BACKGROUND

MEMBRANES AND THE DISEASES WITHIN

Sophie Petit-Zeman
The concept of a true membrane disorder is differs from other disease classes as many diseases involve the membrane to some degree.
Enclosing and compartmentalizing both entire cells and the organelles within them, it is unsurprising that the disruption of membrane function has diverse pathological consequences. From their role in regulating transport in and out of cells or subcellular domains, conferring selective receptivity via protein receptors, anchoring cytoskeletal filaments or components of the extracellular matrix, providing sites for enzyme binding and catalysis, and allowing directed cell or organelle motility, when membranes go wrong, much else can too. In fact, it could be said that many, if not most, diseases involve the membrane in some shape or form. This diversity of nature and function and multiple effects of disruption means that there is no unifying theme by which to classify diseases caused by changes in cell membranes, according to Werner Kramer, Head of Metabolic Diseases at Sanofi-Aventis. And indeed, teasing out membrane-based pathologies, identifying the mechanisms that underlie them and understanding how drugs to treat them work or should best be designed is further complicated by the convergent endpoints for what might appear as distinct disruptions. For example, membrane trafficking can be disrupted by changes to proteins, components of the ubiquitous lipid bilayer, or the cytoskeleton. There are also numerous examples of disease or disorder resulting from alterations in the host of proteins that populate the membrane and function as receptors, transporters, enzymes or structural components. Drugs that target these defects tend therefore to go for more logical targets than the membrane; therapies that target membrane-protein-based diseases, for instance, block, augment or mimic the actions of the protein. There are probably two categories of what can be termed true membrane-based diseases, according to Kramer: the first caused when defects in cytoskeletal components impair membrane function, the second occurring when altered membrane lipid composition disrupts membrane trafficking. Defects in cytoskeleton and membrane function. The cytoskeleton is a microscopic network of actin filaments and microtubules in the cytoplasm that gives the cell shape and coherence. It contains a set of structural proteins linked to the membrane that provide protection from the stresses of many processes, for example, muscle contraction, and also assemble signalling complexes close to cell adhesion molecules. Disruptions in the cytoskeleton can lead to a range of diseases, such as sickle cell anaemia and Duchenne muscular dystrophy (DMD). Sickle cell anaemia is a genetic disease that results in the production of a defective form of haemoglobin, which distorts red blood cells into the classic sickle shape. Red blood cells maintain their shape with a specialized cytoskeleton composed of a network of the proteins actin and spectrin. In sickle cell anaemia, this actin/spectrin lattice locks, making red blood cells much less deformable, and causing them to obstruct the microcirculation. The main drug therapy for sickle cell anaemia, hydroxyurea, is thought to reduce the formation of sickle haemoglobin and hence ameliorate the structural consequences, while also decreasing neutrophil numbers which promote adhesion of sickled cells to blood vessel walls. However, a recent study suggested that hydroxyurea acts directly on the plasma membrane. The drug is known to decrease expression of adhesion molecules on red blood cells, one of which, phosphatidylserine is, unusually, expressed on the outer surface
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BACKGROUND

ER

Golgi Defective glycosylation

Lysosome (GM2-activator deficiency) Substrates 2 Activator protein 1 (NiemannPick A/B)

(Galactosialidosis) (I Cell disease) 6

Multienzyme complex

Lysosomal hydrolase

(Gaucher) 3 7

Products

9 (Danon disease)

8 (Cystinosis)

Figure 1 | The biochemical and cellular basis of lysosomal storage disorders. Most mutations in classic lysosomal storage disorders (LSDs) result in the delivery of a defective enzyme that has a reduced catalytic activity to lysosomes (label 1). In some cases, another protein that is required for optimal hydrolase activity is defective or absent (label 2). An LSD can be caused by the defective transport of a lysosomal hydrolase out of the endoplasmic reticulum (ER) due to a mutation that causes misfolding (label 3). Alternatively, an LSD can be caused by the defective transport of a lysosomal hydrolase out of the ER because a multi-enzyme complex that is required for transport cannot form (label 4). In the Golgi, defective glycosylation could result in an enzyme with reduced catalytic activity (label 5). Alternatively, defective glycosylation in the Golgi could produce an enzyme that cannot reach lysosomes because it cannot bind to mannose-6-phosphate receptors (due to defective glycosylation with mannose-6-phosphate; label 6). Defects in other transport steps from the Golgi could also lead to an LSD (label 7). Several LSDs are caused by defects in integral lysosomal membrane proteins. These include defects in transporters (label 8), or in proteins that are involved in other vital regulatory events of lysosomal function (label 9). In this figure lysosomal hydrolases are shown in various shades of blue, and a relevant LSD example is shown for each defect when one is known. Reproduced from Futerman, A. H. & van Meer, G. The cell biology of lysosomal storage disorders. Nature Rev. Mol. Cell Biol. 5, 554565 (2004). Macmillan Magazines Ltd.

of some red blood cells in sickle cell anaemia. In the study, sickle cell anaemia patients receiving 12 months of hydroxyurea treatment had significantly reduced phosphatidylserine levels in their blood samples. In DMD, a mutation in the dystrophin gene disrupts the ability of the protein product to anchor cytoskeletal elements to the surface membrane. Structural support is lost, the cell membrane becomes permeable, intracellular pressure rises and the cell explodes with the subsequent immune response also adding to the damage. While effective gene therapy for DMD is arguably in sight, it is not expected to benefit those already diagnosed. Mouse models have greatly increased understanding of the mechanisms underlying DMD, the roles of normal and abnormal dystrophin and the possibility that finding ways to increase production of the dystrophin-related protein utrophin could be a step towards effective therapies. Disruption of membrane trafficking. Defects in membranes have particularly profound effects on membrane trafficking, the elaborate system of transport pathways that convey cargo in and out of the cell, from the movement of macromolecules by vesicular transport from donor to acceptor organelles during secretion and endocytosis, to mechanisms of organelle inheritance at cell division. The trafficking machinery requires an extensive part of cellular work: conservative estimates from eukaryotic gene sequencing suggest that 10% of cellular proteins play a role in membrane traffic and protein targeting. However, although much is known about the components of the machinery, much less is known about its regulation.

Prominent candidates for a role in regulating membrane traffic, and whose disruption is implicated in several inherited human disorders, are Rab proteins. More than 50 of these low-molecular-weight GTPbinding proteins have been identified in mammalian cells, all associated with cellular membranes via lipid modification to their carboxyl terminus a process called geranylgeranylation. Researchers at Londons Imperial College, led by Miguel Seabra, are investigating the role of Rab27a in the trafficking disorders choroideremia, HermanskyPudlak and Griscelli syndromes. Choroideremia, an X-linked late-onset retinal degeneration condition characterized by progressive dystrophy of photoreceptors, is caused by a defect in Rab Escort Protein 1, which is required for geranylgeranylation of Rab proteins. In an approach reminiscent to that of unravelling the mysteries of DMD, Seabra is turning to mouse models to understand choroideremia Our main focus is to build a mouse model for choroideremia using conditional gene knockout technology, says Seabra.If successful, we will use the mouse model to investigate the pathogenesis of the disease by trying to understand which cell layer in the retina is primarily involved in triggering the disease and to test gene therapy approaches. Hermansky-Pudlak and Griscelli syndromes are disorders of so-called lysosome-related organelles, such as melanosomes. These diseases are characterized by partial albinism, accompanied with haemorrhagic tendency in Hermansky-Pudlak syndrome, and poorly functioning cytotoxic T-lymphocytes in Griscelli syndrome. Griscelli syndrome is caused by mutations in Rab27a, and there is interest in the possibility that disruption of Rab27a function might in fact underlie all three conditions. If it does, why defects in such a widespread protein apparently

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BACKGROUND
causes conditions that are restricted to a few cell types, such as melanocytes and T-lymphocytes, remains unclear. One suggestion, which has implications for gene therapy, is that a related protein Rab27b compensates for the loss of Rab27a and thereby protects other cell types. Similar to these conditions, Niemann-Pick disease type C (NPC) is a lysosomal storage disorder (LSDs), one of more than 40 rare conditions in which the absence of an enzyme prevents lysosomes in cells from performing their natural recycling function (FIG. 1). This leads to various materials being inappropriately stored in the cell, which results in a host of disorders in which there is progressive deterioration in physical and/or mental states. LSDs are seen as diseases of the membrane in that they can be caused by defects in membrane lipids and proteins, for example, NPC is characterized by lysosomal accumulation of LDL-derived cholesterol. NPC has been linked to malfunction of the NPC1 protein, part of which, the NPC1L1 protein, is enriched in the small intestine where cholesterol is absorbed, and plays a role in its intracellular trafficking. NPC1L1 was recently suggested to be involved in the action of the cholesterol absorption inhibitor ezetimibe (Zetia; Merck/ScheringPlough). This drug is unusual in that it was approved and reached the market without its precise target being known, but recent studies have shown that mice genetically engineered to lack NCP1L1 have reduced efficiency of cholesterol absorption, and the ability of ezetimibe to boost this in such animals is lost. However, there is a twist in the tale, as Kramer explains: NPC1L1 is clearly involved in the ezetimibesensitive pathway of cholesterol absorption, but it does not bind ezetimibe directly making its role as the primary target for ezetimibe unlikely. In a forthcoming paper we demonstrate that binding of ezetimibe from the luminal side of the enterocyte [a type of cell found in the intestine] to a 145 kDa protein is sufficient to block cholesterol absorption, probably by prevention of endocytosis of membrane microdomains. While the cell biology of diseases like NiemannPick and the complexities of cholesterol trafficking and drugs to target them are generating much excitement, developing drugs for membrane-based disorders is hampered by what Kramer describes as the understanding of the mechanisms of membrane trafficking and lipid self-organization being in their infancies. As the ezetimibe story illustrates, identifying drugs which truly interact directly with the plasma membrane, as opposed to the proteins which they house, is complex. The mechanisms of action of an antidiabetic drug similarly illustrate how treatments might have unrecognized actions on the membrane. The oral blood-glucose-lowering drug, glimepiride, as well as stimulating insulin secretion in pancreatic beta-cells, is thought to have insulin-independent extrapancreatic activity. There is debate over the extent to which this reflects the drugs ability to sensitize peripheral tissues to insulin, but there is evidence that it might do so by interacting with membrane microdomains in adipocyte and muscle-cell membranes. Despite these complexities, membranes are generating great interest as more becomes known about their roles in cellular processes. Proving to be much more than cellular wrappers, how membranes function, malfunction and the implications for drug treatment of membrane-based disease are clearly ripe for exploration.
Further reading
Covas, D. T. Effects of hydroxyurea on the membrane of erythrocytes and platelets in sickle cell anemia. Haematologica 89, 273280 (2004). Hong, H.-K. Chakravarti, A. & Takahashi, J. S. The gene for soluble N-ethylmaleimide sensitive factor attachment protein is mutated in hydrocephaly with hop gait (hyh) mice. Proc. Natl Acad. Sci. USA 101, 17481753 (2004). McNeil, P. L. & Steinhardt, R. A. Plasma membrane disruption: repair, prevention, adaptation. Ann. Rev. Cell Dev. Biol. 19, 697731 (2003). Mller, G. Dynamics of plasma membrane microdomains and cross-talk to the insulin signalling cascade. FEBS Lett. 531, 8187 (2002).

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2004 Nature Publishing Group