Introduction and Review of Literature 1.1 Introduction
1) Invasion: This is the first essential step of parasite to start its life cycle inside the RBC
1.3 Proteases of P.falciparum
1.3.4 Threonine proteases
(ii) Egress: Serine protease PfSUB1 is a versatile protease playing various roles in hepatic stage and merozoite invasion of fresh RBCs. Recently it was reported to also have a key role in egress of merozoites in calcium dependent manner which was confirmed by mutagenesis and inhibition study [114, 118, 129-131]. Besides invasion and egress, PfSUB3 was found to have specificity towards profilin which is a multifunctional protein, suggesting PfSUB3 to be involved in the processes of motility, virulence and immune evasion [132, 133]
Figure 1.8. Role of serine protease (PfSUB1) during egress of merozoites. PfSUB1 indirectly mediates RBC rupture by maturing cysteine proteases (SERA family).
Blocking of either PfSUB1 or SERA using specific inhibitors results in blocking of egress.
Therapeutic potentials of serine proteases: Serine proteases in P.falciparum are mostly uncharacterized and not many inhibitors have been synthesized as compared to cysteine and aspartic proteases. However, potent inhibitors have been identified for some serine proteases of essential roles (Table 1.4). PfSUB1 inhibitors result in blocking egress and invasive capabilities of merozoites [113]. Selective PfSUB2 inhibitor also blocks the shedding of MSP1 and AM1 effectively thus hindering invasion. Recent findings on rhomboid inhibitors against PfROM1 and 4 have also shown significant effects on invasion and sporozoite motility which ultimately inhibit sporozoite infection to host cells [127]. PfROM1 is also shown to play important role in formation of parasitophorous vacuole [134]. Owing to these potent effects of inhibitors on parasitic growth and development, serine proteases are also in the limelight to be used as drug targets.
threonine proteases which utilize a particular N-terminal threonine residue and are found in all eukaryotes and archaea bacteria predecessor ClpQ/hslV is present. They represent an important check point for protein quality control by degrading the used or misfolded proteins into amino acids for synthesis of new ones [136]. Proteins that are damaged, misfolded or have fulfilled their physiological role are tagged by a multi-ubiquitin chain which in turn is recognized and bound by subunits of the 19S regulatory particle of the 26S proteasome. The regulatory particle deubiquitinates the target protein unfolds it and threads the linearized polypeptide chain into the 20S core particle of the proteasome where it is degraded (Figure 1.9). These proteasome and its prokaryotic predecessor constitute the T1 family of threonine proteases as T1A and T1B respectively.
Figure 1.9. Schematic representation of proteasome degradation pathway. The 20S and the regulatory cap form the catalytic 26S system. The unused unbiquitinated protein is recognized by regulatory cap and degraded by the 20S core releasing the ubiquitins.
Features of threonine proteases and their roles in P.falciparum: Threonine proteases constitute 14% of the total proteases. Exceptionally, P.falciparum genome possesses both eukaryotic proteasome and prokaryotic predecessor (PfhslV in P.falciparum) which are encoded by 14 genes and one gene respectively [137]. The proteasome is composed of 14α subunits and 14β subunits whereas the PfhslV is composed of two subunits (Table 1.6). The former is expressed throughout the life cycle while the latter is expressed only at schizont and merozoites. Although activity of threonine proteases is detected in
P.falciparum, the individual component of threonine proteases in the proteosome complex is still uncharacterized except for PfhslV [60, 138, 139]. The prokaryotic homologue Pfhs1V protease is found to be involved in growth and development of mitochondria in P.falciparum which is evident from the mutagenesis experiment.
Mutation in the protease blocked normal development and function of mitochondria. This partially characterized PfhslV/PfCIpQ protease is ATP dependent threonine protease and is localized in the cytosol [140]. Recently a ubiquitinin proteasome complex of P.falciparum was reported to be involved in melatonin signaling in co-ordination with atypical kinase PfPK7 [141].
Table 1.6. Members of the threonine proteases in P.falciparum genome*
Threonine protease family
Protease name PlasmoDB ID Characterization status
T1
Proteasome α1 PF14_0716 No
Proteasome α2 MAL6P1.88 No
Proteasome α3 PFC0745c No
Proteasome α4 PF13_0282 No
Proteasome α5 PF07_0112 No
Proteasome α6 MAL8P1.128 No
Proteasome α7 MAL13P1.270 No
Proteasome β1 PFE0915c No
Proteasome β2 MAL8P1.142 No
Proteasome β3 FA0400c No
Proteasome β4 PF14_0676 No
Proteasome β5 PF10_0111 No
Proteasome β6 PFI1545c No
Proteasome β7 PF13_0156 No
PfhslV Not assigned Yes
* Data is compiled from plasmodium database (PlasmoDB.org) and through literature searches.
Therapeutic potentials of threonine proteases: Threonine proteases are new class of protease identified in plasmodium genome and most of which remains uncharacterized.
However, evidences of threonine proteases with multiple functions are identified through inhibitors study showing its potentiality as a drug target [139, 142, 143]. The only characterized PfhslV/PfCIpQ protease is a novel drug target candidate that has no homolog in the human host. Silencing of PfHslV reduced the growth of parasites [144].
Its expression in schizont and merozoite implicates its role in RBC stages. Its roles in hepatic and sexual stages have also been implicated by its response to proteasome inhibitors during these stages [145]. Treatment of parasites with specific threonine protease inhibitor such as epoxomicin, MG132 and lactacystin blocked catalytically active proteasome subunits and delayed parasitic development. This led to the accumulation of ubiquitinated proteins and finally to parasite death suggesting a crucial role played by proteasome [138, 144, 146]. A new class of proteasome inhibitors
peptidosulfonylflorides also showed effective growth inhibition against multi-drug resistant and chloroquine sensitive P.falciparum parasites [147]. All these reasons interest researchers to further study the insights of threonine proteases for development of inhibitors against these proteases for use as antimalarial drugs.