The nepenthesin insert in the Plasmodium falciparum aspartic protease plasmepsin V is necessary for enzyme function

Polino, Alexander J; Miller, Jenna; Bhakat, Soumendranath; Mukherjee, Sumit; Bobba, Suhas; Bowman, Gregory R.; Goldberg, Daniel E.

doi:10.1016/j.jbc.2022.102355

Cited by 3 publications

(1 citation statement)

References 54 publications

(101 reference statements)

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“…Plasmids were isolated from bacterial clones using Nucleobond Xtra Midi (MN) kit and electroporated into the parasite as previously described (53). Successfully integrated clones were selected with media containing 5nM WR99210 from 36h post-transfection onwards as the donor plasmid codes for human dihydrofolate reductase (hDHFR) as the selection marker (54).…”

Section: Plasmid Construction and Transfectionmentioning

confidence: 99%

The mature N termini ofPlasmodiumeffector proteins confer specificity of export

Hasan

Polino

Mukherjee

et al. 2023

Preprint

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The intraerythrocytic malaria parasitePlasmodium falciparumexports hundreds of proteins into the host red blood cell (RBC). Most are targeted to the ER by a stretch of hydrophobic amino acids and cleaved further downstream at a conserved motif called the Protein Export Element (PEXEL) by the ER protease plasmepsin V (PM V). The mature effectors then travel through the secretory pathway to the parasitophorous vacuole (PV) that surrounds the parasite. There, PEXEL proteins are somehow recognized as export-destined proteins, as opposed to PV- resident proteins, and are selectively translocated out into the RBC. The mature N terminus appears to be important for export. There is conflicting data on whether PM V cleavage is needed for proper export, or whether any means of generating the mature N terminus would suffice. We replaced the PEXEL-containing N-terminal sequence of an exported GFP reporter with a signal peptide sequence and showed that precise cleavage by signal peptidase, generating the proper mature N terminus, yields export competence. Expressing a construct with only the native ER targeting signal without the PM V cleavage site dramatically decreased the amount of a mature PEXEL reporter, indicating that the hydrophobic stretch lacks an efficient cleavage signal. Therefore, the PEXEL motif functions as a specialized signal cleavage site when appropriately located after an ER targeting sequence. Our data suggest that PM V cleavage and RBC export are two independent events for PEXEL proteins. We also tested and rejected the hypothesis that an alpha-helical mature N terminus is necessary for export.ImportanceMalaria parasites export hundreds of proteins to the cytoplasm of the host red blood cells for their survival. A five amino acid sequence, called the PEXEL motif, is conserved among many exported proteins and is thought to be a signal for export. However, the motif is cleaved inside the endoplasmic reticulum of the parasite and mature proteins starting from the fourth PEXEL residue travel to the parasite periphery for export. We showed that the PEXEL motif is dispensable for export as long as identical mature proteins can be efficiently produced via alternative means in the ER. We also showed that the exported and non-exported proteins are differentiated at the parasite periphery based on their mature N termini, however, any discernible export signal within that region remained cryptic. Our study resolves a longstanding paradox in PEXEL protein trafficking.

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Section: Plasmid Construction and Transfectionmentioning

confidence: 99%

The mature N termini ofPlasmodiumeffector proteins confer specificity of export

Hasan

Polino

Mukherjee

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

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The mature N-termini of Plasmodium effector proteins confer specificity of export

Hasan,

Polino,

Mukherjee

et al. 2023

mBio

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The intraerythrocytic malaria parasite Plasmodium falciparum exports hundreds of proteins into the host red blood cell (RBC). Most are targeted to the endoplasmic reticulum (ER) by a stretch of hydrophobic amino acids and cleaved further downstream at a conserved motif called the Plasmodium Export Element (PEXEL) by the ER protease plasmepsin V (PM V). The mature effectors then travel through the secretory pathway to the parasitophorous vacuole (PV) that surrounds the parasite. There, PEXEL proteins are somehow recognized as export-destined proteins, as opposed to PV-resident proteins, and are selectively translocated out into the RBC. The mature N-terminus appears to be important for export. There is conflicting data on whether PM V cleavage is needed for proper export or whether any means of generating the mature N-terminus would suffice. We replaced the PEXEL-containing N-terminal sequence of an exported GFP reporter with a signal peptide sequence and showed that precise cleavage by signal peptidase, generating the proper mature N-terminus, yields export competence. Expressing a construct with only the native ER targeting signal without the PM V cleavage site dramatically decreased the amount of a mature PEXEL reporter, indicating that the hydrophobic stretch lacks an efficient cleavage signal. Therefore, the PEXEL motif functions as a specialized signal cleavage site when appropriately located after an ER targeting sequence. Our data suggest that PM V cleavage and RBC export are two independent events for PEXEL proteins. We also tested and rejected the hypothesis that an alpha-helical mature N-terminus is necessary for export. IMPORTANCE Malaria parasites export hundreds of proteins to the cytoplasm of the host red blood cells for their survival. A five amino acid sequence, called the PEXEL motif, is conserved among many exported proteins and is thought to be a signal for export. However, the motif is cleaved inside the endoplasmic reticulum of the parasite, and mature proteins starting from the fourth PEXEL residue travel to the parasite periphery for export. We showed that the PEXEL motif is dispensable for export as long as identical mature proteins can be efficiently produced via alternative means in the ER. We also showed that the exported and non-exported proteins are differentiated at the parasite periphery based on their mature N-termini; however, any discernible export signal within that region remained cryptic. Our study resolves a longstanding paradox in PEXEL protein trafficking.

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In Silico Studies of Four Compounds of Cecropia obtusifolia against Malaria Parasite

Lobato-Tapia,

Moreno-Hernández,

Olivo-Vidal

2023

Molecules

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Malaria is a disease that affects many people in the world. In Mexico, malaria remains an active disease in certain regions, particularly in the states of Chiapas and Chihuahua. While antimalarial effects have been attributed to some species of Cecropia in various countries, no such studies have been conducted in Mexico. Therefore, the objective of this study was to evaluate the in silico antimalarial activity of some active compounds identified according to the literature in the species of Cecropia obtusifolia, belonging to the Cecropiaceae family, such as ursolic acid, α-amyrin, chrysin, and isoorientin. These compounds were evaluated with specific molecular docking and molecular dynamics (MD) studies using three different malarial targets with the PDB codes 1CET, 2BL9, and 4ZL4 as well as the prediction of their pharmacokinetic (Pk) properties. Docking analysis revealed the following best binding energies (kcal/mol): isoorientin–1CET (−9.1), isoorientin–2BL9 (−8.8), and chrysin–4ZL4 (−9.6). MD simulation validated the stability of the complexes. Pharmacokinetics analysis suggested that the compounds would generally perform well if administered. Therefore, these results suggest that these compounds may be used as potential drugs for the treatment of malaria.

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The nepenthesin insert in the Plasmodium falciparum aspartic protease plasmepsin V is necessary for enzyme function

Cited by 3 publications

References 54 publications

The mature N termini ofPlasmodiumeffector proteins confer specificity of export

The mature N termini ofPlasmodiumeffector proteins confer specificity of export

The mature N-termini of Plasmodium effector proteins confer specificity of export

In Silico Studies of Four Compounds of Cecropia obtusifolia against Malaria Parasite

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