RESUMEN

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.

Asunto(s)

Lesión Pulmonar Aguda , Autofagia , Interleucina-6 , Ratones Endogámicos C57BL , Ratones Noqueados , Choque Hemorrágico , Factor de Necrosis Tumoral alfa , Animales , Lesión Pulmonar Aguda/metabolismo , Masculino , Choque Hemorrágico/metabolismo , Choque Hemorrágico/complicaciones , Ratones , Interleucina-6/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Modelos Animales de Enfermedad , Pulmón/metabolismo , Pulmón/patología , Proteínas Asociadas a Microtúbulos/metabolismo

RESUMEN

Microtubule-based vesicle trafficking usually relies upon kinesin and dynein motors and few reports describe microtubule polymerisation driving directional vesicle trafficking. Here we show that Arabidopsis END BINDING1b (EB1b), a microtubule plus-end binding protein, directly interacts with SYP121, a SNARE protein that mediates the trafficking of the K+ channel KAT1 and its distribution to the plasma membrane (PM) in Arabidopsis guard cells. Knockout of AtEB1b and its homologous proteins results in a modest but significant change in the distribution of KAT1 and SYP121 in guard cells and consequently delays light-induced stomatal opening. Live-cell imaging reveals that a portion of SYP121-associated endomembrane compartments co-localise with AtEB1b at the growing ends of microtubules, trafficking along with the growth of microtubules for targeting to the PM. Our study reveals a mechanism of vesicle trafficking driven by microtubule growth, which is involved in the redistribution of PM proteins to modulate guard cell movement.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Membrana Celular , Proteínas Asociadas a Microtúbulos , Microtúbulos , Estomas de Plantas , Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Microtúbulos/metabolismo , Estomas de Plantas/metabolismo , Estomas de Plantas/fisiología , Membrana Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Transporte de Proteínas , Katanina/metabolismo , Katanina/genética , Movimiento Celular , Proteínas de Ciclo Celular

RESUMEN

Cytopathology induced by methamphetamine (METH) is reminiscent of degenerative disorders such as Parkinson's disease, and it is characterized by membrane organelles arranged in tubulo-vesicular structures. These areas, appearing as clusters of vesicles, have never been defined concerning the presence of specific organelles. Therefore, the present study aimed to identify the relative and absolute area of specific membrane-bound organelles following a moderate dose (100 µM) of METH administered to catecholamine-containing PC12 cells. Organelles and antigens were detected by immunofluorescence, and they were further quantified by plain electron microscopy and in situ stoichiometry. This analysis indicated an increase in autophagosomes and damaged mitochondria along with a decrease in lysosomes and healthy mitochondria. Following METH, a severe dissipation of hallmark proteins from their own vesicles was measured. In fact, the amounts of LC3 and p62 were reduced within autophagy vacuoles compared with the whole cytosol. Similarly, LAMP1 and Cathepsin-D within lysosomes were reduced. These findings suggest a loss of compartmentalization and confirm a decrease in the competence of cell clearing organelles during catecholamine degeneration. Such cell entropy is consistent with a loss of energy stores, which routinely govern appropriate subcellular compartmentalization.

Asunto(s)

Autofagosomas , Lisosomas , Metanfetamina , Metanfetamina/farmacología , Animales , Células PC12 , Ratas , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Autofagosomas/metabolismo , Autofagosomas/efectos de los fármacos , Autofagia/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Catepsina D/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo

RESUMEN

Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, microtubule minus ends are sorted outwards away from the chromosomes where they form poles, but then these outward forces must be balanced to form a stable bipolar structure. Simultaneously, microtubule dynamics must be precisely controlled to maintain spindle length and organization. How forces and dynamics are tuned to create a stable bipolar structure is poorly understood. Here, we have gained insight into this question through studies of ZYG-8, a conserved doublecortin-family kinase; the mammalian homolog of this microtubule-associated protein is upregulated in many cancers and has been implicated in cell division, but the mechanisms by which it functions are poorly understood. We found that ZYG-8 depletion from oocytes resulted in overelongated spindles with pole and midspindle defects. Importantly, experiments with monopolar spindles revealed that ZYG-8 depletion led to excess outward forces within the spindle and suggested a potential role for this protein in regulating the force-generating motor BMK-1/kinesin-5. Further, we found that ZYG-8 is also required for proper microtubule dynamics within the oocyte spindle and that kinase activity is required for its function during both meiosis and mitosis. Altogether, our findings reveal new roles for ZYG-8 in oocytes and provide insights into how acentrosomal spindles are stabilized to promote faithful meiosis.

Asunto(s)

Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Microtúbulos , Oocitos , Huso Acromático , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Microtúbulos/metabolismo , Microtúbulos/genética , Huso Acromático/metabolismo , Huso Acromático/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Oocitos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Centrosoma/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética

RESUMEN

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.

Asunto(s)

Familia de las Proteínas 8 Relacionadas con la Autofagia , Autofagia , Glucógeno , Animales , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Autofagia/fisiología , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/química , Sitios de Unión , Cristalografía por Rayos X , Glucógeno/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Modelos Moleculares , Unión Proteica

RESUMEN

Diabetes-related bone loss represents a significant complication that persistently jeopardizes the bone health of individuals with diabetes. Primary cilia proteins have been reported to play a vital role in regulating osteoblast differentiation in diabetes-related bone loss. However, the specific contribution of KIAA0753, a primary cilia protein, in bone loss induced by diabetes remains unclear. In this investigation, we elucidated the pivotal role of KIAA0753 as a promoter of osteoblast differentiation in diabetes. RNA sequencing demonstrated a marked downregulation of KIAA0753 expression in pro-bone MC3T3 cells exposed to a high glucose environment. Diabetes mouse models further validated the downregulation of KIAA0753 protein in the femur. Diabetes was observed to inhibit osteoblast differentiation in vitro, evidenced by downregulating the protein expression of OCN, OPN and ALP, decreasing primary cilia biosynthesis, and suppressing the Hedgehog signalling pathway. Knocking down KIAA0753 using shRNA methods was found to shorten primary cilia. Conversely, overexpression KIAA0753 rescued these changes. Additional insights indicated that KIAA0753 effectively restored osteoblast differentiation by directly interacting with SHH, OCN and Gli2, thereby activating the Hedgehog signalling pathway and mitigating the ubiquitination of Gli2 in diabetes. In summary, we report a negative regulatory relationship between KIAA0753 and diabetes-related bone loss. The clarification of KIAA0753's role offers valuable insights into the intricate mechanisms underlying diabetic bone complications.

Asunto(s)

Diferenciación Celular , Proteínas Asociadas a Microtúbulos , Osteoblastos , Transducción de Señal , Animales , Humanos , Masculino , Ratones , Línea Celular , Cilios/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Ratones Endogámicos C57BL , Osteoblastos/metabolismo , Osteogénesis/genética , Proteínas Asociadas a Microtúbulos/metabolismo

RESUMEN

Family with sequence similarity 161 (Fam161) is an ancient family of microtubule-binding proteins located at the centriole and cilium transition zone (TZ) lumen that exhibit rapid evolution in mice. However, their adaptive role is unclear. Here, we used flies to gain insight into their cell type-specific adaptations. Fam161 is the sole orthologue of FAM161A and FAM161B found in flies. Mutating Fam161 results in reduced male reproduction and abnormal geotaxis behaviour. Fam161 localizes to sensory neuron centrioles and their specialized TZ (the connecting cilium) in a cell type-specific manner, sometimes labelling only the centrioles, sometimes labelling the centrioles and cilium TZ and sometimes labelling the TZ with varying lengths that are longer than other TZ proteins, defining a new ciliary compartment, the extra distal TZ. These findings suggest that Fam161 is an essential centriole and TZ protein with a unique cell type-specific localization in fruit flies that can produce cell type-specific adaptations.

Asunto(s)

Centriolos , Cilios , Proteínas de Drosophila , Animales , Centriolos/metabolismo , Cilios/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Masculino , Drosophila melanogaster/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Especificidad de Órganos

RESUMEN

Autophagy is the primary intracellular degradation system, and it plays an important role in many biological and pathological processes. Studies of autophagy involvement in developmental processes are important for understanding various processes. Among them are fibrosis, degenerative diseases, cancer development, and metastasis formation. Diabetic kidney disease is one of the main causes of chronic kidney disease and end-stage renal failure. The aim of this study was to investigate the immunohistochemical expression patterns of LC3B, LAMP2A, and GRP78 during different developmental stages of early-developing human kidneys and in samples from patients with type II diabetes mellitus. During the 7/8th DW, moderate expression of LC3B and LAMP2A and strong expression of GRP78 were found in the mesonephric glomeruli and tubules. In the 9/10th DW, the expression of LC3B and LAMP2A was even more pronounced in the mesonephric tubules. LC3B, LAMP2A, and GRP78 immunoreactivity was also found in the paramesonephric and mesonephric ducts and was stronger in the 9/10th DW compared with the 7/8th DW. In addition, the expression of LC3B, LAMP2A, and GRP78 also appeared in the mesenchyme surrounding the paramesonephric duct in the 9/10th DW. In the 15/16th DW, the expression of LC3B in the glomeruli was weak, that of LAMP2A was moderate, and that of GRP78 was strong. In the tubuli, the expression of LC3B was moderate, while the expression of LAMP2A and GRP78 was strong. The strongest expression of LC3B, LAMP2A, and GRP78 was observed in the renal medullary structures, including developing blood vessels. In postnatal human kidneys, the most extensive LC3B, LAMP2A, and GRP78 expression in the cortex was found in the epithelium of the proximal convoluted tubules, with weak to moderate expression in the glomeruli. The medullary expression of LC3B was weak, but the expression of LAMP2A and GRP78 was the strongest in the medullary tubular structures. Significantly lower expression of LC3B was found in the glomeruli of the diabetic patients in comparison with the nondiabetic patients, but there was no difference in the expression of LC3B in the tubule-interstitial compartment. The expression of LAMP2A was significantly higher in the tubule-interstitial compartments of the diabetic patients in comparison with the nondiabetic patients, while its expression did not differ in the glomeruli. Extensive expression of GRP78 was found in the glomeruli and the tubule-interstitial compartments, but there was no difference in the expression between the two groups of patients. These data give us new information about the expression of LC3B, LAMP2A, and GRP78 during embryonic, fetal, and early postnatal development. The spatiotemporal expression of LC3B, LAMP2A, and GRP78 indicates the important role of autophagy during the early stages of renal development. In addition, our data suggest a disturbance in autophagy processes in the glomeruli and tubuli of diabetic kidneys as an important factor in the pathogenesis of diabetic kidney disease.

Asunto(s)

Autofagia , Nefropatías Diabéticas , Chaperón BiP del Retículo Endoplásmico , Riñón , Proteína 2 de la Membrana Asociada a los Lisosomas , Proteínas Asociadas a Microtúbulos , Humanos , Chaperón BiP del Retículo Endoplásmico/metabolismo , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Riñón/metabolismo , Riñón/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Biomarcadores/metabolismo , Femenino , Masculino , Proteínas de Choque Térmico/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología

RESUMEN

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease. Its accurate pathogenic mechanisms are incompletely clarified, and effective therapeutic treatments are still inadequate. Autophagy is closely associated with AD and plays multiple roles in eliminating harmful aggregated proteins and maintaining cell homeostasis. This study identified 1191 differentially expressed genes (DEGs) based on the GSE5281 dataset from the GEO database, intersected them with 325 autophagy-related genes from GeneCards, and screened 26 differentially expressed autophagy-related genes (DEAGs). Subsequently, GO and KEGG enrichment analysis was performed and indicated that these DEAGs were primarily involved in autophagy-lysosomal biological process. Further, eight hub genes were determined by PPI construction, and experimental validation was performed by qRT-PCR on a SH-SY5Y cell model. Finally, three hub genes (TFEB, TOMM20, GABARAPL1) were confirmed to have potential application for biomarkers. A multigenic prediction model with good predictability (AUC = 0.871) was constructed in GSE5281 and validated in the GSE132903 dataset. Hub gene-targeted miRNAs closely associated with AD were also retrieved through the miRDB and HDMM database, predicting potential therapeutic agents for AD. This study provides new insights into autophagy-related genes in brain tissues of AD patients and offers more candidate biomarkers for AD mechanistic research as well as clinical diagnosis.

Asunto(s)

Enfermedad de Alzheimer , Autofagia , Biomarcadores , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/diagnóstico , Humanos , Autofagia/genética , MicroARNs/genética , Redes Reguladoras de Genes , Mapas de Interacción de Proteínas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Perfilación de la Expresión Génica , Bases de Datos Genéticas , Línea Celular Tumoral , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice

RESUMEN

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.

Asunto(s)

Aurora Quinasa A , Proteínas Asociadas a Microtúbulos , Neoplasias , Humanos , Aurora Quinasa A/metabolismo , Aurora Quinasa A/genética , Neoplasias/genética , Neoplasias/patología , Neoplasias/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Animales , Mitosis/genética , Aberraciones Cromosómicas , Inestabilidad Cromosómica/genética , Regulación Neoplásica de la Expresión Génica

RESUMEN

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.

Asunto(s)

Familia de las Proteínas 8 Relacionadas con la Autofagia , Autofagia , Humanos , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Animales , Autofagosomas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Membrana Celular/metabolismo

RESUMEN

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.

Asunto(s)

Enfermedad de Alzheimer , Péptidos beta-Amiloides , Proteómica , Humanos , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Proteómica/métodos , Línea Celular Tumoral , Ácido Aspártico Endopeptidasas/metabolismo , Ácido Aspártico Endopeptidasas/genética , Fragmentos de Péptidos/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas tau/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Proteínas Proto-Oncogénicas c-akt/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteoma/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Sistema de Señalización de MAP Quinasas

RESUMEN

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.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Calorimetría , Proteínas Asociadas a Microtúbulos , Unión Proteica , Termodinámica , Calorimetría/métodos , Humanos , Ligandos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Escherichia coli/metabolismo , Péptidos/química , Péptidos/metabolismo

RESUMEN

Autophagosome formation initiated on the endoplasmic reticulum (ER)-associated omegasome requires LC3. Translational regulation of LC3 biosynthesis is unexplored. Here we demonstrate that LC3 mRNA is recruited to omegasomes by directly binding to the ER transmembrane Sigma-1 receptor (S1R). Cell-based and in vitro reconstitution experiments show that S1R interacts with the 3' UTR of LC3 mRNA and ribosomes to promote LC3 translation. Strikingly, the 3' UTR of LC3 is also required for LC3 protein lipidation, thereby linking the mRNA-3' UTR to LC3 function. An autophagy-defective S1R mutant responsible for amyotrophic lateral sclerosis cannot bind LC3 mRNA or induce LC3 translation. We propose a model wherein S1R de-represses LC3 mRNA via its 3' UTR at the ER, enabling LC3 biosynthesis and lipidation. Because several other LC3-related proteins use the same mechanism, our data reveal a conserved pathway for localized translation essential for autophagosome biogenesis with insights illuminating the molecular basis of a neurodegenerative disease.

Asunto(s)

Regiones no Traducidas 3' , Autofagia , Retículo Endoplásmico , Proteínas Asociadas a Microtúbulos , Biosíntesis de Proteínas , ARN Mensajero , Receptores sigma , Receptor Sigma-1 , Receptores sigma/metabolismo , Receptores sigma/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Retículo Endoplásmico/metabolismo , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Regiones no Traducidas 3'/genética , Ribosomas/metabolismo , Animales , Autofagosomas/metabolismo , Células HeLa

RESUMEN

Aurora A kinase, a cell division regulator, is frequently overexpressed in various cancers, provoking genome instability and resistance to antimitotic chemotherapy. Localization and enzymatic activity of Aurora A are regulated by its interaction with the spindle assembly factor TPX2. We have used fragment-based, structure-guided lead discovery to develop small molecule inhibitors of the Aurora A-TPX2 protein-protein interaction (PPI). Our lead compound, CAM2602, inhibits Aurora A:TPX2 interaction, binding Aurora A with 19 nM affinity. CAM2602 exhibits oral bioavailability, causes pharmacodynamic biomarker modulation, and arrests the growth of tumor xenografts. CAM2602 acts by a novel mechanism compared to ATP-competitive inhibitors and is highly specific to Aurora A over Aurora B. Consistent with our finding that Aurora A overexpression drives taxane resistance, these inhibitors synergize with paclitaxel to suppress the outgrowth of pancreatic cancer cells. Our results provide a blueprint for targeting the Aurora A-TPX2 PPI for cancer therapy and suggest a promising clinical utility for this mode of action.

Asunto(s)

Antimitóticos , Aurora Quinasa A , Proteínas de Ciclo Celular , Proteínas Asociadas a Microtúbulos , Humanos , Animales , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Antimitóticos/farmacología , Antimitóticos/química , Línea Celular Tumoral , Proteínas Asociadas a Microtúbulos/metabolismo , Ratones , Proteínas Nucleares/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Ensayos Antitumor por Modelo de Xenoinjerto , Antineoplásicos/farmacología , Antineoplásicos/química , Relación Estructura-Actividad , Paclitaxel/farmacología , Ratones Desnudos

RESUMEN

Statins have evident neuroprotective role in acute ischemic stroke(AIS). The pleiotropic effect by which statin exerts neuroprotective effects, needs to be explored for considering it as one of the future adjunctive therapies in AIS. Endoplasmic reticulum(ER) assists cellular survival by reducing protein aggregates during ischemic conditions. ER-stress mediated apoptosis and autophagy are predominant reasons for neuronal death in AIS. Statin exerts both anti-apoptotic and anti-autophagic effect in neurons under ischemic stress. Although the influence of statin on autophagic neuroprotection has been reported with contradictory results. Thus, in our study we have attempted to understand its influence on autophagic protection while inhibiting upregulation of autophagic death(autosis). Previously we reported, statin can alleviate apoptosis via modulating cardiolipin mediated mitochondrial dysfunction. However, the clearance of damaged mitochondria is essential for prolonged cell survival. In our study, we tried to decipher the mechanism by which statin leads to neuronal survival by the mitophagy mediated cellular clearance. Simvastatin was administered to Sprague Dawley(SD) rats both as prophylaxis and treatment. The safety and efficacy of the statin was validated by assessment of infarct size and functional outcome. A reduction in oxidative and ER-stress were observed in both the prophylactic and treatment groups. The influence of statin on autophagy/apoptosis balance was evaluated by molecular assessment of mitophagy and cellular apoptosis. Statin reduces the post-stroke ER-stress and predominantly upregulated autophagolysosome mediated mitophagy than apoptotic cell death by modulating pAMPK/LC3B/LAMP2 axis. Based on the above findings statin could be explored as an adjunctive therapy for AIS in future.

Asunto(s)

Apoptosis , Autofagia , Estrés del Retículo Endoplásmico , Proteína 2 de la Membrana Asociada a los Lisosomas , Fármacos Neuroprotectores , Ratas Sprague-Dawley , Simvastatina , Animales , Estrés del Retículo Endoplásmico/efectos de los fármacos , Ratas , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Simvastatina/farmacología , Masculino , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Activadas por AMP/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/patología

RESUMEN

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.

Asunto(s)

Polarización de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Proteínas Asociadas a Microtúbulos , Unión Proteica , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Polarización de Fluorescencia/métodos , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia/efectos de los fármacos , Péptidos/metabolismo , Péptidos/química , Ligandos , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo

RESUMEN

Accurate chromosome segregation during cell division relies on coordinated actions of microtubule (MT)-based motor proteins in the mitotic spindle. Kinesin-14 motors play vital roles in spindle assembly and maintenance by crosslinking antiparallel MTs at the spindle midzone and anchoring spindle MTs' minus ends at the poles. In this study, we investigate the force generation and motility of the Kinesin-14 motors HSET and KlpA. Our findings reveal that both motors are non-processive, producing single load-dependent power strokes per MT encounter, with estimated load-free power strokes of ~30 and ~35 nm, respectively. Each homodimeric motor generates forces of ~0.5 pN, but when assembled in teams, they cooperate to generate forces of 1 pN or more. Notably, the cooperative activity among multiple motors leads to increased MT-sliding velocities. These results quantitatively elucidate the structure-function relationship of Kinesin-14 motors and underscore the significance of cooperative behavior in their cellular functions.

Asunto(s)

Cinesinas , Microtúbulos , Huso Acromático , Cinesinas/metabolismo , Microtúbulos/metabolismo , Huso Acromático/metabolismo , Animales , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo

RESUMEN

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.

Asunto(s)

Núcleo Celular , Dineínas , Cinesinas , Proteínas Asociadas a Microtúbulos , Microtúbulos , Proteínas del Tejido Nervioso , Neuronas , Animales , Microtúbulos/metabolismo , Neuronas/metabolismo , Cinesinas/metabolismo , Cinesinas/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Dineínas/metabolismo , Núcleo Celular/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Transporte Activo de Núcleo Celular , Complejo Dinactina/metabolismo , Complejo Dinactina/genética , Movimiento Celular , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Cerebelo/metabolismo , Cerebelo/citología , Sitios de Unión , Humanos

RESUMEN

Microtubules (MTs) are dynamically unstable polar biopolymers switching between periods of polymerization and depolymerization, with the switch from the polymerization to the depolymerization phase termed catastrophe and the reverse transition termed rescue.1 In presence of MT-crosslinking proteins, MTs form parallel or anti-parallel overlaps and self-assemble reversibly into complex networks, such as the mitotic spindle. Differential regulation of MT dynamics in parallel and anti-parallel overlaps is critical for the self-assembly of these networks.2,3 Diffusible MT crosslinkers of the Ase1/MAP65/PRC1 family associate with different affinities to parallel and antiparallel MT overlaps, providing a basis for this differential regulation.4,5,6,7,8,9,10,11 Ase1/MAP65/PRC1 family proteins directly affect MT dynamics12 and recruit other proteins that locally alter MT dynamics, such as CLASP or kinesin-4.7,13,14,15,16 However, how Ase1 differentially regulates MT stability in parallel and antiparallel bundles is unknown. Here, we show that Ase1 selectively promotes antiparallel MT overlap longevity by slowing down the depolymerization velocity and by increasing the rescue frequency, specifically in antiparallelly crosslinked MTs. At the retracting ends of depolymerizing MTs, concomitant with slower depolymerization, we observe retention and accumulation of Ase1 between crosslinked MTs and on isolated MTs. We hypothesize that the ability of Ase1 to reduce the dissociation of tubulin subunits is sufficient to promote its enrichment at MT ends. A mathematical model built on this idea shows good agreement with the experiments. We propose that differential regulation of MT dynamics by Ase1 contributes to mitotic spindle assembly by specifically stabilizing antiparallel overlaps, compared to parallel overlaps or isolated MTs.

Asunto(s)

Proteínas Asociadas a Microtúbulos , Microtúbulos , Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Huso Acromático/metabolismo , Animales , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética