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There are 113 results for Autophagy (displaying 111 to 113).

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.

… with homozygous loss of GRN suffer from the lysosomal storage disease neuronal ceroid lipofuscinosis (Smith et al , ). Vice versa, patients with neuronal ceroid lipofuscinosis show signs of TDP‐43 pathology (Gotzl et al , ). Pathogenic mutations in TBK1 and OPTN suggest that altered autophagy may trigger ALS and FTLD with TDP‐43 pathology (Cirulli et al , ; Freischmidt et al , ). These chronic defects in protein degradation eventually lead to the aggregation of TDP‐43. Here, we asked how loss …

Benjamin M Schwenk et al. The EMBO Journal November 2016

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α‐helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP 8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP 8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMP s, but present in all other VAMP s. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion.

Crystal structure of VAMP8 in an autophagy SNARE complex (PDB: 4WY4). Residues T47, T53, S54, and S61 are shown as sticks‐and‐balls in the middle of the SNARE complex in an orthogonal view. Accessible surface area for each residue in the VAMP8 SNARE domain (calculated from PDB: 4WY4). The 16 SNARE layers are all buried inside the SNARE complex with minimal surface accessibility. The four phosphorylation sites (T47, T53, S54, and S61) all have low accessible surface areas. Sequence of rat …

Seth Malmersjö et al. The EMBO Journal August 2016

Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1

Mutations in the PTEN ‐induced kinase 1 ( PINK 1) are causative of autosomal recessive Parkinson's disease ( PD ). We have previously reported that PINK 1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser 65 ) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK 1‐dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub‐family of Rab GTP ases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser 111 ) in response to PINK 1 activation. Using phospho‐specific antibodies raised against Ser 111 of each of the Rabs, we demonstrate that Rab Ser 111 phosphorylation occurs specifically in response to PINK 1 activation and is abolished in HeLa PINK 1 knockout cells and mutant PINK 1 PD patient‐derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTP ase Ser 111 phosphorylation is not directly regulated by PINK 1 in vitro and demonstrate in cells the time course of Ser 111 phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser 65 . We further show mechanistically that phosphorylation at Ser 111 significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor ( GEF ), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK 1 is able to regulate the phosphorylation of Rab GTP ases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser 111 may represent novel biomarkers of PINK 1 activity in vivo . Our findings also suggest that disruption of Rab GTP ase‐mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.

… that PINK1 and Parkin null flies exhibit significant mitochondrial defects and that PINK1 lies genetically upstream of Parkin (Clark et al , ; Park et al , ). In mammalian cells, PINK1 is activated in response to mitochondrial depolarisation and this stimulates the recruitment of Parkin, a cytosolic protein, to depolarised mitochondria where it ubiquitylates multiple mitochondrial substrates to trigger the removal of mitochondria by autophagy (also known as mitophagy; Narendra et al , , ; Geisler et al …

Yu‐Chiang Lai et al. The EMBO Journal November 2015