A comprehensive review of the interaction between COVID-19 spike proteins with mammalian small and major heat shock proteins.

Coronavirus disease 2019 (COVID-19) is a novel disease that had devastating effects on human lives and the country's economies worldwide. This disease shows similar parasitic traits, requiring the host's biomolecules for its survival and propagation. Spike glycoproteins severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 spike protein) located on the surface of the COVID-19 virus serve as a potential hotspot for antiviral drug development based on their structure. COVID-19 virus calls into action the chaperonin system that assists the attacker, hence favoring infection. To investigate the interaction that occurs between SARS-CoV-2 spike protein and human molecular chaperons (HSPA8 and sHSP27), a series of steps were carried out which included sequence attainment and analysis, followed by multiple sequence alignment, homology modeling, and protein-protein docking which we performed using Cluspro to predict the interactions between SARS-CoV-2 spike protein and human molecular chaperones of interest. Our findings depicted that SARS-CoV-2 spike protein consists of three distinct chains, chains A, B, and C, which interact forming hydrogen bonds, hydrophobic interactions, and electrostatic interactions with both human HSPA8 and HSP27 with -828.3 and -827.9 kcal/mol as binding energies for human HSPA8 and -1166.7 and -1165.9 kcal/mol for HSP27.

Potential for targeting small heat shock protein modifications.

Small heat shock proteins (sHSPs) play key roles in cellular stress and several human diseases. The direct effects of some post-translational modifications (PTMs) on certain sHSPs have been characterized, raising the possibility that small molecules could be used to modulate these modifications and indirectly up- or downregulate sHSP activity.

Interaction of the C-terminal immunoglobulin-like domains (Ig 22-24) of filamin C with human small heat shock proteins.

Small heat shock proteins are the well-known regulators of the cytoskeleton integrity, yet their complexes with actin-binding proteins are underexplored. Filamin C, a dimeric 560 kDa protein, abundant in cardiac and skeletal muscles, crosslinks actin filaments and contributes to Z-disc formation and membrane-cytoskeleton attachment. Here, we analyzed the interaction of a human filamin C fragment containing immunoglobulin-like domains 22-24 (FLNC22-24) with five small heat shock proteins (HspB1, HspB5, HspB6, HspB7, HspB8) and their α-crystallin domains. On size-exclusion chromatography, only HspB7 or its α-crystallin domain formed complexes with FLNC22-24. Despite similar isoelectric points of the small heat shock proteins analyzed, only HspB7 and its α-crystallin domain interacted with FLNC22-24 on native gel electrophoresis. Crosslinking with glutaraldehyde confirmed the formation of complexes between HspB7 (or its α-crystallin domain) and the filamin С fragment, inhibiting intersubunit FLNC crosslinking. These data are consistent with the structure modeling using Alphafold. Thus, the C-terminal fragment (immunoglobulin-like domains 22-24) of filamin C contains the site for HspB7 (or its α-crystallin domain) interaction, which competes with FLNC22-24 dimerization and its probable interaction with different target proteins.

Cryo-EM structure of a 16.5-kDa small heat-shock protein from Methanocaldococcus jannaschii.

The small heat-shock protein (sHSP) from the archaea Methanocaldococcus jannaschii, MjsHSP16.5, functions as a broad substrate ATP-independent holding chaperone protecting misfolded proteins from aggregation under stress conditions. This protein is the first sHSP characterized by X-ray crystallography, thereby contributing significantly to our understanding of sHSPs. However, despite numerous studies assessing its functions and structures, the precise arrangement of the N-terminal domains (NTDs) within this sHSP cage remains elusive. Here we present the cryo-electron microscopy (cryo-EM) structure of MjsHSP16.5 at 2.49-Å resolution. The subunits of MjsHSP16.5 in the cryo-EM structure exhibit lesser compaction compared to their counterparts in the crystal structure. This structural feature holds particular significance in relation to the biophysical properties of MjsHSP16.5, suggesting a close resemblance to this sHSP native state. Additionally, our cryo-EM structure unveils the density of residues 24-33 within the NTD of MjsHSP16.5, a feature that typically remains invisible in the majority of its crystal structures. Notably, these residues show a propensity to adopt a ß-strand conformation and engage in antiparallel interactions with strand ß1, both intra- and inter-subunit modes. These structural insights are corroborated by structural predictions, disulfide bond cross-linking studies of Cys-substitution mutants, and protein disaggregation assays. A comprehensive understanding of the structural features of MjsHSP16.5 expectedly holds the potential to inspire a wide range of interdisciplinary applications, owing to the renowned versatility of this sHSP as a nanoscale protein platform.

Functional Diversity of Mammalian Small Heat Shock Proteins: A Review.

The small heat shock proteins (sHSPs), whose molecular weight ranges from 12∼43 kDa, are members of the heat shock protein (HSP) family that are widely found in all organisms. As intracellular stress resistance molecules, sHSPs play an important role in maintaining the homeostasis of the intracellular environment under various stressful conditions. A total of 10 sHSPs have been identified in mammals, sharing conserved α-crystal domains combined with variable N-terminal and C-terminal regions. Unlike large-molecular-weight HSP, sHSPs prevent substrate protein aggregation through an ATP-independent mechanism. In addition to chaperone activity, sHSPs were also shown to suppress apoptosis, ferroptosis, and senescence, promote autophagy, regulate cytoskeletal dynamics, maintain membrane stability, control the direction of cellular differentiation, modulate angiogenesis, and spermatogenesis, as well as attenuate the inflammatory response and reduce oxidative damage. Phosphorylation is the most significant post-translational modification of sHSPs and is usually an indicator of their activation. Furthermore, abnormalities in sHSPs often lead to aggregation of substrate proteins and dysfunction of client proteins, resulting in disease. This paper reviews the various biological functions of sHSPs in mammals, emphasizing the roles of different sHSPs in specific cellular activities. In addition, we discuss the effect of phosphorylation on the function of sHSPs and the association between sHSPs and disease.

The mRNA binding-mediated self-regulatory function of small heat shock protein IbpA in γ-proteobacteria is conferred by a conserved arginine.

Bacterial small heat shock proteins, such as inclusion body-associated protein A (IbpA) and IbpB, coaggregate with denatured proteins and recruit other chaperones for the processing of aggregates thereby assisting in protein refolding. In addition, as a recently revealed uncommon feature, Escherichia coli IbpA self-represses its own translation through interaction with the 5'-untranslated region of the ibpA mRNA, enabling IbpA to act as a mediator of negative feedback regulation. Although IbpA also suppresses the expression of IbpB, IbpB does not have this self-repression activity despite the two Ibps being highly homologous. In this study, we demonstrate that the self-repression function of IbpA is conserved in other γ-proteobacterial IbpAs. Moreover, we show a cationic residue-rich region in the α-crystallin domain of IbpA, which is not conserved in IbpB, is critical for the self-suppression activity. Notably, we found arginine 93 (R93) located within the α-crystallin domain is an essential residue that cannot be replaced by any of the other 19 amino acids including lysine. We observed that IbpA-R93 mutants completely lost the interaction with the 5' untranslated region of the ibpA mRNA, but retained almost all chaperone activity and were able to sequester denatured proteins. Taken together, we propose the conserved Arg93-mediated translational control of IbpA through RNA binding would be beneficial for a rapid and massive supply of the chaperone on demand.

Role of Small Heat Shock Proteins in the Remodeling of Actin Microfilaments.

Small heat shock proteins (sHsps) play an important role in the maintenance of proteome stability and, particularly, in stabilization of the cytoskeleton and cell contractile apparatus. Cell exposure to different types of stress is accompanied by the translocation of sHsps onto actin filaments; therefore, it is commonly believed that the sHsps are true actin-binding proteins. Investigations of last years have shown that this assumption is incorrect. Stress-induced translocation of sHsp to actin filaments is not the result of direct interaction of these proteins with intact actin, but results from the chaperone-like activity of sHsps and their interaction with various actin-binding proteins. HspB1 and HspB5 interact with giant elastic proteins titin and filamin thus providing an integrity of the contractile apparatus and its proper localization in the cell. HspB6 binds to the universal adapter protein 14-3-3 and only indirectly affects the structure of actin filament. HspB7 interacts with filamin C and controls actin filament assembly. HspB8 forms tight complex with the universal regulatory and adapter protein Bag3 and participates in the chaperone-assisted selective autophagy (CASA) of actin-binding proteins (e.g., filamin), as well as in the actin-depending processes taking place in mitoses. Hence, the mechanisms of sHsp participation in the maintenance of the contractile apparatus and cytoskeleton are much more complicated and diverse than it has been postulated earlier and are not limited to direct interactions of sHsps with actin. The old hypothesis on the direct binding of sHsps to intact actin should be revised and further detailed investigation on the sHsp interaction with minor proteins participating in the formation and remodeling of actin filaments is required.

The Diverse Functions of Small Heat Shock Proteins in the Proteostasis Network.

The protein quality control (PQC) system maintains protein homeostasis by counteracting the accumulation of misfolded protein conformers. Substrate degradation and refolding activities executed by ATP-dependent proteases and chaperones constitute major strategies of the proteostasis network. Small heat shock proteins represent ATP-independent chaperones that bind to misfolded proteins, preventing their uncontrolled aggregation. sHsps share the conserved α-crystallin domain (ACD) and gain functional specificity through variable and largely disordered N- and C-terminal extensions (NTE, CTE). They form large, polydisperse oligomers through multiple, weak interactions between NTE/CTEs and ACD dimers. Sequence variations of sHsps and the large variability of sHsp oligomers enable sHsps to fulfill diverse tasks in the PQC network. sHsp oligomers represent inactive yet dynamic resting states that are rapidly deoligomerized and activated upon stress conditions, releasing substrate binding sites in NTEs and ACDs Bound substrates are usually isolated in large sHsp/substrate complexes. This sequestration activity of sHsps represents a third strategy of the proteostasis network. Substrate sequestration reduces the burden for other PQC components during immediate and persistent stress conditions. Sequestered substrates can be released and directed towards refolding pathways by ATP-dependent Hsp70/Hsp100 chaperones or sorted for degradation by autophagic pathways. sHsps can also maintain the dynamic state of phase-separated stress granules (SGs), which store mRNA and translation factors, by reducing the accumulation of misfolded proteins inside SGs and preventing unfolding of SG components. This ensures SG disassembly and regain of translational capacity during recovery periods.

Multiple nanocages of a cyanophage small heat shock protein with icosahedral and octahedral symmetries.

The structures of a cyanophage small heat shock protein (sHSP) were determined as octahedrons of 24-mers and 48-mers and as icosahedrons of 60-mers. An N-terminal deletion construct of an 18 kDa sHSP of Synechococcus sp. phage S-ShM2 crystallized as a 24-mer and its structure was determined at a resolution of 7 Å. The negative stain electron microscopy (EM) images showed that the full-length protein is a mixture of a major population of larger and a minor population of smaller cage-like particles. Their structures have been determined by electron cryomicroscopy 3D image reconstruction at a resolution of 8 Å. The larger particles are 60-mers with icosahedral symmetry and the smaller ones are 48-mers with octahedral symmetry. These structures are the first of the viral/phage origin and the 60-mer is the largest and the first icosahedral assembly to be reported for sHSPs.

Genome-wide identification of small heat shock protein (HSP20) homologs in three cucurbit species and the expression profiles of CsHSP20s under several abiotic stresses.

Small heat shock protein (HSP20) genes play important roles in biological processes of plants. In this study, a total of 47 CsHSP20 genes, 45 CmHSP20 genes, and 47 ClHSP20 genes were genome-wide identified by 'hmmsearch' and BLASTP using the latest versions of cucumber, melon, and watermelon genomes, respectively. According to the phylogenetic relationships and predicted subcellular localizations, HSP20s of these three cucurbit species were divided into 8 subfamilies (CI-CIV, CP, ER, M, and PX), in which some HSP20s were closely related with each other based on the collinearity analysis. Specific expression patterns of CsHSP20s were checked in 10 different tissues of cucumber plants. RNA-seq analysis of transcript levels, combined with cis-acting elements and GO enrichment analysis suggested that CsHSP20s were responsive to several different types of abiotic stresses, including chilling, temperature and photoperiod, high temperature and high humidity, and salinity. In conclusion, results of this work not only provided valuable information for exploring the regulating mechanisms of CsHSP20s in responding to abiotic stresses in cucumber, but also shed light on the potentially evolutional relations among cucumber, melon, and watermelon from a perspective of comparative genomics that specified on HSP20 gene families.

Two Bacterial Small Heat Shock Proteins, IbpA and IbpB, Form a Functional Heterodimer.

Small heat shock proteins (sHsps) are a conserved class of ATP-independent chaperones which in stress conditions bind to unfolded protein substrates and prevent their irreversible aggregation. Substrates trapped in sHsps-containing aggregates are efficiently refolded into native structures by ATP-dependent Hsp70 and Hsp100 chaperones. Most γ-proteobacteria possess a single sHsp (IbpA), while in a subset of Enterobacterales, as a consequence of ibpA gene duplication event, a two-protein sHsp (IbpA and IbpB) system has evolved. IbpA and IbpB are functionally divergent. Purified IbpA, but not IbpB, stably interacts with aggregated substrates, yet both sHsps are required to be present at the substrate denaturation step for subsequent efficient Hsp70-Hsp100-dependent substrate refolding. IbpA and IbpB interact with each other, influence each other's expression levels and degradation rates. However, the crucial information on how these two sHsps interact and what is the basic building block required for proper sHsps functioning was missing. Here, based on NMR, mass spectrometry and crosslinking studies, we show that IbpA-IbpB heterodimer is a dominating functional unit of the two sHsp system in Enterobacterales. The principle of heterodimer formation is similar to one described for homodimers of single bacterial sHsps. ß-hairpins formed by strands ß5 and ß7 of IbpA or IbpB crystallin domains associate with the other one's ß-sandwich in the heterodimer structure. Relying on crosslinking and molecular dynamics studies, we also propose the orientation of two IbpA-IbpB heterodimers in a higher order tetrameric structure.

Structural basis of substrate recognition and thermal protection by a small heat shock protein.

Small heat shock proteins (sHsps) bind unfolding proteins, thereby playing a pivotal role in the maintenance of proteostasis in virtually all living organisms. Structural elucidation of sHsp-substrate complexes has been hampered by the transient and heterogeneous nature of their interactions, and the precise mechanisms underlying substrate recognition, promiscuity, and chaperone activity of sHsps remain unclear. Here we show the formation of a stable complex between Arabidopsis thaliana plastid sHsp, Hsp21, and its natural substrate 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) under heat stress, and report cryo-electron microscopy structures of Hsp21, DXPS and Hsp21-DXPS complex at near-atomic resolution. Monomeric Hsp21 binds across the dimer interface of DXPS and engages in multivalent interactions by recognizing highly dynamic structural elements in DXPS. Hsp21 partly unfolds its central α-crystallin domain to facilitate binding of DXPS, which preserves a native-like structure. This mode of interaction suggests a mechanism of sHsps anti-aggregation activity towards a broad range of substrates.

Elucidation of Diverse Physico-Chemical Parameters in Mammalian Small Heat Shock Proteins: A Comprehensive Classification and Structural and Functional Exploration Using In Silico Approach.

Small heat shock proteins (sHSPs), often known as molecular chaperones, are most prevalent in nature. Under certain stress-induced conditions, these sHSPs act as an ATP-independent variation and thus prevent the inactivation of various non-native substrate proteins and their aggregation. They also assist other ATP-dependent chaperones in the refolding of these substrates. In the case of prokaryotes and lower eukaryotes, the chaperone functions of sHSPs can bind a wide range of cellular proteins but preferentially protect translation-related proteins and metabolic enzymes. Eukaryotes usually encode a larger number of sHSPs than those of prokaryotes. The chaperone functions of mammalian sHSPs are regulated by phosphorylation in cells and also by temperature. Their sHSPs have different sub-cellular compartments and cell/tissue specificity. The substrate proteins of mammalian sHSPs or eukaryotic sHSPs accordingly reflect their multi-cellular complexity. The sHSPs of animals play roles in different physiological processes as cell differentiation, apoptosis, and longevity. In this work, the characterization, location, tissue specificity, and functional diversity of sHSPs from seven different mammalian species with special emphasis on humans have been studied. Through this extensive work, a novel and significant attempt have been made to classify them based on their omnipresence, tissue specificity, localization, secondary structure, probable mutations, and evolutionary significance.

Analysis of insect nuclear small heat shock proteins and interacting proteins.

The small heat shock proteins (sHsps) are a ubiquitous family of ATP-independent stress proteins found in all domains of life. Drosophila melanogaster Hsp27 (DmHsp27) is the only known nuclear sHsp in insect. Here analyzing sequences from HMMER, we identified 56 additional insect sHsps with conserved arginine-rich nuclear localization signal (NLS) in the N-terminal region. At this time, the exact role of nuclear sHsps remains unknown. DmHsp27 protein-protein interaction analysis from iRefIndex database suggests that this protein, in addition to a putative role of molecular chaperone, is likely involved in other nuclear processes (i.e., chromatin remodeling and transcription). Identification of DmHsp27 interactors should provide key insights on the cellular and molecular functions of this nuclear chaperone.

Classifying the superfamily of small heat shock proteins by using g-gap dipeptide compositions.

Small heat shock protein (sHSP) is a superfamily of molecular chaperone and is found from archaea to human. Recent researches have demonstrated that sHSPs participate in a series of biological processes and are even closely associated with serious diseases. Since sHSP is a very large superfamily and members from different superfamilies exhibit distinct functions, accurate classification of the subfamily of sHSP will be helpful for unrevealing its functions. In the present work, a support vector machine-based method was proposed to classify the subfamily of sHSPs. In the 10-fold cross validation test, an overall accuracy of 93.25% was obtained for classifying the subfamily of sHSPs. The superiority of the proposed method was also demonstrated by comparing it with the other methods. It is anticipated that the proposed method will become a useful tool for classifying the subfamily of sHSPs.

IbpB-bound substrate release in living cells as revealed by unnatural amino acid-mediated photo-crosslinking.

Small heat shock proteins (sHSPs) are known to bind non-native substrates and prevent irreversible aggregation in an ATP-independent manner. However, the dynamic interaction between sHSPs and their substrates in vivo is less studied. Here, by utilizing a genetically incorporated crosslinker, we characterized the interaction between sHSP IbpB and its endogenous substrates in living cells. Through photo-crosslinking analysis of five Bpa variants of IbpB, we found that the substrate binding of IbpB in living cells is reversible upon short-time exposure at 50 °C. Our data provide in vivo evidence that IbpB engages in dynamic substrate release under nonstress conditions and suggest that photo-crosslinking may be a suitable method for investigating dynamic interaction between molecular chaperones and their substrates in living cells.

AgsA oligomer acts as a functional unit.

AgsA (aggregation-suppressing protein) is an ATP-independent molecular chaperone machine belonging to the family of small heat shock proteins (sHSP), and it can prevent the aggregation of non-natural proteins. However, the substrate-binding site of AgsA and the functional unit that captures and binds the substrate remain unknown. In this study, different N-terminal and C-terminal deletion mutants of AgsA were constructed and their effects on AgsA oligomer assembly and chaperone activity were investigated. We found that the IXI motif at the C-terminus and the α-helix at the N-terminus affected the oligomerization and molecular chaperone activity of AgsA. In this work, we obtained a 6.8 Å resolution structure of AgsA using Electron cryo-microscopy (cryo-EM), and found that the functional form of AgsA was an 18-mer with D3 symmetry. Through amino acid mutations, disulfide bonds were introduced into two oligomeric interfaces, namely dimeric interface and non-partner interface. Under oxidation and reduction conditions, the chaperone activity of the disulfide-bonded AgsA did not change significantly, indicating that AgsA would not dissociate to achieve chaperone activity. Therefore, we concluded that the oligomer, especially 18-mer, was the primary functional unit.

Functional analysis of a novel orthologous small heat shock protein (shsp) hsp21.8a and seven species-specific shsps in Tribolium castaneum.

Small heat shock proteins (sHSPs) are important modulators of insect survival. Previous research revealed that there is only one orthologous cluster of shsps in insects. Here, we identified another novel orthologous cluster of shsps in insects by comparative analysis. Multiple stress experiments and function investigation of Tchsp21.8a belonging to this orthologous cluster and seven species-specific shsps were performed in the stored-grain pest Tribolium castaneum. The results indicated that expression of Tchsp21.8a showed weak responses to different stresses. However, expressions of most species-specific shsps exhibited hyper-responses to heat stress, and expressions of all species-specific shsps displayed diverse responses during other stresses to protect beetles in a cooperative manner. Additionally, Tchsp21.8a and species-specific Tcshsp19.7 played important roles in the development of T. castaneum, and all Tcshsps had a certain impact on the fecundity. Our work created a comprehensive reliable scaffold of insect shsps that can further provide instructive insights to pest bio-control.

The Heterooligomerization of Human Small Heat Shock Proteins Is Controlled by Conserved Motif Located in the N-Terminal Domain.

Ubiquitously expressed human small heat shock proteins (sHsps) HspB1, HspB5, HspB6 and HspB8 contain a conserved motif (S/G)RLFD in their N-terminal domain. For each of them, we prepared mutants with a replacement of the conserved R by A (R/A mutants) and a complete deletion of the pentapeptide (Δ mutants) and analyzed their heterooligomerization with other wild-type (WT) human sHsps. We found that WT HspB1 and HspB5 formed heterooligomers with HspB6 only upon heating. In contrast, both HspB1 mutants interacted with WT HspB6 even at low temperature. HspB1/HspB6 heterooligomers revealed a broad size distribution with equimolar ratio suggestive of heterodimers as building blocks, while HspB5/HspB6 heterooligomers had an approximate 2:1 ratio. In contrast, R/A or Δ mutants of HspB6, when mixed with either HspB1 or HspB5, resulted in heterooligomers with a highly variable molar ratio and a decreased HspB6 incorporation. No heterooligomerization of HspB8 or its mutants with either HspB1 or HspB5 could be detected. Finally, R/A or Δ mutations had no effect on heterooligomerization of HspB1 and HspB5 as analyzed by ion exchange chromatography. We conclude that the conserved N-terminal motif plays an important role in heterooligomer formation, as especially pronounced in HspB6 lacking the C-terminal IXI motif.