AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway(1,2). Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis

First synthesis of a polysaccharide-supported lignin model compound and study of its oxidation promoted by lignin peroxidase

Veratrylchitosan, a polysaccharide-supported lignin model compound, has been synthesised by covalently attaching 3-(3,4-dimethoxybenzyloxy)propionic acid to the polysaccharide chitosan through an amide linkage. When this polymer was used as a substrate in the oxidation promoted by lignin peroxidase (LiP), significant decomposition of the lignin model resulted in the formation of veratraldehyde. The oxidation mechanism involves an initial transfer of one electron from chitosan to the active species of UP (LiP I) followed by C-alpha-H deprotonation of an aromatic cation radical. A benzylic radical is then formed which is further oxidised to a benzyl cation. Reaction with water and hydrolysis of the hemiacetal then lead to veratraldehyde formation. An increase in the yields of the oxidation product is observed in the presence of the mediator 2-chloro-1,4-dimethoxybenzene, thus indicating that a more efficient degradation results from the transfer of an electron from the polymer to the radical cation of the mediator. (C) 2003 Elsevier Inc. All rights reserved

Mechanistic and steric issues in the oxidation of phenolic and non-phenolic compounds by laccase or laccase-mediator systems. The case of bifunctional substrates

Steric and redox issues of phenolic and non-phenolic substrates are investigated for a better insight of the reactivity features of the phenoloxidase laccase. Whenever a substrate is endowed with a redox potential too high for direct monoelectronic oxidation by the enzyme, or else is too much encumbered to access the enzymatic pocket, redox mediators overcome the problem, behaving as an interface between enzyme and substrate. For example, the small-sized mediator ABTS, once oxidised by laccase, fruitfully interacts with bulky substrates, 2,4,6-tri(Bu')-phenol providing a significant case. Other mediators, for example HBT, resort to a radical oxidation mechanism precluded to laccase, and can react with non-phenolic substrates which are impossible for the enzyme. The advantages provided by the mediators are discussed, and suitable phenolic compounds, as precursors of phenoxyl radical intermediates, emerge as a new proficient class. They could be the true natural mediators of laccase in the oxidative delignification. In fact, phenoxyl radical fragments generated by laccase from lignin, or from phenolic monomer residuals from the building up of lignin polymer or else deriving from lignin by oxidation with other ligninolytic enzymes, could oxidise non-phenolic residues of lignin thereby causing the breakdown of its alkyl network. The novel mechanistic probe 3,5-di(Bu')-4-OH-benzyl alcohol enables the decoupling of the reactivity channels of a phenolic vs. a benzylic alcohol moiety in the enzymatic oxidation of bifunctional substrates having structural features comparable to portions of lignin. Experimental support is thereby attained for the central role of laccase in biodelignification, in spite of the seemingly lower oxidation power of this enzyme with respect to other and stronger oxidising enzymes excreted by ligninolytic fungi

Towards controlled cationic polymer growth from inorganic oxide defects: directing the mechanism of polystyrene grafting from γ-irradiated silica

Most studies on surface-initiated controlled polymerizations for the synthesis of polymeric covalent organic-inorganic hybrid materials focus on chemical methods requiring specific modifications of the inorganic substrate. Few mechanistically-aware approaches have been undertaken towards exploiting the reactivity of defects induced by physical techniques such as ionizing radiations or UV-Vis light. Within this framework, we take grafted polymerization of styrene from γ-irradiated silica as a mechanistic testing ground where para- and diamagnetic silica defects are present, and polymerization proceeds through both radical and cationic mechanisms, resulting in a bimodal molecular weight distribution. We show that these mechanistic intricacies can be sorted out by resorting to the chemical arsenal developed in the last decades for controlled polymerizations. Specifically, we obtained a silica-polystyrene grafted material by cationic grafting from at 30 °C, a unimodal molecular weight distribution, and a relatively high molecular weight (Mn = 7.4 kDa) with a PDI of 1.68. © 2014 Elsevier Ltd. All rights reserved

A mechanistic survey of the oxidation of alcohols and ethers with the enzyme laccase and mediation by TEMPO

The oxidation of alcohols and ethers by O2 with the enzyme laccase, mediated by the stable N-oxyl radical TEMPO, affords carbonylic products. An ionic mechanism is proposed, where a nucleophilic attack of the oxygen lone-pair of the alcohol (or ether) onto the oxoammonium form of TEMPO (generated by laccase on oxidation) takes place leading to a transient adduct. Subsequent deprotonation of this adduct α to the C−Ο bond leads to the carbonylic product. Additional mechanistic considerations for the laccase-mediated oxidation of ethers and thioethers are offered. The proposed mechanism is supported by: (i) investigating the inter- and intramolecular selectivity of oxidation with appropriate substrates, (ii) thermochemical considerations, and (iii) attempting a Hammett correlation for the oxidation of a series of 4-X-substituted benzyl alcohols, wherein a shift of the rate-determining step as a function of the 4-X-substituent results. Based on the above points, the lack of mediation efficiency of another stable N-oxyl radical (viz., IND-O·) can be explained

A first study on copolymers of a methacrylate containing the 2-(hydroxyimino) aldehyde group and OEGMA. RAFT polymerization and assessment of thermal and photoresponsive polymer behavior

We report the first synthesis of 4-[(hydroxyimino) aldehyde] butyl methacrylate and its RAFT copolymerization with oligo(ethylene glycol) monomethylether methacrylate. For comparison, 6-(hydroxyimino) hexyl methacrylate, containing a free oxime group, and 6-oxohexyl methacrylate, with an aldehyde group, are also synthesized and subjected to RAFT copolymerization with OEGMA. DP(GPC) values of all polymers are in the 18-33 range with PDI < 1.2. Interestingly, the thermal behavior of aqueous solutions of the 2-(hydroxyimino) aldehyde-containing copolymers exhibit a lower cloud point than their analogs, which comprise only a CvN(OH) or CHO functionality. This effect can be ascribed to the simultaneous presence of a hydrogen donor and acceptor in the 2-(hydroxyimino) aldehyde group. Moreover, we present evidence for the thermally reversible E/Z photoisomerism of the 2-(hydroxyimino) aldehyde group (HIA) both in HIABMA and in its copolymers