Ubiquitin-specific protease-like 1 (USPL1) is a SUMO isopeptidase with essential, non-catalytic functions.

Isopeptidases are essential regulators of protein ubiquitination and sumoylation. However, only two families of SUMO isopeptidases are at present known. Here, we report an activity-based search with the suicide inhibitor haemagglutinin (HA)-SUMO-vinylmethylester that led to the identification of a surprising new SUMO protease, ubiquitin-specific protease-like 1 (USPL1). Indeed, USPL1 neither binds nor cleaves ubiquitin, but is a potent SUMO isopeptidase both in vitro and in cells. C13orf22l—an essential but distant zebrafish homologue of USPL1—also acts on SUMO, indicating functional conservation. We have identified invariant USPL1 residues required for SUMO binding and cleavage. USPL1 is a low-abundance protein that colocalizes with coilin in Cajal bodies. Its depletion does not affect global sumoylation, but causes striking coilin mislocalization and impairs cell proliferation, functions that are not dependent on USPL1 catalytic activity. Thus, USPL1 represents a third type of SUMO protease, with essential functions in Cajal body biology

Structural analysis of MDM2 RING separates degradation from regulation of p53 transcription activity

MDM2–MDMX complexes bind the p53 tumor-suppressor protein, inhibiting p53's transcriptional activity and targeting p53 for proteasomal degradation. Inhibitors that disrupt binding between p53 and MDM2 efficiently activate a p53 response, but their use in the treatment of cancers that retain wild-type p53 may be limited by on-target toxicities due to p53 activation in normal tissue. Guided by a novel crystal structure of the MDM2–MDMX–E2(UbcH5B)–ubiquitin complex, we designed MDM2 mutants that prevent E2–ubiquitin binding without altering the RING-domain structure. These mutants lack MDM2's E3 activity but retain the ability to limit p53′s transcriptional activity and allow cell proliferation. Cells expressing these mutants respond more quickly to cellular stress than cells expressing wild-type MDM2, but basal p53 control is maintained. Targeting the MDM2 E3-ligase activity could therefore widen the therapeutic window of p53 activation in tumors

Inhibition of dendritic cell migration by transforming growth factor-β1 increases tumor-draining lymph node metastasis

<p>Abstract</p> <p>Background</p> <p>Transforming growth factor (TGF)-β is known to be produced by progressor tumors and to immobilize dendritic cells (DCs) within those tumors. Moreover, although TGF-β1 has been shown to promote tumor progression, there is still no direct, in vivo evidence as to whether TGF-β1 is able to directly induce distant metastasis.</p> <p>Methods</p> <p>To address that issue and investigate the mechanism by which TGF-β1 suppresses DC activity, we subdermally inoculated mouse ears with squamous cell carcinoma cells stably expressing TGF-β1 or empty vector (mock).</p> <p>Results</p> <p>The numbers of DCs within lymph nodes draining the resultant TGF-β1-expressing tumors was significantly lower than within nodes draining tumors not expressing TGF-β1. We then injected fluorescently labeled bone marrow-derived dendritic cells into the tumors, and subsequent analysis confirmed that the tumors were the source of the DCs within the tumor-draining lymph nodes, and that there were significantly fewer immature DCs within the nodes draining TGF-β1-expressing tumors than within nodes draining tumors not expressing TGF-β1. In addition, 14 days after tumor cell inoculation, lymph node metastasis occurred more frequently in mice inoculated with TGF-β1 transfectants than in those inoculated with the mock transfectants.</p> <p>Conclusions</p> <p>These findings provide new evidence that tumor-derived TGF-β1 inhibits migration of DCs from tumors to their draining lymph nodes, and this immunosuppressive effect of TGF-β1 increases the likelihood of metastasis in the affected nodes.</p

Updates on p53: modulation of p53 degradation as a therapeutic approach

The p53 pathway is aberrant in most human tumours with over 50% expressing mutant p53 proteins. The pathway is critically controlled by protein degradation. Here, we discuss the latest developments in the search for small molecules that can modulate p53 pathway protein stability and restore p53 activity for cancer therapy

TIAF1 self-aggregation in peritumor capsule formation, spontaneous activation of SMAD-responsive promoter in p53-deficient environment, and cell death

Self-aggregation of transforming growth factor β (TGF-β)1-induced antiapoptotic factor (TIAF1) is known in the nondemented human hippocampus, and the aggregating process may lead to generation of amyloid β (Aβ) for causing neurodegeneration. Here, we determined that overexpressed TIAF1 exhibits as aggregates together with Smad4 and Aβ in the cancer stroma and peritumor capsules of solid tumors. Also, TIAF1/Aβ aggregates are shown on the interface between brain neural cells and the metastatic cancer cell mass. TIAF1 is upregulated in developing tumors, but may disappear in established metastatic cancer cells. Growing neuroblastoma cells on the extracellular matrices from other cancer cell types induced production of aggregated TIAF1 and Aβ. In vitro induction of TIAF1 self-association upregulated the expression of tumor suppressors Smad4 and WW domain-containing oxidoreductase (WOX1 or WWOX), and WOX1 in turn increased the TIAF1 expression. TIAF1/Smad4 interaction further enhanced Aβ formation. TIAF1 is known to suppress SMAD-regulated promoter activation. Intriguingly, without p53, self-aggregating TIAF1 spontaneously activated the SMAD-regulated promoter. TIAF1 was essential for p53-, WOX1- and dominant-negative JNK1-induced cell death. TIAF1, p53 and WOX1 acted synergistically in suppressing anchorage-independent growth, blocking cell migration and causing apoptosis. Together, TIAF1 shows an aggregation-dependent control of tumor progression and metastasis, and regulation of cell death

Homeodomain Interacting Protein Kinase 2 Activation Compromises Endothelial Cell Response to Laminar Flow: Protective Role of p21waf1,cip1,sdi1

BACKGROUND: In the cardiovascular system, laminar shear stress (SS) is one of the most important source of endothelial protecting signals. Physical and chemical agents, however, including ionising radiations and anticancer drugs, may injure endothelial cells determining an increase in oxidative stress and genotoxic damage. Whether the SS protective function remains intact in the presence of strong oxidants or DNA damage is currently unclear. METHODS AND RESULTS: To investigate this aspect a series of experiments were performed in which HUVEC were exposed to sub-lethal doses of the radio-mimetic compound Bleomycin (Bleo; 10 microg/ml) which generated free radicals (ROS) without significantly compromising cell survival. Remarkably, the application of a SS of 12 dyne/cm(2) did not protect endothelial cells but markedly accelerated apoptosis compared to controls kept in static culture and in the presence of Bleo. Experiments with the inducible nitric oxide synthase (iNOS) inhibitor GW274150 significantly reduced the SS-dependent apoptosis indicating that the production of NO was relevant for this effect. At molecular level, the ataxia-telangectasia-mutated (ATM) kinase, the homeodomain-interacting protein kinase-2 (HIPK2) and p53 were found activated along a pro-apoptotic signalling pathway while p21(waf1,cip1,sdi1) was prevented from its protective action. RNA interference experiments revealed that HIPK2 and p53 were both important for this process, however, only the forced expression p21(waf1,cip1,sdi1) fully restored the SS-dependent pro-survival function. CONCLUSIONS: This study provides the first evidence that, in the presence of genotoxic damage, laminar flow contributes to endothelial toxicity and death and identifies molecular targets potentially relevant in endothelial dysfunction and cardiovascular disease pathogenesis

The UBA-UIM Domains of the USP25 Regulate the Enzyme Ubiquitination State and Modulate Substrate Recognition

USP25m is the muscle isoform of the deubiquitinating (DUB) enzyme USP25. Similarly to most DUBs, data on USP25 regulation and substrate recognition is scarce. In silico analysis predicted three ubiquitin binding domains (UBDs) at the N-terminus: one ubiquitin-associated domain (UBA) and two ubiquitin-interacting motifs (UIMs), whereas no clear structural homology at the extended C-terminal region outside the catalytic domains were detected. In order to asses the contribution of the UBDs and the C-terminus to the regulation of USP25m catalytic activity, ubiquitination state and substrate interaction, serial and combinatorial deletions were generated. Our results showed that USP25m catalytic activity did not strictly depend on the UBDs, but required a coiled-coil stretch between amino acids 679 to 769. USP25 oligomerized but this interaction did not require either the UBDs or the C-terminus. Besides, USP25 was monoubiquitinated and able to autodeubiquitinate in a possible loop of autoregulation. UBDs favored the monoubiquitination of USP25m at the preferential site lysine 99 (K99). This residue had been previously shown to be a target for SUMO and this modification inhibited USP25 activity. We showed that mutation of K99 clearly diminished USP25-dependent rescue of the specific substrate MyBPC1 from proteasome degradation, thereby supporting a new mechanistic model, in which USP25m is regulated through alternative conjugation of ubiquitin (activating) or SUMO (inhibiting) to the same lysine residue (K99), which may promote the interaction with distinct intramolecular regulatory domains

TRAIL treatment provokes mutations in surviving cells

Chemotherapy and radiotherapy commonly damage DNA and trigger p53-dependent apoptosis through intrinsic apoptotic pathways. Two unfortunate consequences of this mechanism are resistance due to blockade of p53 or intrinsic apoptosis pathways, and mutagenesis of non-malignant surviving cells which can impair cellular function or provoke second malignancies. Death ligand-based drugs, such as tumor necrosis factor-related apoptosis inducing ligand (TRAIL), stimulate extrinsic apoptotic signaling, and may overcome resistance to treatments that induce intrinsic apoptosis. As death receptor ligation does not damage DNA as a primary mechanism of pro-apoptotic action, we hypothesized that surviving cells would remain genetically unscathed, suggesting that death ligand-based therapies may avoid some of the adverse effects associated with traditional cancer treatments. Surprisingly, however, treatment with sub-lethal concentrations of TRAIL or FasL was mutagenic. Mutations arose in viable cells that contained active caspases, and overexpression of the caspase-8 inhibitor crmA or silencing of caspase-8 abolished TRAIL-mediated mutagenesis. Downregulation of the apoptotic nuclease caspase-activated DNAse (CAD)/DNA fragmentation factor 40 (DFF40) prevented the DNA damage associated with TRAIL treatment. Although death ligands do not need to damage DNA in order to induce apoptosis, surviving cells nevertheless incur DNA damage after treatment with these agents

Bilateral inhibition of HAUSP deubiquitinase by a viral interferon regulatory factor protein

Herpesvirus-associated ubiquitin specific protease (HAUSP) regulates the stability of p53 and MDM2, implicating HAUSP as a therapeutic target for tuning p53-mediated anti-tumor activity. Here, we report the structural analysis of HAUSP with Kaposi’s sarcoma-associated herpesvirus vIRF4 and the discovery of two vIRF4-derived peptides, vif1 and vif2, as potent and selective HAUSP antagonists. This analysis reveals a bilateral belt-type interaction resulting in inhibition of HAUSP. The vif1 peptide binds the HAUSP TRAF domain, competitively blocking substrate binding, while the vif2 peptide binds both the HAUSP TRAF and catalytic domains, robustly suppressing its deubiquitination activity. Consequently, peptide treatments comprehensively blocked HAUSP, leading to p53-dependent cell cycle arrest and apoptosis in culture and tumor regression in xenograft mouse model. Thus, the virus has developed a unique molecular strategy to target the HAUSP-MDM2-p53 pathway, and these virus-derived short peptides represent biologically active HAUSP antagonists

Development of Immune-Specific Interaction Potentials and Their Application in the Multi-Agent-System VaccImm

Peptide vaccination in cancer therapy is a promising alternative to conventional methods. However, the parameters for this personalized treatment are difficult to access experimentally. In this respect, in silico models can help to narrow down the parameter space or to explain certain phenomena at a systems level. Herein, we develop two empirical interaction potentials specific to B-cell and T-cell receptor complexes and validate their applicability in comparison to a more general potential. The interaction potentials are applied to the model VaccImm which simulates the immune response against solid tumors under peptide vaccination therapy. This multi-agent system is derived from another immune system simulator (C-ImmSim) and now includes a module that enables the amino acid sequence of immune receptors and their ligands to be taken into account. The multi-agent approach is combined with approved methods for prediction of major histocompatibility complex (MHC)-binding peptides and the newly developed interaction potentials. In the analysis, we critically assess the impact of the different modules on the simulation with VaccImm and how they influence each other. In addition, we explore the reasons for failures in inducing an immune response by examining the activation states of the immune cell populations in detail