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The redox coenzymes NADH and NADPH are broadly required for energy metabolism, biosynthesis and detoxification. Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes, a holistic understanding of the regulation and compartmentalization of NADH- and NADPH-dependent pathways is lacking, partly because of a lack of tools with which to investigate these processes in living cells. We have previously reported the use of the naturally occurring Lactobacillus brevis H2O-forming NADH oxidase (LbNOX) as a genetic tool for manipulation of the NAD+/NADH ratio in human cells. Here, we present triphosphopyridine nucleotide oxidase (TPNOX), a rationally designed and engineered mutant of LbNOX that is strictly specific to NADPH. We characterized the effects of TPNOX expression on cellular metabolism and used it in combination with LbNOX to show how the redox states of mitochondrial NADPH and NADH pools are connected.

A decline in electron transport chain (ETC) activity is associated with many human diseases. Although diminished mitochondrial adenosine triphosphate production is recognized as a source of pathology, the contribution of the associated reduction in the ratio of the amount of oxidized nicotinamide adenine dinucleotide (NAD(+)) to that of its reduced form (NADH) is less clear. We used a water-forming NADH oxidase from Lactobacillus brevis (LbNOX) as a genetic tool for inducing a compartment-specific increase of the NAD(+)/NADH ratio in human cells. We used LbNOX to demonstrate the dependence of key metabolic fluxes, gluconeogenesis, and signaling on the cytosolic or mitochondrial NAD(+)/NADH ratios. Expression of LbNOX in the cytosol or mitochondria ameliorated proliferative and metabolic defects caused by an impaired ETC. The results underscore the role of reductive stress in mitochondrial pathogenesis and demonstrate the utility of targeted LbNOX for direct, compartment-specific manipulation of redox state.

Wiskott-Aldrich syndrome protein (WASP)-homology domain 2 (WH2) is a small and widespread actin-binding motif. In the WASP family, WH2 plays a role in filament nucleation by Arp2/3 complex. Here we describe the crystal structures of complexes of actin with the WH2 domains of WASP, WASP-family verprolin homologous protein, and WASP-interacting protein. Despite low sequence identity, WH2 shares structural similarity with the N-terminal portion of the actin monomer-sequestering thymosin [beta] domain (T[beta]). We show that both domains inhibit nucleotide exchange by targeting the cleft between actin subdomains 1 and 3, a common binding site for many unrelated actin-binding proteins. Importantly, WH2 is significantly shorter than T[beta] but binds actin with [approximately equal to] 10-fold higher affinity. WH2 lacks a C-terminal extension that in T[beta]4 becomes involved in monomer sequestration by interfering with intersubunit contacts in F-actin. Owing to their shorter length, WH2 domains connected in tandem by short linkers can coexist with intersubunit contacts in F-actin and are proposed to function in filament nucleation by lining up actin subunits along a filament strand. The WH2-central region of WASP-family proteins is proposed to function in an analogous way by forming a special class of tandem repeats whose function is to line up actin and Arp2 during Arp2/3 nucleation. The structures also suggest a mechanism for how profilin-binding Pro-rich sequences positioned N-terminal to WH2 could feed actin monomers directly to WH2, thereby playing a role in filament elongation. x-ray crystallography isothermal titration calorimetry | nucleotide exchange

Author(s): Chester L. Drum [1, 2, 3]; Shui-Zhong Yan [1]; Joel Bard [2]; Yue-Quan Shen [1]; Dan Lu [1]; Sandriyana Soelaiman [1]; Zenon Grabarek [2]; Andrew Bohm [2, 4, 5]; [...]

Research has been conducted on the Ca (super)2+ /calmodulin-dependent protein kinase II. The activation mechanism of this kinase has been studied via the steady-state and stopped-flow fluorescent spectroscopies and the results are reported.

The regulation of caldesmon (CaD) by calmodulin (CaM) in relation to the spatial orientation of the proteins was determined through fluorescence energy transfer techniques. Results have shown that the hydrophobic region of Trp chains at positions 659 and 692 determined the CaM-CaD binding. An antiparallel orientation of peptides in the complex binding configuration had a significant effect to the interaction of N-terminal domain of with site B and C-terminal with site A.

Acetylation of lysine residues in rabbit skeletal troponin C (TnC) causes drastic decrease in the overall stability of the protein. The positively charged lysine side chains located in the N-terminal domain moderate TnC's structural stability and interactions with Ca2+ and other troponin components. The lower stability of the acetylated form is due to the intramolecular repulsive forces caused by the increase in the overall negative charge of the molecule.

On the basis of the role in the Ca2+-dependent regulation of vertebrate striated muscle contraction by the interaction between troponin C (TnC) and troponin I (TnI), it was inferred that the inhibitory region on TnI interacts with the N-terminal, regulatory domain of TnC. The characterization of the cross-linked heterodimer (AcCxI) indicated extensive cross-linking between TnC and TnI. This involved both the N-terminal and C-terminal domains of TnC, besides the N-terminal, C-terminal, and inhibitory regions of TnI.

The contraction of cardiac and skeletal muscles is regulated by two proteins, troponin and tropomyosin, which interact with actin filaments (molecules involved in the physical contraction and relaxation of the muscle). Troponin contains three subunits, troponin C, troponin I, and troponin T, which have distinct activities. The troponin C molecule has been isolated, purified and examined by X-ray crystallography so that its three-dimensional structure is known. Using site-directed mutagenesis (in which a particular area of the DNA that encodes the gene is altered), the sites and the mechanism of muscle contraction regulation by troponin can be studied. The regulatory sites have been identified as sites where calcium is bound to one end of the troponin molecule. The calcium binding causes a change in the shape of the molecule, and allows the molecule to bind troponin I. The shape of troponin C was stabilized by bridging the molecule through chemical bonding with two molecules of sulphur. The treponin molecule could no longer bind calcium, which affected the ability of troponin C to bind with troponin I. Consequently, it could not regulate the contraction of muscle fibers. These studies will lead to further understanding of how muscle contraction occurs and how it is regulated. (Consumer Summary produced by Reliance Medical Information, Inc.)

The structural homology and molecular dynamics methods are used to construct various all-atom representation models of smooth muscle myosin light chain kinase (smMLCK) and its two fragments which are a calcium-calmodulin complex-dependent enzyme that activates contraction of smooth muscle. The predicted and observed sedimentation coefficients for models were in agreement with each other for the independently folded catalytic domain, Fn3, and Ig domains which are aligned consecutively on the long axis of the molecule.

The mitochondrial uniporter is a Ca2+-activated Ca2+ channel complex that displays exceptionally high conductance and selectivity. Here, we report cellular metal toxicity screens highlighting the uniporter's role in Mn2+ toxicity. Cells lacking the pore-forming uniporter subunit, MCU, are more resistant to Mn2+ toxicity, while cells lacking the Ca2+-sensing inhibitory subunit, MICU1, are more sensitive than the wild type. Consistent with these findings, Caenorhabditis elegans lacking the uniporter's pore have increased resistance to Mn2+ toxicity. The chemical-genetic interaction between uniporter machinery and Mn2+ toxicity prompted us to hypothesize that Mn2+ can indeed be transported by the uniporter's pore, but this transport is prevented by MICU1. To this end, we demonstrate that, in the absence of MICU1, both Mn2+ and Ca2+ can pass through the uniporter, as evidenced by mitochondrial Mn2+ uptake assays, mitochondrial membrane potential measurements, and mitoplast electrophysiology. We show that Mn2+ does not elicit the conformational change in MICU1 that is physiologically elicited by Ca2+, preventing Mn2+ from inducing the pore opening. Our work showcases a mechanism by which a channel's auxiliary subunit can contribute to its apparent selectivity and, furthermore, may have implications for understanding how manganese contributes to neurodegenerative disease. [ABSTRACT FROM AUTHOR]

The mitochondrial calcium uniporter is a Ca2+-activated Ca2+ channel that is essential for dynamic modulation of mitochondrial function in response to cellular Ca2+ signals. It is regulated by two paralogous EF-hand proteins- MICU1 and MICU2, but the mechanism is unknown. Here, we demonstrate that both MICU1 and MICU2 are stabilized by Ca2+. We reconstitute the MICU1- MICU2 heterodimer and demonstrate that it binds Ca2+ cooperatively with high affinity. We discover that both MICU1 and MICU2 exhibit affinity for the mitochondria-specific lipid cardiolipin. We determine the minimum Ca2+ concentration required for disinhibition of the uniporter in permeabilized cells and report a close match with the Ca2+-binding affinity of MICU1- MICU2. We conclude that cooperative, high-affinity interaction of the MICU1- MICU2 complex with Ca2+ serves as an on-off switch, leading to a tightly controlled channel, capable of responding directly to cytosolic Ca2+ signals. [ABSTRACT FROM AUTHOR]