Crystal Structure of the Retinoblastoma Protein N Domain Provides Insight into Tumor - Assignment Example

Title proposed: The significance of the Crystal Structure of the Retinoblastoma Protein (pRb) N Reviewed Journal: Crystal Structure of the Retinoblastoma Protein N Domain Provides Insight into Tumor Suppression, Ligand Interaction, and Holoprotein Architecture Name: University: Course: Date: Table of Contents Theme Page Number 1.0 Abstract 3 2.0 Introduction 3 3.0 Biological importance of the protein 7 4.0 Significance of the study 8 5.0 Bioinformatics analysis of pRb 6.0. Methodology and Results of pRb N 8 14 7.0 Summary of the Results 22 8.0 Conclusion/ Discussion 9.0 References 23 25 1.0 Abstract Retinoblastoma susceptibility protein is a tumor suppressor protein to a limited number of cancers. Retinoblastoma protein (Rbp or pRb) regulates the cell-cycle, causing cells to divide in a controlled manner. The pRb undergoes degradation through the proteasome-dependent pathways and any inactivation of the pRb results to development of human malignancies (Wang et al., 2001). Understanding the Retinoblastoma protein structural and functional analysis has given an imminent knowledge in Rb N mutation oncogenicity and further categorizes protein distinctive phosphorylation regulated site. However, the pRb degradation processing and regulation is not unclear yet (Boyer et al., 1996; Wang et al., 2001). 2.0 Introduction Retinoblastoma protein (Rbp or pRb) is a tumor suppressor protein to a limited number of cancers such as retinoblastoma and osteosarcoma (Murphree & Benedict, 1984). According to Korenjak & Brehm (2005), the pRb belongs to the ‘pocket’ protein family, with its members containing a pocket like structure. Retinoblastoma protein (pRb) regulates the cell-cycle, causing cells to divide in a controlled manner and undergoes degradation through the proteasome-dependent pathways (Fig 1 and fig 2). The pRb plays a key role in several cellular processes including cell cycle regulation, DNA repair and replication, DNA-damage response, senescence, cell protection against apoptosis, and cell differentiation. The pRb affects gene transcription negatively and positively resulting to regulation of distinct cellular processes. Furthermore, the retinoblastoma protein promotes the differentiation and antiploliferative activity through activation of gene transcription, however, this cell requires gene repression (Wang et al., 2001). According to Classon & Harlow (2002), the retinoblastoma gene is mostly targeted for inactivation at the course human malignancies. Figure 1: the Normal cell cycle (Murphree & Benedict, 1984) The Rb interrelates with the E2F family of transcription factors resulting to active repressor complexes and negative regulation of expression of E2F-dependent genes (Stevaux & Dyson, 2002). The pRb gene product has several roles in cellular processes such as apoptosis and cell cycle (Liu et al., 2004). At the first phase (G0 and G1) of the cell cycle, pRb interacts with E2F family transcription factors, hence, blocking their activity. The E2F proteins activates the expression of genes involved in progression of cell cycle, cyclins A and E, proteins, and enzymes required for DNA replication (Stevaux & Dyson, 2002). The promoters of genes has the binding sites for E2F and their expression take place at G1/S transition, while promoter repression with E2F – binding sites has been demonstrated to form E2F/pRb pocket protein complexes to these sites (Figure 2). Figure 2: How Retinoblastoma protein is normally involved in Cell-cycle control - Activation and in activation (Murphree & Benedict, 1984) The pRb is active in the hypophosphorylation state and inhibit cell cycle hence acting as a tumor suppressor. The pRb is inactivated by phosphorylation during the M- to – G1 transition (Figure 2). Furthermore, PP1 gradually dephosphorylate the pRb, hence returning to the growth-suppressive hypophosphorylated state (Lai et al., 1999). The cell enters the S phase when it complexes with cyclin-dependent kinases (CDK) and cyclins phosphorylate pRb hence, inhibiting its activity. The cyclin D/CDK 4, 6 performs intial phosphorylation, and then followed by additional phosphorylation by Cyclin E/CDK 2 (Figure 2 & Figure 3). Therefore, pRb remains phosphorylated throughout S, G2 and M phases (Lai et al., 1999; Münger & Howley, 2002; Korenjak & Brehm, 2005). The pRb phosphorylation results to the dissociation of E2F- DP from pRb and become active. Free E2F activates Cyclin E and A factors, activating the cyclin-dependent kinases and proliferating cell nuclear antigen (PCNA) which speeds DNA repair and replication through aiding polymerase to attach to DNA (Funk et al., 1997; Korenjak & Brehm, 2005; Das et al., 2005). Figure 3: cell cycle. (An example pathway of RB1gene) The research was carried out to establish the functional understanding of the terminal region of Rb N and the crystal structure of the protease-resistant core. 3.0 Biological importance of the Retinoblastoma protein (pRb) The key function of pRb is seen in preventing disproportionate cell growth through inhibiting cell cycle development until a cell is ready to divide (Münger & Howley, 2002; Korenjak & Brehm, 2005). The RB1 gene, located at 13q14.1- q14.2 encodes the Retinoblastoma protein in human. The retinoblastoma protein is inactivated if both alleles of the RB1 gene are mutated early in life resulting to retinoblastoma cancer (Fig 3). However, little is known as to why an eye cancer results from a gene mutation (Korenjak & Brehm, 2005). Figure 4: Dysregulated Cell Division and Differentiation in Retinoblastoma Retinoblastoma (Rb) is prevalent in early childhood and affects about 1 in 20,000 children. The tumor results from the immature retina. Retinoblastoma forms mainly results either due to hereditary or non-hereditary. Rb gene is a tumor suppressor gene which is located on the long arm of chromosome 13 at the region 14 (mutated/deleted locus on 13q14). The retinoblastoma occurs as a result of mutation that inactivates both the normal alleles. Almost 60% of retinoblastoma is sporadic, meaning that that the mutations occurrence is unilateral and on the other hand, 40% of tumors result from familial, meaning that they occur due to germ line mutation (Du & Pogoriler, 2006). According Shields & Shields (2004), the Rb is assumed to suppress tumor formation in normal cells and its absence is associated to retinoblastoma since a cell can replicate itself as a result uncontrollably, resulting in tumor formation (Figure 3). Rb is found in all body cells and normally they play a role in cell division cycle by preventing DNA replication being triggered by certain regulatory proteins. 4.0 Significance of the study This study reviewed the functional understanding of the Rb N-terminal region, the crystal structure of the Retinoblastoma Protein N and hence, providing insight into Tumor Suppression, ligand interaction, and Holoprotein Architecture. Furthermore, the study identified; (i) the key site of protein interaction in RbN while regulated by phosphorylation, (ii) Rb holoprotein conformation in which the RbN and pocket domains relate and (iii) the regulation of this interaction through ligand binding. Understanding Rb mechanism of tumor suppression at molecular level has provided better into many other cancer types apart from Retinoblastoma (Korenjak & Brehm, 2005). 5.0 Bioinformatics analysis of Retinoblastoma protein (pRb) Figure 5: crystal structure of the RB1 The protein analysis shows that the mutations associated with diseases in most cases occur in conserved genes. Some of these mutations may alter protein function in a deleterious manner occurring in functionally important proteins gene products. The Retinoblastoma protein encoded by RB1 gene has similar sequence and functionality and the gene product is tumor suppressor protein which normally takes part in regulation of cell cycle. Below figure 6 and 7 shows RB1 protein sequence in human and Mus musculus respectively. Retinoblastoma 1 [Homo sapiens] >gi|108773787|ref|NP_000312.2| retinoblastoma 1 [Homosapiens] MPPKTPRKTAATAAAAAAEPPAPPPPPPPEEDPEQDSGPEDLPLVRLEFEETEEPDFTALCQKLKIPDHVRERAWLTWEKVSSVDGVLGGYIQKKKELWGICIFIAAVDLDEMSFTFTELQKNIEISVHKFFNLLKEIDTSTKVDNAMSRLLKKYDVLFALFSKLERTCELIYLTQPSSSISTEINSALVLKVSWITFLLAKGEVLQMEDDLVISFQLMLCVLDYFIKLSPPMLLKEPYKTAVIPINGSPRTPRRGQNRSARIAKQLENDTRIIEVLCKEHECNIDEVKNVYFKNFIPFMNSLGLVTSNGLPEVENLSKRYEEIYLKNKDLDARLFLDHDKTLQTDSIDSFETQRTPRKSNLDEEVNVIPPHTPVRTVMNTIQQLMMILNSASDQPSENLISYFNNCTVNPKESILKRVKDIGYIFKEKFAKAVGQGCVEIGSQRYKLGVRLYYRVMESMLKSEEERLSIQNFSKLLNDNIFHMSLLACALEVVMATYSRSTSQNLDSGTDLSFPWILNVLNLKAFDFYKVIESFIKAEGNLTREMIKHLERCEHRIMESLAWLSDSPLFDLIKQSKDREGPTDHLESACPLNLPLQNNHTAADMYLSPVRSPKKKGSTTRVNSTANAETQATSAFQTQKPLKSTSLSLFYKKVYRLAYLRLNTLCERLLSEHPELEHIIWTLFQHTLQNEYELMRDRHLDQIMMCSMYGICKVKNIDLKFKIIVTAYKDLPHAVQETFKRVLIKEEEYDSIIVFYNSVFMQRLKTNILQYASTRPPTLSPIPHIPRSPYKFPSSPLRIPGGNIYISPLKSPYKISEGLPTPTKMTPRSRILVSIGESFGTSEKFQKINQMVCNSDRVLKRSAEGSNPPKPLKKLRFDIEGSDEADGSKHLPGESKFQQKLAEMTSTRTRMQKQKMNDSMDTSNKEEK Figure 6: RB1 protein sequence in human Retinoblastoma 1 [Mus musculus] >gi|188528630|ref|NP_033055.2| retinoblastoma 1 [Musmusculus] MPPKAPRRAAAAEPPPPPPPPPREDDPAQDSGPEELPLARLEFEEIEEPEFIALCQKLKVPDHVRERAWLTWEKVSSVDGILEGYIQKKKELWGICIFIAAVDLDEMPFTFTELQKSIETSVYKFFDLLKEIDTSTKVDNAMSRLLKKYNVLCALYSKLERTCELIYLTQPSSALSTEINSMLVLKISWITFLLAKGEVLQMEDDLVISFQLMLCVVDYFIKFSPPALLREPYKTAAIPINGSPRTPRRGQNRSARIAKQLENDTRIIEVLCKEHECNIDEVKNVYFKNFIPFINSLGIVSSNGLPEVESLSKRYEEVYLKNKDLDARLFLDHDKTLQTDPIDSFETERTPRKNNPDEEANVVTPHTPVRTVMNTIQQLMVILNSASDQPSENLISYFNNCTVNPKENILKRVKDVGHIFKEKFANAVGQGCVDIGVQRYKLGVRLYYRVMESMLKSEEERLSIQNFSKLLNDNIFHMSLLACALEVVMATYSRSTLQHLDSGTDLSFPWILNVLNLKAFDFYKVIESFIKVEANLTREMIKHLERCEHRIMESLAWLSDSPLFDLIKQSKDGEGPDNLEPACPLSLPLQGNHTAADMYLSPLRSPKKRTSTTRVNSAANTETQAASAFHTQKPLKSTSLALFYKKVYRLAYLRLNTLCARLLSDHPELEHIIWTLFQHTLQNEYELMRDRHLDQIMMCSMYGICKVKNIDLKFKIIVTAYKDLPHAAQETFKRVLIREEEFDSIIVFYNSVFMQRLKTNILQYASTRPPTLSPIPHIPRSPYKFSSSPLRIPGGNIYISPLKSPYKISEGLPTPTKMTPRSRILVSIGESFGTSEKFQKINQMVCNSDRVLKRSAEGGNPPKPLKKLRFDIEGADEADGSKHLPAESKFQQKLAEMTSTRTRMQKQRMNESKDVSNKEEK Figure 7: RB1 protein sequence in Mus musculus When we aligned the human sequence against mouse sequence (Figure 6 and figure 7), the result gave a percentage of 91 identities between the two sequences. On the other hand, there are another 98 sequence with similar domains to RB1 human protein and the most similar domains found in RBL1 protein (retinoblastoma like 1 protein). As it has very similar domains to RB1 domains, it expected to have similar function to the retinoblastoma1 protein (Figure 9). Figure 8 below and overleaf shows Human and Mus musculus gene alignment Figure 8: Human and Mus musculus gene alignment The Human and Mus musculus gene alignment in the figure 8 shows complex interactions between proteins or genes and shows significant conservation of gene expression and prediction function. Gene’s interactions are crucial in understanding biological functions. The alignment of Human and Mus musculus protein is high-scoring, and hence this alignment provides us with a good evaluation of their species deviation. However, this alignment differs from mere sequence homology. Therefore this findings result to network-based predictions of gene functions such as functional innovations like gene displacements. The conserved genes of Eukaryota are shown in figure 9. Figure 9: Gene conserved in Eukaryota The availability of assembled sequences genome for Mus musculus (mouse) and other Eukaryotes as seen in figure 9 allow us to identify regions where there is conservation between the Eukaryotes and human chromosome 13. In general, there is greater similarity seen between human and mouse (Figure 10 and Figure 11) compared to Drosophila Melanogaster, Anopheles gambiae, Arabidopsis thaliana and Oryza sativa. Therefore this low chances of retinoblastoma occurring in organisms with less gene conservation. Figure 10: similar domains architectures had been identified in human Figure 11: Mus musculus has the most similar domain architectures with human domains 6.0 Methodology and Results of Retinoblastoma protein N analysis Analysis 1: RbN Interacts with EID-1 (Markus Hassler et al., 2007) Rationale: characterization of EID-1 binding to RbN Method: In vitro protein-binding assays with purified human proteins. Glutathione-Sepharose-bound proteins were analyzed by immunoblot using antibodies as indicated. GST-p/CAF served as a positive control for EID-1 binding. Isothermal titration calorimetry, 300 μM EID-1 (1-187) was titrated into 30 μM Rb 31- 355 at 9ºC Result: In-vitro results of protein-binding assays amid purified proteins, western blots were antibodies probed. Section A; Rb N aliquots were incubated with equivalent amount of GST-control proteins Section B and B′; EID-1 aliquots were incubated with equivalent amounts of GST-tagged proteins acting as negative control. Section B′ has dashed boxes which indicate regions of interaction in Rb. Section C; aliquots of Rb, polyG, were incubated with GST-EID-1 Section D and D’; the Section D shows the effect of RbN phosphorylation on EID-1 binding by Antibody – FRET – mediated complex detection. Phosphorylation of GST-RbN was done by cyclin A/cdk2 while run with ATP and omitting enzymes as controls. The error bars represent four samples standard deviation. Section D’ illustrates the western blots analysis using phosphorylation site-selective and pan-Rb antibodies. Section E; EID-1 and Rb pocket aliquots incubated with equivalent amounts of GST-tagged proteins (GST-negative control and GST-RbN as a positive control). Conclusion/significance: EID-1 directly Interacts with RbN Analysis 2: Separate EID-1 Regions Interact with RbN and the Rb Pocket (Hassler et al., 2007) Rationale: Analysis of Sequence Supplement by SPOT array Method: A SPOT array of serially overlapping 25-mer EID-1 peptides was probed with RbN WT, RbN polyG, or Rb pocket. Peptide stretches of EID-1 interacting with various Rb constructs are indicated at the bottom right of individual panels Result: Section A; SPOT array in sequence of overlapping 25-mer EID-1 peptides was probed with RbN, RbN polyG and alternatively with Rb pocket. The panels of EID-1 peptides stretches associated with various Rb constructs. Section A′; A 30-mer EID-1 peptide SPOT array identifying EID-1 residues critical for interaction with RbN Section B; the summary of EID-1 peptide interactions based on SPOT array results, the amino acids necessary for interaction with RbN is denoted with colour. Section C and C′; the SPOT array results is based on the association between the C = GST-RbN and C′ = GST-Rb pocket in presence of EID-1 peptides. Conclusion/significance: based on SPOT results, there is an association between GST-RbN and GST-Rb pocket with EID-1 in the presence of various EID-1 peptides Analysis 3: The RbN Domain Interacts with the Rb Pocket Rationale: Protein detection by Antibody- assisted fluorescence resonance energy transfer (FRET) mediated complex. Method: GST-Rb pocket was phosphorylated using purified cyclin A/cdk2 (A/K2) in the presence of 1mM ATP. A control reaction in which ATP was omitted was run and analysed in parallel. Assays were run essentially using 2.5 nM GST-RbN and 200nM Rb pocket-Flag preparation. Error bars represent standard deviation from quadruplet samples. Anti-phospho-Rb western blots 100ng of the respective Rb preparations were resolved by SDS-PAGE and subjected to immunoblot analysis using phosphorylation site-selective and pan Rb antibodies as indicated. Results: Section A and A′; RbN interacts with the Rb pocket. A= RbN were incubated with equivalent amounts of GST acting as a negative control. Schematic A′= dashed boxes indicate regions of Rb large pocket interaction. Section B and B′; RbN interacts with the Rb pocket independently of the pocket spacer region. B= RbN aliquots were incubated with equal amounts of GST- control proteins, B′= Rb pocket modified with the spacer remover by thrombin were incubated with equal amounts of GST-control proteins Illustration C; RbN does not utilize the Arg-rich linker for interaction with the Rb pocket. Section D; the effects of RbN phosphorylation on pocket binding and the Antibody FRET and data presentation were done with phosphorylated RbN. Section E and F′; RbN domain organization with pocket in the presence of EID-1 and affinity precipitation with proteins carried out (Markus Hassler et al., 2007). Conclusion/significance: The RbN Domain Interacts with the Rb Pocket 7.0 Summary of the Results Retinoblastoma protein, pRb, regulates the cell-cycle causing cells to divide in a controlled manner. The finding of the study revealed that two connected cyclin-like folds results to the crystal structure of the Rb N. The structure and functional analysis of the pRb showed that the advancement of the protein pocket family advocating the RbN tumor suppression mechanism. The study identified; a key site of protein interaction in RbN which is controlled by phosphorylation. Furthermore, the findings established a closed Rb holoprotein conformation in which the RbN and pocket domains interacts directly while this interaction is being modulated by ligand binding (Hassler et al., 2007). 8.0 Conclusion and Discussion The RB1 forms a part of four pathways: cell cycle, chronic myeloid leukemia, glioma and pancereatic cancer. This functional activity of pRb, it is regulated by phosphorylation during normal progression through the cell cycle. In the Mus musculus family, the tumour suppressor protein plays a very critical role in controlling cellular proliferation. It does so by majoring regulating and controlling E2F activities. The Rb1 gene also interacts with chemical compounds like the Abl C-terminus. It interacts with the retinoblastoma protein to bind DNA in the cell nucleus, hence, probable growth-inhibitory mediator. Histone H3 lysine and methylation are also known to regulate some euchromatic genes targeted by pRb. The ability of Rb to interact physically with Rb1 gene and bind E2F is associated to its functional role as a growth suppressor. The gene also plays a major role in neuronal cell cycle control and apoptosis, where it is cleaved by caspase. Cyclin dependent kinase (cdk) 4 and cdk6 also are enzymes that interact with pRb to phosphorylate it in the nucleus to regulate G1 phase of the cell cycle. According to Sakanaka et al (1998), there are effects of some drugs on the protein pRb with some difficulties experienced by medical world. The drug ginsenoside Rb 1 is effective in the direct intracerebroventricular infusion; however, it is impractible to use the drug due to problems associated to route of administration in treatment of human transient cerebral ischemic attack (TIA) and cerebral infarction. Culture experiments carried out by Kim (1998) which examined the neuroprotective effect of ginsenoside Rb 1 revealed that high concentrations of ginsenoside ranging from 0.11 to 11 μg/ml reduces glutamate-mediated neurotoxicity hence preventing and reducing neuronal cell death. On the other hand, higher concentrations can possibily be used to prevent apoptosis. It is still not clear as to exactly how the mechanism of neuroprotection affects the protein pRb. The medical world is trying to find drugs which can have a therapeutic effect on patients who have cerebral infarction. They are also trying to find a way to protect cells and ways to effectively administer ginsenoside Rb1 by providing pharmaceutical compositions containing it or its salt to inhibit apoptosis (Sakanaka et al., 1998). The finding of the study shows that two connected cyclin-like folds form the crystal structure of the Rb N. While Rb N boxes A and B of the Rb pocket are related meaning that retinoblastoma originated through duplication. The structural and functional analysis enables better understanding of Rb N mutations and locates unique phosphorylation-regulated site of protein interaction (Hassler et al., 2007). Related studies on Rb pocket domain gave an insight into the fold and properties of Retinoblastoma protein and gave a structural basis for its mode of action and the biological significance. In contrary to the Bioinformatics analysis which suggested the presence of a BRCT domain, the crystal structure of RbN showed a structural design built around tandem cyclin folds which had similarities with Rb pocket domain. Hence, the study finding has revealed that the structural disruption of RbN in vivo affects the stability of the entire protein, while the mutation in RbN results in functional loss (Hassler, et al., 2007). The interaction between the RbN, central positioning unit of C terminus of RbN and pocket regions suggests a compacted arrangement. This is in contrary to the current models, whereby RbN and pocket are considered as separate in their structural and functional entities. 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