Defective Protein Trafficking Research Articles

Mechanisms of Pharmacological Rescue of Trafficking-defective hERG Mutant Channels in Human Long QT Syndrome

Long QT syndrome type 2 is caused by mutations in the human ether-a-go-go-related gene (hERG). We previously reported that the N470D mutation is retained in the endoplasmic reticulum (ER) but can be rescued to the plasma membrane by hERG channel blocker E-4031. The mechanisms of ER retention and how E-4031 rescues the N470D mutant are poorly understood. In this study, we investigated the interaction of hERG channels with the ER chaperone protein calnexin. Using coimmunoprecipitation, we showed that the immature forms of both wild type hERG and N470D associated with calnexin. The association required N-linked glycosylation of hERG channels. Pulse-chase analysis revealed that N470D had a prolonged association with calnexin compared with wild type hERG and E-4031 shortened the time course of calnexin association with N470D. To test whether the prolonged association of N470D with calnexin is due to defective folding of mutant channels, we studied hERG channel folding using the trypsin digestion method. We found that N470D and the immature form of wild type hERG were more sensitive to trypsin digestion than the mature form of wild type hERG. In the presence of E-4031, N470D became more resistant to trypsin even when its ER-to-Golgi transport was blocked by brefeldin A. These results suggest that defective folding of N470D contributes to its prolonged association with calnexin and ER retention and that E-4031 may restore proper folding of the N470D channel leading to its cell surface expression.

Hermansky–Pudlak syndrome: a disease of protein trafficking and organelle function

The Hermansky-Pudlak syndrome (HPS) is a collection of related autosomal recessive disorders which are genetically heterogeneous. There are eight human HPS subtypes, characterized by oculocutaneous albinism and platelet storage disease; prolonged bleeding, congenital neutropenia, pulmonary fibrosis, and granulomatous colitis can also occur. HPS is caused primarily by defects in intracellular protein trafficking that result in the dysfunction of intracellular organelles known as lysosome-related organelles. HPS gene products are all ubiquitously expressed and all associate in various multi-protein complexes, yet HPS has cell type-specific disease expression. Impairment of specialized secretory cells such as melanocytes, platelets, lung alveolar type II epithelial cells and cytotoxic T cells are observed in HPS. This review summarizes recent molecular, biochemical and cell biological analyses together with clinical studies that have led to the correlation of molecular pathology with clinical manifestations and led to insights into such diverse disease processes such as albinism, fibrosis, hemorrhage, and congenital neutropenia.

Ultrastructural Features of Trafficking Defects Are Pronounced in Melanocytic Nevus in Hermansky–Pudlak Syndrome Type 1

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by oculocutaneous albinism, a bleeding disorder, and ceroid lipofuscinosis in the lungs and gut. HPS is genetically heterogeneous and the most common variant, HPS type 1, is caused by mutations in HPS1 gene. The protein encoded by HPS1 is considered to facilitate the trafficking of melanocyte-specific gene products into the premelanosome. We report the ultrastructural findings in a melanocytic nevus seen in a 17-y-old Japanese female patient with HPS1 who is a compound heterozygote of HPS1 mutations, including a novel mutation. Electron microscopy of a pinkish papule corresponding to the melanocytic nevus revealed markedly aberrant, immature melanosomes, large membranous structures, and giant melanosomes in the vicinity of trans-Golgi network, the characteristic abnormalities because of protein trafficking defects in HPS1. These ultrastructural features were far more clearly demonstrated in the nevus cells than in the epidermal melanocytes. Thus, ultrastructural analysis of nevus cells may be an additional diagnostic tool for HPS1 and could give us important clues to further understanding of the pathomechanisms of HPS.

Most LQT-linked KCNH2 mutations are trafficking-defective

Over 100 mutations in the KCNH2 or human ether-a-go-go-related gene, which encodes the α-subunit peptides of the cardiac rapidly activating delayed rectifier K+ channel, have been linked to Long QT syndrome (LQT2). LQT2-linked mutations reduce KCNH2 current (IKCNH2) by altering channel protein trafficking and/or biophysical properties. We tested the hypothesis that the majority of LQT2-linked mutations cause protein trafficking defects. WT and 34 LQT2 missense mutations were heterologously expressed in HEK293 cells.

Degradation of Trafficking-defective Long QT Syndrome Type II Mutant Channels by the Ubiquitin-Proteasome Pathway

Mutations in the human ether-a-go-go-related gene (hERG) cause chromosome 7-linked long QT syndrome type II (LQT2). We have shown previously that LQT2 mutations lead to endoplasmic reticulum (ER) retention and rapid degradation of mutant hERG proteins. In this study we examined the role of the ubiquitin-proteasome pathway in the degradation of the LQT2 mutation Y611H. We showed that proteasome inhibitors N-acetyl-L-leucyl-L-leucyl-L-norleucinal and lactacystin but not lysosome inhibitor leupeptin inhibited the degradation of Y611H mutant channels. In addition, ER mannosidase I inhibitor kifunensine and down-regulation of EDEM (ER degradation-enhancing alpha-mannosidase-like protein) also suppressed the degradation of Y611H mutant channels. Proteasome inhibition but not mannosidase inhibition led to the accumulation of full-length hERG protein in the cytosol. The hERG protein accumulated in the cytosol was deglycosylated. Proteasome inhibition also resulted in the accumulation of polyubiquitinated hERG channels. These results suggest that the degradation of LQT2 mutant channels is mediated by the cytosolic proteasome in a process that involves mannose trimming, polyubiquitination, and deglycosylation of mutant channels.

Diacylglycerol and Its Formation by Phospholipase C Regulate Rab- and SNARE-dependent Yeast Vacuole Fusion

Although diacylglycerol (DAG) can trigger liposome fusion, biological membrane fusion requires Rab and SNARE proteins. We have investigated whether DAG and phosphoinositide-specific phospholipase C (PLC) have a role in the Rab- and SNARE-dependent homo-typic vacuole fusion in Saccharomyces cerevisiae. Vacuole fusion was blocked when DAG was sequestered by a recombinant C1b domain. DAG underwent ATP-dependent turnover during vacuole fusion, but was replenished by the hydrolysis of phosphatidylinositol 4,5-bisphosphate to DAG by PLC. The PLC inhibitors 3-nitrocoumarin and U73122 blocked vacuole fusion in vitro, whereas their inactive homologues did not. Plc1p is the only known PLC in yeast. Yeast cells lacking the PLC1 gene have many small vacuoles, indicating defects in protein trafficking to the vacuole or vacuole fusion, and purified Plc1p stimulates vacuole fusion. Docking-dependent Ca(2+) efflux is absent in plc1Delta vacuoles and was restored only upon the addition of both Plc1p and the Vam7p SNARE. However, vacuoles purified from plc1Delta strains still retain PLC activity and significant 3-nitrocoumarin- and U73122-sensitive fusion, suggesting that there is another PLC in S. cerevisiae with an important role in vacuole fusion.

Pharmacological rescue of trafficking defective HERG channels formed by coassembly of wild-type and long QT mutant N470D subunits

Mutations in the human ether-a-go-go-related gene (HERG) cause long QT syndrome. We previously showed that the HERG N470D mutation expressed as homotetrameric channels causes a protein trafficking defect, and this can be corrected by the HERG channel blocking drug E-4031. The N470D mutant also has been reported to cause dominant negative suppression of HERG current when coexpressed with wild-type channel subunits. The aims of this study were 1). to investigate the molecular mechanism responsible for the dominant negative effect of the N470D mutant coexpressed with wild-type subunits and 2). to test whether the trafficking defective heteromeric channels could be pharmacologically rescued by E-4031. Using a combination of immunoprecipitation and Western blot methods, we showed that N470D mutant and wild-type HERG subunits were physically associated in the endoplasmic reticulum as heteromeric channels. The coassembly resulted in the retention of both wild-type and N470D subunits in the endoplasmic reticulum. Culturing cells in E-4031 increased the cell surface expression of these channels, although with an altered electrophysiological phenotype. These results suggest that the dominant negative effect of the N470D wild-type coassembled channels is caused by retention of heteromeric channels in the endoplasmic reticulum and that the trafficking defect of these channels can be corrected by specific pharmacological strategies.

The ABCA1 Transporter Modulates Late Endocytic Trafficking: INSIGHTS FROM THE CORRECTION OF THE GENETIC DEFECT IN TANGIER DISEASE

We have previously established that the ABCA1 transporter, which plays a critical role in the lipidation of extracellular apolipoprotein acceptors, traffics between late endocytic vesicles and the cell surface (Neufeld, E. B., Remaley, A. T., Demosky, S. J., Jr., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590). The present study provides evidence that ABCA1 in late endocytic vesicles plays a role in cellular lipid efflux. Late endocytic trafficking was defective in Tangier disease fibroblasts that lack functional ABCA1. Consistent with a late endocytic protein trafficking defect, the hydrophobic amine U18666A retained NPC1 in abnormally tubulated, cholesterol-poor, Tangier disease late endosomes, rather than cholesterol-laden lysosomes, as in wild type fibroblasts. Consistent with a lipid trafficking defect, Tangier disease late endocytic vesicles accumulated both cholesterol and sphingomyelin and were immobilized in a perinuclear localization. The excess cholesterol in Tangier disease late endocytic vesicles retained massive amounts of NPC1, which traffics lysosomal cholesterol to other cellular sites. Exogenous apoA-I abrogated the cholesterol-induced retention of NPC1 in wild type but not in Tangier disease late endosomes. Adenovirally mediated ABCA1-GFP expression in Tangier disease fibroblasts corrected the late endocytic trafficking defects and restored apoA-I-mediated cholesterol efflux. ABCA1-GFP expression in wild type fibroblasts also reduced late endosome-associated NPC1, induced a marked uptake of fluorescent apoA-I into ABCA1-GFP-containing endosomes (that shuttled between late endosomes and the cell surface), and enhanced apoA-I-mediated cholesterol efflux. The combined results of this study suggest that ABCA1 converts pools of late endocytic lipids that retain NPC1 to pools that can associate with endocytosed apoA-I, and be released from the cell as nascent high density lipoprotein.

A domain mimic increases DeltaF508 CFTR trafficking and restores cAMP-stimulated anion secretion in cystic fibrosis epithelia.

The major disease-causing mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine 508 (DeltaF508), which adversely affects processing and plasma membrane targeting of CFTR. Under conditions predicted to stabilize protein folding, DeltaF508 CFTR is capable of trafficking to the plasma membrane and retains cAMP-regulated anion channel activity. Overexpression is one factor that increases CFTR trafficking; therefore, we hypothesized that expression of a domain mimic of the first nucleotide-binding fold (NBF1) of CFTR, i.e., the site of F508, may be sufficient to overwhelm the quality control process or otherwise stabilize DeltaF508 CFTR and thereby restore cAMP-stimulated anion secretion. In epithelial cells expressing recombinant DeltaF508 human (h)CFTR, expression of wild-type NBF1 increased the amount of both core-glycosylated and mature protein to a greater extent than expression of DeltaF508 NBF1. Expression of wild-type NBF1 in the DeltaF508 hCFTR cells increased whole cell Cl(-) current density to approximately 50% of that in cells expressing wild-type hCFTR. Expression of NBF1 in polarized epithelial monolayers from a DeltaF508/DeltaF508 cystic fibrosis mouse (MGEF) restored cAMP-stimulated transepithelial anion secretion but not in monolayers from a CFTR-null mouse (MGEN). Restoration of anion secretion was sustained in NBF1-expressing MGEF for >30 passages, whereas MGEN corrected with hCFTR progressively lost anion secretion capability. We conclude that expression of a NBF1 domain mimic may be useful for correction of the DeltaF508 CFTR protein trafficking defect in cystic fibrosis epithelia.

Atrophic macular degeneration mutations in ELOVL4 result in the intracellular misrouting of the protein

Elongation of very long chain fatty acids 4 ( ELOVL4) is a novel member of the ELO family of genes that are involved in fatty acid metabolism. ELOVL4 encodes a putative transmembrane protein of 314 amino acids that carries a possible endoplasmic reticulum (ER) retention/retrieval signal (KXKXX) at the C-terminus. Two distinct mutations, a 5-bp deletion and a complex mutation from the same region in exon 6 of this gene, have been reported so far and are associated with autosomal dominant atrophic macular degeneration (adMD/STGD3). Both of these deletions could result in C-terminal truncation and loss of the ER retention signal in the mutant protein. We expressed the wild-type and mutant proteins in COS-7 and CHO cells to study the intracellular distribution of ELOVL4 and to identify possible implications of the above mutations in its localization. Immunofluorescence analysis of these proteins along with organelle marker antibodies revealed predominant ER localization for wild-type ELOVL4. Targeted deletion of the dilysine motif at the C-terminus of the protein resulted in the loss of ER localization. Immunoelectron microscopy and immunofluorescence analysis revealed a similar ER localization pattern for the protein in human photoreceptors. These data indicate that ELOVL4 is an ER-resident protein, which supports its suggested function in fatty acid elongation. We also demonstrate that the localization of both mutant proteins was dramatically changed from an ER to a Golgi distribution. Our observations suggest that the consequences of defective protein trafficking could underlie the molecular mechanism associated with degeneration of the macula in the patients with adMD/STGD3.

Defective protein trafficking in hERG-associated hereditary long QT syndrome (LQT2): molecular mechanisms and restoration of intracellular protein processing.

Human hereditary long QT syndrome is a cardiac disease characterized by prolongation of the QT interval and increased susceptibility to ventricular arrhythmias and sudden cardiac death. Mutations in the human-ether-a-go-go-related gene (hERG), encoding the protein underlying the repolarizing cardiac I(Kr) potassium current, cause chromosome 7-linked long QT syndrome 2. Loss of function of mutant hERG channels may be caused by several mechanisms, including altered current kinetics, altered ion selectivity, or defective intracellular protein trafficking. Especially the latter category has become a focus of particular interest recently, because some of the mutant subunits display wild type current properties when normal trafficking is restored and channels are inserted in the cell membrane in vitro. This review summarizes the current knowledge on hERG channel trafficking under physiological and pathological conditions. In addition, therapeutic approaches to restore normal hERG trafficking in vitro and in vivo are discussed.

Unusually common cystic fibrosis mutation in Portugal encodes a misprocessed protein

A561E, a novel cystic fibrosis (CF) associated mutation in the first nucleotide binding domain of CFTR, is the second most common CF mutation in Portugal. Properties of the A561E-CFTR protein were studied by immunoblotting, pulse-chase, immunocytochemistry, and MQAE halide-efflux assay in stably transfected BHK cells. Altogether, results presented here suggest that A561E causes protein mislocalization in the endoplasmic reticulum where the mutant protein must be trapped by the quality control mechanism. We conclude that A561E originates a protein trafficking defect, thus belonging to class II of CFTR mutations. As it is the case for F508del-CFTR (the most common CF mutant), low temperature treatment partially rescues a functional A561E-CFTR channel, suggesting that substitution of glutamic acid for alanine at position 561 does not completely abolish CFTR function. Pharmacological strategies previously reported for treatment of CF patients with the F508del mutation could thus be also effective in CF patients bearing the A561E mutation.

Expression of the immunoglobulin superfamily neuroplastin adhesion molecules in adult and developing mouse cerebellum and their localisation to parasagittal stripes.

Neuroplastin (np) 55 and 65 are immunoglobulin superfamily members that arise by alternative splicing of the same gene and have been implicated in long-term activity-dependent synaptic plasticity. Both biochemical and immunocytochemical data suggest that np55 is the predominant isoform (>95% of total neuroplastin) in cerebellum. Neuroplastin immunoreactivity is concentrated in the molecular layer and synaptic glomeruli in the granule cell layer. Expression in the molecular layer appears to be postsynaptic. First, neuroplastin is associated with Purkinje cell dendrites in two mouse granuloprival cerebellar mutants, disabled and cerebellar deficient folia. Second, in an acid sphingomyelinase knockout mouse with widespread protein trafficking defects, neuroplastin accumulates in the Purkinje cell somata. Finally, primary cerebellar cultures show neuroplastin expression in Purkinje cell dendrites and somata lacking normal histotypic organization and synaptic connections, and high-magnification views indicate a preferential association with dendritic spines. In the molecular layer, differences in neuroplastin expression levels present as a parasagittal array of stripes that alternates with that revealed by the expression of another compartmentation antigen, zebrin II/aldolase c. Neuroplastin immunoreactivity is first detected weakly at postnatal day 3 (P3) in the anterior lobe vermis. By P5, parasagittal stripes are already apparent in the immature molecular layer. At this stage, punctate deposits are also localised at the perimeter of the Purkinje cell perikarya; these are no longer detected by P15. The data suggest a role for neuroplastins in the development and maintenance of normal synaptic connections in the cerebellum.

Structure and function of disease-causing missense mutations in the PHEX gene.

The PHEX gene that is mutated in patients with X-linked hypophosphatemia (XLH) encodes a protein homologous to the M13 family of zinc metallopeptidases. The present study was undertaken to assess the impact of nine PHEX missense mutations on cellular trafficking, endopeptidase activity, and protein conformation. Secreted forms of wild-type and mutant PHEX proteins were generated by PCR mutagenesis; these included C85R, D237G, Y317F, G579R, G579V, S711R, A720T, and F731Y identified in XLH patients, and E581V, which in neutral endopeptidase 24.11 abolishes catalytic activity but not plasma membrane localization. The wild-type and D237G, Y317F, E581V, and F731Y proteins were terminally glycosylated and secreted into the medium, whereas the C85R, G579R, G579V, S711R, and A720T proteins were trapped inside the transfected cells. Growing the cells at 26 C permitted the secretion of G579V, S711R, and A720T proteins, although the yield of rescued G579V was insufficient for further analysis. Endopeptidase activity of secreted and rescued PHEX proteins, assessed using a novel internally quenched fluorogenic peptide substrate, revealed that E581V and S711R are completely inactive; D237G and Y317F exhibit 50-60% of wild-type activity; and A720T and F731Y retain full catalytic activity. Conformational analysis by limited proteolysis demonstrated that F731Y is more sensitive to trypsin and D237G is more resistant to endoproteinase Glu-c than the wild-type protein. Thus, defects in protein trafficking, endopeptidase activity, and protein conformation account for loss of PHEX function in XLH patients harboring these missense mutations.

Role of glycosylation in cell surface expression and stability of HERG potassium channels.

The human ether-à-go-go-related gene (HERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel in the heart. We previously showed that HERG channel protein is modified by N-linked glycosylation. HERG protein sequence contains two extracellular consensus sites for N-linked glycosylation (N598, N629). In this study, we used the approaches of site-directed mutagenesis and biochemical modification to inhibit N-linked glycosylation and studied the role of glycosylation in the cell surface expression and turnover of HERG channels. Our results show that N598 is the only site for N-linked glycosylation and that glycosylation is not required for the cell surface expression of functional HERG channels. In contrast, N629 is not used for glycosylation, but mutation of this site (N629Q) causes a protein trafficking defect, which results in its intracellular retention. Pulse-chase experiments show that the turnover rate of nonglycosylated HERG channel is faster than that of the glycosylated form, suggesting that N-linked glycosylation plays an important role in HERG channel stability.

Pharmacological rescue of human K(+) channel long-QT2 mutations: human ether-a-go-go-related gene rescue without block.

Defective protein trafficking is a consequence of gene mutations. Human long-QT (LQT) syndrome results from mutations in several genes, including the human ether-a-go-go-related gene (HERG), which encodes a delayed rectifier K(+) current. Trafficking-defective mutant HERG protein is a mechanism for reduced delayed rectifier K(+) current in LQT2, and high-affinity HERG channel-blocking drugs can result in pharmacological rescue. Methods and Results- We postulated that drug molecules modified to remove high-affinity HERG block may still stabilize mutant proteins in a conformation required for rescue. We tested terfenadine carboxylate (fexofenadine) and terfenadine, structurally similar drugs with markedly different affinities for HERG block, for rescue of trafficking-defective LQT2 mutations. Terfenadine rescued the N470D mutation but blocked the channels. In contrast, fexofenadine rescued N470D with a half-maximal rescue concentration of 177 nmol/L, which is approximately 350-fold lower than the half-maximal channel block concentration. The G601S mutation was also rescued without channel block. Pharmacological rescue can occur without channel block. This could represent a new antiarrhythmic paradigm in the treatment of some trafficking-defective LQT2 mutations.

Intracellular Distribution of Myelin Protein Gene Products Is Altered in Oligodendrocytes of the taiep Rat

Hypomyelination and subsequent demyelination of the taiep rat CNS are thought to result from the abnormal accumulation of microtubules (MTs) in oligodendrocytes that disrupts intracellular transport of components needed to form and maintain the myelin sheath. In this study, myelin gene expression was evaluated in mutant and age-matched controls to determine if MT abnormalities affect the distribution of myelin proteins and their mRNAs. Immunohistochemical analysis of taiep brains and spinal cords revealed a gradual decrease in levels of several myelin proteins including myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and 2′,3′-cyclic nucleotide 3′-phosphodiesterase. Accompanying early declines in MAG and PLP, accumulations of immunoreactive products were detected within oligodendrocytes, consistent with a defect in protein trafficking. Northern blot analysis indicated that diminishing protein levels could not be attributed to changes in transcriptional activity, except for MBP of which mRNA levels decreased with age. Cellular localization of MBP mRNA by in situ hybridization further revealed that transcripts were concentrated within oligodendrocyte cell bodies instead of uniformly distributed throughout processes. These results demonstrate that changes in expression and intracellular localization of myelin gene products are concurrent with increases in MT mass in taiep oligodendrocytes and support our hypothesis that cytoskeletal defects prevent the normal transport of elements required for the formation and maintenance of the myelin sheath.

Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein trafficking.

Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy with serious consequences for the mother and fetus. Two pedigrees have been reported with ICP in the mothers of children with a subtype of autosomal recessive progressive familial intrahepatic cholestasis (PFIC) with raised serum gamma-glutamyl transpeptidase (gamma-GT). Affected children have homozygous mutations in the MDR3 gene (also called ABCB4 ), and heterozygous mothers have ICP. More frequently, however, ICP occurs in women with no known family history of PFIC and the genetic basis of this disorder is unknown. We investigated eight women with ICP and raised serum gamma-GT, but with no known family history of PFIC. DNA sequence analysis revealed a C to A transversion in codon 546 in exon 14 of MDR3 in one patient, which results in the missense substitution of the wild-type alanine with an aspartic acid. We performed functional studies of this mutation introduced into MDR1, a closely related homologue of MDR3. Fluorescence activated cell sorting (FACS) and western analysis indicated that this missense mutation causes disruption of protein trafficking with a subsequent lack of functional protein at the cell surface. The demonstration of a heterozygous missense mutation in the MDR3 gene in a patient with ICP with no known family history of PFIC, analysed by functional studies, is a novel finding. This shows that MDR3 mutations are responsible for the additional phenotype of ICP in a subgroup of women with raised gamma-GT.

Antagonists to the rescue

Antagonists to the rescue

Introduction

Introduction