Limits...
Plasmodium falciparum PfA-M1 aminopeptidase is trafficked via the parasitophorous vacuole and marginally delivered to the food vacuole.

Azimzadeh O, Sow C, Gèze M, Nyalwidhe J, Florent I - Malar. J. (2010)

Bottom Line: Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole.This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole.These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: FRE3206 CNRS/MNHN, Department Regulations, Development, Molecular Diversity, CP52, 61 rue Buffon, F-75005 Paris, France.

ABSTRACT

Background: The Plasmodium falciparum PfA-M1 aminopeptidase, encoded by a single copy gene, displays a neutral optimal activity at pH 7.4. It is thought to be involved in haemoglobin degradation and/or invasion of the host cells. Although a series of inhibitors developed against PfA-M1 suggest that this enzyme is a promising target for therapeutic intervention, the biological function(s) of the three different forms of the enzyme (p120, p96 and p68) are not fully understood. Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole. Alternative destinations, such as the nucleus, have also been proposed.

Methods: By using a combination of techniques, such as cellular and biochemical fractionations, biochemical analysis, mass-spectrometry, immunofluorescence assays and live imaging of GFP fusions to various PfA-M1 domains, evidence is provided for differential localization and behaviour of the three different forms of PfA-M1 in the infected red blood cell which had not been established before.

Results: The high molecular weight p120 form of PfA-M1, the only version of the protein with a hydrophobic transmembrane domain, is detected both inside the parasite and in the parasitophorous vacuole while the processed p68 form is strictly soluble and localized within the parasite. The transient intermediate and soluble p96 form is localized at the border of parasitophorous vacuole and within the parasite in a compartment sensitive to high concentrations of saponin. Upon treatment with brefeldin A, the PfA-M1 maturation is blocked and the enzyme remains in a compartment close to the nucleus.

Conclusions: The PfA-M1 trafficking/maturation scenario that emerges from this data indicates that PfA-M1, synthesized as the precursor p120 form, is targeted to the parasitophorous vacuole via the parasite endoplasmic reticulum/Golgi, where it is converted into the transient p96 form. This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole. These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

Show MeSH

Related in: MedlinePlus

Proposed model for the trafficking and maturation of PfA-M1 in infected erythrocytes. PfA-M1 is targeted to the secretory pathway and the trafficking to the Golgi is sensitive to brefeldin A. The p120 is secreted into the vacuolar space where it undergoes proteolytic processing to yield the p96 form which is endocytosed within the double membrane of the cytostome and taken back into the parasite. The p96 is further processed to the p68 form which is targeted towards the digestive food vacuole, where it is marginally delivered.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2914058&req=5

Figure 9: Proposed model for the trafficking and maturation of PfA-M1 in infected erythrocytes. PfA-M1 is targeted to the secretory pathway and the trafficking to the Golgi is sensitive to brefeldin A. The p120 is secreted into the vacuolar space where it undergoes proteolytic processing to yield the p96 form which is endocytosed within the double membrane of the cytostome and taken back into the parasite. The p96 is further processed to the p68 form which is targeted towards the digestive food vacuole, where it is marginally delivered.

Mentions: In contrast to p120, p96 is within a compartment that is disrupted by higher concentrations of saponin at the periphery of the parasite and the PV. Based on published electron microscopic and ultrastructure data on P. falciparum, p96 could be present either in vesicles being formed at the level of the cytostome or in double membrane structures that have been previously described [38]. A logical hypothesis is that, after its delivery to the PV as the soluble p120 form, PfA-M1 is processed into the soluble p96 that returns into the parasite inside vesicles where the p96 to p68 conversion would occur, since p68 is strictly found within the parasite (Figure 9). Notably, 10,000 g and 100,000 g centrifugations of parasite extracts have no impact on the distribution of the different forms of PfA-M1 between soluble and insoluble fractions (I. Florent, personal communication). As compared to p96, p68 is devoid of the C-terminal part of PfA-M1 that has been mapped from position ~803 to the end of the protein based on Mw predictions from protein sequences (see Additional file 1). Very interestingly, a peptide of about 30 kDa corresponding closely to this C-terminal part (77% coverage from position 807 to 1085 of PfA-M1) was identified in a proteomic study recently performed to identify Plasmodium proteins specifically oxidized after chloroquine treatments of parasites [39]. This experimental result indicates that the C-terminal end of PfA-M1, presumably removed during the p96 to p68 conversion of PfA-M1, is indeed produced in parasites at least as an oxidized form in chloroquine-treated schizonts. The parasite compartment in which this oxidation occurs is not yet identified and will be the focus of future studies. However, this compartment appears distantly related to the FV since none of the known FV haemoglobinases were identified as being oxidized in this proteomic analysis [39]. Another outstanding question is whether the C-terminal part of PfA-M1 that is cleaved from p96 to yield p68 contains signals necessary for the return of p96 into the parasite, before its conversion under the p68 form. C-terminal signals have recently been identified in P. falciparum proteins, such FCP protein [40], and it has been previously noticed that the C-terminal end of PfA-M1 displays the signature of "microbodies targeting signals" [4]. This hypothesis is currently under study in the laboratory using GFP chimeras.


Plasmodium falciparum PfA-M1 aminopeptidase is trafficked via the parasitophorous vacuole and marginally delivered to the food vacuole.

Azimzadeh O, Sow C, Gèze M, Nyalwidhe J, Florent I - Malar. J. (2010)

Proposed model for the trafficking and maturation of PfA-M1 in infected erythrocytes. PfA-M1 is targeted to the secretory pathway and the trafficking to the Golgi is sensitive to brefeldin A. The p120 is secreted into the vacuolar space where it undergoes proteolytic processing to yield the p96 form which is endocytosed within the double membrane of the cytostome and taken back into the parasite. The p96 is further processed to the p68 form which is targeted towards the digestive food vacuole, where it is marginally delivered.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2914058&req=5

Figure 9: Proposed model for the trafficking and maturation of PfA-M1 in infected erythrocytes. PfA-M1 is targeted to the secretory pathway and the trafficking to the Golgi is sensitive to brefeldin A. The p120 is secreted into the vacuolar space where it undergoes proteolytic processing to yield the p96 form which is endocytosed within the double membrane of the cytostome and taken back into the parasite. The p96 is further processed to the p68 form which is targeted towards the digestive food vacuole, where it is marginally delivered.
Mentions: In contrast to p120, p96 is within a compartment that is disrupted by higher concentrations of saponin at the periphery of the parasite and the PV. Based on published electron microscopic and ultrastructure data on P. falciparum, p96 could be present either in vesicles being formed at the level of the cytostome or in double membrane structures that have been previously described [38]. A logical hypothesis is that, after its delivery to the PV as the soluble p120 form, PfA-M1 is processed into the soluble p96 that returns into the parasite inside vesicles where the p96 to p68 conversion would occur, since p68 is strictly found within the parasite (Figure 9). Notably, 10,000 g and 100,000 g centrifugations of parasite extracts have no impact on the distribution of the different forms of PfA-M1 between soluble and insoluble fractions (I. Florent, personal communication). As compared to p96, p68 is devoid of the C-terminal part of PfA-M1 that has been mapped from position ~803 to the end of the protein based on Mw predictions from protein sequences (see Additional file 1). Very interestingly, a peptide of about 30 kDa corresponding closely to this C-terminal part (77% coverage from position 807 to 1085 of PfA-M1) was identified in a proteomic study recently performed to identify Plasmodium proteins specifically oxidized after chloroquine treatments of parasites [39]. This experimental result indicates that the C-terminal end of PfA-M1, presumably removed during the p96 to p68 conversion of PfA-M1, is indeed produced in parasites at least as an oxidized form in chloroquine-treated schizonts. The parasite compartment in which this oxidation occurs is not yet identified and will be the focus of future studies. However, this compartment appears distantly related to the FV since none of the known FV haemoglobinases were identified as being oxidized in this proteomic analysis [39]. Another outstanding question is whether the C-terminal part of PfA-M1 that is cleaved from p96 to yield p68 contains signals necessary for the return of p96 into the parasite, before its conversion under the p68 form. C-terminal signals have recently been identified in P. falciparum proteins, such FCP protein [40], and it has been previously noticed that the C-terminal end of PfA-M1 displays the signature of "microbodies targeting signals" [4]. This hypothesis is currently under study in the laboratory using GFP chimeras.

Bottom Line: Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole.This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole.These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: FRE3206 CNRS/MNHN, Department Regulations, Development, Molecular Diversity, CP52, 61 rue Buffon, F-75005 Paris, France.

ABSTRACT

Background: The Plasmodium falciparum PfA-M1 aminopeptidase, encoded by a single copy gene, displays a neutral optimal activity at pH 7.4. It is thought to be involved in haemoglobin degradation and/or invasion of the host cells. Although a series of inhibitors developed against PfA-M1 suggest that this enzyme is a promising target for therapeutic intervention, the biological function(s) of the three different forms of the enzyme (p120, p96 and p68) are not fully understood. Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole. Alternative destinations, such as the nucleus, have also been proposed.

Methods: By using a combination of techniques, such as cellular and biochemical fractionations, biochemical analysis, mass-spectrometry, immunofluorescence assays and live imaging of GFP fusions to various PfA-M1 domains, evidence is provided for differential localization and behaviour of the three different forms of PfA-M1 in the infected red blood cell which had not been established before.

Results: The high molecular weight p120 form of PfA-M1, the only version of the protein with a hydrophobic transmembrane domain, is detected both inside the parasite and in the parasitophorous vacuole while the processed p68 form is strictly soluble and localized within the parasite. The transient intermediate and soluble p96 form is localized at the border of parasitophorous vacuole and within the parasite in a compartment sensitive to high concentrations of saponin. Upon treatment with brefeldin A, the PfA-M1 maturation is blocked and the enzyme remains in a compartment close to the nucleus.

Conclusions: The PfA-M1 trafficking/maturation scenario that emerges from this data indicates that PfA-M1, synthesized as the precursor p120 form, is targeted to the parasitophorous vacuole via the parasite endoplasmic reticulum/Golgi, where it is converted into the transient p96 form. This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole. These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

Show MeSH
Related in: MedlinePlus