https://minerva-access.unimelb.edu.au/bitstream/handle/11343/194901/Stockwell_Cell_review+2017.pdf

Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease

The Blood-Brain Barrier in Health and Chronic Neurodegenerative Disorders

Macroautophagy mediates the bulk degradation of cytoplasmic components. It accounts for the degradation of most long-lived proteins: cytoplasmic constituents, including organelles, are sequestered into autophagosomes, which subsequently fuse with lysosomes, where degradation occurs. Although the possible involvement of autophagy in homeostasis, development, cell death, and pathogenesis has been repeatedly pointed out, systematic in vivo analysis has not been performed in mammals, mainly because of a limitation of monitoring methods. To understand where and when autophagy occurs in vivo, we have generated transgenic mice systemically expressing GFP fused to LC3, which is a mammalian homologue of yeast Atg8 (Aut7/Apg8) and serves as a marker protein for autophagosomes. Fluorescence microscopic analyses revealed that autophagy is differently induced by nutrient starvation in most tissues. In some tissues, autophagy even occurs actively without starvation treatments. Our results suggest that the regulation of autophagy is organ dependent and the role of autophagy is not restricted to the starvation response. This transgenic mouse model is a useful tool to study mammalian autophagy.

In Vivo Analysis of Autophagy in Response to Nutrient Starvation Using Transgenic Mice Expressing a Fluorescent Autophagosome Marker

ion step follows the decarboxylation, which is consistent with the deuterium isotopic effects observed for thymine 7-hydroxylase which indicate that an irreversible step (or steps) occurs prior to the C-H bond breaking.395 It has also been shown for prolyl 4-hydroxylase that a substrate-derived radical is generated in the reaction, which is consistent with a rebound mechanism.437 It is important to point out that no oxygen intermediate (i.e., bridged superoxo or oxo-ferryl) has been observed for any R-KGdependent enzyme. This warrants future theoretical and experimental study. A detailed molecular mechanism has been proposed for IPNS based on spectroscopic and crystallographic studies.422 Resting IPNS/FeII is also 6C and thus relatively stable toward dioxygen. Substrate ACV binds directly to FeII IPNS through its thiolate group, providing an open coordination position at the FeII. O2 can then react to form an FeIII-superoxo intermediate. This intermediate is suggested422 to perform the first hydrogen-atom abstraction step and close the â-lactam ring, resulting in the formation of the first water molecule and generating an FeIVdO-II intermediate, which completes the second ringclosure process by hydrogen-atom abstraction forming a thiazolidine ring. Previously proposed mechanisms of ACCO involved direct binding of cosubstrate ascorbate to the iron before O2 as part of the oxygen activation process.438,439 The EPR and ESEEM studies of the NO complex of ACCO suggested a quite different molecular mechanism for ACCO.435 An FeIII-superoxo intermediate is proposed. Whether it is preceded by a 6C f 5C process with substrate binding is presently under study.440 This intermediate is thought to initiate a radical process by single hydrogen-atom abstraction or electron-coupled proton transfer (PT)ion or electron-coupled proton transfer (PT) from the bound amino group. The resulting substrate radical may undergo spontaneous conversion into products. The role of cosubstrate ascorbate is proposed to reduce the toxic peroxo byproduct to water. Alternatively, the two-electron reduction of FeIIIsuperoxo by the cosubstrate ascorbate could result in an FeIVdO-II intermediate which initiates the radical reaction.435 4. Rieske-Type Dioxygenases Biochemical Characterization. The Rieske ironsulfur center is a two iron-two sulfur cluster ([2Fe2S]) which has a 2His (on one iron), 2Cys (on the other iron) coordination environment, instead of the 4Cys present in plant ferredoxins. It plays a key role in the electron transport pathway in membranebound cytochrome complexes as well as in some dioxygenases.441 The latter are mainly comprised of two protein components: a reductase containing flavin and a ferredoxin [2Fe-2S], and a terminal oxygenase containing a Rieske [2Fe-2S] cluster and a non-heme iron active site.442 Except for the recently reported alkene monooxygenase that has a binuclear iron site in its terminal oxygenase,10 most of the Rieske-type oxygenases have a mononuclear iron site, which is believed to be the site of dioxygen activation and substrate oxygenation.442,443 The majority of the Rieske-type mononuclear non-heme oxygenases form a family of enzymes which are aromatic-ring-hydroxylating dioxygenases. These catalyze the regioand stereospecific cis-dihydroxylation of an aromatic ring using dioxygen and NAD(P)H (Table 1). Examples include benzene dioxygenase (BDO, EC 1.14.12.3),444 phthalate dioxygenase (PDO, EC 1.14.12.7),445 toluene dioxygenase (EC 1.14.12.11),446 and naphthalene 1,2-dioxygenase (NDO, EC 1.14.12.12),447 which initiate the aerobic degradation of aromatic compounds in the soil bacteria and are targets for bioengineering in bioremediation. This step is the first step in the pathway that ultimately leads to ring cleavage by the intraand extradiol dioxygenases (sections II.B.2 and II.C.1).443 Besides these bacterial dioxygenases, other Rieske-type mononuclear non-heme oxygenases include anthranilate 1,2-dioxygenase (EC 1.14.12.1),448 which deaminates and decarboxylates the substrate to produce catechol; chlorophenylacetate 3,4-dioxygenase (EC 1.14.2.13),449 which converts substrate to catechol with chloride elimination; and 4-methoxybenzoate O-demethylase (putidamonooxin),450 which catalyzes the conversion of 4-methoxybenzoic acid to 4-hydroxybenzoic acid and formaldehyde. The reductase component is usually a monomer (MW ) 12-15 kDa) and utilizes flavin to mediate ET from the two-electron donor NAD(P)H to the oneelectron acceptor [2Fe-2S] cluster and is specific to each terminal oxygenase; other electron donors do not support efficient oxygenation.442 The crystal structure of phthalate dioxygenase reductase is available.451 The terminal oxygenases are large protein aggregates (MW ) 150-200 kDa) containing either multiples of R subunits (BDO R2, PDO R4) or an equimolar combination of R and â subunits (toluene dioxygenase R2â2, NDO R3â3). The R subunits contain a Rieske [2Fe-2S] cluster and a catalytic non-heme FeII center. â subunits do not seem to be involved in the catalytic function (vide infra). Kinetics. Steady-state kinetic studies coupled with various rapid reaction studies of the partial reactions of PDO allowed Ballou et al. to propose a kinetic scheme (Scheme 15).443 On the basis of steady state 278 Chemical Reviews, 2000, Vol. 100, No. 1 Solomon et al.

Geometric and Electronic Structure/Function Correlations in Non-Heme Iron Enzymes

It has been known for many decades that autophagy, a conserved lysosomal degradation pathway, is highly active during differentiation and development. However, until the discovery of the autophagy-related (ATG) genes in the 1990s, the functional significance of this activity was unknown. Initially, genetic knockout studies of ATG genes in lower eukaryotes revealed an essential role for the autophagy pathway in differentiation and development. In recent years, the analyses of systemic and tissue-specific knockout models of ATG genes in mice has led to an explosion of knowledge about the functions of autophagy in mammalian development and differentiation. Here we review the main advances in our understanding of these functions.

Autophagy in mammalian development and differentiation

Mammalian lipoxygenases and their biological relevance.

Membrane-enclosed organelles, a defining characteristic of eukaryotic cells, are lost during differentiation of specific cell types such as reticulocytes (an intermediate in differentiation of erythrocytes), central fibre cells the eye lens, and keratinocytes1. The degradation of these organelles must be tightly regulated with respect to both the time of activation and the specificity of membrane degradation. The expression of 15-lipoxygenase (15-LOX) peaks in reticulocytes immediately before organelle degradation2. Here we show that 15-LOX integrates into the membranes of various organelles, allowing release of proteins from the organelle lumen and access of proteases to both lumenal and integral membrane proteins. In addition, by sparing the plasma membrane, 15-LOX shows the required specificity for organellar membranes. Thus, the action of 15-LOX provides a mechanism by which the natural degradation process can be explained. This conclusion is supported by our finding that lipoxygenase expression in the eye lens is restricted to the region at which organelle degradation occurs.

A function for lipoxygenase in programmed organelle degradation

Historically, the neuron has been the conceptual focus for almost all of neuroscience research. In recent years, however, the concept of the neurovascular unit has emerged as a new paradigm for investigating both physiology and pathology in the CNS. This concept proposes that a purely neurocentric focus is not sufficient, and emphasizes that all cell types in the brain including neuronal, glial and vascular components, must be examined in an integrated context. Cell–cell signaling and coupling between these different compartments form the basis for normal function. Disordered signaling and perturbed coupling form the basis for dysfunction and disease. In this mini-review, we will survey four examples of this phenomenon: hemodynamic neurovascular coupling linking blood flow to brain activity; cellular communications that evoke the blood–brain barrier phenotype; parallel systems that underlie both neurogenesis and angiogenesis in the CNS; and finally, the potential exchange of trophic factors that may link neuronal, glial and vascular homeostasis.

Cell-cell signaling in the neurovascular unit.

Background and Purpose— The natural product baicalein is a specific inhibitor of 12/15-lipoxygenase, but it also has antioxidant properties. The current study was designed to test if the neuroprotective properties of baicalein are related to its lipoxygenase inhibition. Methods— The presence of 12/15-lipoxygenase in the ischemic mouse brain was demonstrated by immunohistochemistry. A mouse model of transient middle cerebral artery occlusion was used to study lipoxygenase-dependent protection of the ischemic brain by baicalein. Rat primary neurons were subjected to oxidative stress in the presence or absence of baicalein. Results— In a mouse model of transient middle cerebral artery occlusion, 12/15-lipoxygenase is increased in the peri-infarct area surrounding the primary infarction, predominantly in neurons. Oxidative toxicity in primary rat neurons is reduced by baicalein. C57Bl6J mice are protected against transient focal ischemia by intraperitoneal injection of baicalein, and a similar degree of prote...

Klaus van Leyen

Papers

Mammalian lipoxygenases and their biological relevance.

A function for lipoxygenase in programmed organelle degradation

Cell-cell signaling in the neurovascular unit.

Baicalein and 12/15-Lipoxygenase in the Ischemic Brain

Structure of the semaphorin-3A receptor binding module.