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Science (New York, N.Y.) Aug 2023Cells use ubiquitin to mark proteins for proteasomal degradation. Although the proteasome also eliminates proteins that are not ubiquitinated, how this occurs...
Cells use ubiquitin to mark proteins for proteasomal degradation. Although the proteasome also eliminates proteins that are not ubiquitinated, how this occurs mechanistically is unclear. Here, we found that midnolin promoted the destruction of many nuclear proteins, including transcription factors encoded by the immediate-early genes. Diverse stimuli induced midnolin, and its overexpression was sufficient to cause the degradation of its targets by a mechanism that did not require ubiquitination. Instead, midnolin associated with the proteasome via an α helix, used its Catch domain to bind a region within substrates that can form a β strand, and used a ubiquitin-like domain to promote substrate destruction. Thus, midnolin contains three regions that function in concert to target a large set of nuclear proteins to the proteasome for degradation.
Topics: Nuclear Proteins; Proteasome Endopeptidase Complex; Ubiquitin; Ubiquitination; Proteolysis; Genes, Immediate-Early; HEK293 Cells; NIH 3T3 Cells; Transcription, Genetic
PubMed: 37616343
DOI: 10.1126/science.adh5021 -
Trends in Biochemical Sciences Jan 2024Post-translational modifications (PTMs) add a major degree of complexity to the proteome and are essential controllers of protein homeostasis. Amongst the hundreds of... (Review)
Review
Post-translational modifications (PTMs) add a major degree of complexity to the proteome and are essential controllers of protein homeostasis. Amongst the hundreds of PTMs identified, ubiquitin and ubiquitin-like (UBL) modifications are recognized as key regulators of cellular processes through their ability to affect protein-protein interactions, protein stability, and thus the functions of their protein targets. Here, we focus on the most recently identified UBL, ubiquitin-fold modifier 1 (UFM1), and the machinery responsible for its transfer to substrates (UFMylation) or its removal (deUFMylation). We first highlight the biochemical peculiarities of these processes, then we develop on how UFMylation and its machinery control various intertwined cellular processes and we highlight some of the outstanding research questions in this emerging field.
Topics: Ubiquitin; Proteins; Ubiquitin-Protein Ligases; Protein Processing, Post-Translational; Cell Communication
PubMed: 37945409
DOI: 10.1016/j.tibs.2023.10.004 -
The FEBS Journal Nov 2023Ubiquitin Fold Modifier-1 (UFM1) is a ubiquitin-like modifier (UBL) that is posttranslationally attached to lysine residues on substrates via a dedicated system of... (Review)
Review
Ubiquitin Fold Modifier-1 (UFM1) is a ubiquitin-like modifier (UBL) that is posttranslationally attached to lysine residues on substrates via a dedicated system of enzymes conserved in most eukaryotes. Despite the structural similarity between UFM1 and ubiquitin, the UFMylation machinery employs unique mechanisms that ensure fidelity. While physiological triggers and consequences of UFMylation are not entirely clear, its biological importance is epitomized by mutations in the UFMylation pathway in human pathophysiology including musculoskeletal and neurodevelopmental diseases. Some of these diseases can be explained by the increased endoplasmic reticulum (ER) stress and disrupted translational homeostasis observed upon loss of UFMylation. The roles of UFM1 in these processes likely stem from its function at the ER where ribosomes are UFMylated in response to translational stalling. In addition, UFMylation has been implicated in other cellular processes including DNA damage response and telomere maintenance. Hence, the study of UFM1 pathway mechanics and its biological function will reveal insights into fundamental cell biology and is likely to afford new therapeutic opportunities for the benefit of human health. To this end, we herein provide a comprehensive guide to the current state of knowledge of UFM1 biogenesis, conjugation, and function with an emphasis on the underlying mechanisms.
Topics: Humans; Proteins; Protein Processing, Post-Translational; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitin-Conjugating Enzymes
PubMed: 36680403
DOI: 10.1111/febs.16730 -
The EMBO Journal Jul 2023Mitophagy is a fundamental quality control mechanism of mitochondria. Its regulatory mechanisms and pathological implications remain poorly understood. Here, via a...
Mitophagy is a fundamental quality control mechanism of mitochondria. Its regulatory mechanisms and pathological implications remain poorly understood. Here, via a mitochondria-targeted genetic screen, we found that knockout (KO) of FBXL4, a mitochondrial disease gene, hyperactivates mitophagy at basal conditions. Subsequent counter screen revealed that FBXL4-KO hyperactivates mitophagy via two mitophagy receptors BNIP3 and NIX. We determined that FBXL4 functions as an integral outer-membrane protein that forms an SCF-FBXL4 ubiquitin E3 ligase complex. SCF-FBXL4 ubiquitinates BNIP3 and NIX to target them for degradation. Pathogenic FBXL4 mutations disrupt SCF-FBXL4 assembly and impair substrate degradation. Fbxl4 mice exhibit elevated BNIP3 and NIX proteins, hyperactive mitophagy, and perinatal lethality. Importantly, knockout of either Bnip3 or Nix rescues metabolic derangements and viability of the Fbxl4 mice. Together, beyond identifying SCF-FBXL4 as a novel mitochondrial ubiquitin E3 ligase restraining basal mitophagy, our results reveal hyperactivated mitophagy as a cause of mitochondrial disease and suggest therapeutic strategies.
Topics: Mice; Animals; Mitophagy; Mitochondria; Mitochondrial Diseases; Ubiquitin-Protein Ligases; Ubiquitins; Mitochondrial Proteins
PubMed: 36896912
DOI: 10.15252/embj.2022113033 -
Autophagy Jul 2023Eukaryotic stress granules (SGs) are highly dynamic assemblies of untranslated mRNAs and proteins that form through liquid-liquid phase separation (LLPS) under cellular...
Eukaryotic stress granules (SGs) are highly dynamic assemblies of untranslated mRNAs and proteins that form through liquid-liquid phase separation (LLPS) under cellular stress. SG formation and elimination process is a conserved cellular strategy to promote cell survival, although the precise regulation of this process is poorly understood. Here, we screened six E3 ubiquitin ligases present in SGs and identified TRIM21 (tripartite motif containing 21) as a central regulator of SG homeostasis that is highly enriched in SGs of cells under arsenite-induced oxidative stress. Knockdown of promotes SG formation whereas overexpression of inhibits the formation of physiological and pathological SGs associated with neurodegenerative diseases. TRIM21 catalyzes K63-linked ubiquitination of the SG core protein, G3BP1 (G3BP stress granule assembly factor 1), and G3BP1 ubiquitination can effectively inhibit LLPS, . Recent reports suggested the involvement of macroautophagy/autophagy, as a stress response pathway, in the regulation of SG homeostasis. We systematically investigated well-defined autophagy receptors and identified SQSTM1/p62 (sequestosome 1) and CALCOCO2/NDP52 (calcium binding and coiled-coil domain 2) as the primary receptors that directly interact with G3BP1 during arsenite-induced stress. Endogenous SQSTM1 and CALCOCO2 localize to the periphery of SGs under oxidative stress and mediate SG elimination, as single knockout of each receptor causes accumulation of physiological and pathological SGs. Collectively, our study broadens the understanding in the regulation of SG homeostasis by showing that TRIM21 and autophagy receptors modulate SG formation and elimination respectively, suggesting the possibility of clinical targeting of these molecules in therapeutic strategies for neurodegenerative diseases. ACTB: actin beta; ALS: amyotrophic lateral sclerosis; BafA1: bafilomycin A; BECN1: beclin 1; C9orf72: C9orf72-SMCR8 complex subunit; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; Co-IP: co-immunoprecipitation; DAPI: 4',6-diamidino-2-phenylindole; FTD: frontotemporal dementia; FUS: FUS RNA binding protein; G3BP1: G3BP stress granule assembly factor 1; GFP: green fluorescent protein; LLPS: liquid-liquid phase separation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NBR1: NBR1 autophagy cargo receptor; NES: nuclear export signal; OPTN: optineurin; RFP: red fluorescent protein; SQSTM1/p62: sequestosome 1; SG: stress granule; TAX1BP1: Tax1 binding protein 1; TOLLIP: toll interacting protein; TRIM21: tripartite motif containing 21; TRIM56: tripartite motif containing 56; UB: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.
Topics: Sequestosome-1 Protein; DNA Helicases; Arsenites; Stress Granules; C9orf72 Protein; Calcium; Autophagy; RNA Helicases; RNA Recognition Motif Proteins; Poly-ADP-Ribose Binding Proteins; Ubiquitination; Carrier Proteins; Apoptosis Regulatory Proteins; Homeostasis; Ubiquitins
PubMed: 36692217
DOI: 10.1080/15548627.2022.2164427 -
Nature Communications Aug 2023Nonalcoholic fatty liver disease (NAFLD) is the most common liver disorder with high morbidity and mortality. The current study aims to explore the role of...
Nonalcoholic fatty liver disease (NAFLD) is the most common liver disorder with high morbidity and mortality. The current study aims to explore the role of Cullin-associated and neddylation-dissociated protein 1 (CAND1) in the development of NAFLD and the underlying mechanisms. CAND1 is reduced in the liver of NAFLD male patients and high fat diet (HFD)-fed male mice. CAND1 alleviates palmitate (PA) induced lipid accumulation in vitro. Hepatocyte-specific knockout of CAND1 exacerbates HFD-induced liver injury in HFD-fed male mice, while hepatocyte-specific knockin of CAND1 ameliorates these pathological changes. Mechanistically, deficiency of CAND1 enhances the assembly of Cullin1, F-box only protein 42 (FBXO42) and acetyl-CoA acyltransferase 2 (ACAA2) complexes, and thus promotes the ubiquitinated degradation of ACAA2. ACAA2 overexpression abolishes the exacerbated effects of CAND1 deficiency on NAFLD. Additionally, androgen receptor binds to the -187 to -2000 promoter region of CAND1. Collectively, CAND1 mitigates NAFLD by inhibiting Cullin1/FBXO42 mediated ACAA2 degradation.
Topics: Male; Animals; Mice; Cullin Proteins; Non-alcoholic Fatty Liver Disease; Acyltransferases; Transcription Factors; Ubiquitin; Diet, High-Fat; Mice, Inbred C57BL; Liver
PubMed: 37528093
DOI: 10.1038/s41467-023-40327-5 -
Molecular Cell Aug 2023Ubiquitin-dependent control of mitochondrial dynamics is important for protein quality and neuronal integrity. Mitofusins, mitochondrial fusion factors, can integrate...
Ubiquitin-dependent control of mitochondrial dynamics is important for protein quality and neuronal integrity. Mitofusins, mitochondrial fusion factors, can integrate cellular stress through their ubiquitylation, which is carried out by multiple E3 enzymes in response to many different stimuli. However, the molecular mechanisms that enable coordinated responses are largely unknown. Here we show that yeast Ufd2, a conserved ubiquitin chain-elongating E4 enzyme, is required for mitochondrial shape adjustments. Under various stresses, Ufd2 translocates to mitochondria and triggers mitofusin ubiquitylation. This elongates ubiquitin chains on mitofusin and promotes its proteasomal degradation, leading to mitochondrial fragmentation. Ufd2 and its human homologue UBE4B also target mitofusin mutants associated with Charcot-Marie-Tooth disease, a hereditary sensory and motor neuropathy characterized by progressive loss of the peripheral nerves. This underscores the pathophysiological importance of E4-mediated ubiquitylation in neurodegeneration. In summary, we identify E4-dependent mitochondrial stress adaptation by linking various metabolic processes to mitochondrial fusion and fission dynamics.
Topics: Humans; Acclimatization; Mitochondria; Saccharomyces cerevisiae; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination; Mitochondrial Proteins
PubMed: 37595558
DOI: 10.1016/j.molcel.2023.07.021 -
Nature Feb 2024Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts...
Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress. Crucial to this process is the silencing factor of the integrated stress response (SIFI), a large E3 ligase complex mutated in ataxia and in early-onset dementia that degrades both unimported mitochondrial precursors and stress response components. By recognizing bifunctional substrate motifs that equally encode protein localization and stability, the SIFI complex turns off a general stress response after a specific stress event has been resolved. Pharmacological stress response silencing sustains cell survival even if stress resolution failed, which underscores the importance of signal termination and provides a roadmap for treating neurodegenerative diseases caused by mitochondrial import defects.
Topics: Apoptosis; Ataxia; Cell Survival; Dementia; Mitochondria; Mitochondrial Proteins; Multiprotein Complexes; Mutation; Neurodegenerative Diseases; Protein Stability; Protein Transport; Proteolysis; Stress, Physiological; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 38297121
DOI: 10.1038/s41586-023-06985-7 -
Nature Jul 2023Break-induced telomere synthesis (BITS) is a RAD51-independent form of break-induced replication that contributes to alternative lengthening of telomeres. This...
Break-induced telomere synthesis (BITS) is a RAD51-independent form of break-induced replication that contributes to alternative lengthening of telomeres. This homology-directed repair mechanism utilizes a minimal replisome comprising proliferating cell nuclear antigen (PCNA) and DNA polymerase-δ to execute conservative DNA repair synthesis over many kilobases. How this long-tract homologous recombination repair synthesis responds to complex secondary DNA structures that elicit replication stress remains unclear. Moreover, whether the break-induced replisome orchestrates additional DNA repair events to ensure processivity is also unclear. Here we combine synchronous double-strand break induction with proteomics of isolated chromatin segments (PICh) to capture the telomeric DNA damage response proteome during BITS. This approach revealed a replication stress-dominated response, highlighted by repair synthesis-driven DNA damage tolerance signalling through RAD18-dependent PCNA ubiquitination. Furthermore, the SNM1A nuclease was identified as the major effector of ubiquitinated PCNA-dependent DNA damage tolerance. SNM1A recognizes the ubiquitin-modified break-induced replisome at damaged telomeres, and this directs its nuclease activity to promote resection. These findings show that break-induced replication orchestrates resection-dependent lesion bypass, with SNM1A nuclease activity serving as a critical effector of ubiquitinated PCNA-directed recombination in mammalian cells.
Topics: Animals; Cell Cycle Proteins; Chromatin; DNA Breaks, Double-Stranded; DNA Polymerase III; DNA Repair; DNA Replication; DNA-Binding Proteins; Exodeoxyribonucleases; Homologous Recombination; Mammals; Proliferating Cell Nuclear Antigen; Proteomics; Rad51 Recombinase; Telomere; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination; Templates, Genetic
PubMed: 37316655
DOI: 10.1038/s41586-023-06177-3 -
Nature Communications Aug 2023UBA1 is the primary E1 ubiquitin-activating enzyme responsible for generation of activated ubiquitin required for ubiquitination, a process that regulates stability and...
UBA1 is the primary E1 ubiquitin-activating enzyme responsible for generation of activated ubiquitin required for ubiquitination, a process that regulates stability and function of numerous proteins. Decreased or insufficient ubiquitination can cause or drive aging and many diseases. Therefore, a small-molecule enhancing UBA1 activity could have broad therapeutic potential. Here we report that auranofin, a drug approved for the treatment of rheumatoid arthritis, is a potent UBA1 activity enhancer. Auranofin binds to the UBA1's ubiquitin fold domain and conjugates to Cys1039 residue. The binding enhances UBA1 interactions with at least 20 different E2 ubiquitin-conjugating enzymes, facilitating ubiquitin charging to E2 and increasing the activities of seven representative E3s in vitro. Auranofin promotes ubiquitination and degradation of misfolded ER proteins during ER-associated degradation in cells at low nanomolar concentrations. It also facilitates outer mitochondrial membrane-associated degradation. These findings suggest that auranofin can serve as a much-needed tool for UBA1 research and therapeutic exploration.
Topics: Ubiquitin; Ubiquitin-Conjugating Enzymes; Auranofin; Ubiquitination; Ubiquitin-Activating Enzymes
PubMed: 37558718
DOI: 10.1038/s41467-023-40537-x