Structural basis of tankyrase activation by polymerization.

The poly-ADP-ribosyltransferase tankyrase (TNKS, TNKS2) controls a wide range of disease-relevant cellular processes, including WNT-ß-catenin signalling, telomere length maintenance, Hippo signalling, DNA damage repair and glucose homeostasis1,2. This has incentivized the development of tankyrase inhibitors. Notwithstanding, our knowledge of the mechanisms that control tankyrase activity has remained limited. Both catalytic and non-catalytic functions of tankyrase depend on its filamentous polymerization3-5. Here we report the cryo-electron microscopy reconstruction of a filament formed by a minimal active unit of tankyrase, comprising the polymerizing sterile alpha motif (SAM) domain and its adjacent catalytic domain. The SAM domain forms a novel antiparallel double helix, positioning the protruding catalytic domains for recurring head-to-head and tail-to-tail interactions. The head interactions are highly conserved among tankyrases and induce an allosteric switch in the active site within the catalytic domain to promote catalysis. Although the tail interactions have a limited effect on catalysis, they are essential to tankyrase function in WNT-ß-catenin signalling. This work reveals a novel SAM domain polymerization mode, illustrates how supramolecular assembly controls catalytic and non-catalytic functions, provides important structural insights into the regulation of a non-DNA-dependent poly-ADP-ribosyltransferase and will guide future efforts to modulate tankyrase and decipher its contribution to disease mechanisms.

Tankyrase Requires SAM Domain-Dependent Polymerization to Support Wnt-ß-Catenin Signaling.

The poly(ADP-ribose) polymerase (PARP) Tankyrase (TNKS and TNKS2) is paramount to Wnt-ß-catenin signaling and a promising therapeutic target in Wnt-dependent cancers. The pool of active ß-catenin is normally limited by destruction complexes, whose assembly depends on the polymeric master scaffolding protein AXIN. Tankyrase, which poly(ADP-ribosyl)ates and thereby destabilizes AXIN, also can polymerize, but the relevance of these polymers has remained unclear. We report crystal structures of the polymerizing TNKS and TNKS2 sterile alpha motif (SAM) domains, revealing versatile head-to-tail interactions. Biochemical studies informed by these structures demonstrate that polymerization is required for Tankyrase to drive ß-catenin-dependent transcription. We show that the polymeric state supports PARP activity and allows Tankyrase to effectively access destruction complexes through enabling avidity-dependent AXIN binding. This study provides an example for regulated signal transduction in non-membrane-enclosed compartments (signalosomes), and it points to novel potential strategies to inhibit Tankyrase function in oncogenic Wnt signaling.

Structural and functional basis of protein phosphatase 5 substrate specificity.

The serine/threonine phosphatase protein phosphatase 5 (PP5) regulates hormone- and stress-induced cellular signaling by association with the molecular chaperone heat shock protein 90 (Hsp90). PP5-mediated dephosphorylation of the cochaperone Cdc37 is essential for activation of Hsp90-dependent kinases. However, the details of this mechanism remain unknown. We determined the crystal structure of a Cdc37 phosphomimetic peptide bound to the catalytic domain of PP5. The structure reveals PP5 utilization of conserved elements of phosphoprotein phosphatase (PPP) structure to bind substrate and provides a template for many PPP-substrate interactions. Our data show that, despite a highly conserved structure, elements of substrate specificity are determined within the phosphatase catalytic domain itself. Structure-based mutations in vivo reveal that PP5-mediated dephosphorylation is required for kinase and steroid hormone receptor release from the chaperone complex. Finally, our data show that hyper- or hypoactivity of PP5 mutants increases Hsp90 binding to its inhibitor, suggesting a mechanism to enhance the efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors.

The iron-sulphur cluster in human DNA2 is required for all biochemical activities of DNA2.

The nuclease/helicase DNA2 plays important roles in DNA replication, repair and processing of stalled replication forks. DNA2 contains an iron-sulphur (FeS) cluster, conserved in eukaryotes and in a related bacterial nuclease. FeS clusters in DNA maintenance proteins are required for structural integrity and/or act as redox-sensors. Here, we demonstrate that loss of the FeS cluster affects binding of human DNA2 to specific DNA substrates, likely through a conformational change that distorts the central DNA binding tunnel. Moreover, we show that the FeS cluster is required for DNA2's nuclease, helicase and ATPase activities. Our data also establish that oxidation of DNA2 impairs DNA binding in vitro, an effect that is reversible upon reduction. Unexpectedly, though, this redox-regulation is independent of the presence of the FeS cluster. Together, our study establishes an important structural role for the FeS cluster in human DNA2 and discovers a redox-regulatory mechanism to control DNA binding.

Profiling tumor-associated markers for early detection of malignant mesothelioma: an epidemiologic study.

Improved detection methods for diagnosis of asymptomatic malignant mesothelioma (MM) are essential for an early and reliable detection and treatment of this type of neoplastic disease. Thus, focus has been on finding tumor markers in the blood that can be used for noninvasive detection of MM. Ninety-four asbestos-exposed subjects defined at high risk, 22 patients with MM, and 54 healthy subjects were recruited for evaluation of the clinical significance of 8-hydroxy-2'-deoxyguanosine (8OHdG) in WBCs and plasma concentrations of soluble mesothelin-related peptides (SMRPs), angiogenic factors [platelet-derived growth factor beta, hepatocyte growth factor, basic fibroblast growth factor, and vascular endothelial growth factor beta (VEGFbeta)], and matrix proteases [matrix metalloproteinase (MMP) 2, MMP9, tissue inhibitor of metalloproteinase (TIMP) 1, and TIMP2] for potential early detection of MM. The area under receiver operating characteristic (ROC) curves indicate that 8OHdG levels can discriminate asbestos-exposed subjects from healthy controls but not from MM patients. Significant area under ROC curve values were found for SMRPs, discriminating asbestos-exposed subjects from MM patients but not from healthy controls. Except for platelet-derived growth factor beta, the hepatocyte growth factor, basic fibroblast growth factor, and VEGFbeta can significantly differentiate high-risk individuals from healthy control and cancer groups. No diagnostic value was observed for MMP2, MMP9, TIMP1, and TIMP2. In addition to the diagnostic performance defined by the ROC analysis, the sensitivity and specificity results of markers with clinical significance were calculated at defined cutoffs. The combination of 8OHdG, VEGFbeta, and SMRPs best distinguished the individual groups, suggesting a potential indicator of early and advanced MM cancers. The combination of blood biomarkers and radiographic findings could be used to stratify the risk of mesothelioma in asbestos-exposed populations.

Assessment of biomarkers in asbestos-exposed workers as indicators of cancer risk.

Epidemiological studies have shown that mortality from malignant mesothelioma (MM) and lung cancer have increased with increasing cumulative exposure to asbestos. To investigate whether tumour-related biomarkers can contribute towards the evaluation of the carcinogenic risk in populations exposed to asbestos, the DNA adduct 8-hydroxy-2'-deoxyguanosine (80HdG), interleukine-6 (IL-6), platelet-derived growth factor (PDGF-BB), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGFbeta) and soluble mesothelin-related peptides (SMRPs) were analysed in a cohort of workers differently exposed to asbestos fibres at the workplace. To document biomarker levels in an unexposed population, 54 age-matched subjects were enrolled. A total of 119 subjects with a history of occupational exposure to asbestos underwent clinical examination and were interviewed by trained personnel, responding to a detailed questionnaire related to duration of asbestos exposure, smoking, and occupational task. According to the occupational tasks, asbestos-exposed subjects were analysed for their asbestos cumulative dose and the association with the biomarkers was evaluated. Among the occupational groups, maintenance workers, pipe fitters and electricians were exposed to a higher cumulative dose of asbestos fibres. Exposure to asbestos significantly increased the steady-state content of 80HdG in DNA. Elevated levels of 80HdG and IL-6 best reflected a high level of SMRPs, which is related to cell transformation. Subjects heavily exposed to asbestos [> 60(ff/cm3) x years] showed also a higher level of angiogenic factors. A combination of angiogenic biomarkers with a specific mesothelioma-biomarker such as SMRPs could be used for close surveillance of workers with a history of asbestos exposure.

Regulation of Wnt/ß-catenin signalling by tankyrase-dependent poly(ADP-ribosyl)ation and scaffolding.

The Wnt/ß-catenin signalling pathway is pivotal for stem cell function and the control of cellular differentiation, both during embryonic development and tissue homeostasis in adults. Its activity is carefully controlled through the concerted interactions of concentration-limited pathway components and a wide range of post-translational modifications, including phosphorylation, ubiquitylation, sumoylation, poly(ADP-ribosyl)ation (PARylation) and acetylation. Regulation of Wnt/ß-catenin signalling by PARylation was discovered relatively recently. The PARP tankyrase PARylates AXIN1/2, an essential central scaffolding protein in the ß-catenin destruction complex, and targets it for degradation, thereby fine-tuning the responsiveness of cells to the Wnt signal. The past few years have not only seen much progress in our understanding of the molecular mechanisms by which PARylation controls the pathway but also witnessed the successful development of tankyrase inhibitors as tool compounds and promising agents for the therapy of Wnt-dependent dysfunctions, including colorectal cancer. Recent work has hinted at more complex roles of tankyrase in Wnt/ß-catenin signalling as well as challenges and opportunities in the development of tankyrase inhibitors. Here we review some of the latest advances in our understanding of tankyrase function in the pathway and efforts to modulate tankyrase activity to re-tune Wnt/ß-catenin signalling in colorectal cancer cells. LINKED ARTICLES: This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc.

Clinical significance of circulating miR-126 quantification in malignant mesothelioma patients.

OBJECTIVES: Aim of this study was to evaluate the accuracy and precision of the detection of individual miRNA as clinical biomarkers in the serum. DESIGN AND METHODS: miRNA-126 was quantified in serum using endogenous and exogenous controls for normalization and the accuracy and precision of the method evaluated. The diagnostic value of serum miRNA-126 was evaluated in malignant mesothelioma (MM) and non-small-cell lung cancer (NSCLC) patients using both relative and absolute qRT-PCR methods. RESULTS: The use of endogenous invariant and exogenous synthetic controls as well sample dilution markedly improves the accuracy and precision of the assay. The inter- and intra-assay analyses revealed that relative qRT-PCR is a more reliable method. Circulating miR-126 detected in the serum by relative qRT-PCRs was found low-expressed in both malignancies, significantly differentiated MM patients from healthy controls and NSCLC from MM, but do not discriminate NSCLC patients from control subjects. Kaplan-Meier analysis revealed that low level of circulating miR-126 in MM patients was strongly associated with worse prognosis. CONCLUSIONS: We propose that this approach can be adopted for accurate analysis of other suitable circulating miRNA markers of different types of cancer.

Relationship of job satisfaction, psychological distress and stress-related biological parameters among healthy nurses: a longitudinal study.

OBJECTIVE: To examine the relationship between job satisfaction, psychological distress, psychosocial processes and stress-related biological factors, and to evaluate whether over time changes of work satisfaction could affect the immunological-inflammatory status of workers. METHODS: One hundred and one nurses were enrolled at the Clinic of Occupational Medicine, Polytechnic University of Marche, Ancona, Italy. Perceived job satisfaction, psychological distress, and social support were assessed every 4 mo over a 1-yr period using 4 self-reported questionnaires. T lymphocytes CD3, CD4(+), CD8(+), CD8(+)-CD57(+), B lymphocyte CD19(+), NK cells CD56(+), and NK cell activity were determined. RESULTS: Job satisfaction was associated with reduced psychological distress and was characterized by low cell numbers of CD8(+) suppressor T cells, CD8(+)-CD57(+) activated T cells, CD56(+) NK cells and low IL-6 levels. Over time changes in psychological parameters were related to changes in the immunological-inflammatory variables. Subjects who increased their job satisfaction showed a reduced psychological stress associated with reduced number of CD8(+)-CD57(+) activated T cells and inflammatory cytokines. CONCLUSIONS: Job (dis)satisfaction is related with psychological mechanisms in stress affecting cellular immune function.