The minimals

As most synthetic biologists and iGEM teams work with Escherichia Coli, the use of other model systems can cause confusion. We hope to ease the legibility of our project descriptions by creating eukaryopedia, an overview about transcription factors and cell lines we used in our studies, as well as general molecular biology issues that affect our work. We hope it can help you find guidance in the jungle that mammalian molecular biology is at the moment.

Contents

Essentials Of Lipid Sensing

Cellular signaling - Nuclear Receptors - Ligand binding domains

Model Organisms

Drosophila Melanogaster - Caenorhabditis elegans - Homo sapiens

Techniques

Apo A IV - CYP1A1 - EGF - HMG CoA synthase - Hsp70 - LDL receptor - PUMA - TNF-alpha

Molecular and Cellular Biology

Post-transcriptional modification / mRNA processing in eukaryotes - Regulation of transcription in eukaryotic organisms

Drugs

Camptothecin - Hygromycin - Zeocin - Neomycin

Cellular components as tools

GPI - Sar-1 - Myrpalm - NLS - GFP

Essentials Of Lipid Sensing

Cellular signaling

Cells have an innate ability to “listen” and correctly react to their local or even distant
environment. Through time it has been observed that a complex systems of communication governs
essential cellular activates and coordinates cell actions.[1] Today, it is well known that processes such as
development, growth, tissue repair or death, metabolic shifts and immunity are all governed, at the
molecular level, by signaling. By understanding cell signaling, diseases may be treated effectively and,
theoretically, artificial tissues may be created. Cells sense information from their local surroundings through a class of proteins known as receptors. Chemicals that activate (or inhibit) receptors are often named hormones, growth factors, cytokines or even neurotransmitters yet their proper term is receptor ligands. Water soluble ligands have cell membrane penetration and thus mostly interact with trans-membranous receptors, whereas ligands with high lipid solubility easily penetrate the cell membrane

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Nuclear Receptos

Nuclear receptors are a class of receptors localized within cells which can sense the presence of lipid soluble ligands (eg steroid hormones). Upon ligand binding these receptors undergo a change in conformation and translocate to the nucleus. A unique feature of these proteins, amongst other receptor classes, is the ability to directly bind to DNA segments known as response elements. These receptors are best viewed as transcription factors which can be activated by extracellular cues[2][3]. The binding ultimately leads to defined changes in gene expression (both activation and repression), thereby controlling the development, homeostasis, and metabolism of the organism. Nuclear receptors bear high homology to each other and are modular into distinct domains: N-terminal regulatory domain, DNA-binding domain, a Hinge region, Ligand binding domain (LBD) and a C-terminal domain.

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Ligand Binding Domains

Ligand binding domain is a conserved sequence and structure amongst the various nuclear receptors whose structure usually referred to as an alpha helical sandwich fold. Three anti parallel alpha helices (the "sandwich filling") are flanked by two alpha helices on one side and three on the other (the "bread"). The ligand cavity has an internal localization just below three anti parallel alpha helical sandwich "filling". The Ligand binding domain together with the DNA binding domain contributes to the interface of the receptor by binding accessory proteins (coactivator and corepressor) and dimerization of receptors. The LBD also contains the activation function 2 (AF-2) whose action is dependent on the presence of bound ligand [8]. The change in receptor configuration which occurs upon ligand binding exposes the AF-2 domain, which promotes transcriptional activity by a wide variety of mechanisms.

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Model Organisms

Drosophila Melanogaster

Drosophila Melanogaster, also known as the common fruit fly, is one of the most frequently used model organisms in biological sciences, including studies in genetics, physiology, microbial pathogenesis and life history evolution.[9] The ecdysone receptor is a nuclear receptor found in D.Melanogaster, where it controls development and contributes to other processes such as reproduction. Its ligands are ecdysteroid which are secreted by the organism’s prothoracic gland.

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Caenorhabditis elegans

Caenorhabditis elegans is a free-living, transparent nematode (roundworm), about 1 mm in length,[10] which lives in temperate soil environments C. elegans is intensively studied as a model organism in biology for a variety of reasons. The developmental fate of every single somatic cell (959 in the adult hermaphrodite; 1031 in the adult male) has been mapped out.[11][12] The C.elegans genome harbors 284 nuclear receptors [10] (a striking figure), which have been shown to control traits such as sex determination, larva development, life span, neuronal growth and identity and much more. As far as nuclear receptors go, they are a gold mine.

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Homo Sapiens

Homo sapiens are the only living species in the Homo genus of bipedal primates in the great ape family. Nuclear receptors number up to 47 in humans, yet only few have been well characterized. They constitute the focus of medicinal reproductive technologies, hormonal medicine (endocrinology), immunology, drug interaction and much more.

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Proteins

LDL receptor

Low-density lipoprotein receptor (LDL receptor) is a cell surface protein which is responsible for the cholesterol supply of the cell. The receptor recognizes the protein B100 which is part of the LDL particles. Binding of the B100 protein to the LDL receptor leads to endocytosis via clathrin coated pits. The vesicle fuses with an endosome, the resulting shift in the pH value leads to the detachment of the LDL and the receptor can be transported back to the plasma membrane (receptor recycling). Through this process the cell takes up the cholesterol which is associated with LDL. LDL accumulation in the blood is responsible for atherosclerosis and many cardiovascular diseases. The LDL gene is regulated by the intracellular level of cholesterol. A low level of cholesterol leads to the activation whereas a high level results in a decrease of transcription [40].

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HMG CoA synthase

3-hydroxy-3-methylglutaryl-CoA synthase (HMG CoA synthase) is an enzyme catalyzing the condensation of Acetyl-CoA and acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). There are two distinct HMG CoA synthases, the cytosolic and the mitochondrial form, encoded by two different genes. The reaction catalyzed by the cytosolic enzyme is a part of the biosynthesis of cholesterol. In mitochondria the same reaction is responsible for keton body formation. Sterol regulatory elements in the promoter region of the gene are responsible for transcriptional regulation [41].

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PUMA

P53 upregulated modulator of apoptosis (PUMA) is protein belonging to the BH3-only family of pro-apoptotic proteins. P53 plays an important role both in p53 dependent and p53 independent apoptosis. The activation of PUMA leads to mitochondrial dysfunction and caspase activation [1]. PUMA is regulated by many transcription factors (TF), these TFs in turn are regulated by extra and intra cellular stimuli like genotoxic stress, toxins, oncogene expression, redox status and growth factors [42].

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Hsp70

Heat shock protein 70 (Hsp70) is an enzyme helping other proteins to fold. Hsp70 proteins are found not only in the cytosol but also in mitochondria and in the endoplasmatic reticulum. The protein binds, together with the cochaperone Hsp40, to newly synthesised (hydrophobic) amino acid residues and prevents the aggregation of those. During cellular stress e.g. oxidative or thermal stress proteins may unfold, Hsp70 binds to the hydrophobic regions of the proteins and prevents further unfolding, aggregation and apoptosis [43].

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Apo A-IV

Apolipoprotein A-IV (Apo A-IV) is a glycoprotein secreted by the small intestine in humans. The production of Apo A-IV is activated through lipid absorption (Chylomicrons). Several studies indicate that Apo A-IV protects against atherosclerosis. It is also thought to be involved in regulation of food intake [44].

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CYP1A1

Cytochrome P450 1A1 (CYP1A1) is an enzyme which is regulated by the aryl hydrocarbon receptor (AhR) signalling pathway. The transcription is also influenced by metal ions and oxidative stress. CYP1A1 catalyzes two of three critical steps in transformation of benz[a]pyren to the carcinogen BP-7,8-dihydrodiol-9,10-epoxide. Furthermore CYP1A1 is involved in processes like xenobiotic metabolism and drug degradation [45].

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EGF

Epidermal Growth Factor (EGF) is a 6045 Da protein discovered by Stanley Cohen in 1986, which won him a Nobel Prize in Physiology and Medicine. EGF regulates cell proliferation by binding to the epidermal growth factor receptors (EGFRs) which are located on the cell surface. Upon binding of EGF to its receptor intrinsic tyrosine kinase activity is stimulated inducing a signaling cascade inside the cell which leads to increased calcium levels, glycolyisis and protein synthesis in the cell. This process ultimately leads to the proliferation of the cell. It was recently shown that c-Jun is one of the targets of EGF action [46], [46].

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TNF-alpha

Tumor Necrosis Factor-alpha (TNF-alpha) is a cytokine involved the cells inflammatory response. TNF-alpha is a homotrimer that binds to one of its to receptors (TNF-R1/ TNF-R2) which then form a trimer themselves. Trimerization of the receptor induces a conformational change and the dissociation of the inhibitory protein SODD (Silencer of Death Domain protein) from the intracellular death domain of the receptor. The adaptor protein TRADD (TNF Receptor-associated Death Domain protein) can bind now to the death domain and allow other protein factors to bind aswell. The three main signaling pathways initiated in this way are: the NF-kB pathway, the MAPK opathway and the death signaling cascade [60].

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Pifithrin-α

Pifithrin-α (PFTα) is proposed to be a specific inhibitor of p53 signaling. It is not yet clear how exactly PFTα inhibits p53, but it seems to act at a stage after p53 translocation to the nucleus. Temporary suppression in vitro of p53 inhibits apoptosis induced by the damage to DNA and thus increases the fraction of cells surviving the stress [61], [61].

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RNA-processing and transcriptional regulation

Post-transcriptional modification / mRNA processing in eukaryotes

To express a gene and successfully synthesize the appropriate protein the gene must firstly transcript into mRNA. Unlike in bacteria, this mRNA molecule is not directly ready for translation; the primary transcript is therefore called precursor-mRNA (pre-mRNA). One of the first modifications is a process referred to as 5’-capping. By means of several biochemical steps a 7-methylguanosine molecule is bound to the 5’ end of the pre-mRNA, via a 5’ to 5’ triphoshpate linkage. This 5’ cap has various functions including prevention of 5’ degradation, export from the nucleus and initiation of translation. Not only the 5’ end but also the 3’ end is modified, this process is called polyadenylation. Therefore a Polyadenylation signal is needed (consensus sequence 5'- AAUAAA-3'), further in the 3’ direction occurs a 5’-CA-3’ element, these both sequences are recognized by the enzymes cleavage and polyadenylation specificity factor and cleavage stimulation factor. Together they are attracting many other proteins including Polyadenylate Polymerase (PAP). The protein complex cuts the pre-mRNA at the CA element and the PAP adds about 200 adenine residues to the 3’ end. The function of the poly-A tail is protection against degradation, marking of the end of the transcript and aid in translation initiation. The pre-mRNA contains not only these sequences coding for the protein, so called exons, but also many sequences which are non-coding. These introns have to be removed, that occurs in a process known as splicing. A protein complex called spliceosom connects all the exons thereby cutting out the introns. Responsible for the recognition of the exon-intron borders are small nuclear RNA within the spliceosom. Many genes can be spliced in several ways, an incident termed alternative splicing [48], [49].

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Regulation of transcription in eukaryotic organisms

Cells have to adapt to changes in their environment and must be able to receive and react to extra cellular signals; cells accomplish these requirements by the up and down regulation of certain proteins. The protein expression in eukaryotic cells can be regulated on many different levels, this article concentrate on the regulation of transcriptions. Only a small percentage of the human genomic DNA is transcribed into mRNA. On the opposite, a huge part of the human genome is involved in regulating the transcription of coding sequences. To initiate transcription of a gene eukaryotic RNA-polymerases have to bind to several general transcription factors to establish the so called initiation complex (IC), which is able to bind to the DNA. The binding occurs upstream of the transcriptional start site (TSS) in a region called core promoter, this part of the promoter often contains specific elements like the TATA-Box (consensus sequence, TATAA/TAA/T, about 30 bp upstream of TSS [50]) and the GC-Box (consensus sequence TGTGGCTNNNAGCCAA) app. 80 bp upstream of the TSS [51] to which the IC can bind. Further upstream is a part of the promoter which is referred to as proximal promoter. Containing specific sequence elements, this part of the promoter is highly important for the transcriptional regulation. Transcription factors can bind to these response elements thereby up regulating or down regulating the gene transcription. Proteins methylating or acetylating the DNA are also involved in gene transcription regulation by remodelling of the chromatin structure.

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Drugs

CPT

Camptothecin (CPT) is a cytotoxic quinoline alkaloid and a topoisomerase I inhibitor isolated from the Camptotheca acuminata (Camptotheca or the Happy tree). It was discovered during a screen for natural anti-cancer drugs in 1966 but it is not not used in cancer therapy due to its severe side effects, but there were various derivatives developed to increase the benefits of this drug while decreasing its negative effects [52]. The two CPT analogues have been approved for cancer chemotherapy today are topotecan and irinotecan. CPT acts by binding to the topoisomerase I-DNA complex using hydrogen bonds and thereby preventing DNA-religation, inducing DNA damage and ultimately causing the cell to die [53].

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Hygromycin

The aminocyclitol antibiotic hygromycin B, that is produced in Streptomyces hygoscopicus, inhibits protein synthesis by interfering into aminoacyl-tRNA recognition and ribosomal translocation. It shows effects in prokaryotes and eukaryotes alike. Hygromycine can be used as a selection marker. The resistance gene encodes for a hygromycin B phosphotransferase, which inactivates the antibiotic by phosphorylation [68]. In the iGEM 2009 project of Heidelberg hygromycin B was used for selection of cells which performed a stable integration of the transfected Plasmid.

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Zeocin

Zeocin shows a high effectiveness in a wide range of organisms. Mammalian, insect and yeast cells are effected as well as prokaryotic cells. It damages DNA by intercalating and causing breaks and therefore cell death. The zeocin resistance gene encodes for protein which binds zeocin and prohibits DNA destruction[71].

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Neomycin

The aminoglycoside antibiotic neomycin is produced by Streptomyces fradiae [69]. Neomycin is a selectionmarker for many different cell types. The resistance gene encodes for a phosphotransferase which inactivates neomycine by phosphorylating it [70].

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Cellular components as tools

GPI

Glycosylphosphatidylinositol (GPI) is a glycolipid. During the posttranslational modification in eukaryotic cells, it becomes attached to hydrophobic C-termini of proteins that have a special singnal peptide on them. This signalpeptide leads their translation into the ER, where the hydrophobic C-terminus will be replaced by a GPI anchor. Because of its hydrophobic nature it attaches the bound protein to the cell membrane [54].

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Sar-1

Sar-1 GTP-binding proteins direct the transport of molecules inside of veiscles from the ER to the golgi and the other way round. Being an anchor for COPII molecules that cause the budding of vesicles off the membranes, it needs a domain to attach to the ER membrane [55]. The C terminus of the Sar-1 protein fullfills this task. Therefore one can use the C terminus as an ER targeting sequence for other proteins.

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Myrpalm

This localization signal is located at the N-terminal end of the amino acid chain. The myrpalm signaling sequence causes a myristilation and palmitolyation of the targeted protein. Both modifications lead to a binding to the cell membrane [56].

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NLS

Nuclear localisation signals are peptidesequences that are able to bind to nuclear import receptors. These cause an import of newly synthesized protein through nuclear pores. This feature is caused by several positively charged amino acids. Nuclear localization signals can be located almost anywhere in the peptide chain [57]. We used a nuclear localization signal at the C-terminal end of the protein.

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GFP

Green Fluorescent Protein (GFP) was first discovered by Shimomura et al. in the Aequorea jellyfish. They described a slightly green colour of a GFP-containing solution that in the sunlight [63]. The same group of scientists investigated the protein in more detail, and have since discovered many characteristics, including the excitation and emission wavelengths . The most important accomplishment was the cloning of the GFP gene into other organisms to make them fluorescent [64], [65]. Many scientists have since worked on GFP and introduced mutations to enhance fluorscence levels or change the spectra. Nowadays flourescent proteins exist in different colours exist increasing their range of application even more.

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References