The Late Endosome and Its Lipid BMP Act as Gateways for Efficient Cytosolic Access of the Delivery Agent dfTAT and Its Macromolecular Cargos.
ABSTRACT: Endosomal entrapment is a severely limiting bottleneck in the delivery of biologics into cells. The compound dfTAT was recently found to circumvent this problem by mediating endosomal leakage efficiently and without toxicity. Herein, we report on the mechanism of endosomal escape of this cell-penetrating peptide. By modulating the trafficking of the peptide within the endocytic pathway, we identify late endosomes as the organelles rendered leaky by dfTAT. We establish that dfTAT binds bis(monoacylglycero)phosphate (BMP), a lipid found in late endosomes, and that the peptide causes the fusion and leakage of bilayers containing BMP. Together, these data identify late endosomes as desirable gateways for cell penetration and BMP as a cellular factor that can be exploited for the development of future delivery agents.
Project description:Many cellular delivery reagents enter the cytosolic space of cells by escaping the lumen of endocytic organelles and, more specifically, late endosomes. The mechanisms involved in endosomal membrane permeation remain largely unresolved, which impedes the improvement of delivery agents. Here, we investigate how 3TAT, a branched analog of the cell-penetrating peptide (CPP) TAT, achieves the permeabilization of bilayers containing bis(monoacylglycero)phosphate (BMP), a lipid found in late endosomes. We establish that the peptide does not induce the leakage of individual lipid bilayers. Instead, leakage requires contact between membranes. Peptide-driven bilayer contacts lead to fusion, lipid mixing, and, critically, peptide encapsulation within proximal bilayers. Notably, this encapsulation is a distinctive property of BMP that explains the specificity of CPP's membrane leakage activity. These results therefore support a model of cell penetration that requires both BMP and the vicinity between bilayers, two features unique to BMP-rich and multivesicular late endosomes.
Project description:Cationic cell-penetrating peptides (CPPs) are a promising vehicle for the delivery of macromolecular drugs. Although many studies have indicated that CPPs enter cells by endocytosis, the mechanisms by which they cross endosomal membranes remain elusive. On the basis of experiments with liposomes, we propose that CPP escape into the cytosol is based on leaky fusion (i.e., fusion associated with the permeabilization of membranes) of the bis(monoacylglycero)phosphate (BMP)-enriched membranes of late endosomes. In our experiments, prototypic CPP HIV-1 TAT peptide did not interact with liposomes mimicking the outer leaflet of the plasma membrane, but it did induce lipid mixing and membrane leakage as it translocated into liposomes mimicking the lipid composition of late endosome. Both membrane leakage and lipid mixing depended on the BMP content and were promoted at acidic pH, which is characteristic of late endosomes. Substitution of BMP with its structural isomer, phosphatidylglycerol (PG), significantly reduced both leakage of the aqueous probe from liposomes and lipid mixing between liposomes. Although affinity of binding to TAT was similar for BMP and PG, BMP exhibited a higher tendency to support the inverted hexagonal phase than PG. Finally, membrane leakage and peptide translocation were both inhibited by inhibitors of lipid mixing, further substantiating the hypothesis that cationic peptides cross BMP-enriched membranes by inducing leaky fusion between them.
Project description:α/β Hydrolase domain-containing 6 (ABHD6) can act as monoacylglycerol hydrolase and is believed to play a role in endocannabinoid signaling as well as in the pathogenesis of obesity and liver steatosis. However, the mechanistic link between gene function and disease is incompletely understood. Here we aimed to further characterize the role of ABHD6 in lipid metabolism. We show that mouse and human ABHD6 degrade bis(monoacylglycero)phosphate (BMP) with high specific activity. BMP, also known as lysobisphosphatidic acid, is enriched in late endosomes/lysosomes, where it plays a key role in the formation of intraluminal vesicles and in lipid sorting. Up to now, little has been known about the catabolism of this lipid. Our data demonstrate that ABHD6 is responsible for ∼ 90% of the BMP hydrolase activity detected in the liver and that knockdown of ABHD6 increases hepatic BMP levels. Tissue fractionation and live-cell imaging experiments revealed that ABHD6 co-localizes with late endosomes/lysosomes. The enzyme is active at cytosolic pH and lacks acid hydrolase activity, implying that it degrades BMP exported from acidic organelles or de novo-formed BMP. In conclusion, our data suggest that ABHD6 controls BMP catabolism and is therefore part of the late endosomal/lysosomal lipid-sorting machinery.
Project description:Many enveloped viruses invade cells via endocytosis and use different environmental factors as triggers for virus-endosome fusion that delivers viral genome into cytosol. Intriguingly, dengue virus (DEN), the most prevalent mosquito-borne virus that infects up to 100 million people each year, fuses only in late endosomes, while activation of DEN protein fusogen glycoprotein E is triggered already at pH characteristic for early endosomes. Are there any cofactors that time DEN fusion to virion entry into late endosomes? Here we show that DEN utilizes bis(monoacylglycero)phosphate, a lipid specific to late endosomes, as a co-factor for its endosomal acidification-dependent fusion machinery. Effective virus fusion to plasma- and intracellular- membranes, as well as to protein-free liposomes, requires the target membrane to contain anionic lipids such as bis(monoacylglycero)phosphate and phosphatidylserine. Anionic lipids act downstream of low-pH-dependent fusion stages and promote the advance from the earliest hemifusion intermediates to the fusion pore opening. To reach anionic lipid-enriched late endosomes, DEN travels through acidified early endosomes, but we found that low pH-dependent loss of fusogenic properties of DEN is relatively slow in the presence of anionic lipid-free target membranes. We propose that anionic lipid-dependence of DEN fusion machinery protects it against premature irreversible restructuring and inactivation and ensures viral fusion in late endosomes, where the virus encounters anionic lipids for the first time during entry. Currently there are neither vaccines nor effective therapies for DEN, and the essential role of the newly identified DEN-bis(monoacylglycero)phosphate interactions in viral genome escape from the endosome suggests a novel target for drug design.
Project description:Many phleboviruses (family Bunyaviridae) are emerging as medically important viruses. These viruses enter target cells by endocytosis and low pH-dependent membrane fusion in late endosomes. However, the necessary and sufficient factors for fusion have not been fully characterized. We have studied the minimal fusion requirements of a prototypic phlebovirus, Uukuniemi virus, in an in vitro virus-liposome assay. We show that efficient lipid mixing between viral and liposome membranes requires close to physiological temperatures and phospholipids with negatively charged headgroups, such as the late endosomal phospholipid bis(monoacylglycero)phosphate. We further demonstrate that bis(monoacylglycero)phosphate increases Uukuniemi virus fusion beyond the lipid mixing stage. By using electron cryotomography of viral particles in the presence or absence of liposomes, we observed that the conformation of phlebovirus glycoprotein capsomers changes from the native conformation toward a more elongated conformation at a fusion permissive pH. Our results suggest a rationale for phlebovirus entry in late endosomes.
Project description:During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747-1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 220.127.116.11) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion.
Project description:Arenavirus entry into host cells occurs through a low pH-dependent fusion with late endosomes that is mediated by the viral glycoprotein complex (GPC). The mechanisms of GPC-mediated membrane fusion and of virus targeting to late endosomes are not well understood. To gain insights into arenavirus fusion, we examined cell-cell fusion induced by the Old World Lassa virus (LASV) GPC complex. LASV GPC-mediated cell fusion is more efficient and occurs at higher pH with target cells expressing human LAMP1 compared to cells lacking this cognate receptor. However, human LAMP1 is not absolutely required for cell-cell fusion or LASV entry. We found that GPC-induced fusion progresses through the same lipid intermediates as fusion mediated by other viral glycoproteins-a lipid curvature-sensitive intermediate upstream of hemifusion and a hemifusion intermediate downstream of acid-dependent steps that can be arrested in the cold. Importantly, GPC-mediated fusion and LASV pseudovirus entry are specifically augmented by an anionic lipid, bis(monoacylglycero)phosphate (BMP), which is highly enriched in late endosomes. This lipid also specifically promotes cell fusion mediated by Junin virus GPC, an unrelated New World arenavirus. We show that BMP promotes late steps of LASV fusion downstream of hemifusion-the formation and enlargement of fusion pores. The BMP-dependence of post-hemifusion stages of arenavirus fusion suggests that these viruses evolved to use this lipid as a cofactor to selectively fuse with late endosomes.
Project description:The late endosomes/endo-lysosomes of vertebrates contain an atypical phospholipid, lysobisphosphatidic acid (LBPA) (also termed bis[monoacylglycero]phosphate [BMP]), which is not detected elsewhere in the cell. LBPA is abundant in the membrane system present in the lumen of this compartment, including intralumenal vesicles (ILVs). In this review, the current knowledge on LBPA and LBPA-containing membranes will be summarized, and their role in the control of endosomal cholesterol will be outlined. Some speculations will also be made on how this system may be overwhelmed in the cholesterol storage disorder Niemann-Pick C. Then, the roles of intralumenal membranes in endo-lysosomal dynamics and functions will be discussed in broader terms. Likewise, the mechanisms that drive the biogenesis of intralumenal membranes, including ESCRTs, will also be discussed, as well as their diverse composition and fate, including degradation in lysosomes and secretion as exosomes. This review will also discuss how intralumenal membranes are hijacked by pathogenic agents during intoxication and infection, and what is the biochemical composition and function of the intra-endosomal lumenal milieu. Finally, this review will allude to the size limitations imposed on intralumenal vesicle functions and speculate on the possible role of LBPA as calcium chelator in the acidic calcium stores of endo-lysosomes.
Project description:Endosomal entrapment is a common bottleneck in various macromolecular delivery approaches. Recently, the polycationic peptide dfTAT was identified as a reagent that induces the efficient leakage of late endosomes and, thereby, enhances the penetration of macromolecules into the cytosol of live human cells. To gain further insights into the features that lead to this activity, the role of peptide sequence was investigated. We establish that the leakage activity of dfTAT can be recapitulated by polyarginine analogs but not by polylysine counterparts. Efficiencies of peptide endocytic uptake increase linearly with the number of arginine residues present. In contrast, peptide cytosolic penetration displays a threshold behavior, indicating that a minimum number of arginines is required to induce endosomal escape. Increasing arginine content above this threshold further augments delivery efficiencies. Yet, it also leads to increasing the toxicity of the delivery agents. Together, these data reveal a relatively narrow arginine-content window for the design of optimally active endosomolytic agents.
Project description:Endosomal maturation is critical for accurate and efficient cargo transport through endosomal compartments. Here we identify a mutation of the novel Drosophila gene, ema (endosomal maturation defective) in a screen for abnormal synaptic overgrowth and defective protein trafficking. Ema is an endosomal membrane protein required for trafficking of fluid-phase and receptor-mediated endocytic cargos. In the ema mutant, enlarged endosomal compartments accumulate as endosomal maturation fails, with early and late endosomes unable to progress into mature degradative late endosomes and lysosomes. Defective endosomal down-regulation of BMP signaling is responsible for the abnormal synaptic overgrowth. Ema binds to and genetically interacts with Vps16A, a component of the class C Vps-HOPS complex that promotes endosomal maturation. The human orthologue of ema, Clec16A, is a candidate susceptibility locus for autoimmune disorders, and its expression rescues the Drosophila mutant demonstrating conserved function. Characterizing this novel gene family identifies a new component of the endosomal pathway and provides insights into class C Vps-HOPS complex function.