RESUMEN

The correction of protein folding is fundamental for cellular functionality and its failure can lead to severe diseases. In this context, molecular chaperones are crucial players involved in the tricky process of assisting in protein folding, stabilization, and degradation. Chaperones, such as heat shock proteins (HSP) 90, 70, and 60, operate within complex systems, interacting with co-chaperones both to prevent protein misfolding and direct to the correct folding. Chaperone targeting drugs could represent a challenging approach for the treatment of cystic fibrosis (CF), an autosomal recessive genetic disease caused by mutations in the CFTR gene, encoding for the CFTR chloride channel. In this review, we discuss the potential role of molecular chaperones as proteostasis modulators affecting CFTR biogenesis. In particular, we focused on HSP90 and HSP70, for their key role in CFTR folding and trafficking, as well as on HSP60 for its involvement in the inflammation process.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Humanos , Chaperonas Moleculares/metabolismo , Pliegue de Proteína/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Animales , Chaperonina 60/metabolismo , Chaperonina 60/química , Chaperonina 60/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/metabolismo

RESUMEN

BACKGROUND: Mycobacterium abscessus complex (MABSc) causes chronic infection in patients with concomitant structural changes in the respiratory tract, which is especially important for patients with cystic fibrosis. To isolate an MABSc culture from clinical material, a variety of nutrient media are used. For species determination of microorganisms isolated on these media, additional identification methods are used, for example, polymerase chain reaction, sequencing, or mass spectrometry. The latter method is relatively easy to implement but requires improvement, due to the identification inaccuracy of nontuberculosis mycobacterias in general. Consequently, a set of nutrient media may be important for subsequent identification by mass spectrometry. METHODS: The study was conducted on 64 strains of MABSc representatives: 56 strains were obtained from patients with cystic fibrosis and 8 strains from patients with pulmonary pathology unrelated to cystic fibrosis. The obtained MABSc strains were transplanted to the universal chromogenic medium and the selective medium for the Burkholderia cepacia complex (BCC) isolation. Species identification was carried out by mass spectrometry based on matrix-activated laser time-of-flight desorption/ionization (MALDI-ToF MS). Microbial identification is based on a comparison of the obtained mass spectra with reference spectra from the database. Microorganisms were identified based on the coincidence degree (Score value). Sample preparation for microbial identification by mass spectrometry was carried out by an extended direct application method. Fragments of the rpoB and hsp65 genes with lengths of 752 bp and 441 bp, respectively, were used as molecular markers for subspecific identification of MABSc strains. RESULTS: A comparison of the peaks obtained after mass spectrometry of MABSc strains isolated on the studied nutrient media showed significant differences between these indicators selective medium for the BCC isolation with the supplement of iron polymaltose hydroxide (III) and universal chromogenic medium (P < 0.001) and selective medium for the BCC isolation with universal chromogenic medium (P < 0.001). Twenty-five strains of MABSc representatives were sequenced: results of subspecies determination in strains isolated on the universal chromogenic medium coincided with the results sequencing in 13 (86.6%) strains out of 15. CONCLUSION: MALDI-ToF mass spectrometry allows microbial identification in a short time and with minimal cost, but it does not yet allow the proper identification of the subspecies of certain microbial groups, such as MABSc. Cultivation methods need optimization and new approaches to the extraction process of the bacterial protein fraction.

Asunto(s)

Medios de Cultivo , Fibrosis Quística , Infecciones por Mycobacterium no Tuberculosas , Mycobacterium abscessus , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Mycobacterium abscessus/aislamiento & purificación , Mycobacterium abscessus/clasificación , Mycobacterium abscessus/genética , Humanos , Medios de Cultivo/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Fibrosis Quística/microbiología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/análisis , Chaperonina 60/genética

RESUMEN

Immune checkpoint blockade (ICB) has emerged as a promising therapeutic option for hepatocellular carcinoma (HCC), but resistance to ICB occurs and patient responses vary. Here, we uncover protein arginine methyltransferase 3 (PRMT3) as a driver for immunotherapy resistance in HCC. We show that PRMT3 expression is induced by ICB-activated T cells via an interferon-gamma (IFNγ)-STAT1 signaling pathway, and higher PRMT3 expression levels correlate with reduced numbers of tumor-infiltrating CD8+ T cells and poorer response to ICB. Genetic depletion or pharmacological inhibition of PRMT3 elicits an influx of T cells into tumors and reduces tumor size in HCC mouse models. Mechanistically, PRMT3 methylates HSP60 at R446 to induce HSP60 oligomerization and maintain mitochondrial homeostasis. Targeting PRMT3-dependent HSP60 methylation disrupts mitochondrial integrity and increases mitochondrial DNA (mtDNA) leakage, which results in cGAS/STING-mediated anti-tumor immunity. Lastly, blocking PRMT3 functions synergize with PD-1 blockade in HCC mouse models. Our study thus identifies PRMT3 as a potential biomarker and therapeutic target to overcome immunotherapy resistance in HCC.

Asunto(s)

Carcinoma Hepatocelular , Chaperonina 60 , Neoplasias Hepáticas , Proteínas de la Membrana , Nucleotidiltransferasas , Proteína-Arginina N-Metiltransferasas , Transducción de Señal , Animales , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Humanos , Ratones , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Chaperonina 60/metabolismo , Chaperonina 60/genética , Línea Celular Tumoral , Metilación , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Mitocondrias/metabolismo , Ratones Endogámicos C57BL , ADN Mitocondrial/genética , ADN Mitocondrial/inmunología , ADN Mitocondrial/metabolismo , Interferón gamma/metabolismo , Interferón gamma/inmunología , Masculino

RESUMEN

GroEL, a chaperone protein responsible for peptide and denatured protein folding, undergoes substantial conformational changes driven by ATP binding and hydrolysis during folding. Utilizing these conformational changes, we demonstrated the GroEL-mediated regioselective photocyclodimerization of 2-anthracenecarboxylic acid (AC) using ATP hydrolysis as an external stimulus. We designed and prepared an optimal GroEL mutant to employ in a docking simulation that has been actively used in recent years. Based on the large difference in the motif of hydrogen bonds between AC and GroEL mutant compared with the wild-type, we predicted that GroELMEL, in which the 307‒309th amino acid residues were mutated to Ala, could alter the orientation of bound AC in GroEL. The GroELMEL-mediated photocyclodimerization of AC can be used for regioselective inversion upon ATP addition to a moderate extent.

Asunto(s)

Adenosina Trifosfato , Antracenos , Chaperonina 60 , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/química , Hidrólisis , Antracenos/química , Antracenos/metabolismo , Chaperonina 60/química , Chaperonina 60/genética , Chaperonina 60/metabolismo , Simulación del Acoplamiento Molecular , Conformación Proteica , Mutación , Enlace de Hidrógeno , Pliegue de Proteína , Ácidos Carboxílicos

RESUMEN

Microglia, essential immune cells in the brain, play crucial roles in neuroinflammation by performing various functions such as neurogenesis, synaptic pruning, and pathogen defense. These cells are activated by inflammatory factors like ß-amyloid (Aß) and oxysterols, leading to morphological and functional changes, including the secretion of inflammatory cytokines and the upregulation of MHC class II molecules. This study focused on identifying specific markers for microglial activation, with a particular emphasis on the roles of oxysterols in this process. We used the HMC3 human microglial cell line to investigate the induction of heat shock protein 60 (HSP60), a chaperonin protein by oxysterols, specifically in the presence of 25-hydroxycholesterol (25OHChol) and 27-hydroxycholesterol (27OHChol). Our findings obtained by the proteomics approach revealed that these oxysterols significantly increased HSP60 expression on microglial cells. This induction was further confirmed using Western blot analysis and immunofluorescence microscopy. Additionally, Aß1-42 also promoted HSP60 expression, indicating its role as a microglial activator. HSP60 involved in protein folding and immune modulation was identified as a potential marker for microglial activation. This study underscores the importance of HSP60 in the inflammatory response of microglia, suggesting its utility as a target for new therapeutic approaches in neuroinflammatory diseases such as Alzheimer's disease (AD).

Asunto(s)

Chaperonina 60 , Hidroxicolesteroles , Microglía , Oxiesteroles , Microglía/metabolismo , Microglía/efectos de los fármacos , Humanos , Chaperonina 60/metabolismo , Oxiesteroles/metabolismo , Oxiesteroles/farmacología , Línea Celular , Hidroxicolesteroles/farmacología , Hidroxicolesteroles/metabolismo , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/farmacología , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , Proteínas Mitocondriales

RESUMEN

This study aims to investigate the potential association between serum levels of cytokines, HSP60, HSP70 and IR (HOMA-IR) in postmenopausal women. We conducted a cross-sectional study involving 381 postmenopausal women, including 94 with a breast cancer diagnosis and 278 without. We analyzed anthropometric and laboratory measurements. Immunoassays were used to measure cytokines (TNF-α, IL-10, and IL-6) as well as heat shock proteins (HSP) 60 and 70 in the serum using the ELISA technique. Women diagnosed with breast cancer showed higher levels of HOMA-IR, IL-6, TNF, and HSP60, and lower levels of IL-10 and HSP70 compared to women without cancer. An association was found between HSP70 and HOMA-IR only in women with breast cancer (ß = 0.22, p = .030; without cancer: ß = 0.04, p = .404), regardless of age, waist circumference, smoking, and physical activity. No associations were observed between cytokines, HSP60, and HOMA-IR in both groups of women. HSP70 is positively associated with IR in women diagnosed with breast cancer.

Asunto(s)

Neoplasias de la Mama , Chaperonina 60 , Proteínas HSP70 de Choque Térmico , Resistencia a la Insulina , Posmenopausia , Humanos , Femenino , Neoplasias de la Mama/sangre , Estudios Transversales , Posmenopausia/sangre , Persona de Mediana Edad , Proteínas HSP70 de Choque Térmico/sangre , Chaperonina 60/sangre , Anciano , Citocinas/sangre , Interleucina-6/sangre , Interleucina-10/sangre , Factor de Necrosis Tumoral alfa/sangre

RESUMEN

Chaperonins are large barrel-shaped complexes that mediate ATP-dependent protein folding1-3. The bacterial chaperonin GroEL forms juxtaposed rings that bind unfolded protein and the lid-shaped cofactor GroES at their apertures. In vitro analyses of the chaperonin reaction have shown that substrate protein folds, unimpaired by aggregation, while transiently encapsulated in the GroEL central cavity by GroES4-6. To determine the functional stoichiometry of GroEL, GroES and client protein in situ, here we visualized chaperonin complexes in their natural cellular environment using cryo-electron tomography. We find that, under various growth conditions, around 55-70% of GroEL binds GroES asymmetrically on one ring, with the remainder populating symmetrical complexes. Bound substrate protein is detected on the free ring of the asymmetrical complex, defining the substrate acceptor state. In situ analysis of GroEL-GroES chambers, validated by high-resolution structures obtained in vitro, showed the presence of encapsulated substrate protein in a folded state before release into the cytosol. Based on a comprehensive quantification and conformational analysis of chaperonin complexes, we propose a GroEL-GroES reaction cycle that consists of linked asymmetrical and symmetrical subreactions mediating protein folding. Our findings illuminate the native conformational and functional chaperonin cycle directly within cells.

Asunto(s)

Chaperonina 10 , Chaperonina 60 , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Proteínas de Escherichia coli , Escherichia coli , Sitios de Unión , Chaperonina 10/metabolismo , Chaperonina 10/química , Chaperonina 10/ultraestructura , Chaperonina 60/metabolismo , Chaperonina 60/química , Chaperonina 60/ultraestructura , Citosol/química , Citosol/metabolismo , Citosol/ultraestructura , Escherichia coli/química , Escherichia coli/citología , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Modelos Moleculares , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Reproducibilidad de los Resultados , Especificidad por Sustrato , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestructura

RESUMEN

Cholinergic circuit defects have been linked to various neurological abnormalities, yet the precise mechanisms underlying the impact of cholinergic signaling on cognitive functions, particularly in the context of neuroinflammation-associated, remain poorly understood. Similarly, while the dopamine receptor (D2R) has been implicated in the pausing of cholinergic interneurons (CIN), its relationship with behavior remains inadequately elucidated. In this study, we aimed to investigate whether D2R plays a role in the regulation of fear and memory in the Hsp60 knockout condition, given the non-canonical involvement of Hsp60 in inflammation. Using a CRE-floxed system, we selectively generated cholinergic neurons specific to Hsp60 knockout mice and subjected them to memory tests. Our results revealed a significant increase in freezing levels during recall and contextual tests in Hsp60-deprived mice. We also observed dysregulation of neurotransmitters and D2R in the hippocampus of Hsp60 knockout mice, along with enhanced impairments in cytokine levels and synaptic protein dysregulations. These changes were accompanied by alterations in PI3K/eIF4E/Jak/ERK/CREB signaling pathways. Notably, D2R agonism via Quinpirole led to a decrease in freezing levels during recall and contextual tests, alongside an increase in IBA-1 expression and improvements in inflammatory response-linked signaling pathways, including JAK/STAT/P38/JNK impairments. Given that these pathways are well-known downstream signaling cascades of D2R, our findings suggest that D2R signaling may contribute to the neuroinflammation induced by Hsp60 deprivation, potentially exacerbating memory impairments.

Asunto(s)

Chaperonina 60 , Neuronas Colinérgicas , Memoria , Ratones Noqueados , Enfermedades Neuroinflamatorias , Animales , Neuronas Colinérgicas/metabolismo , Enfermedades Neuroinflamatorias/inmunología , Chaperonina 60/metabolismo , Chaperonina 60/genética , Ratones , Hipocampo/metabolismo , Transducción de Señal , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D2/genética , Masculino , Miedo , Ratones Endogámicos C57BL , Citocinas/metabolismo

RESUMEN

OBJECTIVE: Mitochondrial proteostasis is critical for cellular function. The molecular chaperone HSP60 is essential for cell function and dysregulation of HSP60 expression has been implicated in cancer and diabetes. The few reported patients carrying HSP60 gene variants show neurodevelopmental delay and brain hypomyelination. Hsp60 interacts with more than 260 mitochondrial proteins but the mitochondrial proteins and functions affected by HSP60 deficiency are poorly characterized. METHODS: We studied two model systems for HSP60 deficiency: (1) engineered HEK cells carrying an inducible dominant negative HSP60 mutant protein, (2) zebrafish HSP60 knockout larvae. Both systems were analyzed by RNASeq, proteomics, and targeted metabolomics, and several functional assays relevant for the respective model. In addition, skin fibroblasts from patients with disease-associated HSP60 variants were analyzed by proteomics. RESULTS: We show that HSP60 deficiency leads to a differentially downregulated mitochondrial matrix proteome, transcriptional activation of stress responses, and dysregulated cholesterol biosynthesis. This leads to lipid accumulation in zebrafish knockout larvae. CONCLUSIONS: Our data provide a compendium of the effects of HSP60 deficiency on the mitochondrial matrix proteome. We show that HSP60 is a master regulator and modulator of mitochondrial functions and metabolic pathways. HSP60 dysfunction also affects cellular metabolism and disrupts the integrated stress response. The effect on cholesterol synthesis explains the effect of HSP60 dysfunction on myelination observed in patients carrying genetic variants of HSP60.

Asunto(s)

Chaperonina 60 , Colesterol , Mitocondrias , Proteínas Mitocondriales , Proteoma , Pez Cebra , Chaperonina 60/metabolismo , Chaperonina 60/genética , Animales , Humanos , Proteoma/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Colesterol/metabolismo , Colesterol/biosíntesis , Mitocondrias/metabolismo , Células HEK293 , Fibroblastos/metabolismo , Larva/metabolismo

RESUMEN

In this study, a tick intracellular symbiont, Candidatus Midichloria mitochondrii, was detected in Hyalomma anatolicum from Xinjiang, China. Morphological identification and cytochrome oxidase subunit I sequence alignment were used for molecular identification of the tick species. PCR detection further revealed the presence of endosymbiont C. M. mitochondrii in the tick. Specific primers were designed for Groel and 16S rRNA genes of C. M. mitochondrii for PCR amplification and phylogenetic analysis. To further investigate the vertical transmission characteristics of C. M. mitochondrii, specific primers were designed based on the FabⅠ gene fragment to detect C. M. mitochondrii in different developmental stages and organs of the tick using qPCR. Of the 336 tick specimens collected from the field, 266 samples were identified as H. anatolicum on the basis of morphological characteristics. The gene fragment alignment results of COI confirmed that these ticks were H. anatolicum. The phylogenetic analysis showed that Groel gene of C. M. mitochondrii clustered with Midichloria strains detected in Ixodes ricinus ticks from Italy and Ixodes holocyclus ticks from Australia, with 100% sequence similarity. Furthermore, the 16S rRNA gene of C. M. mitochondrii clusters with the strains isolated from Hyalomma rufipes ticks in Italy, exhibiting the highest degree of homology. qPCR results showed that C. M. mitochondrii was present at all developmental stages of H. anatolicum, with the highest relative abundance in eggs, and lower relative abundance in nymphs and unfed males. With female tick blood feeding, the relative abundance of C. M. mitochondrii increased, and a particularly high relative abundance was detected in the ovaries of engorged female ticks. This study provides information for studying the survival adaptability of H. anatolicum, and provides data for further investigation of the mechanisms regulating tick endosymbionts in ticks, enriching the reference materials for comprehensive prevention and control of tick-borne diseases.

Asunto(s)

Ixodidae , Filogenia , ARN Ribosómico 16S , Simbiosis , Animales , Ixodidae/microbiología , Ixodidae/crecimiento & desarrollo , ARN Ribosómico 16S/genética , Femenino , Masculino , China , Chaperonina 60/genética , Ninfa/microbiología , Ninfa/crecimiento & desarrollo , Alineación de Secuencia , Complejo IV de Transporte de Electrones/genética , Infestaciones por Garrapatas/parasitología , Infestaciones por Garrapatas/veterinaria , Reacción en Cadena de la Polimerasa , ADN Bacteriano , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

The molecular chaperone GroEL, commonly found in various bacterial species, exhibits heightened expression levels in response to high temperatures and increased levels of oxygen free radicals. Limited literature currently exists on the probiotic role of GroEL in invertebrates. This study sought to explore how the surface protein GroEL from Lactobacillus plantarum Ep-M17 impacts the intestinal barrier function of Penaeus vannamei. Through pull-down and immunofluorescence assays, the interaction between GroEL and Act1 in the gastrointestinal tract of P. vannamei was confirmed. Results from bacterial binding assays demonstrated that rGroEL can bind to pathogens like Vibrio parahaemolyticus E1 (V. p-E1). In vitro experiments revealed that the administration of rGroEL significantly decreased the levels of inflammatory cytokines induced by pathogens while preserving the integrity of tight junctions between intestinal epithelial cells and reducing bacteria-induced apoptosis. Additionally, rGroEL notably lessened the intestinal loading of V. p-E1 in P. vannamei, downregulated immune-related gene expression, and upregulated BCL/BAX expression in the intestines following V. p-E1 challenge. Mechanistic investigations further showed that rGroEL treatment effectively suppressed the expression and phosphorylation of proteins involved in the NF-κB and PI3K-AKT-mTOR signalling pathways in the intestines of bacteria-infected P. vannamei. Furthermore, GroEL reinforces protection against bacterial infections by enhancing the phagocytic and anti-apoptotic capabilities of P. vannamei hemocytes. These results suggest that GroEL may impede the interaction between pathogens and the intestinal mucosa through its competitive binding characteristics, ultimately reducing bacterial infections.

Asunto(s)

Chaperonina 60 , Mucosa Intestinal , Penaeidae , Vibrio parahaemolyticus , Animales , Chaperonina 60/metabolismo , Penaeidae/microbiología , Penaeidae/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Transducción de Señal/efectos de los fármacos , Intestinos/microbiología , Lactobacillus plantarum/metabolismo , Apoptosis/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Lactobacillales/metabolismo

RESUMEN

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.

Asunto(s)

Gardnerella , Vaginosis Bacteriana , Humanos , Vaginosis Bacteriana/microbiología , Femenino , Adulto , Gardnerella/aislamiento & purificación , Gardnerella/genética , Adulto Joven , Vagina/microbiología , Washingtón/epidemiología , Gardnerella vaginalis/aislamiento & purificación , Gardnerella vaginalis/genética , Infecciones por Bacterias Grampositivas/microbiología , Adolescente , Prevalencia , Persona de Mediana Edad , ADN Bacteriano/genética , Chaperonina 60/genética , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

We recently reported on small-molecule inhibitors of the GroES/GroEL chaperone system as potential antibiotics against Escherichia coli and the ESKAPE pathogens but were unable to establish GroES/GroEL as the cellular target, leading to cell death. In this study, using two of our most potent bis-sulfonamido-2-phenylbenzoxazoles (PBZs), we established the binding site of the PBZ molecules using cryo-EM and found that GroEL was the cellular target responsible for the mode of action. Cryo-EM revealed that PBZ1587 binds at the GroEL ring-ring interface (RRI). A cellular reporter assay confirmed that PBZ1587 engaged GroEL in cells, but cellular rescue experiments showed potential off-target effects. This prompted us to explore a closely related analogue, PBZ1038, which is also bound to the RRI. Biochemical characterization showed potent inhibition of Gram-negative chaperonins but much lower potency of chaperonin from a Gram-positive organism, Enterococcus faecium. A cellular reporter assay showed that PBZ1038 also engaged GroEL in cells and that the cytotoxic phenotype could be rescued by a chromosomal copy of E. faecium GroEL/GroES or by expressing a recalcitrant RRI mutant. These data argue that PBZ1038's antimicrobial action is exerted through inhibition of GroES/GroEL, validating this chaperone system as an antibiotic target.

Asunto(s)

Antibacterianos , Chaperonina 10 , Escherichia coli , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Chaperonina 10/metabolismo , Chaperonina 10/antagonistas & inhibidores , Chaperonina 10/química , Escherichia coli/efectos de los fármacos , Chaperonina 60/metabolismo , Chaperonina 60/antagonistas & inhibidores , Chaperonina 60/química , Benzoxazoles/química , Benzoxazoles/farmacología , Benzoxazoles/síntesis química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/química

RESUMEN

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 (Hsp60Δ/ΔIEC). This metabolic perturbation causes self-resolving tissue injury. Regeneration is disrupted in the absence of the aryl hydrocarbon receptor (Hsp60Δ/ΔIEC;AhR-/-) involved in intestinal homeostasis or inflammatory regulator interleukin (IL)-10 (Hsp60Δ/ΔIEC;Il10-/-), causing IBD-like pathology. Injury is absent in the distal colon of germ-free (GF) Hsp60Δ/ΔIEC mice, highlighting bacterial control of metabolic injury. Colonizing GF Hsp60Δ/ΔIEC mice with the synthetic community OMM12 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.

Asunto(s)

Chaperonina 60 , Microbioma Gastrointestinal , Mitocondrias , Animales , Ratones , Mitocondrias/metabolismo , Humanos , Chaperonina 60/genética , Chaperonina 60/metabolismo , Enfermedades Inflamatorias del Intestino/microbiología , Mucosa Intestinal/microbiología , Mucosa Intestinal/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Estrés Oxidativo , Bacteroides/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Hidrocarburo de Aril/genética , Perfilación de la Expresión Génica , Intestinos/microbiología , Intestinos/patología , Modelos Animales de Enfermedad , Enfermedad de Crohn/microbiología

RESUMEN

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.

Asunto(s)

Artritis Reumatoide , Autoantígenos , Chaperonina 60 , Centro Germinal , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Animales , Humanos , Ratones , Autoantígenos/inmunología , Autoantígenos/genética , Centro Germinal/inmunología , Centro Germinal/patología , Chaperonina 60/inmunología , Chaperonina 60/genética , Autoanticuerpos/inmunología , Autoinmunidad , Masculino , Sinoviocitos/inmunología , Sinoviocitos/patología , Sinoviocitos/metabolismo , Artritis Experimental/inmunología , Artritis Experimental/patología , Femenino , Linfocitos B/inmunología , Linfocitos B/patología , Estructuras Linfoides Terciarias/inmunología , Estructuras Linfoides Terciarias/patología

RESUMEN

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.

Asunto(s)

Chaperonina 60 , Codón , Escherichia coli , Pliegue de Proteína , Tetrahidrofolato Deshidrogenasa , Chaperonina 60/genética , Chaperonina 60/química , Chaperonina 60/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/metabolismo , Animales , Codón/genética , Codón/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ratones , Mutación Silenciosa

RESUMEN

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.

Asunto(s)

Chaperonina 60 , Lisina , Macrófagos , Mycobacterium tuberculosis , Proteómica , Sirtuina 3 , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Acetilación , Lisina/metabolismo , Sirtuina 3/metabolismo , Sirtuina 3/genética , Chaperonina 60/metabolismo , Chaperonina 60/genética , Macrófagos/microbiología , Macrófagos/inmunología , Macrófagos/metabolismo , Humanos , Tuberculosis/microbiología , Tuberculosis/inmunología , Tuberculosis/metabolismo , Interacciones Huésped-Patógeno , Procesamiento Proteico-Postraduccional , Apoptosis , Proteínas Mitocondriales

RESUMEN

Photothermal therapy (PTT) is a promising clinical antitumor strategy. However, local hyperthermia inevitably induces heat damage to adjacent normal tissues, while alternative mild-temperature therapy (MPTT, T < 45 °C) is also inefficient due to the overexpressed hyperthermia-induced heat shock proteins (HSPs) by cancer cells. Therefore, developing PTT strategies with minimizing damage to healthy tissues with improved cellular temperature sensitivity is extremely valuable for clinical application. Herein, we proposed the strategy of disrupting the intracellular redox environment via destroying the ROS-defending systems to promote MPTT. The gold(III) porphyrin-Fe3+-tannic acid nanocomplexes (AuTPP@TA-Fe NPs) were achieved via interfacial cohesion and supramolecular assembly of bioadhesive species, which could trigger the Fenton reaction to produce ·OH radicals and downregulation of reductive TrxR enzyme and mitochondrial chaperone protein Hsp60. The aggravation of oxides and the inactivation of Hsp60 provide favorable pathways for impeding the heat shock-induced self-repair mechanism of cancer cells, which strengthens AuTPP@TA-Fe NPs mediated MPTT.

Asunto(s)

Oro , Oxidación-Reducción , Terapia Fototérmica , Humanos , Oro/química , Porfirinas/química , Porfirinas/efectos de la radiación , Porfirinas/farmacología , Animales , Chaperonina 60/química , Chaperonina 60/metabolismo , Ratones , Línea Celular Tumoral , Metaloporfirinas/química , Metaloporfirinas/farmacología , Neoplasias/terapia , Nanopartículas del Metal/química , Nanopartículas del Metal/uso terapéutico

RESUMEN

It has been reported that Mizoribine is an immunosuppressant used to suppress rejection in renal transplantation, nephrotic syndrome, lupus nephritis, and rheumatoid arthritis. The molecular chaperone HSP60 alone induces inflammatory cytokine IL-6 and the co-chaperone HSP10 alone inhibits IL-6 induction. HSP60 and HSP10 form a complex in the presence of ATP. We analyzed the effects of Mizoribine, which is structurally similar to ATP, on the structure and physiological functions of HSP60-HSP10 using Native/PAGE and transmission electron microscopy. At low concentrations of Mizoribine, no complex formation of HSP60-HSP10 was observed, nor was the expression of IL-6 affected. On the other hand, high concentrations of Mizoribine promoted HSP60-HSP10 complex formation and consequently suppressed IL-6 expression. Here, we propose a novel mechanism of immunosuppressive action of Mizoribine.

Asunto(s)

Chaperonina 60 , Interleucina-6 , Ribonucleósidos , Ribonucleósidos/farmacología , Interleucina-6/metabolismo , Chaperonina 60/metabolismo , Humanos , Inmunosupresores/farmacología , Animales , Ratones

RESUMEN

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.

Asunto(s)

Proteínas Bacterianas , Frío , Escherichia coli , Pliegue de Proteína , Pseudoalteromonas , Pseudoalteromonas/genética , Pseudoalteromonas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Chaperonina 60/metabolismo , Chaperonina 60/genética , Chaperonina 60/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Chaperoninas/metabolismo , Chaperoninas/genética , Chaperoninas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/genética