Kinetics of G-protein-coupled receptor endosomal trafficking pathways revealed by single quantum dots

G-protein-coupled receptors (GPCRs) are the largest protein superfamily in the human genome; they comprise 30% of current drug targets and regulate diverse cellular signaling responses. The role of endosomal trafficking in GPCR signaling regulation is gaining substantial consideration. However, this process remains difficult to study due to the inability to distinguish among many individual receptors, simultaneously trafficking within multiple endosomal pathways. Here we show accurate measurement of the internalization and endosomal trafficking of single groups of serotonin (5-hydroxytryptamine, 5-HT) receptors using single quantum dot (QD) probes and quantitative colocalization. We demonstrate that the presence of a QD tag does not interfere with 5-HT receptor internalization or endosomal recycling. Direct measurements show simultaneous trafficking of the 5-HT1A receptor in two distinct endosomal recycling pathways. Single-molecule imaging of endosomal trafficking will significantly impact the understanding of cellular signaling and provide powerful tools to elucidate the actions of GPCR-targeted therapeutics.     

Common pathways in Crohn's disease and other inflammatory diseases revealed by genomics

Genetics may not provide all the answers but it will, in highlighting the pathways relevant to the pathogenesis of Crohn's disease and other inflammatory conditions, at least indicate which questions need answering.     

Mitochondrial iron trafficking and the integration of iron metabolism between the mitochondrion and cytosol

The mitochondrion is well known for its key role in energy transduction. However, it is less well appreciated that it is also a focal point of iron metabolism. Iron is needed not only for heme and iron sulfur cluster (ISC)-containing proteins involved in electron transport and oxidative phosphorylation, but also for a wide variety of cytoplasmic and nuclear functions, including DNA synthesis. The mitochondrial pathways involved in the generation of both heme and ISCs have been characterized to some extent. However, little is known concerning the regulation of iron uptake by the mitochondrion and how this is coordinated with iron metabolism in the cytosol and other organelles (e.g., lysosomes). In this article, we discuss the burgeoning field of mitochondrial iron metabolism and trafficking that has recently been stimulated by the discovery of proteins involved in mitochondrial iron storage (mitochondrial ferritin) and transport (mitoferrin-1 and -2). In addition, recent work examining mitochondrial diseases (e.g., Friedreich''s ataxia) has established that communication exists between iron metabolism in the mitochondrion and the cytosol. This finding has revealed the ability of the mitochondrion to modulate whole-cell iron-processing to satisfy its own requirements for the crucial processes of heme and ISC synthesis. Knowledge of mitochondrial iron-processing pathways and the interaction between organelles and the cytosol could revolutionize the investigation of iron metabolism.     

狼疮肾炎患者肾组织基因表达的研究

目的 应用基因芯片技术对狼疮肾炎(LN)患者肾组织基因的表达进行分析,进一步探讨LN的发病机制.方法 15例LN患者B超定位下肾活检,抽取肾组织RNA,经过mRNA线性扩增、芯片杂交、扫描信号值.采用Rank invariant方法标化,Cluster 3．0软件聚类分析,Crsd软件进行t检验.结果 Toll样受体、干扰素(IFN)-α通路、JAK／STAT信号通路相关基因在弥漫增生型LN,活动指数≥7分和慢性指数＞4分以及系统性红斑狼疮疾病活动指数(SLEDAI)＞10分的患者中高表达.结论 LN存在Toll样受体-IFN-α-JAK／STAT的异常表达,随着肾脏病理活动指数、慢性指数和SLEDAI积分的增加而趋于明显.针对该通路上的关键分子、下游基因产物、作用靶细胞等可能作为将来临床筛选药物的靶点.     

Rosai-Dorfman disease revealed by renal failure: case report

We report a case of Rosa?-Dorfman Disease revealed by renal failure in a 43 years old patient. Clinical presentation included abdominal lymphadenopathy and general status deterioration. Diagnosis was established by histopathological examination of the node which revealed sinusal lymphohistiocytosis. Treatment combined prednisone and cyclophosphamide and was effective with regression of renal failure. We will review the diagnostic criteria and the prognosis of this disorder of unknown etiology.     

Fabry's disease revealed by stroke: a case report

We report a 35-year-old man with a stroke as the presenting feature of Fabry's disease. Cerebrovascular manifestation can be the first manifestation of this disease and must be systematically evoked by the neurologist. The neurological follow-up of these patients must be systematic.     

Selective inhibitor of endosomal trafficking pathways exploited by multiple toxins and viruses

Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease.The success of a broad array of microbial pathogens depends on their ability to gain entry into and/or transport proteins into the cytosol of host cells. Intracellular-acting bacterial toxins have evolved to take advantage of numerous host-mediated entry mechanisms (1), making these toxins ideal tools for studying endocytosis and vesicular trafficking. Indeed, the use of bacterial toxins has contributed to many key discoveries, including membrane recycling, clathrin-independent endocytosis, and retrograde transport (2). Compounds that inhibit entry of ricin, Shiga toxin, and Pseudomonas aeruginosa exotoxin A (ExoA) into host cells have been identified (3–5). These small molecules exhibit varied mechanisms of action, including blockade of retrograde toxin trafficking at the early endosome–trans Golgi network (TGN) junction, morphological disruption of the Golgi apparatus, and inhibition of the toxin active site. Small molecules that disrupt toxin binding, entry, trafficking, and host response can serve not only as probes to dissect such eukaryotic cellular pathways, but also are potential therapeutics for infectious and genetic diseases.Bacillus anthracis, the causative agent of the disease anthrax, secretes binary toxins that enter host cells and disrupt physiological processes. Lethal factor (LF) is a Zn²⁺-dependent metalloprotease that cleaves mitogen-activated protein kinase kinases (MAPKKs) 1–4, 6, and 7 (6, 7) and Nlrp1b (8–10) and reproduces many pathologies of anthrax when injected into laboratory animals (11, 12). The cellular entry of LF is dependent on a cell-binding and translocation subunit known as protective antigen (PA). PA is an 83-kDa protein that is cleaved by host proteases into 63- and 20-kDa fragments, allowing oligomerization of the toxin into a prepore (13). The PA oligomer can then bind up to four monomers of LF, forming a holotoxin complex (14, 15). Two cellular toxin receptors, TEM8 and CMG2, mediate toxin binding and endocytic uptake (16, 17). Acidification of the lumen of the late endosome drives a conformational change in the prepore, resulting in insertion into the endosomal membrane and translocation of LF into the cytosol (18–20). Alternatively, LF may be translocated to the interior of intraluminal vesicles and transported to the late endosome via multivesicular bodies in a process dependent on COPI and ALIX (21). The vesicular membranes then fuse with the limiting endosomal membrane and thereby deliver LF to the cytosol (21).Despite substantial effort to define binding and entry mechanisms used by lethal toxin (LT), much is still unknown about how it, and indeed many other toxins and viruses, gain access to the host cytosol. To address this, we performed a high-throughput screen to identify small molecules that block cellular entry of LT. Here, we report the identification and characterization of a compound that blocks trafficking of various toxins and viruses to acidified endosomes.     

Spatial dynamics of receptor-mediated endocytic trafficking in budding yeast revealed by using fluorescent alpha-factor derivatives

Much progress defining the order and timing of endocytic internalization events has come as a result of real-time, live-cell fluorescence microscopy. Although the availability of numerous endocytic mutants makes yeast an especially valuable organism for functional analysis of endocytic dynamics, a serious limitation has been the lack of a fluorescent cargo for receptor-mediated endocytosis. We have now synthesized biologically active fluorescent mating-pheromone derivatives and demonstrated that receptor-mediated endocytosis in budding yeast occurs via the clathrin- and actin-mediated endocytosis pathway. We found that endocytic proteins first assemble into patches on the plasma membrane, and then alpha-factor associates with the patches. Internalization occurs next, concomitant with actin assembly at patches. Additionally, endocytic vesicles move toward early endosomes on actin cables. Early endosomes also associate with actin cables, and they actively move toward endocytic sites to capture vesicles being released from the plasma membrane. Thus, endocytic vesicle formation and capture of the newly released vesicles by early endosomes occur in a highly concerted manner, mediated by the actin cytoskeleton.     

Interorgan ammonia trafficking in liver disease

Patients with liver disease have reduced urea synthesis capacity resulting in reduced capacity to detoxify ammonia in the liver. The contribution of the gut to the hyperammonemic state observed during liver failure is mainly due to portacaval shunting and not the result of changes in the metabolism of ammonia in the gut. Small intestinal synthesis of ammonia is related to amino acid breakdown, predominantly glutamine, whereas large bowel ammonia production is caused by bacterial breakdown of amino acids and urea. The kidneys produce ammonia but adapt to liver failure in experimental portacaval shunting by reducing ammonia release into the systemic circulation. The kidneys have the ability to switch from net ammonia production to net ammonia excretion. Data from recent studies in patients with cirrhosis of the liver show that the kidneys have a major role in post upper gastrointestinal bleeding hyperammonemia. During hyperammonemia muscle takes up ammonia and plays a major role in (temporarily) detoxifying ammonia to glutamine. Net uptake of ammonia by the brain occurs in patients and experimental animals with acute and chronic liver failure. Insight will be given in recent developments on ammonia lowering therapies which are based on the information of interorgan ammonia trafficking.     

Adult-onset Still's disease revealed by a pleuropericarditis

We report a case of adult-onset Still's disease (AOSD) revealed by pleuropericardial manifestations. A 40 yr old black woman was admitted for flu-like syndrome with pharyngitis, hectic fever, polymorphonuclear hyperleucocytosis and pleuropericarditis. The diagnosis of AOSD was supported by 3 major and 3 minor criteria after exclusion of infectious, haematological and connective tissue diseases. Pulmonary involvement is infrequent in AOSD, and consists of transient pulmonary infiltrates and chronic restrictive pattern. However, pleuritis, like pericarditis, is present in 25% of cases. Initial onset of pleuritis, associated with fever and hyperleucocytosis preceding articular manifestations could be responsible for a delay in diagnosis and a subsequent worsening in the prognosis of the disease. A rapid improvement is usually observed under nonsteroidal anti-inflammatory drug or corticosteroid treatment.     

Cytokine release from innate immune cells: association with diverse membrane trafficking pathways

Cytokines released from innate immune cells play key roles in the regulation of the immune response. These intercellular messengers are the source of soluble regulatory signals that initiate and constrain inflammatory responses to pathogens and injury. Although numerous studies describe detailed signaling pathways induced by cytokines and their specific receptors, there is little information on the mechanisms that control the release of cytokines from different cell types. Indeed, the pathways, molecules, and mechanisms of cytokine release remain a "black box" in immunology. Here, we review research findings and new approaches that have begun to generate information on cytokine trafficking and release by innate immune cells in response to inflammatory or infectious stimuli. Surprisingly complex machinery, multiple organelles, and specialized membrane domains exist in these cells to ensure the selective, temporal, and often polarized release of cytokines in innate immunity.     

Distinct phenotype of a Wilson disease mutation reveals a novel trafficking determinant in the copper transporter ATP7B

Wilson disease (WD) is a monogenic autosomal-recessive disorder of copper accumulation that leads to liver failure and/or neurological deficits. WD is caused by mutations in ATP7B, a transporter that loads Cu(I) onto newly synthesized cupro-enzymes in the trans-Golgi network (TGN) and exports excess copper out of cells by trafficking from the TGN to the plasma membrane. To date, most WD mutations have been shown to disrupt ATP7B activity and/or stability. Using a multidisciplinary approach, including clinical analysis of patients, cell-based assays, and computational studies, we characterized a patient mutation, ATP7B^S653Y, which is stable, does not disrupt Cu(I) transport, yet renders the protein unable to exit the TGN. Bulky or charged substitutions at position 653 mimic the phenotype of the patient mutation. Molecular modeling and dynamic simulation suggest that the S653Y mutation induces local distortions within the transmembrane (TM) domain 1 and alter TM1 interaction with TM2. S653Y abolishes the trafficking-stimulating effects of a secondary mutation in the N-terminal apical targeting domain. This result indicates a role for TM1/TM2 in regulating conformations of cytosolic domains involved in ATP7B trafficking. Taken together, our experiments revealed an unexpected role for TM1/TM2 in copper-regulated trafficking of ATP7B and defined a unique class of WD mutants that are transport-competent but trafficking-defective. Understanding the precise consequences of WD-causing mutations will facilitate the development of advanced mutation-specific therapies.Copper is essential for the normal development and function of human cells because it serves as a cofactor for many important metabolic enzymes. However, intracellular levels of copper must be tightly regulated (1, 2) because excess copper is toxic. Inborn mutations in the Cu(I)-ATPases, ATP7A [Online Mendelian Inheritance in Man (OMIM) accession no.*606882] or ATP7B (OMIM *300011) result in either systemic copper deficiency or copper accumulation in several tissues, causing Menkes disease or Wilson disease (WD), respectively. WD (OM#277900) is an autosomal-recessive disorder with a heterogeneous clinical presentation; in the absence of family history, diagnosis of WD requires multiple clinical and laboratory studies (3). The large number of rare mutations in the ATP7B gene [>500, (www.hgmd.cf.ac.uk/ac/gene.php?gene=ATP7B)] contribute to the difficulty in making genotype-phenotype associations. Presently, about two dozen mutations found in WD patients have been characterized in detail (4–7). These studies revealed that the most common effect of a WD mutation is ATP7B misfolding, which results in retention of newly synthesized ATP7B in the endoplasmic reticulum (ER), a marked decrease in protein stability, and loss of Cu(I)-transport activity (8, 9). Destabilization and inactivation of ATP7B explain the common phenotypic manifestations in WD, such as impaired copper export from the liver and the lack of copper incorporation into secreted cuproenzymes, such as ceruloplasmin (CPN).ATP7B and the highly homologous ATP7A (Menkes disease protein) play central roles in maintaining copper levels in cells. These proteins belong to the evolutionarily conserved family of P_1B-ATPases, which use the energy of ATP hydrolysis to transport copper from the cytosol across cellular membranes (Fig. 1A). ATP7A and ATP7B load copper onto newly synthesized cupro-proteins in the late Golgi and remove excess copper from the cytosol after relocating to vesicles, which in turn traffic to the plasma membrane to release copper into the extracellular milieu. In low and basal copper, ATP7A and ATP7B are located predominantly in a subcompartment of the trans-Golgi network (TGN) marked by syntaxin 6 (10). When copper levels increase, ATP7A and ATP7B exit the TGN in distinct vesicles; ATP7B vesicles move to the apical region in polarized epithelia, whereas ATP7A vesicles move to the basolateral region. Because copper-dependent ATP7B trafficking is a complex process, the precise sequence of events and the function of trafficking determinants in ATP7B’s structure are yet to be fully understood.Open in a separate windowFig. 1.Hypothetical ATP7B model and multiple species alignment of the conserved regions, amino acids 621–668, in the two Cu-ATPases. (A) A hypothetical ATP7B ribbon model, generated by UCSF Chimera, showing the conserved core organization (20). The two large cytoplasmic loops in the core structure are: the A domain (actuator, green), between TM4 and TM5, which contains the phosphatase activity; and the N and P domains (nucleotide binding and phosphorylation, red) between TM6 and TM7, which bind ATP (N), catalyzing formation of a phosphorylated intermediate (P) as part of the catalytic cycle. The eight TMs (yellow are): TM1 (amino acids 645–670), TM2 (including the platform helix, amino acids 694–722), TM3 (amino acids 729–749), TM4 (amino acids 765–786), TM5 (amino acids 916–942), TM6 (amino acids 967–1004), TM7 (amino acids 1307–1345), and TM8 (amino acids 1352–1373). The six N-terminal MBDs (blue, N-MBDs, also referred to as the N-terminal domain of ATP7B, N-ATP7B) (61, 62) were manually positioned onto the published model. The box approximates the region of the multiple species alignment shown in B. (B) A multiple species alignment of human ATP7B sequence 621–668 (Upper) and ATP7A sequence 621–668 (Lower). WD patient mutations are underlined and in bold. The ATP7B S653 position is marked with an asterisk (bold). Portions of MBD6 and TMD 1 are bracketed. In ATP7A, the bracketed sequence shows the deleted region that is replaced with two amino acids (IR) in a patient with Occipital Horn Syndrome. The deleted sequence of ATP7A is underlined and in bold (35). Alignments were obtained using ClustalW (63). Amino acids that are identical (*), conserved (:), and semiconserved (.) are shown.We previously developed a comprehensive set of cell-based assays that use both polarized hepatic cells and fibroblasts lacking ATP7B and ATP7A (derived from a Menkes disease patient) to evaluate the activity, stability, and trafficking of ATP7B and its mutants (11, 12). In this study, we combined these assays with additional mutational analysis and computational studies to dissect the molecular phenotype of WD mutations found in a highly conserved region of ATP7B, G⁶²¹-S⁶⁶⁸. We demonstrate that the S653Y mutation has a distinct “transport-competent/trafficking-defective” phenotype. We also show that the transmembrane segment (TM) that harbors S653 has an important and previously unanticipated role in regulating exit of ATP7B from the TGN in response to copper elevation.