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1.
Mol Cell ; 81(16): 3275-3293.e12, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34245671

RESUMO

Cells communicate with their environment via surface proteins and secreted factors. Unconventional protein secretion (UPS) is an evolutionarily conserved process, via which distinct cargo proteins are secreted upon stress. Most UPS types depend upon the Golgi-associated GRASP55 protein. However, its regulation and biological role remain poorly understood. Here, we show that the mechanistic target of rapamycin complex 1 (mTORC1) directly phosphorylates GRASP55 to maintain its Golgi localization, thus revealing a physiological role for mTORC1 at this organelle. Stimuli that inhibit mTORC1 cause GRASP55 dephosphorylation and relocalization to UPS compartments. Through multiple, unbiased, proteomic analyses, we identify numerous cargoes that follow this unconventional secretory route to reshape the cellular secretome and surfactome. Using MMP2 secretion as a proxy for UPS, we provide important insights on its regulation and physiological role. Collectively, our findings reveal the mTORC1-GRASP55 signaling hub as the integration point in stress signaling upstream of UPS and as a key coordinator of the cellular adaptation to stress.


Assuntos
Proteínas da Matriz do Complexo de Golgi/genética , Proteoma/genética , Proteômica , Estresse Fisiológico/genética , Matriz Extracelular/genética , Complexo de Golgi/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas de Membrana/genética , Transporte Proteico/genética , Transdução de Sinais/genética
2.
EMBO J ; 40(20): e107766, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34516001

RESUMO

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.


Assuntos
Glicoesfingolipídeos/metabolismo , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Brefeldina A/farmacologia , Ceramidas/metabolismo , Toxina da Cólera/farmacologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Expressão Gênica , Glicosilação/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/genética , Proteínas da Matriz do Complexo de Golgi/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Toxina Shiga/farmacologia
3.
Am J Hum Genet ; 107(5): 989-999, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33053334

RESUMO

Osteogenesis imperfecta (OI) is characterized primarily by susceptibility to fractures with or without bone deformation. OI is genetically heterogeneous: over 20 genetic causes are recognized. We identified bi-allelic pathogenic KDELR2 variants as a cause of OI in four families. KDELR2 encodes KDEL endoplasmic reticulum protein retention receptor 2, which recycles ER-resident proteins with a KDEL-like peptide from the cis-Golgi to the ER through COPI retrograde transport. Analysis of patient primary fibroblasts showed intracellular decrease of HSP47 and FKBP65 along with reduced procollagen type I in culture media. Electron microscopy identified an abnormal quality of secreted collagen fibrils with increased amount of HSP47 bound to monomeric and multimeric collagen molecules. Mapping the identified KDELR2 variants onto the crystal structure of G. gallus KDELR2 indicated that these lead to an inactive receptor resulting in impaired KDELR2-mediated Golgi-ER transport. Therefore, in KDELR2-deficient individuals, OI most likely occurs because of the inability of HSP47 to bind KDELR2 and dissociate from collagen type I. Instead, HSP47 remains bound to collagen molecules extracellularly, disrupting fiber formation. This highlights the importance of intracellular recycling of ER-resident molecular chaperones for collagen type I and bone metabolism and a crucial role of HSP47 in the KDELR2-associated pathogenic mechanism leading to OI.


Assuntos
Osso e Ossos/metabolismo , Colágeno Tipo I/metabolismo , Proteínas de Choque Térmico HSP47/metabolismo , Osteogênese Imperfeita/genética , Proteínas de Transporte Vesicular/metabolismo , Adulto , Alelos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Osso e Ossos/patologia , Galinhas , Pré-Escolar , Colágeno Tipo I/química , Colágeno Tipo I/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Proteínas de Choque Térmico HSP47/química , Proteínas de Choque Térmico HSP47/genética , Humanos , Lactente , Masculino , Osteogênese Imperfeita/diagnóstico , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/patologia , Linhagem , Cultura Primária de Células , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Transporte Proteico , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
4.
Int J Mol Sci ; 24(2)2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36674416

RESUMO

The binding of nitric oxide (NO) to heme in the ß1 subunit of soluble guanylyl cyclase (sGC) activates both the heterodimeric α1ß1 and α2ß1 isoforms of the enzyme, leading to the increased production of cGMP from GTP. In cultured human mast cells, exogenous NO is able to inhibit mast cell degranulation via NO-cGMP signaling. However, under inflammatory oxidative or nitrosative stress, sGC becomes insensitive to NO. The occurrence of mast cells in healthy and inflamed human tissues and the in vivo expression of the α1 and ß1 subunits of sGC in human mast cells during inflammation remain largely unresolved and were investigated here. Using peroxidase and double immunohistochemical incubations, no mast cells were found in healthy dental pulp, whereas the inflammation of dental pulp initiated the occurrence of several mast cells expressing the α1 and ß1 subunits of sGC. Since inflammation-induced oxidative and nitrosative stress oxidizes Fe2+ to Fe3+ in the ß1 subunit of sGC, leading to the desensitization of sGC to NO, we hypothesize that the NO- and heme-independent pharmacological activation of sGC in mast cells may be considered as a regulatory strategy for mast cell functions in inflamed human dental pulp.


Assuntos
Polpa Dentária , Guanilato Ciclase , Humanos , Guanilil Ciclase Solúvel/genética , Guanilil Ciclase Solúvel/metabolismo , Guanilato Ciclase/metabolismo , Polpa Dentária/metabolismo , Óxido Nítrico/metabolismo , Inflamação , Heme , GMP Cíclico/metabolismo
5.
J Cell Sci ; 129(4): 706-16, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26746240

RESUMO

Cartilage oligomeric matrix protein (COMP) is an abundant component in the extracellular matrix (ECM) of load-bearing tissues such as tendons and cartilage. It provides adaptor functions by bridging different ECM structures. We have previously shown that COMP is also a constitutive component of healthy human skin and is strongly induced in fibrosis. It binds directly and with high affinity to collagen I and to collagen XII that decorates the surface of collagen I fibrils. We demonstrate here that lack of COMP-collagen interaction in the extracellular space leads to changes in collagen fibril morphology and density, resulting in altered skin biomechanical properties. Surprisingly, COMP also fulfills an important intracellular function in assisting efficient secretion of collagens, which were retained in the endoplasmic reticulum of COMP-null fibroblasts. Accordingly, COMP-null mice showed severely attenuated fibrotic responses in skin. Collagen secretion was fully restored by introducing wild-type COMP. Hence, our work unravels a new, non-structural and intracellular function of the ECM protein COMP in controlling collagen secretion.


Assuntos
Proteína de Matriz Oligomérica de Cartilagem/genética , Colágenos Fibrilares/metabolismo , Pele/metabolismo , Animais , Proteína de Matriz Oligomérica de Cartilagem/metabolismo , Células Cultivadas , Estresse do Retículo Endoplasmático , Feminino , Fibroblastos/metabolismo , Fibrose , Camundongos Endogâmicos C57BL , Pele/patologia
6.
FASEB J ; 31(9): 3978-3990, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28550045

RESUMO

Nephrin is a core component of podocyte (glomerular epithelial cell) slit diaphragm and is required for kidney ultrafiltration. Down-regulation or mislocalization of nephrin has been observed in diabetic kidney disease (DKD), characterized by albuminuria. Here, we investigate the role of protein kinase C and casein kinase 2 substrate in neurons 2 (PACSIN2), a regulator of endocytosis and recycling, in the trafficking of nephrin and development of DKD. We observe that PACSIN2 is up-regulated and nephrin mislocalized in podocytes of obese Zucker diabetic fatty (ZDF) rats that have altered renal function. In cultured podocytes, PACSIN2 and nephrin colocalize and interact. We show that nephrin is endocytosed in PACSIN2-positive membrane regions and that PACSIN2 overexpression increases both nephrin endocytosis and recycling. We identify rabenosyn-5, which is involved in early endosome maturation and endosomal sorting, as a novel interaction partner of PACSIN2. Interestingly, rabenosyn-5 expression is increased in podocytes in obese ZDF rats, and, in vitro, its overexpression enhances the association of PACSIN2 and nephrin. We also show that palmitate, which is elevated in diabetes, enhances this association. Collectively, PACSIN2 is up-regulated and nephrin is abnormally localized in podocytes of diabetic ZDF rats. In vitro, PACSIN2 enhances nephrin turnover apparently via a mechanism involving rabenosyn-5. The data suggest that elevated PACSIN2 expression accelerates nephrin trafficking and associates with albuminuria.-Dumont, V., Tolvanen, T. A., Kuusela, S., Wang, H., Nyman, T. A., Lindfors, S., Tienari, J., Nisen, H., Suetsugu, S., Plomann, M., Kawachi, H., Lehtonen, S. PACSIN2 accelerates nephrin trafficking and is up-regulated in diabetic kidney disease.


Assuntos
Proteínas de Transporte/metabolismo , Nefropatias Diabéticas/metabolismo , Proteínas de Membrana/metabolismo , Podócitos/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Proteínas do Citoesqueleto , Diabetes Mellitus , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Obesidade , Transporte Proteico/fisiologia , Proteínas/genética , Ratos Zucker , Regulação para Cima , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
7.
Pharmacol Res ; 128: 200-210, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29107716

RESUMO

The Pacsin proteins (Pacsin 1, 2 and 3) play an important role in intracellular trafficking and thereby signal transduction in many cells types. This study was designed to examine the role of Pacsin 2 in cardiac development and function. We investigated the development and electrophysiological properties of Pacsin 2 knockout (P2KO) hearts and single cardiomyocytes isolated from 11.5 and 15.5days old fetal mice. Immunofluorescence experiments confirmed the lack of Pacsin 2 protein expression in P2KO cardiac myocytes in comparison to wildtype (WT). Western blotting demonstrates low expression levels of connexin 43 and T-box 3 proteins in P2KO compared to wildtype (WT). Electrophysiology measurements including online Multi-Electrode Array (MEA) based field potential (FP) recordings on isolated whole heart of P2KO mice showed a prolonged AV-conduction time. Patch clamp measurements of P2KO cardiomyocytes revealed differences in action potential (AP) parameters and decreased pacemaker funny channel (If), as well as L-type Ca2+ channel (ICaL), and sodium channel (INa). These findings demonstrate that Pacsin 2 is necessary for cardiac development and function in mouse embryos, which will enhance our knowledge to better understand the genesis of cardiovascular diseases.


Assuntos
Desenvolvimento Embrionário/fisiologia , Coração/fisiologia , Proteínas/fisiologia , Potenciais de Ação , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas do Citoesqueleto , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout
8.
Blood ; 126(1): 80-8, 2015 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-25838348

RESUMO

Bin-Amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) proteins generate tubular membrane invaginations reminiscent of the megakaryocyte (MK) demarcation membrane system (DMS), which provides membranes necessary for future platelets. The F-BAR protein PACSIN2 is one of the most abundant BAR/F-BAR proteins in platelets and the only one reported to interact with the cytoskeletal and scaffold protein filamin A (FlnA), an essential regulator of platelet formation and function. The FlnA-PACSIN2 interaction was therefore investigated in MKs and platelets. PACSIN2 associated with FlnA in human platelets. The interaction required FlnA immunoglobulin-like repeat 20 and the tip of PACSIN2 F-BAR domain and enhanced PACSIN2 F-BAR domain membrane tubulation in vitro. Most human and wild-type mouse platelets had 1 to 2 distinct PACSIN2 foci associated with cell membrane GPIbα, whereas Flna-null platelets had 0 to 4 or more foci. Endogenous PACSIN2 and transfected enhanced green fluorescent protein-PACSIN2 were concentrated in midstage wild-type mouse MKs in a well-defined invagination of the plasma membrane reminiscent of the initiating DMS and dispersed in the absence of FlnA binding. The DMS appeared less well defined, and platelet territories were not readily visualized in Flna-null MKs. We conclude that the FlnA-PACSIN2 interaction regulates membrane tubulation in MKs and platelets and likely contributes to DMS formation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Plaquetas , Membrana Celular/ultraestrutura , Filaminas/metabolismo , Megacariócitos , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Membrana Celular/metabolismo , Células Cultivadas , Filaminas/fisiologia , Células HEK293 , Humanos , Megacariócitos/metabolismo , Megacariócitos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Pseudópodes/metabolismo
9.
Hum Mol Genet ; 23(10): 2769-79, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24385601

RESUMO

How epithelial cells form a tubule with defined length and lumen diameter remains a fundamental question in cell and developmental biology. Loss of control of tubule lumen size in multiple organs including the kidney, liver and pancreas features polycystic kidney disease (PKD). To gain insights into autosomal dominant polycystic kidney disease, we performed yeast two-hybrid screens using the C-terminus of polycystin-1 (PC1) as bait. Here, we report that PC1 interacts with Pacsin 2, a cytoplasmic phosphoprotein that has been implicated in cytoskeletal organization, vesicle trafficking and more recently in cell intercalation during gastrulation. PC1 binds to a 107-residue fragment containing the α3 helix of the F-BAR domain of Pacsin 2 via a coiled-coil domain in its C-tail. PC1 and Pacsin 2 co-localize on the lamellipodia of migrating kidney epithelial cells. PC1 and Pacsin 2-deficient kidney epithelial cells migrate at a slower speed with reduced directional persistency. We further demonstrate that PC1, Pacsin 2 and N-Wasp are in the same protein complex, and both PC1 and Pacsin 2 are required for N-Wasp/Arp2/3-dependent actin remodeling. We propose that PC1 modulates actin cytoskeleton rearrangements and directional cell migration through the Pacsin 2/N-Wasp/Arp2/3 complex, which consequently contributes to the establishment and maintenance of the sophisticated tubular architecture. Disruption of this complex contributes to cyst formation in PKD.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Canais de Cátion TRPP/fisiologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Animais , Linhagem Celular , Células Epiteliais/fisiologia , Humanos , Camundongos Transgênicos , Complexos Multiproteicos/metabolismo , Transporte Proteico , Pseudópodes/metabolismo , Técnicas do Sistema de Duplo-Híbrido
10.
Proc Natl Acad Sci U S A ; 110(34): 13976-81, 2013 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-23918399

RESUMO

The dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses is crucial for synaptic transmission, plasticity, learning, and memory. The protein interacting with C-kinase 1 (PICK1) directly interacts with GluA2/3 subunits of the AMPARs. Although the role of PICK1 in regulating AMPAR trafficking and multiple forms of synaptic plasticity is known, the exact molecular mechanisms underlying this process remain unclear. Here, we report a unique interaction between PICK1 and all three members of the protein kinase C and casein kinase II substrate in neurons (PACSIN) family and show that they form a complex with AMPARs. Our results reveal that knockdown of the neuronal-specific protein, PACSIN1, leads to a significant reduction in AMPAR internalization following the activation of NMDA receptors in hippocampal neurons. The interaction between PICK1 and PACSIN1 is regulated by PACSIN1 phosphorylation within the variable region and is required for AMPAR endocytosis. Similarly, the binding of PICK1 to the ubiquitously expressed PACSIN2 is also regulated by the homologous phosphorylation sites within the PACSIN2-variable region. Genetic deletion of PACSIN2, which is highly expressed in Purkinje cells, eliminates cerebellar long-term depression. This deficit can be fully rescued by overexpressing wild-type PACSIN2, but not by a PACSIN2 phosphomimetic mutant, which does not bind PICK1 efficiently. Taken together, our data demonstrate that the interaction of PICK1 and PACSIN is required for the activity-dependent internalization of AMPARs and for the expression of long-term depression in the cerebellum.


Assuntos
Proteínas de Transporte/metabolismo , Hipocampo/citologia , Proteínas Nucleares/metabolismo , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Células Cultivadas , Proteínas do Citoesqueleto , Escherichia coli , Células HEK293 , Hipocampo/metabolismo , Humanos , Imuno-Histoquímica , Imunoprecipitação , RNA Interferente Pequeno/genética , Ratos
11.
Proc Natl Acad Sci U S A ; 109(11): 4116-21, 2012 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-22371566

RESUMO

Synaptic transmission is mediated by a complex set of molecular events that must be coordinated in time and space. While many proteins that function at the synapse have been identified, the signaling pathways regulating these molecules are poorly understood. Pak5 (p21-activated kinase 5) is a brain-specific isoform of the group II Pak kinases whose substrates and roles within the central nervous system are largely unknown. To gain insight into the physiological roles of Pak5, we engineered a Pak5 mutant to selectively radiolabel its substrates in murine brain extract. Using this approach, we identified two novel Pak5 substrates, Pacsin1 and Synaptojanin1, proteins that directly interact with one another to regulate synaptic vesicle endocytosis and recycling. Pacsin1 and Synaptojanin1 were phosphorylated by Pak5 and the other group II Paks in vitro, and Pak5 phosphorylation promoted Pacsin1-Synaptojanin1 binding both in vitro and in vivo. These results implicate Pak5 in Pacsin1- and Synaptojanin1-mediated synaptic vesicle trafficking and may partially account for the cognitive and behavioral deficits observed in group II Pak-deficient mice.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Neurônios/enzimologia , Neuropeptídeos/metabolismo , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Vesículas Sinápticas/enzimologia , Quinases Ativadas por p21/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Transporte Biológico , Encéfalo/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Modelos Biológicos , Fosforilação , Ligação Proteica , Especificidade por Substrato
12.
J Biol Chem ; 288(13): 9303-12, 2013 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-23420842

RESUMO

The PACSIN (protein kinase C and casein kinase 2 substrate in neurons) adapter proteins couple components of the clathrin-mediated endocytosis machinery with regulators of actin polymerization and thereby regulate the surface expression of specific receptors. The brain-specific PACSIN 1 is enriched at synapses and has been proposed to affect neuromorphogenesis and the formation and maturation of dendritic spines. In studies of how phosphorylation of PACSIN 1 contributes to neuronal function, we identified serine 358 as a specific site used by casein kinase 2 (CK2) in vitro and in vivo. Phosphorylated PACSIN 1 was found in neuronal cytosol and membrane fractions. This localization could be modulated by trophic factors such as brain-derived neurotrophic factor (BDNF). We further show that expression of a phospho-negative PACSIN 1 mutant, S358A, or inhibition of CK2 drastically reduces spine formation in neurons. We identified a novel protein complex containing the spine regulator Rac1, its GTPase-activating protein neuron-associated developmentally regulated protein (NADRIN), and PACSIN 1. CK2 phosphorylation of PACSIN 1 leads to a dissociation of the complex upon BDNF treatment and induces Rac1-dependent spine formation in dendrites of hippocampal neurons. These findings suggest that upon BDNF signaling PACSIN 1 is phosphorylated by CK2 which is essential for spine formation.


Assuntos
Caseína Quinase II/metabolismo , Neuropeptídeos/metabolismo , Fosfoproteínas/metabolismo , Proteína Quinase C/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Encéfalo/metabolismo , Clatrina/metabolismo , Dendritos , Inativação Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Microscopia de Fluorescência/métodos , Modelos Biológicos , Mutação , Plasticidade Neuronal , Neurônios/metabolismo , Fosforilação , Serina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sinapses/metabolismo , Transmissão Sináptica
13.
Cell Commun Signal ; 12: 37, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24930026

RESUMO

BACKGROUND: The receptor for activated C-kinase 1 (RACK1) is a conserved protein belonging to the WD40 repeat family of proteins. It folds into a beta propeller with seven blades which allow interactions with many proteins. Thus it can serve as a scaffolding protein and have roles in several cellular processes. RESULTS: We identified the product of the Dictyostelium discoideum gpbB gene as the Dictyostelium RACK1 homolog. The protein is mainly cytosolic but can also associate with cellular membranes. DdRACK1 binds to phosphoinositides (PIPs) in protein-lipid overlay and liposome-binding assays. The basis of this activity resides in a basic region located in the extended loop between blades 6 and 7 as revealed by mutational analysis. Similar to RACK1 proteins from other organisms DdRACK1 interacts with G protein subunits alpha, beta and gamma as shown by yeast two-hybrid, pulldown, and immunoprecipitation assays. Unlike the Saccharomyces cerevisiae and Cryptococcus neoformans RACK1 proteins it does not appear to take over Gß function in D. discoideum as developmental and other defects were not rescued in Gß null mutants overexpressing GFP-DdRACK1. Overexpression of GFP-tagged DdRACK1 and a mutant version (DdRACK1mut) which carried a charge-reversal mutation in the basic region in wild type cells led to changes during growth and development. CONCLUSION: DdRACK1 interacts with heterotrimeric G proteins and can through these interactions impact on processes specifically regulated by these proteins.


Assuntos
Dictyostelium/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Dictyostelium/crescimento & desenvolvimento , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Dados de Sequência Molecular , Especificidade de Órgãos , Fosfatidilinositóis/metabolismo , Ligação Proteica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
14.
Kidney Int ; 83(3): 426-37, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23235565

RESUMO

The protein kinase C and casein kinase 2 substrate in neurons (Pacsin) is a subfamily of membrane-binding proteins that participates in vesicle trafficking and cytoskeleton organization. Here, we studied Pacsin 2 in kidney development and repair following injury. In the postnatal developing kidneys, Pacsin 2 was found to be expressed in both ureteric bud- and mesenchyme-derived structures including proximal and distal tubules, Bowman's capsule, and the glomerular tuft. In the adult kidney, its expression was decreased in proximal tubules but increased in glomerular tuft when compared to that in the developing kidneys. Interestingly, Pacsin 2 expression was significantly upregulated during the repair phase after ischemia-reperfusion injury, especially on the apical brush border of proximal tubules that experienced massive damage. Pacsin 2 localized to the primary cilia of renal epithelial cells. Knockdown of Pacsin 2 by shRNA did not affect the cell cycle or cell polarity; however, it increased the length of primary cilia, and resulted in significant tubulogenic defects in three-dimensional cell culture. Thus, we propose that Pacsin 2 contributes to kidney development and repair in a nephron-specific manner.


Assuntos
Rim/embriologia , Proteínas/fisiologia , Traumatismo por Reperfusão/fisiopatologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Ciclo Celular , Polaridade Celular , Proliferação de Células , Proteínas do Citoesqueleto , Células Epiteliais/química , Rim/química , Túbulos Renais Coletores/citologia , Camundongos , Proteínas/análise
15.
J Cell Sci ; 124(Pt 14): 2375-88, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21693584

RESUMO

The Rac1 GTPase controls cytoskeletal dynamics and is a key regulator of cell spreading and migration mediated by signaling through effector proteins, such as the PAK kinases and the Scar and WAVE proteins. We previously identified a series of regulatory proteins that associate with Rac1 through its hypervariable C-terminal domain, including the Rac1 activator ß-Pix (also known as Rho guanine-nucleotide-exchange factor 7) and the membrane adapter caveolin-1. Here, we show that Rac1 associates, through its C-terminus, with the F-BAR domain protein PACSIN2, an inducer of membrane tubulation and a regulator of endocytosis. We show that Rac1 localizes with PACSIN2 at intracellular tubular structures and on early endosomes. Active Rac1 induces a loss of PACSIN2-positive tubular structures. By contrast, Rac1 inhibition results in an accumulation of PACSIN2-positive tubules. In addition, PACSIN2 appears to regulate Rac1 signaling; siRNA-mediated loss of PACSIN2 increases the levels of Rac1-GTP and promotes cell spreading and migration in a wound healing assay. Moreover, ectopic expression of PACSIN2 reduces Rac1-GTP levels in a fashion that is dependent on the PACSIN2-Rac1 interaction, on the membrane-tubulating capacity of PACSIN2 and on dynamin. These data identify the BAR-domain protein PACSIN2 as a Rac1 interactor that regulates Rac1-mediated cell spreading and migration.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Células Jurkat , Microtúbulos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Transfecção , Proteínas rac1 de Ligação ao GTP/genética , Domínios de Homologia de src
16.
Cell Commun Signal ; 11: 54, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23915312

RESUMO

BACKGROUND: Dictyostelium harbors several paralogous Sec7 genes that encode members of three subfamilies of the Sec7 superfamily of guanine nucleotide exchange factors. One of them is the cytohesin family represented by three members in D. discoideum, SecG, Sec7 and a further protein distinguished by several transmembrane domains. Cytohesins are characterized by a Sec7-PH tandem domain and have roles in cell adhesion and migration. RESULTS: We study here Sec7. In vitro its PH domain bound preferentially to phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). When following the distribution of GFP-Sec7 in vivo we observed the protein in the cytosol and at the plasma membrane. Strikingly, when cells formed pseudopods, macropinosomes or phagosomes, GFP-Sec7 was conspicuously absent from areas of the plasma membrane which were involved in these processes. Mutant cells lacking Sec7 exhibited an impaired phagocytosis and showed significantly reduced speed and less persistence during migration. Cellular properties associated with mammalian cytohesins like cell-cell and cell-substratum adhesion were not altered. Proteins with roles in membrane trafficking and signal transduction have been identified as putative interaction partners consistent with the data obtained from mutant analysis. CONCLUSIONS: Sec7 is a cytosolic component and is associated with the plasma membrane in a pattern distinctly different from the accumulation of PI(3,4,5)P3. Mutant analysis reveals that loss of the protein affects cellular processes that involve membrane flow and the actin cytoskeleton.


Assuntos
Dictyostelium/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Proteínas de Protozoários/fisiologia , Sequência de Aminoácidos , Adesão Celular/fisiologia , Quimiotaxia , Fatores de Troca do Nucleotídeo Guanina/química , Dados de Sequência Molecular , Fagocitose , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
17.
J Thromb Haemost ; 21(12): 3619-3632, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37678551

RESUMO

BACKGROUND: Upon vessel injury, platelets adhere to exposed matrix constituents via specific membrane receptors, including the von Willebrand factor receptor glycoprotein (GP)Ib-IX-V complex and integrins ß1 and ß3. In platelets, the Fes/CIP4-homology Bin-Amphiphysin-Rvs protein PACSIN2 associates with the cytoskeletal and scaffolding protein filamin A (FlnA), linking GPIbα and integrins to the cytoskeleton. OBJECTIVES: Here we investigated the role of PACSIN2 in platelet function. METHODS: Platelet parameters were evaluated in mice lacking PACSIN2 and platelet integrin ß1. RESULTS: Pacsin2-/- mice displayed mild thrombocytopenia, prolonged bleeding time, and delayed thrombus formation in a ferric chloride-mediated carotid artery injury model, which was normalized by injection of control platelets. Pacsin2-/- platelets formed unstable thrombi that embolized abruptly in a laser-induced cremaster muscle injury model. Pacsin2-/- platelets had hyperactive integrin ß1, as evidenced by increased spreading onto surfaces coated with the collagen receptor α2ß1-specific peptide GFOGER and increased binding of the antibody 9EG7 directed against active integrin ß1. By contrast, Pacsin2-/- platelets had normal integrin αIIbß3 function and expressed P-selectin normally following stimulation through the collagen receptor GPVI or with thrombin. Deletion of platelet integrin ß1 in Pacsin2-/- mice normalized platelet count, hemostasis, and thrombus formation. A PACSIN2 peptide mimicking the FlnA-binding site mediated the pull-down of a FlnA rod 2 construct by integrin ß7, a model for integrin ß-subunits. CONCLUSIONS: Pacsin2-/- mice displayed severe thrombus formation defects due to hyperactive platelet integrin ß1. The data suggest that PACSIN2 binding to FlnA negatively regulates platelet integrin ß1 hemostatic function.


Assuntos
Integrina beta1 , Ativação Plaquetária , Trombose , Animais , Camundongos , Plaquetas/metabolismo , Hemostasia , Hemostáticos/metabolismo , Integrina beta1/metabolismo , Peptídeos/farmacologia , Adesividade Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores de Colágeno/metabolismo , Trombose/metabolismo
18.
Brain Commun ; 4(1): fcac039, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35233527

RESUMO

A deficient transport of amyloid-ß across the blood-brain barrier, and its diminished clearance from the brain, contribute to neurodegenerative and vascular pathologies, such as Alzheimer's disease and cerebral amyloid angiopathy, respectively. At the blood-brain barrier, amyloid-ß efflux transport is associated with the low-density lipoprotein receptor-related protein 1. However, the precise mechanisms governing amyloid-ß transport across the blood-brain barrier, in health and disease, remain to be fully understood. Recent evidence indicates that the low-density lipoprotein receptor-related protein 1 transcytosis occurs through a tubulation-mediated mechanism stabilized by syndapin-2. Here, we show that syndapin-2 is associated with amyloid-ß clearance via low-density lipoprotein receptor-related protein 1 across the blood-brain barrier. We further demonstrate that risk factors for Alzheimer's disease, amyloid-ß expression and ageing, are associated with a decline in the native expression of syndapin-2 within the brain endothelium. Our data reveals that syndapin-2-mediated pathway, and its balance with the endosomal sorting, are important for amyloid-ß clearance proposing a measure to evaluate Alzheimer's disease and ageing, as well as a target for counteracting amyloid-ß build-up. Moreover, we provide evidence for the impact of the avidity of amyloid-ß assemblies in their trafficking across the brain endothelium and in low-density lipoprotein receptor-related protein 1 expression levels, which may affect the overall clearance of amyloid-ß across the blood-brain barrier.

19.
Cell Stress ; 5(11): 173-175, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34782889

RESUMO

Cellular adaptation to stress is a crucial homeostatic process for survival, metabolism, physiology, and disease. Cells respond to stress stimuli (e.g., nutrient starvation, growth factor deprivation, hypoxia, low energy, etc.) by changing the activity of signaling pathways, and interact with their environment by qualitatively and quantitatively modifying their intracellular, surface, and extracellular proteomes. How this delicate communication takes place is a hot topic in cell biological research, and has important implications for human disease.

20.
Nat Commun ; 12(1): 2610, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972531

RESUMO

Angiogenic sprouting relies on collective migration and coordinated rearrangements of endothelial leader and follower cells. VE-cadherin-based adherens junctions have emerged as key cell-cell contacts that transmit forces between cells and trigger signals during collective cell migration in angiogenesis. However, the underlying molecular mechanisms that govern these processes and their functional importance for vascular development still remain unknown. We previously showed that the F-BAR protein PACSIN2 is recruited to tensile asymmetric adherens junctions between leader and follower cells. Here we report that PACSIN2 mediates the formation of endothelial sprouts during angiogenesis by coordinating collective migration. We show that PACSIN2 recruits the trafficking regulators EHD4 and MICAL-L1 to the rear end of asymmetric adherens junctions to form a recycling endosome-like tubular structure. The junctional PACSIN2/EHD4/MICAL-L1 complex controls local VE-cadherin trafficking and thereby coordinates polarized endothelial migration and angiogenesis. Our findings reveal a molecular event at force-dependent asymmetric adherens junctions that occurs during the tug-of-war between endothelial leader and follower cells, and allows for junction-based guidance during collective migration in angiogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Oxigenases de Função Mista/metabolismo , Neovascularização Patológica/metabolismo , Proteínas Nucleares/metabolismo , Junções Aderentes/genética , Junções Aderentes/metabolismo , Animais , Cateninas/metabolismo , Movimento Celular/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/genética , Transdução de Sinais/genética , Esferoides Celulares/metabolismo
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