Summary The APOBEC (Apolipoprotein B mRNA Editing Catalytic Polypeptide-like) family of proteins has diverse and important functions in human health and disease. These proteins have an intrinsic ability to bind to both RNA and single-stranded (ss)DNA. Both function and tissue-specific expression varies widely for each APOBEC protein. We are beginning to understand that the activity of APOBEC proteins is regulated through genetic alterations, changes in their transcription and mRNA processing, and through their interactions with other macromolecules in the cell. Loss of cellular control of APOBEC activities leads to DNA hypermutation and promiscuous RNA editing associated with the development of cancer or viral drug resistance, underscoring the importance of understanding how APOBEC proteins are regulated.

The hairpin ribozyme requires functional group contributions from G8 to assist in phosphodiester bond cleavage. Previously, replacement of G8 by a series of nucleobase variants showed little effect on interdomain docking, but a 3-to 250-fold effect on catalysis. To identify G8 features that contribute to catalysis within the hairpin ribozyme active site, structures for five base variants were solved by X-ray crystallography in a resolution range between 2.3 to 2.7 Å. For comparison, a native all-RNA "G8" hairpin ribozyme structure was refined to 2.05 Å resolution. The native structure revealed a scissile bond angle (τ) of 158°, which is close to the requisite 180° 'in-line' geometry. Mutations G8(inosine), G8(diaminopurine), G8(aminopurine), G8(adenosine) and G8(uridine) folded properly, but exhibited non-ideal scissile bond geometries (τ ranging from 118° to 93°) that paralleled their diminished solution activities. A superposition ensemble of all structures, including a previously described hairpin ribozyme-vanadate complex, indicated the scissile bond can adopt a variety of conformations resulting from perturbation of the chemical environment, and provided a rationale for how the exocyclic amine of nucleobase 8 promotes productive, in-line geometry. Changes at position 8 also caused variations in the A−1 sugar pucker. In this regard, variants A8 and U8 appeared to represent non-productive ground-states in which their 2'-OH groups mimicked the pro-R, non-bridging oxygen of the vanadate transition-state complex. Finally, the results indicated that ordered water molecules bind near the 2'-hydroxyl of A−1, lending support to the hypothesis that solvent may play an important role in the reaction. † This work was supported by NIH Grant GM63162 to J.E.W. ‡ Protein Data Bank Codes for the reported structures: 1ZFR (G8), 1ZFT (G8I), 1ZFV (G8A), 1ZFX (G8U), 2BCY (G8AP), 2BB1 (G8DAP), 2BCZ (G8I/dA−1). § Present address: Rosalind Franklin School of Science and Medicine, Dept. Biochem. and Mol. Biol., 3333 Green Bay Road Rd., N. Chicago, IL 60064, USA * To whom correspondence should be addressed: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave, Box 712, Rochester, New York 14642 USA. Phone: 585 273-4516; Fax: 585 275-6007; Email: Joseph_Wedekind@URMC.Rochester.edu ∥ These authors contributed equally to this work.Supporting Information Available A stereo diagram of representative electron density maps is provided for the native and position 8 variants of this study (Figure 1). A diagram comparing the G8I/2'-OMe A−1 and G8I/2'-deoxy A−1 variants is also available (Figure 2). The method for HPLC composition analysis of AP8 and DAP8 crystals is reported, as well as elution profiles for the separated hairpin ribozyme strands (Figure 3). This information is provided free of charge at http://pubs.acs.org NIH Public Access The hairpin ribozyme is a small ribozyme whose family members catalyze a reversible, sitespecific phosphodiester bond cleavage reacti...

The literature suggests that the physiological functions for which mitochondria sequester Ca(2+) are (1). to stimulate and control the rate of oxidative phosphorylation, (2). to induce the mitochondrial permeability transition (MPT) and perhaps apoptotic cell death, and (3). to modify the shape of cytosolic Ca(2+) pulses or transients. There is strong evidence that intramitochondrial Ca(2+) controls both the rate of ATP production by oxidative phosphorylation and induction of the MPT. Since the results of these processes are so divergent, the signals inducing them must not be ambiguous. Furthermore, as pointed out by Balaban [J. Mol. Cell. Cardiol. 34 (2002 ) 11259-11271], for any repetitive physiological process dependent on intramitochondrial free Ca(2+) concentration ([Ca(2+)](m)), a kind of intramitochondrial homeostasis must exist so that Ca(2+) influx during the pulse is matched by Ca(2+) efflux during the period between pulses to avoid either Ca(2+) buildup or depletion. In addition, mitochondrial Ca(2+) transport modifies both spatial and temporal aspects of cytosolic Ca(2+) signaling. Here, we look at the amounts of Ca(2+) necessary to mediate the functions of mitochondrial Ca(2+) transport and at the mechanisms of transport themselves in order to set up a hypothesis about how the mechanisms carry out their roles. The emphasis here is on isolated mitochondria and on general mitochondrial properties in order to focus on how mitochondria alone may function to fulfill their physiological roles even though the interactions of mitochondria with other organelles, particularly with endoplasmic and sarcoplasmic reticulum [Sci. STKE re1 (2004) 1-9], may also influence this story.

Human APOBEC3G (hA3G) is a cytidine deaminase that restricts human immunodeficiency virus (HIV)-1 infection in a vif(the virion infectivity factor from HIV)-dependent manner. hA3G from HIV-permissive activated CD4؉ T-cells exists as an inactive, high molecular mass (HMM) complex that can be transformed in vitro into an active, low molecular mass (LMM) variant comparable with that of HIV-non-permissive CD4؉ T-cells. Here we present low resolution structures of hA3G in HMM and LMM forms determined by small angle x-ray scattering and advanced shape reconstruction methods. The results show that LMM particles have an extended shape, dissimilar to known cytidine deaminases, featuring novel tail-to-tail dimerization. Shape analysis of LMM and HMM structures revealed how symmetric association of dimers could lead to minimal HMM variants. These observations imply that the disruption of cellular HMM particles may require regulation of protein-RNA, as well as protein-protein interactions, which has implications for therapeutic development.

Hoffman DL, Salter JD, Brookes PS. Response of mitochondrial reactive oxygen species generation to steady-state oxygen tension: implications for hypoxic cell signaling. Am J Physiol Heart Circ Physiol 292: H101-H108, 2007. First published September 8, 2006; doi:10.1152/ajpheart.00699.2006.-Mitochondria are proposed to play an important role in hypoxic cell signaling. One currently accepted signaling paradigm is that the mitochondrial generation of reactive oxygen species (ROS) increases in hypoxia. This is paradoxical, because oxygen is a substrate for ROS generation. Although the response of isolated mitochondrial ROS generation to [O 2] has been examined previously, such investigations did not apply rigorous control over [O2] within the hypoxic signaling range. With the use of open-flow respirometry and fluorimetry, the current study determined the response of isolated rat liver mitochondrial ROS generation to defined steady-state [O2] as low as 0.1 M. In mitochondria respiring under state 4 (quiescent) or state 3 (ATP turnover) conditions, decreased ROS generation was always observed at low [O 2]. It is concluded that the biochemical mechanism to facilitate increased ROS generation in response to hypoxia in cells is not intrinsic to the mitochondrial respiratory chain alone but may involve other factors. The implications for hypoxic cell signaling are discussed.hypoxia-inducible factor; superoxide; free radicals; mitochondria; metabolism REACTIVE OXYGEN SPECIES (ROS) generated by mitochondria are key intermediates in a diverse array of cell signaling events, including regulation of the cell cycle (68), proliferation (51), metalloproteinases (65), apoptosis (54), protein kinases (8,16,54,64), phosphatases (62), growth factor signaling (78), and transcription factors (44, 52) (see Ref. 31 for review). One area of investigation that has been the focus of much recent interest is the potential role of mitochondrial ROS in the signaling events that occur during hypoxia, including the regulation of hypoxia-inducible factors such as HIF-1␣ (2, 4, 7, 10, 25, 32, 48, 56, 59, 67, 84 -86, 88, 89). Whereas the downstream effects of mitochondrially derived ROS are relatively well established (vide supra), the mechanisms and the directionality (i.e., increased or decreased) of mitochondrial ROS generation in hypoxia are currently under debate (59,86). This is an important area of study, since it appears that not only can mitochondria regulate HIF, but HIF and its downstream target genes (e.g., heme oxygenase) are important pathophysiological regulators of mitochondrial function (5, 42, 77). Thus, perturbing the mitochondria/HIF signaling loop may contribute to disease pathogenesis (10).A widely accepted model of hypoxic cell signaling is that mitochondrial ROS generation increases in hypoxia (7,25,32,67,85,86,88,89). This is proposed to occur via O 2 limitation at the terminal enzyme in the mitochondrial respiratory chain, cytochrome c oxidase (complex IV), causing a backup of electrons in the proximal chain and increase...

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Manganese is both an essential element and a potential toxicant. It is essential for the function of manganese superoxide dismutase, pyruvate carboxylase, and arginase (Greger 1999) and can substitute for magnesium in many kinases. However, excessive brain manganese can produce a neurological syndrome associated with loss of dopamine in the striatum and death of non-dopaminergic neurons in the globus pallidus. X-ray absorbance near edge structure (XANES) spectroscopy represents a powerful tool for investigation of relatively small amounts of essential elements or toxicants in biological systems. It can be used not only to determine the oxidation states of these elements, but also to help identify complexes or pools of the element in the biological system by comparing spectra derived from the biological system with those of relevant model compounds. P i , manganese(II) phosphate; Mnpin, manganese protophoryrin IX chloride; Mnpor, manganese meso-tetra(4-sulfonatophenyl) porphine; NMR, nuclear magnetic resonance; PHPA, 2,2¢-dihydroxy-biphenol-5,5¢-diacetate; ROS, reactive oxygen species; SOD, superoxide dismutase; TPP, tetraphenyl phosphonium; XANES, X-ray absorption near edge structure.

The hairpin ribozyme requires functional groups from Ade38 to achieve efficient bond cleavage or ligation. To identify molecular features that contribute to catalysis, structures of position 38 base variants 2,6-diaminopurine (DAP), 2-aminopurine (AP), cytosine (Cyt), and guanine (Gua) were determined between 2.2 and 2.8 Å resolution. For each variant, two substrate modifications were compared: (1) a 29-O-methyl-substituent at Ade-1 was used in lieu of the nucleophile to mimic the precatalytic state, and (2) a 39-deoxy-29,59-phosphodiester linkage between Ade-1 and Gua+1 was used to mimic a reactionintermediate conformation. While the global fold of each variant remained intact, the results revealed the importance of Ade38 N1 and N6 groups. Absence of N6 resulting from AP38 coincided with failure to localize the precatalytic scissile phosphate. Cyt38 severely impaired catalysis in a prior study, and its structures here indicated an anti base conformation that sequesters the imino moiety from the scissile bond. Gua38 was shown to be even more deleterious to activity. Although the precatalytic structure was nominally affected, the reaction-intermediate conformation indicated a severe electrostatic clash between the Gua38 keto oxygen and the pro-Rp oxygen of the scissile bond. Overall, position 38 modifications solved in the presence of 29-OMe Ade-1 deviated from in-line geometry, whereas variants with a 29,59 linkage exhibited S-turn destabilization, as well as base conformational changes from syn to anti. These findings demonstrate the importance of the Ade38 Watson-Crick face in attaining a reaction-intermediate state and the sensitivity of the RNA fold to restructuring when electrostatic and shape features fail to complement.