Unfolded protein response (UPR) is an intercellular signalling pathway that accumulates of misfolded proteins in the ER lumen. Mitochondria and the endoplasmic reticulum work together to form a structural and functional network, that is essential to maintain a cellular homeostasis and to determine the cell fate under different pathophysiological conditions. To maintain control of pro-survival pathway, regulated Ca2+ from the endoplasmic reticulum to the mitochondria (Malhotra & Kaufman, 2011).
The purpose of the ER UPR is to control the response to the accumulated of unfolded or misfolded proteins in the lumen. There are three aims, first is to initially restore the normal function of the cell by slowing translation, activating the signalling pathway which leads to increasing the production of the molecular chaperones that are involved in the protein folding, and degrading the misfolded proteins. If these objectives are not achieved within a certain span or the disruption is prolonged, the UPR will activate apoptosis (Malhotra and Kaufman, 2011).
Figure 1. Signalling unfolded protein response. IRE1, PERK and ATF6 are three proximal sensors that regulate the unfolded protein response through their respective signalling cascade. (Malhotra and Kaufman, 2011).
– IRF1: It is an ER membrane protein with RNase and kinase activity. Under ER stress, IRF1 phosphorylates itself and activates RNase activity.
– PERK: It is a ER transmembrane protein kinase that phosphorylates eIF2a, it’s also a translation factor that inhibits protein
– ATF6: Under normal conditions, ATF6 is an ER transmembrane protein. It is translocated into Golgi under stress conditions. It is cleaved and able to translocate to the nucleus and able to regulate gene transcription.
In mitochondria, mitochondrial unfolded protein response (UPRmt) is a cellular stress response (Zhao, Wang, et al, 2002). Apoptosis does not occur in the UPRmt because the protease is managed to clean up the aggregates. All the proteins within the mitochondria start to degrade during UPRmt as it is beyond the capacity for the chaperone proteins to handle them, this results in the mitochondria to become less functional. The UPRmt can happen in the mitochondrial matrix or the inner membrane (Pellegrino, Nargund and Haynes, 2013).
I. Discuss the similarities and differences between the UPR in the endoplasmic reticulum and the UPR in the mitochondria.
Mitochondria’s classical concept the powerhouse of the cells and an isolated organelle has been challenged over time with the realization that the mitochondria function continually remodels by both fusion and fission events. Both the organelles ER and mitochondria are both dynamic and capable of modifying the structure and function in response to the environmental conditions. They both interact functionally and physically with each other, and one of the important aspects of this interaction is calcium signalling between the two organelles (shown in figure 1). The ER and mitochondria have a 20% close contact in between (Kornmann et al. 2009), this contact which the ER connects with the mitochondria are referred to as (MAM) mitochondrial associated membrane (Vance 1990). This interaction has pivotal roles in numerous cellular function with lipid transport, calcium signalling, energy metabolism and the cell survival (Stone and Vance 2000).
The endoplasmic reticulum and mitochondria are both exposed to nascent polypeptides, both need dedicated protein-folding machinery, constituting each organelle’s protein folding capacity (Pellegrino, Nargund and Haynes, 2013). There are different signalling mechanisms for ER and mitochondria due to the different shapes of the organelle. The ER-localized membrane spanning Kinase (lre1) in the luminal domain, directly senses the unfolded proteins and transmits the signal information to the cytosolic domain of lre1. The signal directly activates the transcription factor Xbp1 (Walter & Ron, 2011). The mitochondria have a different stress responses that respond to perturbations in the matrix. The current shape of the UPRmt signalling suggests that the unfolded or misfolded proteins are being detected in the matrix by ClpP a quality control, which degrades the proteins into peptides. The peptides are then transported into the inner membrane, this leads the activation of ATFS-1 through an unknown mechanism (Pellegrino, Nargund and Haynes, 2013).
II. Describe the experiments which led to the discoveries of the UPR in the endoplasmic reticulum and the UPR in the mitochondria.
Mitochondrial UPR was first discovered in mammalian cultured cells, it was expressing a misfolded mitochondrial protein that resulted in expression mitochondrial protein quality control genes in the nucleus to increase (6).
Recent experiments about the UPR in the mitochondria have indicated that several damaged or energetically dead mitochondria have been cleared from the cell via autophagic degradation, which is through a pathway known as mitophagy (Pellegrino, Nargund and Haynes, 2013).
III. Discuss how the UPR has been implicated in human disease.
Previous evidence proposes that cancer cells are more exposed to higher levels of mitochondrial stress than cells that are normal, this suggests that a dependence on cellular pathways and components that protect the mitochondrial protein folding environment
I. Describe the fluid-mosaic model of the membrane bilayer as initially theorized by Singer and Nicholson (1972). Discuss how our understanding of the membrane bilayer has changed over time. In your answer describe the experiments and new techniques in cell biology which have led to our current understanding.
Lateral diffusion experiment – In the 1970s, a mouse and a human cell were fused together, distribution of proteins was then observed several hours later and compared to time zero. Lowering the temperature slows the process considerably. Does not require ATP or protein synthesis, simply relies on diffusion. Led to the development of the ‘fluid mosaic model’ of membrane structure.
Fluid mosaic model – devised by SJ Singer and GL Nicolson in 1972. • Phospholipids for a continuous lipid bilayer • the proteins float freely in the ‘sea of lipids’ • proteins may interact with the surface (peripheral) or be embedded in the bilayer (integral) • proteins are asymmetrically organised
II. Provide an overview of how aberrant signalling in the MAPK pathway can lead to cancer.
Cancer can be seemed as a disease that communicates between and within cells. Mitogen-activated protein kinases (MAPK) are mediate intracellular signalling which are associated with different types of cellular activities, including cell purification, survival, differentiation and death (McCubrey, Lahair & Franklin, 2006). The extracellular signal-regulated kinase (ERK) pathway is the best studied of the family of mammalian MAPK. It is deregulated in approximately one-third of all human cancers. Here, it will be discussed recent findings on the role of MAPK pathways in cancer.
Most of the cancer-associated mutations of the components of MAPK signalling pathways have been in Ras and B-Raf, which are both participating in the ERK signalling pathway (Dhillon, Hagan, et.al, 2007). The ERK pathway plays a major role in different steps of tumorigenesis including cancer cell purification, invasion and migration.
Most cancer-associated lesions that lead to constitutive activation of ERK signalling occur at early steps of the pathway:
• overexpression of receptor tyrosine kinases
• activating mutations in receptor tyrosine kinases
• sustained autocrine or paracrine production of activating ligands
• Ras mutations and B-Raf mutation
Describe our current understanding of apoptosis. In your answer:
I. Outline the key features of both the intrinsic and extrinsic pathways of apoptosis.
The initiation of apoptosis is tightly regulated by activation mechanisms, once apoptosis begins, the cell leads to death (Albert, Johnson, et.al 2008). The two best understood activation mechanisms are the two pathways intrinsic or extrinsic. The extrinsic apoptosis begins through the stimulation of the transmembrane death receptor, located on the cell membrane, for example, the FAS receptor. In comparison, the intrinsic pathway begins through the release of a signal factor by the mitochondria, which is in the cell.
Extrinsic apoptosis is Mediated by TNF and death receptors (Sprick & Walczak, 2004). Signal molecules such as ligands, which are released by other cells, bind to the transmembrane death receptors, which triggers apoptosis Csipo, Montel, et al, 1998). Binding death receptors such as TRAIL to FAS on a target cell will induce multiple receptors to aggregate together on the surface of the target cell. The aggregation of TRAIL and FAS receptors recruits an adaptor protein known as Fas-associated death domain protein (FADD) on the cytoplasmic side of the receptor (Adrain, Creagh & Martin, 2002). Pro-caspases-8 and Procaspase-10 are inactive proteins until they interact with an activated FADD. The procaspases then become caspase-8 and caspase-10 triggers changes to several other molecules throughout the cell, including messengers that start the breakdown of DNA after being activated by the caspases.
Intrinsic apoptosis is induced by cellular stress, mitochondrial stress specifically. It is caused by DNA damage, heat shock and other stresses that impair a cell’s ability to function (Adrain, Creagh & Martin, 2002). In response to the damage or stress, the cell decides that the existence might be in danger. It activates a set of proteins called BH3-only proteins. Once stress signals are received, the pro-apoptotic proteins in the cytoplasm (BAX and BID), bind to the outer membrane of the mitochondria so that it can release internal content. However, it requires another pro-apoptotic protein (BAK) that resides within the mitochondria, since BAX and BID are not enough to trigger full release (Hague & Paraskeva, 2004). Activated BAX and BAK cause a condition known as MOMP (Mitochondrial outer membrane permeability). MOMP is a point of no return for apoptosis. The steps that lead to MOMP can be stopped by inhibitor molecules, however, once MOMP has reached, the cell will complete the death process. MOMP allows the release of cytochrome C into the cytoplasm. During MOMP, cytochrome C can escape the mitochondria and act as a signalling molecule in the cell cytoplasm. In the cell, cytochrome C promotes the formation of the apoptosome, which performs the final step to beginning cellular breakdown. Once apoptosome is formed, it turns procaspase-9 into caspase-9, this triggers further changes throughout the cell. Among the most important, activation of caspases-3 and -7. At this stage, the breakdown of the cellular materials begins. Caspase-3 shrinks and breaks down the cell’s DNA.
II. Explain how these pathways converge and describe the effectors of apoptotic cell death.
III. Explain how apoptosis contributes to innate cell defence and how viruses have evolved mechanisms to circumvent apoptosis of infected cells.
In attempting viral infection, the host needs to develop a formidable and integrated defence system. It also needs to comprise the innate and adaptive immune response. It became clear in recent years that the attempt to prevent viral infection, replication, dissemination or persistent viral infection of the cell, these protective measures involve the start of the programmed cell death (Apoptosis) (Barber, 2001). Once a virus has invaded a cell, a second host defence -mediated response is also triggered known as (IFNs) a family of cytokines. The IFN is responsible for coordinating and initiating a successful antiviral response, its function is to stimulate the adaptive arm of immunity, which involves (CTLs) cytotoxic T cell, also inducing many intercellular genes that directly prevent cytolysis (Barber, 2001).
Biron (1999) defines the innate arm of immunity as a critical role in the early control of the viral infection and the direction of the adaptive immune response. The viral infection starts off with local invasion, for example, it could start off with an epithetical surface, which results in the target organ such as skin, the pulmonary tract, the nervous system and immune system. After the entry, the viruses will go through a variety of circuits, including cellular host defence which is a mechanism that protects the organism and eradicates the infectious agent. Neutralizing antibody is the first line of defence that is confronted with invading the virus before it infects the cells. The neutralizing antibody binds to a capsid protein of the pathogen which results in preventing viral attachment so that it enters the host (Biron, 1999). These antibodies can be made of natural antibodies, representing a spontaneous repertoire of circulating immunoglobulins, which binds to a virus than influences the infection of a vital target organs as well as direct the virus to secondary lymphatic organs to accelerate and enhance immune responses (Ochsenbein, Fehr, et.al, 1999).