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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 GTP–binding 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 London's Imperial College, led by Miguel Seabra, are investigating the role of Rab27a in the trafficking disorders choroideremia, Hermansky–Pudlak 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 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 (figure below). 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.
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. 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/Schering-Plough). 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 ezetimibe-sensitive 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 Niemann-Pick 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 drug's 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.
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