Coordinated regulation of the ubiquitin-proteasome system (UPS) is crucial for the cell to adjust its protein degradation capacity to changing proteolytic requirements. We have shown previously that ...mammalian cells upregulate proteasome gene expression in response to proteasome inhibition. Here, we report the identification of the transcription factor TCF11 (long isoform of Nrf1) as a key regulator for 26S proteasome formation in human cells to compensate for reduced proteolytic activity. Under noninducing conditions, TCF11 resides in the endoplasmic reticulum (ER) membrane. There, TCF11 is targeted to ER-associated protein degradation requiring the E3 ubiquitin ligase HRD1 and the AAA ATPase p97. Proteasome inhibitors trigger the accumulation of oxidant-damaged proteins and promote the nuclear translocation of TCF11 from the ER, permitting activation of proteasome gene expression by binding to antioxidant response elements in their promoter regions. Thus, we uncovered the transcriptional control loop regulating human proteasome-dependent protein degradation to counteract proteotoxic stress caused by proteasome inhibition.
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► TCF11 (long isoform of Nrf1) regulates proteasome gene transcription in human cells ► TCF11 interacts with ARE sequences in proteasome subunit promoters ► TCF11 levels are regulated by ERAD requiring HRD1 and p97 ► ER transit of TCF11 is important for its translocation into the nucleus
SCL25A46 is a mitochondrial carrier protein that surprisingly localizes to the outer membrane and is distantly related to Ugo1. Here we show that a subset of SLC25A46 interacts with mitochondrial ...dynamics components and the MICOS complex. Decreased expression of SLC25A46 results in increased stability and oligomerization of MFN1 and MFN2 on mitochondria, promoting mitochondrial hyperfusion. A mutation at L341P causes rapid degradation of SLC25A46, which manifests as a rare disease, pontocerebellar hypoplasia. The E3 ubiquitin ligases MULAN and MARCH5 coordinate ubiquitylation of SLC25A46 L341P, leading to degradation by organized activities of P97 and the proteasome. Whereas outer mitochondrial membrane-associated degradation is typically associated with apoptosis or a specialized type of autophagy termed mitophagy, SLC25A46 degradation operates independently of activation of outer membrane stress pathways. Thus SLC25A46 is a new component in mitochondrial dynamics that serves as a regulator for MFN1/2 oligomerization. Moreover, SLC25A46 is selectively degraded from the outer membrane independently of mitophagy and apoptosis, providing a framework for mechanistic studies in the proteolysis of outer membrane proteins.
Significance The lethal disorder, primary hyperoxaluria 1 (PH1), is caused by mutations in peroxisomal-localized alanine:glyoxylate aminotransferase (AGT). AGT contains a C-terminal peroxisomal ...targeting sequence, but mutations generate a strong N-terminal mitochondrial targeting sequence that directs AGT to mitochondria. Although mutant AGT is functional, the enzyme must be in the peroxisome to detoxify glyoxylate and prevent oxalate accumulation. We have identified a Food and Drug Administration-approved drug, dequalinium chloride (DECA), from a chemical genetic screen to identify probes that attenuate mitochondrial protein import. DECA treatment restores trafficking of mutant AGT from mitochondria to peroxisomes with a subsequent reduction in oxalate levels. Thus, repurposing DECA has potential in therapeutic strategies for PH1 because current clinical trials have not produced an effective treatment, short of organ transplant.
Primary hyperoxaluria 1 (PH1; Online Mendelian Inheritance in Man no. 259900), a typically lethal biochemical disorder, may be caused by the AGT ᴾ¹¹ᴸᴳ¹⁷⁰ᴿ allele in which the alanine:glyoxylate aminotransferase (AGT) enzyme is mistargeted from peroxisomes to mitochondria. AGT contains a C-terminal peroxisomal targeting sequence, but mutations generate an N-terminal mitochondrial targeting sequence that directs AGT from peroxisomes to mitochondria. Although AGT ᴾ¹¹ᴸᴳ¹⁷⁰ᴿ is functional, the enzyme must be in the peroxisome to detoxify glyoxylate by conversion to alanine; in disease, amassed glyoxylate in the peroxisome is transported to the cytosol and converted to oxalate by lactate dehydrogenase, leading to kidney failure. From a chemical genetic screen, we have identified small molecules that inhibit mitochondrial protein import. We tested whether one promising candidate, Food and Drug Administration (FDA)-approved dequalinium chloride (DECA), could restore proper peroxisomal trafficking of AGT ᴾ¹¹ᴸᴳ¹⁷⁰ᴿ. Indeed, treatment with DECA inhibited AGT ᴾ¹¹ᴸᴳ¹⁷⁰ᴿ translocation into mitochondria and subsequently restored trafficking to peroxisomes. Previous studies have suggested that a mitochondrial uncoupler might work in a similar manner. Although the uncoupler carbonyl cyanide m-chlorophenyl hydrazone inhibited AGT ᴾ¹¹ᴸᴳ¹⁷⁰ᴿ import into mitochondria, AGT ᴾ¹¹ᴸᴳ¹⁷⁰ᴿ aggregated in the cytosol, and cells subsequently died. In a cellular model system that recapitulated oxalate accumulation, exposure to DECA reduced oxalate accumulation, similar to pyridoxine treatment that works in a small subset of PH1 patients. Moreover, treatment with both DECA and pyridoxine was additive in reducing oxalate levels. Thus, repurposing the FDA-approved DECA may be a pharmacologic strategy to treat PH1 patients with mutations in AGT because an additional 75 missense mutations in AGT may also result in mistrafficking.
Disturbed mitochondrial fusion and fission have been linked to various neurodegenerative disorders. In siblings from two unrelated families who died soon after birth with a profound ...neurodevelopmental disorder characterized by pontocerebellar hypoplasia and apnoea, we discovered a missense mutation and an exonic deletion in the SLC25A46 gene encoding a mitochondrial protein recently implicated in optic atrophy spectrum disorder. We performed functional studies that confirmed the mitochondrial localization and pro-fission properties of SLC25A46. Knockdown of slc24a46 expression in zebrafish embryos caused brain malformation, spinal motor neuron loss, and poor motility. At the cellular level, we observed abnormally elongated mitochondria, which was rescued by co-injection of the wild-type but not the mutant slc25a46 mRNA. Conversely, overexpression of the wild-type protein led to mitochondrial fragmentation and disruption of the mitochondrial network. In contrast to mutations causing non-lethal optic atrophy, missense mutations causing lethal congenital pontocerebellar hypoplasia markedly destabilize the protein. Indeed, the clinical severity appears inversely correlated with the relative stability of the mutant protein. This genotype-phenotype correlation underscores the importance of SLC25A46 and fine tuning of mitochondrial fission and fusion in pontocerebellar hypoplasia and central neurodevelopment in addition to optic and peripheral neuropathy across the life span.
The mitochondrial fission protein Drp1 was proposed to promote NAFLD, as inhibition of hepatocyte Drp1 early in life prevents liver steatosis induced by high-fat diet in mice. However, whether ...Drp1-knockdown in older mice can reverse established NASH is unknown.
N-acetylgalactosamine-siRNA conjugates, an FDA approved method to deliver siRNA selectively to hepatocytes, were used to knockdown hepatocyte-Drp1 in mice (NAG-Drp1si). NASH was induced in C57BL/6NTac mice by Gubra-Amylin-NASH diet (D09100310, 40% fat, 22% fructose and 2% cholesterol) and treatment with NAG-Drp1si was started at week 24 of diet. Circulating transaminases, liver histology, gene expression of fibrosis and inflammation markers, and hydroxyproline synthesis determined NASH severity. Liver NEFA and triglycerides were quantified by GC/MS. Mitochondrial function was determined by respirometry. Western blots of Oma1, Opa1, p-eIf2α, as well as transcriptional analyses of Atf4-regulated genes determined ISR engagement.
NAG-Drp1si treatment decreased body weight and induced liver inflammation in adult healthy mice. Increased hepatic Gdf15 production was the major contributor to body-weight loss caused by NAG-Drp1si treatment, as Gdf15 receptor deletion (Gfral KO) prevented the decrease in food intake and mitigated weight loss. NAG-Drp1si activated the Atf4-controlled integrated stress response (ISR) to increase hepatic Gdf15 expression. NAG-Drp1si in healthy mice caused ER stress and activated the mitochondrial protease Oma1, which are the ER and mitochondrial triggers that activate the Atf4-controlled ISR. Remarkably, induction of NASH was not sufficient to activate Oma1 in liver. However, NAG-Drp1si treatment was sufficient to activate Oma1 in adult mice with NASH, as well as exacerbating NASH-induced ER stress. Consequently, NAG-Drp1si treatment in mice with NASH led to higher ISR activation, exacerbated inflammation, fibrosis and necrosis.
Drp1 mitigates NASH by decreasing ER stress, preventing Oma1 activation and ISR exacerbation. The elevation in Gdf15 actions induced by NAG-Drp1si might represent an adaptive response decreasing the nutrient load to liver when mitochondria are misfunctional. Our study argues against blocking Drp1 in hepatocytes to combat NASH.
•The FDA-approved NAG-siRNA conjugates were used to knock down Drp1 expression in hepatocytes from older mice.•Drp1 knock down in control mice results in liver damage, decreasing food intake via Gdf15 actions.•Drp1 knock down decreases respiration and elevates intrahepatic NEFA, activating Oma1 and the mitochondrial ISR.•IISR, fibrosis, inflammation and hepatocyte cell death are exacerbated by Drp1 knockdown in mice with established NASH.
It is well established that mutual relations exist between the oxidative status of a cell and the ubiquitin proteasome system (UPS). Oxidation of proteins leads to misfolding and thus claims a higher ...proteolytic activity of the UPS to dispose these proteins. Any disturbance of the proteotoxic-protection machinery may result in protein aggregation and thus in disorders like neurodegenerative diseases or cancer. In this article we describe the molecular mechanisms regulating the ubiquitin proteasome system, thereby focussing on Nrf2 as well as on the recently identified transcription factor (TF) TCF11. In response to proteasome inhibition TCF11 plays a central role in upregulating the proteasome system via an ERAD-dependent feedback loop.
The quality control of proteins mediated by the plasticity of the proteasome system is regulated by the timely and flexible formation of this multisubunit proteolytic enzyme complex. Adaptable ...biogenesis of the 20S proteasome core complex is therefore of vital importance for adjusting to changing proteolytic requirements. However, the molecular mechanism and the cellular sites of mammalian proteasome formation are still unresolved. By using precursor complex‐specific antibodies, we now show that the main steps in 20S core complex formation take place at the endoplasmic reticulum (ER). Thereby, the proteasome maturation protein (POMP)—an essential factor of mammalian proteasome biogenesis—interacts with ER membranes, binds to α1–7 rings, recruits β‐subunits stepwise and mediates the association of mammalian precursor complexes with the ER. Thus, POMP facilitates the main steps in 20S core complex formation at the ER to coordinate the assembly process and to provide cells with freshly formed proteasomes at their site of function.
The formin-like protein INF2 is an important player in the polymerization of actin filaments. In this issue, Chakrabarti et al. (2018.
https://doi.org/10.1083/jcb.201709111) demonstrate that INF2 ...mediates actin polymerization at the endoplasmic reticulum (ER), resulting in increased ER-mitochondria contacts, calcium uptake by mitochondria, and mitochondrial division.
Interferon (IFN)-induced immunoproteasomes (i-proteasomes) have been associated with improved processing of major histocompatibility complex (MHC) class I antigens. Here, we show that i-proteasomes ...function to protect cell viability under conditions of IFN-induced oxidative stress. IFNs trigger the production of reactive oxygen species, which induce protein oxidation and the formation of nascent, oxidant-damaged proteins. We find that the ubiquitylation machinery is concomitantly upregulated in response to IFNs, functioning to target defective ribosomal products (DRiPs) for degradation by i-proteasomes. i-proteasome-deficiency in cells and in murine inflammation models results in the formation of aggresome-like induced structures and increased sensitivity to apoptosis. Efficient clearance of these aggregates by the enhanced proteolytic activity of the i-proteasome is important for the preservation of cell viability upon IFN-induced oxidative stress. Our findings suggest that rather than having a specific role in the production of class I antigens, i-proteasomes increase the peptide supply for antigen presentation as part of a more general role in the maintenance of protein homeostasis.
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► IFNs trigger ROS production and the formation of oxidant damaged proteins ► i-proteosome-deficient cells accumulate oxidant-damaged proteins ► Enhanced proteolytic activity of the i-proteasome results in efficient clearance of aggregates ► i-proteasomes prevent cellular damage and apoptosis in murine inflammation models
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