Tutor profile: Marco C.
Subject: Biomedical Science
How cancer cells reprogram their energy metabolism?
* Under aerobic conditions, normal cells process glucose first to pyruvate via glycolysis in the cytosol and thereafter to carbon oxide in the mitochondria. * Under anaerobic conditions, glycolysis is favored, and relatively little pyruvate is dispatched to the oxygen-consuming mitochondria. Cancer cells, even in the presence of oxygen, reprogram their glucose metabolism, and their energy production, by limiting their metabolism largely to glycolysis, leading to a state of “aerobic glycolysis”. They cooperate the efficiency lost by increasing glucose uptake. That’s why cancer cells upregulate GLUT, glucose transporters. Increase uptake and utilization of glucose is used by the tumor noninvasive visualization technique, Positron Emission Tomography (PET) with a radiolabeled analog of glucose as a reporter 18F-fluorodeoxyglucose,FDG Glycolytic fueling has been associated with activation of oncogenes ( RAS and MYC), and mutant tumor suppressors (TP53). Increased glycolysis allows the diversion of glycolytic intermediates into various biosynthetic pathways * Generation of nucleosides and amino acids * Biosynthesis of macromolecules * Biosynthetic of organelles All of which is required for the assemble of new cells. The metabolism of cancer cells differs from normal cells. This trait was first reported by Otto Warburg in 1942. Warburg observed that cancer cells shift their metabolism from Oxidative Phosphorylation (OXPHOS) generation of ATP to Glycolytic ATP production. This metabolic shift is now recognized as the Warburg effect. Current knowledge about metabolism of cancer cells has expanded the Warburg effect beyond the glycolytic shift. Cancer cell shift their metabolism not only to increase glycolytic ATP production, but also to increase the rate of pentose phosphate pathway (PPP), glutaminolysis, and lipid biosynthesis. Further analysis revealed that this metabolic shift is also important for the production of anabolic building blocks to aid increased cell proliferation. In cancer cells, GLUT1 and other glucose transporters import large amounts of glucose to the cytosol where it is broken down by glycolysis. Hexokinase (HK) catalyzes the first reaction of glucose metabolism. HK1 is constitutively expressed in most cells, while HK2 is inducible and commonly expressed in cancer. HK2 is believed to channel glucose flux into anabolic pathways. The last step of glycolysis is the conversion of phosphoenolpyruvate (PEP) to pyruvate, which is catalyzed by Pyruvate kinase (PK). Cancer cells express PK isoform M2, which diverges pyruvate to lactate production instead of Acetyl-CoA generation. This process contributes to increased lactate generation, which was the endpoint observed by Otto Warburg back in 1942. Additionally, the reaction that converts pyruvate to lactate is reversible, while the reaction that converts pyruvate to acetyl-coA is irreversible and commits acetyl-coA to be broken down in the TCA cycle. Pyruvate undergoes oxidative decarboxylation and is converted to acety-coA by Pyruvate dehydrogenase (PDH). This enzyme is regulated by post-translational modification, where the addition of a phosphate group to its amino acid residues will inhibit its activity. In several cancers, pyruvate dehydrogenase kinase (PDHK1) expression is increased, directly contributing to PDH inhibition and further reduction of acetyl-coA generated from pyruvate. Therefore, in cancer cells mitochondrial glucose metabolism is markedly reducePositrod, which allows many of glycolytic intermediates to be diverted into biosynthetic reactions. The oxidation of glucose-6-phosphate to pentose phosphates by the pentose phosphate pathway (PPP) is very important in some tissues. Rapidly diving cells use the pentose ribose-5-phosphate to make RNA, DNA, and coenzymes such as ATP, NADPH, FADH2, and coenzyme A. Other cells use NADPH as the essential product of PPP. NADPH is needed for reductive biosynthesis or to counter the damage of oxidative stress. The oxidation of glucose-6-phosphate by glucose-6-phosphate dehydrogenase (G6PD) is the first reaction of PPP. Glucose-6-phosphate is oxidized to form an intramolecular ester, 6-phosphoglucono-δ-lactone. NADP+ is the electron acceptor and the reaction forms NADPH. In cancer cells, PPP flux is used to provide ribose moieties and reducing equivalents needed for nucleotide and nucleic acid biosynthesis, PPP flux is also coupled with reduced glutathione (GSH) generation. GSH is required for glutathione peroxidase (GPx) detoxification of both organic and inorganic peroxides. G6PD catalyzes the rate-limiting step in PPP oxidative brunch. It has been observed that several oncogenes and tumor suppressors regulate expression and activity of G6PD. Although the Warburg effect has dominated our understanding of cancer metabolic reprogramming, glucose alone cannot supply all the necessary building blocks for a proliferative cell. Most cells are composed of carbon, hydrogen, oxygen, phosphorus, nitrogen and sulfur. Therefore, alternative sources for nitrogen, phosphorus, sulfur, and certain ions are essential to maintain increased cell proliferation. Most cells depend on amino acids as their source for nitrogen. Some cells express glutamine synthetase (GLUL), which is an enzyme able to convert free nitrogen in the form of ammonia by conversion of glutamate to glutamine. Thus, Glutamine is considered to be a non-essential amino acid for humans because of the ability of GLUL to produce glutamine from glutamate and ammonia. Ammonia is not the major source of nitrogen to most cells because it can be toxic and not all cells express GLUL. In tissues that do not express GLUL glutamine can be essential for cell survival. Glutamine can be used for protein synthesis, synthesis of glucosamine, and nucleotide biosynthesis. It can also be lysed by mitochondrial glutaminase (GLS) into glutamate and ammonia. Glutaminase catalyzes the first reaction of the glutaminolysis pathway. This pathway breaks down glutamine producing α-ketoglutarate and ATP. Recent evidence has also highlighted the importance of the glutaminolysis pathways in providing anabolic carbons through the tricarboxylic acid (TCA) cycle for the synthesis of amino acids, nucleotides and lipids in cancer cells. Lipid metabolism is also altered in cancer cells. Increased lipogenesis is considered a hallmark of many cancers, and it is associated with aggressive cancers and poor prognosis. De novo fatty acid synthesis supports membrane biogenesis and energetic demands of increased cell proliferation in cancer cells. Acetyl-coA is an essential metabolite for de novo fatty acids synthesis. Acetyl-coA is generated from pyruvate by intramitochondrial PDH, which, as mentioned before, irreversibly directs glycolytic products to be broken down in the TCA cycle. Citrate derived from TCA cycle is transported to cytosol where it is converted into acetyl-coA by adenosine triphosphate (ATP)-citrate lyase (ACLy). Following this conversion, fatty acid synthase (FASN) catalyzes the condensation of malonyl-coA and acetyl-coA to form a long-chain fatty acid. Malonyl-coA is generated by acetyl-coA carboxylase (ACC). Both ACC and FASN are key rate-limiting enzymes of de novo fatty acids synthesis and are overexpressed in cancer. Increased glycolytic flux is important for fatty acid synthesis since it provides acetyl-coA through pyruvate, and NADPH via the PPP flux. Additionally, other anaplerotic inputs such as glutamine-derived α-ketoglutarate also helps to support de novo lipids biosynthesis. Overall, it is understood that altered metabolism is essential for tumor growth. Metabolic reprogramming is responsible for providing energy, reducing equivalents, and building blocks to maintain rapid cell proliferation and cancer cell survival. Additionally, several metabolites exert signaling function, promoting tumor growth and progression. Cellular stress response is a balance between the amount and nature of stressors and the corresponding coping cellular mechanisms. Theoretically, metabolic reprogramming can contribute to cancer cell development and cancer prevention via metabolic promotion of cell survival or alleviation of stress.
Describe the process of DNA replication
Replication proceeds in stages: initiation, elongation and termination. Initiation The origin of replication in Escherichia coli starts at the oriC site. oriC has two types of sequences that are of special interest: R sites, DNA unwinding element (DUE). R sites are 5 repeats of 9bp sequence that serve as binding site for the Key initiator protein DnaA DNA unwinding element (DUE) a region rich in A=T base pairs. At least 10 different enzymes or proteins participate in the initiation phase of replication. DnaA is a member of the AAA+ ATPases. They form oligomers and slowly hydrolyze ATP. ATP hydrolyzes act as a switch mediating interconversion of two protein states. DnaA bound to ATP is active, while DnaA bound to ADP is inactive. 8 DnaA protein molecules, all in ATP-bound state, assemble to form a helical complex encompassing R and I sites in the oriC DnaA has higher affinity for the R sites than I sites, and it binds to R sites equally well in its ATP or ADP-bound form. DnaA binds to the I site only in the ATP-bound state, which allows discrimination between the active and inactive forms of DnaA. The tight right-handed wrapping of the DNA around DnaA complex introduces a positive supercoil, which leads to the denaturation in the A=T rich DUE region in the associated strain in the nearby DNA. DnaC, another AAA+ ATPase, loads the DnaB protein on to the separated DNA strands in the denaturated region. One hexamer of ATP-bound DnaC, forms a tight complex with the ring shaped DnaB helicase. Two DnaB are added, one to each DNA strand. Following DnaB addition to the DNA strands, ATP bound to DnaC is hydrolyzed releasing DnaC and leaving DnaB bound to DNA. Loading of DnaB is a key step in replication initiation. DnaB migrates along the single-stranded DNA in the 5’-3’ direction, unwinding DNA as it travels. DnaB helicases travel in opposite direction, creating two potential replication forks. All other protein at the replication fork are linked directly or indirectly to DnaB. As replication begins and the strands are separated at the fork, many molecules of single-stranded DNA binding proteins (SSB) bind to stabilize the separated strands. Additionally, DNA gyrase (DNA topoisomerase II) relieves the topological stress induced ahead of the fork by the unwinding reaction. DNA polymerase III is added to the DNA interacting with the DnaB through one of its subunits. Once DNA pol III has been added to the DNA, along with b subunits, the protein Hda binds to the b subunits and interacts with DnaA complex to stimulate hydrolysis of its bound ATP. Hda is another AAA+ ATPase. This ATP hydrolysis leads to disassembly of the DnaA complex at the origin of replication and signals the end of the initiation step. Elongation Includes two distinct operations: leading strand synthesis and lagging strand synthesis. Leading strand synthesis is the more straight forward one. It starts with primase synthesis of a short RNA primer (10 to 60 nucleotides) at the replication origin. DnaG carries out this reaction. DnaG interacts with DnaB helicase and synthesize primers in the opposite direction to that in which the DnaB helicase if moving. DnaB moves along the strand that will become the lagging strand. However, the first primer laid down in the first DnaG/DnaB interaction serves to prime the leading strand DNA synthesis in the opposite direction. Deoxyribonucleotides are added to this primer by DNA pol III complex linked to DnaB. Synthesis proceeds continuously, keeping pace with the unwinding of DNA. Lagging strand synthesis is accomplished in short Okazaki fragments. The synthesis follows two steps: 1. RNA primer is added; 2. DNA pol III binds to primer and adds DNA. This seems simples and fairly similar to the leading strand synthesis. However, the complexity lies in the coordination of leading and lagging strand synthesis. Both strands are produced by a single asymmetric DNA polymerase III dimer; this is accomplished by looping DNA of the lagging strand. The synthesis of the lagging strand Okazaki fragments entails an elegant enzymatic choreography. DnaB helicase and DnaG primase constitute a functional unit within the replication complex, called the primosome. DNA pol III uses one set of its core subunits to synthesize the leading strand continuously, while the other set of core subunits cycles from one Okazaki fragment to the next on the looped lagging strand. While the DnaB helicase travels along the lagging strand template in the 5’-3’ direction DnaG primase occasionally associates with DnaB helicase and synthesis a short RNA primer. When synthesis of an Okazaki fragment has been completed, replication halts, the core subunit of DNA pol III dissociate from their b sliding clamp and associates with a new clamp. Initiating the synthesis of a new Okazaki fragment. Termination The two replication forks of the circular E. coli chromosome meet at ta terminus region containing multiple copies of a 20 bp sequence called Ter. Ter sequences are arranged to create a trap that a replication fork can enter but cannot leave. They act as a binding site for the protein Tus. The Tus-Ter complex can arrest a replication fork from only one direction. Only one Tus-Ter functions per replication cycle, the complex first found by either replication fork.
Describe the functions of DNA.
DNA has four major roles. 1. Replication. DNA bases complementarity create a double helix structure. This tridimensional structure of DNA play a important role in replicating the genetic information stored in the DNA molecule during cell division. The replication of the DNA allows each cell to receive a identical copy of DNA, therefore maintaining the genetic information from cell to cell. 2. Encoding information The sequence of bases of DNA ( A, T, C and G) are organized into genes. Each three consecutive bases form a codon, which specifies a specific amino acid in proteins. To "read" the genetic information containing in the DNA the cell must first transcribe the DNA sequence into an RNA molecule. The RNA sequence can be translated into proteins afterwards. ( IT IS VERY IMPORTANT TO NOTE HERE THAT NOT ALL GENES CODE FOR PROTEINS). 3. Mutation and Recombination Both Mutations and Recombinations of DNA play a role in the evolution of species. Chromosomal DNA helices can swap places with each other creating new sequence of genetic material during recombination. If this recombination happens to occur in the DNA sequences of sex cells the changes generated by DNA recombination can be inherited by the next generation. Mutation happens when the sequence of the DNA molecule is modified, most times without control and at random. If this modification happens in the chromosome of sex cells the change can be inherited by the next generation. In both cases we see modification of the DNA sequence. These modifications might generate different RNA and protein products. If this new products are beneficial to the organism this characteristic might evolve over time in order to make the organism more fit for survival and reproduction. 4. Gene expression in theory, all cells from an organism have the same genetic code ( same DNA sequence). However, cells from different tissues and organs behave and look very different. The main reason behind these differences is the regulation of gene expression. Despite having the same DNA sequence not all genes are expressed at all times. DNA is able to regulate which genes are to be expressed at any given time or space. Several proteins interactive with DNA sequence and help DNA to regulate gene expression.
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