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Mutations in the IER3IP1 (Immediate Early Response-3 Interacting Protein 1) gene can give rise to MEDS1 (Microcephaly with Simplified Gyral Pattern, Epilepsy, and Permanent Neonatal Diabetes Syndrome-1), a severe condition leading to early childhood mortality. The small endoplasmic reticulum (ER)-membrane protein IER3IP1 plays a non-essential role in ER-Golgi transport. Here, we employed secretome and cell-surface proteomics to demonstrate that the absence of IER3IP1 results in the mistrafficking of proteins crucial for neuronal development and survival, including FGFR3, UNC5B and SEMA4D. This phenomenon correlates with the distension of ER membranes and increased lysosomal activity. Notably, the trafficking of cargo receptor ERGIC53 and KDEL-receptor 2 are compromised, with the latter leading to the anomalous secretion of ER-localized chaperones. Our investigation extended to in-utero knock-down of Ier3ip1 in mouse embryo brains, revealing a morphological phenotype in newborn neurons. In summary, our findings provide insights into how the loss or mutation of a 10 kDa small ER-membrane protein can cause a fatal syndrome.
Assuntos
Retículo Endoplasmático , Complexo de Golgi , Microcefalia , Retículo Endoplasmático/metabolismo , Animais , Microcefalia/genética , Microcefalia/metabolismo , Microcefalia/patologia , Camundongos , Complexo de Golgi/metabolismo , Humanos , Mutação , Transporte Proteico , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Neurônios/metabolismo , Neurônios/patologiaRESUMO
Proteins of the secretory pathway undergo glycosylation in the endoplasmic reticulum (ER) and the Golgi apparatus. Altered protein glycosylation can manifest in serious, sometimes fatal malfunctions. We recently showed that mutations in GDP-mannose pyrophosphorylase A (GMPPA) can cause a syndrome characterized by alacrima, achalasia, mental retardation, and myopathic alterations (AAMR syndrome). GMPPA acts as a feedback inhibitor of GDP-mannose pyrophosphorylase B (GMPPB), which provides GDP-mannose as a substrate for protein glycosylation. Loss of GMPPA thus enhances the incorporation of mannose into glycochains of various proteins, including α-dystroglycan (α-DG), a protein that links the extracellular matrix with the cytoskeleton. Here, we further characterized the consequences of loss of GMPPA for the secretory pathway. This includes a fragmentation of the Golgi apparatus, which comes along with a regulation of the abundance of several ER- and Golgi-resident proteins. We further show that the activity of the Golgi-associated endoprotease furin is reduced. Moreover, the fraction of α-DG, which is retained in the ER, is increased. Notably, WT cells cultured at a high mannose concentration display similar changes with increased retention of α-DG, altered structure of the Golgi apparatus, and a decrease in furin activity. In summary, our data underline the importance of a balanced mannose homeostasis for the secretory pathway.
RESUMO
An entirely different mechanism and localization were recently proposed for the COPII coat complex, challenging its well-accepted function to select and concentrate cargo into small COPII-coated spherical transport vesicles. Instead, the COPII complex is suggested to form a dynamic yet stationary collar that forms a boundary between the ER and the ER export membrane domain. This membrane domain, the ER exit site (ERES), is the site of COPII-mediated sorting and concentration of transport competent proteins. Subsequently, the ERES is implicated to mature and bud to form a sizeable pleiomorphic transport carrier that translocate on microtubules to fuse with the Golgi apparatus. Despite this drastic mechanistic dogma shift, most of the underlying protein-protein and protein-membrane interactions remain unchanged. Here, we attempt to provide a detailed description of the newly proposed model of how ER to Golgi transport works by describing the role of several essential proteins of the transport machinery.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório , Complexo de Golgi , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Transporte ProteicoRESUMO
ß-Site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1) is the major described ß-secretase to generate Aß peptides in Alzheimer's disease (AD). However, all therapeutic attempts to block BACE1 activity and to improve AD symptoms have so far failed. A potential candidate for alternative Aß peptides generation is the metalloproteinase meprin ß, which cleaves APP predominantly at alanine in p2 and in this study we can detect an increased meprin ß expression in AD brain. Here, we report the generation of the transgenic APP/lon mouse model of AD lacking the functional Mep1b gene (APP/lon × Mep1b-/-). We examined levels of canonical and truncated Aß species using urea-SDS-PAGE, ELISA and immunohistochemistry in brains of APP/lon mouse × Mep1b-/-. Additionally, we investigated the cognitive abilities of these mice during the Morris water maze task. Aß1-40 and 1-42 levels are reduced in APP/lon mice when meprin ß is absent. Immunohistochemical staining of mouse brain sections revealed that N-terminally truncated Aß2-x peptide deposition is decreased in APP/lon × Mep1b-/- mice. Importantly, loss of meprin ß improved cognitive abilities and rescued learning behavior impairments in APP/lon mice. These observations indicate an important role of meprin ß within the amyloidogenic pathway and Aß production in vivo.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Aprendizagem , Transtornos da Memória/patologia , Metaloendopeptidases/deficiência , Idoso , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Astrócitos/metabolismo , Encéfalo/patologia , Cruzamentos Genéticos , Modelos Animais de Doenças , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Metaloendopeptidases/metabolismo , Camundongos Knockout , Peptídeos/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Gene duplication enables the emergence of new functions by lowering the evolutionary pressure that is posed on the ancestral genes. Previous studies have highlighted the role of specific paralog genes during cell differentiation, for example, in chromatin remodeling complexes. It remains unexplored whether similar mechanisms extend to other biological functions and whether the regulation of paralog genes is conserved across species. Here, we analyze the expression of paralogs across human tissues, during development and neuronal differentiation in fish, rodents and humans. Whereas â¼80% of paralog genes are co-regulated, a subset of paralogs shows divergent expression profiles, contributing to variability of protein complexes. We identify 78 substitutions of paralog pairs that occur during neuronal differentiation and are conserved across species. Among these, we highlight a substitution between the paralogs SEC23A and SEC23B members of the COPII complex. Altering the ratio between these two genes via RNAi-mediated knockdown is sufficient to influence neuron differentiation. We propose that remodeling of the vesicular transport system via paralog substitutions is an evolutionary conserved mechanism enabling neuronal differentiation.
Assuntos
Evolução Biológica , Duplicação Gênica , AnimaisRESUMO
Mutations in the ER-network forming GTPase atlastin3 (ATL3) can cause axon degeneration of sensory neurons by not fully understood mechanisms. We here show that the hereditary sensory and autonomous neuropathy (HSAN)-causing ATL3 Y192C or P338R are excluded from distal axons by a barrier at the axon initial segment (AIS). This barrier is selective for mutated ATL3, but not wildtype ATL3 or unrelated ER-membrane proteins. Actin-depolymerization partially restores the transport of ATL3 Y192C into distal axons. The results point to the existence of a selective diffusion barrier in the ER membrane at the AIS, analogous to the AIS-based barriers for plasma membrane and cytosolic proteins. Functionally, the absence of ATL3 at the distal axon reduces axonal autophagy and the ER network deformation in the soma causes a reduction in axonal lysosomes. Both could contribute to axonal degeneration and eventually to HSAN.
Assuntos
Autofagia/fisiologia , Axônios/fisiologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Mutação/fisiologia , Animais , Axônios/patologia , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BLRESUMO
COPII and COPI mediate the formation of membrane vesicles translocating in opposite directions within the secretory pathway. Live-cell and electron microscopy revealed a novel mode of function for COPII during cargo export from the ER. COPII is recruited to membranes defining the boundary between the ER and ER exit sites, facilitating selective cargo concentration. Using direct observation of living cells, we monitored cargo selection processes, accumulation, and fission of COPII-free ERES membranes. CRISPR/Cas12a tagging, the RUSH system, and pharmaceutical and genetic perturbations of ER-Golgi transport demonstrated that the COPII coat remains bound to the ER-ERES boundary during protein export. Manipulation of the cargo-binding domain in COPII Sec24B prohibits cargo accumulation in ERES. These findings suggest a role for COPII in selecting and concentrating exported cargo rather than coating Golgi-bound carriers. These findings transform our understanding of coat proteins' role in ER-to-Golgi transport.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Células HeLa , Humanos , Transporte ProteicoRESUMO
GDP-mannose-pyrophosphorylase-B (GMPPB) facilitates the generation of GDP-mannose, a sugar donor required for glycosylation. GMPPB defects cause muscle disease due to hypoglycosylation of α-dystroglycan (α-DG). Alpha-DG is part of a protein complex, which links the extracellular matrix with the cytoskeleton, thus stabilizing myofibers. Mutations of the catalytically inactive homolog GMPPA cause alacrima, achalasia, and mental retardation syndrome (AAMR syndrome), which also involves muscle weakness. Here, we showed that Gmppa-KO mice recapitulated cognitive and motor deficits. As structural correlates, we found cortical layering defects, progressive neuron loss, and myopathic alterations. Increased GDP-mannose levels in skeletal muscle and in vitro assays identified GMPPA as an allosteric feedback inhibitor of GMPPB. Thus, its disruption enhanced mannose incorporation into glycoproteins, including α-DG in mice and humans. This increased α-DG turnover and thereby lowered α-DG abundance. In mice, dietary mannose restriction beginning after weaning corrected α-DG hyperglycosylation and abundance, normalized skeletal muscle morphology, and prevented neuron degeneration and the development of motor deficits. Cortical layering and cognitive performance, however, were not improved. We thus identified GMPPA defects as the first congenital disorder of glycosylation characterized by α-DG hyperglycosylation, to our knowledge, and we have unraveled underlying disease mechanisms and identified potential dietary treatment options.
Assuntos
Distroglicanas , Guanosina Difosfato Manose , Músculo Esquelético/metabolismo , Doenças Neuromusculares , Nucleotidiltransferases/deficiência , Animais , Distroglicanas/genética , Distroglicanas/metabolismo , Glicosilação , Guanosina Difosfato Manose/genética , Guanosina Difosfato Manose/metabolismo , Humanos , Camundongos , Camundongos Knockout , Doenças Neuromusculares/dietoterapia , Doenças Neuromusculares/genética , Doenças Neuromusculares/metabolismo , Nucleotidiltransferases/metabolismoRESUMO
An efficient synthesis for silicon-rhodamines was developed, enabling the preparation and evaluation of silicon-rhodamine isothiocyanate (SITC) as a novel tool for facile fluorescent labeling. Ease of use in conjugation to amino groups, high stability and excellent photophysical properties are demonstrated. SITC-actin was found to be neutral to F-actin polymerization induction and well suited for high resolution fluorescence microscopy.
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NBS1 is a critical component of the MRN (MRE11/RAD50/NBS1) complex, which regulates ATM- and ATR-mediated DNA damage response (DDR) pathways. Mutations in NBS1 cause the human genomic instability syndrome Nijmegen Breakage Syndrome (NBS), of which neuronal deficits, including microcephaly and intellectual disability, are classical hallmarks. Given its function in the DDR to ensure proper proliferation and prevent death of replicating cells, NBS1 is essential for life. Here we show that, unexpectedly, Nbs1 deletion is dispensable for postmitotic neurons, but compromises their arborization and migration due to dysregulated Notch signaling. We find that Nbs1 interacts with NICD-RBPJ, the effector of Notch signaling, and inhibits Notch activity. Genetic ablation or pharmaceutical inhibition of Notch signaling rescues the maturation and migration defects of Nbs1-deficient neurons in vitro and in vivo. Upregulation of Notch by Nbs1 deletion is independent of the key DDR downstream effector p53 and inactivation of each MRN component produces a different pattern of Notch activity and distinct neuronal defects. These data indicate that neuronal defects and aberrant Notch activity in Nbs1-deficient cells are unlikely to be a direct consequence of loss of MRN-mediated DDR function. This study discloses a novel function of NBS1 in crosstalk with the Notch pathway in neuron development.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neurogênese , Neurônios/metabolismo , Receptores Notch/metabolismo , Hidrolases Anidrido Ácido/metabolismo , Animais , Células Cultivadas , Dano ao DNA , Reparo do DNA , Embrião de Mamíferos , Fibroblastos , Proteína Homóloga a MRE11/metabolismo , Camundongos , Neurônios/citologiaRESUMO
The quote "bring it back, bring it back, don't take it away from me" from Queen's Love of my life describes the function of the sorting receptor RER1, a 23â kDa protein with four transmembrane domains (TMDs) that localizes to the intermediate compartment and the cis-Golgi. From there it returns escaped proteins that are not supposed to leave the endoplasmic reticulum (ER) back to it. Unique about RER1 is its ability to recognize its ligands through binding motifs in TMDs. Among its substrates are ER-resident proteins, as well as unassembled subunits of multimeric complexes that are retrieved back into the ER, this way guarding the full assembly of their respective complexes. The basic mechanisms for RER1-dependent retrieval have been already elucidated some years ago in yeast. More recently, several important cargoes of RER1 have been described in mammalian cells, and the in vivo role of RER1 is being unveiled by using mouse models. In this Review, we give an overview of the cell biology of RER1 in different models, discuss its controversial role in the brain and provide an outlook on future directions for RER1 research.
Assuntos
Complexo de Golgi , Glicoproteínas de Membrana , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Human selenium-binding protein 1 (SELENBP1) was originally identified as a protein binding selenium, most likely as selenite. SELENBP1 is associated with cellular redox and thiol homeostasis in several respects, including its established role as a methanethiol oxidase that is involved in degradation of methanethiol, a methionine catabolite, generating hydrogen sulfide (H2S) and hydrogen peroxide (H2O2). As both H2S and reactive oxygen species (such as H2O2) are major regulators of Caenorhabditis elegans lifespan and stress resistance, we hypothesized that a SELENBP1 ortholog in C. elegans would likely be involved in regulating these aspects. Here we characterize Y37A1B.5, a putative selenium-binding protein 1 ortholog in C. elegans with 52% primary structure identity to human SELENBP1. While conferring resistance to toxic concentrations of selenite, Y37A1B.5 also attenuates resistance to oxidative stress and lowers C. elegans lifespan: knockdown of Y37A1B.5 using RNA interference resulted in an approx. 10% increase of C. elegans lifespan and an enhanced resistance against the redox cycler paraquat, as well as enhanced motility. Analyses of transgenic reporter strains suggest hypodermal expression and cytoplasmic localization of Y37A1B.5, whose expression decreases with worm age. We identify the transcriptional coregulator MDT-15 and transcription factor EGL-27 as regulators of Y37A1B.5 levels and show that the lifespan extending effect elicited by downregulation of Y37A1B.5 is independent of known MDT-15 interacting factors, such as DAF-16 and NHR-49. In summary, Y37A1B.5 is an ortholog of SELENBP1 that shortens C. elegans lifespan and lowers resistance against oxidative stress, while allowing for a better survival under toxic selenite concentrations.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ácido Selenioso/efeitos adversos , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Citoplasma/metabolismo , Resistência a Medicamentos , Regulação da Expressão Gênica , Humanos , Longevidade , Proteínas de Membrana/química , Estresse Oxidativo , Paraquat/efeitos adversos , Proteínas de Ligação a Selênio/química , Proteínas de Ligação a Selênio/genética , Proteínas de Ligação a Selênio/metabolismo , Homologia Estrutural de ProteínaRESUMO
The Notch receptor is a key mediator of developmental programs and cell-fate decisions. Imbalanced Notch signaling leads to developmental disorders and cancer. To fully characterize the Notch signaling pathway and exploit it in novel therapeutic interventions, a comprehensive view on the regulation and requirements of Notch signaling is needed. Notch is regulated at different levels, ranging from ligand binding, stability to endocytosis. Using an array of different techniques, including reporter gene assays, immunocytochemistry, and ChIP-qPCR we show here, to the best of our knowledge for the first time, regulation of Notch signaling at the level of the nuclear pore. We found that the nuclear pore protein Nup214 (nucleoporin 214) and its interaction partner Nup88 negatively regulate Notch signaling in vitro and in vivo in zebrafish. In mammalian cells, loss of Nup88/214 inhibited nuclear export of recombination signal-binding protein for immunoglobulin κJ region (RBP-J), the DNA-binding component of the Notch pathway. This inhibition increased binding of RBP-J to its cognate promoter regions, resulting in increased downstream Notch signaling. Interestingly, we also found that NUP214 fusion proteins, causative for certain cases of T-cell acute lymphatic leukemia, potentially contribute to tumorigenesis via a Notch-dependent mechanism. In summary, the nuclear pore components Nup88/214 suppress Notch signaling in vitro, and in zebrafish, nuclear RBP-J levels are rate-limiting factors for Notch signaling in mammalian cells, and regulation of nucleocytoplasmic transport of RBP-J may contribute to fine-tuning Notch activity in cells.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular Tumoral , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Morfolinos/genética , Morfolinos/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Notch/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição HES-1/antagonistas & inibidores , Fatores de Transcrição HES-1/genética , Fatores de Transcrição HES-1/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Atlastins (ATLs) are membrane-bound GTPases involved in shaping of the endoplasmic reticulum (ER). Mutations in ATL1 and ATL3 cause spastic paraplegia and hereditary sensory neuropathy. We here show that the sensory neuropathy causing ATL3 Y192C mutation reduces the complexity of the tubular ER-network. ATL3 Y192C delays ER-export by reducing the number of ER exit sites, reduces autophagy, fragments the Golgi and causes malformation of the nucleus. In cultured primary neurons, ATL3 Y192C does not localize to the growing axon, resulting in axon growth deficits. Patient-derived fibroblasts possess a tubular ER with reduced complexity and have a reduced number of autophagosomes. The data suggest that the disease-causing ATL3 Y192C mutation affects multiple ER-related pathways, possibly as a consequence of the distorted ER morphology.
Assuntos
Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Animais , Autofagossomos , Autofagia , Axônios/metabolismo , Células Cultivadas , Retículo Endoplasmático/patologia , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Neurônios/citologia , Neurônios/metabolismoRESUMO
BACKGROUND: Klotho is a well-known anti-aging hormone, which serves as a suppressor of aging through a variety of mechanisms. Aging of skeletal muscle is concomitant with a decrease in muscle stem cell function resulting in impaired regeneration. METHODS: Here we investigate the functional role of the anti-aging hormone Klotho for muscle stem cell function after cardiotoxin-induced injury of skeletal muscle using a klotho hypomorphic mouse line, which is characterized by a premature aging phenotype. Furthermore, we perform floating single myofiber cultures with their adjacent muscle stem cells to investigate the interplay between canonical Wnt signaling and Klotho function. RESULTS: We demonstrate that muscle stem cell numbers are significantly decreased in klotho hypomorphic mice. Furthermore, we show that muscle stem cell function is also severely impaired upon loss of klotho expression, in culture and during regeneration in vivo. Moreover, we demonstrate that addition of recombinant Klotho protein inhibits aberrant excessive Wnt signaling in aged muscle stem cells thereby restoring their functionality. CONCLUSIONS: The anti-aging hormone Klotho counteracts aberrant canonical Wnt signaling in muscle stem cells and might be one of the naturally occurring inhibitors of canonical Wnt signaling in skeletal muscle.
Assuntos
Glucuronidase/fisiologia , Músculo Esquelético/fisiologia , Mioblastos Esqueléticos/fisiologia , Regeneração/fisiologia , Envelhecimento/patologia , Envelhecimento/fisiologia , Senilidade Prematura/patologia , Senilidade Prematura/fisiopatologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Glucuronidase/antagonistas & inibidores , Glucuronidase/deficiência , Glucuronidase/genética , Proteínas Klotho , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Desenvolvimento Muscular/fisiologia , Mioblastos Esqueléticos/efeitos dos fármacos , Mioblastos Esqueléticos/patologia , RNA Mensageiro/genética , Via de Sinalização Wnt/fisiologia , Proteína Wnt3A/farmacologiaRESUMO
Thyroid hormone (TH) transporters are required for the transmembrane passage of TH in target cells. In humans, inactivating mutations in the TH transporter MCT8 cause the Allan-Herndon-Dudley syndrome, characterized by severe neuromuscular symptoms and an abnormal TH serum profile, which is fully replicated in Mct8 knockout mice and Mct8/Oatp1c1 double-knockout (M/O DKO) mice. Analysis of tissue TH content and expression of TH-regulated genes indicate a thyrotoxic state in Mct8-deficient skeletal muscles. Both TH transporters are upregulated in activated satellite cells (SCs). In M/O DKO mice, we observed a strongly reduced number of differentiated SCs, suggesting an impaired stem cell function. Moreover, M/O DKO mice and mice lacking both transporters exclusively in SCs showed impaired skeletal muscle regeneration. Our data provide solid evidence for a unique gate-keeper function of MCT8 and OATP1C1 in SC activation, underscoring the importance of a finely tuned TH signaling during myogenesis.
Assuntos
Proteínas de Membrana Transportadoras/genética , Músculo Esquelético/fisiologia , Proteínas de Transporte de Cátions Orgânicos/genética , Regeneração , Animais , Biomarcadores , Diferenciação Celular , Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Transportadores de Ácidos Monocarboxílicos , Desenvolvimento Muscular/genética , Músculo Esquelético/citologia , Mutação , SimportadoresRESUMO
Rer1 is a sorting receptor in the early secretory pathway that controls the assembly and the cell surface transport of selected multimeric membrane protein complexes. Mice with a Purkinje cell (PC) specific deletion of Rer1 showed normal polarization and differentiation of PCs and normal development of the cerebellum. However, PC-specific loss of Rer1 led to age-dependent motor deficits in beam walk, ladder climbing and gait. Analysis of brain sections revealed a specific degeneration of PCs in the anterior cerebellar lobe in old animals. Electrophysiological recordings demonstrated severe deficits in spontaneous action potential generation. Measurements of resurgent currents indicated decreased surface densities of voltage-gated sodium channels (Nav), but not changes in individual channels. Analysis of mice with a whole brain Rer1-deletion demonstrated a strong down-regulation of Nav1.6 and 1.1 in the absence of Rer1, whereas protein levels of the related Cav2.1 and of Kv3.3 and 7.2 channels were not affected. The data suggest that Rer1 controls the assembly and transport of Nav1.1 and 1.6, the principal sodium channels responsible for recurrent firing, in PCs.
Assuntos
Células de Purkinje/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Potenciais de Ação , Proteínas Adaptadoras de Transporte Vesicular , Envelhecimento/metabolismo , Animais , Axônios/metabolismo , Diferenciação Celular , Proliferação de Células , Cerebelo/metabolismo , Cerebelo/patologia , Cerebelo/fisiopatologia , Deleção de Genes , Camundongos Knockout , Atividade Motora , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Receptores Citoplasmáticos e Nucleares/deficiênciaRESUMO
The γ-secretase complex comprises presenilin (PS), nicastrin (NCT), anterior pharynx-defective 1 (Aph1), and presenilin enhancer 2 (Pen2). PS has two homologues, PS1 and PS2. Aph1 has two isoforms, Aph1a and Aph1b, with the former existing as two splice variants Aph1aL and Aph1aS. Each complex consists of one subunit each, resulting in six different γ-secretases. To better understand the functional differences among the γ-secretases, we reconstituted them using a yeast system and compared Notch1-cleavage and amyloid precursor protein (APP)-cleavage activities. Intriguingly, PS2/Aph1b had a clear substrate specificity: APP-Gal4, but not Notch-Gal4, was cleaved. In HEK cell lines expressing defined γ-secretase subunits, we showed that PS1/Aph1b, PS2/Aph1aL, PS2/Aph1aS and PS2/Aph1b γ-secretase produced amyloid ß peptide (Aß) with a higher Aß42+Aß43-to-Aß40 (Aß42(43)/Aß40) ratio than the other γ-secretases. In addition, PS2/Aph1aS γ-secretase produced less Notch intracellular domain (NICD) than did the other 5 γ-secretases. Considering that the Aß42(43)/Aß40 ratio is relevant in the pathogenesis of Alzheimer's disease (AD), and that inhibition of Notch cleavage causes severe side effect, these results suggest that the PS2/Aph1aS γ-secretase complex is a potential therapeutic target in AD.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas de Membrana/metabolismo , Peptídeo Hidrolases/metabolismo , Presenilina-1/metabolismo , Presenilina-2/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/genética , Western Blotting , Endopeptidases , Células HEK293 , Humanos , Proteínas de Membrana/genética , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/genética , Presenilina-1/genética , Presenilina-2/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A)+ RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A)+ RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors.
Assuntos
Chaperonas de Histonas/metabolismo , Corpos de Inclusão Intranuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Poli A/metabolismo , RNA Mensageiro/metabolismo , Proteína Sequestossoma-1/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas de Ligação a DNA , Chaperonas de Histonas/genética , Humanos , Corpos de Inclusão Intranuclear/genética , Carioferinas/genética , Carioferinas/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/genética , Poli A/genética , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína Sequestossoma-1/genética , Fatores de Transcrição/genética , Proteína Exportina 1RESUMO
Export out of the endoplasmic reticulum (ER) involves the Sar1 and COPII machinery acting at ER exit sites (ERES). Whether and how cargo proteins are recruited upstream of Sar1 and COPII is unclear. Two models are conceivable, a recruitment model where cargo is actively transported through a transport factor and handed over to the Sar1 and COPII machinery in ERES, and a capture model, where cargo freely diffuses into ERES where it is captured by the Sar1 and COPII machinery. Using the novel secretion inhibitor FLI-06, we show that recruitment of the cargo VSVG to ERES is an active process upstream of Sar1 and COPII. Applying FLI-06 before concentration of VSVG in ERES completely abolishes its recruitment. In contrast, applying FLI-06 after VSVG concentration in ERES does not lead to dispersal of the concentrated VSVG, arguing that it inhibits recruitment to ERES as opposed to capture in ERES. FLI-06 also inhibits export out of the trans-Golgi network (TGN), suggesting that similar mechanisms might orchestrate cargo selection and concentration at the ER and TGN. FLI-06 does not inhibit autophagosome biogenesis and the ER-peroxisomal transport route, suggesting that these rely on different mechanisms.