FAT10, a ubiquitin-independent signal for proteasomal degradation

FAT10 is a small ubiquitin-like modifier that is encoded in the major histocompatibility complex and is synergistically inducible by tumor necrosis factor alpha and gamma interferon. It is composed of two ubiquitin-like domains and possesses a free C-terminal diglycine motif that is required for the formation of FAT10 conjugates. Here we show that unconjugated FAT10 and a FAT10 conjugate were rapidly degraded by the proteasome at a similar rate. Fusion of FAT10 to the N terminus of very long-lived proteins enhanced their degradation rate as potently as fusion with ubiquitin did. FAT10-green fluorescent protein fusion proteins were not cleaved but entirely degraded, suggesting that FAT10-specific deconjugating enzymes were not present in the analyzed cell lines. Interestingly, the prevention of ubiquitylation of FAT10 by mutation of all lysines or by expression in ubiquitylation-deficient cells did not affect FAT10 degradation. Thus, conjugation with FAT10 is an alternative and ubiquitin-independent targeting mechanism for degradation by the proteasome, which, in contrast to polyubiquitylation, is cytokine inducible and irreversible.     

The ubiquitin-like modifier FAT10 in cancer development

During the last years it has emerged that the ubiquitin-like modifier FAT10 is directly involved in cancer development. FAT10 expression is highly up-regulated by pro-inflammatory cytokines IFN-γ and TNF-α in all cell types and tissues and it was also found to be up-regulated in many cancer types such as glioma, colorectal, liver or gastric cancer. While pro-inflammatory cytokines within the tumor microenvironment probably contribute to FAT10 overexpression, an increasing body of evidence argues that pro-malignant capacities of FAT10 itself largely underlie its broad and intense overexpression in tumor tissues. FAT10 thereby regulates pathways involved in cancer development such as the NF-κB- or Wnt-signaling. Moreover, FAT10 directly interacts with and influences downstream targets such as MAD2, p53 or β-catenin, leading to enhanced survival, proliferation, invasion and metastasis formation of cancer cells but also of non-malignant cells. In this review we will provide an overview of the regulation of FAT10 expression as well as its function in carcinogenesis.     

FAT10 protein binds to polyglutamine proteins and modulates their solubility

Expansion of polyglutamine (pQ) chain by expanded CAG repeat causes dominantly inherited neurodegeneration such as Huntington disease, dentatorubral-pallidoluysian atrophy (DRPLA), and numbers of other spinocerebellar ataxias. Expanded pQ disrupts the stability of the pQ-harboring protein and increases its susceptibility to aggregation. Aggregated pQ protein is recognized by the ubiquitin proteasome system, and the enzyme ubiquitin ligase covalently attaches ubiquitin, which serves as a degradation signal by the proteasome. However, accumulation of the aggregated proteins in the diseased brain suggests insufficient degradation machinery. Ubiquitin has several functionally related proteins that are similarly attached to target proteins through its C terminus glycine residue. They are called ubiquitin-like molecules, and some of them are similarly related to the protein degradation pathway. One of the ubiquitin-like molecules, FAT10, is known to accelerate protein degradation through a ubiquitin-independent manner, but its role in pQ aggregate degradation is completely unknown. Thus we investigated its role in a Huntington disease cellular model and found that FAT10 molecules were covalently attached to huntingtin through their C terminus glycine. FAT10 binds preferably to huntingtin with a short pQ chain and completely aggregated huntingtin was FAT10-negative. In addition, ataxin-1,3 and DRPLA proteins were both positive for FAT10, and aggregation enhancement was observed upon FAT10 knockdown. These findings were similar to those for huntingtin. Our new finding will provide a new role for FAT10 in the pathogenesis of polyglutamine diseases.     

Pupylation as a signal for proteasomal degradation in bacteria

Posttranslational modifications in the form of covalently attached proteins like ubiquitin (Ub), were long considered an exclusive feature of eukaryotic organisms. The discovery of pupylation, the modification of lysine residues with a prokaryotic, ubiquitin-like protein (Pup), demonstrated that certain bacteria use a tagging pathway functionally related to ubiquitination in order to target proteins for proteasomal degradation. However, functional analogies do not translate into structural or mechanistic relatedness. Bacterial Pup, unlike eukaryotic Ub, does not adopt a β-grasp fold, but is intrinsically disordered. Furthermore, isopeptide bond formation in the pupylation process is carried out by enzymes evolutionary descendent from glutamine synthetases. While in eukaryotes, the proteasome is the main energy-dependent protein degradation machine, bacterial proteasomes exist in addition to other architecturally related degradation complexes, and their specific role along with the role of pupylation is still poorly understood. In Mycobacterium tuberculosis (Mtb), the Pup–proteasome system contributes to pathogenicity by supporting the bacterium's persistence within host macrophages. Here, we describe the mechanism and structural framework of pupylation and the targeting of pupylated proteins to the proteasome complex. Particular attention is given to the comparison of the bacterial Pup–proteasome system and the eukaryotic ubiquitin–proteasome system. Furthermore, the involvement of pupylation and proteasomal degradation in Mtb pathogenesis is discussed together with efforts to establish the Pup–proteasome system as a drug target. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

Ubiquitin is degraded by the ubiquitin system as a monomer and as part of its conjugated target

An important problem concerning regulation of the ubiquitin-proteasome system (UPS) relates to the stability of its own components and the mechanisms of their degradation. It has been demonstrated that monomeric ubiquitin is relatively stable and is probably degraded by the proteasome. It has also been shown that it is destabilized following inactivation of deubiquitinating enzymes, suggesting that failure to release it, results in its concomitant degradation along with its target. Here, we demonstrate that conjugation of monomeric ubiquitin requires both its internal lysines and N-terminal residue. Interestingly however, the degradation of the monomeric species requires also a short C-terminal extension, implying that unlike conjugation, entry into the proteasomal chamber requires a tail that can be generated in the cell via several distinct mechanisms. We further show that accelerated intracellular degradation induced by stress results in depletion of ubiquitin, supporting the notion that ubiquitin is also degraded as part of the chain conjugated to its target substrate.     

Interaction of NUB1 with the proteasome subunit S5a

NUB1 interacts with a ubiquitin-like protein NEDD8 to target the NEDD8 monomer and neddylated proteins to the proteasome for degradation. Therefore, NUB1 is thought to be a potent downregulator of NEDD8 conjugation system. Since NUB1 possesses a UBL domain, which was previously shown to be an S5a-interacting motif in RAD23/HHR23, we initially hypothesized that NUB1 interacts with the S5a subunit of the proteasome through its UBL domain. To examine this, we performed an in vitro GST pull-down assay and a yeast two-hybrid assay. Unexpectedly, our studies revealed that NUB1 directly interacts with the S5a subunit through its C-terminal region between amino acid residues 536 and 584, not through its UBL domain. Although the UBL domain was not an S5a-interacting motif in NUB1, our further studies revealed that the UBL domain is required for the function of NUB1.     

Proteostasis and neurodegeneration: The roles of proteasomal degradation and autophagy

All proteins in a cell continuously turn over, each at its own rate, contributing to a cell's development, differentiation, or aging. Of course, unnecessary protein(s), or those synthesized in excess, that hamper cellular homeostasis should be discarded rapidly. Furthermore, cells that have been subjected to various environmental stresses, e.g., reactive oxygen species (ROS) and UV irradiation, may incur various types of protein damage, which vitiate normal and homeostatic functions in the cell. Thereby, the prompt elimination of impaired proteins is essential for cell viability. This housekeeping is accomplished by two major catabolic routes—proteasomal digestion and autophagy. Strict maintenance of proteostasis is particularly important in non-proliferative cells, especially neurons, and it is plausible that its failure leads to a number of the neurodegenerative diseases becoming prominent in the growing elderly population. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

Identification of a polymorphism in the RING finger of human Bmi-1 that causes its degradation by the ubiquitin-proteasome system

Bmi-1 is a polycomb protein that plays an important role in tumor cell development and maintaining stem cell populations of many cell lineages. Here we identify a polymorphism in human Bmi-1 that changes a cysteine within its RING domain to tyrosine. This C18Y polymorphism is associated with a significant decrease in Bmi-1 level and its elevated ubiquitination, suggesting that it is being destroyed by the ubiquitin-proteasome system. Consistent with this, treating cells with the proteasome inhibitor MG-132 significantly increases C18Y Bmi-1 levels. This is the first example of a polymorphism in Bmi-1 that reduces levels of this important protein.

Structured summary

MINT-6948574: Bmi-1 (uniprotkb:P35226) physically interacts (MI:0218) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)     

Ubiquitin-independent proteasomal degradation of endoplasmic reticulum-localized connexin43 mediated by CIP75

Connexin43 (Cx43) is a transmembrane protein that forms gap junction channels. Regulation of Cx43 turnover is one mechanism to control the level of intercellular communication that occurs through gap junction channels. Proteasomal degradation of Cx43 is regulated in part through CIP75, a ubiquitin-like and ubiquitin-associated domain containing protein. CIP75 interacts with endoplasmic reticulum-localized Cx43, as demonstrated through co-immunoprecipitation and immunofluorescence microscopy experiments. CIP75 also binds to free monoubiquitin and lysine 48-linked tetraubiquitin chains in vitro and binds to ubiquitinated proteins in cellular lysates. However, analysis of Cx43 that immunoprecipitated with CIP75 demonstrated that the Cx43 associated with CIP75 was not ubiquitinated, and a mutant form of Cx43 that lacked lysines capable of ubiquitination retained the capacity to interact with CIP75. These results suggest that although CIP75 can interact with ubiquitinated cellular proteins, its interaction with Cx43 and stimulation of Cx43 proteasomal degradation does not require the ubiquitination of Cx43.     

Evidence for proteasomal degradation of Kv1.5 channel protein

BACKGROUND: The voltage-gated potassium channel Kv1.5 plays a critical role in the maintenance of the membrane potential. While protein degradation is one of the major mechanisms for the regulation of channel functions, little is known on the degradation mechanism of Kv1.5. METHODS AND RESULTS: Kv1.5 was expressed in COS cells and its degradation, intracellular localization, and channel activities were assessed by pulse-chase analysis, immunofluorescence, and patch clamp techniques, respectively. Expressed Kv1.5 had a half-life time of approximately 6.7 h, which was prolonged by the proteasome inhibitors of MG132, ALLN, proteasomal inhibitor 1, or lactacystine, but not by a lysosomal inhibitor chloroquine. MG132 increased the protein level of Kv1.5, as well as the level of its ubiquitinated form in a dose-dependent manner. Similar effects of MG132 on endogenous Kv1.5 were seen in cultured rat atrial cells. Within a cell, Kv1.5 was mainly localized in both the endoplasmic reticulum and Golgi apparatus. MG132 increased the immunoreactivity of Kv1.5 in these compartments and also increased Ik(ur) currents through the cell-surface Kv1.5. Pretreatment with either brefeldin A or colchicine abolished MG132-induced increase in Ik(ur) currents. CONCLUSION: Kv1.5 is degraded by the proteasome. The inhibition of the proteasome increased Ik(ur) currents secondary to stabilization of the channel protein in the endoplasmic reticulum/Golgi apparatus.     

Lipid droplet formation is dispensable for endoplasmic reticulum-associated degradation

Proteins that fail to fold or assemble in the endoplasmic reticulum (ER) are destroyed by cytoplasmic proteasomes through a process known as ER-associated degradation. Substrates of this pathway are initially sequestered within the ER lumen and must therefore be dislocated across the ER membrane to be degraded. It has been proposed that generation of bicellar structures during lipid droplet formation may provide an "escape hatch" through which misfolded proteins, toxins, and viruses can exit the ER. We have directly tested this hypothesis by exploiting yeast strains defective in lipid droplet formation. Our data demonstrate that lipid droplet formation is dispensable for the dislocation of a plant toxin and the degradation of both soluble and integral membrane glycoproteins.     

Regulation of LC3B levels by ubiquitination and proteasomal degradation

ABSTRACT

Like other biological processes, macroautophagy/autophagy must be tightly controlled for maintenance of cellular homeostasis and for proper response to changing cellular conditions. To gain insights into the regulation of autophagy, we recently conducted a genome-wide CRISPR-Cas9 knockout screen using cells expressing endogenous LC3B tagged with GFP-mCherry as a reporter. This approach allowed us to identify the ubiquitin-activating enzyme UBA6 and the hybrid ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 as novel autophagy regulators. We found that these enzymes cooperate to mediate monoubiquitination and proteasomal degradation of LC3B, thus limiting the pool of LC3B available for autophagy. Depletion of UBA6 or BIRC6 increased the level of cytosolic LC3B, enhancing the degradation of autophagy adaptors and the clearance of intracellular proteins aggregates. This finding could be the basis for the development of pharmacological inhibitors of UBA6 or BIRC6 for the treatment of protein aggregation disorders. Recent work by another group showed that BIRC6 itself is subject to ubiquitination and proteasomal degradation, highlighting the existence of a complex regulatory network for the control of LC3B levels.