RESUMO
Dendrites, branched structures extending from neuronal cell soma, are specialized for processing information from other neurons. The morphogenesis of dendritic structures is spatiotemporally regulated by well-orchestrated signaling cascades. Dysregulation of these processes impacts the wiring of neuronal circuit and efficacy of neurotransmission, which contribute to the pathogeneses of neurological disorders. While Cdk5 (cyclin-dependent kinase 5) plays a critical role in neuronal dendritic development, its underlying molecular control is not fully understood. In this study, we show that p39, one of the two neuronal Cdk5 activators, is a key regulator of dendritic morphogenesis. Pyramidal neurons deficient in p39 exhibit aberrant dendritic morphology characterized by shorter length and reduced arborization, which is comparable to dendrites in Cdk5-deficient neurons. RNA sequencing analysis shows that the adaptor protein, WDFY1 (WD repeat and FYVE domain-containing 1), acts downstream of Cdk5/p39 to regulate dendritic morphogenesis. While WDFY1 is elevated in p39-deficient neurons, suppressing its expression rescues the impaired dendritic arborization. Further phosphoproteomic analysis suggests that Cdk5/p39 mediates dendritic morphogenesis by modulating various downstream signaling pathways, including PI3K/Akt-, cAMP-, or small GTPase-mediated signaling transduction pathways, thereby regulating cytoskeletal organization, protein synthesis, and protein trafficking.
Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Dendritos/metabolismo , Proteínas Ligadas a Lipídeos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Western Blotting , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , AMP Cíclico/metabolismo , Quinase 5 Dependente de Ciclina/genética , Proteínas do Citoesqueleto/genética , Células HEK293 , Humanos , Proteínas Ligadas a Lipídeos/genética , Espectrometria de Massas , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Morfogênese/genética , Morfogênese/fisiologia , Sistema Nervoso/citologia , Sistema Nervoso/metabolismo , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologiaRESUMO
Anchorage of a subset of cell surface proteins in eukaryotic cells is mediated by a glycosylphosphatidylinositol (GPI) moiety covalently attached to the carboxy-terminus of the protein moiety. Experimental evidence for the potential of GPI-anchored proteins (GPI-AP) of being released from cells into the extracellular environment has been accumulating, which involves either the loss or retention of the GPI anchor. Release of GPI-AP from donor cells may occur spontaneously or in response to endogenous or environmental signals. The experimental evidence for direct insertion of exogenous GPI-AP equipped with the complete anchor structure into the outer plasma membrane bilayer leaflets of acceptor cells is reviewed as well as the potential underlying molecular mechanisms. Furthermore, promiscuous transfer of certain GPI-AP between plasma membranes of different cells in vivo under certain (patho)physiological conditions has been reported. Engineering of target cell surfaces using chimeric GPI-AP with complete GPI anchor may be useful for therapeutic applications.
Assuntos
Engenharia Celular/métodos , Glicosilfosfatidilinositóis/metabolismo , Proteínas Ligadas a Lipídeos/uso terapêutico , Doenças Metabólicas/terapia , Neoplasias/terapia , Doenças Priônicas/terapia , Transtornos da Coagulação Sanguínea/terapia , Membrana Celular/química , Membrana Celular/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Glicosilfosfatidilinositóis/química , Hemoglobinúria Paroxística/terapia , Humanos , Imunoterapia/métodos , Proteínas Ligadas a Lipídeos/química , Proteínas Ligadas a Lipídeos/metabolismo , Transporte Proteico , Técnicas de Reprodução Assistida , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologiaRESUMO
The membrane skeletal complex, protein 4.1G-membrane palmitoylated protein 6 (MPP6), is localized in spermatogonia and early spermatocytes of mouse seminiferous tubules. In this study, we investigated the Lin7 family of scaffolding proteins, which interact with MPP6. By immunohistochemistry, Lin7a and Lin7c were localized in germ cells, and Lin7c had especially strong staining in spermatogonia and early spermatocytes, characterized by staging of seminiferous tubules. By immunoelectron microscopy, Lin7 localization appeared under cell membranes in germ cells. The Lin7 staining pattern in seminiferous tubules was partially similar to that of 4.1G, cell adhesion molecule 1 (CADM1), and melanoma cell adhesion molecule (MCAM). Lin7-positive cells included type A spermatogonia, as revealed by double staining for Lin28a. Lin7 staining became weaker in MPP6-deficient mice by immunohistochemistry and western blotting, indicating that MPP6 transports and maintains Lin7 in germ cells. The histology of seminiferous tubules was unchanged in MPP6-deficient mice compared to that of wild-type mice. In cultured spermatogonial stem cells maintained with glial cell line-derived neurotropic factor (GDNF), Lin7 was clearly expressed and immunolocalized along cell membranes, especially at cell-cell junctions. Thus, Lin7 protein is expressed in germ cells, and Lin7, particularly Lin7c, is a useful marker for early spermatogenesis.
Assuntos
Guanilato Quinases/análise , Proteínas Ligadas a Lipídeos/análise , Túbulos Seminíferos/química , Proteínas de Transporte Vesicular/análise , Animais , Células Cultivadas , Guanilato Quinases/deficiência , Guanilato Quinases/metabolismo , Proteínas Ligadas a Lipídeos/deficiência , Proteínas Ligadas a Lipídeos/metabolismo , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Túbulos Seminíferos/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
Intraflagellar transport (IFT), which is essential for the formation and function of cilia in most organisms, is the trafficking of IFT trains (i.e. assemblies of IFT particles) that carry cargo within the cilium. Defects in IFT cause several human diseases. IFT trains contain the complexes IFT-A and IFT-B. To dissect the functions of these complexes, we studied a Chlamydomonas mutant that is null for the IFT-A protein IFT140. The mutation had no effect on IFT-B but destabilized IFT-A, preventing flagella assembly. Therefore, IFT-A assembly requires IFT140. Truncated IFT140, which lacks the N-terminal WD repeats of the protein, partially rescued IFT and supported formation of half-length flagella that contained normal levels of IFT-B but greatly reduced amounts of IFT-A. The axonemes of these flagella had normal ultrastructure and, as investigated by SDS-PAGE, normal composition. However, composition of the flagellar 'membrane+matrix' was abnormal. Analysis of the latter fraction by mass spectrometry revealed decreases in small GTPases, lipid-anchored proteins and cell signaling proteins. Thus, IFT-A is specialized for the import of membrane-associated proteins. Abnormal levels of the latter are likely to account for the multiple phenotypes of patients with defects in IFT140.This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteínas de Algas/genética , Membrana Celular/metabolismo , Chlamydomonas reinhardtii/genética , Cílios/metabolismo , Flagelos/metabolismo , Proteínas Ligadas a Lipídeos/genética , Proteínas de Algas/química , Proteínas de Algas/metabolismo , Axonema/metabolismo , Axonema/ultraestrutura , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/ultraestrutura , Ataxia Cerebelar/genética , Ataxia Cerebelar/metabolismo , Ataxia Cerebelar/patologia , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/ultraestrutura , Cílios/ultraestrutura , Síndrome de Ellis-Van Creveld/genética , Síndrome de Ellis-Van Creveld/metabolismo , Síndrome de Ellis-Van Creveld/patologia , Flagelos/ultraestrutura , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Ligadas a Lipídeos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Organismos Geneticamente Modificados , Transporte Proteico , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Transdução de Sinais , Proteína Vermelha FluorescenteRESUMO
BACKGROUND: The intracellular bacterial pathogen Legionella pneumophila proliferates in human alveolar macrophages, resulting in a severe pneumonia termed Legionnaires' disease. Throughout the course of infection, L. pneumophila remains enclosed in a specialized membrane compartment that evades fusion with lysosomes. The pathogen delivers over 300 effector proteins into the host cell, altering host pathways in a manner that sets the stage for efficient pathogen replication. The L. pneumophila effector protein AnkX targets host Rab GTPases and functions in preventing fusion of the Legionella-containing vacuole with lysosomes. However, the current understanding of AnkX's interaction with host proteins and the means through which it exerts its cellular function is limited. RESULTS: Here, we investigated the protein interaction network of AnkX by using the nucleic acid programmable protein array (NAPPA), a high-density platform comprising 10,000 unique human ORFs. This approach facilitated the discovery of PLEKHN1 as a novel interaction partner of AnkX. We confirmed this interaction through multiple independent in vitro pull-down, co-immunoprecipitation, and cell-based assays. Structured illumination microscopy revealed that endogenous PLEKHN1 is found in the nucleus and on vesicular compartments, whereas ectopically produced AnkX co-localized with lipid rafts at the plasma membrane. In mammalian cells, HaloTag-AnkX co-localized with endogenous PLEKHN1 on vesicular compartments. A central fragment of AnkX (amino acids 491-809), containing eight ankyrin repeats, extensively co-localized with endogenous PLEKHN1, indicating that this region may harbor a new function. Further, we found that PLEKHN1 associated with multiple proteins involved in the inflammatory response. CONCLUSIONS: Altogether, our study provides evidence that in addition to Rab GTPases, the L. pneumophila effector AnkX targets nuclear host proteins and suggests that AnkX may have novel functions related to manipulating the inflammatory response.
Assuntos
Repetição de Anquirina/fisiologia , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Legionella pneumophila/metabolismo , Doença dos Legionários/metabolismo , Proteínas Ligadas a Lipídeos/metabolismo , Repetição de Anquirina/genética , Membrana Celular/metabolismo , Endocitose/fisiologia , Células HEK293 , Células HeLa , Humanos , Legionella pneumophila/patogenicidade , Lisossomos/metabolismo , Macrófagos/microbiologia , Proteínas Nucleares , Proteínas Recombinantes , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Bacillus subtilis responds to environmental stress cues and develops endospores for survival. In the process of endospore formation, sporulation initiation is a vital stage and this stage is governed by autophosphorylation of the sensor histidine kinases. The second major sensor kinase KinB perceives the intracellular changes of GTP and ATP during sporulation. However, determination of the environmental signals as well as its related signaling pathway of KinB requires further elucidation. Our current study found that, contrary to the sporulation failure induced by ΔkinA in the nutrient-rich 2× SG medium, the sensor kinase KinB sensed the environmental cues in the nutrient-poor MM medium. Two other membrane proteins, KapB and KbaA, also responded similarly to the same external signal as KinB. Both KapB and KbaA acted upstream of KinB, but they exerted their regulation upon KinB independently. Furthermore, we demonstrated that both the SH3 domain and the α-helix structure in KapB are required for sensing or transducing the signal of sporulation initiation. Collectively, our work here supplied the direct evidences that KinB and its pathway sense the external signal of nutrient starvation in MM medium, and further analyzes the interrelationship among KinB, KbaA, and KapB.
Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Metabolismo , Fosfotransferases/metabolismo , Transdução de Sinais , Esporos Bacterianos/enzimologia , Esporos Bacterianos/crescimento & desenvolvimento , Trifosfato de Adenosina/metabolismo , Meios de Cultura/química , Guanosina Trifosfato/metabolismo , Proteínas Ligadas a Lipídeos/metabolismo , Proteínas de Membrana/metabolismo , Estresse FisiológicoRESUMO
Cardiolipin and phosphatidic acid-binding protein (CLPABP) is a pleckstrin homology domain-containing protein and is localized on the surface of mitochondria of cultured cells as a large protein-RNA complex. To analyze the physiological functions of CLPABP, we established and characterized a CLPABP knockout (KO) mouse. Although expression levels of CLPABP transcripts in the developmental organs were high, CLPABP KO mice were normal at birth and grew normally when young. However, old male mice presented a fatty phenotype, similar to that seen in metabolic syndrome, in parallel with elevated male- and age-dependent CLPABP gene expression. One of the reasons for this obesity in CLPABP KO mice is dependence on increases in leptin concentration in plasma. The leptin transcripts were also upregulated in the adipose tissue of KO mice compared with wild-type (WT) mice. To understand the difference in levels of the transcriptional product, we focused on the effect of CLPABP on the stability of mRNA involving an AU-rich element (ARE) in its 3'UTR dependence on the RNA stabilizer, human antigen R (HuR), which is one of the CLPABP-binding proteins. Increase in stability of ARE-containing mRNAs of leptin by HuR was antagonized by the expression of CLPABP in cultured cells. Depletion of CLPABP disturbed the normal subcellular localization of HuR to stress granules, and overexpression of CLPABP induced instability of leptin mRNA by inhibiting HuR function. Consequently, leptin levels in old male mice might be regulated by CLPABP expression, which might lead to body weight control.
Assuntos
Elementos Ricos em Adenilato e Uridilato/genética , Envelhecimento/genética , Proteínas ELAV/metabolismo , Leptina/genética , Proteínas Ligadas a Lipídeos/metabolismo , Obesidade/genética , Estabilidade de RNA/genética , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Deleção de Genes , Regulação da Expressão Gênica , Leptina/metabolismo , Proteínas Ligadas a Lipídeos/genética , Masculino , Metaboloma , Camundongos Knockout , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , Proteína da Região Y Determinante do Sexo/genética , Proteína da Região Y Determinante do Sexo/metabolismo , Frações Subcelulares/metabolismo , Transcrição GênicaRESUMO
The regulation of oligodendrocyte development and myelin formation in the CNS is poorly defined. Multiple signals influence the rate and extent of CNS myelination, including the noncanonical cyclin-dependent kinase 5 (Cdk5) whose functions are regulated by its activators p35 and p39. Here we show that selective loss of either p35 or p39 perturbed specific aspects of oligodendrocyte development, whereas loss of both p35 and p39 completely inhibited the development of mature oligodendrocytes and myelination. In the absence of p35, oligodendrocyte differentiation was delayed, process outgrowth was truncated in vitro, and the patterning and extent of myelination were perturbed in the CNS of p35(-/-) mice. In the absence of p39, oligodendrocyte maturation was transiently affected both in vitro and in vivo. However, loss of both p35 and p39 in oligodendrocyte lineage cells completely inhibited oligodendrocyte progenitor cell differentiation and myelination both in vitro and after transplantation into shiverer slice cultures. Loss of p35 and p39 had a more profound effect on oligodendrocyte development than simply the loss of Cdk5 and could not be rescued by Cdk5 overexpression. These data suggest p35 and p39 have specific and overlapping roles in oligodendrocyte development, some of which may be independent of Cdk5 activation.
Assuntos
Diferenciação Celular/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas Ligadas a Lipídeos/metabolismo , Proteína Básica da Mielina/metabolismo , Oligodendroglia/fisiologia , Fosfotransferases/metabolismo , Animais , Células Cultivadas , Cerebelo/citologia , Proteínas do Citoesqueleto/genética , Ativadores de Enzimas , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Técnicas In Vitro , Proteínas Ligadas a Lipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Antígenos O/metabolismo , Proteína Oncogênica v-akt/metabolismo , Técnicas de Cultura de Órgãos , Fosfotransferases/genética , TransfecçãoRESUMO
UNLABELLED: By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals--electrostatic, hydrophobic, and lipid-specific-to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE: Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane-bounded protein lattice that recruits genomic RNA into the virus and forms the shell of a budding immature viral capsid. In binding studies of HIV-1 Gag MA to model membranes with well-controlled lipid composition, we dissect the multiple interactions of the MA domain with its target membrane. This results in a detailed understanding of the thermodynamic aspects that determine membrane association, preferential lipid recruitment to the viral shell, and those aspects of Gag assembly into the membrane-bound protein lattice that are determined by MA.
Assuntos
Membrana Celular/metabolismo , Antígenos HIV/metabolismo , HIV-1/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Membrana Celular/química , Colesterol/química , Colesterol/metabolismo , Humanos , Cinética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas Ligadas a Lipídeos/metabolismo , Lipídeos/química , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ligação Proteica , Produtos do Gene gag do Vírus da Imunodeficiência Humana/químicaRESUMO
SNARE proteins catalyze many forms of biological membrane fusion, including Ca(2+)-triggered exocytosis. Although fusion mediated by SNAREs generally involves proteins anchored to each fusing membrane by a transmembrane domain (TMD), the role of TMDs remains unclear, and previous studies diverge on whether SNAREs can drive fusion without a TMD. This issue is important because it relates to the question of the structure and composition of the initial fusion pore, as well as the question of whether SNAREs mediate fusion solely by creating close proximity between two membranes versus a more active role in transmitting force to the membrane to deform and reorganize lipid bilayer structure. To test the role of membrane attachment, we generated four variants of the synaptic v-SNARE synaptobrevin-2 (syb2) anchored to the membrane by lipid instead of protein. These constructs were tested for functional efficacy in three different systems as follows: Ca(2+)-triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca(2+)-synaptotagmin-enhanced SNARE-mediated liposome fusion. Lipid-anchoring motifs harboring one or two lipid acylation sites completely failed to support fusion in any of these assays. Only the lipid-anchoring motif from cysteine string protein-α, which harbors many lipid acylation sites, provided support for fusion but at levels well below that achieved with wild type syb2. Thus, lipid-anchored syb2 provides little or no support for exocytosis, and anchoring syb2 to a membrane by a TMD greatly improves its function. The low activity seen with syb2-cysteine string protein-α may reflect a slower alternative mode of SNARE-mediated membrane fusion.
Assuntos
Membrana Celular/metabolismo , Exocitose/fisiologia , Proteínas Ligadas a Lipídeos/metabolismo , Fusão de Membrana/fisiologia , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Motivos de Aminoácidos , Animais , Cálcio/metabolismo , Membrana Celular/genética , Proteínas Ligadas a Lipídeos/genética , Lipossomos , Camundongos , Camundongos Knockout , Proteína 2 Associada à Membrana da Vesícula/genéticaRESUMO
The membrane protein palmitoylated (MPP) family belongs to the membrane-associated guanylate kinase (MAGUK) family. MPP1 interacts with the protein 4.1 family member, 4.1R, as a membrane skeletal protein complex in erythrocytes. We previously described the interaction of another MPP family, MPP6, with 4.1G in the mouse peripheral nervous system. In the present study, the immunolocalization of MPP6 in the mouse small intestine was examined and compared with that of E-cadherin, zonula occludens (ZO)-1, and 4.1B, which we previously investigated in intestinal epithelial cells. The immunolocalization of MPP6 was also assessed in the small intestines of 4.1B-deficient (-/-) mice. In the small intestine, Western blotting revealed that the molecular weight of MPP6 was approximately 55-kDa, and MPP6 was immunostained under the cell membranes in the basolateral portions of almost all epithelial cells from the crypts to the villi. The immunostaining pattern of MPP6 in epithelial cells was similar to that of E-cadherin, but differed from that of ZO-1. In intestinal epithelial cells, the immunostained area of MPP6 was slightly different from that of 4.1B, which was restricted to the intestinal villi. The immunolocalization of MPP6 in small intestinal epithelial cells was similar between 4.1B(-/-) mice and 4.1B(+/+) mice. In the immunoprecipitation study, another MAGUK family protein, calcium/calmodulin-dependent serine protein kinase (CASK), was shown to molecularly interact with MPP6. Thus, we herein showed the immunolocalization and interaction proteins of MPP6 in the mouse small intestine, and also that 4.1B in epithelial cells was not essential for the sorting of MPP6.
Assuntos
Guanilato Quinases/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Proteínas Ligadas a Lipídeos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Caderinas/metabolismo , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Guanilato Quinases/genética , Mucosa Intestinal/citologia , Proteínas Ligadas a Lipídeos/genética , Proteínas de Membrana , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Pleckstrin homology (PH) domains are lipid-binding modules present in peripheral membrane proteins which interact with phosphatidyl-inositol phosphates (PIPs) in cell membranes. We use multiscale molecular dynamics simulations to characterize the localization and anomalous dynamics of the DAPP1 PH domain on the surface of a PIP-containing lipid bilayer. Both translational and rotational diffusion of the PH domain on the lipid membrane surface exhibit transient subdiffusion, with an exponent α ≈ 0.5 for times of less than 10 ns. In addition to a PIP3 molecule at the canonical binding site of the PH domain, we observe additional PIP molecules in contact with the protein. Fluctuations in the number of PIPs associated with the PH domain exhibit 1/f noise. We suggest that the anomalous diffusion and long-term correlated interaction of the PH domain with the membrane may contribute to an enhanced probability of encounter with target complexes on cell membrane surfaces.
Assuntos
Membrana Celular/metabolismo , Proteínas Ligadas a Lipídeos/química , Proteínas Ligadas a Lipídeos/metabolismo , Modelos Moleculares , Algoritmos , Bicamadas Lipídicas , Conformação Molecular , Simulação de Dinâmica Molecular , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Domínios e Motivos de Interação entre ProteínasRESUMO
The organization of proteins and lipids in the plasma membrane has been the subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored-mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase nor result in any enrichment of nanoscopic-ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane.
Assuntos
Membrana Celular/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Proteínas Ligadas a Lipídeos/metabolismo , Antígenos CD59/química , Antígenos CD59/metabolismo , Carcinoma/metabolismo , Linhagem Celular Tumoral , Glicosilfosfatidilinositóis/química , Proteínas de Fluorescência Verde , Humanos , Fragmentos Fab das Imunoglobulinas , Proteínas Ligadas a Lipídeos/química , Estrutura Terciária de ProteínaRESUMO
SecA is an essential multifunctional protein for the translocation of proteins across bacterial membranes. Though SecA is known to function in the membrane, the detailed mechanism for this process remains unclear. In this study we constructed a series of SecA N-terminal deletions and identified two specific domains crucial for initial SecA/membrane interactions. The first small helix, the linker and part of the second helix (Δ2-22) were found to be dispensable for SecA activity in complementing the growth of a SecA ts mutant. However, deletions of N-terminal aminoacyl residues 23-25 resulted in severe progressive retardation of growth. Moreover, a decrease of SecA activity caused by N-terminal deletions correlated to the loss of SecA membrane binding, formation of lipid-specific domains and channel activity. All together, the results indicate that the N-terminal aminoacyl residues 23-25 play a critical role for SecA binding to membranes and that the N-terminal limit of SecA for activity is at the 25th amino acid.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Genes Bacterianos , Teste de Complementação Genética , Canais Iônicos/química , Canais Iônicos/genética , Canais Iônicos/metabolismo , Proteínas Ligadas a Lipídeos/química , Proteínas Ligadas a Lipídeos/genética , Proteínas Ligadas a Lipídeos/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Membranas/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Canais de Translocação SEC , Proteínas SecA , Deleção de SequênciaRESUMO
The majority of ER-targeted tail-anchored (TA) proteins are inserted into membranes by the Guided Entry of Tail-anchored protein (GET) system. Disruption of this system causes a subset of TA proteins to mislocalize to mitochondria. We show that the AAA+ ATPase Msp1 limits the accumulation of mislocalized TA proteins on mitochondria. Deletion of MSP1 causes the Pex15 and Gos1 TA proteins to accumulate on mitochondria when the GET system is impaired. Likely as a result of failing to extract mislocalized TA proteins, yeast with combined mutation of the MSP1 gene and the GET system exhibit strong synergistic growth defects and severe mitochondrial damage, including loss of mitochondrial DNA and protein and aberrant mitochondrial morphology. Like yeast Msp1, human ATAD1 limits the mitochondrial mislocalization of PEX26 and GOS28, orthologs of Pex15 and Gos1, respectively. GOS28 protein level is also increased in ATAD1(-/-) mouse tissues. Therefore, we propose that yeast Msp1 and mammalian ATAD1 are conserved members of the mitochondrial protein quality control system that might promote the extraction and degradation of mislocalized TA proteins to maintain mitochondrial integrity.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Ligadas a Lipídeos/metabolismo , Mitocôndrias/fisiologia , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Animais , Células Hep G2 , Humanos , Immunoblotting , Imunoprecipitação , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Camundongos , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Consumo de Oxigênio/fisiologia , Fosfoproteínas/metabolismo , Plasmídeos/genética , Transporte Proteico , RNA Interferente Pequeno/genética , Proteínas SNARE/metabolismo , Saccharomyces cerevisiaeRESUMO
The secretins are a family of large multimeric channels in the outer membrane of Gram-negative bacteria that are involved in protein export. In Dickeya dadantii and many other pathogenic bacteria, the lipoprotein pilotin targets the secretin subunits to the outer membrane, allowing a functional type II secretion system to be assembled. Here, the crystal structure of the C-terminal peptide of the secretin subunit bound to its cognate pilotin is reported. In solution, this C-terminal region of the secretin is nonstructured. The secretin peptide folds on binding to the pilotin to form just under four turns of α-helix which bind tightly up against the first helix of the pilotin so that the hydrophobic residues of the secretin helix can bind to the hydrophobic surface of the pilotin. The secretin helix binds parallel to the first part of the fourth helix of the pilotin. An N-capping aspartate encourages helix formation and binding by interacting favourably with the helix dipole of the helical secretin peptide. The structure of the secretin-pilotin complex of the phytopathogenic D. dadantii described here is a paradigm for this interaction in the OutS-PulS family of pilotins, which is essential for the correct assembly of the type II secretion system of several potent human adversaries, including enterohaemorrhagic Escherichia coli and Klebsiella oxytoca.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Enterobacteriaceae/metabolismo , Proteínas Ligadas a Lipídeos/química , Proteínas Ligadas a Lipídeos/metabolismo , Secretina/química , Secretina/metabolismo , Sequência de Aminoácidos , Sistemas de Secreção Bacterianos/fisiologia , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Enterobacteriaceae/patogenicidade , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de ProteínaRESUMO
The conjugation of drug or molecular recognition motif to a hydrophobic fatty entity, for purpose of drug-membrane localization, has been a molecular strategy utilized for targeted inhibition of pathways involved in diseased cells. In general, membrane-anchored inhibitor structures have been composed of either a lipid or sterol group coupled via a broad range of inert linkers to either a peptide or small molecule protein recognition agent. Whilst not adhering to the molecular paradigms of modern medicinal chemistry, this approach has afforded peptidic-based therapeutics with improved cellular and in vivo efficacy, leading to more selective targeting of membrane associated protein targets and the effective immobilization of cytosolic signaling proteins through membrane anchorage. The evidence suggests that membrane-anchored peptidic inhibitors are more selective, potent, structurally rigid, and possess enhanced cell permeability profiles as compared to their non-lipidated precursors. This perspectives article will review the application of lipid or sterol conjugation to peptide inhibitors (lipo-molecules) to circumvent the poor cell permeability and metabolic labilities associated with peptidic therapeutics. In addition, the concept of protein-membrane anchorage as a novel drug modality for inhibiting cytosolic signaling protein motility in cells will be reviewed and its merits as an approach to inhibiting protein complexation, protein nuclear translocation and their potential for more effective targeting of membrane associated targets.
Assuntos
Lipídeos/química , Peptídeos/química , Endossomos/metabolismo , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Proteínas Ligadas a Lipídeos/antagonistas & inibidores , Proteínas Ligadas a Lipídeos/química , Proteínas Ligadas a Lipídeos/metabolismo , Fusão de Membrana/efeitos dos fármacos , Paramyxovirinae/efeitos dos fármacos , Paramyxovirinae/fisiologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Estabilidade Proteica , Transporte Proteico/efeitos dos fármacos , Proteínas/metabolismo , Esteróis/químicaRESUMO
Schmidt-Lanterman incisures (SLIs) are a specific feature of myelinated nerve fibers in the peripheral nervous system (PNS). In this study, we report localization of a signal transduction protein, Src, in the SLIs of mouse sciatic nerves, and its phosphorylation states in Y527 and Y418 (P527 and P418, respectively) under normal conditions or deletion of a membrane skeletal protein, 4.1G. In adult mouse sciatic nerves, Src was immunolocalized in SLIs as a cone-shape, as well as in paranodes and some areas of structures reminiscent of Cajal bands. By immunostaining in normal nerves, P527-Src was strongly detected in SLIs, whereas P418-Src was much weaker. Developmentally, P418-Src was detected in SLIs of early postnatal mouse sciatic nerves. The staining patterns for P527 and P418 in normal adult nerve fibers were opposite to those in primary culture Schwann cells and a Schwannoma cell line, RT4-D6P2T. In 4.1G-deficient nerve fibers, which had neither 4.1G nor the membrane protein palmitoylated 6 (MPP6) in SLIs, the P418-Src immunoreactivity in SLIs was clearly detected at a stronger level than that in the wild type. An immunoprecipitation study revealed Src interaction with MPP6. These findings indicate that the Src-MPP6-4.1G protein complex in SLIs has a role in signal transduction in the PNS.
Assuntos
Guanilato Quinases/metabolismo , Proteínas Ligadas a Lipídeos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fibras Nervosas Mielinizadas/metabolismo , Sistema Nervoso Periférico/citologia , Quinases da Família src/metabolismo , Animais , Guanilato Quinases/análise , Proteínas Ligadas a Lipídeos/análise , Proteínas de Membrana , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/deficiência , FosforilaçãoRESUMO
Lipid composition can differ widely among organelles and even between leaflets of a membrane. Lipid homeostasis is critical because disequilibrium can have disease outcomes. Despite their importance, mechanisms maintaining lipid homeostasis remain poorly understood. Here, we establish a model system to study the global effects of lipid imbalance. Quantitative lipid profiling was integral to monitor changes to lipid composition and for system validation. Applying global transcriptional and proteomic analyses, a dramatically altered biochemical landscape was revealed from adaptive cells. The resulting composite regulation we term the "membrane stress response" (MSR) confers compensation, not through restoration of lipid composition, but by remodeling the protein homeostasis network. To validate its physiological significance, we analyzed the unfolded protein response (UPR), one facet of the MSR and a key regulator of protein homeostasis. We demonstrate that the UPR maintains protein biogenesis, quality control, and membrane integrity-functions otherwise lethally compromised in lipid dysregulated cells.