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Mitochondrial dysfunction is associated with inflammatory bowel diseases (IBDs). To understand how microbial-metabolic circuits contribute to intestinal injury, we disrupt mitochondrial function in the epithelium by deleting the mitochondrial chaperone, heat shock protein 60 (Hsp60 Δ/ΔIEC ). This metabolic perturbation causes self-resolving tissue injury. Regeneration is disrupted in the absence of the aryl hydrocarbon receptor (Hsp60 Δ/ΔIEC ;AhR -/- ) involved in intestinal homeostasis or inflammatory regulator interleukin (IL)-10 (Hsp60 Δ/ΔIEC ;Il10 -/- ), causing IBD-like pathology. Injury is absent in the distal colon of germ-free (GF) Hsp60 Δ/ΔIEC mice, highlighting bacterial control of metabolic injury. Colonizing GF Hsp60 Δ/ΔIEC mice with the synthetic community OMM 12 reveals expansion of metabolically flexible Bacteroides, and B. caecimuris mono-colonization recapitulates the injury. Transcriptional profiling of the metabolically impaired epithelium reveals gene signatures involved in oxidative stress (Ido1, Nos2, Duox2). These signatures are observed in samples from Crohn's disease patients, distinguishing active from inactive inflammation. Thus, mitochondrial perturbation of the epithelium causes microbiota-dependent injury with discriminative inflammatory gene profiles relevant for IBD., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein acetylation and deacetylation are key epigenetic modifications that regulate the initiation and development of several diseases. In the context of infection with Mycobacterium tuberculosis ( M. tb ), these processes are essential for host-pathogen interactions and immune responses. However, the specific effects of acetylation and deacetylation on cellular functions during M. tb infection are not fully understood. This study employed Tandem Mass Tag (TMT) labeling for quantitative proteomic profiling to examine the acetylproteome (acetylome) profiles of noninfected and M. tb -infected macrophages. We identified 715 acetylated peptides from 1,072 proteins and quantified 544 lysine acetylation sites (Kac) in 402 proteins in noninfected and M. tb -infected macrophages. Our research revealed a link between acetylation events and metabolic changes during M. tb infection. Notably, the deacetylation of heat shock protein 60 (HSP60), a key chaperone protein, was significantly associated with this process. Specifically, the deacetylation of HSP60 at K96 by sirtuin3 (SIRT3) enhances macrophage apoptosis, leading to the elimination of intracellular M. tb . These findings underscore the pivotal role of the SIRT3-HSP60 axis in the host immune response to M. tb . This study offers a new perspective on host protein acetylation and suggests that targeting host-directed therapies could be a promising approach for tuberculosis immunotherapy., Importance: Protein acetylation is crucial for the onset, development, and outcome of tuberculosis (TB). Our study comprehensively investigated the dynamics of lysine acetylation during M. tb infection, shedding light on the intricate host-pathogen interactions that underlie the pathogenesis of tuberculosis. Using an advanced quantitative lysine proteomics approach, different profiles of acetylation sites and proteins in macrophages infected with M. tb were identified. Functional enrichment and protein-protein network analyses revealed significant associations between acetylated proteins and key cellular pathways, highlighting their critical role in the host response to M. tb infection. Furthermore, the deacetylation of HSP60 and its influence on macrophage-mediated clearance of M. tb underscore the functional significance of acetylation in tuberculosis pathogenesis. In conclusion, this study provides valuable insights into the regulatory mechanisms governing host immune responses to M. tb infection and offers promising avenues for developing novel therapeutic interventions against TB., Competing Interests: The authors declare no conflict of interest.

The Escherichia coli GroEL/ES chaperonin system facilitates protein folding in an ATP-driven manner. There are <100 obligate clients of this system in E. coli although GroEL can interact and assist the folding of a multitude of proteins in vitro. It has remained unclear, however, which features distinguish obligate clients from all the other proteins in an E. coli cell. To address this question, we established a system for selecting mutations in mouse dihydrofolate reductase (mDHFR), a GroEL interactor, that diminish its dependence on GroEL for folding. Strikingly, both synonymous and non-synonymous codon substitutions were found to reduce mDHFR's dependence on GroEL. The non-synonymous substitutions increase the rate of spontaneous folding whereas computational analysis indicates that the synonymous substitutions appear to affect translation rates at specific sites., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Background: Bacterial vaginosis (BV) is a condition marked by high vaginal bacterial diversity. Gardnerella vaginalis has been implicated in BV but is also detected in healthy women. The Gardnerella genus has been expanded to encompass 6 validly named species and several genomospecies. We hypothesized that particular Gardnerella species may be more associated with BV., Methods: Quantitative polymerase chain reaction (PCR) assays were developed targeting the cpn60 gene of species groups including G. vaginalis, G. piotii/pickettii, G. swidsinskii/greenwoodii, and G. leopoldii. These assays were applied to vaginal swabs from individuals with (n = 101) and without BV (n = 150) attending a sexual health clinic in Seattle, Washington. Weekly swabs were collected from 42 participants for up to 12 weeks., Results: Concentrations and prevalence of each Gardnerella species group were significantly higher in participants with BV; 91.1% of BV-positive participants had 3 or more Gardnerella species groups detected compared to 32.0% of BV-negative participants (P < .0001). BV-negative participants with 3 or more species groups detected were more likely to develop BV within 100 days versus those with fewer (60.5% vs 3.7%, P < .0001)., Conclusions: These results suggest that BV reflects a state of high Gardnerella species diversity. No Gardnerella species group was a specific marker for BV., Competing Interests: Potential conflicts of interest. D. N. F. and T. L. F. receive a royalty from BD related to detection of bacterial vaginosis-associated bacteria and diagnosis of bacterial vaginosis. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Chaperonins from psychrophilic bacteria have been shown to exist as single-ring complexes. This deviation from the standard double-ring structure has been thought to be a beneficial adaptation to the cold environment. Here we show that Cpn60 from the psychrophile Pseudoalteromonas haloplanktis (Ph) maintains its double-ring structure also in the cold. A strongly reduced ATPase activity keeps the chaperonin in an energy-saving dormant state, until binding of client protein activates it. Ph Cpn60 in complex with co-chaperonin Ph Cpn10 efficiently assists in protein folding up to 55 °C. Moreover, we show that recombinant expression of Ph Cpn60 can provide its host Escherichia coli with improved viability under low temperature growth conditions. These properties of the Ph chaperonin may make it a valuable tool in the folding and stabilization of psychrophilic proteins., (© 2024. The Author(s).)

Purpose: Rapid, reliable identification of mycobacteria from positive cultures is essential for patient management, particularly for the differential diagnosis of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) species. The aim of the present study was to evaluate a new "In-Vitro-Diagnostic"-certified PCR kit, FluoroType®-Mycobacteria VER 1.0 (Hain Lifescience GmbH) for NTM and MTBC identification from cultures., Methods: Mycobacteria identification isolated from positive cultures during routine practice at the Lyon university hospital mycobacteria laboratory obtained by hsp65 amplification/sequencing were compared retrospectively and prospectively to those obtained by and the FluoroType®-Mycobacteria VER 1.0 kit., Results: The overall agreement between hsp65 amplification/sequencing and the FluoroType®-Mycobacteria VER 1.0 kit was 88.4% (84/95); 91.2% (52/57) for the retrospective period and 84.2% (32/38) for the prospective period. There were 9 (9.5%) minor discrepancies (species in the FluoroType®-Mycobacteria VER 1.0 database and identified at genus level): 4 during the retrospective period, 5 during the prospective period; and 2 (2.1%) major discrepancies (species in the FluoroType®-Mycobacteria VER 1.0 database and identified incorrectly to species level): 1 during the retrospective period (M. kumamotonense identified as M. abscessus subsp massiliense by the kit) and 1 during the prospective period (M. chimaera identified as M. smegmatis by the kit). Including concordant results at genus level and minor discrepancies, 17.9% (17/95) of strains were identified as Mycobacterium sp. by the FluoroType®-Mycobacteria-VER 1.0 kit., Conclusion: The good performance of the FluoroType®-Mycobacteria-VER 1.0 kit with few major discrepancies could enable its use for first-line identification of positive mycobacteria cultures. However, an alternative identification method at least for reference laboratories is needed owing to the non-negligible proportion of NTM strains were identified at genus level., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Heat shock proteins (HSP) are a group of highly conserved molecular chaperones found in various organisms and have been associated with tumorigenesis, tumor progression, and metastasis. However, the relationship between HSP60 and apoptosis remains elusive. The aim of this study was to explore the role and regulatory mechanisms of apoptosis in response to altered HSP60 expression. We generated DF-1 cell lines of both HSP60 overexpression and knockdown and assessed their impact on apoptosis levels using ELISA and flow cytometry analyses. Additionally, we examined the transcription and protein expression levels of apoptosis-related signaling factors using fluorescence quantitative PCR (qPCR) and Western blotting analyses. Heat shock proteins 60 overexpression led to a significant decrease in apoptosis levels in DF-1 cells, which could be attributed to the downregulation of BAX and BAK expression, the upregulation of Bcl-2, and the decreased expression of Caspase 3. Conversely, HSP60 knockdown led to a substantial increase in apoptosis levels in DF-1 cells, facilitated by the downregulation of BAX and Bcl-2 expression, and the upregulation of BAK expression, which increased Caspase 3 levels, thereby promoting apoptosis. The findings of our study provide the first evidence of the inhibitory effect of HSP60 on apoptosis in DF-1 cells. These observations have significant implications for disease progression and cancer research, with potential medical applications., (Copyright © 2024. Published by Elsevier Inc.)

In the densely populated intracellular milieu, polypeptides are at constant risk of nonspecific interactions and aggregation, posing a threat to essential cellular functions. Cells rely on a network of protein folding factors to deal with this challenge. The Hsp60 family of molecular chaperones, which depend on ATP for function, stands out in the proteostasis network by a characteristic structure comprising two multimeric rings arranged back to back. This review provides an updated overview of GroEL, the bacterial Hsp60, and its GroES (Hsp10) cofactor. Specifically, we highlight recent breakthroughs in understanding the intricate folding mechanisms of the GroEL-GroES nanomachine and explore the newly discovered interaction between GroEL and the chaperedoxin CnoX. Despite considerable research on the GroEL-GroES system, numerous questions remain to be explored., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Congenital heart diseases are the most common birth defects around the world. Emerging evidence suggests that mitochondrial homeostasis is required for normal heart development. In mitochondria, a series of molecular chaperones including heat shock protein 60 (HSP60) are engaged in assisting the import and folding of mitochondrial proteins. However, it remains largely obscure whether and how these mitochondrial chaperones regulate cardiac development. Here, we generated a cardiac-specific Hspd1 deletion mouse model by αMHC-Cre and investigated the role of HSP60 in cardiac development. We observed that deletion of HSP60 in embryonic cardiomyocytes resulted in abnormal heart development and embryonic lethality, characterized by reduced cardiac cell proliferation and thinner ventricular walls, highlighting an essential role of cardiac HSP60 in embryonic heart development and survival. Our results also demonstrated that HSP60 deficiency caused significant downregulation of mitochondrial ETC subunits and induced mitochondrial stress. Analysis of gene expression revealed that P21 that negatively regulates cell proliferation is significantly upregulated in HSP60 knockout hearts. Moreover, HSP60 deficiency induced activation of eIF2α-ATF4 pathway, further indicating the underlying mitochondrial stress in cardiomyocytes after HSP60 deletion. Taken together, our study demonstrated that regular function of mitochondrial chaperones is pivotal for maintaining normal mitochondrial homeostasis and embryonic heart development., Competing Interests: Declaration of competing interest All authors declared no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Molecular chaperones are highly conserved across evolution and play a crucial role in preserving protein homeostasis. The 60 kDa heat shock protein (HSP60), also referred to as chaperonin 60 (Cpn60), resides within mitochondria and is involved in maintaining the organelle's proteome integrity and homeostasis. The HSP60 family, encompassing Cpn60, plays diverse roles in cellular processes, including protein folding, cell signaling, and managing high-temperature stress. In prokaryotes, HSP60 is well understood as a GroEL/GroES complex, which forms a double-ring cavity and aids in protein folding. In eukaryotes, HSP60 is implicated in numerous biological functions, like facilitating the folding of native proteins and influencing disease and development processes. Notably, research highlights its critical involvement in sustaining oxidative stress and preserving mitochondrial integrity. HSP60 perturbation results in the loss of the mitochondria integrity and activates apoptosis. Currently, numerous clinical investigations are in progress to explore targeting HSP60 both in vivo and in vitro across various disease models. These studies aim to enhance our comprehension of disease mechanisms and potentially harness HSP60 as a therapeutic target for various conditions, including cancer, inflammatory disorders, and neurodegenerative diseases. This review delves into the diverse functions of HSP60 in regulating proteo-homeostasis, oxidative stress, ROS, apoptosis, and its implications in diseases like cancer and neurodegeneration.

Chaperonins Hsp60s are required for cellular vitality by assisting protein folding in an ATP-dependent mechanism. Although conserved, the human mitochondrial mHsp60 exhibits molecular characteristics distinct from the E. coli GroEL, with different conformational assembly and higher subunit association dynamics, suggesting a different mechanism. We previously found that the pathological mutant mHsp60 V72I exhibits enhanced subunit association stability and ATPase activity. To provide structural explanations for the V72I mutational effects, here we determined a cryo-EM structure of mHsp60 V72I . Our structural analysis combined with molecular dynamic simulations showed mHsp60 V72I with increased inter-subunit interface, binding free energy, and dissociation force, all contributing to its enhanced subunit association stability. The gate to the nucleotide-binding (NB) site in mHsp60 V72I mimicked the open conformation in the nucleotide-bound state with an additional open channel leading to the NB site, both promoting the mutant's ATPase activity. Our studies highlight the importance of mHsp60's characteristics in its biological function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ectopic lymphoid structures (ELSs) in the rheumatoid synovial joints sustain autoreactivity against locally expressed autoantigens. We recently identified recombinant monoclonal antibodies (RA-rmAbs) derived from single, locally differentiated rheumatoid arthritis (RA) synovial B cells, which specifically recognize fibroblast-like synoviocytes (FLSs). Here, we aimed to identify the specificity of FLS-derived autoantigens fueling local autoimmunity and the functional role of anti-FLS antibodies in promoting chronic inflammation. A subset of anti-FLS RA-rmAbs reacting with a 60 kDa band from FLS extracts demonstrated specificity for HSP60 and partial cross-reactivity to other stromal autoantigens (i.e., calreticulin/vimentin) but not to citrullinated fibrinogen. Anti-FLS RA-rmAbs, but not anti-neutrophil extracellular traps rmAbs, exhibited pathogenic properties in a mouse model of collagen-induced arthritis. In patients, anti-HSP60 antibodies were preferentially detected in RA versus osteoarthritis (OA) synovial fluid. Synovial HSPD1 and CALR gene expression analyzed using bulk RNA-Seq and GeoMx-DSP closely correlated with the lympho-myeloid RA pathotype, and HSP60 protein expression was predominantly observed around ELS. Moreover, we observed a significant reduction in synovial HSP60 gene expression followed B cell depletion with rituximab that was strongly associated with the treatment response. Overall, we report that synovial stromal-derived autoantigens are targeted by pathogenic autoantibodies and are associated with specific RA pathotypes, with potential value for patient stratification and as predictors of the response to B cell-depleting therapies.

Background: Previous observational studies have demonstrated a connection between the risk of Type 2 diabetes mellitus (T2DM) and gastrointestinal problems brought on by Helicobacter pylori (H. pylori) infection. However, little is understood about how these factors impact on T2DM., Method: This study used data from the GWAS database on H. pylori antibodies, gastroduodenal ulcers, chronic gastritis, gastric cancer, T2DM and information on potential mediators: obesity, glycosylated hemoglobin (HbA1c) and blood glucose levels. Using univariate Mendelian randomization (MR) and multivariate MR (MVMR) analyses to evaluate the relationship between H. pylori and associated gastrointestinal diseases with the risk of developing of T2DM and explore the presence of mediators to ascertain the probable mechanisms., Results: Genetic evidence suggests that H. pylori IgG antibody (P = 0.006, b = 0.0945, OR = 1.0995, 95% CI = 1.023-1.176), H. pylori GroEL antibody (P = 0.028, OR = 1.033, 95% CI = 1.004-1.064), gastroduodenal ulcers (P = 0.019, OR = 1.036, 95% CI = 1.006-1.068) and chronic gastritis (P = 0.005, OR = 1.042, 95% CI = 1.012-1.074) are all linked to an increased risk of T2DM, additionally, H. pylori IgG antibody is associated with obesity (P = 0.034, OR = 1.03, 95% CI = 1.002-1.055). The results of MVMR showed that the pathogenic relationship between H. pylori GroEL antibody and gastroduodenal ulcer in T2DM is mediated by blood glucose level and obesity, respectively., Conclusion: Our study found that H. pylori IgG antibody, H. pylori GroEL antibody, gastroduodenal ulcer and chronic gastritis are all related to t T2DM, and blood glucose level and obesity mediate the development of H. pylori GroEL antibody and gastroduodenal ulcer on T2DM, respectively. These findings may inform new prevention and intervention strategies for T2DM., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Sun et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

We found that GroEL in Porphyromonas gingivalis accelerated tumor growth and increased mortality in tumor-bearing mice; GroEL promoted proangiogenic function, which may be the reason for promoting tumor growth. To understand the regulatory mechanisms by which GroEL increases the proangiogenic function of endothelial progenitor cells (EPCs), we explored in this study. In EPCs, MTT assay, wound-healing assay, and tube formation assay were performed to analyze its activity. Western blot and immunoprecipitation were used to study the protein expression along with next-generation sequencing for miRNA expression. Finally, a murine tumorigenesis animal model was used to confirm the results of in vitro. The results indicated that thrombomodulin (TM) direct interacts with PI 3 K/Akt to inhibit the activation of signaling pathways. When the expression of TM is decreased by GroEL stimulation, molecules in the PI 3 K/Akt signaling axis are released and activated, resulting in increased migration and tube formation of EPCs. In addition, GroEL inhibits TM mRNA expression by activating miR-1248, miR-1291, and miR-5701. Losing the functions of miR-1248, miR-1291, and miR-5701 can effectively alleviate the GroEL-induced decrease in TM protein levels and inhibit the proangiogenic abilities of EPCs. These results were also confirmed in animal experiments. In conclusion, the intracellular domain of the TM of EPCs plays a negative regulatory role in the proangiogenic capabilities of EPCs, mainly through direct interaction between TM and PI 3 K/Akt to inhibit the activation of signaling pathways. The effects of GroEL on tumor growth can be reduced by inhibiting the proangiogenic properties of EPCs through the inhibition of the expression of specific miRNAs., (© 2023 The Authors. Molecular Oral Microbiology published by John Wiley & Sons Ltd.)

This paper characterizes the heat stress response (HSR) and explores the impact of temperatures on the immune response of larvae from two chironomid species, Prodiamesa olivacea and Chironomus riparius. Genes involved in crucial metabolic pathways were de novo identified in P. olivacea: Hsp27, Hsp60, Hsp70, Hsc70, Cdc37, and HSF for the heat stress response (HSR) and TOLL, PGRP, C-type lectin, and JAK/hopscotch for the immune system response (ISR). Quantitative real-time PCR was used to evaluate the expression levels of the selected genes in short-term treatments (up to 120') at high temperatures (35 °C and 39 °C). Exposing P. olivacea to elevated temperatures resulted in HSR induction with increased expression of specific heat shock genes, suggesting the potential of HSPs as early indicators of acute thermal stress. Surprisingly, we found that heat shock represses multiple immune genes, revealing the antagonist relation between the heat shock response and the innate immune response in P. olivacea. Our results also showed species-dependent gene responses, with more significant effects in P. olivacea, for most of the biomarkers studied, demonstrating a higher sensitivity in this species to environmental stress conditions than that of C. riparius. This work shows a multi-species approach that enables a deeper understanding of the effects of heat stress at the molecular level in aquatic dipterans., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Heat shock protein 60 (HSP60) is essential for the folding and assembly of newly imported proteins to the mitochondria. HSP60 is overexpressed in most types of cancer, but its association with ovarian cancer is still in dispute. SKOV3 and OVCAR3 were used as experimental models after comparing the expression level of mitochondrial HSP60 in a normal human ovarian epithelial cell line and four ovarian cancer cell lines., Results: Low HSPD1 (Heat Shock Protein Family D (HSP60) Member 1) expression was associated with unfavorable prognosis in ovarian cancer patients. Knockdown of HSPD1 significantly promoted the proliferation and migration of ovarian cancer cells. The differentially expressed proteins after HSPD1 knockdown were enriched in the lipoic acid (LA) biosynthesis and metabolism pathway, in which mitochondrial 3-oxoacyl-ACP synthase (OXSM) was the most downregulated protein and responsible for lipoic acid synthesis. HSP60 interacted with OXSM and overexpression of OXSM or LA treatment could reverse proliferation promotion mediated by HSPD1 knockdown., Conclusions: HSP60 interacted with OXSM and maintained its stability. Knockdown of HSPD1 could promote the proliferation and migration of SKOV3 and OVCAR3 via lowering the protein level of OXSM and LA synthesis., (© 2023. The Author(s).)

The generation of deoxyinosine (dI) in DNA is one of the most important sources of genetic mutations, which may lead to cancer and other human diseases. A further understanding of the biological consequences of dI necessitates the identification and functional characterizations of dI-binding proteins. Herein, we employed a mass spectrometry-based proteomics approach to detect the cellular proteins that may sense the presence of dI in DNA. Our results demonstrated that human mitochondrial heat shock protein 60 (HSPD1) can interact with dI-bearing DNA. We further demonstrated the involvement of HSPD1 in the sodium nitrite-induced DNA damage response and in the modulation of dI levels in vitro and in human cells. Together, these findings revealed HSPD1 as a novel dI-binding protein that may play an important role in the mitochondrial DNA damage control in human cells.

The chaperonins CCT and Hsp60 are molecular chaperones, members of the chaperone system (CS). Chaperones are cytoprotective but if abnormal in quantity or quality they may cause diseases, the chaperonopathies. Here, recent advances in the understanding of CCT and Hsp60 in cancerology are briefly discussed, focusing on breast and brain cancers. CCT subunits, particularly CCT2, were increased in breast cancer cells and this correlated with tumor progression. Experimental induction of CCT2 increase was accompanied by an increase of CCT3, 4, and 5, providing another evidence for the interconnection between the members of the CS and the difficulties expected while manipulating one member with therapeutic purposes. Another in silico study demonstrated a direct correlation between the increase in the tumor tissue of the mRNA levels of all CCT subunits, except CCTB6, with bad prognosis. Studies with glioblastomas demonstrated an increase in the CCT subunits in the tumor tissue and in extracellular vesicles (EVs) derived from them. Expression levels of CCT1, 2, 6A, and 7 were the most increased and markers of bad prognosis, particularly CCT6A. A method for measuring Hsp60 and related miRNA in exosomes from blood of patients with glioblastomas or other brain tumors was discussed, and the results indicate that the triad Hsp60-related miRNAs-exosomes has potential regarding diagnosis and patient monitoring. All these data provide a strong foundation for future studies on the role played by chaperonins in carcinogenesis and for fully developing their theranostics applications along with exosomes., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) has long been studied from many perspectives. As a multisubunit (large subunits [LSUs] and small subunits[SSUs]) protein encoded by genes residing in the chloroplast ( rbcL ) and nuclear ( rbcS ) genomes, RuBisCo also is a model for cytonuclear coevolution following allopolyploid speciation in plants. Here, we studied the genomic and transcriptional cytonuclear coordination of auxiliary chaperonin and chaperones that facilitate RuBisCo biogenesis across multiple natural and artificially synthesized plant allopolyploids. We found similar genomic and transcriptional cytonuclear responses, including respective paternal-to-maternal conversions and maternal homeologous biased expression, in chaperonin/chaperon-assisted folding and assembly of RuBisCo in different allopolyploids. One observation is about the temporally attenuated genomic and transcriptional cytonuclear evolutionary responses during early folding and later assembly process of RuBisCo biogenesis, which were established by long-term evolution and immediate onset of allopolyploidy, respectively. Our study not only points to the potential widespread and hitherto unrecognized features of cytonuclear evolution but also bears implications for the structural interaction interface between LSU and Cpn60 chaperonin and the functioning stage of the Raf2 chaperone.

Growth hormone (GH) plays important roles in growth and development of mammalian animals and is valuable for many applications. This study aimed to express and purify biological active recombinant ovine growth hormone (roGH) through prokaryotic expression system. The roGH coding sequence was ligated into the prokaryotic expression vector and transformed into Escherichia coli (E. coli) for protein expression. Factors that influence the roGH expression were examined and the appropriate culture temperature (20 °C) and inducer (IPTG) concentration (25 μM) were determined. To enhance the soluble expression of the protein, co-expression with the molecular chaperone GroEL-GroES was utilized and eventually achieved a high yield of soluble roGH expressed in E. coli. Further, the fusion tag in expressed target protein could be efficiently removed through thrombin-specific cleavage. The expressed roGH was identified by Western blotting and the LC-MS spectrum confirmed its molecular weight of 22749.22 Da. Finally, the purified roGH had an expected biological activity when assayed in cell models in vitro and experimental mouse in vivo. In conclusion, the present study established an efficient and simple approach to produce recombinant GH, and facilitate relevant research and applications., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Natural evolution is driven by random mutations that improve fitness. In vitro evolution mimics this process, however, on a short time-scale and is driven by the given bait. Here, we used directed in vitro evolution of a random mutant library of Uracil glycosylase (eUNG) displayed on yeast surface to select for binding to chaperones GroEL, DnaK + DnaJ + ATP (DnaKJ) or E. coli cell extract (CE), using binding to the eUNG inhibitor Ugi as probe for native fold. The CE selected population was further divided to Ugi binders (+U) or non-binders (-U). The aim here was to evaluate the sequence space and physical state of the evolved protein binding the different baits. We found that GroEL, DnaKJ and CE-U select and enrich for mutations causing eUNG to misfold, with the three being enriched in mutations in buried and conserved positions, with a tendency to increase positive charge. Still, each selection had its own trajectory, with GroEL and CE-U selecting mutants highly sensitive to protease cleavage while DnaKJ selected partially structured misfolded species with a tendency to refold, making them less sensitive to proteases. More general, our results show that GroEL has a higher tendency to purge promiscuous misfolded protein mutants from the system, while DnaKJ binds misfolding-prone mutant species that are, upon chaperone release, more likely to natively refold. CE-U shares some of the properties of GroEL- and DnaKJ-selected populations, while harboring also unique properties that can be explained by the presence of additional chaperones in CE, such as Trigger factor, HtpG and ClpB., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author contributions Oran Melanker: Conceptualization, Investigation, Methodology, Resources, Visualization, Software, Writing. Pierre Goloubinoff: Conceptualization, Investigation, Methodology, Resources, Visualization, Project administration, Funding acquisition, Supervision, Writing. Gideon Schreiber: Conceptualization, Investigation, Methodology, Resources, Visualization, Project administration, Funding acquisition, Supervision, Writing. Classification Protein evolution., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Mitochondrial proteostasis, regulated by the mitochondrial unfolded protein response (UPRmt), is crucial for maintenance of cellular functions and survival. Elevated oxidative and proteotoxic stress in mitochondria must be attenuated by the activation of a ubiquitous UPRmt to promote prostate cancer (PCa) growth. Here we show that the 2 key components of the UPRmt, heat shock protein 60 (HSP60, a mitochondrial chaperonin) and caseinolytic protease P (ClpP, a mitochondrial protease), were required for the development of advanced PCa. HSP60 regulated ClpP expression via c-Myc and physically interacted with ClpP to restore mitochondrial functions that promote cancer cell survival. HSP60 maintained the ATP-producing functions of mitochondria, which activated the β-catenin pathway and led to the upregulation of c-Myc. We identified a UPRmt inhibitor that blocked HSP60's interaction with ClpP and abrogated survival signaling without altering HSP60's chaperonin function. Disruption of HSP60-ClpP interaction with the UPRmt inhibitor triggered metabolic stress and impeded PCa-promoting signaling. Treatment with the UPRmt inhibitor or genetic ablation of Hsp60 inhibited PCa growth and progression. Together, our findings demonstrate that the HSP60-ClpP-mediated UPRmt is essential for prostate tumorigenesis and the HSP60-ClpP interaction represents a therapeutic vulnerability in PCa.

The molecular chaperone protein HSP60 is mainly distributed in mitochondria and assists protein folding under physiological and pathological conditions. Accumulating evidence suggests abnormally expressed HSP60 in cancer is associated with clinicopathological features and prognosis of cancer patients. HSP60 could be used as a new biomarker for both diagnostic and prognostic purpose and tumor therapy. In this review article, we briefly described the structure, functional cycle, and regulatory mechanism of HSP60, and summarized its functional diversity in cancer as well as recent progress related to the diagnostic application of HSP60 and inhibitors against HSP60, which could provide us a comprehensive understanding about the value of HSP60 in tumor management., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Human pluripotent stem cells (hPSCs) exist in at least two distinct states in mammals: naïve pluripotency that represents several molecular characteristics in pre-implantation epiblast and primed pluripotency that corresponds to cells poised for differentiation in post-implantation epiblast. To identify and characterize the surface molecules that are necessary for the maintenance of naïve hPSCs, we generated a panel of murine monoclonal antibodies (MAbs) specific to the naïve state of hPSCs. Flow cytometry showed that N1-A4, one of the MAbs, bound to naïve hPSCs but not to primed hPSCs. Cell surface biotinylation and immunoprecipitation analysis identified that N1-A4 recognized heat shock protein 60 (HSP60) expressed on the surface of naïve hPSCs. Quantitative polymerase chain reaction (qPCR) analysis showed that HSP60 expression was rapidly downregulated during the embryoid body (EB) differentiation of primed hPSCs. HSP60 knockdown led to a decrease in the expression of pluripotency genes in primed hPSCs. HSP60 depletion also led to a decrease in the expression of pluripotency genes and representative naïve-state-specific genes in naïve hPSCs. Taken together, the results suggest that HSP60 is downregulated during differentiation of hPSCs and is required for the maintenance of pluripotency genes in both primed and naïve hPSCs, suggesting that HSP60 is a regulator of hPSC pluripotency and differentiation., Competing Interests: The authors have declared that no competing interests exist.

Chaperones are proteins that help other proteins fold. They also affect the adaptive evolution of their client proteins by buffering the effect of deleterious mutations and increasing the genetic diversity of evolving proteins. We study how the bacterial chaperone GroE (GroEL+GroES) affects the evolution of green fluorescent protein (GFP). To this end, we subjected GFP to multiple rounds of mutation and selection for its color phenotype in four replicate Escherichia coli populations, and studied its evolutionary dynamics through high-throughput sequencing and mutant engineering. We evolved GFP both under stabilizing selection for its ancestral (green) phenotype, and to directional selection for a new (cyan) phenotype. We did so both under low and high expression of the chaperone GroE. In contrast to previous work, we observe that GroE does not just buffer but also helps purge deleterious (fluorescence reducing) mutations from evolving populations. In doing so, GroE helps reduce the genetic diversity of evolving populations. In addition, it causes phenotypic heterogeneity in mutants with the same genotype, helping to enhance their fluorescence in some cells, and reducing it in others. Our observations show that chaperones can affect adaptive evolution in more than one way., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The cpn 60 barcode sequence has been established as an informative target for microbial species identification. Applications of cpn 60 barcode sequencing are supported by the availability of "universal" PCR primers for amplification and a curated reference database of cpn 60 sequences, cpnDB. A recent reclassification of lactobacilli involving the definition of 23 new genera provided an opportunity to update cpnDB and to determine if the cpn 60 barcode could be used for accurate identification of species consistent with the new framework. Analysis of 275 cpn 60 sequences representing 258/269 of the validly named species in Lactobacillus , Paralactobacillus , and the 23 newer genera showed that cpn 60-based sequence relationships were generally consistent with whole-genome-based phylogeny. Aligning or mapping full-length barcode sequences or a 150 bp subsequence resulted in accurate and unambiguous species identification in almost all cases. Taken together, our results show that the combination of available reference sequence data, "universal" barcode amplification primers, and the inherent sequence diversity within the cpn 60 barcode makes it a useful target for the detection and identification of lactobacilli, as defined by the latest taxonomic framework.

Chaperones play a vital role in the life of cells by facilitating the correct folding of other proteins and maintaining them in a functional state, being themselves, as a rule, more stable than the rest of cell proteins. Their functional properties naturally tempt investigators to actively adapt them for biotechnology needs. This review will mostly focus on the applications found for the bacterial chaperonin GroE and its counterparts from other organisms, in biotechnology or for research purposes, both in their engineered or intact versions.

Background/aim: Heat shock proteins (HSP) play a crucial role in the cellular responses during stressful conditions. In addition, HSP are involved in the regulation of a variety of important signaling pathways and processes as well as many pathological conditions, including cancer. In prostate cancer (PC), HSP60 is associated with poor differentiation and prognostic clinical parameters, such as high Gleason score, initial serum prostate-specific antigen levels, and lower cancer-specific survival. In this study, we investigated the regulation of HSP60 protein in PC., Materials and Methods: LNCaP or PC3 cells were treated with androgens or transfected with vectors containing microRNA-1 (miR-1), HSP60, HSP60-specific short-hairpin RNA (shHSP60), or a miR-1 inhibitor. The change in HSP60 protein levels was examined using Western blot., Results: Treatment of PC cells with androgens did not alter the HSP60 protein levels. Modulation of miR-1 levels in LNCaP cells also did not affect the HSP60 protein. Furthermore, HSP60 levels could not be modified by overexpression or short hairpin RNA., Conclusion: It was found that neither physiological factors, such as androgens and the HSP60-specific miR-1, nor overexpression and knockdown systems could influence the HSP60 protein levels. These results suggest an essential role of HSP60 in PC cells, as its protein expression status is regulated very precisely., (Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

As a powerful and attractive method for detecting gene expression, qRT-PCR has been broadly used in aquaculture research. Understanding the biology of taimen (Hucho taimen) has drawn increasing interest because of its ecological and economic value. Stable reference genes are required for the reliable quantification of gene expression, but such genes have not yet been optimized for taimen. In this study, the stability levels of 10 commonly used candidate reference genes were evaluated using geNorm, NormFinder, BestKeeper, and RefFinder. The expression levels of the 10 genes were detected using 240 samples from 48 experimental groups consisting of 40 individuals treated under four heat-stress conditions (18, 20, 22, and 24 °C) for 24 h and 26 °C for 4, 24, 48, and 72 h. Six tissues (blood, heart, brain, gill, skin, and liver) were collected from each individual. Ribosomal protein S29 (RPS29) and ribosomal protein L19 (RPL19) were the most stable genes among all of the samples, whereas 28S ribosomal RNA (28S rRNA), attachment region binding protein (ARBP), and 18S ribosomal RNA (18S rRNA) were the least stable. These results were verified by an expression analysis of taimen heat-stress genes (heat shock protein 60, hsp60, and heat shock protein 70, hsp70). In conclusion, RPS29 and RPL19 are the optimal reference genes for qRT-PCR analyses of taimen, irrespective of the tissue and experimental conditions. These results allow the reliable study of gene expression in taimen., (© 2022. The Author(s).)

Non-alcoholic fatty liver disease (NAFLD), the most common cause of chronic liver disease, consists of fat deposited (steatosis) in the liver due to causes besides excessive alcohol use. The folding activity of heat shock protein 60 (HSP60) has been shown to protect mitochondria from proteotoxicity under various types of stress. In this study, we investigated whether HSP60 could ameliorate experimental high-fat diet (HFD)-induced obesity and hepatitis and explored the potential mechanism in mice. The results uncovered that HSP60 gain not only alleviated HFD-induced body weight gain, fat accumulation, and hepatocellular steatosis, but also glucose tolerance and insulin resistance according to intraperitoneal glucose tolerance testing and insulin tolerance testing in HSP60 transgenic (HSP60Tg) compared to wild-type (WT) mice by HFD. Furthermore, overexpression of HSP60 in the HFD group resulted in inhibited release of mitochondrial dsRNA (mt-dsRNA) compared to WT mice. In addition, overexpression of HSP60 also inhibited the activation of toll-like receptor 3 (TLR3), melanoma differentiation-associated gene 5 (MDA5), and phosphorylated-interferon regulatory factor 3 (p-IRF3), as well as inflammatory biomarkers such as mRNA of il-1β and il-6 expression in the liver in response to HFD. The in vitro study also confirmed that the addition of HSP-60 mimics in HepG2 cells led to upregulated expression level of HSP60 and restricted release of mt-dsRNA, as well as downregulated expression levels of TLR3, MDA5, and pIRF3. This study provides novel insight into a hepatoprotective effect, whereby HSP60 inhibits the release of dsRNA to repress the TLR3/MDA5/pIRF3 pathway in the context of NAFLD or hepatic inflammation. Therefore, HSP60 may serve as a possible therapeutic target for improving NAFLD.

The pioneering studies carried out on heat shock-induced synthesis of specific proteins in the early 1970s did not identify any Hsp60 family protein in Drosophila . By the early 1980s, although the members of Hsp60 family of heat shock proteins (Hsp) were identified in a wide range of eukaryotes as homologs of the bacterial GroEL, none was known in Drosophila . The existence of the Hsp60 family protein was serendipitously revealed in Drosophila in my laboratory in 1989. Contrary to the earlier reports that all tissues in flies display the canonical heat shock response, the larval Malpighian tubules (MT) did not show induction of any of the major Hsps but synthesis of a putative Hsp60 family protein was found to be the most abundant in this tissue. A few years later, we identified this MTspecific heat shock-induced protein to indeed be a member of the Hsp60/chaperonin family. The Drosophila genome sequence projects subsequently revealed four putative Hsp60 gene sequences in the D . melanogaster genome. The present historical perspective chronicles contributions from my and other laboratories that unraveled several aspects of intriguing biology of the multiple Hsp60 genes in D . melanogaster , and highlights challenging questions awaiting future studies.

Group I chaperonins are a highly conserved family of essential proteins that self-assemble into molecular nanoboxes that mediate the folding of cytoplasmic proteins in bacteria and organelles. GroEL, the chaperonin of Escherichia coli , is the archetype of the family. Protein folding-independent functions have been described for numerous chaperonins, including HtpB, the chaperonin of the bacterial pathogen Legionella pneumophila . Several protein folding-independent functions attributed to HtpB are not shared by GroEL, suggesting that differences in the amino acid (aa) sequence between these two proteins could correlate with functional differences. GroEL and HtpB differ in 137 scattered aa positions. Using the Evolutionary Trace (ET) bioinformatics method, site-directed mutagenesis, and a functional reporter test based upon a yeast-two-hybrid interaction with the eukaryotic protein ECM29, it was determined that out of those 137 aa, ten (M68, M212, S236, K298, N507 and the cluster AEHKD in positions 471-475) were involved in the interaction of HtpB with ECM29. GroEL was completely unable to interact with ECM29, but when GroEL was modified at those 10 aa positions, to display the HtpB aa, it acquired a weak ability to interact with ECM29. This constitutes proof of concept that the unique functional abilities of HtpB can be mapped to specific aa positions.

Background: GroEL, which is a chaperone, plays a key role in maintaining protein homeostasis and, among other functions, serves to prevent protein misfolding and aggregation. In addition, the GroEL protein also has a significant effect on enhancing plant resistance and inhibiting plant diseases. However, the function of the GroEL protein in the inhibition of rice blast remains unknown., Results: Field experiment results show that photosynthetic bacteria PSB-06 have a good control effect on Magnaporthe oryzae. PSB-06 also can promote rice growth and enhance stress resistance. A GroEL protein which was separated and purified from photosynthetic bacteria had a significant antagonistic effect on appressorial formation and pathogenicity of Magnaporthe oryzae, meanwhile transcriptional analysis demonstrated that the GroEL protein could improve the expression of defense gene of rice., Conclusion: Our results show that the photosynthetic bacteria Rhodopseudomonas palustris significantly controls rice blast disease. Its action involves an extracellular GroEL protein, which inhibits appressoria formation, antagonizes the pathogenicity of Magnaporthe oryzae and promotes a host defense response. The research results provide evidence of the potential of this photosynthetic bacterium as a biocontrol agent at least for rice blast control. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Blueberry stunt phytoplasma (BBSP; 'Candidatus Phytoplasma asteris') is an insect-vectored plant pathogen that causes severe yield losses in blueberry (Vaccinium corymbosum), which is the most valuable fruit crop in Canada. Rapid, field-based diagnostic assays are desirable tools for the control of BBSP, as part of an integrated, proactive approach to production management termed biovigilance. We designed and validated a chaperonin-60 (cpn60)-targeted LAMP assay for detection of BBSP, providing a rapid, low cost, field-deployable diagnostic option. Our validation demonstrates that the assay is reproducible, with high analytical specificity and improved sensitivity when compared with 16S rRNA nested PCR. We applied the validated LAMP assay to nearly 2000 blueberry samples from Québec and Nova Scotia over three growing seasons (2016-2018). Our surveys revealed that BBSP is present in most sites across both provinces, though detection of the pathogen in individual plants varied in different tissues across sampling dates and across years, and evidence of spread between plants was limited. To quantify pathogen load in select plants, we designed additional qPCR and ddPCR assays, also based on cpn60. We found that pathogen load fluctuates in individual plants, both within and between growing seasons. Finally, we designed an interactive map to visualize the results of our surveys. These results provide a validated diagnostic assay that can be used as part of a biovigilance strategy for detecting and controlling infections caused by BBSP., (© 2021. The Author(s).)

The application of next-generation sequencing extends from microbial identification to epidemiologic insight and antimicrobial resistance prediction. Despite this potential, the roadblock for clinical laboratories lies in implementation and validation of such complex technology and data analysis. Herein, a validation study used whole-genome sequencing (WGS) for pan-bacterial identification (ID) in a clinical laboratory, and assessed its clinical relevance. A diverse set of 125 bacterial isolates, including a subset without genus (25) and/or species (10) ID, were analyzed by de novo assembly and reference genome mapping. The 16S rRNA, rpoB, and groEL genes were used for ID. Using WGS, 100% (89 of 89) and 89% (79 of 89) of isolates were identified at the genus and species-levels, respectively. WGS also provided improved results for 71% (25/35) isolates originally reported with genus-only or descriptive IDs. Chart review identified cases in which improved genus and/or species-level ID by WGS may have had a positive impact on patient care. Reasons included the use of an ineffective antibiotic owing to unclear ID, use of antibiotics when not clinically indicated, and help with an outbreak investigation. The implementation of next-generation sequencing in a clinical microbiology setting is a challenging but necessary task. This study provides a model for the validation and implementation of bacterial ID by WGS in such a setting., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Diabetes is a major risk factor for cardiovascular diseases, especially cardiomyopathy, a condition in which the smooth muscles of the heart become thick and rigid, affecting the functioning of cardiomyocytes, the contractile cells of the heart. Uncontrolled elevated glucose levels over time can result in oxidative stress, which could lead to inflammation and altered epigenetic mechanisms. In the current study, we investigated whether hyperglycemia can modify cardiac function by directly affecting these changes in cardiomyocytes. To evaluate the adverse effect of high glucose, we measured the levels of gap junction protein, connexin 43, which is responsible for modulating cardiac electric activities and Troponin I, a part of the troponin complex in the heart muscles, commonly used as cardiac markers of ischemic heart disease. AC16 human cardiomyocyte cells were used in this study. Under hyperglycemic conditions, these cells demonstrated altered levels of connexin 43 and Troponin-I after 24 h of exposure. We also examined hyperglycemia induced changes in epigenetic markers: H3K9me1, Sirtuin-1 (SIRT1), and histone deacetylase (HDAC)-2 as well as in inflammatory and stress-related mediators, such as heat shock protein (HSP)-60, receptor for advanced glycation end products (RAGE), toll-like receptor (TLR)-4, high mobility group box (HMGB)-1 and CXC chemokine receptor (CXCR)-4. Cardiomyocytes exposed to 25mM glucose resulted in the downregulation of HSP60 and SIRT1 after 48 h. We further examined that hyperglycemia mediated the decrease in the gap junction protein CX43, as well as CXC chemokine receptor CXCR4 which may affect the physiological functions of the cardiomyocytes when exposed to high glucose for 24 and 48 h. Upregulated expression of DNA-binding nuclear protein HMGB1, along with changes in histone methylation marker H3K9me1 have demonstrated hyperglycemia-induced damage to cardiomyocyte at 24 h of exposure. Our study established that 24 to 48 h of hyperglycemic exposure could stimulate stress-mediated inflammatory mediators in cardiomyocytes in vitro. These stress-related changes in hyperglycemia-induced cardiomyocytes may further initiate an increase in injury markers which eventually could alter the epigenetic processes. Therefore, epigenetic and inflammatory mechanisms in conjunction with alterations in a downstream signaling pathway could have a direct effect on the functionality of the cardiomyocytes exposed to high glucose during short and long-term exposures.

Anaplasma spp. are among the most recognized arthropod-borne infectious agents. Although the novel A. capra has been isolated from wildlife, livestock, and hard ticks from many parts of the world, there is no report regarding the identification of this pathogen from equines and little is known about the epidemiology of A. capra in Equidae. In this study, A. capra was identified in two out of ten blood specimens of wild onagers (Equus hemionus onager) during a routine health check-up in Semnan, Iran by light microscopy and molecular analyses while other pathogens were not detected. First, inclusions on RBC's were observed in two blood smears by light microscopy. Then, the blood specimens of both animals were analyzed by realtime-PCR for Anaplasma, Ehrlichia, and Theileria infections. A 1400 bp sequence of 16S rRNA belonging to Anaplasmataceae and 874 bp fragment for groEL gene for A. capra were amplified in Anaplasma positive samples and sequenced. Preliminary BLAST analysis of sequenced fragments showed high homology to A. capra strains in GenBank database. Finally, nested PCR and restriction enzyme fragment length polymorphism techniques confirmed the pathogen as A. capra. To the best of our knowledge, this study has reported the occurrence of A. capra in wild onagers for the first time and suggests that equines could be infected with this pathogen and act as reservoirs for A. capra., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The pathogenesis of Parkinson's disease (PD) remains elusive, but mitochondrial dysfunction is believed to be one crucial step in its pathogenesis. The mitochondrial unfolded protein response (UPR mt ) is an important mitochondrial quality control strategy that maintains mitochondrial function in response to disturbances of mitochondrial protein homeostasis. Activation of the UPR mt and the beneficial effect of rescuing mitochondrial proteostasis have been reported in several genetic models of PD. However, the pathogenic relevance of the UPR mt in idiopathic PD is unknown. The present study examined the link between the UPR mt and mitochondrial dysfunction in 1-methyl-4-phenylpyridinium (MPP + )-treated SH-SY5Y cells. Treatment with MPP  +  induced activation of the UPR mt , reflected by an increase in the expression of UPR mt -related chaperones, proteases, and transcription mediators. UPR mt activation that was induced by overexpressing mutant ornithine transcarbamylase significantly reduced the production of mitochondrial reactive oxygen species (ROS) and improved cell survival in SH-SY5Y cells following MPP + treatment. Moreover, the overexpression of activating transcription factor 5 (mammalian UPR mt transcription factor) conferred protection against MPP + -induced ROS production and against cell death in SH-SY5Y cells. Overall, our results demonstrate the beneficial effect of UPR mt activation in MPP  +  -treated cells, shedding new light on the mechanism of mitochondrial dysfunction in the pathogenesis of PD., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objectives: Ameloblastoma is an odontogenic neoplasm of the mandible and maxilla with various histological types and subtypes. It has been reported that some ameloblastomas could arise from dentigerous cyst walls; thus, the development of ameloblastoma from dentigerous cysts may be due to differential protein expression. Our aim was to identify a membrane protein that is differentially expressed in ameloblastomas with respect to dentigerous cysts., Methods: We analyzed the SDS-PAGE profiles of membrane proteins from ameloblastomas and dentigerous cysts. The protein in a band present in the ameloblastoma sample, but apparently absent in the dentigerous cyst sample was identified via mass spectrometry as the chaperonin Hsp60. We used western blotting and immunohistochemistry to analyze its overexpression and localization in ameloblastoma., Results: We found a differential band of 95 kDa in the membrane proteins of ameloblastoma. In this band, the chaperonin Hsp60 was identified, and its overexpression was corroborated using western blotting and immunohistochemistry. Hsp60 was localized in the plasma membrane of all ameloblastoma samples studied; in addition, it was found in the cell nucleus of the plexiform subtype of conventional ameloblastoma., Conclusions: Our results suggest that Hsp60 may be involved in ameloblastoma development, and could therefore be a potential therapeutic target for ameloblastoma treatment., Competing Interests: Conflicts of interest All authors declare that there are no conflicts of interest., (Copyright © 2021 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

Chronic stress is a risk factor for the development of psychiatric illnesses through impairment of the ability to appropriately regulate physiological and behavioral responses, but the molecular events that lead to damage of hippocampal neurons remain unclear. The medicinal herb Spilanthes acmella Murr. has been used as a traditional medicine for various diseases and its extracts exhibit antioxidant activity. The present study explored the molecular signals of mitochondrial dynamics and investigated the beneficial effects of S. acmella Murr. An ethyl acetate extract of this plant was used to assess mitochondrial dynamics in response to chronic restraint stress (CRS) in male Sprague-Dawley rats. The results demonstrated that the S. acmella Murr. extract reduced the expression of mitochondrial fission protein but induced HSP60, MnSOD and ATPsynthase in the hippocampus of the CRS rats. In addition, S. acmella Murr. extract reversed depressive symptoms in the forced swim test. Our findings suggested that S. acmella Murr. extract provides a potential treatment of chronic stress, and that the mechanism is associated with the alleviation of neuronal injury and maintenance of mitochondrial function., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Pepper crops in Israel are infected by poleroviruses, Pepper vein yellows virus 2 (PeVYV-2) and Pepper whitefly-borne vein yellows virus (PeWBVYV). Herein we characterize the transmission of PeWBVYV and the aphid-transmitted PeVYV-2, and show that PeWBVYV is specifically transmitted by MEAM1 species of the whitefly Bemisia tabaci, with a minimum latency period of 120 h, and not by the Mediterranean (MED). PeWBVYV and PeVYV-2 were detected in the hemolymph of MED and MEAM1, respectively, however, amounts of PeWBVYV in the hemolymph of MED or PeVYV-2 in MEAM1 were much lower than PeWBVYV in hemolymph of MEAM1. Moreover, we show that PeWBVYV does not interact with the GroEL protein of the symbiont Hamiltonella and thus does not account for the non-transmissibility by MED. An insect glycoprotein, C1QBP, interacting in vitro with the capsid proteins of both PeWBVYV and PeVYV-2 is reported which suggests a putative functional role in polerovirus transmission., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Heat shock protein 60 (HSP60) is highly conserved from prokaryotic to eukaryotic organisms, acting as molecular chaperone and other vital biological functions. In spite of increasing knowledge of HSP60, its evolutionary mechanism on functional adaption is still far from completely understood. Moreover, analysis of codon usage bias (CUB) is a powerful tool to understand evolutionary association studies. However, so far, as we know, no scientific work on CUB of HSP60 in birds has been reported. In this study, we provide a comprehensive analysis on the codon usage and molecular evolution of HSP60 across birds. The results indicated that HSP60 had a weak codon usage bias with high ENC values (range from 52.66 to 61), low RSCU, and A/T-ending codons were mostly preferred. Meanwhile, it was considered that mutation, natural selection, and genetic drift combined to shape codon usage patterns with different strength proportions among various birds for HSP60. Then, the LRT tests suggested that different lineages of birds might be under similar selective pressures. Besides, the two positive selection sites (151 and 131) were detected and might undergo radical changes. These findings would contribute to understand function diversity and molecular evolution of HSP60 in birds., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cutaneous leishmaniasis caused by L. braziliensis induces a pronounced Th1 inflammatory response characterized by IFN-γ production. Even in the absence of parasites, lesions result from a severe inflammatory response in which inflammatory cytokines play an important role. Different approaches have been used to evaluate the therapeutic potential of orally administrated heat shock proteins (Hsp). These proteins are evolutionarily preserved from bacteria to humans, highly expressed under inflammatory conditions and described as immunodominant antigens. Tolerance induced by the oral administration of Hsp65 is capable of suppressing inflammation and inducing differentiation in regulatory cells, and has been successfully demonstrated in several experimental models of autoimmune and inflammatory diseases. We initially administered recombinant Lactococcus lactis ( L. lactis ) prior to infection as a proof of concept, in order to verify its immunomodulatory potential in the inflammatory response arising from L. braziliensis . Using this experimental approach, we demonstrated that the oral administration of a recombinant L. lactis strain, which produces and secretes Hsp65 from Mycobacterium leprae directly into the gut, mitigated the effects of inflammation caused by L. braziliensis infection in association or not with PAM 3CSK4 ( N -α-Palmitoyl- S -[2,3-bis(palmitoyloxy)-(2 RS )-propyl]-L-cysteine, a TLR2 agonist). This was evidenced by the production of anti-inflammatory cytokines and the expansion of regulatory T cells in the draining lymph nodes of BALB/c mice. Our in vitro experimental results suggest that IL-10, TLR-2 and LAP are important immunomodulators in L. braziliensis infection. In addition, recombinant L. lactis administered 4 weeks after infection was observed to decrease lesion size, as well as the number of parasites, and produced a higher IL-10 production and decrease IFN-γ secretion. Together, these results indicate that Hsp65-producing L. lactis can be considered as an alternative candidate for treatment in both autoimmune diseases, as well as in chronic infections that cause inflammatory disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Guerra, Andrade, Nunes, Gama, Tibúrcio, Santos, Azevedo, Tavares, Rebouças, Maiolii, Faria and Brodskyn.)

Escherichia coli chaperonin GroEL, which is a large cylindrical protein complex comprising two heptameric rings with cavities of 4.5 nm each in the center, assists in intracellular protein folding with the aid of GroES and adenosine triphosphate (ATP). Here, we investigated the possibility that GroEL can also encapsulate metal nanoparticles (NPs) up to ∼5 nm in diameter into the cavities with the aid of GroES and ATP. The slow ATP-hydrolyzing GroELD52A/D398A mutant, which forms extremely stable complexes with GroES (half-time of ∼6 days), made it possible to analyze GroEL/GroES complexes containing metal NPs. Scanning transmission electron microscopy-energy-dispersive X-ray spectroscopy analysis proved distinctly that FePt NPs and Au NPs were encapsulated in the GroEL/GroES complexes. Dynamic light scattering measurements showed that the NPs in the GroEL/GroES complex were able to maintain their dispersibility in solution. We previously described that the incubation of GroEL and GroES in the presence of ATP·BeFx and adenosine diphosphate·BeFx resulted in the formation of symmetric football-shaped and asymmetric bullet-shaped complexes, respectively. Based on this knowledge, we successfully constructed the football-shaped complex in which two compartments were occupied by Pt or Au NPs (first compartment) and FePt NPs (second compartment). This study showed that metal NPs were sequentially encapsulated according to the GroEL reaction in a step-by-step manner. In light of these results, chaperonin can be used as a tool for handling nanomaterials., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The present study was aimed to determine the impact of thermal stress on expression dynamics of heat shock protein 60 (HSP60) mRNA in cultured cardiac fibroblast cells of the goat. The heart tissues ( n  = 6) from different goats were used for the culture study. The cardiac fibroblast cells were cultured and subjected to thermal stress at 42 °C for 0, 20, 60 and 100 min. The relative abundance of HSP60 mRNA was assessed by quantitative real-time PCR (qRT-PCR). The cardiac cells exposed to thermal stress at 42 °C for 0 min was taken as control. The relative abundance of HSP60 mRNA did not change at 20 min of thermal stress as compared to control. Thereafter, the relative abundance of HSP60 mRNA was significantly up-regulated ( p  < 0.05) at 60 min and 100 min of thermal stress. However, the highest mRNA expression of HSP60 was noticed at 100 min of thermal stress. The present study indicates that, thermal stress modulates the mRNA expression HSP60 in cultured caprine cardiac fibroblast cells.

Chlamydia trachomatis infects squamous and columnar epithelia at the mucosal surface. Research on gene expression patterns of C. trachomatis has predominantly focused on non-native host cells, with limited data on growth kinetics and gene expression of chlamydia in keratinocytes. Here, we investigated whether early, mid, and late chlamydial genes observed in HeLa cell line studies were co-ordinately regulated at the transcriptional level even in the keratinized cell line model and whether the expression was stage-specific during the developmental cycle. HaCaT cell lines were infected with chlamydia clinical isolates (US151and serovar E) and reference strain (L2 434). Expression of groEL-1, incB, pyk-F, tal, hctA, and omcB genes was conducted with comparative real-time PCR and transcriptional events during the chlamydial developmental cycle using transmission electron microscopy. The relative expression level of each gene and fold difference were calculated using the 2-ΔΔCT method. The expression of groEL-1 and pyk-F genes was highest at 2 hours post-infection (hpi) in the L2 434 and serovar E. The expression of incB gene increased at 2 hpi in L2 434 and serovar E but peaked at 12 hpi in serovar E. L2 434 and US151 had similar tal expression profiles. Increased expression of hctA and omcB genes were found at 2 and 36 hpi in L2 434. Both clinical isolates and reference strains presented the normal chlamydial replication cycle comprising elementary bodies and reticulate bodies within 36 hpi. We show different gene expression patterns between clinical isolates and reference strain during in vitro infection of keratinocytes, with reference strain-inducing consistent expression of genes. These findings confirm that keratinocytes are appropriate cell lines to interrogate cell differentiation, growth kinetics, and gene expression of C. trachomatis infection. Furthermore, more studies with different clinical isolates and genes are needed to better understand the Chlamydial pathogenesis in keratinocytes., Competing Interests: The authors have declared that no competing interests exist.

Aims: During heart failure, the levels of circulatory heat shock protein family D member 1 (HSP60) increase. However, its underlying mechanism is still unknown. The apical domain of heat shock protein family D member 1 (HSPD1) is conserved throughout evolution. We found a point mutation in HSPD1 in a familial dilated cardiomyopathy (DCM) patient. A similar point mutation in HSPD1 in the zebrafish mutant, nbl, led to loss of its regenerative capacity and development of pericardial oedema under heat stress condition. In this study, we aimed to determine the direct involvement of HSPD1 in the development of DCM., Methods and Results: By Sanger method, we found a point mutation (Thr320Ala) in the apical domain of HSPD1, in one familial DCM patient, which was four amino acids away from the point mutation (Val324Glu) in the nbl mutant zebrafish. The nbl mutants showed atrio-ventricular block and sudden death at 8-month post-fertilization. Histological and microscopic analysis of the nbl mutant hearts showed decreased ventricular wall thickness, elevated level of reactive oxygen species (ROS), increased fibrosis, mitochondrial damage, and increased autophagosomes. mRNA and protein expression of autophagy-related genes significantly increased in nbl mutants. We established HEK293 stable cell lines of wild-type, nbl-type, and DCM-type HSPD1, with tetracycline-dependent expression. Compared to wild-type, both nbl- and DCM-type cells showed decreased cell growth, increased expression of ROS and autophagy-related genes, inhibition of the activity of mitochondrial electron transport chain complexes III and IV, and decreased mitochondrial fission and fusion., Conclusion: Mutations in HSPD1 caused mitochondrial dysfunction and induced mitophagy. Mitochondrial dysfunction caused increased ROS and cardiac atrophy., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Plasmodium falciparum harbors group 1 and group 2 chaperonin systems to mediate the folding of cellular proteins in different cellular locations. Two distinct group 1 chaperonins operate in the organelles of mitochondria and apicoplasts, while group 2 chaperonins function in the cytosol. No structural information has been reported for any chaperonin from plasmodium. In this study, we describe the crystal structure of a double heptameric ring Plasmodium falciparum mitochondrial chaperonin 60 (Cpn60) bound with ATP, which differs significantly from any known crystal structure of chaperonin 60. The structure likely represents a unique intermediate state during conformational conversion from the closed state to the opened state. Three of the seven apical domains are highly dynamic while the equatorial domains form a stable ring. The structure implies large movements of the apical domain in the solution play a role in nucleotide-dependent regulation of substrate binding and folding. A unique 26-27 residue insertion in the equatorial domain of Plasmodium falciparum mitochondrial chaperonin greatly increases both inter-ring and intra-ring subunit-subunit interactions. The present structure provides new insights into the mechanism of Cpn60 in chaperonin assembly and function.

Ethnopharmacological Relevance: Overweight/obesity was mentioned by many countries as an obstacle to good health and long life, which increases risk of diseases and disorders. Previous studies suggested that the chronic low-grade inflammation present in the body was considered as the essential pathogenesis for obesity. Chrysin is extracted from traditional Chinese medicine Oroxylum indicum (Linn.) Kurz and plays a superior anti-obesity role. Chrysin could reduce the lipid depot by inhibiting the obesity-related inflammation in adipose tissue. However, the target protein for chrysin to exert its anti-obesity role are not verified., Aim of Study: The present study aimed to screen and validate the target protein for chrysin to reduce the lipid depot in palmitic acid-induced 3T3-L1 adipocytes., Materials and Methods: Obesity model was established employing 0.5 mmol/L palmitic acid-induced 3T3-L1 adipocytes through "Cocktails" method. Two-dimensional gel electrophoresis (2-DE) combined with liquid chromatography-mass spectrometry (LC-MS) was applied to analyze the differentially expressed proteins for chrysin intervention by lipid formation in adipocytes. Gene silencing was utilized to decrease gene expression of the candidate proteins, then production of triglyceride in 3T3-L1 was detected by triglycerides assay to determine the target proteins. Ultraviolet (UV) absorption together with fluorescence spectra validated the direct target proteins of chrysin. They also computed the correlation constants of combination between chrysin and the target proteins. Molecular docking was further employed to identify the main binding amino acids between chrysin and the target protein., Results: 2-DE combined with LC-MS screened four candidate proteins which were related to metabolism and inflammation. The production of triglycerides in 3T3-L1 was reduced after decreasing gene expression of Annexin A2 (ANXA2), 60 kDa heat shock protein (HSP-60) and succinyl-CoA:3-ketoacid coenzyme A transferase 1 (SCOT-S), respectively. UV spectrum showed that the absorbance spectra of ANXA2 from 260 to 300 nm shifted upwards along with the increase in chrysin concentration, meanwhile the absorbance spectra of HSP-60 from 200 to 220 nm and from 265 to 280 nm shifted slightly upwards along with the increase in chrysin concentrations. The results indicated the conjugated structures between chrysin and ANXA2 or HSP-60. Fluorescence quenching further suggested a spontaneous interaction between chrysin and ANXA2 or HSP-60. Finally, molecular docking identified the main binding amino acids between ANXA2 and chrysin were Ser22, Tyr24, Pro267, Val298, Asp299, and Lys302., Conclusions: Chrysin can reduce the amount of triglycerides by directly downregulating the inflammation-related target proteins ANXA2 and HSP-60, exerting an anti-obesity role., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)