RESUMEN
The small GTPase Rab6a is involved in the regulation of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER) in a coat complex coatomer protein I (COPI)-independent pathway. Here, we used a yeast two-hybrid approach to identify binding partners of Rab6a. In particular, we identified the dynein-dynactin-binding protein Bicaudal-D1 (BICD1), one of the two mammalian homologues of Drosophila Bicaudal-D. BICD1 and BICD2 colocalize with Rab6a on the trans-Golgi network (TGN) and on cytoplasmic vesicles, and associate with Golgi membranes in a Rab6-dependent manner. Overexpression of BICD1 enhances the recruitment of dynein-dynactin to Rab6a-containing vesicles. Conversely, overexpression of the carboxy-terminal domain of BICD, which can interact with Rab6a but not with cytoplasmic dynein, inhibits microtubule minus-end-directed movement of green fluorescent protein (GFP)-Rab6a vesicles and induces an accumulation of Rab6a and COPI-independent ER cargo in peripheral structures. These data suggest that coordinated action between Rab6a, BICD and the dynein-dynactin complex controls COPI-independent Golgi-ER transport.
Asunto(s)
Proteína Coatómero/fisiología , Proteínas de Drosophila/fisiología , Dineínas/fisiología , Retículo Endoplásmico/fisiología , Aparato de Golgi/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Animales , Transporte Biológico/fisiología , Células COS , Proteína Coat de Complejo I/fisiología , Complejo Dinactina , Células HeLa , Humanos , Proteínas Motoras Moleculares/fisiología , Datos de Secuencia Molecular , Proteínas de Unión al GTP rab/fisiologíaRESUMEN
In extension to previously applied techniques like yeast two-hybrid and GST pull-down assays, we successfully established a FACS-based FRET analysis to investigate the interaction of the Mint3 adaptor protein and the small Rab GTPase Rab6A in living mammalian cells. A Mint3 mutant containing only the PTB domain (Mint3Delta6) is able to interact with the constitutively active form of Rab6A. Mint3Delta4, a mutant lacking part of the PTB domain was unable to interact with Rab6A in GST pull-down analysis and did not produce FRET signals, when co-expressed with active Rab6A. We demonstrate that this FACS-based FRET analysis is a suitable method for interaction studies between two proteins in living cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Citometría de Flujo/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Mapeo de Interacción de Proteínas/métodos , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , Separación Celular , Células HeLa , Humanos , Proteínas de Unión al GTP rab/genéticaRESUMEN
Epidermal growth factor receptor (EGFR) is a prototype receptor tyrosine kinase and an oncoprotein in many solid tumors. Cell surface display of EGFR is essential for cellular responses to its ligands. While postactivation endocytic trafficking of EGFR has been well elucidated, little is known about mechanisms of basal/preactivation surface display of EGFR. Here, we identify a novel role of the endocytic regulator EHD1 and a potential EHD1 partner, RUSC2, in cell surface display of EGFR. EHD1 and RUSC2 colocalize with EGFR in vesicular/tubular structures and at the Golgi compartment. Inducible EHD1 knockdown reduced the cell surface EGFR expression with accumulation at the Golgi compartment, a phenotype rescued by exogenous EHD1. RUSC2 knockdown phenocopied the EHD1 depletion effects. EHD1 or RUSC2 depletion impaired the EGF-induced cell proliferation, demonstrating that the novel, EHD1- and RUSC2-dependent transport of unstimulated EGFR from the Golgi compartment to the cell surface that we describe is functionally important, with implications for physiologic and oncogenic roles of EGFR and targeted cancer therapies.
Asunto(s)
Proteínas Portadoras/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Comunicación Celular/fisiología , Línea Celular , Membrana Celular/metabolismo , Proliferación Celular/fisiología , Receptores ErbB/metabolismo , Humanos , Ratones , Transporte de Proteínas/fisiología , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
UNLABELLED: DNA watermarks are used for hiding messages or for authenticating genetically modified organisms. Recently, we presented an algorithm called DNA-Crypt for generating DNA-based watermarks that can be integrated into the genome by using the characteristics of the degenerative genetic code. DNA-Crypt generates the watermark by replacing single bases and thus creating synonymous codons that encrypt the hidden information. Mutations within the integrated DNA sequence can be corrected using several mutation correction codes, to keep the hidden information intact. This method has successfully been tested in asexually replicating organisms like bacteria or yeast, where the watermark is duplicated with every cell division. It has been shown that DNA watermarks produced by DNA-Crypt do not influence the transcription or translation of a protein. In sexually reproducing diploid organisms, additional problems can occur, e.g. recombination events can destroy hidden information. Using population predictions as well as statistical analyses we identified a coupled Y-chromosomal/mitochondrial DNA watermarking procedure as the most appropriate for diploid organisms. We developed a mitochondria adapted version of DNA-Crypt, which is called Project Mito that can be used in combination with the original program. AVAILABILITY: http://www.uni-muenster.de/Biologie.NeuroVer/Tumorbiologie/DNA-Crypt/index.html
Asunto(s)
Algoritmos , ADN Mitocondrial/química , ADN Mitocondrial/genética , Código Genético , Almacenamiento y Recuperación de la Información/métodos , Etiquetado de Productos/métodos , Procesamiento de Señales Asistido por Computador , Programas Informáticos , Secuencia de Bases , Datos de Secuencia Molecular , Análisis de Secuencia de ADN/métodosRESUMEN
The prediction of essential biological features based on a given protein sequence is a challenging task in computational biology. To limit the amount of in vitro verification, the prediction of essential biological activities gives the opportunity to detect so far unknown sequences with similar properties. Besides the application within the identification of proteins being involved in tumorigenesis, other functional classes of proteins can be predicted. The prediction accuracy depends on the selected machine learning approach and even more on the composition of the descriptor set used. A computational approach based on feedforward neural networks was applied for the prediction of small GTPases. Consequently, this was realized by taking secondary structure and hydrophobicity information as a preprocessing architecture and thus, as descriptors for the neural networks. We developed a neural network cluster, which consists of a filter network and four subfamily networks. The filter network was trained to identify small GTPases and the subfamily networks were trained to assign a small GTPase to one of the subfamilies. The accuracy of the prediction, whether a given sequence represents a small GTPase is very high (98.25%). The classifications of the subfamily networks yield comparable accuracy. The high prediction accuracy of the neural network cluster developed, gives the opportunity to suggest the use of hydrophobicity and secondary structure prediction in combination with a neural network cluster, as a promising method for the prediction of essential biological activities.
Asunto(s)
Secuencia de Aminoácidos , Biología Computacional , Proteínas de Unión al GTP Monoméricas/química , Redes Neurales de la Computación , Humanos , Datos de Secuencia Molecular , Curva ROCRESUMEN
The Rab family of small GTPases regulate various aspects of cellular dynamics in eukaryotic cells. Membrane trafficking has emerged as central to the functions of leucine-rich repeat kinase 2 (LRRK2), which is associated with inherited and sporadic forms of Parkinson's disease (PD). Rabs act as both regulators of the catalytic activity and targets for serine/threonine phosphorylation by LRRK2. Rab32, Rab38 and Rab29 have been shown to regulate LRRK2 sub-cellular localization through direct interactions. Recently, Rab29 was shown to escort LRRK2 to the Golgi apparatus and activate the phosphorylation of Rab8 and Rab10. Rab32 is linked to multiple cellular functions including endosomal trafficking, mitochondrial dynamics, and melanosome biogenesis. A missense mutation in Rab32 has also recently been linked to PD. Here, we demonstrate that Rab32 directly interacts with sorting nexin 6 (SNX6). SNX6 is a transient subunit of the retromer, an endosome-Golgi retrieval complex whose Vps35 subunit is strongly associated with PD. We could further show that localization of cation-independent mannose-6-phosphate receptors, which are recycled to the trans-Golgi network (TGN) by the retromer, was affected by both Rab32 and SNX6. These data imply that Rab32 is linked to SNX6/retromer trafficking at the Golgi, and also suggests a possible connection between the retromer and Rab32 in the trafficking and biological functions of LRRK2.
Asunto(s)
Aparato de Golgi/metabolismo , Nexinas de Clasificación/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Western Blotting , Línea Celular , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Unión Proteica , Técnicas del Sistema de Dos Híbridos , Red trans-GolgiRESUMEN
Dendritic transport of (m)RNA molecules and localized translation at post-synaptic sites is connected to synaptic plasticity and memory formation. Brain cytoplasmic RNA, 200nt (BC200 RNA) is a brain-specific, small non-messenger RNA with a somatodendritic distribution in primate neurons. The transcript is a component of a ribonucleoprotein particle that is thought to act as a regulator of decentralized translation in dendrites. To elucidate the cellular function of the BC200 ribonucleoprotein particle, we purified BC200 RNA-binding proteins from human brain. Here, we describe the interaction of human Synaptotagmin-binding cytoplasmic RNA interacting protein (SYNCRIP) with BC200 RNA. SYNCRIP was recently characterized as a component of large mRNA transport granules in neurons and is probably involved in local protein synthesis at post-synaptic sites. Our in vitro binding studies demonstrate that SYNCRIP interacts specifically with BC200 RNA and that binding is mediated through its N-terminal RNA recognition motifs and the internal A-rich region of BC200 RNA, respectively. Furthermore, immunoprecipitation experiments indicate an in vivo association of SYNCRIP and BC200 RNA in human brain. Thus, SYNCRIP may recruit BC200 RNA into mRNA transport complexes involved in the regulation of localized translation in dendrites.
Asunto(s)
Encéfalo/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , ARN/metabolismo , Ribonucleoproteínas Citoplasmáticas Pequeñas/genética , Ribonucleoproteínas/metabolismo , Secuencias de Aminoácidos/fisiología , Animales , Ensayo de Cambio de Movilidad Electroforética/métodos , Humanos , Inmunoprecipitación , Masculino , Ratones , Persona de Mediana Edad , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiología , ARN/genética , Análisis de Secuencia de ProteínaRESUMEN
BACKGROUND: DNA-based watermarks are helpful tools to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. In silico analyses showed that in coding regions synonymous codons can be used to insert encrypted information into the genome of living organisms by using the DNA-Crypt algorithm. RESULTS: We integrated an authenticating watermark in the Vam7 sequence. For our investigations we used a mutant Saccharomyces cerevisiae strain, called CG783, which has an amber mutation within the Vam7 sequence. The CG783 cells are unable to sporulate and in addition display an abnormal vacuolar morphology. Transformation of CG783 with pRS314 Vam7 leads to a phenotype very similar to the wildtype yeast strain CG781. The integrated watermark did not influence the function of Vam7 and the resulting phenotype of the CG783 cells transformed with pRS314 Vam7-TB shows no significant differences compared to the CG783 cells transformed with pRS314 Vam7. CONCLUSION: From our experiments we conclude that the DNA watermarks produced by DNA-Crypt do not influence the translation from mRNA into protein. By analyzing the vacuolar morphology, growth rate and ability to sporulate we confirmed that the resulting Vam7 protein was functionally active.
Asunto(s)
ADN/metabolismo , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Consenso , Genes Fúngicos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Proteínas Qc-SNARE/química , Proteínas Qc-SNARE/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alineación de Secuencia , Programas Informáticos , Esporas Fúngicas/metabolismo , Proteína 25 Asociada a Sinaptosomas , Transformación Genética , Vacuolas/metabolismoRESUMEN
Much of our knowledge of the mechanisms governing vesicular transport has come from the combination of genetic and biochemical approaches that have identified Ypt/Rab guanosine 5'-triphosphatases (GTPases) as key components of transport processes in both yeast and mammalian cells. More recently, research has focused on establishing the complex protein-protein interactions necessary for the regulation of vesicular transport by a variety of methods, including the yeast two-hybrid interaction assay. A central component of the signaling pathway regulated by Ypt/Rab proteins is the GTPase cycle, in which the proteins cycle between an active guanosine 5'-triphosphate (GTP)-bound form and an inactive guanosine 5'-diphosphate (GDP)-bound form. Alterations in the conformation of the Ypt/Rab proteins when either GTP or GDP is bound specify the interaction of effector proteins and influence membrane binding. Our work has focused on identifying interacting partners for the GTPases Rab1 and Rab6 and their isoforms, which regulate transport steps between the endoplasmic reticulum and Golgi in mammalian cells. We have employed both active (GTP-bound) and inactive (GDP-bound) Rab1 and Rab6 mutants to identify potential new interacting proteins using the yeast two-hybrid system and have verified these interactions using alternative methods.
Asunto(s)
Proteínas Fúngicas/metabolismo , Transducción de Señal , Vesículas Transportadoras/enzimología , Técnicas del Sistema de Dos Híbridos , Levaduras/enzimología , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab1/metabolismo , Proteínas Fúngicas/genética , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Mutación , Unión Proteica , Levaduras/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab1/genéticaRESUMEN
BACKGROUND: The aim of this paper is to demonstrate the application of watermarks based on DNA sequences to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. Predicted mutations in the genome can be corrected by the DNA-Crypt program leaving the encrypted information intact. Existing DNA cryptographic and steganographic algorithms use synthetic DNA sequences to store binary information however, although these sequences can be used for authentication, they may change the target DNA sequence when introduced into living organisms. RESULTS: The DNA-Crypt algorithm and image steganography are based on the same watermark-hiding principle, namely using the least significant base in case of DNA-Crypt and the least significant bit in case of the image steganography. It can be combined with binary encryption algorithms like AES, RSA or Blowfish. DNA-Crypt is able to correct mutations in the target DNA with several mutation correction codes such as the Hamming-code or the WDH-code. Mutations which can occur infrequently may destroy the encrypted information, however an integrated fuzzy controller decides on a set of heuristics based on three input dimensions, and recommends whether or not to use a correction code. These three input dimensions are the length of the sequence, the individual mutation rate and the stability over time, which is represented by the number of generations. In silico experiments using the Ypt7 in Saccharomyces cerevisiae shows that the DNA watermarks produced by DNA-Crypt do not alter the translation of mRNA into protein. CONCLUSION: The program is able to store watermarks in living organisms and can maintain the original information by correcting mutations itself. Pairwise or multiple sequence alignments show that DNA-Crypt produces few mismatches between the sequences similar to all steganographic algorithms.
Asunto(s)
Algoritmos , Seguridad Computacional , ADN/química , ADN/genética , Almacenamiento y Recuperación de la Información/métodos , Organismos Modificados Genéticamente/genética , Etiquetado de Productos/métodos , Análisis de Secuencia de ADN/métodos , Computadores MolecularesRESUMEN
BACKGROUND: The small GTPase rab1a and its isoform rab1b are essential regulating components in the vesicle transport between the ER and the Golgi apparatus. Rab1 is thought to act as a molecular switch and can change between an active GTP-bound and an inactive GDP-bound conformation. To elucidate the function of rab1, several approaches have been established to isolate effector proteins, which interact with the activated conformation of rab1. To date p115, GM130, golgin-84 and MICAL have been identified as direct interacting partners. Together with rab1, these molecules are components of a protein complex, which mediates and regulates intracellular vesicle transport. RESULTS: Here, we report the characterization of Iporin, which is similar to KIAA0375 as a novel rab1-interacting protein. It was initially identified by yeast two-hybrid screening experiments with the active mutant of rab1b (rab1b Q67R) as bait. Iporin contains a SH3 domain and two polyproline stretches, which are known to play a role in protein/protein interactions. In addition, Iporin encloses a RUN domain, which seems to be a major part of the rab1binding domain (R1BD). Iporin is ubiquitously expressed and immunofluorescence staining displays a cytosolic punctual distribution. Interestingly, we also show that Iporin interacts with another rab1 interacting partner, the GM130 protein. CONCLUSION: Our results demonstrate that Iporin is a potential new interacting partner of rab1. Iporin is different from already identified rab1 interacting proteins concerning protein structure and cellular localization. We conclude that Iporin might function as a link between the targeting of ER derived vesicles, triggered by the rab1 GTPase and a signaling pathway regulated by molecules containing SH3 and/or poly-proline regions. The characterization of this novel intermolecular relation could help to elucidate how vesicles find their way from ER to the Golgi apparatus.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión al GTP rab1/metabolismo , Autoantígenos , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/química , Citosol , Retículo Endoplásmico/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación Missense , Péptidos , Unión Proteica , Distribución Tisular , Técnicas del Sistema de Dos Híbridos , Proteínas de Unión al GTP rab1/genética , Dominios Homologos srcRESUMEN
BC1 RNA and BC200 RNA are two non-homologous, small non-messenger RNAs (snmRNAs) that were generated, evolutionarily, quite recently by retroposition. This process endowed the RNA polymerase III transcripts with central adenosine-rich regions. Both RNAs are expressed almost exclusively in neurons, where they are transported into dendritic processes as ribonucleoprotein particles (RNPs). Here, we demonstrate with a variety of experimental approaches that poly(A)-binding protein (PABP1), a regulator of translation initiation, binds to both RNAs in vitro and in vivo. We identified the association of PABP with BC200 RNA in a tri-hybrid screen and confirmed this binding in electrophoretic mobility-shift assays and via anti-PABP immunoprecipitation of BC1 and BC200 RNAs from crude extracts, immunodepleted extracts, partially purified RNPs and cells transfected with naked RNA. Furthermore, PABP immunoreactivity was localized to neuronal dendrites. Competition experiments using variants of BC1 and BC200 RNAs demonstrated that the central adenosine-rich region of both RNAs mediates binding to PABP. These findings lend support to the hypothesis that the BC1 and BC200 RNPs are involved in protein translation in neuronal dendrites.
Asunto(s)
Neuronas/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Ribonucleoproteínas Citoplasmáticas Pequeñas/genética , Ribonucleoproteínas/metabolismo , Secuencia de Bases , Encéfalo/embriología , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas de Unión a Poli(A) , Pruebas de Precipitina , ARN/genética , Proteínas de Unión al ARN/genética , TransfecciónRESUMEN
Vasopressin (VP) mRNA and the non-coding BC200 RNA are sorted to neuronal dendrites. Among proteins interacting specifically with both RNAs is the multifunctional poly(A)-binding protein (PABP) consisting of four RNA recognition motifs (RRMs) and a C-terminal auxiliary domain. The protein/RNA interaction studies presented here reveal that PABPs association with VP- and BC200 RNA is exclusively mediated by RRMs 3+4. Quantitative binding studies with PABP deletion mutants demonstrate preferential binding of RRMs 3+4 even to poly(A)-homopolymers, while RRMs 1+2 exhibit a lower affinity for those sequences. An optimal interaction with both poly(A)- and non-poly(A) sequences is only achieved by full-size PABP.
Asunto(s)
Poli A/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , ARN/química , ARN/metabolismo , Animales , Sitios de Unión , Línea Celular , Cinética , Datos de Secuencia Molecular , Mutagénesis , Ratas , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Especificidad por Sustrato , TransfecciónRESUMEN
Muscarinic acetylcholine receptors (mAChR) are involved in learning and memory but their molecular function in these processes is not fully understood. In this study, the signal transduction pathway coupling mAChR activation to induction of the activity-regulated cytoskeleton-associated gene (ARC) was examined. ARC was first identified as an effector immediate early gene induced by neuronal activity and ARC protein is thought to play a role in synaptic plasticity. In rats, intraperitoneal injection of pilocarpine, a potent agonist of mAChR, led to increased ARC expression in the brain. In human SH-SY5Y neuroblastoma cells mAChR stimulation with carbachol caused a rapid and robust induction of ARC expression. This effect was inhibited by atropine, a nonselective muscarinic receptor antagonist as well as by M1/M3 subtype-specific antagonists. Analysis of mAChR downstream effectors revealed that protein kinase C (PKC) and tyrosine kinases of the src family are key molecules in the signal cascade leading to ARC expression. Our data suggest, for the first time, that a correlation exists among mAChR-controlled signal cascades, the induction of the effector immediate early gene ARC and synaptic plasticity.
Asunto(s)
Proteínas Inmediatas-Precoces/metabolismo , Proteínas del Tejido Nervioso , Receptores Muscarínicos/fisiología , Animales , Northern Blotting/métodos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular Tumoral , Colinérgicos/farmacología , Proteína 61 Rica en Cisteína , Proteínas del Citoesqueleto , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Inmediatas-Precoces/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Agonistas Muscarínicos/farmacología , Neuroblastoma , Pilocarpina/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
We examined the effect of insulin on the expression of the activity-regulated cytoskeleton-associated gene (ARC), an effector immediate early gene with a proposed role in memory formation. In human SH-SY5Y neuroblastoma cells, application of insulin leads to dramatic increase in ARC mRNA and protein levels. Inhibition experiments reveal, that p21(ras), mitogen-activated protein kinase/extracellular regulated kinase and tyrosine kinase (src) activity are required for the insulin-induced ARC expression in SH-SY5Y cells, whereas protein kinase C is not involved in the signal transduction pathway. Our data indicated for the first time a correlation of the insulin-controlled signal cascade and the induction of synaptic plasticity-associated immediate early genes.
Asunto(s)
Proteínas del Citoesqueleto/genética , Insulina/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas del Tejido Nervioso/genética , Neuronas/enzimología , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Familia-src Quinasas/metabolismo , Sistema Nervioso Central/enzimología , Sistema Nervioso Central/crecimiento & desarrollo , Proteínas del Citoesqueleto/efectos de los fármacos , Proteínas del Citoesqueleto/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Insulina/farmacología , Memoria/fisiología , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma , Plasticidad Neuronal/genética , Neuronas/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Transducción de Señal/genética , Sinapsis/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiología , Células Tumorales CultivadasRESUMEN
Environmental enrichment is intended to improve the welfare of laboratory animals. However, regarding male mice, numerous studies indicate an increase in aggressive behavior due to cage structuring. On the one hand, this might be a problem concerning animal welfare. On the other hand, enrichment is though to hamper environmental standardization and to increase variability of data. Furthermore, increasing fights, arousal, and/or injury in enriched housed animals might superimpose other (positive) environmental effects on behavior and physiology. Therefore, the present study investigated effects of environmental enrichment on behavioral, endocrinological, and immunological parameters in male mice of the docile inbred strain ABG. From weaning until day 77+/-3 of life, animals were kept in stable sibling groups of four under three different housing conditions: (A) nonstructured Makrolon type III laboratory cages ("standard housing"=S); (B) equivalent laboratory cages that were enriched with a box and scaffolding ("enriched housing"=E); and (C) spacious terrariums that were structured richly ("super-enriched housing"=SE). No differences in agonistic behavior, levels of plasma corticosterone (CORT), and activities of adrenal tyrosine hydroxylase (TH) existed among S-, E-, and SE-housed ABG males. Play behavior and general activity increased significantly with increasing enrichment. Concerning immunological parameters, males of both forms of enriched housing showed significantly lower percentages of CD4 and CD8 cells compared to S-housed mice. However, regarding the ratio of CD4/CD8 cells, IL-2, IL-4, IL-10, IFN-gamma, IgG1, and IgG2a, no significant housing-dependent differences were found. Enrichment did neither hamper standardization nor negatively influence the variability of physiological parameters. In summary, using a docile strain of mice revealed the positive effects of environmental enrichment also on male mice. The lack of adverse effects on behavior, physiology, standardization, and variability of data defuses these arguments against providing docile male mice with enrichment.
Asunto(s)
Conducta Animal/fisiología , Vivienda para Animales , Sistema Hipófiso-Suprarrenal/metabolismo , Conducta Agonística , Animales , Corticosterona/sangre , Citocinas/sangre , Conducta Exploratoria , Inmunoglobulinas/sangre , Masculino , Ratones , Ratones Endogámicos , Actividad Motora , Radioinmunoensayo/métodos , Conducta Social , Estadísticas no Paramétricas , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Leucine-rich repeat kinase 2 (LRRK2) is a multi-domain 280 kDa protein that is linked to Parkinson's disease (PD). Mutations especially in the GTPase and kinase domains of LRRK2 are the most common causes of heritable PD and are also found in sporadic forms of PD. Although the cellular function of LRRK2 is largely unknown there is increasing evidence that these mutations cause cell death due to autophagic dysfunction and mitochondrial damage. Here, we demonstrate a novel mechanism of LRRK2 binding and transport, which involves the small GTPases Rab32 and Rab38. Rab32 and its closest homologue Rab38 are known to organize the trans-Golgi network and transport of key enzymes in melanogenesis, whereas their function in non-melanogenic cells is still not well understood. Cellular processes such as autophagy, mitochondrial dynamics, phagocytosis or inflammatory processes in the brain have previously been linked to Rab32. Here, we demonstrate that Rab32 and Rab38, but no other GTPase tested, directly interact with LRRK2. GFP-Trap analyses confirmed the interaction of Rab32 with the endogenous LRRK2. In yeast two-hybrid experiments we identified a predicted coiled-coil motif containing region within the aminoterminus of LRRK2 as the possible interacting domain. Fluorescence microscopy demonstrated a co-localization of Rab32 and LRRK2 at recycling endosomes and transport vesicles, while overexpression of a constitutively active mutant of Rab32 led to an increased co-localization with Rab7/9 positive perinuclear late endosomes/MVBs. Subcellular fractionation experiments supported the novel role of Rab32 in LRRK2 late endosomal transport and sorting in the cell. Thus, Rab32 may regulate the physiological functions of LRRK2.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Secuencias de Aminoácidos , Animales , Biomarcadores/metabolismo , Compartimento Celular , Endosomas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunoprecipitación , Espacio Intracelular/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Ratones , Proteínas Mutantes/metabolismo , Células 3T3 NIH , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/química , Transporte de Proteínas , Fracciones Subcelulares/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Junction-mediating and regulatory protein (JMY) was originally identified as a transcriptional co-factor in the p53-response to DNA damage. Aside from this nuclear function, recent years have uncovered an additional function of JMY, namely in cytoskeleton remodelling and actin assembly. The C-terminus of JMY comprises a canonical VCA-module, the sequence signature of Arp2/3 complex activators. Furthermore, tandem repeats of 3 WH2 (V, or more recently also W) domains render JMY capable of Arp2/3 independent actin assembly. The motility promoting cytoplasmic function of JMY is abrogated upon DNA-damage and nuclear translocation of JMY. To address the precise cellular function of JMY in cellular actin rearrangements, we have searched for potential new interaction partners by mass spectrometry. We identified several candidates and correlated their localization with the subcellular dynamics of JMY. JMY is localized to dynamic vesiculo-tubular structures throughout the cytoplasm, which are decorated with actin and Arp2/3 complex. Moreover, JMY partially colocalizes and interacts with VAP-A, which is involved in vesicle-based transport processes. Finally, overexpression of JMY results in Golgi dispersal by loss from the trans-site and affects VSV-G transport. These analyses, together with biochemical experiments, indicate that JMY drives vesicular trafficking in the trans-Golgi region and at ER-membrane contact sites (MCS), distinct from other Arp2/3 activators involved in vesicle transport processes such as the related WHAMM or WASH.
Asunto(s)
Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Red trans-Golgi/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Animales , Células COS , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Chlorocebus aethiops , Células HeLa , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Nucleares/genética , Multimerización de Proteína , Transporte de Proteínas , Transactivadores/genética , Proteínas de Transporte Vesicular , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Small GTPases of the Rab family are important regulators of a large variety of different cellular functions such as membrane organization and vesicle trafficking. They have been shown to play a role in several human diseases. One prominent member, Rab6, is thought to be involved in the development of Alzheimer's Disease, the most prevalent mental disorder worldwide. Previous studies have shown that Rab6 impairs the processing of the amyloid precursor protein (APP), which is cleaved to ß-amyloid in brains of patients suffering from Alzheimer's Disease. Additionally, all three members of the Mint adaptor family are implied to participate in the amyloidogenic pathway. Here, we report the identification of a new Mint1 isoform in a yeast two-hybrid screening, Mint1 826, which lacks an eleven amino acid (aa) sequence in the conserved C-terminal region. Mint1 826, but not the conventional Mint1, interacts with Rab6 via the PTB domain. This interaction is nucleotide-dependent, Rab6-specific and influences the subcellular localization of Mint1 826. We were able to detect and sequence a corresponding proteolytic peptide derived from cellular Mint1 826 by mass spectrometry proving the absence of aa 495-505 and could show that the deletion does not influence the ability of this adaptor protein to interact with APP. Taking into account that APP interacts and co-localizes with Mint1 826 and is transported in Rab6 positive vesicles, our data suggest that Mint1 826 bridges APP to the small GTPase at distinct cellular sorting points, establishing Mint1 826 as an important player in regulation of APP trafficking and processing.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Studies of the role of tuberous sclerosis complex (TSC) proteins (TSC1/TSC2) in pathology have focused mainly on their capacity to regulate translation and cell growth, but their relationship with alterations of cellular structures and the cell cycle is not yet fully understood. The transforming acidic coiled-coil (TACC) domain-containing proteins are central players in structures and processes connected to the centrosome. Here, TACC3 interactome mapping identified TSC2 and 15 other physical interactors, including the evolutionary conserved interactions with ch-TOG/CKAP5 and FAM161B. TACC3 and TSC2 co-localize and co-purify with components of the nuclear envelope, and their deficiency causes morphological alterations of this structure. During cell division, TACC3 is necessary for the proper localization of phospho-Ser939 TSC2 at spindle poles and cytokinetic bridges. Accordingly, abscission alterations and increased frequency of binucleated cells were observed in Tacc3- and Tsc2-deficient cells relative to controls. In regulating cell division, TSC2 acts epistatically to TACC3 and, in addition to canonical TSC/mTOR signaling and cytokinetic associations, converges to the early mitotic checkpoint mediated by CHFR, consistently with nuclear envelope associations. Our findings link TACC3 to novel structural and cell division functions of TSC2, which may provide additional explanations for the clinical and pathological manifestations of lymphangioleiomyomatosis (LAM) disease and TSC syndrome, including the greater clinical severity of TSC2 mutations compared to TSC1 mutations.