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Tumor cells often exhibit augmented capacity to maintain endoplasmic reticulum (ER) homeostasis under adverse conditions, yet the underlying mechanisms are not well defined. Here, through the evaluation of all human TRIM proteins, we find that TRIM25 is significantly induced upon ER stress. Upregulation of TRIM25 ameliorates oxidative stress, promotes ER-associated degradation (ERAD), and reduces IRE1 signaling in the UPR pathway. In contrast, depletion of TRIM25 leads to ER stress and attenuates tumor cell growth in vitro and in vivo. Mechanistically, TRIM25 directly targets Keap1 by ubiquitination and degradation. This leads to Nrf2 activation, which bolsters anti-oxidant defense and cell survival. TRIM25 expression is positively associated with Nrf2 expression and negatively with Keap1 expression in hepatocellular carcinoma (HCC) xenografts and specimens. Moreover, high TRIM25 expression correlates with poor patient survival in HCC. These findings reveal TRIM25 as a regulator of ER homeostasis and a potential target for tumor therapy. The unfolded protein response allows tumour cells to adapt to ER stress, and aberrant activation of Nrf2 confers cancer progression. Here, the authors show that TRIM25 is induced during ER stress and promotes tumour cell survival by targeting Keap1 for degradation, leading to Nrf2 activation.

Age is the dominant risk factor for most chronic human diseases, but the mechanisms through which ageing confers this risk are largely unknown 1 . The age-related acquisition of somatic mutations that lead to clonal expansion in regenerating haematopoietic stem cell populations has recently been associated with both haematological cancer 2 – 4 and coronary heart disease 5 —this phenomenon is termed clonal haematopoiesis of indeterminate potential (CHIP) 6 . Simultaneous analyses of germline and somatic whole-genome sequences provide the opportunity to identify root causes of CHIP. Here we analyse high-coverage whole-genome sequences from 97,691 participants of diverse ancestries in the National Heart, Lung, and Blood Institute Trans-omics for Precision Medicine (TOPMed) programme, and identify 4,229 individuals with CHIP. We identify associations with blood cell, lipid and inflammatory traits that are specific to different CHIP driver genes. Association of a genome-wide set of germline genetic variants enabled the identification of three genetic loci associated with CHIP status, including one locus at TET2 that was specific to individuals of African ancestry. In silico-informed in vitro evaluation of the TET2 germline locus enabled the identification of a causal variant that disrupts a TET2 distal enhancer, resulting in increased self-renewal of haematopoietic stem cells. Overall, we observe that germline genetic variation shapes haematopoietic stem cell function, leading to CHIP through mechanisms that are specific to clonal haematopoiesis as well as shared mechanisms that lead to somatic mutations across tissues. Analysis of 97,691 high-coverage human blood DNA-derived whole-genome sequences enabled simultaneous identification of germline and somatic mutations that predispose individuals to clonal expansion of haematopoietic stem cells, indicating that both inherited and acquired mutations are linked to age-related cancers and coronary heart disease.

The host factor and interferon (IFN)-stimulated gene (ISG) product, zinc-finger antiviral protein (ZAP), inhibits a number of diverse viruses by usurping and intersecting with multiple cellular pathways. To elucidate its antiviral mechanism, we perform a loss-of-function genome-wide RNAi screen to identify cellular cofactors required for ZAP antiviral activity against the prototype alphavirus, Sindbis virus (SINV). In order to exclude off-target effects, we carry out stringent confirmatory assays to verify the top hits. Important ZAP-liaising partners identified include proteins involved in membrane ion permeability, type I IFN signaling, and post-translational protein modification. The factor contributing most to the antiviral function of ZAP is TRIM25, an E3 ubiquitin and ISG15 ligase. We demonstrate here that TRIM25 interacts with ZAP through the SPRY domain, and TRIM25 mutants lacking the RING or coiled coil domain fail to stimulate ZAP's antiviral activity, suggesting that both TRIM25 ligase activity and its ability to form oligomers are critical for its cofactor function. TRIM25 increases the modification of both the short and long ZAP isoforms by K48- and K63-linked polyubiquitin, although ubiquitination of ZAP does not directly affect its antiviral activity. However, TRIM25 is critical for ZAP's ability to inhibit translation of the incoming SINV genome. Taken together, these data uncover TRIM25 as a bona fide ZAP cofactor that leads to increased ZAP modification enhancing its translational inhibition activity.

Radiotherapy is the primary treatment for patients with nasopharyngeal carcinoma (NPC), and approximately 20% of patients experience treatment failure due to tumour radioresistance. However, the exact regulatory mechanism remains poorly understood. Here, we show that the deubiquitinase USP44 is hypermethylated in NPC, which results in its downregulation. USP44 enhances the sensitivity of NPC cells to radiotherapy in vitro and in vivo. USP44 recruits and stabilizes the E3 ubiquitin ligase TRIM25 by removing its K48-linked polyubiquitin chains at Lys439, which further facilitates the degradation of Ku80 and inhibits its recruitment to DNA double-strand breaks (DSBs), thus enhancing DNA damage and inhibiting DNA repair via non-homologous end joining (NHEJ). Knockout of TRIM25 reverses the radiotherapy sensitization effect of USP44. Clinically, low expression of USP44 indicates a poor prognosis and facilitates tumour relapse in NPC patients. This study suggests the USP44-TRIM25-Ku80 axis provides potential therapeutic targets for NPC patients. Radiotherapy is the mainstay treatment for nasopharyngeal carcinoma (NPC). Here the authors show that the deubiquitinase, USP44, increases radiosensitivity of NPC cells by promoting the degradation of Ku80, and thus enhancing the levels of DNA damage.

NF-κB signaling plays a critical role in tumor growth and treatment resistance in GBM as in many other cancers. However, the molecular mechanisms underlying high, constitutive NF-κB activity in GBM remains to be elucidated. Here, we screened a panel of tripartite motif (TRIM) family proteins and identified TRIM22 as a potential activator of NF-κB using an NF-κB driven luciferase reporter construct in GBM cell lines. Knockout of TRIM22 using Cas9-sgRNAs led to reduced GBM cell proliferation, while TRIM22 overexpression enhanced proliferation of cell populations, in vitro and in an orthotopic xenograft model. However, two TRIM22 mutants, one with a critical RING-finger domain deletion and the other with amino acid changes at two active sites of RING E3 ligase (C15/18A), were both unable to promote GBM cell proliferation over controls, thus implicating E3 ligase activity in the growth-promoting properties of TRIM22. Co-immunoprecipitations demonstrated that TRIM22 bound a negative regulator of NF-κB, NF-κB inhibitor alpha (IκBα), and accelerated its degradation by inducing K48-linked ubiquitination. TRIM22 also formed a complex with the NF-κB upstream regulator IKKγ and promoted K63-linked ubiquitination, which led to the phosphorylation of both IKKα/β and IκBα. Expression of a non-phosphorylation mutant, srIκBα, inhibited the growth-promoting properties of TRIM22 in GBM cell lines. Finally, TRIM22 was increased in a cohort of primary GBM samples on a tissue microarray, and high expression of TRIM22 correlated with other clinical parameters associated with progressive gliomas, such as wild-type IDH1 status. In summary, our study revealed that TRIM22 activated NF-κB signaling through posttranslational modification of two critical regulators of NF-κB signaling in GBM cells.

Hydrocephalus, characterized by cerebral ventricular dilatation, is routinely attributed to primary defects in cerebrospinal fluid (CSF) homeostasis. This fosters CSF shunting as the leading reason for brain surgery in children despite considerable disease heterogeneity. In this study, by integrating human brain transcriptomics with whole-exome sequencing of 483 patients with congenital hydrocephalus (CH), we found convergence of CH risk genes in embryonic neuroepithelial stem cells. Of all CH risk genes, TRIM71/lin-41 harbors the most de novo mutations and is most specifically expressed in neuroepithelial cells. Mice harboring neuroepithelial cell-specific Trim71 deletion or CH-specific Trim71 mutation exhibit prenatal hydrocephalus. CH mutations disrupt TRIM71 binding to its RNA targets, causing premature neuroepithelial cell differentiation and reduced neurogenesis. Cortical hypoplasia leads to a hypercompliant cortex and secondary ventricular enlargement without primary defects in CSF circulation. These data highlight the importance of precisely regulated neuroepithelial cell fate for normal brain–CSF biomechanics and support a clinically relevant neuroprogenitor-based paradigm of CH.Duy and Weise et al. combined human functional integrative genomics with mouse experimental biology to reveal a neuroprogenitor-based genetic subtype of human hydrocephalus with defective neurogenesis and altered brain–fluid biomechanics.

Defects in plasma membrane repair can lead to muscle and heart diseases in humans. Tripartite motif-containing protein (TRIM)72 (mitsugumin 53; MG53) has been determined to rapidly nucleate vesicles at the site of membrane damage, but the underlying molecular mechanisms remain poorly understood. Here we present the structure of Mus musculus TRIM72, a complete model of a TRIM E3 ubiquitin ligase. We demonstrated that the interaction between TRIM72 and phosphatidylserine-enriched membranes is necessary for its oligomeric assembly and ubiquitination activity. Using cryogenic electron tomography and subtomogram averaging, we elucidated a higher-order model of TRIM72 assembly on the phospholipid bilayer. Combining structural and biochemical techniques, we developed a working molecular model of TRIM72, providing insights into the regulation of RING-type E3 ligases through the cooperation of multiple domains in higher-order assemblies. Our findings establish a fundamental basis for the study of TRIM E3 ligases and have therapeutic implications for diseases associated with membrane repair. The authors present the full-length dimeric TRIM72 E3 ubiquitin ligase and the architecture of its high-order assembly bound to a phosphatidylserine-enriched membrane, providing insights into its role in membrane repair and ubiquitylation.

Cisplatin resistance is the main cause of treatment failure in patients with non-small-cell lung cancer (NSCLC). Autophagy is a key mechanism of resistance to chemotherapy. Given that tripartite motif (TRIM)-containing proteins are involved in the regulation of autophagy and chemoresistance, we aimed to study the functions of TRIM protein members in autophagy-mediated chemoresistance of NSCLC. We found that TRIM65 was significantly increased in cisplatin-resistant NSCLC cell line (A549/DDP) as compared to the parental cell line (A549). Knockdown of TRIM65 can enhance cisplatin-induced apoptosis and inhibit autophagy in A549/DDP cells, as indicated by Annexin V/PI staining, caspase3 activity test, and LC3-II immunofluorescence staining. Additionally, knockdown of TRIM65 significantly decreased the expression of an important autophagy mediator, ATG7, which was a potential target of miR-138-5p. miR-138-5p inhibitor significantly abolished the effects of TRIM65 knockdown on autophagy and cisplatin-induced apoptosis. Moreover, TRIM65 induced the ubiquitination and degradation of TNRC6A, resulting in the suppressed expression of miR-138-5p. TRIM65 knockdown inhibited the growth of tumors derived from A549/DDP cells. Furthermore, cisplatin-resistant NSCLC tissues displayed higher expression of TRIM65 mRNA and lower expression of miR-138-5p as compared to cisplatin non-resistant ones. miR-138-5p expression was negatively correlated with TRIM65 mRNA in NSCLC tissues. Collectively, the present study indicates that TRIM65 knockdown attenuates autophagy and cisplatin resistance in A549/DDP cells via regulating miR-138-5p.

Genomic instability is a hallmark of cancer, and has a central role in the initiation and development of breast cancer 1 , 2 . The success of poly-ADP ribose polymerase inhibitors in the treatment of breast cancers that are deficient in homologous recombination exemplifies the utility of synthetically lethal genetic interactions in the treatment of breast cancers that are driven by genomic instability 3 . Given that defects in homologous recombination are present in only a subset of breast cancers, there is a need to identify additional driver mechanisms for genomic instability and targeted strategies to exploit these defects in the treatment of cancer. Here we show that centrosome depletion induces synthetic lethality in cancer cells that contain the 17q23 amplicon, a recurrent copy number aberration that defines about 9% of all primary breast cancer tumours and is associated with high levels of genomic instability 4 – 6 . Specifically, inhibition of polo-like kinase 4 (PLK4) using small molecules leads to centrosome depletion, which triggers mitotic catastrophe in cells that exhibit amplicon-directed overexpression of TRIM37 . To explain this effect, we identify TRIM37 as a negative regulator of centrosomal pericentriolar material. In 17q23-amplified cells that lack centrosomes, increased levels of TRIM37 block the formation of foci that comprise pericentriolar material—these foci are structures with a microtubule-nucleating capacity that are required for successful cell division in the absence of centrosomes. Finally, we find that the overexpression of TRIM37 causes genomic instability by delaying centrosome maturation and separation at mitotic entry, and thereby increases the frequency of mitotic errors. Collectively, these findings highlight TRIM37- dependent genomic instability as a putative driver event in 17q23-amplified breast cancer and provide a rationale for the use of centrosome-targeting therapeutic agents in treating these cancers. TRIM37 overexpression promotes centrosome dysfunction that drives genomic instability in breast cancer cell lines containing the recurrent 17q23 amplicon, revealing a vulnerability that can be targeted to eliminate cancer cells.

Sarcopenia due to loss of skeletal muscle mass and strength leads to physical inactivity and decreased quality of life. The number of individuals with sarcopenia is rapidly increasing as the number of older people increases worldwide, making this condition a medical and social problem. Some patients with sarcopenia exhibit accumulation of peri-muscular adipose tissue (PMAT) as ectopic fat deposition surrounding atrophied muscle. However, an association of PMAT with muscle atrophy has not been demonstrated. Here, we show that PMAT is associated with muscle atrophy in aged mice and that atrophy severity increases in parallel with cumulative doses of PMAT. We observed severe muscle atrophy in two different obese model mice harboring significant PMAT relative to respective control non-obese mice. We also report that denervation-induced muscle atrophy was accelerated in non-obese young mice transplanted around skeletal muscle with obese adipose tissue relative to controls transplanted with non-obese adipose tissue. Notably, transplantation of obese adipose tissue into peri-muscular regions increased nuclear translocation of FoxO transcription factors and upregulated expression FoxO targets associated with proteolysis (Atrogin1 and MuRF1) and cellular senescence (p19 and p21) in muscle. Conversely, in obese mice, PMAT removal attenuated denervation-induced muscle atrophy and suppressed upregulation of genes related to proteolysis and cellular senescence in muscle. We conclude that PMAT accumulation accelerates age- and obesity-induced muscle atrophy by increasing proteolysis and cellular senescence in muscle.