Biological membranes are selective barriers that separate cells from the environment, and also partition intracellular organelles. They are important in the selective transport of biological molecules such as ions, glucose, carbon dioxide in and out of the cell and its organelles (Brown, 1996). Biomembranes, are in common, made up of lipid, protein and sugar molecules, with lipids forming the highest proportion (Saiz et al., 2002). Phospholipid, a component of the lipid molecule, gives membranes their amphipathic property – which forms the primary structure of all biomembranes. Other components of the lipid moiety of membranes include cholesterols and glycolipids that are involved in the regulation of membrane permeability and cell-cell communication, respectively (Crockett, 1998; Malhotra, 2012). Membrane proteins are also a structural component of the biomembranes. They play a number of crucial roles within biomembranes, ranging from binding to extracellular molecules such as ions, solutes and signalling proteins (receptor function), to membrane attachment of cytoskeletal proteins and their involvement also in the intracellular signalling pathway mechanism (Lodish et al., 2013). For example, the transmembrane protein, cystic fibrosis transmembrane-conductance regulator (CFTR), that normally localises in the apical cell membrane of epithelial cells is an ion channel involved in the transport of chloride ions across the epithelial membrane (Riordan, 2008). In addition to selectively transport molecules into and out of the cell, biological membranes are important cellular component that function to compartmentalise organelles, protect the cell from environmental damages, synthesise proteins and energy, and to form a cell-cell communication link (Fagone and Jackowski, 2009). This shows the physiological relevance of membranes, and why it’s important that we study their interactions with other micro- and macro-molecules.
A defect in the functioning of one or more of these membrane components might lead to a membrane or cellular disease. Knowledge of the different cellular mechanisms by which these diseased conditions results is necessary for the proper understanding of how drugs could be used to treat or alleviate the symptoms of the disease (Howell et al., 2006; Ashrafuzzaman and Tuszynski, 2013). Presently, there is no general consensus of a membrane-based disease classification due to the fact that many pathways are implicated for a particular disease (Ashrafuzzaman and Tuszynski, 2013). For example, a defect in membrane transport could be attributed to malfunctioning in several factors, including membrane proteins, lipid bilayers and others factors. Despite this classification problem, Petit-Zeman and Ashrafuzzaman and his colleague argued that there are two classification of membrane-based diseases: defects in cytoskeletal components and alteration of membrane lipid composition, each of which affect membrane function and trafficking, respectively (Petit-Zeman, 2004; Ashrafuzzaman and Tuszynski, 2013). Hyaline membrane disease, Alzheimer’s disease, cystic fibrosis (CF), Duchenne muscular dystrophy (DMD), Hermansky-pudlack syndrome and Neimann-pick disease type C have all been identified as being caused by abnormalities in membrane functioning (Ashrafuzzaman and Tuszynski, 2013).
This essay aims to describe the molecular pathology of two of these diseases: CF and DMD to emphasise the roles membranes play in the cell. It will also suggest some recent treatment options available.
Duchenne Muscular Dystrophy
Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive neuromuscular disease that frequently affects 1 in 3500 liveborn males (Hayes et al., 2008). Anderson et al. (2002) earlier reported that the disease is genetically inherited, and is the second most occurring inherited disease. Clinical manifestations in affected people include a progressive lost of smooth, cardiac and skeletal muscles from around 3 years of age (Hayes et al., 2008). Afflicted patients die at adolescent as a result of prolonged muscle weakness, coupled with respiratory insufficiency.
A major cause of the disease has been linked to the absence (or insufficiency) of a cytoskeletal protein called dystrophin, encoded by the dystrophin gene (Kristen and Kay, 2004). The dystrophin gene is said to be the largest gene of all the human genes, comprising an estimated 2.6 million base pairs of DNA that encodes 79 exons (Kristen and Kay, 2004; Walmsley et al., 2010). Defective (or loss) of dystrophin protein is as a result of insertion or deletion mutation in about 60% of most cases, whereas about 40% of point mutation in the same gene has been attributed to the loss-of-function mutation of the dystrophin gene (Kristen and Kay, 2004). Walmsley et al. (2010) added that the axons at position 45-53 are ”hotspot” for deletion-type frameshift mutation. Research shows that dystrophin gene is also expressed in other tissues apart from cardiac and skeletal tissues. Muntoni et al. (2003) in a review article stated that isoforms of the dystrophin gene is expressed in the brain and the retina, and that mutation in these isoforms, the brain isoform, for example, causes mental retardation and low intelligence quotient in the affected individuals. The brain isoforms are called Dp140 and Dp71 (D’Angelo et al., 2011). Consequently, it’s been theorised that there is a link between DMD and impairments of the central nervous system (CNS) caused by mutation of the dystrophin gene. Insights into these DMD/CNS impairments have come from studies in an mdx mouse model and in Golden Retriever Muscular Dystrophy (gmrd) dogs, both deficient in dystrophin protein (Campbell, 1995; Kristen and Kay, 2004). Becker Muscular Dystrophy is another form of muscular dystrophy, though with a milder phenotype as a result of insignificant level of synthesised dystrophin at the sarcolemma or a small length of the protein, or both (Campbell, 1995). Other types of muscular dystrophies that have been identified and studied are congenital muscular dystrophy (CMD), fukuyama-type congenital MD, limb-girdle MD and finally, severe childhood autosomal recessive muscular dystrophy (SCARMD) that affects both males and females at the same rate (Campbell, 1995).
This 427 kDa dystrophin protein has 4 functional domains that are structurally different: a cysteine-rich domain, an N-terminal domain, a C-terminal coiled coil rod (containing repeats that are spectrin-like) (Lapidos et al., 2004). Dystrophin is associated with other membrane-localised glycoproteins via at least two of its four domains. Its N- and C-terminal domains each bind F-actin and the dystrophin-associated protein complex (DAPC, which comprise proteins such as sarcoglycans, dystroglycans, integrins, caveolin, biglycans, and sarcospan), respectively at the sarcolemma (Kristen and Kay, 2004). As depicted in figure 1 below, the extracellular protein (α-dystroglycan), via its interaction with the G domain of merosin (laminin-2 in figure 1), connects the sarcolemma to the extracellular matrix, a process that depends on the presence of Ca+2(Campbell, 1995). Again, the DAP-complex interacts with the α-dystroglycan and hence, links it to the sarcolemma and finally, the N- and C-terminal domains of dystrophin associate the subsarcolemma cytoskeleton to the membrane by interacting with F-actin and the DAP-complex (Fig. 1) (Campbell, 1995). The carboxyl terminal of dystrophin also directly binds dystrobrevin, and syntrophin triplets, however, only two subunits are shown in the illustration below.
The DAP-complex is vital as it stabilises the sarcolemma during periods of muscle contractions and relaxations (Ehmsen et al., 2002). Ehmsen and colleagues further stated that that DAPC does this by forming a link between the N-terminal bound actin and the extracellular matrix. It’s also reported that the DAPC plays an important part in cell signalling by interacting with Ca+2-bound calmodulin, neuronal nitric oxide synthase (nNOS) and Grb2 (Rando, 2001). These membrane interactions by these transmembrane and membrane-associated proteins are illustrated and summarised in figure 1 below.
Pathophysiology Of DMD: The mechanism underlying the pathogenesis of the disease is the absence (or insufficient synthesis) of the dystrophin protein as a result of frameshift mutations (Muntoni et al., 2003) that lead to the absence of the C-terminal and cysteine-rich domain of the dystrophin protein (Campbell, 1995). The absence of the dystrophin protein causes a disruption of the DAP-complex (and therefore, affects costameric formation), and consequently, there is a damage to fibre and a rapid increase in membrane permeability to ions, and other substances (Kristen and Kay, 2004; Deconinck and Dan, 2007). Initially, there is an improved regeneration of muscle cells, however, progressive damage by necrosis leads to degeneration of the muscles (Perkins and Davies, 2012). Deconinck and Dan (2007) in a review article further added that other secondary events that occur in response to a disruption of the DAP-complex include muscle degeneration, impaired vascular adaptation, apoptosis, high intracellular Ca+2 levels and fibrosis. Diagnosis of the condition is often done by measuring the level of serum creatine kinase – a marker of necrosis in the muscle – which always increase rapidly in DMD patients (Perkins and Davies, 2012). A number of techniques have been employed in the study of the mutations that lead to DMD; these include techniques such as multiplex PCR, quantitative PCR, multiplex amplifiable probe hybridization and studies using model animals such as mdx mice and gmrd dogs (Muntoni et al., 2003).
Treatments For DMD: A incisive knowledge of the pathogenesis of DMD would avail scientists and drug researchers the opportunity to develop drugs or techniques to manage the clinical symptoms of this devastating disease (Deconinck and Dan, 2007). Although there is currently no effective cure for DMD (Deconinck and Dan, 2007), DMD gene therapy holds a promising therapeutic treatment for the afflicted patients (Duan, 2011). In fact, the introduction a an autosomal homologue of dystrophin gene called utrophin witnessed the localisation of the DAP-complex to the sarcolemma and subsequently restores effective muscle contractions and relaxations in mdx mice (Kristen and Kay, 2004). Duan (2011) added that challenges facing the gene therapy approach to treating DMD are the design of a vector to contain the large size of the dystrophin gene and any subsequent immune response that might results. Other therapeutic approach include the use of protease inhibitors to regulate the DAPC pathway, increasing alternative gene expression, use of aminoglycans and chimaeraplasts and antisense oligonucleotides (Kristen and Kay, 2004). Analysing the points above, it can be said that studies in model animals and a knowledge of the causative mechanisms, coupled with the identification of the gene responsible for the dystrophin protein have helped scientists in the search for possible treatment options.
Another membrane-related disease that, compared to DMD, affects males (but also females) is cystic fibrosis (CF). It is described as an autosomal recessive inherited disease that frequently populate among the Caucasian descent, of which 1 in 25 people are carriers and 1 in 2500 are affected also (Davidson and Porteous, 1998). it’s estimated that CF affects about 70,000 individuals worldwide, albeit no cure for the condition despite intensive research in the field (Ramsey et al., 2011). However, insights into research and therapeutic approach to treating CF patients have improved the CF median survival rate from 11 years to 37 years (Shane and Graeme, 2008; Davis, 2011). The disease is caused by defect in a single gene located on chromosome 7 called ”cystic fibrosis transmembrane conductance regulator”, CFTR gene for short, which encodes a 1480 amino acids sequence called CFTR protein (William and Bruce, 2006). Oxford University GeneMedicine (2012) states that this 250,000 base pair CFTR gene was identified in 1989 using the technique of Restriction Fragment Length Polymorphism, RFLP analysis. six different classes of mutations that impair the normal function of this chloride-selective channel protein have been identified (Wilschanski, 2013) and are presented in the table below.
The CF Mutation Database, which is a repository that collects and stores mutations associated with the CF gene currently holds 1938 mutations associated with the CFTR gene (CF Mutation Database, 2011; Wilschanski, 2013). These mutations have given rise to the classification identified in the above table. However, the most common CF mutation, in about 70-90% of CF cases, is the ΔF508 mutation (William and Bruce, 2006). This mutation results from a phenylalanine deletion in exon 10 (of the 27 exons) in the CFTR gene which codes for the foremost nucleotide binding domain (NBD) of the CFTR protein (Wilschanski, 2013).
The CFTR protein is a member of the ABC transporter superfamily that is primarily expressed in epithelial cells of the kidney, pancreas, heart, intestine, vas deferens, sweat glands and lungs (William and Bruce, 2006). It can therefore, be concluded that it’s a multi-organ protein, and any defects in it could become generalised. It’s composed of 12 membrane-spanning alpha-helices that is mainly involved in the selective transport of chloride ions (Tector and Hartl, 1999); 2 NBDs that bind and hydrolyse ATP and are also involved in the regulation of channel opening and closing; and finally, a regulatory (R) domain that binds protein kinase A and protein kinase C to activate the channel (William and Bruce, 2006). It is this opening and closing that mediates the transepithelial movement of salt and water across the apical cell membrane in organs where CFTR protein plays physiological roles. Moreover, the CFTR protein is important in the regulation of other ions such as Na+ (via ENAC channels) and bicarbonate ions (Cohen and Prince, 2012). The CFTR protein associated with the cell membrane is shown in figure II below, together with other proteins that interact to bring about the physiological role of CFTR in the cell.
Pathophysiology Of CF: The class II mutation in delta-F508 of the CFTR gene product is identified by the ER Quality Control (QC) machinery as a misfolded protein and is targeted for proteasomal degradation, and so the protein never reaches the epithelial cell membrane where it functions normally as a selective chloride channel (Riordan et al., 2001; William and Bruce, 2006; Cohen and Prince, 2012). Lodish et al (2013, Pg. 642) added that the three bp phenylalanine deletion stop the usual transport of the CFTR protein to the apical cell membrane by preventing its loading into COPII coated vesicles that usually bud off from the ER to the Golgi apparatus. However, small quantity of the proteins that escape the ER QC machinery are able to reach the cell surface, but are less active and are quickly degraded from the cell surface (Davis, 2011). This is in contrast with wild-type CFTR protein that are sufficiently retained in the apical cell membrane before being targeted for degradation (Davis, 2011). Some CFTR gene mutations encode full-length CFTR proteins that are processed normally in the ER, but are defective in ion-channel activity (Rowe et al., 2005). For example, G551D mutation in table 1 above.
The absence (or insufficient quantity) of CFTR channels at the apical cell membrane brings about a complex, multi-organ symptoms that are characteristic of CF phenotype, although the severity vary among the affected organs (Wilschanski, 2013). Due to the disruption of chloride transport and dysregulation of other ion channels in epithelial tissues of the lungs, mucus hypersecretion and dehydration of the lungs develop in affected individuals (Davidson and Porteous, 1998). As a result, mucocilliary clearance of the mucus in the lungs is impaired, and therefore, pathogens such as P. aeruginosa, S. aereus, Burkolderia apacia and H. influenza find this microenvironment suitable to live and infect the host’s lungs (Rowe et al., 2005). Colonisation by these pathogens and continuous influx of neutrophils lead to a more severe or chronic inflammatory response (Davidson and Porteous, 1998). Subsequently, the affected patient develops respiratory problems (including airway and submucosal gland obstructions), a major cause of death in affected people, and which also determine the quality of life lived by CF patients (Cohen and Prince, 2012). As a multi-organ disease, some patients also suffer chronic fibrosis of the pancreatic duct and infertility in affected male individuals (Rowe et al., 2005). Again, there is high salt concentration in the sweat of CF patients because of the dysfunction effects that CFTR mutations have on the regulation of ENAC channels (Roweet al., 2005). Rowe et al. (2005) also added that the transepithelial potential difference across the sweat gland is higher in CF patients than it’s in unaffected people.
Newborn screening, and genetic, lung function and sweat tests and sputum cultures are some of the diagnostic measures currently in use to detect CF in affected persons (NHLBI (National Heart Lung and Blood Institute), 2011)).
Treatments for CF: Insights into the molecular and genetic basis of CF have open ways for new therapeutic approaches to be developed. Like DMD, gene therapy approach for treating CF patients have received much public and research interest ever since the first CF clinical trials in 1993 (Burney and Davies, 2012). Topical administration of gene targeting agents (GTA) to epithelial cells of the lungs have been the aim of recent clinical trials (Griesenbach et al., 2002; Burney and Davies, 2012). However, Griesenbach, Burney and their colleagues reported that this approach has been faced with challenges owing to immune response and some extracellular physical barriers. Again, certain pharmacological agents have been exploited for the treatment of CF. For example, on January 31, 2012, it was announced that FDA approved the use of Kaledeco (Ivacaftor), that works by increasing the open state probability of CFTR channels in certain CFTR mutations such as G551D and delta-F508 (Song, 2012). Other drugs such as PTC124 (ataluren), and Vx-809 are both in their phase-III clinical trial (CF Foundation, 2013). PTC124 and VX-809 each work by producing a full-length CFTR protein (thereby correcting the premature-stop codon mutation of class I) and by targeting class II delta-F508 CFTR protein to the apical cell surface, respectively (Shane and Graeme, 2008). Other therapeutic approaches aimed at correcting defects in CF and improving the performance of the lungs and at eradicating bacteria and mucus clogging in the lungs and control of inflammatory responses include the use of pulmozymes, nebulised salt solution, and an anti-ineffective drug called tobramycin (Shane and Graeme, 2008).
The important physiological roles played by membranes in normal cellular functioning and diseases have been given much in-depth touch in this essay. Some diseases associated with these membrane dysfunctions have also been elucidated, with particular emphasis on CF and DMD. In all, model organisms have been of immense help in the study of the disease mechanisms and how modern treatment options could be employed to alleviate the symptoms or to completely eradicate the disease. It’s therefore, important to understand disease mechanisms, factors involved, and the many tissues and organs that it affects in order to search for the best possible treatment options.
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