Browsing Posts published in September, 2011

    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.

    However, short-term ADT is not without quality-of-life consequences, including hot flashes and higher rates of erectile dysfunction than with radiotherapy alone. Furthermore, erectile dysfunction may be less responsive to interventions after combined therapy than after radiotherapy alone. In prospective studies, short-term ADT caused measurable muscle loss, fat accumulation, decreased insulin sensitivity, and increased cholesterol and triglyceride levels. In the current study, the 10-year disease-specific mortality in the radiotherapy-alone group was 1%, a finding that does not provide support for the addition of short-term ADT in patients with low-risk prostate cancer. A total of 395 black men participated in this study, allowing evaluation according to racial subgroups. Similar benefits from short-term ADT were seen in the white and black populations with respect to the 10-year rate of overall survival, 10-year disease-specific mortality, and biochemical failure. Overall survival among black men was worse than that among white men, but disease-specific mortality was similar.

    The results of our trial show that the addition of short-term ADT provides a survival benefit for men with intermediate-risk prostate cancer who receive conventional doses of radiotherapy. In addition, our findings suggest a biologic interaction between short-term ADT and radiotherapy, in contrast to several randomized trials of surgery combined with short-term ADT, which did not show a benefit with respect to outcome. The adoption of current radiotherapy techniques such as intensity-modulated radiation therapy, intensity-guided radiation therapy, and lowdose-rate and high-dose-rate brachytherapy now permits the safe delivery of higher doses of radiation than was possible when this study was conducted. These techniques have also been associated with improved efficacy,36-39 bringing into question the value of adding short-term ADT in men with intermediate-risk cancers treated with current irradiation methods. The RTOG has opened a successor study, RTOG 08-15 (NCT00936390), to address this question.

    The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute. Supported by grants (U10 CA21661, to the Radiation Therapy Oncology Group [RTOG]; U10 CA37422, to the Community Clinical Oncology Program; and U10 CA32115, to the RTOG Statistical Center) from the National Cancer Institute. Dr. Chetner reports receiving lecture fees from and serving on the advisory boards of Amgen, Ferring, GlaxoSmithKline, and Eli Lilly and receiving fees for the development of educational presentations from Amgen and GlaxoSmithKline; and Dr. Sandler, consulting fees from Calypso Medical and Varian. No other potential conflict of interest relevant to this article was reported.

    This phase 3 clinical trial evaluated whether the addition of short-term ADT to radiotherapy improved outcomes in patients who had early, localized prostate cancer and a PSA level of 20 ng per milliliter or less — the subgroup of patients with prostate cancer who were known to have the most favorable prognosis at the time the study was initiated. Because of the indolent nature of the disease, a median follow-up period of more than 9 years for surviving patients and vigilant PSA monitoring were required to obtain meaningful results in a patient cohort in which most deaths were due to other causes.

    The study showed that the addition of shortterm ADT to radiotherapy conferred a modest but significant increase in the 10-year rate of overall survival, from 57 to 62%. This increase was accompanied by a significant reduction in 10-year disease-specific mortality from 8% to 4% as well as reductions in the secondary end points of biochemical failure, distant metastases, and the rate of positive findings on repeat prostate biopsy at 2 years. The Gleason score was the only independent prognostic predictor for all end points measured. The lack of surgical staging for regional lymph nodes did not predict poor outcomes, validating the current practice of clinical staging in patients receiving radiotherapy. The efficacy gains were achieved with minimal temporary acute hepatic toxic effects and some decreased erectile function at 1 year, but with no increased risk of death from intercurrent disease, serious cardiovascular toxic effects, or acute or long-term gastrointestinal or genitourinary complications of radiotherapy. The rate of erectile dysfunction observed in this study is similar to that reported in previous studies that involved the use of similar doses of radiotherapy.

    Reanalysis of the data according to risk subgroups showed that the gains in overall survival and reductions in disease-specific mortality were mainly limited to men in the intermediate-risk subgroup, with a number needed to treat of 14 based on the difference in overall survival seen at 10 years. Although the addition of short-term ADT to radiotherapy also appeared to be beneficial in the high-risk patients, the persistent significant increase in 10-year disease-specific mortality provides support for observations from other clinical trials showing that more than 4 months of ADT is required for maximum benefit.

    Among men with low-risk disease, the addition of short-term ADT did not significantly increase the 10-year rate of overall survival or decrease the 10-year rate of disease-specific mortality but did significantly lower the incidence of biochemical failure and positive findings on repeat prostate biopsy at 2 years. It is conceivable that in patients with indolent disease, longer follow-up is required to show a benefit with respect to the diseasespecific mortality and overall survival rates.