RÉSUMÉ
Mitophagy selectively eliminates damaged or dysfunctional mitochondria, playing a crucial role in maintaining mitochondrial quality control. However, it remains unclear whether mitophagy can be fully activated and how it evolves after SCI. Our RNA-seq analysis of animal samples from sham and 1, 3, 5, and 7 days post-SCI indicated that mitophagy was indeed inhibited during the acute and subacute early stages. In vitro experiments showed that this inhibition was closely related to excessive production of reactive oxygen species (ROS) and the downregulation of BNIP3. Excessive ROS led to the blockage of mitophagy flux, accompanied by further mitochondrial dysfunction and increased neuronal apoptosis. Fortunately, ligustilide (LIG) was found to have the ability to reverse the oxidative stress-induced downregulation of BNIP3 and enhance mitophagy through BNIP3-LC3 interaction, alleviating mitochondrial dysfunction and ultimately reducing neuronal apoptosis. Further animal experiments demonstrated that LIG alleviated oxidative stress and mitophagy inhibition, rescued neuronal apoptosis, and promoted tissue repair, ultimately leading to improved motor function. In summary, this study elucidated the state of mitophagy inhibition following SCI and its potential mechanisms, and confirmed the effects of LIG-enhanced mitophagy through BNIP3-LC3, providing new therapeutic targets and strategies for repairing SCI.
Sujet(s)
4-Butyrolactone , Apoptose , Protéines membranaires , Mitophagie , Neurones , Stress oxydatif , Rat Sprague-Dawley , Traumatismes de la moelle épinière , Animaux , Protéines membranaires/métabolisme , Protéines membranaires/génétique , Neurones/métabolisme , Traumatismes de la moelle épinière/métabolisme , 4-Butyrolactone/analogues et dérivés , 4-Butyrolactone/pharmacologie , Rats , Espèces réactives de l'oxygène/métabolisme , Mâle , Mitochondries/métabolisme , Protéines mitochondriales/métabolisme , Protéines associées aux microtubulesRÉSUMÉ
OBJECTIVE: To study the biological role and related mechanism of autophagy in acute lung injury (ALI) of hemorrhagic shock mice. METHODS: According to random number table method, wild-type male C57BL/6 mice were divided into control group, ALI group, rapamycin group and 3-methyladenine (3-MA) group, with 8 mice in each group. Light chain 3 (LC3) gene knockout mice with C57BL/6 background were divided into LC3 knockout group and LC3 knockout+ALI group, with 8 mice in each group. Control group, ALI group, LC3 knockout group, LC3 knockout+ALI group were intraperitoneally injected with 2 mL/kg normal saline, rapamycin group was intraperitoneally injected with 3 mg/kg autophagy activator rapamycin, 3-MA group was intraperitoneally injected with 15 mg/kg autophagy inhibitor 3-MA, all of which were given for 3 consecutive days. 2 hours after the last administration, the hemorrhagic shock induced ALI model was established. 24 hours after modeling, the lung index was calculated. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of lung tissue and lung injury score was performed. The expressions of autophagy genes LC3- II/LC3- I and Beclin-1 in lung tissue were detected by Western blotting. The contents of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and malondialdehyde (MDA) in lung tissue were detected according to the steps of the kit. RESULTS: Compared with the control group, the lung tissue structure was destroyed and exudation increased, lung index, lung injury score, the expressions of LC3- II/LC3- I, Beclin-1, and the contents of TNF-α, IL-6 and MDA in lung tissue significantly increased in the ALI group. Compared with the ALI group, the structural damage and exudation of lung tissue were reduced in the rapamycin group, lung index, lung injury score and the contents of TNF-α, IL-6 and MDA in lung tissue decreased, while the expressions of LC3- II/LC3- I and Beclin-1 in lung tissue increased [lung index: (7.56±0.39)% vs. (9.12±0.59)%, lung injury score: 3.04±0.58 vs. 9.32±2.14, TNF-α (ng/mg): 1.85±0.32 vs. 3.51±0.62, IL-6 (ng/mg): 1.61±0.32 vs. 2.52±0.44, MDA (nmol/mg): 1.03±0.16 vs. 1.88±0.24, LC3- II/LC3- I: 1.21±0.12 vs. 0.39±0.05, Beclin-1/ß-actin: 1.10±0.12 vs. 0.58±0.06, all P < 0.05], while lung tissue structure damage was aggravated and exudation was further increased in the 3-MA group, lung index, lung injury score and the contents of TNF-α, IL-6 and MDA in lung tissue increased, the expressions of LC3- II/LC3- I and Beclin-1 in lung tissue decreased [lung index: (10.44±0.62)% vs. (9.12±0.59)%, lung injury score: 11.59±2.28 vs. 9.32±2.14, TNF-α (ng/mg): 4.77±0.71 vs. 3.51±0.62, IL-6 (ng/mg): 3.44±0.52 vs. 2.52±0.44, MDA (nmol/mg): 2.71±0.42 vs. 1.88±0.24, LC3- II/LC3- I: 0.25±0.04 vs. 0.39±0.05, Beclin-1/ß-actin: 0.21±0.03 vs. 0.58±0.06, all P < 0.05]. Lung index, lung injury score and the contents of TNF-α, IL-6 and MDA in lung tissue of LC3 knockout ALI mice were higher than those of wild-type ALI mice [lung index: (10.44±0.75)% vs. (9.12±0.59)%, lung injury score: 12.41±2.86 vs. 9.32±2.14, TNF-α (ng/mg): 4.85±0.72 vs. 3.51±0.62, IL-6 (ng/mg): 3.28±0.51 vs. 2.52±0.44, MDA (nmol/mg): 2.75±0.41 vs. 1.88±0.24, all P < 0.05]. CONCLUSIONS: Autophagy plays a protective role in ALI of hemorrhagic shock mice, and the related molecular mechanism is the inhibition of inflammatory response and oxidative stress response.
Sujet(s)
Lésion pulmonaire aigüe , Autophagie , Interleukine-6 , Souris de lignée C57BL , Souris knockout , Choc hémorragique , Facteur de nécrose tumorale alpha , Animaux , Lésion pulmonaire aigüe/métabolisme , Mâle , Choc hémorragique/métabolisme , Choc hémorragique/complications , Souris , Interleukine-6/métabolisme , Facteur de nécrose tumorale alpha/métabolisme , Modèles animaux de maladie humaine , Poumon/métabolisme , Poumon/anatomopathologie , Protéines associées aux microtubules/métabolismeRÉSUMÉ
Background: This study investigated the genetic characteristics of five Chinese families with keratoconus (KC). Methods: In the five families affected by KC, medical records, clinical observations, and blood samples were collected from all individuals. All KC family members (n = 20) underwent both whole exome sequencing of genomic DNA and Sanger sequencing to confirm the variants. Online software was utilized to analyze all variants, and the online server I-TASSER was employed for in silico predictions of the three-dimensional protein structures of the variants. The newly discovered variants and single nucleotide polymorphisms were further examined in 322 sporadic KC patients. Results: The Pentacam tomographic composite index in those affected first-degree family members of the probands showed a pathological change. Five new variants were detected in the five probands and other affected members in their families: a heterozygous missense variant g.19043832C>T (p.Ser145Asn) in the homer scaffolding protein 3 (HOMER3) gene; a heterozygous missense variant g.99452113G>A (p.Gly483Arg) in the insulin-like growth factor 1 receptor (IGF1R) gene; a heterozygous missense variant g.55118280G>T (p.Trp843Leu) in the echinoderm microtubule-associated protein like 6 (EML6) gene; a heterozygous frameshift variant c. 1226_1227del (p.Gln410Glufs*17) in the DOP1 leucine zipper-like protein B (DOP1B) gene; and a heterozygous splice-site variant c.7776+2T>A in the neurobeachin-like protein 2 (NBEAL2) gene. These variations were predicted to be potentially pathogenic and associated with KC. Conclusion: Five novel variants in HOMER3, IGF1R, EML6, DOP1B, and NBEAL2 genes were identified in this study and may be associated with the pathogenesis of KC. This study provides new information about the gene variants and their protein changes in KC patients. The findings should be explored further and could potentially be applied to the early diagnosis of KC before clinical onset.
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Asiatiques , Kératocône , Pedigree , Polymorphisme de nucléotide simple , Humains , Kératocône/génétique , Mâle , Femelle , Adulte , Chine , Asiatiques/génétique , Récepteur IGF de type 1/génétique , Protéines à homéodomaine/génétique , , Mutation faux-sens , Adulte d'âge moyen , Jeune adulte , Protéines associées aux microtubules/génétique , Prédisposition génétique à une maladie/génétique , Adolescent , Peuples d'Asie de l'EstRÉSUMÉ
Autophagy of glycogen (glycophagy) is crucial for the maintenance of cellular glucose homeostasis and physiology in mammals. STBD1 can serve as an autophagy receptor to mediate glycophagy by specifically recognizing glycogen and relevant key autophagic factors, but with poorly understood mechanisms. Here, we systematically characterize the interactions of STBD1 with glycogen and related saccharides, and determine the crystal structure of the STBD1 CBM20 domain with maltotetraose, uncovering a unique binding mode involving two different oligosaccharide-binding sites adopted by STBD1 CBM20 for recognizing glycogen. In addition, we demonstrate that the LC3-interacting region (LIR) motif of STBD1 can selectively bind to six mammalian ATG8 family members. We elucidate the detailed molecular mechanism underlying the selective interactions of STBD1 with ATG8 family proteins by solving the STBD1 LIR/GABARAPL1 complex structure. Importantly, our cell-based assays reveal that both the STBD1 LIR/GABARAPL1 interaction and the intact two oligosaccharide binding sites of STBD1 CBM20 are essential for the effective association of STBD1, GABARAPL1, and glycogen in cells. Finally, through mass spectrometry, biochemical, and structural modeling analyses, we unveil that STBD1 can directly bind to the Claw domain of RB1CC1 through its LIR, thereby recruiting the key autophagy initiation factor RB1CC1. In all, our findings provide mechanistic insights into the recognitions of glycogen, ATG8 family proteins, and RB1CC1 by STBD1 and shed light on the potential working mechanism of STBD1-mediated glycophagy.
Sujet(s)
Famille de la protéine-8 associée à l'autophagie , Autophagie , Glycogène , Autophagie/physiologie , Humains , Glycogène/métabolisme , Famille de la protéine-8 associée à l'autophagie/métabolisme , Famille de la protéine-8 associée à l'autophagie/génétique , Famille de la protéine-8 associée à l'autophagie/composition chimique , Sites de fixation , Protéines adaptatrices de la transduction du signal/métabolisme , Protéines adaptatrices de la transduction du signal/composition chimique , Protéines adaptatrices de la transduction du signal/génétique , Liaison aux protéines , Cristallographie aux rayons X , Animaux , Protéines associées aux microtubules/métabolisme , Protéines associées aux microtubules/composition chimique , Protéines associées aux microtubules/génétique , Modèles moléculairesRÉSUMÉ
Importance: Data on the performance of traumatic brain injury (TBI) biomarkers within minutes of injury are lacking. Objectives: To examine the performance of glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and microtubule-associated protein 2 (MAP-2) within 30 and 60 minutes of TBI in identifying intracranial lesions on computed tomography (CT) scan, need for neurosurgical intervention (NSI), and clinically important early outcomes (CIEO). Design, Setting, and Participants: This cohort study is a biomarker analysis of a multicenter prehospital TBI cohort from the Prehospital Tranexamic Acid Use for TBI clinical trial conducted across 20 centers and 39 emergency medical systems in North America from May 2015 to March 2017. Prehospital hemodynamically stable adult patients with traumatic injury and suspected moderate to severe TBI were included. Blood samples were measured for GFAP, UCH-L1, and MAP-2. Data were analyzed from December 1, 2023, to March 15, 2024. Main Outcomes and Measures: The presence of CT lesions, diffuse injury severity on CT, NSI within 24 hours of injury, and CIEO (composite outcome including early death, neurosurgery, or prolonged mechanical ventilation ≥7 days) within 7 days of injury. Results: Of 966 patients enrolled, 804 patients (mean [SD] age, 41 [19] years; 418 [74.2%] male) had blood samples, including 563 within 60 minutes and 375 within 30 minutes of injury. Among patients with blood drawn within 30 minutes of injury, 212 patients (56.5%) had CT lesions, 61 patients (16.3%) had NSI, and 112 patients (30.0%) had CIEO. Among those with blood drawn within 60 minutes, 316 patients (56.1%) had CT lesions, 95 patients (16.9%) had NSI, and 172 patients (30.6%) had CIEO. All biomarkers showed significant elevations with worsening diffuse injury on CT within 30 and 60 minutes of injury. Among blood samples taken within 30 minutes, GFAP had the highest area under the receiver operating characteristic curve (AUC) to detect CT lesions, at 0.88 (95% CI, 0.85-0.92), followed by MAP-2 (AUC, 0.78; 95% CI, 0.73-0.83) and UCH-L1 (AUC, 0.75; 95% CI, 0.70-0.80). Among blood samples taken within 60 minutes, AUCs for CT lesions were 0.89 (95% CI, 0.86-0.92) for GFAP, 0.76 (95% CI, 0.72-0.80) for MAP-2, and 0.73 (95% CI, 0.69-0.77) for UCH-L1. Among blood samples taken within 30 minutes, AUCs for NSI were 0.78 (95% CI, 0.72-0.84) for GFAP, 0.75 (95% CI, 0.68-0.81) for MAP-2, and 0.69 (95% CI, 0.63-0.75) for UCH-L1; and for CIEO, AUCs were 0.89 (95% CI, 0.85-0.93) for GFAP, 0.83 (95% CI, 0.78-0.87) for MAP-2, and 0.77 (95% CI, 0.72-0.82) for UCH-L1. Combining the biomarkers was no better than GFAP alone for all outcomes. At GFAP of 30 pg/mL within 30 minutes, sensitivity for CT lesions was 98.1% (95% CI, 94.9%-99.4%) and specificity was 34.4% (95% CI, 27.2%-42.2%). GFAP levels greater than 6200 pg/mL were associated with high risk of NSI and CIEO. Conclusions and Relevance: In this cohort study of prehospital patients with TBI, GFAP, UCH-L1, and MAP-2 measured within 30 and 60 minutes of injury were significantly associated with traumatic intracranial lesions and diffuse injury severity on CT scan, 24-hour NSI, and 7-day CIEO. GFAP was the strongest independent marker associated with all outcomes. This study sets a precedent for the early utility of GFAP in the first 30 minutes from injury in future clinical and research endeavors.
Sujet(s)
Marqueurs biologiques , Lésions traumatiques de l'encéphale , Protéine gliofibrillaire acide , Protéines associées aux microtubules , Ubiquitin thiolesterase , Humains , Lésions traumatiques de l'encéphale/sang , Ubiquitin thiolesterase/sang , Mâle , Femelle , Adulte , Protéine gliofibrillaire acide/sang , Adulte d'âge moyen , Marqueurs biologiques/sang , Protéines associées aux microtubules/sang , Tomodensitométrie , Études de cohortes , Facteurs tempsRÉSUMÉ
With the rapid progress in deciphering the pathogenesis of Alzheimer's disease (AD), it has been widely accepted that the accumulation of misfolded amyloid ß (Aß) in the brain could cause the neurodegeneration in AD. Although much evidence demonstrates the neurotoxicity of Aß, the role of Aß in the nervous system are complex. However, more comprehensive studies are needed to understand the physiological effect of Aß40 monomers in depth. To explore the physiological mechanism of Aß, we employed mass spectrometry to investigate the altered proteomic events induced by a lower submicromolar concentration of Aß. Human neuroblastoma SH-SY5Y cells were exposed to five different concentrations of Aß1-40 monomers and collected at four time points. The proteomic analysis revealed the time-course behavior of proteins involved in biological processes, such as RNA splicing, nuclear transport and protein localization. Further biological studies indicated that Aß40 monomers may activate PI3K/AKT signaling to regulate p-Tau, Ezrin and MAP2. These three proteins are associated with dendritic morphogenesis, neuronal polarity, synaptogenesis, axon establishment and axon elongation. Moreover, Aß40 monomers may regulate their physiological forms by inhibiting the expression of BACE1 and APP via activation of the ERK1/2 pathway. A comprehensive exploration of pathological and physiological mechanisms of Aß is beneficial for exploring novel treatment.
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Maladie d'Alzheimer , Peptides bêta-amyloïdes , Protéomique , Humains , Peptides bêta-amyloïdes/métabolisme , Maladie d'Alzheimer/métabolisme , Maladie d'Alzheimer/anatomopathologie , Protéomique/méthodes , Lignée cellulaire tumorale , Aspartic acid endopeptidases/métabolisme , Aspartic acid endopeptidases/génétique , Fragments peptidiques/métabolisme , Amyloid precursor protein secretases/métabolisme , Protéines du cytosquelette/métabolisme , Protéines du cytosquelette/génétique , Protéines tau/métabolisme , Phosphatidylinositol 3-kinases/métabolisme , Transduction du signal , Protéines proto-oncogènes c-akt/métabolisme , Précurseur de la protéine bêta-amyloïde/métabolisme , Protéome/métabolisme , Protéines associées aux microtubules/métabolisme , Système de signalisation des MAP kinasesRÉSUMÉ
INTRODUCTION: To explore the mechanisms of labor by investigating the autophagy of placental and fetal membranes tissue in normal pregnant women. METHODS: Placenta and fetal membranes were collected from women with singleton pregnancies without any medical complications and from women who delivered vaginally (labor-initiated group; L group) or by caesarean section (labor-noninitiated group; NL group). Autophagosomes were observed by transmission electron microscopy (TEM). Immunofluorescence and western blotting (WB) were used to detect protein levels of the autophagy markers LC3A and LC3B. TEM, immunohistochemistry (IHC), and WB were used to compare autophagy in different parts of the placenta and fetal membranes in the L and NL groups. The expression of LC3B/LC3A, ROCK1, and ROCK2 in the placenta of nonpregnant and pregnant rats was detected by WB and IHC. RESULTS: TEM and IHC results showed an increase in the number of autophagosomes and autophagic lysosomes in the L group, and WB results indicated an increase in the LC3B/A ratio between the placenta and fetal membranes in the L group. Autophagy was significantly increased on the maternal side of the placenta in the L group, and the level of autophagy became higher near rupture in the fetal membranes and near the point where the umbilical cord joins the placenta in the L group. The LC3B/A ratio increased and ROCK1 and ROCK2 levels decreased in postnatal rats. DISCUSSION: Autophagy can occur in the placenta and fetal membranes and its activity is higher at the onset of labor, suggesting a role in labor.
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Autophagie , Protéines associées aux microtubules , Placenta , rho-Associated Kinases , Femelle , Grossesse , Humains , Autophagie/physiologie , Placenta/métabolisme , Placenta/ultrastructure , rho-Associated Kinases/métabolisme , Protéines associées aux microtubules/métabolisme , Animaux , Rats , Adulte , Début du travail , Autophagosomes/métabolisme , Autophagosomes/ultrastructure , Membranes extraembryonnaires/métabolisme , Travail obstétrical/métabolisme , Rat Sprague-DawleyRÉSUMÉ
Microtubule organization in cells relies on targeting mechanisms. Cytoplasmic linker proteins (CLIPs) and CLIP-associated proteins (CLASPs) are key regulators of microtubule organization, yet the underlying mechanisms remain elusive. Here, we reveal that the C-terminal domain of CLASP2 interacts with a common motif found in several CLASP-binding proteins. This interaction drives the dynamic localization of CLASP2 to distinct cellular compartments, where CLASP2 accumulates in protein condensates at the cell cortex or the microtubule plus end. These condensates physically contact each other via CLASP2-mediated competitive binding, determining cortical microtubule targeting. The phosphorylation of CLASP2 modulates the dynamics of the condensate-condensate interaction and spatiotemporally navigates microtubule growth. Moreover, we identify additional CLASP-interacting proteins that are involved in condensate contacts in a CLASP2-dependent manner, uncovering a general mechanism governing microtubule targeting. Our findings not only unveil a tunable multiphase system regulating microtubule organization, but also offer general mechanistic insights into intricate protein-protein interactions at the mesoscale level.
Sujet(s)
Protéines associées aux microtubules , Microtubules , Liaison aux protéines , Microtubules/métabolisme , Protéines associées aux microtubules/métabolisme , Protéines associées aux microtubules/génétique , Humains , Phosphorylation , Fixation compétitive , Cellules HeLa , Condensats biomoléculaires/métabolisme , Cellules HEK293 , AnimauxRÉSUMÉ
The hormone leptin is critical for regulation of food intake, energy expenditure and overall metabolism. However, the mechanisms that promote leptin secretion from adipocytes in response to nutrient surplus and limit its secretion during nutrient scarcity are unclear. New work reveals that the autophagy protein Atg8/LC3 has a bidirectional role in leptin secretion, both facilitating and limiting its release.
Sujet(s)
Autophagie , Leptine , Autophagie/physiologie , Animaux , Leptine/métabolisme , Nutriments/métabolisme , Adipocytes/métabolisme , Humains , Protéines associées aux microtubules/métabolisme , Protéines associées aux microtubules/génétique , Métabolisme énergétique , Famille de la protéine-8 associée à l'autophagie/métabolisme , Famille de la protéine-8 associée à l'autophagie/génétiqueRÉSUMÉ
Nuclear migration is critical for the proper positioning of neurons in the developing brain. It is known that bidirectional microtubule motors are required for nuclear transport, yet the mechanism of the coordination of opposing motors is still under debate. Using mouse cerebellar granule cells, we demonstrate that Nesprin-2 serves as a nucleus-motor adaptor, coordinating the interplay of kinesin-1 and dynein. Nesprin-2 recruits dynein-dynactin-BicD2 independently of the nearby kinesin-binding LEWD motif. Both motor binding sites are required to rescue nuclear migration defects caused by the loss of function of Nesprin-2. In an intracellular cargo transport assay, the Nesprin-2 fragment encompassing the motor binding sites generates persistent movements toward both microtubule minus and plus ends. Nesprin-2 drives bidirectional cargo movements over a prolonged period along perinuclear microtubules, which advance during the migration of neurons. We propose that Nesprin-2 keeps the nucleus mobile by coordinating opposing motors, enabling continuous nuclear transport along advancing microtubules in migrating cells.
Sujet(s)
Noyau de la cellule , Dynéines , Kinésine , Protéines associées aux microtubules , Microtubules , Protéines de tissu nerveux , Neurones , Animaux , Microtubules/métabolisme , Neurones/métabolisme , Kinésine/métabolisme , Kinésine/génétique , Protéines de tissu nerveux/métabolisme , Protéines de tissu nerveux/génétique , Dynéines/métabolisme , Noyau de la cellule/métabolisme , Souris , Protéines associées aux microtubules/métabolisme , Protéines associées aux microtubules/génétique , Transport nucléaire actif , Complexe dynactine/métabolisme , Complexe dynactine/génétique , Mouvement cellulaire , Protéines des microfilaments/métabolisme , Protéines des microfilaments/génétique , Protéines nucléaires/métabolisme , Protéines nucléaires/génétique , Cervelet/métabolisme , Cervelet/cytologie , Sites de fixation , HumainsRÉSUMÉ
The characterization of interactions between autophagy modifiers (Atg8-family proteins) and their natural ligands (peptides and proteins) or small molecules is important for a detailed understanding of selective autophagy mechanisms and for the design of potential Atg8 inhibitors that affect the autophagy processes in cells. The fluorescence polarization (FP) assay is a rapid, cost-effective, and robust method that provides affinity and selectivity information for small molecules and peptide ligands targeting human Atg8 proteins.This chapter introduces the basic principles of FP assays. In addition, a case study on peptide interaction with human Atg8 proteins (LC3/GABARAPs) is described. Finally, data analysis and quality control of FP assays are discussed for the proper calculation of Ki values for the measured compounds.
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Polarisation de fluorescence , Tests de criblage à haut débit , Protéines associées aux microtubules , Liaison aux protéines , Humains , Protéines associées aux microtubules/métabolisme , Tests de criblage à haut débit/méthodes , Polarisation de fluorescence/méthodes , Protéines régulatrices de l'apoptose/métabolisme , Protéines adaptatrices de la transduction du signal/métabolisme , Autophagie/effets des médicaments et des substances chimiques , Peptides/métabolisme , Peptides/composition chimique , Ligands , Famille de la protéine-8 associée à l'autophagie/métabolismeRÉSUMÉ
Isothermal titration calorimetry (ITC) is a widely used technique for the characterization of protein-protein and protein-ligand interactions. It provides information on the stoichiometry, affinity, and thermodynamic driving forces of interactions. This chapter exemplifies the use of ITC to investigate interactions between human autophagy modifiers (LC3/GABARAP proteins) and their interaction partners, the LIR motif-containing sequences. The purpose of this report is to present a detailed protocol for the production of LC3/GABARAP-interacting LIR peptides using E. coli expression systems. In addition, we outline the design of ITC experiments using the LC3/GABARAP:peptide interactions as an example. Comprehensive troubleshooting notes are provided to facilitate the adaptation of these protocols to different ligand-receptor systems. The methodology outlined for studying protein-ligand interactions will help to avoid common errors and misinterpretations of experimental results.
Sujet(s)
Protéines adaptatrices de la transduction du signal , Protéines régulatrices de l'apoptose , Calorimétrie , Protéines associées aux microtubules , Liaison aux protéines , Thermodynamique , Calorimétrie/méthodes , Humains , Ligands , Protéines associées aux microtubules/métabolisme , Protéines associées aux microtubules/composition chimique , Protéines régulatrices de l'apoptose/métabolisme , Protéines régulatrices de l'apoptose/composition chimique , Protéines adaptatrices de la transduction du signal/métabolisme , Protéines adaptatrices de la transduction du signal/composition chimique , Escherichia coli/métabolisme , Peptides/composition chimique , Peptides/métabolismeRÉSUMÉ
Purpose: Homozygous hypomorphic variants of the RP1 gene, including c.5797C>T, p.Arg1933Ter, have traditionally been considered non-pathogenic. This study aimed to elucidate the clinical manifestations of late-onset, slowly progressive cone/macular dystrophy in patients homozygous for p.Arg1933Ter in the RP1 gene. Methods: Five patients with biallelic p.Arg1933Ter in RP1 were retrospectively recruited, and their clinical profiles were analyzed. Copy number variation analysis and Alu insertion assessment of genes associated with inherited retinal diseases were conducted. The results of comprehensive ophthalmological examinations, multimodal imaging, and full-field electroretinogram tests were analyzed. Results: No specific sequencing errors or structural variations associated with the clinical phenotypes were identified. Alu element insertion in RP1 was not detected. The mean ± SD age at the first visit was 62.2 ± 9.8 years, with symptoms typically starting between 45 and 50 years of age. Two patients exhibited a mild form of cone/macular dystrophy, characterized by a relatively preserved fundus appearance and blurring of the ellipsoid zone on optical coherence tomography. Three patients had late-onset cone/macular dystrophy with significant atrophy. Conclusions: To our knowledge, this study is the first to report that a homozygous hypomorphic variant of RP1, previously considered non-pathogenic, leads to cone/macular dystrophy. Translational Relevance: The study introduces novel possibilities suggesting that the homozygous hypomorphic variant of RP1 may be linked to variant pathogenicity.
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Électrorétinographie , Protéines de l'oeil , Tomographie par cohérence optique , Humains , Mâle , Femelle , Adulte d'âge moyen , Études rétrospectives , Sujet âgé , Protéines de l'oeil/génétique , Acuité visuelle , Variations de nombre de copies de segment d'ADN/génétique , Évolution de la maladie , Dystrophie des cônes/génétique , Dystrophie des cônes/imagerie diagnostique , Dégénérescence maculaire/génétique , Dégénérescence maculaire/anatomopathologie , Dégénérescence maculaire/imagerie diagnostique , Dégénérescence maculaire/congénital , Pedigree , Homozygote , Phénotype , Mutation , Adulte , Âge de début , Protéines associées aux microtubulesRÉSUMÉ
BACKGROUND: Occult Macular Dystrophy (OMD), primarily caused by retinitis pigmentosa 1-like 1 (RP1L1) variants, is a complex retinal disease characterised by progressive vision loss and a normal fundus appearance. This study aims to investigate the diverse phenotypic expressions and genotypic correlations of OMD in Chinese patients, including a rare case of Vitelliform Macular Dystrophy (VMD) associated with RP1L1. METHODS: We analysed seven OMD patients and one VMD patient, all with heterozygous pathogenic RP1L1 variants. Clinical assessments included Best Corrected Visual Acuity (BCVA), visual field testing, Spectral Domain Optical Coherence Tomography (SD-OCT), multifocal Electroretinograms (mfERGs), and microperimetry. Next-generation sequencing was utilised for genetic analysis. RESULTS: The OMD patients displayed a range of phenotypic variability. Most (5 out of 7) had the RP1L1 variant c.133 C > T; p.R45W, associated with central vision loss and specific patterns in SD-OCT and mfERG. Two patients exhibited different RP1L1 variants (c.3599G > T; p.G1200V and c.2880G > C; p.W960C), presenting milder phenotypes. SD-OCT revealed photoreceptor layer changes, with most patients showing decreased mfERG responses in the central rings. Interestingly, a unique case of VMD linked to the RP1L1 variant was observed, distinct from traditional OMD presentations. CONCLUSIONS: This study highlights the phenotypic diversity within OMD and the broader spectrum of RP1L1-associated macular dystrophies, including a novel association with VMD. The findings emphasise the complexity of RP1L1 variants in determining clinical manifestations, underscoring the need for comprehensive genetic and clinical evaluations in macular dystrophies.
Sujet(s)
Électrorétinographie , Protéines de l'oeil , Protéines associées aux microtubules , Tomographie par cohérence optique , Acuité visuelle , Dystrophie maculaire vitelliforme , Humains , Mâle , Femelle , Tomographie par cohérence optique/méthodes , Adulte , Adulte d'âge moyen , Protéines de l'oeil/génétique , Acuité visuelle/physiologie , Dystrophie maculaire vitelliforme/génétique , Dystrophie maculaire vitelliforme/physiopathologie , Dystrophie maculaire vitelliforme/diagnostic , Protéines associées aux microtubules/génétique , Champs visuels/physiologie , Chine/épidémiologie , Jeune adulte , Tests du champ visuel , Pedigree , Adolescent , Phénotype , Mutation , Dégénérescence maculaire/génétique , Dégénérescence maculaire/diagnostic , Dégénérescence maculaire/physiopathologie , Asiatiques/génétique , Sujet âgé , Peuples d'Asie de l'EstRÉSUMÉ
Sex differences may play a role in the etiopathogenesis and severity of metabolic dysfunction-associated steatotic liver disease (MASLD), a disorder characterized by excessive fat accumulation associated with increased inflammation and oxidative stress. We previously observed the development of steatosis specifically in female rats fed a high-fat diet enriched with liquid fructose (HFHFr) for 12 weeks. The aim of this study was to better characterize the observed sex differences by focusing on the antioxidant and cytoprotective pathways related to the KEAP1/NRF2 axis. The KEAP1/NRF2 signaling pathway, autophagy process (LC3B and LAMP2), and endoplasmic reticulum stress response (XBP1) were analyzed in liver homogenates in male and female rats that were fed a 12-week HFHFr diet. In females, the HFHFr diet resulted in the initial activation of the KEAP1/NRF2 pathway, which was not followed by the modulation of downstream molecular targets; this was possibly due to the increase in KEAP1 levels preventing the nuclear translocation of NRF2 despite its cytosolic increase. Interestingly, while in both sexes the HFHFr diet resulted in an increase in the levels of LC3BII/LC3BI, a marker of autophagosome formation, only males showed a significant upregulation of LAMP2 and XBP1s; this did not occur in females, suggesting impaired autophagic flux in this sex. Overall, our results suggest that males are characterized by a greater ability to cope with an HFHFr metabolic stimulus mainly through an autophagic-mediated proteostatic process while in females, this is impaired. This might depend at least in part upon the fine modulation of the cytoprotective and antioxidant KEAP1/NRF2 pathway resulting in sex differences in the occurrence and severity of MASLD. These results should be considered to design effective therapeutics for MASLD.
Sujet(s)
Alimentation riche en graisse , Fructose , Protéine-1 de type kelch associée à ECH , Facteur-2 apparenté à NF-E2 , Caractères sexuels , Transduction du signal , Animaux , Facteur-2 apparenté à NF-E2/métabolisme , Femelle , Mâle , Alimentation riche en graisse/effets indésirables , Transduction du signal/effets des médicaments et des substances chimiques , Rats , Protéine-1 de type kelch associée à ECH/métabolisme , Autophagie/effets des médicaments et des substances chimiques , Protéine-1 liant la boite X/métabolisme , Protéine-1 liant la boite X/génétique , Modèles animaux de maladie humaine , Stéatose hépatique/métabolisme , Stéatose hépatique/anatomopathologie , Foie/métabolisme , Foie/anatomopathologie , Foie/effets des médicaments et des substances chimiques , Stress du réticulum endoplasmique/effets des médicaments et des substances chimiques , Rat Wistar , Stress oxydatif/effets des médicaments et des substances chimiques , Protéines associées aux microtubulesRÉSUMÉ
The AurkA serine/threonine kinase is a key regulator of cell division controlling mitotic entry, centrosome maturation, and chromosome segregation. The microtubule-associated protein TPX2 controls spindle assembly and is the main AurkA regulator, contributing to AurkA activation, localisation, and stabilisation. Since their identification, AurkA and TPX2 have been described as being overexpressed in cancer, with a significant correlation with highly proliferative and aneuploid tumours. Despite the frequent occurrence of AurkA/TPX2 co-overexpression in cancer, the investigation of their involvement in tumorigenesis and cancer therapy resistance mostly arises from studies focusing only on one at the time. Here, we review the existing literature and discuss the mitotic phenotypes described under conditions of AurkA, TPX2, or AurkA/TPX2 overexpression, to build a picture that may help clarify their oncogenic potential through the induction of chromosome instability. We highlight the relevance of the AurkA/TPX2 complex as an oncogenic unit, based on which we discuss recent strategies under development that aim at disrupting the complex as a promising therapeutic perspective.
Sujet(s)
Aurora kinase A , Protéines associées aux microtubules , Tumeurs , Humains , Aurora kinase A/métabolisme , Aurora kinase A/génétique , Tumeurs/génétique , Tumeurs/anatomopathologie , Tumeurs/métabolisme , Protéines associées aux microtubules/métabolisme , Protéines associées aux microtubules/génétique , Protéines du cycle cellulaire/métabolisme , Protéines du cycle cellulaire/génétique , Animaux , Mitose/génétique , Aberrations des chromosomes , Instabilité des chromosomes/génétique , Régulation de l'expression des gènes tumorauxRÉSUMÉ
Autophagy refers to a set of degradative mechanisms whereby cytoplasmic contents are targeted to the lysosome. This is best described for macroautophagy, where a double-membrane compartment (autophagosome) is generated to engulf cytoplasmic contents. Autophagosomes are decorated with ubiquitin-like ATG8 molecules (ATG8s), which are recruited through covalent lipidation, catalysed by the E3-ligase-like ATG16L1 complex. LC3 proteins are ATG8 family members that are often used as a marker for autophagosomes. In contrast to canonical macroautophagy, conjugation of ATG8s to single membranes (CASM) describes a group of non-canonical autophagy processes in which ATG8s are targeted to pre-existing single-membrane compartments. CASM occurs in response to disrupted intracellular pH gradients, when the V-ATPase proton pump recruits ATG16L1 in a process called V-ATPase-ATG16L1-induced LC3 lipidation (VAIL). Recent work has demonstrated a parallel, alternative axis for CASM induction, triggered when the membrane recruitment factor TECPR1 recognises sphingomyelin exposed on the cytosolic face of a membrane and forms an alternative E3-ligase-like complex. This sphingomyelin-TECPR1-induced LC3 lipidation (STIL) is independent of the V-ATPase and ATG16L1. In light of these discoveries, this Cell Science at a Glance article summarises these two mechanisms of CASM to highlight how they differ from canonical macroautophagy, and from each other.
Sujet(s)
Famille de la protéine-8 associée à l'autophagie , Autophagie , Humains , Famille de la protéine-8 associée à l'autophagie/métabolisme , Famille de la protéine-8 associée à l'autophagie/génétique , Animaux , Autophagosomes/métabolisme , Protéines associées aux microtubules/métabolisme , Protéines associées à l'autophagie/métabolisme , Membrane cellulaire/métabolismeRÉSUMÉ
BACKGROUND: Radiotherapy is an important strategy for the treatment of advanced gastric cancer (GC), while the radioresistance limits its effectiveness. Nucleolar and spindle associated protein 1 (NUSAP1) was implicated in cancer progression and chemoresistance. However, the underlying mechanisms of NUSAP1 influencing GC radioresistance remain largely unknown. METHODS: Meta-analysis was conducted to systematically evaluate the prognostic value of NUSAP1 in human cancers. Gene set enrichment analysis (GSEA) was conducted using The Cancer Genome Atlas (TCGA) and gene expression omnibus (GEO) datasets. MRNA and protein expressions were detected by qRT-PCR and western blot, respectively. The radiosensitivity of GC cells was observed by colony formation, flow cytometry, comet, immunofluorescence, and animal assays. Immunoprecipitation assay and mass spectrometry were utilized to identify protein associations. MiRNAs binding with NUSAP1 were determined by starbase prediction, luciferase reporter, and RNA immunoprecipitation (RIP) assays. RESULTS: NUSAP1 high expression predicted worse overall survival (OS) and disease-free survival (DFS) with no statistical heterogeneity through the meta-analysis. Downregulation of NUSAP1 significantly increased GC radiosensitivity by inhibiting colony formation, DNA damage repair, and promoting apoptosis following irradiation. Additionally, NUSAP1 silencing combined with radiation resulted in a synergistic anti-tumor effect in xenograft mouse model. Mechanistically, NUSAP1 interacted with ANXA2, protecting it against protein degradation via impeding its ubiquitination process. NUSAP1 was confirmed as a target of miR-129-5p and negatively regulated by it. CONCLUSION: Our results suggested that NUSAP1 enhanced the radioresistance of GC cells. NUSAP1 could be a promising target to increase GC radiosensitivity.
Sujet(s)
Souris nude , microARN , Radiotolérance , Tumeurs de l'estomac , Ubiquitination , Humains , Tumeurs de l'estomac/génétique , Tumeurs de l'estomac/anatomopathologie , Tumeurs de l'estomac/radiothérapie , Tumeurs de l'estomac/métabolisme , microARN/génétique , Radiotolérance/génétique , Animaux , Souris , Pronostic , Lignée cellulaire tumorale , Régulation de l'expression des gènes tumoraux , Tests d'activité antitumorale sur modèle de xénogreffe , Souris de lignée BALB C , Femelle , Apoptose , Mâle , Protéines associées aux microtubules , Annexine A2RÉSUMÉ
Cancer is the leading cause of disease-related death among children. Vincristine (VCR), a key component of childhood cancer treatment protocols, is associated with the risk of peripheral neuropathy (PN), a condition that may be reversible upon drug discontinuation but can also leave lasting sequelae. Single nucleotide polymorphism (SNP) in genes involved in VCR pharmacokinetics and pharmacodynamics have been investigated in relation to an increased risk of PN. However, the results of these studies have been inconsistent. A retrospective cohort study was conducted to investigate the potential association of drug transporter genes from the ATP-binding cassette (ABC) family and the centrosomal protein 72 (CEP72) gene with the development of PN in 88 Caucasian children diagnosed with cancer and treated with VCR. Genotyping was performed using real-time PCR techniques for the following SNPs: ABCB1 rs1128503, ABCC1 rs246240, ABCC2 rs717620, and CEP72 rs924607. The results indicated that age at diagnosis (OR = 1.33; 95% CI = 1.07-1.75) and the ABCC1 rs246240 G allele (OR = 12.48; 95% CI = 2.26-100.42) were associated with vincristine-induced peripheral neuropathy (VIPN). No association was found between this toxicity and CEP72 rs924607. Our study provides insights that may contribute to optimizing childhood cancer therapy in the future by predicting the risk of VIPN.
Sujet(s)
Protéine-2 associée à la multirésistance aux médicaments , Protéines associées à la multirésistance aux médicaments , Tumeurs , Neuropathies périphériques , Polymorphisme de nucléotide simple , Médecine de précision , Vincristine , Humains , Vincristine/effets indésirables , Vincristine/usage thérapeutique , Neuropathies périphériques/induit chimiquement , Neuropathies périphériques/génétique , Enfant , Femelle , Mâle , Protéines associées à la multirésistance aux médicaments/génétique , Enfant d'âge préscolaire , Médecine de précision/méthodes , Tumeurs/traitement médicamenteux , Tumeurs/génétique , Adolescent , Études rétrospectives , Protéines du cycle cellulaire/génétique , Nourrisson , Antinéoplasiques d'origine végétale/effets indésirables , Antinéoplasiques d'origine végétale/usage thérapeutique , Prédisposition génétique à une maladie , Génotype , Allèles , Sous-famille B de transporteurs à cassette liant l'ATP/génétique , Protéines associées aux microtubulesRÉSUMÉ
BACKGROUND: Gastric cancer (GC) is a prevalent malignancy with limited therapeutic options for advanced stages. This study aimed to identify novel therapeutic targets for GC by profiling HSP90 client kinases. METHODS: We used mass spectrometry-based activity-based protein profiling (ABPP) with a desthiobiotin-ATP probe, combined with sensitivity analysis of HSP90 inhibitors, to profile kinases in a panel of GC cell lines. We identified kinases regulated by HSP90 in inhibitor-sensitive cells and investigated the impact of MASTL knockdown on GC cell behavior. Global proteomic analysis following MASTL knockdown was performed, and bioinformatics tools were used to analyze the resulting data. RESULTS: Four kinases-MASTL, STK11, CHEK1, and MET-were identified as HSP90-regulated in HSP90 inhibitor-sensitive cells. Among these, microtubule-associated serine/threonine kinase-like (MASTL) was upregulated in GC and associated with poor prognosis. MASTL knockdown decreased migration, invasion, and proliferation of GC cells. Global proteomic profiling following MASTL knockdown revealed NEDD4-1 as a potential downstream mediator of MASTL in GC progression. NEDD4-1 was also upregulated in GC and associated with poor prognosis. Similar to MASTL inhibition, NEDD4-1 knockdown suppressed migration, invasion, and proliferation of GC cells. CONCLUSIONS: Our multi-proteomic analyses suggest that targeting MASTL could be a promising therapy for advanced gastric cancer, potentially through the reduction of tumor-promoting proteins including NEDD4-1. This study enhances our understanding of kinase signaling pathways in GC and provides new insights for potential treatment strategies.