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

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

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

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

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

RESUMEN

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.

Asunto(s)

Chaperonina 10 , Chaperonina 60 , Pliegue de Proteína , Chaperonina 60/metabolismo , Chaperonina 60/química , Chaperonina 60/genética , Chaperonina 10/metabolismo , Chaperonina 10/química , Unión Proteica , Bacterias/metabolismo , Bacterias/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética

RESUMEN

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.

Asunto(s)

Chaperonina 60 , Mitocondrias , Estrés Oxidativo , Chaperonina 60/metabolismo , Chaperonina 60/genética , Humanos , Animales , Mitocondrias/metabolismo , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Apoptosis , Enfermedades Neurodegenerativas/metabolismo , Pliegue de Proteína , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

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.

Asunto(s)

Micobacterias no Tuberculosas , Humanos , Estudios Retrospectivos , Estudios Prospectivos , Micobacterias no Tuberculosas/aislamiento & purificación , Micobacterias no Tuberculosas/clasificación , Micobacterias no Tuberculosas/genética , Francia , Proteínas Bacterianas/genética , Mycobacterium/aislamiento & purificación , Mycobacterium/genética , Mycobacterium/clasificación , Reacción en Cadena de la Polimerasa/métodos , Infecciones por Mycobacterium no Tuberculosas/microbiología , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Chaperonina 60/genética , Técnicas de Diagnóstico Molecular/métodos , Sensibilidad y Especificidad

RESUMEN

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.

Asunto(s)

Diabetes Mellitus Tipo 2 , Infecciones por Helicobacter , Helicobacter pylori , Análisis de la Aleatorización Mendeliana , Humanos , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/microbiología , Diabetes Mellitus Tipo 2/genética , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/microbiología , Anticuerpos Antibacterianos/sangre , Enfermedades Gastrointestinales/microbiología , Enfermedades Gastrointestinales/complicaciones , Obesidad/complicaciones , Obesidad/microbiología , Estudio de Asociación del Genoma Completo , Úlcera Péptica/microbiología , Úlcera Péptica/epidemiología , Gastritis/microbiología , Gastritis/complicaciones , Chaperonina 60/genética , Factores de Riesgo

RESUMEN

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.

Asunto(s)

Apoptosis , Chaperonina 60 , Chaperonina 60/genética , Chaperonina 60/metabolismo , Línea Celular , Animales , Pollos , Fibroblastos/fisiología , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen

RESUMEN

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.

Asunto(s)

Chaperonina 60 , Cardiopatías Congénitas , Animales , Ratones , Chaperonina 60/genética , Chaperonina 60/metabolismo , Cardiopatías Congénitas/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Miocitos Cardíacos/metabolismo

RESUMEN

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 mHsp60V72I 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 mHsp60V72I. Our structural analysis combined with molecular dynamic simulations showed mHsp60V72I 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 mHsp60V72I 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.

Asunto(s)

Adenosina Trifosfato , Chaperonina 60 , Microscopía por Crioelectrón , Simulación de Dinámica Molecular , Humanos , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/química , Chaperonina 60/metabolismo , Chaperonina 60/química , Chaperonina 60/genética , Unión Proteica , Sitios de Unión , Estabilidad Proteica , Mutación , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Conformación Proteica

RESUMEN

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.

Asunto(s)

Chironomidae , Animales , Chironomidae/genética , Respuesta al Choque Térmico/genética , Larva/genética , Chaperonina 60/genética , Proteínas HSP70 de Choque Térmico/genética