Mechanism of chaperone coordination during cotranslational protein folding in bacteria.

Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally distinct classes of chaperones promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavored close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor recognizes compact folding intermediates that expose an extensive unfolded surface, and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ, nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to protect incipient structure in the nascent polypeptide well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.

HSP70 binds to specific non-coding RNA and regulates human RNA polymerase III.

Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here, we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone heat shock protein 70 (HSP70) binds to non-coding RNA transcribed by RNA polymerase III (RNA Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by RNA Pol III. Detailed biochemical analyses showed that HSP70 alleviates the inhibitory effect of cognate tRNA transcript on tRNA gene transcription. Thus, our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in cancer therapeutics.

Toxicity of the model protein 3×GFP arises from degradation overload, not from aggregate formation.

Although protein aggregation can cause cytotoxicity, such aggregates can also form to mitigate cytotoxicity from misfolded proteins, although the nature of these contrasting aggregates remains unclear. We previously found that overproduction (op) of a three green fluorescent protein-linked protein (3×GFP) induces giant aggregates and is detrimental to growth. Here, we investigated the mechanism of growth inhibition by 3×GFP-op using non-aggregative 3×MOX-op as a control in Saccharomyces cerevisiae. The 3×GFP aggregates were induced by misfolding, and 3×GFP-op had higher cytotoxicity than 3×MOX-op because it perturbed the ubiquitin-proteasome system. Static aggregates formed by 3×GFP-op dynamically trapped Hsp70 family proteins (Ssa1 and Ssa2 in yeast), causing the heat-shock response. Systematic analysis of mutants deficient in the protein quality control suggested that 3×GFP-op did not cause a critical Hsp70 depletion and aggregation functioned in the direction of mitigating toxicity. Artificial trapping of essential cell cycle regulators into 3×GFP aggregates caused abnormalities in the cell cycle. In conclusion, the formation of the giant 3×GFP aggregates itself is not cytotoxic, as it does not entrap and deplete essential proteins. Rather, it is productive, inducing the heat-shock response while preventing an overload to the degradation system.

HSP70A promotes the photosynthetic activity of marine diatom Phaeodactylum tricornutum under high temperature.

With global climate change, the high-temperature environment has severely impacted the community structure and phenotype of marine diatoms. Phaeodactylum tricornutum, a model species of marine diatom, is sensitive to high temperature, which grow slowly under high temperature. However, the regulatory mechanism of P. tricornutum in response to high-temperature is still unclear. In this study, we found that the expression level of the HSP70A in the wild type (WT) increased 28 times when exposed to high temperature (26°C) for 1 h, indicating that HSP70A plays a role in high temperature in P. tricornutum. Furthermore, overexpression and interference of HSP70A have great impact on the exponential growth phase of P. tricornutum under 26°C. Moreover, the results of Co-immunoprecipitation (Co-IP) suggested that HSP70A potentially involved in the correct folding of the photosynthetic system-related proteins (D1/D2), preventing aggregation. The photosynthetic activity results demonstrated that overexpression of HSP70A improves non-photochemical quenching (NPQ) activity under high-temperature stress. These results reveal that HSP70A regulates the photosynthetic activity of P. tricornutum under high temperatures. This study not only helps us to understand the photosynthetic activity of marine diatoms to high temperature but also provides a molecular mechanism for HSP70A in P. tricornutum under high-temperature stress.

Characterization of activating cis-regulatory elements from the histone genes of Chlamydomonas reinhardtii.

Regulation of gene expression in eukaryotes is controlled by cis-regulatory modules (CRMs). A major class of CRMs are enhancers which are composed of activating cis-regulatory elements (CREs) responsible for upregulating transcription. To date, most enhancers and activating CREs have been studied in angiosperms; in contrast, our knowledge about these key regulators of gene expression in green algae is limited. In this study, we aimed at characterizing putative activating CREs/CRMs from the histone genes of the unicellular model alga Chlamydomonas reinhardtii. To test the activity of four candidates, reporter constructs consisting of a tetramerized CRE, an established promoter, and a gene for the mCerulean3 fluorescent protein were incorporated into the nuclear genome of C. reinhardtii, and their activity was quantified by flow cytometry. Two tested candidates, Eupstr and Ehist cons, significantly upregulated gene expression and were characterized in detail. Eupstr, which originates from highly expressed genes of C. reinhardtii, is an orientation-independent CRE capable of activating both the RBCS2 and ß2-tubulin promoters. Ehist cons, which is a CRM from histone genes of angiosperms, upregulates the ß2-tubulin promoter in C. reinhardtii over a distance of at least 1.5 kb. The octamer motif present in Ehist cons was identified in C. reinhardtii and the related green algae Chlamydomonas incerta, Chlamydomonas schloesseri, and Edaphochlamys debaryana, demonstrating its high evolutionary conservation. The results of this investigation expand our knowledge about the regulation of gene expression in green algae. Furthermore, the characterized activating CREs/CRMs can be applied as valuable genetic tools.

Chaperones Hsc70 and Hsp70 play distinct roles in the replication of bocaparvovirus minute virus of canines.

Minute virus of canines (MVC) belongs to the genus Bocaparvovirus (formerly Bocavirus) within the Parvoviridae family and causes serious respiratory and gastrointestinal symptoms in neonatal canines worldwide. A productive viral infection relies on the successful recruitment of host factors for various stages of the viral life cycle. However, little is known about the MVC-host cell interactions. In this study, we identified that two cellular proteins (Hsc70 and Hsp70) interacted with NS1 and VP2 proteins of MVC, and both two domains of Hsc70/Hsp70 were mediated for their interactions. Functional studies revealed that Hsp70 was induced by MVC infection, knockdown of Hsc70 considerably suppressed MVC replication, whereas the replication was dramatically promoted by Hsp70 knockdown. It is interesting that low amounts of overexpressed Hsp70 enhanced viral protein expression and virus production, but high amounts of Hsp70 overexpression weakened them. Upon Hsp70 overexpressing, we observed that the ubiquitination of viral proteins changed with Hsp70 overexpression, and proteasome inhibitor (MG132) restored an accumulation of viral proteins. In addition, we verified that Hsp70 family inhibitors remarkably decreased MVC replication. Overall, we identified Hsc70 and Hsp70 as interactors of MVC NS1 and VP2 proteins and were involved in MVC replication, which may provide novel targets for anti-MVC approach.

The interaction between the import carrier Hikeshi and HSP70 is modulated by heat, facilitating the nuclear import of HSP70 under heat stress conditions.

Heat stress strongly triggers the nuclear localization of the molecular chaperone HSP70. Hikeshi functions as a unique nuclear import carrier of HSP70. However, how the nuclear import of HSP70 is activated in response to heat stress remains unclear. Here, we investigated the effects of heat on the nuclear import of HSP70. In vitro transport assays revealed that pretreatment of the test samples with heat facilitated the nuclear import of HSP70. Furthermore, binding of Hikeshi to HSP70 increased when temperatures rose. These results indicated that heat is one of the factors that activates the nuclear import of HSP70. Previous studies showed that the F97A mutation in Hikeshi in an extended loop induced an opening in the hydrophobic pocket and facilitated the translocation of Hikeshi through the nuclear pore complex. We found that nuclear accumulation of HSP70 occurred at a lower temperature in cells expressing the Hikeshi-F97A mutant than in cells expressing wild-type Hikeshi. Collectively, our results show that the movement of the extended loop may play an important role in the interaction of Hikeshi with both FG (phenylalanine-glycine)-nucleoporins and HSP70 in a temperature-dependent manner, resulting in the activation of nuclear import of HSP70 in response to heat stress.

Small molecule inhibitor targeting the Hsp70-Bim protein-protein interaction in estrogen receptor-positive breast cancer overcomes tamoxifen resistance.

INTRODUCTION: Estrogen receptor (ER) positive patients compromise about 70% of breast cancers. Tamoxifen, an antagonist of ERα66 (the classic ER), is the most effective and the standard first-line drug. However, its efficacy is limited by the development of acquired resistance. METHODS: A specific inhibitor of Hsp70-Bim protein-protein interaction (PPI), S1g-2, together with an inhibitor of Hsp70-Bag3 PPI, MKT-077 and an ATP-competitive inhibitor VER155008, were used as chemical tools. Cell viability assays, co-immunoprecipitation and gene knockdown were used to investigate the role of Hsp70 in tamoxifen resistance. A xenograft model was established in which tamoxifen-resistant breast cancer (MCF-7/TAM-R) cells maintained in the presence of 5 µM tamoxifen were subcutaneously inoculated. The anti-tumor efficiency of S1g-2 was measured after a daily injection of 0.8 mg/kg for 14 days. RESULTS: It was revealed that Hsp70-Bim PPI protects ERα-positive breast cancer from tamoxifen-induced apoptosis through binding and stabilizing ERα36, rather than ERα66, resulting in sustained EGFR mRNA and protein expression. Disruption of Hsp70-Bim PPI and downregulation of ERα36 expression in tumor samples are consistent with the in vitro functions of S1g-2, resulting in about a three-fold reduction in tumor volume. CONCLUSIONS: The in vivo activity and safety of S1g-2 illustrated that it is a potential strategy for Hsp70-Bim disruption to overcome tamoxifen-resistant ER-positive breast cancer.

Systematic analysis of Heat Shock Protein 70 (HSP70) gene family in radish and potential roles in stress tolerance.

The 70 kD heat shock proteins (HSP70s) represent a class of molecular chaperones that are widely distributed in all kingdoms of life, which play important biological roles in plant growth, development, and stress resistance. However, this family has not been systematically characterized in radish (Raphanus sativus L.). In this study, we identified 34 RsHSP70 genes unevenly distributed within nine chromosomes of R. sativus. Phylogenetic and multiple sequence alignment analyses classified the RsHSP70 proteins into six distinct groups (Group A-F). The characteristics of gene structures, motif distributions, and corresponding cellular compartments were more similar in closely linked groups. Duplication analysis revealed that segmental duplication was the major driving force for the expansion of RsHSP70s in radish, particularly in Group C. Synteny analysis identified eight paralogs (Rs-Rs) in the radish genome and 19 orthologs (Rs-At) between radish and Arabidopsis, and 23 orthologs (Rs-Br) between radish and Chinese cabbage. RNA-seq analysis showed that the expression change of some RsHSP70s were related to responses to heat, drought, cadmium, chilling, and salt stresses and Plasmodiophora brassicae infection, and the expression patterns of these RsHSP70s were significantly different among 14 tissues. Furthermore, we targeted a candidate gene, RsHSP70-23, the product of which is localized in the cytoplasm and involved in the responses to certain abiotic stresses and P. brassicae infection. These findings provide a reference for further molecular studies to improve yield and stress tolerance of radish.

Yeast Bxi1/Ybh3 mediates conserved mitophagy and apoptosis in yeast and mammalian cells: convergence in Bcl-2 family.

The process of degrading unwanted or damaged mitochondria by autophagy, called mitophagy, is essential for mitochondrial quality control together with mitochondrial apoptosis. In mammalian cells, pan-Bcl-2 family members including conical Bcl-2 members and non-conical ones are involved in and govern the two processes. We have illustrated recently the BH3 receptor Hsp70 interacts with Bim to mediate both apoptosis and mitophagy. However, whether similar pathways exist in lower eukaryotes where conical Bcl-2 members are absent remained unclear. Here, a specific inhibitor of the Hsp70-Bim PPI, S1g-10 and its analogs were used as chemical tools to explore the role of yeast Bxi1/Ybh3 in regulating mitophagy and apoptosis. Using Om45-GFP processing assay, we illustrated that yeast Ybh3 mediates a ubiquitin-related mitophagy pathway in both yeast and mammalian cells through association with Hsp70, which is in the same manner with Bim. Moreover, by using Bax/Bak double knockout MEF cells, Ybh3 was identified to induce apoptosis through forming oligomerization to trigger mitochondrial outer membrane permeabilization (MOMP) like Bax. We not only illustrated a conserved ubiquitin-related mitophagy pathway in yeast but also revealed the multi-function of Ybh3 which combines the function of BH3-only protein and multi-domain Bax protein as one.

HSP70-A key regulator in chondrocyte homeostasis under naturally coupled hydrostatic pressure-thermal stimuli.

OBJECTIVE: During physical activities, chondrocytes experience coupled stimulation of hydrostatic pressure (HP) and a transient increase in temperature (T), with the latter varying within a physiological range from 32.5 °C to 38.7 °C. Previous short-term in vitro studies have demonstrated that the combined hydrostatic pressure-thermal (HP-T) stimuli more significantly enhance chondroinduction and chondroprotection of chondrocytes than isolated applications. Interestingly, this combined benefit is associated with a corresponding increase in HSP70 levels when HP and T are combined. The current study therefore explored the indispensable role of HSP70 in mediating the combined effects of HP-T stimuli on chondrocytes. DESIGN: In this mid-long-term study of in vitro engineered cartilage constructs, we assessed chondrocyte responses to HP-T stimuli using customized bioreactor in standard and HSP70-inhibited cultures. RESULTS: Surprisingly, under HSP70-inhibited conditions, the usually beneficial HP-T stimuli, especially its thermal component, exerted detrimental effects on chondrocyte homeostasis, showing a distinct and unfavorable shift in gene and protein expression patterns compared to non-HSP70-inhibited settings. Such effects were corroborated through mechanical testing and confirmed using a secondary cell source. A proteomic-based mechanistic analysis revealed a disruption in the balance between biosynthesis and fundamental cellular structural components in HSP70-inhibited conditions under HP-T stimuli. CONCLUSIONS: Our results highlight the critical role of sufficient HSP70 induction in mediating the beneficial effects of coupled HP-T stimulation on chondrocytes. These findings help pave the way for new therapeutic approaches to enhance physiotherapy outcomes and potentially shed light on the elusive mechanisms underlying the onset of cartilage degeneration, a long-standing enigma in orthopedics.

HOP stabilizes the HSFA1a and plays a main role in the onset of thermomorphogenesis.

Living organisms have the capacity to respond to environmental stimuli, including warm conditions. Upon sensing mild temperature, plants launch a transcriptional response that promotes morphological changes, globally known as thermomorphogenesis. This response is orchestrated by different hormonal networks and by the activity of different transcription factors, including the heat shock factor A1 (HSFA1) family. Members of this family interact with heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90); however, the effect of this binding on the regulation of HSFA1 activity or of the role of cochaperones, such as the HSP70-HSP90 organizing protein (HOP) on HSFA1 regulation, remains unknown. Here, we show that AtHOPs are involved in the folding and stabilization of the HSFA1a and are required for the onset of the transcriptional response associated to thermomorphogenesis. Our results demonstrate that the three members of the AtHOP family bind in vivo to the HSFA1a and that the expression of multiple HSFA1a-responsive-responsive genes is altered in the hop1 hop2 hop3 mutant under warm temperature. Interestingly, HSFA1a is accumulated at lower levels in the hop1 hop2 hop3 mutant, while control levels are recovered in the presence of the proteasome inhibitor MG132 or the synthetic chaperone tauroursodeoxycholic acid (TUDCA). This uncovers the HSFA1a as a client of HOP complexes in plants and reveals the participation of HOPs in HSFA1a stability.

The role of the co-chaperone HOP in plant homeostasis during development and stress.

Proteins need to acquire their native structure in order to become fully functional. In specific cases, the active conformation is obtained spontaneously; nevertheless, many proteins need the assistance of chaperones and co-chaperones to be properly folded. These proteins help to maintain protein homeostasis under control conditions and under different stresses. HOP (HSP70-HSP90 organizing protein) is a highly conserved family of co-chaperones that assist HSP70 and HSP90 in the folding of specific proteins. In the last few years, findings in mammals and yeast have revealed novel functions of HOP and re-defined the role of HOP in protein folding. Here, we provide an overview of the most important aspects of HOP regulation and function in other eukaryotes and analyse whether these aspects are conserved in plants. In addition, we highlight the HOP clients described in plants and the role of HOP in plant development and stress response.

Degradation of citrate synthase lacking the mitochondrial targeting sequence is inhibited in cells defective in Hsp70/Hsp40 chaperones under heat stress conditions.

Most nucleus-encoded mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post-translational manner. In recent years, the quality control mechanisms of nonimported mitochondrial proteins have been intensively studied. In a previous study, we established that in budding yeast a mutant form of citrate synthase 1 (N∆Cit1) that lacks the N-terminal mitochondrial targeting sequence, and therefore mislocalizes to the cytosol is targeted for proteasomal degradation by the SCFUcc1 ubiquitin ligase complex. Here, we show that Hsp70 and Hsp40 chaperones (Ssa1 and Ydj1 in yeast, respectively) are required for N∆Cit1 degradation under heat stress conditions. In the absence of Hsp70 function, a portion of N∆Cit1-GFP formed insoluble aggregates and cytosolic foci. However, the extent of ubiquitination of N∆Cit1 was unaffected, implying that Hsp70/Hsp40 chaperones are involved in the postubiquitination step of N∆Cit1 degradation. Intriguingly, degradation of cytosolic/peroxisomal gluconeogenic citrate synthase (Cit2), an endogenous substrate for SCFUcc1-mediated proteasomal degradation, was not highly dependent on Hsp70 even under heat stress conditions. These results suggest that mitochondrial citrate synthase is thermally vulnerable in the cytosol, where Hsp70/Hsp40 chaperones are required to facilitate its degradation.

HSP70 positively regulates translation by interacting with the IRES and stabilizes the viral structural proteins VP1 and VP3 to facilitate duck hepatitis A virus type 1 replication.

The maintenance of viral protein homeostasis depends on the interaction between host cell proteins and viral proteins. As a molecular chaperone, heat shock protein 70 (HSP70) has been shown to play an important role in viral infection. Our results showed that HSP70 can affect translation, replication, assembly, and release during the life cycle of duck hepatitis A virus type 1 (DHAV-1). We demonstrated that HSP70 can regulate viral translation by interacting with the DHAV-1 internal ribosome entry site (IRES). In addition, HSP70 interacts with the viral capsid proteins VP1 and VP3 and promotes their stability by inhibiting proteasomal degradation, thereby facilitating the assembly of DHAV-1 virions. This study demonstrates the specific role of HSP70 in regulating DHAV-1 replication, which are helpful for understanding the pathogenesis of DHAV-1 infection and provide additional information about the role of HSP70 in infection by different kinds of picornaviruses, as well as the interaction between picornaviruses and host cells.

Discovery of acetophenone/piperazin-2-one hybrids as selective anti-TNBC cancer agents by causing DNA damage.

Twenty-five acetophenone/piperazin-2-one (APPA) hybrids were designed and synthesized based on key pharmacophores found in anti-breast cancer drugs Neratinib, Palbociclib, and Olaparib. Compound 1j exhibited good in vitro antiproliferative activity (IC50 = 6.50 µM) and high selectivity (SI = 9.2 vs HER2-positive breast cancer cells SKBr3; SI = 7.3 vs normal breast cells MCF-10A) against triple negative breast cancer (TNBC) cells MDA-MB-468. In addition, 1j could selectively cause DNA damage, inducing the accumulation of γH2AX and P53 in MDA-MB-468 cells. It also reduced the phosphorylation level of P38 and the expression of HSP70, which further prevented the repair of DNA damage and caused cells S/G2-arrest leading to MDA-MB-468 cells death.

Nucleotides supplementation (Nucleoforce fish™) in Caspian roach (Rutilus caspicus) diet: Growth performance, skin mucosal immune response, and resistance to salinity stress.

In this study, the growth, epidermal mucosal immunity, expression of growth-related genes, cross-protection, and resistance to salinity stress of Caspian roach were scrutinized in response to dietary levels of nucleotides (NT). Accordingly, 1200 fish (0.51 ± 0.01 g) were fed ad libitum with a basal diet (38.88 % crude protein and 10.04 % crude lipid in dry basis) containing incremental levels of NT at 0 (NT-0; control), 0.3 g kg-1 (NT-0.3), 0.6 g kg-1 (NT-0.6), and 1.2 g kg-1 (NT-1.2) for 8 weeks in triplicates. The growth performance was significantly increased in the fish fed with NT-0.6 and NT-1.2 diets compared to the control group (p < 0.05). A significant elevation in the growth hormone and insulin-like growth factor-I gene expression was recorded in NT-added groups at 0.6 and 1.2 g kg-1 compared to the control group (p < 0.05). In contrast to the control group, feeding on NT-0.6 and NT-1.2 diets had a remarkable effect on the skin mucus soluble protein and immunoglobulin levels (p < 0.05). After the feeding trial, we examined how salinity stress (15 g/l salinity) lonely and salinity stress under non-lethal thermal shock (+10 °C) affected heat shock protein (HSP70). Then, the mRNA expression of HSP70 gene from the gill was analyzed at 0, 2, 8, and 24 h post-challenge tests. The HSP70 gene expression level was approximately up-regulated more than 2-fold in NT-6 and NT-1.2 treatments compared to the control group under the salinity stress. Altogether, this research represents that the addition of NT at 0.6 and 1.2 g kg-1 in Caspian roach diet can improve overall performance and resistance to salinity stress.

Shikonin improves the effectiveness of PD-1 blockade in colorectal cancer by enhancing immunogenicity via Hsp70 upregulation.

BACKGROUND: PD-1 blockade has shown impressive clinical outcomes in colorectal cancers patients with high microsatellite instability (MSI-H). However, the majority of patients with colorectal cancer who present low microsatellite instability (MSI-L) or stable microsatellites (MSS) show little response to PD-1 blockade therapy. Here, we have demonstrated that Shikonin (SK) could induce cell death of CT26 cells via classically programmed and immunogenic pathways. METHODS AND RESULTS: SK promoted the membrane exposure of calreticulin and upregulated the expression of heat shock protein 70 (Hsp70). The upregulation of Hsp70 was dependent on ROS induced by SK and silencing of PKM2 in CT26 cells reverts ROS upregulation. Besides, SK synergizes with PD-1 blockade in CT26 tumor mice model, with the increase of intramural DC cells and CD8+ T cells. The expression of Hsp70 in tumor tissue was also increased in combinational SK plus αPD-1 therapy group. CONCLUSIONS: Our study elucidated the potential role of 'Shikonin-PKM2-ROS-Hsp70' axis in the promotion of efficacy of PD-1 blockade in CRC treatments, providing a potential strategy and targets for improving the efficacy of PD-1 blockade in colorectal cancer.

Bacterial heat shock protein genes during induction chemotherapy in pediatric patients with acute lymphoblastic leukemia.

Background: Heat shock proteins (HSP) protect cancer cells. Gastrointestinal bacteria contain HSP genes and can release extracellular vesicles which act as biological shuttles. Stress from treatment may result in a microbial community with more HSP genes, which could contribute to circulating HSP levels. Methods: The authors examined the abundance of five bacterial HSP genes pre-treatment and during induction in stool sequences from 30 pediatric acute lymphoblastic leukemia patients. Results: Decreased mean HTPG counts (p = 0.0024) pre-treatment versus induction were observed. During induction, HTPG, Shannon diversity and Bacteroidetes decreased (p = 7.5e-4; 1.1e-3; 8.6e-4), while DNAK and Firmicutes increased (p = 6.9e-3; 9.2e-4). Conclusion: Understanding microbial HSP gene community changes with treatment is the first step in determining if bacterial HSPs are important to the tumor microenvironment and leukemia treatment.

The effects of the predictability of acclimatory temperature on the growth and thermal tolerance of juvenile Spinibarbus sinensis.

Heated effluent injection, cold hypolimnetic water inputs from dams, and extreme weather events can lead to unpredictable temperature fluctuations in natural waters, impacting fish performance and fitness. We hypothesized that fish exposed to such unpredictable fluctuations would exhibit weaker growth and enhanced thermal tolerance compared to predictable conditions. Qingbo (Spinibarbus sinensis) was selected as the experimental subject in this study. The qingbo were divided into a constant temperature group (C, 22 ± 0.5 °C), a predictable temperature fluctuation group (PF, 22 ± 4 °C, first warming, then cooling within a day) and an unpredictable temperature fluctuation group (UF, 22 ± 4 °C, the order of warming or cooling is random). After 40 days of temperature acclimation, the growth, metabolic rate, spontaneous activity, thermal tolerance, plasma cortisol concentration and liver hsp70 level of the fish were measured. Unexpectedly, neither the PF nor the UF group showed decreased growth compared to the C group. This could be attributed to the fact that temperature variation did not lead to a substantial increase in basic energy expenditure. Furthermore, feeding rates increased due to temperature fluctuations, although the difference was not significant. Both the PF and UF groups exhibited increased upper thermal tolerance, but only the UF group exhibited improved lower thermal tolerance and higher liver hsp70 levels compared to the C group. The qingbo that experienced unpredictable temperature fluctuations had the best thermal tolerance among the 3 groups, which might have occurred because they had the highest level of hsp70 expression. This may safeguard fish against the potential lethal consequences of extreme temperatures in the future. These findings suggested that qingbo exhibited excellent adaptability to both predictable and unpredictable temperature fluctuations, which may be associated with frequent temperature fluctuations in its natural habitat.