COVID-19 and Cell Stress
10.3 Cell Stress
It is a stressful time! COVID-19 creates a pleth- ora of stresses from the sub-cellular level up to the whole globe. Cells can be exposed to a wide range of environmental challenges that impact different sub-cellular levels. Various stressors like xenobiotics, ionizing radiation, hypoxia, chemical toxins, heat stress, oxidative stress, or infectious agents, mainly viruses, lead to pertur- bation in normal cellular functions and homeo- stasis (Galluzzi et al. 2018). Cells execute various adaptive mechanisms to cope with stress and re- establish homeostasis, which include heat shock response, unfolded protein response, and autophagy.
Cells mount to appropriate defensive response based on stress duration, cell type, and macromo- lecular damage. If the stress exceeds the repair capacity and becomes uncontrollable, the cell will turn to apoptosis or programmed cell death (Mehrbod et al. 2019; Samali et al. 2010). The coordination between all regulatory mechanisms is a decision-making for cell fate whether to repair the damage and restore homeostasis or directed to cell demise.
Heat shock response, in general, is a protec- tive response to handle cell stimuli (e.g., heat shock, heavy metals, and oxidative stress) where cells activate the transient expression of chaper- ones or heat shock proteins (HSP). The chaper- ons alleviate the heat shock damaged consequences by refolding the unfolded or aggre- gated proteins. It confers the cells a thermotoler- ance to gain more resistance for various lethal factors (Samali et al. 2010; Richter et al. 2010).
One member of the heat shock protein 70 (HSP70) is the HSPA5, also termed glucose- regulated protein 78 (GRP78) or binding immu- noglobulin protein (Bip). It can be found in the lumen of the endoplasmic reticulum (ER) in all eukaryotic cells (Ibrahim et al. 2019; Lee 2014).
HSPA5 is a 654 amino acid protein that corrects the folding, assembles, and prevents the transport of misfolded proteins (Hendershot et al. 1994;
Haas 1991; Gething and Sambrook 1992).
HSPA5 is a water-soluble protein with small groups of hydrophobic amino acid patches. These
patches are responsible for their role in recogni- tion of unfolded proteins (Ting and Lee 1988).
HSPA5 has two domains, substrate-binding domain (SBD) at the C-terminal and ATP, or nucleotide, binding domain (ABD or NTP) at the N-terminal (Lindquist and Craig 1988). In the case of cell stress, such as cancer or viral infec- tion, the expression of HSPA5 is increased (Ibrahim et al. 2019).
Autophagy is a multistep process required for the capture and turnover of unfolded proteins or their aggregates, organelles (e.g., endoplasmic reticulum, mitochondria, peroxisome), and inva- sive pathogens (e.g., bacteria, viruses) through delivering them to the lysosome to be degraded and recycled. The clearance of unwanted materi- als is governed through three main routes: macro- autophagy (Feng et al. 2014; Choi et al. 2018), microautophagy (Mijaljica et al. 2011), and chaperone-mediated autophagy (Kaushik and Cuervo 2018).
Microautophagy is the destruction of cyto- plasmic materials through a direct engulfment by the lysosome. During the chaperone-mediated autophagy, the unfolded proteins which have a KFERQ motif are recognized by the cytosolic chaperone (HSPA8). Chaperone translocates the substrates to the lysosome via lysosomal- associated membrane protein 2A (LAMP2A). On the other hand, macroautophagy is the primary route. It begins with the sequestration of cargoes of undesirable cellular constituents by double- membrane vesicle of autophagosome followed by a subsequent fusion with the lysosome to be degraded. The cargoes are degraded into its main components, e.g., sugars, nucleosides, amino acids, and fatty acids, and released into the cyto- sol to enable their recycling (Feng et al. 2014;
Choi et al. 2018). P62 or sequestosome-1 is a critical adaptor molecule that delivers cargoes to autophagosomes; it is a modulator of autophago- some biogenesis. Self-polymerization of p62 is essential for its binding to the cargoes. The bind- ing of its ZZ domain stimulates the polymeriza- tion of p62 to the N-terminal arginine of arginylated substrates. For example, in the case of ER-phagy (autophagic degradation of the endoplasmic reticulum), the heat shock protein
HSPA5 is N terminally arginylated and bound to the ZZ domain of p62 allowing its polymeriza- tion. The complex of polymerized p62 along with the ER transmembrane E3 ligase tripartite motif- containing 13 (TRIM13) drives the ER compart- ment to autophagosome leading to its lysosomal degradation (Cha-Molstad et al. 2017; Ji et al.
2019). Thus, HSPA5 not only reliefs the ER stress by initiating the UPR but also plays a criti- cal role in ER-phagy.
10.3.1 HSPA5 Functions in Healthy Versus Stressed Cells
Typically, the function of HSPA5 is to bind to misfolded and unfolded proteins and start ER-associated degradation (ERAD), which is responsible for unfolded protein response (UPR) (Ibrahim et al. 2019; Little et al. 1994; Pfaffenbach and Lee 2011). It binds to unfolded proteins through its SBD and prevents their aggregation.
This process requires energy, which can be obtained through the hydrolysis of ATP in the NBD domain (Luo et al. 2006). Under normal cell conditions (no stress), HSPA5 is attached to three proteins, which are protein kinase RNA- like endoplasmic reticulum kinase (PERK), inositol- requiring enzyme 1 alpha (IRE1α), and activating transcription factor 6 (ATF6). These three proteins are UPR transmembrane sensors for stress. Under stress conditions such as the accumulation of unfolded proteins in ER, HSPA5 is released from these proteins, activating them, which leads to a reduction in protein translation and enhancement of correct folding (Ibrahim et al. 2019; Pfaffenbach and Lee 2011; Sepulveda et al. 2018).
A more detailed description of what happens is as follows. Activation of PERK requires its dimerization to gain its autophosphorylation activity. After that, it phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). That, in turn, inhibits the initiation of protein translation and prevents protein synthe- sis, leading to a reduction in the entry of newly formed proteins into ER. In the case of the acti- vated form of ATF6, it migrates to the Golgi
apparatus where its cleavage occurs. The cleaved part, then, moves to the nucleus and acts as an active transcription factor leading to the upregu- lation of protein transcription that boosts the folding capacity of the ER, such as HSPA5 (Ibrahim et al. 2019; Wang et al. 2009).
On the other hand, the activated form of IRE1α has an endoribonuclease activity. It breaks an intron of length 26-base from the mRNA of X-box binding protein 1 (XBP-1). This protein is a transcriptional factor, which targets genes involved in ERAD, such as protein disulfide isomerase (PDI), ER degradation-enhancing α-mannosidase-like protein (EDEM), endoplas- mic reticulum-localized DnaJ 4 (ERdj4), p58, and DnaJ (Wang et al. 2009).
10.3.2 HSPA5 over the Cell Membrane
Under stress, HSPA5 can be found on the surface of the cells where it can interact with a bunch of ligands or other proteins. Cell-surface HSPA5 (CS-HSPA5) plays an essential role in migration, invasion, apoptosis, signaling, and immunity.
Some of the ligands that bind to CS-HSPA5 are α2 macroglobulin, Isthmin, Par-4, and plasmino- gen Kringle 5 (Ibrahim et al. 2019). The proposed mechanism of GRP78 translocation from the ER to the cell surface was described by Tsai et al.
(2018). There is a tetra-peptide, KDEL, in the C-terminal of HSPA5 that maintains it inside the ER (Munro and Pelham 1987). Under ER stress conditions, ER chaperones’ surface expression is rapidly increased, while the intracellular levels do not change. Thus, it cannot be described by the oversaturation of KDEL receptors’ retrieval mechanisms (Tsai et al. 2018). SRC is a non- receptor protein-tyrosine kinase that belongs to SRC family kinases and has many roles in cancer cells (Yeatman 2004) and also has a function in the relocalization of ER chaperones to the cell surface (Tsai et al. 2018). IRE1α can activate SRC through phosphorylation of Y419 amino acid (Tsai et al. 2018). Upon SRC activation, it triggers a signaling cascade reducing, partially, the retrograde chaperones’ trafficking from Golgi
to ER, allowing escaping of a subfraction of ER chaperones to the cell surface (Tsai et al. 2018).
10.3.3 HSPA5 in Cancer and Viral Infection
HSPA5 has many roles in different cancer types.
In breast cancer, the overexpression of HSPA5 reduces the sensitivity to gemcitabine by inhibit- ing apoptosis (Xie et al. 2016). Besides, HSPA5 overexpression in ovarian carcinoma protects paclitaxel and cisplatin treatments (Zhang et al.
2015; Chen and Xu 2017). On the other hand, overexpression of HSPA5 in pancreatic ductal adenocarcinoma (PDAC) leads to a decrease in patient survivability (Niu et al. 2015).
In addition to the previous roles of CS-HSPA5, it can also act as a receptor for some viruses (Ni et al. 2011; Ibrahim et al. 2019).
Zika virus (ZIKV), which is a flavivirus related to microcephaly, uses its envelope protein for cellular attachment. One of the receptors that help its entry is HSPA5 (Smit et al. 2011; Ojha et al. 2018; Elfiky and Ibrahim 2020). Another virus that can handle the CS-HSPA5 as its recep- tor is the dengue virus (DENV) (Jindadamrongwech et al. 2004). Moreover, the Japanese encephalitis virus (JEV), which is a neurotropic virus that causes encephalitis in humans with high mortality reaching 30%, can use CS-HSPA5 as its receptor in addition to its two receptors, heparan sulfate proteoglycans (HSPGs) and glycosaminoglycans (GAGs).
CS-HSPA5 can bind to JEV envelope domain III (Nain et al. 2017; Chien et al. 2008; Chiou et al.
2005). The MERS-CoV is a human coronavirus that causes severe lower respiratory tract infec- tion, with mortality reaching 36%. MERS-CoV can utilize CS-HSPA5, which promotes its entry in the presence of its functional receptor dipepti- dyl peptidase 4 (DPP4) (Chu et al. 2018; Chan et al. 2015). Ebola virus (EBOV) belongs to the filovirus family and has very high mortality reaching 90%. When the cell is under stress, the HSPA5 is overexpressed and escapes ER reten- tion reaching the cell surface (Elfiky 2020b). Two EBOV glycoproteins (GP1 and GP2) may accu-
mulate in the ER and cause cell stress (Bhattacharyya and Hope 2011). GP1 is the viral protein required for virus entry. Overexpression of HSPA5 is reported to increase EBOV infectiv- ity. Recently, an in silico study provided a predic- tion of the binding site between GRP78 and GP1.
This would pave the route for the design of drugs to prevent this binding (Elfiky 2020b).
HSPA5 also has a role related to the viruses inside the cell. Human papillomavirus (HPV) is reported as the leading cause of cervical, head, anal, and throat cancers. It has more than 150 different strains with HPV16 as the most com- mon cause of cervical cancer worldwide (Elfiky 2020d). One of the nonstructural proteins in HPV16 is E6, which is reported as the element responsible for cancer cell proliferation (Kaliamurthi et al. 2018). One of the host cell proteins that bind to E6 protein is HSPA5. This binding increases the E6 lifetime in vivo because HSPA5 protects it from degradation through the proteasome degradation pathway (Ajiro and Zheng 2015). E6 destabilization is a strategy to prevent cancer proliferation. An in silico study provided a prediction of the bind- ing site between the HSPA5 and E6 protein aiming to show the possible targeting binding site (Elfiky 2020d).
10.3.4 CS-HSPA5 Targeting
Severe side effects are accompanying the treat- ment for cancer; therefore, searching for other ways for cancer treatment is essential (Fennelly and Schneider 1995; Alexandrescu et al. 2005).
One of the methods is to use specific ligands that can bind to cancer cells only (Landon and Deutscher 2003). These ligands can be used as a vehicle to transport chemotherapeutics specifi- cally to cancer cells. Three different types of vehicles are used to target CS-HSPA5: peptides, monoclonal antibodies, and natural com- pounds Elfiky et al. 2020a). Pep42 is a cyclic peptide of 13 amino acids (CTVALPGGYVRVC).
This peptide binds specifically to CS-HSPA5.
Besides, it is mainly hydrophobic, supporting the primary role of HSPA5, which can bind to
unfolded hydrophobic amino acid patches (Kim et al. 2006).
Since CS-HSPA5 acts as a receptor for many viruses, as indicated above, it may be an excellent strategy to try to decrease or inhibit the CS-HSPA5 expression over the cell membrane to fight the infection. Many natural compounds can reduce the expression of CS-HSPA5. Iridoid glu- coside catapol, which is extracted from Radix rehmannia, is reported to inhibit the expression of HSPA5 in human aortic endothelial cells (Hu et al. 2019). An antitumor flavonoid extracted from Sophora flavescens Aiton named kurari- none decreases the expression of HSPA5 in a non-small cell lung cancer cell line (Yang et al.
2018). One of the most exciting HSPA5 down- regulators is triptolide. It is the bioactive com- pound in Tripterygium wilfordii Hook F, which can decrease the expression of HSPA5 in leuke- mia cells (Li et al. 2016). This reduction of HSPA5 by triptolide causes cell death by either apoptosis or autophagy (Chan et al. 2017).
Polyphenol epigallocatechin-3-gallate (EGCG) (found in green tea) decreases the CS-HSPA5;
however, it increases the HSPA5 expression in the ER (Martinotti et al. 2018).
10.4 COVID-19 Inhibition by