ABSTRACT
Autophagy and cellular metabolism are tightly linked processes, but how individual metabolic enzymes regulate the process of autophagy is not well understood. This study implicates ribose-5-phosphate isomerase (RPIA), a key regulator of the pentose phosphate pathway, in the control of autophagy. We used a dual gene deletion strategy, combining shRNA-mediated knockdown studies with CRISPR/Cas9 genome editing. Knockdown of RPIA by shRNA or genomic deletion by CRISPR/Cas9 genome editing, results in an increase of ATG4B-mediated LC3 processing and in the appearance of LC3-positive autophagosomes in cells. Increased LC3 processing upon knockdown of RPIA can be reversed by treatment with the antioxidant N-acetyl cysteine. The results are consistent with a model in which RPIA suppresses autophagy and LC3 processing by modulation of redox signaling.
Subject(s)
Aldose-Ketose Isomerases/metabolism , Autophagy , Microtubule-Associated Proteins/metabolism , Protein Processing, Post-Translational , Acetylcysteine/pharmacology , Animals , Antioxidants/pharmacology , Autophagosomes/drug effects , Autophagosomes/metabolism , Autophagy/drug effects , Autophagy-Related Proteins/metabolism , Base Sequence , CRISPR-Cas Systems/genetics , Cysteine Endopeptidases/metabolism , Gene Knockdown Techniques , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Mice , Protein Processing, Post-Translational/drug effectsABSTRACT
BACKGROUND: Secreted luciferases are highly useful bioluminescent reporters for cell-based assays and drug discovery. A variety of secreted luciferases from marine organisms have been described that harbor an N-terminal signal peptide for release along the classical secretory pathway. Here, we have characterized the secretion of Gaussia luciferase in more detail. RESULTS: We describe three basic mechanisms by which GLUC can be released from cells: first, classical secretion by virtue of the N-terminal signal peptide; second, internal signal peptide-mediated secretion and third, non-conventional secretion in the absence of an N-terminal signal peptide. Non-conventional release of dNGLUC is not stress-induced, does not require autophagy and can be enhanced by growth factor stimulation. Furthermore, we have identified the golgi-associated, gamma adaptin ear containing, ARF binding protein 1 (GGA1) as a suppressor of release of dNGLUC. CONCLUSIONS: Due to its secretion via multiple secretion pathways GLUC can find multiple applications as a research tool to study classical and non-conventional secretion. As GLUC can also be released from a reporter construct by internal signal peptide-mediated secretion it can be incorporated in a novel bicistronic secretion system.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Bacterial Proteins/metabolism , Biochemistry/methods , Luciferases, Firefly/metabolism , ATP-Binding Cassette Transporters/genetics , Adaptor Proteins, Vesicular Transport/genetics , Bacterial Proteins/genetics , Bodily Secretions , Genes/genetics , Genes, Reporter/genetics , HEK293 Cells , Humans , Luciferases, Firefly/genetics , Protein Sorting Signals/geneticsABSTRACT
Manipulation of gene expression on a genome-wide level is one of the most important systematic tools in the post-genome era. Such manipulations have largely been enabled by expression cloning approaches using sequence-verified cDNA libraries, large-scale RNA interference libraries (shRNA or siRNA) and zinc finger nuclease technologies. More recently, the CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas)9-mediated gene editing technology has been described that holds great promise for future use of this technology in genomic manipulation. It was suggested that the CRISPR system has the potential to be used in high-throughput, large-scale loss of function screening. Here we discuss some of the challenges in engineering of CRISPR/Cas genomic libraries and some of the aspects that need to be addressed in order to use this technology on a high-throughput scale.
