For my final posting, that is coincidently, my 50th post, I would like to share this animation video with you that explains the action of electron transport chain and formation of ATP in the mitochondria via oxidative phosphorylation. It has been a wonderful time people. Hope that you enjoy the video and happy studying!!!
“Parting is such sweet sorrow that I shall say farewell til it be morrow”
The time has finally come and I am sad and a bit nostalgic. When we started back in January, I was a bit uneasy and hesitant about this blogging thing. Now, I am very disappointed that we have completed this task.
Reflecting on the past semester, I had a blast. There were some trials and tribulations and days when I did not fancy Biochem at all. However, these were overshadowed by the exciting, invigorating, challenging activities that Sir always had for us with every week. I could not ask for any more in a course. If I had a choice, I would have liked to continue blogging.
Biochemistry was an enjoyable and interesting course that had me obsessing about the topics and the various avenues to research as the weeks went on. Blogging allowed for consolidation of all the course materials, applications of concepts as well as fostering continuous studying. Sir was very cunning indeed in assigning us this activity. I also developed another unintended skill while undertaking this blogging task, Becoming more tech savvy! Trying to maintain captivating blog postings forced me to learn how to use application, search through sites and format documents to uphold the aesthetic appeal of my blog page.
Additionally, I am truly grateful to have Mr Matthew as a lecturer. In all of my University life, I have met very few teachers that have employed effective methods of relating the course material to the student body. his techniques are adaptable, encouraging and promote a high standard of learning by us, his students. He has taken a seemingly difficult course and showed us that once you put the work in, we can accomplish success. Sir has encouraged me to persist in studies and work and eventually everything will fall in place.
Therefore, I want to thank him for always assisting us when we were stuck on assignments, encouraging us when we were fed up, stimulating our minds when we were bored and entertain us when we were unmotivated.
I am looking forward to new and better things. Moreover, I hope that my experiences in Biochemistry are repeated in other avenues of my academic career and hopefully, I can continue to have fun while learning.
Before I go, I want to send warm thanks and praise to all my followers and commenters to my blog. It has been much appreciated to see your approvals and admiration for the information that I presented. I hope that I did not disappoint and I look forward to your support and love in the future. Thank you, thank you, and thank you.
Well, that is all folks. Until we meet again. It has been a trill and I could not anticipate having enjoyed this experience any more than I did. So keep good, study hard and live right…….
So long, farewell, auf weidersehen, goodbye,
Adieu, adieu, adieu, to you and you and you.
Good-bye, good-bye, good-bye!!!!
Alright my Biochem peeps,
Here is my 2nd review article. It is based on the target therapies of cancer treatment. In particular, this article focuses on the drug known as dichloroacetate which targets a mitochondrial enzyme known as pyruvate dehydrogenase kinase. This enzyme inhibits the action of pyruvate dehydrogenase as well as increase the metabolism of cancer cells that facilitate cancer proliferation. This article is quite interesting and thought-provoking. It will aid your understanding of the concepts of glycolysis, Krebs, ETC and oxidative phosphorylation. It is also marvelous to read the mechanism of action of cancer cells and the intricacies of their metabolism that fosters survival and proliferation. Take a read and see for yourself.
Michelakis, Evangelos D. and Gopinath Sutendra. 2013. “Pyruvate dehydrogenase kinase as a novel therapeutic target in oncology.” Frontiers in Oncology 3(38): 1-11. doi: 10.3389/fonc.2013.00038
Pyruvate dehydrogenase kinase as a novel therapeutic target in oncology.
This article is not a study as in the previous posting, but rather delves into the strategies utilized in cancer treatment in particular targeting more efficient, effective and less destructive methods of cancer therapy. This article deals mainly with investigating the effects of inhibition of an enzyme, pyruvate dehydrogenase kinase (PDK) as well as the applications of similar techniques that promote glucose oxidation as a viable option in metabolic targeting of neoplastic cells. The authors also explore the pre-clinical and clinical evidence of the effectiveness of these inhibitors on different cancers.
Neoplastic cells have a unique metabolic property in which there is both molecular and genetic signalling that converts glucose oxidative phosphorylation in the mitochondria of cells to glycolysis in the cytosol even under normal conditions. This results in the increase in glucose consumption as well as lactate production in cancer cells. This process is referred to as the Warburg effect after Otto Warburg who published this hypothesis in 1956. This means that cancer cells depend on glycolysis as its primary energy source as well as the source of macromolecular building blocks through glycolytic intermediates.
This is initiated by the activation of hypoxia-inducible factor 1α (HIF-1α). HIF-1α is activated in hypoxic-like environments and then stimulates the expression of glycolytic enzymes, glucose transporters and mitochondrial enzymes that increase the glycolytic pathway as well as formation of lactate. One important mitochondrial enzyme that is activated is, PDK. PDK is considered the gatekeeper of the mitochondria since it regulates the flux of pyruvate into the mitochondria to undergo oxidative phosphorylation. PDK does this by inhibition of pyruvate dehydrogenase that performs the link reaction that decarboxylates pyruvate to Acetyl-CoA to enter the Krebs cycle. This inhibition leads to pyruvate not entering the mitochondria and therefore is converted to lactate in the cytosol by lactate dehydrogenase. Oncogenes are responsible for the maintenance of hypoxic state for cancer cells to continue to activate HIF. In cancer cells, there is also a loss of p53 gene expression that was responsible for mitochondrial DNA repair and expression cytochrome c oxidase enzymes that aid in apoptosis of the cell. Loss of p53 directly induces the expression of PDK and increases the stability and transcriptional activity of HIF. Additionally, HIF also increase pro-angiogenic and metastasis-promoting factors such as VEGF and SDF-1.
With increase in glycolytic enzymes, there is an increase in glycolysis as well as these glycolytic enzymes are utilized to aid in anti-apoptotic activities, activation of transcription in neoplastic cells, inhibition of mitochondrial function. Enzymes such include, glyceraldehyde 3-phosphate dehydrogenase and hexokinase. An isoform of hexokinase, hexokinase 2 is up regulated in neoplastic cells and then translocated to the mitochondrial transition pore, a channel responsible for the release of apoptotic factors as well as the regulation of anions the facilitate oxidative phosphorylation. Thus, hexokinase 2 facilitates resistance to apoptosis as well as increase in glycolysis in tumour cells. The glycolytic intermediates may also be funnelled to alternative pathways to produce amino acids, nucleic acids and lipids.
The mechanism of action of cancer cells lies in the alteration of the mitochondria of the cell. The mitochondria are responsible for the intrinsic apoptotic mechanisms of the cells and suppression of mitochondrial function is critical to cancer cell proliferation. One method employed by cancer cells is the alteration of the mitochondrial membrane potential of the inner mitochondrial membrane. This membrane potential is crucial to oxidative phosphorylation and electron transport chain. This membrane potential is akin to the proton motive force that is generated as hydrogen ions are pumped into the inter-membrane space of the mitochondrion during electron transport chain. This membrane potential is also critical to maintenance of intracellular calcium levels since mitochondria act like calcium sinkholes that facilitate the influx of calcium.
Neoplastic cells, inhibit glycogen synthase kinase in the cytosol of the cells that lead to the translocation of hexokinase 2 to the mitochondrial membrane. Hexokinase 2 then binds to the inner membrane and blocks and inhibits the voltage-dependent anion channel found in the mitochondrial transition pore that is responsible for the efflux of anions to maintain membrane potential of the mitochondria that is crucial for oxidative phosphorylation and ETC. This results in the hyperpolarization of the membrane and prevents the opening of the channel in the mitochondrial transition pore that is responsible for release of apoptotic factors such as mitochondrial reactive oxygen species, cytochrome c oxidase as well as apoptotic inducing factor.
The suppression of mitochondrial functioning leads to the decrease in oxidative phosphorylation as well as the release of apoptotic factors. With diminishing functioning, the mitochondria of tumour cells begin to degenerate and further increase the proliferative potential of the neoplastic cell. However, this information can be utilized in targeting specific cancers especially solid tumours.
A drug known as dichloroacetate is able to penetrate most cell membranes and tissues that are not accessible to chemotherapy treatments such as the brain. This molecule is able to activate pyruvate dehydrogenase by inhibition of pyruvate dehydrogenase kinase and its isoforms. Dichloroacetate carboxylic group forms a salt bridge with Arg154 residue of the active site of pyruvate dehydrogenase kinase. Dicholoroacetate is able to decrease the membrane potential of inner mitochondria and aid in normalization of mitochondrial function in cancer cells. However, it does not affect non-cancer cells. It functions to increase apoptotic activity in the cancer cells as well as to increase the intake of pyruvate into the mitochondria and increase more ETC./oxidative phosphorylation. This decreases the production of lactate and the alternate pathways of glycolytic intermediates.
Subsequently, there is a deactivation of HIF since HIF is highly regulated by the TCA intermediate, alpha ketogluterate. Hence, an increase in TCA cycle leads to increase production of alpha ketogluterate that facilitates the inhibition of the action of HIF. This results in decrease tumour perfusion, glucose uptake, resistance to apoptosis as well as decrease production of macromolecules essential for tumour proliferation. DCA has also shown to increase p53 activity in cancer cells leading increase activation of apoptotic action by the mitochondria.
DCA is therefore very effective at selectively targeting cancer metabolism by inhibition of pyruvate dehydrogenase kinase. Clinical evidence of this has been observed in non-small cell lung carcinoma, breast carcinoma, glioblastoma, colon cancer, prostate and endometrial cancer as well as leukaemia.
However, other therapies may mimic the action of DCA and acquire similar results. Some therapies include the inhibition of lactate dehydrogenase to facilitate the channelling of pyruvate into the mitochondria to undergo oxidative phosphorylation. Similarly, decrease activation of the M2 isoform of pyruvate kinase that is expressed in neoplastic cells may prove to be another selective target for cancer treatment.
In concluding this article, the authors note that the aforementioned techniques are not a permanent solution to cancer therapy since there is no absolute action of these inhibitors on the metabolism of cancer.
My initial thoughts of this paper, was that it was long-winded and boring. However, as I continued past the introduction, the content became more interesting and captivating. The explanations and the organization of the information laid a good base level to which anyone reading was able to easily follow. It was greatly surprised at the intricacies of cancer metabolism and the very dynamic alterations made for the survival and proliferation of these cells. I knew that the mitochondria of cells were integral to the optimal functioning of the cell, nevertheless, I had no knowledge of the specific actions involved in mitochondrial functioning.
The authors aptly summarized the pathophysiology of neoplastic cells and then linked this to the action of the small DCA molecules and its efficiency in cancer treatment through selective inhibition. The importance of pyruvate dehydrogenase kinase was also elucidated. I was also intrigued by the importance of glycolysis to cancer metabolism and how effective glycolytic pathway is to the rapid growth and sustainability of the tumour.
However, it must be stated that even though DCA and the other techniques mentioned in this paper would prove highly valuable to oncology, there is no absolute solution since neoplastic cells have the ability to adapt and evolve to avoid the actions of these target drugs. There is no certainty that these therapies will show significant reduction in tumour proliferation in cancer patients and is subjected to individual differences. These therapies also have the potential to produce effects on non-cancer cells with prolonged use and high doses. Therefore, I am hesitant to believe that DCA and similar inhibitory therapies are a permanent solution to reducing the prevalence of cancer.
Overall, I think that this is a groundbreaking and lifesaving research in oncology since it will prove to reduce the mortality and morbidity of cancer. It is quite exciting to note that this method is a good step forward in finding more advance and effective treatment for a serious medical condition. Clinical evidence thus far has been positive and with more research, the methods of cancer treatment may develop into a simple cure like penicillin.
ACETYL-COA ADIPOSE ADP ATP
BROWN CITRATE COMPLEXES COUPLING
CYANIDE CYCLE DECARBOXYLATION DINITROPHENOL ELECTROCHEMICAL ELECTRONS ENERGY FADH
FORCE GRADIENT GTP HYDROGEN
INFANTS INNER INTERMEMBRANE IONS
KETOGLUTERATE LINK LIPOATE MAMMALS
MATRIX MEMBRANE NADH OUTER
OXALOACETATE OXIDATIVE OXYGEN PDH
PHOSPHORYLATION PROTEINS PROTON PUMP
PYRUVATE REACTIONS REDOX RESPIRATION
ROTENONE SUCCINYL SYNTHASE THERMOGENIN
BONUS WORD CLUE: POWERHOUSE
Good day my Biochem peeps,
Enzyme Inhibition by FortuneFavorsPrep
In your revision, you must be like me where you are wondering why you can never remember the enzyme kinetics and the graphs seen in the different reversible inhibition methods. Well, I came across this video where the person explains competitive, non-competitive and uncompetitive inhibition.
The guy on this video gives a good summary of each method and gives the main features of each inhibition, the main effects on the Km and Vmax of the enzyme and the mechanism of action of the inhibitor. He then actively draws both the M-M curve and L-B plot for each of the inhibition described. He is showing you why there is a change in the value, how to draw it and what the plot and curve changes means.
This video is well explained and an incentive to studying the inhibition since it summarizes most of what you need to know on the topic for exams. I felt that this video was an apt summary of the topic and there were little flaws in the explanation or demonstration. However, I felt that explanation of mixed inhibition and irreversible inhibition should also be covered. There were also no examples of each type of inhibitor. Nevertheless, this does not take away from the efficacy of the video and I enjoyed it a lot since above all, it was a short video of about 15 minutes.
The main points of the video has been summarized below, however, I am strongly suggesting that you view this video since it will really help you, if you are still confused on the topic and you wanted a nice summary of the essentials of inhibition.
The video begins with a summary of inhibitors. He then begins to describe competitive inhibition and the fact that the inhibitor binds to the active site of the enzyme and competes with the substrate to bind. He illustrates the equation of the enzyme and substrate interaction and shows the effect of a competitive inhibitor on this process. The guy then goes on to explain the effect of this inhibitor on the affinity of the enzyme for the substrate and hence an increase in the Km value. He also mentions that this inhibition may be reversed or overcome by increasing the substrate concentration and hence does not affect the Vmax. Finally, he uses the M-M curve and L-B plot to illustrate what was explained in the text of the video. He drew the two graphs, which was good since it actually demonstrated the method and reason for the position of the points on the graphs.
Then he moved on to non-competitive and uncompetitive inhibition where he proceeded to explain the main points of these two types of inhibition and compared them to competitive inhibition. In summarizing non-competitive, he mentions that this inhibitor binds to a site other than the active site and changes the conformation of the enzyme. He links this to induced fit model mechanism of action and then compares this method to that of competitive inhibition. He then further details the change in the Vmax and Km. He aptly explains that non-competitive inhibition does not prevent the substrate from binding to the enzyme and therefore does not change the affinity of the enzyme for the substrate and hence the Km is changed. However, since it changes the conformation of the enzyme, it decreases the ability for product formation and hence decreases the rate of reaction of the enzyme. This means that Vmax is decreased. Finally, he ends this section by draw the L-B plot and shows the changes that the non-competitive inhibitor has on the enzyme kinetics.
This video ends with an explanation of uncompetitive inhibition. In this final segment, he illustrates again the graphs and show how there is a decrease in the Vmax and Km due to the inhibitor binding to the enzyme-substrate complex. However, he does not mention that this is a proportional decrease and goes into why there is a decrease in these values. He also does not mention that uncompetitive inhibitor may bind to the free enzyme at another site than the active site. But overall, the explanation and illustration was very helpful in visualizing and conceptualizing the topic.
Well, I really hope that this aids in your revision. A point to note is that this should not be taken as the only source on this topic and you should refer to Sir’s lectures and the textbook for further details. Happy studying until the next one….