Mutant huntingtin protein induces MLH1 degradation, DNA hyperexcision, and cGAS-STING-dependent apoptosis.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene. The repeat-expanded HTT encodes a mutated HTT (mHTT), which is known to induce DNA double-strand breaks (DSBs), activation of the cGAS-STING pathway, and apoptosis in HD. However, the mechanism by which mHTT triggers these events is unknown. Here, we show that HTT interacts with both exonuclease 1 (Exo1) and MutLα (MLH1-PMS2), a negative regulator of Exo1. While the HTT-Exo1 interaction suppresses the Exo1-catalyzed DNA end resection during DSB repair, the HTT-MutLα interaction functions to stabilize MLH1. However, mHTT displays a significantly reduced interaction with Exo1 or MutLα, thereby losing the ability to regulate Exo1. Thus, cells expressing mHTT exhibit rapid MLH1 degradation and hyperactive DNA excision, which causes severe DNA damage and cytosolic DNA accumulation. This activates the cGAS-STING pathway to mediate apoptosis. Therefore, we have identified unique functions for both HTT and mHTT in modulating DNA repair and the cGAS-STING pathway-mediated apoptosis by interacting with MLH1. Our work elucidates the mechanism by which mHTT causes HD.

DNAJA2 deficiency activates cGAS-STING pathway via the induction of aberrant mitosis and chromosome instability.

Molecular chaperone HSP70s are attractive targets for cancer therapy, but their substrate broadness and functional non-specificity have limited their role in therapeutical success. Functioning as HSP70's cochaperones, HSP40s determine the client specificity of HSP70s, and could be better targets for cancer therapy. Here we show that tumors defective in HSP40 member DNAJA2 are benefitted from immune-checkpoint blockade (ICB) therapy. Mechanistically, DNAJA2 maintains centrosome homeostasis by timely degrading key centriolar satellite proteins PCM1 and CEP290 via HSC70 chaperone-mediated autophagy (CMA). Tumor cells depleted of DNAJA2 or CMA factor LAMP2A exhibit elevated levels of centriolar satellite proteins, which causes aberrant mitosis characterized by abnormal spindles, chromosome missegregation and micronuclei formation. This activates the cGAS-STING pathway to enhance ICB therapy response in tumors derived from DNAJA2-deficient cells. Our study reveals a role for DNAJA2 to regulate mitotic division and chromosome stability and suggests DNAJA2 as a potential target to enhance cancer immunotherapy, thereby providing strategies to advance HSPs-based cancer therapy.

Soybean-Oil-Body-Substituted Low-Fat Ice Cream with Different Homogenization Pressure, Pasteurization Condition, and Process Sequence: Physicochemical Properties, Texture, and Storage Stability.

The purpose of this research was to explore the impacts of different homogenization pressures, pasteurization conditions, and process sequence on the physical and chemical properties of soybean oil body (SOB)-substituted low-fat ice cream as well as the storage stability of SOB-substituted ice cream under these process parameters. With the increase of homogenization pressure (10-30 MPa), the increase of pasteurization temperature (65 °C for 30 min-85 °C for 15 min), and the addition of SOB before homogenization, the overrun and apparent viscosity of ice cream increased significantly, and the particle size, hardness, and melting rate decreased significantly. Thus, frozen dairy products of desired quality and condition could be obtained by optimizing process parameters. In addition, the SOB ice cream showed better storage stability, which was reflected in lower melting rate and hardness and more stable microstructure compared with the full-milk-fat ice cream. This study opened up new ideas for the application of SOB and the development of nutritious and healthy ice cream. Meanwhile, this research supplied a conceptual basis for the processing and quality optimization of SOB ice cream.

The mismatch recognition protein MutSα promotes nascent strand degradation at stalled replication forks.

Mismatch repair (MMR) is a replication-coupled DNA repair mechanism and plays multiple roles at the replication fork. The well-established MMR functions include correcting misincorporated nucleotides that have escaped the proofreading activity of DNA polymerases, recognizing nonmismatched DNA adducts, and triggering a DNA damage response. In an attempt to determine whether MMR regulates replication progression in cells expressing an ultramutable DNA polymerase ɛ (Polɛ), carrying a proline-to-arginine substitution at amino acid 286 (Polɛ-P286R), we identified an unusual MMR function in response to hydroxyurea (HU)-induced replication stress. Polɛ-P286R cells treated with hydroxyurea exhibit increased MRE11-catalyzed nascent strand degradation. This degradation by MRE11 depends on the mismatch recognition protein MutSα and its binding to stalled replication forks. Increased MutSα binding at replication forks is also associated with decreased loading of replication fork protection factors FANCD2 and BRCA1, suggesting blockage of these fork protection factors from loading to replication forks by MutSα. We find that the MutSα-dependent MRE11-catalyzed fork degradation induces DNA breaks and various chromosome abnormalities. Therefore, unlike the well-known MMR functions of ensuring replication fidelity, the newly identified MMR activity of promoting genome instability may also play a role in cancer avoidance by eliminating rogue cells.

Interplay between H3K36me3, methyltransferase SETD2, and mismatch recognition protein MutSα facilitates processing of oxidative DNA damage in human cells.

Oxidative DNA damage contributes to aging and the pathogenesis of numerous human diseases including cancer. 8-hydroxyguanine (8-oxoG) is the major product of oxidative DNA lesions. Although OGG1-mediated base excision repair is the primary mechanism for 8-oxoG removal, DNA mismatch repair has also been implicated in processing oxidative DNA damage. However, the mechanism of the latter is not fully understood. Here, we treated human cells defective in various 8-oxoG repair factors with H2O2 and performed biochemical, live cell imaging, and chromatin immunoprecipitation sequencing analyses to determine their response to the treatment. We show that the mismatch repair processing of oxidative DNA damage involves cohesive interactions between mismatch recognition protein MutSα, histone mark H3K36me3, and H3K36 trimethyltransferase SETD2, which activates the ATM DNA damage signaling pathway. We found that cells depleted of MutSα or SETD2 accumulate 8-oxoG adducts and fail to trigger H2O2-induced ATM activation. Furthermore, we show that SETD2 physically interacts with both MutSα and ATM, which suggests a role for SETD2 in transducing DNA damage signals from lesion-bound MutSα to ATM. Consistently, MutSα and SETD2 are highly coenriched at oxidative damage sites. The data presented here support a model wherein MutSα, SETD2, ATM, and H3K36me3 constitute a positive feedback loop to help cells cope with oxidative DNA damage.

Effects of Soybean Oil Body as a Milk Fat Substitute on Ice Cream: Physicochemical, Sensory and Digestive Properties.

Soybean oil body (SOB) has potential as a milk fat substitute due to its ideal emulsification, stability and potential biological activity. In this study, SOB was used as a milk fat substitute to prepare ice cream, expecting to reduce the content of saturated fatty acid and improve the quality defects of ice cream products caused by the poor stability of milk fat at low temperatures. This study investigated the effect of SOB as a milk fat substitute (the substitution amount was 10-50%) on ice cream through apparent viscosity, particle size, overrun, melting, texture, sensory and digestive properties. The results show SOB substitution for milk fat significantly increased the apparent viscosity and droplet uniformity and decreased the particle size of the ice cream mixes, indicating that there were lots of intermolecular interactions to improve ice cream stability. In addition, ice cream with 30% to 50% SOB substitution had better melting properties and texture characteristics. The ice cream with 40% SOB substitution had the highest overall acceptability. Furthermore, SOB substitution for milk fat increased unsaturated fatty acid content in ice cream and fatty acid release during digestion, which had potential health benefits for consumers. Therefore, SOB as a milk fat substitute may be an effective way to improve the nutritional value and quality characteristics of dairy products.

NaCl induces flocculation and lipid oxidation of soybean oil body emulsions recovered by neutral aqueous extraction.

BACKGROUND: Soybean oil bodies (SOB) are naturally pre-emulsified lipid droplets recovered directly from soybean seeds. Almost all food emulsions contain salts. However, it was not clear how the incorporation of salts affected the physicochemical stability of SOB. RESULTS: This study investigated the effect of NaCl (0-1.2%) on the physical and oxidative stability of SOB emulsions under neutral (pH 7) and acidic (pH 3) conditions. In the presence of NaCl, the SOB emulsion (pH 7) showed strong flocculation during storage due to electrostatic screening. The NaCl-induced flocculation of SOB was attenuated at pH 3, which may be due to the difference in conformation or interaction of the protein interfaces covering SOB at different pH values. The increase in ionic strength or acid conditioning treatment resulted in a remarkable increase in the stability of SOB emulsions against coalescence. The confocal laser scanning microscopy images also confirmed the NaCl-induced changes in the flocculation/coalescence properties of SOB. The oxidative behavior tests indicated that SOB emulsions containing NaCl were more susceptible to lipid oxidation but protein oxidation was inhibited due to electrostatic screening, which reduced pro-oxidant accessibility of unadsorbed proteins in the emulsion. This oxidative behavior was attenuated at pH 3. CONCLUSION: The incorporation of NaCl significantly reduced the physical and oxidative stability of the SOB emulsion, and acidic pH mitigated NaCl-induced flocculation and lipid oxidation of SOB. © 2021 Society of Chemical Industry.

OTUB1 stabilizes mismatch repair protein MSH2 by blocking ubiquitination.

DNA mismatch repair (MMR) maintains genome stability primarily by correcting replication errors. MMR deficiency can lead to cancer development and bolsters cancer cell resistance to chemotherapy. However, recent studies have shown that checkpoint blockade therapy is effective in MMR-deficient cancers, thus the ability to identify cancer etiology would greatly benefit cancer treatment. MutS homolog 2 (MSH2) is an obligate subunit of mismatch recognition proteins MutSα (MSH2-MSH6) and MutSß (MSH2-MSH3). Precise regulation of MSH2 is critical, as either over- or underexpression of MSH2 results in an increased mutation frequency. The mechanism by which cells maintain MSH2 proteostasis is unknown. Using functional ubiquitination and deubiquitination assays, we show that the ovarian tumor (OTU) family deubiquitinase ubiquitin aldehyde binding 1 (OTUB1) inhibits MSH2 ubiquitination by blocking the E2 ligase ubiquitin transfer activity. Depleting OTUB1 in cells promotes the ubiquitination and subsequent degradation of MSH2, leading to greater mutation frequency and cellular resistance to genotoxic agents, including the common chemotherapy agents N-methyl-N'-nitro-N-nitrosoguanidine and cisplatin. Taken together, our data identify OTUB1 as an important regulator of MSH2 stability and provide evidence that OTUB1 is a potential biomarker for cancer etiology and therapy.

Mispair-bound human MutS-MutL complex triggers DNA incisions and activates mismatch repair.

DNA mismatch repair (MMR) relies on MutS and MutL ATPases for mismatch recognition and strand-specific nuclease recruitment to remove mispaired bases in daughter strands. However, whether the MutS-MutL complex coordinates MMR by ATP-dependent sliding on DNA or protein-protein interactions between the mismatch and strand discrimination signal is ambiguous. Using functional MMR assays and systems preventing proteins from sliding, we show that sliding of human MutSα is required not for MMR initiation, but for final mismatch removal. MutSα recruits MutLα to form a mismatch-bound complex, which initiates MMR by nicking the daughter strand 5' to the mismatch. Exonuclease 1 (Exo1) is then recruited to the nick and conducts 5' → 3' excision. ATP-dependent MutSα dissociation from the mismatch is necessary for Exo1 to remove the mispaired base when the excision reaches the mismatch. Therefore, our study has resolved a long-standing puzzle, and provided new insights into the mechanism of MMR initiation and mispair removal.

MLH1 Deficiency-Triggered DNA Hyperexcision by Exonuclease 1 Activates the cGAS-STING Pathway.

Tumors with defective mismatch repair (dMMR) are responsive to immunotherapy because of dMMR-induced neoantigens and activation of the cGAS-STING pathway. While neoantigens result from the hypermutable nature of dMMR, it is unknown how dMMR activates the cGAS-STING pathway. We show here that loss of the MutLα subunit MLH1, whose defect is responsible for ~50% of dMMR cancers, results in loss of MutLα-specific regulation of exonuclease 1 (Exo1) during DNA repair. This leads to unrestrained DNA excision by Exo1, which causes increased single-strand DNA formation, RPA exhaustion, DNA breaks, and aberrant DNA repair intermediates. Ultimately, this generates chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, activating the cGAS-STING pathway. In this study, we discovered a hitherto unknown MMR mechanism that modulates genome stability and has implications for cancer therapy.

Erratum: Author Correction: Identification of novel genetic variants predisposing to familial oral squamous cell carcinomas.

[This corrects the article DOI: 10.1038/s41421-019-0126-6.].

Identification of novel genetic variants predisposing to familial oral squamous cell carcinomas.

Oral squamous cell carcinoma (OSCC) is a common subtype of head and neck squamous cell carcinoma (HNSCC), but the pathogenesis underlying familial OSCCs is unknown. Here, we analyzed whole-genome sequences of a family with autosomal dominant expression of oral tongue cancer and identified proto-oncogenes VAV2 and IQGAP1 as the primary factors responsible for oral cancer in the family. These two genes are also frequently mutated in sporadic OSCCs and HNSCCs. Functional analysis revealed that the detrimental variants target tumorigenesis-associated pathways, thus confirming that these novel genetic variants help to establish a predisposition to familial OSCC.

Phosphorylation of proliferating cell nuclear antigen promotes cancer progression by activating the ATM/Akt/GSK3ß/Snail signaling pathway.

Proliferating cell nuclear antigen (PCNA) and its posttranslational modifications regulate DNA metabolic reactions, including DNA replication and repair, at replication forks. PCNA phosphorylation at Tyr-211 (PCNA-Y211p) inhibits DNA mismatch repair and induces misincorporation during DNA synthesis. Here, we describe an unexpected role of PCNA-Y211p in cancer promotion and development. Cells expressing phosphorylation-mimicking PCNA, PCNA-Y211D, show elevated hallmarks specific to the epithelial-mesenchymal transition (EMT), including the up-regulation of the EMT-promoting factor Snail and the down-regulation of EMT-inhibitory factors E-cadherin and GSK3ß. The PCNA-Y211D-expressing cells also exhibited active cell migration and underwent G2/M arrest. Interestingly, all of these EMT-associated activities required the activation of ATM and Akt kinases, as inactivating these protein kinases by gene knockdown or inhibitors blocked EMT-associated signaling and cell migration. We concluded that PCNA phosphorylation promotes cancer progression via the ATM/Akt/GSK3ß/Snail signaling pathway. In conclusion, this study identifies a novel PCNA function and reveals the molecular basis of phosphorylated PCNA-mediated cancer development and progression.

Cancer-driving H3G34V/R/D mutations block H3K36 methylation and H3K36me3-MutSα interaction.

Somatic mutations on glycine 34 of histone H3 (H3G34) cause pediatric cancers, but the underlying oncogenic mechanism remains unknown. We demonstrate that substituting H3G34 with arginine, valine, or aspartate (H3G34R/V/D), which converts the non-side chain glycine to a large side chain-containing residue, blocks H3 lysine 36 (H3K36) dimethylation and trimethylation by histone methyltransferases, including SETD2, an H3K36-specific trimethyltransferase. Our structural analysis reveals that the H3 "G33-G34" motif is recognized by a narrow substrate channel, and that H3G34/R/V/D mutations impair the catalytic activity of SETD2 due to steric clashes that impede optimal SETD2-H3K36 interaction. H3G34R/V/D mutations also block H3K36me3 from interacting with mismatch repair (MMR) protein MutSα, preventing the recruitment of the MMR machinery to chromatin. Cells harboring H3G34R/V/D mutations display a mutator phenotype similar to that observed in MMR-defective cells. Therefore, H3G34R/V/D mutations promote genome instability and tumorigenesis by inhibiting MMR activity.

H3K36me3-mediated mismatch repair preferentially protects actively transcribed genes from mutation.

Histone H3 trimethylation at lysine 36 (H3K36me3) is an important histone mark involved in both transcription elongation and DNA mismatch repair (MMR). It is known that H3K36me3 recruits the mismatch-recognition protein MutSα to replicating chromatin via its physical interaction with MutSα's PWWP domain, but the exact role of H3K36me3 in transcription is undefined. Using ChIP combined with whole-genome DNA sequencing analysis, we demonstrate here that H3K36me3, together with MutSα, is involved in protecting against mutation, preferentially in actively transcribed genomic regions. We found that H3K36me3 and MutSα are much more co-enriched in exons and actively transcribed regions than in introns and nontranscribed regions. The H3K36me3-MutSα co-enrichment correlated with a much lower mutation frequency in exons and actively transcribed regions than in introns and nontranscribed regions. Correspondingly, depleting H3K36me3 or disrupting the H3K36me3-MutSα interaction elevated the spontaneous mutation frequency in actively transcribed genes, but it had little influence on the mutation frequency in nontranscribed or transcriptionally inactive regions. Similarly, H2O2-induced mutations, which mainly cause base oxidations, preferentially occurred in actively transcribed genes in MMR-deficient cells. The data presented here suggest that H3K36me3-mediated MMR preferentially safeguards actively transcribed genes not only during replication by efficiently correcting mispairs in early replicating chromatin but also during transcription by directly or indirectly removing DNA lesions associated with a persistently open chromatin structure.

Regulation of mismatch repair by histone code and posttranslational modifications in eukaryotic cells.

DNA mismatch repair (MMR) protects genome integrity by correcting DNA replication-associated mispairs, modulating DNA damage-induced cell cycle checkpoints and regulating homeologous recombination. Loss of MMR function leads to cancer development. This review describes progress in understanding how MMR is carried out in the context of chromatin and how chromatin organization/compaction, epigenetic mechanisms and posttranslational modifications of MMR proteins influence and regulate MMR in eukaryotic cells.

Arsenic Inhibits DNA Mismatch Repair by Promoting EGFR Expression and PCNA Phosphorylation.

Both genotoxic and non-genotoxic chemicals can act as carcinogens. However, while genotoxic compounds lead directly to mutations that promote unregulated cell growth, the mechanism by which non-genotoxic carcinogens lead to cellular transformation is poorly understood. Using a model non-genotoxic carcinogen, arsenic, we show here that exposure to arsenic inhibits mismatch repair (MMR) in human cells, possibly through its ability to stimulate epidermal growth factor receptor (EGFR)-dependent tyrosine phosphorylation of proliferating cellular nuclear antigen (PCNA). HeLa cells exposed to exogenous arsenic demonstrate a dose- and time-dependent increase in the levels of EGFR and tyrosine 211-phosphorylated PCNA. Cell extracts derived from arsenic-treated HeLa cells are defective in MMR, and unphosphorylated recombinant PCNA restores normal MMR activity to these extracts. These results suggest a model in which arsenic induces expression of EGFR, which in turn phosphorylates PCNA, and phosphorylated PCNA then inhibits MMR, leading to increased susceptibility to carcinogenesis. This study suggests a putative novel mechanism of action for arsenic and other non-genotoxic carcinogens.

Phosphorylation of PCNA by EGFR inhibits mismatch repair and promotes misincorporation during DNA synthesis.

Proliferating cell nuclear antigen (PCNA) plays essential roles in eukaryotic cells during DNA replication, DNA mismatch repair (MMR), and other events at the replication fork. Earlier studies show that PCNA is regulated by posttranslational modifications, including phosphorylation of tyrosine 211 (Y211) by the epidermal growth factor receptor (EGFR). However, the functional significance of Y211-phosphorylated PCNA remains unknown. Here, we show that PCNA phosphorylation by EGFR alters its interaction with mismatch-recognition proteins MutSα and MutSß and interferes with PCNA-dependent activation of MutLα endonuclease, thereby inhibiting MMR at the initiation step. Evidence is also provided that Y211-phosphorylated PCNA induces nucleotide misincorporation during DNA synthesis. These findings reveal a novel mechanism by which Y211-phosphorylated PCNA promotes cancer development and progression via facilitating error-prone DNA replication and suppressing the MMR function.

HDAC6 deacetylates and ubiquitinates MSH2 to maintain proper levels of MutSα.

MutS protein homolog 2 (MSH2) is a key DNA mismatch repair protein. It forms the MSH2-MSH6 (MutSα) and MSH2-MSH3 (MutSß) heterodimers, which help to ensure genomic integrity. MutSα not only recognizes and repairs mismatched nucleotides but also recognizes DNA adducts induced by DNA-damaging agents, and triggers cell-cycle arrest and apoptosis. Loss or depletion of MutSα from cells leads to microsatellite instability (MSI) and resistance to DNA damage. Although the level of MutSα can be reduced by the ubiquitin-proteasome pathway, the detailed mechanisms of this regulation remain elusive. Here we report that histone deacetylase 6 (HDAC6) sequentially deacetylates and ubiquitinates MSH2, leading to MSH2 degradation. In addition, HDAC6 significantly reduces cellular sensitivity to DNA-damaging agents and decreases cellular DNA mismatch repair activities by downregulation of MSH2. Overall, these findings reveal a mechanism by which proper levels of MutSα are maintained.