Francisca Magum Timothy
Tawanda Zininga*- Department of Biochemistry, Stellenbosch University, Stellenbosch, South Africa
Plasmodium falciparum, the predominant cause of severe malaria, thrives within both poikilotherm mosquitoes and homeotherm humans, navigating challenging temperature shifts during its life cycle. Survival in such varying environments necessitate the development of robust mechanisms, including a sophisticated protein folding system to mitigate proteopathy. The parasite needs to control the survival of its host cells which affects its chances of development and propagation. Central to this system are heat shock proteins (Hsps), among which small Hsps (sHsps) play pivotal roles in maintaining proteostasis (protein homeostasis). In both humans and P. falciparum, numerous sHsps have been identified, making them attractive candidates as biomarkers for diagnostic and drug development strategies. Evidence is accumulating suggesting that these sHsps participate in cell death processes, potentially influencing disease pathogenesis. Despite their significance, the precise functions of sHsps in P. falciparum’s adaptation to stress conditions remains largely unknown. Comparative structural analysis of sHsps between human and P. falciparum reveals species-specific variations. Despite conserved tertiary structures, unique motifs are found in parasite sHsps which may modulate specialised chaperone functions. This review discusses the conserved and distinctive motifs of sHsps from the human host and the parasite, offering insights into shared and unique attributes. These findings illuminate the potential for species-specific targeting of sHsps, as players in cell death processes that may foster innovative biomarker identification approaches. As malaria continues to ravage Sub-Saharan Africa, understanding the molecular intricacies guiding parasite survival are essential in the development of interventions with heightened efficacy against this global health crisis.
1 Introduction
The main malaria agent, Plasmodium falciparum, survives between two physiologically distinct environments, in the female Anopheles mosquito and the human hosts. Throughout its complicated life cycle, the parasite encounters stressful conditions as it transitions the diverse environmental conditions, including fluctuations in temperature. Despite, these unfavourable conditions, the parasite has developed a highly efficient protein folding system for survival within the host (Shonhai and Blatch, 2014; Proellocks et al., 2016). Heat shock proteins (Hsps), a conserved and ubiquitous group of molecular chaperones that facilitates protein folding, have been implicated in cellular stress responses in both prokaryotic and eukaryotic organisms (Lindquist and Craig, 2003). Within P. falciparum, Hsps play vital roles in the parasite’s ability to adapt and survive through efficient protein folding, translocation (Pérez-Morales and Espinoza, 2015) and the formation of protein complexes (Botha et al., 2011). The formation of protein complexes by Hsps is significant for facilitating interactions between the host and the parasite (Saha et al., 2018; Zininga and Shonhai, 2019) which are part of the determinants of the pathophysiology of malaria.
In some eukaryotic organisms, Hsps account for approximately 2% of the cell proteome (Blatch, 2022). Among these proteins there are small Hsps (sHsps) which are low molecular weight 15–30 kDa members (Buchner et al., 1998). The sHsps are involved in stress response mechanisms and survival strategies that regulate cell death processes. This positions them as promising biomarker candidates for diagnosis and drug development across a wide array of organisms (Nakamoto and Vígh, 2007; Treweek et al., 2014).
The sHsps are molecular chaperones that function as “holdases” to inhibit the aggregation of denatured proteins in a nucleotide independent manner, thus, without refolding capability. They do this by binding to exposed hydrophobic patches on substrate proteins to suppress unproductive binding to other exposed hydrophobic patches on unfolded protein forming toxic aggregates that result in proteopathy (Gusev et al., 2002; Haslbeck and Vierling, 2015). In response to cellular stress such as heat and oxidative stress, the expression of sHsp is upregulated serving to maintain cellular homeostasis. This phenomenon has been linked to the suppression of cell death in cervical cancer cells (Arrigo and Welch, 1987). It is intriguing to note that the P. falciparum resides within human hepatocytes during the pre-erythrocytic stages, and in the red blood cells during the erythrocytic stages which are cells prone to oxidative stress. It has been reported that cells infected with the parasite have upregulated levels of Hsps (Pires et al., 2023), suggesting that Hsps are involved in the host’s response to the presence of the parasite. Consequently, the modulation of autophagy and apoptosis by sHsps in human cells may also affect the fate of P. falciparum. However, our understanding of the roles of P. falciparum sHsps is currently limited, as they have not been fully characterized. Therefore, comparing the structure and function of these parasite sHsps to their human homologs can provide insights into their functions in the parasite. Additionally, other Hsps have been implicated in parasite survival, drug resistance, and pathogenicity, making them potential targets for drug development (Shonhai and Blatch, 2014; Stofberg et al., 2021). Thus, we utilized bioinformatic tools to examine the structural and functional attributes of P. falciparum sHsps and their human homologues. This knowledge will facilitate the subsequent characterization of P. falciparum sHsps and their potential as biomarkers in cell death.
2 Subcellular localization of P. falciparum small Hsps and their human homolog
In humans, ten isoforms of sHsp, also referred to as HspB1 - 10 have been identified. On the other hand, P. falciparum expresses six isoforms of sHsp which we here refer to as PfHsp20-a (PlasmoDB accession number: PF3D_1304500), PfHsp20-b (PF3D7_0816500), PfHsp20-c (PF3D7_1211100), PfHsp20-d (PF3D7_1448700), PfHsp20-e (PF3D7_1472800), and PfHsp20-f (PF3D7_0314000) (Table 1). As in humans, multiple members of the sHsp exist within the same cellular compartment such as HspB1-10 in cytoplasm, implying functional redundancy. However, they also exhibit tissue-specific expression, such as HspB4 and HspB5 being abundant in the eye lens, HspB9 and HspB10 in the testis and HspB2 in cardiac and skeletal muscles (Tedesco et al., 2022), Additionally, during stress, these cytosolic sHsps translocate to the nucleus where they reside in sub-nuclear structures (Mackay et al., 2003). The P. falciparum sHsps, are also widely distributed in different parasite cell compartments as PfHsp20-a, PfHsp20-b, and PfHsp20-c are predominantly localized within the mitochondria, whilst PfHsp20-d and PfHsp20-f are localized within the nucleus, and PfHsp20-e is resident within the apicoplast, suggesting functional specialisation (Hall et al., 2002). This suggest specialised roles for the sHsps in different organisms.
TABLE 1. Summary of human and P. falciparum sHsps functions and subcellular localization.
3 The general structure of sHsps
The sHsp family members are found across all organisms and have a general structure characterized by a tripartite domain architecture. There are two main groups of sHsps with the first group protomers comprised of a conserved protein-binding domain, resembling the alpha crystallins called the α-crystallin domain (ACD), flanked by a variable N-terminal domain (NTD) and a C-terminal domain (CTD) (Figure 1). The other isoforms have a CHORD and Sgt1 (CS) domain in place of the ACD domain. As a result, there is low sequence conservation between the different sHsp family members. This suggest that the sHsps can be selectively targeted as biomarkers unique in parasites when compared to the human Homologs.
FIGURE 1. General structure of sHsp. (A) Schematic model of the domain organization of parasite ACD containing sHsp isoforms and homo sapiens HspB1. (B) Domain organization of CS domain containing parasite sHsp isoforms. (C) Multiple sequence alignment of human and P. falciparum sHsps. (D) Model interaction model of the C-terminal localized IXI motif with ACD in regulating substrate chaperone binding. In MSA, green line above the alignment indicates the N-terminal domain, grey line shows the ACD, and blue bar shows CTD. Cyan box shows conserved SRLDQxFG sequence on NTD of Human HspBs, Red lines/box indicates IXI/V motif in the NTD/CTD. Orange arrows show known phosphorylation sites. Identical residues are shown in white text on black background and similar residues are shown in black text on grey background, and non-conserved residues are shown in black text on white background. MSA was performed using ClustalW (https://www.ebi.ac.uk/Tools/msa/clustalw2/).
3.1 The sHsp alpha crystallin domain
Generally, the ACD is the most conserved domain among the main members of sHsp family, consisting of approximately 80–100 amino acid residues (Hilton et al., 2013). The ACD is characterized by a significant presence of immunoglobulin G (IgG) like β-pleated sheets (Janowska et al., 2019). This domain forms a compact β-sandwich structure comprising two anti-parallel β-sheets (Figure 1). The ACD of humans forms an antiparallel dimer along the β6–β7 strand. The chaperone functionality of sHsps is attributed to the ACD, which consists of two β-sheets flanked by α-helices.
3.2 The sHsp N-terminal domain
Despite the conserved ACD, sHsps have low amino acid sequence conservation levels on the NTD (Figure 1). The N-terminal domain of sHsps is rich in proline and aromatic residues (Kriehuber et al., 2010). This influences the structural variability which exhibits short α-helices, β-strands, and disordered regions (Van Montfort et al., 2001; Wintrode et al., 2003) (Figure 2). However, this flexibility makes the NTD to play an important role in substrate binding and oligomer assembly, and its truncation leads to a loss of chaperone function (Chaves et al., 2017). In addition, the NTD has higher presence of multiple phosphorylable serine/threonine residues (Strózecka et al., 2012; Heirbaut et al., 2014). The multiple phosphorylation sites identified in the mammalian sHsp NTD facilitate the protein functional roles in cellular protection and stress response. Conversely, a conserved stretch of N-terminal residues from 31 to 35 (residues SRLDQxFG) in HspB1, HspB6, and HspB8 has been identified as a negative regulator of activity (Tedesco et al., 2022). Deletion of this stretch enhanced the chaperoning capability (Heirbaut et al., 2014). In contrast, the SRLFDQxFG was shown to be important in the structure-function regulation of HspB4 and HspB5, where this motif plays a role in the quaternary structure and dynamics of subunit assembly of α-crystallins (Pasta et al., 2003). Both the NTD and the ACD of sHsps have been found to be involved in direct binding to client proteins and functioning as chaperones (Janowska et al., 2019).
FIGURE 2. Comparison of 3-dimensional structure of ACD with CS domain. Superimposed structures of Human and P. falciparum sHsp. (A) shows structural arrangement in ACD, (B) The CS domain fold into similar antiparallel beta sandwich structure that corresponds to the fold of ACD. All structures were retrieved from Alphafold (https://alphafold.ebi.ac.uk/; accessed on 25 September 2023) and visualized using Chimera X (https://www.rbvi.ucsf.edu/chimerax).
3.3 The sHsp C-terminal domain
The CTD of sHsps exhibits significant variation in length and is characterized by a high content of polar and charged residues, rendering it highly disordered (Figure 1). In HspB1, HspB2, HspB4, HspB5, and HspB6, the CTD contains a flexible extension that remains disordered and mobile in solution, which acts as a solubilizing agent during chaperone-mediated interactions with client proteins (Strózecka et al., 2012; Boelens, 2020). Moreover, the CTD regions of HspB1, HspB2, HspB4, and HspB5 feature a conserved motif known as IXI/V (Janoskwa et al., 2019; Mogk et al., 2019). This motif facilitates binding to the hydrophobic groove of a neighbouring ACD, acting as a bridge between dimers and influencing the plasticity and overall structure of sHsp oligomers (Janowska et al., 2019; Boelens, 2020). Interestingly, the flexibility of the CTD enables interactions with various binding partners, regardless of their positioning. This is possibly due to their self-association of the ACD module and IXI/V motif. This contact between the ACD cleft and the IXI motif has the potential to hinder interactions between the sHsp and its client proteins (Figure 1D). For instance, in the HspB1 oligomer and HspB2/HspB3 tetramer, the IXI of one monomer extends to engage with the ACD cleft of an adjacent monomer (Clark et al., 2018; Nappi et al., 2020). Consequently, it is suggested that the dynamic control of this interaction plays an important role in regulating the activity of these sHsps (Balana et al., 2021). In HspB3, HspB6, and HspB8 the absence of the IXI/V motif in the CTD is thought to contribute to their reduced tendency to form higher order oligomers (Boelens, 2020). This is possibly due to their self-association of the ACD module and IXI/V motif. Therefore, absence of the IXI motif in some of the parasite sHsp might as well lead to reduced tendency to form higher order oligomers and the loss of interaction with the ACD. This could impact the protein’s ability to regulate or participate in certain protein-protein interactions.
In, P. falciparum sHsp, the domain architecture comprises of the tripartite, NTD, ACD and CTD as in the human equivalences. Despite possessing these three domains, there is low sequence conservation among the members of the parasite sHsp family (Figure 1). For example, like HspB3, HspB4, HspB5, and HspB6, the parasite PfHsp20-b, PfHsp20-d and PfHsp20-e also possesses the IXI motif in their NTD but lack the conserved SRLDQxFG sequence. Also, PfHsp20-a, PfHsp20-c and PfHsp20-d possesses the IXI motif in their CTD similar to the human HspB1, B2, B4. This implies that this motif may impact the function of PfsHsp in the same way as the human ones.
3.4 The CS domain of P. falciparum and their human homologs
Although sHsp are typically characterized by the presence of a conserved ACD, the three P. falciparum sHsp isoforms PfHsp20-d, PfHsp20-e, and PfHsp20-f possess a conserved CS domain (Figure 2). The CS domain generally share 25% sequence similarities with known human p23 as well as the SGT1protein and Rar1 (Kim et al., 1998; Biebl et al., 2021). The p23 is an established co-chaperone of Hsp90 chaperone family, where it plays an integral role to the chaperone machinery function (Grad et al., 2006). On the other hand, the SGT1 proteins are involved in kinetochore assembly and Rar1 protein implicated in development in animals (Garcia-Ranea et al., 2002). This suggests the CS domain implicates a role for sHsps in developmental processes in animals. It is interesting that structurally, the CS domains fold into a similar antiparallel beta sandwich structural fold of ACD (Figure 2). Thus, there is structural conservation between the ACD and the CS domain suggestive of similarities in function to the human p23 and SGT1 proteins (Singh et al., 2015). Therefore, these CS containing P. falciparum sHsp may have chaperone roles that are specialised for parasite co-chaperone p23 in modulating Hsp90 functional efficiency and in parasite development.
The sHsps chaperone function is regulated by the formation of either large hetero or homo—oligomeric structures of about 150–800 kDa (Mymrikov et al., 2020). The formation of higher order oligomers is a result of ionic and hydrophobic interactions. These interactions are influenced by environmental stressors like temperature fluctuations, pH and protein concentration, which subsequently impact the oligomer size and function with smaller oligomers having enhanced functional efficiency (Janowska et al., 2019).
3.5 Expression patterns of small Hsps
Most Hsps are upregulated in response to stress such as heat shock and oxidative stress due to increased levels of ROS. The sHsps family, known for its heterogeneity, displays temporal and tissue-specific expression patterns, suggesting distinct functions and regulatory mechanisms (Morrow and Tanguay, 2012). For example, increased expression patterns of HspB1 were observed in craniofacial bones during development. It was involved in the balance between differentiation and apoptosis, through modulating the viability of osteoblasts and chondrocytes, which are important in fetal skeletal maturation (Petryszak et al., 2014). In contrast, decreased expression of HspB2 were reported during skeletal muscle cell differentiation which limited the cell’s response to heat stress (Thakur et al., 2019). This resulted in the HspB2 primarily localizing to the cytoplasm of myoblasts and consequently myotubes formed distinct aggregates in the perinuclear spaces of myoblasts after heat shock (Thakur et al., 2019). Thus, sHsps play important roles in limiting cellular stress and maintaining proteostasis during different stages of development.
In P. falciparum, chaperone expression profiles correspond to different physiological states of the malaria parasite (Pallavi et al., 2010). A study by Acharya et al. (2009) observed that organellar chaperones that were targeted to mitochondria or apicoplast were highly upregulated in different physiological states of the parasite. Also, the expression of Hsps in P. falciparum has been implicated in modulating parasite developmental stage and response to oxidative stress (Akide-Ndunge et al., 2009). However, the effect of specific sHsps in parasite survival, and responses when subjected to antimalarials that induce oxidative stress in parasitized RBCs still need to be elucidated. A genome-wide expression profiling shows differential expression of Hsps between trophozoite and schizont stages in a Honduran chloroquine-sensitive HB3 P. falciparum parasite cell line (Bozdech et al., 2003). Other previous studies also reported that the expression profiles for parasite Hsps follows a coordinated and stage-dependent pattern (Rawat et al., 2021). Recently, Hsp20-b was observed to be upregulated in dihydroartemisinin treated parasite cells (Pires et al., 2023). These findings imply that parasite’s Hsp system responds to changes in redox conditions and exhibits significantly increased expression in oxidatively stressed parasites. The simultaneous disruption of the parasite’s antioxidant enzymes and Hsp system could impede parasite growth. Taken together, this suggest that parasite sHsp maybe important players in regulating cell survival under stressful conditions such as drug pressure thereby enhancing drug resistance development.
4 Perspectives of small Hsps in cell death
The existence of three primary types of cell death processes, namely, apoptosis, necrosis, and autophagic cell death, has been well-established in many eukaryotes including humans (Figure 3). Apoptosis functions to eliminate damaged or abnormal cells. Cells depend on checkpoints in the cell cycle to hinder the build-up and propagation of genetic mistakes. These control points in the cell cycle rely on signalling pathways that oversee issues such as DNA damage between cell divisions, the breakdown of DNA replication structures in the synthesis phase, and the inadequate assembly of spindle fibres during the mitosis phase (Beere, 2004). Some of the cell cycle regulatory proteins are controlled by the stress protein responses which influence the progression through the cell cycle influencing survival or death (Tokunaga et al., 2022). As such, several heat shock proteins such as Hsp70, Hsp90 and sHsps have been implicated in cell death processes in humans. For instance, Hsp70 inhibits apoptosis by binding to Apaf-1 and decreasing procaspase-9 recruitment, it also sustains the ability to inhibit apoptosis via other channels other than the cytochrome c/Apaf-1/caspase pathway (Albakova et al., 2020). By binding to anionic phospholipids and maintaining the lysosomal membrane, Hsp70 inhibits stress-induced membrane permeabilization and thus apoptosis (Calvaresi et al., 2021). By extension, this suggest that the role of PfHsp70-1 during intraerythrocytic replication may be involved with inhibition of apoptosis. PfHsp70-1 depletion decreases parasite development, showing its relevance in parasite survival (Lu et al., 2020). Similarly, Hsp90 interacts with proteins such as Bcl-2 to reduce Cytochrome C (Cyt C) release and to regulate the formation of apoptotic bodies. It also has an effect on the cleavage and function of caspases such as caspase 3, 6, and 9 (Peng et al., 2022). The detailed discussion on role of these Hsp90 and Hsp70 family members in cell death processes is beyond the scope of this study. However, it should be mentioned that some sHsps directly interact with various components of the tightly regulated cell death machinery, both upstream and downstream of mitochondrial events (Arrigo, 2005; Cristofani et al., 2019). The protective effects of sHsps are attributed to various biochemical mechanisms, including their role as molecular chaperones for misfolded proteins, regulation of enzyme activities, assistance in cellular detoxification against oxidative stress, and interaction with cell death pathways (Bellyei et al., 2007).
FIGURE 3. Schematic representation of the effect of stress on cell cycle. The cell undergoes the synthesis (S), gap (G1, G2) and mitosis phases during its cycle. However, constant and unmanageable stress events in the mitochondrial or plasma membrane affect this normal cycle leading to cell senescence through either apoptosis, autophagy or necrotic cell death.
4.1 Small heat shock proteins in apoptotic pathways
Apoptosis is the most common type of programmed cell death (PCD) in which plasma membrane and membrane organelles play a pivotal role (Galluzzi et al., 2012). Apoptosis has two main signalling pathways that later converge for its effector mechanism. These pathways include the intrinsic pathway which is a mitochondrial mediated pathway where there is release of apoptogenic factors (Figure 4). The second is the extrinsic pathway which involves signalling through the “death” receptors on the cell surface that function with the specific ligands to effect cell death (Lossi, 2022). Additionally, there is another important mechanism known as perforin/granzyme-mediated apoptosis. This pathway is primarily employed by immune cells to eliminate infected or aberrant cells (Rousalova and Krepela, 2010). It is an essential part of the immune system’s defence against pathogens and cancerous cells. Granzymes are cytotoxic proteases released by cytotoxic T lymphocytes (CD8 T cells) to induce cell death in target cells (Zininga et al., 2018; Hay and Slansky, 2022). They are contained within granules along with perforin. Apart from these, there are also mechanisms that inhibit apoptosis which include the pro-survival pathways that activate the PI3K/Akt (phosphoinositide 3-kinase/protein kinase B pathway (Rane et al., 2001; Song et al., 2005) and the cytoskeletal modulation of cell cycle events. The regulation of the pro- and anti-apoptotic pathways is essential in dictating the fate of the cell. Despite the lack of some members of these apoptotic signalling pathways in P. falciparum, all three forms of cell death have been suggested to occur in liver-stage parasites (Eickel et al., 2013).
FIGURE 4. The schematic representation of the interactions of sHsps with factors on the apoptotic pathways. Interaction of Fas with Daxx triggers apoptosis. Phosphorylated dimers of sHsp interact with Daxx preventing its interaction with ASK1 and Fas, thus inhibiting Daxx mediated apoptosis. sHsp also interacts with the pro-apoptotic molecule, Bax and inhibit its translocation into the mitochondria and subsequent release of cytochrome c. sHsp interacts with cytochrome c and prevent cytochrome c mediated interaction of Apaf-1 with caspase-9 from apoptosome. sHsp inhibits the release of the second mitochondria -derived activator of caspases, Smac, which binds to the inhibitor of apoptosis proteins (IAPs) and promote apoptosis via activation of caspases. Receptor Tyrosine Kinases (RTKs) promote cell survival by activating downstream signalling pathways that inhibit apoptosis. However, suppression of this pathway shifts the balance towards pro-apoptotic signals, leading to cell death (grey background).
4.1.1 Extrinsic and intrinsic apoptotic pathways
Apoptotic signalling through the extrinsic pathway is triggered when specific extracellular ligands bind to transmembrane death receptors on the cell. Some of the receptors involved in signalling include Tumor necrosis factor (TNF) receptor 1 (TNFR1), Fas (also known as CD95/Apo-1), and TNF-related apoptosis-inducing ligand (TRAIL) receptors reviewed in Jan and Chaudhry (2019). The binding of the respective ligands on these death receptors leads to the formation of a complex called the death-inducing signalling complex (DISC) (Ghobrial et al., 2005). This DISC is composed of the Fas-associated death domain (FADD), which acts as an adaptor molecule, along with procaspase-8, procaspase-10, and cellular FLICE inhibitory proteins (c-FLIPs) to activate caspase-8. Caspase-8 is a cysteine—aspartate specific protease that dimerises when activated and dissociates from the DISC to initiate a cascade of caspase activations, which marks the execution phase of apoptosis (Mandal et al., 2020; Orning and Lien, 2021). Several caspases play crucial roles as both initiators and executioners of apoptosis, with their functions tightly linked to their structural characteristics and specific substrate preferences.
The intrinsic apoptotic pathway primarily involves mitochondrial mediation and is activated in response to various cellular stresses like oxidative stress, radiation, and cytotoxic drugs. This pathway includes activation of key apoptotic effector proteins Bax and Bak that form pores onto the mitochondrial membranes (Heimer et al., 2019). These pores lead to the release of cytochrome c to facilitate the formation of the apoptosome formation together with Apaf-1 and procaspase-9. The apoptosome complex then triggers caspase-9 activation, which activates a caspase cascade involving caspase-3, leading to cell breakdown and apoptosis (Unnisa et al., 2023).
The extrinsic and intrinsic pathways converge in the execution phase. The execution phase involves further caspase activation, cytoplasmic endonuclease activity, chromatin condensation, and formation of apoptotic bodies. Caspase-3 is a key player, activated by initiator caspases and activating the caspase-activated DNase (CAD) for DNA breakdown. Executioner caspases also drive cytoskeletal reorganization and apoptotic body formation (Wu and Bratton, 2011; Dorstyn et al., 2018).
4.1.2 Small Hsps and apoptosis
The mechanism by which sHsps regulate apoptosis involves their association with key stress signalling and apoptotic molecules, thereby blocking cell death and promoting cell survival, proliferation, or differentiation (Mehlen et al., 1996; Ikwegbue et al., 2018). Hsps post-translational modifications and/or their presence in a given cellular compartment are important variables in determining the interaction of an sHsp with a given apoptotic partner which influences the outcomes on signalling pathway activation or repression. The role of sHsps in various diseases and their involvement in apoptotic events are summarised in Table 2.
TABLE 2. Role of sHsps in apoptosis.
4.1.2.1 Inhibition of pro-apoptotic proteins
The sHsps play a regulatory role in apoptosis by suppressing the activation of pro-apoptotic proteins through three different strategies. The first strategy is used by HspB5, HspB1, and HspB2 which exert their potent anti-apoptotic effects by directly interacting with pro-apoptotic signalling proteins. sHsp interact with Bcl-2 family of proteins and interfere with their functions thereby preventing permeabilization of the mitochondrial membrane (Bruey et al., 2000) and apoptosome formation (Beere, 2004; Breckenridge and Xue, 2004). By activating survival pathways such as Akt and ERK (Palmieri et al., 2017; Sun et al., 2017), sHsps promote the phosphorylation and inactivation of pro-apoptotic proteins like Bad, effectively neutralizing their apoptotic potential. Furthermore, some sHsps such as HspB5 can enhance the activity of NF-κB, a transcription factor that governs the expression of anti-apoptotic genes (Llambi et al., 2011; Pécot et al., 2016). This leads to an increase in the levels of anti-apoptotic proteins like Bcl-2, further increasing the cell’s resistance to apoptosis.
The second strategy is where some sHsps inhibit both pro-inflammatory and pro-apoptotic caspases by binding directly to them and suppressing caspase activation (Kamradt et al., 2005; Ikwegbue et al., 2018). This regulatory mechanism occurs at multiple levels, each contributing to the modified control of cell survival (Figure 4). HspB1, for instance, interacts with pro-caspase 3, inhibiting its maturation into the active caspase. This inhibition curtails the downstream effector apoptotic events. Similarly, HspB5 has been identified as a negative regulator of the extrinsic apoptotic pathway, specifically inhibiting caspase 3 activation (Kamradt et al., 2002). By disrupting the recruitment of these initiator caspases to death receptor complexes, HspB5 impedes their activation and the subsequent activation of downstream apoptotic effectors.
Another key strategy employed by sHsps is the direct sequestration of cytochrome c, a crucial factor in the initiation of caspase activation (Bruey et al., 2000). Upon release from mitochondria into the cytosol, cytochrome c is inhibited by HspB1, preventing its engagement with caspases. The interaction between HspB1 and cytochrome c is intricate and necessitates the dimerization of HspB1 (Paul et al., 2002). This engagement not only inhibits the direct activation of caspases by cytochrome c but also shields cytochrome c from promoting downstream apoptotic signals. Moreover, HspB1 extends its influence on actively obstructing caspase 3, a key executioner caspase, in its active state. This binding prevents the detrimental impact of active caspase 3 on cellular components and processes crucial for apoptosis execution (Bruey et al., 2000; Paul et al., 2002). For instance, in monocytes undergoing apoptosis, HspB1 binds to and effectively suppresses the activity of caspase 3, providing an additional layer of protection against premature cell death (Paul et al., 2002).
4.1.2.2 Regulation of protein folding and repair
sHsps play a role in protein folding, repair, and refolding of misfolded peptides, which can prevent the accumulation of damaged proteins that may trigger apoptosis. HspB1 as a notable example, it plays a pivotal role in regulating apoptosis by controlling protein folding and repair processes (Shan et al., 2021). HspB1’s interactions with clients guide apoptotic proteins toward correct folding, preventing misfolded proteins from triggering erroneous signals that could lead to cell death (Webster et al., 2019). Additionally, sHsps act as safeguards during cellular stress, forming complexes with malfunctioning proteins to prevent aggregation and disruption of cellular functions (Treweek et al., 2014), thereby averting inadvertent activation of apoptotic pathways. HspB1’s involvement extends to enveloping misfolded proteins, facilitating their restoration to functional states and curbing their accumulation. By facilitating substrate transfer to the main foldases, Hsp70 and Hsp90, sHsps ensure timely repair of misfolded or damaged proteins, particularly crucial in thwarting signals that could initiate cell death (Wegele et al., 2004; van Ommeren et al., 2016). This intricate regulation underscores sHsps’ role in preserving cellular equilibrium and offers potential avenues for modulating apoptotic pathways therapeutically.
4.1.2.3 sHsp in oxidative stress regulation
In reducing oxidative damage, sHsps scavenge reactive oxygen species (ROS) generated during oxidative stress, thereby suppressing apoptosis [reviewed in Ikwegbue et al. (2018)]. sHsps, particularly HspB1 and HspB5, exhibit direct antioxidant activity by scavenging ROS and reactive nitrogen species (RNS) (Arrigo et al., 2005). These harmful molecules, generated as by-products of cellular metabolism or due to environmental factors, can damage lipids, proteins, and DNA, ultimately leading to apoptosis. The main mechanism through which sHsps combat oxidative stress, is by binding to ROS molecules, such as hydrogen peroxide (H2O2), hydroxyl radicals (OH), and superoxide radicals (O2) (Huot et al., 1996; Wu et al., 2015). This interaction helps neutralize the reactivity of these species, reducing their potential to cause cellular damage. By scavenging ROS, sHsps maintain the cellular redox balance and protect cells from oxidative stress-induced apoptosis.
Furthermore, sHsps play a crucial role in regulating apoptosis by modulating the intracellular redox state (Christians et al., 2012). Oxidative stress, characterized by an imbalance between ROS production and the antioxidant defence system, can trigger apoptotic pathways. HspB1 and HspB5 stabilize glutathione levels preventing excessive ROS accumulation, in this way, sHsps help mitigate oxidative damage to cellular components (Préville et al., 1999; Xiong et al., 2020). Additionally, sHsps' ability to interact with and stabilize mitochondrial proteins, such as those involved in the electron transport chain, contributes to reducing ROS generation (Arrigo, 2007). By maintaining a balanced redox environment, sHsps inhibit the activation of pro-apoptotic signalling pathways that often arise from oxidative stress, thereby promoting cell survival and suppressing apoptosis.
Given that the intrinsic pathway of apoptosis is mitochondrial dependent and most of the P. falciparum sHsp are localized within the mitochondria, it is plausible to suggest that these proteins may serve a physiological role similar to their human counterparts in regulating apoptosis. In addition, some of the antimalarial drugs, such as chloroquine, target parasite redox homeostasis generating ROS that ultimately cause parasite death (Akide-Ndunge et al., 2009; Nepveu and Turrini, 2013; Coertzen et al., 2018; Siddiqui et al., 2022). The capability of P. falciparum to regulate the oxidative stress, herald the development of drug resistance development (Lehane et al., 2012; Rosenthal and Ng, 2020). It is tempting to speculate that sHsps could play an important role in antimalarial drug resistance.
4.1.2.4 Phosphorylation of sHsps
The post translational modification of sHsps modulates their function. For instance, phosphorylation of sHsps has been implicated in their regulation of apoptosis and other cellular functions (Janowska et al., 2019). Rapid phosphorylation of sHsps, particularly on serine residues after stress, affect their chaperone function prior to increased protein expression over time (Kostenko and Moens, 2009). Phosphorylation can impact the oligomerization state of sHsps, influencing whether they form large oligomers or exist as smaller oligomers or monomers. The oligomerization state is also a functional regulatory mechanism modulating the chaperone activities of sHsps [reviewed in Haslbeck et al. (2019)]. Phosphorylation-induced changes in oligomerization might alter their affinity for client proteins and their ability to prevent protein aggregation (Janowska et al., 2019; Liu et al., 2020).
In their phosphorylated state, sHsps, exhibit differential capability to interact with other proteins involved in apoptosis regulation. For example, the phosphorylated form of HspB1 directly interacts with Daxx, a protein that connects Fas signalling to apoptosis, inhibiting its interaction with Ask1 and Fas proteins, thus blocking apoptosis triggered by their co-expression (Charette and Landry, 2000). This inhibition is influenced by the phosphorylation and oligomerization status as only dimeric phosphorylated HspB1 is capable of binding Daxx and preventing apoptosis. Interestingly, different triggers like heat, oxidative stress, growth factors, and cytokines causes HspB1 to become phosphorylated by MAPKAP kinase 2, which is activated by MAP kinase SAKP2/p38 (Guay et al., 1997; Kostenko et al., 2009). This phosphorylation changes its structure from large homo-oligomers to dimers and monomers. Stress induced phosphorylation of sHsp provides protection against diverse harmful agents and results in their translocation into the nucleus (Liu et al., 2020). In this state, HspB5 and Hsp25 interact with nuclear components, such as splicing factors and the intracellular lamin A/C (Beere, 2004). This implicates the sHsps’ involvement in maintaining nuclear stability and potentially influencing apoptotic signalling pathways originating from the nucleus.
In addition, phosphorylation of sHsps can also impact signalling pathways involved in apoptosis regulation (Figure 4). For example, phosphorylated HspB1 activates the pro-survival Akt pathway, which in turn affects apoptotic responses by inhibiting caspase activity and phosphorylating pro-apoptotic proteins such as Bad (Song et al., 2005). Moreover, the phosphorylation of HspB1 enhances its interactions with kinases like MK2 and p38-MAPK (Kostenko et al., 2009), leading to activation of these pathways and downstream protective effects. This intricate crosstalk demonstrates how phosphorylated sHsps actively participate in orchestrating key events in cell survival and apoptotic signalling cascades. Furthermore, the chaperone activity of sHsps, which is important for their ability to prevent protein misfolding and aggregation is dependent on their regulation state (Rogalla et al., 1999; Ecroyd et al., 2007). Phosphorylation can either enhance or inhibit the chaperone function of sHsps, depending on the specific phosphorylation sites. Phosphorylated HspB1, for instance, binds to and stabilizes actin, preventing its depolymerization under thermal stress (Ammendola et al., 2021). This preservation of the cytoskeletal structure is crucial for preventing cytochrome c release from mitochondria and subsequent caspase activation. The phosphorylation-induced chaperone activity of sHsps is thus an essential component of their anti-apoptotic function, contributing to the overall cellular response to stress-induced apoptotic stimuli (Mounier and Arrigo, 2002; Povea-Cabello et al., 2017).
Apoptosis has been observed in both multicellular organisms and in unicellular eukaryotes, including P. falciparum. Like mammalian apoptosis, the apoptosis in P. falciparum appears to be an active and protein synthesis-dependent mechanism (Meslin et al., 2011; Sena-Dos-santos et al., 2021) and shows typical apoptotic features such as chromatin condensation, DNA fragmentation, and the formation of apoptotic bodies (Mohapatra et al., 2022). These apoptotic-like features are displayed by the malaria parasite during its lifecycle, particularly during the differentiation of gametocytes into gametes within the mosquito midgut (Kumari et al., 2022). A global proteomic analysis study revealed that P. falciparum increases the production of stress-related proteins, including Hsp70, as well as components of the translation machinery (Rathore et al., 2015). This suggests that the parasite is actively trying to combat the stress, demonstrating its mechanisms to adapt and overcome the challenges posed by proteasome inhibition. However, unlike humans, Plasmodium parasites lack the caspase genes responsible for cell death regulation. Nevertheless, several studies have identified metacaspases (MCA) in P. falciparum parasites, which possess a catalytic domain that bears resemblance to caspases (Meslin et al., 2011; Kumar et al., 2019; Vandana et al., 2020). This similarity has been confirmed that metacaspases are key players involved in cell death through apoptotic-like pathways (Vercammen et al., 2007; Jiang and Wang, 2004; Kumar et al., 2019). Similar to caspases, metacaspases’ activity depends on a functional catalytic domain generated through auto processing, mimicking caspase activation and also function as effectors of cell death (Coll et al., 2010; Zalila et al., 2010). In these organisms, metacaspases have been shown to undergo autoprocessing, leading to the removal of the prodomain, which is likely essential for their cell death function (Meslin et al., 2011). P. falciparum genome contains three metacaspase genes: PfMCA1, PfMCA2, and PfMCA3. PfMCA1 is the only one with the necessary histidine and atypical catalytic dyad with serine in place of cysteine (Meslin et al., 2011; Kumar et al., 2019). When PfMCA1 is expressed in vitro, it undergoes auto-processing, leading to the removal of the pro-domain (Meslin et al., 2011). This auto-processing and release of the catalytic domain of PfMCA1 likely play a vital role in its cell death function in the parasite. However, the PfMCA3 was shown to exhibit a unique activity similar to trypsin and not caspases like activity (Kumar et al., 2019). On the other hand, PfMCA2 was shown that its inhibition promotes apoptosis (Shankar et al., 2020). These findings suggest that parasite metacaspases may modulate cell death processes through unique pathways that still needs to be elucidated.
Interestingly, like other intracellular obligate protozoan, P. falciparum have evolved strategies to avoid direct destruction by the host’s immune system. However, the infected red blood cells that initiate apoptosis as a defence mechanism, express surface receptors such as P. falciparum glutamic-acid-rich protein (PfGARP) that are recognised by macrophages (Mohapatra et al., 2022). These apoptotic red cells are then engulfed by macrophages, potentially eliminating both the parasite and the infected red blood cell (Al-Olayan et al., 2002; Raj et al., 2020). This powerful defence mechanism exerts selective pressure on parasites leading them to develop strategies to manipulate the host red cell’s apoptotic signalling in their favour. This comes at the backdrop that, several protozoan parasites including P. falciparum have been reported to inhibit the apoptotic signalling pathway of the host cell to ensure their survival (Al-Olayan et al., 2002; Viriyavejakul et al., 2014). It still remains to be validated if parasite sHsps modulate host cell death processes such as apoptosis to ensure their survival.
4.2 Small heat shock proteins and autophagy
Autophagy is a fundamental cellular process responsible for the degradation and recycling of cellular constituents. This cellular process known as "self-eating," plays a pivotal role in maintaining cellular homeostasis and supplying energy during periods of nutrient scarcity or stress. Autophagy encompasses three distinct types in eukaryotic cells: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) (Locatelli and Cenci, 2022). While both macroautophagy and microautophagy involve membrane rearrangement for sequestering cytoplasmic material, they differ in the site of sequestration (Figure 5). Macroautophagy forms a double-membrane vesicle, the autophagosome, which likely involves the endoplasmic reticulum (ER), Golgi complex, and/or mitochondria. This autophagosome then fuses with the lysosome or vacuole, delivering its contents (Yorimitsu and Klionsky, 2005). On the other hand, microautophagy directly engulfs cytosolic components into the lysosome or vacuole through invagination and scission of the organelle’s limiting membrane. In contrast, CMA does not entail significant membrane rearrangement, but rather relies on the lipid composition of the lysosome membrane (Yang et al., 2019), Hsps, both in the cytosol and within the lysosome lumen, along with the lysosome-associated membrane protein (LAMP)-2a (Cuervo et al., 2004; Massey et al., 2006). CMA involves the translocation of unfolded proteins across the lysosome’s limiting membrane and requires the involvement of the molecular chaperones such as Hsc70. These Hsc70 identifies misfolded substrates and directs them to the lysosome, where they are unfolded and translocated across the lysosomal membrane for degradation (Baaklini et al., 2020).
FIGURE 5. sHsps are involved in the activation and progression of autophagy. (1) Chaperone-mediated autophagy (CMA), and (2) the ubiquitin/proteasome system are key pathways for oxidized protein degradation. (3) Opposing effects of sHsp in the JNK pathways leads to beclin ubiquitylation and degradation, whilst sHsps activates AKT1 pathway that results in the formation of lysosomal membrane complexes with Hsp70 and LC3-II. (4) Both CMA and JNK pathways converge with effector signalling by fusion to form autophagosome from the fusion of phagophore and lysosome. The formation of the autolysosome is mediated by sHsps.
Increasing evidence has illuminated the multifaceted roles of autophagy in different aspects of human health. The core components of the mammalian autophagic machinery are homologous to the autophagy-related genes (Atg) initially discovered in yeast (Mizushima et al., 2008; Li et al., 2020). Starvation, a potent inducer of autophagy, inhibits the mammalian target of rapamycin (mTOR), a component of the macromolecular complex mTORC1 (Figure 5). mTORC1 negatively regulates another complex involving Unc-51 like autophagy activating kinase 1 (ULK1), ATG13, ATG101, and FAK Family Kinase-Interacting Protein Of 200 kDa (FIP200), which is activated upon nutrient depletion or energy exhaustion, initiating autophagy (Kim et al., 1998; Zhang et al., 2019). The Beclin 1-interacting complex acts as a regulatory platform for autophagy, comprising Beclin 1, BCL-2 family proteins (which inhibit autophagy), class III phosphatidylinositol 3-kinase (VPS34), and ATG14L (He and Levine, 2010). Activation of this complex leads to the production of phosphatidylinositol-3-phosphate, promoting the nucleation of autophagosomal membranes. Autophagosomal elongation requires two ubiquitin-like conjugation systems: ATG5-ATG12 and LC3-ATG8. The conversion of LC3-I to its autophagosomal membrane-associated form, LC3-II, serves as a marker for autophagosome formation (Ravikumar et al., 2010).
Small Hsps have emerged as key regulators of autophagy and protein quality control. Some sHsps in humans, such as HspB1, HspB5, and HspB2, are implicated as significant regulators of autophagic processes in cells by interacting with key autophagy-related proteins (De Maio et al., 2019). For instance, it was reported that over expression of HspB1 induced autophagy and autophagic flux in rat renal (kidney) proximal tubular NRK-LC3 cells (Matsumoto et al., 2015). Hamouda et al. (2014) demonstrated that HspB8 confers resistance to bortezomib, a proteasome inhibitor, by promoting autophagy-mediated clearance of misfolded proteins (Figure 5). Moreover, the enhanced clearance of aggregates through the autophagy pathway was found to re-sensitize bortezomib-resistant myeloma cells to proteasome inhibitors, thereby improving cell viability. The expression levels of sHsps have an effect on the role they play in autophagy (Li et al., 2018). For example, in cardiac myocytes, low HspB8 levels lead to enlarged cell size by activating phosphoglucomutase and inhibiting casein kinase 2, an anti-apoptotic protein (Hamouda et al., 2014). Conversely, elevated HspB8 levels promote apoptosis. HspB8’s interaction with Bag3 forms a complex involving Hsc70 and CHIP, triggering autophagy to clear misfolded proteins (Cheng et al., 2023). This complex also prompts eIF2α phosphorylation, inhibiting protein synthesis and enhancing macroautophagy (Humeau et al., 2020). Depending on the cellular context, autophagy either safeguards against death by clearing damaged organelles and providing energy substrates or leads to self-destructive cell outcomes via excessive autophagic activity. Additionally, autophagy can activate apoptotic or necrotic cell death pathways through shared regulators, like Bcl-2 family proteins.
In P. falciparum, the autophagic pathway has been identified as a process similar to the parasite’s apoptosis under drug pressure. The erythrocytic stages of the parasite were observed to undergo cell death through autophagy-like mechanism (Gaviria et al., 2013). Immunofluorescence and electron microscopic analysis of P. falciparum and P. vivax parasites treated with either chloroquine or artemisinin (Gaviria et al., 2013; Singh and Chakraborty, 2016) showed the presence of vacuoles with single or double membranes, resembling autophagosomes (Ray et al., 2022). However, the canonical autophagy pathway in P. falciparum uses different upstream signalling molecules unlike other organisms, certain upstream kinases responsible for detecting nutrient limitation and inducing autophagy are thought to be absent in the parasite (Cervantes et al., 2014; Usman et al., 2023). Consistent with this, mild nutrient starvation or inhibitors of key upstream kinases did not induce autophagy in P. falciparum. However, downstream proteins involved in autophagy were detected and appear to be functional in the parasite. Several PfATG proteins involved in autophagosome formation, retrieval, and vesicle breakdown were identified (Cervantes et al., 2014). Notably, PfATG8 and PfATG18, critical proteins in the autophagy process, were found to be associated with vesicles throughout the asexual and sexual erythrocytic cycle (Agrawal et al., 2019). These vesicles were also observed within the host red blood cells, suggesting the involvement of a complex export system in transporting parasite proteins to the host cell membrane (Brennand et al., 2011; Duszenko et al., 2011). Despite the accumulating evidence of autophagic processes in P. falciparum, the role of sHsps remains unexplored. Understanding the molecular mechanisms by which sHsps regulate autophagy in the parasite holds the potential for the development of novel malaria therapies.
4.3 Role of small heat shock proteins in necrosis
Necrosis is a form of cell death occurring as a result of prolonged strong cell stress which is associated with the loss of plasma membrane integrity and subsequent leakage of the cytoplasmic material. In some cases, necrosis is secondary to other cell death processes such as late stages of apoptosis and autophagy [reviewed in Green and Llambi (2015)]. Due to these passive processes, necrosis is generally considered as accidental cell death. However, there are some cases where signalling pathways play a role in active necrotic cell death “necroptosis” (Degterev et al., 2008). Necroptosis is not fully understood but is thought to be a receptor-interacting protein (RIP)-kinase dependent cell death process that is triggered by Toll-like receptor (TLR)-3, TRL-4 and/or TNFRI (Llambi et al., 2011; Green and Llambi, 2015; Yu et al., 2021). These death cell receptors activate the kinase activity of RIP3 to recruit mixed lineage kinase domain-like (complex II) facilitated by RIP1 (de Almagro and Vucic, 2015). Complex II when phosphorylated by RIP3 facilitate the plasma membrane disruption and subsequent cell lysis. The increased levels of ROS from mitochondrial damage, NADPH oxidase 1, increase in calcium influx and ATP depletion are thought to be initiating signals for necroptosis. Necroptosis is also thought to initiate inflammatory responses from the released intracellular material and damage associated molecular patterns (DAMP). In addition, RIP3 was shown to induce the cleavage and maturation of highly inflammatory cytokines interleukin –(IL)-1β and IL-18 mediated by NOD–LRR- and pyrin domain containing protein 3 (NLRP3) inflammasome (Vince et al., 2012; Weinlich et al., 2017).
The complications of malaria in the liver, lung, and brain are due to neutrophil infiltration and activation contributing to tissue necrosis (Rocha et al., 2015; Feintuch et al., 2016; de Menezes et al., 2019). In another study, it was observed that during acute P. chabaudi malaria, the liver recruits IL-1α-producing neutrophils as part of the immune response to infected red blood cells in the liver vasculature (de Menezes et al., 2019). This causes the neutrophils in the infected liver to upregulate IL-1α production at both mRNA and protein levels. The recruitment of IL-1α-producing neutrophils to the liver amplifies the inflammatory response through IL-1R signalling, leading to the local secretion of pro-inflammatory cytokines (Gross et al., 2012; de Menezes et al., 2019). The local release of IL-1α promotes TNF-α production, leading to hepatocyte death and the development of necrotic lesions (Basu et al., 2010). This excessive production of pro-inflammatory cytokines, including TNF-α, contributes to the immunopathology of malaria and is associated with disease severity (Nussler et al., 1993). Additionally, TNF-α and NO are involved in the loss of infectivity of circulating gametocytes. TNF-α, together with IFN-γ, optimizes nitric oxide (NO) production for parasite clearance and resolution of fever (Artavanis-Tsakonas and Riley, 2002). Some Hsps play a role in modulating necrotic cell death. For example, HspB1 is involved in regulating necrosis. TNF-α treatment leads to the rapid phosphorylation of HspB2, a homolog of HspB1, independent of C kinase (Rogalla et al., 1999). This phosphorylation is important in mediating HspB1’s interaction with IKK-γ and suppressing TNF-α-induced NF-κB activation (Saklatvala et al., 1991). Also, Hsp90 exerts influence over key proteins within the RIP1/RIP3/MLKL pathway by modulating their stability, phosphorylation, and expression. Hsp90 is involved in the regulation of both RIP3 and MLKL, shaping their roles in necroptosis. The exact effects of Hsp90 inhibitors may differ, and prolonged inhibition can result in the degradation of these proteins (Jacobsen et al., 2016).
When comparing necrosis in mammals to that in Plasmodium infection, it becomes evident that in mammals, necrosis arises under pathological conditions with stronger or prolonged stimuli. However, in Plasmodium infection, tissue necrosis is an unfortunate complication that is primarily driven by the inflammatory response against parasite infection (Hui et al., 2023). This results in the release of pro-inflammatory cytokines such as TNF-α and IL-1α (Porter et al., 2008). These cytokines contribute to cell death. Similarly, P. vivax infection has been reported to cause renal cortical (Nair et al., 2019), intestinal (Pawelka et al., 2021), necrosis. Despite the lack of data on the role of endogenous parasite sHsps in parasite death, the potential role of sHsps in host tissue necrosis highlights the complex interplay between the host’s immune response and tissue damage.
5 Future perspectives
5.1 Small Hsp as immunomodulants
Hsps exhibit immunomodulatory functions, making them attractive candidates for vaccines (Miroshnichenko et al., 2005; Zininga et al., 2018) specifically, sHsps plays a role in immunomodulation through their participation in antigen presentation (AP), a critical step in activating adaptive immune responses (Li et al., 2002; Miroshnichenko et al., 2005). AP facilitates peptide binding and delivery to major histocompatibility complex (MHC) molecules (Li et al., 2002). This interaction enhances antigen processing and presentation, leading to the activation of T cells and subsequent immune responses. Furthermore, sHsps have been found to modulate inflammatory responses by regulating the activity of pro-inflammatory cytokines and chemokines (Van Noort et al., 2012; Powell et al., 2021). For instance, sHsps have been implicated in enhancing the functions of immune cells. There have been reports that Hsp70 enhances the cytotoxic activity of natural killer (NK) cells against tumor cells and parasite infected RBCs through perforin and granzyme B dependent cell lysis (Lv et al., 2012; Zininga and Shonhai, 2019). This highlights the potential of sHsps, although not shown to be surface exposed on RBCs to enhance innate immune responses and contribute to antiparasitic immunity. Due to their immunomodulatory properties, sHsps also have the potential to serve as adjuvants in malaria vaccine formulations to boost vaccine efficacy.
5.2 Small Hsps as diagnostic markers
sHsps have also shown promise as diagnostic markers for various diseases. The unique expression patterns and alterations of sHsps in diseased conditions make them attractive candidates for diagnostic purposes (Yu et al., 2010; Morrow and Tanguay, 2012; Wyciszkiewicz et al., 2019). Aberrant expression of sHsps has been observed in different types of cancer, and their levels can serve as indicators of disease progression and prognosis. High HspB1 expression levels are associated with advanced tumour stages, metastasis, and poor patient outcomes. Moreover, sHsps have been explored as diagnostic markers in cardiovascular diseases (Bleijerveld et al., 2013) and neurodegenerative disorders [reviewed in Navarro-zaragoza et al. (2021)]. Differential life-stage specific expression levels and localization of sHsps in malaria could potentially aid in the early diagnosis and monitoring of malaria. In addition, the low level of conservation between the human and malarial sHsp, offers promise to selective targeting of parasite Hsps as diagnostic markers. There is need for further validation studies to fully establish the clinical utility of sHsps as diagnostic markers in malaria.
5.3 Small Hsps as drug target
Small Hsps also serve as attractive drug targets due to their involvement in various disease processes. The unique structural and functional characteristics of sHsps among the human host and the parasite make them valuable targets for therapeutic intervention. The sHsp potential as drug targets in cancer and other diseases heighten the need for exploration as targets for novel antimalarial drugs. The overexpression of certain Hsps, such as sHsp and Hsp70, has been associated with cancer progression, chemotherapy resistance, and poor patient outcomes (Acunzo et al., 2012). By targeting sHsps, it is possible to disrupt their pro-survival and anti-apoptotic functions, thereby sensitizing cancer cells to chemotherapy or radiation therapy. Several approaches have been investigated, to repurpose some of the anti-Hsp cancer drugs into malaria treatment options including the development of small molecule inhibitors that specifically bind to Hsps and disrupt their interactions with client proteins (Zininga and Shonhai, 2019; Stofberg et al., 2021). Beyond general inhibitors of the heat shock response, there is a need to develop inhibitors that target specific sHsps, particularly HspB1 and HspB5. Peptide aptamers that interact specifically with HspB1 have been proposed as potential inhibitors (Gibert et al., 2013). In cell cultures, these aptamers disrupted the phosphorylation and oligomerization of HspB1, weakening its anti-apoptotic and cytoprotective property (Gibert et al., 2013; Yoon and Rossi, 2018). In addition, the small molecule inhibitor (E)-5-(2-Bromovinyl)-2-Deoxyuridine (RP101) that targets HspB1 in cancer therapy (Seul-Ki et al., 2019), offers promise for repurposing to malaria. RP101 is a compound that counteracts HspB1’s functions, hindering gene amplification and enhancing cell death (Fahrig et al., 2006; Heinrich et al., 2011). Clinical studies involving RP101 have shown promise in extending the survival of pancreatic cancer patients (Heinrich et al., 2011; de Gooijer et al., 2021). RP101 acts as a chemo-sensitizing agent, effectively countering resistance and amplifying the impact of several chemotherapeutic medications such as mitomycin, gemcitabine, cisplatin, and cyclophosphamide (Xiong et al., 2020; Yang et al., 2021). Similarly, another drug, quercetin, a naturally occurring bioflavonoid derived from plants, exhibit chemo sensitising capabilities through inhibition of the expression of heat shock factor-1 (HSF-1) and its binding to the heat shock element in DNA (Wang et al., 2018; Vafadar et al., 2020). Quercetin augments the efficacy of primary chemotherapeutic agents like doxorubicin, gemcitabine, 5-fluorouracil, and cisplatin. Interestingly, beyond inhibiting HspB1 expression, quercetin also inhibits HspB1’s function by impeding its phosphorylation within cancer stem cells (CSCs) (Chen et al., 2012). Considering the WHO guidelines on malaria treatment that combination therapeutics are the first line options in several countries, the chemo sensitization of RP101 and quercetin has the potential to be the “silver bullet” in reversing drug resistance to common antimalarial therapeutics.
6 Conclusion
In conclusion, sHsps play crucial roles in the stress response and survival strategies of P. falciparum, the causative agent of malaria. These proteins are involved in protein folding, translocation, and the formation of protein complexes, facilitating the parasite’s adaptation and interactions with the host. Comparing the structure and function of P. falciparum sHsps to their human counterparts provides valuable insights into their potential as druggable targets. Additionally, the ability of sHsps to modulate antigen presentation, regulate inflammatory responses, and enhance immune cell functions highlights their potential in designing effective vaccines. Furthermore, sHsps show promise as diagnostic markers for malaria due to their altered stage specific expression patterns and unique structure that can be targeted by antibodies used in diagnostic kit development. Lastly, targeting sHsps opens new possibilities for drug development, in antimalarial treatments from repurposed and further improved cancer therapeutics. Disrupting the functions of sHsps can sensitize P. falciparum to current antimalarials in efforts to roll back malaria.
Author contributions
FT: Investigation, Visualization, Writing–original draft. TZ: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Visualization, Writing–review and editing.
Funding
The authors declare financial support was received for the research, authorship, and/or publication of this article. This work is based on the research supported in part by the Department of Science and Technology/National Research Foundation (NRF) of South Africa (Grant Numbers 129401 and 145,405) awarded to TZ; Mandela-Rhodes scholarship awarded to FT.
Acknowledgments
The authors would like to acknowledge Stellenbosch University Sub-Committee B and the ECAD and the Department of Higher Education and Training (DHET) Future Professors Programme fellowship for support to TZ. We thank Karli Bothma for critical reading of this manuscript. We are grateful to Frontiers for waiver of the Article processing charges for this publication.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
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Keywords: small heat shock proteins, molecular chaperones, biomarker candidates, Plasmodium falciparum, malaria
Citation: Timothy FM and Zininga T (2023) Small heat shock proteins as modulators of cell death in Plasmodium falciparum parasites and its human host. Front. Cell. Death 2:1322780. doi: 10.3389/fceld.2023.1322780
Received: 16 October 2023; Accepted: 14 November 2023;
Published: 29 November 2023.
Edited by:
Farhang Alem, George Mason University, United StatesReviewed by:
Yuyao Tian, Boston Children’s Hospital and Harvard Medical School, United StatesMahendra Seervi, All India Institute of Medical Sciences, India
Copyright © 2023 Timothy and Zininga. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Tawanda Zininga, tzininga@sun.ac.za