Specific calpain activity evaluation in Plasmodium parasites Mayrim M. Gomes a,c , Alexandre Budu b , Priscilla D.S. Ventura c , Piero Bagnaresi b , Simone S. Cotrin b , Rodrigo L.O.R. Cunha d , Adriana K. Carmona b , Luiz Juliano b , Marcos L. Gazarini c,⇑ a Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, UNIFESP, São Paulo, SP, Brazil b Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, SP, Brazil c Departamento de Biociências, Universidade Federal de São Paulo, Santos, SP, Brazil d Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, SP, Brazil article info Article history: Received 1 July 2014 Received in revised form 13 August 2014 Accepted 6 September 2014 Available online 2 October 2014 Keywords: Plasmodium Proteases Calpain Calcium signaling Calmodulin abstract In the intraerythrocytic trophozoite stages of Plasmodium falciparum, the calcium-dependent cysteine protease calpain (Pf-calpain) has an important role in the parasite calcium modulation and cell development. We established specific conditions to follow by confocal microscopy and spectrofluorime- try measurements the intracellular activity of Pf-calpain in live cells. The catalytic activity was measured using the fluorogenic Z-Phe-Arg-MCA (where Z is carbobenzoxy and MCA is 4-methylcoumaryl-7-amide). The calmodulin inhibitor calmidazolium and the sarcoplasmic reticulum calcium ATPase inhibitor thapsi- gargin were used for modifications in the cytosolic calcium concentrations that persisted in the absence of extracellular calcium. The observed calcium-dependent peptidase activity was greatly inhibited by specific cysteine protease inhibitor E-64 and by the selective calpain inhibitor ALLN (N-acetyl-L-leucyl- L-leucyl-L-norleucinal). Taken together, we observed that intracellular Pf-calpain can be selectively detected and is the main calcium-dependent protease in the intraerythrocytic stages of the parasite. The method described here can be helpful in cell metabolism studies and antimalarial drug screening. Ó 2014 Elsevier Inc. All rights reserved. Malaria is an aggressive disease that is responsible for millions of deaths annually [1]; therefore, it is urgent to identify new drug targets and develop new treatment strategies. Plasmodium prote- ases participate in essential events for parasite life, including the invasion of and egress from erythrocytes and hemoglobin degrada- tion for the acquisition of amino acids [2–4]. Malaria parasite pro- teases, such as aspartic, cysteine, and serine proteases and metalloproteases, have been considered potential therapeutic tar- gets [5–7]. Falcipain-2 and falcipain-3, which are the best-studied members of this class, are located in the parasite’s food vacuole and are essential for the hydrolysis of hemoglobin [8] and develop- ment [9]. In addition, part of Plasmodium protease activities are regulated by cellular events with release calcium [10], which is an important second messenger that regulates various functions in eukaryotic cells such as protein secretion, gene expression, and cellular development [11,12]. Calpain, a calcium-dependent cysteine protease expressed in mammals and other organisms, has an ortholog identified in P. falciparum that has been associated with the development and invasion and egress of the parasite from the host cell [7,13–18]. The Plasmodium genome encodes a unique calpain [15,19] that is expressed during all intraerythrocytic stages and possesses high sequence similarity to Caenorhabditis elegans calpain, which is classified as an atypical calpain [19]. However, there is a lack of biochemical data related to this enzyme, most likely due to the unusual gene size (6.147 bp) [15]. Studies per- formed on rodent malaria parasite species (Plasmodium chabaudi) and parasites responsible for human malaria (Plasmodium falcipa- rum) have shown that both maintain nanomolar levels of cytosolic calcium during maturation [20] with spontaneous oscillations [21,22] and use specific organelles (endoplasmic reticulum, acidic compartments, and mitochondria) to store this ion [23,24]. In the erythrocyte egress step, the increase in the parasite’s cytoplasmic calcium is essential and most likely is related to calpain activation and erythrocyte cytoskeleton destabilization [25]. The growth of Plasmodium parasites is inhibited by a variety of Ca 2+ ionophores, Ca 2+ channel blockers, calcium chelators, and calmodulin inhibitors [26–28], showing the importance of this ion to parasite survival and development. Here, we report a selective measurement method to evaluate intracellular calpain activity in Plasmodium parasites and the influ- ence of calcium homeostasis disruption by inhibiting calmodulin or endoplasmic calcium pump. http://dx.doi.org/10.1016/j.ab.2014.09.005 0003-2697/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Fax: +55 13 32218058. E-mail addresses: marcos.gazarini@unifesp.br, marcos.gazarini@gmail.com (M.L. Gazarini). Analytical Biochemistry 468 (2015) 22–27 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio