The endoplasmic reticulum chaperone
            <scp>PfGRP170</scp>
            is essential for asexual development and is linked to stress response in malaria parasites

Kudyba, Heather M.; Cobb, David W.; Fierro, Manuel A; Florentin, Anat; Ljolje, Dragan; Singh, Balwan; Lucchi, Naomi W.; Muralidharan, Vasant

doi:10.1111/cmi.13042

Cited by 24 publications

(18 citation statements)

References 78 publications

(193 reference statements)

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“…1F). Similar colocalization has been observed for several other Plasmodium ER proteins such as plasmepsin V, signal peptidase complex 25, signal peptidase complex 21 (28), and glucose-responsive protein 170 (29).…”

Section: Resultssupporting

confidence: 80%

An Endoplasmic Reticulum CREC Family Protein Regulates the Egress Proteolytic Cascade in Malaria Parasites

Fierro

Asady

Brooks

et al. 2020

mBio

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The endoplasmic reticulum (ER) is thought to play an essential role during egress of malaria parasites because the ER is assumed to be required for biogenesis and secretion of egress-related organelles. However, no proteins localized to the parasite ER have been shown to play a role in egress of malaria parasites. In this study, we generated conditional mutants of the Plasmodium falciparum endoplasmic reticulum-resident calcium-binding protein (PfERC), a member of the CREC family. Knockdown of the PfERC gene showed that this gene is essential for asexual growth of P. falciparum. Analysis of the intraerythrocytic life cycle revealed that PfERC is essential for parasite egress but is not required for protein trafficking or calcium storage. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic maturation of the subtilisin-like serine protease SUB1. Using double mutant parasites, we showed that PfERC is required for the proteolytic maturation of the essential aspartic protease plasmepsin X, which is required for SUB1 cleavage. Further, we showed that processing of substrates downstream of the proteolytic cascade is inhibited by PfERC knockdown. Thus, these data establish that the ER-resident CREC family protein PfERC is a key early regulator of the egress proteolytic cascade of malaria parasites. IMPORTANCE The divergent eukaryotic parasites that cause malaria grow and divide within a vacuole inside a host cell, which they have to break open once they finish cell division. The egress of daughter parasites requires the activation of a proteolytic cascade, and a subtilisin-like protease initiates a proteolytic cascade to break down the membranes blocking egress. It is assumed that the parasite endoplasmic reticulum plays a role in this process, but the proteins in this organelle required for egress remain unknown. We have identified an early ER-resident regulator essential for the maturation of the recently discovered aspartic protease in the egress proteolytic cascade, plasmepsin X, which is required for maturation of the subtilisin-like protease. Conditional loss of PfERC results in the formation of immature and inactive egress proteases that are unable to breakdown the vacuolar membrane barring release of daughter parasites.

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Section: Resultssupporting

confidence: 80%

An Endoplasmic Reticulum CREC Family Protein Regulates the Egress Proteolytic Cascade in Malaria Parasites

Fierro

Asady

Brooks

et al. 2020

mBio

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“…For the pull-down proteomic analysis of PfClpP apt , PfClpR apt and PfClpS apt parasites, the parental line (wild type clone 3D7) was used a control and all pulldown experiments were done in duplicates using two different clones. Immunoprecipitation protocols were performed using anti-V5 antibody as previously described 39 . Briefly, pellets from 10 9 parasites were isolated using cold saponin and were lysed and sonicated in Extraction Buffer (40 mM Tris HCL pH 7.6, 150 mM KCL, and 1 mM EDTA) supplemented with 0.5% NP-40 (VWR) and HALT protease inhibitor (Thermo).…”

Section: Methodsmentioning

confidence: 99%

The Clp System in Malaria Parasites Degrades Essential Substrates to Regulate Plastid Biogenesis

Florentin¹,

Stephens²,

Brooks³

et al. 2019

Preprint

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The human malaria parasite, Plasmodium falciparum, contains an essential plastid called the apicoplast. Most of apicoplast proteins are encoded by the nuclear genome and it is unclear how the plastid proteome is regulated. Here, we study an apicoplast-localized caseinolytic-protease (Clp) system and how it regulates organelle proteostasis. Using null and conditional mutants, we demonstrated that the Clp protease (PfClpP) has robust enzymatic activity that is essential for apicoplast biogenesis. We developed a CRISPR/Cas9 based system to express catalytically-dead PfClpP, which showed that PfClpP oligomerizes as a zymogen and matured via trans-autocatalysis. The expression of a Clp chaperone (PfClpC) mutant led to the discovery of a functional chaperone-protease interaction essential for plastid function. Conditional mutants of the substrate-adaptor (PfClpS) demonstrated its essential function in plastid biogenesis. A combination of multiple affinity purification screens identified the Clp complex composition as well as putative Clp substrates. This comprehensive study reveals the molecular composition and interactions influencing the proteolytic function of the apicoplast Clp system and demonstrates its central role in the biogenesis of the plastid in malaria parasites.

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“…For the pulldown proteomic analysis of PfClpP apt , PfClpR apt , and PfClpS apt parasites, the parental line (wild-type clone 3D7) was used a control and for PfClpP DEAD , PfClpC wt the parental line PfClpP apt was used a control. IP protocols were performed using anti-V5 antibody (PfClpP apt , PfClpR apt , and PfClpS apt ) or anti-Ty antibody (PfClpP DEAD and PfClpC wt ) as previously described (40). Detailed IP and co-IP protocols can be found in SI Appendix, Supplemental Methods.…”

Section: Methodsmentioning

confidence: 99%

Plastid biogenesis in malaria parasites requires the interactions and catalytic activity of the Clp proteolytic system

Florentin

Stephens

Brooks

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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The human malaria parasite,Plasmodium falciparum, contains an essential plastid called the apicoplast. Most apicoplast proteins are encoded by the nuclear genome and it is unclear how the plastid proteome is regulated. Here, we study an apicoplast-localized caseinolytic-protease (Clp) system and how it regulates organelle proteostasis. Using null and conditional mutants, we demonstrate that theP. falciparumClp protease (PfClpP) has robust enzymatic activity that is essential for apicoplast biogenesis. We developed a CRISPR/Cas9-based system to express catalytically deadPfClpP, which showed thatPfClpP oligomerizes as a zymogen and is matured via transautocatalysis. The expression of both wild-type and mutant Clp chaperone (PfClpC) variants revealed a functional chaperone–protease interaction. Conditional mutants of the substrate-adaptor (PfClpS) demonstrated its essential function in plastid biogenesis. A combination of multiple affinity purification screens identified the Clp complex composition as well as putative Clp substrates. This comprehensive study reveals the molecular composition and interactions influencing the proteolytic function of the apicoplast Clp system and demonstrates its central role in the biogenesis of the plastid in malaria parasites.

show abstract

The endoplasmic reticulum chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites

Cited by 24 publications

References 78 publications

An Endoplasmic Reticulum CREC Family Protein Regulates the Egress Proteolytic Cascade in Malaria Parasites

An Endoplasmic Reticulum CREC Family Protein Regulates the Egress Proteolytic Cascade in Malaria Parasites

The Clp System in Malaria Parasites Degrades Essential Substrates to Regulate Plastid Biogenesis

Plastid biogenesis in malaria parasites requires the interactions and catalytic activity of the Clp proteolytic system

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