking, DNA repair and translation (Nathan, Kim, Ting, Gygi, Goldberg 2013).

The cell also has an ubiquitin cleavage tool, which is referred to as deubiquitionation. The human genome contains genes for the so-called 79 deubiquitin enzymes (DUBs). It is a group of specialized isopeptides, whose function is to cleave the bond between the C-terminal glycine ubiquitin and the protein to which it binds, and this protein may be another ubiquitin. By their activity they function as antagonists to the action of the E3 ligase.

DUBs are divided into five main families: UCHs (Ubiquitin C-terminal Hydrolases), USPs (Ubiquitin-Specific Proteases), OTUs (Ovary Tumor Proteases), Josephine and JAMMs JAB1 / MPN / MOV3 metalloenzymes). Enzymes from the JAMMs family have metalloprotease activity, enzymes with the other four families function all as cysteine ​​proteases.

Criteria determining the affinity of DUBs for the substrate that cleaves are different: their function may be specific to either a particular protein, a particular type of ubiquitin chain, or may be associated with a proteasome. Proteasome-associated deubiquitinases contain a domain that interacts with proteasome and behaves as its free subunits. Their purpose is to cleave the linked polyubiquitin chains from the proteasome pathway substrate so that they are not degraded along with the substrate in the proteasome. This ensures the recycling of ubiquitin molecules and their adequate supply for the cell-based ubiquitination reactions. In this function, deubiquitinases specific to a certain type of chain are involved, which loose chains in the cytoplasm are further cleaved up to the individual ubiquitin molecules. It also breaks the chains bound to proteins if it is a chain whose cleavage is given deubiquitinase. The latter group of substrate-specific DUBs cleaves chains of different types, provided that they are bound to a particular protein or proteins for which the enzyme is revivified. Their function is primarily regulatory, as their activity slows the degradation of the protein or other cellular action associated with its ubiquitination. As a result, more contradictory factors determine the ubiquitination of the protein, allowing more complex regulation of this key cellular process.

The proteasome complex is responsible for its important role in handling several of protein-related diseases such as neuronal disorders and prions [Nandi D et al 2006]. This complex degrades and break the obsolete proteins which might be toxic to our body. Such type of degradation can be carried out by proteasome ubiquitin pathway in  amore effective manner. This complex consists of four ring like structures piled up like a cylinder to form a hollow central core . Each ring is made up of seven protein molecules where the outer cap is made up of alpha subunits. They act as a barrier molecules to enter and leave the proteasome core. The inner rings consist of beta subunits and make up the actual sites for the degradation.

For centuries, lysosomes were thought be the major cell organelles which aid in protein degradation. But with the onset of the discovery of proteosomes and ubiquitin like molecules, the whole notion was changed. It was found that the proteosome and ubiquitin work in a coordinated fashion in tagging and degrading the obsolete proteins  [Rubinsztein, David C, 2006].

The first step in this process is identification of the target protein which was found abnormal or obsolete. Such type of proteins also called obsolete proteins need to be removed from the environment [Ardley HC, 2004].

Ubiquitin protein has huge functional diversity and is required for precessing variety of cellular processes such as Transcription and DNA repair, Synthesis of ribosomes, Apoptosis and Cell cycle, Immune response and inflammation of the immune cells, Neural and muscular degeneration. It was also found that the ubiquitin pathway holds responsible for the pathogenesis of many of the genetic disorders. The ubiquitin proteasome pathway is required for removing denatured, misfolded, non- functional proteins that may cause disease and short-lived proteins within the cell.

Several proteins are not formed in order or more obsolete proteins are formed in the cell.  The temperature of the cell is maintained at 37°C or higher which denatures the proteins. Such denatured proteins have a hydrophobic area which can be tagged and broken down. Some of the proteins like proteins with an exposed peptide sequence are very unstable  and vulnerable for hydrolysis which need to be labelled. Proteins like cyclins which help in cell division usually process the obsolete to the ubiquitin proteosme system (UPS). There are some within the cells like proteases and kinases which modify and changes the conformational structure of the proteins.  The high salt concentration and fatty acid accumulation might also aid in denaturation and degrade the proteins. All of the above show the severity of the toxic levels of the proteins in the cell. With such a high rate of errors, the misfolded proteins need to be broken down at a high speed.

Proteins are first unfolded and refolded at the receptors of the Ubiquitin proteins. They label the proteins which have to be degraded. If such labelling or tagging is not done, proteosome cannot distinguish which proteins have to be degraded [Helmuth, Laura 2001]. This could eventually leads to serious accumulation of the malfunctioned protein and increase the cell toxicity and cell death. If such a ubiquitin system is not present within all the cells, some of the insoluble  protein clusters formed might block the proteasome channel and as such the defective proteins might be accumulated. Proteosomes also get aged and may slow down in functioning during the infection with sporadic diseases. This also might be responsible for the accumulation of toxic clusters within the cell.

Method:

Protein Expression on small scale:

 

Luria Broth media was used in this experiment to growth the bacteria. 5ml of LB broth (sterile) and 5μL of Kanamycin (50g/mL) was added and a loopful of inoculating culture was inoculated at 37°C shaker/incubator for overnight. The overnight media was added with the respective antibiotic and incoluated with 250μL (1:100) of the starter culture. The culture was incubated at 37°C shaker/incubator for overnight.

Determination of Bacterial growth:

The bacterial growth was determined using the spectrophotometer. Briefly,1ml of sample was taken in 1.5ml of cuvette and the optical density at 600nm (OD600nm) was recorded. Sterile LB media was used as control. OD values were recorded before and after induction with IPTG (0.5mM). Induction was carried only to the culture OD 0.613. The flasks were incubated in a shaker/incubator at 20°C overnight.

Both the tubes contained before induction and after induction were collected and centrifuged at 3200xg for 20min in a cooling centrifuge.

The pellets were resuspended in BugBuster Master Mix (1gm/5ml) by pipetting up and down for several times and incubating them on roller for about 15 min at room temperature.  BugBuster master mix breaks the cell membrane of bacteria and aids in degrading the DNA and RNA. It helps in releasing the soluble material. 1μL of sample from before induction whole cells and after induction whole cells were used for SDSPAGE analysis.

The contents were centrifuged at 4°C for 20min at maximum speed. The supernatant was collected before induction cell extracts and after induction cell extracts were used for SDS PAGE.

Bacterial protein extraction:

The bacterial strains (E.coli BL21 Star) were transformed with pRSF_Duet1 Ube2t, using kanamycin (50µg/mL) as the natural marker. The protein of interest cloned was MCS1 which is His6-Ube2t (His tagged).

Macintosh HD:Users:ubcg49ac:Desktop:Ube2tmap.png

Figure 2.  Plasmid map showing the recombinant vector pRSF-Duet with Ube2t sequence which was used to express the Ube2t protein (E2).

The molecular mass of the given sequence was calculated suing the online calculator tool (http://protcalc.sourceforge.net/). The molecular mass was found to be 24638.0410. The pH of the sequence at the isoelectric point was found to be 7.70 and the charge at pH 7 was found to be 2.8.

The bacterial pellet was collected which expresses E2.  The pellet was thawed and added with 1X EDTA-free protease inhibitor followed by 1mL of buffer E-A (50 mM Hepes pH7, 150 mM NaCl, 2mM MgCl2, 1mM DTT, 25 mM imidazole and 5% (v/v) glycerol). The contents were mixed thoroughly and incubated for 5min at 37°C before vortexing. The contents were added to defrosted bacterial pellet lysed with 1mL of lysozyme (30mg/mL) solution and 3μL Nuclease solution (25units/mL). 28mL of Buffer E-A (50 mM Hepes pH7, 150 mM NaCl, 2mM MgCl2, 1mM DTT, 25 mM imidazole and 5% (v/v) glycerol) was added. The contents were incubated on a tube roller initially for half and hour at room temperature, then 30minutes on a tube roller at 4°C.

Supernatant collection: The contents left over are transferred to a fresh 30mL centrifuge tube and centrifuged at 48000 x g for 30min using a floor standing centrifuge at 4C. The supernatant was collected. A 5µL aliquot of the cell extracts was and the samples were stored in freezer until further use.

SDS PAGE:

In this experiment the protein profile unidirectional SDS-PAGE was performed on 10% separating gel and 5% stacking gel. This was done in a mini vertical gel system. About 100µg of protein sample was loaded in each sample well. 10µl of sample loading buffer with bromophenol blue was added as the tracking dye. The first well was used for protein ladder. The gels were run at a voltage of about 300V for 17mins. Following the run, the gels were placed in tray containing coomassie brilliant blue and incubated on the shaker or roller for overnight. The gel was photographed and stored for further use.

Immobilized metal ion affinity chromatography (IMAC) charged with Ni2+ is used for the purification of histidine-tagged proteins and the samples were pooled for Ion exchange chromatography.

Ion exchange chromatography:

An ion-chromatography resin which binds a positively charged species is known as a cation exchanger. Most of the proteins which are negatively charged or neutral at pH 7 will not bind to the resin. E2 is usually positively charged at pH 7 from its isoeletric point therefore a commercial cation exchanger 1mL SP Sepharose (GE Healthcare) was used in this experiment.

Procedure: 1mL SP Sepharose fast flow column was used with 200mL Buffer E-C (50mM Hepes pH 7 / 5% glycerol / 1mM DTT). The column was equilibrated with buffers E-E (50 mM Hepes pH 7 / 150 mM NaCl / 1 mM DTT / 5% (v/v) glycerol) for 4 hours. The samples were eluted with100mL Buffer E-D (50mM Hepes pH 7 / 1000mM NaCl / 5% glycerol / 1mM DTT). All the fractions were pooled from the affinity step into a 50mL tube. Only the purest fractions which contains proteins were collected. The sample obtained was further concentrated to 15ml in an Amicon MWCO 10kDa concentrator and 30 mM of NaCl was used to adjust the salt concentration.

Western Blotting: Western blotting is used to detect specific proteins of interest. The experiment was aimed at observing the proteins from the whole cells, extracts of cells and protein samples. The blotted gels were transferred onto nitrocellulose membrane. An anti-His antibody is used in the protocol.

Materials and reagents required: SDS-PAGE apparatus; TransBlot Turbo Transfer system and Gel DocTM EZ System were used in the study.

 

2 x SDS-PAGE precast gels were used in the protocol and Trans-Blot Turbo Mini PVDF Transfer units were used for the blotting process. Monoclonal antibodies of Anti-polyHistidine-Peroxidase clone HIS-1 was ordered from Sigma (cat no:-A7058).  The transferred blots were washed with buffer (1x PBS + 0.05% TWEEN®20). Blocking solution (1x PBS + 5% non-fat dry milk) was used. The membrane was incubated with Anti-polyHistidine Peroxidase conjugate antibody (1:2000). Development of the blot was made using a substrate (SIGMAFAST™ 3,3ʹ- Diaminobenzidine). The reaction was also stopped by removing the substrate solution.

Determination of protein concentration by direct UV absorption:

The supernatant collected was subjected to estimation of concentration. The concentration of the protein sample can be calculated directly by UV absorption at 280nm. 1ml of supernatant was added to 1.5ml cuvette and placed in the UV spectrophotometer and observed for the optical density at 280nm. Readings are noted at both 260nm and 280nm. Sterile distilled water was used as the blank. The readings were tabulated and calculated using the formula. Concentration (mg/ml) = (1.55 x A280) – 0.76 x A260).

Dynamic Light Scattering

Dynamic Light Scattering, DLS is visible laser light. The Avid Nano W130i dynamic light scattering system was used in this experiment to assess aggregation of the E2 protein as well as to calculate the hydrodynamic radius of the protein. The E2 protein sample was loaded into Avid Nano blade cell cuvette and the samples was analyzed by DLS

Ubiquitination Assay: Ubiquitin a small signalling molecule present ubiquitously in the cellular locations. As such it is called ubiquitin. It is the most versatile signalling molecule as it can be linked to chains. The cascade is a 3 step process which requires three enzymes for priming, transporting and linking the ubiquitin molecules to the target protein. E2 protein is specilaised protein for activation of Fanconi Anemia DNA repair pathway. It aids in repairing the interstrand crosslinks (ICL’s) in DNA. In the absence of this function, the cells become susceptible for carcinogenic affects. Ube2t self or autoubiquitinate in vitro and in vivo conditions. This is due to the close vicinity of a Lysine residue to the cysteine though other sites. Hence it is possible for carrying out simple assays to demonstrate the autoubiquination. Autoubiquitination is a reversible process which requires the use of a deubiquitinising protein known as a DUB. These are endopeptidases which bind Zn2+. In the present assay, we will add ubiquitin to E2 and in deubiquitination we add DUB to remove the ubiquitin. This returns the E2 to its native state.

Three different proteins namely E1, E2 and ubiquitin are mixed in the presence of ATP and incubated at 37 °C. The samples treated are then analysed on SDS gel for further validation.

Two tubes labelled as +ATP (with ATP) and the second –ATP without ATP are added with the specified reagents in the order as shown in the Table 1. At time T=0 a 10μL aliquot was removed from the tubes and 3.3μL of gel loading buffer was added in order to stop the reaction. The process was repeated at 30min and 60min of time. Finally the aliquot at 60min was added with 1μL of apyrase (0.5units/μL) to both the reaction tubes. The reaction mixtures were freezed and used for deubiquitination assay. The 6 samples obtained were on SDS PAGE for further validation along with the protein ladder.

Table 1 :Use the volumes indicated in Table 1 below.

+ATP assay Volume /μL –ATP assay Volume /μL
Buffer 54 57
MgCl2 3 3
E2 9 9
E1 3 3
Ubiquitin 3 3
ATP 3 0

Deubiquitination Assay: The assay was carried basin on the result analysis of the ubiquitination assay.Sample of ubiquitinated protein, DUB solution, 4X SDS-PAGE loading buffer with β-mercaptoethanol and SDS-PAGE gel apparatus are required for the experiment. 45μL sample (+ATP run) from the ubiquitination assay was defrosted. A 10μL of aliquot was added to 3.3μL of SDS-PAGE loading buffer and stored at room temperature. To the contents 1μL of DUB (0.5μM) was added and incubated at room temperature for 15 min. 3.3μL of SDS-PAGE loading buffer was used in order to stop  the reaction. The prosses was repeat the same at 30min. The two samples obtained (15min, 30min) were run on SDS-PAGE gel along with the protein ladder. Sample without DUB serves as the control.

Results:

SDS-PAGE of Small Scale Protein Expression:

The SDS PAGE gel of the proteins before and after induction showed significant results. Before induction, in both the whole cells and cell extracts, there was no significant protein expression. On induction, there was an ample amount of protein expression [Figure 1] observed in both the whole cell and cell extracts. This proves of the positive induction which expressed the desired protein. The bands were found to be approximately of 24Kda from figure 1. Both lane 4 and 5 showed expression of the protein. The protein was found to be expressed after induction with the IPTG. Lane 2 and 3 showed no expression (i.e in both whole cell and cell extract).

Figure 1: SDS-PAGE of Small Scale Protein Expression. Protein ladder is lane 1. Before Whole cell is lane 2. Before Cell extract is lane 3. After Whole cell is lane 4 and After Cell extract is lane 5.

ION exchange chromatography:

Ion exchange chromatography was done to purify the protein of interest. The protein of interest was E2 which was found to be expressed in the bacterial cells (E.coli BL21 Star)  using the plasmid pRSF_Duet1 Ube2t. The protein of interest cloned was MCS1 which is His6-Ube2t (His tagged).

On separation of protein fractionation, the supernatant was allowed for ion exchange to elute the protein of interest. The protein of interest was found to be approximately around 24KDa. As shown in the figure 2, the protein of interest was found at a peak of molecular weight 24KDa between the 35-40ml of elute volume. The sample component was collected and later used for the SDS PAGE analysis for validation.

Figure 2: Ion exchange chromatogram showing the peak value of the desired protein. The protein of interest peak was shown to be at 25KDa. The volume of the elute between 35-40ml.

SDS PAGE analysis of ion exchange gel fractions:

The gel shows the purest samples containing the protein fractions only. From the figure 3, it is clearly understood that sample from the pooled fraction after affinity chromatography was showing same response after the ion exchange. The protein band of interest was found to be approximately 25 KDa which was correlating to the hypothetical calculated value (24638.0410 ) from the online calculator. The protein bands confirm of the presence of the desired protein. All the samples were pooled and used for the Ubiquitination assay. Fraction 6, 7, 8 and 9 were pooled together and used for purification on gel exclusion chromatography.

Figure 3: SDS- PAGE of samples after ion exchange. Protein ladder is lane 1. Pooled sample after affinity chromatography is lane 2. Flow through peak that was collected after the start of the injection is lane 3,4 and 5. Wash peak that was collected at the start of the wash is lane 6. Fraction in the 0-100% gradient that contained significant increase in absorbance at 280nm is lane 7,8,9 and 10.

Figure 1: SEC profiling of His tagged proteins. Absorbance greater than 260nm was recorded. The fitted curve was represented as a solid line. The UV absorption was measured by Ultraspec 4300 pro UV/Visible spectrophotometer (path length: 1 cm). All the samples used in the experiment were prepared in buffer A. The SEC patern was performed on 16/60 Sephacryl S100 HR column on an AKTA system.

Figure 2: SDS-Page Gel showing the fractions from the gel exclusion chromatography. Protein ladder is lane 1. Pooled sample of E2 protein after affinity chromatography is lane 2. Flow through peak that was collected after the start of the injection (t2, T3 and T4) is lane 3,4 and 5. Wash peak that was collected at the start of the wash (T7) is lane 6. Fraction in the 0-100% gradient that contained significant increase in absorbance at 280nm (T12, T13, T14 and T15) is lane 7,8,9 and 10

Fraction from the affinity chromatography contained protein of interest. Lane 7, 8, and 10 showed the bands at specific length saying the presence of the proteins. All these fractions showed an increase in the absorbance at 280nm. These fractions were collected and used for the western blot analysis. These fractions pooled were found to be pure.

Western blotting: The blot resulted in sharp and distinct bands at the respective molecular weights. The sharp and distinct bands at 25Kda were observed in all the fractions. This demonstrates that all of the fractions contained protein fractions of interest. Fraction 8 and fraction 12 showed little or no bands at 25kDa. This confirms that the proteins in study contained the desired fractions.

Figure 4: Western blot of the samples before and after pooling from the purification procedures. Protein marker is lane 1.The whole cell is lane 2. Cell extracts is lane 3  Pooled sample after affinity chromatography is lane 4. Pooled sample after ion exchange chromatography is lane 5. Fractions from size exclusion chromatography; is lane 6. The concentrated fractions from size exclusion chromatography is lane lane 7. Pooled sample after ion exchange chromatography is lane 8,9,10,11 and 12.

The sample proteins obtained were estimated for the concentration at 260 and 280nm. The concentration of the sample from the given spectrophotometer report was found to be 0.01647. The absorbance at 280nm and 260nm was found to be 0.013 and 0.017 respectively [Figure 4]. The protein concentration was calculated [Layne, E, 1957] using the formula Concentration (mg/ml) = (1.55 x A280) – 0.76 x A260).

Figure 4: Spectrophotometer report of the protein sample. All the values were average of triplicates.

Ubiquitination: The E2 conjugating enzyme tested in the reaction catalysed the substrate ubiquitylation assay. From the data it seems likely that E2 conjugation with the substrate was possible at 30min and thereafter. In lane 2, the substrate was free in nature, whereas in lane 4 the substrate conjugation was seen clearly. This clearly indicates the role of E2 in conjugation. As you can see from the figure 5, the conjugation increased with the time. In lane 6 and 7 the conjugation of E2 can be clearly depicted. Conjugated enzyme in lane 7 was seen approximately at 25KDa.

Figure 5: Correlation function graph from the dynamic light scattering experiment. Estimated moleculer weight of the sample E2 obtained was 128.20Kda. all the results were average ± sd.

The correlogram obtained from the experiment proves that the sample is containing very small particles. From the graph it is also evident that the correlation  of the signal will decay more fastly. Correlation Curve or correlation function can be measured in a dynamic light scattering (DLS) experiment. The curve obtained was smooth and depicts of the single exponential decay function. This suggests of the possibility that the sample was of mono-size particle in dispersion. Usually in monodisperse spheres, the correlation function will be a single solid line with a single decay on top of the baseline. This confirms that DLS is absolute [figure 5].

Figure 6: Graph showing the mass distribution properties of DLS experiment. Top left: mass distribution curve; Top right: intensity distribution; Bottom left: Volume distribution; Bottom right: Number distribution.

Polydispersity can be calculated using the graphs obtained from the percent intensity, number, volume and distribution. From the graphs it was obtained that the mass distribution,  intensity distribution, Volume distribution and Number distribution all were found to be 100%. As shown in the graph all the peaks were occupied in only a single range. They were found between 100 to 101 only. This confirms that the particles were monodisperse in nature.


Figure 7: SDS-PAGE showing Ube2t Ubiquitination. Protein ladder is lane 1. 0 minutes samples with ATP is lane 2. 0 minutes samples without ATP is lane 3. 30 minutes samples with ATP is lane 4. 30 minutes samples without ATP is lane 5. 60 minutes samples with ATP is lane 6 and 60 minutes samples without ATP is lane 7

Deubiquitination: From the figure 6, it is clearly understood that the E2 conjugation was decreased as the time increased. In lane 12, the conjugated sample was found to be deconjugated. In lane 13 and 14  the deconjugated substrate can be seen away from the E2. This clearly depicts of the role of E2 in conjugation.

Figure 8: SDS-PAGE of Deubiquitination. Protein ladder is lane 1. 0 minutes with DUB is lane 2. 30 minutes with DUB is lane 3 and 30 minutes with DUB is lane 14. The samples were run on 10% gel.

In the figure 6, E2 protein was observed at about 25KDa. Once DUB was added initially, the deconjugation of the protein can be seen clearly. In lane 13 and 14, the deconjugated substrate was found to be separated at about 50KDa.

Discussion:

 

Ubiquitination is a post-translational modification which is reversible in nature and extensively used during regulation. The attachment of ubiquitin to some of the cellular proteins influences many such pathways and inturn disrupts the protein localization and proteosome degradation. This regulation is linked to many diseases in humans like cancer, neuronal dysfunction and muscle wasting diseases. The E2 conjugating enzyme can either directly bind to E3 or directly transfer the ubiquitin to the target protein. In order to carry this function, E2 binds to E1 and activated ubiquitin. Humans possess about 35 different types of E2 enzymes which are highly characterised and highly conserved in nature.

The results obtained from the small trial protein extraction were found to be successful. The proteins isolated were quantified using the UV spectrophotometry and was found to be pure. The concentration of the protein sample was found to be promising for the filtration techniques.  The dynamic light scattering experiment confirms that the sample in the study was monodisperse in nature. From the instrument report it was observed that the PDindex was 1.264 (which is greater than 1). If the polymer or sample with a PDI>1 means that is has monomer units all arranged equally in chains but of varying lengths. Most probably the sample would be a natural polymer. And this PDI concludes of the nature of the sample, whether it is monodisperse or polydisperse. If the PDI value is one or greater, it indicates mono dispersity and the experimental sample was thus fund to be monodisperse in nature.

The supernatant was subjected to affinity chromatography for purification of desired protein. The samples which contained only the protein fraction were pooled and run on ion exchange column for further purification. The gel shows the purest samples containing the protein fractions only. From the figure 3, it was observed that the band was our protein of interest with an approximate molecular weight of 25Kda. The sample obtained was confirmed of the E2. This sample was further purified on gel exclusion chromatography. The elutes were pooled for the total fraction and used for the ubiquitination assay and deubiquitination assay. To test whether the samples from the whole cell extracts and pooled fractions contained the protein of interest, western blot analysis was used for confirmation. Anti polyHistidine-Peroxidase antibodies were sued for the confirmatory studies. All the proteins of interest were tagged with Histidine residues. The antibodies used in the study was used during the hybridization step. The western blot confirmed that all of the fractions contained protein of interest. The blots resulted or showed distinct bands at about 25KDa which confirms of the presence of protein of our interest.

The main approach of the study was to confirm of the assertive role of E2 in autoubiquitination. As the experiment was done in the absence of E3, the role of E2 was possible defined and understood. The data clearly shows the role of the E2 in directing the ubiquitination. From Ube2t ubiquitination assay , it was understood that the E2 aids in ubiquitination. In Ube2t ubiquitination assay, in the presence of ATP.

The experiment as designed was mainly to confirm the role of E2 in ubiquitination as an individual or to conjugate with the E1. As the study was done without using E3, the study confirms the role of E2 alone in ubiquitination. From the data presented in the Ube2t ubiquitination assay, the E2 protein conjugated with the substrate. The conjugation with the substrate was shown clearly in figure 7. In this case, as the time increased with addition of ATP, the E2 protein conjugation with the substrate was seen. This confirmation alone cannot validate the results of the role of E2. Hence a deubiquitination assay was designed in parallel. According to deubiquitination  assay, an enzyme called DUB (Deubiquitinating enzymes ) was used. This enzyme returns the state of the protein to its natural state.  In other words, the effect produced by the E2 can be reversed back by the DUB [Elsasser S, 2004]. The role of DUBs in human diseases still remains unpredicted. But it has been very useful in treating or bringing back to natural conditions in terms of cancer and other neuronal disorders. These DUBs are very crucial in treating some malignant tumors. And significantly, in the deubiquitinating assay, the DUB showed its cleaving activity. The conjugated proteins in the presence of E2 were deubiquitinated and that can be seen from the figure 8.

In lane 12 the conjugated protein can be seen moving at 30kDa. And the activity was found to be time dependant, and as the incubation time increased, the ubiquitinated protein was deconjugated by the DUB. The gel clearly shows the presence of two distinctive bands at approximately 50KDa. And from the present study our findings confirm the possible individual role of E2 in conjugation. As E2 is thought to act lonely or in conjunction with E1, the activity was mainly to confirm the role of E2. Hence as the study was without E2 and the results also shows of its nature.

Ubiquitin gets covalently attached to itself or to other proteins either individually as a molecule or in the form of poly-ubiquitin chains. This poly chain formation requires the series of ATP dependant enzymes namely, E1, E2 and E3 [Reyes-Turcu FE, 2009]. E3 binds to substrate and also to an E2 to form a complex called E2-E3. This complex formation is very crucial for the development and extension of the cascade [Finley D. 2009]. At the same time ubiquitin can be conjugated to itself to form diverse types of chain linkages. And our study is in relation to this literature. E3 is normally required for the substrate complex formation, but in contrast from our findings, we found that in the absence of E3, conjugating activity was found. Usually in the absence of E3, E2 should not aid in conjugation [Vierstra RD, 2009; Elsasser S, 2004]. But our results show that E2 alone was responsible for the polyubiquitination. This finding was confirmed on the western blot in both the ubiquitination and deubiquitination assays.

On summing up, the E2 protein can be studied in detail about its mechanism and its action. This can unravel many questions of genetic disorders in humans. This peculiar feature of E2 conjugating enzyme to complete the cascade in the absence of E3 would surely be of interest for the scientific field. Moreover, we can expect a better understanding of the prognosis of the diseases and in developing diagnostic tools.



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