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There are 47 results for Insulin (displaying 41 to 47).

Role of Angiomotin‐like 2 mono‐ubiquitination on YAP inhibition

LATS1/2 (large tumor suppressor) kinases and the Angiomotin family proteins are potent inhibitors of the YAP (yes‐associated protein) oncoprotein, but the underlying molecular mechanism is not fully understood. Here, we report for the first time that USP9X is a deubiquitinase of Angiomotin‐like 2 (AMOTL2) and that AMOTL2 mono‐ubiquitination is required for YAP inhibition. USP9X knockdown increased the LATS‐mediated phosphorylation of YAP and decreased the transcriptional output of YAP. Conversely, over‐expression of USP9X reactivated YAP in densely cultured cells. Both genetic and biochemical approaches identified AMOTL2 as a target of USP9X. AMOTL2 was found to be ubiquitinated at K347 and K408, which both reside in the protein's coiled‐coil domain. The AMOTL2 K347/408R mutant, which cannot be ubiquitinated, was impaired in its ability to inhibit YAP. Furthermore, ubiquitinated AMOTL2 can bind to the UBA domain of LATS kinase, and this domain is required for the function of LATS. Our results provide novel insights into the activation mechanisms of core Hippo pathway components.

… , and suggest many interesting new questions that warrant future investigation. RPE, MCF10A and 293T cells were purchased from ATCC. MCF10A cells were cultured in DMEM/F12 supplemented with 5% horse serum (Invitrogen), 20 ng/ml EGF (Peprotech), 10 μg/ml insulin (Sigma), 0.5 μg/ml hydrocortisone (Sigma), 100 ng/ml cholera toxin (Sigma) and penicillin/streptomycin (Invitrogen). 293T and RPE cells were cultured in DMEM or DMEM/F12 supplemented with 10% FBS and penicillin/streptomycin. Immortalized …

Miju Kim et al. EMBO Reports January 2016

Activation of serum/glucocorticoid‐induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy

Maintaining skeletal muscle mass is essential for general health and prevention of disease progression in various neuromuscular conditions. Currently, no treatments are available to prevent progressive loss of muscle mass in any of these conditions. Hibernating mammals are protected from muscle atrophy despite prolonged periods of immobilization and starvation. Here, we describe a mechanism underlying muscle preservation and translate it to non‐hibernating mammals. Although Akt has an established role in skeletal muscle homeostasis, we find that serum‐ and glucocorticoid‐inducible kinase 1 (SGK1) regulates muscle mass maintenance via downregulation of proteolysis and autophagy as well as increased protein synthesis during hibernation. We demonstrate that SGK1 is critical for the maintenance of skeletal muscle homeostasis and function in non‐hibernating mammals in normal and atrophic conditions such as starvation and immobilization. Our results identify a novel therapeutic target to combat loss of skeletal muscle mass associated with muscle degeneration and atrophy.

… ) signalling as well as epigenetically mediated pathways have been suggested to play an important role (Lee et al, ; Morin & Storey, ; Nowell et al, ; Shavlakadze & Grounds, ). In non‐hibernating mammals, current paradigm dictates that muscle cell strength, growth and survival are dependent upon the PI3K/Akt/mTOR pathway, which is activated in response to several growth factors including insulin‐growth factor‐I (IGF‐1; Glass, ). The binding of IGF‐1 to its receptor triggers the activation …

Eva Andres‐Mateos et al. EMBO molecular medicine January 2013

The mitochondrial calcium uniporter regulates breast cancer progression via HIF‐1α

Triple‐negative breast cancer ( TNBC ) represents the most aggressive breast tumor subtype. However, the molecular determinants responsible for the metastatic TNBC phenotype are only partially understood. We here show that expression of the mitochondrial calcium uniporter ( MCU ), the selective channel responsible for mitochondrial Ca 2+ uptake, correlates with tumor size and lymph node infiltration, suggesting that mitochondrial Ca 2+ uptake might be instrumental for tumor growth and metastatic formation. Accordingly, MCU downregulation hampered cell motility and invasiveness and reduced tumor growth, lymph node infiltration, and lung metastasis in TNBC xenografts. In MCU ‐silenced cells, production of mitochondrial reactive oxygen species ( mROS ) is blunted and expression of the hypoxia‐inducible factor‐1α ( HIF ‐1α) is reduced, suggesting a signaling role for mROS and HIF ‐1α, downstream of mitochondrial Ca 2+ . Finally, in breast cancer mRNA samples, a positive correlation of MCU expression with HIF ‐1α signaling route is present. Our results indicate that MCU plays a central role in TNBC growth and metastasis formation and suggest that mitochondrial Ca 2+ uptake is a potential novel therapeutic target for clinical intervention.

… bovine serum (FBS) (Life Technologies). MDA‐MB‐231 cells were cultured in DMEM/F12 medium (1:1) (Life Technologies), supplemented with 10% FBS. MCF10AT1k.cl2 and MCF10CA1a.cl1 cells were cultured in DMEM/F12 supplemented with 5% horse serum (HS), 10 μg/ml insulin, 20 ng/ml EGF, 8.5 ng/ml cholera toxin, 500 ng/ml hydrocortisone. All media were supplemented with 1% penicillin G‐streptomycin sulfate (Euroclone) and 1% l ‐glutamine (Euroclone). Cells were maintained in culture at 37°C, with 5% CO 2 …

Anna Tosatto et al. EMBO molecular medicine May 2016

Mitochondria are required for pro‐ageing features of the senescent phenotype

Cell senescence is an important tumour suppressor mechanism and driver of ageing. Both functions are dependent on the development of the senescent phenotype, which involves an overproduction of pro‐inflammatory and pro‐oxidant signals. However, the exact mechanisms regulating these phenotypes remain poorly understood. Here, we show the critical role of mitochondria in cellular senescence. In multiple models of senescence, absence of mitochondria reduced a spectrum of senescence effectors and phenotypes while preserving ATP production via enhanced glycolysis. Global transcriptomic analysis by RNA sequencing revealed that a vast number of senescent‐associated changes are dependent on mitochondria, particularly the pro‐inflammatory phenotype. Mechanistically, we show that the ATM , Akt and mTORC 1 phosphorylation cascade integrates signals from the DNA damage response ( DDR ) towards PGC ‐1β‐dependent mitochondrial biogenesis, contributing to a ROS ‐mediated activation of the DDR and cell cycle arrest. Finally, we demonstrate that the reduction in mitochondrial content in vivo , by either mTORC 1 inhibition or PGC ‐1β deletion, prevents senescence in the ageing mouse liver. Our results suggest that mitochondria are a candidate target for interventions to reduce the deleterious impact of senescence in ageing tissues.

… content impacts on DDR and our data revealing a role for PGC‐1β in senescence in vitro , we found reduced senescent markers such as TAF (Fig  K) in PGC‐1β −/− mice. The absence of PGC‐1β also ameliorated age‐dependent decline in glucose and insulin tolerance ( ). Similar to rapamycin‐fed mice, we did not find any changes in MnSOD expression in PGC‐1β −/− mice liver tissues ( ). Altogether, our data strongly support a causal link between the DDR, mTORC1 and mitochondrial mass increase …

Clara Correia‐Melo et al. The EMBO Journal March 2016

Combined Nurr1 and Foxa2 roles in the therapy of Parkinson's disease

Use of the physiological mechanisms promoting midbrain DA ( mDA ) neuron survival seems an appropriate option for developing treatments for Parkinson's disease ( PD ). mDA neurons are specifically marked by expression of the transcription factors Nurr1 and Foxa2. We show herein that Nurr1 and Foxa2 interact to protect mDA neurons against various toxic insults, but their expression is lost during aging and degenerative processes. In addition to their proposed cell‐autonomous actions in mDA neurons, forced expression of these factors in neighboring glia synergistically protects degenerating mDA neurons in a paracrine mode. As a consequence of these bimodal actions, adeno‐associated virus ( AAV )‐mediated gene delivery of Nurr1 and Foxa2 in a PD mouse model markedly protected mDA neurons and motor behaviors associated with nigrostriatal DA neurotransmission. The effects of the combined gene delivery were dramatic, highly reproducible, and sustained for at least 1 year, suggesting that expression of these factors is a promising approach in PD therapy.

… , transforming growth factor‐β (TGF‐β), sonic hedgehog (SHH) from astrocytes (Trendelenburg & Dirnagl, ), and insulin‐like growth factor 1/2 (IGF1/2) and BDNF from microglia (Suh et al , ). The observation of Nrf2 activation and expression of antioxidant genes in mDA neurons after Nurr1 + Foxa2‐CM treatment (Fig  F and G) suggest that the Nurr1‐ and Foxa2‐mediated paracrine actions involve the release of neurotrophic factors, because these factors are major cytokines activating Nrf2 via the MAPK …

Sang‐Min Oh et al. EMBO molecular medicine May 2015

TDP‐43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons

Nuclear clearance of TDP ‐43 into cytoplasmic aggregates is a key driver of neurodegeneration in amyotrophic lateral sclerosis ( ALS ) and frontotemporal lobar degeneration ( FTLD ), but the mechanisms are unclear. Here, we show that TDP ‐43 knockdown specifically reduces the number and motility of RAB11‐positive recycling endosomes in dendrites, while TDP‐43 overexpression has the opposite effect. This is associated with delayed transferrin recycling in TDP‐43‐knockdown neurons and decreased β2‐transferrin levels in patient CSF . Whole proteome quantification identified the upregulation of the ESCRT component VPS 4B upon TDP ‐43 knockdown in neurons. Luciferase reporter assays and chromatin immunoprecipitation suggest that TDP ‐43 represses VPS 4B transcription. Preventing VPS4B upregulation or expression of its functional antagonist ALIX restores trafficking of recycling endosomes. Proteomic analysis revealed the broad reduction in surface expression of key receptors upon TDP ‐43 knockdown, including ErbB4, the neuregulin 1 receptor. TDP ‐43 knockdown delays the surface delivery of ErbB4. ErbB4 overexpression, but not neuregulin 1 stimulation, prevents dendrite loss upon TDP ‐43 knockdown. Thus, impaired recycling of ErbB4 and other receptors to the cell surface may contribute to TDP ‐43‐induced neurodegeneration by blocking trophic signaling.

… storage. After thawing, neurons were plated on PDL‐coated glass‐bottom dishes in HN media (DMEM/F12 and Neurobasal 1:1, 0.5% N2, 1% B27, 0.1 % glucose, 2 μg/ml insulin, 10 ng/ml GDNF, 10 ng/ml BDNF, 10 μM Y‐27632). Every two days half of the media were exchanged. Neurons were transduced with lentivirus seven days after thawing and imaged three days later. Astrocyte feeder cells were prepared from embryonic day 18 CD rats (Charles River) as described previously (Kaech & Banker, ), plated onto …

Benjamin M Schwenk et al. The EMBO Journal November 2016

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

Membrane‐less organelles in cells are large, dynamic protein/protein or protein/ RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher‐order assemblies influences the recruitment of the speckle‐type POZ protein ( SPOP ) to nuclear speckles. SPOP , a cullin‐3‐ RING ubiquitin ligase ( CRL 3) substrate adaptor, self‐associates into higher‐order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild‐type SPOP localizes to liquid nuclear speckles, self‐association‐deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB ‐mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration‐dependent populations of the resulting oligomeric species. Higher‐order oligomerization of SPOP stimulates CRL 3 SPOP ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher‐order protein self‐association may be a general mechanism to concentrate functional components in membrane‐less cellular bodies.

… the experimental CG‐MALS data without prior knowledge of the dissociation constants and oligomeric states of the individual domains, demonstrating the analytical power of CG‐MALS. Careful biophysical characterization has previously allowed the elucidation of isodemic self‐association behavior for several other proteins, including insulin (Jeffrey et al , ), FtsZ (Rivas et al , ), Phage ϕ29 protein p6 (Abril et al , ), chicken deoxy hemoglobin D (Rana & Riggs, ), human apolipoprotein C‐II (Yang …

Melissa R Marzahn et al. The EMBO Journal June 2016