Protein Synthesis and Degradation
The prohibitin-binding compound fluorizoline affects multiple components of the translational machinery and inhibits protein synthesis
Fluorizoline (FLZ) binds to prohibitin-1 and -2 (PHB1/2), which are pleiotropic scaffold proteins known to affect signaling pathways involved in several intracellular processes. However, it is not yet clear how FLZ exerts its effect. Here, we show that exposure of three different human cancer cell lines to FLZ increases the phosphorylation of key translation factors, particularly of initiation factor 2 (eIF2) and elongation factor 2 (eEF2), modifications that inhibit their activities. FLZ also impaired signaling through mTOR complex 1, which also regulates the translational machinery, e.g.The kinase PERK represses translation of the G-protein–coupled receptor LGR5 and receptor tyrosine kinase ERBB3 during ER stress in cancer cells
As a branch of the unfolded protein response, protein kinase R-like endoplasmic reticulum kinase (PERK) represses global translation in response to endoplasmic reticulum (ER) stress. This pathophysiological condition is associated with the tumor microenvironment in cancer. Previous findings in our lab have suggested that PERK selectively represses translation of some mRNAs, but this possibility awaits additional investigation. In this study, we show that a stem-cell marker protein, leucine-rich repeat-containing G-protein–coupled receptor 5 (LGR5), is rapidly depleted in colon cancer cells during ER stress, an effect that depended on the PERK-mediated translational repression.Endoplasmic reticulum stress differentially inhibits endoplasmic reticulum and inner nuclear membrane protein quality control degradation pathways
Endoplasmic reticulum (ER) stress occurs when the abundance of unfolded proteins in the ER exceeds the capacity of the folding machinery. Despite the expanding cadre of characterized cellular adaptations to ER stress, knowledge of the effects of ER stress on cellular physiology remains incomplete. We investigated the impact of ER stress on ER and inner nuclear membrane protein quality control mechanisms in Saccharomyces cerevisiae. We analyzed the turnover of substrates of four ubiquitin ligases (Doa10, Rkr1/Ltn1, Hrd1, and the Asi complex) and the metalloprotease Ste24 in induced models of ER stress.Ribosome depurination by ricin leads to inhibition of endoplasmic reticulum stress–induced HAC1 mRNA splicing on the ribosome
Ricin undergoes retrograde transport to the endoplasmic reticulum (ER), and ricin toxin A chain (RTA) enters the cytosol from the ER. Previous reports indicated that RTA inhibits activation of the unfolded protein response (UPR) in yeast and in mammalian cells. Both precursor (preRTA) and mature form of RTA (mRTA) inhibited splicing of HAC1u (u for uninduced) mRNA, suggesting that UPR inhibition occurred on the cytosolic face of the ER. Here, we examined the role of ribosome binding and depurination activity on inhibition of the UPR using mRTA mutants.Lysine trimethylation regulates 78-kDa glucose-regulated protein proteostasis during endoplasmic reticulum stress
The up-regulation of chaperones such as the 78-kDa glucose-regulated protein (GRP78, also referred to as BiP or HSPA5) is part of the adaptive cellular response to endoplasmic reticulum (ER) stress. GRP78 is widely used as a marker of the unfolded protein response, associated with sustained ER stress. Here we report the discovery of a proteostatic mechanism involving GRP78 trimethylation in the context of ER stress. Using mass spectrometry–based proteomics, we identified two GRP78 fractions, one homeostatic and one induced by ER stress.Endoplasmic reticulum stress–induced degradation of DNAJB12 stimulates BOK accumulation and primes cancer cells for apoptosis
DNAJB12 (JB12) is an endoplasmic reticulum (ER)–associated Hsp40 family protein that recruits Hsp70 to the ER surface to coordinate the function of ER-associated and cytosolic chaperone systems in protein quality control. Hsp70 is stress-inducible, but paradoxically, we report here that JB12 was degraded by the proteasome during severe ER stress. Destabilized JB12 was degraded by ER-associated degradation complexes that contained HERP, Sel1L, and gp78. JB12 was the only ER-associated chaperone that was destabilized by reductive stress.Nuclear Matrix Protein 4 Is a Novel Regulator of Ribosome Biogenesis and Controls the Unfolded Protein Response via Repression of Gadd34 Expression
The unfolded protein response (UPR) maintains protein homeostasis by governing the processing capacity of the endoplasmic reticulum (ER) to manage ER client loads; however, key regulators within the UPR remain to be identified. Activation of the UPR sensor PERK (EIFAK3/PEK) results in the phosphorylation of the α subunit of eIF2 (eIF2α-P), which represses translation initiation and reduces influx of newly synthesized proteins into the overloaded ER. As part of this adaptive response, eIF2α-P also induces a feedback mechanism through enhanced transcriptional and translational expression of Gadd34 (Ppp1r15A),which targets type 1 protein phosphatase for dephosphorylation of eIF2α-P to restore protein synthesis.Translation Regulation of the Glutamyl-prolyl-tRNA Synthetase Gene EPRS through Bypass of Upstream Open Reading Frames with Noncanonical Initiation Codons
In the integrated stress response, phosphorylation of eIF2α (eIF2α-P) reduces protein synthesis while concomitantly promoting preferential translation of specific transcripts associated with stress adaptation. Translation of the glutamyl-prolyl-tRNA synthetase gene EPRS is enhanced in response to eIF2α-P. To identify the underlying mechanism of translation control, we employed biochemical approaches to determine the regulatory features by which upstream ORFs (uORFs) direct downstream translation control and expression of the EPRS coding region.Site-specific Proteolysis Mobilizes TorsinA from the Membrane of the Endoplasmic Reticulum (ER) in Response to ER Stress and B Cell Stimulation
Torsin ATPases are the only representatives of the AAA+ ATPase family that reside in the lumen of the endoplasmic reticulum (ER) and nuclear envelope. Two of these, TorsinA and TorsinB, are anchored to the ER membrane by virtue of an N-terminal hydrophobic domain. Here we demonstrate that the imposition of ER stress leads to a proteolytic cleavage event that selectively removes the hydrophobic domain from the AAA+ domain of TorsinA, which retains catalytic activity. Both the pharmacological inhibition profile and the identified cleavage site between two juxtaposed cysteine residues are distinct from those of presently known proteases, suggesting that a hitherto uncharacterized, membrane-associated protease accounts for TorsinA processing.Ribosome Elongation Stall Directs Gene-specific Translation in the Integrated Stress Response
Upon exposure to environmental stress, phosphorylation of the α subunit of eIF2 (eIF2α-P) represses global protein synthesis, coincident with preferential translation of gene transcripts that mitigate stress damage or alternatively trigger apoptosis. Because there are multiple mammalian eIF2 kinases, each responding to different stress arrangements, this translational control scheme is referred to as the integrated stress response (ISR). Included among the preferentially translated mRNAs induced by eIF2α-P is that encoding the transcription factor CHOP (DDIT3/GADD153).Selenoprotein S-dependent Selenoprotein K Binding to p97(VCP) Protein Is Essential for Endoplasmic Reticulum-associated Degradation
Cytosolic valosin-containing protein (p97(VCP)) is translocated to the ER membrane by binding to selenoprotein S (SelS), which is an ER membrane protein, during endoplasmic reticulum-associated degradation (ERAD). Selenoprotein K (SelK) is another known p97(VCP)-binding selenoprotein, and the expression of both SelS and SelK is increased under ER stress. To understand the regulatory mechanisms of SelS, SelK, and p97(VCP) during ERAD, the interaction of the selenoproteins with p97(VCP) was investigated using N2a cells and HEK293 cells.Ribosome Reinitiation Directs Gene-specific Translation and Regulates the Integrated Stress Response
Background: eIF2α-P induced GADD34 and constitutively expressed CReP target PP1c to dephosphorylate eIF2α-P to dictate translation control of the ISR.Results: Differential expression of GADD34 and CReP is regulated by upstream ORF (uORF)-mediated ribosome reinitiation.Conclusion: uORFs regulate differential expression of GADD34 and CReP and are important for cell adaptation to stress.Significance: Regulation of eIF2α-P is central for protein homeostasis and cell viability.Evidence That Base-pairing Interaction between Intron and mRNA Leader Sequences Inhibits Initiation of HAC1 mRNA Translation in Yeast
Background: Hac1 protein, encoded by a cytoplasmically spliced mRNA, activates the unfolded protein response to maintain cellular protein homeostasis and alleviate endoplasmic reticulum stress.Results: Under non-stress conditions, translation initiation on the HAC1 mRNA is repressed.Conclusion: Base-pairing interaction between the 5′ leader and intron represses translation initiation on the HAC1 mRNA.Significance: A unique mechanism of intron-mediated inhibition of ribosomal scanning.
