RESUMO
Located at the inner leaflet of the plasma membrane (PM), phosphatidyl-inositol 4,5-bisphosphate [PI(4,5)P2] composes only 1-2 mol% of total PM lipids. With its synthesis and turnover both spatially and temporally regulated, PI(4,5)P2 recruits and interacts with hundreds of cellular proteins to support a broad spectrum of cellular functions. Several factors contribute to the versatile and dynamic distribution of PI(4,5)P2 in membranes. Physiological multivalent cations such as Ca2+ and Mg2+ can bridge between PI(4,5)P2 headgroups, forming nanoscopic PI(4,5)P2-cation clusters. The distinct lipid environment surrounding PI(4,5)P2 affects the degree of PI(4,5)P2 clustering. In addition, diverse cellular proteins interacting with PI(4,5)P2 can further regulate PI(4,5)P2 lateral distribution and accessibility. This review summarizes the current understanding of PI(4,5)P2 behavior in both cells and model membranes, with emphasis on both multivalent cation- and protein-induced PI(4,5)P2 clustering. Understanding the nature of spatially separated pools of PI(4,5)P2 is fundamental to cell biology.
Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Replicação Viral/fisiologia , Animais , Membrana Celular/metabolismo , Humanos , Micelas , Fosfatidilinositol 4,5-Difosfato/química , Proteínas Virais/metabolismoRESUMO
Phosphoinositides (PIs) are membrane phospholipids produced through the local activity of PI kinases and phosphatases that selectively add or remove phosphate groups from the inositol head group. PIs control membrane composition and play key roles in many cellular processes including actin dynamics, endosomal trafficking, autophagy, and nuclear functions. Mutations in phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] phosphatases cause a broad spectrum of neurodevelopmental disorders such as Lowe and Joubert syndromes and congenital muscular dystrophy with cataracts and intellectual disability, which are thus associated with increased levels of PI(4,5)P2. Here, we describe a neurodevelopmental disorder associated with an increase in the production of PI(4,5)P2 and with PI-signaling dysfunction. We identified three de novo heterozygous missense variants in PIP5K1C, which encodes an isoform of the phosphatidylinositol 4-phosphate 5-kinase (PIP5KIγ), in nine unrelated children exhibiting intellectual disability, developmental delay, acquired microcephaly, seizures, visual abnormalities, and dysmorphic features. We provide evidence that the PIP5K1C variants result in an increase of the endosomal PI(4,5)P2 pool, giving rise to ectopic recruitment of filamentous actin at early endosomes (EEs) that in turn causes dysfunction in EE trafficking. In addition, we generated an in vivo zebrafish model that recapitulates the disorder we describe with developmental defects affecting the forebrain, including the eyes, as well as craniofacial abnormalities, further demonstrating the pathogenic effect of the PIP5K1C variants.
Assuntos
Deficiência Intelectual , Fosfatidilinositóis , Animais , Síndrome , Actinas , Peixe-Zebra/genética , Deficiência Intelectual/genética , Monoéster Fosfórico Hidrolases/genética , Fosfatos de FosfatidilinositolRESUMO
Ebola virus (EBOV) is a filamentous negative-sense RNA virus, which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work, we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane-binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine and phosphatidylinositol 4,5-bisphosphate in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles. In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together, our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.
Assuntos
Membrana Celular , Ebolavirus , Montagem de Vírus , Liberação de Vírus , Humanos , Substituição de Aminoácidos , Membrana Celular/metabolismo , Ebolavirus/metabolismo , Ebolavirus/genética , Células HEK293 , Doença pelo Vírus Ebola/metabolismo , Doença pelo Vírus Ebola/virologia , Mutação , Nucleoproteínas , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilserinas/metabolismo , Fosfatidilserinas/química , Ligação Proteica , Eletricidade Estática , Proteínas do Core Viral/metabolismo , Proteínas do Core Viral/química , Proteínas do Core Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/química , Vírion/metabolismo , Vírion/genéticaRESUMO
Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a key enzyme producing the signaling lipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 ] in eukaryotes. Although PIP5K genes are reported to be involved in pollen tube germination and growth, the essential roles of PIP5K in these processes remain unclear. Here, we performed a comprehensive genetic analysis of the Arabidopsis thaliana PIP5K4, PIP5K5, and PIP5K6 genes and revealed that their redundant function is essential for pollen germination. Pollen with the pip5k4pip5k5pip5k6 triple mutation was sterile, while pollen germination efficiency and pollen tube growth were reduced in the pip5k6 single mutant and further reduced in the pip5k4pip5k6 and pip5k5pip5k6 double mutants. YFP-fusion proteins, PIP5K4-YFP, PIP5K5-YFP, and PIP5K6-YFP, which could rescue the sterility of the triple mutant pollen, preferentially localized to the tricolpate aperture area and the future germination site on the plasma membrane prior to germination. Triple mutant pollen grains under the germination condition, in which spatiotemporal localization of the PtdIns(4,5)P2 fluorescent marker protein 2xmCHERRY-2xPHPLC as seen in the wild type was abolished, exhibited swelling and rupture of the pollen wall, but neither the conspicuous protruding site nor site-specific deposition of cell wall materials for germination. These data indicate that PIP5K4-6 and their product PtdIns(4,5)P2 are essential for pollen germination, possibly through the establishment of the germination polarity in a pollen grain.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Germinação/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Tubo Polínico/metabolismo , PólenRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is one of the deadliest and most aggressive hematological malignancies, but its pathological mechanism in controlling cell survival is not fully understood. Oculocerebrorenal syndrome of Lowe is a rare X-linked recessive disorder characterized by cataracts, intellectual disability, and proteinuria. This disease has been shown to be caused by mutation of oculocerebrorenal syndrome of Lowe 1 (OCRL1; OCRL), encoding a phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] 5-phosphatase involved in regulating membrane trafficking; however, its function in cancer cells is unclear. Here, we uncovered that OCRL1 is overexpressed in T-ALL cells, and knockdown of OCRL1 results in cell death, indicating the essential role of OCRL in controlling T-ALL cell survival. We show OCRL is primarily localized in the Golgi and can translocate to plasma membrane (PM) upon ligand stimulation. We found OCRL interacts with oxysterol-binding protein-related protein 4L, which facilitates OCRL translocation from the Golgi to the PM upon cluster of differentiation 3 stimulation. Thus, OCRL represses the activity of oxysterol-binding protein-related protein 4L to prevent excessive PI(4,5)P2 hydrolysis by phosphoinositide phospholipase C ß3 and uncontrolled Ca2+ release from the endoplasmic reticulum. We propose OCRL1 deletion leads to accumulation of PI(4,5)P2 in the PM, disrupting the normal Ca2+ oscillation pattern in the cytosol and leading to mitochondrial Ca2+ overloading, ultimately causing T-ALL cell mitochondrial dysfunction and cell death. These results highlight a critical role for OCRL in maintaining moderate PI(4,5)P2 availability in T-ALL cells. Our findings also raise the possibility of targeting OCRL1 to treat T-ALL disease.
Assuntos
Membrana Celular , Fosfatidilinositol 4,5-Difosfato , Monoéster Fosfórico Hidrolases , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Linfócitos T , Humanos , Membrana Celular/metabolismo , Sobrevivência Celular , Hidrólise , Síndrome Oculocerebrorrenal/enzimologia , Síndrome Oculocerebrorrenal/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Linfócitos T/citologia , Linfócitos T/imunologia , Monoéster Fosfórico Hidrolases/biossíntese , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Complexo de Golgi/metabolismo , Ligantes , Transporte Proteico , Sinalização do Cálcio , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Citosol/metabolismoRESUMO
Focal adhesion kinase (FAK) is a key component of the membrane proximal signaling layer in focal adhesion complexes, regulating important cellular processes, including cell migration, proliferation, and survival. In the cytosol, FAK adopts an autoinhibited state but is activated upon recruitment into focal adhesions, yet how this occurs or what induces structural changes is unknown. Here, we employ cryo-electron microscopy to reveal how FAK associates with lipid membranes and how membrane interactions unlock FAK autoinhibition to promote activation. Intriguingly, initial binding of FAK to the membrane causes steric clashes that release the kinase domain from autoinhibition, allowing it to undergo a large conformational change and interact itself with the membrane in an orientation that places the active site toward the membrane. In this conformation, the autophosphorylation site is exposed and multiple interfaces align to promote FAK oligomerization on the membrane. We show that interfaces responsible for initial dimerization and membrane attachment are essential for FAK autophosphorylation and resulting cellular activity including cancer cell invasion, while stable FAK oligomerization appears to be needed for optimal cancer cell proliferation in an anchorage-independent manner. Together, our data provide structural details of a key membrane bound state of FAK that is primed for efficient autophosphorylation and activation, hence revealing the critical event in integrin mediated FAK activation and signaling at focal adhesions.
Assuntos
Proteínas Aviárias/química , Proteína-Tirosina Quinases de Adesão Focal/química , Membranas/química , Multimerização Proteica , Animais , Proteínas Aviárias/metabolismo , Galinhas , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Células HEK293 , Humanos , Membranas/enzimologia , Relação Estrutura-AtividadeRESUMO
The inward rectifier potassium channel Kir7.1, encoded by the KCNJ13 gene, is a tetramer composed of two-transmembrane domain-spanning monomers, closer in homology to Kir channels associated with potassium transport such as Kir1.1, 1.2, and 1.3. Compared with other channels, Kir7.1 exhibits small unitary conductance and low dependence on external potassium. Kir7.1 channels also show a phosphatidylinositol 4,5-bisphosphate (PIP2) dependence for opening. Accordingly, retinopathy-associated Kir7.1 mutations mapped at the binding site for PIP2 resulted in channel gating defects leading to channelopathies such as snowflake vitreoretinal degeneration and Leber congenital amaurosis in blind patients. Lately, this channel's role in energy homeostasis was reported due to the direct interaction with the melanocortin type 4 receptor (MC4R) in the hypothalamus. As this channel seems to play a multipronged role in potassium homeostasis and neuronal excitability, we will discuss what is predicted from a structural viewpoint and its possible implications for hunger control.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização , Humanos , Mutação , Neurônios/metabolismo , Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Domínios ProteicosRESUMO
Ion channels are integral membrane proteins whose gating has been increasingly shown to depend on the presence of the low-abundance membrane phospholipid, phosphatidylinositol (4,5) bisphosphate. The expression and function of ion channels is tightly regulated via protein phosphorylation by specific kinases, including various PKC isoforms. Several channels have further been shown to be regulated by PKC through altered surface expression, probability of channel opening, shifts in voltage dependence of their activation, or changes in inactivation or desensitization. In this review, we survey the impact of phosphorylation of various ion channels by PKC isoforms and examine the dependence of phosphorylated ion channels on phosphatidylinositol (4,5) bisphosphate as a mechanistic endpoint to control channel gating.
Assuntos
Canais Iônicos , Fosfatidilinositol 4,5-Difosfato , Proteína Quinase C , Canais Iônicos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosforilação , Proteína Quinase C/metabolismoRESUMO
Besides its protective role in the maintenance of cell homeostasis, the plasma membrane is the site of exchanges between the cell interior and the extracellular medium. To circumvent the hydrophobic barrier formed by the acyl chains of the lipid bilayer, protein channels and transporters are key players in the exchange of small hydrophilic compounds such as ions or nutrients, but they hardly account for the transport of larger biological molecules. Exchange of proteins usually relies on membrane-fusion events between vesicles and the plasma membrane. In recent years, several alternative unconventional protein secretion (UPS) pathways across the plasma membrane have been characterised for a specific set of secreted substrates, some of them excluding any membrane-fusion events (Dimou and Nickel, Curr Biol 28:R406-R410, 2018). One of thesbe pathways, referred as type I UPS, relies on the direct translocation of the protein across the plasma membrane and not surprisingly, lipids are essential players in this process. In this chapter, we discuss the roles of phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and cholesterol in unconventional pathways involving Engrailed-2 homeoprotein and fibroblast growth factor 2.
Assuntos
Bicamadas Lipídicas , Proteínas de Membrana Transportadoras , Bicamadas Lipídicas/metabolismo , Membrana Celular/metabolismo , Transporte Proteico , Proteínas de Membrana Transportadoras/metabolismo , Colesterol/metabolismo , Fosfatidilinositol 4,5-Difosfato/análise , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismoRESUMO
Cholesterol (Chol) is an essential component of all eukaryotic cell membranes that affects the function of numerous peripheral as well as integral membrane proteins. Chol is synthesized in the ER, but it is selectively enriched within the plasma membrane (PM) and other endomembranes, which requires Chol to cross the aqueous phase of the cytoplasm. In addition to the classical vesicular trafficking pathways that are known to facilitate the bulk transport of membrane intermediates, Chol is also transported via non-vesicular lipid transfer proteins that work primarily within specialized membrane contact sites. Some of these transport pathways work against established concentration gradients and hence require energy. Recent studies highlight the unique role of phosphoinositides (PPIns), and phosphatidylinositol 4-phosphate (PI4P) in particular, for the control of non-vesicular Chol transport. In this chapter, we will review the emerging connection between Chol, PPIns, and lipid transfer proteins that include the important family of oxysterol-binding protein related proteins, or ORPs.
Assuntos
Colesterol , Fosfatos de Fosfatidilinositol , Fosforilação , Colesterol/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Transporte Biológico , Proteínas de Membrana/metabolismo , Membrana Celular/metabolismoRESUMO
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is the most abundant membrane phosphoinositide and cholesterol is an essential component of the plasma membrane (PM). Both lipids play key roles in a variety of cellular functions including as signaling molecules and major regulators of protein function. This chapter provides an overview of these two important lipids. Starting from a brief description of their structure, synthesis, and regulation, the chapter continues to describe the primary functions and signaling processes in which PI(4,5)P2 and cholesterol are involved. While PI(4,5)P2 and cholesterol can act independently, they often act in concert or affect each other's impact. The chapters in this volume on "Cholesterol and PI(4,5)P2 in Vital Biological Functions: From Coexistence to Crosstalk" focus on the emerging relationship between cholesterol and PI(4,5)P2 in a variety of biological systems and processes. In this chapter, the next section provides examples from the ion channel field demonstrating that PI(4,5)P2 and cholesterol can act via common mechanisms. The chapter ends with a discussion of future directions.
Assuntos
Fosfatidilinositol 4,5-Difosfato , Fosfatidilinositóis , Fosfatidilinositol 4,5-Difosfato/metabolismo , Membrana Celular/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas/metabolismo , Colesterol/metabolismoRESUMO
Plakophilin-3 is a ubiquitously expressed protein found widely in epithelial cells and is a critical component of desmosomes. The plakophilin-3 carboxy-terminal domain harbors nine armadillo repeat motifs with largely unknown functions. Here, we report the 5 Å cryogenic electron microscopy (cryoEM) structure of the armadillo repeat motif domain of plakophilin-3, one of the smaller cryoEM structures reported to date. We find that this domain is a monomer or homodimer in solution. In addition, using an in vitro actin co-sedimentation assay, we show that the armadillo repeat domain of plakophilin-3 directly interacts with F-actin. This feature, through direct interactions with actin filaments, could be responsible for the observed association of extra-desmosomal plakophilin-3 with the actin cytoskeleton directly attached to the adherens junctions in A431 epithelial cells. Further, we demonstrate, through lipid binding analyses, that plakophilin-3 can effectively be recruited to the plasma membrane through phosphatidylinositol-4,5-bisphosphate-mediated interactions. Collectively, we report on novel properties of plakophilin-3, which may be conserved throughout the plakophilin protein family and may be behind the roles of these proteins in cell-cell adhesion.
Assuntos
Actinas , Placofilinas , Citoesqueleto de Actina , Actinas/metabolismo , Desmossomos/metabolismo , Placofilinas/metabolismoRESUMO
Functional hyperemia-activity-dependent increases in local blood perfusion-underlies the on-demand delivery of blood to regions of enhanced neuronal activity, a process that is crucial for brain health. Importantly, functional hyperemia deficits have been linked to multiple dementia risk factors, including aging, chronic hypertension, and cerebral small vessel disease (cSVD). We previously reported crippled functional hyperemia in a mouse model of genetic cSVD that was likely caused by depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) in capillary endothelial cells (EC) downstream of impaired epidermal growth factor receptor (EGFR) signaling. Here, using EC-specific EGFR-knockout (KO) mice, we directly examined the role of endothelial EGFR signaling in functional hyperemia, assessed by measuring increases in cerebral blood flow in response to contralateral whisker stimulation using laser Doppler flowmetry. Molecular characterizations showed that EGFR expression was dramatically decreased in freshly isolated capillaries from EC-EGFR-KO mice, as expected. Notably, whisker stimulation-induced functional hyperemia was significantly impaired in these mice, an effect that was rescued by administration of PIP2, but not by the EGFR ligand, HB-EGF. These data suggest that the deletion of the EGFR specifically in ECs attenuates functional hyperemia, likely via depleting PIP2 and subsequently incapacitating Kir2.1 channel functionality in capillary ECs. Thus, our study underscores the role of endothelial EGFR signaling in functional hyperemia of the brain.
Assuntos
Células Endoteliais , Hiperemia , Camundongos , Animais , Células Endoteliais/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Encéfalo/metabolismo , Família de Proteínas EGF/metabolismo , Família de Proteínas EGF/farmacologia , Fator de Crescimento Epidérmico/metabolismoRESUMO
ATP-sensitive potassium (KATP ) channels couple the intracellular ATP concentration to insulin secretion. KATP channel activity is inhibited by ATP binding to the Kir6.2 tetramer and activated by phosphatidylinositol 4,5-bisphosphate (PIP2 ). Here, we use molecular dynamics simulation, electrophysiology and fluorescence spectroscopy to show that ATP and PIP2 occupy different binding pockets that share a single amino acid residue, K39. When both ligands are present, simulations suggest that K39 shows a greater preference to co-ordinate with PIP2 than with ATP. They also predict that a neonatal diabetes mutation at K39 (K39R) increases the number of hydrogen bonds formed between K39 and PIP2 , potentially accounting for the reduced ATP inhibition observed in electrophysiological experiments. Our work suggests that PIP2 and ATP interact allosterically to regulate KATP channel activity. KEY POINTS: The KATP channel is activated by the binding of phosphatidylinositol 4,5-bisphosphate (PIP2 ) lipids and inactivated by the binding of ATP. K39 has the potential to bind to both PIP2 and ATP. A mutation to this residue (K39R) results in neonatal diabetes. This study uses patch-clamp fluorometry, electrophysiology and molecular dynamics simulation. We show that PIP2 competes with ATP for K39, and this reduces channel inhibition by ATP. We show that K39R increases channel affinity to PIP2 by increasing the number of hydrogen bonds with PIP2 , when compared with the wild-type K39. This therefore decreases KATP channel inhibition by ATP.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização , Trifosfato de Adenosina/metabolismo , Aminoácidos , Humanos , Recém-Nascido , Fosfatidilinositol 4,5-Difosfato/fisiologia , Fosfatidilinositóis , Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/fisiologiaRESUMO
Marburg virus (MARV) is a lipid-enveloped virus harboring a negative-sense RNA genome, which has caused sporadic outbreaks of viral hemorrhagic fever in sub-Saharan Africa. MARV assembles and buds from the host cell plasma membrane where MARV matrix protein (mVP40) dimers associate with anionic lipids at the plasma membrane inner leaflet and undergo a dynamic and extensive self-oligomerization into the structural matrix layer. The MARV matrix layer confers the virion filamentous shape and stability but how host lipids modulate mVP40 oligomerization is mostly unknown. Using in vitro and cellular techniques, we present a mVP40 assembly model highlighting two distinct oligomerization interfaces: the (N-terminal domain [NTD] and C-terminal domain [CTD]) in mVP40. Cellular studies of NTD and CTD oligomerization interface mutants demonstrate the importance of each interface in matrix assembly. The assembly steps include protein trafficking to the plasma membrane, homo-multimerization that induced protein enrichment, plasma membrane fluidity changes, and elongations at the plasma membrane. An ascorbate peroxidase derivative (APEX)-transmission electron microscopy method was employed to closely assess the ultrastructural localization and formation of viral particles for wildtype mVP40 and NTD and CTD oligomerization interface mutants. Taken together, these studies present a mechanistic model of mVP40 oligomerization and assembly at the plasma membrane during virion assembly that requires interactions with phosphatidylserine for NTD-NTD interactions and phosphatidylinositol-4,5-bisphosphate for proper CTD-CTD interactions. These findings have broader implications in understanding budding of lipid-enveloped viruses from the host cell plasma membrane and potential strategies to target protein-protein or lipid-protein interactions to inhibit virus budding.
Assuntos
Doença do Vírus de Marburg/virologia , Marburgvirus/fisiologia , Lipídeos de Membrana/metabolismo , Proteínas da Matriz Viral/metabolismo , Vírion/metabolismo , Animais , Células COS , Membrana Celular/química , Membrana Celular/metabolismo , Chlorocebus aethiops , Células HEK293 , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Doença do Vírus de Marburg/metabolismo , Marburgvirus/química , Lipídeos de Membrana/química , Modelos Moleculares , Multimerização Proteica , Proteínas da Matriz Viral/química , Vírion/química , Montagem de VírusRESUMO
Transient receptor potential canonical type 5 (TRPC5) ion channels are expressed in the brain and kidney and have been identified as promising therapeutic targets whose selective inhibition can protect against diseases driven by a leaky kidney filter, such as focal segmental glomerular sclerosis. TRPC5 channels are activated not only by elevated levels of extracellular Ca2+or lanthanide ions but also by G protein (Gq/11) stimulation. Phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by phospholipase C enzymes leads to PKC-mediated phosphorylation of TRPC5 channels and their subsequent desensitization. However, the roles of PIP2 in activation and maintenance of TRPC5 channel activity via its hydrolysis product diacyl glycerol (DAG), as well as the mechanism of desensitization of TRPC5 activity by DAG-stimulated PKC activity, remain unclear. Here, we designed experiments to distinguish between the processes underlying channel activation and inhibition. Employing whole-cell patch-clamp, we used an optogenetic tool to dephosphorylate PIP2 and assess channel-PIP2 interactions influenced by activators, such as DAG, or inhibitors, such as PKC phosphorylation. Using total internal reflection microscopy, we assessed channel cell surface density. We show that PIP2 controls both the PKC-mediated inhibition and the DAG- and lanthanide-mediated activation of TRPC5 currents via control of gating rather than channel cell surface density. These mechanistic insights promise to aid in the development of more selective and precise inhibitors to block TRPC5 channel activity and illuminate new opportunities for targeted therapies for a group of chronic kidney diseases for which there is currently a great unmet need.
Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Cátion TRPC/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
During the late phase of HIV-1 infection, viral Gag polyproteins are targeted to the plasma membrane (PM) for assembly. Gag localization at the PM is a prerequisite for the incorporation of the envelope protein (Env) into budding particles. Gag assembly and Env incorporation are mediated by the N-terminal myristoylated matrix (MA) domain of Gag. Nonconservative mutations in the trimer interface of MA (A45E, T70R, and L75G) were found to impair Env incorporation and infectivity, leading to the hypothesis that MA trimerization is an obligatory step for Env incorporation. Conversely, Env incorporation can be rescued by a compensatory mutation in the MA trimer interface (Q63R). The impact of these MA mutations on the structure and trimerization properties of MA is not known. In this study, we employed NMR spectroscopy, X-ray crystallography, and sedimentation techniques to characterize the structure and trimerization properties of HIV-1 MA A45E, Q63R, T70R, and L75G mutant proteins. NMR data revealed that these point mutations did not alter the overall structure and folding of MA but caused minor structural perturbations in the trimer interface. Analytical ultracentrifugation data indicated that mutations had a minimal effect on the MA monomer-trimer equilibrium. The high-resolution X-ray structure of the unmyristoylated MA Q63R protein revealed hydrogen bonding between the side chains of adjacent Arg-63 and Ser-67 on neighboring MA molecules, providing the first structural evidence for an additional intermolecular interaction in the trimer interface. These findings advance our knowledge of the interplay of MA trimerization and Env incorporation into HIV-1 particles.
Assuntos
Produtos do Gene gag/genética , Infecções por HIV/genética , HIV-1/genética , Proteínas da Matriz Viral/genética , Membrana Celular/genética , Membrana Celular/ultraestrutura , Membrana Celular/virologia , Produtos do Gene gag/ultraestrutura , Infecções por HIV/virologia , HIV-1/patogenicidade , Humanos , Mutação/genética , Ligação Proteica/genética , Multimerização Proteica/genética , Proteínas da Matriz Viral/ultraestrutura , Vírion/genética , Vírion/ultraestrutura , Montagem de Vírus/genética , Replicação Viral/genéticaRESUMO
Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) produces phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2 ), a signaling phospholipid critical for various cellular processes in eukaryotes. The Arabidopsis thaliana genome encodes 11 PIP5K genes. Of these, three type B PIP5K genes, PIP5K7, PIP5K8, and PIP5K9, constitute a subgroup highly conserved in land plants, suggesting that they retain a critical function shared by land plants. In this study, we comprehensively investigated the biological functions of the PIP5K7-9 subgroup genes. Reporter gene analyses revealed their preferential expression in meristematic and vascular tissues. Their YFP-fusion proteins localized primarily to the plasma membrane in root meristem epidermal cells. We selected a mutant line that was considered to be null for each gene. Under normal growth conditions, neither single mutants nor multiple mutants of any combination exhibited noticeable phenotypic changes. However, stress conditions with mannitol or NaCl suppressed main root growth and reduced proximal root meristem size to a greater extent in the pip5k7pip5k8pip5k9 triple mutant than in the wild type. In root meristem epidermal cells of the triple mutant, where plasma membrane localization of the PtdIns(4,5)P2 marker P24Y is impaired to a large extent, brefeldin A body formation is retarded compared with the wild type under hyperosmotic stress. These results indicate that PIP5K7, PIP5K8, and PIP5K9 are not required under normal growth conditions, but are redundantly involved in root growth adaptation to hyperosmotic conditions, possibly through the PtdIns(4,5)P2 function promoting plasma membrane recycling in root meristem cells.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais , Adaptação Fisiológica , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Membrana Celular/enzimologia , Genes Reporter , Mutação , Pressão Osmótica , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plântula/enzimologia , Plântula/genética , Plântula/fisiologia , Plântula/ultraestruturaRESUMO
Staphylococcus aureus, a Gram-positive pathogen, invades cells mainly in an integrin-dependent manner. As the activity or conformation of several integrin-associated proteins can be regulated by phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2 ), we investigated the roles of PI-4,5-P2 and PI-4,5-P2 -producing enzymes in cellular invasion by S. aureus. PI-4,5-P2 accumulated upon contact of S. aureus with the host cell, and targeting of an active PI-4,5-P2 phosphatase to the plasma membrane reduced bacterial invasion. Knockdown of individual phosphatidylinositol-4-phosphate 5-kinases revealed that phosphatidylinositol-4-phosphate 5-kinase γ (PIP5KIγ) plays an important role in bacterial internalization. Specific ablation of the talin and FAK-binding motif in PIP5KIγ90 reduced bacterial invasion, which could be rescued by reexpression of an active, but not inactive PIP5KIγ90. Furthermore, PIP5KIγ90-deficient cells showed normal basal PI-4,5-P2 levels in the plasma membrane but reduced the accumulation of PI-4,5-P2 and talin at sites of S. aureus attachment and overall lower levels of FAK phosphorylation. These results highlight the importance of local synthesis of PI-4,5-P2 by a focal adhesion-associated lipid kinase for integrin-mediated internalization of S. aureus.
Assuntos
Aderência Bacteriana , Interações Hospedeiro-Patógeno , Integrinas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Células HEK293 , Humanos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ligação Proteica , Transdução de SinaisRESUMO
The clathrin coat assembly protein AP180 drives endocytosis, which is crucial for numerous physiological events, such as the internalization and recycling of receptors, uptake of neurotransmitters and entry of viruses, including SARS-CoV-2, by interacting with clathrin. Moreover, dysfunction of AP180 underlies the pathogenesis of Alzheimer's disease. Therefore, it is important to understand the mechanisms of assembly and, especially, disassembly of AP180/clathrin-containing cages. Here, we identified AP180 as a novel phosphatidic acid (PA)-binding protein from the mouse brain. Intriguingly, liposome binding assays using various phospholipids and PA species revealed that AP180 most strongly bound to 1-stearoyl-2-docosahexaenoyl-PA (18:0/22:6-PA) to a comparable extent as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which is known to associate with AP180. An AP180 N-terminal homology domain (1-289 aa) interacted with 18:0/22:6-PA, and a lysine-rich motif (K38-K39-K40) was essential for binding. The 18:0/22:6-PA in liposomes in 100 nm diameter showed strong AP180-binding activity at neutral pH. Notably, 18:0/22:6-PA significantly attenuated the interaction of AP180 with clathrin. However, PI(4,5)P2 did not show such an effect. Taken together, these results indicate the novel mechanism by which 18:0/22:6-PA selectively regulates the disassembly of AP180/clathrin-containing cages.