Development of a novel assay for human tyrosyl DNA phosphodiesterase 2

Tyrosyl DNA phosphodiesterase 2 (TDP2), a newly discovered enzyme that cleaves 5′-phosphotyrosyl bonds, is a potential target for chemotherapy. TDP2 possesses both 3′- and 5′-tyrosyl-DNA phosphodiesterase activity, which is generally measured in a gel-based assay using 3′- and 5′-phosphotyrosyl linkage at the 3′ and 5′ ends of an oligonucleotide. To understand the enzymatic mechanism of this novel enzyme, the gel-based assay is useful, but this technique is cumbersome for TDP2 inhibitor screening. For this reason, we have designed a novel assay using p-nitrophenyl-thymidine-5′-phosphate (T5PNP) as a substrate. This assay can be used in continuous colorimetric assays in a 96-well format. We compared the salt and pH effect on product formation with the colorimetric and gel-based assays and showed that they behave similarly. Steady-state kinetic studies showed that the 5′ activity of TDP2 is 1000-fold more efficient than T5PNP. Tyrosyl DNA phosphodiesterase 1 (TDP1) and human AP-endonuclease 1 (APE1) could not hydrolyze T5PNP. Sodium orthovanadate, a known inhibitor of TDP2, inhibits product formation from T5PNP by TDP2 (IC₅₀ = 40 mM). Our results suggest that this novel assay system with this new TDP2 substrate can be used for inhibitor screening in a high-throughput manner.     

Galactose‐modified duocarmycin prodrugs as senolytics

Senescence is a stable growth arrest that impairs the replication of damaged, old or preneoplastic cells, therefore contributing to tissue homeostasis. Senescent cells accumulate during ageing and are associated with cancer, fibrosis and many age‐related pathologies. Recent evidence suggests that the selective elimination of senescent cells can be effective on the treatment of many of these senescence‐associated diseases. A universal characteristic of senescent cells is that they display elevated activity of the lysosomal β‐galactosidase, and this has been exploited as a marker for senescence (senescence‐associated β‐galactosidase activity). Consequently, we hypothesized that galactose‐modified cytotoxic prodrugs will be preferentially processed by senescent cells, resulting in their selective killing. Here, we show that different galactose‐modified duocarmycin (GMD) derivatives preferentially kill senescent cells. GMD prodrugs induce selective apoptosis of senescent cells in a lysosomal β‐galactosidase (GLB1)‐dependent manner. GMD prodrugs can eliminate a broad range of senescent cells in culture, and treatment with a GMD prodrug enhances the elimination of bystander senescent cells that accumulate upon whole‐body irradiation treatment of mice. Moreover, taking advantage of a mouse model of adamantinomatous craniopharyngioma (ACP), we show that treatment with a GMD prodrug selectively reduced the number of β‐catenin‐positive preneoplastic senescent cells. In summary, the above results make a case for testing the potential of galactose‐modified duocarmycin prodrugs to treat senescence‐related pathologies.     

Environmental plasticity and colonisation history in the Atlantic salmon microbiome: A translocation experiment

Microbial communities associated with the gut and the skin are strongly influenced by environmental factors, and can rapidly adapt to change. Historical processes may also affect the microbiome. In particular, variation in microbial colonisation in early life has the potential to induce lasting effects on microbial assemblages. However, little is known about the relative extent of microbiome plasticity or the importance of historical colonisation effects following environmental change, especially for nonmammalian species. To investigate this we performed a reciprocal translocation of Atlantic salmon between artificial and semi‐natural conditions. Wild and hatchery‐reared fry were transferred to three common garden experimental environments for 6 weeks: standard hatchery conditions, hatchery conditions with an enriched diet, and simulated wild conditions. We characterized the faecal and skin microbiome of individual fish before and after the environmental translocation, using a BACI (before‐after‐control‐impact) design. We found evidence of extensive microbiome plasticity for both the gut and skin, with the greatest changes in alpha and beta diversity associated with the largest changes in environment and diet. Microbiome richness and diversity were entirely determined by environment, with no detectable effects of fish origin, and there was also a near‐complete turnover in microbiome structure. However, we also identified, for the first time in fish, evidence of historical colonisation effects reflecting early‐life experience, including ASVs characteristic of captive rearing. These results have important implications for host adaptation to local selective pressures, and highlight how conditions experienced during early life can have a long‐term influence on the microbiome and, potentially, host health.     

A quantitative analysis of the interplay of environment,neighborhood, and cell state in 3D spheroids

Cells react to their microenvironment by integrating external stimuli into phenotypic decisions via an intracellular signaling network. To analyze the interplay of environment, local neighborhood, and internal cell state effects on phenotypic variability, we developed an experimental approach that enables multiplexed mass cytometric imaging analysis of up to 240 pooled spheroid microtissues. We quantified the contributions of environment, neighborhood, and intracellular state to marker variability in single cells of the spheroids. A linear model explained on average more than half of the variability of 34 markers across four cell lines and six growth conditions. The contributions of cell‐intrinsic and environmental factors to marker variability are hierarchically interdependent, a finding that we propose has general implications for systems‐level studies of single‐cell phenotypic variability. By the overexpression of 51 signaling protein constructs in subsets of cells, we also identified proteins that have cell‐intrinsic and cell‐extrinsic effects. Our study deconvolves factors influencing cellular phenotype in a 3D tissue and provides a scalable experimental system, analytical principles, and rich multiplexed imaging datasets for future studies.     

N-terminal extension of N-methylpurine DNA glycosylase is required for turnover in hypoxanthine excision reaction

N-Methylpurine DNA glycosylase (MPG) initiates base excision repair in DNA by removing a wide variety of alkylated, deaminated, and lipid peroxidation-induced purine adducts. In this study we tested the role of N-terminal extension on MPG hypoxanthine (Hx) cleavage activity. Our results showed that MPG lacking N-terminal extension excises hypoxanthine with significantly reduced efficiency, one-third of that exhibited by full-length MPG under similar conditions. Steady-state kinetics showed full-length MPG has higher V(max) and lower K(m) than NDelta100 MPG. Real time binding experiments by surface plasmon resonance spectroscopy suggested that truncation can substantially increase the equilibrium binding constant of MPG toward Hx, but under single-turnover conditions there is apparently no effect on catalytic chemistry; however, the truncation of the N-terminal tail affected the turnover of the enzyme significantly under multiple turnover conditions. Real time binding experiments by surface plasmon resonance spectroscopy further showed that NDelta100 MPG binds approximately six times more tightly toward its product apurinic/apyrimidinic site than the substrate, whereas full-length MPG similarly binds to both the substrate and the product. We thereby conclude that the N-terminal tail in MPG plays a critical role in overcoming the product inhibition, which is achieved by reducing the differences of MPG binding affinity toward Hx and apurinic/apyrimidinic sites and thus is essential for the Hx cleavage reaction of MPG. The results from this study also affirm the need for reinvestigation of full-length MPG for its enzymatic and structural properties, which are currently available mostly for the truncated protein.     

The E6 oncoprotein from HPV16 enhances the canonical Wnt/β-catenin pathway in skin epidermis in vivo

The contribution of the Wnt signaling pathway to human papilloma virus (HPV)-induced carcinogenesis is poorly understood. In high-grade dysplastic lesions that are caused by high-risk HPVs (HR-HPV), β-catenin is often located in the cell nucleus, which suggests that Wnt pathway may be involved in the development of HPV-related carcinomas. Most of the oncogenic potential of HR-HPVs resides on the PDZ-binding domain of E6 protein. We hypothesized that the PDZ-binding domain of the HPV16-E6 oncoprotein induces the nuclear accumulation of β-catenin due to its capacity to degrade PDZ-containing cellular targets. To test this hypothesis, we evaluated the staining pattern of β-catenin in the skin epidermis of transgenic mice expressing the full-length E6 oncoprotein (K14E6 mice) and measured LacZ gene expression in K14E6 mice that were crossed with a strain expressing LacZ that was knocked into the Axin2 locus (Axin2(+/LacZ) mice). Here, we show that the E6 oncoprotein enhances the nuclear accumulation of β-catenin, the accumulation of cellular β-catenin-responsive genes, and the expression of LacZ. None of these effects were observed when a truncated E6 oncoprotein that lacks the PDZ-binding domain was expressed alone (K14E6ΔPDZ mice) or in combination with Axin2(+/LacZ). Conversely, cotransfection with either E6 or E6ΔPDZ similarly enhanced canonical Wnt signaling in short-term in vitro assays that used a luciferase Wnt/β-catenin/TCF-dependent promoter. We propose that the activation of canonical Wnt signaling could be induced by the HPV16-E6 oncoprotein; however, the participation of the E6 PDZ-binding domain seems to be important in in vivo models only.     

GFAP-Cre-mediated transgenic activation of Bmi1 results in pituitary tumors

Bmi1 is a member of the polycomb repressive complex 1 and plays different roles during embryonic development, depending on the developmental context. Bmi1 over expression is observed in many types of cancer, including tumors of astroglial and neural origin. Although genetic depletion of Bmi1 has been described to result in tumor inhibitory effects partly through INK4A/Arf mediated senescence and apoptosis and also through INK4A/Arf independent effects, it has not been proven that Bmi1 can be causally involved in the formation of these tumors. To see whether this is the case, we developed two conditional Bmi1 transgenic models that were crossed with GFAP-Cre mice to activate transgenic expression in neural and glial lineages. We show here that these mice generate intermediate and anterior lobe pituitary tumors that are positive for ACTH and beta-endorphin. Combined transgenic expression of Bmi1 together with conditional loss of Rb resulted in pituitary tumors but was insufficient to induce medulloblastoma therefore indicating that the oncogenic function of Bmi1 depends on regulation of p16(INK4A)/Rb rather than on regulation of p19(ARF)/p53. Human pituitary adenomas show Bmi1 overexpression in over 50% of the cases, which indicates that Bmi1 could be causally involved in formation of these tumors similarly as in our mouse model.     

Dipole-dipole interaction stabilizes the transition state of apurinic/apyrimidinic endonuclease--abasic site interaction

Human apurinic/apyrimidinic (AP) endonuclease (hAPE) initiates the repair of an abasic site (AP site). To gain insight into the mechanisms of damage recognition of hAPE, we conducted surface plasmon resonance spectroscopy to study the thermodynamics and kinetics of its interaction with substrate DNA containing an abasic site (AP DNA). The affinity of hAPE binding toward DNA increased as much as 6-fold after replacing a single adenine (equilibrium dissociation constant, K(D), 5.3 nm) with an AP site (K(D), 0.87 nm). The enzyme-substrate complex formation appears to be thermodynamically stabilized and favored by a large change in Gibbs free energy, DeltaG degrees (-50 kJ/mol). The latter is supported by a high negative change in enthalpy, DeltaH degrees (-43 kJ/mol) and also positive change in entropy, DeltaS degrees (24 J/(K mol)), and thus the binding process is spontaneous at all temperatures. Analysis of kinetic parameters reveals small enthalpy of activation for association, DeltaH degrees++(ass) (-17 kJ/mol), and activation energy for association (E(a), -14 kJ/mol) when compared with the enthalpy of activation for dissociation, DeltaH degrees++(diss) (26 kJ/mol), and activation energy in the reverse direction (E(d), 28 kJ/mol). Furthermore, varying concentration of KCl showed an increase in binding affinity at low concentration but complete abrogation of the binding at higher concentration, implying the importance of hydrophobic, but predominantly ionic, forces in the Michaelis-Menten complex formation. Thus, low activation energy and the enthalpy of activation, which are perhaps a result of dipole-dipole interactions, play critical roles in AP site binding of APE.