The role of chaperonin proteins in health and illness

Alzheimer’s disease (AD)​ is a progressive neurodegenerative disorder of the central nervous system characterized by cognitive dysfunction and behavioral impairments. The diagnosis of AD has shifted from traditional symptom-dependent criteria to biomarker-dependent criteria. Chaperonin dysfunction is directly linked to key neuropathological features of AD, including Aβ deposition, tau protein hyperphosphorylation, and loss of the mitochondrial membrane potential. HSP60 expression is elevated in the lymphocytes of patients with early-stage AD, and urinary CCT1 levels can help distinguish mild cognitive impairment (the prodromal stage of AD) patients from normal controls ​​ [102–104].

HSP60 has a dual pathogenic mechanism in AD pathogenesis. HSP60 is significantly upregulated in the lymphocytes and brain tissues of AD patients, likely representing a stress response to Aβ and tau pathology, which may exert a neuroprotective effect. However, once HSP60 function is impaired within neuronal mitochondria, it hinders the clearance of Aβ from mitochondria and the inhibition of Aβ oligomerization, exacerbating intramitochondrial oxidative stress and leading to Aβ-induced mitochondrial toxicity and neurotoxicity ​​ [57, 103]. On the other hand, extracellular overexpression of HSP60 can activate microglia (the key immune cells involved in neuroinflammation), modulate the expression of TNF-α and IL-1β, and engage the NLRP3 inflammasome pathway, thereby promoting neuroinflammatory responses, as shown in Fig. 3b [102, 105].

The pathogenic mechanism of TRiC in AD primarily involves impaired clearance of neuropathological aggregates due to its downregulation. For example, decreased expression of CCT2 and CCT4 in the AD brain may reduce the capacity to fold or clear tau and Aβ. CCT1 and CCT8 act as toxic inhibitors of Aβ, alleviating Aβ-induced paralysis phenotypes. The apical domains of CCT3 and CCT7 (ApiCCT3 and ApiCCT7) can also inhibit tau aggregation in a dose-dependent manner. In AD mouse models, the upregulation of CCT4 via the oral administration of enzyme-digested phycocyanin reduces Aβ aggregation and improves cognitive deficits [47, 106–108]. Beyond simple downregulation, oxidative modifications of TRiC also play a significant role in AD pathogenesis. Compared with that in control mice, the CCT5 protein in AD mice exhibited a greater degree of oxidation. Oxidation of CCT5 can disrupt chaperonin function, leading to the accumulation and aggregation of misfolded proteins. Furthermore, it can impair the folding of actin and tubulin, thereby damaging the neuronal cytoskeleton, as shown in Fig. 3b [109].

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive degeneration and death of motor neurons. Significant elevation of HSP60 levels has been observed in the cerebrospinal fluid (CSF) of ALS patients. Given that HSP60 is a key regulator of microglial activation, its upregulation in ALS-CSF may contribute to pathogenesis by promoting chronic microglial activation and subsequent microglia-mediated neuroinflammation [110]. In ALS models in which motor neuron-like cell lines express mutant SOD1 genes, the CCT5, CCT7, and CCT8 subunits accumulate from the nucleus to the cytoplasm in neurons. This alteration in subcellular localization may represent an adaptive cellular response aimed at facilitating the clearance of misfolded proteins and aggregates generated during the disease process, as shown in Fig. 3c [111].

Huntington’s disease (HD) is an autosomal dominant movement disorder that is clinically characterized by choreiform movements. The primary cause of HD is a mutation in the Huntingtin gene on chromosome 4, leading to the production of a mutant Huntingtin protein (mHTT) containing an expanded polyglutamine (PolyQ) repeat sequence. The aggregation of mHTT in cortical and striatal neurons results in progressive atrophy of the striatum and cerebral cortex, manifesting as motor dysfunction. mHTT can suppress HSP60 expression, which leads to the collapse of mitochondrial proteostasis and the accumulation of ROS, thereby accelerating neuronal degeneration, as shown in Fig. 3d [112].

TRiC and its subunits are involved in regulating mHTT aggregation through the following three mechanisms, as shown in Fig. 3d. First, the CCT5 homo-oligomeric complex can physically isolate mHTT by capping the ends of mHTT fibrils and encapsulating oligomers, thereby preventing further aggregation [113]. Second, CCT1, CCT4, and CCT5 can inhibit mHTT aggregation by facilitating the refolding of misfolded proteins [47, 114, 115]. Third, TRiC regulates autophagy by maintaining lysosomal function and the cytoskeleton, contributing to the degradation of mHTT aggregates [116]. Aberrant expression of TRiC and its subunits is associated with key neuropathological features of HD, including mHTT protein aggregation and corticostriatal atrophy. Therefore, maintaining normal TRiC expression has therapeutic value for ameliorating HD symptoms. Specifically, CCT3 and ApiCCT1 (the apical domain of CCT1) significantly reduce mHTT aggregate formation, improve both anterograde and retrograde transport of brain-derived neurotrophic factor, restore the trophic status of striatal and cortical neurons, and consequently exert neuroprotective effects [117–120]​​.

The primary pathological hallmarks of PD are progressive degeneration, apoptosis, and loss of dopaminergic neurons in the substantia nigra pars compacta. HSP60 is significantly lower in PD neurons, which coincides with defects in mitophagy and an impaired oxidative stress response, thereby rendering dopaminergic neurons more vulnerable to oxidative damage and protein aggregation [121, 122] However, in the context of BDNF-modified stem cell therapy, upregulated expression of HSP60 can modulate microglial function by activating the TLR4‒MyD88 signaling pathway, promoting anti-inflammatory responses (such as polarization to the M2 phenotype) and releasing anti-inflammatory factors (e.g., IL-10 and TGF-β), consequently alleviating neuroinflammation, as shown in Fig. 3e [122].

Dysregulated redox homeostasis, which involves oxidative and nitrative stress, is a significant contributor to dopaminergic neuronal loss in PD. Altered expression of TRiC and its subunits in PD often represents a manifestation of the cellular stress response. In an SH-SY5Y cell model of oxidative stress and neurodegeneration induced by the neurotoxin MPP +, the expression of CCT2 is upregulated [123]. Similarly, in an N27 cell model of H₂O₂-induced apoptotic cell death, CCT7 expression is increased [124]. As oxidative stress can lead to the misfolding or damage of cytoskeletal proteins, the upregulation of CCT2 and CCT7 is likely an adaptive response to assist in the correct folding or repair of these critical proteins, which is important for counteracting oxidative stress-triggered neuronal apoptosis. However, other studies have identified CCT2 and CCT7 as key proteins whose expression is downregulated in D2-type medium spiny neurons (D2-MSNs) of Parkinson’s disease patients [125]. In healthy individuals, dopamine inhibits D2-MSNs to facilitate movement. In PD, reduced dopamine secretion leads to decreased inhibition of D2-MSNs, resulting in overexcitation and the manifestation of the hypokinetic motor phenotype of PD. Since neuronal activity in D2-MSNs is highly energy-consuming and the synthesis and folding operations of TRiC are also energy-intensive processes, under conditions of severe energy depletion, cells must prioritize the allocation of limited energy resources to sustain the most fundamental vital activities to avoid immediate cell death. Therefore, the observed downregulation of CCT2 and CCT7 in D2-MSNs may represent a decompensation of the TRiC stress response in the context of excessive metabolic and proteotoxic pressure caused by the hyperexcited state of these neurons, as shown in Fig. 3e. Beyond oxidative stress, nitrative modifications of proteins by reactive nitrogen species are also implicated in PD. Since CCT1 has been identified as a target of protein nitration in the rat brain, aberrant nitrative modification of CCT1 may contribute to the progression of neurodegenerative diseases such as PD [126].

Owing to the constant contractile work of the heart, its energy demands are enormous, making it highly susceptible to mitochondrial energy deficiency caused by HSP60 defects, potentially leading to cardiomyopathy and even heart failure. In a cardiomyocyte-specific Hspd1 knockout mouse model, deletion of Hspd1 altered the activity of mitochondrial complexes, the mitochondrial membrane potential, and ROS production, ultimately resulting in dilated cardiomyopathy (DCM), heart failure, and death [132]. Similarly, an HSPD1 T320A missense mutation was identified in patients with familial DCM. This mutation causes a loss of HSP60 chaperone function, leading to reduced activity of mitochondrial complexes III/IV, increased ROS production, and activation of the Pink1/Parkin pathway, which enhances mitophagy and upregulates FOXO3/MuRF1 expression, consequently causing cardiomyocyte atrophy/fibrosis and impaired cardiac contractile function, as shown in Fig. 4b [133]. When patients with ischemic cardiomyopathy (ICM) or DCM were stratified into low and high-expression groups based on the median myocardial HSP60 expression level, the low HSP60 expression group required heart transplantation or LVAD implantation at a significantly younger age (average reduction of 4.9 years), suggesting that reduced HSP60 expression may accelerate the progression of heart failure [134].

Chaperonins are associated with key pathological processes in coronary artery disease, including atherosclerotic plaque formation, coronary injury, and thrombus formation. HSP60 is directly involved in cardiovascular pathology, and elevated serum HSP60 levels are positively correlated with cardiovascular risk [135]. Serum HSP60 levels are significantly greater in chronic periodontitis patients who also have coronary heart disease than in those with periodontitis alone [136]. Furthermore, increased concentrations of anti-HSP60 antibodies are associated with greater coronary artery calcification in asymptomatic adults [137]. TRiC and its subunits also hold significant diagnostic value and contribute to the development of coronary artery disease. Kawasaki disease (KD), an acute systemic vasculitis syndrome primarily affecting children under five years of age, has coronary arteritis as its most severe complication. CCT1 is specifically expressed in coronary artery vascular smooth muscle cells, and its protein level is inversely correlated with the risk of coronary artery disease. Thus, CCT1 may serve as a marker for KD disease activity [138, 139]. Additionally, the heterozygous genetic mutation combination of CCT7 Ser525Leu/GUCY1A3 Leu163Phefs*24 significantly increases the risk of myocardial infarction. The CCT7 Ser525Leu mutation leads to defective TRiC folding, severely impairing the protein levels and activity of soluble guanylyl cyclase (sGC) and cyclic guanosine monophosphate (cGMP). This results in dysfunction of the NO-sGC-cGMP signaling pathway, which normally exerts protective effects in the cardiovascular system, such as vasodilation and inhibition of platelet activation. The impaired NO-sGC-cGMP pathway accelerates thrombus formation, thereby significantly increasing the genetic susceptibility to myocardial infarction in carriers of these mutations [140, 141].

The upregulation of TRiC and its subunits can be beneficial in coronary artery disease. An endothelial cell hypoxia/reoxygenation (H/R) model was used to study myocardial ischemia‒reperfusion injury. Scutellarin has been shown to protect human cardiac microvascular endothelial cells from H/R injury by upregulating CCT6A, exerting a cardioprotective effect [142]. Additionally, Zhang et al. compared lncRNA expression profiles in endothelial microvesicles (EMVs) derived from human umbilical vein endothelial cells (HUVECs) after H/R injury (HR-EMVs) versus EMVs from HUVECs subjected to H/R with propofol post-conditioning (HR + P)-EMVs and reported that (HR + P)-EMVs highly expressed lncCCT4-2. HR-EMVs induce apoptosis and oxidative stress in cardiomyocytes, and the lncCCT4-2 from (HR + P)-EMVs, upon uptake by cardiomyocytes, bind to and stabilize CCT4 mRNA, slowing its degradation and enhancing CCT4 protein expression, significantly reducing oxidative stress and apoptosis in cardiomyocytes, improving cardiac function, and diminishing infarct size, as shown in Fig. 4c [143].

Coronary atherosclerotic heart disease, the most common form of coronary artery disease, is significantly driven by HSP60 during the atherogenic process. Under stress conditions such as smoking, hypertension, or oxLDL accumulation, HSP60 expression increases within arterial wall cells, and HSP60 is secreted extracellularly. Extracellular HSP60 acts as a damage-associated molecular pattern (DAMP), activating Toll-like receptors (TLR2/TLR4) and the NF-κB pathway, thereby promoting the secretion of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6, exacerbating systemic inflammation and activating the vascular endothelium [144]. Concurrently, extracellular HSP60 can induce the production of anti-HSP60 antibodies, which mount an autoimmune attack against vascular endothelial cells, promoting endothelial injury and plaque formation, as shown in Fig. 4c [145, 146]. Furthermore, anti-HSP60 antibody levels are positively correlated with pulse wave velocity (an indicator of arterial stiffness) and negatively correlated with flow-mediated vasodilation (a measure of endothelial function) [147]. Therefore, reestablishing immune tolerance to HSP60 represents an important therapeutic direction for atherosclerotic plaque management.

Within mitochondria, the pro- or anti-inflammatory effects of HSP60 are influenced by the intensity and duration of stress, as shown in Fig. 4d. The mitochondrial unfolded protein response (mtUPR) is a protective mechanism activated in response to mitochondrial stress. When misfolded or unfolded proteins accumulate within mitochondria, the mtUPR signaling pathway is initiated, upregulating the expression of mitochondrial chaperonins such as HSP60 to restore mitochondrial proteostasis and mitigate inflammation. In non-alcoholic fatty liver disease, HSP60 overexpression can suppress the release of mitochondrial double-stranded RNA (mtdsRNA), thereby alleviating inflammation mediated by the mt-dsRNA/TLR3/MDA5 pathway [149, 150]. In acute drug-induced liver injury caused by acetaminophen overdose, the mtUPR is activated, and mitochondrial HSP60 expression increases in a time-dependent manner, whereas serum ALT/AST levels decrease significantly over time. The upregulation of HSP60 coincides with the control of liver inflammation [151].

However, if excessive stress leads to mitochondrial HSP60 aberrant expression or impaired function, misfolded proteins cannot be cleared promptly, which exacerbates mitochondrial damage and initiates or exacerbates inflammation through mechanisms such as energy deficiency, excessive ROS production, and the release of mitochondrial DNA (mtDNA) [152, 153]. Under the pathological conditions of osteoarthritis, sustained or excessive activation of the mtUPR can exhaust the protective capacity of intra-mitochondrial HSP60, leading to a significant downregulation of HSP60 expression in knee joint tissue. When HSP60 function is insufficient, the collapse of mitochondrial proteostasis directly causes mitochondrial dysfunction and massive production of ROS, which activate inflammatory signaling pathways (e.g., NF-κB) and promote the release of inflammatory factors (e.g., IL-1β and TNF-α). These cytokines, in turn, further attack chondrocytes and mitochondria, creating a vicious pro-inflammatory cycle that accelerates cartilage degeneration ​​​​ [154, 155].

Within the cell, the pro- or anti-inflammatory effects of HSP60 are influenced by the specific type of stress or pharmacological agent, as shown in Fig. 4e. In alcohol-related tissue injury, treatment with the probiotic L. fermentum upregulates cytosolic HSP60, which subsequently inhibits IKK and stabilizes IκB-α, thereby blocking the activation of the NF-κB signaling pathway and ultimately reducing the production of downstream pro-inflammatory factors such as TNF-α, IL-6, and MMP-9 [156]. Conversely, in lead-induced liver injury, cytosolic HSP60 released due to mitochondrial dysfunction can activate the HSP60/NLRP3/NF-κB signaling pathway, which promotes caspase-1 activation, leading to the expression of pro-inflammatory factors such as IL-1β, IL-18, and TNF-α, thereby amplifying the inflammatory response [157].

Extracellularly, HSP60 can promote the progression of inflammation by initiating innate immune responses, as shown in Fig. 4f. Under abnormal stress conditions, HSP60 can translocate from the inside of the cell to the cytomembrane, or be released as a free molecule due to cell death, or be secreted from living cells via non-classical pathways (e.g., lysosomal vesicles) into the extracellular environment [158, 159]. Extracellular HSP60 is upregulated in various conditions, including smoking-related pneumonia [159, 160], chronic calculous cholecystitis [161], atopic dermatitis [162], splenic inflammation [163], adipose inflammation [164, 165], and neuroinflammation [57, 103]. As a DAMP, extracellular HSP60 can be recognized by pattern recognition receptors on immune cells (e.g., macrophages, monocytes, microglia, and dendritic cells), such as Toll-like receptors (TLR2 and TLR4) or TREM-2 (triggering receptor expressed on myeloid cells 2), which can activate downstream signaling pathways (e.g., MyD88/NF-κB). Furthermore, extracellular HSP60 promotes dendritic cell maturation and activation, enhances macrophage infiltration and polarization toward a pro-inflammatory (M1) phenotype, stimulates the production of pro-inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6, IL-12, and IFN-γ), and contributes to NLRP3 inflammasome activation, thereby exacerbating inflammation [166, 167]. In blood vessels, increased HSP60 expression may also induce COX-2 and NOS-2, promoting endothelial cell activation, increasing vascular permeability and inflammatory cell infiltration, and aggravating tissue edema and fibrosis [161].

Extracellularly, HSP60 can further modulate inflammation by influencing the adaptive immune response, as shown in Fig. 4f. HSP60 directly induces the proliferation of naive B cells and upregulates the expression of MHC class II molecules and co-stimulatory molecules on the B-cell surface, thereby increasing their capacity as antigen-presenting cells. It promotes the differentiation of activated B cells toward an immunoregulatory phenotype characterized by the secretion of IL-10 and IL-6. HSP60 indirectly regulates T-cell responses through B cells. T cells stimulated by HSP60-activated B cells secrete increased levels of IFN-γ and IL-10, skewing the T-cell response toward the Th0 phenotype or exerting a balancing/regulatory effect [167]. In patients with rheumatoid arthritis, self-HSP60, upon uptake by antigen-presenting cells, is presented via MHC class II molecules to low-affinity autoreactive CD4 + T cells. These T cells tend to exhibit a Th2 phenotype, secrete anti-inflammatory cytokines such as IL-4 and IL-10, and inhibit inflammatory progression [168]. In cholecystitis, a positive correlation exists between the expression of HSP60 and IL-13 in the gallbladder wall, indicating that HSP60 may promote Th2 immune bias, thereby influencing the inflammatory process [161].

In addition to pathogenic autoantibodies, dysregulated expression of natural autoantibodies (nAAbs) also contributes to the pathogenesis of autoimmune diseases, as shown in Fig. 5a. In pregnant women with HT, nAAbs targeting HSP60 are produced. In healthy individuals, nAAbs recognize evolutionarily conserved antigens (e.g., heat shock proteins and mitochondrial enzymes) without causing pathological damage and are involved in maintaining immune tolerance. However, compared with healthy pregnant women, HT patients present elevated serum levels of anti-HSP60 IgM nAAbs and decreased levels of anti-HSP60 IgG nAAbs during both early and late pregnancy. Excess HSP60 IgM nAAbs may promote inflammation via complement activation, whereas a reduction in HSP60 IgG nAAbs could impair the maintenance of immune tolerance, adversely affecting the immune environment at the maternal‒fetal interface. Together, these imbalances may increase the risk of pregnancy complications such as miscarriage and preterm birth [176, 177].

Exogenous chaperonin-induced cross-reactive chaperonin antibodies also contribute to the pathogenesis of autoimmune diseases, as shown in Fig. 5a. The heat shock protein HSPB from Helicobacter pylori (Hp) is highly immunogenic and shares homology with human HSP60. Consequently, the cross-reactive immune response triggered by Hp may lead to gastric mucosal damage [178]. In a mouse model of SLE, subcutaneous injection of bacterial HSP65 from Mycobacterium leprae—which shares high homology with self-HSP60—can accelerate SLE progression by cross-reactively activating autoreactive T and B cells and by activating TLR4 to promote the release of inflammatory cytokines [179]. The archaeon Methanobrevibacter oralis (M. oralis), a significant oral archaeon, expresses a Group II chaperonin, Cpn, which shares 28.8–40.0% sequence homology with human CCT subunits (CCT1–8). Antibodies generated against M. oralis Cpn can cross-react with human CCT proteins, potentially inducing autoimmunity and contributing to the pathogenesis of periodontitis [49, 50]. The chaperonin GroEL from the periodontal pathogen Porphyromonas gingivalis shares approximately 60% sequence homology with human HSP60 in immunodominant epitope regions. Therefore, anti-GroEL antibodies produced by the host immune system can attack human HSP60, particularly those that target cardiovascular tissues, leading to endothelial dysfunction and amplification of an inflammatory cascade, thereby participating in the formation and progression of atherosclerotic plaques [144]. Concurrently, periodontitis-induced tissue damage also releases HSP60 into the circulation and induces the production of anti-HSP60 antibodies, promoting plaque formation [145].

Chaperonins contribute to the formation of a multi-layered autoimmune environment by influencing the infiltration of autoimmune cells, as shown in Fig. 5a. In patients with ankylosing spondylitis (AS), CCT2 expression is decreased in the blood. Given that CCT2 levels are negatively correlated with those of neutrophils and NKT cells, its low expression may promote the infiltration of these pro-inflammatory cells, thereby driving the chronic inflammatory process in AS [172]. Lupus nephritis (LN) is a severe complication of SLE. In LN patients, renal tubular epithelial cells express CCT6A, and γδ T cells can specifically bind and activate CCT6A, promoting the secretion of IFN-γ and IL-2, which causes cytotoxic damage to the renal tubules. Subsequent renal tissue injury releases soluble CCT6A, which in turn persistently activates more γδ T cells and creates a vicious cycle leading to more severe pathological damage in the kidney, thereby worsening LN [174]. In a rat model of RA, a wild-type peptide of the human HSP60 protein induced pro-inflammatory CD4 + FoxP3- T cells (such as Th1 and Th17 cells) but failed to induce regulatory T cells (Tregs), which led to a Th17/Treg imbalance, promoting the secretion of pro-inflammatory cytokines such as IFN-γ and IL-17 and exacerbating inflammation [180]. In type 1 diabetes, HSP60 acts as an autoantigen to activate pathogenic Th1 cells and concurrently engages the innate immune system via TLR-4 pathways. Together, these responses drive islet inflammation, culminating in β-cell destruction and insulin secretory failure [181].

Beyond their roles in viral diseases and host antiviral immunity, chaperonins also play critical roles in the pathogenesis of infections caused by bacteria (Helicobacter pylori, Clostridium difficile, Bordetella pertussis, Salmonella enterica, Shigella flexneri, Listeria monocytogenes), chlamydia (Chlamydia trachomatis), fungi (Histoplasma spp., Paracoccidioides spp., Candida albicans, Trichophyton indotineae), and parasites (Plasmodium falciparum, nematode Heligmosomoides polygyrus).

HSP60 and TRiC exhibit upregulated expression and enhanced chaperonin activity in the majority of cancers and/or their extracellular vesicles. This has been reported in a wide range of cancers, including glioma [215], neuroblastoma [216], medulloblastoma [217], sinonasal adenocarcinoma [218, 219], nasopharyngeal carcinoma [219], oral squamous cell carcinoma [220–222], ameloblastoma [223], head and neck squamous cell carcinoma [224, 225], papillary thyroid carcinoma [226–229], laryngeal cancer [230], esophageal squamous cell carcinoma (ESCC) [231–239], lung cancer [240–253], breast cancer [25, 35, 254–272], gastric cancer [19, 223, 273–277], hepatocellular carcinoma (HCC) [34, 36, 223, 278–292], gallbladder cancer [293], pancreatic cancer [153, 294, 295], renal cell carcinoma [296], colon cancer [223, 297–308], endometrial cancer [309], cervical cancer [310–314], ovarian cancer [315–321], bladder cancer [322, 323], prostate cancer [223, 324–329], testicular cancer [330, 331], acute myelocytic leukemia [332], B-cell non-Hodgkin’s lymphoma [333–335], multiple myeloma [336–338], leiomyosarcoma [339], melanoma [340], osteosarcoma [341–343], and Ewing’s sarcoma [344]. In summary, the expression levels of these chaperonins are closely correlated with malignant biological phenotypes of cancer cells—such as proliferation, migration, and invasion—as well as malignant clinical phenotypes, including advanced tumor stage, higher pathological grade, metastasis, recurrence, shortened survival, and treatment resistance.

However, exceptions exist. For example, HSP60 expression is downregulated in ovarian cancer cells derived from ascites [345], in some cases of advanced colorectal cancer [346], and in certain HCCs [347]. Similarly, CCT3 expression is decreased in patients with testicular cancer exhibiting asthenospermia [348], and CCT4 expression is downregulated in Wilms’ tumor tissue [349]. In these specific contexts, the downregulation of chaperonin expression is associated with more malignant tumor phenotypes.

Aberrantly expressed chaperonins contribute to tumorigenesis and progression by participating in various biological processes, including cytoskeletal remodeling, cell cycle regulation, apoptosis, metabolic reprogramming, and modulation of the tumor immune microenvironment, as shown in Fig. 5d.

Regarding cytoskeleton, chaperonins, particularly TRiC, promote the occurrence and development of cancer by regulating cytoskeletal dynamics. For example, CCT2 in neuroblastoma [216] and breast cancer [350], CCT4 in pre-B cell acute lymphoblastic leukemia [351] and ESCC [233, 234, 236], and CCT8 in glioma [215] can facilitate the folding of cytoskeletal proteins (actin and tubulin), thereby stabilizing the cytoskeleton, which drives malignant phenotypes that depend on the cytoskeleton, such as the proliferation, migration, and invasion of cancer cells.

Regarding cell cycle, chaperonins promote cancer proliferation by regulating the cycle-related proteins [352]. For example, CCT2 and CCT3 in breast cancer [266, 267, 350], CCT3 in gastric cancer [19] and melanoma [340], CCT8 in non-Hodgkin’s lymphoma [333], and CCT8 in HCC [288, 353–356] can all modulate the expression and activity of cell cycle-related proteins such as PCNA, Cyclin D1, Cyclin E, CDK2, CDK4, and CDK6, which drive cell cycle progression and enhance cancer cell proliferation. Additionally, chaperonins contribute to cancer proliferation by regulating the expression of telomere-associated proteins and modulating telomerase activity. For example, CCT4 in acute myeloid leukemia (AML) ​​ [357] and CCT6A in cervical cancer [312, 313] can maintain telomere length stability, thereby increasing the proliferative capacity of cancer cells and promoting cancer survival and progression.

Regarding apoptosis, chaperonins inhibit cancer apoptosis and promote carcinogenesis and progression by regulating apoptosis-related proteins such as Bax, Bak, Bcl-2, Bim, Caspase-3, Survivin and p53. As a mitochondrial chaperonin, HSP60 participates in maintaining mitochondrial homeostasis. HSP60 deficiency leads to dysfunctional mitochondrial metabolic enzymes, mtDNA leakage, decreased mitochondrial membrane potential, increased ROS production, and ultimately induces cancer apoptosis [358]. In the mitochondria of cancers, HSP60 is overexpressed and can directly bind to cyclophilin D to antagonize cyclophilin D-induced opening of the mitochondrial permeability transition pore, preventing cytochrome c release and caspase activation and thereby inhibiting cancer apoptosis [358]. In ameloblastomas, HSP60 overexpression helps cancer cells maintain mitochondrial homeostasis, limits excessive ROS production, and enables cancer cells to evade ROS-triggered apoptotic signals, thereby promoting cancer progression and recurrence [223]. In oral squamous cell carcinoma, HSP60 interacts with Survivin to form a complex, stabilizing mitochondrial Survivin and subsequently inhibiting caspase activation, thus suppressing cancer apoptosis [222]. Chaperonins also participate in regulating apoptosis by interfering with the cancer suppressive function of p53. In colon cancer cells, CCT8 binds to wild-type p53 in the cytoplasm, preventing its nuclear translocation [304]. CCT8 can also directly bind to the ribosomal protein RPL4 in the cytoplasm of colon cancer cells, disrupting the RPL4-MDM2 interaction and enhancing MDM2-mediated ubiquitination and degradation of p53 [303]. In cervical cancer, CCT8 forms a stable complex with the HPV E6 oncoprotein, increasing its ability to degrade p53 [314]. The obstruction of nuclear translocation or degradation of p53 can drive the expression of downstream cyclins, accelerate genomic instability, and promote tumor cell escape from apoptosis, thereby facilitating tumor proliferation, invasion, and metastasis.

Regarding cancer metabolic reprogramming, most malignant cancers require nutrients and energy, such as glucose, lipids, amino acids, and nucleotides, to support cell growth and proliferation. To survive under stressful conditions such as hypoxia and nutrient limitation, cancer cells initiate metabolic reprogramming. Mitochondrial metabolic reprogramming is a core component of cancer metabolic reprogramming. HSP60, a key molecule in maintaining mitochondrial metabolic function, promotes ATP synthesis through its involvement in folding mitochondrial metabolic enzymes and maintaining mitochondrial homeostasis, thereby supplying energy for cancer cells [253, 359, 360]. Chaperonins also regulate the metabolic reprogramming of glucose, amino acid, and lipid metabolism, thereby promoting cancer progression. In glucose metabolism, for example, the CCT6A/CCT8/THOC3/PFKFB4 axis in lung squamous cell carcinoma [361], the CCT6A/STAT1/HK2 axis in lung adenocarcinoma [248], CCT3 in lung adenocarcinoma [243], and CCT8 in cervical cancer [314] all regulate glycolytic enzymes, promoting cancer cell glycolysis and providing more energy for cancer cell proliferation. In amino acid metabolism, the activated CCT6A/RPS3/serine axis can provide material and energy for the rapid proliferation of HCC cells by mediating one-carbon metabolism [362]. Lipid metabolism, for example, the CCT3-LINC00326 axis in HCC [363] and CCT8 in cervical cancer [314], can influence oxidative stress levels and the energy supply within cancer cells by regulating lipid metabolism, thereby promoting cancer progression.

Regarding cancer immune microenvironment, the expression of chaperonin is associated with the extent of infiltration and polarization of anti-tumor immune cells. In lung adenocarcinoma, patients with high HSP60 expression levels show lower levels of infiltration by activated and immature B cells and CD4 + T cells within cancers [364]. In colon cancer, HSP60 can activate the TLR2‒MYD88 pathway, induce the differentiation of monocytic myeloid-derived suppressor cells, and inhibit T-cell activity [365]. In medulloblastoma, CCT2 expression is negatively correlated with the level of CD4 + T-cell infiltration [217]. In breast cancer, CCT2 can suppress CD4 + T-cell activation by restricting Ca2 + -NFAT1 signaling [366]. In lung adenocarcinoma, CCT3 can promote Th2 cell infiltration while inhibiting the activity of immature dendritic cells and mast cells [367]. In Wilms’ tumor, low expression of CCT4 can activate the ErbB pathway, increase the secretion of the inflammatory factors IL-6/IL-8, and create a pro-tumor inflammatory microenvironment [349]. In colorectal cancer, CCT6A can lead to CD8⁺ T-cell exhaustion by upregulating TUBA1B expression and is associated with reduced infiltration of CD4⁺ T cells, B cells, and dendritic cells in the tumor microenvironment [298, 368]. In pancreatic ductal adenocarcinoma, the key exosomal protein CCT6A derived from tumors can drive the polarization of tumor-associated macrophages toward the M2 phenotype ​​ [294]. Therefore, chaperonins can promote cancer cell immune escape by weakening anti-cancer immune responses and forming an immunosuppressive cancer microenvironment.

The expression level of chaperonins is directly correlated with chemosensitivity. In most cancers, high expression of chaperonins is closely associated with cancer drug resistance [233, 234, 242, 317, 369–375]. Chaperonins mediate cancer drug resistance through mechanisms such as modulating the cancer immune microenvironment, regulating mitochondrial function, affecting drug accumulation, stabilizing the cytoskeletal structure, and interfering with cell death pathways.

Regarding cancer immune microenvironment, the expression level of HSP60 is significantly greater in colon cancer patients who are insensitive to the mFOLFOX chemotherapy regimen than in those in the sensitive group. High HSP60 expression is positively correlated with the infiltration of immunosuppressive cells (e.g., DCs, FOXP3 + Tregs, and CD4 + T cells) and negatively correlated with the infiltration of cytotoxic cells (CD8 + T cells). HSP60 may contribute to drug resistance by recruiting immunosuppressive cells to weaken anti-cancer immune responses [369]. In HCC, the methylation of HSP60 by protein arginine methyltransferase 3 can inhibit the cGAS/STING innate immune pathway, impair T-cell-mediated anti-cancer immunity, and is consequently associated with resistance to immune checkpoint blockade therapy [360].

Regarding mitochondrial function, HSP60 can increase oxidative phosphorylation capacity by activating the mtUPRmt and upregulating β-catenin signaling, thereby providing energy for cancer cells and leading to drug resistance. Conversely, chemotherapy drugs can induce mitochondrial stress and ROS production. The upregulation of HSP60 helps to counteract chemotherapy-induced mitochondrial stress by stabilizing mitochondria and inhibiting ROS-dependent apoptosis, thus promoting drug resistance [253]. Therefore, inhibiting HSP60 can sensitize neuroendocrine prostate cancer cells to cisplatin and increase the sensitivity of glioblastoma to resveratrol [329, 359].

Regarding drug accumulation, chaperonins primarily affect the intracellular concentration of chemotherapeutic agents in cancer cells by promoting drug efflux and reducing drug influx. In AML, CCT1 upregulates the expression of drug efflux pumps (MRP1 and P-gp) by activating the PI3K/AKT signaling pathway, thereby reducing the intracellular accumulation of doxorubicin [376]. In ESCC, overexpression of CCT8 increases the expression of cytoskeletal proteins to maintain cell membrane integrity, which can reduce the influx of cisplatin [236].

Regarding cytoskeleton, chaperonins can directly affect the chemosensitivity of chemotherapeutic agents to microtubule polymerization/depolymerization by stabilizing the cytoskeleton. In acute lymphoblastic leukemia, CCT2 and CCT5 assist in the correct folding of actin and tubulin, stabilizing microtubules and counteracting the microtubule-depolymerizing effect of vincristine [377]. Similarly, loss of CCT3 leads to aberrant microtubule structure, impaired microtubule polymerization, and defective kinetochore‒microtubule attachment. Consequently, CCT3-deficient HCC cells become more sensitive to vincristine (which inhibits microtubule polymerization) but resistant to paclitaxel (which promotes microtubule polymerization) [378].

Regarding cell death pathways, chaperonins can resist chemotherapy-induced cell death by inhibiting apoptosis, autophagy and ferroptosis, thereby enhancing pro-survival signaling. In AML, CCT1 inhibits apoptosis and autophagy by activating the AKT/mTOR signaling pathway, increasing resistance to doxorubicin [371, 379]. In non-small cell lung cancer, CCT3 inhibits apoptosis by activating the JAK2/STAT3 pathway, increasing resistance to cisplatin [242]. The overexpression of CCT6A in colorectal cancer also inhibits apoptosis and autophagy, thereby increasing resistance to cisplatin [373]. In HCC, CCT3 binds to ACTN4, impeding the recycling of transferrin receptor protein 1 and inhibiting iron endocytosis, thereby interfering with sorafenib-induced ferroptosis and leading to drug resistance to sorafenib [380].

For diseases driven by chaperonin upregulation, inhibitors targeting HSP60 and TRiC are under investigation. HSP60 inhibitors, such as KHS101, Myrtucommulone A, 6-Shogaol, Mizoribine, and Epolactaene, function by blocking ATP binding/hydrolysis or through covalent reactions with specific cysteine residues in HSP60, thereby inhibiting its function [252, 409, 412, 414]. TRiC inhibition​ can be achieved via non-subunit-specific inhibitors or subunit-specific inhibitors. Non-subunit-specific TRiC inhibitors​ include HSF1A, CT20p, Cytochalasin D, Latrunculin A, and Cucurbitacin E [205, 216, 241, 415]. Subunit-specific TRiC inhibitors​ target specific TRiC subunits for degradation and include I-Trp, Anticarin-β, DCAF12, PCV2 Cap, PARK2, TRIM21, and TRIM38 [219, 341, 416, 419–423].

For diseases driven by chaperonin downregulation, therapeutic approaches aim to increase chaperonin levels, either indirectly or directly. Indirect upregulators​ include compounds such as Curcumin, DLN-BDNF, FHPRO for Men antioxidant capsules, Enzyme-digested phycocyanin, and Scutellarin, which can increase the expression of chaperonins [60, 107, 122, 142, 424]. Direct supplementation strategies​ involve administering the deficient chaperonin protein itself. Examples are Human recombinant mitochondrial HSP60, Human recombinant CCT, ApiCCT1, ApiCCT3, ApiCCT7, the Tat-CCT2 fusion protein, CCT2-UC-MSC-EVs, and (HR + P)-EMV-lncCCT4-2 [108, 117, 143, 426–428].

Beyond the straightforward strategies of supplementing or reducing chaperonin levels, the development of immune attack therapy and immune tolerance therapy based on the antigenicity of chaperonins has gained significant attention in cancers and autoimmune diseases, respectively. For diseases characterized by upregulated chaperonin expression, therapeutics have been developed that exploit chaperonins as antigenic targets. An example is the antibody‒drug conjugate (ADC) McAb-A6-Au, which consists of a cytotoxic drug linked to an antibody targeting a chaperonin [432, 433]. Additionally, T cells expressing two additional T-cell receptors (TETARs) can significantly increase the killing specificity of T cells. For example, personalized adoptive T-cell therapy, which simultaneously targets a common melanoma antigen (gp100) and the individual’s mutant antigen (CCT6A), has been developed for melanoma patients with CCT6A mutations [434, 435].

In autoimmune diseases triggered by aberrant chaperonin upregulation, immune tolerance vaccines containing chaperonin components aim to induce immune tolerance to the chaperonin antigen. Several HSP60 vaccine studies have shown promise. For example, oral HSP60 vaccines [460], intranasal HSP60 vaccines [461], multi-HSP60 epitope vaccines [462], and the injection of the complete tolerance-inducing adjuvant 8206 (which forms an active vaccine in situ with the endogenous pathogenic autoantigen HSP60) [463] have been shown in animal models to significantly reduce the size of atherosclerotic plaques [338, 436–438].

Simvastatin may indirectly reduce anti-HSP antibody levels by inhibiting the activation of NF-κB, suppressing T-cell activation, and downregulating CD40 expression. In a phase II clinical trial involving 102 dyslipidemic patients, treatment with simvastatin was associated with significant reductions in serum anti-HSP60 titers [464].

HSP65, which is present in both DNA-hsp65 (a plasmid DNA backbone carrying the specific Mycobacterium HSP65 gene) and HspE7 (a recombinant fusion protein comprising Hsp65 from Mycobacterium bovis BCG and the E7 protein from human papillomavirus type 16), also belongs to the HSP60 family. The roles of HSP65 in these drugs are to either 1) express the HSP65 protein to activate innate or adaptive immunity, or 2) serve as an adjuvant to enhance the immunogenicity of the HPV E7 antigen, thereby targeting cancer tissues or oncogenic proteins. In a phase I clinical trial involving 21 patients with advanced head and neck carcinoma, ultrasound-guided intratumoral injection of naked DNA-hsp65 plasmid induced partial tumor volume regression in 4 patients and stable disease in 1 patient [465]. In a phase II clinical trial involving 27 pediatric recurrent respiratory papillomatosis (RRP) patients, treatment with HspE7 improved the clinical course as it reduces the frequency of required surgeries [457]. In a phase II clinical trial involving 58 patients with cervical intraepithelial neoplasia III (CIN III), treatment with HspE7 promoted the regression of CIN III lesions, with 22.5% of patients achieving a pathologic complete response and 55% achieving a clinical partial response [467]. In a phase I/II clinical trial involving 15 HIV patients with high-grade anal intraepithelial neoplasia (HG-AIN), treatment with HspE7 resulted in the regression of lesions to AIN 1 or ASC-US in five patients [468].

DiaPep277, p336-351-CTB, and CIGB‑814 all contain peptide segments derived from HSP60. These peptides, either alone or in conjunction with adjuvants, can modulate T-cell responses by promoting a shift from a pro-inflammatory Th1 phenotype toward an anti-inflammatory Th2 or regulatory T-cell (Treg) phenotype. In a phase II clinical trial (NCT01103284↗) involving 35 adult patients with type 1 diabetes, DiaPep277 was demonstrated to modulate immune responses, effectively delay the loss of β-cell function, and improve glycemic control [469]. However, in another phase II clinical trial involving 30 pediatric patients with type 1 diabetes, DiaPep277 showed no beneficial effects in preserving β-cell function or improving metabolic control [470]. Unfortunately, the research results related to DiaPep277 in a phase III clinical trial (NCT00615264↗) were retracted due to allegations of academic misconduct [474]. In a phase I/II clinical trial involving 8 cases of Behcet’s uveitis, oral p336-351-CTB prevented the recurrence of uveitis [471]. In a phase I clinical trial (RPCEC00000238) involving 20 patients with moderate active rheumatoid arthritis, subcutaneous administration of CIGB‑814 significantly reduced IL‑17 and IFN‑γ levels, promoted clinical improvement and radiographic improvement [472]. In a phase II clinical trial (RPCEC00000313) involving 24 patients with severe and critical COVID‑19, intravenous administration of CIGB‑814 effectively controlled hyperinflammation and facilitated patient recovery [473].