Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor

Tan, Michele S. Y.; Koussis, Konstantinos; Withers‐Martinez, Chrislaine; Howell, Steven; Thomas, J A; Hackett, Fiona; Knuepfer, Ellen; Shen, Min; Hall, Matthew D.; Snijders, Ambrosius P; Blackman, Michael J.

doi:10.15252/embj.2020107226

Cited by 12 publications

(9 citation statements)

References 55 publications

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“…Moreover, autocatalytic maturation of SERA6 needs a PVlocated protein cofactor, named merozoite surface antigen 180, which is also a SUB1 substrate. This multi-step proteolytic process is required for dismantling the host RBC cytoskeleton facilitating the parasite egress (Tan et al, 2021). Therefore, it remains to be further described how Ca 2+ may modulate actors in these proteolytic cascade events.…”

Section: Other Effectors Involved In Ca 2+ Signalingmentioning

confidence: 99%

Calcium in the Backstage of Malaria Parasite Biology

Oliveira

Alborghetti

Carneiro

et al. 2021

Front. Cell. Infect. Microbiol.

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The calcium ion (Ca2+) is a ubiquitous second messenger involved in key biological processes in prokaryotes and eukaryotes. In Plasmodium species, Ca2+ signaling plays a central role in the parasite life cycle. It has been associated with parasite development, fertilization, locomotion, and host cell infection. Despite the lack of a canonical inositol-1,4,5-triphosphate receptor gene in the Plasmodium genome, pharmacological evidence indicates that inositol-1,4,5-triphosphate triggers Ca2+ mobilization from the endoplasmic reticulum. Other structures such as acidocalcisomes, food vacuole and mitochondria are proposed to act as supplementary intracellular Ca2+ reservoirs. Several Ca2+-binding proteins (CaBPs) trigger downstream signaling. Other proteins with no EF-hand motifs, but apparently involved with CaBPs, are depicted as playing an important role in the erythrocyte invasion and egress. It is also proposed that a cross-talk among kinases, which are not members of the family of Ca2+-dependent protein kinases, such as protein kinases G, A and B, play additional roles mediated indirectly by Ca2+ regulation. This statement may be extended for proteins directly related to invasion or egress, such as SUB1, ERC, IMC1I, IMC1g, GAP45 and EBA175. In this review, we update our understanding of aspects of Ca2+-mediated signaling correlated to the developmental stages of the malaria parasite life cycle.

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Section: Other Effectors Involved In Ca 2+ Signalingmentioning

confidence: 99%

Calcium in the Backstage of Malaria Parasite Biology

Oliveira

Alborghetti

Carneiro

et al. 2021

Front. Cell. Infect. Microbiol.

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“…Among others, the exonemes contain the subtilisin‐like protease SUB1 and the aspartic protease plasmepsin X (PMX). Once activated by PMX, SUB1 processes a variety of targets that are present in the PV, like the serine‐repeat antigen proteins SERA5 and SERA6 as well as the merozoite surface protein MSP1, and these proteins are later required for destabilizing the RBC cytoskeleton prior to RBCM destruction as well as for mediating the invasion of a new RBC by the freshly released merozoites (Arastu‐Kapur et al, 2008; Collins et al, 2017; Mukherjee et al, 2023; Nasamu et al, 2017; Ruecker et al, 2012; Silmon de Monerri et al, 2011; Stallmach et al, 2015; Tan et al, 2021; Thomas et al, 2018; Yeoh et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Comparative proteomics of vesicles essential for the egress of Plasmodium falciparum gametocytes from red blood cells

Sassmannshausen

Bennink

Distler

et al. 2023

Molecular Microbiology

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Transmission of malaria parasites to the mosquito is mediated by sexual precursor cells, the gametocytes. Upon entering the mosquito midgut, the gametocytes egress from the enveloping erythrocyte while passing through gametogenesis. Egress follows an inside‐out mode during which the membrane of the parasitophorous vacuole (PV) ruptures prior to the erythrocyte membrane. Membrane rupture requires exocytosis of specialized egress vesicles of the parasites; that is, osmiophilic bodies (OBs) involved in rupturing the PV membrane, and vesicles that harbor the perforin‐like protein PPLP2 (here termed P‐EVs) required for erythrocyte lysis. While some OB proteins have been identified, like G377 and MDV1/Peg3, the majority of egress vesicle‐resident proteins is yet unknown. Here, we used high‐resolution imaging and BioID methods to study the two egress vesicle types in Plasmodium falciparum gametocytes. We show that OB exocytosis precedes discharge of the P‐EVs and that exocytosis of the P‐EVs, but not of the OBs, is calcium sensitive. Both vesicle types exhibit distinct proteomes with the majority of proteins located in the OBs. In addition to known egress‐related proteins, we identified novel components of OBs and P‐EVs, including vesicle‐trafficking proteins. Our data provide insight into the immense molecular machinery required for the inside‐out egress of P. falciparum gametocytes.

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“…Phenotypic screening of chemical libraries against P. falciparum blood-stage parasites cultured in vitro (Gamo et al, 2010) and target-based approaches (recently reviewed in Guerra & Winzeler, 2022) have been used to identify and develop different drug candidates, particularly under the supervision of the Medicines for Malaria Venture (https:// www.mmv.org/). Among the many new drug targets identified, Plasmodium subtilisin-like serine protease 1 (SUB1) has emerged as a promising candidate because it meets several of the expected prerequisites: (i) the sequence and organization of the SUB1 active site are highly related to bacterial subtilisins and are significantly distant from human subtilisins (Barale et al, 1999;Giganti et al, 2014), suggesting that highly specific SUB1 inhibitors could be produced without unwanted off-target effects, (ii) the sub1 gene is required for the Plasmodium life cycle (Yeoh et al, 2007;Thomas et al, 2018), thus excluding biological redundancy, and (iii) SUB1 is a key player in the finely regulated cascade of proteases that govern egress of the parasite from the host red blood cells (Thomas et al, 2018;Yeoh et al, 2007;Pino et al, 2017;Nasamu et al, 2017;Collins et al, 2013;Tan et al, 2021;Mukherjee et al, 2022;Dvorin & Goldberg, 2022), a key step in the asexual intraerythrocytic cycle of Plasmodium sp. These repeated cycles of parasite growth and egress cause the symptoms of malaria.…”

Section: Introductionmentioning

confidence: 99%

3D structures of the Plasmodium vivax subtilisin-like drug target SUB1 reveal conformational changes to accommodate a substrate-derived α-ketoamide inhibitor

Martinez,

Batista,

Maurel

et al. 2023

Acta Crystallogr D Struct Biol

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The constant selection and propagation of multi-resistant Plasmodium sp. parasites require the identification of new antimalarial candidates involved in as-yet untargeted metabolic pathways. Subtilisin-like protease 1 (SUB1) belongs to a new generation of drug targets because it plays a crucial role during egress of the parasite from infected host cells at different stages of its life cycle. SUB1 is characterized by an unusual pro-region that tightly interacts with its cognate catalytic domain, thus precluding 3D structural analysis of enzyme–inhibitor complexes. In the present study, to overcome this limitation, stringent ionic conditions and controlled proteolysis of recombinant full-length P. vivax SUB1 were used to obtain crystals of an active and stable catalytic domain (PvS1Cat) without a pro-region. High-resolution 3D structures of PvS1Cat, alone and in complex with an α-ketoamide substrate-derived inhibitor (MAM-117), showed that, as expected, the catalytic serine of SUB1 formed a covalent bond with the α-keto group of the inhibitor. A network of hydrogen bonds and hydrophobic interactions stabilized the complex, including at the P1′ and P2′ positions of the inhibitor, although P′ residues are usually less important in defining the substrate specificity of subtilisins. Moreover, when associated with a substrate-derived peptidomimetic inhibitor, the catalytic groove of SUB1 underwent significant structural changes, particularly in its S4 pocket. These findings pave the way for future strategies for the design of optimized SUB1-specific inhibitors that may define a novel class of antimalarial candidates.

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Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor

Cited by 12 publications

References 55 publications

Calcium in the Backstage of Malaria Parasite Biology

Calcium in the Backstage of Malaria Parasite Biology

Comparative proteomics of vesicles essential for the egress of Plasmodium falciparum gametocytes from red blood cells

3D structures of the Plasmodium vivax subtilisin-like drug target SUB1 reveal conformational changes to accommodate a substrate-derived α-ketoamide inhibitor

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