CELL BIOLOGY 2 (CBG) SBOBINE, A.Y. 2021-2022-pagine-4.pdf

10.12.2021 RETTA L.3 CCM DISEASE PART 3 Last time we were talking about cerebral cavernous malformation and we just mentioned the major discoveries including the find...

10.12.2021 RETTA L.3 CCM DISEASE PART 3 Last time we were talking about cerebral cavernous malformation and we just mentioned the major discoveries including the findings of the CCM gene and we also mentioned the next steps, so, from the identification of genes, how can you define the molecular mechanism underlying a genetic disease including CCM disease. We mentioned the progress in the field including the identification of some interactors binding proteins of the protein of our interest, so the protein of our interest in this case is CCM1, also known as KRIT1, the structure of the protein is represented in the picture. (fig.1) fig.1 Basically when you characterize a protein you can define some structural motifs, the structural motifs or structural domains are important in order to define the function of a protein because the structural domains are involved in protein-protein interaction, for instance, so they are involved in the function of the protein of our interest because of the interaction with binding parts with other proteins that cooperate with the protein of our interest in some molecular function. By identifying some binding patterns, you can infer some functions. The identification of the binding patterns and the molecular characterization of this interaction is helpful in order to define the function of a protein and the function of mechanisms involved in a human disease. In this case it was demonstrated that KRIT1 protein was a protein of 736 amino acids and contains different domains including the FERM domain at the C-terminal. The protein, then, contains other domains including ankyrin repeats, in the middle, and NPXY motif at the N-terminal. All this motifs are involved in protein-protein interaction, for instance, the NPXY motif is involved in the interaction with ICAP-1 while the FERM domain is involved in interaction with RAP1A. fig.2 Last time we mentioned the initial hypothesis related to the function of the newly identified protein KRIT1, so because of its interaction it was possible to hypothesize that KRIT1 was involved in the regulation of cell adhesion to extracellular matrix mediated by proteins that are called integrins. (fig.2) Opera song “là ci darem la mano” After the identification of the major interaction that I mentioned, so ICAP1 and RAP1, other proteins were identified as molecular partners of KRIT1 including CCM2, that is another protein involved in CCM disease. Is encoded by the second CCM 2 gene that we mentioned last time so the protein encoded by the CCM 2 gene that is also mutated in patient affected by CCM disease interacts with KRIT1. In addition, also the third protein encoded by the third gene involved in CCM disease, so namely CCM3, participates in the formation of 15  10.12.2021 RETTA L.3 molecular complexes that play a role in the pathogenesis of this disease. The picture is becoming more clear, because we can see more molecular pieces that are part of the molecular puzzle; we are defying a molecular puzzle that plays a role in the pathogenesis of a specific disease, by adding molecular pieces we can have a more clear picture of this puzzle. Now we are in 2007, so almost 8 years after the discovery of the first gene involved in CCM disease, so in the paper published in 2007 Angela Glading made a fundamental discovery, she found that CCM1 gene is involved in regulation of cell-cell adhesion, besides cell-matrix adhesion. Cell-cell adhesion is fundamental for the stability of endothelial layer that form blood vessels. If cell-cell junctions brake up hemorrhages can take place, so it’s important that cell-cell junction are formed and are stable. So this was a fundamental discover because researchers started to understand what can happen when there is a mutation to the gene encoding for the protein involved in CCM disease. So mutation in such a gene could cause the braking up of cell junction, and this in turn can cause hemorrhages. So because of this discoveries it was possible to start to think about specific mechanisms that could underly the pathogenesis of the disease. Later on other information were provided including this fundamental discover, so now we are in 2010, Rebecca Stockton that was working in the same lab as Angela Glading, demonstrated that the loss of function of CCM proteins caused alteration of the actin cytoskeleton and in particular cause the formation of structure called stressed fibers, so later on we will discuss more in detail actin cytoskeleton and stress fibers. CCM gene plays a major role in the dynamic of the actin cytoskeleton, so because of this fundamental discovery it was possible to start to think about potential treatment and therapeutic approaches because the loss of function of CCM gene cause the braking up of cell-cell junction and the alteration of the active cytoskeleton, let’s try to test a drug that is able to counteract this molecular process including the alteration of the cytoskeleton, there were at that time drugs able to do this, so they were tested in cellular model of CCM disease, that are cells that carry mutation of the gene involved in the disease, by testing some drugs able to act on the regulation of the dynamics of the actin cytoskeleton was possible to demonstrate that this drugs were effective in reverting the molecular phenotype, at least in vitro, at least in a cellular model. This was very important because researcher started to see a prospective related to the treatment of the disease. ANIMAL MODELS How can you add more information and find more in detail the molecular mechanism of a disease and so improve the therapeutic approaches? You can take advantage of animal models of human diseases, that are models that develop the disease of your interest maybe because you cause the alteration of the genes involved in such diseases. You can make a specific alteration in a specific target of the gene and you can in such a manner develop a so called animal model of a disease of your interest. This has been done in case of CCM disease so different groups started to develop such animal models and then they tested the function of the CCM genes in vivo (animal model). What are the function of a CCM gene in vivo? By inducing the so called knock out of CCM gene by targeting a project procedure. It was possible to demonstrate that the loss of function of a gene , for instance CCM1 is located on the long arm of chromosome 7, if you inactivate both alleles of this gene. For a gene we have two alleles one on the maternal origin of the homologous chromosome and the other on the paternal origin. By inducing the homozygous knockout of CCM1 on both alleles, it was possible to demonstrate that CCM1 is fundamental for life. If you cause such mutation in a gene the embryo will develop until a specific point during the embryonal life and then it will die before life. So there are not people carrying the homozygous mutation for CCM1 because this is not compatible with life, this has been demonstrated in animal models. So this is also a fundamental discover because indicates to you that you are studying a gene that plays a fundamental role in embryo development and the formation of the organism. The mutation of just one of the two gene was vital with no apparent phenotypes, so people carrying this disease are heterozygote. For such a mutation so they carry one allele mutated and the other one normal because if both alleles were mutated ,they could not be born so there is no possibility that our patient is affected by the homozygous mutation. What is the advantage of such a model? Besides allowing us the identify the molecular mechanisms underlying the pathogenesis, such a model allows us to develop also a therapeutical approach. So before I mentioned the effectiveness of some potential database compounds in cellular models, so why not test it in animal models. The animal model is available so you can test this compound in animal models. But let me show you details of the progress into this field. Now you can see that the first animal model was produced in 16 10.12.2021 RETTA L.3 2004 and later on we also developed an animal model that is still available in the lab. ANIMAL MODELS: ZEBRAFISH Others developed another animal model: the zebrafish (Fig.3). It is also useful for defining the function of human genes, in particular the genes involved in the development of the cardiovascular system, because this models are very convenient and during the embryo development are transparent so you can look at the embryo development under a microscope and see whether there are some alteration in some organs. The loss of function of KRIT1 in zebrafish caused a vascular phenotype including a dilated heart, so it is related to vascular disfunction. That’s why death can occur. So other researchers provided additional contributes, so now we have in 2011, almost 10 years after the discovery of CCM1 gene so in this specific paper, also provided by the group of Mark Ginsberg in the United States, it was demonstrated that the interaction between KRIT1 and RAP1 is fundamental for the correct function of KRIT1. So KRIT1 can be present but if its interaction with RAP1 is altered and maybe even because of a point mutation, you see this is just a point mutation (the change of an amino acid with another), any specific fig.3 mutation so this mutation is sufficient to cause this phenotype, so this disease can take place because of alterations of interactions between for instance KRIT1 and RAP1 so this interaction is fundamental for the function of KRIT1. (Inaudible question ) – so you don’t understand how you can make a point mutation – (inaudible question ) – oh , this is an amazing amino acid that is arginine that locate in the position 452- you remember I told you there are 736 amino acids – this amino acid ( arginine ) is located after position 450 so you can make a substitution of arginine with glutamic acid because of a point mutation of the nucleotide sequence of the DNA.( inaudible question ) – yes you can make a target mutation where you want. So this is a very simple mutation it’s sufficient to cause the disease. What is the information that you can derive from such results? The information is that disease is caused because of the lack of interaction between KRIT1 and its major interactor RAP1. so this interaction is fundamental. It is not sufficient that a protein is expressed, its fundamental that its molecular interaction work perfectly otherwise , despite the expression of the protein , disfunctions can take place. The conclusion is: this interaction is required for normal cardiovascular development in vivo. This was demonstrated in zebrafish but it is possible to infer that this takes place also in humans. This was an information that made more clear the information about the mechanisms underlying the disease. MOUSE MODELS: MOUSE MODEL 17  10.12.2021 RETTA L.3 Zebrafish model is very convenient because you can monitor the embryonal development under the microscope and you can label some cells with fluorescence proteins and it was possible to label the cell that from the vasculature. With green fluorescent proteins, for instance, you could see in such case the blood vessels emitting green light and you can monitor the development of cardiovascular system by monitoring the emission of green lights by cells that form the vasculature. So, it’s a very simple organism but can provide very useful information. Then you have to use an organism that is closer to humans, like a mouse. So, in the mouse model, as I mentioned before, the heterozygous mutation of CCM1 was not sufficient to cause the disease, so the beginning this was quite frustration, because the situation in the human was that patient must carry a heterozygous mutation of the CMM gene but this mutation do not seem to cause apparent phenotype. How can we solve this problem? Researcher started to think that additional events could be required. The mutation of CCM gene predisposed to the development of the disease, but in order to develop the disease it requires additional events. What are theses additional events? They started to think about the double heat mechanism, also called two heat mechanism. The two heat refers to the fact that maybe in order to develop a pathological phenotype maybe is required to have a first event that could be the mutation of the gene of your interest and a second heat, so what could be a second heat? The second heat could be the mutation of the second allele. The homozygous mutation of both alleles is not compatible with life if this mutation takes place in all the cells during embryo development, but if the second mutation of the second allele takes place in just few cells of the adult, all the other cells are normal, maybe the cells involved with the CCM lesions. This second type of mutation is called somatic mutation. What is a somatic mutation? It’s a mutation that occurs in somatic cells, so one or fig.4 few cells in the adult. The germ line mutation is present in all the cells because you inherit it from your parents, that’s why you are predisposed to develop this genetic disease. CCM lesion develop in cells in brain where the second heat took place, this is possible, think about cancer, it is caused by somatic mutation during adult life. Some cells can undergo mutations in specific genes and this can cause disease so somatic mutation is a mutation that can happen in adult life in one or few cells. - This was the hypothesis: the heterozygous wasn’t enough to induce the disease, while the homozygous is lethal during the embryonal development. So this allows to think about the mechanism mentioned, so the potential involvement of the second heat. CCM lesions are focal, and you could think that maybe in the region where the lesion takes place maybe the second heat could take place and this would cause the development of such a lesion. That also explain the difference between the familial and the sporadic form of the disease. In the sporadic form of CCM disease you can imagine that a somatic mutation takes place in the first allele during the adult life and rarely the second heat takes place in the second allele and just there the CCM lesion develop. This can be a possibility because you can see that a patient did not have the germ line mutation, so it was not predisposed to develop a disease but it was unlucky to develop a somatic mutation on one allele and later on second on the 2nd allele in same cells and that’s explain the sporadic cases maybe carry only one lesion, because the combination of the 2 heat in the same cells is less likely. Statistically that two mutations occur on the same gene of 2 allele of the same cell is unlikely, while in the familial cases, because all the cells already carried one mutation of one allele, that the second allele could be mutated in the adult life is more probable. This is way they carry a lot of lesions because the mechanism could take place in a lot of cells. This is very stimulating because then you can say, okay, what can I do now as a researcher to define the mechanism, first of all, is it true that this is right hypothesis? Are there other possible hypothesis? We have to be careful, the hypothesis that we mentioned Is plausible, but it is not the only one. Is it possible that the second heat could be related to environmental aspect? Including some microenvironmental heats that take place locally? So if there is a cell 18 10.12.2021 RETTA L.3 that carries the mutation of one allele of CMM1, then this is sensitive to the second heat but this second heat could also be an environmental heat that occurs locally. If the effect of the first mutation is to make more sensitive the cell that carries this mutation to stressful events that occur locally in the microenvironment surrounding the cells. So now there are 2 possibility: one is the genetic second heat, the second one is the environmental second heat, a second heat is required otherwise we could not explain the focal formation of the lesions. A second heat is required but this could be either genetic , affecting the second allele in a somatic manner or an environmental second heat that takes place locally. So the question was still open, these are 2 major possibility, both of them are plausible. ANIMAL MODELS: CONDITIONAL KNOCKOUT OF A CCM GENE How is it possible to proceed? What is possible to do to understand which hypothesis is true? It is possible by developing another animal model. So the homozygous mutation of CCM genes at the embryonic level is lethal, the embryo dies after 9.5 days after fertilization, so up to 9 day the embryo can develop, so it’s true that CCM1 is fundamental for life but it’s not fundamental for the first 9 days of embryonal development, in these 9 days the embryo can live despite the mutation of CCM1. fig.5 This means that this gene is important after 9 days, when the cardiovascular system forms, then the CCM1 gene is fundamental and in the absence of CCM1 the cardiovascular system does not develop and causes death. This is a problem, how can we study the function of a problem in the adult life, even if the genetic double heat is the true hypothesis how can we test this? We can develop a mouse model that is called conditional knock out. I cannot induce the homozygous knockout in the embryo, but if I would be able to allow the mouse to develop and be born and induce a knockout during its adult life, it would be different. This is called conditional knockout, I can induce the knockout when I want. In addition I can induce the knockout in a tissue specific manner, so I can mutate that gene in a homozygous manner only in endothelial cells, while all the other tissues are normal. I can induce the knockout when I want and in a tissue-specific manner,in the tissue that is dealing with the disease , what happens is that the mouse develop CCM lesion. In this case the experiment was on CCM2, you can have a similar situation in the experiment on CCM1. The mouse develop somatic lesion, but notice that the lesions are not everywhere, they are only in the cerebellum. They develop also in the retina. So why this? This was crucial in allowing us to decide whether the genetic double heat or the environmental double heat hypothesis where involved, what does this result tell you? ( answer from one students : it is maybe because it is active in that part of brain and not in every part.) - So you said that such result can suggest that CCM2 plays a major role only in the cells that are located in the cerebellum, but last time I showed you that lesions can develop also in other parts of brain. P14 means 14 days after birth, p means post another. You induce the knockout everywhere, but later on the lesion form on other parts of the brain, so your hypothesis is not supported as otherwise you should not see lesions in other parts of the brain – your explanation was perfect if the gene only played a role in the cerebellum but this is not the case. Maybe the second heat is not enough, such result indicates that even the double heat is not sufficient, that’s the only possibility. Maybe the second heat is required but it’s not sufficient, maybe not even required if there is another type of heat that plays a fundamental role. What other information can you derive from such a result? You have to interpret correctly the results. Maybe in the 19  10.12.2021 RETTA L.3 cerebellum there is the additional event that can make the difference, can trigger the development of the lesion. what can it be? Angiogenesis, if you check you can see that even after birth , in the cerebellum , there is angiogenic activity, that is the formation of blood vessels which is not in other parts of the brain. After some years, this was in 2011, it took maybe 7 years that this was proved to be true. When there are angiogenic events there are stressful condition: there is oxidative stress, there is inflammation. They can serve us the additional trigger. Basically after ten years we are sure that inflammatory factors and oxidants that form locally because of local events are fundamental for triggering the lesion formation. If you can control such environmental factors maybe you can limit the progression of disease. Is that possible? Yes, in such a manner you can prevent the sever phenotypes associated with the disease in some patients. Because of such results it was possible to make some hypothesis including the 2 that I mentioned, and in particular the second one so the environmental stressful events could be more plausible, it could explain everything, including the different susceptibility of different members of the same family carrying the same mutation, if it is true that environmental stressful event must take place in order to trigger the development of lesion, then you can think about the possibility that different people can have a different sensibility to stressful event, and this can explain the interindividual differences in the severity of disease , there are some people that develop more severe phenotypes, other people, including member of the same family, do not develop even symptoms. If it is true that the second heat is required the possibility is basically the same but the difference could be that maybe because of different genetic backgrounds, even in the same family they can have different genetical background, only identical twins have the same genetic background, Other members of the family can develop different phenotypes, starting from asympthomic lesions or no lesions up to very severe lesions. The most severe phenotype is death, because lesions can cause haemorrhages, and this can be lethal. There are different example of famous people that died from this disease. Such results suggest us molecular mechanism and risk factors so suggests us also some approaches either for therapy or prevention. Still because of this result we are starting to see some light. “It is not enough to look, it is necessary to look with eyes that want to see and believe in what they see” Interpretation make the difference. You should remember this aphorism for your life. You should be careful to interpret the results. Let’s go on, what are the additional results that provided more information about pathogenetic mechanism underlying this disease. We started to work in the early 2000 and we discovered an alternative splicing isoform. The isoforms are named with letter, this was the first isoform beside the canonical form that we called KIRT1A, but then there were also contributes regarding the structures of the protein involved, so this group provided the structural information about the relation between RAP1 of KRIT1, because there is a correlation between structure and function. There is a tight structure-function relationship. So defying the structure of a protein and the structure of proteins that are interacting each other can be helpful. So structural characterization of crystallographic x-ray allowed to define the structure of this proteins and this protein-protein interaction. IDENTIFICATION OF THE MURINE KRIT1B ISOFORM 20 10.12.2021 RETTA L.3 fig.6 The KRIT1B isoform is an alternative splicing isoform and the alternative splicing is referred to the 15 coding exons. (I told you KRIT1 has 16 coding exons, there are totally 20 exons which 16 are coding exons) , the 15th one can be alternatively spliced. The removing of this coding exon will cause the deletion of 39 amino acid, this occurs in the C-terminal region. It is a three lobes domains, F1-F2-F3.(fig.6) The FIRM domain is affected by the splicing, but what is the functional difference between the two isoforms You can compere the isoforms in cellular models. fig.7 fig.8 This is the structure of the FERM domain (fig.7), you can recognize the three lobes that contains maybe both α helix and β sheets or only alfa helix. What about the deletion? We were able to demonstrate that the effect of the deletion alter the domain. You imagine that because of the alternative splicing over the 15 coding exon this yellow beta sheet is missing. What is the effect? I used to refer to this figure (fig.8) as a butterfly. In the alternative splicing isoform one wing is missing and without that wing the interaction with the MPXY motif that used to take place in this region cannot occur, and because of this function, we were able to demonstrate this: (fig.9) By labelling proteins we performed florescence microscopy and you can recognize the parts of the cell, the difference is the presence or the absence of the 39 amino acids of the FIRM domain. What is the effect of this? It is clear, it is like white and black, the lack of the 39 amino acids in the F3 lobe of the FIRM domain prevents nuclear localization. No doubts about the interpretation. 21  10.12.2021 RETTA L.3 fig.9 You can see that the 39 amino acids are fundamental for the nuclear localization of KRIT1. This information is fundamental, first of all, you are relating the function of a protein to a specific region, including the possibility to translocate to the nucleus and undergo what is called the nucleus to cytoplasmic shuttle, there are proteins that can shuttle between cytoplasm and nucleus depending on the stimuli. That’s also important for the their function, also there are proteins in the nucleus to activate gene expression. Understanding the mechanism that undergo the nucleus transfer localization of a protein can be relevant in order to understand its function. In conclusion, because of the interaction of the F3 lob of the FERM domain and the NPXY motif present in the N-terminal region, the KRIT1A can undergo an intramolecular interaction between the C-terminal part of the protein and the N-terminal part of the protein (fig.10). Some proteins close, acquire the closed conformation where the N-terminal part interact with the C-terminal part. In other condition this protein ca open up, this also take part in the function. It is amazing to know that a protein can close by making intramolecular interaction between a region and another region over the same protein. KRIT1A can shuttle between the nucleus and the cytoplasm because of specific mechanisms, while KRIT1B cannot enter the nucleus because can not acquire the closed conformation. F3 LOBE is altered , so this interaction can not take place anymore. So this isoform stays always in the cytoplasm. 22 10.12.2021 RETTA L.3 fig.10 Opera song “la donna è mobile” It was possible to demonstrate how a protein can translocate into the nucleus and can undergo nucleus- endoplasmic shuttling, this allows you to characterize the functions of the protein. Later on it was possible to make a more clear picture about interaction and molecular function. 23