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Retrograde signalling is a very important mechanism when it comes to altering the behaviour of a cell due to stress. Stress expressed towards the mitochondria causes for the inactivation of its ability to regulate calcium levels. It is calcium that is involved in the retrograde signalling resulting in the activation of specific transcription factors, such as NFκB. Ultimately leading to the changes in a cells behaviour, determined by which genes are expressed by the specific transcription factor.
To describe the generation of reactive oxygen species (ROS) by mitochondrial electron transport chain (ETC).Also study the importance of mitochondria in calcium haemostasis.
To explain the interaction of these signalling molecules, in particular calcium as a signalling molecule, with targets within the nucleus such as NF-KB and NFAT
To understand the impact of signal reception on expression of nuclear genes. In particular those coding for protective antioxidative proteins in response to an increase in ROS levels is the mitochondria
The generation of the signalling molecules within organelles
Generation of ROS within Mitochondria:
Mitochondrial retrograde signalling pathway is for communication from mitochondria to the nucleus that influences many cellular and organismal activities under both normal and pathophysiological conditions.
Mitochondria is the main production of ATP in cells however mitochondria is also the main major sources of Reaction oxygen species (ROS) for example superoxide anion and H2O2 in cells. ROS generation by mitochondria is linked with ATP production by mitochondria. The movement of electrons along respiratory chain are responsible for the generation of ROS in mitochondria.
Major mechanisms for ROS production is that reduced substrates synthesized in metabolic pathways provide electrons to complex I and II of the electron transport chain. The main production for superoxide (O2−•) occur at complex I and III, but small amounts can be formed at complex II and IV. Ubiquinone (Q) which is the electron carrier of complex III is reduced to ubiquinol (QH2). This transfers an electron to cytochrome c (Cy C) via an iron protein inhibited by myxothiazol (Myx).The resulting semiubiquinone (Q*) is oxidized back to ubiquinone by cytochrome b (Cy b). This can also transfer electrons to oxygen to form O2−•. Myxothiazol reduces O2−• formation since it blocks Q* production. On the other hand, antimycin A (Ant A) enhances O2−• formation by increasing Q* levels. Rotenone (Rot) blocks electron flow to O2−• formation, initiating its production.
Reactive oxygen species can cause damages within the cell such as, nucleobase oxidation by hydroxyl radical (ROS). In this process, Hydroxyl damages the DNA base pairs by oxidising. Also, Hydroxyl radicals are involved in cross linking of proteins. This process, alters the functions of proteins (Murphy P.M. ,2009).
Homeostasis of calcium level by mitochondria:
Mitochondria takes place in in Ca2+ homeostasis.Ca2+ concentration plays role in cell death and this is well studied area whereas normal cytosolic calcium concentration signals is barely investigated and understood. Few Ca2+ transport systems are adjusted by oxidation. Oxidation elevates the activity of inositol 1,4,5-trisphosphate and ryanodine receptors. These are the major channels for releasing Ca2+ from intracellular stocks in response to cellular stimulation.
Mitochondria controls the calcium concentrations [Ca2+]i signals by Ca2+ uptake and release during cytosolic calcium mobilization, especially in mitochondria situated close to Ca2+ release channels. Mitochondrial inhibitors adjust calcium signals in various cell types. Despite, these inhibitors decrease mitochondrial Ca2+ influx; they also damage ROS generation in few systems. In addition, mitochondria generate ROS in response to cell stimulation, an effect that abolished by mitochondrial inhibitors that at the same time inhibits [Ca2+]i signals(Almaraz C.C.,2006).
Interaction takes place between the mitochondria and Ca2+ release channels. When cell is stimulated with inositol 1,4,5-trisphosphate (IP3) and cADP ribose (cADPr) ;calcium release is initiated from endoplasmic reticulum (ER) via their specific receptors which are the IP3R and ryanodine receptors (RyR). Local cytosolic calcium concentration ([Ca2+]i) elevated levels are buffered by mitochondrial. Then; mitochondria uptakes calcium via uniporter (MU) or by other possible alternative routes which are not indicated in the figure. Intra mitochondrial calcium initiates production of reduced level substrates such as NADH, through metabolic pathways and speeds up the electron transport chain, elevating the ROS generation, which makes the Ca2+ release easier via sensitization of IP3R and RyR.
Other known mechanisms that elevates ROS production are via modification of the electron transport complexes by mitochondrial calcium and blockage by nitric oxide (NO*). These can be formed by Ca2+ and by mitochondria. In addition to this, mitochondria can support Ca2+ release by efflux through Na+/Ca2+ exchange (NCX) and by elevated levels of cADP ribose. Outer mitochondrial membrane is not involved in the figure 1 (Almaraz C.C.,2006) .
Retrograde signalling is stimulated through increased cytosolic calcium levels. This increased cytosolic calcium is achieved through disruption to the membrane potential of the mitochondria. Many factors can cause for this disruption as described earlier, the generation of ROS is huge factor in altering this membrane potential. Thus causing mitochondria to be unable to take part the uptake of free calcium.
Through the alteration in this cytosolic calcium levels in terms of increasing, it goes onto directly increase the levels of Calcineurin and various calcium dependent kinases. Ultimately these two molecules go onto activate many targets within the nucleus. Calcineurin directly activates the molecule we will focus on within this poster, NFκB. These calcium dependent kinases go onto activate nuclear targets such as: CHOP, ATF2, CEBP/delta, Egr-1 and CREB. (Looking at figure 2, showing the overall retrograde pathway involved in the activation of NFκB).
Figure 2) - Is illustrating the overall retrograde signalling pathway, in terms of the stress put onto the mitochondria, which then has the effect of altering the mitochondrial membrane potential, which causes inactivation of the mitochondria's ability to regulate calcium levels, in turn causing an increase in cytosolic calcium. This then causing the activation of calcineurin and calcium dependent kinases, which go onto activate specific nuclear targets, (Butow R. A., 2004)
As earlier discussed through mitochondrial stress (ROS) there is an alteration in the mitochondrial membrane potential, thus causing an increase in cytosolic calcium levels. This increase in cytosolic calcium proceeds to cause the inactivation of IKBβ.This inactivation is achieved through dephosphorylation, mediated by calcineurin. The function of IKBβ is to act as an inhibitory molecule to the NFκB, through binding to it and keeping it inactivated in the cytosol. Therefore if this is dephosphorylated and inactivated then it can cause for the NFκB to become activated in the cytosol, once activated the NFκB will translocate to the nucleus. Upon entering the nucleus it will perform its task as a transcription factor and regulate the expression of various genes. (Looking at figure 3, showing an outline to the pathway involved in the activation of NFκB).
Figure 3) - Is illustrating the main steps involved in the retrograde signalling pathway between the mitochondria and the nucleus,
as the main focus being on the activation of this nuclear target NFκB.
Expression of antioxidative proteins in response to increased ROS levels
Maintenance of mitochondrial function depends on contributions from the nuclear genome, which codes for the majority of mitochondrial proteins. Retrograde signalling is a process that ensures mitochondrial function is maintained by expression of nuclear genes which counteract changes in the functional state of mitochondria including: impaired oxidative phosphorylation, uncoupling and hypoxia.
Retrograde pathways concerning oxidative stress are activated by mitochondrial or cytoplasmic proteins which detect an increase in ROS. Reactive Oxygen Species is term used to describe a group of species produced as a by-product of the TCA cycle. An increase in ROS generates a retrograde signal which leads to the expression of nuclear genes which code for proteins involved in cellular and mitochondrial detoxification. These proteins include: SOD enzymes (MnSOD, CuZnSOD, and extracellular SOD) which reduce superoxide to H2O2. H2O2 is the substrate for peroxiredoxins (Prx), which reduce H2O2 to water, as well as for other enzymes such as catalase and glutathione peroxidase (GPX). (Fig. 3)
The mitochondrial matrix protein MnSOD is an example of an antioxidant produced in relation to oxidative stress. The SOD2 gene is tightly regulated by the transcription factors FOXO3a and NF-kB, whose activity is tightly regulated as well.
FOXO3a regulates activation of this gene in quiescent cells (a cell which is not dividing or in G phase) in response to H2O2 (a ROS). NF-kB regulates it in normal growing cells in response to mitochondria-generated superoxide (a ROS). These sensor systems and their mechanisms of activation are not well characterised.
NFAT proteins play various roles in the nucleus. Although not all are well understood, studies have suggested that NFAT regulates transcription of proteins involved in cell apoptosis (Lui et al 2006).
Fig 4) - Proposed Model of POX-induced Apoptosis in cancer cells. Relevant sections highlighted in red. From Lui et. al 2006
Mitochondria is an important molecule, it participates in the signalling to nucleus as well is being the major energy production organelle. It generates reactive oxygen (ROS) species that can lead to damages within the cells. Mitochondria also control the calcium levels which calcium is very important molecule in determination cell death.
To summarise, it clear through the activation of this particular retrograde signalling pathway it causes for the activation of specific nuclear targets (transcription factors). From activation of these, they go onto regulate expression of specific genes, ultimately changing the behaviour of a particular cell.
In response to increased cytosolic calcium transcriptional factors NF-kB and NFAT are activated. In conjunction with FOXO3a, NF-kB regulates expression of the SOD2 gene in different cellular conditions. This SOD2 gene codes for an SOD involved in detoxification of the superoxide by converting it to H2O2. This is the substrate for another reaction which finally converts it into water. The function of NFAT is more varied but thought to have a role in activation of genes involved in cell apoptosis.