Structural basis for the wobbler mouse neurodegenerative disorder caused by mutation in the Vps54 subunit of the GARP complex.

The multisubunit Golgi-associated retrograde protein (GARP) complex is required for tethering and fusion of endosome-derived transport vesicles to the trans-Golgi network. Mutation of leucine-967 to glutamine in the Vps54 subunit of GARP is responsible for spinal muscular atrophy in the wobbler mouse, an animal model of amyotrophic lateral sclerosis. The crystal structure at 1.7 A resolution of the mouse Vps54 C-terminal fragment harboring leucine-967, in conjunction with comparative sequence analysis, reveals that Vps54 has a continuous alpha-helical bundle organization similar to that of other multisubunit tethering complexes. The structure shows that leucine-967 is buried within the alpha-helical bundle through predominantly hydrophobic interactions that are critical for domain stability and folding in vitro. Mutation of this residue to glutamine does not prevent integration of Vps54 into the GARP complex but greatly reduces the half-life and levels of the protein in vivo. Severely reduced levels of mutant Vps54 and, consequently, of the whole GARP complex underlie the phenotype of the wobbler mouse.

Multilayered mechanism of CD4 downregulation by HIV-1 Vpu involving distinct ER retention and ERAD targeting steps.

A key function of the Vpu protein of HIV-1 is the targeting of newly-synthesized CD4 for proteasomal degradation. This function has been proposed to occur by a mechanism that is fundamentally distinct from the cellular ER-associated degradation (ERAD) pathway. However, using a combination of genetic, biochemical and morphological methodologies, we find that CD4 degradation induced by Vpu is dependent on a key component of the ERAD machinery, the VCP-UFD1L-NPL4 complex, as well as on SCF(beta-TrCP)-dependent ubiquitination of the CD4 cytosolic tail on lysine and serine/threonine residues. When degradation of CD4 is blocked by either inactivation of the VCP-UFD1L-NPL4 complex or prevention of CD4 ubiquitination, Vpu still retains the bulk of CD4 in the ER mainly through transmembrane domain interactions. Addition of a strong ER export signal from the VSV-G protein overrides this retention. Thus, Vpu exerts two distinct activities in the process of downregulating CD4: ER retention followed by targeting to late stages of ERAD. The multiple levels at which Vpu engages these cellular quality control mechanisms underscore the importance of ensuring profound suppression of CD4 to the life cycle of HIV-1.

Low plasma membrane expression of the miltefosine transport complex renders Leishmania braziliensis refractory to the drug.

Miltefosine (hexadecylphosphocholine, MLF) is the first oral drug with recognized efficacy against both visceral and cutaneous leishmaniasis. However, some clinical studies have suggested that MLF shows significantly less efficiency against the cutaneous leishmaniasis caused by Leishmania braziliensis. In this work, we have determined the cellular and molecular basis for the natural MLF resistance observed in L. braziliensis. Four independent L. braziliensis clinical isolates showed a marked decrease in MLF sensitivity that was due to their inability to internalize the drug. MLF internalization in the highly sensitive L. donovani species requires at least two proteins in the plasma membrane, LdMT, a P-type ATPase involved in phospholipid translocation, and its beta subunit, LdRos3. Strikingly, L. braziliensis parasites showed highly reduced levels of this MLF translocation machinery at the plasma membrane, mainly because of the low expression levels of the beta subunit, LbRos3. Overexpression of LbRos3 induces increased MLF sensitivity not only in L. braziliensis promastigotes but also in intracellular amastigotes. These results further highlight the importance of the MLF translocation machinery in determining MLF potency and point toward the development of protocols to routinely monitor MLF susceptibility in geographic areas where L. braziliensis might be prevalent.

Inactivation of the miltefosine transporter, LdMT, causes miltefosine resistance that is conferred to the amastigote stage of Leishmania donovani and persists in vivo.

Miltefosine (hexadecylphosphocholine) is the first oral antileishmanial drug. In this study, we addressed the question whether miltefosine-resistant Leishmania donovani promastigotes transform to miltefosine-resistant amastigotes. A promastigote line, M-mutR, showed defective internalisation of miltefosine owing to mutations in LdMT, similar to previously described resistant lines. M-mutR parasites were infective to macrophages in vitro as well as in BALB/c mice in vivo. There was good correlation of in vitro resistance indices between promastigotes and intracellular amastigotes. Most importantly, M-mutR parasites retained the resistant phenotype in vivo, with no decrease of hepatic burden in BALB/c mice following miltefosine treatment up to 30 mg/kg (ca. 90% inhibition in wild-type infections). No cross-resistance to other antileishmanial drugs was observed in M-mutR amastigotes.

Ang2/fat-free is a conserved subunit of the Golgi-associated retrograde protein complex.

The Golgi-associated retrograde protein (GARP) complex mediates tethering and fusion of endosome-derived transport carriers to the trans-Golgi network (TGN). In the yeast Saccharomyces cerevisiae, GARP comprises four subunits named Vps51p, Vps52p, Vps53p, and Vps54p. Orthologues of the GARP subunits, except for Vps51p, have been identified in all other eukaryotes. A yeast two-hybrid screen of a human cDNA library yielded a phylogenetically conserved protein, Ang2/Fat-free, which interacts with human Vps52, Vps53 and Vps54. Human Ang2 is larger than yeast Vps51p, but exhibits significant homology in an N-terminal coiled-coil region that mediates assembly with other GARP subunits. Biochemical analyses show that human Ang2, Vps52, Vps53 and Vps54 form an obligatory 1:1:1:1 complex that strongly interacts with the regulatory Habc domain of the TGN SNARE, Syntaxin 6. Depletion of Ang2 or the GARP subunits similarly impairs protein retrieval to the TGN, lysosomal enzyme sorting, endosomal cholesterol traffic¤ and autophagy. These findings indicate that Ang2 is the missing component of the GARP complex in most eukaryotes.

Dual roles of the mammalian GARP complex in tethering and SNARE complex assembly at the trans-golgi network.

Tethering factors and SNAREs control the last two steps of vesicular trafficking: the initial interaction and the fusion, respectively, of transport vesicles with target membranes. The Golgi-associated retrograde protein (GARP) complex regulates retrograde transport from endosomes to the trans-Golgi network (TGN). Although GARP has been proposed to function as a tethering factor at the TGN, direct evidence for such a role is still lacking. Herein we report novel and specific interactions of the mammalian GARP complex with SNAREs that participate in endosome-to-TGN transport, namely, syntaxin 6, syntaxin 16, and Vamp4. These interactions depend on the N-terminal regions of Vps53 and Vps54 and the SNARE motif of the SNAREs. We show that GARP functions upstream of the SNAREs, regulating their localization and assembly into SNARE complexes. However, interactions of GARP with SNAREs are insufficient to promote retrograde transport, because deletion of the C-terminal region of Vps53 precludes GARP function without affecting GARP-SNARE interactions. Finally, we present in vitro data consistent with a tethering role for GARP, which is disrupted by deletion of the Vps53 C-terminal region. These findings indicate that GARP orchestrates retrograde transport from endosomes to the TGN by promoting vesicle tethering and assembly of SNARE complexes in consecutive, independent steps.

Requirement of the human GARP complex for mannose 6-phosphate-receptor-dependent sorting of cathepsin D to lysosomes.

The biosynthetic sorting of acid hydrolases to lysosomes relies on transmembrane, mannose 6-phosphate receptors (MPRs) that cycle between the TGN and endosomes. Herein we report that maintenance of this cycling requires the function of the mammalian Golgi-associated retrograde protein (GARP) complex. Depletion of any of the three GARP subunits, Vps52, Vps53, or Vps54, by RNAi impairs sorting of the precursor of the acid hydrolase, cathepsin D, to lysosomes and leads to its secretion into the culture medium. As a consequence, lysosomes become swollen, likely due to a buildup of undegraded materials. Missorting of cathepsin D in GARP-depleted cells results from accumulation of recycling MPRs in a population of light, small vesicles downstream of endosomes. These vesicles might correspond to intermediates in retrograde transport from endosomes to the TGN. Depletion of GARP subunits also blocks the retrograde transport of the TGN protein, TGN46, and the B subunit of Shiga toxin. These observations indicate that the mammalian GARP complex plays a general role in the delivery of retrograde cargo into the TGN. We also report that a Vps54 mutant protein in the Wobbler mouse strain is active in retrograde transport, thus explaining the viability of these mutant mice.

Phospholipid translocation and miltefosine potency require both L. donovani miltefosine transporter and the new protein LdRos3 in Leishmania parasites.

The antitumor drug miltefosine has been recently approved as the first oral drug active against visceral leishmaniasis. We have previously identified the L. donovani miltefosine transporter (LdMT) as a P-type ATPase involved in phospholipid translocation at the plasma membrane of Leishmania parasites. Here we show that this protein is essential but not sufficient for the phospholipid translocation activity and, thus, for the potency of the drug. Based on recent findings in yeast, we have identified the putative beta subunit of LdMT, named LdRos3, as another protein factor required for the translocation activity. LdRos3 belongs to the CDC50/Lem3 family, proposed as likely beta subunits for P4-ATPases. The phenotype of LdRos3-defective parasites was identical to that of the LdMT-/-, including a defect in the uptake of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)-phosphatidylserine, generally considered as not affected in Lem3p-deficient yeast. Both LdMT and LdRos3 normally localized to the plasma membrane but were retained inside the endoplasmic reticulum in the absence of the other protein or when inactivating point mutations were introduced in LdMT. Modulating the expression levels of either protein independently, we show that any one of them could behave as the protein limiting the level of flippase activity. Thus, LdMT and LdRos3 seem to form part of the same translocation machinery that determines flippase activity and miltefosine sensitivity in Leishmania, further supporting the consideration of CDC50/Lem3 proteins as beta subunits required for the normal functioning of P4-ATPases.

Mechanisms of experimental resistance of Leishmania to miltefosine: Implications for clinical use.

Miltefosine (hexadecylphosphocholine, MIL), registered as Impavido((R)), has become the first oral drug for the treatment of visceral and cutaneous leishmanasis. MIL is a simple molecule, very stable, relatively safe and highly efficient in clinical trials. However, MIL requires a long treatment course (28 days) and has a long half-life (around 150h), which might accelerate the emergence of drug resistance in case of inadequate use. The mechanisms of MIL resistance have been studied in vitro with experimental resistant lines. Resistance was shown to develop quickly in Leishmania promastigotes. Interestingly, a decreased MIL accumulation has always accounted for the resistance phenotype. The lower MIL accumulation can be achieved by two independent mechanisms: (i) an increase in drug efflux, mediated by the overexpression of the ABC transporter P-glycoprotein, and (ii) a decrease in drug uptake, which is easily achieved by the inactivation of any one of the two proteins known to be responsible for the MIL uptake, the MIL transporter LdMT and its beta subunit LdRos3. Policies concerning a proper use of this drug should be followed and supervised by health authorities of endemic areas to minimalize the risk for the appearance of drug failures and to ensure a long life span for this effective oral drug.

Combination of suboptimal doses of inhibitors targeting different domains of LtrMDR1 efficiently overcomes resistance of Leishmania spp. to Miltefosine by inhibiting drug efflux.

Miltefosine (hexadecylphosphocholine) is the first orally active drug approved for the treatment of leishmaniasis. We have previously shown the involvement of LtrMDR1, a P-glycoprotein-like transporter belonging to the ATP-binding cassette superfamily, in miltefosine resistance in Leishmania. Here we show that overexpression of LtrMDR1 increases miltefosine efflux, leading to a decrease in drug accumulation in the parasites. Although LtrMDR1 modulation might be an efficient way to overcome this resistance, a main drawback associated with the use of P-glycoprotein inhibitors is related to their intrinsic toxicity. In order to diminish possible side effects, we have combined suboptimal doses of modulators targeting both the cytosolic and transmembrane domains of LtrMDR1. Preliminary structure-activity relationships have allowed us to design a new and potent flavonoid derivative with high affinity for the cytosolic nucleotide-binding domains. As modulators directed to the transmembrane domains, we have selected one of the most potent dihydro-beta-agarofuran sesquiterpenes described, and we have also studied the effects of two of the most promising, latest-developed modulators of human P-glycoprotein, zosuquidar (LY335979) and elacridar (GF120918). The results show that this combinatorial strategy efficiently overcomes P-glycoprotein-mediated parasite miltefosine resistance by increasing intracellular miltefosine accumulation without any side effect in the parental, sensitive, Leishmania line and in different mammalian cell lines.

Leishmania donovani resistance to miltefosine involves a defective inward translocation of the drug.

Miltefosine (hexadecylphosphocholine [HePC]) is the first drug approved for the oral treatment of visceral leishmaniasis. As part of a study on the mechanisms of action of this drug and on the rates of resistance to this drug, we have been working in vitro with an Leishmania donovani line that was previously shown to be 15-fold more resistant to HePC. We have studied the accumulation of [(14)C]HePC by L. donovani promastigotes and have found a drastic reduction (>95%) in the ability of the resistant line to internalize the drug. Binding of HePC to the plasma membrane and drug efflux from preloaded cells were similar in both drug-sensitive and -resistant lines, and no [(14)C]HePC metabolism was evident in either line. Resistant parasites were also unable to take up other short-chain phospholipid analogs, independently of their polar head group, even though endocytosis remained unaltered. Finally, HePC uptake was temperature and energy dependent and sensitive to the thiol-reactive agent N-ethylmaleimide. We propose that inward translocation of a short-chain phospholipid across the plasma membrane may exist in Leishmania promastigotes and that such activity is defective in the resistant line.

Characterization of an ABCA-like transporter involved in vesicular trafficking in the protozoan parasite Trypanosoma cruzi.

Protozoan parasites are responsible of important healthy problems, among others malaria, leishmaniasis and trypanosomiasis. The present work reports the characterization of the first mammalian ATP-binding cassette transporter, subfamily A (ABCA)-like in Trypanosoma cruzi. TcABC1 is a single copy gene differentially expressed along the life cycle of the parasite, being absent in its infective form. TcABC1 localizes to the plasma membrane, flagellar pocket and intracellular vesicles. Functional studies of TcABC1 in transfected parasites suggest that the protein is implicated in intracellular trafficking, as determined by the analysis of endocytosis and exocytosis events. The accumulation of the endocytic markers FM4-64 and NBD-SM is increased in transfected parasites. Similarly, ectophosphatase and ectoATPase activities are increased in TcABC1 overproducers. Indeed, transmission electronic microscopy analysis showed a higher number of intracellular vesicles in TcABC1 transfectants. Taken together, these results suggest that the protein is involved in the endocytic and exocytic pathways of T. cruzi.

Functional cloning of the miltefosine transporter. A novel P-type phospholipid translocase from Leishmania involved in drug resistance.

The antitumor drug miltefosine (hexadecylphosphocholine, MIL) has recently been approved as the first oral agent for the treatment of visceral leishmaniasis. Little is known about the mechanisms of action and uptake of MIL in either parasites or tumor cell lines. We have cloned a putative MIL transporter (LdMT) by functional rescue, using a Leishmania donovani-resistant line defective in the inward-directed translocation of both MIL and glycerophospholipids. LdMT is a novel P-type ATPase belonging to the partially characterized aminophospholipid translocase subfamily. Resistant parasites transfected with LdMT regain their sensitivity to MIL and edelfosine and the ability to normally take up [14C]MIL and fluorescent-labeled glycerophospholipids. Moreover, LdMT localizes to the plasma membrane, and its overexpression in Leishmania tarentolae, a species non-sensitive to MIL, significantly increases the uptake of [14C]MIL, strongly suggesting that this protein behaves as a true translocase. Finally, both LdMT-resistant alleles encompass single but distinct point mutations, each of which impairs transport function, explaining the resistant phenotype. These results demonstrate biochemically and genetically the direct involvement of LdMT in MIL and phospholipids translocation in Leishmania and describe for the first time a P-type ATPase involved in MIL uptake and potency in eukaryotic cells.