The classification of hspC proteins is based on their compartmentalization. For example, cytoplasmic proteins are grouped as hspC1 (hsp 90-alpha), hspC2 (hsp 90-alpha A2), and hspC3 (hsp 90-beta), ER-resident member; hsoC4 or GRP94 (GP96), and mitochondrial localized member; hspC5 (TRAP1) (Figure 1). Other hspC proteins are classified based on their expression pattern, where hspCβ is constitutively expressed, while hspCα is induced in stressful conditions. The structural, biochemical, and molecular characteristics of hsp90 have been extensively reviewed. The molecular structure of hspC homologs is made of domains, namely: the N-terminal domain (NTD), C-terminal domain (CTD), and middle domain (MD). In eukaryotes, a variable linker domain exists that connects the N-terminal domain (NTD) to the middle domain (MD) (Borges and Ramos, 2005). Each domain within the hspC90 structure serves a specific function. The NTD binds to ATP, hence it is referred to as the nucleotide-binding site. The dimerization of protein occurs in the CTD. The CTD consists of critical motifs such as MEEVD or KDEL which are solely on the hspC structure and regulates its localization in the cellular system be it ER or cytoplasm. Although the charged linker domain has a divergent sequence among many eukaryotic organisms, it is crucial for the chaperone function, interaction, and flexibility (Kayastha et al., 2024). HspC is responsible for numerous functions, such as the maturation, structural maintenance, and proper regulation of specific target proteins involved in cell cycle control and signal transduction. ATP plays a key role in the functional activities of HspC in the cellular system. This functional cycle could promote structural changes in the substrates, leading to their activation. It interacts dynamically with various co-chaperones that modulate its substrate recognition, ATPase cycle, and chaperone function.

The STRING database for known protein-protein interactions was used to identify proteins interacting with human hspC. The protein sequences of hspC and the selected interacting proteins were obtained from the protein data bank via STRING. Among the selected proteins, E3 ubiquitin-protein ligase was found to have close interactions with hspC. This ligase collaborates with ATXN3 in the degradation of misfolded chaperone substrates. ATXN3 restricts the length of the ubiquitin chain attached to STUB1/CHIP substrates and prevents further chain extension. Additionally, it ubiquitinates NOS1 in concert with hspA and DnaJC, thereby modulating the activity of several chaperone complexes, including hspA and hspC (Figure 2) (Chavez et al., 2016). Cellular tumor antigen p53, which acts as a tumor suppressor in many tumor types, was also identified. It induces growth arrest or apoptosis and is involved in cell cycle regulation as a trans-activator negatively regulating cell division. Furthermore, DnaJ homolog subfamily B member 1 was found to interact with hspA and stimulate ATPase activity. Moreover, heat shock protein hspC-alpha, a molecular chaperone, plays a vital role in promoting the maturation, structural maintenance, and proper regulation of specific target proteins involved in cell cycle control and signal transduction (Lindquist and Craig, 1988). By inducing conformational changes in client proteins and interacting with different co-chaperones to modulate its substrate recognition, ATPase cycle, and chaperone function, this chaperone goes through a functional cycle that is linked to its ATPase activity.

Heat shock protein 70 comprises of three important subdomains, namely: the N-terminal ATPase domain, which is 44 kDa and binds and hydrolyzes ATP to ADP, generating energy for chaperone activity (Sharma and Masison, 2009). This crucial domain contains subdomains (IA, IB, IIA, IIB) which surround the ATPase binding pocket. This domain is followed by the 28 kDa substrate binding domain (SBD), which is responsible for binding newly synthesized client proteins for proper folding (Figure 3). This domain consists of the 15 kDa beta-sheet and the 10 kDa helical subdomains. The beta-sheet subdomain has a groove that binds to hydrophobic and neutral amino acids. The C-terminal domain (CTD), which is 10 kDa, is the last domain structure found in the HSP70 structure and forms a lid-like structure over the SBD to assist in trapping the substrate.

Heat shock proteins (HSPs) are highly conserved proteins present in all types of cells. Among the 10 identified families, hsp60 stands out as a crucial player. Functioning as ATP-dependent molecular chaperones in the cytoplasm, hsp60 aids in the folding of newly synthesized polypeptides and the assemblingy of multiprotein complexes (Figure 4). Notably, hsp60 is expressed on the cell surface, particularly in bacteria, where its expression increases significantly during host infection and in biofilm formation (MaleKi et al., 2016). In the extracellular environment, hsp60 can act as a crucial link between immune cells, impacting the immune system’s activity in various ways. Furthermore, HSPs have demonstrated their potential as potent activators of the immune system and are considered strong candidates for subunit vaccines or adjuvants (Hong et al., 2024).

The consistent synthesis of polypeptides within the cellular system is crucial for proper cellular function. However, this process occurs in a complex and crowded environment. Molecular chaperones, particularly heat shock proteins (hsps), play a pivotal role in ensuring the quality and functionality of newly synthesized proteins (Qiu et al., 2006). HspA is a key player in maintaining protein homeostasis. Its adaptability and specificity in substrate binding depend significantly on its interaction with co-chaperones such as DnaJCs, JDPs, and J-proteins (Figure 5). These interactions are essential for coordinating HspA’s two major domains, the substrate binding domain (SBD) and the nucleotide-binding domain (NBD) (Qiu et al., 2006). The transient interaction of the SBD with target proteins is modulated by an allosteric mechanism involving ATP hydrolysis and ADP/ATP exchange in the NBD, highlighting the sophisticated regulatory processes involved in protein synthesis and function.

The JDPs have a dual role: they help substrate proteins bind to hspA in its ATP-bound state. At the same time, a direct interaction between a JDP and hspA triggers the hydrolysis of ATP and causes hspA to transition to its ADP-bound state, which has a high affinity for the specific polypeptide brought over by a particular JDP. Another important factor is the nucleotide exchange factor (NEF), which promotes the dissociation of ADP and the binding of a new ATP molecule. This modification changes the hspA structure to a low-affinity state, leading to the release of the substrate (Qiu et al., 2006; Boston et al., 1996).

Molecular chaperones cooperate in the folding process of the newly synthesized polypeptide. For example, dnaJC binds the client proteins as a cochaperone and hands them over to hspA to assist newly synthesized proteins in completing their folding to a three-dimensional structure. This structure is fully functional, however, if the protein has not fully achieved its functional activities it is handed over to hspC for further folding (Figure 6). The hspA and hspC molecular chaperone systems are essential for regulating protein homeostasis in eukaryotic cells under normal and stressful conditions. Both hspA and hspC work together to maintain proteostasis in the cell. Their functions are activated by cochaperones, which interact with either hspA or hspC (Olivier et al., 2019). The human heat shock organizing protein (Hop) and its yeast ortholog Stip1 are eukaryotic cochaperones that play a critical role in transferring client proteins from hspA to hspC. This transfer is regulated by Hop, as it can interact with both hspA and hspC simultaneously, forming a ternary complex. Unlike eukaryotes, prokaryotes do not have a Hop ortholog, so the hspA and hspC directly interact. It is believed that eukaryotic hspA and hspC can also form a prokaryote-like binary chaperone complex in the absence of Hop (Pedrola and Rüdiger, 2021). This binary complex exhibits enhanced protein folding and anti-aggregation activities.

Small heat shock proteins are low molecular weight proteins ranging between 12 and 43 kDa. There are various types of sHSPs, which include HSPB1-8. Alpha crystallin 6 is the most studied family due to its major presence in the human eye lens (Figure 7). sHSPs form large oligomeric complexes under physiological conditions. The rate of disassembly influences their chaperoning activities. Both heat and other environmental conditions contribute to changes in their structure (Tedesco et al., 2022). The main role of small heat shock proteins is to bind to partially denatured polypeptides to prevent irreversible aggregation under stressful conditions. Small heat shock proteins form part of the cellular protein quality control system. The figure below shows the crucial domains that constitute the small heat shock proteins, with the alpha-crystallin domain (ACD) in the middle, followed by a flexible N-terminal region (NTR) at one end and a short C-terminal region (CTR) on the other side (Tedesco et al., 2022). About 90 amino acids are found in the ACD, a conserved domain made up of beta strands that form dimers, the basic building blocks of sHSPs. Both NTR and CTR play crucial roles in oligomerization.

Multiple techniques exist to detect and measure the expression patterns of heat shock proteins in both normal and pathological conditions. Mass spectrometry (MS) is crucial for identifying and quantifying these proteins, offering insights into their structural and chemical properties, which is essential for proteomics research and biomarker discovery. In cell and tissue extracts, the enzyme-linked immunosorbent assay (ELISA) is another prominent method used to quantify heat shock proteins, including HSP90α and HSP27 (Lee et al., 2015). This highly sensitive technique can identify low protein levels. The Western blot method is also valuable for protein detection and measurement; it involves gel electrophoresis of proteins followed by transferring them to a membrane to detect specific heat shock proteins using antibodies. This method yields information on the molecular weight and expression patterns of these proteins. Immunohistochemistry is a technique applied to detect proteins in tissue samples by staining them with specific antibodies targeting heat shock proteins, enabling visualization of protein localization and expression within tissues. Lastly, bead-based immunoassays provide high sensitivity and specificity, allowing for multiplexing to detect multiple heat shock proteins simultaneously (Xia, 2017; Njemini et al., 2008).

The proteins of interest, SARS−CoV−2 nsp7/8 complex (PDB ID 7JLT), were attained from the Protein Data Bank. The molecular chaperone hsp40 was modelled using sequences obtained from UniProtKB (ID P25685), and the SWISS−MODEL tool was used to perform the homology modelling using the crystallized structure (PDB ID YDJ1) as a template. The chaperon HSP70’s 3D structure was obtained from Protein Data Bank (PDB ID 4IO8). The modelled proteins were validated using PDBsum (Negahdary et al., 2023).

Before protein−protein docking, all three proteins were prepared using the “Protein Preparation Workflow” of the Maestro Schrodinger suite. Some of the parameters employed in the protein preparation included using a pH of 7.4 and removal of water, to mention a few. The protein nsp7/8 complex consisted of four chains (A, B, C and D). The chain names were modified to ensure the chains were treated individually and not incorporated as a single chain. That is, the chain name of hsp70 was changed to chain L, and that of hsp40 was changed to chain H (Yamkela et al., 2023).

Protein-protein docking was studied to understand better the dynamics between two protein molecules. HADDOCK v2.4 (Anas et al., 2019b) was used for the docking analysis. This server was selected because it uses experimental or bioinformatic-derived data (ambiguity restraints) to drive the docking process. These data include NMR-derived intermolecular distances, residual dipolar couplings, or site-specific mutagenesis data, which help improve the accuracy of the predictions. Both proteins’ (HSP70 and HSP90) active residues were identified using information on the protein data bank and structural data (Protein Data Bank, https://www.rcsb.org/). The conformational space of the protein-protein complex was sampled by executing the docking in unbound, rigid-body, and flexible refinement phases. The HADDOCK v2.4 server was programmed to generate 1,000 dock poses; the top two hundred were chosen for further improvement. The docked complexes were analyzed using LigPlot++ (v1.4.2), a tool designed to visualize protein-ligand interactions (Olivier et al., 2019), and their binding affinities were analyzed using the AREA AFFINITY server (https://affinity.cuhk.edu.cn/index accessed on 17 May 2023). This server allows for the pasting of the docked proteins and no parameters were set on the server. Molecular dynamic simulations were conducted to evaluate the stability of the docked complexes (Sahel et al., 2024; Sesethu et al., 2023b).

The interaction analysis of the selected docking pose was performed using the “Protein Interaction Analysis” tool of BioLuminate v4.6. A two−dimensional image was generated using the specific interactions highlighted in the same Excel file for both instances. An already 2D image is available in the folder under results in the Protein−Protein docking folder.

The ligands used in this study were attained from PubChem using the following IDs: NSC11926 and NSC44175. These small molecules are naturally occurring compounds that have been used to target the SARS−CoV−2 membrane protease and the HSPA8 − Spike protein complexAutoDockTools (v1.5.7) was used in the preparation of the protein complexes (hsp40 − nsp complex and hsp70 − nsp complex. Using AutoDockTools (v1.5.7), water molecules were removed, non−polar hydrogens were merged into the protein complexes, and Kollman charges were added. The prepared structures were saved in “pdbqt” format. Before grid box generation, the SiteMap tool from Maestro as used to identify potential binding sites that were used to generate a grid box to which docking of the ligands was to occur. The following grid box information was generated: for hsp70 − nsp complex,the following coordinates were generated: npts 44; 46; 42; and centre 28.060; 20.005; 15.800 and for hsp40 − nsp complex the following were generated: npts 46; 44; 42 and centre −38.643; −1.426; 12.196. Open Babel GUI (v3.0.0) was used to convert the protonated ligands NSC11926 and NSC44175 (pH7.4 using Avogadro) from “sdf” format to “pdbqt” format in preparation for docking. AutoDockTools (v1.5.7) was used to prepare the ligand similarly to that of the protein complexes. AutoDock Vina was then used to perform protein−ligand docking, and the results were analysed using PyMOL and Ligplot⁺ (Acharya et al., 2021).

Heat shock proteins (HSPs) are activated by various external or foreign substances such as bacteria and viruses. When SARS-CoV-2 enters the human body, the expression levels of heat shock proteins increase. For example, a study conducted by Negahdary in 2023 on COVID-19 patients using a sandwich-like system sensitive dectection for heat shock protein 70, suggested that hsp70 levels were found to be increased by approximately 50%. Therefore, making the expression of hsp70 is correlated to poor treatment of SARS-CoV-2 (Anandakrishnan et al., 2012). This increased production of heat shock proteins during a viral invasion is considered a potential biomarker. hspA8 is an important biomarker with significant potential for diagnosing and predicting the prognosis of COVID-19 (Anandakrishnan et al., 2012). Studies have shown that hspA8 exhibits varying concentration levels in COVID-19, cancer, diabetes, and certain heart diseases, making it a potential diagnostic biomarker. In a clinical study, the concentrations of hspA in the plasma of COVID-19 patients admitted to the intensive care unit (ICU) were assessed. The increased levels of hspA8 were confirmed as a biomarker predicting mortality for COVID-19 patients. This biomarker was reported to remain relatively stable at 200 ng mL−1 during a 1-week evaluation (Pattar et al., 2020; Muddagoni et al., 2021). In our laboratory, we are currently testing the role of HspA8 in its interaction with selected SARS-CoV-2 proteins. We aim to develop an alternative treatment for COVID-19. Recently, we published a comprehensive review on the interaction between the spike proteins of SARS-CoV-2 and mammalian small and major heat shock proteins. This review demonstrates how the virus hijacks the human folding machinery.

SARS-CoV-2 has various proteins that play a crucial role in its development and replication. Spike protein and ACE-2 proteins have attracted a lot of research interest due to their role in viral entry into the human system. In addition, non-structural proteins have been identified as important molecules in the development and replication of SARS-CoV-2 (Ikwegbue et al., 2017), with more than 16 non-structural proteins from the viral genome being observed. Non-structural protein complexes have drawn much interest in the research industry, and scientists want to understand their role in the viral replication and developmental stages of the coronavirus. The SARS-CoV-2 NSP7/8 complex was sampled as proteins to understand how they interact with human host molecular folding machinery and with selected compounds to block their interaction (Figure 11).

Coronavirus replication and transcription involve multiple virus-encoded non-structural proteins (NSPs). Like other polypeptides, NSPs require assistance from molecular chaperones to form a three-dimensional structure for proper function (Figure 12). Viruses rely on human hosts to produce viral proteins by hijacking human ribosomes and heat shock proteins as folding partners. To establish the potential involvement of selected molecular chaperones in the folding stages of the viral complex protein SARS-CoV-2 NSP7/8, they were docked with the complex in a review paper. The results showed an interaction between human heat shock proteins, such as hsp10, and SARS-CoV-2 NSP7/8, indicating their role in achieving the three-dimensional structure essential for protein function. These findings suggest that viruses like SARS-CoV-2 use the human host’s folding machinery for replication (Kuramitsu, 2012). Moreover, human heat shock proteins could serve as biomarkers and potential drug targets for COVID-19 treatment. Increased levels of heat shock proteins in the presence of the virus could indicate danger within the human cellular system, while these proteins also act as molecular chaperones guiding the folding of newly produced polypeptides. This interaction between client proteins can be targeted and disrupted using drugs to interfere with the folding process.

Molecular docking was carried out to assess the binding of small molecules to the selected heat shock proteins (dnaJC and hspA) and SARS-CoV-2 NSP7/8 complex. Interactions were observed as shown in Figure 13 with Leucine 122, Serine 54, and Leucine 35 being the noticeable amino acids involved both in the dnaJC complex and hspA complex with 2,5-Dihydroxy-3,6-diphenyl-p-benzoquinone compound. This suggests that both folding pieces of machinery recognize this amino acid during the folding processes of the SARS-CoV-2 NSP7/8 complex. These amino acids are thus, interesting drug targets in the treatment of COVID-19. We are currently in the process of testing this treatment technique in my laboratory (Hwang et al., 2020). Also, Figure 14 shows that the hsp70 interaction was established, and certain amino acids were observed as interesting candidates in the study that is currently being conducted. It is important to note that the ligand was docked at the binding site, which was identified at the interaction location. The ligand was docked in the binding pocket of hsp70, demonstrating the most favorable binding affinity of −9.1 kcal mol−1, in comparison to the binding affinity of −6.6 kcal mol−1 (the highlighted modes) achieved when the ligand binds at the identified binding site as determined by SiteMap (Table 7). The in vitro study is underway in my laboratory to confirm this data in mouse models and cell culture.