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1.
PLoS One ; 18(8): e0290002, 2023.
Article in English | MEDLINE | ID: mdl-37578983

ABSTRACT

The ubiquitin-like modifier FAT10 is highly upregulated under inflammatory conditions and targets its conjugation substrates to the degradation by the 26S proteasome. This process termed FAT10ylation is mediated by an enzymatic cascade and includes the E1 activating enzyme ubiquitin-like modifier activating enzyme 6 (UBA6), the E2 conjugating enzyme UBA6-specific E2 enzyme 1 (USE1) and E3 ligases, such as Parkin. In this study, the function of the HECT-type ubiquitin E3 ligase HUWE1 was investigated as a putative E3 ligase and/or conjugation substrate of FAT10. Our data provide strong evidence that HUWE1 is FAT10ylated in a UBA6 and FAT10 diglycine-dependent manner in vitro and in cellulo and that the HUWE1-FAT10 conjugate is targeted to proteasomal degradation. Since the mutation of all relevant cysteine residues within the HUWE1 HECT domain did not abolish FAT10 conjugation, a role of HUWE1 as E3 ligase for FAT10ylation is rather unlikely. Moreover, we have identified the autophagy-related protein AMBRA1 as a new FAT10 interaction partner. We show that the HUWE1-FAT10 conjugate formation is diminished in presence of AMBRA1, while the interaction between AMBRA1 and HUWE1 is strengthened in presence of FAT10. This implies a putative interplay of all three proteins in cellular processes such as mitophagy.


Subject(s)
Ubiquitin , Ubiquitins , Ubiquitin/metabolism , Ubiquitins/genetics , Ubiquitins/metabolism , Ubiquitin-Protein Ligases/genetics
2.
Life Sci Alliance ; 6(11)2023 11.
Article in English | MEDLINE | ID: mdl-37604583

ABSTRACT

The ubiquitin-like modifier FAT10 is up-regulated in many different cell types by IFNγ and TNFα (TNF) and directly targets proteins for proteasomal degradation. FAT10 gets covalently conjugated to its conjugation substrates by the E1 activating enzyme UBA6, the E2 conjugating enzyme USE1, and E3 ligases including Parkin. To date, USE1 was supposed to be the only E2 enzyme for FAT10ylation, and we show here that a knockout of USE1 strongly diminished FAT10 conjugation. Remarkably, under inflammatory conditions in the presence of TNF, FAT10 conjugation appears to be independent of USE1. We report on the identification of additional E2 conjugating enzymes, which were previously not associated with FAT10. We confirm their capacity to be charged with FAT10 onto their active site cysteine, and to rescue FAT10 conjugation in the absence of USE1. This finding strongly widens the field of FAT10 research by pointing to multiple, so far unknown pathways for the conjugation of FAT10, disclosing novel possibilities for pharmacological interventions to regulate FAT10 conjugation under inflammatory conditions and/or viral infections.


Subject(s)
Inflammation , Tumor Necrosis Factor-alpha , Ubiquitin , Cysteine , Ubiquitin/metabolism , Humans , Inflammation/metabolism
3.
Life Sci Alliance ; 6(8)2023 08.
Article in English | MEDLINE | ID: mdl-37188463

ABSTRACT

The interaction of the 19S regulatory particle of the 26S proteasome with ubiquitylated proteins leads to gate opening of the 20S core particle and increases its proteolytic activity by binding of the ubiquitin chain to the inhibitory deubiquitylation enzyme USP14 on the 19S regulatory subunit RPN1. Covalent modification of proteins with the cytokine inducible ubiquitin-like modifier FAT10 is an alternative signal for proteasomal degradation. Here, we report that FAT10 and its interaction partner NUB1L facilitate the gate opening of the 20S proteasome in an ubiquitin- and USP14-independent manner. We also show that FAT10 is capable to activate all peptidolytic activities of the 26S proteasome, however only together with NUB1L, by binding to the UBA domains of NUB1L and thereby interfering with NUB1L dimerization. The binding of FAT10 to NUB1L leads to an increased affinity of NUB1L for the subunit RPN1. In conclusion, the herein described cooperation of FAT10 and NUB1L is a substrate-induced mechanism to activate the 26S proteasome.


Subject(s)
Proteasome Endopeptidase Complex , Ubiquitins , Humans , Mice , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Ubiquitin/metabolism , Ubiquitins/genetics , Ubiquitins/metabolism , Animals
4.
Cell Rep ; 34(11): 108857, 2021 03 16.
Article in English | MEDLINE | ID: mdl-33730565

ABSTRACT

Parkin is an E3 ubiquitin ligase belonging to the RING-between-RING family. Mutations in the Parkin-encoding gene PARK2 are associated with familial Parkinson's disease. Here, we investigate the interplay between Parkin and the inflammatory cytokine-induced ubiquitin-like modifier FAT10. FAT10 targets hundreds of proteins for degradation by the 26S proteasome. We show that FAT10 gets conjugated to Parkin and mediates its degradation in a proteasome-dependent manner. Parkin binds to the E2 enzyme of FAT10 (USE1), auto-FAT10ylates itself, and facilitates FAT10ylation of the Parkin substrate Mitofusin2 in vitro and in cells, thus identifying Parkin as a FAT10 E3 ligase. On mitochondrial depolarization, FAT10ylation of Parkin inhibits its activation and ubiquitin-ligase activity causing impairment of mitophagy progression and aggravation of rotenone-mediated death of dopaminergic neuronal cells. In conclusion, FAT10ylation inhibits Parkin and mitophagy rendering FAT10 a likely inflammation-induced exacerbating factor and potential drug target for Parkinson's disease.


Subject(s)
Mitophagy , Ubiquitin-Protein Ligases/metabolism , Ubiquitins/metabolism , Cell Death , Cytosol/metabolism , GTP Phosphohydrolases/metabolism , HEK293 Cells , HeLa Cells , Humans , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Neurons/metabolism , Neurons/pathology , Proteasome Endopeptidase Complex/metabolism , Protein Transport , Proteolysis , Reactive Oxygen Species/metabolism , Substrate Specificity , Ubiquitination
5.
J Biol Chem ; 295(42): 14402-14418, 2020 10 16.
Article in English | MEDLINE | ID: mdl-32817338

ABSTRACT

The retina-specific chaperone aryl hydrocarbon interacting protein-like 1 (AIPL1) is essential for the correct assembly of phosphodiesterase 6 (PDE6), which is a pivotal effector enzyme for phototransduction and vision because it hydrolyzes cGMP. AIPL1 interacts with the cytokine-inducible ubiquitin-like modifier FAT10, which gets covalently conjugated to hundreds of proteins and targets its conjugation substrates for proteasomal degradation, but whether FAT10 affects PDE6 function or turnover is unknown. Here, we show that FAT10 mRNA is expressed in human retina and identify rod PDE6 as a retina-specific substrate of FAT10 conjugation. We found that AIPL1 stabilizes the FAT10 monomer and the PDE6-FAT10 conjugate. Additionally, we elucidated the functional consequences of PDE6 FAT10ylation. On the one hand, we demonstrate that FAT10 targets PDE6 for proteasomal degradation by formation of a covalent isopeptide linkage. On the other hand, FAT10 inhibits PDE6 cGMP hydrolyzing activity by noncovalently interacting with the PDE6 GAFa and catalytic domains. Therefore, FAT10 may contribute to loss of PDE6 and, as a consequence, degeneration of retinal cells in eye diseases linked to inflammation and inherited blindness-causing mutations in AIPL1.


Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Proteasome Endopeptidase Complex/metabolism , Retina/metabolism , Ubiquitins/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Motifs , Animals , Catalytic Domain , Cyclic GMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 6/chemistry , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mutagenesis, Site-Directed , Proteasome Endopeptidase Complex/chemistry , Proteasome Inhibitors/pharmacology , Protein Binding , Proteolysis/drug effects , RNA, Messenger/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Ubiquitin/metabolism , Ubiquitins/chemistry , Ubiquitins/genetics
6.
Nat Commun ; 10(1): 4452, 2019 10 01.
Article in English | MEDLINE | ID: mdl-31575873

ABSTRACT

The covalent attachment of the cytokine-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) to hundreds of substrate proteins leads to their rapid degradation by the 26 S proteasome independently of ubiquitylation. Here, we identify another function of FAT10, showing that it interferes with the activation of SUMO1/2/3 in vitro and down-regulates SUMO conjugation and the SUMO-dependent formation of promyelocytic leukemia protein (PML) bodies in cells. Mechanistically, we show that FAT10 directly binds to and impedes the activity of the heterodimeric SUMO E1 activating enzyme AOS1/UBA2 by competing very efficiently with SUMO for activation and thioester formation. Nevertheless, activation of FAT10 by AOS1/UBA2 does not lead to covalent conjugation of FAT10 with substrate proteins which relies on its cognate E1 enzyme UBA6. Hence, we report that one ubiquitin-like modifier (FAT10) inhibits the conjugation and function of another ubiquitin-like modifier (SUMO) by impairing its activation.


Subject(s)
Promyelocytic Leukemia Protein/metabolism , Protein Processing, Post-Translational/physiology , SUMO-1 Protein/metabolism , Ubiquitins/metabolism , Down-Regulation , Gene Knockdown Techniques , HEK293 Cells , Humans , Proteasome Endopeptidase Complex/metabolism , Recombinant Proteins , Small Ubiquitin-Related Modifier Proteins/metabolism , Transcription Factors/metabolism , Ubiquitin-Activating Enzymes/genetics , Ubiquitin-Activating Enzymes/metabolism , Ubiquitination , Ubiquitins/genetics
7.
Methods Enzymol ; 618: 229-256, 2019.
Article in English | MEDLINE | ID: mdl-30850054

ABSTRACT

The ubiquitin-like modifier FAT10 (also called ubiquitin D (UBD)) interacts noncovalently with a substantial number of proteins and also gets covalently conjugated to many substrate proteins, leading to their degradation by the 26S proteasome. FAT10 comprises two loosely folded ubiquitin-like domains that are connected by a flexible linker, and this unusual structure makes it highly prone to aggregation. Here, we report methods to purify high amounts of soluble recombinant FAT10 for various uses, such as in vitro FAT10ylation assays. In addition, we describe how to generate and handle overexpressed as well as endogenous FAT10 in cellulo for use in immunoprecipitations, Western blot analyses, and FAT10 degradation studies.


Subject(s)
Ubiquitins/metabolism , Blotting, Western , Cell Line , Gene Expression , Humans , Immunoprecipitation , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Transfection/methods , Ubiquitins/genetics , Ubiquitins/isolation & purification , Up-Regulation
8.
J Biol Chem ; 294(12): 4315-4330, 2019 03 22.
Article in English | MEDLINE | ID: mdl-30718280

ABSTRACT

The deubiquitylation of target proteins is mediated by deubiquitylating enzymes (DUB) such as OTUB1, which plays an important role in immune response, cell cycle progression, and DNA repair. Within these processes, OTUB1 reduces the ubiquitylation of target proteins in two distinct ways, either by using its catalytic DUB activity or in a noncatalytic manner by inhibiting the E2-conjugating enzyme. Here, we show that the ubiquitin-like modifier FAT10 regulates OTUB1 stability and functionality in different ways. Covalent FAT10ylation of OTUB1 resulted in its proteasomal degradation, whereas a noncovalent interaction stabilized OTUB1. We provide evidence that OTUB1 interacts directly with FAT10 and the E2-conjugating enzyme USE1. This interaction strongly stimulated OTUB1 DUB activity toward Lys-48-linked diubiquitin. Furthermore, the noncovalent interaction between FAT10 and OTUB1 not only enhanced its isopeptidase activity toward Lys-48-linked ubiquitin moieties but also strengthened its noncatalytic activity in reducing Lys-63 polyubiquitylation of its target protein TRAF3 (TNF receptor-associated factor 3). Additionally, the cellular clearance of overall polyubiquitylation by OTUB1 was strongly stimulated through the presence of FAT10. The addition of FAT10 also led to an increased interaction between OTUB1 and its cognate E2 UbcH5B, implying a function of FAT10 in the inhibition of polyubiquitylation. Overall, these data indicate that FAT10 not only plays a role in covalent modification, leading its substrates to proteasomal degradation, but also regulates the stability and functionality of target proteins by interacting in a noncovalent manner. FAT10 is thereby able to exert a major influence on ubiquitylation processes.


Subject(s)
Cysteine Endopeptidases/metabolism , Ubiquitin/metabolism , Ubiquitins/metabolism , Deubiquitinating Enzymes , HEK293 Cells , Humans , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Proteolysis , SNARE Proteins , Ubiquitination , Vesicular Transport Proteins
9.
Nat Commun ; 9(1): 4646, 2018 11 02.
Article in English | MEDLINE | ID: mdl-30389928

ABSTRACT

The original version of the Supplementary Information associated with this Article inadvertently omitted Supplementary Table 3. The HTML version of the Article has been updated to include a corrected version of the Supplementary Information.

10.
Nat Commun ; 9(1): 3321, 2018 08 20.
Article in English | MEDLINE | ID: mdl-30127417

ABSTRACT

FAT10 is a ubiquitin-like modifier that directly targets proteins for proteasomal degradation. Here, we report the high-resolution structures of the two individual ubiquitin-like domains (UBD) of FAT10 that are joined by a flexible linker. While the UBDs of FAT10 show the typical ubiquitin-fold, their surfaces are entirely different from each other and from ubiquitin explaining their unique binding specificities. Deletion of the linker abrogates FAT10-conjugation while its mutation blocks auto-FAT10ylation of the FAT10-conjugating enzyme USE1 but not bulk conjugate formation. FAT10- but not ubiquitin-mediated degradation is independent of the segregase VCP/p97 in the presence but not the absence of FAT10's unstructured N-terminal heptapeptide. Stabilization of the FAT10 UBDs strongly decelerates degradation suggesting that the intrinsic instability of FAT10 together with its disordered N-terminus enables the rapid, joint degradation of FAT10 and its substrates without the need for FAT10 de-conjugation and partial substrate unfolding.


Subject(s)
Proteasome Endopeptidase Complex/metabolism , Proteolysis , Ubiquitins/metabolism , Amino Acid Sequence , Amino Acid Substitution , Cysteine , HEK293 Cells , HeLa Cells , Humans , Models, Molecular , Protein Domains , Protein Stability , Ubiquitin/chemistry , Ubiquitins/chemistry , Valosin Containing Protein/metabolism
11.
FEBS J ; 281(7): 1848-59, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24528925

ABSTRACT

The cytokine-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) targets its substrates for degradation by the proteasome. FAT10 is conjugated to its substrates via the bispecific, ubiquitin-activating and FAT10-activating enzyme UBA6, the likewise bispecific conjugating enzyme UBA6-specific E2 enzyme 1 (USE1), and possibly E3 ligases. By MS analysis, we found that USE1 undergoes self-FAT10ylation in cis, mainly at Lys323. Mutation of Lys323 to an arginine did not abolish auto-FAT10ylation of USE1, but every other lysine could instead be modified with FAT10. Similarly to bulk FAT10 substrates, FAT10ylation of USE1 accelerated its proteasomal degradation. Interestingly, the USE1-FAT10 conjugate continued to be an active E2 enzyme, because both FAT10 and ubiquitin could still be thioester-linked to the USE1-FAT10 conjugate. We therefore suggest that the major function of USE1 auto-FAT10ylation is to serve as a negative feedback mechanism to limit the conjugation of FAT10 upon its cytokine-mediated induction by reducing the amount of USE1 through proteasomal degradation of the USE1-FAT10 conjugate. STRUCTURED DIGITAL ABSTRACT: •USE1 physically interacts with FAT10 by anti bait coimmunoprecipitation (View interaction) •USE1 physically interacts with FAT10 by anti tag coimmunoprecipitation (View interaction).


Subject(s)
Ubiquitination , Ubiquitins/metabolism , Feedback, Physiological , HEK293 Cells , Humans , Mutation , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Proteolysis , SNARE Proteins , Ubiquitins/genetics , Vesicular Transport Proteins
12.
J Cell Sci ; 125(Pt 19): 4576-85, 2012 Oct 01.
Article in English | MEDLINE | ID: mdl-22797925

ABSTRACT

FAT10 is a ubiquitin-like modifier proposed to function in apoptosis induction, cell cycle control and NF-κB activation. Upon induction by pro-inflammatory cytokines, hundreds of endogenous substrates become covalently conjugated to FAT10 leading to their proteasomal degradation. Nevertheless, only three substrates have been identified so far to which FAT10 becomes covalently attached through a non-reducible isopeptide bond, and these are the FAT10-conjugating enzyme USE1 which auto-FAT10ylates itself in cis, the tumor suppressor p53 and the ubiquitin-activating enzyme UBE1 (UBA1). To identify additional FAT10 substrates and interaction partners, we used a new monoclonal FAT10-specific antibody to immunopurify endogenous FAT10 conjugates from interferon (IFN)γ-and tumor necrosis factor (TNF)α-stimulated cells for identification by mass spectrometry. In addition to two already known FAT10-interacting proteins, histone deacetylase 6 and UBA6, we identified 569 novel FAT10-interacting proteins involved in different functional pathways such as autophagy, cell cycle regulation, apoptosis and cancer. Thirty-one percent of all identified proteins were categorized as putative covalently linked substrates. One of the identified proteins, the autophagosomal receptor p62/SQSTM1, was further investigated. p62 becomes covalently mono-FAT10ylated at several lysines, and FAT10 colocalizes with p62 in p62 bodies. Strikingly, FAT10ylation of p62 leads to its proteasomal degradation, and prolonged induction of endogenous FAT10 expression by pro-inflammatory cytokines leads to a decrease of endogenous p62. The elucidation of the FAT10 degradome should enable a better understanding of why FAT10 has evolved as an additional transferable tag for proteasomal degradation.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Proteomics , Ubiquitins/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Amino Acid Motifs , Autophagy/drug effects , HEK293 Cells , HeLa Cells , Humans , Immunoprecipitation , Interferon-gamma/pharmacology , Lysine/metabolism , Mass Spectrometry , Protein Binding/drug effects , Protein Structure, Tertiary , Protein Transport/drug effects , Sequestosome-1 Protein , Substrate Specificity/drug effects , Tumor Necrosis Factor-alpha/pharmacology
13.
Int J Cancer ; 128(12): 2803-14, 2011 Jun 15.
Article in English | MEDLINE | ID: mdl-20726000

ABSTRACT

Cytokines play a crucial role in tumor initiation and progression. Here, we demonstrate that interleukin (IL)-6 is a key factor by driving tumor progression from benign to malignant, invasive tumors in the HaCaT-model of human skin carcinoma. IL-6 activates STAT3 and directly stimulates proliferation and migration of the benign noninvasive HaCaT-ras A-5 cells in vitro. Furthermore, IL-6 induces a complex, reciprocally regulated cytokine network in the tumor cells that includes inflammatory and angiogenic factors such as IL-8, GM-CSF, VEGF and MCP-1. These IL-6 effects lead to tumor cell invasion in organotypic cultures in vitro and to the formation of malignant and invasive s.c. tumors in vivo. Tumor invasion is supported by the IL-6 induced overexpression of MMP-1 in vitro and in vivo. These data demonstrate a key function of IL-6 in the progression of skin SCCs by regulating a complex cytokine and protease network and suggest new therapeutic approaches to target this central player in skin carcinogenesis.


Subject(s)
Carcinoma, Squamous Cell/pathology , Cytokines/physiology , Interleukin-6/physiology , Skin Neoplasms/pathology , Base Sequence , Blotting, Western , Cell Proliferation , DNA Primers , Disease Progression , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique, Indirect , Humans , In Situ Hybridization , Neoplasm Invasiveness , Neoplasm Metastasis , STAT3 Transcription Factor/antagonists & inhibitors
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