The dimeric deubiquitinase USP28 integrates 53BP1 and MYC functions to limit DNA damage.

DNA replication is a major source of endogenous DNA damage in tumor cells and a key target of cellular response to genotoxic stress. DNA replication can be deregulated by oncoproteins, such as transcription factor MYC, aberrantly activated in many human cancers. MYC is stringently regulated by the ubiquitin system - for example, ubiquitination controls recruitment of the elongation factor PAF1c, instrumental in MYC activity. Curiously, a key MYC-targeting deubiquitinase USP28 also controls cellular response to DNA damage via the mediator protein 53BP1. USP28 forms stable dimers, but the biological role of USP28 dimerization is unknown. We show here that dimerization limits USP28 activity and restricts recruitment of PAF1c by MYC. Expression of monomeric USP28 stabilizes MYC and promotes PAF1c recruitment, leading to ectopic DNA synthesis and replication-associated DNA damage. USP28 dimerization is stimulated by 53BP1, which selectively binds USP28 dimers. Genotoxic stress diminishes 53BP1-USP28 interaction, promotes disassembly of USP28 dimers and stimulates PAF1c recruitment by MYC. This triggers firing of DNA replication origins during early response to genotoxins and exacerbates DNA damage. We propose that dimerization of USP28 prevents ectopic DNA replication at transcriptionally active chromatin to maintain genome stability.

Trim33 masks a non-transcriptional function of E2f4 in replication fork progression.

Replicative stress promotes genomic instability and tumorigenesis but also presents an effective therapeutic endpoint, rationalizing detailed analysis of pathways that control DNA replication. We show here that the transcription factor E2f4 recruits the DNA helicase Recql to facilitate progression of DNA replication forks upon drug- or oncogene-induced replicative stress. In unperturbed cells, the Trim33 ubiquitin ligase targets E2f4 for degradation, limiting its genomic binding and interactions with Recql. Replicative stress blunts Trim33-dependent ubiquitination of E2f4, which stimulates transient Recql recruitment to chromatin and facilitates recovery of DNA synthesis. In contrast, deletion of Trim33 induces chronic genome-wide recruitment of Recql and strongly accelerates DNA replication under stress, compromising checkpoint signaling and DNA repair. Depletion of Trim33 in Myc-overexpressing cells leads to accumulation of replication-associated DNA damage and delays Myc-driven tumorigenesis. We propose that the Trim33-E2f4-Recql axis controls progression of DNA replication forks along transcriptionally active chromatin to maintain genome integrity.

RNAPII-dependent ATM signaling at collisions with replication forks.

Deregulation of RNA Polymerase II (RNAPII) by oncogenic signaling leads to collisions of RNAPII with DNA synthesis machinery (transcription-replication conflicts, TRCs). TRCs can result in DNA damage and are thought to underlie genomic instability in tumor cells. Here we provide evidence that elongating RNAPII nucleates activation of the ATM kinase at TRCs to stimulate DNA repair. We show the ATPase WRNIP1 associates with RNAPII and limits ATM activation during unperturbed cell cycle. WRNIP1 binding to elongating RNAPII requires catalytic activity of the ubiquitin ligase HUWE1. Mutation of HUWE1 induces TRCs, promotes WRNIP1 dissociation from RNAPII and binding to the replisome, stimulating ATM recruitment and activation at RNAPII. TRCs and translocation of WRNIP1 are rapidly induced in response to hydroxyurea treatment to activate ATM and facilitate subsequent DNA repair. We propose that TRCs can provide a controlled mechanism for stalling of replication forks and ATM activation, instrumental in cellular response to replicative stress.

USP28 controls SREBP2 and the mevalonate pathway to drive tumour growth in squamous cancer.

SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A, ubiquinone and cholesterol and also provides substrates for protein prenylation. Here, we identify SREBP2 as a novel substrate for USP28, a deubiquitinating enzyme that is frequently upregulated in squamous cancers. Our results show that silencing of USP28 reduces expression of MVP enzymes and lowers metabolic flux into this pathway. We also show that USP28 binds to mature SREBP2, leading to its deubiquitination and stabilisation. USP28 depletion rendered cancer cells highly sensitive to MVP inhibition by statins, which was rescued by the addition of geranyl-geranyl pyrophosphate. Analysis of human tissue microarrays revealed elevated expression of USP28, SREBP2 and MVP enzymes in lung squamous cell carcinoma (LSCC) compared to lung adenocarcinoma (LADC). Moreover, CRISPR/Cas-mediated deletion of SREBP2 selectively attenuated tumour growth in a KRas/p53/LKB1 mutant mouse model of lung cancer. Finally, we demonstrate that statins synergise with a dual USP28/25 inhibitor to reduce viability of SCC cells. Our findings suggest that combinatorial targeting of MVP and USP28 could be a therapeutic strategy for the treatment of squamous cell carcinomas.

LXRα activation and Raf inhibition trigger lethal lipotoxicity in liver cancer.

The success of molecular therapies targeting specific metabolic pathways in cancer is often limited by the plasticity and adaptability of metabolic networks. Here we show that pharmacologically induced lipotoxicity represents a promising therapeutic strategy for the treatment of hepatocellular carcinoma (HCC). LXRα-induced liponeogenesis and Raf-1 inhibition are synthetic lethal in HCC owing to a toxic accumulation of saturated fatty acids. Raf-1 was found to bind and activate SCD1, and conformation-changing DFG-out Raf inhibitors could disrupt this interaction, thereby blocking fatty acid desaturation and inducing lethal lipotoxicity. Studies in genetically engineered and nonalcoholic steatohepatitis-induced HCC mouse models and xenograft models of human HCC revealed that therapies comprising LXR agonists and Raf inhibitors were well tolerated and capable of overcoming therapy resistance in HCC. Conceptually, our study suggests pharmacologically induced lipotoxicity as a new mode for metabolic targeting of liver cancer.

Human collectin-11 (COLEC11) and its synergic genetic interaction with MASP2 are associated with the pathophysiology of Chagas Disease.

Chagas Disease (CD) is an anthropozoonosis caused by Trypanosoma cruzi. With complex pathophysiology and variable clinical presentation, CD outcome can be influenced by parasite persistence and the host immune response. Complement activation is one of the primary defense mechanisms against pathogens, which can be initiated via pathogen recognition by pattern recognition molecules (PRMs). Collectin-11 is a multifunctional soluble PRM lectin, widely distributed throughout the body, with important participation in host defense, homeostasis, and embryogenesis. In complex with mannose-binding lectin-associated serine proteases (MASPs), collectin-11 may initiate the activation of complement, playing a role against pathogens, including T. cruzi. In this study, collectin-11 plasma levels and COLEC11 variants in exon 7 were assessed in a Brazilian cohort of 251 patients with chronic CD and 108 healthy controls. Gene-gene interactions between COLEC11 and MASP2 variants were analyzed. Collectin-11 levels were significantly decreased in CD patients compared to controls (p<0.0001). The allele rs7567833G, the genotypes rs7567833AG and rs7567833GG, and the COLEC11*GGC haplotype were related to T. cruzi infection and clinical progression towards symptomatic CD. COLEC11 and MASP2*CD risk genotypes were associated with cardiomyopathy (p = 0.014; OR 9.3, 95% CI 1.2-74) and with the cardiodigestive form of CD (p = 0.005; OR 15.2, 95% CI 1.7-137), suggesting that both loci act synergistically in immune modulation of the disease. The decreased levels of collectin-11 in CD patients may be associated with the disease process. The COLEC11 variant rs7567833G and also the COLEC11 and MASP2*CD risk genotype interaction were associated with the pathophysiology of CD.

The systemin receptor SYR1 enhances resistance of tomato against herbivorous insects.

The discovery in tomato of systemin, the first plant peptide hormone1,2, was a fundamental change for the concept of plant hormones. Numerous other peptides have since been shown to play regulatory roles in many aspects of the plant life, including growth, development, fertilization and interactions with symbiotic organisms3-6. Systemin, an 18 amino acid peptide derived from a larger precursor protein 7 , was proposed to act as the spreading signal that triggers systemic defence responses observed in plants after wounding or attack by herbivores1,7,8. Further work culminated in the identification of a leucine-rich repeat receptor kinase (LRR-RK) as the systemin receptor 160 (SR160)9,10. SR160 is a tomato homologue of Brassinosteroid Insensitive 1 (BRI1), which mediates the regulation of growth and development in response to the steroid hormone brassinolide11-13. However, a role of SR160/BRI1 as systemin receptor could not be corroborated by others14-16. Here, we demonstrate that perception of systemin depends on a pair of distinct LRR-RKs termed SYR1 and SYR2. SYR1 acts as a genuine systemin receptor that binds systemin with high affinity and specificity. Further, we show that presence of SYR1, although not decisive for local and systemic wound responses, is important for defence against insect herbivory.

Author Correction: The pattern-recognition receptor CORE of Solanaceae detects bacterial cold-shock protein.

In the version of this Article originally published, Fig. 6b, which is composed of individual pictures of six plants, inadvertently and erroneously displayed the same image of one Col-0 wt plant twice. This has been corrected so that Fig. 6b now shows two different representative plants for the Col-0 wt control.

The pattern-recognition receptor CORE of Solanaceae detects bacterial cold-shock protein.

Plants and animals recognize microbial invaders by detecting microbe-associated molecular patterns (MAMPs) by cell surface receptors. Many plant species of the Solanaceae family detect the highly conserved nucleic acid binding motif RNP-1 of bacterial cold-shock proteins (CSPs), represented by the peptide csp22, as a MAMP. Here, we exploited the natural variation in csp22 perception observed between cultivated tomato (Solanum lycopersicum) and Solanum pennellii to map and identify the leucine-rich repeat (LRR) receptor kinase CORE (cold shock protein receptor) of tomato as the specific, high-affinity receptor site for csp22. Corroborating its function as a genuine receptor, heterologous expression of CORE in Arabidopsis thaliana conferred full sensitivity to csp22 and, importantly, it also rendered these plants more resistant to infection by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Our study also confirms the biotechnological potential of enhancing plant immunity by interspecies transfer of highly effective pattern-recognition receptors such as CORE to different plant families.