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Sample records for mutation impairs trafficking

  1. Mutations in the D1 domain of von Willebrand factor impair their propeptide-dependent multimerization, intracellular trafficking and secretion.

    PubMed

    Yin, Jie; Ma, Zhenni; Su, Jian; Wang, Jiong-Wei; Zhao, Xiaojuan; Ling, Jing; Bai, Xia; Ouyang, Wanyan; Wang, Zhaoyue; Yu, Ziqiang; Ruan, Changgeng

    2015-01-01

    We identified three novel mutations (p.Gly39Arg, p.Lys157Glu, p.Cys379Gly) and one previously known mutation (p.Asp141Asn) in the von Willebrand factor propeptide from three von Willebrand disease patients. All four mutations impaired multimerization of von Willebrand factor, due to reduced oxidoreductase activity of isomeric propeptide. These mutations resulted in the endothelial reticulum retention and impaired basal and stimulated secretions of von Willebrand factor. Our results support that the mutations in the D1 domain lead to defective multimerization, intracellular trafficking, and secretion of von Willebrand factor and result in bleeding of patients. PMID:26088471

  2. AP1S3 mutations are associated with pustular psoriasis and impaired Toll-like receptor 3 trafficking.

    PubMed

    Setta-Kaffetzi, Niovi; Simpson, Michael A; Navarini, Alexander A; Patel, Varsha M; Lu, Hui-Chun; Allen, Michael H; Duckworth, Michael; Bachelez, Hervé; Burden, A David; Choon, Siew-Eng; Griffiths, Christopher E M; Kirby, Brian; Kolios, Antonios; Seyger, Marieke M B; Prins, Christa; Smahi, Asma; Trembath, Richard C; Fraternali, Franca; Smith, Catherine H; Barker, Jonathan N; Capon, Francesca

    2014-05-01

    Adaptor protein complex 1 (AP-1) is an evolutionary conserved heterotetramer that promotes vesicular trafficking between the trans-Golgi network and the endosomes. The knockout of most murine AP-1 complex subunits is embryonically lethal, so the identification of human disease-associated alleles has the unique potential to deliver insights into gene function. Here, we report two founder mutations (c.11T>G [p.Phe4Cys] and c.97C>T [p.Arg33Trp]) in AP1S3, the gene encoding AP-1 complex subunit σ1C, in 15 unrelated individuals with a severe autoinflammatory skin disorder known as pustular psoriasis. Because the variants are predicted to destabilize the 3D structure of the AP-1 complex, we generated AP1S3-knockdown cell lines to investigate the consequences of AP-1 deficiency in skin keratinocytes. We found that AP1S3 silencing disrupted the endosomal translocation of the innate pattern-recognition receptor TLR-3 (Toll-like receptor 3) and resulted in a marked inhibition of downstream signaling. These findings identify pustular psoriasis as an autoinflammatory phenotype caused by defects in vesicular trafficking and demonstrate a requirement of AP-1 for Toll-like receptor homeostasis. PMID:24791904

  3. Impaired intracellular trafficking defines early Parkinson's disease

    PubMed Central

    Hunn, Benjamin H.M.; Cragg, Stephanie J.; Bolam, J. Paul; Spillantini, Maria-Grazia; Wade-Martins, Richard

    2015-01-01

    Parkinson's disease (PD) is an insidious and incurable neurodegenerative disease, and represents a significant cost to individuals, carers, and ageing societies. It is defined at post-mortem by the loss of dopamine neurons in the substantia nigra together with the presence of Lewy bodies and Lewy neurites. We examine here the role of α-synuclein and other cellular transport proteins implicated in PD and how their aberrant activity may be compounded by the unique anatomy of the dopaminergic neuron. This review uses multiple lines of evidence from genetic studies, human tissue, induced pluripotent stem cells, and refined animal models to argue that prodromal PD can be defined as a disease of impaired intracellular trafficking. Dysfunction of the dopaminergic synapse heralds trafficking impairment. PMID:25639775

  4. Mechanism of impaired microtubule-dependent peroxisome trafficking and oxidative stress in SPAST-mutated cells from patients with Hereditary Spastic Paraplegia.

    PubMed

    Wali, Gautam; Sutharsan, Ratneswary; Fan, Yongjun; Stewart, Romal; Tello Velasquez, Johana; Sue, Carolyn M; Crane, Denis I; Mackay-Sim, Alan

    2016-01-01

    Hereditary spastic paraplegia (HSP) is an inherited neurological condition that leads to progressive spasticity and gait abnormalities. Adult-onset HSP is most commonly caused by mutations in SPAST, which encodes spastin a microtubule severing protein. In olfactory stem cell lines derived from patients carrying different SPAST mutations, we investigated microtubule-dependent peroxisome movement with time-lapse imaging and automated image analysis. The average speed of peroxisomes in patient-cells was slower, with fewer fast moving peroxisomes than in cells from healthy controls. This was not because of impairment of peroxisome-microtubule interactions because the time-dependent saltatory dynamics of movement of individual peroxisomes was unaffected in patient-cells. Our observations indicate that average peroxisome speeds are less in patient-cells because of the lower probability of individual peroxisome interactions with the reduced numbers of stable microtubules: peroxisome speeds in patient cells are restored by epothilone D, a tubulin-binding drug that increases the number of stable microtubules to control levels. Patient-cells were under increased oxidative stress and were more sensitive than control-cells to hydrogen peroxide, which is primarily metabolised by peroxisomal catalase. Epothilone D also ameliorated patient-cell sensitivity to hydrogen-peroxide. Our findings suggest a mechanism for neurodegeneration whereby SPAST mutations indirectly lead to impaired peroxisome transport and oxidative stress. PMID:27229699

  5. Mechanism of impaired microtubule-dependent peroxisome trafficking and oxidative stress in SPAST-mutated cells from patients with Hereditary Spastic Paraplegia

    PubMed Central

    Wali, Gautam; Sutharsan, Ratneswary; Fan, Yongjun; Stewart, Romal; Tello Velasquez, Johana; Sue, Carolyn M; Crane, Denis I.; Mackay-Sim, Alan

    2016-01-01

    Hereditary spastic paraplegia (HSP) is an inherited neurological condition that leads to progressive spasticity and gait abnormalities. Adult-onset HSP is most commonly caused by mutations in SPAST, which encodes spastin a microtubule severing protein. In olfactory stem cell lines derived from patients carrying different SPAST mutations, we investigated microtubule-dependent peroxisome movement with time-lapse imaging and automated image analysis. The average speed of peroxisomes in patient-cells was slower, with fewer fast moving peroxisomes than in cells from healthy controls. This was not because of impairment of peroxisome-microtubule interactions because the time-dependent saltatory dynamics of movement of individual peroxisomes was unaffected in patient-cells. Our observations indicate that average peroxisome speeds are less in patient-cells because of the lower probability of individual peroxisome interactions with the reduced numbers of stable microtubules: peroxisome speeds in patient cells are restored by epothilone D, a tubulin-binding drug that increases the number of stable microtubules to control levels. Patient-cells were under increased oxidative stress and were more sensitive than control-cells to hydrogen peroxide, which is primarily metabolised by peroxisomal catalase. Epothilone D also ameliorated patient-cell sensitivity to hydrogen-peroxide. Our findings suggest a mechanism for neurodegeneration whereby SPAST mutations indirectly lead to impaired peroxisome transport and oxidative stress. PMID:27229699

  6. Association of nonsense mutation in GABRG2 with abnormal trafficking of GABAA receptors in severe epilepsy.

    PubMed

    Ishii, Atsushi; Kanaumi, Takeshi; Sohda, Miwa; Misumi, Yoshio; Zhang, Bo; Kakinuma, Naoto; Haga, Yoshiko; Watanabe, Kazuyoshi; Takeda, Sen; Okada, Motohiro; Ueno, Shinya; Kaneko, Sunao; Takashima, Sachio; Hirose, Shinichi

    2014-03-01

    Mutations in GABRG2, which encodes the γ2 subunit of GABAA receptors, can cause both genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. Most GABRG2 truncating mutations associated with Dravet syndrome result in premature termination codons (PTCs) and are stably translated into mutant proteins with potential dominant-negative effects. This study involved search for mutations in candidate genes for Dravet syndrome, namely SCN1A, 2A, 1B, 2B, GABRA1, B2, and G2. A heterozygous nonsense mutation (c.118C>T, p.Q40X) in GABRG2 was identified in dizygotic twin girls with Dravet syndrome and their apparently healthy father. Electrophysiological studies with the reconstituted GABAA receptors in HEK cells showed reduced GABA-induced currents when mutated γ2 DNA was cotransfected with wild-type α1 and β2 subunits. In this case, immunohistochemistry using antibodies to the α1 and γ2 subunits of GABAA receptor showed granular staining in the soma. In addition, microinjection of mutated γ2 subunit cDNA into HEK cells severely inhibited intracellular trafficking of GABAA receptor subunits α1 and β2, and retention of these proteins in the endoplasmic reticulum. The mutated γ2 subunit-expressing neurons also showed impaired axonal transport of the α1 and β2 subunits. Our findings suggested that different phenotypes of epilepsy, e.g., GEFS+ and Dravet syndrome (which share similar abnormalities in causative genes) are likely due to impaired axonal transport associated with the dominant-negative effects of GABRG2. PMID:24480790

  7. First missense mutation outside of SERAC1 lipase domain affecting intracellular cholesterol trafficking.

    PubMed

    Rodríguez-García, María Elena; Martín-Hernández, Elena; de Aragón, Ana Martínez; García-Silva, María Teresa; Quijada-Fraile, Pilar; Arenas, Joaquín; Martín, Miguel A; Martínez-Azorín, Francisco

    2016-01-01

    We report the clinical and genetic findings in a Spanish boy who presented MEGDEL syndrome, a very rare inborn error of metabolism. Whole-exome sequencing uncovered a new homozygous mutation in the serine active site containing 1 (SERAC1) gene, which is essential for both mitochondrial function and intracellular cholesterol trafficking. Functional studies in patient fibroblasts showed that p.D224G mutation affects the intracellular cholesterol trafficking. Only three missense mutations in this gene have been described before, being p.D224G the first missense mutation outside of the SERAC1 serine-lipase domain. Therefore, we conclude that the defect in cholesterol trafficking is not limited to alterations in this specific part of the protein. PMID:26445863

  8. M-CSF receptor mutations in hereditary diffuse leukoencephalopathy with spheroids impair not only kinase activity but also surface expression

    SciTech Connect

    Hiyoshi, Masateru; Hashimoto, Michihiro; Yukihara, Mamiko; Bhuyan, Farzana; Suzu, Shinya

    2013-11-01

    Highlights: •Many mutations were identified in Fms as a putative genetic cause of HDLS. •All of the mutations tested severely impair the kinase activity. •Most of the mutations also impair the trafficking to the cell surface. •These defects further suggest that HDLS is caused by a loss of Fms function. -- Abstract: The tyrosine kinase Fms, the cell surface receptor for M-CSF and IL-34, is critical for microglial proliferation and differentiation in the brain. Recently, a number of mutations have been identified in Fms as a putative genetic cause of hereditary diffuse leukoencephalopathy with spheroids (HDLS), implying an important role of microglial dysfunction in HDLS pathogenesis. In this study, we initially confirmed that 11 mutations, which reside within the ATP-binding or major tyrosine kinase domain, caused a severe impairment of ligand-induced Fms auto-phosphorylation. Intriguingly, we found that 10 of the 11 mutants also showed a weak cell surface expression, which was associated with a concomitant increase in the low molecular weight hypo-N-glycosylated immature gp130Fms-like species. Indeed, the mutant proteins heavily accumulated to the Golgi-like perinuclear regions. These results indicate that all of the Fms mutations tested severely impair the kinase activity and most of the mutations also impair the trafficking to the cell surface, further suggesting that HDLS is caused by the loss of Fms function.

  9. The AQP2 mutation V71M causes nephrogenic diabetes insipidus in humans but does not impair the function of a bacterial homolog

    PubMed Central

    Klein, Noreen; Kümmerer, Nadine; Hobernik, Dominika; Schneider, Dirk

    2015-01-01

    Several point mutations have been identified in human aquaporins, but their effects on the function of the respective aquaporins are mostly enigmatic. We analyzed the impact of the aquaporin 2 mutation V71M, which causes nephrogenic diabetes insipidus in humans, on aquaporin structure and activity, using the bacterial aquaglyceroporin GlpF as a model. Importantly, the sequence and structure around the V71M mutation is highly conserved between aquaporin 2 and GlpF. The V71M mutation neither impairs substrate flux nor oligomerization of the aquaglyceroporin. Therefore, the human aquaporin 2 mutant V71M is most likely active, but cellular trafficking is probably impaired. PMID:26442203

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

    PubMed Central

    Braiterman, Lelita T.; Murthy, Amrutha; Jayakanthan, Samuel; Nyasae, Lydia; Tzeng, Eric; Gromadzka, Grazyna; Woolf, Thomas B.; Lutsenko, Svetlana; Hubbard, Ann L.

    2014-01-01

    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, ATP7BS653Y, 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. PMID:24706876

  11. Mutations of Vasopressin Receptor 2 Including Novel L312S Have Differential Effects on Trafficking.

    PubMed

    Tiulpakov, Anatoly; White, Carl W; Abhayawardana, Rekhati S; See, Heng B; Chan, Audrey S; Seeber, Ruth M; Heng, Julian I; Dedov, Ivan; Pavlos, Nathan J; Pfleger, Kevin D G

    2016-08-01

    Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a genetic disease first described in 2 unrelated male infants with severe symptomatic hyponatremia. Despite undetectable arginine vasopressin levels, patients have inappropriately concentrated urine resulting in hyponatremia, hypoosmolality, and natriuresis. Here, we describe and functionally characterize a novel vasopressin type 2 receptor (V2R) gain-of-function mutation. An L312S substitution in the seventh transmembrane domain was identified in a boy presenting with water-induced hyponatremic seizures at the age of 5.8 years. We show that, compared with wild-type V2R, the L312S mutation results in the constitutive production of cAMP, indicative of the gain-of-function NSIAD profile. Interestingly, like the previously described F229V and I130N NSIAD-causing mutants, this appears to both occur in the absence of notable constitutive β-arrestin2 recruitment and can be reduced by the inverse agonist Tolvaptan. In addition, to understand the effect of various V2R substitutions on the full receptor "life-cycle," we have used and further developed a bioluminescence resonance energy transfer intracellular localization assay using multiple localization markers validated with confocal microscopy. This allowed us to characterize differences in the constitutive and ligand-induced localization and trafficking profiles of the novel L312S mutation as well as for previously described V2R gain-of-function mutants (NSIAD; R137C and R137L), loss-of-function mutants (nephrogenic diabetes insipidus; R137H, R181C, and M311V), and a putative silent V266A V2R polymorphism. In doing so, we describe differences in trafficking between unique V2R substitutions, even at the same amino acid position, therefore highlighting the value of full and thorough characterization of receptor function beyond simple signaling pathway analysis. PMID:27355191

  12. Mutations of Vasopressin Receptor 2 Including Novel L312S Have Differential Effects on Trafficking

    PubMed Central

    Tiulpakov, Anatoly; White, Carl W.; Abhayawardana, Rekhati S.; See, Heng B.; Chan, Audrey S.; Seeber, Ruth M.; Heng, Julian I.; Dedov, Ivan; Pavlos, Nathan J.

    2016-01-01

    Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a genetic disease first described in 2 unrelated male infants with severe symptomatic hyponatremia. Despite undetectable arginine vasopressin levels, patients have inappropriately concentrated urine resulting in hyponatremia, hypoosmolality, and natriuresis. Here, we describe and functionally characterize a novel vasopressin type 2 receptor (V2R) gain-of-function mutation. An L312S substitution in the seventh transmembrane domain was identified in a boy presenting with water-induced hyponatremic seizures at the age of 5.8 years. We show that, compared with wild-type V2R, the L312S mutation results in the constitutive production of cAMP, indicative of the gain-of-function NSIAD profile. Interestingly, like the previously described F229V and I130N NSIAD-causing mutants, this appears to both occur in the absence of notable constitutive β-arrestin2 recruitment and can be reduced by the inverse agonist Tolvaptan. In addition, to understand the effect of various V2R substitutions on the full receptor “life-cycle,” we have used and further developed a bioluminescence resonance energy transfer intracellular localization assay using multiple localization markers validated with confocal microscopy. This allowed us to characterize differences in the constitutive and ligand-induced localization and trafficking profiles of the novel L312S mutation as well as for previously described V2R gain-of-function mutants (NSIAD; R137C and R137L), loss-of-function mutants (nephrogenic diabetes insipidus; R137H, R181C, and M311V), and a putative silent V266A V2R polymorphism. In doing so, we describe differences in trafficking between unique V2R substitutions, even at the same amino acid position, therefore highlighting the value of full and thorough characterization of receptor function beyond simple signaling pathway analysis. PMID:27355191

  13. A novel mutation in DDR2 causing spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL) results in defective intra-cellular trafficking

    PubMed Central

    2014-01-01

    Background The rare autosomal genetic disorder, Spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL), is reported to be caused by missense or splice site mutations in the human discoidin domain receptor 2 (DDR2) gene. Previously our group has established that trafficking defects and loss of ligand binding are the underlying cellular mechanisms of several SMED-SL causing mutations. Here we report the clinical characteristics of two siblings of consanguineous marriage with suspected SMED-SL and identification of a novel disease-causing mutation in the DDR2 gene. Methods Clinical evaluation and radiography were performed to evaluate the patients. All the coding exons and splice sites of the DDR2 gene were sequenced by Sanger sequencing. Subcellular localization of the mutated DDR2 protein was determined by confocal microscopy, deglycosylation assay and Western blotting. DDR2 activity was measured by collagen activation and Western analysis. Results In addition to the typical features of SMED-SL, one of the patients has an eye phenotype including visual impairment due to optic atrophy. DNA sequencing revealed a novel homozygous dinucleotide deletion mutation (c.2468_2469delCT) on exon 18 of the DDR2 gene in both patients. The mutation resulted in a frameshift leading to an amino acid change at position S823 and a predicted premature termination of translation (p.S823Cfs*2). Subcellular localization of the mutant protein was analyzed in mammalian cell lines, and it was found to be largely retained in the endoplasmic reticulum (ER), which was further supported by its N-glycosylation profile. In keeping with its cellular mis-localization, the mutant protein was found to be deficient in collagen-induced receptor activation, suggesting protein trafficking defects as the major cellular mechanism underlying the loss of DDR2 function in our patients. Conclusions Our results indicate that the novel mutation results in defective trafficking

  14. N-terminal tagging of the dopamine transporter impairs protein expression and trafficking in vivo

    PubMed Central

    Vecchio, Laura M.; Bermejo, M. Kristel; Beerepoot, Pieter; Ramsey, Amy J.

    2014-01-01

    The dopamine transporter (DAT) is the primary protein responsible for the uptake of dopamine from the extracellular space back into presynaptic neurons. As such, it plays an important role in the cessation of dopaminergic neurotransmission and in the maintenance of extracellular dopamine homeostasis. Here, we report the development of a new BAC transgenic mouse line that expresses DAT with an N-terminal HA-epitope (HAD-Tg). In this line, two copies of the HA-DAT BAC are incorporated into the genome, increasing DAT mRNA levels by 47%. Despite the increase in mRNA levels, HAD-Tg mice show no significant increase in the level of DAT protein in the striatum, indicating a defect in protein trafficking or stability. By crossing HAD-Tg mice with DAT knockout mice (DAT-KO), we engineered mice that exclusively express HA-tagged DAT in the absence of endogenous DAT (DAT-KO/HAD-Tg). We show that DAT-KO/HAD-Tg mice express only 8.5% of WT DAT levels in the striatum. Importantly, the HA-tagged DAT that is present in DAT-KO/HAD-Tg mice is functional, as it is able to partially rescue the DAT-KO hyperactive phenotype. Finally, we provide evidence that the HA-tagged DAT is retained in the cell body based on a reduction in the striatum:midbrain protein ratio. These results demonstrate that the presence of the N-terminal tag leads to impaired DAT protein expression in vivo due in part to improper trafficking of the tagged transporter, and highlight the importance of the N-terminus in the transport of DAT to striatal terminals. PMID:24886986

  15. Kinase Impaired BRAF Mutations Confer Lung Cancer Sensitivity to Dasatinib

    PubMed Central

    Sen, Banibrata; Peng, Shaohua; Tang, Ximing; Erickson, Heidi S.; Galindo, Hector; Mazumdar, Tuhina; Stewart, David J.; Wistuba, Ignacio; Johnson, Faye M.

    2013-01-01

    During a clinical trial of the tyrosine kinase inhibitor dasatinib for advanced non–small cell lung cancer (NSCLC) one patient responded dramatically and remains cancer-free 4 years later. A comprehensive analysis of his tumor revealed a previously undescribed, kinase inactivating BRAF mutation (Y472CBRAF); no inactivating BRAF mutations were found in the non-responding tumors taken from other patients. Cells transfected with Y472CBRAF exhibited CRAF, MEK, and ERK activation – characteristics identical to signaling changes that occur with previously known kinase inactivating BRAF mutants. Dasatinib selectively induced senescence in NSCLC cells with inactivating BRAF mutations. Transfection of other NSCLC cells with these BRAF mutations also increased these cells’ dasatinib sensitivity, whereas transfection with an activating BRAF mutation led to their increased dasatinib resistance. The sensitivity induced by Y472CBRAF was reversed by the introduction of a BRAF mutation that impairs RAF dimerization. Dasatinib inhibited CRAF modestly, but concurrently induced RAF dimerization resulting in ERK activation in NSCLC cells with kinase inactivating BRAF mutations. The sensitivity of NSCLC with kinase impaired BRAF to dasatinib suggested synthetic lethality of BRAF and a dasatinib target. Inhibiting BRAF in NSCLC cells expressing wild-type BRAF likewise enhanced these cells’ dasatinib sensitivity. Thus, the patient’s BRAF mutation was likely responsible for his tumor’s marked response to dasatinib, suggesting that tumors bearing kinase impaired BRAF mutations may be exquisitely sensitive to dasatinib. Moreover, the potential synthetic lethality of combination therapy including dasatinib and BRAF inhibitors may lead to additional therapeutic options against cancers with wild-type BRAF. PMID:22649091

  16. Functional Rescue of Trafficking-Impaired ABCB4 Mutants by Chemical Chaperones

    PubMed Central

    Gordo-Gilart, Raquel; Andueza, Sara; Hierro, Loreto; Jara, Paloma; Alvarez, Luis

    2016-01-01

    Multidrug resistance protein 3 (MDR3, ABCB4) is a hepatocellular membrane protein that mediates biliary secretion of phosphatidylcholine. Null mutations in ABCB4 gene give rise to severe early-onset cholestatic liver disease. We have previously shown that the disease-associated mutations p.G68R, p.G228R, p.D459H, and p.A934T resulted in retention of ABCB4 in the endoplasmic reticulum, thus failing to target the plasma membrane. In the present study, we tested the ability of two compounds with chaperone-like activity, 4-phenylbutyrate and curcumin, to rescue these ABCB4 mutants by assessing their effects on subcellular localization, protein maturation, and phospholipid efflux capability. Incubation of transfected cells at a reduced temperature (30°C) or exposure to pharmacological doses of either 4-PBA or curcumin restored cell surface expression of mutants G228R and A934T. The delivery of these mutants to the plasma membrane was accompanied by a switch in the ratio of mature to inmature protein forms, leading to a predominant expression of the mature protein. This effect was due to an improvement in the maturation rate and not to the stabilization of the mature forms. Both mutants were also functionally rescued, displaying bile salt-dependent phospholipid efflux activity after addition of 4-PBA or curcumin. Drug-induced rescue was mutant specific, given neither 4-PBA nor curcumin had an effect on the ABCB4 mutants G68R and A934T. Collectively, these data indicate that the functionality of selected trafficking-defective ABCB4 mutants can be recovered by chemical chaperones through restoration of membrane localization, suggesting a potential treatment for patients carrying such mutations. PMID:26900700

  17. Functional Rescue of Trafficking-Impaired ABCB4 Mutants by Chemical Chaperones.

    PubMed

    Gordo-Gilart, Raquel; Andueza, Sara; Hierro, Loreto; Jara, Paloma; Alvarez, Luis

    2016-01-01

    Multidrug resistance protein 3 (MDR3, ABCB4) is a hepatocellular membrane protein that mediates biliary secretion of phosphatidylcholine. Null mutations in ABCB4 gene give rise to severe early-onset cholestatic liver disease. We have previously shown that the disease-associated mutations p.G68R, p.G228R, p.D459H, and p.A934T resulted in retention of ABCB4 in the endoplasmic reticulum, thus failing to target the plasma membrane. In the present study, we tested the ability of two compounds with chaperone-like activity, 4-phenylbutyrate and curcumin, to rescue these ABCB4 mutants by assessing their effects on subcellular localization, protein maturation, and phospholipid efflux capability. Incubation of transfected cells at a reduced temperature (30°C) or exposure to pharmacological doses of either 4-PBA or curcumin restored cell surface expression of mutants G228R and A934T. The delivery of these mutants to the plasma membrane was accompanied by a switch in the ratio of mature to inmature protein forms, leading to a predominant expression of the mature protein. This effect was due to an improvement in the maturation rate and not to the stabilization of the mature forms. Both mutants were also functionally rescued, displaying bile salt-dependent phospholipid efflux activity after addition of 4-PBA or curcumin. Drug-induced rescue was mutant specific, given neither 4-PBA nor curcumin had an effect on the ABCB4 mutants G68R and A934T. Collectively, these data indicate that the functionality of selected trafficking-defective ABCB4 mutants can be recovered by chemical chaperones through restoration of membrane localization, suggesting a potential treatment for patients carrying such mutations. PMID:26900700

  18. Pathogenic mechanism of an autism-associated neuroligin mutation involves altered AMPA-receptor trafficking.

    PubMed

    Chanda, S; Aoto, J; Lee, S-J; Wernig, M; Südhof, T C

    2016-02-01

    Neuroligins are postsynaptic cell-adhesion molecules that bind to presynaptic neurexins. Although the general synaptic role of neuroligins is undisputed, their specific functions at a synapse remain unclear, even controversial. Moreover, many neuroligin gene mutations were associated with autism, but the pathophysiological relevance of these mutations is often unknown, and their mechanisms of action uninvestigated. Here, we examine the synaptic effects of an autism-associated neuroligin-4 substitution (called R704C), which mutates a cytoplasmic arginine residue that is conserved in all neuroligins. We show that the R704C mutation, when introduced into neuroligin-3, enhances the interaction between neuroligin-3 and AMPA receptors, increases AMPA-receptor internalization and decreases postsynaptic AMPA-receptor levels. When introduced into neuroligin-4, conversely, the R704C mutation unexpectedly elevated AMPA-receptor-mediated synaptic responses. These results suggest a general functional link between neuroligins and AMPA receptors, indicate that both neuroligin-3 and -4 act at excitatory synapses but perform surprisingly distinct functions, and demonstrate that the R704C mutation significantly impairs the normal function of neuroligin-4, thereby validating its pathogenicity. PMID:25778475

  19. RCAN1 links impaired neurotrophin trafficking to aberrant development of the sympathetic nervous system in Down syndrome

    PubMed Central

    Patel, Ami; Yamashita, Naoya; Ascaño, Maria; Bodmer, Daniel; Boehm, Erica; Bodkin-Clarke, Chantal; Ryu, Yun Kyoung; Kuruvilla, Rejji

    2015-01-01

    Down syndrome is the most common chromosomal disorder affecting the nervous system in humans. To date, investigations of neural anomalies in Down syndrome have focused on the central nervous system, although dysfunction of the peripheral nervous system is a common manifestation. The molecular and cellular bases underlying peripheral abnormalities have remained undefined. Here, we report the developmental loss of sympathetic innervation in human Down syndrome organs and in a mouse model. We show that excess regulator of calcineurin 1 (RCAN1), an endogenous inhibitor of the calcineurin phosphatase that is triplicated in Down syndrome, impairs neurotrophic support of sympathetic neurons by inhibiting endocytosis of the nerve growth factor (NGF) receptor, TrkA. Genetically correcting RCAN1 levels in Down syndrome mice markedly improves NGF-dependent receptor trafficking, neuronal survival and innervation. These results uncover a critical link between calcineurin signalling, impaired neurotrophin trafficking and neurodevelopmental deficits in the peripheral nervous system in Down syndrome. PMID:26658127

  20. Mutations in Bruton's tyrosine kinase impair IgA responses.

    PubMed

    Mitsuiki, Noriko; Yang, Xi; Bartol, Sophinus J W; Grosserichter-Wagener, Christina; Kosaka, Yoshiyuki; Takada, Hidetoshi; Imai, Kohsuke; Kanegane, Hirokazu; Mizutani, Shuki; van der Burg, Mirjam; van Zelm, Menno C; Ohara, Osamu; Morio, Tomohiro

    2015-03-01

    X-linked agammaglobulinemia (XLA) is a primary immunodeficiency caused by mutations in Bruton's tyrosine kinase (BTK), and is characterized by markedly decreased numbers of blood B cells and an absence of all immunoglobulin isotypes. We performed whole exome sequencing in a male pediatric patient with dysgammaglobulinemia with IgA deficiency. Genetic analysis revealed a BTK missense mutation (Thr316Ala). To investigate whether a BTK mutation underlay this antibody deficiency with marked decrease of IgA in this patient, we performed functional analyses of B cells and phagocytes, and molecular analyses of somatic hypermutation and class switch recombination. The BTK missense mutation resulted in B cells with reduced BTK and high IgM expression. Equal proportions of CD19(low) and CD19(normal) fractions were observed, and both included naïve and memory B cells. Calcium influx and phospholipase Cγ2 phosphorylation upon IgM stimulation were marginally impaired in CD19(low), but not in CD19(+) B cells. Similar to XLA patients, IgA transcripts showed low SHM levels, whereas IgG transcripts were hardly affected. Our analyses suggest that the BTK mutation likely underlies the disease in this case, and that hypomorphic BTK mutations can result in normal circulating B cell numbers, but specifically impair IgA responses. PMID:25589397

  1. The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking.

    PubMed

    Dumas, Audrey; Lê-Bury, Gabrielle; Marie-Anaïs, Florence; Herit, Floriane; Mazzolini, Julie; Guilbert, Thomas; Bourdoncle, Pierre; Russell, David G; Benichou, Serge; Zahraoui, Ahmed; Niedergang, Florence

    2015-10-26

    Human immunodeficiency virus type 1 (HIV-1) impairs major functions of macrophages but the molecular basis for this defect remains poorly characterized. Here, we show that macrophages infected with HIV-1 were unable to respond efficiently to phagocytic triggers and to clear bacteria. The maturation of phagosomes, defined by the presence of late endocytic markers, hydrolases, and reactive oxygen species, was perturbed in HIV-1-infected macrophages. We showed that maturation arrest occurred at the level of the EHD3/MICAL-L1 endosomal sorting machinery. Unexpectedly, we found that the regulatory viral protein (Vpr) was crucial to perturb phagosome maturation. Our data reveal that Vpr interacted with EB1, p150(Glued), and dynein heavy chain and was sufficient to critically alter the microtubule plus end localization of EB1 and p150(Glued), hence altering the centripetal movement of phagosomes and their maturation. Thus, we identify Vpr as a modulator of the microtubule-dependent endocytic trafficking in HIV-1-infected macrophages, leading to strong alterations in phagolysosome biogenesis. PMID:26504171

  2. The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking

    PubMed Central

    Dumas, Audrey; Lê-Bury, Gabrielle; Marie-Anaïs, Florence; Herit, Floriane; Mazzolini, Julie; Guilbert, Thomas; Bourdoncle, Pierre; Russell, David G.; Benichou, Serge; Zahraoui, Ahmed

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) impairs major functions of macrophages but the molecular basis for this defect remains poorly characterized. Here, we show that macrophages infected with HIV-1 were unable to respond efficiently to phagocytic triggers and to clear bacteria. The maturation of phagosomes, defined by the presence of late endocytic markers, hydrolases, and reactive oxygen species, was perturbed in HIV-1–infected macrophages. We showed that maturation arrest occurred at the level of the EHD3/MICAL-L1 endosomal sorting machinery. Unexpectedly, we found that the regulatory viral protein (Vpr) was crucial to perturb phagosome maturation. Our data reveal that Vpr interacted with EB1, p150Glued, and dynein heavy chain and was sufficient to critically alter the microtubule plus end localization of EB1 and p150Glued, hence altering the centripetal movement of phagosomes and their maturation. Thus, we identify Vpr as a modulator of the microtubule-dependent endocytic trafficking in HIV-1–infected macrophages, leading to strong alterations in phagolysosome biogenesis. PMID:26504171

  3. Analysis of Trafficking, Stability and Function of Human Connexin 26 Gap Junction Channels with Deafness-Causing Mutations in the Fourth Transmembrane Helix

    PubMed Central

    Ambrosi, Cinzia; Walker, Amy E.; DePriest, Adam D.; Cone, Angela C.; Lu, Connie; Badger, John; Skerrett, I. Martha; Sosinsky, Gina E.

    2013-01-01

    Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased

  4. Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus

    PubMed Central

    del Toro, Daniel; Alberch, Jordi; Lázaro-Diéguez, Francisco; Martín-Ibáñez, Raquel; Xifró, Xavier; Egea, Gustavo

    2009-01-01

    Huntingtin regulates post-Golgi trafficking of secreted proteins. Here, we studied the mechanism by which mutant huntingtin impairs this process. Colocalization studies and Western blot analysis of isolated Golgi membranes showed a reduction of huntingtin in the Golgi apparatus of cells expressing mutant huntingtin. These findings correlated with a decrease in the levels of optineurin and Rab8 in the Golgi apparatus that can be reverted by overexpression of full-length wild-type huntingtin. In addition, immunoprecipitation studies showed reduced interaction between mutant huntingtin and optineurin/Rab8. Cells expressing mutant huntingtin produced both an accumulation of clathrin adaptor complex 1 at the Golgi and an increase of clathrin-coated vesicles in the vicinity of Golgi cisternae as revealed by electron microscopy. Furthermore, inverse fluorescence recovery after photobleaching analysis for lysosomal-associated membrane protein-1 and mannose-6-phosphate receptor showed that the optineurin/Rab8-dependent post-Golgi trafficking to lysosomes was impaired in cells expressing mutant huntingtin or reducing huntingtin levels by small interfering RNA. Accordingly, these cells showed a lower content of cathepsin D in lysosomes, which led to an overall reduction of lysosomal activity. Together, our results indicate that mutant huntingtin perturbs post-Golgi trafficking to lysosomal compartments by delocalizing the optineurin/Rab8 complex, which, in turn, affects the lysosomal function. PMID:19144827

  5. Inherited human sex reversal due to impaired nucleocytoplasmic trafficking of SRY defines a male transcriptional threshold

    PubMed Central

    Chen, Yen-Shan; Racca, Joseph D.; Phillips, Nelson B.; Weiss, Michael A.

    2013-01-01

    Human testis determination is initiated by SRY (sex determining region on Y chromosome). Mutations in SRY cause gonadal dysgenesis with female somatic phenotype. Two subtle variants (V60L and I90M in the high-mobility group box) define inherited alleles shared by an XY sterile daughter and fertile father. Whereas specific DNA binding and bending are unaffected in a rat embryonic pre-Sertoli cell line, the variants exhibited selective defects in nucleocytoplasmic shuttling due to impaired nuclear import (V60L; mediated by Exportin-4) or export (I90M; mediated by chromosome region maintenance 1). Decreased shuttling limits nuclear accumulation of phosphorylated (activated) SRY, in turn reducing occupancy of DNA sites regulating Sertoli-cell differentiation [the testis-specific SRY-box 9 (Sox9) enhancer]. Despite distinct patterns of biochemical and cell-biological perturbations, V60L and I90M each attenuated Sox9 expression in transient transfection assays by twofold. Such attenuation was also observed in studies of V60A, a clinical variant associated with ovotestes and hence ambiguity between divergent cell fates. This shared twofold threshold is reminiscent of autosomal syndromes of transcription-factor haploinsufficiency, including XY sex reversal associated with mutations in SOX9. Our results demonstrate that nucleocytoplasmic shuttling of SRY is necessary for robust initiation of testicular development. Although also characteristic of ungulate orthologs, such shuttling is not conserved among rodents wherein impaired nuclear export of the high-mobility group box and import-dependent phosphorylation are compensated by a microsatellite-associated transcriptional activation domain. Human sex reversal due to subtle defects in the nucleocytoplasmic shuttling of SRY suggests that its transcriptional activity lies near the edge of developmental ambiguity. PMID:24003159

  6. A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

    PubMed Central

    Zhang, Michael L.; Sinner, Moritz F.; Dolmatova, Elena V.; Tucker, Nathan R.; McLellan, Micheal; Shea, Marisa A.; Milan, David J.; Lunetta, Kathryn L.; Benjamin, Emelia J.; Ellinor, Patrick T.

    2014-01-01

    Background Atrial fibrillation (AF) is the most common arrhythmia, and a recent genome-wide association study identified HCN4 as a novel AF susceptibility locus. HCN4 encodes for the cardiac pacemaker channel and HCN4 mutations are associated with familial sinus bradycardia and AF. Objective To determine whether novel variants in the coding region of HCN4 contribute to the susceptibility for AF. Methods We sequenced the coding region of HCN4 for novel variants from 527 cases with early-onset AF from the Massachusetts General Hospital AF Study and 443 referents from the Framingham Heart Study. We used site-directed mutagenesis, cellular electrophysiology, immunocytochemistry and confocal microscopy to functionally characterize novel variants. Results We found the frequency of novel coding HCN4 variants was 2-fold greater for individuals with AF (seven variants) compared to the referents (three variants). We determined that one, (p.Pro257Ser, located in the amino-terminus adjacent to the first transmembrane spanning domain) of the seven novel HCN4 variants in our AF cases did not traffick to cell membrane while the remaining six were not functionally different from wild type. Also, the three novel variants in our referents did not alter function compared to wild type. Co-expression studies showed that the p.Pro257Ser mutant channel failed to co-localize with the wild type HCN4 channel on the cell membrane. Conclusion Our findings are consistent with HCN4 haploinsufficiency as the likely mechanism for early-onset AF in the p.Pro257Ser carrier. PMID:24607718

  7. Visual impairment in FOXG1-mutated individuals and mice.

    PubMed

    Boggio, E M; Pancrazi, L; Gennaro, M; Lo Rizzo, C; Mari, F; Meloni, I; Ariani, F; Panighini, A; Novelli, E; Biagioni, M; Strettoi, E; Hayek, J; Rufa, A; Pizzorusso, T; Renieri, A; Costa, M

    2016-06-01

    The Forkead Box G1 (FOXG1 in humans, Foxg1 in mice) gene encodes for a DNA-binding transcription factor, essential for the development of the telencephalon in mammalian forebrain. Mutations in FOXG1 have been reported to be involved in the onset of Rett Syndrome, for which sequence alterations of MECP2 and CDKL5 are known. While visual alterations are not classical hallmarks of Rett syndrome, an increasing body of evidence shows visual impairment in patients and in MeCP2 and CDKL5 animal models. Herein we focused on the functional role of FOXG1 in the visual system of animal models (Foxg1(+/Cre) mice) and of a cohort of subjects carrying FOXG1 mutations or deletions. Visual physiology of Foxg1(+/Cre) mice was assessed by visually evoked potentials, which revealed a significant reduction in response amplitude and visual acuity with respect to wild-type littermates. Morphological investigation showed abnormalities in the organization of excitatory/inhibitory circuits in the visual cortex. No alterations were observed in retinal structure. By examining a cohort of FOXG1-mutated individuals with a panel of neuro-ophthalmological assessments, we found that all of them exhibited visual alterations compatible with high-level visual dysfunctions. In conclusion our data show that Foxg1 haploinsufficiency results in an impairment of mouse and human visual cortical function. PMID:27001178

  8. Prion-impairing mutations in Hsp70 chaperone Ssa1

    PubMed Central

    Needham, Patrick G.; Masison, Daniel C.

    2008-01-01

    We previously described many Hsp70 Ssa1p mutants that impair [PSI+] prion propagation in yeast without affecting cell growth. To determine how the mutations alter Hsp70 we analyzed biochemically the substrate-binding domain (SBD) mutant L483W and the nucleotide-binding domain (NBD) mutants A17V and R34K. Ssa1L483W ATPase activity was elevated 10-fold and was least stimulated by substrates or Hsp40 co-chaperones. Ssa1A17V and Ssa1R34K ATPase activities were nearly wild type but both showed increased stimulation by substrates. Peptide binding and reactivation of denatured luciferase were enhanced in Ssa1A17V and Ssa1R34K but compromised in Ssa1L483W. The nucleotide exchange factor Fes1 influenced ATPase of wild type Ssa1 and each mutant differently. Partial protease digestion uncovered similar and distinct conformational changes of the substrate-binding domain among the three mutants. Our data suggest that prion-impairing mutations of Ssa1 can increase or decrease substrate interactions, alter the Hsp70 reaction cycle at different points and impair normal NBD-SBD cooperation. PMID:18706386

  9. KCNC3R420H, a K+ Channel Mutation Causative in Spinocerebellar Ataxia 13 Displays Aberrant Intracellular Trafficking

    PubMed Central

    Gallego-Iradi, Carolina; Bickford, Justin S.; Khare, Swati; Hall, Alexis; Nick, Jerelyn A.; Salmasinia, Donya; Wawrowsky, Kolja; Bannykh, Serguei; Huynh, Duong P.; Rincon-Limas, Diego E.; Pulst, Stefan M.; Nick, Harry S.; Fernandez-Funez, Pedro; Waters, Michael F.

    2014-01-01

    Spinocerebellar ataxia 13 (SCA13) is an autosomal dominant disease resulting from mutations in KCNC3 (Kv3.3), a voltage-gated potassium channel. The KCNC3R420H mutation was first identified as causative for SCA13 in a four-generation Filipino kindred with over 20 affected individuals. Electrophysiological analyses in oocytes previously showed that this mutation did not lead to a functional channel and displayed a dominant negative phenotype. In an effort to identify the molecular basis of this allelic form of SCA13, we first determined that human KCNC3WT and KCNC3R420H display disparate posttranslational modifications, and the mutant protein has reduced complex glycan adducts. Immunohistochemical analyses demonstrated that KCNC3R420H was not properly trafficking to the plasma membrane and surface biotinylation demonstrated that KCNC3R420H exhibited only 24% as much surface expression as KCNC3WT. KCNC3R420H trafficked through the ER but was retained in the Golgi. KCNC3R420H expression results in altered Golgi and cellular morphology. Electron microscopy of KCNC3R420H localization further supports retention in the Golgi. These results are specific to the KCNC3R420H allele and provide new insight into the molecular basis of disease manifestation in SCA13. PMID:25152487

  10. Catecholamine stress alters neutrophil trafficking and impairs wound healing by β2-adrenergic receptor-mediated upregulation of IL-6.

    PubMed

    Kim, Min-Ho; Gorouhi, Farzam; Ramirez, Sandra; Granick, Jennifer L; Byrne, Barbara A; Soulika, Athena M; Simon, Scott I; Isseroff, R Rivkah

    2014-03-01

    Stress-induced hormones can alter the inflammatory response to tissue injury; however, the precise mechanism by which epinephrine influences inflammatory response and wound healing is not well defined. Here we demonstrate that epinephrine alters the neutrophil (polymorphonuclear leukocyte (PMN))-dependent inflammatory response to a cutaneous wound. Using noninvasive real-time imaging of genetically tagged PMNs in a murine skin wound, chronic, epinephrine-mediated stress was modeled by sustained delivery of epinephrine. Prolonged systemic exposure of epinephrine resulted in persistent PMN trafficking to the wound site via an IL-6-mediated mechanism, and this in turn impaired wound repair. Further, we demonstrate that β2-adrenergic receptor-dependent activation of proinflammatory macrophages is critical for epinephrine-mediated IL-6 production. This study expands our current understanding of stress hormone-mediated impairment of wound healing and provides an important mechanistic link to explain how epinephrine stress exacerbates inflammation via increased number and lifetime of PMNs. PMID:24121404

  11. Neurodegeneration mutations in dynactin impair dynein-dependent nuclear migration

    PubMed Central

    Moore, Jeffrey K.; Sept, David; Cooper, John A.

    2009-01-01

    Neurodegenerative disease in humans and mice can be caused by mutations affecting the microtubule motor dynein or its biochemical regulator, dynactin, a multiprotein complex required for dynein function (1–4). A single amino acid change, G59S, in the conserved cytoskeletal-associated protein glycine-rich (CAP-Gly) domain of the p150glued subunit of dynactin can cause motor neuron degeneration in humans and mice, which resembles ALS (2, 5–8). The molecular mechanism by which G59S impairs the function of dynein is not understood. Also, the relevance of the CAP-Gly domain for dynein motility has not been demonstrated in vivo. Here, we generate a mutant that is analogous to G59S in budding yeast, and show that this mutation produces a highly specific phenotype related to dynein function. The effect of the point mutation is identical to that of complete loss of the CAP-Gly domain. Our results demonstrate that the CAP-Gly domain has a critical role in the initiation and persistence of dynein-dependent movement of the mitotic spindle and nucleus, but it is otherwise dispensable for dynein-based movement. The need for this function appears to be context-dependent, and we speculate that CAP-Gly activity may only be necessary when dynein needs to overcome high force thresholds to produce movement. PMID:19279216

  12. ATP7A trafficking and mechanisms underlying the distal motor neuropathy induced by mutations in ATP7A.

    PubMed

    Yi, Ling; Kaler, Stephen

    2014-05-01

    Diverse mutations in the gene encoding the copper transporter ATP7A lead to X-linked recessive Menkes disease or occipital horn syndrome. Recently, two unique ATP7A missense mutations, T994I and P1386S, were shown to cause isolated adult-onset distal motor neuropathy. These mutations induce subtle defects in ATP7A intracellular trafficking resulting in preferential accumulation at the plasma membrane compared to wild-type ATP7A. Immunoprecipitation assays revealed abnormal interaction between ATP7A(T994I) and p97/VCP, a protein mutated in two autosomal dominant forms of motor neuron disease. Small-interfering RNA knockdown of valosin-containing protein corrected ATP7A(T994I) mislocalization. For ATP7A(P1386S) , flow cytometry documented that nonpermeabilized fibroblasts bound a C-terminal ATP7A antibody, suggesting unstable insertion of the eighth transmembrane segment due to a helix-breaker effect of the amino acid substitution. This could sabotage interaction of ATP7A(P1386S) with adaptor protein complexes. These molecular events appear to selectively disturb normal motor neuron function and lead to neurologic illness that takes years and sometimes decades to develop. PMID:24754450

  13. Age-Dependent Cell Trafficking Defects in Draining Lymph Nodes Impair Adaptive Immunity and Control of West Nile Virus Infection

    PubMed Central

    Richner, Justin M.; Gmyrek, Grzegorz B.; Govero, Jennifer; Tu, Yizheng; van der Windt, Gerritje J. W.; Metcalf, Talibah U.; Haddad, Elias K.; Textor, Johannes; Miller, Mark J.; Diamond, Michael S.

    2015-01-01

    Impaired immune responses in the elderly lead to reduced vaccine efficacy and increased susceptibility to viral infections. Although several groups have documented age-dependent defects in adaptive immune priming, the deficits that occur prior to antigen encounter remain largely unexplored. Herein, we identify novel mechanisms for compromised adaptive immunity that occurs with aging in the context of infection with West Nile virus (WNV), an encephalitic flavivirus that preferentially causes disease in the elderly. An impaired IgM and IgG response and enhanced vulnerability to WNV infection during aging was linked to delayed germinal center formation in the draining lymph node (DLN). Adoptive transfer studies and two-photon intravital microscopy revealed a decreased trafficking capacity of donor naïve CD4+ T cells from old mice, which manifested as impaired T cell diapedesis at high endothelial venules and reduced cell motility within DLN prior to antigen encounter. Furthermore, leukocyte accumulation in the DLN within the first few days of WNV infection or antigen-adjuvant administration was diminished more generally in old mice and associated with a second aging-related defect in local cytokine and chemokine production. Thus, age-dependent cell-intrinsic and environmental defects in the DLN result in delayed immune cell recruitment and antigen recognition. These deficits compromise priming of early adaptive immune responses and likely contribute to the susceptibility of old animals to acute WNV infection. PMID:26204259

  14. Fast track, dynein-dependent nuclear targeting of human immunodeficiency virus Vpr protein; impaired trafficking in a clinical isolate.

    PubMed

    Caly, Leon; Kassouf, Vicki T; Moseley, Gregory W; Diefenbach, Russell J; Cunningham, Anthony L; Jans, David A

    2016-02-12

    Nuclear import of the accessory protein Vpr is central to infection by human immunodeficiency virus (HIV). We previously identified the Vpr F72L mutation in a HIV-infected, long-term non-progressor, showing that it resulted in reduced Vpr nuclear accumulation and altered cytoplasmic localisation. Here we demonstrate for the first time that the effects of nuclear accumulation of the F72L mutation are due to impairment of microtubule-dependent-enhancement of Vpr nuclear import. We use high resolution imaging approaches including fluorescence recovery after photobleaching and other approaches to document interaction between Vpr and the dynein light chain protein, DYNLT1, and impaired interaction of the F72L mutant with DYNLT1. The results implicate MTs/DYNLT1 as drivers of Vpr nuclear import and HIV infection, with important therapeutic implications. PMID:26792716

  15. Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain.

    PubMed

    Poirier, Steve; Hamouda, Hocine Ait; Villeneuve, Louis; Demers, Annie; Mayer, Gaétan

    2016-01-01

    PCSK9 is a secreted ligand and negative post-translational regulator of low-density lipoprotein receptor (LDLR) in hepatocytes. Gain-of-function (GOF) or loss-of-function (LOF) mutations in PCSK9 are directly correlated with high or low plasma LDL-cholesterol levels, respectively. Therefore, PCSK9 is a prevailing lipid-lowering target to prevent coronary heart diseases and stroke. Herein, we fused monomeric fluorescent proteins to PCSK9 and LDLR to visualize their intra- and extracellular trafficking dynamics by live confocal microscopy. Fluorescence recovery after photobleaching (FRAP) showed that PCSK9 LOF R46L mutant and GOF mutations S127R and D129G, but not the LDLR high-affinity mutant D374Y, significantly accelerate PCSK9 exit from the endoplasmic reticulum (ER). Quantitative analysis of inverse FRAP revealed that only R46L presented a much slower trafficking from the trans-Golgi network (TGN) to the plasma membrane and a lower mobile fraction likely suggesting accumulation or delayed exit at the TGN as an underlying mechanism. While not primarily involved in LDLR binding, PCSK9 C-terminal domain (CTD) was found to be essential to induce LDLR degradation both upon its overexpression in cells or via the extracellular pathway. Our data revealed that PCSK9 CTD is required for the localization of PCSK9 at the TGN and increases its LDLR-mediated endocytosis. Interestingly, intracellular lysosomal targeting of PCSK9-ΔCTD was able to rescue its capacity to induce LDLR degradation emphasizing a role of the CTD in the sorting of PCSK9-LDLR complex towards late endocytic compartments. Finally, we validated our dual fluorescence system as a cell based-assay by preventing PCSK9 internalization using a PCSK9-LDLR blocking antibody, which may be expended to identify protein, peptide or small molecule inhibitors of PCSK9. PMID:27280970

  16. Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain

    PubMed Central

    Villeneuve, Louis; Demers, Annie; Mayer, Gaétan

    2016-01-01

    PCSK9 is a secreted ligand and negative post-translational regulator of low-density lipoprotein receptor (LDLR) in hepatocytes. Gain-of-function (GOF) or loss-of-function (LOF) mutations in PCSK9 are directly correlated with high or low plasma LDL-cholesterol levels, respectively. Therefore, PCSK9 is a prevailing lipid-lowering target to prevent coronary heart diseases and stroke. Herein, we fused monomeric fluorescent proteins to PCSK9 and LDLR to visualize their intra- and extracellular trafficking dynamics by live confocal microscopy. Fluorescence recovery after photobleaching (FRAP) showed that PCSK9 LOF R46L mutant and GOF mutations S127R and D129G, but not the LDLR high-affinity mutant D374Y, significantly accelerate PCSK9 exit from the endoplasmic reticulum (ER). Quantitative analysis of inverse FRAP revealed that only R46L presented a much slower trafficking from the trans-Golgi network (TGN) to the plasma membrane and a lower mobile fraction likely suggesting accumulation or delayed exit at the TGN as an underlying mechanism. While not primarily involved in LDLR binding, PCSK9 C-terminal domain (CTD) was found to be essential to induce LDLR degradation both upon its overexpression in cells or via the extracellular pathway. Our data revealed that PCSK9 CTD is required for the localization of PCSK9 at the TGN and increases its LDLR-mediated endocytosis. Interestingly, intracellular lysosomal targeting of PCSK9-ΔCTD was able to rescue its capacity to induce LDLR degradation emphasizing a role of the CTD in the sorting of PCSK9-LDLR complex towards late endocytic compartments. Finally, we validated our dual fluorescence system as a cell based-assay by preventing PCSK9 internalization using a PCSK9-LDLR blocking antibody, which may be expended to identify protein, peptide or small molecule inhibitors of PCSK9. PMID:27280970

  17. MiR-17-5p Impairs Trafficking of H-ERG K+ Channel Protein by Targeting Multiple ER Stress-Related Chaperones during Chronic Oxidative Stress

    PubMed Central

    Wang, Qi; Hu, Weina; Lei, Mingming; Wang, Yong; Yan, Bing; Liu, Jun; Zhang, Ren; Jin, Yuanzhe

    2013-01-01

    Background To investigate if microRNAs (miRNAs) play a role in regulating h-ERG trafficking in the setting of chronic oxidative stress as a common deleterious factor for many cardiac disorders. Methods We treated neonatal rat ventricular myocytes and HEK293 cells with stable expression of h-ERG with H2O2 for 12 h and 48 h. Expression of miR-17-5p seed miRNAs was quantified by real-time RT-PCR. Protein levels of chaperones and h-ERG trafficking were measured by Western blot analysis. Luciferase reporter gene assay was used to study miRNA and target interactions. Whole-cell patch-clamp techniques were employed to record h-ERG K+ current. Results H-ERG trafficking was impaired by H2O2 after 48 h treatment, accompanied by reciprocal changes of expression between miR-17-5p seed miRNAs and several chaperones (Hsp70, Hsc70, CANX, and Golga2), with the former upregulated and the latter downregulated. We established these chaperones as targets for miR-17-5p. Application miR-17-5p inhibitor rescued H2O2-induced impairment of h-ERG trafficking. Upregulation of endogenous by H2O2 or forced miR-17-5p expression either reduced h-ERG current. Sequestration of AP1 by its decoy molecule eliminated the upregulation of miR-17-5p, and ameliorated impairment of h-ERG trafficking. Conclusions Collectively, deregulation of the miR-17-5p seed family miRNAs can cause severe impairment of h-ERG trafficking through targeting multiple ER stress-related chaperones, and activation of AP1 likely accounts for the deleterious upregulation of these miRNAs, in the setting of prolonged duration of oxidative stress. These findings revealed the role of miRNAs in h-ERG trafficking, which may contribute to the cardiac electrical disturbances associated with oxidative stress. PMID:24386440

  18. A Mutation in the Vesicle-Trafficking Protein VAPB Causes Late-Onset Spinal Muscular Atrophy and Amyotrophic Lateral Sclerosis

    PubMed Central

    Nishimura, Agnes L.; Mitne-Neto, Miguel; Silva, Helga C. A.; Richieri-Costa, Antônio; Middleton, Susan; Cascio, Duilio; Kok, Fernando; Oliveira, João R. M.; Gillingwater, Tom; Webb, Jeanette; Skehel, Paul; Zatz, Mayana

    2004-01-01

    Motor neuron diseases (MNDs) are a group of neurodegenerative disorders with involvement of upper and/or lower motor neurons, such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), progressive bulbar palsy, and primary lateral sclerosis. Recently, we have mapped a new locus for an atypical form of ALS/MND (atypical amyotrophic lateral sclerosis [ALS8]) at 20q13.3 in a large white Brazilian family. Here, we report the finding of a novel missense mutation in the vesicle-associated membrane protein/synaptobrevin-associated membrane protein B (VAPB) gene in patients from this family. Subsequently, the same mutation was identified in patients from six additional kindreds but with different clinical courses, such as ALS8, late-onset SMA, and typical severe ALS with rapid progression. Although it was not possible to link all these families, haplotype analysis suggests a founder effect. Members of the vesicle-associated proteins are intracellular membrane proteins that can associate with microtubules and that have been shown to have a function in membrane transport. These data suggest that clinically variable MNDs may be caused by a dysfunction in intracellular membrane trafficking. PMID:15372378

  19. Human Trafficking

    MedlinePlus

    ... TRAFFICKING (English) Listen < Back to Search FACT SHEET: HUMAN TRAFFICKING (English) Published: August 2, 2012 Topics: Public Awareness , ... organizations that protect and serve trafficking victims. National Human Trafficking Resource Center at 1.888.373.7888 Last ...

  20. How mutational epistasis impairs predictability in protein evolution and design.

    PubMed

    Miton, Charlotte M; Tokuriki, Nobuhiko

    2016-07-01

    There has been much debate about the extent to which mutational epistasis, that is, the dependence of the outcome of a mutation on the genetic background, constrains evolutionary trajectories. The degree of unpredictability introduced by epistasis, due to the non-additivity of functional effects, strongly hinders the strategies developed in protein design and engineering. While many studies have addressed this issue through systematic characterization of evolutionary trajectories within individual enzymes, the field lacks a consensus view on this matter. In this work, we performed a comprehensive analysis of epistasis by analyzing the mutational effects from nine adaptive trajectories toward new enzymatic functions. We quantified epistasis by comparing the effect of mutations occurring between two genetic backgrounds: the starting enzyme (for example, wild type) and the intermediate variant on which the mutation occurred during the trajectory. We found that most trajectories exhibit positive epistasis, in which the mutational effect is more beneficial when it occurs later in the evolutionary trajectory. Approximately half (49%) of functional mutations were neutral or negative on the wild-type background, but became beneficial at a later stage in the trajectory, indicating that these functional mutations were not predictable from the initial starting point. While some cases of strong epistasis were associated with direct interaction between residues, many others were caused by long-range indirect interactions between mutations. Our work highlights the prevalence of epistasis in enzyme adaptive evolution, in particular positive epistasis, and suggests the necessity of incorporating mutational epistasis in protein engineering and design to create highly efficient catalysts. PMID:26757214

  1. Rare Mutations in Renal Sodium and Potassium Transporter Genes Exhibit Impaired Transport Function

    PubMed Central

    Welling, Paul A.

    2014-01-01

    Purpose of review Recent efforts to explore the genetic underpinnings of hypertension revealed rare mutations in kidney salt transport genes contribute to blood pressure variation and hypertension susceptibility in the general population. The current review focuses on these latest findings, highlighting a discussion about the rare mutations and how they affect transport function. Recent findings Rare mutations that confer a low blood pressure trait and resistance to hypertension have recently been extensively studied. Physiological and biochemical analyses of the effected renal salt transport molecules (NKCC2 (SLC12A1), ROMK (KCNJ1), and NCC (SLC12A3)) revealed that most of the mutations do, in fact, cause a loss of transport function. The mutations disrupt transport by many different mechanisms, including altering biosynthetic processing, trafficking, ion transport, and regulation. Summary New insights into the genetic basis of hypertension have recently emerged, supporting a major role of rare, rather than common, gene variants. Many different rare mutations have been found to affect the functions of different salt transporter genes by different mechanisms, yet all confer the same blood pressure phenotype. These studies reinforce the critical roles of the kidney, and renal salt transport in blood pressure regulation and hypertension. PMID:24253496

  2. ATRX mutations and glioblastoma: Impaired DNA damage repair, alternative lengthening of telomeres, and genetic instability.

    PubMed

    Koschmann, Carl; Lowenstein, Pedro R; Castro, Maria G

    2016-05-01

    Alpha thalassemia/mental retardation syndrome X-linked (ATRX) is mutated in nearly a third of pediatric glioblastoma (GBM) patients. We developed an animal model of ATRX-deficient GBM. Using this model combined with analysis of multiple human glioma genome-wide datasets, we determined that ATRX mutation leads to genetic instability, impaired non-homologous end joining, and alternate lengthening of telomeres (ALT). PMID:27314101

  3. Homeostatic regulation of T cell trafficking by a B cell derived peptide is impaired in autoimmune and chronic inflammatory disease

    PubMed Central

    Apta, Bonita; Kuravi, Sahithi J.; Yates, Clara M.; Kennedy, Amy; Odedra, Arjun; Alassiri, Mohammed; Harrison, Matthew; Martin, Ashley; Barone, Francesca; Nayar, Saba; Hitchcock, Jessica R.; Cunningham, Adam F.; Raza, Karim; Filer, Andrew; Copland, David A.; Dick, Andrew D.; Robinson, Joseph; Kalia, Neena; Walker, Lucy S. K.; Buckley, Christopher D.; Nash, Gerard B.; Narendran, Parth; Rainger, G. Ed.

    2015-01-01

    During an inflammatory response, lymphocyte recruitment into tissue must be tightly controlled because dysregulated trafficking contributes to the pathogenesis of chronic disease. Here we show that during inflammation and in response to adiponectin, B cells tonically inhibit T cell trafficking by secreting a peptide (PEPITEM) proteolytically derived from 14.3.3.ζδ protein. PEPITEM binds cadherin-15 on endothelial cells, promoting synthesis and release of sphingosine-1 phosphate, which inhibits trafficking of T cells without affecting recruitment of other leukocytes. Expression of adiponectin receptors on B cells and adiponectin induced PEPITEM secretion wanes with age, implying immune senescence of the pathway. Additionally, these changes are evident in individuals with type-1-diabetes or rheumatoid arthritis, and circulating PEPITEM in patient serum is reduced compared to healthy age matched donors. In both diseases, tonic inhibition of T cell trafficking across inflamed endothelium is lost. Importantly, control of patient T cell trafficking is re-established by exogenous PEPITEM. Moreover, in animal models of peritonitis, hepatic I/R injury, Salmonella infection, Uveitis and Sjögren’s Syndrome, PEPITEM could reduce T cell recruitment into inflamed tissues. PMID:25894827

  4. Clinical findings in nondemented mutation carriers predisposed to Alzheimer's disease: a model of mild cognitive impairment.

    PubMed

    Almkvist, Ove; Axelman, Karin; Basun, Hans; Jensen, Malene; Viitanen, Matti; Wahlund, Lars-Olof; Lannfelt, Lars

    2003-01-01

    Individuals carrying a mutation associated with Alzheimer's disease (AD) may serve as a model of mild cognitive impairment (MCI). Nondemented individuals from these families can be subdivided into asymptomatic and symptomatic groups. Four families were studied. Two families are associated with APP mutations (KN670/671ML, E693G) and two with PS1 mutation (M146V, H163Y). Clinical symptoms, level of global cognitive functioning as evaluated by Mini-Mental State Examination, neuropsychological test results, neuroradiological examinations (magnetic resonance imaging (MRI) and single-photon emission tomography (SPECT)), as well as cerebrospinal fluid (CSF) measurements of tau and beta-amyloid are reported. Nondemented mutation carriers did not report any symptoms indicating cognitive decline. In addition, no clinical signs of dementia or marked cognitive impairment in neuropsychological tests were found. A reduction of temporal blood flow with SPECT was indicated in 5/13 nondemented mutation carriers. Two of these 13 individuals had moderate hyperintensities in deep white matter as observed on MRI. CSF measurements of A beta 42/43 were inconclusive because of large biological variation. A nonsignificant elevation of tau was detected in mutation carriers. In conclusion, clinical examinations of relatively young individuals carrying an AD mutation did not reveal any marked abnormalities before the clinical onset of dementia. PMID:12603253

  5. Mutations in the carboxyl-terminal SEC24 binding motif of the serotonin transporter impair folding of the transporter.

    PubMed

    El-Kasaby, Ali; Just, Herwig; Malle, Elisabeth; Stolt-Bergner, Peggy C; Sitte, Harald H; Freissmuth, Michael; Kudlacek, Oliver

    2010-12-10

    The serotonin transporter (SERT) is a member of the SLC6 family of solute carriers. SERT plays a crucial role in synaptic neurotransmission by retrieving released serotonin. The intracellular carboxyl terminus of various neurotransmitter transporters has been shown to be important for the correct delivery of SLC6 family members to the cell surface. Here we studied the importance of the C terminus in trafficking and folding of human SERT. Serial truncations followed by mutagenesis identified sequence spots (PG(601,602), RII(607-609)) within the C terminus relevant for export of SERT from the endoplasmic reticulum (ER). RI(607,608) is homologous to the RL-motif that in other SLC6 family members provides a docking site for the COPII component Sec24D. The primary defect resulting from mutation at PG(601,602) and RI(607,608) was impaired folding, because mutated transporters failed to bind the inhibitor [(3)H]imipramine. In contrast, when retained in the ER (e.g. by dominant negative Sar1) the wild type transporter bound [(3)H]imipramine with an affinity comparable to that of the surface-expressed transporter. SERT-RI(607,608)AA and SERT-RII(607-609)AAA were partially rescued by treatment of cells with the nonspecific chemical chaperone DMSO or the specific pharmacochaperone ibogaine (which binds to the inward facing conformation of SERT) but not by other classes of ligands (inhibitors, substrates, amphetamines). These observations (i) demonstrate an hitherto unappreciated role of the C terminus in the folding of SERT, (ii) indicates that the folding trajectory proceeds via an inward facing intermediate, and (iii) suggest a model where the RI-motif plays a crucial role in preventing premature Sec24-recruitment and export of incorrectly folded transporters. PMID:20889976

  6. Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation.

    PubMed

    Valentin-Vega, Yasmine A; Wang, Yong-Dong; Parker, Matthew; Patmore, Deanna M; Kanagaraj, Anderson; Moore, Jennifer; Rusch, Michael; Finkelstein, David; Ellison, David W; Gilbertson, Richard J; Zhang, Jinghui; Kim, Hong Joo; Taylor, J Paul

    2016-01-01

    DDX3X is a DEAD-box RNA helicase that has been implicated in multiple aspects of RNA metabolism including translation initiation and the assembly of stress granules (SGs). Recent genomic studies have reported recurrent DDX3X mutations in numerous tumors including medulloblastoma (MB), but the physiological impact of these mutations is poorly understood. Here we show that a consistent feature of MB-associated mutations is SG hyper-assembly and concomitant translation impairment. We used CLIP-seq to obtain a comprehensive assessment of DDX3X binding targets and ribosome profiling for high-resolution assessment of global translation. Surprisingly, mutant DDX3X expression caused broad inhibition of translation that impacted DDX3X targeted and non-targeted mRNAs alike. Assessment of translation efficiency with single-cell resolution revealed that SG hyper-assembly correlated precisely with impaired global translation. SG hyper-assembly and translation impairment driven by mutant DDX3X were rescued by a genetic approach that limited SG assembly and by deletion of the N-terminal low complexity domain within DDX3X. Thus, in addition to a primary defect at the level of translation initiation caused by DDX3X mutation, SG assembly itself contributes to global translation inhibition. This work provides mechanistic insights into the consequences of cancer-related DDX3X mutations, suggesting that globally reduced translation may provide a context-dependent survival advantage that must be considered as a possible contributor to tumorigenesis. PMID:27180681

  7. Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation

    PubMed Central

    Valentin-Vega, Yasmine A.; Wang, Yong-Dong; Parker, Matthew; Patmore, Deanna M.; Kanagaraj, Anderson; Moore, Jennifer; Rusch, Michael; Finkelstein, David; Ellison, David W.; Gilbertson, Richard J.; Zhang, Jinghui; Kim, Hong Joo; Taylor, J. Paul

    2016-01-01

    DDX3X is a DEAD-box RNA helicase that has been implicated in multiple aspects of RNA metabolism including translation initiation and the assembly of stress granules (SGs). Recent genomic studies have reported recurrent DDX3X mutations in numerous tumors including medulloblastoma (MB), but the physiological impact of these mutations is poorly understood. Here we show that a consistent feature of MB-associated mutations is SG hyper-assembly and concomitant translation impairment. We used CLIP-seq to obtain a comprehensive assessment of DDX3X binding targets and ribosome profiling for high-resolution assessment of global translation. Surprisingly, mutant DDX3X expression caused broad inhibition of translation that impacted DDX3X targeted and non-targeted mRNAs alike. Assessment of translation efficiency with single-cell resolution revealed that SG hyper-assembly correlated precisely with impaired global translation. SG hyper-assembly and translation impairment driven by mutant DDX3X were rescued by a genetic approach that limited SG assembly and by deletion of the N-terminal low complexity domain within DDX3X. Thus, in addition to a primary defect at the level of translation initiation caused by DDX3X mutation, SG assembly itself contributes to global translation inhibition. This work provides mechanistic insights into the consequences of cancer-related DDX3X mutations, suggesting that globally reduced translation may provide a context-dependent survival advantage that must be considered as a possible contributor to tumorigenesis. PMID:27180681

  8. Study of modifiers factors associated to mitochondrial mutations in individuals with hearing impairment

    SciTech Connect

    Sousa de Moraes, Vanessa Cristine; Alexandrino, Fabiana; Andrade, Paula Baloni; Camara, Marilia Fontenele; Sartorato, Edi Lucia

    2009-04-03

    Hearing impairment is the most prevalent sensorial deficit in the general population. Congenital deafness occurs in about 1 in 1000 live births, of which approximately 50% has hereditary cause in development countries. Non-syndromic deafness can be caused by mutations in both nuclear and mitochondrial genes. Mutations in mtDNA have been associated with aminoglycoside-induced and non-syndromic deafness in many families worldwide. However, the nuclear background influences the phenotypic expression of these pathogenic mutations. Indeed, it has been proposed that nuclear modifier genes modulate the phenotypic manifestation of the mitochondrial A1555G mutation in the MTRNR1 gene. The both putative nuclear modifiers genes TRMU and MTO1 encoding a highly conserved mitochondrial related to tRNA modification. It has been hypothesizes that human TRMU and also MTO1 nuclear genes may modulate the phenotypic manifestation of deafness-associated mitochondrial mutations. The aim of this work was to elucidate the contribution of mitochondrial mutations, nuclear modifier genes mutations and aminoglycoside exposure in the deafness phenotype. Our findings suggest that the genetic background of individuals may play an important role in the pathogenesis of deafness-associated with mitochondrial mutation and aminoglycoside-induced.

  9. A human CCT5 gene mutation causing distal neuropathy impairs hexadecamer assembly in an archaeal model

    PubMed Central

    Min, Wonki; Angileri, Francesca; Luo, Haibin; Lauria, Antonino; Shanmugasundaram, Maruda; Almerico, Anna Maria; Cappello, Francesco; de Macario, Everly Conway; Lednev, Igor K.; Macario, Alberto J. L.; Robb, Frank T.

    2014-01-01

    Chaperonins mediate protein folding in a cavity formed by multisubunit rings. The human CCT has eight non-identical subunits and the His147Arg mutation in one subunit, CCT5, causes neuropathy. Knowledge is scarce on the impact of this and other mutations upon the chaperone's structure and functions. To make progress, experimental models must be developed. We used an archaeal mutant homolog and demonstrated that the His147Arg mutant has impaired oligomeric assembly, ATPase activity, and defective protein homeostasis functions. These results establish for the first time that a human chaperonin gene defect can be reproduced and studied at the molecular level with an archaeal homolog. The major advantage of the system, consisting of rings with eight identical subunits, is that it amplifies the effects of a mutation as compared with the human counterpart, in which just one subunit per ring is defective. Therefore, the slight deficit of a non-lethal mutation can be detected and characterized. PMID:25345891

  10. R152C DNA Pol β mutation impairs base excision repair and induces cellular transformation

    PubMed Central

    Zhao, Jing; Sun, Hongfang; Zhou, Xiaolong; Wu, Xuping; He, Lingfeng; Hu, Zhigang; Chen, Haoyan; Shen, Binghui; Guo, Zhigang

    2016-01-01

    DNA polymerase β (Pol β) is a key enzyme in DNA base excision repair (BER), a pathway that maintains genome integrity and stability. Pol β mutations have been detected in various types of cancers, suggesting a possible linkage between Pol β mutations and cancer. However, it is not clear whether and how Pol β mutations cause cancer onset and progression. In the current work, we show that a substitution mutation, R152C, impairs Pol β polymerase activity and BER efficiency. Cells harboring Pol β R152C are sensitive to the DNA damaging agents methyl methanesulfonate (MMS) and H2O2. Moreover, the mutant cells display a high frequency of chromatid breakages and aneuploidy and also form foci. Taken together, our data indicate that Pol β R152C can drive cellular transformation. PMID:26760506

  11. A Novel Splice-Site Mutation in the GJB2 Gene Causing Mild Postlingual Hearing Impairment

    PubMed Central

    Gandía, Marta; del Castillo, Francisco J.; Rodríguez-Álvarez, Francisco J.; Garrido, Gema; Villamar, Manuela; Calderón, Manuela; Moreno-Pelayo, Miguel A.; Moreno, Felipe; del Castillo, Ignacio

    2013-01-01

    The DFNB1 subtype of autosomal recessive, nonsyndromic hearing impairment, caused by mutations affecting the GJB2 (connection-26) gene, is highly prevalent in most populations worldwide. DFNB1 hearing impairment is mostly severe or profound and usually appears before the acquisition of speech (prelingual onset), though a small number of hypomorphic missense mutations result in mild or moderate deafness of postlingual onset. We identified a novel GJB2 splice-site mutation, c. -22-2A>C, in three siblings with mild postlingual hearing impairment that were compound heterozygous for c. -22-2A>C and c.35delG. Reverse transcriptase-PCR experiments performed on total RNA extracted from saliva samples from one of these siblings confirmed that c. -22-2A>C abolished the acceptor splice site of the single GJB2 intron, resulting in the absence of normally processed transcripts from this allele. However, we did isolate transcripts from the c. -22-2A>C allele that keep an intact GJB2 coding region and that were generated by use of an alternative acceptor splice site previously unknown. The residual expression of wild-type connection-26 encoded by these transcripts probably underlies the mild severity and late onset of the hearing impairment of these subjects. PMID:24039984

  12. PROKR2 missense mutations associated with Kallmann syndrome impair receptor signalling activity.

    PubMed

    Monnier, Carine; Dodé, Catherine; Fabre, Ludovic; Teixeira, Luis; Labesse, Gilles; Pin, Jean-Philippe; Hardelin, Jean-Pierre; Rondard, Philippe

    2009-01-01

    Kallmann syndrome (KS) combines hypogonadism due to gonadotropin-releasing hormone deficiency, and anosmia or hyposmia, related to defective olfactory bulb morphogenesis. In a large series of KS patients, ten different missense mutations (p.R85C, p.R85H, p.R164Q, p.L173R, p.W178S, p.Q210R, p.R268C, p.P290S, p.M323I, p.V331M) have been identified in the gene encoding the G protein-coupled receptor prokineticin receptor-2 (PROKR2), most often in the heterozygous state. Many of these mutations were, however, also found in clinically unaffected individuals, thus raising the question of their actual implication in the KS phenotype. We reproduced each of the ten mutations in a recombinant murine Prokr2, and tested their effects on the signalling activity in transfected HEK-293 cells, by measuring intracellular calcium release upon ligand-activation of the receptor. We found that all mutated receptors except one (M323I) had decreased signalling activities. These could be explained by different defective mechanisms. Three mutations (L173R, W178S, P290S) impaired cell surface-targeting of the receptor. One mutation (Q210R) abolished ligand-binding. Finally, five mutations (R85C, R85H, R164Q, R268C, V331M) presumably impaired G protein-coupling of the receptor. In addition, when wild-type and mutant receptors were coexpressed in HEK-293 cells, none of the mutant receptors that were retained within the cells did affect cell surface-targeting of the wild-type receptor, and none of the mutant receptors properly addressed at the plasma membrane did affect wild-type receptor signalling activity. This argues against a dominant negative effect of the mutations in vivo. PMID:18826963

  13. PROKR2 missense mutations associated with Kallmann syndrome impair receptor signalling activity

    PubMed Central

    Monnier, Carine; Dodé, Catherine; Fabre, Ludovic; Teixeira, Luis; Labesse, Gilles; Pin, Jean-Philippe; Hardelin, Jean-Pierre; Rondard, Philippe

    2009-01-01

    Kallmann syndrome (KS) combines hypogonadism due to gonadotropin-releasing hormone deficiency, and anosmia or hyposmia, related to defective olfactory bulb morphogenesis. In a large series of KS patients, ten different missense mutations (p.R85C, p.R85H, p.R164Q, p.L173R, p.W178S, p.Q210R, p.R268C, p.P290S, p.M323I, p.V331M) have been identified in the gene encoding the G protein-coupled receptor prokineticin receptor-2 (PROKR2), most often in the heterozygous state. Many of these mutations were, however, also found in clinically unaffected individuals, thus raising the question of their actual implication in the KS phenotype. We reproduced each of the ten mutations in a recombinant murine Prokr2, and tested their effects on the signalling activity in transfected HEK-293 cells, by measuring intracellular calcium release upon ligand-activation of the receptor. We found that all mutated receptors except one (M323I) had decreased signalling activities. These could be explained by different defective mechanisms. Three mutations (L173R, W178S, P290S) impaired cell surface-targeting of the receptor. One mutation (Q210R) abolished ligand-binding. Finally, five mutations (R85C, R85H, R164Q, R268C, V331M) presumably impaired G protein-coupling of the receptor. In addition, when wild-type and mutant receptors were coexpressed in HEK-293 cells, none of the mutant receptors that were retained within the cells did affect cell surface-targeting of the wild-type receptor, and none of the mutant receptors properly addressed at the plasma membrane did affect wild-type receptor signalling activity. This argues against a dominant negative effect of the mutations in vivo. PMID:18826963

  14. PLEKHM2 mutation leads to abnormal localization of lysosomes, impaired autophagy flux and associates with recessive dilated cardiomyopathy and left ventricular noncompaction.

    PubMed

    Muhammad, Emad; Levitas, Aviva; Singh, Sonia R; Braiman, Alex; Ofir, Rivka; Etzion, Sharon; Sheffield, Val C; Etzion, Yoram; Carrier, Lucie; Parvari, Ruti

    2015-12-20

    Gene mutations, mostly segregating with a dominant mode of inheritance, are important causes of dilated cardiomyopathy (DCM), a disease characterized by enlarged ventricular dimensions, impaired cardiac function, heart failure and high risk of death. Another myocardial abnormality often linked to gene mutations is left ventricular noncompaction (LVNC) characterized by a typical diffuse spongy appearance of the left ventricle. Here, we describe a large Bedouin family presenting with a severe recessive DCM and LVNC. Homozygosity mapping and exome sequencing identified a single gene variant that segregated as expected and was neither reported in databases nor in Bedouin population controls. The PLEKHM2 cDNA2156_2157delAG variant causes the frameshift p.Lys645AlafsTer12 and/or the skipping of exon 11 that results in deletion of 30 highly conserved amino acids. PLEKHM2 is known to interact with several Rabs and with kinesin-1, affecting endosomal trafficking. Accordingly, patients' primary fibroblasts exhibited abnormal subcellular distribution of endosomes marked by Rab5, Rab7 and Rab9, as well as the Golgi apparatus. In addition, lysosomes appeared to be concentrated in the perinuclear region, and autophagy flux was impaired. Transfection of wild-type PLEKHM2 cDNA into patient's fibroblasts corrected the subcellular distribution of the lysosomes, supporting the causal effect of PLEKHM2 mutation. PLEKHM2 joins LAMP-2 and BAG3 as a disease gene altering autophagy resulting in an isolated cardiac phenotype. The association of PLEKHM2 mutation with DCM and LVNC supports the importance of autophagy for normal cardiac function. PMID:26464484

  15. Trafficking defects and loss of ligand binding are the underlying causes of all reported DDR2 missense mutations found in SMED-SL patients.

    PubMed

    Ali, Bassam R; Xu, Huifang; Akawi, Nadia A; John, Anne; Karuvantevida, Noushad S; Langer, Ruth; Al-Gazali, Lihadh; Leitinger, Birgit

    2010-06-01

    Spondylo-meta-epiphyseal dysplasia (SMED) with short limbs and abnormal calcifications (SMED-SL) is a rare, autosomal recessive human growth disorder, characterized by disproportionate short stature, short limbs, short broad fingers, abnormal metaphyses and epiphyses, platyspondyly and premature calcifications. Recently, three missense mutations and one splice-site mutation in the DDR2 gene were identified as causative genetic defects for SMED-SL, but the underlying cellular and biochemical mechanisms were not explored. Here we report a novel DDR2 missense mutation, c.337G>A (p.E113K), that causes SMED-SL in two siblings in the United Arab Emirates. Another DDR2 missense mutation, c.2254C>T (p.R752C), matching one of the previously reported SMED-SL mutations, was found in a second affected family. DDR2 is a plasma membrane receptor tyrosine kinase that functions as a collagen receptor. We expressed DDR2 constructs with the identified point mutations in human cell lines and evaluated their localization and functional properties. We found that all SMED-SL missense mutants were defective in collagen-induced receptor activation and that the three previously reported mutants (p.T713I, p.I726R and p.R752C) were retained in the endoplasmic reticulum. The novel mutant (p.E113K), in contrast, trafficked normally, like wild-type DDR2, but failed to bind collagen. This finding is in agreement with our recent structural data identifying Glu113 as an important amino acid in the DDR2 ligand-binding site. Our data thus demonstrate that SMED-SL can result from at least two different loss-of-function mechanisms: namely defects in DDR2 targeting to the plasma membrane or the loss of its ligand-binding activity. PMID:20223752

  16. Recessive mutations in POLR1C cause a leukodystrophy by impairing biogenesis of RNA polymerase III

    PubMed Central

    Thiffault, Isabelle; Wolf, Nicole I.; Forget, Diane; Guerrero, Kether; Tran, Luan T.; Choquet, Karine; Lavallée-Adam, Mathieu; Poitras, Christian; Brais, Bernard; Yoon, Grace; Sztriha, Laszlo; Webster, Richard I.; Timmann, Dagmar; van de Warrenburg, Bart P.; Seeger, Jürgen; Zimmermann, Alíz; Máté, Adrienn; Goizet, Cyril; Fung, Eva; van der Knaap, Marjo S.; Fribourg, Sébastien; Vanderver, Adeline; Simons, Cas; Taft, Ryan J.; Yates III, John R.; Coulombe, Benoit; Bernard, Geneviève

    2015-01-01

    A small proportion of 4H (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism) or RNA polymerase III (POLR3)-related leukodystrophy cases are negative for mutations in the previously identified causative genes POLR3A and POLR3B. Here we report eight of these cases carrying recessive mutations in POLR1C, a gene encoding a shared POLR1 and POLR3 subunit, also mutated in some Treacher Collins syndrome (TCS) cases. Using shotgun proteomics and ChIP sequencing, we demonstrate that leukodystrophy-causative mutations, but not TCS mutations, in POLR1C impair assembly and nuclear import of POLR3, but not POLR1, leading to decreased binding to POLR3 target genes. This study is the first to show that distinct mutations in a gene coding for a shared subunit of two RNA polymerases lead to selective modification of the enzymes' availability leading to two different clinical conditions and to shed some light on the pathophysiological mechanism of one of the most common hypomyelinating leukodystrophies, POLR3-related leukodystrophy. PMID:26151409

  17. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation.

    PubMed

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease. PMID:27570485

  18. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation

    PubMed Central

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease. PMID:27570485

  19. Mutation of Dcdc2 in mice leads to impairments in auditory processing and memory ability.

    PubMed

    Truong, D T; Che, A; Rendall, A R; Szalkowski, C E; LoTurco, J J; Galaburda, A M; Holly Fitch, R

    2014-11-01

    Dyslexia is a complex neurodevelopmental disorder characterized by impaired reading ability despite normal intellect, and is associated with specific difficulties in phonological and rapid auditory processing (RAP), visual attention and working memory. Genetic variants in Doublecortin domain-containing protein 2 (DCDC2) have been associated with dyslexia, impairments in phonological processing and in short-term/working memory. The purpose of this study was to determine whether sensory and behavioral impairments can result directly from mutation of the Dcdc2 gene in mice. Several behavioral tasks, including a modified pre-pulse inhibition paradigm (to examine auditory processing), a 4/8 radial arm maze (to assess/dissociate working vs. reference memory) and rotarod (to examine sensorimotor ability and motor learning), were used to assess the effects of Dcdc2 mutation. Behavioral results revealed deficits in RAP, working memory and reference memory in Dcdc2(del2/del2) mice when compared with matched wild types. Current findings parallel clinical research linking genetic variants of DCDC2 with specific impairments of phonological processing and memory ability. PMID:25130614

  20. Impaired replication stress response in cells from immunodeficiency patients carrying Cernunnos/XLF mutations.

    PubMed

    Schwartz, Michal; Oren, Yifat S; Bester, Assaf C; Rahat, Ayelet; Sfez, Ruthy; Yitzchaik, Shlomo; de Villartay, Jean-Pierre; Kerem, Batsheva

    2009-01-01

    Non-Homologous End Joining (NHEJ) is one of the two major pathways of DNA Double Strand Breaks (DSBs) repair. Mutations in human NHEJ genes can lead to immunodeficiency due to its role in V(D)J recombination in the immune system. In addition, most patients carrying mutations in NHEJ genes display developmental anomalies which are likely the result of a general defect in repair of endogenously induced DSBs such as those arising during normal DNA replication. Cernunnos/XLF is a recently identified NHEJ gene which is mutated in immunodeficiency with microcephaly patients. Here we aimed to investigate whether Cernunnos/XLF mutations disrupt the ability of patient cells to respond to replication stress conditions. Our results demonstrate that Cernunnos/XLF mutated cells and cells downregulated for Cernunnos/XLF have increased sensitivity to conditions which perturb DNA replication. In addition, under replication stress, these cells exhibit impaired DSB repair and increased accumulation of cells in G2/M. Moreover Cernunnos/XLF mutated and down regulated cells display greater chromosomal instability, particularly at fragile sites, under replication stress conditions. These results provide evidence for the role of Cernunnos/XLF in repair of DSBs and maintenance of genomic stability under replication stress conditions. This is the first study of a NHEJ syndrome showing association with impaired cellular response to replication stress conditions. These findings may be related to the clinical features in these patients which are not due to the V(D)J recombination defect. Additionally, in light of the emerging important role of replication stress in the early stages of cancer development, our findings may provide a mechanism for the role of NHEJ in preventing tumorigenesis. PMID:19223975

  1. Disease-associated Mutations in the Prion Protein Impair Laminin-induced Process Outgrowth and Survival*

    PubMed Central

    Machado, Cleiton F.; Beraldo, Flavio H.; Santos, Tiago G.; Bourgeon, Dominique; Landemberger, Michele C.; Roffé, Martin; Martins, Vilma R.

    2012-01-01

    Prions, the agents of transmissible spongiform encephalopathies, require the expression of prion protein (PrPC) to propagate disease. PrPC is converted into an abnormal insoluble form, PrPSc, that gains neurotoxic activity. Conversely, clinical manifestations of prion disease may occur either before or in the absence of PrPSc deposits, but the loss of normal PrPC function contribution for the etiology of these diseases is still debatable. Prion disease-associated mutations in PrPC represent one of the best models to understand the impact of PrPC loss-of-function. PrPC associates with various molecules and, in particular, the interaction of PrPC with laminin (Ln) modulates neuronal plasticity and memory formation. To assess the functional alterations associated with PrPC mutations, wild-type and mutated PrPC proteins were expressed in a neural cell line derived from a PrPC-null mouse. Treatment with the laminin γ1 chain peptide (Ln γ1), which mimics the Ln binding site for PrPC, increased intracellular calcium in cells expressing wild-type PrPC, whereas a significantly lower response was observed in cells expressing mutated PrPC molecules. The Ln γ1 did not promote process outgrowth or protect against staurosporine-induced cell death in cells expressing mutated PrPC molecules in contrast to cells expressing wild-type PrPC. The co-expression of wild-type PrPC with mutated PrPC molecules was able to rescue the Ln protective effects, indicating the lack of negative dominance of PrPC mutated molecules. These results indicate that PrPC mutations impair process outgrowth and survival mediated by Ln γ1 peptide in neural cells, which may contribute to the pathogenesis of genetic prion diseases. PMID:23132868

  2. De Novo Mutations in CHAMP1 Cause Intellectual Disability with Severe Speech Impairment

    PubMed Central

    Hempel, Maja; Cremer, Kirsten; Ockeloen, Charlotte W.; Lichtenbelt, Klaske D.; Herkert, Johanna C.; Denecke, Jonas; Haack, Tobias B.; Zink, Alexander M.; Becker, Jessica; Wohlleber, Eva; Johannsen, Jessika; Alhaddad, Bader; Pfundt, Rolph; Fuchs, Sigrid; Wieczorek, Dagmar; Strom, Tim M.; van Gassen, Koen L.I.; Kleefstra, Tjitske; Kubisch, Christian; Engels, Hartmut; Lessel, Davor

    2015-01-01

    CHAMP1 encodes a protein with a function in kinetochore-microtubule attachment and in the regulation of chromosome segregation, both of which are known to be important for neurodevelopment. By trio whole-exome sequencing, we have identified de novo deleterious mutations in CHAMP1 in five unrelated individuals affected by intellectual disability with severe speech impairment, motor developmental delay, muscular hypotonia, and similar dysmorphic features including short philtrum and a tented upper and everted lover lip. In addition to two frameshift and one nonsense mutations, we found an identical nonsense mutation, c.1192C>T (p.Arg398∗), in two affected individuals. All mutations, if resulting in a stable protein, are predicted to lead to the loss of the functionally important zinc-finger domains in the C terminus of the protein, which regulate CHAMP1 localization to chromosomes and the mitotic spindle, thereby providing a mechanistic understanding for their pathogenicity. We thus establish deleterious de novo mutations in CHAMP1 as a cause of intellectual disability. PMID:26340335

  3. Mutations in zinc finger 407 [ZNF407] cause a unique autosomal recessive cognitive impairment syndrome

    PubMed Central

    2014-01-01

    Background A consanguineous Arab family is affected by an apparently novel autosomal recessive disorder characterized by cognitive impairment, failure-to-thrive, hypotonia and dysmorphic features including bilateral ptosis and epicanthic folds, synophrys, midface hypoplasia, downturned mouth corners, thin upper vermillion border and prominent ears, bilateral 5th finger camptodactyly, bilateral short 4th metatarsal bones, and limited knee mobility bilaterally. Methods The family was studied by homozygosity mapping, candidate gene mutation screening and whole Exome Next Generation Sequencing of a single affected member to identify the offending gene and mutation. The mutated gene product was studied by structural bioinformatics methods. Results A damaging c.C5054G mutation affecting an evolutionary highly conserved amino acid p.S1685W was identified in the ZNF407 gene at 18q23. The Serine to Tryptophane mutation affects two of the three ZNF407 isoforms and is located in the last third of the protein, in a linker peptide adjoining two zinc-finger domains. Structural analyses of this mutation shows disruption of an H-bond that locks the relative spatial position of the two fingers, leading to a higher flexibility of the linker and thus to a decreased probability of binding to the target DNA sequence essentially eliminating the functionality of downstream domains and interfering with the expression of various genes under ZNF407 control during fetal brain development. Conclusions ZNF407 is a transcription factor with an essential role in brain development. When specific and limited in number homozygosity intervals exist that harbor the offending gene in consanguineous families, Whole Exome Sequencing of a single affected individual is an efficient approach to gene mapping and mutation identification. PMID:24907849

  4. CNNM2 Mutations Cause Impaired Brain Development and Seizures in Patients with Hypomagnesemia

    PubMed Central

    Lameris, Anke L. L.; van Wijk, Erwin; Flik, Gert; Regele, Sabrina; Korenke, G. Christoph; Neophytou, Birgit; Rust, Stephan; Reintjes, Nadine; Konrad, Martin; Bindels, René J. M.; Hoenderop, Joost G. J.

    2014-01-01

    Intellectual disability and seizures are frequently associated with hypomagnesemia and have an important genetic component. However, to find the genetic origin of intellectual disability and seizures often remains challenging because of considerable genetic heterogeneity and clinical variability. In this study, we have identified new mutations in CNNM2 in five families suffering from mental retardation, seizures, and hypomagnesemia. For the first time, a recessive mode of inheritance of CNNM2 mutations was observed. Importantly, patients with recessive CNNM2 mutations suffer from brain malformations and severe intellectual disability. Additionally, three patients with moderate mental disability were shown to carry de novo heterozygous missense mutations in the CNNM2 gene. To elucidate the physiological role of CNNM2 and explain the pathomechanisms of disease, we studied CNNM2 function combining in vitro activity assays and the zebrafish knockdown model system. Using stable Mg2+ isotopes, we demonstrated that CNNM2 increases cellular Mg2+ uptake in HEK293 cells and that this process occurs through regulation of the Mg2+-permeable cation channel TRPM7. In contrast, cells expressing mutated CNNM2 proteins did not show increased Mg2+ uptake. Knockdown of cnnm2 isoforms in zebrafish resulted in disturbed brain development including neurodevelopmental impairments such as increased embryonic spontaneous contractions and weak touch-evoked escape behaviour, and reduced body Mg content, indicative of impaired renal Mg2+ absorption. These phenotypes were rescued by injection of mammalian wild-type Cnnm2 cRNA, whereas mammalian mutant Cnnm2 cRNA did not improve the zebrafish knockdown phenotypes. We therefore concluded that CNNM2 is fundamental for brain development, neurological functioning and Mg2+ homeostasis. By establishing the loss-of-function zebrafish model for CNNM2 genetic disease, we provide a unique system for testing therapeutic drugs targeting CNNM2 and for

  5. PICK1 and ICA69 control insulin granule trafficking and their deficiencies lead to impaired glucose tolerance.

    PubMed

    Cao, Mian; Mao, Zhuo; Kam, Chuen; Xiao, Nan; Cao, Xiaoxing; Shen, Chong; Cheng, Kenneth K Y; Xu, Aimin; Lee, Kwong-Man; Jiang, Liwen; Xia, Jun

    2013-01-01

    Diabetes is a metabolic disorder characterized by hyperglycemia. Insulin, which is secreted by pancreatic beta cells, is recognized as the critical regulator of blood glucose, but the molecular machinery responsible for insulin trafficking remains poorly defined. In particular, the roles of cytosolic factors that govern the formation and maturation of insulin granules are unclear. Here we report that PICK1 and ICA69, two cytosolic lipid-binding proteins, formed heteromeric BAR-domain complexes that associated with insulin granules at different stages of their maturation. PICK1-ICA69 heteromeric complexes associated with immature secretory granules near the trans-Golgi network (TGN). A brief treatment of Brefeldin A, which blocks vesicle budding from the Golgi, increased the amount of PICK1 and ICA69 at TGN. On the other hand, mature secretory granules were associated with PICK1 only, not ICA69. PICK1 deficiency in mice caused the complete loss of ICA69 and led to increased food and water intake but lower body weight. Glucose tolerance tests demonstrated that these mutant mice had high blood glucose, a consequence of insufficient insulin. Importantly, while the total insulin level was reduced in PICK1-deficient beta cells, proinsulin was increased. Lastly, ICA69 knockout mice also displayed similar phenotype as the mice deficient in PICK1. Together, our results indicate that PICK1 and ICA69 are key regulators of the formation and maturation of insulin granules. PMID:23630453

  6. Hypomorphic mutation in mouse Nppc gene causes retarded bone growth due to impaired endochondral ossification

    SciTech Connect

    Tsuji, Takehito Kondo, Eri; Yasoda, Akihiro; Inamoto, Masataka; Kiyosu, Chiyo; Nakao, Kazuwa; Kunieda, Tetsuo

    2008-11-07

    Long bone abnormality (lbab/lbab) is a spontaneous mutant mouse characterized by dwarfism with shorter long bones. A missense mutation was reported in the Nppc gene, which encodes C-type natriuretic peptide (CNP), but it has not been confirmed whether this mutation is responsible for the dwarf phenotype. To verify that the mutation causes the dwarfism of lbab/lbab mice, we first investigated the effect of CNP in lbab/lbab mice. By transgenic rescue with chondrocyte-specific expression of CNP, the dwarf phenotype in lbab/lbab mice was completely compensated. Next, we revealed that CNP derived from the lbab allele retained only slight activity to induce cGMP production through its receptor. Histological analysis showed that both proliferative and hypertrophic zones of chondrocytes in the growth plate of lbab/lbab mice were markedly reduced. Our results demonstrate that lbab/lbab mice have a hypomorphic mutation in the Nppc gene that is responsible for dwarfism caused by impaired endochondral ossification.

  7. TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport.

    PubMed

    Schmidts, Miriam; Hou, Yuqing; Cortés, Claudio R; Mans, Dorus A; Huber, Celine; Boldt, Karsten; Patel, Mitali; van Reeuwijk, Jeroen; Plaza, Jean-Marc; van Beersum, Sylvia E C; Yap, Zhi Min; Letteboer, Stef J F; Taylor, S Paige; Herridge, Warren; Johnson, Colin A; Scambler, Peter J; Ueffing, Marius; Kayserili, Hulya; Krakow, Deborah; King, Stephen M; Beales, Philip L; Al-Gazali, Lihadh; Wicking, Carol; Cormier-Daire, Valerie; Roepman, Ronald; Mitchison, Hannah M; Witman, George B

    2015-01-01

    The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas, accompanied by destabilization of the retrograde IFT dynein motor. We thus define TCTEX1D2 as an integral component of the evolutionarily conserved retrograde IFT machinery. In complex with several IFT dynein light chains, it is required for correct vertebrate skeletal formation but may be functionally redundant under certain conditions. PMID:26044572

  8. TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport

    PubMed Central

    Schmidts, Miriam; Hou, Yuqing; Cortés, Claudio R.; Mans, Dorus A.; Huber, Celine; Boldt, Karsten; Patel, Mitali; van Reeuwijk, Jeroen; Plaza, Jean-Marc; van Beersum, Sylvia E. C.; Yap, Zhi Min; Letteboer, Stef J. F.; Taylor, S. Paige; Herridge, Warren; Johnson, Colin A.; Scambler, Peter J.; Ueffing, Marius; Kayserili, Hulya; Krakow, Deborah; King, Stephen M.; Beales, Philip L.; Al-Gazali, Lihadh; Wicking, Carol; Cormier-Daire, Valerie; Roepman, Ronald; Mitchison, Hannah M.; Witman, George B.; Al-Turki, Saeed; Anderson, Carl; Anney, Richard; Antony, Dinu; Asimit, Jennifer; Ayub, Mohammad; Barrett, Jeff; Barroso, Inês; Bentham, Jamie; Bhattacharya, Shoumo; Blackwood, Douglas; Bobrow, Martin; Bochukova, Elena; Bolton, Patrick; Boustred, Chris; Breen, Gerome; Brion, Marie-Jo; Brown, Andrew; Calissano, Mattia; Carss, Keren; Chatterjee, Krishna; Chen, Lu; Cirak, Sebhattin; Clapham, Peter; Clement, Gail; Coates, Guy; Collier, David; Cosgrove, Catherine; Cox, Tony; Craddock, Nick; Crooks, Lucy; Curran, Sarah; Daly, Allan; Danecek, Petr; Smith, George Davey; Day-Williams, Aaron; Day, Ian; Durbin, Richard; Edkins, Sarah; Ellis, Peter; Evans, David; Farooqi, I. Sadaf; Fatemifar, Ghazaleh; Fitzpatrick, David; Flicek, Paul; Floyd, Jamie; Foley, A. Reghan; Franklin, Chris; Futema, Marta; Gallagher, Louise; Gaunt, Tom; Geschwind, Daniel; Greenwood, Celia; Grozeva, Detelina; Guo, Xiaosen; Gurling, Hugh; Hart, Deborah; Hendricks, Audrey; Holmans, Peter; Huang, Jie; Humphries, Steve E.; Hurles, Matt; Hysi, Pirro; Jackson, David; Jamshidi, Yalda; Jewell, David; Chris, Joyce; Kaye, Jane; Keane, Thomas; Kemp, John; Kennedy, Karen; Kent, Alastair; Kolb-Kokocinski, Anja; Lachance, Genevieve; Langford, Cordelia; Lee, Irene; Li, Rui; Li, Yingrui; Ryan, Liu; Lönnqvist, Jouko; Lopes, Margarida; MacArthur, Daniel G.; Massimo, Mangino; Marchini, Jonathan; Maslen, John; McCarthy, Shane; McGuffin, Peter; McIntosh, Andrew; McKechanie, Andrew; McQuillin, Andrew; Memari, Yasin; Metrustry, Sarah; Min, Josine; Moayyeri, Alireza; Morris, James; Muddyman, Dawn; Muntoni, Francesco; Northstone, Kate; O'Donovan, Michael; O'Rahilly, Stephen; Onoufriadis, Alexandros; Oualkacha, Karim; Owen, Michael; Palotie, Aarno; Panoutsopoulou, Kalliope; Parker, Victoria; Parr, Jeremy; Paternoster, Lavinia; Paunio, Tiina; Payne, Felicity; Perry, John; Pietilainen, Olli; Plagnol, Vincent; Quail, Michael A.; Quaye, Lydia; Raymond, Lucy; Rehnström, Karola; Brent Richards, J.; Ring, Sue; Ritchie, Graham R S; Savage, David B.; Schoenmakers, Nadia; Semple, Robert K.; Serra, Eva; Shihab, Hashem; Shin, So-Youn; Skuse, David; Small, Kerrin; Smee, Carol; Soler, Artigas María; Soranzo, Nicole; Southam, Lorraine; Spector, Tim; St Pourcain, Beate; St. Clair, David; Stalker, Jim; Surdulescu, Gabriela; Suvisaari, Jaana; Tachmazidou, Ioanna; Tian, Jing; Timpson, Nic; Tobin, Martin; Valdes, Ana; van Kogelenberg, Margriet; Vijayarangakannan, Parthiban; Wain, Louise; Walter, Klaudia; Wang, Jun; Ward, Kirsten; Wheeler, Ellie; Whittall, Ros; Williams, Hywel; Williamson, Kathy; Wilson, Scott G.; Wong, Kim; Whyte, Tamieka; ChangJiang, Xu; Zeggini, Eleftheria; Zhang, Feng; Zheng, Hou-Feng

    2015-01-01

    The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas, accompanied by destabilization of the retrograde IFT dynein motor. We thus define TCTEX1D2 as an integral component of the evolutionarily conserved retrograde IFT machinery. In complex with several IFT dynein light chains, it is required for correct vertebrate skeletal formation but may be functionally redundant under certain conditions. PMID:26044572

  9. Autosomal recessive PGM3 mutations link glycosylation defects to atopy, immune deficiency, autoimmunity, and neurocognitive impairment

    PubMed Central

    Zhang, Yu; Yu, Xiaomin; Ichikawa, Mie; Lyons, Jonathan J.; Datta, Shrimati; Lamborn, Ian T.; Jing, Huie; Kim, Emily S.; Biancalana, Matthew; Wolfe, Lynne A.; DiMaggio, Thomas; Matthews, Helen F.; Kranick, Sarah M.; Stone, Kelly D.; Holland, Steven M.; Reich, Daniel S.; Hughes, Jason D.; Mehmet, Huseyin; McElwee, Joshua; Freeman, Alexandra F.; Freeze, Hudson H.; Su, Helen C.; Milner, Joshua D.

    2014-01-01

    Background Identifying genetic syndromes that lead to significant atopic disease can open new pathways for investigation and intervention in allergy. Objective To define a genetic syndrome of severe atopy, elevated serum IgE, immune deficiency, autoimmunity, and motor and neurocognitive impairment. Methods Eight patients from two families who had similar syndromic features were studied. Thorough clinical evaluations, including brain MRI and sensory evoked potentials, were performed. Peripheral lymphocyte flow cytometry, antibody responses, and T cell cytokine production were measured. Whole exome sequencing was performed to identify disease-causing mutations. Immunoblotting, qRT-PCR, enzymatic assays, nucleotide sugar and sugar phosphate analyses along with MALDI-TOF mass spectrometry of glycans were used to determine the molecular consequences of the mutations. Results Marked atopy and autoimmunity were associated with increased TH2 and TH17 cytokine production by CD4+ T cells. Bacterial and viral infection susceptibility were noted along with T cell lymphopenia, particularly of CD8+ T cells, and reduced memory B cells. Apparent brain hypomyelination resulted in markedly delayed evoked potentials and likely contributed to neurological abnormalities. Disease segregated with novel autosomal recessive mutations in a single gene, phosphoglucomutase 3 (PGM3). Although PGM3 protein expression was variably diminished, impaired function was demonstrated by decreased enzyme activity and reduced UDP-GlcNAc, along with decreased O- and N-linked protein glycosylation in patients’ cells. These results define a new Congenital Disorder of Glycosylation. Conclusions Autosomal recessive, hypomorphic PGM3 mutations underlie a disorder of severe atopy, immune deficiency, autoimmunity, intellectual disability and hypomyelination. PMID:24589341

  10. A Mutation in the Mouse Ttc26 Gene Leads to Impaired Hedgehog Signaling

    PubMed Central

    Swiderski, Ruth E.; Nakano, Yoko; Mullins, Robert F.; Seo, Seongjin; Bánfi, Botond

    2014-01-01

    The phenotype of the spontaneous mutant mouse hop-sterile (hop) is characterized by a hopping gait, polydactyly, hydrocephalus, and male sterility. Previous analyses of the hop mouse revealed a deficiency of inner dynein arms in motile cilia and a lack of sperm flagella, potentially accounting for the hydrocephalus and male sterility. The etiology of the other phenotypes and the location of the hop mutation remained unexplored. Here we show that the hop mutation is located in the Ttc26 gene and impairs Hedgehog (Hh) signaling. Expression analysis showed that this mutation led to dramatically reduced levels of the Ttc26 protein, and protein-protein interaction assays demonstrated that wild-type Ttc26 binds directly to the Ift46 subunit of Intraflagellar Transport (IFT) complex B. Although IFT is required for ciliogenesis, the Ttc26 defect did not result in a decrease in the number or length of primary cilia. Nevertheless, Hh signaling was reduced in the hop mouse, as revealed by impaired activation of Gli transcription factors in embryonic fibroblasts and abnormal patterning of the neural tube. Unlike the previously characterized mutations that affect IFT complex B, hop did not interfere with Hh-induced accumulation of Gli at the tip of the primary cilium, but rather with the subsequent dissociation of Gli from its negative regulator, Sufu. Our analysis of the hop mouse line provides novel insights into Hh signaling, demonstrating that Ttc26 is necessary for efficient coupling between the accumulation of Gli at the ciliary tip and its dissociation from Sufu. PMID:25340710

  11. Mutation in E1, the Ubiquitin Activating Enzyme, Reduces Drosophila Lifespan and Results in Motor Impairment

    PubMed Central

    Liu, Hsiu-Yu; Pfleger, Cathie M.

    2013-01-01

    Neurodegenerative diseases cause tremendous suffering for those afflicted and their families. Many of these diseases involve accumulation of mis-folded or aggregated proteins thought to play a causal role in disease pathology. Ubiquitinated proteins are often found in these protein aggregates, and the aggregates themselves have been shown to inhibit the activity of the proteasome. These and other alterations in the Ubiquitin Pathway observed in neurodegenerative diseases have led to the question of whether impairment of the Ubiquitin Pathway on its own can increase mortality or if ongoing neurodegeneration alters Ubiquitin Pathway function as a side-effect. To address the role of the Ubiquitin Pathway in vivo, we studied loss-of-function mutations in the Drosophila Ubiquitin Activating Enzyme, Uba1 or E1, the most upstream enzyme in the Ubiquitin Pathway. Loss of only one functional copy of E1 caused a significant reduction in adult lifespan. Rare homozygous hypomorphic E1 mutants reached adulthood. These mutants exhibited further reduced lifespan and showed inappropriate Ras activation in the brain. Removing just one functional copy of Ras restored the lifespan of heterozygous E1 mutants to that of wild-type flies and increased the survival of homozygous E1 mutants. E1 homozygous mutants also showed severe motor impairment. Our findings suggest that processes that impair the Ubiquitin Pathway are sufficient to cause early mortality. Reduced lifespan and motor impairment are seen in the human disease X-linked Infantile Spinal Muscular Atrophy, which is associated with mutation in human E1 warranting further analysis of these mutants as a potential animal model for study of this disease. PMID:23382794

  12. Disease-associated mutations in the prion protein impair laminin-induced process outgrowth and survival.

    PubMed

    Machado, Cleiton F; Beraldo, Flavio H; Santos, Tiago G; Bourgeon, Dominique; Landemberger, Michele C; Roffé, Martin; Martins, Vilma R

    2012-12-21

    Prions, the agents of transmissible spongiform encephalopathies, require the expression of prion protein (PrP(C)) to propagate disease. PrP(C) is converted into an abnormal insoluble form, PrP(Sc), that gains neurotoxic activity. Conversely, clinical manifestations of prion disease may occur either before or in the absence of PrP(Sc) deposits, but the loss of normal PrP(C) function contribution for the etiology of these diseases is still debatable. Prion disease-associated mutations in PrP(C) represent one of the best models to understand the impact of PrP(C) loss-of-function. PrP(C) associates with various molecules and, in particular, the interaction of PrP(C) with laminin (Ln) modulates neuronal plasticity and memory formation. To assess the functional alterations associated with PrP(C) mutations, wild-type and mutated PrP(C) proteins were expressed in a neural cell line derived from a PrP(C)-null mouse. Treatment with the laminin γ1 chain peptide (Ln γ1), which mimics the Ln binding site for PrP(C), increased intracellular calcium in cells expressing wild-type PrP(C), whereas a significantly lower response was observed in cells expressing mutated PrP(C) molecules. The Ln γ1 did not promote process outgrowth or protect against staurosporine-induced cell death in cells expressing mutated PrP(C) molecules in contrast to cells expressing wild-type PrP(C). The co-expression of wild-type PrP(C) with mutated PrP(C) molecules was able to rescue the Ln protective effects, indicating the lack of negative dominance of PrP(C) mutated molecules. These results indicate that PrP(C) mutations impair process outgrowth and survival mediated by Ln γ1 peptide in neural cells, which may contribute to the pathogenesis of genetic prion diseases. PMID:23132868

  13. MARVELD2 (DFNB49) Mutations in the Hearing Impaired Central European Roma Population - Prevalence, Clinical Impact and the Common Origin

    PubMed Central

    Mátyás, Petra; Ficek, Andrej; Hučková, Miloslava; Sůrová, Martina; Šafka-Brožková, Dana; Anwar, Saima; Bene, Judit; Straka, Slavomír; Janicsek, Ingrid; Ahmed, Zubair M.; Seeman, Pavel; Melegh, Béla; Profant, Milan; Klimeš, Iwar; Riazuddin, Saima; Kádasi, Ľudevít; Gašperíková, Daniela

    2015-01-01

    Background In the present study we aimed: 1) To establish the prevalence and clinical impact of DFNB49 mutations in deaf Roma from 2 Central European countries (Slovakia and Hungary), and 2) to analyze a possible common origin of the c.1331+2T>C mutation among Roma and Pakistani mutation carriers identified in the present and previous studies. Methods We sequenced 6 exons of the MARVELD2 gene in a group of 143 unrelated hearing impaired Slovak Roma patients. Simultaneously, we used RFLP to detect the c.1331+2T>C mutation in 85 Hungarian deaf Roma patients, control groups of 702 normal hearing Romanies from both countries and 375 hearing impaired Slovak Caucasians. We analyzed the haplotype using 21 SNPs spanning a 5.34Mb around the mutation c.1331+2T>C. Results One pathogenic mutation (c.1331+2T>C) was identified in 12 homozygous hearing impaired Roma patients. Allele frequency of this mutation was higher in Hungarian (10%) than in Slovak (3.85%) Roma patients. The identified common haplotype in Roma patients was defined by 18 SNP markers (3.89 Mb). Fourteen common SNPs were also shared among Pakistani and Roma homozygotes. Biallelic mutation carriers suffered from prelingual bilateral moderate to profound sensorineural hearing loss. Conclusions We demonstrate different frequencies of the c.1331+2T>C mutation in hearing impaired Romanies from 3 Central European countries. In addition, our results provide support for the hypothesis of a possible common ancestor of the Slovak, Hungarian and Czech Roma as well as Pakistani deaf patients. Testing for the c.1331+2T>C mutation may be recommended in GJB2 negative Roma cases with early-onset sensorineural hearing loss. PMID:25885414

  14. Human Trafficking

    ERIC Educational Resources Information Center

    Wilson, David McKay

    2011-01-01

    The shadowy, criminal nature of human trafficking makes evaluating its nature and scope difficult. The U.S. State Department and anti-trafficking groups estimate that worldwide some 27 million people are caught in a form of forced servitude today. Public awareness of modern-day slavery is gaining momentum thanks to new abolitionist efforts. Among…

  15. Persistent Hepatitis C Virus Infection Impairs Ribavirin Antiviral Activity through Clathrin-Mediated Trafficking of Equilibrative Nucleoside Transporter 1

    PubMed Central

    Panigrahi, Rajesh; Chandra, Partha K.; Ferraris, Pauline; Kurt, Ramazan; Song, Kyoungsub; Garry, Robert F.; Reiss, Krzysztof; Coe, Imogen R.; Furihata, Tomomi; Balart, Luis A.; Wu, Tong

    2014-01-01

    ABSTRACT Ribavirin (RBV) continues to be an important component of interferon-free hepatitis C treatment regimens, as RBV alone does not inhibit hepatitis C virus (HCV) replication effectively; the reason for this ineffectiveness has not been established. In this study, we investigated the RBV resistance mechanism using a persistently HCV-infected cell culture system. The antiviral activity of RBV against HCV was progressively impaired in the persistently infected culture, whereas interferon lambda 1 (IFN-λ1), a type III IFN, showed a strong antiviral response and induced viral clearance. We found that HCV replication in persistently infected cultures induces an autophagy response that impairs RBV uptake by preventing the expression of equilibrative nucleoside transporter 1 (ENT1). The Huh-7.5 cell line treated with an autophagy inducer, Torin 1, downregulated membrane expression of ENT1 and terminated RBV uptake. In contrast, the autophagy inhibitors hydroxychloroquine (HCQ), 3-methyladenine (3-MA), and bafilomycin A1 (BafA1) prevented ENT1 degradation and enhanced RBV antiviral activity. The HCV-induced autophagy response, as well as treatment with Torin 1, degrades clathrin heavy chain expression in a hepatoma cell line. Reduced expression of the clathrin heavy chain by HCV prevents ENT1 recycling to the plasma membrane and forces ENT1 to the lysosome for degradation. This study provides a potential mechanism for the impairment of RBV antiviral activity in persistently HCV-infected cell cultures and suggests that inhibition of the HCV-induced autophagy response could be used as a strategy for improving RBV antiviral activity against HCV infection. IMPORTANCE The results from this work will allow a review of the competing theories of antiviral therapy development in the field of HCV virology. Ribavirin (RBV) remains an important component of interferon-free hepatitis C treatment regimens. The reason why RBV alone does not inhibit HCV replication effectively has

  16. PICK1 and ICA69 Control Insulin Granule Trafficking and Their Deficiencies Lead to Impaired Glucose Tolerance

    PubMed Central

    Kam, Chuen; Xiao, Nan; Cao, Xiaoxing; Shen, Chong; Cheng, Kenneth K. Y.; Xu, Aimin; Lee, Kwong-Man; Jiang, Liwen; Xia, Jun

    2013-01-01

    Diabetes is a metabolic disorder characterized by hyperglycemia. Insulin, which is secreted by pancreatic beta cells, is recognized as the critical regulator of blood glucose, but the molecular machinery responsible for insulin trafficking remains poorly defined. In particular, the roles of cytosolic factors that govern the formation and maturation of insulin granules are unclear. Here we report that PICK1 and ICA69, two cytosolic lipid-binding proteins, formed heteromeric BAR-domain complexes that associated with insulin granules at different stages of their maturation. PICK1-ICA69 heteromeric complexes associated with immature secretory granules near the trans-Golgi network (TGN). A brief treatment of Brefeldin A, which blocks vesicle budding from the Golgi, increased the amount of PICK1 and ICA69 at TGN. On the other hand, mature secretory granules were associated with PICK1 only, not ICA69. PICK1 deficiency in mice caused the complete loss of ICA69 and led to increased food and water intake but lower body weight. Glucose tolerance tests demonstrated that these mutant mice had high blood glucose, a consequence of insufficient insulin. Importantly, while the total insulin level was reduced in PICK1-deficient beta cells, proinsulin was increased. Lastly, ICA69 knockout mice also displayed similar phenotype as the mice deficient in PICK1. Together, our results indicate that PICK1 and ICA69 are key regulators of the formation and maturation of insulin granules. Author Summary Insulin is a key regulator of blood glucose and insufficient insulin leads to diabetes. Insulin is synthesized as proinsulin, processed in endoplasmic reticulum and Golgi, and eventually packaged into insulin granules, a type of dense core vesicles. Despite its importance, the molecular mechanisms governing the biogenesis and maturation of insulin granules are not fully understood. In this study, we identified two cytosolic proteins, PICK1 and ICA69, as important regulators of insulin granule

  17. Two distinct clinical features and cognitive impairment in amyotrophic lateral sclerosis patients with TARDBP gene mutations in the Chinese population.

    PubMed

    Ju, XiaoDong; Liu, WenChao; Li, XiaoGang; Liu, Na; Zhang, Nan; Liu, Tao; Deng, Min

    2016-02-01

    Mutations in the TARDBP gene have been identified as a major causative factor in amyotrophic lateral sclerosis (ALS). However, few reports have analyzed the relationship of genotype-phenotype, especially in Chinese ALS patients. Our study investigated the presence and frequency of TARDBP mutations in Chinese patients with ALS. Additionally, we investigated correlations among clinical features and TARDBP gene mutations in a large ALS family with the p.M337 V mutation and one sporadic ALS (SALS) patient with the p.S393 L mutation. The pedigree with the p.M337 V mutation showed variable clinical features with a long lifespan, particularly cognitive impairment. One patient carrying the p.S393 L mutation experienced ALS with cognitive impairment; the patient also had a family history of frontotemporal dementia (FTD). This is the first report of detailed genetic and clinical characterizations of the TARDBP gene in a Chinese population. This research is also the first to demonstrate that the p.M337 V and the p.S393 L mutations are related to cognitive impairment in ALS patients. The mutation frequency of TARDBP was 5.6% in Chinese, SOD1-negative familial ALS (FALS), which was much higher than that reported in previous studies conducted with Caucasian populations, whereas the TARDBP mutation frequency was lower in the Chinese population with regard to SALS patients. Our results emphasize the importance of the genetic and clinical characterization of TARDBP mutations in ALS, which allows us to understand the genotype-phenotype relationship and relative frequencies in different populations. PMID:26639158

  18. Migraine mutations impair hippocampal learning despite enhanced long-term potentiation.

    PubMed

    Dilekoz, Ergin; Houben, Thijs; Eikermann-Haerter, Katharina; Balkaya, Mustafa; Lenselink, A Mariette; Whalen, Michael J; Spijker, Sabine; Ferrari, Michel D; van den Maagdenberg, Arn M J M; Ayata, Cenk

    2015-02-25

    To explain cognitive and memory difficulties observed in some familial hemiplegic migraine (FHM) patients, we examined hippocampal neurotransmission and plasticity in knock-in mice expressing the FHM type 1 (FHM1) R192Q gain-of function mutation in the CACNA1A gene that encodes the α1A subunit of neuronal CaV2.1 channels. We determined stimulus intensity-response curves for anterior commissure-evoked hippocampal CA1 field potentials in strata pyramidale and radiatum and assessed neuroplasticity by inducing long-term potentiation (LTP) and long-term depression (LTD) in anesthetized mice in vivo. We also studied learning and memory using contextual fear-conditioning, Morris water maze, and novel object recognition tests. Hippocampal field potentials were significantly enhanced in R192Q mice compared with wild-type controls. Stimulus intensity-response curves were shifted to the left and displayed larger maxima in the mutants. LTP was augmented by twofold in R192Q mice, whereas LTD was unchanged compared with wild-type mice. R192Q mice showed significant spatial memory deficits in contextual fear-conditioning and Morris water maze tests compared with wild-type controls. Novel object recognition was not impaired in R192Q mice; however, mice carrying the more severe S218L CACNA1A mutation showed marked deficits in this test, suggesting a genotype-phenotype relationship. Thus, whereas FHM1 gain-of-function mutations enhance hippocampal excitatory transmission and LTP, learning and memory are paradoxically impaired, providing a possible explanation for cognitive changes detected in FHM. Data suggest that abnormally enhanced plasticity can be as detrimental to efficient learning as reduced plasticity and highlight how genetically enhanced neuronal excitability may impact cognitive function. PMID:25716839

  19. Compound heterozygous NOTCH1 mutations underlie impaired cardiogenesis in a patient with hypoplastic left heart syndrome.

    PubMed

    Theis, Jeanne L; Hrstka, Sybil C L; Evans, Jared M; O'Byrne, Megan M; de Andrade, Mariza; O'Leary, Patrick W; Nelson, Timothy J; Olson, Timothy M

    2015-09-01

    Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect (CHD) that necessitates staged, single ventricle surgical palliation. An increased frequency of bicuspid aortic valve (BAV) has been observed among relatives. We postulated number of mutant alleles as a molecular basis for variable CHD expression in an extended family comprised of an HLHS proband and four family members who underwent echocardiography and whole-genome sequencing (WGS). Dermal fibroblast-derived induced pluripotent stem cells (iPSC) were procured from the proband-parent trio and bioengineered into cardiomyocytes. Cardiac phenotyping revealed aortic valve atresia and a slit-like left ventricular cavity in the HLHS proband, isolated bicuspid pulmonary valve in his mother, BAV in a maternal 4° relative, and no CHD in his father or sister. Filtering of WGS for rare, functional variants that segregated with CHD and were compound heterozygous in the HLHS proband identified NOTCH1 as the sole candidate gene. An unreported missense mutation (P1964L) in the cytoplasmic domain, segregating with semilunar valve malformation, was maternally inherited and a rare missense mutation (P1256L) in the extracellular domain, clinically silent in the heterozygous state, was paternally inherited. Patient-specific iPSCs exhibited diminished transcript levels of NOTCH1 signaling pathway components, impaired myocardiogenesis, and a higher prevalence of heterogeneous myofilament organization. Extended, phenotypically characterized families enable WGS-derived variant filtering for plausible Mendelian modes of inheritance, a powerful strategy to discover molecular underpinnings of CHD. Identification of compound heterozygous NOTCH1 mutations and iPSC-based functional modeling implicate mutant allele burden and impaired myogenic potential as mechanisms for HLHS. PMID:26164125

  20. Ciliopathies: The Trafficking Connection

    PubMed Central

    Madhivanan, Kayalvizhi; Aguilar, R. Claudio

    2014-01-01

    The primary cilium (PC) is a very dynamic hair-like membrane structure that assembles/disassembles in a cell-cycle dependent manner and is present in almost every cell type. Despite being continuous with the plasma membrane, a diffusion barrier located at the ciliary base confers the PC properties of a separate organelle with very specific characteristics and membrane composition. Therefore, vesicle trafficking is the major process by which components are acquired for cilium formation and maintenance. In fact, a system of specific sorting signals controls the right of cargo admission into the cilia. Disruption to the ciliary structure or its function leads to multi-organ diseases known as ciliopathies. These illnesses arise from a spectrum of mutations in any of the more than 50 loci linked to these conditions. Therefore, it is not surprising that symptom variability (specific manifestations and severity) among and within ciliopathies seems to be an emerging characteristic. Nevertheless, one can speculate that mutations occurring in genes whose products contribute to the overall vesicle trafficking to the PC (i.e., affecting cilia assembly) will lead to more severe symptoms, while those involved in the transport of specific cargoes will result in milder phenotypes. In this review, we summarize the trafficking mechanisms to the cilia and also provide a description of the trafficking defects observed in some ciliopathies which can be correlated to the severity of the pathology. PMID:25040720

  1. Biallelic mutations in CAD, impair de novo pyrimidine biosynthesis and decrease glycosylation precursors

    PubMed Central

    Ng, Bobby G.; Wolfe, Lynne A.; Ichikawa, Mie; Markello, Thomas; He, Miao; Tifft, Cynthia J.; Gahl, William A.; Freeze, Hudson H.

    2015-01-01

    In mitochondria, carbamoyl-phosphate synthetase 1 activity produces carbamoyl phosphate for urea synthesis, and deficiency results in hyperammonemia. Cytoplasmic carbamoyl-phosphate synthetase 2, however, is part of a tri-functional enzyme encoded by CAD; no human disease has been attributed to this gene. The tri-functional enzyme contains carbamoyl-phosphate synthetase 2 (CPS2), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities, which comprise the first three of six reactions required for de novo pyrimidine biosynthesis. Here we characterize an individual who is compound heterozygous for mutations in different domains of CAD. One mutation, c.1843-1G>A, results in an in-frame deletion of exon 13. The other, c.6071G>A, causes a missense mutation (p.Arg2024Gln) in a highly conserved residue that is essential for carbamoyl-phosphate binding. Metabolic flux studies showed impaired aspartate incorporation into RNA and DNA through the de novo synthesis pathway. In addition, CTP, UTP and nearly all UDP-activated sugars that serve as donors for glycosylation were decreased. Uridine supplementation rescued these abnormalities, suggesting a potential therapy for this new glycosylation disorder. PMID:25678555

  2. Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity.

    PubMed

    Ledoux, Sarah; Guthrie, Christine

    2016-06-01

    Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (Δ247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The Δ247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type Δ247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of Δ247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases Δ247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the Δ247-Brr2 RP mutants compared with the wild type Δ247-Brr2. When stimulated by Prp8, wild type Δ247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices. PMID:27072132

  3. Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway.

    PubMed

    Antunes, Ana T; Goos, Yvonne J; Pereboom, Tamara C; Hermkens, Dorien; Wlodarski, Marcin W; Da Costa, Lydie; MacInnes, Alyson W

    2015-07-01

    Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes. PMID:26132763

  4. Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway

    PubMed Central

    Hermkens, Dorien; Wlodarski, Marcin W.; Da Costa, Lydie; MacInnes, Alyson W.

    2015-01-01

    Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes. PMID:26132763

  5. Mutation of a single threonine in the cytoplasmic NH2 terminus disrupts trafficking of renal betaine-GABA transporter 1 during hypertonic stress.

    PubMed

    Schweikhard, Eva S; Kempson, Stephen A; Ziegler, Christine; Burckhardt, Birgitta C

    2014-07-01

    Betaine is an important osmolyte and is, compared with other organs, much more abundant in the kidneys, where it enters cells in the medulla by betaine-GABA transporter 1 (BGT1) to balance osmoregulation in the countercurrent system. In wild-type (wt-)BGT1-expressing oocytes, GABA-mediated currents were diminished by preincubation of oocytes with 100 nM PMA or 5 μM dioctanoyl-sn-glycerol, activators of PKC, whereas the application of staurosporine before the application of dioctanoyl-sn-glycerol restored the response to GABA. Four potential phosphorylation sites on BGT1 were mutated to alanine by site-directed mutagenesis. Three mutants (T235A, S428A, and S564A) evoked GABA currents comparable in magnitude to currents observed in wt-BGT1-expressing oocytes, whereas GABA currents in T40A were barely detectable. Uptake of [(3)H]GABA was also determined in human embryonic kidney-293 cells expressing enhanced green fluorescent protein (EGFP)-tagged BGT1 with the same mutations. T235A, S428A, and S564A showed upregulation of GABA uptake after hypertonic stress and downregulation by PMA similar to EGFP-wt-BGT1. In contrast, T40A did not respond to either hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in Madin-Darby canine kidney cells revealed that T40A was present in the cytoplasm after 24 h of hypertonic stress. whereas the other mutants and EGFP-wt-BGT1 were in the plasma membrane. All mutants, including T40A, comigrated with wt-BGT1 on Western blots, suggesting that they are full-length proteins. T40A, however, cannot be phosphorylated, as revealed using a specific anti-phosphoantibody, and, therefore, T40 may be important for the trafficking and insertion of BGT1 in the plasma membrane. PMID:24829506

  6. PX-RICS-deficient mice mimic autism spectrum disorder in Jacobsen syndrome through impaired GABAA receptor trafficking

    PubMed Central

    Nakamura, Tsutomu; Arima-Yoshida, Fumiko; Sakaue, Fumika; Nasu-Nishimura, Yukiko; Takeda, Yasuko; Matsuura, Ken; Akshoomoff, Natacha; Mattson, Sarah N.; Grossfeld, Paul D.; Manabe, Toshiya; Akiyama, Tetsu

    2016-01-01

    Jacobsen syndrome (JBS) is a rare congenital disorder caused by a terminal deletion of the long arm of chromosome 11. A subset of patients exhibit social behavioural problems that meet the diagnostic criteria for autism spectrum disorder (ASD); however, the underlying molecular pathogenesis remains poorly understood. PX-RICS is located in the chromosomal region commonly deleted in JBS patients with autistic-like behaviour. Here we report that PX-RICS-deficient mice exhibit ASD-like social behaviours and ASD-related comorbidities. PX-RICS-deficient neurons show reduced surface γ-aminobutyric acid type A receptor (GABAAR) levels and impaired GABAAR-mediated synaptic transmission. PX-RICS, GABARAP and 14-3-3ζ/θ form an adaptor complex that interconnects GABAAR and dynein/dynactin, thereby facilitating GABAAR surface expression. ASD-like behavioural abnormalities in PX-RICS-deficient mice are ameliorated by enhancing inhibitory synaptic transmission with a GABAAR agonist. Our findings demonstrate a critical role of PX-RICS in cognition and suggest a causal link between PX-RICS deletion and ASD-like behaviour in JBS patients. PMID:26979507

  7. PX-RICS-deficient mice mimic autism spectrum disorder in Jacobsen syndrome through impaired GABAA receptor trafficking.

    PubMed

    Nakamura, Tsutomu; Arima-Yoshida, Fumiko; Sakaue, Fumika; Nasu-Nishimura, Yukiko; Takeda, Yasuko; Matsuura, Ken; Akshoomoff, Natacha; Mattson, Sarah N; Grossfeld, Paul D; Manabe, Toshiya; Akiyama, Tetsu

    2016-01-01

    Jacobsen syndrome (JBS) is a rare congenital disorder caused by a terminal deletion of the long arm of chromosome 11. A subset of patients exhibit social behavioural problems that meet the diagnostic criteria for autism spectrum disorder (ASD); however, the underlying molecular pathogenesis remains poorly understood. PX-RICS is located in the chromosomal region commonly deleted in JBS patients with autistic-like behaviour. Here we report that PX-RICS-deficient mice exhibit ASD-like social behaviours and ASD-related comorbidities. PX-RICS-deficient neurons show reduced surface γ-aminobutyric acid type A receptor (GABAAR) levels and impaired GABAAR-mediated synaptic transmission. PX-RICS, GABARAP and 14-3-3ζ/θ form an adaptor complex that interconnects GABAAR and dynein/dynactin, thereby facilitating GABAAR surface expression. ASD-like behavioural abnormalities in PX-RICS-deficient mice are ameliorated by enhancing inhibitory synaptic transmission with a GABAAR agonist. Our findings demonstrate a critical role of PX-RICS in cognition and suggest a causal link between PX-RICS deletion and ASD-like behaviour in JBS patients. PMID:26979507

  8. Human Trafficking

    MedlinePlus

    ... to debt bondage or peonage in which traffickers demand labor as a means repayment for a real ... human smuggling are two separate crimes under federal law. There are several important differences between them. Human ...

  9. Microcephaly-dystonia due to mutated PLEKHG2 with impaired actin polymerization.

    PubMed

    Edvardson, Simon; Wang, Haibo; Dor, Talya; Atawneh, Osamah; Yaacov, Barak; Gartner, Jutta; Cinnamon, Yuval; Chen, Songhai; Elpeleg, Orly

    2016-01-01

    Rearrangement of the actin cytoskeleton is controlled by RhoGTPases which are activated by RhoGEFs. We identified homozygosity for Arg204Trp mutation in the Rho guanidine exchange factor (RhoGEF) PLEKHG2 gene in five patients with profound mental retardation, dystonia, postnatal microcephaly, and distinct neuroimaging pattern. The activity of the mutant PLEKHG2 was significantly decreased, both in basal state and when Gβγ- or lysophosphatidic acid (LPA)-stimulated. SDF1a-stimulated actin polymerization was significantly impaired in patient cells, and this abnormality was duplicated in control cells when PLEKHG2 expression was downregulated. These results underscore the role of PLEKHG2 in actin polymerization and delineate the clinical and radiological findings in PLEKHG2 deficiency. PMID:26573021

  10. KSR2 Mutations Are Associated with Obesity, Insulin Resistance, and Impaired Cellular Fuel Oxidation

    PubMed Central

    Pearce, Laura R.; Atanassova, Neli; Banton, Matthew C.; Bottomley, Bill; van der Klaauw, Agatha A.; Revelli, Jean-Pierre; Hendricks, Audrey; Keogh, Julia M.; Henning, Elana; Doree, Deon; Jeter-Jones, Sabrina; Garg, Sumedha; Bochukova, Elena G.; Bounds, Rebecca; Ashford, Sofie; Gayton, Emma; Hindmarsh, Peter C.; Shield, Julian P.H.; Crowne, Elizabeth; Barford, David; Wareham, Nick J.; O’Rahilly, Stephen; Murphy, Michael P.; Powell, David R.; Barroso, Ines; Farooqi, I. Sadaf

    2013-01-01

    Summary Kinase suppressor of Ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Targeted deletion of Ksr2 leads to obesity in mice, suggesting a role in energy homeostasis. We explored the role of KSR2 in humans by sequencing 2,101 individuals with severe early-onset obesity and 1,536 controls. We identified multiple rare variants in KSR2 that disrupt signaling through the Raf-MEK-ERK pathway and impair cellular fatty acid oxidation and glucose oxidation in transfected cells; effects that can be ameliorated by the commonly prescribed antidiabetic drug, metformin. Mutation carriers exhibit hyperphagia in childhood, low heart rate, reduced basal metabolic rate and severe insulin resistance. These data establish KSR2 as an important regulator of energy intake, energy expenditure, and substrate utilization in humans. Modulation of KSR2-mediated effects may represent a novel therapeutic strategy for obesity and type 2 diabetes. PaperFlick PMID:24209692

  11. Impairment of immunity to Candida and Mycobacterium in humans with bi-allelic RORC mutations*

    PubMed Central

    Halwani, Rabih; Ma, Cindy S.; Wong, Natalie; Soudais, Claire; Henderson, Lauren A.; Marzouqa, Hiyam; Shamma, Jamal; Gonzalez, Marcela; Martinez-Barricarte, Rubén; Okada, Chizuru; Avery, Danielle T.; Latorre, Daniela; Deswarte, Caroline; Jabot-Hanin, Fabienne; Torrado, Egidio; Fountain, Jeffrey; Belkadi, Aziz; Itan, Yuval; Boisson, Bertrand; Migaud, Mélanie; Arlehamn, Cecilia S. Lindestam; Sette, Alessandro; Breton, Sylvain; McCluskey, James; Rossjohn, Jamie; de Villartay, Jean-Pierre; Moshous, Despina; Hambleton, Sophie; Latour, Sylvain; Arkwright, Peter D.; Picard, Capucine; Lantz, Olivier; Engelhard, Dan; Kobayashi, Masao; Abel, Laurent; Casanova, Jean-Laurent

    2015-01-01

    Human inborn errors of immunity mediated by the cytokines interleukin (IL)-17A/F underlie mucocutaneous candidiasis, whereas inborn errors of interferon (IFN)-γ immunity underlie mycobacterial disease. We report the discovery of bi-allelic RORC loss-of-function mutations in seven individuals from three kindreds of different ethnic origins with both candidiasis and mycobacteriosis. The lack of functional RORγ and RORγT isoforms resulted in the absence of IL-17A/F-producing T cells in these individuals, probably accounting for their chronic candidiasis. Unexpectedly, leukocytes from RORγ- and RORγT-deficient individuals also displayed an impaired IFN-γ response to Mycobacterium. This principally reflected profoundly defective IFN-γ production by circulating γδ T cells and CD4+CCR6+ CXCR3+ αβ T cells. In humans, both mucocutaneous immunity to Candida and systemic immunity to Mycobacterium require RORγ, or RORγT, or both. PMID:26160376

  12. Munc18-1 mutations that strongly impair SNARE-complex binding support normal synaptic transmission

    PubMed Central

    Meijer, Marieke; Burkhardt, Pawel; de Wit, Heidi; Toonen, Ruud F; Fasshauer, Dirk; Verhage, Matthijs

    2012-01-01

    Synaptic transmission depends critically on the Sec1p/Munc18 protein Munc18-1, but it is unclear whether Munc18-1 primarily operates as a integral part of the fusion machinery or has a more upstream role in fusion complex assembly. Here, we show that point mutations in Munc18-1 that interfere with binding to the free Syntaxin1a N-terminus and strongly impair binding to assembled SNARE complexes all support normal docking, priming and fusion of synaptic vesicles, and normal synaptic plasticity in munc18-1 null mutant neurons. These data support a prevailing role of Munc18-1 before/during SNARE-complex assembly, while its continued association to assembled SNARE complexes is dispensable for synaptic transmission. PMID:22446389

  13. Tyrosinase processing and intracellular trafficking is disrupted in mouse primary melanocytes carrying the underwhite (uw) mutation. A model for oculocutaneous albinism (OCA) type 4.

    PubMed

    Costin, Gertrude-E; Valencia, Julio C; Vieira, Wilfred D; Lamoreux, M Lynn; Hearing, Vincent J

    2003-08-01

    Oculocutaneous albinism (OCA) type 4 is a newly identified human autosomal recessive hypopigmentary disorder that disrupts pigmentation in the skin, hair and eyes. Three other forms of OCA have been previously characterized, each resulting from the aberrant processing and/or sorting of tyrosinase, the enzyme critical to pigment production in mammals. The disruption of tyrosinase trafficking occurs at the level of the endoplasmic reticulum (ER) in OCA1 and OCA3, but at the post-Golgi level in OCA2. The gene responsible for OCA4 is the human homologue of the mouse underwhite (uw) gene, which encodes the membrane-associated transporter protein (MATP). To characterize OCA4, we investigated the processing and sorting of melanogenic proteins in primary melanocytes derived from uw/uw mice and from wild-type mice. OCA4 melanocytes were found to be constantly secreted into the medium dark vesicles that contain tyrosinase and two other melanogenic enzymes, Tyrp1 (tyrosinase-related protein 1) and Dct (DOPAchrome tautomerase); this secretory process is not seen in wild-type melanocytes. Although tyrosinase was synthesized at comparable rates in wild-type and in uw-mutant melanocytes, tyrosinase activity in uw-mutant melanocytes was only about 20% of that found in wild-type melanocytes, and was enriched only about threefold in melanosomes compared with the ninefold enrichment in wild-type melanocytes. OCA4 melanocytes showed a marked difference from wild-type melanocytes in that tyrosinase was abnormally secreted from the cells, a process similar to that seen in OCA2 melanocytes, which results from a mutation of the pink-eyed dilution (P) gene. The P protein and MATP have 12 transmembrane regions and are predicted to function as transporters. Ultrastructural analysis shows that the vesicles secreted from OCA4 melanocytes are mostly early stage melanosomes. Taken together, our results show that in OCA4 melanocytes, tyrosinase processing and intracellular trafficking to the

  14. Effects of wild type tau and disease-linked tau mutations on microtubule organization and intracellular trafficking.

    PubMed

    Yu, Dezhi; Feinstein, Stuart C; Valentine, Megan T

    2016-05-24

    We investigate the effects of transient expression of wild type (WT) and disease-linked mutations of tau (R406W, P301L, ΔN296) on cytoskeletal organization and cargo transport in COS-7 cells, which are natively tau-free. The introduction of tau proteins (either WT or mutant forms) leads to a dramatic restructuring of the microtubule cytoskeleton, as observed using immunofluorescence microscopy. Yet, this microtubule bundling and aggregation has a modest effect on the speed and travel distance of motor-driven cargo transport, as measured by the motions of fluorescently-labeled lysosomes. This suggests that localized transport events are insensitive to the global structure of the microtubule cytoskeleton. Importantly, we also found no evidence that the disease-linked tau mutants were particularly toxic; in fact we found that expression of mutant and WT tau had similar effects on overall microtubule structure and transport phenotypes. PMID:26674472

  15. Mutations in Alström protein impair terminal differentiation of cardiomyocytes.

    PubMed

    Shenje, Lincoln T; Andersen, Peter; Halushka, Marc K; Lui, Cecillia; Fernandez, Laviel; Collin, Gayle B; Amat-Alarcon, Nuria; Meschino, Wendy; Cutz, Ernest; Chang, Kenneth; Yonescu, Raluca; Batista, Denise A S; Chen, Yan; Chelko, Stephen; Crosson, Jane E; Scheel, Janet; Vricella, Luca; Craig, Brian D; Marosy, Beth A; Mohr, David W; Hetrick, Kurt N; Romm, Jane M; Scott, Alan F; Valle, David; Naggert, Jürgen K; Kwon, Chulan; Doheny, Kimberly F; Judge, Daniel P

    2014-01-01

    Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole-exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognize homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at 2 weeks postnatal compared with wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest. PMID:24595103

  16. Mutations in LTBP4 Cause a Syndrome of Impaired Pulmonary, Gastrointestinal, Genitourinary, Musculoskeletal, and Dermal Development

    PubMed Central

    Urban, Zsolt; Hucthagowder, Vishwanathan; Schürmann, Nura; Todorovic, Vesna; Zilberberg, Lior; Choi, Jiwon; Sens, Carla; Brown, Chester W.; Clark, Robin D.; Holland, Kristen E.; Marble, Michael; Sakai, Lynn Y.; Dabovic, Branka; Rifkin, Daniel B.; Davis, Elaine C.

    2009-01-01

    We report recessive mutations in the gene for the latent transforming growth factor-β binding protein 4 (LTBP4) in four unrelated patients with a human syndrome disrupting pulmonary, gastrointestinal, urinary, musculoskeletal, craniofacial, and dermal development. All patients had severe respiratory distress, with cystic and atelectatic changes in the lungs complicated by tracheomalacia and diaphragmatic hernia. Three of the four patients died of respiratory failure. Cardiovascular lesions were mild, limited to pulmonary artery stenosis and patent foramen ovale. Gastrointestinal malformations included diverticulosis, enlargement, tortuosity, and stenosis at various levels of the intestinal tract. The urinary tract was affected by diverticulosis and hydronephrosis. Joint laxity and low muscle tone contributed to musculoskeletal problems compounded by postnatal growth delay. Craniofacial features included microretrognathia, flat midface, receding forehead, and wide fontanelles. All patients had cutis laxa. Four of the five identified LTBP4 mutations led to premature termination of translation and destabilization of the LTBP4 mRNA. Impaired synthesis and lack of deposition of LTBP4 into the extracellular matrix (ECM) caused increased transforming growth factor-β (TGF-β) activity in cultured fibroblasts and defective elastic fiber assembly in all tissues affected by the disease. These molecular defects were associated with blocked alveolarization and airway collapse in the lung. Our results show that coupling of TGF-β signaling and ECM assembly is essential for proper development and is achieved in multiple human organ systems by multifunctional proteins such as LTBP4. PMID:19836010

  17. TREM2 mutations implicated in neurodegeneration impair cell surface transport and phagocytosis.

    PubMed

    Kleinberger, Gernot; Yamanishi, Yoshinori; Suárez-Calvet, Marc; Czirr, Eva; Lohmann, Ebba; Cuyvers, Elise; Struyfs, Hanne; Pettkus, Nadine; Wenninger-Weinzierl, Andrea; Mazaheri, Fargol; Tahirovic, Sabina; Lleó, Alberto; Alcolea, Daniel; Fortea, Juan; Willem, Michael; Lammich, Sven; Molinuevo, José L; Sánchez-Valle, Raquel; Antonell, Anna; Ramirez, Alfredo; Heneka, Michael T; Sleegers, Kristel; van der Zee, Julie; Martin, Jean-Jacques; Engelborghs, Sebastiaan; Demirtas-Tatlidede, Asli; Zetterberg, Henrik; Van Broeckhoven, Christine; Gurvit, Hakan; Wyss-Coray, Tony; Hardy, John; Colonna, Marco; Haass, Christian

    2014-07-01

    Genetic variants in the triggering receptor expressed on myeloid cells 2 (TREM2) have been linked to Nasu-Hakola disease, Alzheimer's disease (AD), Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia (FTD), and FTD-like syndrome without bone involvement. TREM2 is an innate immune receptor preferentially expressed by microglia and is involved in inflammation and phagocytosis. Whether and how TREM2 missense mutations affect TREM2 function is unclear. We report that missense mutations associated with FTD and FTD-like syndrome reduce TREM2 maturation, abolish shedding by ADAM proteases, and impair the phagocytic activity of TREM2-expressing cells. As a consequence of reduced shedding, TREM2 is virtually absent in the cerebrospinal fluid (CSF) and plasma of a patient with FTD-like syndrome. A decrease in soluble TREM2 was also observed in the CSF of patients with AD and FTD, further suggesting that reduced TREM2 function may contribute to increased risk for two neurodegenerative disorders. PMID:24990881

  18. GIGYF2 mutation in late-onset Parkinson’s disease with cognitive impairment

    PubMed Central

    Ruiz-Martinez, Javier; Krebs, Catharine E.; Makarov, Vladimir; Gorostidi, Ana; Martí-Massó, Jose Félix; Paisán-Ruiz, Coro

    2015-01-01

    Although in the last two decades there has been considerable progress in understanding the genetic basis of Parkinson’s disease (PD), the majority of PD is sporadic and its genetic causes are largely unknown. In an attempt to identify novel genetic causes of PD, whole exome sequencing and subsequent analyses were performed in a family featuring late-onset PD with cognitive impairment. A novel genetic variant (p.Arg610Gly) in the GIGYF2 gene, previously known to be associated with PD, was identified as potential disease-causing mutation. The GIGYF2 p.Arg610Gly mutation situated in the GYF domain of the encoding protein was predicted to be pathogenic and to disrupt the GYF’s ligand–binding abilities. While further research is still required, this finding may shed light on the GIGYF2-associated mechanisms that lead to PD and suggests insulin dysregulation as a disease-specific mechanism for both PD and cognitive dysfunction. PMID:26134514

  19. Disease-Causing SDHAF1 Mutations Impair Transfer of Fe-S Clusters to SDHB.

    PubMed

    Maio, Nunziata; Ghezzi, Daniele; Verrigni, Daniela; Rizza, Teresa; Bertini, Enrico; Martinelli, Diego; Zeviani, Massimo; Singh, Anamika; Carrozzo, Rosalba; Rouault, Tracey A

    2016-02-01

    SDHAF1 mutations cause a rare mitochondrial complex II (CII) deficiency, which manifests as infantile leukoencephalopathy with elevated levels of serum and white matter succinate and lactate. Here, we demonstrate that SDHAF1 contributes to iron-sulfur (Fe-S) cluster incorporation into the Fe-S subunit of CII, SDHB. SDHAF1 transiently binds to aromatic peptides of SDHB through an arginine-rich region in its C terminus and specifically engages a Fe-S donor complex, consisting of the scaffold, holo-ISCU, and the co-chaperone-chaperone pair, HSC20-HSPA9, through an LYR motif near its N-terminal domain. Pathogenic mutations of SDHAF1 abrogate binding to SDHB, which impairs biogenesis of holo-SDHB and results in LONP1-mediated degradation of SDHB. Riboflavin treatment was found to ameliorate the neurologic condition of patients. We demonstrate that riboflavin enhances flavinylation of SDHA and reduces levels of succinate and Hypoxia-Inducible Factor (HIF)-1α and -2α, explaining the favorable response of patients to riboflavin. PMID:26749241

  20. Identification of a point mutation impairing the binding between aquaporin-4 and neuromyelitis optica autoantibodies.

    PubMed

    Pisani, Francesco; Mola, Maria Grazia; Simone, Laura; Rosito, Stefania; Alberga, Domenico; Mangiatordi, Giuseppe Felice; Lattanzi, Gianluca; Nicolotti, Orazio; Frigeri, Antonio; Svelto, Maria; Nicchia, Grazia Paola

    2014-10-31

    Neuromyelitis optica (NMO) is characterized by the presence of pathogenic autoantibodies (NMO-IgGs) against supra-molecular assemblies of aquaporin-4 (AQP4), known as orthogonal array of particles (OAPs). NMO-IgGs have a polyclonal origin and recognize different conformational epitopes involving extracellular AQP4 loops A, C, and E. Here we hypothesize a pivotal role for AQP4 transmembrane regions (TMs) in epitope assembly. On the basis of multialignment analysis, mutagenesis, NMO-IgG binding, and cytotoxicity assay, we have disclosed the key role of aspartate 69 (Asp(69)) of TM2 for NMO-IgG epitope assembly. Mutation of Asp(69) to histidine severely impairs NMO-IgG binding for 85.7% of the NMO patient sera analyzed here. Although Blue Native-PAGE, total internal reflection fluorescence microscopy, and water transport assays indicate that the OAP Asp(69) mutant is similar in structure and function to the wild type, molecular dynamic simulations have revealed that the D(69)H mutation has the effect of altering the structural rearrangements of extracellular loop A. In conclusion, Asp(69) is crucial for the spatial control of loop A, the particular molecular conformation of which enables the assembly of NMO-IgG epitopes. These findings provide additional clues for new strategies for NMO treatment and a wealth of information to better approach NMO pathogenesis. PMID:25239624

  1. Spontaneous mutation 7B-1 in tomato impairs blue light-induced stomatal opening.

    PubMed

    Hlavinka, Jan; Nauš, Jan; Fellner, Martin

    2013-08-01

    It was reported earlier that 7B-1 mutant in tomato (Solanum lycopersicum L.), an ABA overproducer, is defective in blue light (BL) signaling leading to BL-specific resistance to abiotic and biotic stresses. In this work, we examine responses of stomata to blue, red and white lights, fusicoccin, anion channel blockers (anthracene-9-carboxylic acid; 9-AC and niflumic acid; NIF) and ABA. Our results showed that the aperture of 7B-1 stomata does not increase in BL, suggesting that 7B-1 mutation impairs an element of BL signaling pathway involved in stomatal opening. Similar stomatal responses of 7B-1 and wild type (WT) to fusicoccin or 9-AC points out that activity of H(+)-ATPase and 9-AC-sensitive anion channels per se is not likely affected by the mutation. Since 9-AC restored stomatal opening of 7B-1 in BL, it seems that 9-AC and BL could block similar type of anion channels. The stomata of both genotypes did not respond to NIF neither in darkness nor in any light conditions tested. In light, 9-AC but not NIF restored stomatal opening inhibited by ABA in WT and 7B-1. We suggest that in comparison to WT, the activity of S-type anion channels in 7B-1 is more promoted by increased ABA content, and less reduced by BL, because of the mutant resistance to BL. PMID:23759105

  2. A novel de novo dominant negative mutation in DNM1L impairs mitochondrial fission and presents as childhood epileptic encephalopathy.

    PubMed

    Fahrner, Jill A; Liu, Raymond; Perry, Michael Scott; Klein, Jessica; Chan, David C

    2016-08-01

    DNM1L encodes dynamin-related protein 1 (DRP1/DLP1), a key component of the mitochondrial fission machinery that is essential for proper functioning of the mammalian brain. Previously reported probands with de novo missense mutations in DNM1L presented in the first year of life with severe encephalopathy and refractory epilepsy, with several dying within the first several weeks after birth. In contrast, we report identical novel missense mutations in DNM1L in two unrelated probands who experienced normal development for several years before presenting with refractory focal status epilepticus and subsequent rapid neurological decline. We expand the phenotype of DNM1L-related mitochondrial fission defects, reveal common unique clinical characteristics and imaging findings, and compare the cellular impact of this novel mutation to the previously reported A395D lethal variant. We demonstrate that our R403C mutation, which resides in the assembly region of DRP1, acts by a dominant-negative mechanism and reduces oligomerization, mitochondrial fission activity, and mitochondrial recruitment of DRP1, but to a lesser extent compared to the A395D mutation. In contrast to the initial report of neonatal lethality resulting from DNM1L mutation and DRP1 dysfunction, our results show that milder DRP1 impairment is compatible with normal early development and subsequently results in a distinct set of neurological findings. In addition, we identify a common pathogenic mechanism whereby DNM1L mutations impair mitochondrial fission. © 2016 Wiley Periodicals, Inc. PMID:27145208

  3. Gain-of-Function Mutations in RARB Cause Intellectual Disability with Progressive Motor Impairment.

    PubMed

    Srour, Myriam; Caron, Véronique; Pearson, Toni; Nielsen, Sarah B; Lévesque, Sébastien; Delrue, Marie-Ange; Becker, Troy A; Hamdan, Fadi F; Kibar, Zoha; Sattler, Shannon G; Schneider, Michael C; Bitoun, Pierre; Chassaing, Nicolas; Rosenfeld, Jill A; Xia, Fan; Desai, Sonal; Roeder, Elizabeth; Kimonis, Virginia; Schneider, Adele; Littlejohn, Rebecca Okashah; Douzgou, Sofia; Tremblay, André; Michaud, Jacques L

    2016-08-01

    Retinoic acid (RA) signaling plays a key role in the development and function of several systems in mammals. We previously discovered that the de novo mutations c.1159C>T (p.Arg387Cys) and c.1159C>A (p.Arg387Ser) in the RA Receptor Beta (RARB) gene cause microphthalmia and diaphragmatic hernia. However, the natural history of affected subjects beyond the prenatal or neonatal period was unknown. Here, we describe nine additional subjects with microphthalmia who have de novo mutations in RARB, including the previously described p.Arg387Cys as well as the novel c.887G>C (p.Gly296Ala) and c.638T>C (p.Leu213Pro). Moreover, we review the information on four previously reported cases. All subjects who survived the neonatal period (n = 10) displayed severe global developmental delay with progressive motor impairment due to spasticity and/or dystonia (with or without chorea). The majority of subjects also showed Chiari type I malformation and severe feeding difficulties. We previously found that p.Arg387Cys and p.Arg387Ser induce a gain-of-function. We show here that the p.Gly296Ala and p.Leu213Pro RARB mutations further promote the RA ligand-induced transcriptional activity by twofold to threefold over the wild-type receptor, also indicating a gain-of-function mechanism. These observations suggest that precise regulation of RA signaling is required for brain development and/or function in humans. PMID:27120018

  4. ECEL1 mutation implicates impaired axonal arborization of motor nerves in the pathogenesis of distal arthrogryposis.

    PubMed

    Nagata, Kenichi; Kiryu-Seo, Sumiko; Tamada, Hiromi; Okuyama-Uchimura, Fumi; Kiyama, Hiroshi; Saido, Takaomi C

    2016-07-01

    The membrane-bound metalloprotease endothelin-converting enzyme-like 1 (ECEL1) has been newly identified as a causal gene of a specific type of distal arthrogryposis (DA). In contrast to most causal genes of DA, ECEL1 is predominantly expressed in neuronal cells, suggesting a unique neurogenic pathogenesis in a subset of DA patients with ECEL1 mutation. The present study analyzed developmental motor innervation and neuromuscular junction formation in limbs of the rodent homologue damage-induced neuronal endopeptidase (DINE)-deficient mouse. Whole-mount immunostaining was performed in DINE-deficient limbs expressing motoneuron-specific GFP to visualize motor innervation throughout the limb. Although DINE-deficient motor nerves displayed normal trajectory patterns from the spinal cord to skeletal muscles, they indicated impaired axonal arborization in skeletal muscles in the forelimbs and hindlimbs. Systematic examination of motor innervation in over 10 different hindlimb muscles provided evidence that DINE gene disruption leads to insufficient arborization of motor nerves after arriving at the skeletal muscle. Interestingly, the axonal arborization defect in foot muscles appeared more severe than in other hindlimb muscles, which was partially consistent with the proximal-distal phenotypic discordance observed in DA patients. Additionally, the number of innervated neuromuscular junction was significantly reduced in the severely affected DINE-deficient muscle. Furthermore, we generated a DINE knock-in (KI) mouse model with a pathogenic mutation, which was recently identified in DA patients. Axonal arborization defects were clearly detected in motor nerves of the DINE KI limb, which was identical to the DINE-deficient limb. Given that the encoded sequences, as well as ECEL1 and DINE expression profiles, are highly conserved between mouse and human, abnormal arborization of motor axons and subsequent failure of NMJ formation could be a primary cause of DA with ECEL1

  5. A novel biallelic splice site mutation of TECTA causes moderate to severe hearing impairment in an Algerian family.

    PubMed

    Behlouli, Asma; Bonnet, Crystel; Abdi, Samia; Hasbellaoui, Mokhtar; Boudjenah, Farid; Hardelin, Jean-Pierre; Louha, Malek; Makrelouf, Mohamed; Ammar-Khodja, Fatima; Zenati, Akila; Petit, Christine

    2016-08-01

    Congenital deafness is certainly one of the most common monogenic diseases in humans, but it is also one of the most genetically heterogeneous, which makes molecular diagnosis challenging in most cases. Whole-exome sequencing in two out of three Algerian siblings affected by recessively-inherited, moderate to severe sensorineural deafness allowed us to identify a novel splice donor site mutation (c.5272+1G > A) in the gene encoding α-tectorin, a major component of the cochlear tectorial membrane. The mutation was present at the homozygous state in the three affected siblings, and at the heterozygous state in their unaffected, consanguineous parents. To our knowledge, this is the first reported TECTA mutation leading to the DFNB21 form of hearing impairment among Maghrebian individuals suffering from congenital hearing impairment, which further illustrates the diversity of the genes involved in congenital deafness in the Maghreb. PMID:27368438

  6. Trafficking modulator TENin1 inhibits endocytosis, causes endomembrane protein accumulation at the pre-vacuolar compartment and impairs gravitropic response in Arabidopsis thaliana.

    PubMed

    Paudyal, Rupesh; Jamaluddin, Adam; Warren, James P; Doyle, Siamsa M; Robert, Stéphanie; Warriner, Stuart L; Baker, Alison

    2014-06-01

    Auxin gradients are established and maintained by polarized distribution of auxin transporters that undergo constitutive endocytic recycling from the PM (plasma membrane) and are essential for the gravitropic response in plants. The present study characterizes an inhibitor of endomembrane protein trafficking, TE1 (trafficking and endocytosis inhibitor 1/TENin1) that reduces gravitropic root bending in Arabidopsis thaliana seedlings. Short-term TE1 treatment causes accumulation of PM proteins, including the BR (brassinosteroid) receptor BRI1 (BR insensitive 1), PIP2a (PM intrinsic protein 2a) and the auxin transporter PIN2 (PIN-FORMED 2) in a PVC (pre-vacuolar related compartment), which is sensitive to BFA (Brefeldin A). This compound inhibits endocytosis from the PM and promotes trafficking to the vacuole, consistent with inhibition of retrieval of proteins to the TGN (trans-Golgi network) from the PVC and the PM. However, trafficking of newly synthesized proteins to the PM is unaffected. The short-term protein trafficking inhibition and long-term effect on plant growth and survival caused by TE1 were fully reversible upon drug washout. Structure-activity relationship studies revealed that only minor modifications were possible without loss of biological activity. Diversity in Arabidopsis ecotypes was also exploited to identify two Arabidopsis accessions that display reduced sensitivity to TE1. This compound and the resistant Arabidopsis accessions may be used as a resource in future studies to better understand endomembrane trafficking in plants. PMID:24654932

  7. Trafficking modulator TENin1 inhibits endocytosis, causes endomembrane protein accumulation at the pre-vacuolar compartment and impairs gravitropic response in Arabidopsis thaliana

    PubMed Central

    Paudyal, Rupesh; Jamaluddin, Adam; Warren, James P.; Doyle, Siamsa M.; Robert, Stéphanie; Warriner, Stuart L.; Baker, Alison

    2014-01-01

    Auxin gradients are established and maintained by polarized distribution of auxin transporters that undergo constitutive endocytic recycling from the PM (plasma membrane) and are essential for the gravitropic response in plants. The present study characterizes an inhibitor of endomembrane protein trafficking, TE1 (trafficking and endocytosis inhibitor 1/TENin1) that reduces gravitropic root bending in Arabidopsis thaliana seedlings. Short-term TE1 treatment causes accumulation of PM proteins, including the BR (brassinosteroid) receptor BRI1 (BR insensitive 1), PIP2a (PM intrinsic protein 2a) and the auxin transporter PIN2 (PIN-FORMED 2) in a PVC (pre-vacuolar related compartment), which is sensitive to BFA (Brefeldin A). This compound inhibits endocytosis from the PM and promotes trafficking to the vacuole, consistent with inhibition of retrieval of proteins to the TGN (trans-Golgi network) from the PVC and the PM. However, trafficking of newly synthesized proteins to the PM is unaffected. The short-term protein trafficking inhibition and long-term effect on plant growth and survival caused by TE1 were fully reversible upon drug washout. Structure–activity relationship studies revealed that only minor modifications were possible without loss of biological activity. Diversity in Arabidopsis ecotypes was also exploited to identify two Arabidopsis accessions that display reduced sensitivity to TE1. This compound and the resistant Arabidopsis accessions may be used as a resource in future studies to better understand endomembrane trafficking in plants. PMID:24654932

  8. Mutations affecting the SAND domain of DEAF1 cause intellectual disability with severe speech impairment and behavioral problems.

    PubMed

    Vulto-van Silfhout, Anneke T; Rajamanickam, Shivakumar; Jensik, Philip J; Vergult, Sarah; de Rocker, Nina; Newhall, Kathryn J; Raghavan, Ramya; Reardon, Sara N; Jarrett, Kelsey; McIntyre, Tara; Bulinski, Joseph; Ownby, Stacy L; Huggenvik, Jodi I; McKnight, G Stanley; Rose, Gregory M; Cai, Xiang; Willaert, Andy; Zweier, Christiane; Endele, Sabine; de Ligt, Joep; van Bon, Bregje W M; Lugtenberg, Dorien; de Vries, Petra F; Veltman, Joris A; van Bokhoven, Hans; Brunner, Han G; Rauch, Anita; de Brouwer, Arjan P M; Carvill, Gemma L; Hoischen, Alexander; Mefford, Heather C; Eichler, Evan E; Vissers, Lisenka E L M; Menten, Björn; Collard, Michael W; de Vries, Bert B A

    2014-05-01

    Recently, we identified in two individuals with intellectual disability (ID) different de novo mutations in DEAF1, which encodes a transcription factor with an important role in embryonic development. To ascertain whether these mutations in DEAF1 are causative for the ID phenotype, we performed targeted resequencing of DEAF1 in an additional cohort of over 2,300 individuals with unexplained ID and identified two additional individuals with de novo mutations in this gene. All four individuals had severe ID with severely affected speech development, and three showed severe behavioral problems. DEAF1 is highly expressed in the CNS, especially during early embryonic development. All four mutations were missense mutations affecting the SAND domain of DEAF1. Altered DEAF1 harboring any of the four amino acid changes showed impaired transcriptional regulation of the DEAF1 promoter. Moreover, behavioral studies in mice with a conditional knockout of Deaf1 in the brain showed memory deficits and increased anxiety-like behavior. Our results demonstrate that mutations in DEAF1 cause ID and behavioral problems, most likely as a result of impaired transcriptional regulation by DEAF1. PMID:24726472

  9. Homozygosity mapping reveals mutations of GRXCR1 as a cause of autosomal-recessive nonsyndromic hearing impairment.

    PubMed

    Schraders, Margit; Lee, Kwanghyuk; Oostrik, Jaap; Huygen, Patrick L M; Ali, Ghazanfar; Hoefsloot, Lies H; Veltman, Joris A; Cremers, Frans P M; Basit, Sulman; Ansar, Muhammad; Cremers, Cor W R J; Kunst, Henricus P M; Ahmad, Wasim; Admiraal, Ronald J C; Leal, Suzanne M; Kremer, Hannie

    2010-02-12

    We identified overlapping homozygous regions within the DFNB25 locus in two Dutch and ten Pakistani families with sensorineural autosomal-recessive nonsyndromic hearing impairment (arNSHI). Only one of the families, W98-053, was not consanguineous, and its sibship pointed toward a reduced critical region of 0.9 Mb. This region contained the GRXCR1 gene, and the orthologous mouse gene was described to be mutated in the pirouette (pi) mutant with resulting hearing loss and circling behavior. Sequence analysis of the GRXCR1 gene in hearing-impaired family members revealed splice-site mutations in two Dutch families and a missense and nonsense mutation, respectively, in two Pakistani families. The splice-site mutations are predicted to cause frameshifts and premature stop codons. In family W98-053, this could be confirmed by cDNA analysis. GRXCR1 is predicted to contain a GRX-like domain. GRX domains are involved in reversible S-glutathionylation of proteins and thereby in the modulation of activity and/or localization of these proteins. The missense mutation is located in this domain, whereas the nonsense and splice-site mutations may result in complete or partial absence of the GRX-like domain or of the complete protein. Hearing loss in patients with GRXCR1 mutations is congenital and is moderate to profound. Progression of the hearing loss was observed in family W98-053. Vestibular dysfunction was observed in some but not all affected individuals. Quantitative analysis of GRXCR1 transcripts in fetal and adult human tissues revealed a preferential expression of the gene in fetal cochlea, which may explain the nonsyndromic nature of the hearing impairment. PMID:20137778

  10. Saccharomyces cerevisiae Hsp70 mutations affect [PSI+] prion propagation and cell growth differently and implicate Hsp40 and tetratricopeptide repeat cochaperones in impairment of [PSI+].

    PubMed Central

    Jones, Gary W; Masison, Daniel C

    2003-01-01

    We previously described an Hsp70 mutant (Ssa1-21p), altered in a conserved residue (L483W), that dominantly impairs yeast [PSI(+)] prion propagation without affecting growth. We generated new SSA1 mutations that impaired [PSI(+)] propagation and second-site mutations in SSA1-21 that restored normal propagation. Effects of mutations on growth did not correlate with [PSI(+)] phenotype, revealing differences in Hsp70 function required for growth and [PSI(+)] propagation and suggesting that Hsp70 interacts differently with [PSI(+)] prion aggregates than with other cellular substrates. Complementary suppression of altered activity between forward and suppressing mutations suggests that mutations that impair [PSI(+)] affect a similar Hsp70 function and that suppressing mutations similarly overcome this effect. All new mutations that impaired [PSI(+)] propagation were located in the ATPase domain. Locations and homology of several suppressing substitutions suggest that they weaken Hsp70's substrate-trapping conformation, implying that impairment of [PSI(+)] by forward mutations is due to altered ability of the ATPase domain to regulate substrate binding. Other suppressing mutations are in residues important for interactions with Hsp40 or TPR-containing cochaperones, suggesting that such interactions are necessary for the impairment of [PSI(+)] propagation caused by mutant Ssa1p. PMID:12618389

  11. Mutations that impair interaction properties of TRIM32 associated with limb-girdle muscular dystrophy 2H.

    PubMed

    Saccone, Valentina; Palmieri, Michela; Passamano, Luigia; Piluso, Giulio; Meroni, Germana; Politano, Luisa; Nigro, Vincenzo

    2008-02-01

    TRIM32 belongs to a large family of proteins characterized by a tripartite motif, possibly involved in the ubiquitination process, acting as an E3 ligase. In addition, TRIM32 has six NHL repeats with putative interaction properties. A homozygous mutation at the third NHL repeat (D487N) has been found in patients with limb girdle muscular dystrophy 2H (LGMD2H). This mutation was only identified in the inbred Manitoba Hutterite or their descendants. Interestingly, a mutation in the B-box domain of TRIM32 cosegregates with Bardet-Biedl syndrome type 11 (BBS11). The signs of BBS11 include obesity, pigmentary and retinal malformations, diabetes, polydactyly, and no muscular dystrophy, suggesting an alternative disease mechanism. We aim to ascertain whether D487N is the only pathological LGMD2H allele, limited to Hutterites. We studied the TRIM32 gene in 310 LGMD patients with no mutations at the other known loci. We identified four patients with novel mutated alleles. Two mutations were homozygous and missing in controls. These mutations also clustered at the NHL domain, suggesting that a specific (interaction) property might be abolished in LGMD2H patients. No mutations were found at the B-box region where the BBS11 mutation is found. We tested TRIM32 and its mutants by yeast-two-hybrid assay, developing an interaction test to validate mutations. All LGMD2H mutants, but not the BBS11, lost their ability to self-interact. The interaction of TRIM32 mutants with E2N, a protein involved in the ubiquitination process, was similarly impaired. In conclusion, the mutations here reported may cause muscular dystrophy by affecting the interaction properties of TRIM32. PMID:17994549

  12. RUNX2 Mutation Impairs 1α,25-Dihydroxyvitamin D3 mediated Osteoclastogenesis in Dental Follicle Cells

    PubMed Central

    Wang, X. Z.; Sun, X. Y.; Zhang, C. Y.; Yang, X.; Yan, W. J.; Ge, L. H.; Zheng, S. G.

    2016-01-01

    Cleidocranial dysplasia (CCD), a skeletal disorder characterized by delayed permanent tooth eruption and other dental abnormalities, is caused by heterozygous RUNX2 mutations. As an osteoblast-specific transcription factor, RUNX2 plays a role in bone remodeling, tooth formation and tooth eruption. To investigate the crosstalk between RUNX2 and 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) in human dental follicle cells (hDFCs) during osteoclast formation, we established a co-culture system of hDFCs from CCD patient and healthy donors with peripheral blood mononuclear cells (PBMCs). Expression of the osteoclast-associated genes and the number of TRAP+ cells were reduced in CCD hDFCs, indicating its suppressed osteoclast-inductive ability, which was reflected by the downregulated RANKL/OPG ratio. In addition, 1α,25-(OH)2D3-stimulation elevated the expression of osteoclast-related genes, as well as RANKL mRNA levels and RANKL/OPG ratios in control hDFCs. Conversely, RUNX2 mutation abolished this 1α,25-(OH)2D3-induced RANKL gene activation and osteoclast formation in CCD hDFCs. Therefore, RUNX2 haploinsufficiency impairs dental follicle-induced osteoclast formation capacity through RANKL/OPG signaling, which may be partially responsible for delayed permanent tooth eruption in CCD patients. Furthermore, this abnormality was not rescued by 1α,25-(OH)2D3 application because 1α,25-(OH)2D3-induced RANKL activation in hDFCs is mediated principally via the RUNX2-dependent pathway. PMID:27068678

  13. Association between idiopathic hearing loss and mitochondrial DNA mutations: A study on 169 hearing-impaired subjects

    PubMed Central

    GUARAN, VALERIA; ASTOLFI, LAURA; CASTIGLIONE, ALESSANDRO; SIMONI, EDI; OLIVETTO, ELENA; GALASSO, MARCO; TREVISI, PATRIZIA; BUSI, MICOL; VOLINIA, STEFANO; MARTINI, ALESSANDRO

    2013-01-01

    Mutations in mitochondrial DNA (mtDNA) have been shown to be an important cause of sensorineural hearing loss (SNHL). In this study, we performed a clinical and genetic analysis of 169 hearing-impaired patients and some of their relatives suffering from idiopathic SNHL, both familial and sporadic. The analysis of four fragments of their mtDNA identified several polymorphisms, the well known pathogenic mutation, A1555G, and some novel mutations in different genes, implying changes in the aminoacidic sequence. A novel sporadic mutation in 12S rRNA (MT-RNR1), not previously reported in the literature, was found in a case of possible aminoglycoside-induced progressive deafness. PMID:23969527

  14. The Novel PMCA2 Pump Mutation Tommy Impairs Cytosolic Calcium Clearance in Hair Cells and Links to Deafness in Mice*

    PubMed Central

    Bortolozzi, Mario; Brini, Marisa; Parkinson, Nick; Crispino, Giulia; Scimemi, Pietro; De Siati, Romolo Daniele; Di Leva, Francesca; Parker, Andrew; Ortolano, Saida; Arslan, Edoardo; Brown, Steve D.; Carafoli, Ernesto; Mammano, Fabio

    2010-01-01

    The mechanotransduction process in hair cells in the inner ear is associated with the influx of calcium from the endolymph. Calcium is exported back to the endolymph via the splice variant w/a of the PMCA2 of the stereocilia membrane. To further investigate the role of the pump, we have identified and characterized a novel ENU-induced mouse mutation, Tommy, in the PMCA2 gene. The mutation causes a non-conservative E629K change in the second intracellular loop of the pump that harbors the active site. Tommy mice show profound hearing impairment from P18, with significant differences in hearing thresholds between wild type and heterozygotes. Expression of mutant PMCA2 in CHO cells shows calcium extrusion impairment; specifically, the long term, non-stimulated calcium extrusion activity of the pump is inhibited. Calcium extrusion was investigated directly in neonatal organotypic cultures of the utricle sensory epithelium in Tommy mice. Confocal imaging combined with flash photolysis of caged calcium showed impairment of calcium export in both Tommy heterozygotes and homozygotes. Immunofluorescence studies of the organ of Corti in homozygous Tommy mice showed a progressive base to apex degeneration of hair cells after P40. Our results on the Tommy mutation along with previously observed interactions between cadherin-23 and PMCA2 mutations in mouse and humans underline the importance of maintaining the appropriate calcium concentrations in the endolymph to control the rigidity of cadherin and ensure the function of interstereocilia links, including tip links, of the stereocilia bundle. PMID:20826782

  15. Mutations in PTPRQ are a cause of autosomal-recessive nonsyndromic hearing impairment DFNB84 and associated with vestibular dysfunction.

    PubMed

    Schraders, Margit; Oostrik, Jaap; Huygen, Patrick L M; Strom, Tim M; van Wijk, Erwin; Kunst, Henricus P M; Hoefsloot, Lies H; Cremers, Cor W R J; Admiraal, Ronald J C; Kremer, Hannie

    2010-04-01

    We identified overlapping homozygous regions within the DFNB84 locus in a nonconsanguineous Dutch family and a consanguineous Moroccan family with sensorineural autosomal-recessive nonsyndromic hearing impairment (arNSHI). The critical region of 3.17 Mb harbored the PTPRQ gene and mouse models with homozygous mutations in the orthologous gene display severe hearing loss. We show that the human PTPRQ gene was not completely annotated and that additional, alternatively spliced exons are present at the 5' end of the gene. Different PTPRQ isoforms are encoded with a varying number of fibronectin type 3 (FN3) domains, a transmembrane domain, and a phosphatase domain. Sequence analysis of the PTPRQ gene in members of the families revealed a nonsense mutation in the Dutch family and a missense mutation in the Moroccan family. The missense mutation is located in one of the FN3 domains. The nonsense mutation results in a truncated protein with only a small number of FN3 domains and no transmembrane or phosphatase domain. Hearing loss in the patients with PTPRQ mutations is likely to be congenital and moderate to profound and most severe in the family with the nonsense mutation. Progression of the hearing loss was observed in both families. The hearing loss is accompanied by vestibular dysfunction in all affected individuals. Although we show that PTPRQ is expressed in many tissues, no symptoms other than deafness were observed in the patients. PMID:20346435

  16. Meta-analysis of SHANK Mutations in Autism Spectrum Disorders: A Gradient of Severity in Cognitive Impairments

    PubMed Central

    Leblond, Claire S.; Nava, Caroline; Polge, Anne; Gauthier, Julie; Huguet, Guillaume; Lumbroso, Serge; Giuliano, Fabienne; Stordeur, Coline; Depienne, Christel; Mouzat, Kevin; Pinto, Dalila; Howe, Jennifer; Lemière, Nathalie; Durand, Christelle M.; Guibert, Jessica; Ey, Elodie; Toro, Roberto; Peyre, Hugo; Mathieu, Alexandre; Amsellem, Frédérique; Rastam, Maria; Gillberg, I. Carina; Rappold, Gudrun A.; Holt, Richard; Monaco, Anthony P.; Maestrini, Elena; Galan, Pilar; Heron, Delphine; Jacquette, Aurélia; Afenjar, Alexandra; Rastetter, Agnès; Brice, Alexis; Devillard, Françoise; Assouline, Brigitte; Laffargue, Fanny; Lespinasse, James; Chiesa, Jean; Rivier, François; Bonneau, Dominique; Regnault, Beatrice; Zelenika, Diana; Delepine, Marc; Lathrop, Mark; Sanlaville, Damien; Schluth-Bolard, Caroline; Edery, Patrick; Perrin, Laurence; Tabet, Anne Claude; Schmeisser, Michael J.; Boeckers, Tobias M.; Coleman, Mary; Sato, Daisuke; Szatmari, Peter; Scherer, Stephen W.; Rouleau, Guy A.; Betancur, Catalina; Leboyer, Marion; Gillberg, Christopher

    2014-01-01

    SHANK genes code for scaffold proteins located at the post-synaptic density of glutamatergic synapses. In neurons, SHANK2 and SHANK3 have a positive effect on the induction and maturation of dendritic spines, whereas SHANK1 induces the enlargement of spine heads. Mutations in SHANK genes have been associated with autism spectrum disorders (ASD), but their prevalence and clinical relevance remain to be determined. Here, we performed a new screen and a meta-analysis of SHANK copy-number and coding-sequence variants in ASD. Copy-number variants were analyzed in 5,657 patients and 19,163 controls, coding-sequence variants were ascertained in 760 to 2,147 patients and 492 to 1,090 controls (depending on the gene), and, individuals carrying de novo or truncating SHANK mutations underwent an extensive clinical investigation. Copy-number variants and truncating mutations in SHANK genes were present in ∼1% of patients with ASD: mutations in SHANK1 were rare (0.04%) and present in males with normal IQ and autism; mutations in SHANK2 were present in 0.17% of patients with ASD and mild intellectual disability; mutations in SHANK3 were present in 0.69% of patients with ASD and up to 2.12% of the cases with moderate to profound intellectual disability. In summary, mutations of the SHANK genes were detected in the whole spectrum of autism with a gradient of severity in cognitive impairment. Given the rare frequency of SHANK1 and SHANK2 deleterious mutations, the clinical relevance of these genes remains to be ascertained. In contrast, the frequency and the penetrance of SHANK3 mutations in individuals with ASD and intellectual disability—more than 1 in 50—warrant its consideration for mutation screening in clinical practice. PMID:25188300

  17. Targeted next-generation sequencing of deafness genes in hearing-impaired individuals uncovers informative mutations

    PubMed Central

    Vona, Barbara; Müller, Tobias; Nanda, Indrajit; Neuner, Cordula; Hofrichter, Michaela A. H.; Schröder, Jörg; Bartsch, Oliver; Läßig, Anne; Keilmann, Annerose; Schraven, Sebastian; Kraus, Fabian; Shehata-Dieler, Wafaa; Haaf, Thomas

    2014-01-01

    Purpose: Targeted next-generation sequencing provides a remarkable opportunity to identify variants in known disease genes, particularly in extremely heterogeneous disorders such as nonsyndromic hearing loss. The present study attempts to shed light on the complexity of hearing impairment. Methods: Using one of two next-generation sequencing panels containing either 80 or 129 deafness genes, we screened 30 individuals with nonsyndromic hearing loss (from 23 unrelated families) and analyzed 9 normal-hearing controls. Results: Overall, we found an average of 3.7 variants (in 80 genes) with deleterious prediction outcome, including a number of novel variants, in individuals with nonsyndromic hearing loss and 1.4 in controls. By next-generation sequencing alone, 12 of 23 (52%) probands were diagnosed with monogenic forms of nonsyndromic hearing loss; one individual displayed a DNA sequence mutation together with a microdeletion. Two (9%) probands have Usher syndrome. In the undiagnosed individuals (10/23; 43%) we detected a significant enrichment of potentially pathogenic variants as compared to controls. Conclusion: Next-generation sequencing combined with microarrays provides the diagnosis for approximately half of the GJB2 mutation–negative individuals. Usher syndrome was found to be more frequent in the study cohort than anticipated. The conditions in a proportion of individuals with nonsyndromic hearing loss, particularly in the undiagnosed group, may have been caused or modified by an accumulation of unfavorable variants across multiple genes. PMID:24875298

  18. IMMUNODEFICIENCIES. Impairment of immunity to Candida and Mycobacterium in humans with bi-allelic RORC mutations.

    PubMed

    Okada, Satoshi; Markle, Janet G; Deenick, Elissa K; Mele, Federico; Averbuch, Dina; Lagos, Macarena; Alzahrani, Mohammed; Al-Muhsen, Saleh; Halwani, Rabih; Ma, Cindy S; Wong, Natalie; Soudais, Claire; Henderson, Lauren A; Marzouqa, Hiyam; Shamma, Jamal; Gonzalez, Marcela; Martinez-Barricarte, Rubén; Okada, Chizuru; Avery, Danielle T; Latorre, Daniela; Deswarte, Caroline; Jabot-Hanin, Fabienne; Torrado, Egidio; Fountain, Jeffrey; Belkadi, Aziz; Itan, Yuval; Boisson, Bertrand; Migaud, Mélanie; Arlehamn, Cecilia S Lindestam; Sette, Alessandro; Breton, Sylvain; McCluskey, James; Rossjohn, Jamie; de Villartay, Jean-Pierre; Moshous, Despina; Hambleton, Sophie; Latour, Sylvain; Arkwright, Peter D; Picard, Capucine; Lantz, Olivier; Engelhard, Dan; Kobayashi, Masao; Abel, Laurent; Cooper, Andrea M; Notarangelo, Luigi D; Boisson-Dupuis, Stéphanie; Puel, Anne; Sallusto, Federica; Bustamante, Jacinta; Tangye, Stuart G; Casanova, Jean-Laurent

    2015-08-01

    Human inborn errors of immunity mediated by the cytokines interleukin-17A and interleukin-17F (IL-17A/F) underlie mucocutaneous candidiasis, whereas inborn errors of interferon-γ (IFN-γ) immunity underlie mycobacterial disease. We report the discovery of bi-allelic RORC loss-of-function mutations in seven individuals from three kindreds of different ethnic origins with both candidiasis and mycobacteriosis. The lack of functional RORγ and RORγT isoforms resulted in the absence of IL-17A/F-producing T cells in these individuals, probably accounting for their chronic candidiasis. Unexpectedly, leukocytes from RORγ- and RORγT-deficient individuals also displayed an impaired IFN-γ response to Mycobacterium. This principally reflected profoundly defective IFN-γ production by circulating γδ T cells and CD4(+)CCR6(+)CXCR3(+) αβ T cells. In humans, both mucocutaneous immunity to Candida and systemic immunity to Mycobacterium require RORγ, RORγT, or both. PMID:26160376

  19. Impaired water maze learning performance without altered dopaminergic function in mice heterozygous for the GDNF mutation.

    PubMed

    Gerlai, R; McNamara, A; Choi-Lundberg, D L; Armanini, M; Ross, J; Powell-Braxton, L; Phillips, H S

    2001-10-01

    Exogenous glial cell line-derived neurotrophic factor (GDNF) exhibits potent survival-promoting effects on dopaminergic neurons of the nigrostriatal pathway that is implicated in Parkinson's disease and also protects neurons in forebrain ischemia of animal models. However, a role for endogenous GDNF in brain function has not been established. Although mice homozygous for a targeted deletion of the GDNF gene have been generated, these mice die within hours of birth because of deficits in kidney morphogenesis, and, thus, the effect of the absence of GDNF on brain function could not be studied. Herein, we sought to determine whether adult mice, heterozygous for a GDNF mutation on two different genetic backgrounds, demonstrate alterations in the nigrostriatal dopaminergic system or in cognitive function. While both neurochemical and behavioural measures suggested that reduction of GDNF gene expression in the mutant mice does not alter the nigrostriatal dopaminergic system, it led to a significant and selective impairment of performance in the spatial version of the Morris water maze. A standard panel of blood chemistry tests and basic pathological analyses did not reveal alterations in the mutants that could account for the observed performance deficit. These results suggest that endogenous GDNF may not be critical for the development and functioning of the nigrostriatal dopaminergic system but it plays an important role in cognitive abilities. PMID:11683907

  20. Biallelic nonsense mutations in the otogelin-like gene (OTOGL) in a child affected by mild to moderate hearing impairment.

    PubMed

    Bonnet, C; Louha, M; Loundon, N; Michalski, N; Verpy, E; Smagghe, L; Hardelin, J-P; Rouillon, I; Jonard, L; Couderc, R; Gherbi, S; Garabedian, E N; Denoyelle, F; Petit, C; Marlin, S

    2013-09-25

    Hearing impairment is characterized by great genetic heterogeneity. We report the identification, by whole exome sequencing, of two different nonsense mutations (c.1558C>T; p.Gln520 and c.2773C>T; p.Arg925) in the otogelin-like gene (OTOGL), in a child affected by mild to moderate isolated deafness. Parental genotypes allowed us to conclude that these mutations are present in the compound heterozygous state in the patient. In addition, our clinical data establish that the tectorial membrane and/or the outer hair cells are defective in this form of deafness. PMID:23850727

  1. Adult-onset focal expression of mutated human tau in the hippocampus impairs spatial working memory of rats

    PubMed Central

    Mustroph, M.L.; King, M.A.; Klein, R.L.; Ramirez, J.J.

    2012-01-01

    Tauopathy in the hippocampus is one of the earliest cardinal features of Alzheimer’s disease (AD), a condition characterized by progressive memory impairments. In fact, density of tau neurofibrillary tangles (NFTs) in the hippocampus strongly correlates with severity of cognitive impairments in AD. In the present study, we employed a somatic cell gene transfer technique to create a rodent model of tauopathy by injecting a recombinant adeno-associated viral vector with a mutated human tau gene (P301L) into the hippocampus of adult rats. The P301L mutation is causal for frontotemporal dementia with parkinsonism-17 (FTDP-17), but it has been used for studying memory effects characteristic of AD in transgenic mice. To ascertain if P301L-induced mnemonic deficits are persistent, animals were tested for 6 months. It was hypothesized that adult-onset, spatially restricted tau expression in the hippocampus would produce progressive spatial working memory deficits on a learned alternation task. Rats injected with the tau vector exhibited persistent impairments on the hippocampal-dependent task beginning at about 6 weeks post-transduction compared to rats injected with a green fluorescent protein vector. Histological analysis of brains for expression of human tau revealed hyperphosphorylated human tau and NFTs in the hippocampus in experimental animals only. Thus, adult-onset, vector-induced tauopathy spatially restricted to the hippocampus progressively impaired spatial working memory in rats. We conclude that the model faithfully reproduces histological and behavioral findings characteristic of dementing tauopathies. The rapid onset of sustained memory impairment establishes a preclinical model particularly suited to the development of potential tauopathy therapeutics. PMID:22561128

  2. Severe congenital neutropenia with neurological impairment due to a homozygous VPS45 p.E238K mutation: A case report suggesting a genotype-phenotype correlation.

    PubMed

    Meerschaut, Ilse; Bordon, Victoria; Dhooge, Catharina; Delbeke, Patricia; Vanlander, Arnaud V; Simon, Amos; Klein, Christoph; Kooy, R Frank; Somech, Raz; Callewaert, Bert

    2015-12-01

    VPS45 mutations cause severe congenital neutropenia (SCN). We report on a girl with SCN and neurological impairment harboring a homozygous p.E238K mutation in VPS45 (vacuolar sorting protein 45). She successfully underwent hematopoietic stem cell transplantation. Our findings delineate the phenotype and indicate a possible genotype-phenotype correlation for neurological involvement. PMID:26358756

  3. A Novel de novo Mutation in CEACAM16 Associated with Postlingual Hearing Impairment

    PubMed Central

    Hofrichter, Michaela A.H.; Nanda, Indrajit; Gräf, Jens; Schröder, Jörg; Shehata-Dieler, Wafaa; Vona, Barbara; Haaf, Thomas

    2015-01-01

    Mutations in CEACAM16 cause autosomal dominant nonsyndromic hearing loss (DFNA4B). So far, 2 families have been reported with segregating missense mutations, both in the immunoglobulin constant domain A of the CEACAM16 protein. In this study, we used the TruSight One panel to investigate a parent-child trio without familial history of hearing loss and one affected child. When filtering for recessive inheritance and de novo events, we discovered a de novo CEACAM16 mutation (c.1094T>G, p.Leu365Arg) as the sole likely pathogenic variant. The de novo mutation was confirmed by Sanger sequencing and STR analysis. The proband's hearing loss closely matches the described onset and severity for DFNA4B. We present the third CEACAM16 variant and the first de novo mutation in CEACAM16. This de novo mutation is robustly described as a pathogenic mutation according to in silico mutation prediction tools and affects a highly conserved amino acid in the most strongly conserved CEACAM16 N2 domain. Our strategy of screening family trios enhances de novo mutation discovery and the exclusion of other variants of potential interest through pedigree filtering. PMID:26648831

  4. Mutations in GABAA receptor subunits associated with genetic epilepsies.

    PubMed

    Macdonald, Robert L; Kang, Jing-Qiong; Gallagher, Martin J

    2010-06-01

    Mutations in inhibitory GABAA receptor subunit genes (GABRA1, GABRB3, GABRG2 and GABRD) have been associated with genetic epilepsy syndromes including childhood absence epilepsy (CAE), juvenile myoclonic epilepsy (JME), pure febrile seizures (FS), generalized epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome (DS)/severe myoclonic epilepsy in infancy (SMEI). These mutations are found in both translated and untranslated gene regions and have been shown to affect the GABAA receptors by altering receptor function and/or by impairing receptor biogenesis by multiple mechanisms including reducing subunit mRNA transcription or stability, impairing subunit folding, stability, or oligomerization and by inhibiting receptor trafficking. PMID:20308251

  5. PKA catalytic subunit mutations in adrenocortical Cushing's adenoma impair association with the regulatory subunit.

    PubMed

    Calebiro, Davide; Hannawacker, Annette; Lyga, Sandra; Bathon, Kerstin; Zabel, Ulrike; Ronchi, Cristina; Beuschlein, Felix; Reincke, Martin; Lorenz, Kristina; Allolio, Bruno; Kisker, Caroline; Fassnacht, Martin; Lohse, Martin J

    2014-01-01

    We recently identified a high prevalence of mutations affecting the catalytic (Cα) subunit of protein kinase A (PKA) in cortisol-secreting adrenocortical adenomas. The two identified mutations (Leu206Arg and Leu199_Cys200insTrp) are associated with increased PKA catalytic activity, but the underlying mechanisms are highly controversial. Here we utilize a combination of biochemical and optical assays, including fluorescence resonance energy transfer in living cells, to analyze the consequences of the two mutations with respect to the formation of the PKA holoenzyme and its regulation by cAMP. Our results indicate that neither mutant can form a stable PKA complex, due to the location of the mutations at the interface between the catalytic and the regulatory subunits. We conclude that the two mutations cause high basal catalytic activity and lack of regulation by cAMP through interference of complex formation between the regulatory and the catalytic subunits of PKA. PMID:25477193

  6. CHCHD10 mutations promote loss of mitochondrial cristae junctions with impaired mitochondrial genome maintenance and inhibition of apoptosis.

    PubMed

    Genin, Emmanuelle C; Plutino, Morgane; Bannwarth, Sylvie; Villa, Elodie; Cisneros-Barroso, Eugenia; Roy, Madhuparna; Ortega-Vila, Bernardo; Fragaki, Konstantina; Lespinasse, Françoise; Pinero-Martos, Estefania; Augé, Gaëlle; Moore, David; Burté, Florence; Lacas-Gervais, Sandra; Kageyama, Yusuke; Itoh, Kie; Yu-Wai-Man, Patrick; Sesaki, Hiromi; Ricci, Jean-Ehrland; Vives-Bauza, Cristofol; Paquis-Flucklinger, Véronique

    2016-01-01

    CHCHD10-related diseases include mitochondrial DNA instability disorder, frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) clinical spectrum, late-onset spinal motor neuropathy (SMAJ), and Charcot-Marie-Tooth disease type 2 (CMT2). Here, we show that CHCHD10 resides with mitofilin, CHCHD3 and CHCHD6 within the "mitochondrial contact site and cristae organizing system" (MICOS) complex. CHCHD10 mutations lead to MICOS complex disassembly and loss of mitochondrial cristae with a decrease in nucleoid number and nucleoid disorganization. Repair of the mitochondrial genome after oxidative stress is impaired in CHCHD10 mutant fibroblasts and this likely explains the accumulation of deleted mtDNA molecules in patient muscle. CHCHD10 mutant fibroblasts are not defective in the delivery of mitochondria to lysosomes suggesting that impaired mitophagy does not contribute to mtDNA instability. Interestingly, the expression of CHCHD10 mutant alleles inhibits apoptosis by preventing cytochrome c release. PMID:26666268

  7. Axonal transport of TDP-43 mRNA granules in neurons is impaired by ALS-causing mutations

    PubMed Central

    Carrasco, Monica A.; Williams, Luis A.; Winborn, Christina S.; Han, Steve S. W.; Kiskinis, Evangelos; Winborn, Brett; Freibaum, Brian D.; Kanagaraj, Anderson; Clare, Alison J.; Badders, Nisha M.; Bilican, Bilada; Chaum, Edward; Chandran, Siddharthan; Shaw, Christopher E.; Eggan, Kevin C.; Maniatis, Tom; Taylor, J. Paul

    2014-01-01

    Summary The RNA binding protein TDP-43 regulates RNA metabolism at multiple levels, including transcription, RNA splicing, and mRNA stability. TDP-43 is a major component of the cytoplasmic inclusions characteristic of amyotrophic lateral sclerosis and some types of frontotemporal lobar degeneration. The importance of TDP-43 in disease is underscored by the fact that dominant missense mutations are sufficient to cause disease, although the role of TDP-43 in pathogenesis is unknown. Here we show that TDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons in vitro and in vivo and facilitate delivery of target mRNA to distal neuronal compartments. TDP-43 mutations impair this mRNA transport function in vivo and in vitro, including in stem cell-derived motor neurons from ALS patients bearing any one of three different TDP-43 ALS-causing mutations. Thus, TDP43 mutations that cause ALS lead to partial loss of a novel cytoplasmic function of TDP-43. PMID:24507191

  8. A mutation in the human Uncoordinated 119 gene impairs TCR signaling and is associated with CD4 lymphopenia

    PubMed Central

    Gorska, Magdalena M.

    2012-01-01

    Idiopathic CD4 lymphopenia (ICL) is an immunodeficiency disorder of unclear etiology. Here we describe a heterozygous dominant-negative missense mutation (codon 22 GGC→GTC; V22G) of the signaling adaptor protein Uncoordinated 119 (Unc119) in an ICL patient. The patient is a 32-year-old female with < 300 CD4 T cells/μL and with a history of recurrent sinusitis/otitis media, frequent episodes of shingles, a widespread fungal nail infection, fungal dermatitis, oral herpetic lesions, and bronchiolitis obliterans organizing pneumonia after 2 episodes of bacterial pneumonia. The patient's cells have reduced response to TCR stimulation, with impairment in both localization and enzymatic activation of the lymphocyte-specific kinase (Lck) resulting in decreased cell proliferation. Transduction of the mutant Unc119 but not wild-type Unc119 into normal T cells reproduces the signaling and proliferation defects. The mutation disrupts the Unc119-Lck interaction which is normally needed for stimulation of the Lck catalytic activity by TCR. The mutant protein also causes mislocalization of Lck to Rab11+ perinuclear endosomes. The mutation is not present in 2 other patients with ICL, patients with secondary CD4 lymphopenia or 60 healthy subjects. The V22G mutation of Unc119 represents a novel genetic defect in ICL. PMID:22184408

  9. A Novel Locus Harbouring a Functional CD164 Nonsense Mutation Identified in a Large Danish Family with Nonsyndromic Hearing Impairment.

    PubMed

    Nyegaard, Mette; Rendtorff, Nanna D; Nielsen, Morten S; Corydon, Thomas J; Demontis, Ditte; Starnawska, Anna; Hedemand, Anne; Buniello, Annalisa; Niola, Francesco; Overgaard, Michael T; Leal, Suzanne M; Ahmad, Wasim; Wikman, Friedrik P; Petersen, Kirsten B; Crüger, Dorthe G; Oostrik, Jaap; Kremer, Hannie; Tommerup, Niels; Frödin, Morten; Steel, Karen P; Tranebjærg, Lisbeth; Børglum, Anders D

    2015-07-01

    Nonsyndromic hearing impairment (NSHI) is a highly heterogeneous condition with more than eighty known causative genes. However, in the clinical setting, a large number of NSHI families have unexplained etiology, suggesting that there are many more genes to be identified. In this study we used SNP-based linkage analysis and follow up microsatellite markers to identify a novel locus (DFNA66) on chromosome 6q15-21 (LOD 5.1) in a large Danish family with dominantly inherited NSHI. By locus specific capture and next-generation sequencing, we identified a c.574C>T heterozygous nonsense mutation (p.R192*) in CD164. This gene encodes a 197 amino acid transmembrane sialomucin (known as endolyn, MUC-24 or CD164), which is widely expressed and involved in cell adhesion and migration. The mutation segregated with the phenotype and was absent in 1200 Danish control individuals and in databases with whole-genome and exome sequence data. The predicted effect of the mutation was a truncation of the last six C-terminal residues of the cytoplasmic tail of CD164, including a highly conserved canonical sorting motif (YXXФ). In whole blood from an affected individual, we found by RT-PCR both the wild-type and the mutated transcript suggesting that the mutant transcript escapes nonsense mediated decay. Functional studies in HEK cells demonstrated that the truncated protein was almost completely retained on the plasma cell membrane in contrast to the wild-type protein, which targeted primarily to the endo-lysosomal compartments, implicating failed endocytosis as a possible disease mechanism. In the mouse ear, we found CD164 expressed in the inner and outer hair cells of the organ of Corti, as well as in other locations in the cochlear duct. In conclusion, we have identified a new DFNA locus located on chromosome 6q15-21 and implicated CD164 as a novel gene for hearing impairment. PMID:26197441

  10. A Novel Locus Harbouring a Functional CD164 Nonsense Mutation Identified in a Large Danish Family with Nonsyndromic Hearing Impairment

    PubMed Central

    Nielsen, Morten S.; Corydon, Thomas J.; Demontis, Ditte; Starnawska, Anna; Hedemand, Anne; Buniello, Annalisa; Niola, Francesco; Overgaard, Michael T.; Leal, Suzanne M.; Ahmad, Wasim; Wikman, Friedrik P.; Petersen, Kirsten B.; Crüger, Dorthe G.; Oostrik, Jaap; Kremer, Hannie; Tommerup, Niels; Frödin, Morten; Steel, Karen P.; Tranebjærg, Lisbeth; Børglum, Anders D.

    2015-01-01

    Nonsyndromic hearing impairment (NSHI) is a highly heterogeneous condition with more than eighty known causative genes. However, in the clinical setting, a large number of NSHI families have unexplained etiology, suggesting that there are many more genes to be identified. In this study we used SNP-based linkage analysis and follow up microsatellite markers to identify a novel locus (DFNA66) on chromosome 6q15-21 (LOD 5.1) in a large Danish family with dominantly inherited NSHI. By locus specific capture and next-generation sequencing, we identified a c.574C>T heterozygous nonsense mutation (p.R192*) in CD164. This gene encodes a 197 amino acid transmembrane sialomucin (known as endolyn, MUC-24 or CD164), which is widely expressed and involved in cell adhesion and migration. The mutation segregated with the phenotype and was absent in 1200 Danish control individuals and in databases with whole-genome and exome sequence data. The predicted effect of the mutation was a truncation of the last six C-terminal residues of the cytoplasmic tail of CD164, including a highly conserved canonical sorting motif (YXXФ). In whole blood from an affected individual, we found by RT-PCR both the wild-type and the mutated transcript suggesting that the mutant transcript escapes nonsense mediated decay. Functional studies in HEK cells demonstrated that the truncated protein was almost completely retained on the plasma cell membrane in contrast to the wild-type protein, which targeted primarily to the endo-lysosomal compartments, implicating failed endocytosis as a possible disease mechanism. In the mouse ear, we found CD164 expressed in the inner and outer hair cells of the organ of Corti, as well as in other locations in the cochlear duct. In conclusion, we have identified a new DFNA locus located on chromosome 6q15-21 and implicated CD164 as a novel gene for hearing impairment. PMID:26197441

  11. Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis

    PubMed Central

    Liu, Luyan; Okada, Satoshi; Kong, Xiao-Fei; Kreins, Alexandra Y.; Cypowyj, Sophie; Abhyankar, Avinash; Toubiana, Julie; Itan, Yuval; Audry, Magali; Nitschke, Patrick; Masson, Cécile; Toth, Beata; Flatot, Jérome; Migaud, Mélanie; Chrabieh, Maya; Kochetkov, Tatiana; Bolze, Alexandre; Borghesi, Alessandro; Toulon, Antoine; Hiller, Julia; Eyerich, Stefanie; Eyerich, Kilian; Gulácsy, Vera; Chernyshova, Ludmyla; Chernyshov, Viktor; Bondarenko, Anastasia; María Cortés Grimaldo, Rosa; Blancas-Galicia, Lizbeth; Madrigal Beas, Ileana Maria; Roesler, Joachim; Magdorf, Klaus; Engelhard, Dan; Thumerelle, Caroline; Burgel, Pierre-Régis; Hoernes, Miriam; Drexel, Barbara; Seger, Reinhard; Kusuma, Theresia; Jansson, Annette F.; Sawalle-Belohradsky, Julie; Belohradsky, Bernd; Jouanguy, Emmanuelle; Bustamante, Jacinta; Bué, Mélanie; Karin, Nathan; Wildbaum, Gizi; Bodemer, Christine; Lortholary, Olivier; Fischer, Alain; Blanche, Stéphane; Al-Muhsen, Saleh; Reichenbach, Janine; Kobayashi, Masao; Rosales, Francisco Espinosa; Lozano, Carlos Torres; Kilic, Sara Sebnem; Oleastro, Matias; Etzioni, Amos; Traidl-Hoffmann, Claudia; Renner, Ellen D.; Abel, Laurent; Picard, Capucine; Maródi, László; Boisson-Dupuis, Stéphanie

    2011-01-01

    Chronic mucocutaneous candidiasis disease (CMCD) may be caused by autosomal dominant (AD) IL-17F deficiency or autosomal recessive (AR) IL-17RA deficiency. Here, using whole-exome sequencing, we identified heterozygous germline mutations in STAT1 in 47 patients from 20 kindreds with AD CMCD. Previously described heterozygous STAT1 mutant alleles are loss-of-function and cause AD predisposition to mycobacterial disease caused by impaired STAT1-dependent cellular responses to IFN-γ. Other loss-of-function STAT1 alleles cause AR predisposition to intracellular bacterial and viral diseases, caused by impaired STAT1-dependent responses to IFN-α/β, IFN-γ, IFN-λ, and IL-27. In contrast, the 12 AD CMCD-inducing STAT1 mutant alleles described here are gain-of-function and increase STAT1-dependent cellular responses to these cytokines, and to cytokines that predominantly activate STAT3, such as IL-6 and IL-21. All of these mutations affect the coiled-coil domain and impair the nuclear dephosphorylation of activated STAT1, accounting for their gain-of-function and dominance. Stronger cellular responses to the STAT1-dependent IL-17 inhibitors IFN-α/β, IFN-γ, and IL-27, and stronger STAT1 activation in response to the STAT3-dependent IL-17 inducers IL-6 and IL-21, hinder the development of T cells producing IL-17A, IL-17F, and IL-22. Gain-of-function STAT1 alleles therefore cause AD CMCD by impairing IL-17 immunity. PMID:21727188

  12. Autism-Associated Insertion Mutation (InsG) of Shank3 Exon 21 Causes Impaired Synaptic Transmission and Behavioral Deficits.

    PubMed

    Speed, Haley E; Kouser, Mehreen; Xuan, Zhong; Reimers, Jeremy M; Ochoa, Christine F; Gupta, Natasha; Liu, Shunan; Powell, Craig M

    2015-07-01

    SHANK3 (also known as PROSAP2) is a postsynaptic scaffolding protein at excitatory synapses in which mutations and deletions have been implicated in patients with idiopathic autism, Phelan-McDermid (aka 22q13 microdeletion) syndrome, and other neuropsychiatric disorders. In this study, we have created a novel mouse model of human autism caused by the insertion of a single guanine nucleotide into exon 21 (Shank3(G)). The resulting frameshift causes a premature STOP codon and loss of major higher molecular weight Shank3 isoforms at the synapse. Shank3(G/G) mice exhibit deficits in hippocampus-dependent spatial learning, impaired motor coordination, altered response to novelty, and sensory processing deficits. At the cellular level, Shank3(G/G) mice also exhibit impaired hippocampal excitatory transmission and plasticity as well as changes in baseline NMDA receptor-mediated synaptic responses. This work identifies clear alterations in synaptic function and behavior in a novel, genetically accurate mouse model of autism mimicking an autism-associated insertion mutation. Furthermore, these findings lay the foundation for future studies aimed to validate and study region-selective and temporally selective genetic reversal studies in the Shank3(G/G) mouse that was engineered with such future experiments in mind. PMID:26134648

  13. Autism-Associated Insertion Mutation (InsG) of Shank3 Exon 21 Causes Impaired Synaptic Transmission and Behavioral Deficits

    PubMed Central

    Speed, Haley E.; Kouser, Mehreen; Xuan, Zhong; Reimers, Jeremy M.; Ochoa, Christine F.; Gupta, Natasha; Liu, Shunan

    2015-01-01

    SHANK3 (also known as PROSAP2) is a postsynaptic scaffolding protein at excitatory synapses in which mutations and deletions have been implicated in patients with idiopathic autism, Phelan–McDermid (aka 22q13 microdeletion) syndrome, and other neuropsychiatric disorders. In this study, we have created a novel mouse model of human autism caused by the insertion of a single guanine nucleotide into exon 21 (Shank3G). The resulting frameshift causes a premature STOP codon and loss of major higher molecular weight Shank3 isoforms at the synapse. Shank3G/G mice exhibit deficits in hippocampus-dependent spatial learning, impaired motor coordination, altered response to novelty, and sensory processing deficits. At the cellular level, Shank3G/G mice also exhibit impaired hippocampal excitatory transmission and plasticity as well as changes in baseline NMDA receptor-mediated synaptic responses. This work identifies clear alterations in synaptic function and behavior in a novel, genetically accurate mouse model of autism mimicking an autism-associated insertion mutation. Furthermore, these findings lay the foundation for future studies aimed to validate and study region-selective and temporally selective genetic reversal studies in the Shank3G/G mouse that was engineered with such future experiments in mind. PMID:26134648

  14. Epileptic encephalopathy-causing mutations in DNM1 impair synaptic vesicle endocytosis

    PubMed Central

    Dhindsa, Ryan S.; Bradrick, Shelton S.; Yao, Xiaodi; Heinzen, Erin L.; Petrovski, Slave; Krueger, Brian J.; Johnson, Michael R.; Frankel, Wayne N.; Petrou, Steven; Boumil, Rebecca M.

    2015-01-01

    Objective: To elucidate the functional consequences of epileptic encephalopathy–causing de novo mutations in DNM1 (A177P, K206N, G359A), which encodes a large mechanochemical GTPase essential for neuronal synaptic vesicle endocytosis. Methods: HeLa and COS-7 cells transfected with wild-type and mutant DNM1 constructs were used for transferrin assays, high-content imaging, colocalization studies, Western blotting, and electron microscopy (EM). EM was also conducted on the brain sections of mice harboring a middle-domain Dnm1 mutation (Dnm1Ftfl). Results: We demonstrate that the expression of each mutant protein decreased endocytosis activity in a dominant-negative manner. One of the G-domain mutations, K206N, decreased protein levels. The G359A mutation, which occurs in the middle domain, disrupted higher-order DNM1 oligomerization. EM of mutant DNM1-transfected HeLa cells and of the Dnm1Ftfl mouse brain revealed vesicle defects, indicating that the mutations likely interfere with DNM1's vesicle scission activity. Conclusion: Together, these data suggest that the dysfunction of vesicle scission during synaptic vesicle endocytosis can lead to serious early-onset epilepsies. PMID:27066543

  15. A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients.

    PubMed

    Sun, Yishan; Paşca, Sergiu P; Portmann, Thomas; Goold, Carleton; Worringer, Kathleen A; Guan, Wendy; Chan, Karen C; Gai, Hui; Vogt, Daniel; Chen, Ying-Jiun J; Mao, Rong; Chan, Karrie; Rubenstein, John Lr; Madison, Daniel V; Hallmayer, Joachim; Froehlich-Santino, Wendy M; Bernstein, Jonathan A; Dolmetsch, Ricardo E

    2016-01-01

    Dravet Syndrome is an intractable form of childhood epilepsy associated with deleterious mutations in SCN1A, the gene encoding neuronal sodium channel Nav1.1. Earlier studies using human induced pluripotent stem cells (iPSCs) have produced mixed results regarding the importance of Nav1.1 in human inhibitory versus excitatory neurons. We studied a Nav1.1 mutation (p.S1328P) identified in a pair of twins with Dravet Syndrome and generated iPSC-derived neurons from these patients. Characterization of the mutant channel revealed a decrease in current amplitude and hypersensitivity to steady-state inactivation. We then differentiated Dravet-Syndrome and control iPSCs into telencephalic excitatory neurons or medial ganglionic eminence (MGE)-like inhibitory neurons. Dravet inhibitory neurons showed deficits in sodium currents and action potential firing, which were rescued by a Nav1.1 transgene, whereas Dravet excitatory neurons were normal. Our study identifies biophysical impairments underlying a deleterious Nav1.1 mutation and supports the hypothesis that Dravet Syndrome arises from defective inhibitory neurons. PMID:27458797

  16. Homozygous SLC6A17 Mutations Cause Autosomal-Recessive Intellectual Disability with Progressive Tremor, Speech Impairment, and Behavioral Problems

    PubMed Central

    Iqbal, Zafar; Willemsen, Marjolein H.; Papon, Marie-Amélie; Musante, Luciana; Benevento, Marco; Hu, Hao; Venselaar, Hanka; Wissink-Lindhout, Willemijn M.; Vulto-van Silfhout, Anneke T.; Vissers, Lisenka E.L.M.; de Brouwer, Arjan P.M.; Marouillat, Sylviane; Wienker, Thomas F.; Ropers, Hans Hilger; Kahrizi, Kimia; Nadif Kasri, Nael; Najmabadi, Hossein; Laumonnier, Frédéric; Kleefstra, Tjitske; van Bokhoven, Hans

    2015-01-01

    We report on Dutch and Iranian families with affected individuals who present with moderate to severe intellectual disability and additional phenotypes including progressive tremor, speech impairment, and behavioral problems in certain individuals. A combination of exome sequencing and homozygosity mapping revealed homozygous mutations c.484G>A (p.Gly162Arg) and c.1898C>G (p.Pro633Arg) in SLC6A17. SLC6A17 is predominantly expressed in the brain, encodes a synaptic vesicular transporter of neutral amino acids and glutamate, and plays an important role in the regulation of glutamatergic synapses. Prediction programs and 3D modeling suggest that the identified mutations are deleterious to protein function. To directly test the functional consequences, we investigated the neuronal subcellular localization of overexpressed wild-type and mutant variants in mouse primary hippocampal neuronal cells. Wild-type protein was present in soma, axons, dendrites, and dendritic spines. p.Pro633Arg altered SLC6A17 was found in soma and proximal dendrites but did not reach spines. p.Gly162Arg altered SLC6A17 showed a normal subcellular distribution but was associated with an abnormal neuronal morphology mainly characterized by the loss of dendritic spines. In summary, our genetic findings implicate homozygous SLC6A17 mutations in autosomal-recessive intellectual disability, and their pathogenic role is strengthened by genetic evidence and in silico and in vitro functional analyses. PMID:25704603

  17. A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

    PubMed Central

    Sun, Yishan; Paşca, Sergiu P; Portmann, Thomas; Goold, Carleton; Worringer, Kathleen A; Guan, Wendy; Chan, Karen C; Gai, Hui; Vogt, Daniel; Chen, Ying-Jiun J; Mao, Rong; Chan, Karrie; Rubenstein, John LR; Madison, Daniel V; Hallmayer, Joachim; Froehlich-Santino, Wendy M; Bernstein, Jonathan A; Dolmetsch, Ricardo E

    2016-01-01

    Dravet Syndrome is an intractable form of childhood epilepsy associated with deleterious mutations in SCN1A, the gene encoding neuronal sodium channel Nav1.1. Earlier studies using human induced pluripotent stem cells (iPSCs) have produced mixed results regarding the importance of Nav1.1 in human inhibitory versus excitatory neurons. We studied a Nav1.1 mutation (p.S1328P) identified in a pair of twins with Dravet Syndrome and generated iPSC-derived neurons from these patients. Characterization of the mutant channel revealed a decrease in current amplitude and hypersensitivity to steady-state inactivation. We then differentiated Dravet-Syndrome and control iPSCs into telencephalic excitatory neurons or medial ganglionic eminence (MGE)-like inhibitory neurons. Dravet inhibitory neurons showed deficits in sodium currents and action potential firing, which were rescued by a Nav1.1 transgene, whereas Dravet excitatory neurons were normal. Our study identifies biophysical impairments underlying a deleterious Nav1.1 mutation and supports the hypothesis that Dravet Syndrome arises from defective inhibitory neurons. DOI: http://dx.doi.org/10.7554/eLife.13073.001 PMID:27458797

  18. Mutations in NALCN Cause an Autosomal-Recessive Syndrome with Severe Hypotonia, Speech Impairment, and Cognitive Delay

    PubMed Central

    Al-Sayed, Moeenaldeen D.; Al-Zaidan, Hamad; Albakheet, AlBandary; Hakami, Hana; Kenana, Rosan; Al-Yafee, Yusra; Al-Dosary, Mazhor; Qari, Alya; Al-Sheddi, Tarfa; Al-Muheiza, Muhammed; Al-Qubbaj, Wafa; Lakmache, Yamina; Al-Hindi, Hindi; Ghaziuddin, Muhammad; Colak, Dilek; Kaya, Namik

    2013-01-01

    Sodium leak channel, nonselective (NALCN) is a voltage-independent and cation-nonselective channel that is mainly responsible for the leaky sodium transport across neuronal membranes and controls neuronal excitability. Although NALCN variants have been conflictingly reported to be in linkage disequilibrium with schizophrenia and bipolar disorder, to our knowledge, no mutations have been reported to date for any inherited disorders. Using linkage, SNP-based homozygosity mapping, targeted sequencing, and confirmatory exome sequencing, we identified two mutations, one missense and one nonsense, in NALCN in two unrelated families. The mutations cause an autosomal-recessive syndrome characterized by subtle facial dysmorphism, variable degrees of hypotonia, speech impairment, chronic constipation, and intellectual disability. Furthermore, one of the families pursued preimplantation genetic diagnosis on the basis of the results from this study, and the mother recently delivered healthy twins, a boy and a girl, with no symptoms of hypotonia, which was present in all the affected children at birth. Hence, the two families we describe here represent instances of loss of function in human NALCN. PMID:24075186

  19. Putative Breast Cancer Driver Mutations in TBX3 Cause Impaired Transcriptional Repression

    PubMed Central

    Fischer, Kathrin; Pflugfelder, Gert O.

    2015-01-01

    The closely related T-box transcription factors TBX2 and TBX3 are frequently overexpressed in melanoma and various types of human cancers, in particular, breast cancer. The overexpression of TBX2 and TBX3 can have several cellular effects, among them suppression of senescence, promotion of epithelial–mesenchymal transition, and invasive cell motility. In contrast, loss of function of TBX3 and most other human T-box genes causes developmental haploinsufficiency syndromes. Stephens and colleagues (1), by exome sequencing of breast tumor samples, identified five different mutations in TBX3, all affecting the DNA-binding T-domain. One in-frame deletion of a single amino acid, p.N212delN, was observed twice. Due to the clustering of these mutations to the T-domain and for statistical reasons, TBX3 was inferred to be a driver gene in breast cancer. Since mutations in the T-domain generally cause loss of function and because the tumorigenic action of TBX3 has generally been attributed to overexpression, we determined whether the putative driver mutations had loss- or gain-of-function properties. We tested two in-frame deletions, one missense, and one frameshift mutant protein for DNA-binding in vitro, and for target gene repression in cell culture. In addition, we performed an in silico analysis of somatic TBX mutations in breast cancer, collected in The Cancer Genome Atlas (TCGA). Both the experimental and the in silico analysis indicate that the observed mutations predominantly cause loss of TBX3 function. PMID:26579496

  20. Mitochondrial impairment observed in fibroblasts from South African Parkinson’s disease patients with parkin mutations

    SciTech Connect

    Merwe, Celia van der; Loos, Ben; Swart, Chrisna; Kinnear, Craig; Merwe, Lize van der; Pillay, Komala; Muller, Nolan; Zaharie, Dan; Engelbrecht, Lize; Carr, Jonathan; and others

    2014-05-02

    Highlights: • Mitochondrial dysfunction observed in patients with parkin-null mutations. • Mitochondrial ATP levels were decreased. • Electron-dense vacuoles were observed in the patients. • Mitochondria from muscle biopsies appeared within normal limits. • One patient did not show these defects possibly due to compensatory mechanisms. - Abstract: Parkinson’s disease (PD), defined as a neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra in the midbrain. Loss-of-function mutations in the parkin gene are a major cause of autosomal recessive, early-onset PD. Parkin has been implicated in the maintenance of healthy mitochondria, although previous studies show conflicting findings regarding mitochondrial abnormalities in fibroblasts from patients harboring parkin-null mutations. The aim of the present study was to determine whether South African PD patients with parkin mutations exhibit evidence for mitochondrial dysfunction. Fibroblasts were cultured from skin biopsies obtained from three patients with homozygous parkin-null mutations, two heterozygous mutation carriers and two wild-type controls. Muscle biopsies were obtained from two of the patients. The muscle fibers showed subtle abnormalities such as slightly swollen mitochondria in focal areas of the fibers and some folding of the sarcolemma. Although no differences in the degree of mitochondrial network branching were found in the fibroblasts, ultrastructural abnormalities were observed including the presence of electron-dense vacuoles. Moreover, decreased ATP levels which are consistent with mitochondrial dysfunction were observed in the patients’ fibroblasts compared to controls. Remarkably, these defects did not manifest in one patient, which may be due to possible compensatory mechanisms. These results suggest that parkin-null patients exhibit features of mitochondrial dysfunction. Involvement of mitochondria as a key role player in PD

  1. The ER-Membrane Transport System Is Critical for Intercellular Trafficking of the NSm Movement Protein and Tomato Spotted Wilt Tospovirus.

    PubMed

    Feng, Zhike; Xue, Fan; Xu, Min; Chen, Xiaojiao; Zhao, Wenyang; Garcia-Murria, Maria J; Mingarro, Ismael; Liu, Yong; Huang, Ying; Jiang, Lei; Zhu, Min; Tao, Xiaorong

    2016-02-01

    Plant viruses move through plasmodesmata to infect new cells. The plant endoplasmic reticulum (ER) is interconnected among cells via the ER desmotubule in the plasmodesma across the cell wall, forming a continuous ER network throughout the entire plant. This ER continuity is unique to plants and has been postulated to serve as a platform for the intercellular trafficking of macromolecules. In the present study, the contribution of the plant ER membrane transport system to the intercellular trafficking of the NSm movement protein and Tomato spotted wilt tospovirus (TSWV) is investigated. We showed that TSWV NSm is physically associated with the ER membrane in Nicotiana benthamiana plants. An NSm-GFP fusion protein transiently expressed in single leaf cells was trafficked into neighboring cells. Mutations in NSm that impaired its association with the ER or caused its mis-localization to other subcellular sites inhibited cell-to-cell trafficking. Pharmacological disruption of the ER network severely inhibited NSm-GFP trafficking but not GFP diffusion. In the Arabidopsis thaliana mutant rhd3 with an impaired ER network, NSm-GFP trafficking was significantly reduced, whereas GFP diffusion was not affected. We also showed that the ER-to-Golgi secretion pathway and the cytoskeleton transport systems were not involved in the intercellular trafficking of TSWV NSm. Importantly, TSWV cell-to-cell spread was delayed in the ER-defective rhd3 mutant, and this reduced viral infection was not due to reduced replication. On the basis of robust biochemical, cellular and genetic analysis, we established that the ER membrane transport system serves as an important direct route for intercellular trafficking of NSm and TSWV. PMID:26863622

  2. The ER-Membrane Transport System Is Critical for Intercellular Trafficking of the NSm Movement Protein and Tomato Spotted Wilt Tospovirus

    PubMed Central

    Feng, Zhike; Xue, Fan; Xu, Min; Chen, Xiaojiao; Zhao, Wenyang; Garcia-Murria, Maria J.; Mingarro, Ismael; Liu, Yong; Huang, Ying; Jiang, Lei; Zhu, Min; Tao, Xiaorong

    2016-01-01

    Plant viruses move through plasmodesmata to infect new cells. The plant endoplasmic reticulum (ER) is interconnected among cells via the ER desmotubule in the plasmodesma across the cell wall, forming a continuous ER network throughout the entire plant. This ER continuity is unique to plants and has been postulated to serve as a platform for the intercellular trafficking of macromolecules. In the present study, the contribution of the plant ER membrane transport system to the intercellular trafficking of the NSm movement protein and Tomato spotted wilt tospovirus (TSWV) is investigated. We showed that TSWV NSm is physically associated with the ER membrane in Nicotiana benthamiana plants. An NSm-GFP fusion protein transiently expressed in single leaf cells was trafficked into neighboring cells. Mutations in NSm that impaired its association with the ER or caused its mis-localization to other subcellular sites inhibited cell-to-cell trafficking. Pharmacological disruption of the ER network severely inhibited NSm-GFP trafficking but not GFP diffusion. In the Arabidopsis thaliana mutant rhd3 with an impaired ER network, NSm-GFP trafficking was significantly reduced, whereas GFP diffusion was not affected. We also showed that the ER-to-Golgi secretion pathway and the cytoskeleton transport systems were not involved in the intercellular trafficking of TSWV NSm. Importantly, TSWV cell-to-cell spread was delayed in the ER-defective rhd3 mutant, and this reduced viral infection was not due to reduced replication. On the basis of robust biochemical, cellular and genetic analysis, we established that the ER membrane transport system serves as an important direct route for intercellular trafficking of NSm and TSWV. PMID:26863622

  3. NADP(+)-IDH Mutations Promote Hypersuccinylation that Impairs Mitochondria Respiration and Induces Apoptosis Resistance.

    PubMed

    Li, Feng; He, Xiadi; Ye, Dingwei; Lin, Yan; Yu, Hongxiu; Yao, Cuifang; Huang, Lei; Zhang, Jianong; Wang, Fang; Xu, Sha; Wu, Xiaohui; Liu, Lixia; Yang, Chen; Shi, Jiaqi; He, Xiaoyang; Liu, Jie; Qu, Yuanyuan; Guo, Fushen; Zhao, Jianyuan; Xu, Wei; Zhao, Shimin

    2015-11-19

    Elucidating the tumorigenic mechanism of R-2-hydroxyglutarate (R-2HG) is critical for determining how NADP(+)-IDH mutations cause cancer. Here we report that R-2HG induces cancerous metabolism and apoptosis resistance through promoting hypersuccinylation. By competitive inhibition of the mitochondrial tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH), R-2HG preferentially induced succinyl-CoA accumulation and hypersuccinylation in the mitochondria. IDH1 mutation-bearing glioma samples and cells were hypersuccinylated in the mitochondria. IDH1 mutation or SDH inactivation resulted in hypersuccinylation, causing respiration inhibition and inducing cancerous metabolism and mitochondrial depolarization. These mitochondrial dysfunctions induced BCL-2 accumulation at the mitochondrial membrane, leading to apoptosis resistance of hypersuccinylated cells. Relief of hypersuccinylation by overexpressing the desuccinylase SIRT5 or supplementing glycine rescued mitochondrial dysfunctions, reversed BCL-2 accumulation, and slowed the oncogenic growth of hypersuccinylated IDH1(R132C)-harboring HT1080 cells. Thus, R-2HG-induced hypersuccinylation contributes to the tumorigenicity of NADP(+)-IDH mutations, suggesting the potential of hypersuccinylation inhibition as an intervention for hypersuccinylation-related tumors. PMID:26585387

  4. Primary microcephaly, impaired DNA replication, and genomic instability caused by compound heterozygous ATR mutations.

    PubMed

    Mokrani-Benhelli, Houda; Gaillard, Laetitia; Biasutto, Patricia; Le Guen, Tangui; Touzot, Fabien; Vasquez, Nadia; Komatsu, Jun; Conseiller, Emmanuel; Pïcard, Capucine; Gluckman, Eliane; Francannet, Christine; Fischer, Alain; Durandy, Anne; Soulier, Jean; de Villartay, Jean-Pierre; Cavazzana-Calvo, Marina; Revy, Patrick

    2013-02-01

    Ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) kinases are two key regulators of DNA-damage responses (DDR) that are mainly activated in response to DNA double-strand breaks and single-stranded DNA damages, respectively. Seckel syndrome, a rare genetic disorder characterized by a microcephaly and a markedly reduced body size, has been associated with defective ATR-dependent DNA damage signaling. However, the only human genetic ATR defect reported so far is a hypomorphic splicing mutation identified in five related individuals with Seckel syndrome. Here, we report the first case of primary microcephaly with compound heterozygous mutations in ATR: a 540 kb genomic deletion on one allele and a missense mutation leading to splice dysregulation on the other, which ultimately lead to a sharp decrease in ATR expression. DNA combing technology revealed a profound spontaneous alteration of several DNA replication parameters in patient's cells and FISH analyses highlighted the genomic instability caused by ATR deficiency. Collectively, our results emphasize the crucial role for ATR in the control of DNA replication, and reinforce the complementary and nonredundant contributions of ATM and ATR in human cells to face DNA damages and warrant genome integrity. PMID:23111928

  5. Dravet Syndrome and "SCN1A" Gene Mutation Related-Epilepsies: Cognitive Impairment and Its Determinants

    ERIC Educational Resources Information Center

    Guerrini, Renzo; Falchi, Melania

    2011-01-01

    Some studies have demonstrated that cognitive decline occurs in Dravet syndrome, starting shortly after the onset of seizures, rapidly progressing and then plateauing within a few years. It is unclear whether children that develop the syndrome had entirely normal cognitive skills before seizure onset, since subtle impairment easily escapes…

  6. D184E mutation in aquaporin-4 gene impairs water permeability and links to deafness.

    PubMed

    Nicchia, G P; Ficarella, R; Rossi, A; Giangreco, I; Nicolotti, O; Carotti, A; Pisani, F; Estivill, X; Gasparini, P; Svelto, M; Frigeri, A

    2011-12-01

    Aquaporins (AQPs) play a physiological role in several organs and tissues, and their alteration is associated with disorders of water regulation. The identification of molecular interactions, which are crucial in determining the rate of water flux through the channel, is of pivotal role for the discovery of molecules able to target those interactions and therefore to be used for pathologies ascribable to an altered AQP-dependent water balance. In the present study, a mutational screening of human aquaporin-4 (AQP4) gene was performed on subjects with variable degrees of hearing loss. One heterozygous missense mutation was identified in a Spanish sporadic case, leading to an Asp/Glu amino acid substitution at position 184 (D184E). A BLAST analysis revealed that the amino acid D184 is conserved across species, consistently with a crucial role in the structure/function of AQP4 water channels. The mutation induces a significant reduction in water permeability as measured by the Xenopus laevis oocytes swelling assay and by the use of mammalian cells by total internal reflection microscopy. By Western blot, immunofluorescence and 2D Blue Native/SDS-PAGE we show that the reduction in water permeability is not ascribable to a reduced expression of AQP4 mutant protein or to its incorrect plasma membrane targeting and aggregation into orthogonal arrays of particles. Molecular dynamics simulation provided a molecular explanation of the mechanism whereby the mutation induces a loss of function of the channel. Substituting glutamate for aspartate affects the mobility of the D loop, which acquires a higher propensity to equilibrate in a "closed conformation", thus affecting the rate of water flux. We speculate that this mutation, combined with other genetic defects or concurrently with certain environmental stimuli, could confer a higher susceptibility to deafness. PMID:21952128

  7. Novel IL1RAPL1 mutations associated with intellectual disability impair synaptogenesis

    PubMed Central

    Ramos-Brossier, Mariana; Montani, Caterina; Lebrun, Nicolas; Gritti, Laura; Martin, Christelle; Seminatore-Nole, Christine; Toussaint, Aurelie; Moreno, Sarah; Poirier, Karine; Dorseuil, Olivier; Chelly, Jamel; Hackett, Anna; Gecz, Jozef; Bieth, Eric; Faudet, Anne; Heron, Delphine; Kooy, Frank; Loeys, Bart; Humeau, Yann; Sala, Carlo; Billuart, Pierre

    2015-01-01

    Mutations in interleukin-1 receptor accessory protein like 1 (IL1RAPL1) gene have been associated with non-syndromic intellectual disability and autism spectrum disorder. This protein interacts with synaptic partners like PSD-95 and PTPδ, regulating the formation and function of excitatory synapses. The aim of this work is to characterize the synaptic consequences of three IL1RAPL1 mutations, two novel causing the deletion of exon 6 (Δex6) and one point mutation (C31R), identified in patients with intellectual disability. Using immunofluorescence and electrophysiological recordings we examined the effects of IL1RAPL1 mutants over-expression on synapse formation and function in cultured rodent hippocampal neurons. Δex6 but not C31R mutation leads to IL1RAPL1 protein instability and mislocalization within dendrites. Analysis of different markers of excitatory synapses and sEPSC recording revealed that both mutants fail to induce pre- and post-synaptic differentiation, contrary to WT IL1RAPL1 protein. Cell aggregation and immunoprecipitation assays in HEK293 cells showed a reduction of the interaction between IL1RAPL1 mutants and PTPδ that could explain the observed synaptogenic defect in neurons. However, these mutants do not affect all cellular signaling since their over-expression still activates JNK pathway. We conclude that both mutations described in this study lead to a partial loss of function of the IL1RAPL1 protein through different mechanisms. Our work highlights the important function of the trans-synaptic PTPδ/ IL1RAPL1 interaction in synaptogenesis and as such, in intellectual disability in the patients. PMID:25305082

  8. Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis

    PubMed Central

    Zhang, Yi; Nikolovski, Nino; Sorieul, Mathias; Vellosillo, Tamara; McFarlane, Heather E.; Dupree, Ray; Kesten, Christopher; Schneider, René; Driemeier, Carlos; Lathe, Rahul; Lampugnani, Edwin; Yu, Xiaolan; Ivakov, Alexander; Doblin, Monika S.; Mortimer, Jenny C.; Brown, Steven P.; Persson, Staffan; Dupree, Paul

    2016-01-01

    As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus. PMID:27277162

  9. Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis.

    PubMed

    Zhang, Yi; Nikolovski, Nino; Sorieul, Mathias; Vellosillo, Tamara; McFarlane, Heather E; Dupree, Ray; Kesten, Christopher; Schneider, René; Driemeier, Carlos; Lathe, Rahul; Lampugnani, Edwin; Yu, Xiaolan; Ivakov, Alexander; Doblin, Monika S; Mortimer, Jenny C; Brown, Steven P; Persson, Staffan; Dupree, Paul

    2016-01-01

    As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus. PMID:27277162

  10. Mechanisms involved in the reduction of GABAA receptor alpha1-subunit expression caused by the epilepsy mutation A322D in the trafficking-competent receptor.

    PubMed

    Bradley, Clarrisa A; Taghibiglou, Changiz; Collingridge, Graham L; Wang, Yu Tian

    2008-08-01

    A mutation in the alpha1-subunit (A322D) of GABA(A)Rs is responsible for juvenile myoclonic epilepsy in a large Canadian family. Previous work has identified that this mutant affects the cell expression and function of recombinant GABA(A)Rs, expressed in HEK293 cells. Here we have extended these observations by showing that the mutation promotes association with the endoplasmic reticulum chaperone calnexin and accelerates the degradation rate of the subunits approximately 2.5-fold. We also find that the mutation causes the subunit to be degraded largely by a lysosomal-dependent process. Furthermore, we find that the mutation results in receptors that are inserted into the plasma membrane but are more rapidly endocytosed by a dynamin and caveolin1-dependent mechanism. These results suggest that the mutant subunit can form functional receptors, but that these have a shorter lifetime on the plasma membrane. PMID:18534981

  11. A Mutation in C2orf64 Causes Impaired Cytochrome c Oxidase Assembly and Mitochondrial Cardiomyopathy

    PubMed Central

    Huigsloot, Merei; Nijtmans, Leo G.; Szklarczyk, Radek; Baars, Marieke J.H.; van den Brand, Mariël A.M.; HendriksFranssen, Marthe G.M.; van den Heuvel, Lambertus P.; Smeitink, Jan A.M.; Huynen, Martijn A.; Rodenburg, Richard J.T.

    2011-01-01

    The assembly of mitochondrial respiratory chain complex IV (cytochrome c oxidase) involves the coordinated action of several assembly chaperones. In Saccharomyces cerevisiae, at least 30 different assembly chaperones have been identified. To date, pathogenic mutations leading to a mitochondrial disorder have been identified in only seven of the corresponding human genes. One of the genes for which the relevance to human pathology is unknown is C2orf64, an ortholog of the S. cerevisiae gene PET191. This gene has previously been shown to be a complex IV assembly factor in yeast, although its exact role is still unknown. Previous research in a large cohort of complex IV deficient patients did not support an etiological role of C2orf64 in complex IV deficiency. In this report, a homozygous mutation in C2orf64 is described in two siblings affected by fatal neonatal cardiomyopathy. Pathogenicity of the mutation is supported by the results of a complementation experiment, showing that complex IV activity can be fully restored by retroviral transduction of wild-type C2orf64 in patient-derived fibroblasts. Detailed analysis of complex IV assembly intermediates in patient fibroblasts by 2D-BN PAGE revealed the accumulation of a small assembly intermediate containing subunit COX1 but not the COX2, COX4, or COX5b subunits, indicating that C2orf64 is involved in an early step of the complex IV assembly process. The results of this study demonstrate that C2orf64 is essential for human complex IV assembly and that C2orf64 mutational analysis should be considered for complex IV deficient patients, in particular those with hypertrophic cardiomyopathy. PMID:21457908

  12. A mutation in FRIZZLED2 impairs Wnt signaling and causes autosomal dominant omodysplasia

    PubMed Central

    Saal, Howard M.; Prows, Cynthia A.; Guerreiro, Iris; Donlin, Milene; Knudson, Luke; Sund, Kristen L.; Chang, Ching-Fang; Brugmann, Samantha A.; Stottmann, Rolf W.

    2015-01-01

    Autosomal dominant omodysplasia is a rare skeletal dysplasia characterized by short humeri, radial head dislocation, short first metacarpals, facial dysmorphism and genitourinary anomalies. We performed next-generation whole-exome sequencing and comparative analysis of a proband with omodysplasia, her unaffected parents and her affected daughter. We identified a de novo mutation in FRIZZLED2 (FZD2) in the proband and her daughter that was not found in unaffected family members. The FZD2 mutation (c.1644G>A) changes a tryptophan residue at amino acid 548 to a premature stop (p.Trp548*). This altered protein is still produced in vitro, but we show reduced ability of this mutant form of FZD2 to interact with its downstream target DISHEVELLED. Furthermore, expressing the mutant form of FZD2 in vitro is not able to facilitate the cellular response to canonical Wnt signaling like wild-type FZD2. We therefore conclude that the FRIZZLED2 mutation is a de novo, novel cause for autosomal dominant omodysplasia. PMID:25759469

  13. Functional link between Rab GTPase-mediated membrane trafficking and PI4,5P2 signaling.

    PubMed

    Li, Cuifang; Kita, Ayako; Hashimoto, Yuuka; Ihara, Misako; Kato, Ayaka; Ogura, Naoya; Doi, Akira; Oku, Masahide; Itoh, Toshiki; Sakai, Yasuyoshi; Sugiura, Reiko

    2014-03-01

    Fission yeast its3(+) encodes an essential phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) that regulates cell integrity and cytokinesis. We performed a genetic screen to identify genes that function in PI4P5K-mediated signaling, and identified gyp10(+) encoding a Rab GTPase-activating protein (GAP), a negative regulator for Rab GTPase signaling. Its3 overproduction caused growth defects and abnormal cytoplasmic accumulation of the Its3 protein, which can be stained by calcofluor. Notably, Its3 overproducing cells displayed abnormal membranous structures, multilamella Golgi and fragmented vacuoles showed by Electron microscopy. Furthermore, the excess cytoplasmic Its3 structure partly colocalized with the fluorescence of FM4-64. Gyp10 rescued both growth defects and abnormal Its3 localization when it was over-expressed. Gyp10 functionally interacted with the Rab GTPases Ypt3 and Ryh1, both of which regulate Golgi membrane trafficking. Consistently, mutation or deletion of Ypt3 and Ryh1 suppressed phenotypes associated with Its3 overproduction. Importantly, the plasma membrane localization of Its3 was also affected by the impairment of the Ypt3/Ryh1 Rab membrane trafficking, thus suggesting that membrane trafficking events regulated by two Rab GTPases functionally interacts with PI4,5P2 signaling. These results suggest a mechanism whereby PI4P5K signaling/localization is affected by Golgi membrane trafficking, thus provide a functional link between the PI4,5P2 signaling and Rab-mediated trafficking. PMID:24350606

  14. The deubiquitinating enzyme USP8 promotes trafficking and degradation of the chemokine receptor 4 at the sorting endosome.

    PubMed

    Berlin, Ilana; Higginbotham, Katherine M; Dise, Rebecca S; Sierra, Maria I; Nash, Piers D

    2010-11-26

    Reversible ubiquitination orchestrated by the opposition of ubiquitin ligases and deubiquitinating enzymes mediates endocytic trafficking of cell surface receptors for lysosomal degradation. Ubiquitin-specific protease 8 (USP8) has previously been implicated in endocytosis of several receptors by virtue of their deubiquitination. The present study explores an indirect role for USP8 in cargo trafficking through its regulation of the chemokine receptor 4 (CXCR4). Contrary to the effects of USP8 loss on enhanced green fluorescent protein, we find that USP8 depletion stabilizes CXCR4 on the cell surface and attenuates receptor degradation without affecting its ubiquitination status. In the presence of ligand, diminished CXCR4 turnover is accompanied by receptor accumulation on enlarged early endosomes and leads to enhancement of phospho-ERK signaling. Perturbation in CXCR4 trafficking, resulting from USP8 inactivation, occurs at the ESCRT-0 checkpoint, and catalytic mutation of USP8 specifically targeted to the ESCRT-0 complex impairs the spatial and temporal organization of the sorting endosome. USP8 functionally opposes the ubiquitin ligase AIP4 with respect to ESCRT-0 ubiquitination, thereby promoting trafficking of CXCR4. Collectively, our findings demonstrate a functional cooperation between USP8, AIP4, and the ESCRT-0 machinery at the early sorting phase of CXCR4 and underscore the versatility of USP8 in shaping trafficking events at the early-to-late endosome transition. PMID:20876529

  15. The Sorting Receptor SorCS1 Regulates Trafficking of Neurexin and AMPA Receptors

    PubMed Central

    Savas, Jeffrey N.; Ribeiro, Luís F.; Wierda, Keimpe D.; Wright, Rebecca; DeNardo, Laura A.; Rice, Heather C.; Chamma, Ingrid; Wang, Yi-Zhi; Zemla, Roland; Lavallée-Adam, Mathieu; Vennekens, Kristel M.; O'Sullivan, Matthew L.; Antonios, Joseph K.; Hall, Elizabeth A.; Thoumine, Olivier; Attie, Alan D.; Ghosh, Anirvan; Yates, John R.; de Wit, Joris

    2015-01-01

    The formation, function, and plasticity of synapses require dynamic changes in synaptic receptor composition. Here we identify the sorting receptor SorCS1 as a key regulator of synaptic receptor trafficking. Four independent proteomic analyses identify the synaptic adhesion molecule neurexin and the AMPA glutamate receptor (AMPAR) as major proteins sorted by SorCS1. SorCS1 localizes to early and recycling endosomes and regulates neurexin and AMPAR surface trafficking. Surface proteome analysis of SorCS1-deficient neurons shows decreased surface levels of these, and additional, receptors. Quantitative in vivo analysis of SorCS1 knockout synaptic proteomes identifies SorCS1 as a global trafficking regulator and reveals decreased levels of receptors regulating adhesion and neurotransmission, including neurexins and AMPARs. Consequently, glutamatergic transmission at SorCS1–deficient synapses is reduced due to impaired AMPAR surface expression. SORCS1 mutations have been associated with autism and Alzheimer's disease, suggesting that perturbed receptor trafficking contributes to defects in synaptic composition and function underlying synaptopathies. PMID:26291160

  16. Juvenile-onset Sporadic Amyotrophic Lateral Sclerosis with a Frameshift FUS Gene Mutation Presenting Unique Neuroradiological Findings and Cognitive Impairment.

    PubMed

    Hirayanagi, Kimitoshi; Sato, Masayuki; Furuta, Natsumi; Makioka, Kouki; Ikeda, Yoshio

    2016-01-01

    A 24-year-old Japanese woman developed anterocollis, weakness of the proximal arms, and subsequent cognitive impairment. A neurological examination revealed amyotrophic lateral sclerosis (ALS) without a family history. Systemic muscle atrophy progressed rapidly. Cerebral MRI clearly exhibited high signal intensities along the bilateral pyramidal tracts. An analysis of the FUS gene revealed a heterozygous two-base pair deletion, c.1507-1508delAG (p.G504WfsX515). A subset of juvenile-onset familial/sporadic ALS cases with FUS gene mutations reportedly demonstrates mental retardation or learning difficulty. Our study emphasizes the importance of conducting a FUS gene analysis in juvenile-onset ALS cases, even when no family occurrence is confirmed. PMID:26984092

  17. β-Amyloid Impairs AMPA Receptor Trafficking and Function by Reducing Ca2+/Calmodulin-dependent Protein Kinase II Synaptic Distribution*

    PubMed Central

    Gu, Zhenglin; Liu, Wenhua; Yan, Zhen

    2009-01-01

    A fundamental feature of Alzheimer disease (AD) is the accumulation of β-amyloid (Aβ), a peptide generated from the amyloid precursor protein (APP). Emerging evidence suggests that soluble Aβ oligomers adversely affect synaptic function, which leads to cognitive failure associated with AD. The Aβ-induced synaptic dysfunction has been attributed to the synaptic removal of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (AMPARs); however, it is unclear how Aβ induces the loss of AMPARs at the synapses. In this study we have examined the potential involvement of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a signaling molecule critical for AMPAR trafficking and function. We found that the synaptic pool of CaMKII was significantly decreased in cortical neurons from APP transgenic mice, and the density of CaMKII clusters at synapses was significantly reduced by Aβ oligomer treatment. In parallel, the surface expression of GluR1 subunit as well as AMPAR-mediated synaptic response and ionic current was selectively decreased in APP transgenic mice and Aβ-treated cultures. Moreover, the reducing effect of Aβ on AMPAR current density was mimicked and occluded by knockdown of CaMKII and blocked by overexpression of CaMKII. These results suggest that the Aβ-induced change in CaMKII subcellular distribution may underlie the removal of AMPARs from synaptic membrane by Aβ. PMID:19240035

  18. Impaired neuronal KCC2 function by biallelic SLC12A5 mutations in migrating focal seizures and severe developmental delay.

    PubMed

    Saitsu, Hirotomo; Watanabe, Miho; Akita, Tenpei; Ohba, Chihiro; Sugai, Kenji; Ong, Winnie Peitee; Shiraishi, Hideaki; Yuasa, Shota; Matsumoto, Hiroshi; Beng, Khoo Teik; Saitoh, Shinji; Miyatake, Satoko; Nakashima, Mitsuko; Miyake, Noriko; Kato, Mitsuhiro; Fukuda, Atsuo; Matsumoto, Naomichi

    2016-01-01

    Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the early-onset epileptic syndromes characterized by migrating polymorphous focal seizures. Whole exome sequencing (WES) in ten sporadic and one familial case of EIMFS revealed compound heterozygous SLC12A5 (encoding the neuronal K(+)-Cl(-) co-transporter KCC2) mutations in two families: c.279 + 1G > C causing skipping of exon 3 in the transcript (p.E50_Q93del) and c.572 C >T (p.A191V) in individuals 1 and 2, and c.967T > C (p.S323P) and c.1243 A > G (p.M415V) in individual 3. Another patient (individual 4) with migrating multifocal seizures and compound heterozygous mutations [c.953G > C (p.W318S) and c.2242_2244del (p.S748del)] was identified by searching WES data from 526 patients and SLC12A5-targeted resequencing data from 141 patients with infantile epilepsy. Gramicidin-perforated patch-clamp analysis demonstrated strongly suppressed Cl(-) extrusion function of E50_Q93del and M415V mutants, with mildly impaired function of A191V and S323P mutants. Cell surface expression levels of these KCC2 mutants were similar to wildtype KCC2. Heterologous expression of two KCC2 mutants, mimicking the patient status, produced a significantly greater intracellular Cl(-) level than with wildtype KCC2, but less than without KCC2. These data clearly demonstrated that partially disrupted neuronal Cl(-) extrusion, mediated by two types of differentially impaired KCC2 mutant in an individual, causes EIMFS. PMID:27436767

  19. Impaired neuronal KCC2 function by biallelic SLC12A5 mutations in migrating focal seizures and severe developmental delay

    PubMed Central

    Saitsu, Hirotomo; Watanabe, Miho; Akita, Tenpei; Ohba, Chihiro; Sugai, Kenji; Ong, Winnie Peitee; Shiraishi, Hideaki; Yuasa, Shota; Matsumoto, Hiroshi; Beng, Khoo Teik; Saitoh, Shinji; Miyatake, Satoko; Nakashima, Mitsuko; Miyake, Noriko; Kato, Mitsuhiro; Fukuda, Atsuo; Matsumoto, Naomichi

    2016-01-01

    Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the early-onset epileptic syndromes characterized by migrating polymorphous focal seizures. Whole exome sequencing (WES) in ten sporadic and one familial case of EIMFS revealed compound heterozygous SLC12A5 (encoding the neuronal K+-Cl− co-transporter KCC2) mutations in two families: c.279 + 1G > C causing skipping of exon 3 in the transcript (p.E50_Q93del) and c.572 C >T (p.A191V) in individuals 1 and 2, and c.967T > C (p.S323P) and c.1243 A > G (p.M415V) in individual 3. Another patient (individual 4) with migrating multifocal seizures and compound heterozygous mutations [c.953G > C (p.W318S) and c.2242_2244del (p.S748del)] was identified by searching WES data from 526 patients and SLC12A5-targeted resequencing data from 141 patients with infantile epilepsy. Gramicidin-perforated patch-clamp analysis demonstrated strongly suppressed Cl− extrusion function of E50_Q93del and M415V mutants, with mildly impaired function of A191V and S323P mutants. Cell surface expression levels of these KCC2 mutants were similar to wildtype KCC2. Heterologous expression of two KCC2 mutants, mimicking the patient status, produced a significantly greater intracellular Cl− level than with wildtype KCC2, but less than without KCC2. These data clearly demonstrated that partially disrupted neuronal Cl− extrusion, mediated by two types of differentially impaired KCC2 mutant in an individual, causes EIMFS. PMID:27436767

  20. [Auditory neuropathy due to the Q829X mutation in the gene encoding otoferlin (OTOF) in an infant screened for newborn hearing impairment].

    PubMed

    Gallo-Terán, J; Morales-Angulo, C; Sánchez, N; Manrique, M; Rodríguez-Ballesteros, M; Moreno-Pelayo, M A; Moreno, E; del Castillo, I

    2006-01-01

    We report an infant with auditory neuropathy secondary to the Q829X mutation in the gene encoding otoferlin (OTOF). Included in a universal newborn hearing screening program, the subject passed the otoacoustic emission (OAEs) test. Given that the infant had a familial history of deafness auditory brainstem response (ABR) testing was performed, revealing a profound hearing impairment. The genetic study confirmed that the subject was homozygous for the Q829X mutation in OTOF. The patient underwent a cochlear implant, obtaining satisfactory results. The moderately high prevalence of this mutation in the Spanish population could produce a significant false negative rate in newborn hearing screening programs using OAEs. PMID:17036997

  1. Mutations Impairing GSK3-Mediated MAF Phosphorylation Cause Cataract, Deafness, Intellectual Disability, Seizures, and a Down Syndrome-like Facies

    PubMed Central

    Niceta, Marcello; Stellacci, Emilia; Gripp, Karen W.; Zampino, Giuseppe; Kousi, Maria; Anselmi, Massimiliano; Traversa, Alice; Ciolfi, Andrea; Stabley, Deborah; Bruselles, Alessandro; Caputo, Viviana; Cecchetti, Serena; Prudente, Sabrina; Fiorenza, Maria T.; Boitani, Carla; Philip, Nicole; Niyazov, Dmitriy; Leoni, Chiara; Nakane, Takaya; Keppler-Noreuil, Kim; Braddock, Stephen R.; Gillessen-Kaesbach, Gabriele; Palleschi, Antonio; Campeau, Philippe M.; Lee, Brendan H.L.; Pouponnot, Celio; Stella, Lorenzo; Bocchinfuso, Gianfranco; Katsanis, Nicholas; Sol-Church, Katia; Tartaglia, Marco

    2015-01-01

    Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing development. PMID:25865493

  2. Mutations in the NB-ARC Domain of I-2 That Impair ATP Hydrolysis Cause Autoactivation1[OA

    PubMed Central

    Tameling, Wladimir I.L.; Vossen, Jack H.; Albrecht, Mario; Lengauer, Thomas; Berden, Jan A.; Haring, Michel A.; Cornelissen, Ben J.C.; Takken, Frank L.W.

    2006-01-01

    Resistance (R) proteins in plants confer specificity to the innate immune system. Most R proteins have a centrally located NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain. For two tomato (Lycopersicon esculentum) R proteins, I-2 and Mi-1, we have previously shown that this domain acts as an ATPase module that can hydrolyze ATP in vitro. To investigate the role of nucleotide binding and hydrolysis for the function of I-2 in planta, specific mutations were introduced in conserved motifs of the NB-ARC domain. Two mutations resulted in autoactivating proteins that induce a pathogen-independent hypersensitive response upon expression in planta. These mutant forms of I-2 were found to be impaired in ATP hydrolysis, but not in ATP binding, suggesting that the ATP- rather than the ADP-bound state of I-2 is the active form that triggers defense signaling. In addition, upon ADP binding, the protein displayed an increased affinity for ADP suggestive of a change of conformation. Based on these data, we propose that the NB-ARC domain of I-2, and likely of related R proteins, functions as a molecular switch whose state (on/off) depends on the nucleotide bound (ATP/ADP). PMID:16489136

  3. Homozygous NOTCH3 null mutation and impaired NOTCH3 signaling in recessive early-onset arteriopathy and cavitating leukoencephalopathy

    PubMed Central

    Pippucci, Tommaso; Maresca, Alessandra; Magini, Pamela; Cenacchi, Giovanna; Donadio, Vincenzo; Palombo, Flavia; Papa, Valentina; Incensi, Alex; Gasparre, Giuseppe; Valentino, Maria Lucia; Preziuso, Carmela; Pisano, Annalinda; Ragno, Michele; Liguori, Rocco; Giordano, Carla; Tonon, Caterina; Lodi, Raffaele; Parmeggiani, Antonia; Carelli, Valerio; Seri, Marco

    2015-01-01

    Notch signaling is essential for vascular physiology. Neomorphic heterozygous mutations in NOTCH3, one of the four human NOTCH receptors, cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Hypomorphic heterozygous alleles have been occasionally described in association with a spectrum of cerebrovascular phenotypes overlapping CADASIL, but their pathogenic potential is unclear. We describe a patient with childhood-onset arteriopathy, cavitating leukoencephalopathy with cerebral white matter abnormalities presented as diffuse cavitations, multiple lacunar infarctions and disseminated microbleeds. We identified a novel homozygous c.C2898A (p.C966*) null mutation in NOTCH3 abolishing NOTCH3 expression and causing NOTCH3 signaling impairment. NOTCH3 targets acting in the regulation of arterial tone (KCNA5) or expressed in the vasculature (CDH6) were downregulated. Patient's vessels were characterized by smooth muscle degeneration as in CADASIL, but without deposition of granular osmiophilic material (GOM), the CADASIL hallmark. The heterozygous parents displayed similar but less dramatic trends in decrease in the expression of NOTCH3 and its targets, as well as in vessel degeneration. This study suggests a functional link between NOTCH3 deficiency and pathogenesis of vascular leukoencephalopathies. PMID:25870235

  4. An ALS disease mutation in Cdc48/p97 impairs 20S proteasome binding and proteolytic communication.

    PubMed

    Barthelme, Dominik; Jauregui, Ruben; Sauer, Robert T

    2015-09-01

    Cdc48 (also known as p97 or VCP) is an essential and highly abundant, double-ring AAA+ ATPase, which is ubiquitous in archaea and eukaryotes. In archaea, Cdc48 ring hexamers play a direct role in quality control by unfolding and translocating protein substrates into the degradation chamber of the 20S proteasome. Whether Cdc48 and 20S cooperate directly in protein degradation in eukaryotic cells is unclear. Two regions of Cdc48 are important for 20S binding, the pore-2 loop at the bottom of the D2 AAA+ ring and a C-terminal tripeptide. Here, we identify an aspartic acid in the pore-2 loop as an important element in 20S recognition. Importantly, mutation of this aspartate in human Cdc48 has been linked to familial amyotrophic lateral sclerosis (ALS). In archaeal or human Cdc48 variants, we find that mutation of this pore-2 residue impairs 20S binding and proteolytic communication but does not affect the stability of the hexamer or rates of ATP hydrolysis and protein unfolding. These results suggest that human Cdc48 interacts functionally with the 20S proteasome. PMID:26134898

  5. Mutations Impairing GSK3-Mediated MAF Phosphorylation Cause Cataract, Deafness, Intellectual Disability, Seizures, and a Down Syndrome-like Facies.

    PubMed

    Niceta, Marcello; Stellacci, Emilia; Gripp, Karen W; Zampino, Giuseppe; Kousi, Maria; Anselmi, Massimiliano; Traversa, Alice; Ciolfi, Andrea; Stabley, Deborah; Bruselles, Alessandro; Caputo, Viviana; Cecchetti, Serena; Prudente, Sabrina; Fiorenza, Maria T; Boitani, Carla; Philip, Nicole; Niyazov, Dmitriy; Leoni, Chiara; Nakane, Takaya; Keppler-Noreuil, Kim; Braddock, Stephen R; Gillessen-Kaesbach, Gabriele; Palleschi, Antonio; Campeau, Philippe M; Lee, Brendan H L; Pouponnot, Celio; Stella, Lorenzo; Bocchinfuso, Gianfranco; Katsanis, Nicholas; Sol-Church, Katia; Tartaglia, Marco

    2015-05-01

    Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing development. PMID:25865493

  6. R4496C RyR2 mutation impairs atrial and ventricular contractility

    PubMed Central

    Coppini, Raffaele; Scellini, Beatrice; Ferrara, Claudia; Pioner, Josè Manuel; Mazzoni, Luca; Priori, Silvia; Cerbai, Elisabetta; Tesi, Chiara; Poggesi, Corrado

    2016-01-01

    Ryanodine receptor (RyR2) is the major Ca2+ channel of the cardiac sarcoplasmic reticulum (SR) and plays a crucial role in the generation of myocardial force. Changes in RyR2 gating properties and resulting increases in its open probability (Po) are associated with Ca2+ leakage from the SR and arrhythmias; however, the effects of RyR2 dysfunction on myocardial contractility are unknown. Here, we investigated the possibility that a RyR2 mutation associated with catecholaminergic polymorphic ventricular tachycardia, R4496C, affects the contractile function of atrial and ventricular myocardium. We measured isometric twitch tension in left ventricular and atrial trabeculae from wild-type mice and heterozygous transgenic mice carrying the R4496C RyR2 mutation and found that twitch force was comparable under baseline conditions (30°C, 2 mM [Ca2+]o, 1 Hz). However, the positive inotropic responses to high stimulation frequency, 0.1 µM isoproterenol, and 5 mM [Ca2+]o were decreased in R4496C trabeculae, as was post-rest potentiation. We investigated the mechanisms underlying inotropic insufficiency in R4496C muscles in single ventricular myocytes. Under baseline conditions, the amplitude of the Ca2+ transient was normal, despite the reduced SR Ca2+ content. Under inotropic challenge, however, R4496C myocytes were unable to boost the amplitude of Ca2+ transients because they are incapable of properly increasing the amount of Ca2+ stored in the SR because of a larger SR Ca2+ leakage. Recovery of force in response to premature stimuli was faster in R4496C myocardium, despite the unchanged rates of recovery of L-type Ca2+ channel current (ICa-L) and SR Ca2+ content in single myocytes. A faster recovery from inactivation of the mutant R4496C channels could explain this behavior. In conclusion, changes in RyR2 channel gating associated with the R4496C mutation could be directly responsible for the alterations in both ventricular and atrial contractility. The increased RyR2 Po

  7. Mutation in archain 1, a subunit of COPI coatomer complex, causes diluted coat color and Purkinje cell degeneration.

    PubMed

    Xu, Xinjie; Kedlaya, Rajendra; Higuchi, Hitoshi; Ikeda, Sakae; Justice, Monica J; Setaluri, Vijayasaradhi; Ikeda, Akihiro

    2010-05-01

    Intracellular trafficking is critical for delivering molecules and organelles to their proper destinations to carry out normal cellular functions. Disruption of intracellular trafficking has been implicated in the pathogenesis of various neurodegenerative disorders. In addition, a number of genes involved in vesicle/organelle trafficking are also essential for pigmentation, and loss of those genes is often associated with mouse coat-color dilution and human hypopigmentary disorders. Hence, we postulated that screening for mouse mutants with both neurological defects and coat-color dilution will help identify additional factors associated with intracellular trafficking in neuronal cells. In this study, we characterized a mouse mutant with a unique N-ethyl-N-nitrosourea (ENU)-induced mutation, named nur17. nur17 mutant mice exhibit both coat-color dilution and ataxia due to Purkinje cell degeneration in the cerebellum. By positional cloning, we identified that the nur17 mouse carries a T-to-C missense mutation in archain 1 (Arcn1) gene which encodes the delta subunit of the coat protein I (COPI) complex required for intracellular trafficking. Consistent with this function, we found that intracellular trafficking is disrupted in nur17 melanocytes. Moreover, the nur17 mutation leads to common characteristics of neurodegenerative disorders such as abnormal protein accumulation, ER stress, and neurofibrillary tangles. Our study documents for the first time the physiological consequences of the impairment of the ARCN1 function in the whole animal and demonstrates a direct association between ARCN1 and neurodegeneration. PMID:20502676

  8. Human Surfactant Protein A2 Gene Mutations Impair Dimmer/Trimer Assembly Leading to Deficiency in Protein Sialylation and Secretion

    PubMed Central

    Shen, Haitao; Li, Hui; Yang, Wenbing; Pan, Bing; Huang, Guowei; Lin, Guangyu; Ma, Lian; Willard, Belinda; Gu, Jiang; Zheng, Lemin; Wang, Yongyu

    2012-01-01

    Surfactant protein A2 (SP-A2) plays an essential role in surfactant metabolism and lung host defense. SP-A2 mutations in the carbohydrate recognition domain have been related to familial pulmonary fibrosis and can lead to a recombinant protein secretion deficiency in vitro. In this study, we explored the molecular mechanism of protein secretion deficiency and the subsequent biological effects in CHO-K1 cells expressing both wild-type and several different mutant forms of SP-A2. We demonstrate that the SP-A2 G231V and F198S mutants impair the formation of dimmer/trimer SP-A2 which contributes to the protein secretion defect. A deficiency in sialylation, but not N-linked glycosylation, is critical to the observed dimmer/trimer impairment-induced secretion defect. Furthermore, both mutant forms accumulate in the ER and form NP-40-insoluble aggregates. In addition, the soluble mutant SP-A2 could be partially degraded through the proteasome pathway but not the lysosome or autophagy pathway. Intriguingly, 4-phenylbutyrate acid (4-PBA), a chemical chaperone, alleviates aggregate formation and partially rescued the protein secretion of SP-A2 mutants. In conclusion, SP-A2 G231V and F198S mutants impair the dimmer/trimer assembly, which contributes to the protein sialylation and secretion deficiency. The intracellular protein mutants could be partially degraded through the proteasome pathway and also formed aggregates. The treatment of the cells with 4-PBA resulted in reduced aggregation and rescued the secretion of mutant SP-A2. PMID:23056344

  9. Simultaneous impairment of neuronal and metabolic function of mutated gephyrin in a patient with epileptic encephalopathy.

    PubMed

    Dejanovic, Borislav; Djémié, Tania; Grünewald, Nora; Suls, Arvid; Kress, Vanessa; Hetsch, Florian; Craiu, Dana; Zemel, Matthew; Gormley, Padhraig; Lal, Dennis; Myers, Candace T; Mefford, Heather C; Palotie, Aarno; Helbig, Ingo; Meier, Jochen C; De Jonghe, Peter; Weckhuysen, Sarah; Schwarz, Guenter

    2015-12-01

    Synaptic inhibition is essential for shaping the dynamics of neuronal networks, and aberrant inhibition plays an important role in neurological disorders. Gephyrin is a central player at inhibitory postsynapses, directly binds and organizes GABAA and glycine receptors (GABAARs and GlyRs), and is thereby indispensable for normal inhibitory neurotransmission. Additionally, gephyrin catalyzes the synthesis of the molybdenum cofactor (MoCo) in peripheral tissue. We identified a de novo missense mutation (G375D) in the gephyrin gene (GPHN) in a patient with epileptic encephalopathy resembling Dravet syndrome. Although stably expressed and correctly folded, gephyrin-G375D was non-synaptically localized in neurons and acted dominant-negatively on the clustering of wild-type gephyrin leading to a marked decrease in GABAAR surface expression and GABAergic signaling. We identified a decreased binding affinity between gephyrin-G375D and the receptors, suggesting that Gly375 is essential for gephyrin-receptor complex formation. Surprisingly, gephyrin-G375D was also unable to synthesize MoCo and activate MoCo-dependent enzymes. Thus, we describe a missense mutation that affects both functions of gephyrin and suggest that the identified defect at GABAergic synapses is the mechanism underlying the patient's severe phenotype. PMID:26613940

  10. Point mutation impairs centromeric CENH3 loading and induces haploid plants.

    PubMed

    Karimi-Ashtiyani, Raheleh; Ishii, Takayoshi; Niessen, Markus; Stein, Nils; Heckmann, Stefan; Gurushidze, Maia; Banaei-Moghaddam, Ali Mohammad; Fuchs, Jörg; Schubert, Veit; Koch, Kerstin; Weiss, Oda; Demidov, Dmitri; Schmidt, Klaus; Kumlehn, Jochen; Houben, Andreas

    2015-09-01

    The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called "CENP-A") is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923-937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest. PMID:26294252

  11. Desmin Mutation in the C-Terminal Domain Impairs Traction Force Generation in Myoblasts.

    PubMed

    Charrier, Elisabeth E; Asnacios, Atef; Milloud, Rachel; De Mets, Richard; Balland, Martial; Delort, Florence; Cardoso, Olivier; Vicart, Patrick; Batonnet-Pichon, Sabrina; Hénon, Sylvie

    2016-01-19

    The cytoskeleton plays a key role in the ability of cells to both resist mechanical stress and generate force, but the precise involvement of intermediate filaments in these processes remains unclear. We focus here on desmin, a type III intermediate filament, which is specifically expressed in muscle cells and serves as a skeletal muscle differentiation marker. By using several complementary experimental techniques, we have investigated the impact of overexpressing desmin and expressing a mutant desmin on the passive and active mechanical properties of C2C12 myoblasts. We first show that the overexpression of wild-type-desmin increases the overall rigidity of the cells, whereas the expression of a mutated E413K desmin does not. This mutation in the desmin gene is one of those leading to desminopathies, a subgroup of myopathies associated with progressive muscular weakness that are characterized by the presence of desmin aggregates and a disorganization of sarcomeres. We show that the expression of this mutant desmin in C2C12 myoblasts induces desmin network disorganization, desmin aggregate formation, and a small decrease in the number and total length of stress fibers. We finally demonstrate that expression of the E413K mutant desmin also alters the traction forces generation of single myoblasts lacking organized sarcomeres. PMID:26789769

  12. Point mutation impairs centromeric CENH3 loading and induces haploid plants

    PubMed Central

    Karimi-Ashtiyani, Raheleh; Ishii, Takayoshi; Niessen, Markus; Stein, Nils; Heckmann, Stefan; Gurushidze, Maia; Banaei-Moghaddam, Ali Mohammad; Fuchs, Jörg; Schubert, Veit; Koch, Kerstin; Weiss, Oda; Demidov, Dmitri; Schmidt, Klaus; Kumlehn, Jochen; Houben, Andreas

    2015-01-01

    The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called “CENP-A”) is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923–937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest. PMID:26294252

  13. Impaired complex III assembly associated with BCS1L gene mutations in isolated mitochondrial encephalopathy.

    PubMed

    Fernandez-Vizarra, Erika; Bugiani, Marianna; Goffrini, Paola; Carrara, Franco; Farina, Laura; Procopio, Elena; Donati, Alice; Uziel, Graziella; Ferrero, Iliana; Zeviani, Massimo

    2007-05-15

    We investigated two unrelated children with an isolated defect of mitochondrial complex III activity. The clinical picture was characterized by a progressive encephalopathy featuring early-onset developmental delay, spasticity, seizures, lactic acidosis, brain atrophy and MRI signal changes in the basal ganglia. Both children were compound heterozygotes for novel mutations in the human bc1 synthesis like (BCS1L) gene, which encodes an AAA mitochondrial protein putatively involved in both iron homeostasis and complex III assembly. The pathogenic role of the mutations was confirmed by complementation assays, using a DeltaBcs1 strain of Saccharomyces cerevisiae. By investigating complex III assembly and the structural features of the BCS1L gene product in skeletal muscle, cultured fibroblasts and lymphoblastoid cell lines from our patients, we have demonstrated, for the first time in a mammalian system, that a major function of BCS1L is to promote the maturation of complex III and, more specifically, the incorporation of the Rieske iron-sulfur protein into the nascent complex. Defective BCS1L leads to the formation of a catalytically inactive, structurally unstable complex III. We have also shown that BCS1L is contained within a high-molecular-weight supramolecular complex which is clearly distinct from complex III intermediates. PMID:17403714

  14. ATP2C1 gene mutations in Hailey-Hailey disease and possible roles of SPCA1 isoforms in membrane trafficking.

    PubMed

    Micaroni, M; Giacchetti, G; Plebani, R; Xiao, G G; Federici, L

    2016-01-01

    ATP2C1 gene codes for the secretory pathway Ca(2+)/Mn(2+)-ATPase pump type 1 (SPCA1) localizing at the golgi apparatus. Mutations on the human ATP2C1 gene, causing decreased levels of the SPCA1 expression, have been identified as the cause of the Hailey-Hailey disease, a rare skin disorder. In the last few years, several mutations have been described, and here we summarize how they are distributed along the gene and how missense mutations affect protein expression. SPCA1 is expressed in four different isoforms through alternative splicing of the ATP2C1 gene and none of these isoforms is differentially affected by any of these mutations. However, a better understanding of the tissue specific expression of the isoforms, their localization along the secretory pathway, their specific binding partners and the role of the C-terminal tail making isoforms different from each other, will be future goals of the research in this field. PMID:27277681

  15. SLC30A10 Is a Cell Surface-Localized Manganese Efflux Transporter, and Parkinsonism-Causing Mutations Block Its Intracellular Trafficking and Efflux Activity

    PubMed Central

    Leyva-Illades, Dinorah; Chen, Pan; Zogzas, Charles E.; Hutchens, Steven; Mercado, Jonathan M.; Swaim, Caleb D.; Morrisett, Richard A.; Bowman, Aaron B.

    2014-01-01

    Manganese (Mn) is an essential metal, but elevated cellular levels are toxic and may lead to the development of an irreversible parkinsonian-like syndrome that has no treatment. Mn-induced parkinsonism generally occurs as a result of exposure to elevated Mn levels in occupational or environmental settings. Additionally, patients with compromised liver function attributable to diseases, such as cirrhosis, fail to excrete Mn and may develop Mn-induced parkinsonism in the absence of exposure to elevated Mn. Recently, a new form of familial parkinsonism was reported to occur as a result of mutations in SLC30A10. The cellular function of SLC30A10 and the mechanisms by which mutations in this protein cause parkinsonism are unclear. Here, using a combination of mechanistic and functional studies in cell culture, Caenorhabditis elegans, and primary midbrain neurons, we show that SLC30A10 is a cell surface-localized Mn efflux transporter that reduces cellular Mn levels and protects against Mn-induced toxicity. Importantly, mutations in SLC30A10 that cause familial parkinsonism blocked the ability of the transporter to traffic to the cell surface and to mediate Mn efflux. Although expression of disease-causing SLC30A10 mutations were not deleterious by themselves, neurons and worms expressing these mutants exhibited enhanced sensitivity to Mn toxicity. Our results provide novel insights into the mechanisms involved in the onset of a familial form of parkinsonism and highlight the possibility of using enhanced Mn efflux as a therapeutic strategy for the potential management of Mn-induced parkinsonism, including that occurring as a result of mutations in SLC30A10. PMID:25319704

  16. Mutations of Cytosolic Loop Residues Impair Assembly and Maturation of α7 Nicotinic Acetylcholine Receptors

    PubMed Central

    Mukherjee, Jayanta; Kuryatov, Alexander; Moss, Stephen J.; Lindstrom, Jon M.; Anand, Rene

    2009-01-01

    Mechanisms that regulate early events in the biogenesis of the α7 nicotinic acetylcholine receptor (α7 AChR) are not well understood. Data presented here show that single amino acid mutations in the cytoplasmic loop of the α7 AChR, between position 335 and 343, abolish or attenuate expression of mature pentameric α7 AChRs in both human embryonic kidney tsA201 (HEK) and neuronal SH-SY5Y cells. Although the number of mature α7 AChRs is increased significantly in the presence of the chaperone protein RIC-3 in HEK cells, sucrose gradient sedimentation reveals that the vast majority of α7 subunits are aggregated or improperly assembled. Transfection of α7 AChRs in SH-SY5Y cells, which endogenously express the α7 AChR, results in a much larger fraction of subunits assembled into mature AChRs. Thus, efficient assembly of α7 AChRs is influenced by several regions of the large cytoplasmic domain, as well perhaps by other parts of its structure, and requires as yet unknown factors not required by other AChR subtypes. PMID:19627445

  17. Mutation of a diacidic motif in SIV-PBj Nef impairs T-cell activation and enteropathic disease

    PubMed Central

    2011-01-01

    Background The non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM). Results Mutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG) viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP) kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6. Conclusions In sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential. PMID:21366921

  18. Gain-of-Function Mutations in the KATP Channel (KCNJ11) Impair Coordinated Hand-Eye Tracking

    PubMed Central

    McTaggart, James S.; Jenkinson, Ned; Brittain, John-Stuart; Greeley, Siri A. W.; Hattersley, Andrew T.; Ashcroft, Frances M.

    2013-01-01

    Background Gain-of-function mutations in the ATP-sensitive potassium channel can cause permanent neonatal diabetes mellitus (PNDM) or neonatal diabetes accompanied by a constellation of neurological symptoms (iDEND syndrome). Studies of a mouse model of iDEND syndrome revealed that cerebellar Purkinje cell electrical activity was impaired and that the mice exhibited poor motor coordination. In this study, we probed the hand-eye coordination of PNDM and iDEND patients using visual tracking tasks to see if poor motor coordination is also a feature of the human disease. Methods Control participants (n = 14), patients with iDEND syndrome (n = 6 or 7), and patients with PNDM (n = 7) completed three computer-based tasks in which a moving target was tracked with a joystick-controlled cursor. Patients with PNDM and iDEND were being treated with sulphonylurea drugs at the time of testing. Results No differences were seen between PNDM patients and controls. Patients with iDEND syndrome were significantly less accurate than controls in two of the three tasks. The greatest differences were seen when iDEND patients tracked blanked targets, i.e. when predictive tracking was required. In this task, iDEND patients incurred more discrepancy errors (p = 0.009) and more velocity errors (p  = 0.009) than controls. Conclusions These results identify impaired hand-eye coordination as a new clinical feature of iDEND. The aetiology of this feature is likely to involve cerebellar dysfunction. The data further suggest that sulphonylurea doses that control the diabetes of these patients may be insufficient to fully correct their neurological symptoms. PMID:23626843

  19. Adenylate cyclase 1 (ADCY1) mutations cause recessive hearing impairment in humans and defects in hair cell function and hearing in zebrafish

    PubMed Central

    Santos-Cortez, Regie Lyn P.; Lee, Kwanghyuk; Giese, Arnaud P.; Ansar, Muhammad; Amin-Ud-Din, Muhammad; Rehn, Kira; Wang, Xin; Aziz, Abdul; Chiu, Ilene; Hussain Ali, Raja; Smith, Joshua D.; Shendure, Jay; Bamshad, Michael; Nickerson, Deborah A.; Ahmed, Zubair M.; Ahmad, Wasim; Riazuddin, Saima; Leal, Suzanne M.

    2014-01-01

    Cyclic AMP (cAMP) production, which is important for mechanotransduction within the inner ear, is catalyzed by adenylate cyclases (AC). However, knowledge of the role of ACs in hearing is limited. Previously, a novel autosomal recessive non-syndromic hearing impairment locus DFNB44 was mapped to chromosome 7p14.1-q11.22 in a consanguineous family from Pakistan. Through whole-exome sequencing of DNA samples from hearing-impaired family members, a nonsense mutation c.3112C>T (p.Arg1038*) within adenylate cyclase 1 (ADCY1) was identified. This stop-gained mutation segregated with hearing impairment within the family and was not identified in ethnically matched controls or within variant databases. This mutation is predicted to cause the loss of 82 amino acids from the carboxyl tail, including highly conserved residues within the catalytic domain, plus a calmodulin-stimulation defect, both of which are expected to decrease enzymatic efficiency. Individuals who are homozygous for this mutation had symmetric, mild-to-moderate mixed hearing impairment. Zebrafish adcy1b morphants had no FM1-43 dye uptake and lacked startle response, indicating hair cell dysfunction and gross hearing impairment. In the mouse, Adcy1 expression was observed throughout inner ear development and maturation. ADCY1 was localized to the cytoplasm of supporting cells and hair cells of the cochlea and vestibule and also to cochlear hair cell nuclei and stereocilia. Ex vivo studies in COS-7 cells suggest that the carboxyl tail of ADCY1 is essential for localization to actin-based microvilli. These results demonstrate that ADCY1 has an evolutionarily conserved role in hearing and that cAMP signaling is important to hair cell function within the inner ear. PMID:24482543

  20. IL23R (Interleukin 23 Receptor) Variants Protective against Inflammatory Bowel Diseases (IBD) Display Loss of Function due to Impaired Protein Stability and Intracellular Trafficking.

    PubMed

    Sivanesan, Durga; Beauchamp, Claudine; Quinou, Christiane; Lee, Jonathan; Lesage, Sylvie; Chemtob, Sylvain; Rioux, John D; Michnick, Stephen W

    2016-04-15

    Genome-wide association studies as well as murine models have shown that the interleukin 23 receptor (IL23R) pathway plays a pivotal role in chronic inflammatory diseases such as Crohn disease (CD), ulcerative colitis, psoriasis, and type 1 diabetes. Genome-wide association studies and targeted re-sequencing studies have revealed the presence of multiple potentially causal variants of the IL23R. Specifically the G149R, V362I, and R381Q IL23Rα chain variants are linked to protection against the development of Crohn disease and ulcerative colitis in humans. Moreover, the exact mechanism of action of these receptor variants has not been elucidated. We show that all three of these IL23Rα variants cause a reduction in IL23 receptor activation-mediated phosphorylation of the signal-transducing activator of transcription 3 (STAT3) and phosphorylation of signal transducing activator of transcription 4 (STAT4). The reduction in signaling is due to lower levels of cell surface receptor expression. For G149R, the receptor retention in the endoplasmic reticulum is due to an impairment of receptor maturation, whereas the R381Q and V362I variants have reduced protein stability. Finally, we demonstrate that the endogenous expression of IL23Rα protein from V362I and R381Q variants in human lymphoblastoid cell lines exhibited lower expression levels relative to susceptibility alleles. Our results suggest a convergent cause of IL23Rα variant protection against chronic inflammatory disease. PMID:26887945

  1. Impaired interactions between mouse Eyal harboring mutations found in patients with branchio-oto-renal syndrome and Six, Dach, and G proteins.

    PubMed

    Ozaki, Hidenori; Watanabe, Yoko; Ikeda, Keiko; Kawakami, Kiyoshi

    2002-01-01

    Mutations in the EYA1 gene are responsible for branchio-oto-renal (BOR) syndrome as well as for other ocular defects. Most of the mutations are located within or in the vicinity of the EYA domain, which is highly conserved in the EYA protein family. The EYA domain is required for protein-protein interactions, which are important to the biological function of EYA proteins. To determine how EYA1 mutations cause BOR syndrome and/or ocular defects, we tested the effects of Eya1 mutations on interactions with Six. Dach, and G proteins by mammalian two-hybrid and GST-pulldown assays. Defective interactions were noted between BOR-type mutations S486P and L504R of Eya1 and Dach1, G proteins, and some Six proteins. These mutations impaired the activation of transcription from a Six-responsive gene, myogenin, with Six5. S486P and L504R showed an altered digestion pattern with trypsin, and L504R also decreased the sensitivity to V8 protease digestion and produced a peptide fragment with a different M(r). Our results suggest that defective protein-protein interactions of the mutations in the EYA domain underlie BOR syndrome and that SIX, DACH, and/or G proteins are possibly involved in the pathogenic processes. PMID:11950062

  2. [The mutation spectrum of the GJB2 gene in Belarussian patients with hearing loss. Results of pilot genetic screening of hearing impairment in newborns].

    PubMed

    Bliznets, E A; Marcul', D N; Khorov, O G; Markova, T G; Poliakov, A V

    2014-02-01

    A total of 111 unrelated probands and their 8 sibs from Grodno oblast (Belarus) with bilateral isolated sensorineural hearing impairment were studied for the presence of mutations in the connexin 26--GJB2gene. Mutations were detected in 51 probands (46% of the sample). A significantly higher frequency of the GJB2gene mutations was observed in familial cases of the disease with the autosomal recessive type of inheritance (in 78% of families). Detected peculiarities of the GJB2 gene mutation spectrum demonstrated that use of the algorithm, which was developed for Russian patients, is optimal for the molecular study of patients from Be- larus. In the sample of patients with hearing loss, the highest (among other similar samples studied in the world) allele frequency of c.313_326de114 mutation (7% out of all pathological GJB2 alleles) was registered; Polish origin of this deletion was suggested. It was demonstrated that detection of the GJB2 gene mutation on only one patient's chromosome is insufficient to confirm a molecular genetic diagnosis of hearing loss of the DFNB1 genetic type (autosomal recessive hearing loss caused by the GJB2 gene mutations). Pilot screening in the presence of GJB2 gene mutations in newborns from Grodno oblast was conducted. The material from 235 children was studied during the screening; nine heterozygous carriers of the mutation were found. The c.35delG mutation was detected in a homozygous state in a single newborn (hearing loss of moderate severity was subsequently audiologically confirmed in this child). PMID:25711030

  3. Correspondence regarding Ballana et al., "Mitochondrial 12S rRNA gene mutations affect RNA secondary structure and lead to variable penetrance in hearing impairment".

    PubMed

    Abreu-Silva, R S; Batissoco, A C; Lezirovitz, K; Romanos, J; Rincon, D; Auricchio, M T B M; Otto, P A; Mingroni-Netto, R C

    2006-05-12

    Ballana et al. [E. Ballana, E. Morales, R. Rabionet, B. Montserrat, M. Ventayol, O. Bravo, P. Gasparini, X. Estivill, Mitochondrial 12S rRNA gene mutations affect RNA secondary structure and lead to variable penetrance in hearing impairment, Biochem. Biophys. Res. Commun. 341 (2006) 950-957] detected a T1291C mutation segregating in a Cuban pedigree with hearing impairment. They interpreted it as probably pathogenic, based on family history, RNA conformation prediction and its absence in a control group of 95 Spanish subjects. We screened a sample of 203 deaf subjects and 300 hearing controls (110 "European-Brazilians" and 190 "African-Brazilians") for the mitochondrial mutations A1555G and T1291C. Five deaf subjects had the T1291C substitution, three isolated cases and two familial cases. In the latter, deafness was paternally inherited or segregated with the A1555G mutation. This doesn't support the hypothesis of T1291C mutation being pathogenic. Two "African-Brazilian" controls also had the T1291C substitution. Six of the seven T1291C-carriers (five deaf and two controls) had mitochondrial DNA of African origin, belonging to macrohaplogroup L1/L2. Therefore, these data point to T1291C substitution as most probably an African non-pathogenic polymorphism. PMID:16574076

  4. NGF stimulation of erk phosphorylation is impaired by a point mutation in the transmembrane domain of trkA receptor.

    PubMed

    Monshipouri, M; Jiang, H; Lazarovici, P

    2000-01-01

    The nerve growth factor (NGF) trkA receptor is a transmembrane glycoprotein composed of a large extracellular ligand-binding region connected to the cytoplasmic tyrosine kinase region by a single transmembrane domain (TMD). To explore the role of TMD in the process of receptor activation, we substituted the hydrophobic amino-acid residue valine 432 with the charged amino-acid glutamic acid (designated V432E mutant) by utilizing in vitro site-directed mutagenesis. NIH 3T3 cells lacking endogenous NGF receptors were stably transfected with a pRc/CMV vector carrying either wild-type (trkA) or mutated (V432E) receptors. Stable transfectants were shown, using 125I-NGF binding and Western-blot analysis, to express the trkA recombinant receptors. Scatchard analysis revealed similar affinity for NGF in wild-type and V432E receptors. Although the level of basal trkA receptor tyrosine phosphorylation was higher in the mutant than in the wild-type, NGF stimulation of WT 11 and V432E transfectants resulted in a rapid increase in receptor tyrosine phosphorylation and of its intracellular adaptor protein SHC. In contrast to WT 11, V432E mutants showed very low levels of NGF-, and moderate levels of FGF-induced erks phosphorylation, respectively. Collectively, these findings suggest that a single substitution (V432E) in the trkA TMD results in a selective impairment of trkA-mediated erks signaling pathway. PMID:10854038

  5. Lysosomal sorting of amyloid-β by the SORLA receptor is impaired by a familial Alzheimer's disease mutation.

    PubMed

    Caglayan, Safak; Takagi-Niidome, Shizuka; Liao, Fan; Carlo, Anne-Sophie; Schmidt, Vanessa; Burgert, Tilman; Kitago, Yu; Füchtbauer, Ernst-Martin; Füchtbauer, Annette; Holtzman, David M; Takagi, Junichi; Willnow, Thomas E

    2014-02-12

    SORLA/SORL1 is a unique neuronal sorting receptor for the amyloid precursor protein that has been causally implicated in both sporadic and autosomal dominant familial forms of Alzheimer's disease (AD). Brain concentrations of SORLA are inversely correlated with amyloid-β (Aβ) in mouse models and AD patients, suggesting that increasing expression of this receptor could be a therapeutic option for decreasing the amount of amyloidogenic products in affected individuals. We characterize a new mouse model in which SORLA is overexpressed, and show a decrease in Aβ concentrations in mouse brain. We trace the underlying molecular mechanism to the ability of this receptor to direct lysosomal targeting of nascent Aβ peptides. Aβ binds to the amino-terminal VPS10P domain of SORLA, and this binding is impaired by a familial AD mutation in SORL1. Thus, loss of SORLA's Aβ sorting function is a potential cause of AD in patients, and SORLA may be a new therapeutic target for AD drug development. PMID:24523320

  6. A Novel Mutation in Isoform 3 of the Plasma Membrane Ca2+ Pump Impairs Cellular Ca2+ Homeostasis in a Patient with Cerebellar Ataxia and Laminin Subunit 1α Mutations.

    PubMed

    Calì, Tito; Lopreiato, Raffaele; Shimony, Joshua; Vineyard, Marisa; Frizzarin, Martina; Zanni, Ginevra; Zanotti, Giuseppe; Brini, Marisa; Shinawi, Marwan; Carafoli, Ernesto

    2015-06-26

    The particular importance of Ca(2+) signaling to neurons demands its precise regulation within their cytoplasm. Isoform 3 of the plasma membrane Ca(2+) ATPase (the PMCA3 pump), which is highly expressed in brain and cerebellum, plays an important role in the regulation of neuronal Ca(2+). A genetic defect of the PMCA3 pump has been described in one family with X-linked congenital cerebellar ataxia. Here we describe a novel mutation in the ATP2B3 gene in a patient with global developmental delay, generalized hypotonia and cerebellar ataxia. The mutation (a R482H replacement) impairs the Ca(2+) ejection function of the pump. It reduces the ability of the pump expressed in model cells to control Ca(2+) transients generated by cell stimulation and impairs its Ca(2+) extrusion function under conditions of low resting cytosolic Ca(2+) as well. In silico analysis of the structural effect of the mutation suggests a reduced stabilization of the portion of the pump surrounding the mutated residue in the Ca(2+)-bound state. The patient also carries two missense mutations in LAMA1, encoding laminin subunit 1α. On the basis of the family pedigree of the patient, the presence of both PMCA3 and laminin subunit 1α mutations appears to be necessary for the development of the disease. Considering the observed defect in cellular Ca(2+) homeostasis and the previous finding that PMCAs act as digenic modulators in Ca(2+)-linked pathologies, the PMCA3 dysfunction along with LAMA1 mutations could act synergistically to cause the neurological phenotype. PMID:25953895

  7. Deregulation and Station Trafficking.

    ERIC Educational Resources Information Center

    Bates, Benjamin J.

    To test whether the revocation of the Federal Communications Commission's "Anti-Trafficking" rule (requiring television station owners to keep a station for three years before transferring its license to another party) impacted station owner behavior, a study compared the behavior of television station "traffickers" (owners seeking quick turnovers…

  8. A Point Mutation in the Gene for Asparagine-Linked Glycosylation 10B (Alg10b) Causes Nonsyndromic Hearing Impairment in Mice (Mus musculus)

    PubMed Central

    Probst, Frank J.; Corrigan, Rebecca R.; del Gaudio, Daniela; Salinger, Andrew P.; Lorenzo, Isabel; Gao, Simon S.; Chiu, Ilene; Xia, Anping

    2013-01-01

    The study of mouse hearing impairment mutants has led to the identification of a number of human hearing impairment genes and has greatly furthered our understanding of the physiology of hearing. The novel mouse mutant neurological/sensory 5 (nse5) demonstrates a significantly reduced or absent startle response to sound and is therefore a potential murine model of human hearing impairment. Genetic analysis of 500 intercross progeny localized the mutant locus to a 524 kilobase (kb) interval on mouse chromosome 15. A missense mutation in a highly-conserved amino acid was found in the asparagine-linked glycosylation 10B gene (Alg10b), which is within the critical interval for the nse5 mutation. A 20.4 kb transgene containing a wildtype copy of the Alg10b gene rescued the mutant phenotype in nse5/nse5 homozygous animals, confirming that the mutation in Alg10b is responsible for the nse5/nse5 mutant phenotype. Homozygous nse5/nse5 mutants had abnormal auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), and cochlear microphonics (CMs). Endocochlear potentials (EPs), on the other hand, were normal. ABRs and DPOAEs also confirmed the rescue of the mutant nse5/nse5 phenotype by the wildtype Alg10b transgene. These results suggested a defect in the outer hair cells of mutant animals, which was confirmed by histologic analysis. This is the first report of mutation in a gene involved in the asparagine (N)-linked glycosylation pathway causing nonsyndromic hearing impairment, and it suggests that the hearing apparatus, and the outer hair cells in particular, are exquisitely sensitive to perturbations of the N-linked glycosylation pathway. PMID:24303013

  9. Postnatal microcephaly and pain insensitivity due to a de novo heterozygous DNM1L mutation causing impaired mitochondrial fission and function.

    PubMed

    Sheffer, Ruth; Douiev, Liza; Edvardson, Simon; Shaag, Avraham; Tamimi, Khaled; Soiferman, Devorah; Meiner, Vardiella; Saada, Ann

    2016-06-01

    An emerging class of mitochondrial disorders is caused by mutations in nuclear genes affecting mitochondrial dynamics and function. One of these is the DNM1L gene encoding the dynamin-related protein 1 (DRP1), which is pivotal in the mitochondrial fission process. Here, we describe a patient with a novel dominant-negative, de novo DNM1L mutation, which expands the clinical spectrum. The patient reported here exhibits a chronic neurological disorder, characterized by postnatal microcephaly, developmental delay, and pain insensitivity. Muscle biopsy disclosed decreased respiratory chain complex IV activity. Exome sequencing showed a de novo heterozygous c.1084G>A (p.G362S) mutation. Subsequent studies of patient skin fibroblasts showed markedly impaired mitochondrial fission and a partial respiratory chain defect while peroxisomal morphology remained intact. Human foreskin fibroblasts over-expressing the mutant DNM1L gene displayed aberrant mitochondrial morphology. © 2016 Wiley Periodicals, Inc. PMID:26992161

  10. Mitochondrial peptidase IMMP2L mutation causes early onset of age-associated disorders and impairs adult stem cell self-renewal.

    PubMed

    George, Sunil K; Jiao, Yan; Bishop, Colin E; Lu, Baisong

    2011-08-01

    Mitochondrial reactive oxygen species (ROS) are proposed to play a central role in aging and age-associated disorders, although direct in vivo evidence is lacking. We recently generated a mouse mutant with mutated inner mitochondrial membrane peptidase 2-like (Immp2l) gene, which impairs the signal peptide sequence processing of mitochondrial proteins cytochrome c1 and glycerol phosphate dehydrogenase 2. The mitochondria from mutant mice generate elevated levels of superoxide ion and cause impaired fertility in both sexes. Here, we design experiments to examine the effects of excessive mitochondrial ROS generation on health span. We show that Immp2l mutation increases oxidative stress in multiple organs such as the brain and the kidney, although expression of superoxide dismutases in these tissues of the mutants is also increased. The mutants show multiple aging-associated phenotypes, including wasting, sarcopenia, loss of subcutaneous fat, kyphosis, and ataxia, with female mutants showing earlier onset and more severe age-associated disorders than male mutants. The loss of body weight and fat was unrelated to food intake. Adipose-derived stromal cells (ADSC) from mutant mice showed impaired proliferation capability, formed significantly less and smaller colonies in colony formation assays, although they retained adipogenic differentiation capability in vitro. This functional impairment was accompanied by increased levels of oxidative stress. Our data showed that mitochondrial ROS is the driving force of accelerated aging and suggested that ROS damage to adult stem cells could be one of the mechanisms for age-associated disorders. PMID:21332923

  11. Effects of hemochromatosis and transferrin gene mutations on peripheral iron dyshomeostasis in mild cognitive impairment and Alzheimer's and Parkinson's diseases

    PubMed Central

    Mariani, S.; Ventriglia, M.; Simonelli, I.; Spalletta, G.; Bucossi, S.; Siotto, M.; Assogna, F.; Melgari, J. M.; Vernieri, F.; Squitti, R.

    2013-01-01

    Deregulation of iron metabolism has been observed in patients with neurodegenerative diseases. We have carried out a molecular analysis investigating the interaction between iron specific gene variants [transferrin (TF, P589S), hemochromatosis (HFE) C282Y and (H63D)], iron biochemical variables [iron, Tf, ceruloplasmin (Cp), Cp:Tf ratio and % of Tf saturation (% Tf-sat)] and apolipoprotein E (APOE) gene variants in 139 Alzheimer's disease (AD), 27 Mild Cognitive Impairment (MCI), 78 Parkinson's disease (PD) patients and 139 healthy controls to investigate mechanisms of iron regulation or toxicity. No difference in genetic variant distributions between patients and controls was found in our Italian sample, but the stratification for the APOEε4 allele revealed that among the APOEε4 carriers was higher the frequency of those carriers of at least a mutated TF P589S allele. Decreased Tf in both AD and MCI and increased Cp:Tf ratio in AD vs. controls were detected. A multinomial logistic regression model revealed that increased iron and Cp:Tf ratio and being man instead of woman increased the risk of having PD, that increased values of Cp:Tf ratio corresponded to a 4-fold increase of the relative risk of having MCI, while higher Cp levels were protective for PD and MCI. Our study has some limitations: the small size of the samples, one ethnic group considered, the rarity of some alleles which prevent the statistical power of some genetic analysis. Even though they need confirmation in larger cohorts, our data suggest the hypothesis that deregulation of iron metabolism, in addition to other factors, has some effect on the PD disease risk. PMID:23935582

  12. Prevalence of the A1555G (12S rRNA) and tRNASer(UCN) mitochondrial mutations in hearing-impaired Brazilian patients.

    PubMed

    Abreu-Silva, R S; Lezirovitz, K; Braga, M C C; Spinelli, M; Pirana, S; Della-Rosa, V A; Otto, P A; Mingroni-Netto, R C

    2006-02-01

    Mitochondrial mutations are responsible for at least 1% of the cases of hereditary deafness, but the contribution of each mutation has not yet been defined in African-derived or native American genetic backgrounds. A total of 203 unselected hearing-impaired patients were screened for the presence of the mitochondrial mutation A1555G in the 12S rRNA gene and mutations in the tRNASer(UCN) gene in order to assess their frequency in the ethnically admixed Brazilian population. We found four individuals with A1555G mutation (2%), which is a frequency similar to those reported for European-derived populations in unselected samples. On the other hand, complete sequencing of the tRNASer(UCN) did not reveal reported pathogenic substitutions, namely A7445G, 7472insC, T7510C, or T7511C. Instead, other rare substitutions were found such as T1291C, A7569G, and G7444A. To evaluate the significance of these findings, 110 "European-Brazilians" and 190 "African-Brazilians" unrelated hearing controls were screened. The T1291C, A7569G and G7444A substitutions were each found in about 1% (2/190) of individuals of African ancestry, suggesting that they are probably polymorphic. Our results indicate that screening for the A1555G mutation is recommended among all Brazilian deaf patients, while testing for mutations in the tRNASer(UCN) gene should be considered only when other frequent deafness-causing mutations have been excluded or in the presence of a maternal transmission pattern. PMID:16470309

  13. Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

    PubMed Central

    Zhou, Xiaolai; Sun, Lirong; Bastos de Oliveira, Francisco; Qi, Xiaoyang; Brown, William J.; Smolka, Marcus B.; Sun, Ying

    2015-01-01

    Mutations in the progranulin (PGRN) gene have been linked to two distinct neurodegenerative diseases, frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL). Accumulating evidence suggests a critical role of PGRN in lysosomes. However, how PGRN is trafficked to lysosomes is still not clear. Here we report a novel pathway for lysosomal delivery of PGRN. We found that prosaposin (PSAP) interacts with PGRN and facilitates its lysosomal targeting in both biosynthetic and endocytic pathways via the cation-independent mannose 6-phosphate receptor and low density lipoprotein receptor-related protein 1. PSAP deficiency in mice leads to severe PGRN trafficking defects and a drastic increase in serum PGRN levels. We further showed that this PSAP pathway is independent of, but complementary to, the previously identified PGRN lysosomal trafficking mediated by sortilin. Collectively, our results provide new understanding on PGRN trafficking and shed light on the molecular mechanisms behind FTLD and NCL caused by PGRN mutations. PMID:26370502

  14. ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

    PubMed Central

    Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier

    2015-01-01

    Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. PMID:26342016

  15. NADf chip, a two-color microarray for simultaneous screening of multigene mutations associated with hearing impairment in North African Mediterranean countries.

    PubMed

    Chakchouk, Imen; Ben Said, Mariem; Jbeli, Fida; Benmarzoug, Riadh; Loukil, Salma; Smeti, Ibtihel; Chakroun, Amine; Gibriel, Abdullah Ahmed; Ghorbel, Abdelmonem; Hadjkacem, Hassen; Masmoudi, Saber

    2015-03-01

    Hearing impairment (HI) is the most frequent sensory defect. Genetic causes are involved in two thirds of prelingual cases. Moreover, the autosomal recessive HI frequency is increased in countries where there is a high rate of consanguinity, such as in North African Mediterranean countries. This population shares several features, including history and social behavior, that promote the spread of founder mutations. HI is characterized by tremendous heterogeneity in both the genetic and clinical aspects. The identification of the causal mutation is important for early diagnosis, clinical follow-up, and genetic counseling. Addressing the extreme genetic heterogeneity of HI using classic molecular methods would be expensive and time-consuming. We designed a cost-effective North African Deafness chip for rapid and simultaneous analysis of 58 mutations using multiplex PCR coupled with dual-color arrayed primer extension. These mutations are found in North African HI patients and are distributed over 31 exons and five introns in 21 distinct genes. Assay specificity was initially optimized using 103 archived DNA samples of known genotypes. Blind validation of HI-unrelated patients revealed mutant alleles in 13 samples, and these mutations were confirmed by Sanger sequencing. The North African Deafness chip allows for simultaneous genotyping of eight different samples, at a minimal cost and in a single day, and is therefore amenable to large-scale molecular screening of HI in North Africa. PMID:25560255

  16. Phenotypic variability in a seven-generation Swedish family segregating autosomal dominant hearing impairment due to a novel EYA4 frameshift mutation.

    PubMed

    Frykholm, Carina; Klar, Joakim; Arnesson, Hanna; Rehnman, Anna-Carin; Lodahl, Marianne; Wedén, Ulla; Dahl, Niklas; Tranebjærg, Lisbeth; Rendtorff, Nanna D

    2015-05-25

    Linkage to an interval overlapping the DFNA10 locus on chromosome 6q22-23 was found through genome wide linkage analysis in a seven-generation Swedish family segregating postlingual, autosomal dominant nonsyndromic sensorineural hearing impairment. A novel heterozygous frame-shift mutation (c.579_580insTACC, p.(Asp194Tyrfs*52)) in EYA4 was identified that truncates the so-called variable region of the protein. The mutation is predicted to result in haploinsufficiency of the EYA4 product. No evidence for dilated cardiomyopathy was found in the family, contrasting to a previous family with a deletion resulting in a similar truncation in the variable region. A highly variable age of onset was seen in the mutation carriers. For assessment of the aetiology of this variability, clinical and audiometric data analyses were performed. The affected family members all had similar cross-sectional and longitudinal deterioration of pure tone average (PTA) once the process of hearing deterioration had started, and no gender, parent-of-origin or family branch differences on PTA could be found. Age at onset varied between the family branches. In summary, this is the ninth published genetically verified DFNA10 family. The results imply that unidentified factors, genetic or environmental, other than the EYA4 mutation, are of importance for the age at onset of DFNA10, and that mutation early in the variable region of the EYA4 protein can occur in the absence of dilated cardiomyopathy. PMID:25681523

  17. Altered trafficking and stability of polycystins underlie polycystic kidney disease

    PubMed Central

    Cai, Yiqiang; Fedeles, Sorin V.; Dong, Ke; Anyatonwu, Georgia; Onoe, Tamehito; Mitobe, Michihiro; Gao, Jian-Dong; Okuhara, Dayne; Tian, Xin; Gallagher, Anna-Rachel; Tang, Zhangui; Xie, Xiaoli; Lalioti, Maria D.; Lee, Ann-Hwee; Ehrlich, Barbara E.; Somlo, Stefan

    2014-01-01

    The most severe form of autosomal dominant polycystic kidney disease occurs in patients with mutations in the gene (PKD1) encoding polycystin-1 (PC1). PC1 is a complex polytopic membrane protein expressed in cilia that undergoes autoproteolytic cleavage at a G protein–coupled receptor proteolytic site (GPS). A quarter of PKD1 mutations are missense variants, though it is not clear how these mutations promote disease. Here, we established a cell-based system to evaluate these mutations and determined that GPS cleavage is required for PC1 trafficking to cilia. A common feature among a subset of pathogenic missense mutations is a resulting failure of PC1 to traffic to cilia regardless of GPS cleavage. The application of our system also identified a missense mutation in the gene encoding polycystin-2 (PC2) that prevented this protein from properly trafficking to cilia. Using a Pkd1-BAC recombineering approach, we developed murine models to study the effects of these mutations and confirmed that only the cleaved form of PC1 exits the ER and can rescue the embryonically lethal Pkd1-null mutation. Additionally, steady-state expression levels of the intramembranous COOH-terminal fragment of cleaved PC1 required an intact interaction with PC2. The results of this study demonstrate that PC1 trafficking and expression require GPS cleavage and PC2 interaction, respectively, and provide a framework for functional assays to categorize the effects of missense mutations in polycystins. PMID:25365220

  18. Exploring the link between glucocerebrosidase mutations and parkinsonism

    PubMed Central

    Westbroek, Wendy; Gustafson, Ann Marie; Sidransky, Ellen

    2012-01-01

    Clinical, genetic and pathological studies all demonstrate that mutations in glucocerebrosidase (GBA), which encodes the lysosomal enzyme deficient in Gaucher disease (GD), are an important and common risk factor for Parkinson disease (PD) and related disorders. Some patients with GD and Gaucher carriers develop parkinsonism. Furthermore, subjects with PD have a greatly increased frequency of GBA mutations. GBA mutation carriers exhibit diverse parkinsonian phenotypes, and have glucocerebrosidase-positive Lewy bodies. Although the mechanism for this association is unknown, we present several theories, including enhanced protein aggregation, prion transmission, lipid accumulation, and impaired autophagy, mitophagy or trafficking. Each has inherent limitations, and an unknown “second hit” might be essential. Elucidating the basis for this link will have important consequences and should provide new insights into lysosomal pathways and potential treatment strategies. PMID:21723784

  19. Impaired Acid Catalysis by Mutation of a Protein Loop Hinge Residue in a YopH Mutant Revealed by Crystal Structures

    SciTech Connect

    Brandao, T.; Robinson, H; Johnson, S; Hengge, A

    2009-01-01

    Catalysis by the Yersinia protein-tyrosine phosphatase YopH is significantly impaired by the mutation of the conserved Trp354 residue to Phe. Though not a catalytic residue, this Trp is a hinge residue in a conserved flexible loop (the WPD-loop) that must close during catalysis. To learn why this seemingly conservative mutation reduces catalysis by 2 orders of magnitude, we have solved high-resolution crystal structures for the W354F YopH in the absence and in the presence of tungstate and vanadate. Oxyanion binding to the P-loop in W354F is analogous to that observed in the native enzyme. However, the WPD-loop in the presence of oxyanions assumes a half-closed conformation, in contrast to the fully closed state observed in structures of the native enzyme. This observation provides an explanation for the impaired general acid catalysis observed in kinetic experiments with Trp mutants. A 1.4 Angstroms structure of the W354F mutant obtained in the presence of vanadate reveals an unusual divanadate species with a cyclic [VO]2 core, which has precedent in small molecules but has not been previously reported in a protein crystal structure.

  20. Loss-of-Function Mutations in SERPINB8 Linked to Exfoliative Ichthyosis with Impaired Mechanical Stability of Intercellular Adhesions.

    PubMed

    Pigors, Manuela; Sarig, Ofer; Heinz, Lisa; Plagnol, Vincent; Fischer, Judith; Mohamad, Janan; Malchin, Natalia; Rajpopat, Shefali; Kharfi, Monia; Lestringant, Giles G; Sprecher, Eli; Kelsell, David P; Blaydon, Diana C

    2016-08-01

    SERPINS comprise a large and functionally diverse family of serine protease inhibitors. Here, we report three unrelated families with loss-of-function mutations in SERPINB8 in association with an autosomal-recessive form of exfoliative ichthyosis. Whole-exome sequencing of affected individuals from a consanguineous Tunisian family and a large Israeli family revealed a homozygous frameshift mutation, c.947delA (p.Lys316Serfs(∗)90), and a nonsense mutation, c.850C>T (p.Arg284(∗)), respectively. These two mutations are located in the last exon of SERPINB8 and, hence, would not be expected to lead to nonsense-mediated decay of the mRNA; nonetheless, both mutations are predicted to lead to loss of the reactive site loop of SERPINB8, which is crucial for forming the SERPINB8-protease complex. Using Sanger sequencing, a homozygous missense mutation, c.2T>C (p.Met1?), predicted to result in an N-terminal truncated protein, was identified in an additional family from UAE. Histological analysis of a skin biopsy from an individual homozygous for the variant p.Arg284(∗) showed disadhesion of keratinocytes in the lower epidermal layers plus decreased SERPINB8 levels compared to control. In vitro studies utilizing siRNA-mediated knockdown of SERPINB8 in keratinocytes demonstrated that in the absence of the protein, there is a cell-cell adhesion defect, particularly when cells are subjected to mechanical stress. In addition, immunoblotting and immunostaining revealed an upregulation of desmosomal proteins. In conclusion, we report mutations in SERPINB8 that are associated with exfoliative ichthyosis and provide evidence that SERPINB8 contributes to the mechanical stability of intercellular adhesions in the epidermis. PMID:27476651

  1. Age-Related Hearing Impairment (ARHI) Associated with GJB2 Single Mutation IVS1+1G>A in the Yakut Population Isolate in Eastern Siberia

    PubMed Central

    Pshennikova, Vera G.; Solovyev, Aisen V.; Klarov, Leonid A.; Solovyeva, Natalya A.; Kozhevnikov, Andrei A.; Vasilyeva, Lena M.; Fedotova, Elvira E.; Pak, Maria V.; Lekhanova, Sargylana N.; Zakharova, Elena V.; Savvinova, Kyunney E.; Gotovtsev, Nyurgun N.; Rafailo, Adyum M.; Luginov, Nikolay V.; Alexeev, Anatoliy N.; Posukh, Olga L.; Dzhemileva, Lilya U.; Khusnutdinova, Elza K.; Fedorova, Sardana A.

    2014-01-01

    Age-Related Hearing Impairment (ARHI) is one of the frequent sensory disorders registered in 50% of individuals over 80 years. ARHI is a multifactorial disorder due to environmental and poor-known genetic components. In this study, we present the data on age-related hearing impairment of 48 heterozygous carriers of mutation IVS1+1G>A (GJB2 gene) and 97 subjects with GJB2 genotype wt/wt in the Republic of Sakha/Yakutia (Eastern Siberia, Russia). This subarctic territory was found as the region with the most extensive accumulation of mutation IVS1+1G>A in the world as a result of founder effect in the unique Yakut population isolate. The GJB2 gene resequencing and detailed audiological analysis in the frequency range 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 kHz were performed in all examined subjects that allowed to investigate genotype-phenotype correlations between the presence of single mutation IVS1+1G>A and hearing of subjects from examined groups. We revealed the linear correlation between increase of average hearing thresholds at speech frequencies (PTA0.5,1.0,2.0,4.0 kHz) and age of individuals with GJB2 genotype IVS1+1G>A/wt (rs = 0.499, p = 0.006860 for males and rs = 0.427, p = 0.000277 for females). Moreover, the average hearing thresholds on high frequency (8.0 kHz) in individuals with genotype IVS1+1G>A/wt (both sexes) were significantly worse than in individuals with genotype wt/wt (p<0.05). Age of hearing loss manifestation in individuals with genotype IVS1+1G>A/wt was estimated to be ∼40 years (rs = 0.504, p = 0.003). These findings demonstrate that the single IVS1+1G>A mutation (GJB2) is associated with age-related hearing impairment (ARHI) of the IVS1+1G>A carriers in the Yakuts. PMID:24959830

  2. Impairment of autophagosome-lysosome fusion in the buff mutant mice with the VPS33A(D251E) mutation.

    PubMed

    Zhen, Yuanli; Li, Wei

    2015-01-01

    The HOPS (homotypic fusion and protein sorting) complex functions in endocytic and autophagic pathways in both lower eukaryotes and mammalian cells through its involvement in fusion events between endosomes and lysosomes or autophagosomes and lysosomes. However, the differential molecular mechanisms underlying these fusion processes are largely unknown. Buff (bf) is a mouse mutant that carries an Asp251-to-Glu point mutation (D251E) in the VPS33A protein, a tethering protein and a core subunit of the HOPS complex. Bf mice showed impaired spontaneous locomotor activity, motor learning, and autophagic activity. Although the gross anatomy of the brain was apparently normal, the number of Purkinje cells was significantly reduced. Furthermore, we found that fusion between autophagosomes and lysosomes was defective in bf cells without compromising the endocytic pathway. The direct association of mutant VPS33A(D251E) with the autophagic SNARE complex, STX17 (syntaxin 17)-VAMP8-SNAP29, was enhanced. In addition, the VPS33A(D251E) mutation enhanced interactions with other HOPS subunits, namely VPS41, VPS39, VPS18, and VPS11, except for VPS16. Reduction of the interactions between VPS33A(Y440D) and several other HOPS subunits led to decreased association with STX17. These results suggest that the VPS33A(D251E) mutation plays dual roles by increasing the HOPS complex assembly and its association with the autophagic SNARE complex, which selectively affects the autophagosome-lysosome fusion that impairs basal autophagic activity and induces Purkinje cell loss. PMID:26259518

  3. ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function.

    PubMed

    Murakami, Tetsuro; Qamar, Seema; Lin, Julie Qiaojin; Schierle, Gabriele S Kaminski; Rees, Eric; Miyashita, Akinori; Costa, Ana R; Dodd, Roger B; Chan, Fiona T S; Michel, Claire H; Kronenberg-Versteeg, Deborah; Li, Yi; Yang, Seung-Pil; Wakutani, Yosuke; Meadows, William; Ferry, Rodylyn Rose; Dong, Liang; Tartaglia, Gian Gaetano; Favrin, Giorgio; Lin, Wen-Lang; Dickson, Dennis W; Zhen, Mei; Ron, David; Schmitt-Ulms, Gerold; Fraser, Paul E; Shneider, Neil A; Holt, Christine; Vendruscolo, Michele; Kaminski, Clemens F; St George-Hyslop, Peter

    2015-11-18

    The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins. PMID:26526393

  4. ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function

    PubMed Central

    Murakami, Tetsuro; Qamar, Seema; Lin, Julie Qiaojin; Schierle, Gabriele S. Kaminski; Rees, Eric; Miyashita, Akinori; Costa, Ana R.; Dodd, Roger B.; Chan, Fiona T.S.; Michel, Claire H.; Kronenberg-Versteeg, Deborah; Li, Yi; Yang, Seung-Pil; Wakutani, Yosuke; Meadows, William; Ferry, Rodylyn Rose; Dong, Liang; Tartaglia, Gian Gaetano; Favrin, Giorgio; Lin, Wen-Lang; Dickson, Dennis W.; Zhen, Mei; Ron, David; Schmitt-Ulms, Gerold; Fraser, Paul E.; Shneider, Neil A.; Holt, Christine; Vendruscolo, Michele; Kaminski, Clemens F.; St George-Hyslop, Peter

    2015-01-01

    Summary The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins. PMID:26526393

  5. A novel HSD17B10 mutation impairing the activities of the mitochondrial RNase P complex causes X-linked intractable epilepsy and neurodevelopmental regression.

    PubMed

    Falk, Marni J; Gai, Xiaowu; Shigematsu, Megumi; Vilardo, Elisa; Takase, Ryuichi; McCormick, Elizabeth; Christian, Thomas; Place, Emily; Pierce, Eric A; Consugar, Mark; Gamper, Howard B; Rossmanith, Walter; Hou, Ya-Ming

    2016-05-01

    We report a Caucasian boy with intractable epilepsy and global developmental delay. Whole-exome sequencing identified the likely genetic etiology as a novel p.K212E mutation in the X-linked gene HSD17B10 for mitochondrial short-chain dehydrogenase/reductase SDR5C1. Mutations in HSD17B10 cause the HSD10 disease, traditionally classified as a metabolic disorder due to the role of SDR5C1 in fatty and amino acid metabolism. However, SDR5C1 is also an essential subunit of human mitochondrial RNase P, the enzyme responsible for 5'-processing and methylation of purine-9 of mitochondrial tRNAs. Here we show that the p.K212E mutation impairs the SDR5C1-dependent mitochondrial RNase P activities, and suggest that the pathogenicity of p.K212E is due to a general mitochondrial dysfunction caused by reduction in SDR5C1-dependent maturation of mitochondrial tRNAs. PMID:26950678

  6. Engineered mutations in fibrillin-1 leading to Marfan syndrome act at the protein, cellular and organismal levels.

    PubMed

    Zeyer, Karina A; Reinhardt, Dieter P

    2015-01-01

    Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. They are multi-domain proteins, containing primarily calcium binding epidermal growth factor-like (cbEGF) domains and 8-cysteine/transforming growth factor-beta binding protein-like (TB) domains. Mutations in the fibrillin-1 gene give rise to Marfan syndrome, a connective tissue disorder with clinical complications in the cardiovascular, skeletal, ocular and other organ systems. Here, we review the consequences of engineered Marfan syndrome mutations in fibrillin-1 at the protein, cellular and organismal levels. Representative point mutations associated with Marfan syndrome in affected individuals have been introduced and analyzed in recombinant fibrillin-1 fragments. Those mutations affect fibrillin-1 on a structural and functional level. Mutations which impair folding of cbEGF domains can affect protein trafficking. Protein folding disrupted by some mutations can lead to defective secretion in mutant fibrillin-1 fragments, whereas fragments with other Marfan mutations are secreted normally. Many Marfan mutations render fibrillin-1 more susceptible to proteolysis. There is also evidence that some mutations affect heparin binding. Few mutations have been further analyzed in mouse models. An extensively studied mouse model of Marfan syndrome expresses mouse fibrillin-1 with a missense mutation (p.C1039G). The mice display similar characteristics to human patients with Marfan syndrome. Overall, the analyses of engineered mutations leading to Marfan syndrome provide important insights into the pathogenic molecular mechanisms exerted by mutated fibrillin-1. PMID:26281765

  7. Environment and drug trafficking.

    PubMed

    Bryson, L O

    1992-01-01

    Illicit drug trafficking is a very complex matter, not only because it causes serious and pernicious problems in the socio-economic sphere, but because drug-taking can lead to personal degradation. To this situation, lamentable enough in itself, must be added the immense ecological and environmental damage, which presents grave and serious dangers for the planet. PMID:1302599

  8. Impaired hair follicle morphogenesis and cycling with abnormal epidermal differentiation in nackt mice, a cathepsin L-deficient mutation.

    PubMed

    Benavides, Fernando; Starost, Matthew F; Flores, Mónica; Gimenez-Conti, Irma B; Guénet, Jean-Louis; Conti, Claudio J

    2002-08-01

    We previously described an autosomal-recessive mutation named nackt (nkt) exhibiting partial alopecia associated with CD4(+) T-cell deficiency. Also, we recently reported that nkt (now Ctsl(nkt)) comprises a deletion in the cathepsin L (Ctsl) gene. Another recent study reported that Ctsl knockout mice have CD4(+) T-cell deficiency and periodic shedding of hair, which recapitulate the nkt mutation and the old furless (fs) mutation. The current study focuses on the dermatological aspects of the nkt mutation. Careful histological analysis of skin development of homozygous nkt mice revealed a delayed hair follicle morphogenesis and late onset of the first catagen stage. The skin of Ctsl(nkt)/Ctsl(nkt) mice showed mild epidermal hyperplasia and hyperkeratosis, severe hyperplasia of the sebaceous glands, and structural alterations of hair follicles. Epidermal differentiation seems to be affected in nkt skin, with overexpression of involucrin and profilaggrin/filaggrin along with focal areas of keratin 6 expression in the interfollicular epidermis. Severe epidermal hyperplasia, acanthosis, orthokeratosis, and hyperkeratosis were only observed in mice maintained in nonpathogen-free environments. The analysis of Rag2-/- Ctsl(nkt)/Ctsl(nkt) double-mutant mice indicates that the skin defect remains under the absence of T and B cells. This animal model provides in vivo evidence that cysteine protease cathepsin L plays a critical role in hair follicle morphogenesis and cycling, as well as epidermal differentiation. PMID:12163394

  9. Impaired Hair Follicle Morphogenesis and Cycling with Abnormal Epidermal Differentiation in nackt Mice, a Cathepsin L-Deficient Mutation

    PubMed Central

    Benavides, Fernando; Starost, Matthew F.; Flores, Mónica; Gimenez-Conti, Irma B.; Guénet, Jean-Louis; Conti, Claudio J.

    2002-01-01

    We previously described an autosomal-recessive mutation named nackt (nkt) exhibiting partial alopecia associated with CD4+ T-cell deficiency. Also, we recently reported that nkt (now Ctslnkt) comprises a deletion in the cathepsin L (Ctsl) gene. Another recent study reported that Ctsl knockout mice have CD4+ T-cell deficiency and periodic shedding of hair, which recapitulate the nkt mutation and the old furless (fs) mutation. The current study focuses on the dermatological aspects of the nkt mutation. Careful histological analysis of skin development of homozygous nkt mice revealed a delayed hair follicle morphogenesis and late onset of the first catagen stage. The skin of Ctslnkt/Ctslnkt mice showed mild epidermal hyperplasia and hyperkeratosis, severe hyperplasia of the sebaceous glands, and structural alterations of hair follicles. Epidermal differentiation seems to be affected in nkt skin, with overexpression of involucrin and profilaggrin/filaggrin along with focal areas of keratin 6 expression in the interfollicular epidermis. Severe epidermal hyperplasia, acanthosis, orthokeratosis, and hyperkeratosis were only observed in mice maintained in nonpathogen-free environments. The analysis of Rag2−/− Ctslnkt/Ctslnkt double-mutant mice indicates that the skin defect remains under the absence of T and B cells. This animal model provides in vivo evidence that cysteine protease cathepsin L plays a critical role in hair follicle morphogenesis and cycling, as well as epidermal differentiation. PMID:12163394

  10. Impaired surface membrane insertion of homo- and heterodimeric human muscle chloride channels carrying amino-terminal myotonia-causing mutations

    PubMed Central

    Ronstedt, Katharina; Sternberg, Damien; Detro-Dassen, Silvia; Gramkow, Thomas; Begemann, Birgit; Becher, Toni; Kilian, Petra; Grieschat, Matthias; Machtens, Jan-Philipp; Schmalzing, Günther; Fischer, Martin; Fahlke, Christoph

    2015-01-01

    Mutations in the muscle chloride channel gene (CLCN1) cause myotonia congenita, an inherited condition characterized by muscle stiffness upon sudden forceful movement. We here studied the functional consequences of four disease-causing mutations that predict amino acid substitutions Q43R, S70L, Y137D and Q160H. Wild-type (WT) and mutant hClC-1 channels were heterologously expressed as YFP or CFP fusion protein in HEK293T cells and analyzed by whole-cell patch clamp and fluorescence recordings on individual cells. Q43R, Y137D and Q160H, but not S70L reduced macroscopic current amplitudes, but left channel gating and unitary current amplitudes unaffected. We developed a novel assay combining electrophysiological and fluorescence measurements at the single-cell level in order to measure the probability of ion channel surface membrane insertion. With the exception of S70L, all tested mutations significantly reduced the relative number of homodimeric hClC-1 channels in the surface membrane. The strongest effect was seen for Q43R that reduced the surface insertion probability by more than 99% in Q43R homodimeric channels and by 92 ± 3% in heterodimeric WT/Q43R channels compared to homodimeric WT channels. The new method offers a sensitive approach to investigate mutations that were reported to cause channelopathies, but display only minor changes in ion channel function. PMID:26502825

  11. Mutations in the exocyst component Sec5 disrupt neuronal membrane traffic, but neurotransmitter release persists.

    PubMed

    Murthy, Mala; Garza, Dan; Scheller, Richard H; Schwarz, Thomas L

    2003-02-01

    The exocyst (Sec6/8) complex is necessary for secretion in yeast and has been postulated to establish polarity by directing vesicle fusion to specific sites along the plasma membrane. The complex may also function in the nervous system, but its precise role is unknown. We have investigated exocyst function in Drosophila with mutations in one member of the complex, sec5. Null alleles die as growth-arrested larvae, whose neuromuscular junctions fail to expand. In culture, neurite outgrowth fails in sec5 mutants once maternal Sec5 is exhausted. Using a trafficking assay, we found impairments in the membrane addition of newly synthesized proteins. In contrast, synaptic vesicle fusion was not impaired. Thus, Sec5 differentiates between two forms of vesicle trafficking: trafficking for cell growth and membrane protein insertion depend on sec5, whereas transmitter secretion does not. In this regard, sec5 differs from the homologs of other yeast exocytosis genes that are required for both neuronal trafficking pathways. PMID:12575951

  12. ZC4H2 Mutations Are Associated with Arthrogryposis Multiplex Congenita and Intellectual Disability through Impairment of Central and Peripheral Synaptic Plasticity

    PubMed Central

    Hirata, Hiromi; Nanda, Indrajit; van Riesen, Anne; McMichael, Gai; Hu, Hao; Hambrock, Melanie; Papon, Marie-Amélie; Fischer, Ute; Marouillat, Sylviane; Ding, Can; Alirol, Servane; Bienek, Melanie; Preisler-Adams, Sabine; Grimme, Astrid; Seelow, Dominik; Webster, Richard; Haan, Eric; MacLennan, Alastair; Stenzel, Werner; Yap, Tzu Ying; Gardner, Alison; Nguyen, Lam Son; Shaw, Marie; Lebrun, Nicolas; Haas, Stefan A.; Kress, Wolfram; Haaf, Thomas; Schellenberger, Elke; Chelly, Jamel; Viot, Géraldine; Shaffer, Lisa G.; Rosenfeld, Jill A.; Kramer, Nancy; Falk, Rena; El-Khechen, Dima; Escobar, Luis F.; Hennekam, Raoul; Wieacker, Peter; Hübner, Christoph; Ropers, Hans-Hilger; Gecz, Jozef; Schuelke, Markus; Laumonnier, Frédéric; Kalscheuer, Vera M.

    2013-01-01

    Arthrogryposis multiplex congenita (AMC) is caused by heterogeneous pathologies leading to multiple antenatal joint contractures through fetal akinesia. Understanding the pathophysiology of this disorder is important for clinical care of the affected individuals and genetic counseling of the families. We thus aimed to establish the genetic basis of an AMC subtype that is associated with multiple dysmorphic features and intellectual disability (ID). We used haplotype analysis, next-generation sequencing, array comparative genomic hybridization, and chromosome breakpoint mapping to identify the pathogenic mutations in families and simplex cases. Suspected disease variants were verified by cosegregation analysis. We identified disease-causing mutations in the zinc-finger gene ZC4H2 in four families affected by X-linked AMC plus ID and one family affected by cerebral palsy. Several heterozygous females were also affected, but to a lesser degree. Furthermore, we found two ZC4H2 deletions and one rearrangement in two female and one male unrelated simplex cases, respectively. In mouse primary hippocampal neurons, transiently produced ZC4H2 localized to the postsynaptic compartment of excitatory synapses, and the altered protein influenced dendritic spine density. In zebrafish, antisense-morpholino-mediated zc4h2 knockdown caused abnormal swimming and impaired α-motoneuron development. All missense mutations identified herein failed to rescue the swimming defect of zebrafish morphants. We conclude that ZC4H2 point mutations, rearrangements, and small deletions cause a clinically variable broad-spectrum neurodevelopmental disorder of the central and peripheral nervous systems in both familial and simplex cases of both sexes. Our results highlight the importance of ZC4H2 for genetic testing of individuals presenting with ID plus muscle weakness and minor or major forms of AMC. PMID:23623388

  13. ZC4H2 mutations are associated with arthrogryposis multiplex congenita and intellectual disability through impairment of central and peripheral synaptic plasticity.

    PubMed

    Hirata, Hiromi; Nanda, Indrajit; van Riesen, Anne; McMichael, Gai; Hu, Hao; Hambrock, Melanie; Papon, Marie-Amélie; Fischer, Ute; Marouillat, Sylviane; Ding, Can; Alirol, Servane; Bienek, Melanie; Preisler-Adams, Sabine; Grimme, Astrid; Seelow, Dominik; Webster, Richard; Haan, Eric; MacLennan, Alastair; Stenzel, Werner; Yap, Tzu Ying; Gardner, Alison; Nguyen, Lam Son; Shaw, Marie; Lebrun, Nicolas; Haas, Stefan A; Kress, Wolfram; Haaf, Thomas; Schellenberger, Elke; Chelly, Jamel; Viot, Géraldine; Shaffer, Lisa G; Rosenfeld, Jill A; Kramer, Nancy; Falk, Rena; El-Khechen, Dima; Escobar, Luis F; Hennekam, Raoul; Wieacker, Peter; Hübner, Christoph; Ropers, Hans-Hilger; Gecz, Jozef; Schuelke, Markus; Laumonnier, Frédéric; Kalscheuer, Vera M

    2013-05-01

    Arthrogryposis multiplex congenita (AMC) is caused by heterogeneous pathologies leading to multiple antenatal joint contractures through fetal akinesia. Understanding the pathophysiology of this disorder is important for clinical care of the affected individuals and genetic counseling of the families. We thus aimed to establish the genetic basis of an AMC subtype that is associated with multiple dysmorphic features and intellectual disability (ID). We used haplotype analysis, next-generation sequencing, array comparative genomic hybridization, and chromosome breakpoint mapping to identify the pathogenic mutations in families and simplex cases. Suspected disease variants were verified by cosegregation analysis. We identified disease-causing mutations in the zinc-finger gene ZC4H2 in four families affected by X-linked AMC plus ID and one family affected by cerebral palsy. Several heterozygous females were also affected, but to a lesser degree. Furthermore, we found two ZC4H2 deletions and one rearrangement in two female and one male unrelated simplex cases, respectively. In mouse primary hippocampal neurons, transiently produced ZC4H2 localized to the postsynaptic compartment of excitatory synapses, and the altered protein influenced dendritic spine density. In zebrafish, antisense-morpholino-mediated zc4h2 knockdown caused abnormal swimming and impaired α-motoneuron development. All missense mutations identified herein failed to rescue the swimming defect of zebrafish morphants. We conclude that ZC4H2 point mutations, rearrangements, and small deletions cause a clinically variable broad-spectrum neurodevelopmental disorder of the central and peripheral nervous systems in both familial and simplex cases of both sexes. Our results highlight the importance of ZC4H2 for genetic testing of individuals presenting with ID plus muscle weakness and minor or major forms of AMC. PMID:23623388

  14. A Homozygous [Cys25]PTH(1-84) Mutation That Impairs PTH/PTHrP Receptor Activation Defines a Novel Form of Hypoparathyroidism.

    PubMed

    Lee, Sihoon; Mannstadt, Michael; Guo, Jun; Kim, Seul Min; Yi, Hyon-Seung; Khatri, Ashok; Dean, Thomas; Okazaki, Makoto; Gardella, Thomas J; Jüppner, Harald

    2015-10-01

    Hypocalcemia and hyperphosphatemia are encountered in idiopathic hypoparathyroidism (IHP) and pseudohypoparathyroidism type Ib (PHP1B). In contrast to PHP1B, which is caused by resistance toward parathyroid hormone (PTH), the genetic defects leading to IHP impair production of this important regulator of mineral ion homeostasis. So far, only five PTH mutations were shown to cause IHP, each of which is located in the hormone's pre-pro leader segment and thus impair hormone secretion. In three siblings affected by IHP, we now identified a homozygous arginine-to-cysteine mutation at position 25 (R25C) of the mature PTH(1-84) polypeptide; heterozygous family members are healthy. Depending on the assay used for evaluating these patients, plasma PTH levels were either low or profoundly elevated, thus leading to ambiguities regarding the underlying diagnosis, namely IHP or PHP1B. Consistent with increased PTH levels, recombinant [Cys25]PTH(1-84) and wild-type PTH(1-84) were secreted equally well by transfected COS-7 cells. However, synthetic [Cys25]PTH(1-34) was found to have a lower binding affinity for the PTH receptor type-1 (PTH1R) than PTH(1-34) and consequently a lower efficiency for stimulating cAMP formation in cells expressing this receptor. Consistent with these in vitro findings, long-term infusion of [Cys25]PTH(1-34) resulted only in minimal calcemic and phosphaturic responses, despite readily detectable levels of [Cys25]PTH(1-34) in plasma. The mineral ion abnormalities observed in the three IHP patients are thus most likely caused by the inherited homozygous missense PTH mutation, which reduces bioactivity of the secreted hormone. Based on these findings, screening for PTH(1-84) mutations should be considered when clinical and laboratory findings are consistent with PHP1B, but GNAS methylation changes have been excluded. Differentiating between IHP and PHP1B has considerable implications for genetic counseling, therapy, and long-term outcome because

  15. Molecular mechanism of sulphonylurea block of K(ATP) channels carrying mutations that impair ATP inhibition and cause neonatal diabetes.

    PubMed

    Proks, Peter; de Wet, Heidi; Ashcroft, Frances M

    2013-11-01

    Sulphonylurea drugs are the therapy of choice for treating neonatal diabetes (ND) caused by mutations in the ATP-sensitive K(+) channel (KATP channel). We investigated the interactions between MgATP, MgADP, and the sulphonylurea gliclazide with KATP channels expressed in Xenopus oocytes. In the absence of MgATP, gliclazide block was similar for wild-type channels and those carrying the Kir6.2 ND mutations R210C, G334D, I296L, and V59M. Gliclazide abolished the stimulatory effect of MgATP on all channels. Conversely, high MgATP concentrations reduced the gliclazide concentration, producing a half-maximal block of G334D and R201C channels and suggesting a mutual antagonism between nucleotide and gliclazide binding. The maximal extent of high-affinity gliclazide block of wild-type channels was increased by MgATP, but this effect was smaller for ND channels; channels that were least sensitive to ATP inhibition showed the smallest increase in sulphonylurea block. Consequently, G334D and I296L channels were not fully blocked, even at physiological MgATP concentrations (1 mmol/L). Glibenclamide block was also reduced in β-cells expressing Kir6.2-V59M channels. These data help to explain why patients with some mutations (e.g., G334D, I296L) are insensitive to sulphonylurea therapy, why higher drug concentrations are needed to treat ND than type 2 diabetes, and why patients with severe ND mutations are less prone to drug-induced hypoglycemia. PMID:23835339

  16. Impaired riboflavin transport due to missense mutations in SLC52A2 causes Brown-Vialetto-Van Laere syndrome.

    PubMed

    Haack, Tobias B; Makowski, Christine; Yao, Yoshiaki; Graf, Elisabeth; Hempel, Maja; Wieland, Thomas; Tauer, Ulrike; Ahting, Uwe; Mayr, Johannes A; Freisinger, Peter; Yoshimatsu, Hiroki; Inui, Ken; Strom, Tim M; Meitinger, Thomas; Yonezawa, Atsushi; Prokisch, Holger

    2012-11-01

    Brown-Vialetto-Van Laere syndrome (BVVLS [MIM 211530]) is a rare neurological disorder characterized by infancy onset sensorineural deafness and ponto-bulbar palsy. Mutations in SLC52A3 (formerly C20orf54), coding for riboflavin transporter 2 (hRFT2), have been identified as the molecular genetic correlate in several individuals with BVVLS. Exome sequencing of just one single case revealed that compound heterozygosity for two pathogenic mutations in the SLC52A2 gene coding for riboflavin transporter 3 (hRFT3), another member of the riboflavin transporter family, is also associated with BVVLS. Overexpression studies confirmed that the gene products of both mutant alleles have reduced riboflavin transport activities. While mutations in SLC52A3 cause decreased plasma riboflavin levels, concordant with a role of SLC52A3 in riboflavin uptake from food, the SLC52A2-mutant individual had normal plasma riboflavin concentrations, a finding in line with a postulated function of SLC52A2 in riboflavin uptake from blood into target cells. Our results contribute to the understanding of human riboflavin metabolism and underscore its role in the pathogenesis of BVVLS, thereby providing a rational basis for a high-dose riboflavin treatment. PMID:22864630

  17. Ras trafficking, localization and compartmentalized signalling

    PubMed Central

    Prior, Ian A.; Hancock, John F.

    2012-01-01

    Ras proteins are proto-oncogenes that are frequently mutated in human cancers. Three closely related isoforms, HRAS, KRAS and NRAS, are expressed in all cells and have overlapping but distinctive functions. Recent work has revealed how differences between the Ras isoforms in their trafficking, localization and protein-membrane orientation enable signalling specificity to be determined. We review the various strategies used to characterize compartmentalized Ras localization and signalling. Localization is an important contextual modifier of signalling networks and insights from the Ras system are of widespread relevance for researchers interested in signalling initiated from membranes. PMID:21924373

  18. Lysosomal Trafficking Regulator (LYST).

    PubMed

    Ji, Xiaojie; Chang, Bo; Naggert, Jürgen K; Nishina, Patsy M

    2016-01-01

    Regulation of vesicle trafficking to lysosomes and lysosome-related organelles (LROs) as well as regulation of the size of these organelles are critical to maintain their functions. Disruption of the lysosomal trafficking regulator (LYST) results in Chediak-Higashi syndrome (CHS), a rare autosomal recessive disorder characterized by oculocutaneous albinism, prolonged bleeding, severe immunodeficiency, recurrent bacterial infection, neurologic dysfunction and hemophagocytic lympohistiocytosis (HLH). The classic diagnostic feature of the syndrome is enlarged LROs in all cell types, including lysosomes, melanosomes, cytolytic granules and platelet dense bodies. The most striking CHS ocular pathology observed is an enlargement of melanosomes in the retinal pigment epithelium (RPE), which leads to aberrant distribution of eye pigmentation, and results in photophobia and decreased visual acuity. Understanding the molecular function of LYST and identification of its interacting partners may provide therapeutic targets for CHS and other diseases associated with the regulation of LRO size and/or vesicle trafficking, such as asthma, urticaria and Leishmania amazonensis infections. PMID:26427484

  19. CYP2R1 Mutations Impair Generation of 25-hydroxyvitamin D and Cause an Atypical Form of Vitamin D Deficiency

    PubMed Central

    Fischer, Philip R.; Singh, Ravinder J.; Roizen, Jeffrey; Levine, Michael A.

    2015-01-01

    Context: Production of the active vitamin D hormone 1,25-dihydroxyvitamin D requires hepatic 25-hydroxylation of vitamin D. The CYP2R1 gene encodes the principal vitamin D 25-hydroxylase in humans. Objective: This study aimed to determine the prevalence of CYP2R1 mutations in Nigerian children with familial rickets and vitamin D deficiency and assess the functional effect on 25-hydroxylase activity. Design and Participants: We sequenced the CYP2R1 gene in subjects with sporadic rickets and affected subjects from families in which more than one member had rickets. Main Outcome Measures: Function of mutant CYP2R1 genes as assessed in vivo by serum 25-hydroxyvitamin D values after administration of vitamin D and in vitro by analysis of mutant forms of the CYP2R1. Results: CYP2R1 sequences were normal in 27 children with sporadic rickets, but missense mutations were identified in affected members of 2 of 12 families, a previously identified L99P, and a novel K242N. In silico analyses predicted that both substitutions would have deleterious effects on the variant proteins, and in vitro studies showed that K242N and L99P had markedly reduced or complete loss of 25-hydroxylase activity, respectively. Heterozygous subjects were less affected than homozygous subjects, and oral administration of vitamin D led to significantly lower increases in serum 25-hydroxyvitamin D in heterozygous than in control subjects, whereas homozygous subjects showed negligible increases. Conclusion: These studies confirm that CYP2R1 is the principal 25-hydroxylase in humans and demonstrate that CYP2R1 alleles have dosage-dependent effects on vitamin D homeostasis. CYP2R1 mutations cause a novel form of genetic vitamin D deficiency with semidominant inheritance. PMID:25942481

  20. Effects of Curculigoside on Memory Impairment and Bone Loss via Anti-Oxidative Character in APP/PS1 Mutated Transgenic Mice.

    PubMed

    Zhao, Lu; Liu, Sha; Wang, Yin; Zhang, Qiaoyan; Zhao, Wenjuan; Wang, Zejian; Yin, Ming

    2015-01-01

    Alzheimer's disease (AD) and osteoporosis are two closely related multifactorial progressively degenerative diseases that predominantly affect aged people. These two diseases share many common risk factors, including old age, being female, smoking, excessive drinking, low estrogen, and vitamin D3 levels. Additionally, oxidative damage and the dysfunction of the antioxidant system play important roles in the pathogenesis of osteoporosis and AD. Aβ not only leads to impaired memory but also plays a crucial role in the demineralization process of bone tissues of older people and women with menopause. Curculigoside can promote calcium deposition and increase the levels of ALP and Runx2 in osteoblasts under oxidative stress via anti-oxidative character. Therefore, we investigated the effects of CUR on the spatial learning and memory by the Morris water maze and brain immunohistochemistry, and bone microstructure and material properties of femurs by micro-computed tomography and mechanical testing in APP/PS1 mutated transgenic mice. Oral administration of CUR can significantly enhance learning performance and ameliorate bone loss in APP/PS1 mutated transgenic mice, and the mechanism may be related to its antioxidant effect. Based on these results, CUR has real potential as a new natural resource for developing medicines or dietary supplements for the prevention and treatment of the two closely linked multifactorial progressive degenerative disorders, AD and osteoporosis. PMID:26186010

  1. The embryonic myosin R672C mutation that underlies Freeman-Sheldon syndrome impairs cross-bridge detachment and cycling in adult skeletal muscle.

    PubMed

    Racca, Alice W; Beck, Anita E; McMillin, Margaret J; Korte, F Steven; Bamshad, Michael J; Regnier, Michael

    2015-06-15

    Distal arthrogryposis is the most common known heritable cause of congenital contractures (e.g. clubfoot) and results from mutations in genes that encode proteins of the contractile complex of skeletal muscle cells. Mutations are most frequently found in MYH3 and are predicted to impair the function of embryonic myosin. We measured the contractile properties of individual skeletal muscle cells and the activation and relaxation kinetics of isolated myofibrils from two adult individuals with an R672C substitution in embryonic myosin and distal arthrogryposis syndrome 2A (DA2A) or Freeman-Sheldon syndrome. In R672C-containing muscle cells, we observed reduced specific force, a prolonged time to relaxation and incomplete relaxation (elevated residual force). In R672C-containing muscle myofibrils, the initial, slower phase of relaxation had a longer duration and slower rate, and time to complete relaxation was greatly prolonged. These observations can be collectively explained by a small subpopulation of myosin cross-bridges with greatly reduced detachment kinetics, resulting in a slower and less complete deactivation of thin filaments at the end of contractions. These findings have important implications for selecting and testing directed therapeutic options for persons with DA2A and perhaps congenital contractures in general. PMID:25740846

  2. Effects of Curculigoside on Memory Impairment and Bone Loss via Anti-Oxidative Character in APP/PS1 Mutated Transgenic Mice

    PubMed Central

    Zhang, Qiaoyan; Zhao, Wenjuan; Wang, Zejian; Yin, Ming

    2015-01-01

    Alzheimer's disease (AD) and osteoporosis are two closely related multifactorial progressively degenerative diseases that predominantly affect aged people. These two diseases share many common risk factors, including old age, being female, smoking, excessive drinking, low estrogen, and vitamin D3 levels. Additionally, oxidative damage and the dysfunction of the antioxidant system play important roles in the pathogenesis of osteoporosis and AD. Aβ not only leads to impaired memory but also plays a crucial role in the demineralization process of bone tissues of older people and women with menopause. Curculigoside can promote calcium deposition and increase the levels of ALP and Runx2 in osteoblasts under oxidative stress via anti-oxidative character. Therefore, we investigated the effects of CUR on the spatial learning and memory by the Morris water maze and brain immunohistochemistry, and bone microstructure and material properties of femurs by micro-computed tomography and mechanical testing in APP/PS1 mutated transgenic mice. Oral administration of CUR can significantly enhance learning performance and ameliorate bone loss in APP/PS1 mutated transgenic mice, and the mechanism may be related to its antioxidant effect. Based on these results, CUR has real potential as a new natural resource for developing medicines or dietary supplements for the prevention and treatment of the two closely linked multifactorial progressive degenerative disorders, AD and osteoporosis. PMID:26186010

  3. The V499G/Y501H Mutation Impairs Fast Motor Kinetics of Prestin and Has Significance for Defining Functional Independence of Individual Prestin Subunits*

    PubMed Central

    Homma, Kazuaki; Duan, Chongwen; Zheng, Jing; Cheatham, Mary Ann; Dallos, Peter

    2013-01-01

    Outer hair cells (OHCs) are a mammalian innovation for mechanically amplifying sound energy to overcome the viscous damping of the cochlear partition. Although the voltage-dependent OHC membrane motor, prestin, has been demonstrated to be essential for mammalian cochlear amplification, the molecular mechanism by which prestin converts electrical energy into mechanical displacement/force remains elusive. Identifying mutations that alter the motor function of prestin provides vital information for unraveling the energy transduction mechanism of prestin. We show that the V499G/Y501H mutation does not deprive prestin of its voltage-induced motor activity, but it does significantly impair the fast motor kinetics and voltage operating range. Furthermore, mutagenesis studies suggest that Val-499 is the primary site responsible for these changes. We also show that V499G/Y501H prestin forms heteromers with wild-type prestin and that the fast motor kinetics of wild-type prestin is not affected by heteromer formation with V499G/Y501H prestin. These results suggest that prestin subunits are individually functional within a given multimer. PMID:23212912

  4. Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway.

    PubMed

    Yoo, Jin-San; Moyer, Bryan D; Bannykh, Sergei; Yoo, Hyeon-Mi; Riordan, John R; Balch, William E

    2002-03-29

    The mechanism(s) of cystic fibrosis transmembrane conductance regulator (CFTR) trafficking from the endoplasmic reticulum (ER) through the Golgi apparatus, the step impaired in individuals afflicted with the prevalent CFTR-DeltaF508 mutation leading to cystic fibrosis, is largely unknown. Recent morphological observations suggested that CFTR is largely absent from the Golgi in situ (Bannykh, S. I., Bannykh, G. I., Fish, K. N., Moyer, B. D., Riordan, J. R., and Balch, W. E. (2000) Traffic 1, 852-870), raising the possibility of a novel trafficking pathway through the early secretory pathway. We now report that export of CFTR from the ER is regulated by the conventional coat protein complex II (COPII) in all cell types tested. Remarkably, in a cell type-specific manner, processing of CFTR from the core-glycosylated (band B) ER form to the complex-glycosylated (band C) isoform followed a non-conventional pathway that was insensitive to dominant negative Arf1, Rab1a/Rab2 GTPases, or the SNAp REceptor (SNARE) component syntaxin 5, all of which block the conventional trafficking pathway from the ER to the Golgi. Moreover, CFTR transport through the non-conventional pathway was potently blocked by overexpression of the late endosomal target-SNARE syntaxin 13, suggesting that recycling through a late Golgi/endosomal system was a prerequisite for CFTR maturation. We conclude that CFTR transport in the early secretory pathway can involve a novel pathway between the ER and late Golgi/endosomal compartments that may influence developmental expression of CFTR on the cell surface in polarized epithelial cells. PMID:11799116

  5. Mutation of DNA Polymerase β R137Q Results in Retarded Embryo Development Due to Impaired DNA Base Excision Repair in Mice

    PubMed Central

    Pan, Feiyan; Zhao, Jing; Zhou, Ting; Kuang, Zhihui; Dai, Huifang; Wu, Huan; Sun, Hongfang; Zhou, Xiaolong; Wu, Xuping; Hu, Zhigang; He, Lingfeng; Shen, Binghui; Guo, Zhigang

    2016-01-01

    DNA polymerase β (Pol β), a key enzyme in the DNA base excision repair (BER) pathway, is pivotal in maintaining the integrity and stability of genomes. One Pol β mutation that has been identified in tumors, R137Q (arginine to glutamine substitution), has been shown to lower polymerase activity, and impair its DNA repair capacity. However, the exact functional deficiency associated with this polymorphism in living organisms is still unknown. Here, we constructed Pol β R137Q knock-in mice, and found that homozygous knock-in mouse embryos were typically small in size and had a high mortality rate (21%). These embryonic abnormalities were caused by slow cell proliferation and increased apoptosis. In R137Q knock-in mouse embryos, the BER efficiency was severely impaired, which subsequently resulted in double-strand breaks (DSBs) and chromosomal aberrations. Furthermore, R137Q mouse embryo fibroblasts (MEFs) were more sensitive to DNA-damaging reagents, such as methyl methanesulfonate (MMS) and H2O2. They displayed a higher percentage of DSBs, and were more likely to undergo apoptosis. Our results indicate that R137 is a key amino acid site that is essential for proper Pol β functioning in maintaining genomic stability and embryo development. PMID:27358192

  6. Mutation of DNA Polymerase β R137Q Results in Retarded Embryo Development Due to Impaired DNA Base Excision Repair in Mice.

    PubMed

    Pan, Feiyan; Zhao, Jing; Zhou, Ting; Kuang, Zhihui; Dai, Huifang; Wu, Huan; Sun, Hongfang; Zhou, Xiaolong; Wu, Xuping; Hu, Zhigang; He, Lingfeng; Shen, Binghui; Guo, Zhigang

    2016-01-01

    DNA polymerase β (Pol β), a key enzyme in the DNA base excision repair (BER) pathway, is pivotal in maintaining the integrity and stability of genomes. One Pol β mutation that has been identified in tumors, R137Q (arginine to glutamine substitution), has been shown to lower polymerase activity, and impair its DNA repair capacity. However, the exact functional deficiency associated with this polymorphism in living organisms is still unknown. Here, we constructed Pol β R137Q knock-in mice, and found that homozygous knock-in mouse embryos were typically small in size and had a high mortality rate (21%). These embryonic abnormalities were caused by slow cell proliferation and increased apoptosis. In R137Q knock-in mouse embryos, the BER efficiency was severely impaired, which subsequently resulted in double-strand breaks (DSBs) and chromosomal aberrations. Furthermore, R137Q mouse embryo fibroblasts (MEFs) were more sensitive to DNA-damaging reagents, such as methyl methanesulfonate (MMS) and H2O2. They displayed a higher percentage of DSBs, and were more likely to undergo apoptosis. Our results indicate that R137 is a key amino acid site that is essential for proper Pol β functioning in maintaining genomic stability and embryo development. PMID:27358192

  7. Impaired peroxisome proliferator-activated receptor gamma function through mutation of a conserved salt bridge (R425C) in familial partial lipodystrophy.

    PubMed

    Jeninga, Ellen H; van Beekum, Olivier; van Dijk, Aalt D J; Hamers, Nicole; Hendriks-Stegeman, Brenda I; Bonvin, Alexandre M J J; Berger, Ruud; Kalkhoven, Eric

    2007-05-01

    The nuclear receptor peroxisome proliferator-activated receptor (PPAR) gamma plays a key role in the regulation of glucose and lipid metabolism in adipocytes by regulating their differentiation, maintenance, and function. A heterozygous mutation in the PPARG gene, which changes an arginine residue at position 425 into a cysteine (R425C), has been reported in a patient with familial partial lipodystrophy subtype 3 (FPLD3). The strong conservation of arginine 425 among nuclear receptors that heterodimerize with retinoic acid X receptor prompted us to investigate the functional consequences of the R425C mutation on PPARgamma function. Here we show that this mutant displayed strongly reduced transcriptional activity compared with wild-type PPARgamma, irrespective of cell type, promoter context, or ligand, whereas transrepression of nuclear factor-kappaB activity remained largely intact. Our data indicate that the reduced transcriptional activity of PPARgamma R425C is not caused by impaired corepressor release, but due to reduced dimerization with retinoic acid X receptor alpha in combination with reduced ligand binding and subsequent coactivator binding. As a consequence of these molecular defects, the R425C mutant was less effective in inducing adipocyte differentiation. PPARgamma R425C did not inhibit its wild-type counterpart in a dominant-negative manner, suggesting a haploinsufficiency mechanism in at least some FPLD3 patients. Using molecular dynamics simulations, substitution of R425 with cysteine is predicted to cause the formation of an alternative salt bridge. This structural change provides a likely explanation of how mutation of a single conserved residue in a patient with FPLD3 can disrupt the function of the adipogenic transcription factor PPARgamma on multiple levels. PMID:17312272

  8. The R740S mutation in the V-ATPase a3 subunit increases lysosomal pH, impairs NFATc1 translocation, and decreases in vitro osteoclastogenesis.

    PubMed

    Voronov, Irina; Ochotny, Noelle; Jaumouillé, Valentin; Owen, Celeste; Manolson, Morris F; Aubin, Jane E

    2013-01-01

    Vacuolar H(+) -ATPase (V-ATPase), a multisubunit enzyme located at the ruffled border and in lysosomes of osteoclasts, is necessary for bone resorption. We previously showed that heterozygous mice with an R740S mutation in the a3 subunit of V-ATPase (+/R740S) have mild osteopetrosis resulting from an ∼90% reduction in proton translocation across osteoclast membranes. Here we show that lysosomal pH is also higher in +/R740S compared with wild-type (+/+) osteoclasts. Both osteoclast number and size were decreased in cultures of +/R740S compared with +/+ bone marrow cells, with concomitant decreased expression of key osteoclast markers (TRAP, cathepsin K, OSCAR, DC-STAMP, and NFATc1), suggesting that low lysosomal pH plays an important role in osteoclastogenesis. To elucidate the molecular mechanism of this inhibition, NFATc1 activation was assessed. NFATc1 nuclear translocation was significantly reduced in +/R740S compared with +/+ cells; however, this was not because of impaired enzymatic activity of calcineurin, the phosphatase responsible for NFATc1 dephosphorylation. Protein and RNA expression levels of regulator of calcineurin 1 (RCAN1), an endogenous inhibitor of NFATc1 activation and a protein degraded in lysosomes, were not significantly different between +/R740S and +/+ osteoclasts, but the RCAN1/NFATc1 ratio was significantly higher in +/R740S versus +/+ cells. The lysosomal inhibitor chloroquine significantly increased RCAN1 accumulation in +/+ cells, consistent with the hypothesis that higher lysosomal pH impairs RCAN1 degradation, leading to a higher RCAN1/NFATc1 ratio and consequently NFATc1 inhibition. Our data indicate that increased lysosomal pH in osteoclasts leads to decreased NFATc1 signaling and nuclear translocation, resulting in a cell autonomous impairment of osteoclastogenesis in vitro. PMID:22865292

  9. Mutations in Mll2, an H3K4 Methyltransferase, Result in Insulin Resistance and Impaired Glucose Tolerance in Mice

    PubMed Central

    Schröter, David; Matthews, Helen C.; Bogani, Debora; Moir, Lee; Long, Anna; Church, Christopher; Hugill, Alison; Anstee, Quentin M.; Goldin, Rob; Thursz, Mark; Hollfelder, Florian; Cox, Roger D.

    2013-01-01

    We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5–14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level. PMID:23826075

  10. Health implications of human trafficking.

    PubMed

    Richards, Tiffany A

    2014-01-01

    Freedom is arguably the most cherished right in the United States. But each year, approximately 14,500 to 17,500 women, men and children are trafficked into the United States for the purposes of forced labor or sexual exploitation. Human trafficking has significant effects on both physical and mental health. This article describes the features of human trafficking, its physical and mental health effects and the vital role nurses can play in providing care to this vulnerable population. PMID:24750655

  11. Role of adaptor proteins and clathrin in the trafficking of human kidney anion exchanger 1 (kAE1) to the cell surface.

    PubMed

    Junking, Mutita; Sawasdee, Nunghathai; Duangtum, Natapol; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

    2014-07-01

    Kidney anion exchanger 1 (kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl-/HCO3-) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells. PMID:24698155

  12. Structure of Human B12 Trafficking Protein CblD Reveals Molecular Mimicry and Identifies a New Subfamily of Nitro-FMN Reductases.

    PubMed

    Yamada, Kazuhiro; Gherasim, Carmen; Banerjee, Ruma; Koutmos, Markos

    2015-12-01

    In mammals, B12 (or cobalamin) is an essential cofactor required by methionine synthase and methylmalonyl-CoA mutase. A complex intracellular pathway supports the assimilation of cobalamin into its active cofactor forms and delivery to its target enzymes. MMADHC (the methylmalonic aciduria and homocystinuria type D protein), commonly referred to as CblD, is a key chaperone involved in intracellular cobalamin trafficking, and mutations in CblD cause methylmalonic aciduria and/or homocystinuria. Herein, we report the first crystal structure of the globular C-terminal domain of human CblD, which is sufficient for its interaction with MMADHC (the methylmalonic aciduria and homocystinuria type C protein), or CblC, and for supporting the cytoplasmic cobalamin trafficking pathway. CblD contains an α+β fold that is structurally reminiscent of the nitro-FMN reductase superfamily. Two of the closest structural relatives of CblD are CblC, a multifunctional enzyme important for cobalamin trafficking, and the activation domain of methionine synthase. CblD, CblC, and the activation domain of methionine synthase share several distinguishing features and, together with two recently described corrinoid-dependent reductive dehalogenases, constitute a new subclass within the nitro-FMN reductase superfamily. We demonstrate that CblD enhances oxidation of cob(II)alamin bound to CblC and that disease-causing mutations in CblD impair the kinetics of this reaction. The striking structural similarity of CblD to CblC, believed to be contiguous in the cobalamin trafficking pathway, suggests the co-option of molecular mimicry as a strategy for achieving its function. PMID:26364851

  13. Alteration of the proteostasis network of plant cells promotes the post-endoplasmic reticulum trafficking of recombinant mutant (L444P) human β-glucocerebrosidase

    PubMed Central

    Babajani, Gholamreza; Kermode, Allison R

    2014-01-01

    Gaucher disease is a prevalent lysosomal storage disease characterized by a deficiency in the activity of lysosomal acid β-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45). One of the most prevalent disease-causing mutations in humans is a L444P missense mutation in the GCase protein, which results in its disrupted folding in the endoplasmic reticulum (ER) and impaired post-ER trafficking. To determine whether the post-ER trafficking of this severely malfolded protein can be restored, we expressed the mutant L444P GCase as a recombinant protein in transgenic tobacco (Nicotiana tabacum L. cv Bright Yellow 2 [BY2]) cells, in which the GCase variant was equipped with a plant signal peptide to allow for secretion upon rescued trafficking out of the ER. The recombinant L444P mutant GCase was retained in the plant endoplasmic reticulum (ER). Kifunensine and Eeyarestatin I, both inhibitors of ER-associated degradation (ERAD), and the proteostasis regulators, celastrol and MG-132, increased the steady-state levels of the mutant protein inside the plant cells and further promoted the post-ER trafficking of L444P GCase, as indicated by endoglycosidase-H sensitivity- and secretion- analyses. Transcript profiling of genes encoding ER-molecular chaperones, ER stress responsive proteins, and cytoplasmic heat shock response proteins, revealed insignificant or only very modest changes in response to the ERAD inhibitors and proteostasis regulators. An exception was the marked response to celastrol which reduced the steady-state levels of cytoplasmic HSP90 transcripts and protein. As HSP90 participates in the targeting of misfolded proteins to the proteasome pathway, its down-modulation in response to celastrol may partly account for the mechanism of improved homeostasis of L444P GCase mediated by this triterpene. PMID:24713615

  14. SLC6A3 coding variant Ala559Val found in two autism probands alters dopamine transporter function and trafficking.

    PubMed

    Bowton, E; Saunders, C; Reddy, I A; Campbell, N G; Hamilton, P J; Henry, L K; Coon, H; Sakrikar, D; Veenstra-VanderWeele, J M; Blakely, R D; Sutcliffe, J; Matthies, H J G; Erreger, K; Galli, A

    2014-01-01

    Emerging evidence associates dysfunction in the dopamine (DA) transporter (DAT) with the pathophysiology of autism spectrum disorder (ASD). The human DAT (hDAT; SLC6A3) rare variant with an Ala to Val substitution at amino acid 559 (hDAT A559V) was previously reported in individuals with bipolar disorder or attention-deficit hyperactivity disorder (ADHD). We have demonstrated that this variant is hyper-phosphorylated at the amino (N)-terminal serine (Ser) residues and promotes an anomalous DA efflux phenotype. Here, we report the novel identification of hDAT A559V in two unrelated ASD subjects and provide the first mechanistic description of its impaired trafficking phenotype. DAT surface expression is dynamically regulated by DAT substrates including the psychostimulant amphetamine (AMPH), which causes hDAT trafficking away from the plasma membrane. The integrity of DAT trafficking directly impacts DA transport capacity and therefore dopaminergic neurotransmission. Here, we show that hDAT A559V is resistant to AMPH-induced cell surface redistribution. This unique trafficking phenotype is conferred by altered protein kinase C β (PKCβ) activity. Cells expressing hDAT A559V exhibit constitutively elevated PKCβ activity, inhibition of which restores the AMPH-induced hDAT A559V membrane redistribution. Mechanistically, we link the inability of hDAT A559V to traffic in response to AMPH to the phosphorylation of the five most distal DAT N-terminal Ser. Mutation of these N-terminal Ser to Ala restores AMPH-induced trafficking. Furthermore, hDAT A559V has a diminished ability to transport AMPH, and therefore lacks AMPH-induced DA efflux. Pharmacological inhibition of PKCβ or Ser to Ala substitution in the hDAT A559V background restores AMPH-induced DA efflux while promoting intracellular AMPH accumulation. Although hDAT A559V is a rare variant, it has been found in multiple probands with neuropsychiatric disorders associated with imbalances in DA neurotransmission

  15. Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy

    PubMed Central

    Shibata, Tatsuhiro; Ohta, Tsutomu; Tong, Kit I.; Kokubu, Akiko; Odogawa, Reiko; Tsuta, Koji; Asamura, Hisao; Yamamoto, Masayuki; Hirohashi, Setsuo

    2008-01-01

    The nuclear factor E2-related factor 2 (Nrf2) is a master transcriptional activator of genes encoding numerous cytoprotective enzymes that are induced in response to environmental and endogenously derived oxidative/electrophilic agents. Under normal, nonstressed circumstances, low cellular concentrations of Nrf2 are maintained by proteasomal degradation through a Keap1-Cul3-Roc1-dependent mechanism. A model for Nrf2 activation has been proposed in which two amino-terminal motifs, DLG and ETGE, promote efficient ubiquitination and rapid turnover; known as the two-site substrate recognition/hinge and latch model. Here, we show that in human cancer, somatic mutations occur in the coding region of NRF2, especially among patients with a history of smoking or suffering from squamous cell carcinoma; in the latter case, this leads to poor prognosis. These mutations specifically alter amino acids in the DLG or ETGE motifs, resulting in aberrant cellular accumulation of Nrf2. Mutant Nrf2 cells display constitutive induction of cytoprotective enzymes and drug efflux pumps, which are insensitive to Keap1-mediated regulation. Suppression of Nrf2 protein levels by siRNA knockdown sensitized cancer cells to oxidative stress and chemotherapeutic reagents. Our results strongly support the contention that constitutive Nrf2 activation affords cancer cells with undue protection from their inherently stressed microenvironment and anti-cancer treatments. Hence, inactivation of the Nrf2 pathway may represent a therapeutic strategy to reinforce current treatments for malignancy. Congruously, the present study also provides in vivo validation of the two-site substrate recognition model for Nrf2 activation by the Keap1-Cul3-based E3 ligase. PMID:18757741

  16. The DNA Ligase IV Syndrome R278H Mutation Impairs B Lymphopoiesis via Error-Prone Nonhomologous End-Joining.

    PubMed

    Park, Jihye; Welner, Robert S; Chan, Mei-Yee; Troppito, Logan; Staber, Philipp B; Tenen, Daniel G; Yan, Catherine T

    2016-01-01

    Hypomorphic mutations in the nonhomologous end-joining (NHEJ) DNA repair protein DNA ligase IV (LIG4) lead to immunodeficiency with varying severity. In this study, using a murine knock-in model, we investigated the mechanisms underlying abnormalities in class switch recombination (CSR) associated with the human homozygous Lig4 R278H mutation. Previously, we found that despite the near absence of Lig4 end-ligation activity and severely reduced mature B cell numbers, Lig4(R278H/R278H) (Lig4(R/R)) mice exhibit only a partial CSR block, producing near normal IgG1 and IgE but substantially reduced IgG3, IgG2b, and IgA serum levels. In this study, to address the cause of these abnormalities, we assayed CSR in Lig4(R/R) B cells generated via preassembled IgH and IgK V region exons (HL). This revealed that Lig4(R278H) protein levels while intact exhibited a higher turnover rate during activation of switching to IgG3 and IgG2b, as well as delays in CSR kinetics associated with defective proliferation during activation of switching to IgG1 and IgE. Activated Lig4(R/R)HL B cells consistently accumulated high frequencies of activation-induced cytidine deaminase-dependent IgH locus chromosomal breaks and translocations and were more prone to apoptosis, effects that appeared to be p53-independent, as p53 deficiency did not markedly influence these events. Importantly, NHEJ instead of alternative end-joining (A-EJ) was revealed as the predominant mechanism catalyzing robust CSR. Defective CSR was linked to failed NHEJ and residual A-EJ access to unrepaired double-strand breaks. These data firmly demonstrate that Lig4(R278H) activity renders NHEJ to be more error-prone, and they predict increased error-prone NHEJ activity and A-EJ suppression as the cause of the defective B lymphopoiesis in Lig4 patients. PMID:26608917

  17. Loss-of-function mutations in MGME1 impair mtDNA replication and cause multi-systemic mitochondrial disease

    PubMed Central

    Kornblum, Cornelia; Nicholls, Thomas J; Haack, Tobias B.; Schöler, Susanne; Peeva, Viktoriya; Danhauser, Katharina; Hallmann, Kerstin; Zsurka, Gábor; Rorbach, Joanna; Iuso, Arcangela; Wieland, Thomas; Sciacco, Monica; Ronchi, Dario; Comi, Giacomo P.; Moggio, Maurizio; Quinzii, Catarina M.; DiMauro, Salvatore; Calvo, Sarah E.; Mootha, Vamsi K.; Klopstock, Thomas; Strom, Tim M.; Meitinger, Thomas; Minczuk, Michal; Kunz, Wolfram S.; Prokisch, Holger

    2013-01-01

    Known disease mechanisms in mitochondrial DNA (mtDNA) maintenance disorders alter either the mitochondrial replication machinery (POLG1, POLG22 and C10orf23) or the biosynthesis pathways of deoxyribonucleoside 5′-triphosphates for mtDNA synthesis4–11. However, in many of these disorders, the underlying genetic defect has not yet been discovered. Here, we identified homozygous nonsense and missense mutations in the orphan gene C20orf72 in three families with a mitochondrial syndrome characterized by external ophthalmoplegia, emaciation, and respiratory failure. Muscle biopsies showed mtDNA depletion and multiple mtDNA deletions. C20orf72, hereafter MGME1 (mitochondrial genome maintenance exonuclease 1), encodes a mitochondrial RecB-type exonuclease belonging to the PD-(D/E)XK nuclease superfamily. We demonstrate that MGME1 cleaves single-stranded DNA and processes DNA flap substrates. Upon chemically induced mtDNA depletion, patient fibroblasts fail to repopulate. They also accumulate intermediates of stalled replication and show increased levels of 7S DNA, as do MGME1-depleted cells. Hence, we show that MGME1-mediated mtDNA processing is essential for mitochondrial genome maintenance. PMID:23313956

  18. Mutation of a single residue in the ba3 oxidase specifically impairs protonation of the pump site

    PubMed Central

    von Ballmoos, Christoph; Gonska, Nathalie; Lachmann, Peter; Gennis, Robert B.; Ädelroth, Pia; Brzezinski, Peter

    2015-01-01

    The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative “pump site” was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba3 cytochrome c oxidase. PMID:25733886

  19. Nonsense mutations in the COX1 subunit impair the stability of respiratory chain complexes rather than their assembly

    PubMed Central

    Hornig-Do, Hue-Tran; Tatsuta, Takashi; Buckermann, Angela; Bust, Maria; Kollberg, Gittan; Rötig, Agnes; Hellmich, Martin; Nijtmans, Leo; Wiesner, Rudolf J

    2012-01-01

    Respiratory chain (RC) complexes are organized into supercomplexes forming ‘respirasomes'. The mechanism underlying the interdependence of individual complexes is still unclear. Here, we show in human patient cells that the presence of a truncated COX1 subunit leads to destabilization of complex IV (CIV) and other RC complexes. Surprisingly, the truncated COX1 protein is integrated into subcomplexes, the holocomplex and even into supercomplexes, which however are all unstable. Depletion of the m-AAA protease AFG3L2 increases stability of the truncated COX1 and other mitochondrially encoded proteins, whereas overexpression of wild-type AFG3L2 decreases their stability. Both full-length and truncated COX1 proteins physically interact with AFG3L2. Expression of a dominant negative AFG3L2 variant also promotes stabilization of CIV proteins as well as the assembled complex and rescues the severe phenotype in heteroplasmic cells. Our data indicate that the mechanism underlying pathogenesis in these patients is the rapid clearance of unstable respiratory complexes by quality control pathways, rather than their impaired assembly. PMID:22252130

  20. A dominant mutation in the light-oxygen and voltage2 domain vicinity impairs phototropin1 signaling in tomato.

    PubMed

    Sharma, Sulabha; Kharshiing, Eros; Srinivas, Ankanagari; Zikihara, Kazunori; Tokutomi, Satoru; Nagatani, Akira; Fukayama, Hiroshi; Bodanapu, Reddaiah; Behera, Rajendra K; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2014-04-01

    In higher plants, blue light (BL) phototropism is primarily controlled by the phototropins, which are also involved in stomatal movement and chloroplast relocation. These photoresponses are mediated by two phototropins, phot1 and phot2. Phot1 mediates responses with higher sensitivity than phot2, and phot2 specifically mediates chloroplast avoidance and dark positioning responses. Here, we report the isolation and characterization of a Nonphototropic seedling1 (Nps1) mutant of tomato (Solanum lycopersicum). The mutant is impaired in low-fluence BL responses, including chloroplast accumulation and stomatal opening. Genetic analyses show that the mutant locus is dominant negative in nature. In dark-grown seedlings of the Nps1 mutant, phot1 protein accumulates at a highly reduced level relative to the wild type and lacks BL-induced autophosphorylation. The mutant harbors a single glycine-1484-to-alanine transition in the Hinge1 region of a phot1 homolog, resulting in an arginine-to-histidine substitution (R495H) in a highly conserved A'α helix proximal to the light-oxygen and voltage2 domain of the translated gene product. Significantly, the R495H substitution occurring in the Hinge1 region of PHOT1 abolishes its regulatory activity in Nps1 seedlings, thereby highlighting the functional significance of the A'α helix region in phototropic signaling of tomato. PMID:24515830

  1. Rapid mineralocorticoid receptor trafficking.

    PubMed

    Gekle, M; Bretschneider, M; Meinel, S; Ruhs, S; Grossmann, C

    2014-03-01

    The mineralocorticoid receptor (MR) is a ligand-dependent transcription factor that physiologically regulates water-electrolyte homeostasis and controls blood pressure. The MR can also elicit inflammatory and remodeling processes in the cardiovascular system and the kidneys, which require the presence of additional pathological factors like for example nitrosative stress. However, the underlying molecular mechanism(s) for pathophysiological MR effects remain(s) elusive. The inactive MR is located in the cytosol associated with chaperone molecules including HSP90. After ligand binding, the MR monomer rapidly translocates into the nucleus while still being associated to HSP90 and after dissociation from HSP90 binds to hormone-response-elements called glucocorticoid response elements (GREs) as a dimer. There are indications that rapid MR trafficking is modulated in the presence of high salt, oxidative or nitrosative stress, hypothetically by induction or posttranslational modifications. Additionally, glucocorticoids and the enzyme 11beta hydroxysteroid dehydrogenase may also influence MR activation. Because MR trafficking and its modulation by micro-milieu factors influence MR cellular localization, it is not only relevant for genomic but also for nongenomic MR effects. PMID:24252381

  2. A lysine-to-arginine mutation on NEDD8 markedly reduces the activity of cullin RING E3 ligase through the impairment of neddylation cascades

    SciTech Connect

    Sui, Yiyan; Liu, Yaobin; Xu, Guoqiang

    2015-06-12

    Neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) is a ubiquitin-like modifier, which forms covalent conjugates on lysines of its substrates. This post-translational modification, neddylation, plays important roles in tumor cell proliferation and viability. Ubiquitin can form diverse polyubiquitin chains, on its seven lysines, which play important functions in various biological processes. However, the roles of lysines in NEDD8 have not been explored. Here, we generated nine NEDD8 point mutants, each with one lysine replaced by an arginine, to study the putative function of lysines in NEDD8. Our experiments discover that Lys27 in NEDD8 is a critical residue for protein neddylation. Replacement of this residue with arginine almost completely eliminates the conjugation of NEDD8 to its substrates. Furthermore, we find that the K27R mutant impairs NEDD8 conjugation to the E2 enzyme, which normally forms thioester bonds for further transferring NEDD8 to its ligases and substrates. Therefore, this mutation completely inhibits global protein neddylation, including neddylation of cullin family proteins, resulting in decreased activity of cullin-RING E3 ligases. This work sheds new light on the roles of NEDD8 lysines on neddylation cascades and provides a dominant negative mutant for the study of neddylation and its biological functions. - Highlights: • Lys27 in NEDD8 is critical for protein neddylation. • NEDD8 K27R mutant impairs the NEDD8 conjugation. • NEDD8 K27R mutant significantly reduces the activity of cullin-RING E3 ligases.

  3. Host Resistance to Intracellular Infection: Mutation of Natural Resistance-associated Macrophage Protein 1 (Nramp1) Impairs Phagosomal Acidification

    PubMed Central

    Hackam, David J.; Rotstein, Ori D.; Zhang, Wei-jian; Gruenheid, Samantha; Gros, Philippe; Grinstein, Sergio

    1998-01-01

    The mechanisms underlying the survival of intracellular parasites such as mycobacteria in host macrophages remain poorly understood. In mice, mutations at the Nramp1 gene (for natural resistance-associated macrophage protein), cause susceptibility to mycobacterial infections. Nramp1 encodes an integral membrane protein that is recruited to the phagosome membrane in infected macrophages. In this study, we used microfluorescence ratio imaging of macrophages from wild-type and Nramp1 mutant mice to analyze the effect of loss of Nramp1 function on the properties of phagosomes containing inert particles or live mycobacteria. The pH of phagosomes containing live Mycobacterium bovis was significantly more acidic in Nramp1- expressing macrophages than in mutant cells (pH 5.5 ± 0.06 versus pH 6.6 ± 0.05, respectively; P <0.005). The enhanced acidification could not be accounted for by differences in proton consumption during dismutation of superoxide, phagosomal buffering power, counterion conductance, or in the rate of proton “leak”, as these were found to be comparable in wild-type and Nramp1-deficient macrophages. Rather, after ingestion of live mycobacteria, Nramp1-expressing cells exhibited increased concanamycin-sensitive H+ pumping across the phagosomal membrane. This was associated with an enhanced ability of phagosomes to fuse with vacuolar-type ATPase–containing late endosomes and/or lysosomes. This effect was restricted to live M. bovis and was not seen in phagosomes containing dead M. bovis or latex beads. These data support the notion that Nramp1 affects intracellular mycobacterial replication by modulating phagosomal pH, suggesting that Nramp1 plays a central role in this process. PMID:9670047

  4. Mutation of SPOTTED LEAF3 (SPL3) impairs abscisic acid-responsive signalling and delays leaf senescence in rice

    PubMed Central

    Wang, Seung-Hyun; Lim, Jung-Hyun; Kim, Sang-Sook; Cho, Sung-Hwan; Yoo, Soo-Cheul; Koh, Hee-Jong; Sakuraba, Yasuhito; Paek, Nam-Chon

    2015-01-01

    Lesion mimic mutants commonly display spontaneous cell death in pre-senescent green leaves under normal conditions, without pathogen attack. Despite molecular and phenotypic characterization of several lesion mimic mutants, the mechanisms of the spontaneous formation of cell death lesions remain largely unknown. Here, the rice lesion mimic mutant spotted leaf3 (spl3) was examined. When grown under a light/dark cycle, the spl3 mutant appeared similar to wild-type at early developmental stages, but lesions gradually appeared in the mature leaves close to heading stage. By contrast, in spl3 mutants grown under continuous light, severe cell death lesions formed in developing leaves, even at the seedling stage. Histochemical analysis showed that hydrogen peroxide accumulated in the mutant, likely causing the cell death phenotype. By map-based cloning and complementation, it was shown that a 1-bp deletion in the first exon of Oryza sativa Mitogen-Activated Protein Kinase Kinase Kinase1 (OsMAPKKK1)/OsEDR1/OsACDR1 causes the spl3 mutant phenotype. The spl3 mutant was found to be insensitive to abscisic acid (ABA), showing normal root growth in ABA-containing media and delayed leaf yellowing during dark-induced and natural senescence. Expression of ABA signalling-associated genes was also less responsive to ABA treatment in the mutant. Furthermore, the spl3 mutant had lower transcript levels and activities of catalases, which scavenge hydrogen peroxide, probably due to impairment of ABA-responsive signalling. Finally, a possible molecular mechanism of lesion formation in the mature leaves of spl3 mutant is discussed. PMID:26276867

  5. The G115S mutation associated with maturity-onset diabetes of the young impairs hepatocyte nuclear factor 4alpha activities and introduces a PKA phosphorylation site in its DNA-binding domain.

    PubMed

    Oxombre, Bénédicte; Kouach, Mostafa; Moerman, Ericka; Formstecher, Pierre; Laine, Bernard

    2004-11-01

    HNF4alpha (hepatocyte nuclear factor 4alpha) belongs to a complex transcription factor network that is crucial for the function of hepatocytes and pancreatic beta-cells. In these cells, it activates the expression of a very large number of genes, including genes involved in the transport and metabolism of glucose and lipids. Mutations in the HNF4alpha gene correlate with MODY1 (maturity-onset diabetes of the young 1), a form of type II diabetes characterized by an impaired glucose-induced insulin secretion. The MODY1 G115S (Gly115-->Ser) HNF4alpha mutation is located in the DNA-binding domain of this nuclear receptor. We show here that the G115S mutation failed to affect HNF4alpha-mediated transcription on apolipoprotein promoters in HepG2 cells. Conversely, in pancreatic beta-cell lines, this mutation resulted in strong impairments of HNF4alpha transcriptional activity on the promoters of LPK (liver pyruvate kinase) and HNF1alpha, with this transcription factor playing a key role in endocrine pancreas. We show as well that the G115S mutation creates a PKA (protein kinase A) phosphorylation site, and that PKA-mediated phosphorylation results in a decreased transcriptional activity of the mutant. Moreover, the G115E (Gly115-->Glu) mutation mimicking phosphorylation reduced HNF4alpha DNA-binding and transcriptional activities. Our results may account for the 100% penetrance of diabetes in human carriers of this mutation. In addition, they suggest that introduction of a phosphorylation site in the DNA-binding domain may represent a new mechanism by which a MODY1 mutation leads to loss of HNF4alpha function. PMID:15233628

  6. The G115S mutation associated with maturity-onset diabetes of the young impairs hepatocyte nuclear factor 4α activities and introduces a PKA phosphorylation site in its DNA-binding domain

    PubMed Central

    2004-01-01

    HNF4α (hepatocyte nuclear factor 4α) belongs to a complex transcription factor network that is crucial for the function of hepatocytes and pancreatic β-cells. In these cells, it activates the expression of a very large number of genes, including genes involved in the transport and metabolism of glucose and lipids. Mutations in the HNF4α gene correlate with MODY1 (maturity-onset diabetes of the young 1), a form of type II diabetes characterized by an impaired glucose-induced insulin secretion. The MODY1 G115S (Gly115→Ser) HNF4α mutation is located in the DNA-binding domain of this nuclear receptor. We show here that the G115S mutation failed to affect HNF4α-mediated transcription on apolipoprotein promoters in HepG2 cells. Conversely, in pancreatic β-cell lines, this mutation resulted in strong impairments of HNF4α transcriptional activity on the promoters of LPK (liver pyruvate kinase) and HNF1α, with this transcription factor playing a key role in endocrine pancreas. We show as well that the G115S mutation creates a PKA (protein kinase A) phosphorylation site, and that PKA-mediated phosphorylation results in a decreased transcriptional activity of the mutant. Moreover, the G115E (Gly115→Glu) mutation mimicking phosphorylation reduced HNF4α DNA-binding and transcriptional activities. Our results may account for the 100% penetrance of diabetes in human carriers of this mutation. In addition, they suggest that introduction of a phosphorylation site in the DNA-binding domain may represent a new mechanism by which a MODY1 mutation leads to loss of HNF4α function. PMID:15233628

  7. β-III spectrin underpins ankyrin R function in Purkinje cell dendritic trees: protein complex critical for sodium channel activity is impaired by SCA5-associated mutations.

    PubMed

    Clarkson, Yvonne L; Perkins, Emma M; Cairncross, Callum J; Lyndon, Alastair R; Skehel, Paul A; Jackson, Mandy

    2014-07-15

    Beta III spectrin is present throughout the elaborate dendritic tree of cerebellar Purkinje cells and is required for normal neuronal morphology and cell survival. Spinocerebellar ataxia type 5 (SCA5) and spectrin associated autosomal recessive cerebellar ataxia type 1 are human neurodegenerative diseases involving progressive gait ataxia and cerebellar atrophy. Both disorders appear to result from loss of β-III spectrin function. Further elucidation of β-III spectrin function is therefore needed to understand disease mechanisms and identify potential therapeutic options. Here, we report that β-III spectrin is essential for the recruitment and maintenance of ankyrin R at the plasma membrane of Purkinje cell dendrites. Two SCA5-associated mutations of β-III spectrin both reduce ankyrin R levels at the cell membrane. Moreover, a wild-type β-III spectrin/ankyrin-R complex increases sodium channel levels and activity in cell culture, whereas mutant β-III spectrin complexes fail to enhance sodium currents. This suggests impaired ability to form stable complexes between the adaptor protein ankyrin R and its interacting partners in the Purkinje cell dendritic tree is a key mechanism by which mutant forms of β-III spectrin cause ataxia, initially by Purkinje cell dysfunction and exacerbated by subsequent cell death. PMID:24603075

  8. 31 CFR 536.311 - Narcotics trafficking.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Narcotics trafficking. 536.311 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any activity...

  9. 31 CFR 536.311 - Narcotics trafficking.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false Narcotics trafficking. 536.311 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any activity...

  10. 31 CFR 536.311 - Narcotics trafficking.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 31 Money and Finance: Treasury 3 2010-07-01 2010-07-01 false Narcotics trafficking. 536.311... OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any...

  11. 31 CFR 536.311 - Narcotics trafficking.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false Narcotics trafficking. 536.311 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any activity...

  12. 31 CFR 536.311 - Narcotics trafficking.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false Narcotics trafficking. 536.311 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any activity...

  13. Women trafficking: causes, concerns, care!

    PubMed

    Khowaja, Shaneela Sadaruddin; Tharani, Ambreen Jawed; Agha, Ajmal; Karamaliani, Rozina Sherali

    2012-08-01

    Pakistan is both a country of origin and destination as far as women trafficking is concerned. Poverty, gender discrimination, lack of education, and ignorance about legal rights are some of the underlying causes. Available data suggest several areas of concern, like, for instance: direct health effects, maladaptive coping leading to the use of illicit drugs, and inaccessibility to healthcare facilities. Therefore, numerous interventions would be required at three levels: the prevention of trafficking, the protection of victims and the prosecution of the traffickers. PMID:23862261

  14. Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons.

    PubMed

    Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, C L; Kaul, M; Singh, K K

    2014-09-01

    Human immunodeficiency virus-1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons. PMID:24825317

  15. Intracellular Mannose Binding Lectin Mediates Subcellular Trafficking of HIV-1 gp120 in Neurons

    PubMed Central

    Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, CL; Kaul, M; Singh, KK

    2014-01-01

    Human immunodeficiency virus -1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons. PMID:24825317

  16. A novel recurrent CHEK2 Y390C mutation identified in high-risk Chinese breast cancer patients impairs its activity and is associated with increased breast cancer risk.

    PubMed

    Wang, N; Ding, H; Liu, C; Li, X; Wei, L; Yu, J; Liu, M; Ying, M; Gao, W; Jiang, H; Wang, Y

    2015-10-01

    Certain predisposition factors such as BRCA1/2 and CHEK2 mutations cause familial breast cancers that occur early. In China, breast cancers are diagnosed at relatively younger age, and higher percentage of patients are diagnosed before 40 years, than that in Caucasians. However, the prevalence for BRCA1/2 mutations and reported CHEK2 germline mutations is much lower or absent in Chinese population, arguing for the need to study other novel risk alleles among Chinese breast cancer patients. In this study, we searched for CHEK2 mutations in young, high-risk breast cancer patients in China and detected a missense variant Y390C (1169A > G) in 12 of 150 patients (8.0%) and 2 in 250 healthy controls (0.8%, P = 0.0002). Four of the Y390C carriers have family history of breast and/or ovarian cancer. In patients without family history, Y390C carriers tend to develop breast cancer early, before 35 years of age. The codon change at Y390, a highly conserved residue located in CHEK2's kinase domain, appeared to significantly impair CHEK2 activity. Functional analysis suggested that the CHEK2 Y390C mutation is deleterious as judged by the mutant protein's inability to inactivate CDC25A or to activate p53 after DNA damage. Cells expressing the CHEK2 Y390C variant showed impaired p21 and Puma expression after DNA damage, and the deregulated cell cycle checkpoint and apoptotic response may help conserve mutations and therefore contribute to tumorigeneisis. Taken together, our results not only identified a novel CHEK2 allele that is associated with cancer families and confers increased breast cancer risk, but also showed that this allele significantly impairs CHEK2 function during DNA damage response. Our results provide further insight on how the function of such an important cancer gene may be impaired by existing mutations to facilitate tumorigenesis. It also offers a new subject for breast cancer monitoring, prevention and management. PMID:25619829

  17. Illicit Trafficking of Natural Radionuclides

    NASA Astrophysics Data System (ADS)

    Friedrich, Steinhäusler; Lyudmila, Zaitseva

    2008-08-01

    Natural radionuclides have been subject to trafficking worldwide, involving natural uranium ore (U 238), processed uranium (yellow cake), low enriched uranium (<20% U 235) or highly enriched uranium (>20% U 235), radium (Ra 226), polonium (Po 210), and natural thorium ore (Th 232). An important prerequisite to successful illicit trafficking activities is access to a suitable logistical infrastructure enabling an undercover shipment of radioactive materials and, in case of trafficking natural uranium or thorium ore, capable of transporting large volumes of material. Covert en route diversion of an authorised uranium transport, together with covert diversion of uranium concentrate from an operating or closed uranium mines or mills, are subject of case studies. Such cases, involving Israel, Iran, Pakistan and Libya, have been analyzed in terms of international actors involved and methods deployed. Using international incident data contained in the Database on Nuclear Smuggling, Theft and Orphan Radiation Sources (DSTO) and international experience gained from the fight against drug trafficking, a generic Trafficking Pathway Model (TPM) is developed for trafficking of natural radionuclides. The TPM covers the complete trafficking cycle, ranging from material diversion, covert material transport, material concealment, and all associated operational procedures. The model subdivides the trafficking cycle into five phases: (1) Material diversion by insider(s) or initiation by outsider(s); (2) Covert transport; (3) Material brokerage; (4) Material sale; (5) Material delivery. An Action Plan is recommended, addressing the strengthening of the national infrastructure for material protection and accounting, development of higher standards of good governance, and needs for improving the control system deployed by customs, border guards and security forces.

  18. Illicit Trafficking of Natural Radionuclides

    SciTech Connect

    Friedrich, Steinhaeusler; Lyudmila, Zaitseva

    2008-08-07

    Natural radionuclides have been subject to trafficking worldwide, involving natural uranium ore (U 238), processed uranium (yellow cake), low enriched uranium (<20% U 235) or highly enriched uranium (>20% U 235), radium (Ra 226), polonium (Po 210), and natural thorium ore (Th 232). An important prerequisite to successful illicit trafficking activities is access to a suitable logistical infrastructure enabling an undercover shipment of radioactive materials and, in case of trafficking natural uranium or thorium ore, capable of transporting large volumes of material. Covert en route diversion of an authorised uranium transport, together with covert diversion of uranium concentrate from an operating or closed uranium mines or mills, are subject of case studies. Such cases, involving Israel, Iran, Pakistan and Libya, have been analyzed in terms of international actors involved and methods deployed. Using international incident data contained in the Database on Nuclear Smuggling, Theft and Orphan Radiation Sources (DSTO) and international experience gained from the fight against drug trafficking, a generic Trafficking Pathway Model (TPM) is developed for trafficking of natural radionuclides. The TPM covers the complete trafficking cycle, ranging from material diversion, covert material transport, material concealment, and all associated operational procedures. The model subdivides the trafficking cycle into five phases: (1) Material diversion by insider(s) or initiation by outsider(s); (2) Covert transport; (3) Material brokerage; (4) Material sale; (5) Material delivery. An Action Plan is recommended, addressing the strengthening of the national infrastructure for material protection and accounting, development of higher standards of good governance, and needs for improving the control system deployed by customs, border guards and security forces.

  19. ENU-mutagenesis mice with a non-synonymous mutation in Grin1 exhibit abnormal anxiety-like behaviors, impaired fear memory, and decreased acoustic startle response

    PubMed Central

    2013-01-01

    Background The Grin1 (glutamate receptor, ionotropic, NMDA1) gene expresses a subunit of N-methyl-D-aspartate (NMDA) receptors that is considered to play an important role in excitatory neurotransmission, synaptic plasticity, and brain development. Grin1 is a candidate susceptibility gene for neuropsychiatric disorders, including schizophrenia, bipolar disorder, and attention deficit/hyperactivity disorder (ADHD). In our previous study, we examined an N-ethyl-N-nitrosourea (ENU)-generated mutant mouse strain (Grin1Rgsc174/Grin1+) that has a non-synonymous mutation in Grin1. These mutant mice showed hyperactivity, increased novelty-seeking to objects, and abnormal social interactions. Therefore, Grin1Rgsc174/Grin1+ mice may serve as a potential animal model of neuropsychiatric disorders. However, other behavioral characteristics related to these disorders, such as working memory function and sensorimotor gating, have not been fully explored in these mutant mice. In this study, to further investigate the behavioral phenotypes of Grin1Rgsc174/Grin1+ mice, we subjected them to a comprehensive battery of behavioral tests. Results There was no significant difference in nociception between Grin1Rgsc174/Grin1+ and wild-type mice. The mutants did not display any abnormalities in the Porsolt forced swim and tail suspension tests. We confirmed the previous observations that the locomotor activity of these mutant mice increased in the open field and home cage activity tests. They displayed abnormal anxiety-like behaviors in the light/dark transition and the elevated plus maze tests. Both contextual and cued fear memory were severely deficient in the fear conditioning test. The mutant mice exhibited slightly impaired working memory in the eight-arm radial maze test. The startle amplitude was markedly decreased in Grin1Rgsc174/Grin1+ mice, whereas no significant differences between genotypes were detected in the prepulse inhibition (PPI) test. The mutant mice showed no obvious

  20. Sex trafficking in South Asia.

    PubMed

    Huda, S

    2006-09-01

    Economic and social inequalities and political conflicts have led to the movement of persons within each country and across the borders in South Asia. Globalization has encouraged free mobility of capital, technology, experts and sex tourism. Illiteracy, dependency, violence, social stigma, cultural stereotypes, gender disparity and endemic poverty, among other factors, place women and children in powerless, non-negotiable situations that have contributed to the emergence and breeding of the cavernous problem of sex trafficking in the entire region. This alarming spread of sex trafficking has fuelled the spread of HIV infection in South Asia, posing a unique and serious threat to community health, poverty alleviation and other crucial aspects of human development. Although the SAARC (South Asian Association for Regional Cooperation) Convention on Trafficking in Women and Children has been an important breakthrough, most of the countries in the region do not have anti-trafficking legislation or means to protect the victims. Countries of the region should make a concerted effort to treat trafficking victims as "victims" of human rights violations in all anti-trafficking strategies and actions. PMID:16846602

  1. Characterization of the Role of a Highly Conserved Sequence in ATP Binding Cassette Transporter G (ABCG) Family in ABCG1 Stability, Oligomerization, and Trafficking

    PubMed Central

    2013-01-01

    ATP-binding cassette transporter G1 (ABCG1) mediates cholesterol and oxysterol efflux onto lipidated lipoproteins and plays an important role in macrophage reverse cholesterol transport. Here, we identified a highly conserved sequence present in the five ABCG transporter family members. The conserved sequence is located between the nucleotide binding domain and the transmembrane domain and contains five amino acid residues from Asn at position 316 to Phe at position 320 in ABCG1 (NPADF). We found that cells expressing mutant ABCG1, in which Asn316, Pro317, Asp319, and Phe320 in the conserved sequence were replaced with Ala simultaneously, showed impaired cholesterol efflux activity compared with wild type ABCG1-expressing cells. A more detailed mutagenesis study revealed that mutation of Asn316 or Phe 320 to Ala significantly reduced cellular cholesterol and 7-ketocholesterol efflux conferred by ABCG1, whereas replacement of Pro317 or Asp319 with Ala had no detectable effect. To confirm the important role of Asn316 and Phe320, we mutated Asn316 to Asp (N316D) and Gln (N316Q), and Phe320 to Ile (F320I) and Tyr (F320Y). The mutant F320Y showed the same phenotype as wild type ABCG1. However, the efflux of cholesterol and 7-ketocholesterol was reduced in cells expressing ABCG1 mutant N316D, N316Q, or F320I compared with wild type ABCG1. Further, mutations N316Q and F320I impaired ABCG1 trafficking while having no marked effect on the stability and oligomerization of ABCG1. The mutant N316Q and F320I could not be transported to the cell surface efficiently. Instead, the mutant proteins were mainly localized intracellularly. Thus, these findings indicate that the two highly conserved amino acid residues, Asn and Phe, play an important role in ABCG1-dependent export of cellular cholesterol, mainly through the regulation of ABCG1 trafficking. PMID:24320932

  2. To discuss illicit nuclear trafficking

    SciTech Connect

    Balatsky, Galya I; Severe, William R; Wallace, Richard K

    2010-01-01

    The Illicit nuclear trafficking panel was conducted at the 4th Annual INMM workshop on Reducing the Risk from Radioactive and Nuclear Materials on February 2-3, 2010 in Washington DC. While the workshop occurred prior to the Nuclear Security Summit, April 12-13 2010 in Washington DC, some of the summit issues were raised during the workshop. The Communique of the Washington Nuclear Security Summit stated that 'Nuclear terrorism is one of the most challenging threats to international security, and strong nuclear security measures are the most effective means to prevent terrorists, criminals, or other unauthorized actors from acquiring nuclear materials.' The Illicit Trafficking panel is one means to strengthen nuclear security and cooperation at bilateral, regional and multilateral levels. Such a panel promotes nuclear security culture through technology development, human resources development, education and training. It is a tool which stresses the importance of international cooperation and coordination of assistance to improve efforts to prevent and respond to incidents of illicit nuclear trafficking. Illicit trafficking panel included representatives from US government, an international organization (IAEA), private industry and a non-governmental organization to discuss illicit nuclear trafficking issues. The focus of discussions was on best practices and challenges for addressing illicit nuclear trafficking. Terrorism connection. Workshop discussions pointed out the identification of terrorist connections with several trafficking incidents. Several trafficking cases involved real buyers (as opposed to undercover law enforcement agents) and there have been reports identifying individuals associated with terrorist organizations as prospective plutonium buyers. Some specific groups have been identified that consistently search for materials to buy on the black market, but no criminal groups were identified that specialize in nuclear materials or isotope smuggling

  3. Insulin Granule Biogenesis, Trafficking and Exocytosis

    PubMed Central

    Hou, June Chunqiu; Min, Le; Pessin, Jeffrey E.

    2015-01-01

    It is becoming increasingly apparent that beta cell dysfunction resulting in abnormal insulin secretion is the essential element in the progression of patients from a state of impaired glucose tolerance to frank type 2 diabetes (Del Prato, 2003; Del Prato and Tiengo, 2001). Although extensive studies have examined the molecular, cellular and physiologic mechanisms of insulin granule biogenesis, sorting, and exocytosis the precise mechanisms controlling these processes and their dysregulation in the developed of diabetes remains an area of important investigation. We now know that insulin biogenesis initiates with the synthesis of preproinsulin in rough endoplastic reticulum and conversion of preproinsulin to proinsulin. Proinsulin begins to be packaged in the Trans-Golgi Network and is sorting into immature secretory granules. These immature granules become acidic via ATP-dependent proton pump and proinsulin undergoes proteolytic cleavage resulting the formation of insulin and C-peptide. During the granule maturation process, insulin is crystallized with zinc and calcium in the form of dense-core granules and unwanted cargo and membrane proteins undergo selective retrograde trafficking to either the constitutive trafficking pathway for secretion or to degradative pathways. The newly formed mature dense-core insulin granules populate two different intracellular pools, the readily releasable pools (RRP) and the reserved pool. These two distinct populations are thought to be responsible for the biphasic nature of insulin release in which the RRP granules are associated with the plasma membrane and undergo an acute calcium-dependent release accounting for first phase insulin secretion. In contrast, second phase insulin secretion requires the trafficking of the reserved granule pool to the plasma membrane. The initial trigger for insulin granule fusion with the plasma membrane is a rise in intracellular calcium and in the case of glucose stimulation results from

  4. Mutation for Nonsyndromic Mental Retardation in the trans-2-Enoyl-CoA Reductase TER Gene Involved in Fatty Acid Elongation Impairs the Enzyme Activity and Stability, Leading to Change in Sphingolipid Profile*

    PubMed Central

    Abe, Kensuke; Ohno, Yusuke; Sassa, Takayuki; Taguchi, Ryo; Çalışkan, Minal; Ober, Carole; Kihara, Akio

    2013-01-01

    Very long-chain fatty acids (VLCFAs, chain length >C20) exist in tissues throughout the body and are synthesized by repetition of the fatty acid (FA) elongation cycle composed of four successive enzymatic reactions. In mammals, the TER gene is the only gene encoding trans-2-enoyl-CoA reductase, which catalyzes the fourth reaction in the FA elongation cycle. The TER P182L mutation is the pathogenic mutation for nonsyndromic mental retardation. This mutation substitutes a leucine for a proline residue at amino acid 182 in the TER enzyme. Currently, the mechanism by which the TER P182L mutation causes nonsyndromic mental retardation is unknown. To understand the effect of this mutation on the TER enzyme and VLCFA synthesis, we have biochemically characterized the TER P182L mutant enzyme using yeast and mammalian cells transfected with the TER P182L mutant gene and analyzed the FA elongation cycle in the B-lymphoblastoid cell line with the homozygous TER P182L mutation (TERP182L/P182L B-lymphoblastoid cell line). We have found that TER P182L mutant enzyme exhibits reduced trans-2-enoyl-CoA reductase activity and protein stability, thereby impairing VLCFA synthesis and, in turn, altering the sphingolipid profile (i.e. decreased level of C24 sphingomyelin and C24 ceramide) in the TERP182L/P182L B-lymphoblastoid cell line. We have also found that in addition to the TER enzyme-catalyzed fourth reaction, the third reaction in the FA elongation cycle is affected by the TER P182L mutation. These findings provide new insight into the biochemical defects associated with this genetic mutation. PMID:24220030

  5. Impaired Mitochondrial Energy Production Causes Light-Induced Photoreceptor Degeneration Independent of Oxidative Stress.

    PubMed

    Jaiswal, Manish; Haelterman, Nele A; Sandoval, Hector; Xiong, Bo; Donti, Taraka; Kalsotra, Auinash; Yamamoto, Shinya; Cooper, Thomas A; Graham, Brett H; Bellen, Hugo J

    2015-07-01

    Two insults often underlie a variety of eye diseases including glaucoma, optic atrophy, and retinal degeneration--defects in mitochondrial function and aberrant Rhodopsin trafficking. Although mitochondrial defects are often associated with oxidative stress, they have not been linked to Rhodopsin trafficking. In an unbiased forward genetic screen designed to isolate mutations that cause photoreceptor degeneration, we identified mutations in a nuclear-encoded mitochondrial gene, ppr, a homolog of human LRPPRC. We found that ppr is required for protection against light-induced degeneration. Its function is essential to maintain membrane depolarization of the photoreceptors upon repetitive light exposure, and an impaired phototransduction cascade in ppr mutants results in excessive Rhodopsin1 endocytosis. Moreover, loss of ppr results in a reduction in mitochondrial RNAs, reduced electron transport chain activity, and reduced ATP levels. Oxidative stress, however, is not induced. We propose that the reduced ATP level in ppr mutants underlies the phototransduction defect, leading to increased Rhodopsin1 endocytosis during light exposure, causing photoreceptor degeneration independent of oxidative stress. This hypothesis is bolstered by characterization of two other genes isolated in the screen, pyruvate dehydrogenase and citrate synthase. Their loss also causes a light-induced degeneration, excessive Rhodopsin1 endocytosis and reduced ATP without concurrent oxidative stress, unlike many other mutations in mitochondrial genes that are associated with elevated oxidative stress and light-independent photoreceptor demise. PMID:26176594

  6. Impaired Mitochondrial Energy Production Causes Light-Induced Photoreceptor Degeneration Independent of Oxidative Stress

    PubMed Central

    Jaiswal, Manish; Haelterman, Nele A.; Sandoval, Hector; Xiong, Bo; Donti, Taraka; Kalsotra, Auinash; Yamamoto, Shinya; Cooper, Thomas A.; Graham, Brett H.; Bellen, Hugo J.

    2015-01-01

    Two insults often underlie a variety of eye diseases including glaucoma, optic atrophy, and retinal degeneration—defects in mitochondrial function and aberrant Rhodopsin trafficking. Although mitochondrial defects are often associated with oxidative stress, they have not been linked to Rhodopsin trafficking. In an unbiased forward genetic screen designed to isolate mutations that cause photoreceptor degeneration, we identified mutations in a nuclear-encoded mitochondrial gene, ppr, a homolog of human LRPPRC. We found that ppr is required for protection against light-induced degeneration. Its function is essential to maintain membrane depolarization of the photoreceptors upon repetitive light exposure, and an impaired phototransduction cascade in ppr mutants results in excessive Rhodopsin1 endocytosis. Moreover, loss of ppr results in a reduction in mitochondrial RNAs, reduced electron transport chain activity, and reduced ATP levels. Oxidative stress, however, is not induced. We propose that the reduced ATP level in ppr mutants underlies the phototransduction defect, leading to increased Rhodopsin1 endocytosis during light exposure, causing photoreceptor degeneration independent of oxidative stress. This hypothesis is bolstered by characterization of two other genes isolated in the screen, pyruvate dehydrogenase and citrate synthase. Their loss also causes a light-induced degeneration, excessive Rhodopsin1 endocytosis and reduced ATP without concurrent oxidative stress, unlike many other mutations in mitochondrial genes that are associated with elevated oxidative stress and light-independent photoreceptor demise. PMID:26176594

  7. A Novel Domain-Specific Mutation in a Sclerosteosis Patient Suggests a Role of LRP4 as an Anchor for Sclerostin in Human Bone.

    PubMed

    Fijalkowski, Igor; Geets, Ellen; Steenackers, Ellen; Van Hoof, Viviane; Ramos, Feliciano J; Mortier, Geert; Fortuna, Ana Maria; Van Hul, Wim; Boudin, Eveline

    2016-04-01

    Mutations in the LRP4 gene, coding for a Wnt signaling coreceptor, have been found to cause several allelic conditions. Among these, two are characterized by a strong skeletal involvement, namely sclerosteosis and Cenani-Lenz syndrome. In this work, we evaluated the role of LRP4 in the pathophysiology of these diseases. First, we report a novel LRP4 mutation, leading to the substitution of arginine at position 1170 in glutamine, identified in a patient with sclerosteosis. This mutation is located in the central cavity of the third β-propeller domain, which is in line with two other sclerosteosis mutations we previously described. Reporter assays demonstrate that this mutation leads to impaired sclerostin inhibition of Wnt signaling. Moreover, we compared the effect of this novel variant to mutations causing Cenani-Lenz syndrome and show that impaired membrane trafficking of the LRP4 protein is the likely mechanism underlying Cenani-Lenz syndrome. This is in contrast to sclerosteosis mutations, previously shown to impair the binding between LRP4 and sclerostin. In addition, to better understand the biology of LRP4, we investigated the circulating sclerostin levels in the serum of a patient suffering from sclerosteosis owing to a LRP4 mutation. We demonstrate that impaired sclerostin binding to the mutated LRP4 protein leads to dramatic increase in circulating sclerostin in this patient. With this study, we provide the first evidence suggesting that LRP4 is responsible for the retention of sclerostin in the bone environment in humans. These findings raise potential concerns about the utility of determining circulating sclerostin levels as a marker for other bone-related parameters. Although more studies are needed to fully understand the mechanism whereby LRP4 facilitates sclerostin action, it is clear that this protein represents a potent target for future osteoporosis therapies and an interesting alternative for the antisclerostin treatment currently under study

  8. [Progress in research on defective protein trafficking and functional restoration in HERG-associated long QT syndrome].

    PubMed

    Fang, Peiliang; Lian, Jiangfang

    2016-02-01

    The human ether-a-go-go related gene (HERG) encodes the α -subunit of the rapid component of the delayed rectifier K(+) channel, which is essential for the third repolarization of the action potential of human myocardial cells. Mutations of the HERG gene can cause type II hereditary long QT syndrome (LQT2), characterized by prolongation of the QT interval, abnormal T wave, torsade de pointes, syncope and sudden cardiac death. So far more than 300 HERG mutations have been identified, the majority of which can cause LQT2 due to HERG protein trafficking defect. It has been reported that certain drugs can induce acquired long QT syndrome through directly blocking the pore and/or affecting the HERG trafficking. The trafficking defects and K(+) currents can be restored with low temperature and certain drugs. However, the mechanisms underlying defective trafficking caused by HERG mutations and the inhibition/restoration of HERG trafficking by drugs are still unknown. This review summarizes the current understanding of the molecular mechanisms including HERG trafficking under physiological and pathological conditions, and the effects of drugs on the HERG trafficking, in order to provide theoretical evidence for the diagnosis and treatment of long QT syndrome. PMID:26829745

  9. Trafficking in persons: a health concern?

    PubMed

    Zimmerman, Cathy; Kiss, Ligia; Houssain, Mazeda; Watts, Charlotte

    2009-01-01

    Human trafficking is a phenomenon that has now been documented in most regions in the world. Although trafficking of women and girls for sexual exploitation is the most commonly recognised form of trafficking, it is widely acknowledged that human trafficking also involves men, women and children who are trafficked for various forms of labour exploitation and into other abusive circumstances. Despite the violence and harm inherent in most trafficking situations, there remains extremely little evidence on the individual and public health implications of any form of human trafficking. The Brazilian government has recently launched a national plan to combat human trafficking. However, because the health risks associated with human trafficking have not been well-recognised or documented, there is extremely limited reliable data on the health needs of trafficked persons to inform policy and practices.. Brazilian policy-makers and service providers should be encouraged to learn about the likely range of health impacts of trafficking, and incorporate this into anti-trafficking protection and response strategies. As well as prevention activities, the government, international and local organisations should work together with the public health research community to study the health needs of trafficked persons and explore opportunities to provide safe and appropriate services to victims in need of care. PMID:19721944

  10. POPDC1(S201F) causes muscular dystrophy and arrhythmia by affecting protein trafficking.

    PubMed

    Schindler, Roland F R; Scotton, Chiara; Zhang, Jianguo; Passarelli, Chiara; Ortiz-Bonnin, Beatriz; Simrick, Subreena; Schwerte, Thorsten; Poon, Kar-Lai; Fang, Mingyan; Rinné, Susanne; Froese, Alexander; Nikolaev, Viacheslav O; Grunert, Christiane; Müller, Thomas; Tasca, Giorgio; Sarathchandra, Padmini; Drago, Fabrizio; Dallapiccola, Bruno; Rapezzi, Claudio; Arbustini, Eloisa; Di Raimo, Francesca Romana; Neri, Marcella; Selvatici, Rita; Gualandi, Francesca; Fattori, Fabiana; Pietrangelo, Antonello; Li, Wenyan; Jiang, Hui; Xu, Xun; Bertini, Enrico; Decher, Niels; Wang, Jun; Brand, Thomas; Ferlini, Alessandra

    2016-01-01

    The Popeye domain-containing 1 (POPDC1) gene encodes a plasma membrane-localized cAMP-binding protein that is abundantly expressed in striated muscle. In animal models, POPDC1 is an essential regulator of structure and function of cardiac and skeletal muscle; however, POPDC1 mutations have not been associated with human cardiac and muscular diseases. Here, we have described a homozygous missense variant (c.602C>T, p.S201F) in POPDC1, identified by whole-exome sequencing, in a family of 4 with cardiac arrhythmia and limb-girdle muscular dystrophy (LGMD). This allele was absent in known databases and segregated with the pathological phenotype in this family. We did not find the allele in a further screen of 104 patients with a similar phenotype, suggesting this mutation to be family specific. Compared with WT protein, POPDC1(S201F) displayed a 50% reduction in cAMP affinity, and in skeletal muscle from patients, both POPDC1(S201F) and WT POPDC2 displayed impaired membrane trafficking. Forced expression of POPDC1(S201F) in a murine cardiac muscle cell line (HL-1) increased hyperpolarization and upstroke velocity of the action potential. In zebrafish, expression of the homologous mutation (popdc1(S191F)) caused heart and skeletal muscle phenotypes that resembled those observed in patients. Our study therefore identifies POPDC1 as a disease gene causing a very rare autosomal recessive cardiac arrhythmia and LGMD, expanding the genetic causes of this heterogeneous group of inherited rare diseases. PMID:26642364

  11. POPDC1S201F causes muscular dystrophy and arrhythmia by affecting protein trafficking

    PubMed Central

    Schindler, Roland F.R.; Scotton, Chiara; Zhang, Jianguo; Passarelli, Chiara; Ortiz-Bonnin, Beatriz; Simrick, Subreena; Schwerte, Thorsten; Poon, Kar-Lai; Fang, Mingyan; Rinné, Susanne; Froese, Alexander; Nikolaev, Viacheslav O.; Grunert, Christiane; Müller, Thomas; Tasca, Giorgio; Sarathchandra, Padmini; Drago, Fabrizio; Dallapiccola, Bruno; Rapezzi, Claudio; Arbustini, Eloisa; Di Raimo, Francesca Romana; Neri, Marcella; Selvatici, Rita; Gualandi, Francesca; Fattori, Fabiana; Pietrangelo, Antonello; Li, Wenyan; Jiang, Hui; Xu, Xun; Bertini, Enrico; Decher, Niels; Wang, Jun; Brand, Thomas; Ferlini, Alessandra

    2015-01-01

    The Popeye domain–containing 1 (POPDC1) gene encodes a plasma membrane–localized cAMP-binding protein that is abundantly expressed in striated muscle. In animal models, POPDC1 is an essential regulator of structure and function of cardiac and skeletal muscle; however, POPDC1 mutations have not been associated with human cardiac and muscular diseases. Here, we have described a homozygous missense variant (c.602C>T, p.S201F) in POPDC1, identified by whole-exome sequencing, in a family of 4 with cardiac arrhythmia and limb-girdle muscular dystrophy (LGMD). This allele was absent in known databases and segregated with the pathological phenotype in this family. We did not find the allele in a further screen of 104 patients with a similar phenotype, suggesting this mutation to be family specific. Compared with WT protein, POPDC1S201F displayed a 50% reduction in cAMP affinity, and in skeletal muscle from patients, both POPDC1S201F and WT POPDC2 displayed impaired membrane trafficking. Forced expression of POPDC1S201F in a murine cardiac muscle cell line (HL-1) increased hyperpolarization and upstroke velocity of the action potential. In zebrafish, expression of the homologous mutation (popdc1S191F) caused heart and skeletal muscle phenotypes that resembled those observed in patients. Our study therefore identifies POPDC1 as a disease gene causing a very rare autosomal recessive cardiac arrhythmia and LGMD, expanding the genetic causes of this heterogeneous group of inherited rare diseases. PMID:26642364

  12. The golgin GMAP-210 is required for efficient membrane trafficking in the early secretory pathway

    PubMed Central

    Roboti, Peristera; Sato, Keisuke; Lowe, Martin

    2015-01-01

    Golgins are coiled-coil proteins that participate in membrane-tethering events at the Golgi complex. Golgin-mediated tethering is thought to be important for vesicular trafficking and Golgi organization. However, the degree to which individual golgins contribute to these processes is poorly defined, and it has been proposed that golgins act in a largely redundant manner. Previous studies on the golgin GMAP-210 (also known as TRIP11), which is mutated in the rare skeletal disorder achondrogenesis type 1A, have yielded conflicting results regarding its involvement in trafficking. Here, we re-investigated the trafficking role of GMAP-210, and found that it is indeed required for efficient trafficking in the secretory pathway. GMAP-210 acts at both the endoplasmic reticulum (ER)-to-Golgi intermediate compartment (ERGIC) and Golgi complex during anterograde trafficking, and is also required for retrograde trafficking to the ER. Using co-depletion experiments, we also found that GMAP-210 acts in a partially redundant manner with the golgin GM130 to ensure efficient anterograde cargo delivery to the cis-Golgi. In summary, our results indicate a role for GMAP-210 in several trafficking steps at the ER–Golgi interface, some of which are partially redundant with another golgin, namely GM130 (also known as GOLGA2). PMID:25717001

  13. The golgin GMAP-210 is required for efficient membrane trafficking in the early secretory pathway.

    PubMed

    Roboti, Peristera; Sato, Keisuke; Lowe, Martin

    2015-04-15

    Golgins are coiled-coil proteins that participate in membrane-tethering events at the Golgi complex. Golgin-mediated tethering is thought to be important for vesicular trafficking and Golgi organization. However, the degree to which individual golgins contribute to these processes is poorly defined, and it has been proposed that golgins act in a largely redundant manner. Previous studies on the golgin GMAP-210 (also known as TRIP11), which is mutated in the rare skeletal disorder achondrogenesis type 1A, have yielded conflicting results regarding its involvement in trafficking. Here, we re-investigated the trafficking role of GMAP-210, and found that it is indeed required for efficient trafficking in the secretory pathway. GMAP-210 acts at both the endoplasmic reticulum (ER)-to-Golgi intermediate compartment (ERGIC) and Golgi complex during anterograde trafficking, and is also required for retrograde trafficking to the ER. Using co-depletion experiments, we also found that GMAP-210 acts in a partially redundant manner with the golgin GM130 to ensure efficient anterograde cargo delivery to the cis-Golgi. In summary, our results indicate a role for GMAP-210 in several trafficking steps at the ER-Golgi interface, some of which are partially redundant with another golgin, namely GM130 (also known as GOLGA2). PMID:25717001

  14. ECHIDNA-mediated post-Golgi trafficking of auxin carriers for differential cell elongation.

    PubMed

    Boutté, Yohann; Jonsson, Kristoffer; McFarlane, Heather E; Johnson, Errin; Gendre, Delphine; Swarup, Ranjan; Friml, Jirí; Samuels, Lacey; Robert, Stéphanie; Bhalerao, Rishikesh P

    2013-10-01

    The plant hormone indole-acetic acid (auxin) is essential for many aspects of plant development. Auxin-mediated growth regulation typically involves the establishment of an auxin concentration gradient mediated by polarly localized auxin transporters. The localization of auxin carriers and their amount at the plasma membrane are controlled by membrane trafficking processes such as secretion, endocytosis, and recycling. In contrast to endocytosis or recycling, how the secretory pathway mediates the localization of auxin carriers is not well understood. In this study we have used the differential cell elongation process during apical hook development to elucidate the mechanisms underlying the post-Golgi trafficking of auxin carriers in Arabidopsis. We show that differential cell elongation during apical hook development is defective in Arabidopsis mutant echidna (ech). ECH protein is required for the trans-Golgi network (TGN)-mediated trafficking of the auxin influx carrier AUX1 to the plasma membrane. In contrast, ech mutation only marginally perturbs the trafficking of the highly related auxin influx carrier LIKE-AUX1-3 or the auxin efflux carrier PIN-FORMED-3, both also involved in hook development. Electron tomography reveals that the trafficking defects in ech mutant are associated with the perturbation of secretory vesicle genesis from the TGN. Our results identify differential mechanisms for the post-Golgi trafficking of de novo-synthesized auxin carriers to plasma membrane from the TGN and reveal how trafficking of auxin influx carriers mediates the control of differential cell elongation in apical hook development. PMID:24043780

  15. In Vitro Mutational Analysis of the β2 Adrenergic Receptor, an In Vivo Surrogate Odorant Receptor

    PubMed Central

    Pfister, Patrick; Tomoiaga, Delia; Rogers, Matthew E.; Feinstein, Paul

    2015-01-01

    Many G-protein coupled receptors (GPCRs), such as odorant receptors (ORs), cannot be characterized in heterologous cells because of their difficulty in trafficking to the plasma membrane. In contrast, a surrogate OR, the GPCR mouse β2-adrenergic-receptor (mβ2AR), robustly traffics to the plasma membrane. We set out to characterize mβ2AR mutants in vitro for their eventual use in olfactory axon guidance studies. We performed an extensive mutational analysis of mβ2AR using a Green Fluorescent Protein-tagged mβ2AR (mβ2AR::GFP) to easily assess the extent of its plasma membrane localization. In order to characterize mutants for their ability to successfully transduce ligand-initiated signal cascades, we determined the half maximal effective concentrations (EC50) and maximal response to isoprenaline, a known mβ2AR agonist. Our analysis reveals that removal of amino terminal (Nt) N-glycosylation sites and the carboxy terminal (Ct) palmitoylation site of mβ2AR do not affect its plasma membrane localization. By contrast, when both the Nt and Ct of mβ2AR are replaced with those of M71 OR, plasma membrane trafficking is impaired. We further analyze three mβ2AR mutants (RDY, E268A, and C327R) used in olfactory axon guidance studies and are able to decorrelate their plasma membrane trafficking with their capacity to respond to isoprenaline. A deletion of the Ct prevents proper trafficking and abolishes activity, but plasma membrane trafficking can be selectively rescued by a Tyrosine to Alanine mutation in the highly conserved GPCR motif NPxxY. This new loss-of-function mutant argues for a model in which residues located at the end of transmembrane domain 7 can act as a retention signal when unmasked. Additionally, to our surprise, amongst our set of mutations only Ct mutations appear to lower mβ2AR EC50s revealing their critical role in G-protein coupling. We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs

  16. In Vitro Mutational Analysis of the β2 Adrenergic Receptor, an In Vivo Surrogate Odorant Receptor.

    PubMed

    Jamet, Sophie; Bubnell, Jaclyn; Pfister, Patrick; Tomoiaga, Delia; Rogers, Matthew E; Feinstein, Paul

    2015-01-01

    Many G-protein coupled receptors (GPCRs), such as odorant receptors (ORs), cannot be characterized in heterologous cells because of their difficulty in trafficking to the plasma membrane. In contrast, a surrogate OR, the GPCR mouse β2-adrenergic-receptor (mβ2AR), robustly traffics to the plasma membrane. We set out to characterize mβ2AR mutants in vitro for their eventual use in olfactory axon guidance studies. We performed an extensive mutational analysis of mβ2AR using a Green Fluorescent Protein-tagged mβ2AR (mβ2AR::GFP) to easily assess the extent of its plasma membrane localization. In order to characterize mutants for their ability to successfully transduce ligand-initiated signal cascades, we determined the half maximal effective concentrations (EC50) and maximal response to isoprenaline, a known mβ2AR agonist. Our analysis reveals that removal of amino terminal (Nt) N-glycosylation sites and the carboxy terminal (Ct) palmitoylation site of mβ2AR do not affect its plasma membrane localization. By contrast, when both the Nt and Ct of mβ2AR are replaced with those of M71 OR, plasma membrane trafficking is impaired. We further analyze three mβ2AR mutants (RDY, E268A, and C327R) used in olfactory axon guidance studies and are able to decorrelate their plasma membrane trafficking with their capacity to respond to isoprenaline. A deletion of the Ct prevents proper trafficking and abolishes activity, but plasma membrane trafficking can be selectively rescued by a Tyrosine to Alanine mutation in the highly conserved GPCR motif NPxxY. This new loss-of-function mutant argues for a model in which residues located at the end of transmembrane domain 7 can act as a retention signal when unmasked. Additionally, to our surprise, amongst our set of mutations only Ct mutations appear to lower mβ2AR EC50s revealing their critical role in G-protein coupling. We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs

  17. Understanding human trafficking in the United States.

    PubMed

    Logan, T K; Walker, Robert; Hunt, Gretchen

    2009-01-01

    The topic of modern-day slavery or human trafficking has received increased media and national attention. However, to date there has been limited research on the nature and scope of human trafficking in the United States. This article describes and synthesizes nine reports that assess the U.S. service organizations' legal representative knowledge of, and experience with, human trafficking cases, as well as information from actual cases and media reports. This article has five main goals: (a) to define what human trafficking is, and is not; (b) to describe factors identified as contributing to vulnerability to being trafficked and keeping a person entrapped in the situation; (c) to examine how the crime of human trafficking differs from other kinds of crimes in the United States; (d) to explore how human trafficking victims are identified; and, (e) to provide recommendations to better address human trafficking in the United States. PMID:19056686

  18. Common variable immunodeficiency, impaired neurological development and reduced numbers of T regulatory cells in a 10-year-old boy with a STAT1 gain-of-function mutation.

    PubMed

    Kobbe, Robin; Kolster, Manuela; Fuchs, Sebastian; Schulze-Sturm, Ulf; Jenderny, Jutta; Kochhan, Lothar; Staab, Julia; Tolosa, Eva; Grimbacher, Bodo; Meyer, Thomas

    2016-07-25

    Recently, gain-of-function (GOF) mutations in the gene encoding signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis (CMC). This case report describes a 10-year-old boy presenting with signs of common variable immunodeficiency (CVID), failure to thrive, impaired neurological development, and a history of recurrent mucocutaneous Candida infections. Sequencing of the STAT1 gene identified a heterozygous missense mutation in exon 7 encoding the STAT1 coiled-coil domain (c.514T>C, p.Phe172Leu). In addition to hypogammaglobulinemia with B-cell deficiency, and a low percentage of Th17 cells, immunological analysis of the patient revealed a marked depletion of forkhead-box P3(+)-expressing regulatory T cells (Tregs). In vitro stimulation of T cells from the patient with interferon-α (IFNα) and/or IFNɣ resulted in a significantly increased expression of STAT1-regulated target genes such as MIG1, IRF1, MX1, MCP1/CCL2, IFI-56K, and CXCL10 as compared to IFN-treated cells from a healthy control, while no IFNα/ɣ-mediated up-regulation of the FOXP3 gene was found. These data demonstrate that the STAT1 GOF mutation F172L, which results in impaired stability of the antiparallel STAT1 dimer conformation, is associated with inhibited Treg cell development and neurological symptoms. PMID:27063510

  19. Sex trafficking and the exploitation of adolescents.

    PubMed

    McClain, Natalie M; Garrity, Stacy E

    2011-01-01

    Human trafficking affects a surprisingly large number of adolescents around the globe. Women and girls make up the majority of sex trafficking victims. Nurses must be aware of sex trafficking as a form of sexual violence in the adolescent population. Nurses can play a role in identifying, intervening, and advocating for victims of human trafficking as they currently do for patients that are the victims of other types of violent crimes. PMID:21284727

  20. Identification of TMEM230 mutations in familial Parkinson's disease.

    PubMed

    Deng, Han-Xiang; Shi, Yong; Yang, Yi; Ahmeti, Kreshnik B; Miller, Nimrod; Huang, Cao; Cheng, Lijun; Zhai, Hong; Deng, Sheng; Nuytemans, Karen; Corbett, Nicola J; Kim, Myung Jong; Deng, Hao; Tang, Beisha; Yang, Ziquang; Xu, Yanming; Chan, Piu; Huang, Bo; Gao, Xiao-Ping; Song, Zhi; Liu, Zhenhua; Fecto, Faisal; Siddique, Nailah; Foroud, Tatiana; Jankovic, Joseph; Ghetti, Bernardino; Nicholson, Daniel A; Krainc, Dimitri; Melen, Onur; Vance, Jeffery M; Pericak-Vance, Margaret A; Ma, Yong-Chao; Rajput, Ali H; Siddique, Teepu

    2016-07-01

    Parkinson's disease is the second most common neurodegenerative disorder without effective treatment. It is generally sporadic with unknown etiology. However, genetic studies of rare familial forms have led to the identification of mutations in several genes, which are linked to typical Parkinson's disease or parkinsonian disorders. The pathogenesis of Parkinson's disease remains largely elusive. Here we report a locus for autosomal dominant, clinically typical and Lewy body-confirmed Parkinson's disease on the short arm of chromosome 20 (20pter-p12) and identify TMEM230 as the disease-causing gene. We show that TMEM230 encodes a transmembrane protein of secretory/recycling vesicles, including synaptic vesicles in neurons. Disease-linked TMEM230 mutants impair synaptic vesicle trafficking. Our data provide genetic evidence that a mutant transmembrane protein of synaptic vesicles in neurons is etiologically linked to Parkinson's disease, with implications for understanding the pathogenic mechanism of Parkinson's disease and for developing rational therapies. PMID:27270108

  1. 31 CFR 598.310 - Narcotics trafficking.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false Narcotics trafficking. 598.310 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY FOREIGN NARCOTICS KINGPIN SANCTIONS REGULATIONS General Definitions § 598.310 Narcotics trafficking. The term narcotics trafficking means any illicit activity...

  2. 31 CFR 598.310 - Narcotics trafficking.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 31 Money and Finance: Treasury 3 2010-07-01 2010-07-01 false Narcotics trafficking. 598.310... OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY FOREIGN NARCOTICS KINGPIN SANCTIONS REGULATIONS General Definitions § 598.310 Narcotics trafficking. The term narcotics trafficking means any...

  3. 31 CFR 598.310 - Narcotics trafficking.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false Narcotics trafficking. 598.310 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY FOREIGN NARCOTICS KINGPIN SANCTIONS REGULATIONS General Definitions § 598.310 Narcotics trafficking. The term narcotics trafficking means any illicit activity...

  4. 31 CFR 598.310 - Narcotics trafficking.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false Narcotics trafficking. 598.310 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY FOREIGN NARCOTICS KINGPIN SANCTIONS REGULATIONS General Definitions § 598.310 Narcotics trafficking. The term narcotics trafficking means any illicit activity...

  5. 31 CFR 598.310 - Narcotics trafficking.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Narcotics trafficking. 598.310 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY FOREIGN NARCOTICS KINGPIN SANCTIONS REGULATIONS General Definitions § 598.310 Narcotics trafficking. The term narcotics trafficking means any illicit activity...

  6. A Congenital Neutrophil Defect Syndrome Associated with Mutations in VPS45

    PubMed Central

    Vilboux, Thierry; Lev, Atar; Malicdan, May Christine V.; Simon, Amos J.; Järvinen, Päivi; Racek, Tomas; Puchalka, Jacek; Sood, Raman; Carrington, Blake; Bishop, Kevin; Mullikin, James; Huizing, Marjan; Garty, Ben Zion; Eyal, Eran; Wolach, Baruch; Gavrieli, Ronit; Toren, Amos; Soudack, Michalle; Atawneh, Osama M.; Babushkin, Tatiana; Schiby, Ginette; Cullinane, Andrew; Avivi, Camila; Polak-Charcon, Sylvie; Barshack, Iris; Amariglio, Ninette; Rechavi, Gideon; van der Werff ten Bosch, Jutte; Anikster, Yair; Klein, Christoph; Gahl, William A.; Somech, Raz

    2013-01-01

    Background Neutrophils are the predominant phagocytes that provide protection against bacterial and fungal infections. Genetically determined neutrophil disorders confer a predisposition to severe infections and reveal novel mechanisms that control vesicular trafficking, hematopoiesis, and innate immunity. Methods We clinically evaluated seven children from five families who had neutropenia, neutrophil dysfunction, bone marrow fibrosis, and nephromegaly. To identify the causative gene, we performed homozygosity mapping using single-nucleotide polymorphism arrays, whole-exome sequencing, immunoblotting, immunofluorescence, electron microscopy, a real-time quantitative polymerase–chain-reaction assay, immunohistochemistry, flow cytometry, fibroblast motility assays, measurements of apoptosis, and zebrafish models. Correction experiments were performed by transfecting mutant fibroblasts with the nonmutated gene. Results All seven affected children had homozygous mutations (Thr224Asn or Glu238Lys, depending on the child's ethnic origin) in VPS45, which encodes a protein that regulates membrane trafficking through the endosomal system. The level of VPS45 protein was reduced, as were the VPS45 binding partners rabenosyn-5 and syntaxin-16. The level of β1 integrin was reduced on the surface of VPS45-deficient neutrophils and fibroblasts. VPS45-deficient fibroblasts were characterized by impaired motility and increased apoptosis. A zebrafish model of vps45 deficiency showed a marked paucity of myeloperoxidase-positive cells (i.e., neutrophils). Transfection of patient cells with nonmutated VPS45 corrected the migration defect and decreased apoptosis. Conclusions Defective endosomal intracellular protein trafficking due to biallelic mutations in VPS45 underlies a new immunodeficiency syndrome involving impaired neutrophil function. (Funded by the National Human Genome Research Institute and others.) PMID:23738510

  7. Diacylglycerol kinases in membrane trafficking

    PubMed Central

    Xie, Shuwei; Naslavsky, Naava; Caplan, Steve

    2015-01-01

    Diacylglycerol kinases (DGKs) belong to a family of cytosolic kinases that regulate the phosphorylation of diacylglycerol (DAG), converting it into phosphatidic acid (PA). There are 10 known mammalian DGK isoforms, each with a different tissue distribution and substrate specificity. These differences allow regulation of cellular responses by fine-tuning the delicate balance of cellular DAG and PA. DGK isoforms are best characterized as mediators of signal transduction and immune function. However, since recent studies reveal that DAG and PA are also involved in the regulation of endocytic trafficking, it is therefore anticipated that DGKs also plays an important role in membrane trafficking. In this review, we summarize the literature discussing the role of DGK isoforms at different stages of endocytic trafficking, including endocytosis, exocytosis, endocytic recycling, and transport from/to the Golgi apparatus. Overall, these studies contribute to our understanding of the involvement of PA and DAG in endocytic trafficking, an area of research that is drawing increasing attention in recent years. PMID:27057419

  8. Human trafficking and the healthcare professional.

    PubMed

    Barrows, Jeffrey; Finger, Reginald

    2008-05-01

    Despite the legislation passed in the 19th century outlawing human slavery, it is more widespread today than at the conclusion of the civil war. Modern human slavery, termed human trafficking, comes in several forms. The most common type of human trafficking is sex trafficking, the sale of women and children into prostitution. Labor trafficking is the sale of men, women, and children into hard labor for which they receive little or no compensation. Other forms of trafficking include child soldiering, war brides, and organ removal. Healthcare professionals play a critical role in both finding victims of human trafficking while they are still in captivity, as well as caring for their mental and physical needs upon release. Those working in the healthcare profession need to be educated regarding how a trafficking victim may present, as well as their unique healthcare needs. PMID:18414161

  9. Novel AE1 mutations in recessive distal renal tubular acidosis. Loss-of-function is rescued by glycophorin A.

    PubMed

    Tanphaichitr, V S; Sumboonnanonda, A; Ideguchi, H; Shayakul, C; Brugnara, C; Takao, M; Veerakul, G; Alper, S L

    1998-12-15

    The AE1 gene encodes band 3 Cl-/HCO3- exchangers that are expressed both in the erythrocyte and in the acid-secreting, type A intercalated cells of the kidney. Kidney AE1 contributes to urinary acidification by providing the major exit route for HCO3- across the basolateral membrane. Several AE1 mutations cosegregate with dominantly transmitted nonsyndromic renal tubular acidosis (dRTA). However, the modest degree of in vitro hypofunction exhibited by these dRTA-associated mutations fails to explain the disease phenotype in light of the normal urinary acidification associated with the complete loss-of-function exhibited by AE1 mutations linked to dominant spherocytosis. We report here novel AE1 mutations linked to a recessive syndrome of dRTA and hemolytic anemia in which red cell anion transport is normal. Both affected individuals were triply homozygous for two benign mutations M31T and K56E and for the loss-of-function mutation, G701D. AE1 G701D loss-of-function was accompanied by impaired trafficking to the Xenopus oocyte surface. Coexpression with AE1 G701D of the erythroid AE1 chaperonin, glycophorin A, rescued both AE1-mediated Cl- transport and AE1 surface expression in oocytes. The genetic and functional data both suggest that the homozygous AE1 G701D mutation causes recessively transmitted dRTA in this kindred with apparently normal erythroid anion transport. PMID:9854053

  10. CLIC4 regulates apical exocytosis and renal tube luminogenesis through retromer- and actin-mediated endocytic trafficking

    PubMed Central

    Chou, Szu-Yi; Hsu, Kuo-Shun; Otsu, Wataru; Hsu, Ya-Chu; Luo, Yun-Cin; Yeh, Celine; Shehab, Syed S.; Chen, Jie; Shieh, Vincent; He, Guo-an; Marean, Michael B.; Felsen, Diane; Ding, Aihao; Poppas, Dix P.; Chuang, Jen-Zen; Sung, Ching-Hwa

    2016-01-01

    Chloride intracellular channel 4 (CLIC4) is a mammalian homologue of EXC-4 whose mutation is associated with cystic excretory canals in nematodes. Here we show that CLIC4-null mouse embryos exhibit impaired renal tubulogenesis. In both developing and developed kidneys, CLIC4 is specifically enriched in the proximal tubule epithelial cells, in which CLIC4 is important for luminal delivery, microvillus morphogenesis, and endolysosomal biogenesis. Adult CLIC4-null proximal tubules display aberrant dilation. In MDCK 3D cultures, CLIC4 is expressed on early endosome, recycling endosome and apical transport carriers before reaching its steady-state apical membrane localization in mature lumen. CLIC4 suppression causes impaired apical vesicle coalescence and central lumen formation, a phenotype that can be rescued by Rab8 and Cdc42. Furthermore, we show that retromer- and branched actin-mediated trafficking on early endosome regulates apical delivery during early luminogenesis. CLIC4 selectively modulates retromer-mediated apical transport by negatively regulating the formation of branched actin on early endosomes. PMID:26786190

  11. Mutations in the Tight-Junction Gene Claudin 19 (CLDN19) Are Associated with Renal Magnesium Wasting, Renal Failure, and Severe Ocular Involvement

    PubMed Central

    Konrad, Martin; Schaller, André; Seelow, Dominik; Pandey, Amit V.; Waldegger, Siegfried; Lesslauer, Annegret; Vitzthum, Helga; Suzuki, Yoshiro; Luk, John M.; Becker, Christian; Schlingmann, Karl P.; Schmid, Marcel; Rodriguez-Soriano, Juan; Ariceta, Gema; Cano, Francisco; Enriquez, Ricardo; Jüppner, Harald; Bakkaloglu, Sevcan A.; Hediger, Matthias A.; Gallati, Sabina; Neuhauss, Stephan C. F.; Nürnberg, Peter; Weber, Stefanie

    2006-01-01

    Claudins are major components of tight junctions and contribute to the epithelial-barrier function by restricting free diffusion of solutes through the paracellular pathway. We have mapped a new locus for recessive renal magnesium loss on chromosome 1p34.2 and have identified mutations in CLDN19, a member of the claudin multigene family, in patients affected by hypomagnesemia, renal failure, and severe ocular abnormalities. CLDN19 encodes the tight-junction protein claudin-19, and we demonstrate high expression of CLDN19 in renal tubules and the retina. The identified mutations interfere severely with either cell-membrane trafficking or the assembly of the claudin-19 protein. The identification of CLDN19 mutations in patients with chronic renal failure and severe visual impairment supports the fundamental role of claudin-19 for normal renal tubular function and undisturbed organization and development of the retina. PMID:17033971

  12. Trafficking of the Follitropin Receptor

    PubMed Central

    Ulloa-Aguirre, Alfredo; Dias, James A.; Bousfield, George; Huhtaniemi, Ilpo; Reiter, Eric

    2015-01-01

    The follitropin or follicle-stimulating hormone receptor (FSHR) belongs to a highly conserved subfamily of the G protein-coupled receptor (GPCR) superfamily and is mainly expressed in specific cells in the gonads. As any other GPCR, the newly synthesized FSHR has to be correctly folded and processed in order to traffic to the cell surface plasma membrane and interact with its cognate ligand. In this chapter, we describe in detail the conditions and procedures used to study outward trafficking of the FSHR from the endoplasmic reticulum to the plasma membrane. We also describe some methods to analyze phosphorylation, β-arrestin recruitment, internalization, and recycling of this particular receptor, which have proved useful in our hands for dissecting its downward trafficking and fate following agonist stimulation. PMID:23351732

  13. Biopolitical management, economic calculation and "trafficked women".

    PubMed

    Berman, Jacqueline

    2010-01-01

    Narratives surrounding human trafficking, especially trafficking in women for sex work, employ gendered and racialized tropes that have among their effects, a shrouding of women's economic decision-making and state collusion in benefiting from their labour. This paper explores the operation of these narratives in order to understand the ways in which they mask the economics of trafficking by sensationalizing the sexual and criminal aspects of it, which in turn allows the state to pursue political projects under the guise of a benevolent concern for trafficked women and/or protection of its own citizens. This paper will explore one national example: Article 18 of Italian Law 40 (1998). I argue that its passage has led to an increase in cooperation with criminal prosecution of traffickers largely because it approaches trafficked women as capable of making decisions about how and what they themselves want to do. This paper will also consider a more global approach to trafficking embedded in the concept of "migration management", an International Organization for Migration (IOM) framework that is now shaping EU, US and other national immigration laws and policies that impact trafficking. It will also examine the inherent limitations of both the national and global approach as an occasion to unpack how Article 18 and Migration Management function as forms of biopolitical management that participate in the production of "trafficking victims" into a massified population to be managed, rather than engender a more engaged discussion of what constitutes trafficking and how to redress it. PMID:20645471

  14. Functional rescue of a kidney anion exchanger 1 trafficking mutant in renal epithelial cells.

    PubMed

    Chu, Carmen Y S; King, Jennifer C; Berrini, Mattia; Alexander, R Todd; Cordat, Emmanuelle

    2013-01-01

    Mutations in the SLC4A1 gene encoding the anion exchanger 1 (AE1) can cause distal renal tubular acidosis (dRTA), a disease often due to mis-trafficking of the mutant protein. In this study, we investigated whether trafficking of a Golgi-retained dRTA mutant, G701D kAE1, or two dRTA mutants retained in the endoplasmic reticulum, C479W and R589H kAE1, could be functionally rescued to the plasma membrane of Madin-Darby Canine Kidney (MDCK) cells. Treatments with DMSO, glycerol, the corrector VX-809, or low temperature incubations restored the basolateral trafficking of G701D kAE1 mutant. These treatments had no significant rescuing effect on trafficking of the mis-folded C479W or R589H kAE1 mutants. DMSO was the only treatment that partially restored G701D kAE1 function in the plasma membrane of MDCK cells. Our experiments show that trafficking of intracellularly retained dRTA kAE1 mutants can be partially restored, and that one chemical treatment rescued both trafficking and function of a dRTA mutant. These studies provide an opportunity to develop alternative therapeutic solutions for dRTA patients. PMID:23460825

  15. Functional Rescue of a Kidney Anion Exchanger 1 Trafficking Mutant in Renal Epithelial Cells

    PubMed Central

    Chu, Carmen Y. S.; King, Jennifer C.; Berrini, Mattia; Alexander, R. Todd; Cordat, Emmanuelle

    2013-01-01

    Mutations in the SLC4A1 gene encoding the anion exchanger 1 (AE1) can cause distal renal tubular acidosis (dRTA), a disease often due to mis-trafficking of the mutant protein. In this study, we investigated whether trafficking of a Golgi-retained dRTA mutant, G701D kAE1, or two dRTA mutants retained in the endoplasmic reticulum, C479W and R589H kAE1, could be functionally rescued to the plasma membrane of Madin-Darby Canine Kidney (MDCK) cells. Treatments with DMSO, glycerol, the corrector VX-809, or low temperature incubations restored the basolateral trafficking of G701D kAE1 mutant. These treatments had no significant rescuing effect on trafficking of the mis-folded C479W or R589H kAE1 mutants. DMSO was the only treatment that partially restored G701D kAE1 function in the plasma membrane of MDCK cells. Our experiments show that trafficking of intracellularly retained dRTA kAE1 mutants can be partially restored, and that one chemical treatment rescued both trafficking and function of a dRTA mutant. These studies provide an opportunity to develop alternative therapeutic solutions for dRTA patients. PMID:23460825

  16. Computational analysis of the mutations in BAP1, PBRM1 and SETD2 genes reveals the impaired molecular processes in renal cell carcinoma

    PubMed Central

    Occhipinti, Giulia; Santoni, Matteo; Massari, Francesco; Sotte, Valeria; Iacovelli, Roberto; Burattini, Luciano; Santini, Daniele; Montironi, Rodolfo; Cascinu, Stefano; Principato, Giovanni

    2015-01-01

    Clear cell Renal Cell Carcinoma (ccRCC) is due to loss of von Hippel–Lindau (VHL) gene and at least one out of three chromatin regulating genes BRCA1-associated protein-1 (BAP1), Polybromo-1 (PBRM1) and Set domain-containing 2 (SETD2). More than 350, 700 and 500 mutations are known respectively for BAP1, PBRM1 and SETD2 genes. Each variation damages these genes with different severity levels. Unfortunately for most of these mutations the molecular effect is unknown, so precluding a severity classification. Moreover, the huge number of these gene mutations does not allow to perform experimental assays for each of them. By bioinformatic tools, we performed predictions of the molecular effects of all mutations lying in BAP1, PBRM1 and SETD2 genes. Our results allow to distinguish whether a mutation alters protein function directly or by splicing pattern destruction and how much severely. This classification could be useful to reveal correlation with patients’ outcome, to guide experiments, to select the variations that are worth to be included in translational/association studies, and to direct gene therapies. PMID:26452128

  17. Pathogenic Mutations in the Valosin-containing Protein/p97(VCP) N-domain Inhibit the SUMOylation of VCP and Lead to Impaired Stress Response.

    PubMed

    Wang, Tao; Xu, Wangchao; Qin, Meiling; Yang, Yi; Bao, Puhua; Shen, Fuxiao; Zhang, Zhenlin; Xu, Jin

    2016-07-01

    Valosin-containing protein/p97(VCP) is a hexameric ATPase vital to protein degradation during endoplasmic reticulum stress. It regulates diverse cellular functions including autophagy, chromatin remodeling, and DNA repair. In addition, mutations in VCP cause inclusion body myopathy, Paget disease of the bone, and frontotemporal dementia (IBMPFD), as well as amyotrophic lateral sclerosis. Nevertheless, how the VCP activities were regulated and how the pathogenic mutations affect the function of VCP during stress are not unclear. Here we show that the small ubiquitin-like modifier (SUMO)-ylation of VCP is a normal stress response inhibited by the disease-causing mutations in the N-domain. Under oxidative and endoplasmic reticulum stress conditions, the SUMOylation of VCP facilitates the distribution of VCP to stress granules and nucleus, and promotes the VCP hexamer assembly. In contrast, pathogenic mutations in the VCP N-domain lead to reduced SUMOylation and weakened VCP hexamer formation upon stress. Defective SUMOylation of VCP also causes altered co-factor binding and attenuated endoplasmic reticulum-associated protein degradation. Furthermore, SUMO-defective VCP fails to protect against stress-induced toxicity in Drosophila Therefore, our results have revealed SUMOylation as a molecular signaling switch to regulate the distribution and functions of VCP during stress response, and suggest that deficiency in VCP SUMOylation caused by pathogenic mutations will render cells vulnerable to stress insults. PMID:27226613

  18. LKB1/AMPK and PKA control ABCB11 trafficking and polarization in hepatocytes.

    PubMed

    Homolya, László; Fu, Dong; Sengupta, Prabuddha; Jarnik, Michal; Gillet, Jean-Pierre; Vitale-Cross, Lynn; Gutkind, J Silvio; Lippincott-Schwartz, Jennifer; Arias, Irwin M

    2014-01-01

    Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation. PMID:24643070

  19. LKB1/AMPK and PKA Control ABCB11 Trafficking and Polarization in Hepatocytes

    PubMed Central

    Homolya, László; Fu, Dong; Sengupta, Prabuddha; Jarnik, Michal; Gillet, Jean-Pierre; Vitale-Cross, Lynn; Gutkind, J. Silvio; Lippincott-Schwartz, Jennifer; Arias, Irwin M.

    2014-01-01

    Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation. PMID:24643070

  20. Agonist-trafficking and hallucinogens.

    PubMed

    González-Maeso, Javier; Sealfon, Stuart C

    2009-01-01

    Seven transmembrane domain receptors, also termed G protein-coupled receptors (GPCRs), represent the most common molecular target for therapeutic drugs. The generally accepted pharmacological model for GPCR activation is the ternary complex model, in which GPCRs exist in a dynamic equilibrium between the active and inactive conformational states. However, the demonstration that different agonists sometimes elicit a different relative activation of two signaling pathways downstream of the same receptor has led to a revision of the ternary complex model. According to this agonist- trafficking model, agonists stabilize distinct activated receptor conformations that preferentially activate specific signaling pathways. Hallucinogenic drugs and non-hallucinogenic drugs represent an attractive experimental system with which to study agonist-trafficking of receptor signaling. Thus many of the behavioral responses induced by hallucinogenic drugs, such as lysergic acid diethylamide (LSD), psilocybin or mescaline, depend on activation of serotonin 5-HT(2A) receptors (5-HT2ARs). In contrast, this neuropsychological state in humans is not induced by closely related chemicals, such as lisuride or ergotamine, despite their similar in vitro activity at the 5-HT2AR. In this review, we summarize the current knowledge, as well as unresolved questions, regarding agonist-trafficking and the mechanism of action of hallucinogenic drugs. PMID:19275609

  1. Viral Subversion of Nucleocytoplasmic Trafficking

    PubMed Central

    Yarbrough, Melanie L.; Mata, Miguel A.; Sakthivel, Ramanavelan; Fontoura, Beatriz M. A.

    2014-01-01

    Trafficking of proteins and RNA into and out of the nucleus occurs through the nuclear pore complex (NPC). Due to its critical function in many cellular processes, the NPC and transport factors are common targets of several viruses that disrupt key constituents of the machinery to facilitate viral replication. Many viruses such as poliovirus and severe acute respiratory syndrome (SARS) virus inhibit protein import into the nucleus, while viruses such as influenza A virus target and disrupt host mRNA nuclear export. Current evidence indicates that these viruses may employ such strategies to avert the host immune response. Conversely, many viruses co-opt nucleocytoplasmic trafficking to facilitate transport of viral RNAs. Since viral proteins interact with key regulators of the host nuclear transport machinery, viruses have served as invaluable tools of discovery that led to the identification of novel constituents of nuclear transport pathways. In addition, this review explores the importance of nucleocytoplasmic trafficking to viral pathogenesis as these studies revealed new antiviral therapeutic strategies and exposed previously unknown cellular mechanisms. Further understanding of nuclear transport pathways will determine whether such therapeutics will be useful treatments for important human pathogens. PMID:24289861

  2. Adenosine receptor desensitization and trafficking.

    PubMed

    Mundell, Stuart; Kelly, Eamonn

    2011-05-01

    As with the majority of G-protein-coupled receptors, all four of the adenosine receptor subtypes are known to undergo agonist-induced regulation in the form of desensitization and trafficking. These processes can limit the ability of adenosine receptors to couple to intracellular signalling pathways and thus reduce the ability of adenosine receptor agonists as well as endogenous adenosine to produce cellular responses. In addition, since adenosine receptors couple to multiple signalling pathways, these pathways may desensitize differentially, while the desensitization of one pathway could even trigger signalling via another. Thus, the overall picture of adenosine receptor regulation can be complex. For all adenosine receptor subtypes, there is evidence to implicate arrestins in agonist-induced desensitization and trafficking, but there is also evidence for other possible forms of regulation, including second messenger-dependent kinase regulation, heterologous effects involving G proteins, and the involvement of non-clathrin trafficking pathways such as caveolae. In this review, the evidence implicating these mechanisms is summarized for each adenosine receptor subtype, and we also discuss those issues of adenosine receptor regulation that remain to be resolved as well as likely directions for future research in this field. PMID:20550943

  3. Protein trafficking in kinetoplastid protozoa.

    PubMed Central

    Clayton, C; Häusler, T; Blattner, J

    1995-01-01

    The kinetoplastid protozoa infect hosts ranging from invertebrates to plants and mammals, causing diseases of medical and economic importance. They are the earliest-branching organisms in eucaryotic evolution to have either mitochondria or peroxisome-like microbodies. Investigation of their protein trafficking enables us to identify characteristics that have been conserved throughout eucaryotic evolution and also reveals how far variations, or alternative mechanisms, are possible. Protein trafficking in kinetoplastids is in many respects similar to that in higher eucaryotes, including mammals and yeasts. Differences in signal sequence specificities exist, however, for all subcellular locations so far examined in detail--microbodies, mitochondria, and endoplasmic reticulum--with signals being more degenerate, or shorter, than those of their higher eucaryotic counterparts. Some components of the normal array of trafficking mechanisms may be missing in most (if not all) kinetoplastids: examples are clathrin-coated vesicles, recycling receptors, and mannose 6-phosphate-mediated lysosomal targeting. Other aspects and structures are unique to the kinetoplastids or are as yet unexplained. Some of these peculiarities may eventually prove to be weak points that can be used as targets for chemotherapy; others may turn out to be much more widespread than currently suspected. PMID:7565409

  4. The R215W mutation in NBS1 impairs {gamma}-H2AX binding and affects DNA repair: molecular bases for the severe phenotype of 657del5/R215W Nijmegen breakage syndrome patients

    SciTech Connect

    Masi, Alessandra di Viganotti, Mara; Polticelli, Fabio; Ascenzi, Paolo; Tanzarella, Caterina; Antoccia, Antonio

    2008-05-09

    Nijmegen breakage syndrome (NBS) is a genetic disorder characterized by chromosomal instability and hypersensitivity to ionising radiation. Compound heterozygous 657del5/R215W NBS patients display a clinical phenotype more severe than the majority of NBS patients homozygous for the 657del5 mutation. The NBS1 protein, mutated in NBS patients, contains a FHA/BRCT domain necessary for the DNA-double strand break (DSB) damage response. Recently, a second BRCT domain has been identified, however, its role is still unknown. Here, we demonstrate that the R215W mutation in NBS1 impairs histone {gamma}-H2AX binding after induction of DNA damage, leading to a delay in DNA-DSB rejoining. Molecular modelling reveals that the 215 residue of NBS1 is located between the two BRCT domains, affecting their relative orientation that appears critical for {gamma}-H2AX binding. Present data represent the first evidence for the role of NBS1 tandem BRCT domains in {gamma}-H2AX recognition, and could explain the severe phenotype observed in 657del5/R215W NBS patients.

  5. Reconstitution of defective protein trafficking rescues Long-QT syndrome in zebrafish.

    PubMed

    Meder, Benjamin; Scholz, Eberhard P; Hassel, David; Wolff, Christoph; Just, Steffen; Berger, Ina M; Patzel, Eva; Karle, Christoph; Katus, Hugo A; Rottbauer, Wolfgang

    2011-05-01

    Inherited cardiac arrhythmias are caused by genetic defects in ion channels and associated proteins. Mutations in these channels often do not affect their biophysical properties, but rather interfere with their trafficking to the cell membrane. Accordingly, strategies that could reroute the mutated channels to the membrane should be sufficient to restore the electrical properties of the affected cells, thereby suppressing the underlying arrhythmia. We identified here both, embryonic and adult zebrafish breakdance (bre) as a valuable model for human Long-QT syndrome. Electrocardiograms of adult homozygous bre mutants exhibit significant QT prolongation caused by delayed repolarization of the ventricle. We further show that the bre mutation (zERG(I59S)) disrupts ERG protein trafficking, thereby reducing the amount of active potassium channels on the cell membrane. Interestingly, improvement of channel trafficking by cisapride or dimethylsulfoxid is sufficient to reconstitute ERG channels on the cell membrane in a manner that suffices to suppress the Long-QT induced arrhythmia in breakdance mutant zebrafish. In summary, we show for the first time that therapeutic intervention can cure protein trafficking defects and the associated cardiac arrhythmia in vivo. PMID:21458413

  6. Mutations in the PCNA-binding site of CDKN1C inhibit cell proliferation by impairing the entry into S phase.

    PubMed

    Borges, Kleiton S; Arboleda, Valerie A; Vilain, Eric

    2015-01-01

    CDKN1C (also known as P57 (kip2) ) is a cyclin-dependent kinase inhibitor that functions as a negative regulator of cell proliferation through G1 phase cell cycle arrest. Recently, our group described gain-of-function mutations in the PCNA-binding site of CDKN1C that result in an undergrowth syndrome called IMAGe Syndrome (Intrauterine Growth Restriction, Metaphyseal dysplasia, Adrenal hypoplasia, and Genital anomalies), with life-threatening consequences. Loss-of-function mutations in CDKN1C have been identified in 5-10% of individuals with Beckwith-Wiedemann syndrome (BWS), an overgrowth disorder with features that are the opposite of IMAGe syndrome. Here, we investigate the effects of IMAGe-associated mutations on protein stability, cell cycle progression and cell proliferation. Mutations in the PCNA-binding site of CDKN1C significantly increase CDKN1C protein stability and prevent cell cycle progression into the S phase. Overexpression of either wild-type or BWS-mutant CDKN1C inhibited cell proliferation. However, the IMAGe-mutant CDKN1C protein decreased cell growth significantly more than both the wild-type or BWS protein. These findings bring new insights into the molecular events underlying IMAGe syndrome. PMID:25861374

  7. The localization of FGFR3 mutations causing thanatophoric dysplasia type I differentially affects phosphorylation, processing and ubiquitylation of the receptor.

    PubMed

    Bonaventure, Jacky; Gibbs, Linda; Horne, William C; Baron, Roland

    2007-06-01

    Recurrent missense fibroblast growth factor receptor 3 (FGFR3) mutations have been ascribed to skeletal dysplasias of variable severity including the lethal neonatal thanatophoric dysplasia types I (TDI) and II (TDII). To elucidate the role of activating mutations causing TDI on receptor trafficking and endocytosis, a series of four mutants located in different domains of the receptor were generated and transiently expressed. The putatively elongated X807R receptor was identified as three isoforms. The fully glycosylated mature isoform was constitutively but mildly phosphorylated. Similarly, mutations affecting the extracellular domain (R248C and Y373C) induced moderate constitutive receptor phosphorylation. By contrast, the K650M mutation affecting the tyrosine kinase 2 (TK2) domain produced heavy phosphorylation of the nonglycosylated and mannose-rich isoforms that impaired receptor trafficking through the Golgi network. This resulted in defective expression of the mature isoform at the cell surface. Normal processing was rescued by tyrosine kinase inhibitor treatment. Internalization of the R248C and Y373C mutant receptors, which form stable disulfide-bonded dimers at the cell surface was less efficient than the wild-type, whereas ubiquitylation was markedly increased but apparently independent of the E3 ubiquitin-ligase casitas B-lineage lymphoma (c-Cbl). Constitutive phosphorylation of c-Cbl by the K650M mutant appeared to be related to the intracellular retention of the receptor. Therefore, although mutation K650M affecting the TK2 domain induces defective targeting of the overphosphorylated receptor, a different mechanism characterized by receptor retention at the plasma membrane, excessive ubiquitylation and reduced degradation results from mutations that affect the extracellular domain and the stop codon. PMID:17509076

  8. Mutations in BICD2 Cause Dominant Congenital Spinal Muscular Atrophy and Hereditary Spastic Paraplegia

    PubMed Central

    Oates, Emily C.; Rossor, Alexander M.; Hafezparast, Majid; Gonzalez, Michael; Speziani, Fiorella; MacArthur, Daniel G.; Lek, Monkol; Cottenie, Ellen; Scoto, Mariacristina; Foley, A. Reghan; Hurles, Matthew; Houlden, Henry; Greensmith, Linda; Auer-Grumbach, Michaela; Pieber, Thomas R.; Strom, Tim M.; Schule, Rebecca; Herrmann, David N.; Sowden, Janet E.; Acsadi, Gyula; Menezes, Manoj P.; Clarke, Nigel F.; Züchner, Stephan; Muntoni, Francesco; North, Kathryn N.; Reilly, Mary M.

    2013-01-01

    Dominant congenital spinal muscular atrophy (DCSMA) is a disorder of developing anterior horn cells and shows lower-limb predominance and clinical overlap with hereditary spastic paraplegia (HSP), a lower-limb-predominant disorder of corticospinal motor neurons. We have identified four mutations in bicaudal D homolog 2 (Drosophila) (BICD2) in six kindreds affected by DCSMA, DCSMA with upper motor neuron features, or HSP. BICD2 encodes BICD2, a key adaptor protein that interacts with the dynein-dynactin motor complex, which facilitates trafficking of cellular cargos that are critical to motor neuron development and maintenance. We demonstrate that mutations resulting in amino acid substitutions in two binding regions of BICD2 increase its binding affinity for the cytoplasmic dynein-dynactin complex, which might result in the perturbation of BICD2-dynein-dynactin-mediated trafficking, and impair neurite outgrowth. These findings provide insight into the mechanism underlying both the static and the slowly progressive clinical features and the motor neuron pathology that characterize BICD2-associated diseases, and underscore the importance of the dynein-dynactin transport pathway in the development and survival of both lower and upper motor neurons. PMID:23664120

  9. A Founder Mutation in VPS11 Causes an Autosomal Recessive Leukoencephalopathy Linked to Autophagic Defects

    PubMed Central

    Schaffner, Adam; Fedick, Anastasia; Kaye, Lauren E.; Liao, Jun; Yachelevich, Naomi; Chu, Mary-Lynn; Boles, Richard G.; Moran, Ellen; Tokita, Mari; Gorman, Elizabeth; Zhang, Wei; Xia, Fan; Leduc, Magalie; Yang, Yaping; Eng, Christine; Wong, Lee-Jun; Schiffmann, Raphael; Diaz, George A.; Kornreich, Ruth; Thummel, Ryan; Wasserstein, Melissa; Yue, Zhenyu; Edelmann, Lisa

    2016-01-01

    Genetic leukoencephalopathies (gLEs) are a group of heterogeneous disorders with white matter abnormalities affecting the central nervous system (CNS). The causative mutation in ~50% of gLEs is unknown. Using whole exome sequencing (WES), we identified homozygosity for a missense variant, VPS11: c.2536T>G (p.C846G), as the genetic cause of a leukoencephalopathy syndrome in five individuals from three unrelated Ashkenazi Jewish (AJ) families. All five patients exhibited highly concordant disease progression characterized by infantile onset leukoencephalopathy with brain white matter abnormalities, severe motor impairment, cortical blindness, intellectual disability, and seizures. The carrier frequency of the VPS11: c.2536T>G variant is 1:250 in the AJ population (n = 2,026). VPS11 protein is a core component of HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering) protein complexes involved in membrane trafficking and fusion of the lysosomes and endosomes. The cysteine 846 resides in an evolutionarily conserved cysteine-rich RING-H2 domain in carboxyl terminal regions of VPS11 proteins. Our data shows that the C846G mutation causes aberrant ubiquitination and accelerated turnover of VPS11 protein as well as compromised VPS11-VPS18 complex assembly, suggesting a loss of function in the mutant protein. Reduced VPS11 expression leads to an impaired autophagic activity in human cells. Importantly, zebrafish harboring a vps11 mutation with truncated RING-H2 domain demonstrated a significant reduction in CNS myelination following extensive neuronal death in the hindbrain and midbrain. Thus, our study reveals a defect in VPS11 as the underlying etiology for an autosomal recessive leukoencephalopathy disorder associated with a dysfunctional autophagy-lysosome trafficking pathway. PMID:27120463

  10. A Founder Mutation in VPS11 Causes an Autosomal Recessive Leukoencephalopathy Linked to Autophagic Defects.

    PubMed

    Zhang, Jinglan; Lachance, Véronik; Schaffner, Adam; Li, Xianting; Fedick, Anastasia; Kaye, Lauren E; Liao, Jun; Rosenfeld, Jill; Yachelevich, Naomi; Chu, Mary-Lynn; Mitchell, Wendy G; Boles, Richard G; Moran, Ellen; Tokita, Mari; Gorman, Elizabeth; Bagley, Kaytee; Zhang, Wei; Xia, Fan; Leduc, Magalie; Yang, Yaping; Eng, Christine; Wong, Lee-Jun; Schiffmann, Raphael; Diaz, George A; Kornreich, Ruth; Thummel, Ryan; Wasserstein, Melissa; Yue, Zhenyu; Edelmann, Lisa

    2016-04-01

    Genetic leukoencephalopathies (gLEs) are a group of heterogeneous disorders with white matter abnormalities affecting the central nervous system (CNS). The causative mutation in ~50% of gLEs is unknown. Using whole exome sequencing (WES), we identified homozygosity for a missense variant, VPS11: c.2536T>G (p.C846G), as the genetic cause of a leukoencephalopathy syndrome in five individuals from three unrelated Ashkenazi Jewish (AJ) families. All five patients exhibited highly concordant disease progression characterized by infantile onset leukoencephalopathy with brain white matter abnormalities, severe motor impairment, cortical blindness, intellectual disability, and seizures. The carrier frequency of the VPS11: c.2536T>G variant is 1:250 in the AJ population (n = 2,026). VPS11 protein is a core component of HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering) protein complexes involved in membrane trafficking and fusion of the lysosomes and endosomes. The cysteine 846 resides in an evolutionarily conserved cysteine-rich RING-H2 domain in carboxyl terminal regions of VPS11 proteins. Our data shows that the C846G mutation causes aberrant ubiquitination and accelerated turnover of VPS11 protein as well as compromised VPS11-VPS18 complex assembly, suggesting a loss of function in the mutant protein. Reduced VPS11 expression leads to an impaired autophagic activity in human cells. Importantly, zebrafish harboring a vps11 mutation with truncated RING-H2 domain demonstrated a significant reduction in CNS myelination following extensive neuronal death in the hindbrain and midbrain. Thus, our study reveals a defect in VPS11 as the underlying etiology for an autosomal recessive leukoencephalopathy disorder associated with a dysfunctional autophagy-lysosome trafficking pathway. PMID:27120463

  11. Multiple Dileucine-like Motifs Direct VGLUT1 Trafficking

    PubMed Central

    Foss, Sarah M.; Li, Haiyan; Santos, Magda S.; Edwards, Robert H.

    2013-01-01

    The vesicular glutamate transporters (VGLUTs) package glutamate into synaptic vesicles, and the two principal isoforms VGLUT1 and VGLUT2 have been suggested to influence the properties of release. To understand how a VGLUT isoform might influence transmitter release, we have studied their trafficking and previously identified a dileucine-like endocytic motif in the C terminus of VGLUT1. Disruption of this motif impairs the activity-dependent recycling of VGLUT1, but does not eliminate its endocytosis. We now report the identification of two additional dileucine-like motifs in the N terminus of VGLUT1 that are not well conserved in the other isoforms. In the absence of all three motifs, rat VGLUT1 shows limited accumulation at synaptic sites and no longer responds to stimulation. In addition, shRNA-mediated knockdown of clathrin adaptor proteins AP-1 and AP-2 shows that the C-terminal motif acts largely via AP-2, whereas the N-terminal motifs use AP-1. Without the C-terminal motif, knockdown of AP-1 reduces the proportion of VGLUT1 that responds to stimulation. VGLUT1 thus contains multiple sorting signals that engage distinct trafficking mechanisms. In contrast to VGLUT1, the trafficking of VGLUT2 depends almost entirely on the conserved C-terminal dileucine-like motif: without this motif, a substantial fraction of VGLUT2 redistributes to the plasma membrane and the transporter's synaptic localization is disrupted. Consistent with these differences in trafficking signals, wild-type VGLUT1 and VGLUT2 differ in their response to stimulation. PMID:23804088

  12. Analysis of GPCR Localization and Trafficking

    PubMed Central

    Hislop, James N.; von Zastrow, Mark

    2016-01-01

    Localization and trafficking of G protein-coupled receptors (GPCRs) is increasingly recognized to play a fundamental role in receptor-mediated signaling and its regulation. Individual receptors, including closely homologous subtypes with otherwise similar functional properties, can differ considerably in their membrane trafficking properties. In this chapter, we describe several approaches for experimentally assessing the subcellular localization and trafficking of selected GPCRs. Firstly, we describe a flexible method for receptor localization using fluorescence microscopy. We then describe two complementary approaches, using fluorescence flow cytometry and surface biotinylation, for examining receptor internalization and trafficking in the endocytic pathway. PMID:21607873

  13. Human anion exchanger1 mutations and distal renal tubular acidosis.

    PubMed

    Yenchitsomanus, Pa-thai

    2003-09-01

    The human anion exchanger 1 (AE1 or SLC4A1) gene encodes anion exchanger 1 (or band 3) protein in erythrocytes and in alpha-intercalated cells of the kidney. Thus, AE1 mutations show pleiotrophic effects resulting in two distinct and seemingly unrelated defects, an erythrocyte abnormality and distal renal tubular acidosis (dRTA). Southeast Asian ovalocytosis (SAO), a well-known red blood cell (RBC) defect, which is widespread in Southeast Asian regions, is caused by AE1 mutation due to a deletion of 27 base pairs in codons 400-408 (delta400-408) leading to an in-frame 9 amino-acid loss in the protein. Co-existence of SAO and dRTA is usually not seen in the same individual. However, the two conditions can co-exist as the result of compound heterozygosities between delta400-408 and other mutations. The reported genotypes include delta400-408/G701D, delta400-408/R602H, delta400-408/deltaV850, and delta400-408/A858D. The presence of dRTA, with or without RBC abnormalities, may occur from homozygous or compound heterozygous conditions of recessive AE1 mutations (eg G701D/G701D, V488M/V488M, deltaV850/deltaV850, deltaV850/A858D, G701D/S773P) or heterozygous dominant AE1 mutations (eg R598H, R589C, R589S, S613F, R901X). Codon 589 of this gene seems to be a 'mutational hot-spot' since repeated mutations at this codon occurring in different ethnic groups and at least two de novo (R589H and R589C) mutations have been observed. Therefore, AE1 mutations can result in both recessive and dominant dRTA, possibly depending on the position of the amino acid change in the protein. As several mutant AE1 proteins still maintain a significant anion transport function but are defective in targeting to the cell surface, impaired intracellular trafficking of the mutant AE1 is an important molecular mechanism involved in the pathogenesis of dRTA associated with AE1 mutations. PMID:15115146

  14. Etomidate blocks LTP and impairs learning but does not enhance tonic inhibition in mice carrying the N265M point mutation in the beta3 subunit of the GABA(A) receptor.

    PubMed

    Zarnowska, E D; Rodgers, F C; Oh, I; Rau, V; Lor, C; Laha, K T; Jurd, R; Rudolph, U; Eger, E I; Pearce, R A

    2015-06-01

    Enhancement of tonic inhibition mediated by extrasynaptic α5-subunit containing GABAA receptors (GABAARs) has been proposed as the mechanism by which a variety of anesthetics, including the general anesthetic etomidate, impair learning and memory. Since α5 subunits preferentially partner with β3 subunits, we tested the hypothesis that etomidate acts through β3-subunit containing GABAARs to enhance tonic inhibition, block LTP, and impair memory. We measured the effects of etomidate in wild type mice and in mice carrying a point mutation in the GABAAR β3-subunit (β3-N265M) that renders these receptors insensitive to etomidate. Etomidate enhanced tonic inhibition in CA1 pyramidal cells of the hippocampus in wild type but not in mutant mice, demonstrating that tonic inhibition is mediated by β3-subunit containing GABAARs. However, despite its inability to enhance tonic inhibition, etomidate did block LTP in brain slices from mutant mice as well as in those from wild type mice. Etomidate also impaired fear conditioning to context, with no differences between genotypes. In studies of recombinant receptors expressed in HEK293 cells, α5β1γ2L GABAARs were insensitive to amnestic concentrations of etomidate (1 μM and below), whereas α5β2γ2L and α5β3γ2L GABAARs were enhanced. We conclude that etomidate enhances tonic inhibition in pyramidal cells through its action on α5β3-containing GABAA receptors, but blocks LTP and impairs learning by other means - most likely by modulating α5β2-containing GABAA receptors. The critical anesthetic targets underlying amnesia might include other forms of inhibition imposed on pyramidal neurons (e.g. slow phasic inhibition), or inhibitory processes on non-pyramidal cells (e.g. interneurons). PMID:25680234

  15. Mutation in the splicing factor Hprp3p linked to retinitis pigmentosa impairs interactions within the U4/U6 snRNP complex

    PubMed Central

    Gonzalez-Santos, Juana Maria; Cao, Huibi; Duan, Rongqi Cathleen; Hu, Jim

    2015-01-01

    Mutations in PRPF3, a gene encoding the essential pre-mRNA splicing factor Hprp3p, have been identified in patients with autosomal dominant retinitis pigmentosa type 18 (RP18). Patients with RP18 have one of two single amino acid substitutions, Pro493Ser or Thr494Met, at the highly conserved Hprp3p C-terminal region. Pro493Ser occurs sporadically, whereas Thr494Met is observed in several unlinked RP families worldwide. The latter mutation also alters a potential recognition motif for phosphorylation by casein kinase II (CKII). To understand the molecular basis of RP18, we examined the consequences of Thr494Met mutation on Hprp3p molecular interactions with components of the U4/U6.U5 small nuclear ribonucleoprotein particles (snRNPs) complex. Since numerous mutations causing human diseases change pre-mRNA splice sites, we investigated whether Thr494Met substitution affects the processing of PRPF3 mRNA. We found that Thr494Met does not affect PRPF3 mRNA processing, indicating that the mutation may exert its effect primarily at the protein level. We used small hairpin RNAs to specifically silence the endogenous PRPF3 while simultaneously expressing HA-tagged Thr494Met. We demonstrated that the C-but not N-terminal region of Hprp3p is indeed phosphorylated by CKII in vitro and in cells. CKII-mediated Hprp3p phosphorylation was significantly reduced by Thr494Met mutation. Consequently, the Hprp3p C-terminal region is rendered partially defective in its association with itself, Hprp4p, and U4/U6 snRNA. Our findings provide new insights into the biology of Hprp3p and suggest that the loss of Hprp3p phosphorylation at Thr494 is a key step for initiating Thr494Met aberrant interactions within U4/U6 snRNP complex and that these are likely linked to the RP18 phenotype. PMID:17932117

  16. Optical Control of Peroxisomal Trafficking.

    PubMed

    Spiltoir, Jessica I; Strickland, Devin; Glotzer, Michael; Tucker, Chandra L

    2016-07-15

    The blue-light-responsive LOV2 domain of Avena sativa phototropin1 (AsLOV2) has been used to regulate activity and binding of diverse protein targets with light. Here, we used AsLOV2 to photocage a peroxisomal targeting sequence, allowing light regulation of peroxisomal protein import. We generated a protein tag, LOV-PTS1, that can be appended to proteins of interest to direct their import to the peroxisome with light. This method provides a means to inducibly trigger peroxisomal protein trafficking in specific cells at user-defined times. PMID:26513473

  17. A Novel IFITM5 Mutation in Severe Atypical Osteogenesis Imperfecta Type VI Impairs Osteoblast Production of Pigment Epithelium-Derived Factor

    PubMed Central

    Farber, Charles R; Reich, Adi; Barnes, Aileen M; Becerra, Patricia; Rauch, Frank; Cabral, Wayne A; Bae, Alison; Quinlan, Aaron; Glorieux, Francis H; Clemens, Thomas L; Marini, Joan C

    2015-01-01

    Osteogenesis imperfecta (OI) types V and VI are caused, respectively, by a unique dominant mutation in IFITM5, encoding BRIL, a transmembrane ifitm-like protein most strongly expressed in the skeletal system, and recessive null mutations in SERPINF1, encoding pigment epithelium-derived factor (PEDF). We identified a 25-year-old woman with severe OI whose dermal fibroblasts and cultured osteoblasts displayed minimal secretion of PEDF, but whose serum PEDF level was in the normal range. SERPINF1 sequences were normal despite bone histomorphometry consistent with type VI OI and elevated childhood serum alkaline phosphatase. We performed exome sequencing on the proband, both parents, and an unaffected sibling. IFITM5 emerged as the candidate gene from bioinformatics analysis, and was corroborated by membership in a murine bone co-expression network module containing all currently known OI genes. The de novo IFITM5 mutation was confirmed in one allele of the proband, resulting in a p.S40L substitution in the intracellular domain of BRIL but was absent in unaffected family members. IFITM5 expression was normal in proband fibroblasts and osteoblasts, and BRIL protein level was similar to control in differentiated proband osteoblasts on Western blot and in permeabilized mutant osteoblasts by microscopy. In contrast, SERPINF1 expression was decreased in proband osteoblasts; PEDF was barely detectable in conditioned media of proband cells. Expression and secretion of type I collagen was similarly decreased in proband osteoblasts; the expression pattern of several osteoblast markers largely overlapped reported values from cells with a primary PEDF defect. In contrast, osteoblasts from a typical case of type V OI, with an activating mutation at the 5′-terminus of BRIL, have increased SERPINF1 expression and PEDF secretion during osteoblast differentiation. Together, these data suggest that BRIL and PEDF have a relationship that connects the genes for types V and VI OI and

  18. Impaired Action Potential Initiation in GABAergic Interneurons Causes Hyperexcitable Networks in an Epileptic Mouse Model Carrying a Human NaV1.1 Mutation

    PubMed Central

    Hedrich, Ulrike B.S.; Liautard, Camille; Kirschenbaum, Daniel; Pofahl, Martin; Lavigne, Jennifer; Liu, Yuanyuan; Theiss, Stephan; Slotta, Johannes; Escayg, Andrew; Dihné, Marcel; Beck, Heinz

    2014-01-01

    Mutations in SCN1A and other ion channel genes can cause different epileptic phenotypes, but the precise mechanisms underlying the development of hyperexcitable networks are largely unknown. Here, we present a multisystem analysis of an SCN1A mouse model carrying the NaV1.1-R1648H mutation, which causes febrile seizures and epilepsy in humans. We found a ubiquitous hypoexcitability of interneurons in thalamus, cortex, and hippocampus, without detectable changes in excitatory neurons. Interestingly, somatic Na+ channels in interneurons and persistent Na+ currents were not significantly changed. Instead, the key mechanism of interneuron dysfunction was a deficit of action potential initiation at the axon initial segment that was identified by analyzing action potential firing. This deficit increased with the duration of firing periods, suggesting that increased slow inactivation, as recorded for recombinant mutated channels, could play an important role. The deficit in interneuron firing caused reduced action potential-driven inhibition of excitatory neurons as revealed by less frequent spontaneous but not miniature IPSCs. Multiple approaches indicated increased spontaneous thalamocortical and hippocampal network activity in mutant mice, as follows: (1) more synchronous and higher-frequency firing was recorded in primary neuronal cultures plated on multielectrode arrays; (2) thalamocortical slices examined by field potential recordings revealed spontaneous activities and pathological high-frequency oscillations; and (3) multineuron Ca2+ imaging in hippocampal slices showed increased spontaneous neuronal activity. Thus, an interneuron-specific generalized defect in action potential initiation causes multisystem disinhibition and network hyperexcitability, which can well explain the occurrence of seizures in the studied mouse model and in patients carrying this mutation. PMID:25378155

  19. A mutation in the mouse Amelx tri-tyrosyl domain results in impaired secretion of amelogenin and phenocopies human X-linked amelogenesis imperfecta

    PubMed Central

    Barron, Martin J.; Brookes, Steven J.; Kirkham, Jennifer; Shore, Roger C.; Hunt, Charlotte; Mironov, Aleksandr; Kingswell, Nicola J.; Maycock, Joanne; Shuttleworth, C. Adrian; Dixon, Michael J.

    2010-01-01

    Amelogenesis imperfecta (AI) describes a broad group of clinically and genetically heterogeneous inherited defects of dental enamel bio-mineralization. Despite identification of a number of genetic mutations underlying AI, the precise causal mechanisms have yet to be determined. Using a multi-disciplinary approach, we describe here a mis-sense mutation in the mouse Amelx gene resulting in a Y → H substitution in the tri-tyrosyl domain of the enamel extracellular matrix protein amelogenin. The enamel in affected animals phenocopies human X-linked AI where similar mutations have been reported. Animals affected by the mutation have severe defects of enamel bio-mineralization associated with absence of full-length amelogenin protein in the developing enamel matrix, loss of ameloblast phenotype, increased ameloblast apoptosis and formation of multi-cellular masses. We present evidence to demonstrate that affected ameloblasts express but fail to secrete full-length amelogenin leading to engorgement of the endoplasmic reticulum/Golgi apparatus. Immunohistochemical analysis revealed accumulations of both amelogenin and ameloblastin in affected cells. Co-transfection of Ambn and mutant Amelx in a eukaryotic cell line also revealed intracellular abnormalities and increased cytotoxicity compared with cells singly transfected with wild-type Amelx, mutant Amelx or Ambn or co-transfected with both wild-type Amelx and Ambn. We hypothesize that intracellular protein–protein interactions mediated via the amelogenin tri-tyrosyl motif are a key mechanistic factor underpinning the molecular pathogenesis in this example of AI. This study therefore successfully links phenotype with underlying genetic lesion in a relevant murine model for human AI. PMID:20067920

  20. Autism-associated mutations in ProSAP2/Shank3 impair synaptic transmission and neurexin-neuroligin-mediated transsynaptic signaling.

    PubMed

    Arons, Magali H; Thynne, Charlotte J; Grabrucker, Andreas M; Li, Dong; Schoen, Michael; Cheyne, Juliette E; Boeckers, Tobias M; Montgomery, Johanna M; Garner, Craig C

    2012-10-24

    Mutations in several postsynaptic proteins have recently been implicated in the molecular pathogenesis of autism and autism spectrum disorders (ASDs), including Neuroligins, Neurexins, and members of the ProSAP/Shank family, thereby suggesting that these genetic forms of autism may share common synaptic mechanisms. Initial studies of ASD-associated mutations in ProSAP2/Shank3 support a role for this protein in glutamate receptor function and spine morphology, but these synaptic phenotypes are not universally penetrant, indicating that other core facets of ProSAP2/Shank3 function must underlie synaptic deficits in patients with ASDs. In the present study, we have examined whether the ability of ProSAP2/Shank3 to interact with the cytoplasmic tail of Neuroligins functions to coordinate pre/postsynaptic signaling through the Neurexin-Neuroligin signaling complex in hippocampal neurons of Rattus norvegicus. Indeed, we find that synaptic levels of ProSAP2/Shank3 regulate AMPA and NMDA receptor-mediated synaptic transmission and induce widespread changes in the levels of presynaptic and postsynaptic proteins via Neurexin-Neuroligin transsynaptic signaling. ASD-associated mutations in ProSAP2/Shank3 disrupt not only postsynaptic AMPA and NMDA receptor signaling but also interfere with the ability of ProSAP2/Shank3 to signal across the synapse to alter presynaptic structure and function. These data indicate that ASD-associated mutations in a subset of synaptic proteins may target core cellular pathways that coordinate the functional matching and maturation of excitatory synapses in the CNS. PMID:23100419

  1. Kidney function and influence of sunlight exposure in patients with impaired 24-hydroxylation of vitamin D due to CYP24A1 mutations.

    PubMed

    Figueres, Marie-Lucile; Linglart, Agnès; Bienaime, Frank; Allain-Launay, Emma; Roussey-Kessler, Gwenaelle; Ryckewaert, Amélie; Kottler, Marie-Laure; Hourmant, Maryvonne

    2015-01-01

    Loss-of-function mutations of CYP24A1, the enzyme that converts the major circulating and active forms of vitamin D to inactive metabolites, recently have been implicated in idiopathic infantile hypercalcemia. Patients with biallelic mutations in CYP24A1 present with severe hypercalcemia and nephrocalcinosis in infancy or hypercalciuria, kidney stones, and nephrocalcinosis in adulthood. We describe a cohort of 7 patients (2 adults, 5 children) presenting with severe hypercalcemia who had homozygous or compound heterozygous mutations in CYP24A1. Acute episodes of hypercalcemia in infancy were the first symptom in 6 of 7 patients; in all patients, symptoms included nephrocalcinosis, hypercalciuria, low parathyroid hormone (PTH) levels, and higher than expected 1,25-dihydroxyvitamin D levels. Longitudinal data suggested that in most patients, periods of increased sunlight exposure tended to correlate with decreases in PTH levels and increases in calcemia and calciuria. Follow-up of the 2 adult patients showed reduced glomerular filtration rate and extrarenal manifestations, including calcic corneal deposits and osteoporosis. Cases of severe PTH-independent hypercalcemia associated with hypercalciuria in infants should prompt genetic analysis of CYP24A1. These patients should be monitored carefully throughout life because they may be at increased risk for developing chronic kidney disease. PMID:25446019

  2. Luminal expression of cubilin is impaired in Imerslund-Gräsbeck syndrome with compound AMN mutations in intron 3 and exon 7

    PubMed Central

    Namour, Fares; Dobrovoljski, Gabriele; Chery, Celine; Audonnet, Sandra; Feillet, François; Sperl, Wolfgang; Gueant, Jean-Louis

    2011-01-01

    Juvenile megaloblastic anaemia 1 (OMIM # 261100) is a rare autosomic disorder characterized by selective cobalamin mal-absorption and inconstant proteinuria produced by mutations in either CUBN or AMN genes. Amnionless, the gene product of AMN, is a transmembrane protein that binds tightly to the N-terminal end of cubilin, the gene product of CUBN. Cubilin binds to intrinsic factor-cobalamin complex and is expressed in the distal intestine and the proximal renal tubule. We report a compound AMN heterozygosity with c.742C>T, p.Gln248X and c.208-2A>G mutations in 2 siblings that led to premature termination codon in exon 7 and exon 6, respectively. It produced a dramatic decrease in receptor activity in urine, despite absence of CUBN mutation and normal affinity of the receptor for intrinsic factor binding. Heterozygous carriers for c.742T and c.208-2G had no pathological signs. These results indicate that amnionless is essential for the correct luminal expression of cubilin in humans. PMID:21750092

  3. An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes

    PubMed Central

    Barbon, Elena; Pignani, Silvia; Branchini, Alessio; Bernardi, Francesco; Pinotti, Mirko; Bovolenta, Matteo

    2016-01-01

    Tailored approaches to restore defective transcription responsible for severe diseases have been poorly explored. We tested transcription activator-like effectors fused to an activation domain (TALE-TFs) in a coagulation factor VII (FVII) deficiency model. In this model, the deficiency is caused by the −94C > G or −61T > G mutation, which abrogate the binding of Sp1 or HNF-4 transcription factors. Reporter assays in hepatoma HepG2 cells naturally expressing FVII identified a single TALE-TF (TF4) that, by targeting the region between mutations, specifically trans-activated both the variant (>100-fold) and wild-type (20–40-fold) F7 promoters. Importantly, in the genomic context of transfected HepG2 and transduced primary hepatocytes, TF4 increased F7 mRNA and protein levels (2- to 3-fold) without detectable off-target effects, even for the homologous F10 gene. The ectopic F7 expression in renal HEK293 cells was modestly affected by TF4 or by TALE-TF combinations. These results provide experimental evidence for TALE-TFs as gene-specific tools useful to counteract disease-causing promoter mutations. PMID:27341548

  4. Neuron Membrane Trafficking and Protein Kinases Involved in Autism and ADHD

    PubMed Central

    Kitagishi, Yasuko; Minami, Akari; Nakanishi, Atsuko; Ogura, Yasunori; Matsuda, Satoru

    2015-01-01

    A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention. PMID:25647412

  5. Preliminary Validation of the Sex Trafficking Attitudes Scale.

    PubMed

    Houston-Kolnik, Jaclyn D; Todd, Nathan R; Wilson, Midge

    2016-09-01

    This study presents the Sex Trafficking Attitudes Scale (STAS), assessing cognitive, behavioral, and affective attitudes toward the sex trafficking of women and girls. Across two studies, exploratory and confirmatory factor analyses revealed and confirmed six subscales: (a) Knowledge About Sex Trafficking, (b) Awareness of Sex Trafficking, (c) Attitudes Toward Ability to Leave Sex Trafficking, (d) Attitudes Toward Helping Survivors, (e) Empathic Reactions Toward Sex Trafficking, and (f) Efficacy to Reduce Sex Trafficking. Results showed support for convergent validity as the subscales were associated with related measures. The STAS holds promise to expand research and inform efforts to support trafficking survivors. PMID:26834147

  6. G Protein-coupled Receptor Kinase-mediated Phosphorylation Regulates Post-endocytic Trafficking of the D2 Dopamine Receptor*S⃞

    PubMed Central

    Namkung, Yoon; Dipace, Concetta; Javitch, Jonathan A.; Sibley, David R.

    2009-01-01

    We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D2 dopamine receptor (DAR). Agonist activation of D2 DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D2 DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [35S]GTPγS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D2 DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor. PMID:19332542

  7. Trafficking activity of myosin XXI is required in assembly of Leishmania flagellum.

    PubMed

    Katta, Santharam S; Tammana, Trinadh V Satish; Sahasrabuddhe, Amogh A; Bajpai, Virendra K; Gupta, Chhitar M

    2010-06-15

    Actin-based myosin motors have a pivotal role in intracellular trafficking in eukaryotic cells. The parasitic protozoan organism Leishmania expresses a novel class of myosin, myosin XXI (Myo21), which is preferentially localized at the proximal region of the flagellum. However, its function in this organism remains largely unknown. Here, we show that Myo21 interacts with actin, and its expression is dependent of the growth stage. We further reveal that depletion of Myo21 levels results in impairment of the flagellar assembly and intracellular trafficking. These defects are, however, reversed by episomal complementation. Additionally, it is shown that deletion of the Myo21 gene leads to generation of ploidy, suggesting an essential role of Myo21 in survival of Leishmania cells. Together, these results indicate that actin-dependent trafficking activity of Myo21 is essentially required during assembly of the Leishmania flagellum. PMID:20501700

  8. Recessive TRAPPC11 Mutations Cause a Disease Spectrum of Limb Girdle Muscular Dystrophy and Myopathy with Movement Disorder and Intellectual Disability

    PubMed Central

    Bögershausen, Nina; Shahrzad, Nassim; Chong, Jessica X.; von Kleist-Retzow, Jürgen-Christoph; Stanga, Daniela; Li, Yun; Bernier, Francois P.; Loucks, Catrina M.; Wirth, Radu; Puffenberger, Eric G.; Hegele, Robert A.; Schreml, Julia; Lapointe, Gabriel; Keupp, Katharina; Brett, Christopher L.; Anderson, Rebecca; Hahn, Andreas; Innes, A. Micheil; Suchowersky, Oksana; Mets, Marilyn B.; Nürnberg, Gudrun; McLeod, D. Ross; Thiele, Holger; Waggoner, Darrel; Altmüller, Janine; Boycott, Kym M.; Schoser, Benedikt; Nürnberg, Peter; Ober, Carole; Heller, Raoul; Parboosingh, Jillian S.; Wollnik, Bernd; Sacher, Michael; Lamont, Ryan E.

    2013-01-01

    Myopathies are a clinically and etiologically heterogeneous group of disorders that can range from limb girdle muscular dystrophy (LGMD) to syndromic forms with associated features including intellectual disability. Here, we report the identification of mutations in transport protein particle complex 11 (TRAPPC11) in three individuals of a consanguineous Syrian family presenting with LGMD and in five individuals of Hutterite descent presenting with myopathy, infantile hyperkinetic movements, ataxia, and intellectual disability. By using a combination of whole-exome or genome sequencing with homozygosity mapping, we identified the homozygous c.2938G>A (p.Gly980Arg) missense mutation within the gryzun domain of TRAPPC11 in the Syrian LGMD family and the homozygous c.1287+5G>A splice-site mutation resulting in a 58 amino acid in-frame deletion (p.Ala372_Ser429del) in the foie gras domain of TRAPPC11 in the Hutterite families. TRAPPC11 encodes a component of the multiprotein TRAPP complex involved in membrane trafficking. We demonstrate that both mutations impair the binding ability of TRAPPC11 to other TRAPP complex components and disrupt the Golgi apparatus architecture. Marker trafficking experiments for the p.Ala372_Ser429del deletion indicated normal ER-to-Golgi trafficking but dramatically delayed exit from the Golgi to the cell surface. Moreover, we observed alterations of the lysosomal membrane glycoproteins lysosome-associated membrane protein 1 (LAMP1) and LAMP2 as a consequence of TRAPPC11 dysfunction supporting a defect in the transport of secretory proteins as the underlying pathomechanism. PMID:23830518

  9. Recessive TRAPPC11 mutations cause a disease spectrum of limb girdle muscular dystrophy and myopathy with movement disorder and intellectual disability.

    PubMed

    Bögershausen, Nina; Shahrzad, Nassim; Chong, Jessica X; von Kleist-Retzow, Jürgen-Christoph; Stanga, Daniela; Li, Yun; Bernier, Francois P; Loucks, Catrina M; Wirth, Radu; Puffenberger, Eric G; Hegele, Robert A; Schreml, Julia; Lapointe, Gabriel; Keupp, Katharina; Brett, Christopher L; Anderson, Rebecca; Hahn, Andreas; Innes, A Micheil; Suchowersky, Oksana; Mets, Marilyn B; Nürnberg, Gudrun; McLeod, D Ross; Thiele, Holger; Waggoner, Darrel; Altmüller, Janine; Boycott, Kym M; Schoser, Benedikt; Nürnberg, Peter; Ober, Carole; Heller, Raoul; Parboosingh, Jillian S; Wollnik, Bernd; Sacher, Michael; Lamont, Ryan E

    2013-07-11

    Myopathies are a clinically and etiologically heterogeneous group of disorders that can range from limb girdle muscular dystrophy (LGMD) to syndromic forms with associated features including intellectual disability. Here, we report the identification of mutations in transport protein particle complex 11 (TRAPPC11) in three individuals of a consanguineous Syrian family presenting with LGMD and in five individuals of Hutterite descent presenting with myopathy, infantile hyperkinetic movements, ataxia, and intellectual disability. By using a combination of whole-exome or genome sequencing with homozygosity mapping, we identified the homozygous c.2938G>A (p.Gly980Arg) missense mutation within the gryzun domain of TRAPPC11 in the Syrian LGMD family and the homozygous c.1287+5G>A splice-site mutation resulting in a 58 amino acid in-frame deletion (p.Ala372_Ser429del) in the foie gras domain of TRAPPC11 in the Hutterite families. TRAPPC11 encodes a component of the multiprotein TRAPP complex involved in membrane trafficking. We demonstrate that both mutations impair the binding ability of TRAPPC11 to other TRAPP complex components and disrupt the Golgi apparatus architecture. Marker trafficking experiments for the p.Ala372_Ser429del deletion indicated normal ER-to-Golgi trafficking but dramatically delayed exit from the Golgi to the cell surface. Moreover, we observed alterations of the lysosomal membrane glycoproteins lysosome-associated membrane protein 1 (LAMP1) and LAMP2 as a consequence of TRAPPC11 dysfunction supporting a defect in the transport of secretory proteins as the underlying pathomechanism. PMID:23830518

  10. Sequence and structure-specific elements of HERG mRNA determine channel synthesis and trafficking efficiency

    PubMed Central

    Sroubek, Jakub; Krishnan, Yamini; McDonald, Thomas V.

    2013-01-01

    Human ether-á-gogo-related gene (HERG) encodes a potassium channel that is highly susceptible to deleterious mutations resulting in susceptibility to fatal cardiac arrhythmias. Most mutations adversely affect HERG channel assembly and trafficking. Why the channel is so vulnerable to missense mutations is not well understood. Since nothing is known of how mRNA structural elements factor in channel processing, we synthesized a codon-modified HERG cDNA (HERG-CM) where the codons were synonymously changed to reduce GC content, secondary structure, and rare codon usage. HERG-CM produced typical IKr-like currents; however, channel synthesis and processing were markedly different. Translation efficiency was reduced for HERG-CM, as determined by heterologous expression, in vitro translation, and polysomal profiling. Trafficking efficiency to the cell surface was greatly enhanced, as assayed by immunofluorescence, subcellular fractionation, and surface labeling. Chimeras of HERG-NT/CM indicated that trafficking efficiency was largely dependent on 5′ sequences, while translation efficiency involved multiple areas. These results suggest that HERG translation and trafficking rates are independently governed by noncoding information in various regions of the mRNA molecule. Noncoding information embedded within the mRNA may play a role in the pathogenesis of hereditary arrhythmia syndromes and could provide an avenue for targeted therapeutics.—Sroubek, J., Krishnan, Y., McDonald, T V. Sequence- and structure-specific elements of HERG mRNA determine channel synthesis and trafficking efficiency. PMID:23608144

  11. Examining the Risk of Nuclear Trafficking

    SciTech Connect

    Balatsky, Galya; Severe, William R; Schoeneck, Jeffery

    2009-01-01

    The need to stop illicit trafficking of nuclear and radioactive materials around the world is undeniable and urgent. This issue is particularly evident due to the highly dangerous consequences of the risks involved, the known interest of terrorist groups in acquiring such materials and the vulnerability of theft and diversion of such materials. Yet the phenomenon of nuclear trafficking remains a subject where the unknown dominates what is known on the subject. The trafficking panel at the Institute for Nuclear Materials Management (INMM) Workshop on Reducing the Risk of Radioactive and Nuclear Materials that took place in Albuquerque, New Mexico, March 10-11, 2009, dealt with some of the issues associated with nuclear trafficking. Different points of view on how to better address trafficking and thwart perpetrator efforts were discussed. This paper presents some of these views and addresses practical measures that should be considered to improve the situation.

  12. A point mutation at tyrosine-809 in the human colony-stimulating factor 1 receptor impairs mitogenesis without abrogating tyrosine kinase activity, association with phosphatidylinositol 3-kinase, or induction of c-fos and junB genes

    SciTech Connect

    Roussel, M.F. ); Shurtleff, S.A.; Downing, J.R. ); Sherr, C.J. Saint Jude Children's Research Hospital, Memphis, TN )

    1990-09-01

    Substitution of phenylalanine for tyrosine-809 in the human colony-stimulating factor 1 receptor (CSF-1R) inhibited its ability to transduce ligand-dependent mitogenic signals in mouse NIH 3T3 cells. When combined with an activating mutation at codon 301 that induces constitutive CSF-1R tyrosine kinase activity, the codon 809 mutation suppressed ligand-independent cell transformation. Comparative mapping tryptic phosphopeptides from mutant and wild-type CSF-1R indicated that tyrosine-809 is a site of ligand-dependent receptor phosphorylation in vivo. The mutant receptor was active as a tyrosine kinase in vitro and in vivo, underwent CSF-1-dependent association with a phosphatidylinositol 3-kinase, and induced expression of the protooncogenes c-fos and junB, underscoring its ability to trigger some of the known cellular responses to CSF-1. The mutant receptor is likely to be impaired in its ability to interact with critical cellular effectors whose activity is required for mitogenesis.

  13. Selective expression of Parkinson's disease-related Leucine-rich repeat kinase 2 G2019S missense mutation in midbrain dopaminergic neurons impairs dopamine release and dopaminergic gene expression.

    PubMed

    Liu, Guoxiang; Sgobio, Carmelo; Gu, Xinglong; Sun, Lixin; Lin, Xian; Yu, Jia; Parisiadou, Loukia; Xie, Chengsong; Sastry, Namratha; Ding, Jinhui; Lohr, Kelly M; Miller, Gary W; Mateo, Yolanda; Lovinger, David M; Cai, Huaibin

    2015-09-15

    Preferential dysfunction/degeneration of midbrain substantia nigra pars compacta (SNpc) dopaminergic (DA) neurons contributes to the main movement symptoms manifested in Parkinson's disease (PD). Although the Leucine-rich repeat kinase 2 (LRRK2) G2019S missense mutation (LRRK2 G2019S) is the most common causative genetic factor linked to PD, the effects of LRRK2 G2019S on the function and survival of SNpc DA neurons are poorly understood. Using a binary gene expression system, we generated transgenic mice expressing either wild-type human LRRK2 (WT mice) or the LRRK2 G2019S mutation (G2019S mice) selectively in the midbrain DA neurons. Here we show that overexpression of LRRK2 G2019S did not induce overt motor abnormalities or substantial SNpc DA neuron loss. However, the LRRK2 G2019S mutation impaired dopamine homeostasis and release in aged mice. This reduction in dopamine content/release coincided with the degeneration of DA axon terminals and decreased expression of DA neuron-enriched genes tyrosine hydroxylase (TH), vesicular monoamine transporter 2, dopamine transporter and aldehyde dehydrogenase 1. These factors are responsible for dopamine synthesis, transport and degradation, and their expression is regulated by transcription factor paired-like homeodomain 3 (PITX3). Levels of Pitx3 mRNA and protein were similarly decreased in the SNpc DA neurons of aged G2019S mice. Together, these findings suggest that PITX3-dependent transcription regulation could be one of the many potential mechanisms by which LRRK2 G2019S acts in SNpc DA neurons, resulting in downregulation of its downstream target genes critical for dopamine homeostasis and release. PMID:26123485

  14. Heterozygous Mutation in IκBNS Leads to Reduced Levels of Natural IgM Antibodies and Impaired Responses to T-Independent Type 2 Antigens

    PubMed Central

    Pedersen, Gabriel K.; Ádori, Monika; Stark, Julian M.; Khoenkhoen, Sharesta; Arnold, Carrie; Beutler, Bruce; Karlsson Hedestam, Gunilla B.

    2016-01-01

    Mice deficient in central components of classical NF-κB signaling have low levels of circulating natural IgM antibodies and fail to respond to immunization with T-independent type 2 (TI-2) antigens. A plausible explanation for these defects is the severely reduced numbers of B-1 and marginal zone B (MZB) cells in such mice. By using an ethyl-N-nitrosourea mutagenesis screen, we identified a role for the atypical IκB protein IκBNS in humoral immunity. IκBNS-deficient mice lack B-1 cells and have severely reduced numbers of MZB cells, and thus resemble several other strains with defects in classical NF-κB signaling. We analyzed mice heterozygous for the identified IκBNS mutation and demonstrate that these mice have an intermediary phenotype in terms of levels of circulating IgM antibodies and responses to TI-2 antigens. However, in contrast to mice that are homozygous for the IκBNS mutation, the heterozygous mice had normal frequencies of B-1 and MZB cells. These results suggest that there is a requirement for IκBNS expression from two functional alleles for maintaining normal levels of circulating natural IgM antibodies and responses to TI-2 antigens. PMID:26973645

  15. The mechanism by which P250L mutation impairs flavivirus-NS1 dimerization: an investigation based on molecular dynamics simulations.

    PubMed

    Oliveira, Edson R A; de Alencastro, Ricardo B; Horta, Bruno A C

    2016-09-01

    The flavivirus non-structural protein 1 (NS1) is a conserved glycoprotein with as yet undefined biological function. This protein dimerizes when inside infected cells or associated to cell membranes but also forms lipid-associated hexamers when secreted to the extracellular space. A single amino acid substitution (P250L) is capable of preventing the dimerization of NS1 resulting in lower virulence and slower virus replication. In this work, based on molecular dynamics simulations of the dengue-2 virus NS1 [Formula: see text]-ladder monomer as a core model, we found that this mutation can induce several conformational changes that importantly affect critical monomer-monomer interactions. Based on additional simulations, we suggest a mechanism by which a highly orchestrated sequence of events propagate the local perturbations around the mutation site towards the dimer interface. The elucidation of such a mechanism could potentially support new strategies for rational production of live-attenuated vaccines and highlights a step forward in the development of novel anti-flavivirus measures. PMID:27324799

  16. Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair.

    PubMed

    Nakazawa, Yuka; Sasaki, Kensaku; Mitsutake, Norisato; Matsuse, Michiko; Shimada, Mayuko; Nardo, Tiziana; Takahashi, Yoshito; Ohyama, Kaname; Ito, Kosei; Mishima, Hiroyuki; Nomura, Masayo; Kinoshita, Akira; Ono, Shinji; Takenaka, Katsuya; Masuyama, Ritsuko; Kudo, Takashi; Slor, Hanoch; Utani, Atsushi; Tateishi, Satoshi; Yamashita, Shunichi; Stefanini, Miria; Lehmann, Alan R; Yoshiura, Koh-ichiro; Ogi, Tomoo

    2012-05-01

    UV-sensitive syndrome (UV(S)S) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma. Despite mild clinical features, cells from individuals with UV(S)S, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER), which removes DNA damage in actively transcribed genes. Three of the seven known UV(S)S cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively). The remaining four individuals with UVSS , one of whom is described for the first time here, formed a separate UV(S)S-A complementation group; however, the responsible gene was unknown. Using exome sequencing, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UV(S)S-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER–deficient disorders. PMID:22466610

  17. Disease-associated mutations in the extracytoplasmic loops of cystic fibrosis transmembrane conductance regulator do not impede biosynthetic processing but impair chloride channel stability.

    PubMed

    Hämmerle, M M; Aleksandrov, A A; Riordan, J R

    2001-05-01

    Consistent with its function as a chloride channel regulated entirely from the cytoplasmic side of the plasma membrane, the cystic fibrosis transmembrane conductance regulator (CFTR) glycoprotein exposes little of its mass on the exterior surface of cells. The first and fourth extracytoplasmic loops (ELs) contain approximately 15 and 30 residues, respectively; the other four ELs are extremely short. To examine the influence of missense mutants in ELs detected in patients with cystic fibrosis, we have expressed them in mammalian (baby hamster kidney (BHK21)) cells and assessed their biosynthetic processing and chloride channel activity. In contrast to previous findings that 18 of 30 disease-associated missense mutations in cytoplasmic loops caused retention of the nascent polypeptides in the endoplasmic reticulum, all the EL mutants studied matured and were transported to the cell surface. This pronounced asymmetry is consistent with the notion that endoplasmic reticulum quality control of nascent CFTR is exerted primarily on the cytoplasmic side of the membrane. Although this set of EL mutations has little effect on CFTR maturation, most of them seriously compromise its chloride channel activity. Substitutions at six different positions in EL1 and single positions in EL2 and EL4 all destabilized the open state, some of them severely, indicating that the ELs contribute to the stability of the CFTR ion pore. PMID:11278813

  18. Regulation of membrane trafficking by signalling on endosomal and lysosomal membranes

    PubMed Central

    Li, Xinran; Garrity, Abigail G; Xu, Haoxing

    2013-01-01

    Endosomal and lysosomal membrane trafficking requires the coordination of multiple signalling events to control cargo sorting and processing, and endosome maturation. The initiation and termination of signalling events in endosomes and lysosomes is not well understood, but several key regulators have been identified, which include small GTPases, phosphoinositides, and Ca2+. Small GTPases act as master regulators and molecular switches in a GTP-dependent manner, initiating signalling cascades to regulate the direction and specificity of endosomal trafficking. Phosphoinositides are membrane-bound lipids that indicate vesicular identities for recruiting specific cytoplasmic proteins to endosomal membranes, thus allowing specificity of membrane fusion, fission, and cargo sorting to occur within and between specific vesicle compartments. In addition, phosphoinositides regulate the function of membrane proteins such as ion channels and transporters in a compartment-specific manner to mediate transport and signalling. Finally, Ca2+, a locally acting second messenger released from intracellular ion channels, may provide precise spatiotemporal regulation of endosomal signalling and trafficking events. Small GTPase signalling can regulate phosphoinositide conversion during endosome maturation, and electrophysiological studies on isolated endosomes have shown that endosomal and lysosomal Ca2+ channels are directly modulated by endosomal lipids. Thus trafficking and maturation of endosomes and lysosomes can be precisely regulated by dynamic changes in GTPases and membrane lipids, as well as Ca2+ signalling. Importantly, impaired phosphoinositide and Ca2+ signalling can cause endosomal and lysosomal trafficking defects at the cellular level, and a spectrum of lysosome storage diseases. PMID:23878375

  19. Toll-like receptor 9 trafficking and signaling for type I interferons requires PIKfyve activity.

    PubMed

    Hayashi, Kachiko; Sasai, Miwa; Iwasaki, Akiko

    2015-09-01

    Toll-like receptors (TLRs) traffic to distinct membranes for signaling. TLR7 and TLR9 recognize viral nucleic acids in the endosomes and induce robust anti-viral program. Signaling from these TLRs bifurcate at the level of distinct endosomal compartments, namely VAMP3(+) and LAMP(+) endosomes, to mediate the induction of cytokine and type I interferon (IFN) genes, respectively. The formation of the TLR9 endosome competent for IFNs induction requires AP-3. Phosphoinositides (PIs) mark distinct subcellular membranes and control membrane trafficking. However, their role in TLR trafficking and signaling in different dendritic cell (DC) subsets remains unclear. Here, we examined the role of phosphatidylinositol 3P 5-kinase, PIKfyve, in TLR9 trafficking and signaling. We demonstrate that inhibition of PIKfyve activity preferentially blocks TLR9 signaling for type I IFN induction in FLT3L-bone marrow-derived DCs. By confocal microscopy using RAW264.7 cells, we show that trafficking of both TLR9 and CpG to the LAMP1(+) compartment was blocked by PIKfyve inhibitor treatment, whereas their trafficking to the VAMP3(+) endosome remained intact. Further, AP-3 recruitment to TLR9 endosomes was impaired by PIKfyve inhibition. These data indicate that PIKfyve provides critical PIs necessary for the formation of endosome from which TLR9 signals to induce type I IFNs. PMID:25925170

  20. Chemokine receptor internalization and intracellular trafficking.

    PubMed

    Neel, Nicole F; Schutyser, Evemie; Sai, Jiqing; Fan, Guo-Huang; Richmond, Ann

    2005-12-01

    The internalization and intracellular trafficking of chemokine receptors have important implications for the cellular responses elicited by chemokine receptors. The major pathway by which chemokine receptors internalize is the clathrin-mediated pathway, but some receptors may utilize lipid rafts/caveolae-dependent internalization routes. This review discusses the current knowledge and controversies regarding these two different routes of endocytosis. The functional consequences of internalization and the regulation of chemokine receptor recycling will also be addressed. Modifications of chemokine receptors, such as palmitoylation, ubiquitination, glycosylation, and sulfation, may also impact trafficking, chemotaxis and signaling. Finally, this review will cover the internalization and trafficking of viral and decoy chemokine receptors. PMID:15998596

  1. Female sex trafficking: conceptual issues, current debates, and future directions.

    PubMed

    Meshkovska, Biljana; Siegel, Melissa; Stutterheim, Sarah E; Bos, Arjan E R

    2015-01-01

    Female sex trafficking is a pressing concern. In this article, we provide a comprehensive overview of relevant issues regarding the concept of female sex trafficking and research in the field of human trafficking, drawing on a variety of disciplines, including economics, gender and sexuality studies, psychology, sociology, law, and social work. We discuss the debates surrounding the definition of human trafficking, compare and contrast it with human smuggling, and outline connections between female sex trafficking and the issue of sex work and prostitution. We further discuss the history and current estimations of female sex trafficking. We then outline the main actors in female sex trafficking, including trafficked persons, traffickers, clients, and service providers, and we overview the trafficking process from recruitment to identification, recovery, and (re)integration. Finally, we conclude with recommendations for future research that tie together the concepts of vulnerability, exploitation, and long-term recovery and (re)integration. PMID:25897567

  2. Sex Trafficking of Women and Girls

    PubMed Central

    Deshpande, Neha A; Nour, Nawal M

    2013-01-01

    Sex trafficking involves some form of forced or coerced sexual exploitation that is not limited to prostitution, and has become a significant and growing problem in both the United States and the larger global community. The costs to society include the degradation of human and women’s rights, poor public health, disrupted communities, and diminished social development. Victims of sex trafficking acquire adverse physical and psychological health conditions and social disadvantages. Thus, sex trafficking is a critical health issue with broader social implications that requires both medical and legal attention. Healthcare professionals can work to improve the screening, identification, and assistance of victims of sex trafficking in a clinical setting and help these women and girls access legal and social services. PMID:23687554

  3. Committee opinion no. 507: human trafficking.

    PubMed

    2011-09-01

    Human trafficking is a widespread problem with estimates ranging from 14,000 to 50,000 individuals trafficked into the United States annually. This hidden population involves the commercial sex industry, agriculture, factories, hotel and restaurant businesses, domestic workers, marriage brokers, and some adoption firms. Because 80% of trafficked individuals are women and girls, women’s health care providers may better serve their diverse patient population by increasing their awareness of this problem. The exploitation of people of any race, gender, sexual orientation, or ethnicity is unacceptable at any time, in any place. The members of the American College of Obstetricians and Gynecologists should be aware of this problem and strive to recognize and assist their patients who are victims or who have been victims of human trafficking. PMID:21860320

  4. Ovarian Cystadenoma in a Trafficked Patient.

    PubMed

    Titchen, Kanani E; Katz, Douglas; Martinez, Kidian; White, Krishna

    2016-05-01

    The topic of child sex trafficking is receiving increased attention both in the lay press and in research articles. Recently, a number of physician organizations have issued policy statements calling for the education and involvement of physicians in combating this form of "modern-day slavery." Primary care and emergency medicine physicians have led these efforts, but a number of these victims may present to surgeons. Surgeons are in a unique position to identify trafficked patients; during the process of undraping, intubation, and surgical preparation, signs of trafficking such as tattoos, scars, dental injuries, and bruising may be evident. In addition, these patients may have specific needs in terms of anesthesia and postoperative care due to substance abuse. Here, we report the case of an 18-year-old girl with a history of sexual exploitation who presents for cystadenoma excision. To our knowledge, this is the first report of a sex-trafficked pediatric patient presenting for surgery. PMID:27244785

  5. Human trafficking law and social structures.

    PubMed

    Wooditch, Alese

    2012-08-01

    Human trafficking has only recently emerged at the forefront of policy reform, even in developed nations. Yet, heightened awareness of the issue has not translated into effective policy as the majority of nations have ineffective antitrafficking practices; many countries have failed to criminalize human trafficking, whereas others do not actively enforce statutes in place. By applying Black's theory of law, this study offers a preliminary understanding into the variation of global prosecutorial efforts in human trafficking and adequacy of antitrafficking law. To isolate this relationship, the effects of trafficking markets are controlled. As with prior research, the study finds limited support for the theory. The article concludes with a discussion on the implications of the quantity of antitrafficking law and morphology association for policy development. PMID:21948250

  6. Sex trafficking of women and girls.

    PubMed

    Deshpande, Neha A; Nour, Nawal M

    2013-01-01

    Sex trafficking involves some form of forced or coerced sexual exploitation that is not limited to prostitution, and has become a significant and growing problem in both the United States and the larger global community. The costs to society include the degradation of human and women's rights, poor public health, disrupted communities, and diminished social development. Victims of sex trafficking acquire adverse physical and psychological health conditions and social disadvantages. Thus, sex trafficking is a critical health issue with broader social implications that requires both medical and legal attention. Healthcare professionals can work to improve the screening, identification, and assistance of victims of sex trafficking in a clinical setting and help these women and girls access legal and social services. PMID:23687554

  7. Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules.

    PubMed

    Pulipparacharuvil, Suprabha; Akbar, Mohammed Ali; Ray, Sanchali; Sevrioukov, Evgueny A; Haberman, Adam S; Rohrer, Jack; Krämer, Helmut

    2005-08-15

    Mutations that disrupt trafficking to lysosomes and lysosome-related organelles cause multiple diseases, including Hermansky-Pudlak syndrome. The Drosophila eye is a model system for analyzing such mutations. The eye-color genes carnation and deep orange encode two subunits of the Vps-C protein complex required for endosomal trafficking and pigment-granule biogenesis. Here we demonstrate that dVps16A (CG8454) encodes another Vps-C subunit. Biochemical experiments revealed a specific interaction between the dVps16A C-terminus and the Sec1/Munc18 homolog Carnation but not its closest homolog, dVps33B. Instead, dVps33B interacted with a related protein, dVps16B (CG18112). Deep orange bound both Vps16 homologs. Like a deep orange null mutation, eye-specific RNAi-induced knockdown of dVps16A inhibited lysosomal delivery of internalized ligands and interfered with biogenesis of pigment granules. Ubiquitous knockdown of dVps16A was lethal. Together, these findings demonstrate that Drosophila Vps16A is essential for lysosomal trafficking. Furthermore, metazoans have two types of Vps-C complexes with non-redundant functions. PMID:16046475

  8. A Dominant Mutation in the Light-Oxygen and Voltage2 Domain Vicinity Impairs Phototropin1 Signaling in Tomato1[C][W][OPEN

    PubMed Central

    Sharma, Sulabha; Kharshiing, Eros; Srinivas, Ankanagari; Zikihara, Kazunori; Tokutomi, Satoru; Nagatani, Akira; Fukayama, Hiroshi; Bodanapu, Reddaiah; Behera, Rajendra K.; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2014-01-01

    In higher plants, blue light (BL) phototropism is primarily controlled by the phototropins, which are also involved in stomatal movement and chloroplast relocation. These photoresponses are mediated by two phototropins, phot1 and phot2. Phot1 mediates responses with higher sensitivity than phot2, and phot2 specifically mediates chloroplast avoidance and dark positioning responses. Here, we report the isolation and characterization of a Nonphototropic seedling1 (Nps1) mutant of tomato (Solanum lycopersicum). The mutant is impaired in low-fluence BL responses, including chloroplast accumulation and stomatal opening. Genetic analyses show that the mutant locus is dominant negative in nature. In dark-grown seedlings of the Nps1 mutant, phot1 protein accumulates at a highly reduced level relative to the wild type and lacks BL-induced autophosphorylation. The mutant harbors a single glycine-1484-to-alanine transition in the Hinge1 region of a phot1 homolog, resulting in an arginine-to-histidine substitution (R495H) in a highly conserved A′α helix proximal to the light-oxygen and voltage2 domain of the translated gene product. Significantly, the R495H substitution occurring in the Hinge1 region of PHOT1 abolishes its regulatory activity in Nps1 seedlings, thereby highlighting the functional significance of the A′α helix region in phototropic signaling of tomato. PMID:24515830

  9. Transmembrane protein OSTA-1 shapes sensory cilia morphology via regulation of intracellular membrane trafficking in C. elegans

    PubMed Central

    Olivier-Mason, Anique; Wojtyniak, Martin; Bowie, Rachel V.; Nechipurenko, Inna V.; Blacque, Oliver E.; Sengupta, Piali

    2013-01-01

    The structure and function of primary cilia are critically dependent on intracellular trafficking pathways that transport ciliary membrane and protein components. The mechanisms by which these trafficking pathways are regulated are not fully characterized. Here we identify the transmembrane protein OSTA-1 as a new regulator of the trafficking pathways that shape the morphology and protein composition of sensory cilia in C. elegans. osta-1 encodes an organic solute transporter alpha-like protein, mammalian homologs of which have been implicated in membrane trafficking and solute transport, although a role in regulating cilia structure has not previously been demonstrated. We show that mutations in osta-1 result in altered ciliary membrane volume, branch length and complexity, as well as defects in localization of a subset of ciliary transmembrane proteins in different sensory cilia types. OSTA-1 is associated with transport vesicles, localizes to a ciliary compartment shown to house trafficking proteins, and regulates both retrograde and anterograde flux of the endosome-associated RAB-5 small GTPase. Genetic epistasis experiments with sensory signaling, exocytic and endocytic proteins further implicate OSTA-1 as a crucial regulator of ciliary architecture via regulation of cilia-destined trafficking. Our findings suggest that regulation of transport pathways in a cell type-specific manner contributes to diversity in sensory cilia structure and might allow dynamic remodeling of ciliary architecture via multiple inputs. PMID:23482491

  10. The Intracellular Trafficking Pathway of Transferrin

    PubMed Central

    Mayle, Kristine M.; Le, Alexander M.; Kamei, Daniel T.

    2011-01-01

    Background Transferrin (Tf) is an iron-binding protein that facilitates iron-uptake in cells. Iron-loaded Tf first binds to the Tf receptor (TfR) and enters the cell through clathrin-mediated endocytosis. Inside the cell, Tf is trafficked to early endosomes, delivers iron, and then is subsequently directed to recycling endosomes to be taken back to the cell surface. Scope of Review We aim to review the various methods and techniques that researchers have employed for elucidating the Tf trafficking pathway and the cell-machinery components involved. These experimental methods can be categorized as microscopy, radioactivity, and surface plasmon resonance (SPR). Major Conclusions Qualitative experiments, such as total internal reflectance fluorescence (TIRF), electron, laser-scanning confocal, and spinning-disk confocal microscopy, have been utilized to determine the roles of key components in the Tf trafficking pathway. These techniques allow temporal resolution and are useful for imaging Tf endocytosis and recycling, which occur on the order of seconds to minutes. Additionally, radiolabeling and SPR methods, when combined with mathematical modeling, have enabled researchers to estimate quantitative kinetic parameters and equilibrium constants associated with Tf binding and trafficking. General Significance Both qualitative and quantitative data can be used to analyze the Tf trafficking pathway. The valuable information that is obtained about the Tf trafficking pathway can then be combined with mathematical models to identify design criteria to improve the ability of Tf to deliver anticancer drugs. PMID:21968002

  11. RNA trafficking in parasitic plant systems.

    PubMed

    Leblanc, Megan; Kim, Gunjune; Westwood, James H

    2012-01-01

    RNA trafficking in plants contributes to local and long-distance coordination of plant development and response to the environment. However, investigations of mobile RNA identity and function are hindered by the inherent difficulty of tracing a given molecule of RNA from its cell of origin to its destination. Several methods have been used to address this problem, but all are limited to some extent by constraints associated with accurately sampling phloem sap or detecting trafficked RNA. Certain parasitic plant species form symplastic connections to their hosts and thereby provide an additional system for studying RNA trafficking. The haustorial connections of Cuscuta and Phelipanche species are similar to graft junctions in that they are able to transmit mRNAs, viral RNAs, siRNAs, and proteins from the host plants to the parasite. In contrast to other graft systems, these parasites form connections with host species that span a wide phylogenetic range, such that a high degree of nucleotide sequence divergence may exist between host and parasites and allow confident identification of most host RNAs in the parasite system. The ability to identify host RNAs in parasites, and vice versa, will facilitate genomics approaches to understanding RNA trafficking. This review discusses the nature of host-parasite connections and the potential significance of host RNAs for the parasite. Additional research on host-parasite interactions is needed to interpret results of RNA trafficking studies, but parasitic plants may provide a fascinating new perspective on RNA trafficking. PMID:22936942

  12. Folding and Rescue of a Cystic Fibrosis Transmembrane Conductance Regulator Trafficking Mutant Identified Using Human-Murine Chimeric Proteins*

    PubMed Central

    Da Paula, Ana Carina; Sousa, Marisa; Xu, Zhe; Dawson, Elizabeth S.; Boyd, A. Christopher; Sheppard, David N.; Amaral, Margarida D.

    2010-01-01

    Impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel causes cystic fibrosis, a fatal genetic disease. Here, to gain insight into CFTR structure and function, we exploited interspecies differences between CFTR homologues using human (h)-murine (m) CFTR chimeras containing murine nucleotide-binding domains (NBDs) or regulatory domain on an hCFTR backbone. Among 15 hmCFTR chimeras analyzed, all but two were correctly processed, one containing part of mNBD1 and another containing part of mNBD2. Based on physicochemical distance analysis of divergent residues between human and murine CFTR in the two misprocessed hmCFTR chimeras, we generated point mutations for analysis of respective CFTR processing and functional properties. We identified one amino acid substitution (K584E-CFTR) that disrupts CFTR processing in NBD1. No single mutation was identified in NBD2 that disrupts protein processing. However, a number of NBD2 mutants altered channel function. Analysis of structural models of CFTR identified that although Lys584 interacts with residue Leu581 in human CFTR Glu584 interacts with Phe581 in mouse CFTR. Introduction of the murine residue (Phe581) in cis with K584E in human CFTR rescued the processing and trafficking defects of K584E-CFTR. Our data demonstrate that human-murine CFTR chimeras may be used to validate structural models of full-length CFTR. We also conclude that hmCFTR chimeras are a valuable tool to elucidate interactions between different domains of CFTR. PMID:20551307

  13. LULL1 Retargets TorsinA to the Nuclear Envelope Revealing an Activity That Is Impaired by the DYT1 Dystonia Mutation

    PubMed Central

    Vander Heyden, Abigail B.; Naismith, Teresa V.; Snapp, Erik L.; Hodzic, Didier

    2009-01-01

    TorsinA (TorA) is an AAA+ ATPase in the endoplasmic reticulum (ER) lumen that is mutated in early onset DYT1 dystonia. TorA is an essential protein in mice and is thought to function in the nuclear envelope (NE) despite localizing throughout the ER. Here, we report that transient interaction of TorA with the ER membrane protein LULL1 targets TorA to the NE. FRAP and Blue Native PAGE indicate that TorA is a stable, slowly diffusing oligomer in either the absence or presence of LULL1. Increasing LULL1 expression redistributes both wild-type and disease-mutant TorA to the NE, while decreasing LULL1 with shRNAs eliminates intrinsic enrichment of disease-mutant TorA in the NE. When concentrated in the NE, TorA displaces the nuclear membrane proteins Sun2, nesprin-2G, and nesprin-3 while leaving nuclear pores and Sun1 unchanged. Wild-type TorA also induces changes in NE membrane structure. Because SUN proteins interact with nesprins to connect nucleus and cytoskeleton, these effects suggest a new role for TorA in modulating complexes that traverse the NE. Importantly, once concentrated in the NE, disease-mutant TorA displaces Sun2 with reduced efficiency and does not change NE membrane structure. Together, our data suggest that LULL1 regulates the distribution and activity of TorA within the ER and NE lumen and reveal functional defects in the mutant protein responsible for DYT1 dystonia. PMID:19339278

  14. A mutation deleting sequences encoding the amino terminus of human cytomegalovirus UL84 impairs interaction with UL44 and capsid localization.

    PubMed

    Strang, Blair L; Bender, Brian J; Sharma, Mayuri; Pesola, Jean M; Sanders, Rebecca L; Spector, Deborah H; Coen, Donald M

    2012-10-01

    Protein-protein interactions are required for many biological functions. Previous work has demonstrated an interaction between the human cytomegalovirus DNA polymerase subunit UL44 and the viral replication factor UL84. In this study, glutathione S-transferase pulldown assays indicated that residues 1 to 68 of UL84 are both necessary and sufficient for efficient interaction of UL84 with UL44 in vitro. We created a mutant virus in which sequences encoding these residues were deleted. This mutant displayed decreased virus replication compared to wild-type virus. Immunoprecipitation assays showed that the mutation decreased but did not abrogate association of UL84 with UL44 in infected cell lysate, suggesting that the association in the infected cell can involve other protein-protein interactions. Further immunoprecipitation assays indicated that IRS1, TRS1, and nucleolin are candidates for such interactions in infected cells. Quantitative real-time PCR analysis of viral DNA indicated that the absence of the UL84 amino terminus does not notably affect viral DNA synthesis. Western blotting experiments and pulse labeling of infected cells with [(35)S]methionine demonstrated a rather modest downregulation of levels of multiple proteins and particularly decreased levels of the minor capsid protein UL85. Electron microscopy demonstrated that viral capsids assemble but are mislocalized in nuclei of cells infected with the mutant virus, with fewer cytoplasmic capsids detected. In sum, deletion of the sequences encoding the amino terminus of UL84 affects interaction with UL44 and virus replication unexpectedly, not viral DNA synthesis. Mislocalization of viral capsids in infected cell nuclei likely contributes to the observed decrease in virus replication. PMID:22855486

  15. Clathrin Light Chains Regulate Clathrin-Mediated Trafficking, Auxin Signaling, and Development in Arabidopsis[C][W][OA

    PubMed Central

    Wang, Chao; Yan, Xu; Chen, Qian; Jiang, Nan; Fu, Wei; Ma, Bojun; Liu, Jianzhong; Li, Chuanyou; Bednarek, Sebastian Y.; Pan, Jianwei

    2013-01-01

    Plant clathrin-mediated membrane trafficking is involved in many developmental processes as well as in responses to environmental cues. Previous studies have shown that clathrin-mediated endocytosis of the plasma membrane (PM) auxin transporter PIN-FORMED1 is regulated by the extracellular auxin receptor AUXIN BINDING PROTEIN1 (ABP1). However, the mechanisms by which ABP1 and other factors regulate clathrin-mediated trafficking are poorly understood. Here, we applied a genetic strategy and time-resolved imaging to dissect the role of clathrin light chains (CLCs) and ABP1 in auxin regulation of clathrin-mediated trafficking in Arabidopsis thaliana. Auxin was found to differentially regulate the PM and trans-Golgi network/early endosome (TGN/EE) association of CLCs and heavy chains (CHCs) in an ABP1-dependent but TRANSPORT INHIBITOR RESPONSE1/AUXIN-BINDING F-BOX PROTEIN (TIR1/AFB)-independent manner. Loss of CLC2 and CLC3 affected CHC membrane association, decreased both internalization and intracellular trafficking of PM proteins, and impaired auxin-regulated endocytosis. Consistent with these results, basipetal auxin transport, auxin sensitivity and distribution, and root gravitropism were also found to be dramatically altered in clc2 clc3 double mutants, resulting in pleiotropic defects in plant development. These results suggest that CLCs are key regulators in clathrin-mediated trafficking downstream of ABP1-mediated signaling and thus play a critical role in membrane trafficking from the TGN/EE and PM during plant development. PMID:23424247

  16. Mutation of the gene encoding the ribonuclease P RNA in the hyperthermophilic archaeon Thermococcus kodakarensis causes decreased growth rate and impaired processing of tRNA precursors.

    PubMed

    Ueda, Toshifumi; Ishino, Sonoko; Suematsu, Kotaro; Nakashima, Takashi; Kakuta, Yoshimitsu; Kawarabayasi, Yutaka; Ishino, Yoshizumi; Kimura, Makoto

    2015-12-25

    Ribonuclease P (RNase P) catalyzes the processing of 5' leader sequences of tRNA precursors in all three phylogenetic domains. RNase P also plays an essential role in non-tRNA biogenesis in bacterial and eukaryotic cells. For archaeal RNase Ps, additional functions, however, remain poorly understood. To gain insight into the biological function of archaeal RNase Ps in vivo, we prepared archaeal mutants KUWΔP3, KUWΔP8, and KUWΔP16, in which the gene segments encoding stem-loops containing helices, respectively, P3, P8 and P16 in RNase P RNA (TkopRNA) of the hyperthermophilic archaeon Thermococcus kodakarensis were deleted. Phenotypic analysis showed that KUWΔP3 and KUWΔP16 grew slowly compared with wild-type T. kodakarensis KUW1, while KUWΔP8 displayed no difference from T. kodakarensis KUW1. RNase P isolated using an affinity-tag from KUWΔP3 had reduced pre-tRNA cleavage activity compared with that from T. kodakarensis KUW1. Moreover, quantitative RT-PCR (qRT-PCR) and Northern blots analyses of KUWΔP3 showed greater accumulation of unprocessed transcripts for pre-tRNAs than that of T. kodakarensis KUW1. The current study represents the first attempt to prepare mutant T. kodakarensis with impaired RNase P for functional investigation. Comparative whole-transcriptome analysis of T. kodakarensis KUW1 and KUWΔP3 should allow for the comprehensive identification of RNA substrates for archaeal RNase Ps. PMID:26551464

  17. Single Point Mutation in Bin/Amphiphysin/Rvs (BAR) Sequence of Endophilin Impairs Dimerization, Membrane Shaping, and Src Homology 3 Domain-mediated Partnership*

    PubMed Central

    Gortat, Anna; San-Roman, Mabel Jouve; Vannier, Christian; Schmidt, Anne A.

    2012-01-01

    Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are essential players in the dynamics of intracellular compartments. The BAR domain is an evolutionarily conserved dimeric module characterized by a crescent-shaped structure whose intrinsic curvature, flexibility, and ability to assemble into highly ordered oligomers contribute to inducing the curvature of target membranes. Endophilins, diverging into A and B subgroups, are BAR and SH3 domain-containing proteins. They exert activities in membrane dynamic processes such as endocytosis, autophagy, mitochondrial dynamics, and permeabilization during apoptosis. Here, we report on the involvement of the third α-helix of the endophilin A BAR sequence in dimerization and identify leucine 215 as a key residue within a network of hydrophobic interactions stabilizing the entire BAR dimer interface. With the combination of N-terminal truncation retaining the high dimerization capacity of the third α-helices of endophilin A and leucine 215 substitution by aspartate (L215D), we demonstrate the essential role of BAR sequence-mediated dimerization on SH3 domain partnership. In comparison with wild type, full-length endophilin A2 heterodimers with one protomer bearing the L215D substitution exhibit very significant changes in membrane binding and shaping activities as well as a dramatic decrease of SH3 domain partnership. This suggests that subtle changes in the conformation and/or rigidity of the BAR domain impact both the control of membrane curvature and downstream binding to effectors. Finally, we show that expression, in mammalian cells, of endophilin A2 bearing the L215D substitution impairs the endocytic recycling of transferrin receptors. PMID:22167186

  18. Sex work and sex trafficking.

    PubMed

    Ditmore, M; Saunders, P

    1998-01-01

    Preventing HIV infection and other sexually transmitted diseases (STDs), as well as sexual and physical violence, are major occupational health and safety concerns for prostitutes. Considerable evidence shows that anti-prostitution laws facilitate violence and abuse against prostitutes and may increase their risk of contracting HIV/STDs. For example, police often take advantage of existing laws against prostitution to demand money or sex. In general, the strict enforcement of anti-prostitution laws marginalizes prostitutes from services which could help them avoid abuse and promotes an environment in which prostitutes must take risks to avoid detection and arrest. One strategy to improve prostitutes' lives would therefore be to remove laws which prevent them from working safely and from travelling abroad to work legally. Projects in which prostitutes are actively involved have helped break down stereotypes against prostitutes, while police-sex worker liaison projects in Scotland and Australia have led to higher levels of reporting of crimes against prostitutes. The Network of Sex Work Projects (NSWP), an organization which links sex worker health programs around the world, has found that the incidence of HIV/STDs among prostitutes is lowest when they have control over their work conditions; access to condoms, lubricants, and other safe sex materials; and respect of their basic human and legal rights. People need to understand that consensual involvement in sex work is different from forced sex trafficking. PMID:12348692

  19. The hidden crime: human trafficking.

    PubMed

    Clause, Kristen J; Lawler, Kate Byrnes

    2013-01-01

    As the primary contact in the health care system, nurses can play a role in combating this crime and assisting the victims. Assessment for abuse, neglect, trauma, recurrent sexually transmitted infections (STIs) and fear of a controlling partner is critical. Following up on "red flags" and understanding methods of safe questioning can make the difference between slavery and recovery for victims. Nurses must also know the professional referrals in their areas once a potential victim has been identified. This may be a very dangerous undertaking and must be handled by experienced personnel. Referrals to forensic nurses or physicians, domestic violence professionals or law enforcement may be indicated. Initially, a nurse may want to consult with the agency social worker for guidance. Human trafficking is a human rights crime. Unfortunately, it is more prevalent in all types of communities than most people suspect. Nurses can be heroes to the victims through understanding of this crime and vigilance in the assessment and care of all people they encounter in their practices. PMID:24218718

  20. The hidden crime: human trafficking.

    PubMed

    Clause, Kristen J; Lawler, Kate Byrnes

    2013-01-01

    As the primary contact in the health care system, nurses can play a role in combating this crime and assisting the victims. Assessment for abuse, neglect, trauma, recurrent sexually transmitted infections (STIs) and fear of a controlling partner is critical. Following up on "red flags" and understanding methods of safe questioning can make the difference between slavery and recovery for victims. Nurses must also know the professional referrals in their areas once a potential victim has been identified. This may be a very dangerous undertaking and must be handled by experienced personnel. Referrals to forensic nurses or physicians, domestic violence professionals or law enforcement may be indicated. Initially, a nurse may want to consult with the agency social worker for guidance. Human trafficking is a human rights crime. Unfortunately, it is more prevalent in all types of communities than most people suspect. Nurses can be heroes to the victims through understanding of this crime and vigilance in the assessment and care of all people they encounter in their practices. To learn more or to help with this cause, visit the Somaly Mam Foundation at www.somaly.org or the U.S. Department of State at www. state.gov. PMID:23977773

  1. Mutation of FIG4 causes a rapidly progressive, asymmetric neuronal degeneration

    PubMed Central

    Zhang, Xuebao; Chow, Clement Y.; Sahenk, Zarife; Shy, Michael E.; Meisler, Miriam H.

    2008-01-01

    Recessive Charcot-Marie-Tooth disease type-4J (CMT4J) and its animal model, the pale tremor mouse (plt), are caused by mutations of the FIG4 gene encoding a PI(3,5)P2 5-phosphatase. We describe the 9-year clinical course of CMT4J, including asymmetric, rapidly progressive paralysis, in two siblings. Sensory symptoms were absent despite reduced numbers of sensory axons. Thus, the phenotypic presentation of CMT4J clinically resembles motor neuron disease. Time-lapse imaging of fibroblasts from CMT4J patients demonstrates impaired trafficking of intracellular organelles because of obstruction by vacuoles. Further characterization of plt mice identified axonal degeneration in motor and sensory neurons, limited segmental demyelination, lack of TUNEL staining and lack of accumulation of ubiquitinated protein in vacuoles of motor and sensory neurons. This study represents the first documentation of the natural history of CMT4J. Physical obstruction of organelle trafficking by vacuoles is a potential novel cellular mechanism of neurodegeneration. PMID:18556664

  2. Valproic Acid Affects Membrane Trafficking and Cell-Wall Integrity in Fission Yeast

    PubMed Central

    Miyatake, Makoto; Kuno, Takayoshi; Kita, Ayako; Katsura, Kosaku; Takegawa, Kaoru; Uno, Satoshi; Nabata, Toshiya; Sugiura, Reiko

    2007-01-01

    Valproic acid (VPA) is widely used to treat epilepsy and manic-depressive illness. Although VPA has been reported to exert a variety of biochemical effects, the exact mechanisms underlying its therapeutic effects remain elusive. To gain further insights into the molecular mechanisms of VPA action, a genetic screen for fission yeast mutants that show hypersensitivity to VPA was performed. One of the genes that we identified was vps45+, which encodes a member of the Sec1/Munc18 family that is implicated in membrane trafficking. Notably, several mutations affecting membrane trafficking also resulted in hypersensitivity to VPA. These include ypt3+ and ryh1+, both encoding a Rab family protein, and apm1+, encoding the μ1 subunit of the adaptor protein complex AP-1. More importantly, VPA caused vacuolar fragmentation and inhibited the glycosylation and the secretion of acid phosphatase in wild-type cells, suggesting that VPA affects membrane trafficking. Interestingly, the cell-wall-damaging agents such as micafungin or the inhibition of calcineurin dramatically enhanced the sensitivity of wild-type cells to VPA. Consistently, VPA treatment of wild-type cells enhanced their sensitivity to the cell-wall-digesting enzymes. Altogether, our results suggest that VPA affects membrane trafficking, which leads to the enhanced sensitivity to cell-wall damage in fission yeast. PMID:17287531

  3. Sterol dynamics during endocytic trafficking in Arabidopsis.

    PubMed

    Stanislas, Thomas; Grebe, Markus; Boutté, Yohann

    2014-01-01

    Sterols are lipids found in membranes of eukaryotic cells. Functions of sterols have been demonstrated for various cellular processes including endocytic trafficking in animal, fungal, and plant cells. The ability to visualize sterols at the subcellular level is crucial to understand sterol distribution and function during endocytic trafficking. In plant cells, the polyene antibiotic filipin is the most extensively used tool for the specific detection of fluorescently labeled 3-β-hydroxysterols in situ. Filipin can to some extent be used to track sterol internalization in live cells, but this application is limited, due to the inhibitory effects filipin exerts on sterol-dependent endocytosis. Nevertheless, filipin-sterol labeling can be performed on aldehyde-fixed cells which allows for sterol detection in endocytic compartments. This approach can combine studies correlating sterol distribution with experimental manipulations of endocytic trafficking pathways. Here, we describe step-by-step protocols and troubleshooting for procedures on live and fixed cells to visualize sterols during endocytic trafficking. We also provide a detailed discussion of advantages and limitations of both methods. Moreover, we illustrate the use of the endocytic recycling inhibitor brefeldin A and a genetically modified version of one of its target molecules for studying endocytic sterol trafficking. PMID:25117272

  4. KIF13B regulates angiogenesis through Golgi to plasma membrane trafficking of VEGFR2

    PubMed Central

    Yamada, Kaori H.; Nakajima, Yuki; Geyer, Melissa; Wary, Kishore K.; Ushio-Fukai, Masuko; Komarova, Yulia; Malik, Asrar B.

    2014-01-01

    ABSTRACT Although the trafficking of newly synthesized VEGFR2 to the plasma membrane is a key determinant of angiogenesis, the molecular mechanisms of Golgi to plasma membrane trafficking are unknown. Here, we have identified a key role of the kinesin family plus-end molecular motor KIF13B in delivering VEGFR2 cargo from the Golgi to the endothelial cell surface. KIF13B is shown to interact directly with VEGFR2 on microtubules. We also observed that overexpression of truncated versions of KIF13B containing the binding domains that interact with VEGFR2 inhibited VEGF-induced capillary tube formation. KIF13B depletion prevented VEGF-mediated endothelial migration, capillary tube formation and neo-vascularization in mice. Impairment in trafficking induced by knockdown of KIF13B shunted VEGFR2 towards the lysosomal degradation pathway. Thus, KIF13B is an essential molecular motor required for the trafficking of VEGFR2 from the Golgi, and its delivery to the endothelial cell surface mediates angiogenesis. PMID:25128562

  5. RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border.

    PubMed

    Marcos-Ramiro, Beatriz; García-Weber, Diego; Barroso, Susana; Feito, Jorge; Ortega, María C; Cernuda-Morollón, Eva; Reglero-Real, Natalia; Fernández-Martín, Laura; Durán, Maria C; Alonso, Miguel A; Correas, Isabel; Cox, Susan; Ridley, Anne J; Millán, Jaime

    2016-05-01

    Endothelial barrier dysfunction underlies chronic inflammatory diseases. In searching for new proteins essential to the human endothelial inflammatory response, we have found that the endosomal GTPase RhoB is up-regulated in response to inflammatory cytokines and expressed in the endothelium of some chronically inflamed tissues. We show that although RhoB and the related RhoA and RhoC play additive and redundant roles in various aspects of endothelial barrier function, RhoB specifically inhibits barrier restoration after acute cell contraction by preventing plasma membrane extension. During barrier restoration, RhoB trafficking is induced between vesicles containing RhoB nanoclusters and plasma membrane protrusions. The Rho GTPase Rac1 controls membrane spreading and stabilizes endothelial barriers. We show that RhoB colocalizes with Rac1 in endosomes and inhibits Rac1 activity and trafficking to the cell border during barrier recovery. Inhibition of endosomal trafficking impairs barrier reformation, whereas induction of Rac1 translocation to the plasma membrane accelerates it. Therefore, RhoB-specific regulation of Rac1 trafficking controls endothelial barrier integrity during inflammation. PMID:27138256

  6. A rare cause of fatal pulmonary alveolar proteinosis: Niemann-Pick disease type C2 and a novel mutation.

    PubMed

    Yaman, Ayhan; Eminoğlu, Fatma T; Kendirli, Tanıl; Ödek, Çağlar; Ceylaner, Serdar; Kansu, Aydan; İnce, Elif; Deda, Gülhis

    2015-09-01

    Niemann-Pick disease type C (NPC) is a fatal autosomal recessive lipid storage disease associated with impaired trafficking of unesterified cholesterol and glycolipids in lysosomes and late endosomes. This disease is commonly characterized by hepatosplenomegaly and severe progressive neurological dysfunction. There are two defective genes that cause this illness. One of these genes is NPC1 gene which is the cause of illness in 95% of the patients. The other gene is the rare type NPC2 which is the cause of illness in 5% of the patients. Patients with NPC2 usually present with respiratory distress in early infancy, which is rather unusual with NPC1. This article discusses about a patient who died at an early age from pulmonary involvement and who subsequently was found to have a novel homozygous mutation of NPC2 gene. PMID:26024245

  7. Delineation of the Clinical, Molecular and Cellular Aspects of Novel JAM3 Mutations Underlying the Autosomal Recessive Hemorrhagic Destruction of the Brain, Subependymal Calcification and Congenital Cataracts

    PubMed Central

    Akawi, Nadia A.; Canpolat, Fuat E.; White, Susan M.; Quilis-Esquerra, Josep; Sanchez, Martin Morales; Gamundi, Maria José; Mochida, Ganeshwaran H.; Walsh, Christopher A.; Ali, Bassam R.; Al-Gazali, Lihadh

    2014-01-01

    We have recently shown that the hemorrhagic destruction of the brain, subependymal calcification and congenital cataracts is caused by biallelic mutations in the gene encoding junctional adhesion molecule 3 (JAM3) protein. Affected members from three new families underwent detailed clinical examination including imaging of the brain. Affected individuals presented with a distinctive phenotype comprising hemorrhagic destruction of the brain, subependymal calcification and congenital cataracts. All patients had a catastrophic clinical course resulting in death in 7 out of 10 affected individuals. Sequencing the coding exons of JAM3 revealed three novel homozygous mutations: c.2T>G (p.M1R), c.346G>A (p.E116K) and c.656G>A (p.C219Y). The p.M1R mutation affects the start codon and therefore is predicted to impair protein synthesis. Cellular studies showed that the p.C219Y mutation resulted in a significant retention of the mutated protein in the endoplasmic reticulum, suggesting a trafficking defect. The p.E116K mutant traffics normally to the plasma membrane as the wild type and may have lost its function due to the lack of interaction with an interacting partner. Our data further support the importance of JAM3 in the development and function of the vascular system and the brain. PMID:23255084

  8. Vesicle trafficking and cell surface membrane patchiness.

    PubMed

    Tang, Q; Edidin, M

    2001-07-01

    Membrane proteins and lipids often appear to be distributed in patches on the cell surface. These patches are often assumed to be membrane domains, arising from specific molecular associations. However, a computer simulation (Gheber and Edidin, 1999) shows that membrane patchiness may result from a combination of vesicle trafficking and dynamic barriers to lateral mobility. The simulation predicts that the steady-state patches of proteins and lipids seen on the cell surface will decay if vesicle trafficking is inhibited. To test this prediction, we compared the apparent sizes and intensities of patches of class I HLA molecules, integral membrane proteins, before and after inhibiting endocytic vesicle traffic from the cell surface, either by incubation in hypertonic medium or by expression of a dominant-negative mutant dynamin. As predicted by the simulation, the apparent sizes of HLA patches increased, whereas their intensities decreased after endocytosis and vesicle trafficking were inhibited. PMID:11423406

  9. Chloride in vesicular trafficking and function.

    PubMed

    Stauber, Tobias; Jentsch, Thomas J

    2013-01-01

    Luminal acidification is of pivotal importance for the physiology of the secretory and endocytic pathways and its diverse trafficking events. Acidification by the proton-pumping V-ATPase requires charge compensation by counterion currents that are commonly attributed to chloride. The molecular identification of intracellular chloride transporters and the improvement of methodologies for measuring intraorganellar pH and chloride have facilitated the investigation of the physiology of vesicular chloride transport. New data question the requirement of chloride for pH regulation of various organelles and furthermore ascribe functions to chloride that are beyond merely electrically shunting the proton pump. This review surveys the currently established and proposed intracellular chloride transporters and gives an overview of membrane-trafficking steps that are affected by the perturbation of chloride transport. Finally, potential mechanisms of membrane-trafficking modulation by chloride are discussed and put into the context of organellar ion homeostasis in general. PMID:23092411

  10. Endocytosis and Endosomal Trafficking in Plants.

    PubMed

    Paez Valencia, Julio; Goodman, Kaija; Otegui, Marisa S

    2016-04-29

    Endocytosis and endosomal trafficking are essential processes in cells that control the dynamics and turnover of plasma membrane proteins, such as receptors, transporters, and cell wall biosynthetic enzymes. Plasma membrane proteins (cargo) are internalized by endocytosis through clathrin-dependent or clathrin-independent mechanism and delivered to early endosomes. From the endosomes, cargo proteins are recycled back to the plasma membrane via different pathways, which rely on small GTPases and the retromer complex. Proteins that are targeted for degradation through ubiquitination are sorted into endosomal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery for degradation in the vacuole. Endocytic and endosomal trafficking regulates many cellular, developmental, and physiological processes, including cellular polarization, hormone transport, metal ion homeostasis, cytokinesis, pathogen responses, and development. In this review, we discuss the mechanisms that mediate the recognition and sorting of endocytic and endosomal cargos, the vesiculation processes that mediate their trafficking, and their connection to cellular and physiological responses in plants. PMID:27128466

  11. Purinergic Signaling During Immune Cell Trafficking.

    PubMed

    Ferrari, Davide; McNamee, Eóin N; Idzko, Marco; Gambari, Roberto; Eltzschig, Holger K

    2016-06-01

    Migration and positioning of immune cells is fundamental for their differentiation and recruitment at sites of infection. Besides the fundamental role played by chemokines and their receptors, recent studies demonstrate that a complex network of purinergic signaling events plays a key role in these trafficking events. This process includes the release of nucleotides (such as ATP and ADP) and subsequent autocrine and paracrine signaling events through nucleotide receptors. At the same time, surface-expressed ectoapyrases and nucleotidases convert extracellular nucleotides to adenosine, and adenosine signaling events play additional functional roles in leucocyte trafficking. In this review we revisit classical paradigms of inflammatory cell trafficking in the context of recent studies implicating purinergic signaling events in this process. PMID:27142306

  12. Regulation of GPCR Trafficking by Ubiquitin

    PubMed Central

    Kennedy, Justine E.; Marchese, Adriano

    2015-01-01

    G protein-coupled receptor (GPCR)-promoted signaling mediates cellular responses to a variety of stimuli involved in diverse physiological processes. In addition, GPCRs are also the largest class of target for many drugs used to treat a variety of diseases. Despite the role of GPCR signaling in health and disease, the molecular mechanisms governing GPCR signaling remain poorly understanding. Classically, GPCR signaling is tightly regulated by GPCR kinases and β-arrestins, which act in a concerted fashion to govern GPCR desensitization and also GPCR trafficking. Ubiquitination has now emerged as an important posttranslational modification that has multiple roles, either directly or indirectly, in governing GPCR trafficking. Recent studies have revealed a mechanistic link between GPCR phosphorylation, β-arrestins, and ubiquitination. Here, we review recent developments in our understanding of how ubiquitin regulates GPCR trafficking within the endocytic pathway. PMID:26055053

  13. A role of the sulfonylurea receptor 1 in endocytic trafficking of ATP-sensitive potassium channels

    PubMed Central

    Bruederle, Cathrin E.; Gay, Joel; Shyng, Show-Ling

    2011-01-01

    The ATP-sensitive potassium (KATP) channel consisting of sulfonylurea receptor 1 (SUR1) and inward rectifier potassium channel 6.2 (Kir6.2) has a well-established role in insulin secretion. Mutations in either subunit can lead to disease due to aberrant channel gating, altered channel density at the cell surface or a combination of both. Endocytic trafficking of channels at the plasma membrane is one way to influence surface channel numbers. It has been previously reported that channel endocytosis is dependent on a tyrosine-based motif in Kir6.2 while SUR1 alone is unable to internalize. In this study, we followed endocytic trafficking of surface channels in real time by live cell imaging of channel subunits tagged with an extracellular minimal α-bungarotoxin binding peptide labeled with a fluorescent dye. We demonstrate that SUR1 undergoes endocytosis independent of Kir6.2. Moreover, mutations in the putative endocytosis motif of Kir6.2, Y330C, Y330A and F333I are unable to prevent channel endocytosis. These findings challenge the notion that Kir6.2 bears the sole endocytic signal for KATP channels and support a role of SUR1 in this trafficking process. PMID:21649805

  14. Trafficking in persons and development: towards greater policy coherence.

    PubMed

    Danailova-Trainor, Gergana; Laczko, Frank

    2010-01-01

    Poverty is often regarded as the "root cause" of trafficking, but the linkages between poverty, a lack of development and trafficking are complex. For example, there is some evidence to suggest that victims of cross-border trafficking are more likely to originate from middle-income rather than lower-income countries. Trafficking and development have tended to be treated as very separate policy areas and the assessment of the development impact of counter-trafficking programmes is still at an early stage. This paper outlines a possible framework for a more evidence-based approach to understanding the linkages between trafficking, trafficking policy and human development. The paper argues that the human development gains from greater mobility could be significantly enhanced if there was greater coherence between policies to combat trafficking and policies to promote development. PMID:20645470

  15. Organ trading, tourism, and trafficking within Europe.

    PubMed

    Pattinson, Shaun D

    2008-03-01

    This article argues for a regulatory and institutional response towards organ trading, tourism and trafficking that differs from extant approaches. European countries have hitherto adopted blanket prohibitions on organ trading (i.e. the buying or selling of human organs). This article advances the view that policy makers have thereby overreacted to legitimate public health concerns and the evils of organ trafficking (i.e. organ trading and tourism involving coercion or deception). It argues for a trial of a very tightly regulated system of organ trading that could eventually lead to a limited system of organ tourism (i.e. organ trading involving more than one jurisdiction). PMID:18592891

  16. Defects in optineurin- and myosin VI-mediated cellular trafficking in amyotrophic lateral sclerosis.

    PubMed

    Sundaramoorthy, Vinod; Walker, Adam K; Tan, Vanessa; Fifita, Jennifer A; Mccann, Emily P; Williams, Kelly L; Blair, Ian P; Guillemin, Gilles J; Farg, Manal A; Atkin, Julie D

    2015-07-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder primarily affecting motor neurons. Mutations in optineurin cause a small proportion of familial ALS cases, and wild-type (WT) optineurin is misfolded and forms inclusions in sporadic ALS patient motor neurons. However, it is unknown how optineurin mutation or misfolding leads to ALS. Optineurin acts an adaptor protein connecting the molecular motor myosin VI to secretory vesicles and autophagosomes. Here, we demonstrate that ALS-linked mutations p.Q398X and p.E478G disrupt the association of optineurin with myosin VI, leading to an abnormal diffuse cytoplasmic distribution, inhibition of secretory protein trafficking, endoplasmic reticulum (ER) stress and Golgi fragmentation in motor neuron-like NSC-34 cells. We also provide further insight into the role of optineurin as an autophagy receptor. WT optineurin associated with lysosomes and promoted autophagosome fusion to lysosomes in neuronal cells, implying that it mediates trafficking of lysosomes during autophagy in association with myosin VI. However, either expression of ALS mutant optineurin or small interfering RNA-mediated knockdown of endogenous optineurin blocked lysosome fusion to autophagosomes, resulting in autophagosome accumulation. Together these results indicate that ALS-linked mutations in optineurin disrupt myosin VI-mediated intracellular trafficking processes. In addition, in control human patient tissues, optineurin displayed its normal vesicular localization, but in sporadic ALS patient tissues, vesicles were present in a significantly decreased proportion of motor neurons. Optineurin binding to myosin VI was also decreased in tissue lysates from sporadic ALS spinal cords. This study therefore links several previously described pathological mechanisms in ALS, including defects in autophagy, fragmentation of the Golgi and induction of ER stress, to disruption of optineurin function. These findings also indicate that

  17. Human Trafficking: A Review for Mental Health Professionals

    ERIC Educational Resources Information Center

    Yakushko, Oksana

    2009-01-01

    This article provides a review of current research on human trafficking for mental health practitioners and scholars. In addition to an overview of definitions, causes and processes of trafficking, the article highlights mental health consequences of trafficking along with suggestions for treatment of survivors. Directions for counseling services,…

  18. Trafficking of Children in Albania: Patterns of Recruitment and Reintegration

    ERIC Educational Resources Information Center

    Gjermeni, Eglantina; Van Hook, Mary P.; Gjipali, Saemira; Xhillari, Lindita; Lungu, Fatjon; Hazizi, Anila

    2008-01-01

    Problem: Many children in Albania and other countries of Eastern Europe are being trafficked as part of the global business of human trafficking. Objectives: The study sought to identify the patterns of child trafficking involving Albanian children, and especially children's views of the role of family issues and the nature of the trafficking…

  19. 78 FR 70571 - Advisory Council on Wildlife Trafficking; Rescheduled Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-26

    ... Fish and Wildlife Service Advisory Council on Wildlife Trafficking; Rescheduled Meeting AGENCY: Fish... Service (Service), announce a public meeting of the Advisory Council on Wildlife Trafficking (Council... announce that the Advisory Council on Wildlife Trafficking (Council) will hold a meeting to...

  20. 78 FR 59950 - Advisory Council on Wildlife Trafficking

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-30

    ... Fish and Wildlife Service Advisory Council on Wildlife Trafficking AGENCY: Fish and Wildlife Service... public meeting of the Advisory Council on Wildlife Trafficking (Council). DATES: The meeting will be held... announce that the Advisory Council on Wildlife Trafficking (Council) will hold a meeting to...

  1. Child organ trafficking: global reality and inadequate international response.

    PubMed

    Bagheri, Alireza

    2016-06-01

    In organ transplantation, the demand for human organs has grown far faster than the supply of organs. This has opened the door for illegal organ trade and trafficking including from children. Organized crime groups and individual organ brokers exploit the situation and, as a result, black markets are becoming more numerous and organized organ trafficking is expanding worldwide. While underprivileged and vulnerable men and women in developing countries are a major source of trafficked organs, and may themselves be trafficked for the purpose of illegal organ removal and trade, children are at especial risk of exploitation. With the confirmed cases of children being trafficked for their organs, child organ trafficking, which once called a "modern urban legend", is a sad reality in today's world. By presenting a global picture of child organ trafficking, this paper emphasizes that child organ trafficking is no longer a myth but a reality which has to be addressed. It argues that the international efforts against organ trafficking and trafficking in human beings for organ removal have failed to address child organ trafficking adequately. This chapter suggests that more orchestrated international collaboration as well as development of preventive measure and legally binding documents are needed to fight child organ trafficking and to support its victims. PMID:26612382

  2. Alternative paradigms for ion channelopathies: disorders of ion channel membrane trafficking and posttranslational modification.

    PubMed

    Curran, Jerry; Mohler, Peter J

    2015-01-01

    Channelopathies are a diverse set of disorders associated with defects in ion channel (and transporter) function. Although the vast majority of channelopathies are linked with inherited mutations that alter ion channel biophysical properties, another group of similar disorders has emerged that alter ion channel synthesis, membrane trafficking, and/or posttranslational modifications. In fact, some electrical and episodic disorders have now been identified that are not defects in the ion channel but instead reflect dysfunction in an ion channel (or transporter) regulatory protein. This review focuses on alternative paradigms for physiological disorders associated with protein biosynthesis, folding, trafficking, and membrane retention. Furthermore, the review highlights the role of aberrant posttranslational modifications in acquired channelopathies. PMID:25293528

  3. Gating characteristics control glutamate receptor distribution and trafficking in vivo.

    PubMed

    Petzoldt, Astrid G; Lee, Yü-Hien; Khorramshahi, Omid; Reynolds, Eric; Plested, Andrew J R; Herzel, Hanspeter; Sigrist, Stephan J

    2014-09-01

    Glutamate-releasing synapses dominate excitatory release in the brain. Mechanisms governing their assembly are of major importance for circuit development and long-term plasticity underlying learning and memory. AMPA/Kainate-type glutamate receptors (GluRs) are tetrameric ligand-gated ion channels that open their ion-conducting pores in response to binding of the neurotransmitter. Changes in subunit composition of postsynaptic GluRs are highly relevant for plasticity and development of glutamatergic synapses [1-4]. To date, posttranslational modifications, mostly operating via the intracellular C-terminal domains (CTDs) of GluRs, are presumed to be the major regulator of trafficking [5]. In recent years, structural and electrophysiological analyses have improved our understanding of GluR gating mechanism [6-11]. However, whether conformational changes subsequent to glutamate binding may per se be able to influence GluR trafficking has remained an unaddressed question. Using a Drosophila system allowing for extended visualization of GluR trafficking in vivo, we here provide evidence that mutations changing the gating behavior alter GluR distribution and trafficking. GluR mutants associated with reduced charge transfer segregated from coexpressed wild-type GluRs on the level of individual postsynaptic densities. Segregation was lost upon blocking of evoked glutamate release. Photobleaching experiments suggested increased mobility of mutants with reduced charge transfer, which accumulated prematurely during early steps of synapse assembly, but failed to further increase their level in accordance with assembly of the presynaptic scaffold. In summary, gating characteristics seem to be a new variable for the understanding of GluR trafficking relevant to both development and plasticity. PMID:25131677

  4. The SNARE VAMP7 Regulates Exocytic Trafficking of Interleukin-12 in Dendritic Cells.

    PubMed

    Chiaruttini, Giulia; Piperno, Giulia M; Jouve, Mabel; De Nardi, Francesca; Larghi, Paola; Peden, Andrew A; Baj, Gabriele; Müller, Sabina; Valitutti, Salvatore; Galli, Thierry; Benvenuti, Federica

    2016-03-22

    Interleukin-12 (IL-12), produced by dendritic cells in response to activation, is central to pathogen eradication and tumor rejection. The trafficking pathways controlling spatial distribution and intracellular transport of IL-12 vesicles to the cell surface are still unknown. Here, we show that intracellular IL-12 localizes in late endocytic vesicles marked by the SNARE VAMP7. Dendritic cells (DCs) from VAMP7-deficient mice are partially impaired in the multidirectional release of IL-12. Upon encounter with antigen-specific T cells, IL-12-containing vesicles rapidly redistribute at the immune synapse and release IL-12 in a process entirely dependent on VAMP7 expression. Consistently, acquisition of effector functions is reduced in T cells stimulated by VAMP7-null DCs. These results provide insights into IL-12 intracellular trafficking pathways and show that VAMP7-mediated release of IL-12 at the immune synapse is a mechanism to transmit innate signals to T cells. PMID:26972013

  5. The SNARE VAMP7 Regulates Exocytic Trafficking of Interleukin-12 in Dendritic Cells

    PubMed Central

    Chiaruttini, Giulia; Piperno, Giulia M.; Jouve, Mabel; De Nardi, Francesca; Larghi, Paola; Peden, Andrew A.; Baj, Gabriele; Müller, Sabina; Valitutti, Salvatore; Galli, Thierry; Benvenuti, Federica

    2016-01-01

    Summary Interleukin-12 (IL-12), produced by dendritic cells in response to activation, is central to pathogen eradication and tumor rejection. The trafficking pathways controlling spatial distribution and intracellular transport of IL-12 vesicles to the cell surface are still unknown. Here, we show that intracellular IL-12 localizes in late endocytic vesicles marked by the SNARE VAMP7. Dendritic cells (DCs) from VAMP7-deficient mice are partially impaired in the multidirectional release of IL-12. Upon encounter with antigen-specific T cells, IL-12-containing vesicles rapidly redistribute at the immune synapse and release IL-12 in a process entirely dependent on VAMP7 expression. Consistently, acquisition of effector functions is reduced in T cells stimulated by VAMP7-null DCs. These results provide insights into IL-12 intracellular trafficking pathways and show that VAMP7-mediated release of IL-12 at the immune synapse is a mechanism to transmit innate signals to T cells. PMID:26972013

  6. Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7.

    PubMed

    D'Costa, Vanessa M; Braun, Virginie; Landekic, Marija; Shi, Rong; Proteau, Ariane; McDonald, Laura; Cygler, Miroslaw; Grinstein, Sergio; Brumell, John H

    2015-09-01

    Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target. PMID:26299973

  7. Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders

    PubMed Central

    Nakazawa, Takanobu; Hashimoto, Ryota; Sakoori, Kazuto; Sugaya, Yuki; Tanimura, Asami; Hashimotodani, Yuki; Ohi, Kazutaka; Yamamori, Hidenaga; Yasuda, Yuka; Umeda-Yano, Satomi; Kiyama, Yuji; Konno, Kohtarou; Inoue, Takeshi; Yokoyama, Kazumasa; Inoue, Takafumi; Numata, Shusuke; Ohnuma, Tohru; Iwata, Nakao; Ozaki, Norio; Hashimoto, Hitoshi; Watanabe, Masahiko; Manabe, Toshiya; Yamamoto, Tadashi; Takeda, Masatoshi; Kano, Masanobu

    2016-01-01

    Intracellular trafficking of receptor proteins is essential for neurons to detect various extracellular factors during the formation and refinement of neural circuits. However, the precise mechanisms underlying the trafficking of neurotrophin receptors to synapses remain elusive. Here, we demonstrate that a brain-enriched sorting nexin, ARHGAP33, is a new type of regulator for the intracellular trafficking of TrkB, a high-affinity receptor for brain-derived neurotrophic factor. ARHGAP33 knockout (KO) mice exhibit reduced expression of synaptic TrkB, impaired spine development and neuropsychiatric disorder-related behavioural abnormalities. These deficits are rescued by specific pharmacological enhancement of TrkB signalling in ARHGAP33 KO mice. Mechanistically, ARHGAP33 interacts with SORT1 to cooperatively regulate TrkB trafficking. Human ARHGAP33 is associated with brain phenotypes and reduced SORT1 expression is found in patients with schizophrenia. We propose that ARHGAP33/SORT1-mediated TrkB trafficking is essential for synapse development and that the dysfunction of this mechanism may be a new molecular pathology of neuropsychiatric disorders. PMID:26839058

  8. Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders.

    PubMed

    Nakazawa, Takanobu; Hashimoto, Ryota; Sakoori, Kazuto; Sugaya, Yuki; Tanimura, Asami; Hashimotodani, Yuki; Ohi, Kazutaka; Yamamori, Hidenaga; Yasuda, Yuka; Umeda-Yano, Satomi; Kiyama, Yuji; Konno, Kohtarou; Inoue, Takeshi; Yokoyama, Kazumasa; Inoue, Takafumi; Numata, Shusuke; Ohnuma, Tohru; Iwata, Nakao; Ozaki, Norio; Hashimoto, Hitoshi; Watanabe, Masahiko; Manabe, Toshiya; Yamamoto, Tadashi; Takeda, Masatoshi; Kano, Masanobu

    2016-01-01

    Intracellular trafficking of receptor proteins is essential for neurons to detect various extracellular factors during the formation and refinement of neural circuits. However, the precise mechanisms underlying the trafficking of neurotrophin receptors to synapses remain elusive. Here, we demonstrate that a brain-enriched sorting nexin, ARHGAP33, is a new type of regulator for the intracellular trafficking of TrkB, a high-affinity receptor for brain-derived neurotrophic factor. ARHGAP33 knockout (KO) mice exhibit reduced expression of synaptic TrkB, impaired spine development and neuropsychiatric disorder-related behavioural abnormalities. These deficits are rescued by specific pharmacological enhancement of TrkB signalling in ARHGAP33 KO mice. Mechanistically, ARHGAP33 interacts with SORT1 to cooperatively regulate TrkB trafficking. Human ARHGAP33 is associated with brain phenotypes and reduced SORT1 expression is found in patients with schizophrenia. We propose that ARHGAP33/SORT1-mediated TrkB trafficking is essential for synapse development and that the dysfunction of this mechanism may be a new molecular pathology of neuropsychiatric disorders. PMID:26839058

  9. Excess sphingomyelin disturbs ATG9A trafficking and autophagosome closure.

    PubMed

    Corcelle-Termeau, Elisabeth; Vindeløv, Signe Diness; Hämälistö, Saara; Mograbi, Baharia; Keldsbo, Anne; Bräsen, Jan Hinrich; Favaro, Elena; Adam, Dieter; Szyniarowski, Piotr; Hofman, Paul; Krautwald, Stefan; Farkas, Thomas; Petersen, Nikolaj H T; Rohde, Mikkel; Linkermann, Andreas; Jäättelä, Marja

    2016-05-01

    Sphingomyelin is an essential cellular lipid that traffics between plasma membrane and intracellular organelles until directed to lysosomes for SMPD1 (sphingomyelin phosphodiesterase 1)-mediated degradation. Inactivating mutations in the SMPD1 gene result in Niemann-Pick diseases type A and B characterized by sphingomyelin accumulation and severely disturbed tissue homeostasis. Here, we report that sphingomyelin overload disturbs the maturation and closure of autophagic membranes. Niemann-Pick type A patient fibroblasts and SMPD1-depleted cancer cells accumulate elongated and unclosed autophagic membranes as well as abnormally swollen autophagosomes in the absence of normal autophagosomes and autolysosomes. The immature autophagic membranes are rich in WIPI2, ATG16L1 and MAP1LC3B but display reduced association with ATG9A. Contrary to its normal trafficking between plasma membrane, intracellular organelles and autophagic membranes, ATG9A concentrates in transferrin receptor-positive juxtanuclear recycling endosomes in SMPD1-deficient cells. Supporting a causative role for ATG9A mistrafficking in the autophagy defect observed in SMPD1-deficient cells, ectopic ATG9A effectively reverts this phenotype. Exogenous C12-sphingomyelin induces a similar juxtanuclear accumulation of ATG9A and subsequent defect in the maturation of autophagic membranes in healthy cells while the main sphingomyelin metabolite, ceramide, fails to revert the autophagy defective phenotype in SMPD1-deficient cells. Juxtanuclear accumulation of ATG9A and defective autophagy are also evident in tissues of smpd1-deficient mice with a subsequent inability to cope with kidney ischemia-reperfusion stress. These data reveal sphingomyelin as an important regulator of ATG9A trafficking and maturation of early autophagic membranes. PMID:27070082

  10. Excess sphingomyelin disturbs ATG9A trafficking and autophagosome closure

    PubMed Central

    Corcelle-Termeau, Elisabeth; Vindeløv, Signe Diness; Hämälistö, Saara; Mograbi, Baharia; Keldsbo, Anne; Bräsen, Jan Hinrich; Favaro, Elena; Adam, Dieter; Szyniarowski, Piotr; Hofman, Paul; Krautwald, Stefan; Farkas, Thomas; Petersen, Nikolaj H.T.; Rohde, Mikkel; Linkermann, Andreas; Jäättelä, Marja

    2016-01-01

    ABSTRACT Sphingomyelin is an essential cellular lipid that traffics between plasma membrane and intracellular organelles until directed to lysosomes for SMPD1 (sphingomyelin phosphodiesterase 1)-mediated degradation. Inactivating mutations in the SMPD1 gene result in Niemann-Pick diseases type A and B characterized by sphingomyelin accumulation and severely disturbed tissue homeostasis. Here, we report that sphingomyelin overload disturbs the maturation and closure of autophagic membranes. Niemann-Pick type A patient fibroblasts and SMPD1-depleted cancer cells accumulate elongated and unclosed autophagic membranes as well as abnormally swollen autophagosomes in the absence of normal autophagosomes and autolysosomes. The immature autophagic membranes are rich in WIPI2, ATG16L1 and MAP1LC3B but display reduced association with ATG9A. Contrary to its normal trafficking between plasma membrane, intracellular organelles and autophagic membranes, ATG9A concentrates in transferrin receptor-positive juxtanuclear recycling endosomes in SMPD1-deficient cells. Supporting a causative role for ATG9A mistrafficking in the autophagy defect observed in SMPD1-deficient cells, ectopic ATG9A effectively reverts this phenotype. Exogenous C12-sphingomyelin induces a similar juxtanuclear accumulation of ATG9A and subsequent defect in the maturation of autophagic membranes in healthy cells while the main sphingomyelin metabolite, ceramide, fails to revert the autophagy defective phenotype in SMPD1-deficient cells. Juxtanuclear accumulation of ATG9A and defective autophagy are also evident in tissues of smpd1-deficient mice with a subsequent inability to cope with kidney ischemia-reperfusion stress. These data reveal sphingomyelin as an important regulator of ATG9A trafficking and maturation of early autophagic membranes. PMID:27070082

  11. Three families with autosomal dominant nephrogenic diabetes insipidus caused by aquaporin-2 mutations in the C-terminus.

    PubMed

    Kuwahara, M; Iwai, K; Ooeda, T; Igarashi, T; Ogawa, E; Katsushima, Y; Shinbo, I; Uchida, S; Terada, Y; Arthus, M F; Lonergan, M; Fujiwara, T M; Bichet, D G; Marumo, F; Sasaki, S

    2001-10-01

    The vasopressin-regulated water channel aquaporin-2 (AQP2) is known to tetramerize in the apical membrane of the renal tubular cells and contributes to urine concentration. We identified three novel mutations, each in a single allele of exon 4 of the AQP2 gene, in three families showing autosomal dominant nephrogenic diabetes insipidus (NDI). These mutations were found in the C-terminus of AQP2: a deletion of G at nucleotide 721 (721 delG), a deletion of 10 nucleotides starting at nucleotide 763 (763-772del), and a deletion of 7 nucleotides starting at nucleotide 812 (812-818del). The wild-type AQP2 is predicted to be a 271-amino acid protein, whereas these mutant genes are predicted to encode proteins that are 330-333 amino acids in length, because of the frameshift mutations. Interestingly, these three mutant AQP2s shared the same C-terminal tail of 61 amino acids. In Xenopus oocytes injected with mutant AQP2 cRNAs, the osmotic water permeability (Pf) was much smaller than that of oocytes with the AQP2 wild-type (14%-17%). Immunoblot analysis of the lysates of the oocytes expressing the mutant AQP2s detected a band at 34 kD, whereas the immunoblot of the plasma-membrane fractions of the oocytes and immunocytochemistry failed to show a significant surface expression, suggesting a defect in trafficking of these mutant proteins. Furthermore, coinjection of wild-type cRNAs with mutant cRNAs markedly decreased the oocyte Pf in parallel with the surface expression of the wild-type AQP2. Immunoprecipitation with antibodies against wild-type and mutant AQP2 indicated the formation of mixed oligomers composed of wild-type and mutant AQP2 monomers. Our results suggest that the trafficking of mutant AQP2 is impaired because of elongation of the C-terminal tail, and the dominant-negative effect is attributed to oligomerization of the wild-type and mutant AQP2s. Segregation of the mutations in the C-terminus of AQP2 with dominant-type NDI underlies the importance of this

  12. Trafficking Mechanisms Underlying Neuronal Voltage-gated Ion Channel Localization at the Axon Initial Segment

    PubMed Central

    Vacher, Helene; Trimmer, James S.

    2012-01-01

    Summary Voltage-gated ion channels are diverse and fundamental determinants of neuronal intrinsic excitability. Voltage-gated K+ (Kv) and Na+ (Nav) channels play complex yet fundamentally important roles in determining intrinsic excitability. The Kv and Nav channels located at the axon initial segment (AIS) play a unique and especially important role in generating neuronal output in the form of anterograde axonal and backpropagating action potentials, Aberrant intrinsic excitability in individual neurons within networks contributes to synchronous neuronal activity leading to seizures. Mutations in ion channel genes gives rise to a variety of seizure-related “Channelopathies”, and many of the ion channel subunits associated with epilepsy mutations are localized at the AIS, making this a hotspot for epileptogenesis. Here we review the cellular mechanisms that underlie the trafficking of Kv and Nav channels found at the AIS, and how Kv and Nav channel mutations associated with epilepsy can alter these processes. PMID:23216576

  13. Dysregulation of protein trafficking in neurodegeneration

    PubMed Central

    2014-01-01

    Intracellular protein trafficking plays an important role in neuronal function and survival. Protein misfolding is a common theme found in many neurodegenerative diseases, and intracellular trafficking machinery contributes to the pathological accumulation and clearance of misfolded proteins. Although neurodegenerative diseases exhibit distinct pathological features, abnormal endocytic trafficking is apparent in several neurodegenerative diseases, such as Alzheimer’s disease (AD), Down syndrome (DS) and Parkinson’s disease (PD). In this review, we will focus on protein sorting defects in three major neurodegenerative diseases, including AD, DS and PD. An important pathological feature of AD is the presence of extracellular senile plaques in the brain. Senile plaques are composed of β-amyloid (Aβ) peptide aggregates. Multiple lines of evidence demonstrate that over-production/aggregation of Aβ in the brain is a primary cause of AD and attenuation of Aβ generation has become a topic of extreme interest in AD research. Aβ is generated from β-amyloid precursor protein (APP) through sequential cleavage by β-secretase and the γ-secretase complex. Alternatively, APP can be cleaved by α-secretase within the Aβ domain to release soluble APPα which precludes Aβ generation. DS patients display a strikingly similar pathology to AD patients, including the generation of neuronal amyloid plaques. Moreover, all DS patients develop an AD-like neuropathology by their 40 s. Therefore, understanding the metabolism/processing of APP and how these underlying mechanisms may be pathologically compromised is crucial for future AD and DS therapeutic strategies. Evidence accumulated thus far reveals that synaptic vesicle regulation, endocytic trafficking, and lysosome-mediated autophagy are involved in increased susceptibility to PD. Here we review current knowledge of endosomal trafficking regulation in AD, DS and PD. PMID:25152012

  14. Domestic minor sex trafficking: what the PNP needs to know.

    PubMed

    Hornor, Gail

    2015-01-01

    Human trafficking is a major global public health problem and represents a substantial human rights violation. Human trafficking has been receiving attention in both the lay media and professional literature. Human trafficking can include commercial sex, forced labor, child soldiers, and stealing of human organs. One form of human trafficking represents a significant American pediatric health problem: domestic minor sex trafficking (DMST). DMST is the commercial sexual abuse of children by selling, buying, or trading their sexual service. This continuing education article will define DMST and discuss it in terms of prevalence, risk factors, and practice implications for the pediatric nurse practitioner. PMID:25497135

  15. Drug-induced Inhibition and Trafficking Disruption of ion Channels: Pathogenesis of QT Abnormalities and Drug-induced Fatal Arrhythmias.

    PubMed

    Cubeddu, Luigi X

    2016-01-01

    Risk of severe and fatal ventricular arrhythmias, presenting as Torsade de Pointes (TdP), is increased in congenital and acquired forms of long QT syndromes (LQTS). Drug-induced inhibition of K+ currents, IKs, IKr, IK1, and/or Ito, delay repolarization, prolong QT, and increase the risk of TdP. Drug-induced interference with IKr is the most common cause of acquired LQTS/TdP. Multiple drugs bind to KNCH2-hERG-K+ channels affecting IKr, including antiarrythmics, antibiotics, antivirals, azole-antifungals, antimalarials, anticancer, antiemetics, prokinetics, antipsychotics, and antidepressants. Azithromycin has been recently added to this list. In addition to direct channel inhibition, some drugs interfere with the traffic of channels from the endoplasmic reticulum to the cell membrane, decreasing mature channel membrane density; e.g., pentamidine, geldalamicin, arsenic trioxide, digoxin, and probucol. Other drugs, such as ketoconazole, fluoxetine, norfluoxetine, citalopram, escitalopram, donepezil, tamoxifen, endoxifen, atazanavir, and roxitromycin, induce both direct channel inhibition and impaired channel trafficking. Although many drugs prolong the QT interval, TdP is a rare event. The following conditions increase the risk of drug-induced TdP: a) Disease states/electrolyte levels (heart failure, structural cardiac disease, bradycardia, hypokalemia); b) Pharmacogenomic variables (presence of congenital LQTS, subclinical ion-channel mutations, history of or having a relative with history of drug-induced long QT/TdP); c) Pharmacodynamic and kinetic factors (high doses, women, elderly, metabolism inhibitors, combining two or more QT prolonging drugs, drugs that prolong the QT and increase QT dispersion, and drugs with multiple actions on ion channels). Because most of these conditions are preventable, careful evaluation of risk factors and increased knowledge of drug use associated with repolarization abnormalities are strongly recommended. PMID:26926294

  16. Physical health symptoms reported by trafficked women receiving post-trafficking support in Moldova: prevalence, severity and associated factors

    PubMed Central

    2012-01-01

    Background Many trafficked people suffer high levels of physical, sexual and psychological abuse. Yet, there has been limited research on the physical health problems associated with human trafficking or how the health needs of women in post-trafficking support settings vary according to socio-demographic or trafficking characteristics. Methods We analysed the prevalence and severity of 15 health symptoms reported by 120 trafficked women who had returned to Moldova between December 2007 and December 2008 and were registered with the International Organisation for Migration Assistance and Protection Programme. Women had returned to Moldova an average of 5.9 months prior to interview (range 2-12 months). Results Headaches (61.7%), stomach pain (60.9%), memory problems (44.2%), back pain (42.5%), loss of appetite (35%), and tooth pain (35%) were amongst the most commonly reported symptoms amongst both women trafficked for sexual exploitation and women trafficked for labour exploitation. The prevalence of headache and memory problems was strongly associated with duration of exploitation. Conclusions Trafficked women who register for post-trafficking support services after returning to their country of origin are likely to have long-term physical and dental health needs and should be provided with access to comprehensive medical services. Health problems among women who register for post-trafficking support services after returning to their country of origin are not limited to women trafficked for sexual exploitation but are also experienced by victims of labour exploitation. PMID:22834807

  17. miRNA expression patterns in normal breast tissue and invasive breast cancers of BRCA1 and BRCA2 germ-line mutation carriers

    PubMed Central

    Vos, Shoko; Vesuna, Farhad; Raman, Venu; van Diest, Paul J.; van der Groep, Petra

    2015-01-01

    miRNA deregulation has been found to promote carcinogenesis. Little is known about miRNA deregulation in hereditary breast tumors as no miRNA expression profiling studies have been performed in normal breast tissue of BRCA1 and BRCA2 mutation carriers. miRNA profiles of 17 BRCA1- and 9 BRCA2-associated breast carcinomas were analyzed using microarrays. Normal breast tissues from BRCA1 and BRCA2 mutation carriers (both n = 5) and non-mutation carriers (n = 10) were also included. Candidate miRNAs were validated by qRT-PCR. Breast carcinomas showed extensive miRNA alteration compared to normal breast tissues in BRCA1 and BRCA2 mutation carriers. Moreover, normal breast tissue from BRCA1 mutation carriers already showed miRNA alterations compared to non-mutation carriers. Chromosomal distribution analysis showed several hotspots containing down- or up-regulated miRNAs. Pathway analysis yielded many similarities between the BRCA1 and BRCA2 axes with miRNAs involved in cell cycle regulation, proliferation and apoptosis. Lesser known pathways were also affected, including cellular movement and protein trafficking. This study provides a comprehensive insight into the potential role of miRNA deregulation in BRCA1/2-associated breast carcinogenesis. The observed extensive miRNA deregulation is likely the result of genome-wide effects of chromosomal instability caused by impaired BRCA1 or BRCA2 function. This study's results also suggest the existence of common pathways driving breast carcinogenesis in both BRCA1 and BRCA2 germ-line mutation carriers. PMID:26378051

  18. Loss-of-function mutations in the IL-21 receptor gene cause a primary immunodeficiency syndrome

    PubMed Central

    Kotlarz, Daniel; Ziętara, Natalia; Uzel, Gulbu; Weidemann, Thomas; Braun, Christian J.; Diestelhorst, Jana; Krawitz, Peter M.; Robinson, Peter N.; Hecht, Jochen; Puchałka, Jacek; Gertz, E. Michael; Schäffer, Alejandro A.; Lawrence, Monica G.; Kardava, Lela; Pfeifer, Dietmar; Baumann, Ulrich; Pfister, Eva-Doreen; Hanson, Eric P.; Schambach, Axel; Jacobs, Roland; Kreipe, Hans; Moir, Susan; Milner, Joshua D.; Schwille, Petra; Mundlos, Stefan

    2013-01-01

    Primary immunodeficiencies (PIDs) represent exquisite models for studying mechanisms of human host defense. In this study, we report on two unrelated kindreds, with two patients each, who had cryptosporidial infections associated with chronic cholangitis and liver disease. Using exome and candidate gene sequencing, we identified two distinct homozygous loss-of-function mutations in the interleukin-21 receptor gene (IL21R; c.G602T, p.Arg201Leu and c.240_245delCTGCCA, p.C81_H82del). The IL-21RArg201Leu mutation causes aberrant trafficking of the IL-21R to the plasma membrane, abrogates IL-21 ligand binding, and leads to defective phosphorylation of signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5. We observed impaired IL-21–induced proliferation and immunoglobulin class-switching in B cells, cytokine production in T cells, and NK cell cytotoxicity. Our study indicates that human IL-21R deficiency causes an immunodeficiency and highlights the need for early diagnosis and allogeneic hematopoietic stem cell transplantation in affected children. PMID:23440042

  19. Small GTPase regulation of GPCR anterograde trafficking

    PubMed Central

    Wang, Guansong; Wu, Guangyu

    2011-01-01

    The physiological functions of heterotrimeric G protein-coupled receptors (GPCRs) are dictated by their intracellular trafficking and precise targeting to the functional destinations. Over the past decades, most studies on the trafficking of GPCRs have focused on the events involved in endocytosis and recycling. In contrast, the molecular mechanisms underlying anterograde transport of newly synthesized GPCRs from the endoplasmic reticulum (ER) to the cell surface have just begun to be revealed. In this review, we will discuss current advances in understanding the role of Ras-like GTPases, specifically the Rab and Sar1/ARF subfamilies, in regulating cell-surface transport of GPCRs en route from the ER and the Golgi. PMID:22015208

  20. Sex Trafficking: Policies, Programs, and Services.

    PubMed

    Orme, Julie; Ross-Sheriff, Fariyal

    2015-10-01

    Sex trafficking (ST), a contemporary form of female slavery, is a human rights issue of critical concern to social work. The global response to ST has been substantial, and 166 countries have adopted anti-ST legislation. Despite considerable efforts to combat ST, the magnitude is increasing. To date, the majority of anti-ST efforts have focused on criminalization policies that target traffickers or purchasers of sexual services, who are predominantly male; prevention programming and services for predominantly female victims have received less support. Therapeutic services to assist pornography addicts and purchasers of sexual services are also necessary. In this article, authors examine current anti-ST policies, programs, and services, both domestically and globally, and present an innovative paradigm that addresses social inequities and emphasizes prevention programming. They conclude with a discussion of the paradigm's implications for social work policies, practices, and services. PMID:26489349

  1. Endocytic membrane trafficking and neurodegenerative disease.

    PubMed

    Schreij, Andrea M A; Fon, Edward A; McPherson, Peter S

    2016-04-01

    Neurodegenerative diseases are amongst the most devastating of human disorders. New technologies have led to a rapid increase in the identification of disease-related genes with an enhanced appreciation of the key roles played by genetics in the etiology of these disorders. Importantly, pinpointing the normal function of disease gene proteins leads to new understanding of the cellular machineries and pathways that are altered in the disease process. One such emerging pathway is membrane trafficking in the endosomal system. This key cellular process controls the localization and levels of a myriad of proteins and is thus critical for normal cell function. In this review we will focus on three neurodegenerative diseases; Parkinson disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegias, for which a large number of newly discovered disease genes encode proteins that function in endosomal membrane trafficking. We will describe how alterations in these proteins affect endosomal function and speculate on the contributions of these disruptions to disease pathophysiology. PMID:26721251

  2. Technosocial Predictive Analytics for Illicit Nuclear Trafficking

    SciTech Connect

    Sanfilippo, Antonio P.; Butner, R. Scott; Cowell, Andrew J.; Dalton, Angela C.; Haack, Jereme N.; Kreyling, Sean J.; Riensche, Roderick M.; White, Amanda M.; Whitney, Paul D.

    2011-03-29

    Illicit nuclear trafficking networks are a national security threat. These networks can directly lead to nuclear proliferation, as state or non-state actors attempt to identify and acquire nuclear weapons-related expertise, technologies, components, and materials. The ability to characterize and anticipate the key nodes, transit routes, and exchange mechanisms associated with these networks is essential to influence, disrupt, interdict or destroy the function of the networks and their processes. The complexities inherent to the characterization and anticipation of illicit nuclear trafficking networks requires that a variety of modeling and knowledge technologies be jointly harnessed to construct an effective analytical and decision making workflow in which specific case studies can be built in reasonable time and with realistic effort. In this paper, we explore a solution to this challenge that integrates evidentiary and dynamic modeling with knowledge management and analytical gaming, and demonstrate its application to a geopolitical region at risk.

  3. Incunabular immunological events in prion trafficking.

    PubMed

    Michel, Brady; Meyerett-Reid, Crystal; Johnson, Theodore; Ferguson, Adam; Wyckoff, Christy; Pulford, Bruce; Bender, Heather; Avery, Anne; Telling, Glenn; Dow, Steven; Zabel, Mark D

    2012-01-01

    While prions probably interact with the innate immune system immediately following infection, little is known about this initial confrontation. Here we investigated incunabular events in lymphotropic and intranodal prion trafficking by following highly enriched, fluorescent prions from infection sites to draining lymph nodes. We detected biphasic lymphotropic transport of prions from the initial entry site upon peripheral prion inoculation. Prions arrived in draining lymph nodes cell autonomously within two hours of intraperitoneal administration. Monocytes and dendritic cells (DCs) required Complement for optimal prion delivery to lymph nodes hours later in a second wave of prion trafficking. B cells constituted the majority of prion-bearing cells in the mediastinal lymph node by six hours, indicating intranodal prion reception from resident DCs or subcapsulary sinus macrophages or directly from follicular conduits. These data reveal novel, cell autonomous prion lymphotropism, and a prominent role for B cells in intranodal prion movement. PMID:22679554

  4. Clathrin-Independent Trafficking of AMPA Receptors

    PubMed Central

    Tigaret, Cezar M.; Mellor, Jack R.

    2015-01-01

    Membrane trafficking of AMPA receptors (AMPARs) is critical for neuronal function and plasticity. Although rapid forms of AMPAR internalization during long-term depression (LTD) require clathrin and dynamin, the mechanisms governing constitutive AMPAR turnover and internalization of AMPARs during slow homeostatic forms of synaptic plasticity remain unexplored. Here, we show that, in contrast to LTD, constitutive AMPAR internalization and homeostatic AMPAR downscaling in rat neurons do not require dynamin or clathrin function. Instead, constitutive AMPAR trafficking is blocked by a Rac1 inhibitor and is regulated by a dynamic nonstructural pool of F-actin. Our findings reveal a novel role for neuronal clathrin-independent endocytosis controlled by actin dynamics and suggest that the interplay between different modes of receptor endocytosis provides for segregation between distinct modes of neuronal plasticity. PMID:25810514

  5. BEX5/RabA1b Regulates trans-Golgi Network-to-Plasma Membrane Protein Trafficking in Arabidopsis[W

    PubMed Central

    Feraru, Elena; Feraru, Mugurel I.; Asaoka, Rin; Paciorek, Tomasz; De Rycke, Riet; Tanaka, Hirokazu; Nakano, Akihiko; Friml, Jiří

    2012-01-01

    Constitutive endocytic recycling is a crucial mechanism allowing regulation of the activity of proteins at the plasma membrane and for rapid changes in their localization, as demonstrated in plants for PIN-FORMED (PIN) proteins, the auxin transporters. To identify novel molecular components of endocytic recycling, mainly exocytosis, we designed a PIN1-green fluorescent protein fluorescence imaging–based forward genetic screen for Arabidopsis thaliana mutants that showed increased intracellular accumulation of cargos in response to the trafficking inhibitor brefeldin A (BFA). We identified bex5 (for BFA-visualized exocytic trafficking defective), a novel dominant mutant carrying a missense mutation that disrupts a conserved sequence motif of the small GTPase, RAS GENES FROM RAT BRAINA1b. bex5 displays defects such as enhanced protein accumulation in abnormal BFA compartments, aberrant endosomes, and defective exocytosis and transcytosis. BEX5/RabA1b localizes to trans-Golgi network/early endosomes (TGN/EE) and acts on distinct trafficking processes like those regulated by GTP exchange factors on ADP-ribosylation factors GNOM-LIKE1 and HOPM INTERACTOR7/BFA-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1, which regulate trafficking at the Golgi apparatus and TGN/EE, respectively. All together, this study identifies Arabidopsis BEX5/RabA1b as a novel regulator of protein trafficking from a TGN/EE compartment to the plasma membrane. PMID:22773752

  6. The Human Epilepsy Mutation GABRG2(Q390X) Causes Chronic Subunit Accumulation and Neurodegeneration

    PubMed Central

    Kang, Jing-Qiong; Shen, Wangzhen; Zhou, Chengwen; Xu, Dong; Macdonald, Robert L.

    2015-01-01

    Genetic epilepsy and neurodegenerative diseases are two common neurological disorders conventionally viewed as being unrelated. A subset of patients with severe genetic epilepsies with impaired development and often death respond poorly to anticonvulsant drug therapy, suggesting a need for new therapeutic targets. Previously, we reported that multiple GABAA receptor epilepsy mutations caused protein misfolding and abnormal receptor trafficking. Here we establish in a novel model of a severe human genetic epileptic encephalopathy, the Gabrg2+/Q390X knock-in mouse, that in addition to impairing inhibitory neurotransmission, mutant GABAA receptor γ2(Q390X) subunits accumulated and aggregated intracellularly, activated caspase 3 and caused widespread, age-dependent neurodegeneration. These novel findings suggest that the fundamental protein metabolism and cellular consequences of the epilepsy-associated mutant γ2(Q390X) ion channel subunit are not fundamentally different from those associated with neurodegeneration. The study has far-reaching significance for identification of conserved pathological cascades and mechanism-based therapies that overlap genetic epilepsies and neurodegenerative diseases. PMID:26005849

  7. Trafficking of ThermoTRP Channels

    PubMed Central

    Ferrandiz-Huertas, Clotilde; Mathivanan, Sakthikumar; Wolf, Christoph Jakob; Devesa, Isabel; Ferrer-Montiel, Antonio

    2014-01-01

    ThermoTRP channels (thermoTRPs) define a subfamily of the transient receptor potential (TRP) channels that are activated by changes in the environmental temperature, from noxious cold to injurious heat. Acting as integrators of several stimuli and signalling pathways, dysfunction of these channels contributes to several pathological states. The surface expression of thermoTRPs is controlled by both, the constitutive and regulated vesicular trafficking. Modulation of receptor surface density during pathological processes is nowadays considered as an interesting therapeutic approach for management of diseases, such as chronic pain, in which an increased trafficking is associated with the pathological state. This review will focus on the recent advances trafficking of the thermoTRP channels, TRPV1, TRPV2, TRPV4, TRPM3, TRPM8 and TRPA1, into/from the plasma membrane. Particularly, regulated membrane insertion of thermoTRPs channels contributes to a fine tuning of final channel activity, and indeed, it has resulted in the development of novel therapeutic approaches with successful clinical results such as disruption of SNARE-dependent exocytosis by botulinum toxin or botulinomimetic peptides. PMID:25257900

  8. Role of dynamin and clathrin in the cellular trafficking of flotillins.

    PubMed

    Meister, Melanie; Zuk, Alexandra; Tikkanen, Ritva

    2014-07-01

    Flotillin-1 and flotillin-2 are highly conserved, membrane-microdomain-associated proteins that have been shown to be involved in signal transduction, membrane trafficking and cell adhesion. Upon growth factor stimulation, flotillins are tyrosine phosphorylated and become endocytosed from the plasma membrane into endosomes from which they are recycled back to the plasma membrane. Although a role for flotillin-1 in the endocytosis of certain cargo proteins has been suggested, it is not known how the growth-factor-induced endocytosis of flotillins is regulated and which endocytosis pathway is used. However, this is likely to be different from the pathway used by flotillin-dependent cargo. In this study, we have addressed the mechanistic details of flotillin trafficking during growth factor signaling. We show that dynamin-2 activity is required for the uptake of flotillins from the plasma membrane upon epidermal growth factor stimulation, and inhibition of dynamin-2 GTPase activity impairs flotillin endocytosis. Surprisingly, recycling of flotillins from endosomes to the plasma membrane appears to require both dynamin-2 and clathrin. Upon overexpression of dynamin-2 mutants or depletion of clathrin heavy chain, flotillins are permanently trapped in endosomes. These data show that clathrin and dynamin are required for the endosomal sorting of flotillins, and the study provides a mechanistic dissection of the thus far poorly characterized endosomal trafficking of flotillins. PMID:24809731

  9. PACS-1 Mediates Phosphorylation-Dependent Ciliary Trafficking of the CNG Channel in Olfactory Sensory Neurons

    PubMed Central

    Jenkins, Paul M.; Zhang, Lian; Thomas, Gary; Martens, Jeffrey R.

    2009-01-01

    Impaired ciliary protein transport in olfactory sensory neurons (OSNs) leads to anosmia, and is a newly recognized clinical manifestation of a class of human disorders called ciliopathies. Surprisingly little is known regarding the mechanisms controlling trafficking to this unique neuronal compartment. Here, we show a novel role for phosphofurin acidic cluster-sorting protein 1 (PACS-1) in the ciliary trafficking of the olfactory CNG channel. PACS-1 is an intracellular sorting protein that mediates its effects through the binding of acidic clusters on cargo protein. This interaction is dependent on CK2 phosphorylation of both PACS-1 and its cargo. We show that CNGB1b contains two putative PACS-1 binding sites, which are phosphorylated by the serine/threonine protein kinase, CK2. Additionally, we show that PACS-1 is expressed in OSNs and interacts in complex with the CNG channel. CK2 inhibition in native OSNs causes a loss of CNG channel from cilia and subsequent olfactory dysfunction, while adenoviral expression of mutant PACS-1 causes similar mislocalization. These results provide a mechanism for the subunit-dependent ciliary trafficking of the CNG channel and offer insight into the mechanisms of ciliary transport. PMID:19710307

  10. Galectin-3 regulates intracellular trafficking of epidermal growth factor receptor through Alix and promotes keratinocyte migration

    PubMed Central

    Liu, Wei; Hsu, Daniel K.; Chen, Huan-Yuan; Yang, Ri-Yao; Carraway, Kermit L.; Isseroff, Roslyn R.; Liu, Fu-Tong

    2012-01-01

    The epidermal growth factor receptor (EGFR)-mediated signaling pathways are important in a variety of cellular processes, including cell migration and wound re-epithelialization. Intracellular trafficking of EGFR is critical for maintaining EGFR surface expression. Galectin-3, a member of an animal lectin family, has been implicated in a number of physiological and pathological processes. Through studies of galectin-3-deficient mice and cells isolated from these mice, we demonstrated that absence of galectin-3 impairs keratinocyte migration and skin wound re-epithelialization. We have linked this pro-migratory function to a crucial role of cytosolic galectin-3 in controlling intracellular trafficking and cell surface expression of EGFR after EGF stimulation. Without galectin-3, the surface levels of EGFR are dramatically reduced and the receptor accumulates diffusely in the cytoplasm. This is associated with reduced rates of both endocytosis and recycling of the receptor. We have provided evidence that this novel function of galectin-3 may be mediated through interaction with its binding partner Alix, which is a protein component of the endosomal sorting complex required for transport (ESCRT) machinery. Our results suggest that galectin-3 is potentially a critical regulator of a number of important cellular responses through its intracellular control of trafficking of cell surface receptors. PMID:22785133

  11. HAPLESS13-Mediated Trafficking of STRUBBELIG Is Critical for Ovule Development in Arabidopsis.

    PubMed

    Wang, Jia-Gang; Feng, Chong; Liu, Hai-Hong; Ge, Fu-Rong; Li, Sha; Li, Hong-Ju; Zhang, Yan

    2016-08-01

    Planar morphogenesis, a distinct feature of multicellular organisms, is crucial for the development of ovule, progenitor of seeds. Both receptor-like kinases (RLKs) such as STRUBBELIG (SUB) and auxin gradient mediated by PIN-FORMED1 (PIN1) play instructive roles in this process. Fine-tuned intercellular communications between different cell layers during ovule development demands dynamic membrane distribution of these cell-surface proteins, presumably through vesicle-mediated sorting. However, the way it's achieved and the trafficking routes involved are obscure. We report that HAPLESS13 (HAP13)-mediated trafficking of SUB is critical for ovule development. HAP13 encodes the μ subunit of adaptor protein 1 (AP1) that mediates protein sorting at the trans-Golgi network/early endosome (TGN/EE). The HAP13 mutant, hap13-1, is defective in outer integument growth, resulting in exposed nucellus accompanied with impaired pollen tube guidance and reception. SUB is mis-targeted in hap13-1. However, unlike that of PIN2, the distribution of PIN1 is independent of HAP13. Genetic interference of exocytic trafficking at the TGN/EE by specifically downregulating HAP13 phenocopied the defects of hap13-1 in SUB targeting and ovule development, supporting a key role of sporophytically expressed SUB in instructing female gametogenesis. PMID:27541731

  12. HAPLESS13-Mediated Trafficking of STRUBBELIG Is Critical for Ovule Development in Arabidopsis

    PubMed Central

    Wang, Jia-Gang; Feng, Chong; Liu, Hai-Hong; Ge, Fu-Rong; Li, Sha; Zhang, Yan

    2016-01-01

    Planar morphogenesis, a distinct feature of multicellular organisms, is crucial for the development of ovule, progenitor of seeds. Both receptor-like kinases (RLKs) such as STRUBBELIG (SUB) and auxin gradient mediated by PIN-FORMED1 (PIN1) play instructive roles in this process. Fine-tuned intercellular communications between different cell layers during ovule development demands dynamic membrane distribution of these cell-surface proteins, presumably through vesicle-mediated sorting. However, the way it’s achieved and the trafficking routes involved are obscure. We report that HAPLESS13 (HAP13)-mediated trafficking of SUB is critical for ovule development. HAP13 encodes the μ subunit of adaptor protein 1 (AP1) that mediates protein sorting at the trans-Golgi network/early endosome (TGN/EE). The HAP13 mutant, hap13-1, is defective in outer integument growth, resulting in exposed nucellus accompanied with impaired pollen tube guidance and reception. SUB is mis-targeted in hap13-1. However, unlike that of PIN2, the distribution of PIN1 is independent of HAP13. Genetic interference of exocytic trafficking at the TGN/EE by specifically downregulating HAP13 phenocopied the defects of hap13-1 in SUB targeting and ovule development, supporting a key role of sporophytically expressed SUB in instructing female gametogenesis. PMID:27541731

  13. P120-Catenin Regulates Early Trafficking Stages of the N-Cadherin Precursor Complex

    PubMed Central

    Wehrendt, Diana P.; Carmona, Fernando; González Wusener, Ana E.; González, Ángela; Martínez, Juan M. Lázaro; Arregui, Carlos O.

    2016-01-01

    It is well established that binding of p120 catenin to the cytoplasmic domain of surface cadherin prevents cadherin endocytosis and degradation, contributing to cell-cell adhesion. In the present work we show that p120 catenin bound to the N-cadherin precursor, contributes to its anterograde movement from the endoplasmic reticulum (ER) to the Golgi complex. In HeLa cells, depletion of p120 expression, or blocking its binding to N-cadherin, increased the accumulation of the precursor in the ER, while it decreased the localization of mature N-cadherin at intercellular junctions. Reconstitution experiments in p120-deficient SW48 cells with all three major isoforms of p120 (1, 3 and 4) had similar capacity to promote the processing of the N-cadherin precursor to the mature form, and its localization at cell-cell junctions. P120 catenin and protein tyrosine phosphatase PTP1B facilitated the recruitment of the N-ethylmaleimide sensitive factor (NSF), an ATPase involved in vesicular trafficking, to the N-cadherin precursor complex. Dominant negative NSF E329Q impaired N-cadherin trafficking, maturation and localization at cell-cell junctions. Our results uncover a new role for p120 catenin bound to the N-cadherin precursor ensuring its trafficking through the biosynthetic pathway towards the cell surface. PMID:27254316

  14. Nephrocystin proteins NPHP5 and Cep290 regulate BBSome integrity, ciliary trafficking and cargo delivery

    PubMed Central

    Barbelanne, Marine; Hossain, Delowar; Chan, David Puth; Peränen, Johan; Tsang, William Y.

    2015-01-01

    Proper functioning of cilia, hair-like structures responsible for sensation and locomotion, requires nephrocystin-5 (NPHP5) and a multi-subunit complex called the Bardet–Biedl syndrome (BBS)ome, but their precise relationship is not understood. The BBSome is involved in the trafficking of membrane cargos to cilia. While it is known that a loss of any single subunit prevents ciliary trafficking of the BBSome and its cargos, the mechanisms underlying ciliary entry of this complex are not well characterized. Here, we report that a transition zone protein NPHP5 contains two separate BBS-binding sites and interacts with the BBSome to mediate its integrity. Depletion of NPHP5, or expression of NPHP5 mutant missing one binding site, specifically leads to dissociation of BBS2 and BBS5 from the BBSome and loss of ciliary BBS2 and BBS5 without compromising the ability of the other subunits to traffic into cilia. Depletion of Cep290, another transition zone protein that directly binds to NPHP5, causes additional dissociation of BBS8 and loss of ciliary BBS8. Furthermore, delivery of BBSome cargos, smoothened, VPAC2 and Rab8a, to the ciliary compartment is completely disabled in the absence of single BBS subunits, but is selectively impaired in the absence of NPHP5 or Cep290. These findings define a new role of NPHP5 and Cep290 in controlling integrity and ciliary trafficking of the BBSome, which in turn impinge on the delivery of ciliary cargo. PMID:25552655

  15. Norepinephrine Deficiency Is Caused by Combined Abnormal mRNA Processing and Defective Protein Trafficking of Dopamine β-Hydroxylase*

    PubMed Central

    Kim, Chun-Hyung; Leung, Amanda; Huh, Yang Hoon; Yang, Eungi; Kim, Deog-Joong; Leblanc, Pierre; Ryu, Hoon; Kim, Kyungjin; Kim, Dong-Wook; Garland, Emily M.; Raj, Satish R.; Biaggioni, Italo; Robertson, David; Kim, Kwang-Soo

    2011-01-01

    Human norepinephrine (NE) deficiency (or dopamine β-hydroxylase (DBH) deficiency) is a rare congenital disorder of primary autonomic failure, in which neurotransmitters NE and epinephrine are undetectable. Although potential pathogenic mutations, such as a common splice donor site mutation (IVS1+2T→C) and various missense mutations, in NE deficiency patients were identified, molecular mechanisms underlying this disease remain unknown. Here, we show that the IVS1+2T→C mutation results in a non-detectable level of DBH protein production and that all three missense mutations tested lead to the DBH protein being trapped in the endoplasmic reticulum (ER). Supporting the view that mutant DBH induces an ER stress response, exogenous expression of mutant DBH dramatically induced expression of BiP, a master ER chaperone. Furthermore, we found that a pharmacological chaperone, glycerol, significantly rescued defective trafficking of mutant DBH proteins. Taken together, we propose that NE deficiency is caused by the combined abnormal processing of DBH mRNA and defective protein trafficking and that this disease could be treated by a pharmacological chaperone(s). PMID:21209083

  16. Norepinephrine deficiency is caused by combined abnormal mRNA processing and defective protein trafficking of dopamine beta-hydroxylase.

    PubMed

    Kim, Chun-Hyung; Leung, Amanda; Huh, Yang Hoon; Yang, Eungi; Kim, Deog-Joong; Leblanc, Pierre; Ryu, Hoon; Kim, Kyungjin; Kim, Dong-Wook; Garland, Emily M; Raj, Satish R; Biaggioni, Italo; Robertson, David; Kim, Kwang-Soo

    2011-03-18

    Human norepinephrine (NE) deficiency (or dopamine β-hydroxylase (DBH) deficiency) is a rare congenital disorder of primary autonomic failure, in which neurotransmitters NE and epinephrine are undetectable. Although potential pathogenic mutations, such as a common splice donor site mutation (IVS1+2T→C) and various missense mutations, in NE deficiency patients were identified, molecular mechanisms underlying this disease remain unknown. Here, we show that the IVS1+2T→C mutation results in a non-detectable level of DBH protein production and that all three missense mutations tested lead to the DBH protein being trapped in the endoplasmic reticulum (ER). Supporting the view that mutant DBH induces an ER stress response, exogenous expression of mutant DBH dramatically induced expression of BiP, a master ER chaperone. Furthermore, we found that a pharmacological chaperone, glycerol, significantly rescued defective trafficking of mutant DBH proteins. Taken together, we propose that NE deficiency is caused by the combined abnormal processing of DBH mRNA and defective protein trafficking and that this disease could be treated by a pharmacological chaperone(s). PMID:21209083

  17. TLR signals posttranscriptionally regulate the cytokine trafficking mediator sortilin

    PubMed Central

    Yabe-Wada, Toshiki; Matsuba, Shintaro; Takeda, Kazuya; Sato, Tetsuya; Suyama, Mikita; Ohkawa, Yasuyuki; Takai, Toshiyuki; Shi, Haifeng; Philpott, Caroline C.; Nakamura, Akira

    2016-01-01

    Regulating the transcription, translation and secretion of cytokines is crucial for controlling the appropriate balance of inflammation. Here we report that the sorting receptor sortilin plays a key role in cytokine production. We observed interactions of sortilin with multiple cytokines including IFN-α, and sortilin depletion in plasmacytoid dendritic cells (pDCs) led to a reduction of IFN-α secretion, suggesting a pivotal role of sortilin in the exocytic trafficking of IFN-α in pDCs. Moreover, sortilin mRNA was degraded posttranscriptionally upon stimulation with various TLR ligands. Poly-rC-binding protein 1 (PCBP1) recognized the C-rich element (CRE) in the 3′ UTR of sortilin mRNA, and depletion of PCBP1 enhanced the degradation of sortilin transcripts, suggesting that PCBP1 can act as a trans-acting factor to stabilize sortilin transcripts. The nucleotide-binding ability of PCBP1 was impaired by zinc ions and alterations of intracellular zinc affect sortilin expression. PCBP1 may therefore control the stability of sortilin transcripts by sensing intracellular zinc levels. Collectively, our findings provide insights into the posttranslational regulation of cytokine production through the posttranscriptional control of sortilin expression by TLR signals. PMID:27220277

  18. Active Trafficking of Alpha 1 Antitrypsin across the Lung Endothelium

    PubMed Central

    Lockett, Angelia D.; Brown, Mary Beth; Santos-Falcon, Nieves; Rush, Natalia I.; Oueini, Houssam; Oberle, Amber J.; Bolanis, Esther; Fragoso, Miryam A.; Petrusca, Daniela N.; Serban, Karina A.; Schweitzer, Kelly S.; Presson Jr., Robert G.

    2014-01-01

    The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT) may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency. PMID:24743137

  19. TLR signals posttranscriptionally regulate the cytokine trafficking mediator sortilin.

    PubMed

    Yabe-Wada, Toshiki; Matsuba, Shintaro; Takeda, Kazuya; Sato, Tetsuya; Suyama, Mikita; Ohkawa, Yasuyuki; Takai, Toshiyuki; Shi, Haifeng; Philpott, Caroline C; Nakamura, Akira

    2016-01-01

    Regulating the transcription, translation and secretion of cytokines is crucial for controlling the appropriate balance of inflammation. Here we report that the sorting receptor sortilin plays a key role in cytokine production. We observed interactions of sortilin with multiple cytokines including IFN-α, and sortilin depletion in plasmacytoid dendritic cells (pDCs) led to a reduction of IFN-α secretion, suggesting a pivotal role of sortilin in the exocytic trafficking of IFN-α in pDCs. Moreover, sortilin mRNA was degraded posttranscriptionally upon stimulation with various TLR ligands. Poly-rC-binding protein 1 (PCBP1) recognized the C-rich element (CRE) in the 3' UTR of sortilin mRNA, and depletion of PCBP1 enhanced the degradation of sortilin transcripts, suggesting that PCBP1 can act as a trans-acting factor to stabilize sortilin transcripts. The nucleotide-binding ability of PCBP1 was impaired by zinc ions and alterations of intracellular zinc affect sortilin expression. PCBP1 may therefore control the stability of sortilin transcripts by sensing intracellular zinc levels. Collectively, our findings provide insights into the posttranslational regulation of cytokine production through the posttranscriptional control of sortilin expression by TLR signals. PMID:27220277

  20. Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis

    PubMed Central

    Larson, Charles L.; Beare, Paul A.; Howe, Dale; Heinzen, Robert A.

    2013-01-01

    Successful macrophage colonization by Coxiella burnetii, the cause of human Q fever, requires pathogen-directed biogenesis of a large, growth-permissive parasitophorous vacuole (PV) with phagolysosomal characteristics. The vesicular trafficking pathways co-opted by C. burnetii for PV development are poorly defined; however, it is predicted that effector proteins delivered to the cytosol by a defective in organelle trafficking/intracellular multiplication (Dot/Icm) type 4B secretion system are required for membrane recruitment. Here, we describe involvement of clathrin-mediated vesicular trafficking in PV generation and the engagement of this pathway by the C. burnetii type 4B secretion system substrate Coxiella vacuolar protein A (CvpA). CvpA contains multiple dileucine [DERQ]XXXL[LI] and tyrosine (YXXΦ)-based endocytic sorting motifs like those recognized by the clathrin adaptor protein (AP) complexes AP1, AP2, and AP3. A C. burnetii ΔcvpA mutant exhibited significant defects in replication and PV development, confirming the importance of CvpA in infection. Ectopically expressed mCherry-CvpA localized to tubular and vesicular domains of pericentrosomal recycling endosomes positive for Rab11 and transferrin receptor, and CvpA membrane interactions were lost upon mutation of endocytic sorting motifs. Consistent with CvpA engagement of the endocytic recycling system, ectopic expression reduced uptake of transferrin. In pull-down assays, peptides containing CvpA-sorting motifs and full-length CvpA interacted with AP2 subunits and clathrin heavy chain. Furthermore, depletion of AP2 or clathrin by siRNA treatment significantly inhibited C. burnetii replication. Thus, our results reveal the importance of clathrin-coated vesicle trafficking in C. burnetii infection and define a role for CvpA in subverting these transport mechanisms. PMID:24248335

  1. Differential Regulation of Endosomal GPCR/β-Arrestin Complexes and Trafficking by MAPK*

    PubMed Central

    Khoury, Etienne; Nikolajev, Ljiljana; Simaan, May; Namkung, Yoon; Laporte, Stéphane A.

    2014-01-01

    β-Arrestins are signaling adaptors that bind to agonist-occupied G protein-coupled receptors (GPCRs) and target them for endocytosis; however, the mechanisms regulating receptor/β-arrestin complexes and trafficking in endosomes, remain ill defined. Here we show, in live cells, differential dynamic regulation of endosomal bradykinin B2 receptor (B2R) complexes with either β-arrestin-1 or -2. We find a novel role for MAPK in the B2R/β-arrestin-2 complex formation, receptor trafficking and signaling mediated by an ERK1/2 regulatory motif in the hinge domain of the rat β-arrestin-2 (PET178P), but not rat β-arrestin-1 (PER177P). While the ERK1/2 regulatory motif is conserved between rat and mouse β-arrestin-2, it is surprisingly not conserved in human β-arrestin-2 (PEK178P). However, mutation of lysine 178 to threonine is sufficient to confer MAPK sensitivity to the human β-arrestin-2. Furthermore, substitution for a phosphomimetic residue in both the rat and the human β-arrestin-2 (T/K178D) significantly stabilizes B2R/β-arrestin complexes in endosomes, delays receptor recycling to the plasma membrane and maintains intracellular MAPK signaling. Similarly, the endosomal trafficking of β2-adrenergic, angiotensin II type 1 and vasopressin V2 receptors was altered by the β-arrestin-2 T178D mutant. Our findings unveil a novel subtype specific mode of MAPK-dependent regulation of β-arrestins in intracellular trafficking and signaling of GPCRs, and suggest differential endosomal receptor/β-arrestin-2 signaling roles among species. PMID:25016018

  2. Oxidative stress inhibits caveolin-1 palmitoylation and trafficking in endothelial cells

    NASA Technical Reports Server (NTRS)

    Parat, Marie-Odile; Stachowicz, Rafal Z.; Fox, Paul L.

    2002-01-01

    During normal and pathological conditions, endothelial cells (ECs) are subjected to locally generated reactive oxygen species, produced by themselves or by other vessel wall cells. In excess these molecules cause oxidative injury to the cell but at moderate levels they might modulate intracellular signalling pathways. We have investigated the effect of oxidative stress on the palmitoylation and trafficking of caveolin-1 in bovine aortic ECs. Exogenous H2O2 did not alter the intracellular localization of caveolin-1 in ECs. However, metabolic labelling experiments showed that H2O2 inhibited the trafficking of newly synthesized caveolin-1 to membrane raft domains. Several mechanisms potentially responsible for this inhibition were examined. Impairment of caveolin-1 synthesis by H2O2 was not responsible for diminished trafficking. Similarly, the inhibition was independent of H2O2-induced caveolin-1 phosphorylation as shown by the markedly different concentration dependences. We tested the effect of H2O2 on palmitoylation of caveolin-1 by the incorporation of [3H]palmitic acid. Exposure of ECs to H2O2 markedly inhibited the palmitoylation of caveolin-1. Comparable inhibition was observed after treatment of cells with H2O2 delivered either as a bolus or by continuous delivery with glucose and glucose oxidase. Kinetic studies showed that H2O2 did not alter the rate of caveolin-1 depalmitoylation but instead decreased the 'on-rate' of palmitoylation. Together these results show for the first time the modulation of protein palmitoylation by oxidative stress, and suggest a cellular mechanism by which stress might influence caveolin-1-dependent cell activities such as the concentration of signalling proteins and cholesterol trafficking.

  3. ALTERATIONS IN MATERNAL-FETAL CELLULAR TRAFFICKING AFTER FETAL SURGERY

    PubMed Central

    Saadai, Payam; Lee, Tzong-Hae; Bautista, Geoanna; Gonzales, Kelly D.; Nijagal, Amar; Busch, Michael P.; Kim, CJ; Romero, Roberto; Lee, Hanmin; Hirose, Shinjiro; Rand, Larry; Miniati, Douglas; Farmer, Diana L.; MacKenzie, Tippi C.

    2012-01-01

    Background/Purpose Bi-directional trafficking of cells between the mother and the fetus is routine in pregnancy and a component of maternal-fetal tolerance. Changes in fetal-to-maternal cellular trafficking have been reported in prenatal complications, but maternal-to-fetal trafficking has never been studied in the context of fetal intervention. We hypothesized that patients undergoing open fetal surgery would have altered maternal-fetal cellular trafficking. Methods Cellular trafficking was analyzed in patients with myelomeningocele (MMC) who underwent open fetal surgical repair (n=5), MMC patients who had routine postnatal repair (n=6), and normal term patients (n=9). As a control for the fetal operation, trafficking was also analyzed in patients who were delivered by an ex utero intrapartum treatment (EXIT) procedure (n=6). Microchimerism in maternal and cord blood was determined using quantitative real-time PCR for non-shared alleles. Results Maternal-to-fetal trafficking was significantly increased in patients who underwent open fetal surgery for MMC compared to normal controls, postnatal MMC repair, and EXIT patients. There were no differences in fetal-to-maternal cell trafficking between groups. Conclusion Patients undergoing open fetal surgery for MMC have elevated levels of maternal microchimerism. These results suggest altered trafficking and/or increased proliferation of maternal cells in fetal blood and may have important implications for preterm labor. PMID:22703775

  4. Evidence for Ubiquitin-Regulated Nuclear and Subnuclear Trafficking among Paramyxovirinae Matrix Proteins

    PubMed Central

    Pentecost, Mickey; Vashisht, Ajay A.; Beaty, Shannon M.; Park, Arnold; Wang, Yao E.; Yun, Tatyana E; Freiberg, Alexander N.; Wohlschlegel, James A.; Lee, Benhur

    2015-01-01

    The paramyxovirus matrix (M) protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane. Transient nuclear residence of some M proteins hints at non-structural roles. However, little is known regarding the mechanisms that regulate the nuclear sojourn. Previously, we found that the nuclear-cytoplasmic trafficking of Nipah virus M (NiV-M) is a prerequisite for budding, and is regulated by a bipartite nuclear localization signal (NLSbp), a leucine-rich nuclear export signal (NES), and monoubiquitination of the K258 residue within the NLSbp itself (NLSbp-lysine). To define whether the sequence determinants of nuclear trafficking identified in NiV-M are common among other Paramyxovirinae M proteins, we generated the homologous NES and NLSbp-lysine mutations in M proteins from the five major Paramyxovirinae genera. Using quantitative 3D confocal microscopy, we determined that the NES and NLSbp-lysine are required for the efficient nuclear export of the M proteins of Nipah virus, Hendra virus, Sendai virus, and Mumps virus. Pharmacological depletion of free ubiquitin or mutation of the conserved NLSbp-lysine to an arginine, which inhibits M ubiquitination, also results in nuclear and nucleolar retention of these M proteins. Recombinant Sendai virus (rSeV-eGFP) bearing the NES or NLSbp-lysine M mutants rescued at similar efficiencies to wild type. However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread. Recombinant Mumps virus (rMuV-eGFP) bearing the homologous mutations showed similar defects in viral morphogenesis. Finally, shotgun proteomics experiments indicated that the interactomes of Paramyxovirinae M proteins are significantly enriched for components of the nuclear pore complex, nuclear transport receptors, and nucleolar proteins. We then synthesize our functional and proteomics data to propose a working model for the ubiquitin-regulated nuclear

  5. Evidence for ubiquitin-regulated nuclear and subnuclear trafficking among Paramyxovirinae matrix proteins.

    PubMed

    Pentecost, Mickey; Vashisht, Ajay A; Lester, Talia; Voros, Tim; Beaty, Shannon M; Park, Arnold; Wang, Yao E; Yun, Tatyana E; Freiberg, Alexander N; Wohlschlegel, James A; Lee, Benhur

    2015-03-01

    The paramyxovirus matrix (M) protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane. Transient nuclear residence of some M proteins hints at non-structural roles. However, little is known regarding the mechanisms that regulate the nuclear sojourn. Previously, we found that the nuclear-cytoplasmic trafficking of Nipah virus M (NiV-M) is a prerequisite for budding, and is regulated by a bipartite nuclear localization signal (NLSbp), a leucine-rich nuclear export signal (NES), and monoubiquitination of the K258 residue within the NLSbp itself (NLSbp-lysine). To define whether the sequence determinants of nuclear trafficking identified in NiV-M are common among other Paramyxovirinae M proteins, we generated the homologous NES and NLSbp-lysine mutations in M proteins from the five major Paramyxovirinae genera. Using quantitative 3D confocal microscopy, we determined that the NES and NLSbp-lysine are required for the efficient nuclear export of the M proteins of Nipah virus, Hendra virus, Sendai virus, and Mumps virus. Pharmacological depletion of free ubiquitin or mutation of the conserved NLSbp-lysine to an arginine, which inhibits M ubiquitination, also results in nuclear and nucleolar retention of these M proteins. Recombinant Sendai virus (rSeV-eGFP) bearing the NES or NLSbp-lysine M mutants rescued at similar efficiencies to wild type. However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread. Recombinant Mumps virus (rMuV-eGFP) bearing the homologous mutations showed similar defects in viral morphogenesis. Finally, shotgun proteomics experiments indicated that the interactomes of Paramyxovirinae M proteins are significantly enriched for components of the nuclear pore complex, nuclear transport receptors, and nucleolar proteins. We then synthesize our functional and proteomics data to propose a working model for the ubiquitin-regulated nuclear

  6. α-Synuclein-induced lysosomal dysfunction occurs through disruptions in protein trafficking in human midbrain synucleinopathy models.

    PubMed

    Mazzulli, Joseph R; Zunke, Friederike; Isacson, Ole; Studer, Lorenz; Krainc, Dimitri

    2016-02-16

    Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by the accumulation of protein aggregates comprised of α-synuclein (α-syn). A major barrier in treatment discovery for PD is the lack of identifiable therapeutic pathways capable of reducing aggregates in human neuronal model systems. Mutations in key components of protein trafficking and cellular degradation machinery represent important risk factors for PD; however, their precise role in disease progression and interaction with α-syn remains unclear. Here, we find that α-syn accumulation reduced lysosomal degradation capacity in human midbrain dopamine models of synucleinopathies through disrupting hydrolase trafficking. Accumulation of α-syn at the cell body resulted in aberrant association with cis-Golgi-tethering factor GM130 and disrupted the endoplasmic reticulum-Golgi localization of rab1a, a key mediator of vesicular transport. Overexpression of rab1a restored Golgi structure, improved hydrolase trafficking and activity, and reduced pathological α-syn in patient neurons. Our work suggests that enhancement of lysosomal hydrolase trafficking may prove beneficial in synucleinopathies and indicates that human midbrain disease models may be useful for identifying critical therapeutic pathways in PD and related disorders. PMID:26839413

  7. The most common cystic fibrosis-associated mutation destabilizes the dimeric state of the nucleotide-binding domains of CFTR.

    PubMed

    Jih, Kang-Yang; Li, Min; Hwang, Tzyh-Chang; Bompadre, Silvia G

    2011-06-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP binding cassette (ABC) superfamily. The deletion of the phenylalanine 508 (ΔF508-CFTR) is the most common mutation among cystic fibrosis (CF) patients. The mutant channels present a severe trafficking defect, and the few channels that reach the plasma membrane are functionally impaired. Interestingly, an ATP analogue, N6-(2-phenylethyl)-2′-deoxy-ATP (P-dATP), can increase the open probability (Po) to ∼0.7, implying that the gating defect of ΔF508 may involve the ligand binding domains, such as interfering with the formation or separation of the dimeric states of the nucleotide-binding domains (NBDs). To test this hypothesis, we employed two approaches developed for gauging the stability of the NBD dimeric states using the patch-clamp technique. We measured the locked-open time induced by pyrophosphate (PPi), which reflects the stability of the full NBD dimer state, and the ligand exchange time for ATP/N6-(2-phenylethyl)-ATP (P-ATP), which measures the stability of the partial NBD dimer state wherein the head of NBD1 and the tail of NBD2 remain associated. We found that both the PPi-induced locked-open time and the ATP/P-ATP ligand exchange time of ΔF508-CFTR channels are dramatically shortened, suggesting that the ΔF508 mutation destabilizes the full and partial NBD dimer states. We also tested if mutations that have been shown to improve trafficking of ΔF508-CFTR, namely the solubilizing mutation F494N/Q637R and ΔRI (deletion of the regulatory insertion), exert any effects on these newly identified functional defects associated with ΔF508-CFTR. Our results indicate that although these mutations increase the membrane expression and function of ΔF508-CFTR, they have limited impact on the stability of both full and partial NBD dimeric states for ΔF508 channels. The structure-function insights gained from this mechanism may provide clues for future

  8. The most common cystic fibrosis-associated mutation destabilizes the dimeric state of the nucleotide-binding domains of CFTR

    PubMed Central

    Jih, Kang-Yang; Li, Min; Hwang, Tzyh-Chang; Bompadre, Silvia G

    2011-01-01

    Abstract The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP binding cassette (ABC) superfamily. The deletion of the phenylalanine 508 (ΔF508-CFTR) is the most common mutation among cystic fibrosis (CF) patients. The mutant channels present a severe trafficking defect, and the few channels that reach the plasma membrane are functionally impaired. Interestingly, an ATP analogue, N6-(2-phenylethyl)-2′-deoxy-ATP (P-dATP), can increase the open probability (Po) to ∼0.7, implying that the gating defect of ΔF508 may involve the ligand binding domains, such as interfering with the formation or separation of the dimeric states of the nucleotide-binding domains (NBDs). To test this hypothesis, we employed two approaches developed for gauging the stability of the NBD dimeric states using the patch-clamp technique. We measured the locked-open time induced by pyrophosphate (PPi), which reflects the stability of the full NBD dimer state, and the ligand exchange time for ATP/N6-(2-phenylethyl)-ATP (P-ATP), which measures the stability of the partial NBD dimer state wherein the head of NBD1 and the tail of NBD2 remain associated. We found that both the PPi-induced locked-open time and the ATP/P-ATP ligand exchange time of ΔF508-CFTR channels are dramatically shortened, suggesting that the ΔF508 mutation destabilizes the full and partial NBD dimer states. We also tested if mutations that have been shown to improve trafficking of ΔF508-CFTR, namely the solubilizing mutation F494N/Q637R and ΔRI (deletion of the regulatory insertion), exert any effects on these newly identified functional defects associated with ΔF508-CFTR. Our results indicate that although these mutations increase the membrane expression and function of ΔF508-CFTR, they have limited impact on the stability of both full and partial NBD dimeric states for ΔF508 channels. The structure–function insights gained from this mechanism may provide clues

  9. Hearing Impairments

    NASA Astrophysics Data System (ADS)

    Cavender, Anna; Ladner, Richard E.

    For many people with hearing impairments, the degree of hearing loss is only a small aspect of their disability and does not necessarily determine the types of accessibility solutions or accommodations that may be required. For some people, the ability to adjust the audio volume may be sufficient. For others, translation to a signed language may be more appropriate. For still others, access to text alternatives may be the best solution. Because of these differences, it is important for researchers in Web accessibility to understand that people with hearing impairments may have very different cultural-linguistic traditions and personal backgrounds.

  10. 3 CFR - Continuation of the National Emergency With Respect to Significant Narcotics Traffickers Centered...

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... to Significant Narcotics Traffickers Centered in Colombia Presidential Documents Other Presidential... Narcotics Traffickers Centered in Colombia On October 21, 1995, by Executive Order 12978, the President... economy of the United States constituted by the actions of significant narcotics traffickers centered...

  11. 3 CFR - Continuation of the National Emergency With Respect to Significant Narcotics Traffickers Centered...

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... to Significant Narcotics Traffickers Centered in Colombia Presidential Documents Other Presidential... Narcotics Traffickers Centered in Colombia On October 21, 1995, by Executive Order 12978, the President declared a national emergency with respect to significant narcotics traffickers centered in...

  12. 3 CFR - Continuation of the National Emergency With Respect to Significant Narcotics Traffickers Centered...

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... to Significant Narcotics Traffickers Centered in Colombia Presidential Documents Other Presidential... Narcotics Traffickers Centered in Colombia On October 21, 1995, by Executive Order 12978, the President... economy of the United States constituted by the actions of significant narcotics traffickers centered...

  13. 3 CFR - Continuation of the National Emergency With Respect to Significant Narcotics Traffickers Centered...

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... to Significant Narcotics Traffickers Centered in Colombia Presidential Documents Other Presidential... Narcotics Traffickers Centered in Colombia On October 21, 1995, by Executive Order 12978, the President... economy of the United States constituted by the actions of significant narcotics traffickers centered...

  14. 3 CFR - Continuation of the National Emergency With Respect to Significant Narcotics Traffickers Centered...

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... to Significant Narcotics Traffickers Centered in Colombia Presidential Documents Other Presidential... Narcotics Traffickers Centered in Colombia On October 21, 1995, by Executive Order 12978, the President... economy of the United States constituted by the actions of significant narcotics traffickers centered...

  15. Functional Rescue of β1-Adrenoceptor Dimerization and Trafficking by Pharmacological Chaperones

    PubMed Central

    Kobayashi, Hiroyuki; Ogawa, Koji; Yao, Rong; Lichtarge, Olivier; Bouvier, Michel

    2009-01-01

    Site-directed mutagenesis guided by evolutionary trace analysis revealed that substitution of V179 and W183 within a cluster of evolutionarily important residues on the surface of the fourth transmembrane domain of the β1-adrenergic receptor (β1AR) significantly reduced the propensity of the receptor to self-assemble into homodimers as assessed by bioluminescence resonance energy transfer in living cells. These results suggest that mutation of V179 and W183 result in conformational changes that reduce homodimerization either directly by interfering with the dimerization interface or indirectly by causing local misfolding that result in reduced self-assembly. However, the mutations did not cause a general misfolding of the β1AR as they did not prevent heterodimerization with the β2AR. The homodimerization-compromised mutants were significantly retained in the endoplasmic reticulum (ER) and could not be properly matured and trafficked to the cell surface. Lipophilic β-adrenergic ligands acted as pharmacological chaperones by restoring both dimerization and plasma membrane trafficking of the ER-retained dimerization-compromised β1AR mutants. These results clearly indicate that homodimerization occurs early in the biosynthetic process in the ER and that pharmacological chaperones can promote both dimerization and cell surface targeting, most likely by stabilizing receptor conformations compatible with the two processes. PMID:19515093

  16. Ganglioside Regulation of AMPA Receptor Trafficking

    PubMed Central

    Prendergast, Jillian; Umanah, George K.E.; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G.; Cole, Robert N.; Huganir, Richard L.; Dawson, Ted M.; Dawson, Valina L.

    2014-01-01

    Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p < 10−4), three regulate neurotransmitter receptor trafficking: Thorase (ATPase family AAA domain-containing protein 1), soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (γ-SNAP), and the transmembrane protein Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans. PMID:25253868

  17. Plant vacuole morphology and vacuolar trafficking

    PubMed Central

    Zhang, Chunhua; Hicks, Glenn R.; Raikhel, Natasha V.

    2014-01-01

    Plant vacuoles are essential organelles for plant growth and development, and have multiple functions. Vacuoles are highly dynamic and pleiomorphic, and their size varies depending on the cell type and growth conditions. Vacuoles compartmentalize different cellular components such as proteins, sugars, ions and other secondary metabolites and play critical roles in plants response to different biotic/abiotic signaling pathways. In this review, we will summarize the patterns of changes in vacuole morphology in certain cell types, our understanding of the mechanisms of plant vacuole biogenesis, and the role of SNAREs and Rab GTPases in vacuolar trafficking. PMID:25309565

  18. Autophagy and proteins involved in vesicular trafficking.

    PubMed

    Amaya, Celina; Fader, Claudio Marcelo; Colombo, María Isabel

    2015-11-14

    Autophagy is an intracellular degradation system that, as a basic mechanism it delivers cytoplasmic components to the lysosomes in order to maintain adequate energy levels and cellular homeostasis. This complex cellular process is activated by low cellular nutrient levels and other stress situations such as low ATP levels, the accumulation of damaged proteins or organelles, or pathogen invasion. Autophagy as a multistep process involves vesicular transport events leading to tethering and fusion of autophagic vesicles with several intracellular compartments. This review summarizes our current understanding of the autophagic pathway with emphasis in the trafficking machinery (i.e. Rabs GTPases and SNAP receptors (SNAREs)) involved in specific steps of the pathway. PMID:26450776

  19. A neutrophil intrinsic impairment affecting Rab27a and degranulation in cystic fibrosis is corrected by CFTR potentiator therapy

    PubMed Central

    Pohl, Kerstin; Hayes, Elaine; Keenan, Joanne; Henry, Michael; Meleady, Paula; Molloy, Kevin; Jundi, Bakr; Bergin, David A.; McCarthy, Cormac; McElvaney, Oliver J.; White, Michelle M.; Clynes, Martin; McElvaney, Noel G.

    2014-01-01

    Studies have endeavored to reconcile whether dysfunction of neutrophils in people with cystic fibrosis (CF) is a result of the genetic defect or is secondary due to infection and inflammation. In this study, we illustrate that disrupted function of the CF transmembrane conductance regulator (CFTR), such as that which occurs in patients with ∆F508 and/or G551D mutations, correlates with impaired degranulation of antimicrobial proteins. We demonstrate that CF blood neutrophils release less secondary and tertiary granule components compared with control cells and that activation of the low-molecular-mass GTP-binding protein Rab27a, involved in the regulation of granule trafficking, is defective. The mechanism leading to impaired degranulation involves altered ion homeostasis caused by defective CFTR function with increased cytosolic levels of chloride and sodium, yet decreased magnesium measured in CF neutrophils. Decreased magnesium concentration in vivo and in vitro resulted in significantly decreased levels of GTP-bound Rab27a. Treatment of G551D patients with the ion channel potentiator ivacaftor resulted in normalized neutrophil cytosolic ion levels and activation of Rab27a, thereby leading to increased degranulation and bacterial killing. Our results confirm that intrinsic alterations of circulating neutrophils from patients with CF are corrected by ivacaftor, thus illustrating additional clinical benefits for CFTR modulator therapy. PMID:24934256

  20. Loss of cysteine 584 impairs the storage and release, but not the synthesis of von Willebrand factor.

    PubMed

    Daidone, V; Barbon, G; Pontara, E; Cattini, G M; Gallinaro, L; Zampese, E; Pizzo, P; Casonato, A

    2014-12-01

    Cysteines play a key part in von Willebrand factor (VWF) dimerisation and polymerisation, and their loss may severely affect VWF structure and function. We report on three patients with type 3 von Willebrand disease carrying the new c.1751G>T missense mutation that induces the substitution of cysteine 584 by phenylalanine (C584F), and the deletion of seven nucleotides in exon 7 (c.729_735del), producing a premature stop codon at position 454 (E244Lfs*211). VWF was almost undetectable in the patients' plasma and platelets, while a single, poorly represented, oligomer emerged on plasma VWF multimer analysis. No post-DDAVP increase in VWF and factor VIII was observed. Expressing human recombinant C584F-VWF in HEK293T cells showed that C584F-VWF was synthesised and multimerised but not secreted - apart from the first oligomer, which was slightly represented in the conditioned medium, with a pattern similar to the patients' plasma VWF. The in vitro expression of the E244Lfs*211-VWF revealed a defective synthesis of the mutated VWF, with a behavior typical of loss of function mutations. Cellular trafficking, investigated in HEK293 cells, indicated a normal C584F-VWF content in the endoplasmic reticulum and Golgi apparatus, confirming the synthesis and multimerisation of C584F-VWF. No pseudo-Weibel Palade bodies were demonstrable, however, suggesting that C584F mutation impairs the storage of C584F-VWF. These findings point to cysteine 584 having a role in the release of VWF and its targeting to pseudo-Weibel Palade bodies in vitro, as well as in its storage and release by endothelial cells in vivo. PMID:25230768

  1. KRAS Mutation

    PubMed Central

    Franklin, Wilbur A.; Haney, Jerry; Sugita, Michio; Bemis, Lynne; Jimeno, Antonio; Messersmith, Wells A.

    2010-01-01

    Treatment of colon carcinoma with the anti-epidermal growth factor receptor antibody Cetuximab is reported to be ineffective in KRAS-mutant tumors. Mutation testing techniques have therefore become an urgent concern. We have compared three methods for detecting KRAS mutations in 59 cases of colon carcinoma: 1) high resolution melting, 2) the amplification refractory mutation system using a bifunctional self-probing primer (ARMS/Scorpion, ARMS/S), and 3) direct sequencing. We also evaluated the effects of the methods of sectioning and coring of paraffin blocks to obtain tumor DNA on assay sensitivity and specificity. The most sensitive and specific combination of block sampling and mutational analysis was ARMS/S performed on DNA derived from 1-mm paraffin cores. This combination of tissue sampling and testing method detected KRAS mutations in 46% of colon tumors. Four samples were positive by ARMS/S, but initially negative by direct sequencing. Cloned DNA samples were retested by direct sequencing, and in all four cases KRAS mutations were identified in the DNA. In six cases, high resolution melting abnormalities could not be confirmed as specific mutations either by ARMS/S or direct sequencing. We conclude that coring of the paraffin blocks and testing by ARMS/S is a sensitive, specific, and efficient method for KRAS testing. PMID:20007845

  2. Interaction between the Linker, Pre-S1, and TRP Domains Determines Folding, Assembly, and Trafficking of TRPV Channels.

    PubMed

    Garcia-Elias, Anna; Berna-Erro, Alejandro; Rubio-Moscardo, Fanny; Pardo-Pastor, Carlos; Mrkonjić, Sanela; Sepúlveda, Romina V; Vicente, Rubén; González-Nilo, Fernando; Valverde, Miguel A

    2015-08-01

    Functional transient receptor potential (TRP) channels result from the assembly of four subunits. Here, we show an interaction between the pre-S1, TRP, and the ankyrin repeat domain (ARD)-S1 linker domains of TRPV1 and TRPV4 that is essential for proper channel assembly. Neutralization of TRPV4 pre-S1 K462 resulted in protein retention in the ER, defective glycosylation and trafficking, and unresponsiveness to TRPV4-activating stimuli. Similar results were obtained with the equivalent mutation in TRPV1 pre-S1. Molecular dynamics simulations revealed that TRPV4-K462 generated an alternating hydrogen network with E745 (TRP box) and D425 (pre-S1 linker), and that K462Q mutation affected subunit folding. Consistently, single TRPV4-E745A or TRPV4-D425A mutations moderately affected TRPV4 biogenesis while double TRPV4-D425A/E745A mutation resumed the TRPV4-K462Q phenotype. Thus, the interaction between pre-S1, TRP, and linker domains is mandatory to generate a structural conformation that allows the contacts between adjacent subunits to promote correct assembly and trafficking to the plasma membrane. PMID:26146187

  3. Identification of residues in ABCG2 affecting protein trafficking and drug transport, using co-evolutionary analysis of ABCG sequences

    PubMed Central

    Haider, Ameena J.; Cox, Megan H.; Jones, Natalie; Goode, Alice J.; Bridge, Katherine S.; Wong, Kelvin; Briggs, Deborah; Kerr, Ian D.

    2015-01-01

    ABCG2 is an ABC (ATP-binding cassette) transporter with a physiological role in urate transport in the kidney and is also implicated in multi-drug efflux from a number of organs in the body. The trafficking of the protein and the mechanism by which it recognizes and transports diverse drugs are important areas of research. In the current study, we have made a series of single amino acid mutations in ABCG2 on the basis of sequence analysis. Mutant isoforms were characterized for cell surface expression and function. One mutant (I573A) showed disrupted glycosylation and reduced trafficking kinetics. In contrast with many ABC transporter folding mutations which appear to be ‘rescued’ by chemical chaperones or low temperature incubation, the I573A mutation was not enriched at the cell surface by either treatment, with the majority of the protein being retained in the endoplasmic reticulum (ER). Two other mutations (P485A and M549A) showed distinct effects on transport of ABCG2 substrates reinforcing the role of TM helix 3 in drug recognition and transport and indicating the presence of intracellular coupling regions in ABCG2. PMID:26294421

  4. 22 CFR 40.23 - Controlled substance traffickers. [Reserved

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 22 Foreign Relations 1 2013-04-01 2013-04-01 false Controlled substance traffickers. 40.23 Section 40.23 Foreign Relations DEPARTMENT OF STATE VISAS REGULATIONS PERTAINING TO BOTH NONIMMIGRANTS AND... Certain Crimes § 40.23 Controlled substance traffickers....

  5. 22 CFR 40.23 - Controlled substance traffickers. [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Controlled substance traffickers. 40.23 Section 40.23 Foreign Relations DEPARTMENT OF STATE VISAS REGULATIONS PERTAINING TO BOTH NONIMMIGRANTS AND... Certain Crimes § 40.23 Controlled substance traffickers....

  6. 22 CFR 40.23 - Controlled substance traffickers. [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 22 Foreign Relations 1 2011-04-01 2011-04-01 false Controlled substance traffickers. 40.23 Section 40.23 Foreign Relations DEPARTMENT OF STATE VISAS REGULATIONS PERTAINING TO BOTH NONIMMIGRANTS AND... Certain Crimes § 40.23 Controlled substance traffickers....

  7. 22 CFR 40.23 - Controlled substance traffickers. [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 22 Foreign Relations 1 2014-04-01 2014-04-01 false Controlled substance traffickers. 40.23 Section 40.23 Foreign Relations DEPARTMENT OF STATE VISAS REGULATIONS PERTAINING TO BOTH NONIMMIGRANTS AND... Certain Crimes § 40.23 Controlled substance traffickers....

  8. Teaching about Trafficking: Opportunities and Challenges for Critical Engagement

    ERIC Educational Resources Information Center

    Dragiewicz, Molly

    2008-01-01

    The author came to know about trafficking by accident, when she was hired as a research assistant at The Protection Project (TPP) in 1999. As a feminist teacher, the author was very aware of the divisions among feminists on the subject of trafficking, and was interested in communicating these differences to students who were not well versed in the…

  9. Aggression in Sexually Abused Trafficked Girls and Efficacy of Intervention

    ERIC Educational Resources Information Center

    Deb, Sibnath; Mukherjee, Aparna; Mathews, Ben

    2011-01-01

    The broad objective of this study was to understand the incidence and severity of aggression among sexually abused girls who were trafficked and who were then further used for commercial sexual exploitation (referred to subsequently as sexually abused trafficked girls). In addition, the impact of counseling for minimizing aggression in these girls…

  10. Protein kinesis: The dynamics of protein trafficking and stability

    SciTech Connect

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  11. Domestic Minor Sex Trafficking in the United States

    ERIC Educational Resources Information Center

    Kotrla, Kimberly

    2010-01-01

    By now, most social workers are familiar with the issue of human trafficking. However, many are likely unfamiliar with research indicating that youths constitute the most vulnerable group in the United States for becoming victims of sex trafficking and that most women in prostitution actually entered as minors. Some experts are now referring to…

  12. 48 CFR 52.222-50 - Combating Trafficking in Persons.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    .... Commercial sex act means any sex act on account of which anything of value is given to or received by any... trafficking in persons means— (1) Sex trafficking in which a commercial sex act is induced by force, fraud, or coercion, or in which the person induced to perform such act has not attained 18 years of age; or (2)...

  13. 48 CFR 52.222-50 - Combating Trafficking in Persons.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    .... Commercial sex act means any sex act on account of which anything of value is given to or received by any... trafficking in persons means— (1) Sex trafficking in which a commercial sex act is induced by force, fraud, or coercion, or in which the person induced to perform such act has not attained 18 years of age; or (2)...

  14. 48 CFR 52.222-50 - Combating Trafficking in Persons.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    .... Commercial sex act means any sex act on account of which anything of value is given to or received by any... trafficking in persons means— (1) Sex trafficking in which a commercial sex act is induced by force, fraud, or coercion, or in which the person induced to perform such act has not attained 18 years of age; or (2)...

  15. 78 FR 59317 - Federal Acquisition Regulation; Ending Trafficking in Persons

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-26

    ... (77 FR 60029, October 2, 2012), and Title XVII, entitled ``Ending Trafficking in Government... and maintaining compliance plans. (Notice--MVC-2013-01 was published in the Federal Register at 78 FR... Trafficking in Persons in Federal Contracts'', dated September 25, 2012, (77 FR 60029, October 2, 2012),...

  16. 31 CFR 536.312 - Specially designated narcotics trafficker.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... designated narcotics trafficker means: (a) Persons listed in the annex to Executive Order 12978 (3 CFR, 1995... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Specially designated narcotics... (Continued) OFFICE OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING...

  17. 31 CFR 536.312 - Specially designated narcotics trafficker.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... designated narcotics trafficker means: (a) Persons listed in the annex to Executive Order 12978 (3 CFR, 1995... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false Specially designated narcotics... (Continued) OFFICE OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING...

  18. 31 CFR 536.312 - Specially designated narcotics trafficker.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... designated narcotics trafficker means: (a) Persons listed in the annex to Executive Order 12978 (3 CFR, 1995... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false Specially designated narcotics... (Continued) OFFICE OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING...

  19. 31 CFR 536.312 - Specially designated narcotics trafficker.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... designated narcotics trafficker means: (a) Persons listed in the annex to Executive Order 12978 (3 CFR, 1995... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false Specially designated narcotics... (Continued) OFFICE OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING...

  20. 31 CFR 536.312 - Specially designated narcotics trafficker.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... designated narcotics trafficker means: (a) Persons listed in the annex to Executive Order 12978 (3 CFR, 1995... 31 Money and Finance: Treasury 3 2010-07-01 2010-07-01 false Specially designated narcotics... (Continued) OFFICE OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING...

  1. Adolescent Black Males' Drug Trafficking and Addiction: Three Theoretical Perspectives.

    ERIC Educational Resources Information Center

    Moore, Sharon E.

    1995-01-01

    Explains the incidence and nature of drug trafficking and chemical dependency among adolescent black males. The paper also discusses the social science theories of Emile Durkheim, Karl Marx, and Molefi Asante to better understand the behaviors, and the consequences of those behaviors, of young black males who participate in drug trafficking. (GR)

  2. 48 CFR 52.222-50 - Combating Trafficking in Persons.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    .... Commercial sex act means any sex act on account of which anything of value is given to or received by any... an employee of the Contractor directly engaged in the performance of work under the contract who has... trafficking in persons means— (1) Sex trafficking in which a commercial sex act is induced by force, fraud,...

  3. 48 CFR 52.222-50 - Combating Trafficking in Persons.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    .... Commercial sex act means any sex act on account of which anything of value is given to or received by any... an employee of the Contractor directly engaged in the performance of work under the contract who has... trafficking in persons means— (1) Sex trafficking in which a commercial sex act is induced by force, fraud,...

  4. Falling through the Gaps: Safeguarding Children Trafficked into the UK

    ERIC Educational Resources Information Center

    Bokhari, Farrah

    2008-01-01

    An overview of child trafficking in the UK explores the nature and methods of this abuse, as well as the treatment and protection afforded to these particularly vulnerable children. It highlights the shortcomings and inconsistent standards of local authorities, the lack of specialist protection and the uncertainty of a trafficked child's…

  5. A solution NMR investigation into the impaired self-assembly properties of two murine amelogenins containing the point mutations T21→I or P41→T

    SciTech Connect

    Buchko, Garry W.; Lin, Genyao; Tarasevich, Barbara J.; Shaw, Wendy J.

    2013-08-26

    Amelogenesis imperfecta describes a group of inherited disorders