Browsing Posts in Disorders

    The reasons that such different clinical outcomes (Dent 2 and Lowe’s syndrome) can stem from mutations in OCRL1 remain to be defined, with two likely hypotheses being that compensatory genes (e.g., INPP5B, encoding inositol polyphosphate 5-phosphatase) or alternative initiation codons in OCRL downstream of nonsense mutations might be activated in a tissue-specific way in patients with Dent 2.51 However, the overlap of the renal phenotypes caused by OCRL and CLCN5 mutations allows the prediction that these two genes participate in a common molecular pathway that controls endosomal trafficking of the multiligand receptor megalin.

    Summary

    It is reasonable to hope that our basic knowledge of membrane trafficking will continue to provide insights into the pathogenesis of mendelian diseases and that studies of these diseases will continue to enhance our understanding of the membrane-trafficking system. In particular, it will be of great interest in this context to learn how to place the genes that are involved in trafficking-related diseases into coherent pathogenetic pathways.
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    Regrettably, the wealth of new insights into the molecular defects in membrane-trafficking disorders has not yet led to a proportionate availability of effective therapies. However, in the past few years, the potential of mendelian diseases to drive the process of drug development has been recognized. An example in the field of membrane transport is cystic fibrosis. Effective modulators of the folding, trafficking, and activity of CFTR (the chloride channel that is mutated in cystic fibrosis) have been found through high-throughput screening that was aimed at identifying pharmacologic treatments for this disease. Some of these modulators (e.g., VX-809) are now being tested in clinical trials. In addition, interest in the pathways affected in mendelian disorders is being raised further by the recognition that efforts to develop drugs for their treatment might also prove useful in common diseases in which the same pathways might have a pathogenetic role, such as type 2 diabetes and Alzheimer’s disease.

    More than 50% of patients with hereditary spastic paraplegia carry mutations in one of three genes: spastin (SPG4), receptor-expression-enhancing protein 1 (SPG31 or REEP1), or atlastin-1 (SPG3A). Spastin encodes an ATPase with a microtubule-severing activity that has different splice variants with different subcellular localizations, including the endosomes and the endoplasmic reticulum. Notably, spastin interacts with the other hereditary spastic paraplegia protein, REEP1. REEP proteins, and the structurally related reticulon proteins, have a major morphogenetic role at the endoplasmic reticulum45 because of a conserved domain of approximately 200 amino acids with two hydrophobic segments that form a hairpin in the membrane and have membrane-bending properties. Through this domain and its ability to oligomerize, the REEP and reticulon proteins can shape membranes of the endoplasmic reticulum into tubules.45 Intriguingly, spastin also interacts with the third major hereditary spastic paraplegia protein, atlastin. These collective observations led to the hypothesis that atlastin itself might have a role in the morphogenesis of the endoplasmic reticulum. This disease-inspired hypothesis turned out to be correct and revealed that atlastin is involved in the generation of the tubular endoplasmic-reticulum network, since it mediates homotypic fusion of tubules in the endoplasmic reticulum. Finally, in a further tightening of the relationships among atlastin-1, spastin, and REEP1, these three proteins have recently been reported to interact with one another.
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    This emerging scenario supports a convergent mechanism of disease in the many forms of hereditary spastic paraplegia that involve a defect in the formation of the endoplasmic reticulum tubular network. This might be particularly detrimental for long spinal neurons, since the endoplasmic reticulum is a conduit for many important small molecules with signaling or structural roles (e.g., calcium and lipids). Thus, the pervasiveness and continuity of the endoplasmic-reticulum network might well be essential in these extremely elongated cells, whereas such a network may be at least partially dispensable in smaller cells.

    As in such examples, other cases can be identified in which information that is gathered from genetic diseases might reasonably lead to the discovery of converging molecular pathways in the near future. One such case is inherited renal Fanconi’s syndrome, a common clinical manifestation of a heterogeneous group of genetic disorders that are characterized by dysfunction of renal proximal tubular cells. These cells reabsorb more than 90% of nutrients, vitamins, and low-molecular-weight proteins present in the ultrafiltrate. This reabsorption of nutrients and proteins relies on efficient endocytic recycling of the multiligand receptor megalin, which captures its ligands in the ultrafiltrate, internalizes them through clathrin-dependent endocytosis, delivers them to the endolysosomes, and then recycles back to the apical surface of the cell for another round of transport. The endocytic system of these cells is subjected to a very heavy burden, and a drop in its efficiency can cause low-molecular-weight proteinuria, one of the hallmarks of renal Fanconi’s syndrome. Such a decline in efficiency might arise from defects in this endocytic receptor, megalin; in its associated receptor, cubilin; or in the machinery associated with their endocytosis and recycling. For instance, impaired trafficking of megalin has been suggested to occur in Dent’s disease, a proximal renal tubulopathy characterized by low-molecular-weight proteinuria, nephrocalcinosis, and hypercalciuria. This disease is caused by mutations in CLCN5, which encodes the renal chloride–proton antiporter, which in turn controls the acidification and recycling activity of endosomal compartments. Moreover, it has been shown that some forms of Dent’s disease (Dent 2) appear to also derive from mutations in OCRL1, which encodes an endosome-associated phosphatidylinositol 4,5-bisphosphate 5-phosphatase. OCRL1 was originally discovered as the causative gene in Lowe’s syndrome, a more serious disease that is characterized by proximal renal tubular dysfunction and by congenital cataracts and mental retardation.

    Other, more recent examples of this type of molecular lesson involve entire groups of mendelian disorders that share overlapping clinical phenotypes, even though they arise from mutations in different genes. These syndromes have highlighted the existence of complex molecular networks or pathways that include distinct but functionally converging genes. A paradigmatic example has come from a genetically heterogeneous group of inherited neurologic disorders that are characterized by progressive spasticity and weakness of the lower limbs. These disorders, which are caused by corticospinal motor neuron axonopathy, are the hereditary spastic paraplegias. They have autosomal dominant, recessive, and X-linked inheritance. To date, 20 genes have been identified, half of which are involved in membrane trafficking along the exocytic and endocytic pathways. The remainder are involved in mitochondrial functions, myelination, lipid metabolism, and DNA repair.

    More than 50% of patients with hereditary spastic paraplegia carry mutations in one of three genes: spastin (SPG4), receptor-expression-enhancing protein 1 (SPG31 or REEP1), or atlastin-1 (SPG3A). Spastin encodes an ATPase with a microtubule-severing activity that has different splice variants with different subcellular localizations, including the endosomes and the endoplasmic reticulum. Notably, spastin interacts with the other hereditary spastic paraplegia protein, REEP1. REEP proteins, and the structurally related reticulon proteins, have a major morphogenetic role at the endoplasmic reticulum45 because of a conserved domain of approximately 200 amino acids with two hydrophobic segments that form a hairpin in the membrane and have membrane-bending properties. Through this domain and its ability to oligomerize, the REEP and reticulon proteins can shape membranes of the endoplasmic reticulum into tubules. Intriguingly, spastin also interacts with the third major hereditary spastic paraplegia protein, atlastin. These collective observations led to the hypothesis that atlastin itself might have a role in the morphogenesis of the endoplasmic reticulum. This disease-inspired hypothesis turned out to be correct and revealed that atlastin is involved in the generation of the tubular endoplasmic-reticulum network, since it mediates homotypic fusion of tubules in the endoplasmic reticulum. Finally, in a further tightening of the relationships among atlastin-1, spastin, and REEP1, these three proteins have recently been reported to interact with one another.

    This emerging scenario supports a convergent mechanism of disease in the many forms of hereditary spastic paraplegia that involve a defect in the formation of the endoplasmic reticulum tubular network. This might be particularly detrimental for long spinal neurons, since the endoplasmic reticulum is a conduit for many important small molecules with signaling or structural roles (e.g., calcium and lipids). Thus, the pervasiveness and continuity of the endoplasmic-reticulum network might well be essential in these extremely elongated cells, whereas such a network may be at least partially dispensable in smaller cells.

    Another reason for the tissue specificity of symptoms relates to a requirement for very efficient trafficking in tissues that require high transport rates for their function. Here, a defect without consequence for other cells might result in functional collapse, as can be seen in a number of cases: for cells that transport very large amounts of cargo at some stage of their life cycle, such as Schwann cells during myelination, which can selectively express genetic defects of ubiquitous trafficking components, such as MTMR2, MTMR13, FIG4, and SH3TC2, resulting in the demyelinating forms of Charcot–Marie–Tooth disease (CMT4). Also included are cells that require very high rates of internalization and recycling of plasma-membrane components, such as proximal tubular cells in the kidney, which must reabsorb essential components from the ultrafiltrate and which suffer from genetic defects of components of the endosomal system (as in many inherited forms of renal Fanconi’s syndrome, including Lowe’s syndrome), and cells that require very efficient long-range transport and communication, such as motor neurons, which are particularly sensitive to defects in proteins involved in different steps of membrane trafficking (as is the case in hereditary spastic paraplegias).

    Lessons on the Role of Transport Proteins

    Our understanding of mendelian diseases can benefit from knowledge of the transport machinery. However, the reverse is also true: important lessons on the physiological functions of transport proteins can be derived from the study of disease genes. Classic examples are the combined deficiency of coagulation factors V and VIII and mucolipidosis II (also called inclusion-cell disease). Here, studies of the factors V and VIII combined deficiency helped to reveal the physiological role in transport of the protein ERGIC53 (also called lectin mannose-binding 1). After it was discovered that a mutation in this protein is the cause of factors V and VIII deficiency, a series of studies revealed that ERGIC53 functions as a chaperone in protein transport from the endoplasmic reticulum to the Golgi complex for a specific subgroup of secreted proteins that includes these two coagulation factors. As for mucolipidosis II, Hickman and Neufeld observed in 1972 that lysosomal enzymes from patients with inclusion-cell disease “failed to reach their lysosomal destination.” Subsequent studies indicated that this disorder is caused by a defect in the Golgi enzyme that phosphorylates a specific mannose on lysosomal hydrolases. These observations helped in gaining an understanding of the key role of the mannose-6-phosphate receptor in the transport of these hydrolases from the Golgi complex to the lysosomes.

    The distinction between cargo proteins and trafficking-machinery components can also be useful for the analysis of the mechanisms by which defective transport-related genes can lead to clinical manifestations. When a cargo protein is mutated, the pathogenetic chain of events that is set in motion can involve either a loss of function of the mutated cargo protein, because of truncation or early degradation (e.g., a channel protein, cystic fibrosis transmembrane conductance regulator) or a gain of function because of the accumulation of the mutated cargo protein in a given compartment, which would usually be the endoplasmic reticulum. This accumulation can activate the unfolded protein response. If the load of misfolded cargo exceeds the capacity of the compensatory mechanisms activated through the unfolded-protein response, the response becomes maladaptive and triggers cell damage and death. This happens, for instance, in various disorders of myelinating cells, in which mutations in genes encoding the abundant peripheral myelin protein zero are responsible for a dominant form of Charcot–Marie–Tooth disease, called CMT1B, caused by the accumulation of the protein in the endoplasmic reticulum, activation of the unfolded-protein response, and toxicity in Schwann cells.
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    For mutations in the machinery proteins, a central question is how defects in conserved ubiquitous housekeeping components can give rise to manifestations that are often specific to an organ or a tissue. In a few instances, the answer is that the defective genes are predominantly expressed as specific isoforms in the affected tissues (as is the case in muscle dystrophies linked to defects in caveolin 3, the muscle-specific isoform of caveolin). In many other cases, however, the reason for this selective tissue vulnerability appears to lie in the high demand for the defective genes in the tissues that then become damaged. There appear to be two general explanations for this tissue specificity. The first is the presence of special tissue-specific cargoes, which might require high levels and full function of a particular trafficking component to be correctly transported. This occurs, for instance, in cells such as osteocytes or chondrocytes and intestinal cells, which secrete oversized cargoes. These cargoes include procollagen type I or II (rigid protofibrils measuring 300 nm in length) for osteocytes or chondrocytes and chylomicrons (particles measuring up to 1 μm in diameter) for intestinal cells. Here, mutations in the ubiquitous COPII component Sec23a or in the transport protein particle (TRAPP) complex subunit TRAPPC2 (which is involved in trafficking between the endoplasmic reticulum and the Golgi complex) can selectively affect osteocytes and chondrocytes, resulting in cranio-lenticulo-sutural dysplasia38 and spondyloepiphyseal dysplasia tarda, respectively. Along the same lines, mutations in the Sar1B GTPase that controls the COPII cycle can affect the secretion of chylomicrons in enterocytes and cause Anderson’s disease (also called chylomicron retention disease). Presumably, the same molecular defects can be compensated for in other cells and tissues by redundant mechanisms that can handle regular, but not special, cargo types.

    Thus, a conspicuous feature of the mammalian transport apparatus is its great complexity. There are more transport strategies, types of vesicles, and trafficking pathways than was expected until only a few years ago. Also, each anterograde trafficking step is counterbalanced by one or more recycling steps, and most of the various endocytic stations appear to be interconnected bidirectionally.31 Moreover, certain specialized cells host uniquely differentiated organelles (e.g., secretory granules in endocrine and exocrine cells, melanosomes in melanocytes, lytic granules in immune cells, and dense granules in platelets), and at least in some cells (and potentially in all) there are unconventional secretion pathways through which a number of soluble cytosolic proteins can be transported directly to the extracellular space and some transmembrane proteins can be transported to the cell surface without passing through the Golgi complex32.

    A consequence of this multiplicity is a remarkable degree of redundancy and functional plasticity of the transport systems. This redundancy can partially compensate for certain genetic defects, and it can do so more efficiently in some cells than in others, depending on cell-specific requirements, which results in the selective vulnerability of certain tissues.

    Another important issue is how the overall trafficking system maintains its homeostasis in the face of the rapid membrane fluxes that constantly change the size and composition of the transport organelles, or compartments. Among several possible mechanisms, one that has been recently explored relies on signaling circuits located on the trafficking organelles themselves that sense the passage of traffic and rapidly react to restore the balance across the compartments.

    Mechanistic Basis

    During the past decade, the increasingly rapid discovery of genes that are linked to human diseases has revealed that several such genes are involved in membrane trafficking. Efforts are now being more specifically directed toward understanding how disease manifestations can be mechanistically explained through our basic knowledge of the trafficking machinery and toward exploiting this new knowledge of the molecular basis of genetic syndromes to obtain insights into the organization of the trafficking processes.

    Mendelian diseases of membrane trafficking arise from mutations in genes that encode either cargo proteins or components of the biosynthetic and trafficking machinery. Among these genes, those that encode cargo proteins are more widely represented because they are more numerous and because many cargoes are tissue-specific and not essential for the survival of an embryo. On the other hand, mutations in genes that encode ubiquitous transport-machinery proteins are more likely to be lethal. Nevertheless, several of these mutations have been found to be involved in mendelian diseases, and more continue to be reported. Probably some of these mutations can, under favorable conditions, be partially compensated for by the plasticity of the transport systems.

    Once in the Golgi complex, cargo proteins must traverse this organelle, which is composed of a series of interconnected stacks of four to six flat membranous cisternae and of tubular–saccular networks located at the cis and trans poles of the stacks. The main functions of the Golgi complex are to transport and chemically process cargo proteins and lipids, activities that mostly involve glycosylation. The mechanism of cargo transfer through the Golgi complex is composite and appears to involve the process of cisternal progression–maturation for large supramolecular cargoes, as well as other mechanisms for different cargo classes.

    After passing through the Golgi complex and reaching the trans-Golgi network, different cargoes are packaged in specialized membranous carriers, within which they are shipped out to their respective destinations, such as the lysosomes or the plasma membrane.20 Most proteins that are destined for the lysosomes (lysosomal enzymes) contain a mannose-6-phosphate tag and are sorted by the mannose-6-phosphate receptor into vesicles that are coated with a further protein complex, which is based on clathrin.21 Other cargoes move to the plasma membrane (or to their specific basolateral or apical domains in polarized cells) within large, apparently uncoated pleomorphic carriers that form at the trans-Golgi network. Also, in certain specialized cells, selected cargo proteins are greatly condensed into secretory granules that accumulate in the cytoplasm until their secretion is triggered by specific signals. Thus, there are several types of transport vesicles, all of which are formed by the fissioning of membrane buds from donor membranes, undergo translocation by microtubule-based motors, and dock onto and fuse with their acceptor membranes.

    Once at the cell surface, most membrane proteins undergo endocytosis, a fundamental process that is involved in many functions, including control of the composition of the plasma membrane, cell signaling, and uptake of essential nutrients. There are several types of endocytic carriers, which differ in the proteins they transport, in their morphologic features and dynamics, and in their underlying molecular mechanisms.28 The best-characterized carriers are the clathrin-coated vesicles, the caveolin-coated vesicles, and the macropinosomes (pleomorphic carriers that can engulf large volumes of extracellular fluid). Phagosomes are similar to macropinosomes, and in specialized cells (e.g., macrophages) they mediate the internalization of large objects (typically bacteria), which are then digested in the lysosomes.

    Most endocytic carriers then converge in the early endosomes, a vacuolar–tubular sorting station from which cargo proteins are sorted and delivered to several destinations. These destinations include the plasma membrane again; the recycling endosomes, another important sorting station from which cargo proteins can either return to the plasma membrane or move into the trans-Golgi network; and the late endosomes (the last endocytic station), from which some cargoes move to the Golgi complex and others are transferred to lysosomes for degradation. Another organelle that can fuse directly with lysosomes is the autophagosome. Autophagy is a process by which damaged cytosolic and organellar components are enveloped in specialized membranes and targeted for lysosomal degradation.

    Nearly all the molecules that are expressed in mammalian cells reach their correct intracellular locations by virtue of sophisticated transport-and-delivery systems. Central among these is the intracellular membrane-transport apparatus, which is designed to ferry most of the transmembrane proteins and nearly all the secreted proteins — about a third of the human proteome — from their site of synthesis, the endoplasmic reticulum, to their final destinations.

    Membrane transport is responsible for controlling the size, shape, and molecular composition of most cellular organelles, including the plasma membrane, and for mediating the secretion of thousands of cargo species, including hormones, growth factors, antibodies, matrix and serum proteins, digestive enzymes, and many more. To carry out this enormous task, the system relies on a large ensemble of organelles, including the endoplasmic reticulum, the Golgi complex, and the endolysosomal stations, and on an underlying molecular machinery that is estimated to comprise more than 2000 proteins. It is no surprise, then, that alterations to membrane transport, either genetic or otherwise, are associated with many diseases. Here, after a brief overview of the pathways, strategies, and mechanisms of membrane transport, we focus on mendelian disorders that arise from defects of the membrane-transport machinery.

    Pathways of Membrane Trafficking

    The main morphologic and functional features of the secretory and endocytic pathways were initially sketched out by the pioneers of modern cell biology in the 1960s and 1970s. Since then, this picture has grown enormously in richness and complexity, and the underlying molecular machinery has been unraveled through approaches that are based on yeast genetics and biochemical identification of the relevant components in mammals.

    The transport of newly synthesized secretory proteins begins at their site of synthesis, the endoplasmic reticulum, a network of dynamically interconnected membrane tubules and cisternae.

    Proteins are cotranslationally inserted into the lumen of the endoplasmic reticulum, where they are glycosylated and folded by a complex machinery that includes the chaperone proteins. Folding is essential, and when it cannot be completed, proteins are degraded by the degradation system associated with the endoplasmic reticulum. Moreover, if unfolded proteins accumulate in the endoplasmic reticulum, as they do under certain stress conditions, the unfolded-protein response ensues. The unfolded-protein response is a compensatory reaction that results primarily in an increase in the production of the folding-machinery proteins but can also influence different cell functions and lead to cell death or survival.

    After folding, proteins enter the exit sites of the endoplasmic reticulum, where they are sorted into either small or large pleomorphic budding vesicles that are generated through the membrane-bending properties of coat protein complex II (COPII). All vesicles then detach from the endoplasmic reticulum through membrane fission and move to the endoplasmic reticulum–Golgi intermediate compartment (ERGIC). From there, carriers containing secretory cargoes are transported forward to the Golgi complex. This step requires another coat complex, COPI, and includes the translocation of the carriers along microtubules mediated by motor proteins. From the cis pole of the Golgi, the secretory cargoes proceed toward the trans pole, whereas the machinery proteins that participate in the formation of anterograde carriers must be returned to the endoplasmic reticulum for another round of transport. This recycling is the task of COPI-dependent vesicles that form from both the ERGIC and the Golgi complex.