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DNA-protein crosslinks (DPCs) are highly toxic DNA lesions that threaten genomic integrity. Recent findings highlight that SPRTN, a specialized DNA-dependent metalloprotease, is a central player in proteolytic cleavage of DPCs. Previous studies suggest that SPRTN deubiquitination is important for its chromatin association and activation. However, the regulation and consequence of SPRTN deubiquitination remain unclear. Here, we report that in response to DPC induction, the deubiquitinase VCPIP1/VCIP135 is phosphorylated and activated by ATM/ATR. VCPIP1, in turn, deubiquitinates SPRTN and promotes its chromatin relocalization. The deubiquitination of SPRTN is required for its subsequent acetylation, which promotes SPRTN relocation to the site of chromatin damage. Furthermore, Vcpip1 knockout mice are prone to genomic instability and premature aging. We propose a model that two sequential post-translational modifications (PTMs) regulate SPRTN chromatin accessibility to repair DPCs and maintain genomic stability and healthy life span.

Summary The family of bacterial SidE enzymes catalyzes non-canonical phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophila. Here, we describe identification of two bacterial effectors that reverse PR ubiquitination and are thus named deubiquitinases for PR ubiquitination (DUPs; DupA and DupB). Structural analyses revealed that DupA and SidE ubiquitin ligases harbor a highly homologous catalytic phosphodiesterase (PDE) domain. However, unlike SidE ubiquitin ligases, DupA displays increased affinity to PR-ubiquitinated substrates, which allows DupA to cleave PR ubiquitin from substrates. Interfering with DupA-ubiquitin binding switches its activity toward SidE-type ligase. Given the high affinity of DupA to PR-ubiquitinated substrates, we exploited a catalytically inactive DupA mutant to trap and identify more than 180 PR-ubiquitinated host proteins in Legionella-infected cells. Proteins involved in endoplasmic reticulum (ER) fragmentation and membrane recruitment to Legionella-containing vacuoles (LCV) emerged as major SidE targets. The global map of PR-ubiquitinated substrates provides critical insights into host-pathogen interactions during Legionella infection., Graphical Abstract, Highlights • Phosphoribosyl serine ubiquitination can be reverted by DupA and DupB • DUP specifically binds to and cleaves PR-ubiquitin from serine on substrates • Catalytically inactive DupA mutants can capture PR-ubiquitinated proteins • PR ubiquitination on multiple ER structural proteins causes ER fragmentation, Shin et al. show that phosphoribosyl serine ubiquitination can be reversed by two deubiquitinases (DupA and DupB) from Legionella. Two DUPs specifically cleave PR-ubiquitin from serine on substrates. Catalytically inactive DupA-based proteomics approach reveals PR-ubiquitinated proteins and their roles in endoplasmic reticulum (ER) remodeling.

Summary Poly-ubiquitin chains direct protein substrates to the 26S proteasome, where they are removed by the deubiquitinase Rpn11 during ATP-dependent substrate degradation. Rapid deubiquitination is required for efficient degradation but must be restricted to committed substrates that are engaged with the ATPase motor to prevent premature ubiquitin chain removal and substrate escape. Here we reveal the ubiquitin-bound structure of Rpn11 from S. cerevisiae and the mechanisms for mechanochemical coupling of substrate degradation and deubiquitination. Ubiquitin binding induces a conformational switch of Rpn11's Insert-1 loop from an inactive closed state to an active β hairpin. This switch is rate-limiting for deubiquitination and strongly accelerated by mechanical substrate translocation into the AAA+ motor. Deubiquitination by Rpn11 and ubiquitin unfolding by the ATPases are in direct competition. The AAA+ motor-driven acceleration of Rpn11 is therefore important to ensure that poly-ubiquitin chains are removed only from committed substrates and fast enough to prevent their co-degradation.

Many infections and stress signals can rapidly activate the NLRP3 inflammasome to elicit robust inflammatory responses. This activation requires a priming step, which is thought to be mainly for upregulating NLRP3 transcription. However, recent studies report that the NLRP3 inflammasome can be activated independently of transcription, suggesting that the priming process has unknown essential regulatory steps. Here, we report that JNK1-mediated NLRP3 phosphorylation at S194 is a critical priming event and is essential for NLRP3 inflammasome activation. We show that NLRP3 inflammasome activation is disrupted in NLRP3-S194A knockin mice. JNK1-mediated NLRP3 S194 phosphorylation is critical for NLRP3 deubiquitination and facilitates its self-association and the subsequent inflammasome assembly. Importantly, we demonstrate that blocking S194 phosphorylation prevents NLRP3 inflammasome activation in cryopyrin-associated periodic syndromes (CAPS). Thus, our study reveals a key priming molecular event that is a prerequisite for NLRP3 inflammasome activation. Inhibiting NLRP3 phosphorylation could be an effective treatment for NLRP3-related diseases.

Summary In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions., Graphical Abstract, Highlights • Detailed structural studies of the BRCA1-A and BRISC-SHMT2 protein complex • Complex-specific subunits functionalize BRCC36, regulating its activity and targeting • BRCA1-A sequesters BRCA1 with nanomolar affinity • Cancer-associated metabolic enzyme SHMT2 inhibits BRISC deubiquitination activity, Structural studies of the BRCA1-A and BRISC complexes reveal a common enzymatic core that is functionalized by complex-specific proteins. BRCA1-A localizes to DNA double-strand breaks, sequesters BRCA1, and suppresses DNA resection. BRISC regulates protein degradation by the endosomal-lysosomal pathway through deubiquitination and is inhibited by the metabolic enzyme SHMT2.

Nrf2 is a master regulator of the antioxidant response. Under basal conditions, Nrf2 is polyubiquitinated by the Keap1-Cul3 E3 ligase and degraded by the 26S proteasome. In response to Nrf2 inducers there is a switch in polyubiquitination from Nrf2 to Keap1. Currently, regulation of the Nrf2-Keap1 pathway by ubiquitination is largely understood. However, the mechanism responsible for removal of ubiquitin conjugated to Nrf2 or Keap1 remains unknown. Here we report that the deubiquitinating enzyme, USP15, specifically deubiquitinates Keap1, which suppresses the Nrf2 pathway. We demonstrated that deubiquitinated Keap1 incorporates into the Keap1-Cul3-E3 ligase complex more efficiently, enhancing the complex stability and enzymatic activity. Consequently, there is an increase in Nrf2 protein degradation and a reduction in Nrf2 target gene expression. Furthermore, USP15-siRNA enhances chemoresistance of cells through upregulation of Nrf2. These findings further our understanding of how the Nrf2-Keap1 pathway is regulated, which is imperative in targeting this pathway for chemoprevention or chemotherapy.

International audience; NLRP3 is an important pattern recognition receptor involved in mediating inflammasome activation in response to viral and bacterial infections as well as various proinflammatory stimuli associated with tissue damage or malfunction. Upon activation, NLRP3 assembles a multimeric inflammasome complex comprising the adaptor ASC and the effector pro-caspase-1 to mediate the activation of caspase-1. Although NLRP3 expression is induced by the NF-κB pathway, the posttranscriptional molecular mechanism controlling the activation of NLRP3 remains elusive. Using both pharmacological and molecular approaches, we show that the activation of NLRP3 inflammasome is regulated by a deubiquitination mechanism. We further identify the deubiquitinating enzyme, BRCC3, as a critical regulator of NLRP3 activity by promoting its deubiquitination and characterizing NLRP3 as a substrate for the cytosolic BRCC3-containing BRISC complex. Our results elucidate a regulatory mechanism involving BRCC3-dependent NLRP3 regulation and highlight NLRP3 ubiquitination as a potential therapeutic target for inflammatory diseases.

Ubiquitination typically occurs through the sequential action of three enzymes catalyzing ubiquitin activation (E1), conjugation (E2), and ligation (E3) and regulates diverse eukaryotic cellular processes. Although monoubiquitination commonly confers nondegradative activities, mechanisms underlying its temporal and spatial regulation and functional plasticity still remain largely unknown. Here we demonstrate that SETDB1, a major histone H3K9 methyltransferase is monoubiquitinated at the evolutionarily conserved lysine-867 in its SET-Insertion domain. This ubiquitination is directly catalyzed by UBE2E family of E2 enzymes in an E3-independent manner while the conjugated-ubiquitin (Ub) is protected from active deubiquitination. The resulting constitutive lysine-867 monoubiquitination is essential for SETDB1's enzymatic activity and endogenous retrovirus silencing in murine embryonic stem cells. Furthermore, the canonical hydrophobic patch on the conjugated-Ub is critical for Ub protection and function. Together, our findings highlight an E3-independent mechanism for monoubiquitination and reveal mechanistic details of SETDB1's enzymatic activity and the functional significance of its SET-Insertion.

Eukaryotic cells target proteins for degradation by the 26S proteasome by attaching a ubiquitin chain. Using a rapid assay, we analyzed the initial binding of ubiquitinated proteins to purified 26S particles as an isolated process at 4°C. Subunits Rpn10 and Rpn13 contribute equally to the high-affinity binding of ubiquitin chains, but in their absence, ubiquitin conjugates bind to another site with 4-fold lower affinity. Conjugate binding is stimulated 2- to 4-fold by binding of ATP or the nonhydrolyzable analog, ATPγS (but not ADP), to the 19S ATPases. Following this initial, reversible association, ubiquitin conjugates at 37°C become more tightly bound through a step that requires ATP hydrolysis and a loosely folded domain on the protein, but appears independent of ubiquitin. Unfolded or loosely folded polypeptides can inhibit this tighter binding. This commitment step precedes substrate deubiquitination and allows for selection of ubiquitinated proteins capable of being unfolded and efficiently degraded.

In eukaryotic cells, ubiquitination of proteins leads to their degradation by the 26S proteasome. We tested if the ubiquitin (Ub) chain also regulates the proteasome's capacity for proteolysis. After incubation with polyubiquitinated proteins, 26S proteasomes hydrolyzed peptides and proteins 2- to 7-fold faster. Ub conjugates enhanced peptide hydrolysis by stimulating gate opening in the 20S proteasome. This stimulation was seen when this gate was closed or transiently open, but not maximally open. Gate opening requires conjugate association with Usp14/Ubp6 and also occurs if Ub aldehyde occupies this isopeptidase's active site. No stimulation was observed with 26S from Ubp6Delta mutants, but this effect was restored upon addition of Usp14/Ubp6 (even an inactive Ubp6). The stimulation of gate opening by Ub conjugates through Usp14/Ubp6 requires nucleotide binding to the gate-regulatory ATPases. This activation enhances the selectivity of the 26S proteasome for ubiquitinated proteins and links their deubiquitination to their degradation.

Deubiquitinating enzymes (DUBs) are proteases that can antagonize ubiquitin-mediated signaling by disassembling ubiquitin-protein conjugates. How DUBs are regulated in vivo and how their substrate specificities are achieved are largely unknown. The conserved DUB Uch37 is found on proteasomes in organisms ranging from fission yeast to humans. Deubiquitination by Uch37 is activated by proteasomal binding, which enables Uch37 to process polyubiquitin chains. Here we show that in the nucleus Uch37 is also associated with the human Ino80 chromatin-remodeling complex (hINO80). In hINO80, Uch37 is held in an inactive state; however, it can be activated by transient interaction of the Ino80 complex with the proteasome. Thus, DUB activities can be modulated both positively and negatively via dynamic interactions with partner proteins. In addition, our findings suggest that the proteasome and the hINO80 chromatin-remodeling complex may cooperate to regulate transcription or DNA repair, processes in which both complexes have been implicated.

A decline in proteasome function is causally connected to neuronal aging and aging-associated neuropathologies. By using hippocampal neurons in culture and in vivo, we show that aging triggers a reduction and a cytoplasm-to-nucleus redistribution of the E3 ubiquitin ligase mahogunin (MGRN1). Proteasome impairment induces MGRN1 monoubiquitination, the key post-translational modification for its nuclear entry. One potential mechanism for MGRN1 monoubiquitination is via progressive deubiquitination at the proteasome of polyubiquitinated MGRN1. Once in the nucleus, MGRN1 potentiates the transcriptional cellular response to proteotoxic stress. Inhibition of MGRN1 impairs ATF3-mediated neuronal responsiveness to proteosomal stress and increases neuronal stress, while increasing MGRN1 ameliorates signs of neuronal aging, including cognitive performance in old animals. Our results imply that, among others, the strength of neuronal survival in a proteasomal deterioration background, like during aging, depends on the fine-tuning of ubiquitination-deubiquitination.

In this issue of Molecular Cell, Atanassov et al. (2009) show that the GCN5-containing SAGA complex regulates telomere function via deubiquitination and stabilization of the telomere repeat binding factor TRF1.

A20 is a potent anti-inflammatory protein that inhibits NF-κB, and A20 dysfunction is associated with autoimmunity and B-cell lymphoma. A20 harbors a deubiquitination enzyme domain and can employ multiple mechanisms to antagonize ubiquitination upstream of NEMO, a regulatory subunit of the IκB kinase complex (IKK). However, direct evidence of IKK inhibition by A20 is lacking, and the inhibitory mechanism remains poorly understood. Here we show that A20 can directly impair IKK activation without deubiquitination or impairment of ubiquitination enzymes. We find that polyubiquitin binding by A20, which is largely dependent on A20’s seventh zinc finger motif (ZnF7), induces specific binding to NEMO. Remarkably, this ubiquitin-induced recruitment of A20 to NEMO is sufficient to block IKK phosphorylation by its upstream kinase TAK1. Our results suggest a non-catalytic mechanism of IKK inhibition by A20 and a means by which polyubiquitin chains can specify a signaling outcome.

Summary Cdc48p is an abundant and conserved member of the AAA ATPase family of molecular chaperones. Cdc48p performs ubiquitin-selective functions, which are mediated by numerous ubiquitin binding adaptors, including the Npl4p-Ufd1p complex. Previous studies suggest that Cdc48p-containing complexes carry out many biochemical activities, including ubiquitination, deubiquitination, protein complex segregation, and targeting of ubiquitinated substrates to the proteasome. The molecular mechanisms by which Cdc48p-containing complexes participate in these processes remain poorly defined. We show here by using physiologically relevant Cdc48p substrates (i.e., endoplasmic membrane-associated/tethered dimers of Mga2p and Spt23p) and in vitro systems with purified proteins that Cdc48p Npl4p/Ufd1p binds to and promotes segregation of the tethered proteins via a polyubiquitin signal present on the membrane-bound proteins. Mobilization does not involve retrotranslocation of the associated anchors. These results provide biochemical evidence that Cdc48p Npl4p/Ufd1p functions as a polyubiquitin-selective segregase and that a polyubiquitin-Cdc48p pathway modulates protein interactions at cell membranes.

The p53 tumor suppressor protein has a major role in protecting the integrity of the genome. In unstressed cells, p53 is maintained at low levels by the ubiquitin-proteasome pathway. A balance between ubiquitin ligase activity (Hdm2, COP1, and Pirh2) and the ubiquitin protease activity of the Herpes virus-associated ubiquitin-specific protease (HAUSP) determines the half-life of p53. HAUSP also modulates p53 stability indirectly by deubiquitination and stabilization of Hdm2. The Hdmx protein affects p53 stability as well through its interaction with and regulation of Hdm2. Vice versa, Hdmx is a target for Hdm2-mediated ubiquitination and degradation. Here, we show that HAUSP also interacts with Hdmx, resulting in its direct deubiquitination and stabilization. HAUSP activity is required to maintain normal Hdmx protein levels. Therefore, the balance between HAUSP and Hdm2 activity determines Hdmx protein stability. Importantly, impaired deubiquitination of Hdmx/Hdm2 by HAUSP contributes to the DNA damage-induced degradation of Hdmx and transient instability of Hdm2.

Our previous study showed that ubiquitination of p53 is reversible and that the ubiquitin hydrolase HAUSP can stabilize p53 by deubiquitination. Here, we found that partial reduction of endogenous HAUSP levels by RNAi indeed destabilizes endogenous p53; surprisingly, however, nearly complete ablation of HAUSP stabilizes and activates p53. We further show that this phenomenon occurs because HAUSP stabilizes Mdm2 in a p53-independent manner, providing an interesting feedback loop in p53 regulation. Notably, HAUSP is required for Mdm2 stability in normal cells; in HAUSP-ablated cells, self-ubiquitinated-Mdm2 becomes extremely unstable, leading to indirect p53 activation. Furthermore, this feedback regulation is specific to Mdm2; in HeLa cells, where p53 is preferentially degraded by viral E6-dependent ubiquitination, depletion of HAUSP fails to activate p53. This study provides an example of an ubiquitin ligase (Mdm2) that is directly regulated by a deubiquitinase (HAUSP) and also reveals a dynamic role of HAUSP in the p53-Mdm2 pathway.