RESUMEN
Lipid droplets (LDs) are dynamic organelles essential for cellular lipid homeostasis. Assembly of LDs occurs in the endoplasmic reticulum (ER), and the conserved ER membrane protein seipin emerged as a key player in this process. Here, we review recent advances provided by structural, biochemical, and in silico analysis that revealed mechanistic insights into the molecular role of the seipin complexes and led to an updated model for LD biogenesis. We further discuss how other ER components cooperate with seipin during LD biogenesis. Understanding the molecular mechanisms underlying seipin-mediated LD assembly is important to uncover the fundamental aspects of lipid homeostasis and organelle biogenesis and to provide hints on the pathogenesis of lipid storage disorders.
Asunto(s)
Retículo Endoplásmico , Subunidades gamma de la Proteína de Unión al GTP , Gotas Lipídicas , Gotas Lipídicas/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/genética , Humanos , Retículo Endoplásmico/metabolismo , Animales , Metabolismo de los LípidosRESUMEN
The HIV envelope protein gp160 comprises two subunits, gp120 and gp41, responsible for receptor binding and membrane fusion during viral entry, respectively. In the course of the membrane fusion process, gp41 undergoes a conformational change, leading to the formation of a six-helix bundle (SHB), which ultimately drives membrane fusion. The gp41 C-terminal and N-terminal heptad repeats (CHR and NHR) interact with one another to form the SHB, and this step can be targeted by peptide inhibitors, which are used in the clinic to mitigate HIV infection. Here, we discover the calcium interaction motifs (CIMs) in the gp41 CHR and NHR regions via NMR spectroscopy. We find that the assembly of the CHR-NHR SHB is facilitated in Ca2+-containing media and impaired in CIM mutants. Of note, the clinically approved, gp41-derived fusion inhibitor T20, which does not contain the CIM motif, exhibits reduced inhibitory efficiency when challenged with calcium. This finding could have important implications for the development of better fusion inhibitors for HIV.
Asunto(s)
Infecciones por VIH , VIH-1 , Secuencia de Aminoácidos , Calcio/metabolismo , Proteína gp41 de Envoltorio del VIH/química , VIH-1/química , Humanos , Fusión de MembranaRESUMEN
Lipid droplets (LDs) are universal lipid storage organelles with a core of neutral lipids, such as triacylglycerols, surrounded by a phospholipid monolayer. This unique architecture is generated during LD biogenesis at endoplasmic reticulum (ER) sites marked by Seipin, a conserved membrane protein mutated in lipodystrophy. Here structural, biochemical and molecular dynamics simulation approaches reveal the mechanism of LD formation by the yeast Seipin Sei1 and its membrane partner Ldb16. We show that Sei1 luminal domain assembles a homooligomeric ring, which, in contrast to other Seipins, is unable to concentrate triacylglycerol. Instead, Sei1 positions Ldb16, which concentrates triacylglycerol within the Sei1 ring through critical hydroxyl residues. Triacylglycerol recruitment to the complex is further promoted by Sei1 transmembrane segments, which also control Ldb16 stability. Thus, we propose that LD assembly by the Sei1/Ldb16 complex, and likely other Seipins, requires sequential triacylglycerol-concentrating steps via distinct elements in the ER membrane and lumen.
Asunto(s)
Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Gotas Lipídicas/química , Gotas Lipídicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Retículo Endoplásmico/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/genética , Lípidos de la Membrana , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales , Modelos Moleculares , Simulación de Dinámica Molecular , Fosfolípidos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Triglicéridos/metabolismoRESUMEN
Lipid droplets (LDs) are versatile organelles with central roles in lipid and energy metabolism in all eukaryotes. They primarily buffer excess fatty acids by storing them as neutral lipids, mainly triglycerides and steryl esters. The neutral lipids form a core, surrounded by a unique phospholipid monolayer coated with a defined set of proteins. Thus, the architecture of LDs sets them apart from all other membrane-bound organelles. The origin of LDs remained controversial for a long time. However, it has become clear that their biogenesis occurs at the endoplasmic reticulum (ER) and is a lipid driven process. LD formation is intiatied by the demixing of neutral lipids from membrane phospholipids, leading to the formation of a neutral lipid "lens" like structure between the leaflets of the ER bilayer. As this lens grows, it buds out of the membrane towards the cytosol to give rise to a LD. Recent biophysical and cell biological experiments indicate that LD biogenesis occurs at specific ER domains. These domains are enriched in various proteins required for normal LD formation and possibly have a lipid composition distinct from the remaining ER membrane. Here, we describe the prevailing model for LD formation and discuss recent insights on how proteins organize ER domains involved in LD biogenesis.
Asunto(s)
Gotas Lipídicas/metabolismo , Animales , Humanos , Proteínas de la Membrana/metabolismo , Modelos BiológicosRESUMEN
The human immunodeficiency virus enters its host cells by membrane fusion, initiated by the gp41 subunit of its envelope protein. gp41 has also been shown to bind T-cell receptor (TCR) complex components, interfering with TCR signaling leading to reduced T-cell activation. This immunoinhibitory activity is suggested to occur during the membrane fusion process and is attributed to various membranotropic regions of the gp41 ectodomain and to the transmembrane domain. Although extensively studied, the cytosolic region of gp41, termed the cytoplasmic tail (CT), has not been examined in the context of immune suppression. Here we investigated whether the CT inhibits T-cell activation in different T-cell models by utilizing gp41-derived peptides and expressed full gp41 proteins. We found that a conserved region of the CT, termed lentiviral lytic peptide 2 (LLP2), specifically inhibits the activation of mouse, Jurkat, and human primary T-cells. This inhibition resulted in reduced T-cell proliferation, gene expression, cytokine secretion, and cell surface expression of CD69. Differential activation of the TCR signaling cascade revealed that CT-based immune suppression occurs downstream of the TCR complex. Moreover, LLP2 peptide treatment of Jurkat and primary human T-cells impaired Akt but not NFκB and ERK1/2 activation, suggesting that immune suppression occurs through the Akt pathway. These findings identify a novel gp41 T-cell suppressive element with a unique inhibitory mechanism that can take place post-membrane fusion.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Secuencias de Aminoácidos , Animales , Proliferación Celular , Citocinas/genética , Citocinas/metabolismo , Expresión Génica , Proteína gp41 de Envoltorio del VIH/química , Humanos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Fosforilación , Dominios Proteicos , Proteínas Proto-Oncogénicas c-akt/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células RAW 264.7 , Transducción de Señal , Linfocitos T/metabolismo , Linfocitos T/virología , Serina-Treonina Quinasas TOR/química , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The ability of the Lentivirus HIV-1 to inhibit T-cell activation by its gp41 fusion protein is well documented, yet limited data exists regarding other viral fusion proteins. HIV-1 utilizes membrane binding region of gp41 to inhibit T-cell receptor (TCR) complex activation. Here we examined whether this T-cell suppression strategy is unique to the HIV-1 gp41. We focused on T-cell modulation by the gp21 fusion peptide (FP) of the Human T-lymphotropic Virus 1 (HTLV-1), a Deltaretrovirus that like HIV infects CD4+ T-cells. Using mouse and human in-vitro T-cell models together with in-vivo T-cell hyper activation mouse model, we reveal that HTLV-1's FP inhibits T-cell activation and unlike the HIV FP, bypasses the TCR complex. HTLV FP inhibition induces a decrease in Th1 and an elevation in Th2 responses observed in mRNA, cytokine and transcription factor profiles. Administration of the HTLV FP in a T-cell hyper activation mouse model of multiple sclerosis alleviated symptoms and delayed disease onset. We further pinpointed the modulatory region within HTLV-1's FP to the same region previously identified as the HIV-1 FP active region, suggesting that through convergent evolution both viruses have obtained the ability to modulate T-cells using the same region of their fusion protein. Overall, our findings suggest that fusion protein based T-cell modulation may be a common viral trait.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/inmunología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Proteínas Virales de Fusión/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Membrana Celular/metabolismo , Células Cultivadas , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Activación de Linfocitos , Fusión de Membrana , Ratones , Ratones Endogámicos C57BL , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The HIV gp160 envelope fusion protein is situated in the viral membrane and mediates virus entry into its host cell. Increasing evidence suggests that virtually all parts of the HIV envelope are structurally and functionally dependent on membranes. Protein-lipid interactions and membrane properties influence the dynamics of a manifold of gp160 biological activities such as membrane fusion, immune suppression and gp160 incorporation into virions during HIV budding and assembly. In the following we will summarize our current understanding of this interdependence between membrane interaction, structural conformation and functionality of the different gp160 domains. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/química , Proteínas gp160 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/química , VIH-1/química , Microdominios de Membrana/química , Esfingomielinas/química , Secuencia de Aminoácidos , Expresión Génica , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Proteínas gp160 de Envoltorio del VIH/genética , Proteínas gp160 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Interacciones Huésped-Patógeno , Humanos , Fusión de Membrana , Microdominios de Membrana/inmunología , Microdominios de Membrana/virología , Conformación Proteica , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Esfingomielinas/inmunología , Linfocitos T/inmunología , Linfocitos T/virología , Ensamble de Virus/inmunología , Liberación del Virus/inmunologíaRESUMEN
To successfully infect and persist within its host, HIV-1 utilizes several immunosuppressive motifs within its gp41 envelope glycoprotein to manipulate and evade the immune system. The transmembrane domain (TMD) of gp41 downregulates T-cell receptor (TCR) signaling through a hitherto unknown mechanism. Interactions between TMDs within the membrane milieu have been shown to be typically mediated by particular amino acids, such as interactions between basic and acidic residues and dimerization motifs as GxxxG. The HIV-1 TMD exhibits both a polar arginine (Arg(696)) residue and a GxxxG motif, making them ideal candidates for mediators of TMD-TCR interaction. Using a primary T-cell activation assay and biochemical and biophysical methods, we demonstrate that the gp41 TMD directly interacts with TMDs of the TCR and the CD3 coreceptors (δ, γ, and ε) within the membrane, presumably leading to impairment of complex assembly. Additionally, we reveal that although Arg(696) does not affect TMD immunosuppression, the GxxxG motif is crucial in mediating gp41's TMD interaction with the CD3 coreceptors of the TCR. These findings suggest that compared with other gp41 immunosuppressive motifs, the gp41 TMD has multiple targets within the TCR complex, suggesting less susceptibility to evolutionary pressure and consequently being advantageous for the virus over the host immune response. Furthermore, as the GxxxG motif mediates interactions of the gp41 TMD with multiple receptors, it emerges as an attractive drug target. This multitarget inhibitory mechanism might be a strategy utilized by HIV to interfere with the function of additional host receptors.
Asunto(s)
Complejo CD3/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Evasión Inmune , Receptores de Antígenos de Linfocitos T gamma-delta/antagonistas & inhibidores , Linfocitos T/metabolismo , Secuencias de Aminoácidos , Animales , Arginina/química , Complejo CD3/química , Línea Celular , Proliferación Celular , Células Cultivadas , Dimerización , Proteína gp41 de Envoltorio del VIH/química , VIH-1/inmunología , Humanos , Ensayos de Liberación de Interferón gamma , Activación de Linfocitos , Ratones Endogámicos C57BL , Mutación , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Liposomas UnilamelaresRESUMEN
For successful infection and propagation viruses must overcome many obstacles such as the immune system and entry into their host cells. HIV utilizes its trimeric envelope protein gp160, specifically the gp41 subunit, to enter its host cell. During this process, a gp41-central coiled coil is formed from three N- and three C-terminal heptad repeats, termed the six-helix bundle (SHB), which drives membrane fusion. Recently, T-cell suppression has been reported as an additional function for several regions of gp41 by interfering with the T-cell receptor (TCR) signalling cascade. One of these regions encompasses the conserved pocket binding domain (PBD) that is situated in the C-terminal heptad repeat (CHR) and stabilizes SHB formation. This could indicate that the PBD plays a role in T-cell suppression in addition to its role in membrane fusion. To investigate this dual function, we used two independent cell cultures coupled with biophysical techniques. The data reveal that the PBD mediates T-cell suppression by stabilizing a TCR-binding conformation in the membrane. Moreover, we show that the clinically used HIV fusion inhibitor T-20 did not show suppressive abilities, in contrast with the potent fusion inhibitor C34. In addition, by focusing on SHB conformation after its assembly, we shed light on a mechanism by which gp41's function alternates from membrane fusion facilitation to suppression of TCR activation.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Internalización del Virus , Animales , Proteína gp41 de Envoltorio del VIH/genética , Inhibidores de Fusión de VIH/farmacología , VIH-1/genética , Células HeLa , Humanos , Ratones , Péptidos/genética , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/genética , Secuencias Repetitivas de AminoácidoRESUMEN
Lipid-conjugated peptides have advanced the understanding of membrane protein functions and the roles of lipids in the membrane milieu. These lipopeptides modulate various biological systems such as viral fusion. A single function has been suggested for the lipid, binding to the membrane and thus elevating the local concentration of the peptide at the target site. In the present paper, we challenged this argument by exploring in-depth the antiviral mechanism of lipopeptides, which comprise sphinganine, the lipid backbone of DHSM (dihydrosphingomyelin), and an HIV-1 envelope-derived peptide. Surprisingly, we discovered a partnership between the lipid and the peptide that impaired early membrane fusion events by reducing CD4 receptor lateral diffusion and HIV-1 fusion peptide-mediated lipid mixing. Moreover, only the joint function of sphinganine and its conjugate peptide disrupted HIV-1 fusion protein assembly and folding at the later fusion steps. Via imaging techniques we revealed for the first time the direct localization of these lipopeptides to the virus-cell and cell-cell contact sites. Overall, the findings of the present study may suggest lipid-protein interactions in various biological systems and may help uncover a role for elevated DHSM in HIV-1 and its target cell membranes.