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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 -
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 -
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 -
The Plant Cell Sep 2023Ubiquitination modulates protein turnover or activity depending on the number and location of attached ubiquitin (Ub) moieties. Proteins marked by a lysine 48...
Ubiquitination modulates protein turnover or activity depending on the number and location of attached ubiquitin (Ub) moieties. Proteins marked by a lysine 48 (K48)-linked polyubiquitin chain are usually targeted to the 26S proteasome for degradation; however, other polyubiquitin chains, such as those attached to K63, usually regulate other protein properties. Here, we show that 2 PLANT U-BOX E3 ligases, PUB25 and PUB26, facilitate both K48- and K63-linked ubiquitination of the transcriptional regulator INDUCER OF C-REPEAT BINDING FACTOR (CBF) EXPRESSION1 (ICE1) during different periods of cold stress in Arabidopsis (Arabidopsis thaliana), thus dynamically modulating ICE1 stability. Moreover, PUB25 and PUB26 attach both K48- and K63-linked Ub chains to MYB15 in response to cold stress. However, the ubiquitination patterns of ICE1 and MYB15 mediated by PUB25 and PUB26 differ, thus modulating their protein stability and abundance during different stages of cold stress. Furthermore, ICE1 interacts with and inhibits the DNA-binding activity of MYB15, resulting in an upregulation of CBF expression. This study unravels a mechanism by which PUB25 and PUB26 add different polyubiquitin chains to ICE1 and MYB15 to modulate their stability, thereby regulating the timing and degree of cold stress responses in plants.
Topics: Arabidopsis; Arabidopsis Proteins; Cold-Shock Response; Polyubiquitin; Transcription Factors; Ubiquitination; Ubiquitin
PubMed: 37279565
DOI: 10.1093/plcell/koad159 -
Nature Communications Jul 2023Despite advances in cancer treatment, immune checkpoint blockade (ICB) only achieves complete response in some patients, illustrating the need to identify resistance...
Despite advances in cancer treatment, immune checkpoint blockade (ICB) only achieves complete response in some patients, illustrating the need to identify resistance mechanisms. Using an ICB-insensitive tumor model, here we discover cisplatin enhances the anti-tumor effect of PD-L1 blockade and upregulates the expression of Ariadne RBR E3 ubiquitin-protein ligase 1 (ARIH1) in tumors. Arih1 overexpression promotes cytotoxic T cell infiltration, inhibits tumor growth, and potentiates PD-L1 blockade. ARIH1 mediates ubiquitination and degradation of DNA-PKcs to trigger activation of the STING pathway, which is blocked by the phospho-mimetic mutant T68E/S213D of cGAS protein. Using a high-throughput drug screen, we further identify that ACY738, less cytotoxic than cisplatin, effectively upregulates ARIH1 and activates STING signaling, sensitizing tumors to PD-L1 blockade. Our findings delineate a mechanism that tumors mediate ICB resistance through the loss of ARIH1 and ARIH1-DNA-PKcs-STING signaling and indicate that activating ARIH1 is an effective strategy to improve the efficacy of cancer immunotherapy.
Topics: Humans; B7-H1 Antigen; Cisplatin; Immune Checkpoint Inhibitors; Neoplasms; T-Lymphocytes; DNA; Ubiquitin-Protein Ligases
PubMed: 37429863
DOI: 10.1038/s41467-023-39920-5 -
Nucleic Acids Research Sep 2023Histone deacetylase 6 (HDAC6) mediates DNA damage signaling by regulating the mismatch repair and nucleotide excision repair pathways. Whether HDAC6 also mediates DNA...
Histone deacetylase 6 (HDAC6) mediates DNA damage signaling by regulating the mismatch repair and nucleotide excision repair pathways. Whether HDAC6 also mediates DNA double-strand break (DSB) repair is unclear. Here, we report that HDAC6 negatively regulates DSB repair in an enzyme activity-independent manner. In unstressed cells, HDAC6 interacts with H2A/H2A.X to prevent its interaction with the E3 ligase RNF168. Upon sensing DSBs, RNF168 rapidly ubiquitinates HDAC6 at lysine 116, leading to HDAC6 proteasomal degradation and a restored interaction between RNF168 and H2A/H2A.X. H2A/H2A.X is ubiquitinated by RNF168, precipitating the recruitment of DSB repair factors (including 53BP1 and BRCA1) to chromatin and subsequent DNA repair. These findings reveal novel regulatory machinery based on an HDAC6-RNF168 axis that regulates the H2A/H2A.X ubiquitination status. Interfering with this axis might be leveraged to disrupt a key mechanism of cancer cell resistance to genotoxic damage and form a potential therapeutic strategy for cancer.
Topics: Humans; Cell Line, Tumor; DNA Damage; DNA Repair; Histone Deacetylase 6; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 37503842
DOI: 10.1093/nar/gkad631 -
Science Translational Medicine Nov 2023Aberrant DNA methylation has been implicated as a key driver of prostate cancer lineage plasticity and histologic transformation to neuroendocrine prostate cancer...
Aberrant DNA methylation has been implicated as a key driver of prostate cancer lineage plasticity and histologic transformation to neuroendocrine prostate cancer (NEPC). DNA methyltransferases (DNMTs) are highly expressed, and global DNA methylation is dysregulated in NEPC. We identified that deletion of DNMT genes decreases expression of neuroendocrine lineage markers and substantially reduced NEPC tumor development and metastasis in vivo. Decitabine, a pan-DNMT inhibitor, attenuated tumor growth in NEPC patient-derived xenograft models, as well as retinoblastoma gene ()-deficient castration-resistant prostate adenocarcinoma (CRPC) models compared with -proficient CRPC. We further found that DNMT inhibition increased expression of B7 homolog 3 (B7-H3), an emerging druggable target, via demethylation of B7-H3. We tested DS-7300a (i-DXd), an antibody-drug conjugate targeting B7-H3, alone and in combination with decitabine in models of advanced prostate cancer. There was potent single-agent antitumor activity of DS-7300a in both CRPC and NEPC bearing high expression of B7-H3. In B7-H3-low models, combination therapy of decitabine plus DS-7300a resulted in enhanced response. DNMT inhibition may therefore be a promising therapeutic target for NEPC and RB1-deficient CRPC and may sensitize B7-H3-low prostate cancer to DS-7300a through increasing target expression. NEPC and RB1-deficient CRPC represent prostate cancer subgroups with poor prognosis, and the development of biomarker-driven therapeutic strategies for these populations may ultimately help improve patient outcomes.
Topics: Male; Humans; Prostatic Neoplasms, Castration-Resistant; DNA Methylation; Decitabine; Cell Line, Tumor; Prostatic Neoplasms; Neuroendocrine Tumors; Transcription Factors; Antineoplastic Agents; Ubiquitin-Protein Ligases; Retinoblastoma Binding Proteins
PubMed: 37967200
DOI: 10.1126/scitranslmed.adf6732 -
Nature Communications Nov 2023Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors...
Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We present BioE3, a powerful approach for matching substrates to Ub E3 ligases of interest. Using BirA-E3 ligase fusions and bioUb, site-specific biotinylation of Ub-modified substrates of particular E3s facilitates proteomic identification. We show that BioE3 identifies both known and new targets of two RING-type E3 ligases: RNF4 (DNA damage response, PML bodies), and MIB1 (endocytosis, autophagy, centrosome dynamics). Versatile BioE3 identifies targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, BioE3 works with NEDD4, a HECT-type E3, identifying new targets linked to vesicular trafficking. BioE3 detects altered specificity in response to chemicals, opening avenues for targeted protein degradation, and may be applicable for other Ub-likes (UbLs, e.g., SUMO) and E3 types. BioE3 applications shed light on cellular regulation by the complex UbL network.
Topics: Ubiquitin-Protein Ligases; Ubiquitin; Ubiquitination; Proteomics; Proteolysis
PubMed: 37996419
DOI: 10.1038/s41467-023-43326-8 -
International Journal of Molecular... Dec 2023Mitochondrial dysregulation, such as mitochondrial complex I deficiency, increased oxidative stress, perturbation of mitochondrial dynamics and mitophagy, has long been... (Review)
Review
Mitochondrial dysregulation, such as mitochondrial complex I deficiency, increased oxidative stress, perturbation of mitochondrial dynamics and mitophagy, has long been implicated in the pathogenesis of PD. Initiating from the observation that mitochondrial toxins cause PD-like symptoms and mitochondrial DNA mutations are associated with increased risk of PD, many mutated genes linked to familial forms of PD, including , , and , have also been found to affect the mitochondrial features. Recent research has uncovered a much more complex involvement of mitochondria in PD. Disruption of mitochondrial quality control coupled with abnormal secretion of mitochondrial contents to dispose damaged organelles may play a role in the pathogenesis of PD. Furthermore, due to its bacterial ancestry, circulating mitochondrial DNAs can function as damage-associated molecular patterns eliciting inflammatory response. In this review, we summarize and discuss the connection between mitochondrial dysfunction and PD, highlighting the molecular triggers of the disease process, the intra- and extracellular roles of mitochondria in PD as well as the therapeutic potential of mitochondrial transplantation.
Topics: Humans; Parkinson Disease; Ubiquitin-Protein Ligases; Mitochondria; DNA, Mitochondrial; Mitophagy
PubMed: 38069350
DOI: 10.3390/ijms242317027 -
Molecular Cell Dec 2023Reactive aldehydes are abundant endogenous metabolites that challenge homeostasis by crosslinking cellular macromolecules. Aldehyde-induced DNA damage requires repair to...
Reactive aldehydes are abundant endogenous metabolites that challenge homeostasis by crosslinking cellular macromolecules. Aldehyde-induced DNA damage requires repair to prevent cancer and premature aging, but it is unknown whether cells also possess mechanisms that resolve aldehyde-induced RNA lesions. Here, we establish photoactivatable ribonucleoside-enhanced crosslinking (PAR-CL) as a model system to study RNA crosslinking damage in the absence of confounding DNA damage in human cells. We find that such RNA damage causes translation stress by stalling elongating ribosomes, which leads to collisions with trailing ribosomes and activation of multiple stress response pathways. Moreover, we discovered a translation-coupled quality control mechanism that resolves covalent RNA-protein crosslinks. Collisions between translating ribosomes and crosslinked mRNA-binding proteins trigger their modification with atypical K6- and K48-linked ubiquitin chains. Ubiquitylation requires the E3 ligase RNF14 and leads to proteasomal degradation of the protein adduct. Our findings identify RNA lesion-induced translational stress as a central component of crosslinking damage.
Topics: Humans; RNA; Ubiquitination; Ubiquitin; Ribosomes; Ubiquitin-Protein Ligases; Aldehydes; Protein Biosynthesis
PubMed: 37951216
DOI: 10.1016/j.molcel.2023.10.012