Concerted SUMO-targeted ubiquitin ligase activities of TOPORS and RNF4 are essential for stress management and cell proliferation.

Protein SUMOylation provides a principal driving force for cellular stress responses, including DNA-protein crosslink (DPC) repair and arsenic-induced PML body degradation. In this study, using genome-scale screens, we identified the human E3 ligase TOPORS as a key effector of SUMO-dependent DPC resolution. We demonstrate that TOPORS promotes DPC repair by functioning as a SUMO-targeted ubiquitin ligase (STUbL), combining ubiquitin ligase activity through its RING domain with poly-SUMO binding via SUMO-interacting motifs, analogous to the STUbL RNF4. Mechanistically, TOPORS is a SUMO1-selective STUbL that complements RNF4 in generating complex ubiquitin landscapes on SUMOylated targets, including DPCs and PML, stimulating efficient p97/VCP unfoldase recruitment and proteasomal degradation. Combined loss of TOPORS and RNF4 is synthetic lethal even in unstressed cells, involving defective clearance of SUMOylated proteins from chromatin accompanied by cell cycle arrest and apoptosis. Our findings establish TOPORS as a STUbL whose parallel action with RNF4 defines a general mechanistic principle in crucial cellular processes governed by direct SUMO-ubiquitin crosstalk.

Mechanism and function of DNA replication-independent DNA-protein crosslink repair via the SUMO-RNF4 pathway.

DNA-protein crosslinks (DPCs) obstruct essential DNA transactions, posing a serious threat to genome stability and functionality. DPCs are proteolytically processed in a ubiquitin- and DNA replication-dependent manner by SPRTN and the proteasome but can also be resolved via targeted SUMOylation. However, the mechanistic basis of SUMO-mediated DPC resolution and its interplay with replication-coupled DPC repair remain unclear. Here, we show that the SUMO-targeted ubiquitin ligase RNF4 defines a major pathway for ubiquitylation and proteasomal clearance of SUMOylated DPCs in the absence of DNA replication. Importantly, SUMO modifications of DPCs neither stimulate nor inhibit their rapid DNA replication-coupled proteolysis. Instead, DPC SUMOylation provides a critical salvage mechanism to remove DPCs formed after DNA replication, as DPCs on duplex DNA do not activate interphase DNA damage checkpoints. Consequently, in the absence of the SUMO-RNF4 pathway cells are able to enter mitosis with a high load of unresolved DPCs, leading to defective chromosome segregation and cell death. Collectively, these findings provide mechanistic insights into SUMO-driven pathways underlying replication-independent DPC resolution and highlight their critical importance in maintaining chromosome stability and cellular fitness.

SUMOylation promotes protective responses to DNA-protein crosslinks.

DNA-protein crosslinks (DPCs) are highly cytotoxic lesions that obstruct essential DNA transactions and whose resolution is critical for cell and organismal fitness. However, the mechanisms by which cells respond to and overcome DPCs remain incompletely understood. Recent studies unveiled a dedicated DPC repair pathway in higher eukaryotes involving the SprT-type metalloprotease SPRTN/DVC1, which proteolytically processes DPCs during DNA replication in a ubiquitin-regulated manner. Here, we show that chemically induced and defined enzymatic DPCs trigger potent chromatin SUMOylation responses targeting the crosslinked proteins and associated factors. Consequently, inhibiting SUMOylation compromises DPC clearance and cellular fitness. We demonstrate that ACRC/GCNA family SprT proteases interact with SUMO and establish important physiological roles of Caenorhabditis elegans GCNA-1 and SUMOylation in promoting germ cell and embryonic survival upon DPC formation. Our findings provide first global insights into signaling responses to DPCs and reveal an evolutionarily conserved function of SUMOylation in facilitating responses to these lesions in metazoans that may complement replication-coupled DPC resolution processes.

An epidemic of allergic contact dermatitis caused by a new allergen, caprylhydroxamic acid, in moisturizers.

BACKGROUND: In 2016, dermatologists in Finland suspected contact allergy in several patients using moisturizers under the trade name Apobase®. Following a formulation change, Phenostat™, which is a mixture of phenoxyethanol, caprylhydroxamic acid, and methylpropanediol, was used as a preservative in Apobase® moisturizers in Finland. OBJECTIVES: To confirm the suspected contact allergy to Apobase® cream, oily cream, and/or lotion, and to identify the specific contact allergen and define its optimal patch test concentration. METHODS: Thirty-nine patients with suspected contact allergy to Apobase® creams or lotion were patch tested in four Finnish dermatological clinics. The patch tests included old and new Apobase® formulas and their preservative agents: phenoxyethanol, methylpropanediol, and dilution series of Phenostat™ and caprylhydroxamic acid or its potassium salt. RESULTS: The patch tests showed positive reactions to the new Apobase® formulas, Phenostat™, and caprylhydroxamic acid or its potassium salt, but not to the old Apobase® formulas, methylpropanediol, or phenoxyethanol. CONCLUSIONS: We found a new contact allergen, caprylhydroxyamic acid, which caused an epidemic of allergic contact dermatitis in patients using moisturizers containing this preservative. Whether the sensitizing capacity of caprylhydroxamic acid depends on the other chemicals used in Apobase® moisturizers needs further investigation.

E4 ligase-specific ubiquitination hubs coordinate DNA double-strand-break repair and apoptosis.

Multiple protein ubiquitination events at DNA double-strand breaks (DSBs) regulate damage recognition, signaling and repair. It has remained poorly understood how the repair process of DSBs is coordinated with the apoptotic response. Here, we identified the E4 ubiquitin ligase UFD-2 as a mediator of DNA-damage-induced apoptosis in a genetic screen in Caenorhabditis elegans. We found that, after initiation of homologous recombination by RAD-51, UFD-2 forms foci that contain substrate-processivity factors including the ubiquitin-selective segregase CDC-48 (p97), the deubiquitination enzyme ATX-3 (Ataxin-3) and the proteasome. In the absence of UFD-2, RAD-51 foci persist, and DNA damage-induced apoptosis is prevented. In contrast, UFD-2 foci are retained until recombination intermediates are removed by the Holliday-junction-processing enzymes GEN-1, MUS-81 or XPF-1. Formation of UFD-2 foci also requires proapoptotic CEP-1 (p53) signaling. Our findings establish a central role of UFD-2 in the coordination between the DNA-repair process and the apoptotic response.

Ring of Change: CDC48/p97 Drives Protein Dynamics at Chromatin.

The dynamic composition of proteins associated with nuclear DNA is a fundamental property of chromosome biology. In the chromatin compartment dedicated protein complexes govern the accurate synthesis and repair of the genomic information and define the state of DNA compaction in vital cellular processes such as chromosome segregation or transcription. Unscheduled or faulty association of protein complexes with DNA has detrimental consequences on genome integrity. Consequently, the association of protein complexes with DNA is remarkably dynamic and can respond rapidly to cellular signaling events, which requires tight spatiotemporal control. In this context, the ring-like AAA+ ATPase CDC48/p97 emerges as a key regulator of protein complexes that are marked with ubiquitin or SUMO. Mechanistically, CDC48/p97 functions as a segregase facilitating the extraction of substrate proteins from the chromatin. As such, CDC48/p97 drives molecular reactions either by directed disassembly or rearrangement of chromatin-bound protein complexes. The importance of this mechanism is reflected by human pathologies linked to p97 mutations, including neurodegenerative disorders, oncogenesis, and premature aging. This review focuses on the recent insights into molecular mechanisms that determine CDC48/p97 function in the chromatin environment, which is particularly relevant for cancer and aging research.

Multidisciplinary Management of Mastocytosis: Nordic Expert Group Consensus.

Mastocytosis is a heterogeneous group of diseases defined by an increased number and accumulation of mast cells, and often also by signs and symptoms of mast cell activation. Disease subtypes range from indolent to rare aggressive forms. Mastocytosis affects people of all ages and has been considered rare; however, it is probably underdiagnosed with potential severe implications. Diagnosis can be challenging and symptoms may be complex and involve multiple organ-systems. In general it is advised that patients should be referred to centres with experience in the disease offering an individualized, multidisciplinary approach. We present here consensus recommendations from a Nordic expert group for the diagnosis and general management of patients with mastocytosis.

Create and preserve: proteostasis in development and aging is governed by Cdc48/p97/VCP.

The AAA-ATPase Cdc48 (also called p97 or VCP) acts as a key regulator in proteolytic pathways, coordinating recruitment and targeting of substrate proteins to the 26S proteasome or lysosomal degradation. However, in contrast to the well-known function in ubiquitin-dependent cellular processes, the physiological relevance of Cdc48 in organismic development and maintenance of protein homeostasis is less understood. Therefore, studies on multicellular model organisms help to decipher how Cdc48-dependent proteolysis is regulated in time and space to meet developmental requirements. Given the importance of developmental regulation and tissue maintenance, defects in Cdc48 activity have been linked to several human pathologies including protein aggregation diseases. Thus, addressing the underlying disease mechanisms not only contributes to our understanding on the organism-wide function of Cdc48 but also facilitates the design of specific medical therapies. In this review, we will portray the role of Cdc48 in the context of multicellular organisms, pointing out its importance for developmental processes, tissue surveillance, and disease prevention. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

Ubiquitin C-terminal hydrolase-L1 potentiates cancer chemosensitivity by stabilizing NOXA.

The BH3-only protein NOXA represents one of the critical mediators of DNA-damage-induced cell death. In particular, its involvement in cellular responses to cancer chemotherapy is increasingly evident. Here, we identify a strategy of cancer cells to escape genotoxic chemotherapy by increasing proteasomal degradation of NOXA. We show that the deubiquitylating enzyme UCH-L1 is a key regulator of NOXA turnover, which protects NOXA from proteasomal degradation by removing Lys(48)-linked polyubiquitin chains. In the majority of tumors from patients with melanoma or colorectal cancer suffering from high rates of chemoresistance, NOXA fails to accumulate because UCH-L1 expression is epigenetically silenced. Whereas UCH-L1/NOXA-positive tumor samples exhibit increased sensitivity to genotoxic chemotherapy, downregulation of UCH-L1 or inhibition of its deubiquitylase activity resulted in reduced NOXA stability and resistance to genotoxic chemotherapy in both human and C. elegans cells. Our data identify the UCH-L1/NOXA interaction as a therapeutic target for overcoming cancer chemoresistance.

A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure.

The ubiquitin ligases RNF8 and RNF168 orchestrate DNA damage signalling through the ubiquitylation of histone H2A and the recruitment of downstream repair factors. Here, we demonstrate that RNF8, but not RNF168 or the canonical H2A ubiquitin ligase RNF2, mediates extensive chromatin decondensation. Our data show that CHD4, the catalytic subunit of the NuRD complex, interacts with RNF8 and is essential for RNF8-mediated chromatin unfolding. The chromatin remodelling activity of CHD4 promotes efficient ubiquitin conjugation and assembly of RNF168 and BRCA1 at DNA double-strand breaks. Interestingly, RNF8-mediated recruitment of CHD4 and subsequent chromatin remodelling were independent of the ubiquitin-ligase activity of RNF8, but involved a non-canonical interaction with the forkhead-associated (FHA) domain. Our study reveals a new mechanism of chromatin remodelling-assisted ubiquitylation, which involves the cooperation between CHD4 and RNF8 to create a local chromatin environment that is permissive to the assembly of checkpoint and repair machineries at DNA lesions.

The AAA-ATPase VCP/p97 promotes 53BP1 recruitment by removing L3MBTL1 from DNA double-strand breaks.

The accumulation of the human tumor suppressor 53BP1 at DNA damage sites requires the ubiquitin ligases RNF8 and RNF168. As 53BP1 recognizes dimethylated Lys20 in histone H4 (H4K20me2), the requirement for RNF8- and RNF168-mediated ubiquitylation has been unclear. Here we show that RNF8-mediated ubiquitylation facilitates the recruitment of the AAA-ATPase valosin-containing protein (VCP, also known as p97) and its cofactor NPL4 to sites of double-strand breaks. RIDDLE cells, which lack functional RNF168, also show impaired recruitment of VCP to DNA damage. The ATPase activity of VCP promotes the release of the Polycomb protein L3MBTL1 from chromatin, which also binds the H4K20me2 histone mark, thereby facilitating 53BP1 recruitment. Consistent with this, nematodes lacking the VCP orthologs CDC-48.1 or CDC-48.2, or cofactors UFD-1 or NPL-4, are highly sensitive to ionizing radiation. Our data suggest that human RNF8 and RNF168 promote VCP-mediated displacement of L3MBTL1 to unmask 53BP1 chromatin binding sites.

Contact sensitization to methylisothiazolinone in Finland--a multicentre study.

BACKGROUND: Antimicrobials constitute the second most common cause of contact allergy to cosmetics. Methylisothiazolinone (MI), previously always used together with methylchloroisothiazolinone (MCI), has recently been approved in the EU for use on its own in cosmetics and also various industrial products. MCI has been classified as an extreme-strong and MI as a strong-moderate sensitizer. OBJECTIVES: To study the frequency of positive patch test reactions to MI, and its relevance and relation to MCI/MI sensitivity, in Finland. METHODS: Over a period of 3 years (2006-2008), MI 0.1% (1000 ppm) and 0.03% (300 ppm) were patch tested in 10,821 patients at eight Finnish dermatological clinics. During 2008, patients with positive reactions to MI were asked to take part in a repeated open application test (ROAT). RESULTS: Of the patients tested, 1.4% and 0.6% showed positive patch test reactions to 0.1% and 0.03% MI, respectively. Sixty-six per cent of those who were MI-positive were also positive to 100 ppm MCI/MI. Thirty-three agreed to undergo the use test, and 10 of these gave positive results (30%). CONCLUSIONS: Our data show that MI used alone also potentially induces contact allergy. Careful monitoring is needed to determine whether or not this antimicrobial is safe to use in cosmetics.

Sensitization to thiourea derivatives among Finnish patients with suspected contact dermatitis.

BACKGROUND: Thiourea derivatives in rubber products may induce contact sensitization and allergic contact dermatitis. Sensitization is most often from neoprene rubber, but the multitude of possible sensitizing products has remained poorly characterized. OBJECTIVE: The aim of this study was to collect information on the occurrence of thiourea-related contact allergy and to show novel sources of sensitization. PATIENTS AND METHODS: A mixture of dibutyl-, diethyl-, and diphenylthiourea was included in patch test baseline series in five Finnish dermatology clinics during 2002-2007. In addition, an extended series of rubber chemicals was tested in patients with suspected rubber allergy. Sources of sensitization to thioureas were analysed in sensitized patients. RESULTS: Thiourea mix yielded positive patch test reactions in 59 of 15,100 patients (0.39%); 33/59 patients were also tested with individual rubber chemicals. Diethylthiourea was positive in 24/33, diphenylthiourea in 5, and dibutylthiourea in 1 patient. The most common sources of sensitization included various neoprene-containing orthopaedic braces, sports equipment, and foot wear. CONCLUSIONS: The sources of sensitization to thiourea chemicals were detected in most cases. These sources comprise a heterogenous group of products extending from orthopaedic materials to sports equipment.

Occupational allergic contact dermatitis from cinnamon including one case from airborne exposure.

BACKGROUND: The bark of the cinnamon tree is used as a spice; its flavour is from an essential oil containing mainly cinnamal. OBJECTIVE: To report new cases of occupational allergic contact dermatitis (ACD) from cinnamon and a novel case of airborne cinnamon contact allergy. METHODS: We examined the patient material of two dermatological clinics in Helsinki to find cinnamon contact allergic patients and review their clinical records. RESULTS: We found six patients with delayed contact allergy to cinnamon. In four patients, cinnamon was the main cause of occupational ACD. Three of them had dermatitis on their hands and one patient on the face and neck. In the latter case, the exposure was shown to be airborne. In addition, the fifth patient was occupationally sensitized to cinnamon, but it was not the main cause of his hand dermatitis. In the sixth patient, cinnamon allergy was considered to derive from cross-allergy to fragrances. Five of the patients reacted to cinnamal separately and in fragrance mix I. None of the six patients had immediate-type cinnamon allergy. CONCLUSIONS: Occupational contact allergy to cinnamon is rare but needs to be considered in workers handling foods. Cinnamal is possibly the main allergen in cinnamon.

1,2-benzisothiazolin-3-one in disposable polyvinyl chloride gloves for medical use.

BACKGROUND: Benzisothiazolinone is used as a slimicide in the manufacture of disposable powder-free polyvinyl chloride (PVC) gloves. We recently reported 6 patients from dentistry and health care probably sensitized to benzisothiazolinone in PVC gloves. OBJECTIVE: The study aimed to investigate how widely disposable PVC gloves for medical use on the Finnish market in 2006 contain benzisothiazolinone and to report new cases from 2 clinics in Helsinki. METHODS: 31 brands of disposable PVC gloves were analysed for their benzisothiazolinone content. We looked through the patient material of Helsinki University Central Hospital to find benzisothiazolinone allergic patients. We also described 3 previously unpublished benzisothiazolinone allergic patients from Finnish Institute of Occupational Health. RESULTS: 9 (30%) of the 31 glove brands contained 3-26 p.p.m. benzisothiazolinone. From the 2 clinics, we found 5 new benzisothiazolinone allergic patients who had used PVC gloves containing benzisothiazolinone. In addition, 3 patients had used disposable PVC gloves whose benzisothiazolinone content remained unknown. CONCLUSIONS: In Finland, benzisothiazolinone in powder-free PVC gloves has caused a small epidemic of allergic contact dermatitis in dental personnel and other health care workers. 1/3 of the disposable PVC gloves contained some benzisothiazolinone. A concentration of 20 p.p.m. benzisothiazolinone in a glove seems to be enough for sensitization.

Histamine, but not leukotriene C4, is an essential mediator in cold urticaria wheals.

In addition to histamine, leukotriene C4 (LTC4) might also play a role in mediating cold urticaria wheals. To study the significance of LTC4 vs. histamine, 6 patients with cold urticaria were challenged with the ice cube test before and after ingestion of 10 mg cetirizine (antihistamine), 10 mg montelukast (leukotriene antagonist) or a combination of both drugs. Cetirizine diminished the cold-induced wheal by 50+/- 42%. Montelukast had no significant effect, and the combination of both drugs diminished the wheal by 37+/- 33%. Furthermore, a skin microdialysis technique detected the release of histamine in the cold-induced wheal, whereas no LTC4 release was detected. In conclusion, the antihistamine is effective and histamine is released, whereas the leukotriene antagonist is not effective and LTC4 is not released in the cold urticaria wheal.