The Cellular Biochemistry of Cultured Reindeer Cells - Assignment Example

?THE CELLULAR BIOCHEMISTRY OF CULTURED REINDEER CELLS i) The signaling pathway for the degradation of FuIL protein. Cellular signaling pathways are activated by both intracellular and extracellular factors (signals). These act on receptors either on the cell surface, the cytoplasm or the nucleus. They then induce a cascade of events that lead to the cell performing certain activities, such as division, motility, apoptosis, and in this case, protein degradation. In this specific scenario, the signal is extracellular in origin. Due to the rapid response, and nuclear events caused by the factor Santa, it is most probable that it acts on nuclear receptors. The santa factor may bind to its specific receptor in the cytosol or on the nuclear membrane. This causes the formation of a receptor-ligand complex that then acts as an effector molecule on the DNA (transcription factor or co-factor). This is basically transcription regulation. Several pathways can be implicated. They include; acting as specificity factors, repressors, activators or enhancers. Taking that the santa-receptor complex acts as a repressor for gene transcription; this complex may bind to the operator portion of the DNA and prevent the DNA polymerase from transcribing a specific gene, or genes. In the given case, the gene coding for FuIL protein will not be transcribed. The result is that no mRNA is formed, and subsequently no synthesis of the protein. Alternatively, it may act as a post translational modifier. Here, it will modify the folding structure of the FuIL protein, and subsequently lead to the ‘exposure’ of the degron on the protein for action by ubiquitin conjugating enzymes E1, E2 and E3. Combinations of different ubiquitin-conjugating enzymes and other factors constitute other pathways of the ubiquitin system, each of which conjugates a specific subset of proteins. There is some evidence that certain sequence elements and structural motifs of target proteins are degradation signals which mark them for ubiquitination by a particular model of the ubiquitin system and for eventual degradation. When looking at the santa factor as a direct promoter of protein degradation, it may diffuse into the cell and bind to the FuIL protein (like an enzyme-substrate complex) causing a conformational change in its structure that exposes the normally hidden degron. As a ligand, it may also bind to a receptor that catalyzes the phosphorylation forming a phosphodegron which is specific to certain multiunit ubiquitin ligases. An example would be a serpentine receptor. The serpentine receptors are coupled to a plasma membrane phospholipase C that cleaves PIP2 to diacylglycerol and IP3. By opening calcium channels in the endoplasmic reticulum, IP3 raises cytosolic calcium. Diacylglycerol and calcium act to activate protein kinase C, which phosphorylates and changes the conformation of the protein. This may be exposure of the degron. This then leads to facilitated function of ubiquitin and proteasome complexes that target and destroy specific proteins. Polyubiquitin tagging of proteins (ubiquitin attachment site in proteins is commonly a lysine side chain) by specific enzymes (E1,2 and 3) provides the major source of selectivity in the process of degradation, whereas the 26S proteasome complex performs the protein unfolding necessary for cleavage of the ubiquitin tagged proteins. This also provides an avenue for selectivity for the FuIL, as the E3 may bind to the santa, become allosterically modified and thus become specific to the FuIL. Such a mechanism is evident where the growth-regulating plant indole auxin binds to a specific E3 ligase, and forms part of a protein-binding interface that allows high-affinity interaction with specific protein substrates. (Sharon M) ii) Santa Response Element. The santa response element acts as a secure site for binding of RNA polymerase and the required transcription factors such as the SREB. It thus acts as an operator for the operon in question. The SREB molecule requires transport into the nucleus so as to exert its effects on the transcription. For this to occur, it has to be shuttled across by molecules known as importins. The importins have two subunits, importin ? and importin ?. The importin-beta mediates interactions with the pore complex, while importin-alpha acts as an adaptor that binds a NLS on the SREB. (Heijne) An NLS is a sequence of amino acids on a protein that is being tagged for transport into the nucleus. Its function is similar to a degron in some sense, as it marks a protein making it specific to certain cellular activities. There are two main types; Classical NLSs and Non-classical NLSs. Classical ones may be either monopartite or bipartite. Examples; “KKKRKV” that is monopartite. Assuming the receptor was serpentine and trans-membrane; the serpentine receptors are coupled to a plasma membrane phospholipase C that cleaves PIP2 to diacylglycerol and IP3. By opening calcium channels in the endoplasmic reticulum, IP3 raises cytosolic calcium. Diacylglycerol and calcium act to activate protein kinase C, which phosphorylates and changes the activity of certain cellular proteins. Assuming that it phosphorylates RanGDP to RanGTP, there will be importin mediated transport into the nucleus, and thus increase of nuclear RanGTP. The NLS-importin bonding will be facilitated, by expression of more importin, and presence of high GTP/GDP gradient. Thus the SREB will be carried into the nucleus through nuclear pores. Once the SREB-importin complex is inside the nucleus, it binds to RanGTP. This causes dissociation of the protein SREB, and the importin-RanGTP complex is carried back out. It is then hydrolyzed by RanGTPase, yielding RanGDP and free importin. The santa mediated phosphorylation may also phosphorylate serine residues on SREB, causing conformational changes that may lead to exposure of its NLS. This then directly facilitates SREB binding to importin alpha, and subsequent transport of SREB into the nucleus. Assuming that the receptor for santa is of the JAK-STAT type, it will function in the classic manner by activation of a Janus Kinase (intracellular) when bound by the ligand-which in this case is santa. This then leads to phosphorylation of an amino acid residue on the SREB protein (as for tyrosine in STAT), which leads to conformational changes. These changes lead to the expression of the NLS that binds to the importin and RanGDP complex. This is the most probable pathway. Activation of this JAK-STAT pathway requires an external/extracellular ligand that activates the kinases. They then carry out auto phosphorylation and phosphorylation of their related receptors. They hence generate docking sites for the STATs (in this case, SREB). They then phosphorylate and dimerise and activate the cytosolic molecules, which were otherwise inert (singh). They are then shuttled into the nucleus and exert their effects on the DNA. In the illustration below, the STAT molecules have similar functionality to SREB, and the pink molecule, santa factor (normally a cytokine). The SREB molecule then acts as a transcription factor binding to a certain portion of the genome also known as the regulator region. Here, they either facilitate or repress binding of DNA polymerase. They therefore control gene expression and hence protein biosynthesis. These molecules are formed in the cytoplasm as well, and hence require certain pathways and signals for their localization to the nucleus. As has been discussed in the previous paragraphs, expression of NLS is one of the important ways in which this can be achieved. iii) SREB binding. SREB acts as a transcription factor when it enters the nucleus to exert effects on the genomic DNA transcription. As mentioned earlier, the SREB binds to a sequence of six nucleotides, and thus forms part of the transcription factor complex. This complex consists of the TATA box binding protein, activators, co-activators, repressors or enhancers. The SREB acts as a TATA binding protein which is a general transcription factor (locker). This therefore means that the SREB will initiate binding of the other basal factors to finally complete the TF complex facilitating the binding of DNA polymerase II. The entire complex with the TATA forms what is known as Transcription factor IID. TFIID is composed of several subunits called TBP-associated factors (TAFs, 16) and the TATA Binding Protein. It is important to note that in eukaryote cells, transcription factors are transcribed in the nucleus but are translated in the cytoplasm. Many proteins that are active in the nucleus contain nuclear localization signals that direct them into the nucleus. This is a key point in their regulation. Important classes of transcription factors such as some nuclear receptors must first bind a ligand while in the cytoplasm before they can relocate to the nucleus. This is similar to the action of the SREB as seen in the previous section. The HAT and HDAC enzymes are enzymes that act on the DNA strand’s histones by acetylation and de-acetylation. This essentially causes an increase in DNA coiling and inaccessibility (HAT). Thus, HAT is linked to transcriptional activation. The converse applies to the HDAC. The interaction of these enzymes and the DNA portion subjected to the transcription leads to facilitated transcription (A) A bromodomain is a protein domain that recognizes acetylated lysine residues. These are such as those on the N-terminal tails of histones. This recognition is often a prerequisite for protein-histone association and chromatin remodeling. Chromodomains (also called chromatin organization modifier) is a protein structural domain of 40-50 amino acid residues found in proteins associated with the remodeling and manipulation of chromatin (S Messmer) The completion of the complex flags the start of transcription. References Calderon-Villalobos LI ---Estelle M --- Robinson CV ---Sharon M ---Tan X---Zheng C---Zheng N. Mechanism of auxin perception by the TIR1 ubiquitin ligase (640-645). 2007. Print Gunnar Von Heijne. Protein Targetting, Transport, and Translocation (297,298,309). Academic press, 2002, print Rakhesh K.Singh. Novel Role of the JAK-STAT Pathway in Mediating the Effects of Atypical Antipsychotics on 5-HT2A Receptor Signaling (Page 52-55). ProQuest, 2008, print. Locker, Joseph. Transcription factors (22-23). Garland science, 2012, print. Whiteside ST, Goodbourn S. "Signal transduction and nuclear targeting: regulation of transcription factor activity by subcellular localisation". Journal of Cell Science, 1993. (page 949–55) Meyers Robert A. Epigenetic Regulation and Epigenomics (686). John Meyers and sons, 2012, Print S Messmer, A Franke, R Paro. Analysis of the functional role of the Polycomb chromo domain in Drosophila melanogaster. Genes. 1992. (page 1241–1254) Read More