phosphatidylinositol 4-phosphate

磷脂酰肌醇 4 - 磷酸酯
  • 文章类型: Journal Article
    在巨自噬期间,细胞质成分被自噬体吞噬。溶酶体与封闭的自噬体融合,但不与未封闭的中间结构融合。这在一定程度上是通过将自噬体SNARE突触蛋白17(STX17)晚期募集到成熟的自噬体来实现的。然而,STX17如何识别自噬体成熟尚不清楚。这里,我们表明,STX17的这种时间调节的募集取决于STX17的带正电荷的C末端区域。与这一发现一致,成熟的自噬体与未封闭的中间结构相比带负电。自噬体的静电成熟可能是由自噬体膜中磷脂酰肌醇4-磷酸(PI4P)的积累驱动的。因此,自噬体PI4P的去磷酸化阻止了STX17与自噬体的关联。此外,分子动力学模拟支持STX17跨膜螺旋的PI4P依赖性膜插入。基于这些发现,我们提出了一个模型,在该模型中,成熟自噬体的STX17募集受到PI4P驱动的自噬体表面电荷变化的时间调控.
    During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.
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  • 文章类型: Journal Article
    自噬体膜电荷的变化控制SNARE蛋白的募集,以确保膜融合在自噬过程中的正确时间发生。
    A change in the electric charge of autophagosome membranes controls the recruitment of SNARE proteins to ensure that membrane fusion occurs at the right time during autophagy.
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  • 文章类型: Journal Article
    氧化固醇结合蛋白相关蛋白(ORP)通过在内质网(ER)和其他细胞区域之间将甾醇或磷脂酰丝氨酸交换为PI4P,在真核细胞中的脂质分布中起关键作用。然而,目前尚不清楚它们的交换能力如何与PI4P代谢相关.为了定量地解决这个问题,我们分析代表性ORP的活性,Osh4p,在用PI4P磷酸酶Sac1p和磷脂酰肌醇(PI)4-激酶功能化的ER和高尔基体模拟膜重建的ER/高尔基体界面中,分别。使用实时检测,我们证明,在三磷酸腺苷(ATP)添加后,Osh4p在这些膜之间产生甾醇梯度,依靠PI4P的空间距离的合成和水解,并量化此过程需要多少PI4P。然后,我们开发了一个定量精确的动力学模型,通过我们的数据验证,并对此进行推断以估计PI4P代谢在多大程度上可以驱动ORP介导的固醇在细胞中的转移。最后,我们表明Sec14p可以通过在膜之间转移PI来支持PI4P代谢和Osh4p活性。这项研究确定PI4P合成驱动ORP介导的脂质交换,并且需要ATP能量来产生膜间脂质梯度。此外,它定义了ORP可以在细胞中分布脂质的程度,并重新评估了PI转移蛋白在PI4P代谢中的作用。
    Oxysterol-binding protein-related proteins (ORPs) play key roles in the distribution of lipids in eukaryotic cells by exchanging sterol or phosphatidylserine for PI4P between the endoplasmic reticulum (ER) and other cell regions. However, it is unclear how their exchange capacity is coupled to PI4P metabolism. To address this question quantitatively, we analyze the activity of a representative ORP, Osh4p, in an ER/Golgi interface reconstituted with ER- and Golgi-mimetic membranes functionalized with PI4P phosphatase Sac1p and phosphatidylinositol (PI) 4-kinase, respectively. Using real-time assays, we demonstrate that upon adenosine triphosphate (ATP) addition, Osh4p creates a sterol gradient between these membranes, relying on the spatially distant synthesis and hydrolysis of PI4P, and quantify how much PI4P is needed for this process. Then, we develop a quantitatively accurate kinetic model, validated by our data, and extrapolate this to estimate to what extent PI4P metabolism can drive ORP-mediated sterol transfer in cells. Finally, we show that Sec14p can support PI4P metabolism and Osh4p activity by transferring PI between membranes. This study establishes that PI4P synthesis drives ORP-mediated lipid exchange and that ATP energy is needed to generate intermembrane lipid gradients. Furthermore, it defines to what extent ORPs can distribute lipids in the cell and reassesses the role of PI-transfer proteins in PI4P metabolism.
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  • 文章类型: Preprint
    KRAS是一种小的GTPase,在哺乳动物细胞中普遍表达,作为调节细胞增殖和分化的分子开关。使KRAS具有组成活性的致癌突变在人类癌症中频繁发生。KRAS必须定位于质膜(PM)以获得生物活性。KRASPM结合由KRAS膜锚与磷脂酰丝氨酸(PtdSer)的相互作用介导,因此,消耗PMPtdSer含量消除了KRASPM结合和致癌功能。从全基因组siRNA筛选中寻找调节KRASPM定位的基因,我们确定了一组磷脂酰肌醇(PI)3-磷酸酶家族成员:肌管蛋白相关(MTMR)蛋白2,3,4和7。在这里,我们显示了MTMR2/3/4/7表达的敲低会破坏KRASPM相互作用。分子机制涉及PMPI4-磷酸(PI4P)水平的消耗,反过来又破坏了氧固醇结合蛋白相关蛋白(ORP)5的亚细胞定位和操作,该蛋白是PtdSer脂质转移蛋白,可维持PMPtdSer含量。同时,沉默MTMR2/3/4/7表达会提高PI3P的PM水平,并降低PM和PtdSer的总细胞水平。总之,我们建议MTMR蛋白提供的PI3-磷酸酶活性是产生PMPI合成PMPI4P所必需的,反过来,促进PtdSer和KRAS的PM本地化。
    KRAS is a small GTPase, ubiquitously expressed in mammalian cells, that functions as a molecular switch to regulate cell proliferation and differentiation. Oncogenic mutations that render KRAS constitutively active occur frequently in human cancers. KRAS must localize to the plasma membrane (PM) for biological activity. KRAS PM binding is mediated by interactions of the KRAS membrane anchor with phosphatidylserine (PtdSer), therefore, depleting PM PtdSer content abrogates KRAS PM binding and oncogenic function. From a genome-wide siRNA screen to search for genes that regulate KRAS PM localization, we identified a set of phosphatidylinositol (PI) 3-phosphatase family members: myotubularin-related (MTMR) proteins 2, 3, 4 and 7. Here we show that knockdown of MTMR 2/3/4/7 expression disrupts KRAS PM interactions. The molecular mechanism involves depletion of PM PI 4-phosphate (PI4P) levels, which in turn disrupts the subcellular localization and operation of oxysterol-binding protein related protein (ORP) 5, a PtdSer lipid transfer protein that maintains PM PtdSer content. Concomitantly, silencing MTMR 2/3/4/7 expression elevates PM levels of PI3P and reduces PM and total cellular levels of PtdSer. In summary we propose that the PI 3-phosphatase activity provided by MTMR proteins is required to generate PM PI for the synthesis of PM PI4P, which in turn, promotes the PM localization of PtdSer and KRAS.
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  • 文章类型: Journal Article
    几种细胞内病原体,如结核分枝杆菌,损伤内膜以进入细胞质并破坏先天免疫反应。宿主通过招募将病原体保留在液泡内的修复机器来抵消内膜损伤。这里,我们表明,内质网(ER)-高尔基体蛋白氧固醇结合蛋白(OSBP)及其盘基网柄菌同源物OSBP8被募集到含有分枝杆菌的液泡(MCV)依赖于ESX-1分泌系统的存在,表明它们的动员与膜损伤有关。缺乏OSBP8会导致磷脂酰肌醇-4-磷酸(PI4P)在MCV上的过度积累并降低细胞活力。OSBP8耗尽细胞的溶酶体和液泡的降解能力降低,有利于分枝杆菌的生长。与OSBP8在膜修复中的潜在作用一致,感染结核分枝杆菌的人巨噬细胞以ESX-1依赖性方式募集OSBP.这些发现确定了用于恢复MCV的ER依赖性修复机制,其中OSBP8起平衡受损膜上的PI4P水平的作用。重要性结核病仍然是全球负担,是单一病原体的主要传染病之一。结核分枝杆菌,病原体,已经完善了许多在其宿主内复制和持续存在的方法。虽然分枝杆菌诱导液泡损伤以逃避有毒环境并最终逃逸到细胞质中,宿主招募修复机器来恢复MCV膜。然而,脂质是如何递送用于膜修复的,人们知之甚少。使用先进的荧光成像和体积相关方法,我们证明,这涉及到内质网(ER)-高尔基脂质转移蛋白OSBP8在盘基网柄菌/分枝杆菌系统中的募集。引人注目的是,OSBP8的消耗影响溶酶体功能加速分枝杆菌生长。这表明ER依赖性修复途径构成了针对细胞内病原体如结核分枝杆菌的宿主防御机制。
    OBJECTIVE: Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. Mycobacterium tuberculosis, the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the endoplasmic reticulum (ER)-Golgi lipid transfer protein OSBP8 in the Dictyostelium discoideum/Mycobacterium marinum system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defense mechanism against intracellular pathogens such as M. tuberculosis.
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  • 文章类型: Preprint
    真核细胞中的脂质分布取决于脂质转移和脂质代谢之间的紧密耦合。然而,这些联轴器的描述仍然很差。值得注意的是,目前尚不清楚OSBP相关蛋白(ORP)家族的脂质交换剂在多大程度上,与PI(4)P代谢相结合,有助于在内质网(ER)和其他细胞区域之间形成甾醇和磷脂酰丝氨酸梯度。为了解决这个问题,我们在体外检测了Osh4p的活性,代表ORP,在由PI4激酶产生PI(4)P的高尔基体模拟膜和由磷酸酶Sac1p水解PI(4)P的ER模拟膜之间。使用定量,实时分析,我们证明了Osh4p通过甾醇/PI(4)P交换在两个膜之间产生甾醇梯度,一旦PI(4)P梯度在ATP添加后在该界面处产生,并定义该过程必须合成多少PI(4)P。然后,使用我们体外数据支持的动力学模型,我们估计PI(4)P代谢在多大程度上可以驱动细胞中的脂质转移。最后,我们展示了Sec14p,通过在膜之间转移磷脂酰肌醇,可以支持PI(4)P的合成和Osh4p生成甾醇梯度。这些结果表明ORP在多大程度上,在PI(4)P代谢的控制下,可以在细胞中分布脂质。
    Lipid distribution in the eukaryotic cells depends on tight couplings between lipid transfer and lipid metabolism. Yet these couplings remain poorly described. Notably, it is unclear to what extent lipid exchangers of the OSBP-related proteins (ORPs) family, coupled to PI(4)P metabolism, contribute to the formation of sterol and phosphatidylserine gradient between the endoplasmic reticulum (ER) and other cell regions. To address this question, we have examined in vitro the activity of Osh4p, a representative ORP, between Golgi mimetic membranes in which PI(4)P is produced by a PI 4-kinase and ER mimetic membranes in which PI(4)P is hydrolyzed by the phosphatase Sac1p. Using quantitative, real-time assays, we demonstrate that Osh4p creates a sterol gradient between the two membranes by sterol/PI(4)P exchange as soon as a PI(4)P gradient is generated at this interface following ATP addition, and define how much PI(4)P must be synthesized for this process. Then, using a kinetic model supported by our in vitro data, we estimate to what extent PI(4)P metabolism can drive lipid transfer in cells. Finally, we show that Sec14p, by transferring phosphatidylinositol between membranes, can support the synthesis of PI(4)P and the creation of a sterol gradient by Osh4p. These results indicate to what extent ORPs, under the control of PI(4)P metabolism, can distribute lipids in the cell.
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  • 文章类型: Preprint
    慢性升高的神经体液驱动,特别是肾上腺素能张力升高导致心肌细胞β-肾上腺素能受体(β-AR)过度刺激,是心力衰竭进展的关键机制。β1-AR和β2-AR是存在于人类心脏中的两种主要的β-AR亚型,然而,它们对心脏功能和肥大产生不同甚至相反的影响。例如,β1ARs的慢性激活导致有害的心脏重塑,而β2AR信号传导具有保护作用.通过β2ARs保护心脏的潜在分子机制仍不清楚。在这里,我们表明β2-AR通过抑制高尔基体的PLCε信号传导来防止肥大。β2AR介导的PLC抑制的机制需要β2AR的内化,核内体Gi和Gβγ亚基信号的激活和ERK激活。该途径抑制血管紧张素II和高尔基体-β1-AR介导的高尔基体上的磷酸肌醇水解的刺激,最终导致降低的PKD和HDAC5磷酸化和对心脏肥大的保护。这揭示了PLCs途径的β2-AR拮抗机制,该机制可能有助于β2-AR信号传导对心力衰竭发展的已知保护作用。
    Chronically elevated neurohumoral drive, and particularly elevated adrenergic tone leading to β-adrenergic receptor (β-AR) overstimulation in cardiac myocytes, is a key mechanism involved in the progression of heart failure. β1-AR and β2-ARs are the two major subtypes of β-ARs present in the human heart, however, they elicit different or even opposite effects on cardiac function and hypertrophy. For example, chronic activation of β1ARs drives detrimental cardiac remodeling while β2AR signaling is protective. The underlying molecular mechanisms for cardiac protection through β2ARs remain unclear. Here we show that β2-AR protects against hypertrophy through inhibition of PLCε signaling at the Golgi apparatus. The mechanism for β2AR-mediated PLC inhibition requires internalization of β2AR, activation of Gi and Gβγ subunit signaling at endosomes and ERK activation. This pathway inhibits both angiotensin II and Golgi-β1-AR-mediated stimulation of phosphoinositide hydrolysis at the Golgi apparatus ultimately resulting in decreased PKD and HDAC5 phosphorylation and protection against cardiac hypertrophy. This reveals a mechanism for β2-AR antagonism of the PLCε pathway that may contribute to the known protective effects of β2-AR signaling on the development of heart failure.
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  • 文章类型: Journal Article
    胆固醇(Chol)是所有真核细胞膜的重要组成部分,它影响许多外周以及完整的膜蛋白的功能。Chol在ER中合成,但它选择性地富集在质膜(PM)和其他内膜中,这需要Chol穿过细胞质的水相。除了已知有助于膜中间体批量运输的经典囊泡运输途径外,Chol还通过主要在专门的膜接触位点内起作用的非囊泡脂质转移蛋白运输。这些运输途径中的一些对已建立的浓度梯度起作用,因此需要能量。最近的研究强调了磷酸肌醇(PPIns)的独特作用,特别是磷脂酰肌醇4-磷酸酯(PI4P),用于控制非囊泡胆汁转运。在这一章中,我们将回顾Chol之间的新联系,PPIns,和脂质转移蛋白,包括氧固醇结合蛋白相关蛋白的重要家族,或ORP。
    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.
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  • 文章类型: Journal Article
    Osh6是氧固醇结合蛋白相关蛋白(ORP)家族的成员,是一种脂质转运蛋白,参与磷脂酰丝氨酸(PS)在内质网(ER)和质膜(PM)之间的转运。我们使用生物物理方法来详细表征其运输机制。我们检查了Osh6的所有潜在配体的转运。PI4P和PS是描述最好的脂质货物分子;此外,我们证明了PIP2也可以被Osh6运输。到目前为止,这是两个货物分子之间的交换,PS和PI4P,在Osh6的脂质结合袋中,被认为是PS转运的重要驱动力。然而,我们表明,Osh6可以在没有PI4P的帮助下有效地沿着梯度运输PS,并且PI4P抑制了沿着其梯度的PS运输。这一观察结果突出表明,PS和PI4P之间的交换确实至关重要,但是PI4P与蛋白质结合而不是增强PS运输会抑制蛋白质。我们认为这对于传输方向性很重要,因为它可以防止PS从浓度较高的PM返回到合成的ER。我们的结果还强调了ER常驻Sac1磷酸酶的重要性,该磷酸酶可实现PS的运输并通过PI4P消耗确保其方向性。此外,我们表明,在ER膜的情况下,Sac1活性受膜的负电荷调节,该负电荷可以由PS或PI阴离子提供。
    Osh6, a member of the oxysterol-binding protein-related protein (ORP) family, is a lipid transport protein that is involved in the transport of phosphatidylserine (PS) between the endoplasmic reticulum (ER) and the plasma membrane (PM). We used a biophysical approach to characterize its transport mechanism in detail. We examined the transport of all potential ligands of Osh6. PI4P and PS are the best described lipid cargo molecules; in addition, we showed that PIP2 can be transported by Osh6 as well. So far, it was the exchange between the two cargo molecules, PS and PI4P, in the lipid-binding pocket of Osh6 that was considered an essential driving force for the PS transport. However, we showed that Osh6 can efficiently transport PS along the gradient without the help of PI4P and that PI4P inhibits the PS transport along its gradient. This observation highlights that the exchange between PS and PI4P is indeed crucial, but PI4P bound to the protein rather than intensifying the PS transport suppresses it. We considered this to be important for the transport directionality as it prevents PS from returning back from the PM where its concentration is high to the ER where it is synthesized. Our results also highlighted the importance of the ER resident Sac1 phosphatase that enables the PS transport and ensures its directionality by PI4P consumption. Furthermore, we showed that the Sac1 activity is regulated by the negative charge of the membrane that can be provided by PS or PI anions in the case of the ER membrane.
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  • 文章类型: Journal Article
    已知带有细菌病原体嗜肺军团菌的吞噬体富含磷脂酰肌醇4-磷酸(PtdIns4P),这对于锚定其毒力因子的子集以及可能与支持细胞内细菌生长的含军团菌液泡(LCV)的生物发生有关的信号事件很重要。在这里,我们证明了效应子MavQ是一种磷酸肌醇3激酶,可特异性催化磷脂酰肌醇(PtdIns)转化为PtdIns3P。MavQ的产物随后被效应物LepB磷酸化以产生PtdIns(3,4)P2,其3-磷酸随后被另一个效应物SidF去除以产生PtdIns4P。我们还表明,MavQ与LCV相关,并且ΔmavQ突变体在PtdIns4P结合效应子的锚定中显示出表型,类似于ΔlepB或ΔsidF突变体。我们的结果建立了嗜肺乳杆菌通过由MavQ组成的催化轴从头生物合成PtdIns4P的机制,LepB,和其吞噬体表面的SidF。
    The phagosome harboring the bacterial pathogen Legionella pneumophila is known to be enriched with phosphatidylinositol 4-phosphate (PtdIns4P), which is important for anchoring a subset of its virulence factors and potentially for signaling events implicated in the biogenesis of the Legionella-containing vacuole (LCV) that supports intracellular bacterial growth. Here we demonstrate that the effector MavQ is a phosphoinositide 3-kinase that specifically catalyzes the conversion of phosphatidylinositol (PtdIns) into PtdIns3P. The product of MavQ is subsequently phosphorylated by the effector LepB to yield PtdIns(3,4)P2, whose 3-phosphate is then removed by another effector SidF to generate PtdIns4P. We also show that MavQ is associated with the LCV and the ∆mavQ mutant displays phenotypes in the anchoring of a PtdIns4P-binding effector similar to those of ∆lepB or ∆sidF mutants. Our results establish a mechanism of de novo PtdIns4P biosynthesis by L. pneumophila via a catalysis axis comprised of MavQ, LepB, and SidF on the surface of its phagosome.
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