The essence of life resides in the cell, and maintaining the proper function and survival of cells depends on internal protein homeostasis. To navigate ever-changing environmental conditions, cells must effectively manage protein folding, transport, and degradation. This is where Heat Shock Proteins (HSPs) come into play, acting as crucial regulators of protein homeostasis within cells.

HSPs represent a highly conserved family of molecular chaperones present across all organisms. They oversee the entire lifecycle of proteins—from their synthesis to their degradation—through a complex network of interactions. HSPs assist newly synthesized polypeptides in folding into their correct structures, refold misfolded proteins, prevent aggregation, and direct irreparably damaged proteins toward degradation. These roles are vital for cells to respond to various stresses, such as heat shock, hypoxia, and oxidative stress, thereby maintaining cell survival and function.

Although HSPs originated in prokaryotes, their roles in eukaryotes are far more intricate and extensive. Different HSP family members possess distinct characteristics in terms of cellular localization and substrate selection, forming a sophisticated network for maintaining protein homeostasis. As research into HSP structure and function deepens, their significant roles in numerous diseases are becoming increasingly apparent.

Figure 1. The proteostasis network of HSPs. (Hu C, et al., 2022)

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Overview of the HSP Family

The HSP family comprises several members, primarily categorized into six major classes: HSP70, HSP90, HSP60, HSP40, small HSPs, and HSP110. These members vary in cellular localization, substrate selection, and molecular mechanisms, but all contribute to maintaining intracellular protein homeostasis.

HSP70 Family: Guardians of Newly Synthesized Chains

The HSP70 family, one of the most critical members of the HSPs, plays a pivotal role in protein folding, transport, and degradation. HSP70's core structure includes two main domains: the N-terminal nucleotide-binding domain (NBD) and the C-terminal substrate-binding domain (SBD). ATP binding and hydrolysis drive conformational changes in the SBD, regulating its affinity for substrates and facilitating the binding-release cycle.

HSP70's chaperone function relies on interactions with co-chaperones. J-domain proteins (JDP/HSP40) recognize unfolded or misfolded substrate proteins and guide them to HSP70, enhancing their ATPase activity and substrate binding capabilities. The nucleotide exchange factor (NEF) is another crucial co-chaperone that regulates HSP70's nucleotide-binding state, facilitating substrate release and subsequent folding processes.

HSP70 is present in various cellular compartments, including the cytoplasm, endoplasmic reticulum, and mitochondria. It is involved in post-translational modification, protein folding, transport, and degradation. HSP70 binds to newly synthesized polypeptides to prevent premature aggregation and directs them to downstream folding systems like HSP60. For already folded proteins with unstable conformations, HSP70 can refold them to restore function. When proteins cannot be properly folded, HSP70 directs them to proteasomes or autophagosomes for degradation.

This comprehensive regulation makes HSP70 a crucial defense mechanism for cells under stress. During heat shock, oxidative stress, and other conditions, HSP70 expression is significantly upregulated, providing protective benefits. Additionally, HSP70 participates in signaling pathways to maintain normal physiological functions, acting as a constant "molecular nanny" within the cell.

HSP90 Family: Stabilizers of Key Client Proteins

Unlike HSP70, which primarily manages newly synthesized polypeptides, the HSP90 family specializes in stabilizing key "client" proteins, such as transcription factors, kinases, and signaling proteins. These client proteins are essential for various cellular functions, and their stability and activity rely on HSP90's chaperone activity.

HSP90's core structure includes an N-terminal nucleotide-binding domain and a C-terminal substrate-binding domain. ATP binding and hydrolysis drive HSP90's transition between open and closed conformations, regulating its affinity for client proteins. HSP90 interacts with various co-chaperones, including HSP70, HOP, and AHA1, forming a complex regulatory network.

HOP acts as a bridge between HSP70 and HSP90, facilitating their interaction and client protein transfer. AHA1 enhances HSP90's ATPase activity, speeding up client protein folding and maturation. HSP90 also recognizes and binds client proteins, providing stable binding sites for them.

Through these intricate molecular interactions, HSP90 maintains the conformational stability of client proteins, preventing misfolding or excessive degradation, and ensuring their functional persistence. This function is crucial for many cellular processes, such as signal transduction, cell cycle regulation, and transcription regulation.

HSP90 family members include cytosolic HSP90, endoplasmic reticulum HSP90, and mitochondrial HSP90. These members differ in their cellular localization and client protein selection but collectively maintain the stability of key cellular proteins. Dysregulation of HSP90 functions is often linked to major diseases, such as cancer and neurodegenerative disorders, making it a potential therapeutic target.

HSP60 Family: Folding Machines for Newly Synthesized Polypeptides

The HSP60 family is found in various cellular compartments, including the cytoplasm, mitochondria, and chloroplasts. It is primarily responsible for assisting newly synthesized polypeptides in folding into their native conformations.

The core structure of HSP60 consists of two ring-shaped oligomers, each made up of seven HSP60 subunits, forming a complex chamber. Within this chamber, newly synthesized polypeptides are temporarily stored and folded into their native state with the help of HSP10, an auxiliary factor. HSP10 forms a "cap" over the chamber's entrance, working with HSP60 to create a dynamic protein folding machine.

The HSP60 folding process involves ATP binding and hydrolysis. ATP binding expands the HSP60 chamber, allowing substrates to enter, while subsequent ATP hydrolysis drives conformational changes in HSP60, releasing the folded substrates. This ATP-driven dynamic regulation ensures the precise and orderly folding of substrates.

HSP60 plays a critical role in maintaining correct protein folding after synthesis, both in the cytoplasm and within mitochondria and chloroplasts. Deficiencies in HSP60 function are associated with mitochondrial diseases, cancer, and neurodegenerative disorders.

Small HSPs: Preventing Aggregation and Maintaining Homeostasis

Small HSPs (sHSPs) are a class of HSP family members with smaller molecular weights that are typically upregulated in response to various stresses. sHSPs are present in the cytoplasm, mitochondria, and endoplasmic reticulum, and their primary role is to maintain cellular protein homeostasis by preventing protein aggregation.

sHSPs can form stable dimers and oligomers. These complexes bind to misfolded or denatured substrate proteins, preventing further aggregation and supporting downstream folding systems like HSP70 and HSP60. sHSPs can also promote the disaggregation and refolding of aggregates, aiding in the elimination of harmful protein deposits.

In addition to their direct interaction with substrates, sHSPs can regulate cellular signaling pathways. For example, HSP27 modulates the Akt and ERK pathways, enhancing the cell's antioxidant and anti-apoptotic capabilities, thus providing a protective role in stress responses.

Members of the sHSP family, such as HSP27, HSPB2, and HSPB3, are expressed in various cell types and organelles. They collectively form a robust barrier against protein aggregation, contributing to stress resistance, anti-apoptosis, and overall protein quality control.

Key Regulatory Roles of the HSP Family in Diseases

Members of the HSP family not only play critical roles in normal cellular physiology but also have significant implications in the development and progression of various diseases. Below, we explore the important regulatory functions of HSPs in cancer, neurodegenerative diseases, cardiovascular diseases, and autoimmune diseases.

HSPs in Cancer

HSPs are often overexpressed in malignancies and are associated with poor prognosis. They can promote tumor cell proliferation, metastasis, and invasion.

For example, HSP70 is highly expressed in acute leukemia cells, and patients with lower HSP70 expression tend to have better outcomes. HSP70 stabilizes the oncogenic fusion protein FLT3-ITD in leukemia cells, advancing tumor progression. In prostate cancer, HSP27 enhances metastasis-related gene expression by regulating the Hippo pathway, thereby facilitating tumor spread. HSP90 maintains the stability of tumor-associated proteins like kinases, transcription factors, and signaling proteins, activating tumor-related pathways and driving malignant traits.

HSPs also influence tumor angiogenesis and the immune microenvironment. HSP90 stabilizes key transcription factors such as HIF-1α and NF-κB, promoting angiogenesis and epithelial-mesenchymal transition (EMT), which enhances tumor invasion and metastasis. In colorectal cancer, HSP90 promotes angiogenesis by stabilizing the CD24 protein and activating the STAT3 pathway.

Extracellular HSPs, such as HSP60 and HSP70, can be released from tumor cells and act as "immune advantage molecules," activating antigen-presenting cells, inducing inflammatory responses, and potentially triggering autoimmune reactions. HSPs also regulate tumor-associated macrophages and myeloid-derived suppressor cells, impacting the body's antitumor immune response.

In summary, HSPs are integral to key tumor processes like proliferation, invasion, metastasis, and the construction of the tumor microenvironment. They are valuable as biomarkers and therapeutic targets, with interventions aiming at HSPs showing promise in cancer treatment and prognosis improvement.

HSPs in Neurodegenerative Diseases

Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), are characterized by the accumulation of misfolded or aggregated proteins. HSPs play a significant role in preventing or mitigating the damage caused by these protein deposits.

In AD, amyloid-beta (Aβ) and tau protein aggregation lead to neurodegeneration. HSP70 and HSP90 contribute to protein degradation pathways and refold misfolded proteins. For example, HSP70 can prevent tau hyperphosphorylation and Aβ aggregation, reducing neurotoxic effects and promoting neuronal survival. The increased expression of HSP70 and HSP90 is associated with decreased Aβ plaque formation and improved cognitive function in animal models.

HSP90 also regulates the aggregation of alpha-synuclein in PD. By stabilizing key signaling proteins like HIF-1α and regulating alpha-synuclein levels, HSP90 impacts neuroinflammation and neuronal cell survival. In HD, HSP70 assists in preventing huntingtin protein aggregation and reduces neuronal cell death.

Enhancing HSP expression or function through small molecules or gene therapy could provide new therapeutic strategies for neurodegenerative diseases. For example, pharmacological agents targeting HSP70 or HSP90 are being investigated for their potential to alleviate neurodegenerative symptoms and improve cognitive function.

HSPs in Cardiovascular Diseases

HSPs also have significant roles in cardiovascular health and disease. They influence the progression of heart diseases, including heart failure, ischemia, and atherosclerosis.

HSP70 and HSP90 can protect against ischemia-reperfusion injury by stabilizing proteins involved in cellular stress responses and apoptosis. In heart failure, HSPs play roles in preventing cardiomyocyte apoptosis, improving contractile function, and reducing oxidative stress.

In atherosclerosis, HSP60 and HSP70 are involved in plaque formation and progression. HSP60 can promote inflammation by activating immune cells and enhancing the production of pro-inflammatory cytokines. HSP70, on the other hand, has protective effects by reducing oxidative stress and inflammation in endothelial cells.

HSPs in Autoimmune Diseases

In autoimmune diseases, HSPs can act as autoantigens, triggering immune responses against self-proteins. For instance, HSP60 is recognized as an autoantigen in rheumatoid arthritis, with antibodies against HSP60 contributing to joint inflammation and damage.

In systemic lupus erythematosus (SLE), elevated levels of circulating HSPs, such as HSP70, are associated with disease activity and the presence of autoantibodies. HSPs may influence immune system activation and contribute to disease exacerbation.

Future Directions and Therapeutic Potential

The significant roles of HSPs in disease highlight their potential as therapeutic targets. Research is ongoing to develop HSP-targeted therapies, including inhibitors, activators, and gene therapies, to modulate their functions and improve disease outcomes.

For example, HSP90 inhibitors, such as geldanamycin, are being tested in clinical trials for cancer treatment due to their ability to disrupt oncogenic pathways and reduce tumor progression. HSP70 activators, like geranylgeranylacetone, are being investigated for their neuroprotective effects in neurodegenerative diseases.

Overall, the HSP family represents a complex network of molecular chaperones with essential roles in maintaining protein homeostasis and responding to cellular stress. Continued research into their mechanisms and functions will advance our understanding of disease processes and lead to novel therapeutic approaches for a variety of conditions.