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1.
Cell ; 179(6): 1306-1318.e18, 2019 11 27.
Article in English | MEDLINE | ID: mdl-31761535

ABSTRACT

Cells have evolved complex mechanisms to maintain protein homeostasis, such as the UPRER, which are strongly associated with several diseases and the aging process. We performed a whole-genome CRISPR-based knockout (KO) screen to identify genes important for cells to survive ER-based protein misfolding stress. We identified the cell-surface hyaluronidase (HAase), Transmembrane Protein 2 (TMEM2), as a potent modulator of ER stress resistance. The breakdown of the glycosaminoglycan, hyaluronan (HA), by TMEM2 within the extracellular matrix (ECM) altered ER stress resistance independent of canonical UPRER pathways but dependent upon the cell-surface receptor, CD44, a putative HA receptor, and the MAPK cell-signaling components, ERK and p38. Last, and most surprisingly, ectopic expression of human TMEM2 in C. elegans protected animals from ER stress and increased both longevity and pathogen resistance independent of canonical UPRER activation but dependent on the ERK ortholog mpk-1 and the p38 ortholog pmk-1.


Subject(s)
Caenorhabditis elegans/physiology , Endoplasmic Reticulum/metabolism , Hyaluronoglucosaminidase/metabolism , Longevity/physiology , Membrane Proteins/metabolism , Unfolded Protein Response , Animals , Caenorhabditis elegans/immunology , Cell Line , Cell Proliferation , Disease Resistance , Endoplasmic Reticulum Stress , Fibroblasts/metabolism , Humans , Immunity, Innate , Models, Biological , Molecular Weight , Signal Transduction
2.
Genome Res ; 29(8): 1322-1328, 2019 08.
Article in English | MEDLINE | ID: mdl-31239279

ABSTRACT

Genome editing tools have simplified the generation of knock-in gene fusions, yet the prevalent use of gene-specific homology-directed repair (HDR) templates still hinders scalability. Consequently, realization of large-scale gene tagging requires further development of approaches to generate knock-in protein fusions via generic donors that do not require locus-specific homology sequences. Here, we combine intron-based protein trapping with homology-independent repair-based integration of a generic donor and demonstrate precise, scalable, and efficient gene tagging. Because editing is performed in introns using a synthetic exon, this approach tolerates mutations in the unedited allele, indels at the integration site, and the addition of resistance genes that do not disrupt the target gene coding sequence, resulting in easy and flexible gene tagging.


Subject(s)
Gene Editing/methods , Genome, Human , Introns , Mutagenesis, Insertional , Recombinant Fusion Proteins/genetics , Base Sequence , CRISPR-Associated Protein 9/genetics , CRISPR-Associated Protein 9/metabolism , Calnexin/genetics , Calnexin/metabolism , Cell Line, Tumor , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Exons , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression , HEK293 Cells , HeLa Cells , Humans , Plasmids/chemistry , Plasmids/metabolism , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism , Recombinant Fusion Proteins/biosynthesis , Vimentin/genetics , Vimentin/metabolism
3.
PLoS One ; 6(6): e20501, 2011.
Article in English | MEDLINE | ID: mdl-21695208

ABSTRACT

Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid ß peptide (Aß) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aß(1-42). Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aß pathology. Systemic PADK injections in APP(SwInd) and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aß immunostaining as well as Aß(x-42) sandwich ELISA measures in APP(SwInd) mice of 10-11 months. More extensive Aß deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aß(1-38) occurs as Aß(1-42) levels decrease in the mouse models, indicating that PADK treatment leads to Aß truncation. Associated with Aß clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aß(1-42) accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aß pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders.


Subject(s)
Alzheimer Disease/pathology , Lysosomes/drug effects , Protective Agents/pharmacology , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/metabolism , Animals , Behavior, Animal/drug effects , Biomarkers/metabolism , Cathepsin B/metabolism , Cathepsin D/metabolism , Disease Models, Animal , Hippocampus/drug effects , Hippocampus/pathology , Intracellular Space/drug effects , Intracellular Space/metabolism , Ketones/pharmacology , Lysosomes/enzymology , Mice , Mice, Transgenic , Neurons/drug effects , Neurons/metabolism , Peptide Fragments/metabolism , Synapses/drug effects , Synapses/metabolism , rab GTP-Binding Proteins/metabolism
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