Tuning the Reactivity of Cofacial Porphyrin Prisms for Oxygen Reduction Using Modular Building Blocks.

We assembled eight cofacial porphyrin prisms using MTPyP (M = Co(II) or Zn(II), TPyP = 4-tetrapyridylporphyrin) and functionalized ruthenium-based "molecular clips" using coordination-driven self-assembly. Our approach allows for the rapid synthesis of these architectures in isolated yields as high as 98% for the assembly step. Structural and reactivity studies provided a deeper understanding of the role of the building blocks on the oxygen reduction reaction (ORR). Catalytic efficacy was probed by using cyclic and hydrodynamic voltammetry on heterogeneous catalyst inks in aqueous media. The reported prisms showed outstanding selectivity (>98%) for the kinetically hindered 4e-/4H+ reduction of O2 to H2O over the kinetically more accessible 2e-/2H+ reduction to H2O2. Furthermore, we have demonstrated significant cofacial enhancement in the observed catalytic rate constant ks (∼5 orders of magnitude) over the mononuclear analogue. We conclude that the steric bulk of the clip plays an important role in the structural dynamics of these prisms, which in turn modulates the ORR reactivity with respect to selectivity and kinetics.

Coordination-Driven Self-Assembly of Cyclometalated Iridium Squares Using Linear Aromatic Diisocyanides.

Here, we demonstrate facile [4 + 4] coordination-driven self-assembly of cyclometalated iridium(III) using linear aryldiisocyanide bridging ligands (BLs). A family of nine new [Ir(C^N)2(µ-BL)]44+ coordination cages is described, where C^N is the cyclometalating ligand-2-phenylpyridine (ppy), 2-phenylbenzothiazole (bt), or 1-phenylisoquinoline (piq)-and BL is the diisocyanide BL, with varying spacer lengths between the isocyanide binding sites. These supramolecular coordination compounds are prepared via a one-pot synthesis, with isolated yields of 40-83%. 1H NMR spectroscopy confirms the selective isolation of a single product, which is affirmed to be the M4L4 square by high-resolution mass spectrometry. Detailed photophysical studies were carried out to reveal the nature of the luminescent triplet states in these complexes. In most cases, phosphorescence arises from the [Ir(C^N)2]+ nodes, with the emission color determined by the cyclometalating ligand. However, in two cases, the lowest-energy triplet state resides on the aromatic core of the BL, and weak phosphorescence from that state is observed. This work shows that aromatic diisocyanide ligands enable coordination-driven assembly of inert iridium(III) nodes under mild conditions, producing supramolecular coordination complexes with desirable photophysical properties.

DNA mutagenic activity and capacity for HIV-1 restriction of the cytidine deaminase APOBEC3G depend on whether DNA or RNA binds to tyrosine 315.

APOBEC3G (A3G) belongs to the AID/APOBEC protein family of cytidine deaminases (CDA) that bind to nucleic acids. A3G mutates the HIV genome by deamination of dC to dU, leading to accumulation of virus-inactivating mutations. Binding to cellular RNAs inhibits A3G binding to substrate single-stranded (ss) DNA and CDA activity. Bulk RNA and substrate ssDNA bind to the same three A3G tryptic peptides (amino acids 181-194, 314-320, and 345-374) that form parts of a continuously exposed protein surface extending from the catalytic domain in the C terminus of A3G to its N terminus. We show here that the A3G tyrosines 181 and 315 directly cross-linked ssDNA. Binding experiments showed that a Y315A mutation alone significantly reduced A3G binding to both ssDNA and RNA, whereas Y181A and Y182A mutations only moderately affected A3G nucleic acid binding. Consistent with these findings, the Y315A mutant exhibited little to no deaminase activity in an Escherichia coli DNA mutator reporter, whereas Y181A and Y182A mutants retained ∼50% of wild-type A3G activity. The Y315A mutant also showed a markedly reduced ability to assemble into viral particles and had reduced antiviral activity. In uninfected cells, the impaired RNA-binding capacity of Y315A was evident by a shift of A3G from high-molecular-mass ribonucleoprotein complexes to low-molecular-mass complexes. We conclude that Tyr-315 is essential for coordinating ssDNA interaction with or entry to the deaminase domain and hypothesize that RNA bound to Tyr-315 may be sufficient to competitively inhibit ssDNA deaminase-dependent antiviral activity.

Highly Emissive Self-Assembled BODIPY-Platinum Supramolecular Triangles.

Light-emitting supramolecular coordination complexes (SCCs) have been widely studied for applications in the chemical and biological sciences. Herein, we report the coordination-driven self-assembly of two highly emissive platinum(II) supramolecular triangles (1 and 2) containing BODIPY-based bridging ligands. The metallacycles exhibit favorable anticancer activities against HeLa cells (IC50 of 6.41 and 2.11 µM). The characteristic ∼570 nm fluorescence of the boron dipyrromethene (BODIPY) moieties in the metallacycles permits their intracellular visualization using confocal microscopy. Additionally, the BODIPY fluorophore is an excellent photodynamic agent, making the metallacycles as ideal therapeutics for photodynamic therapy (PDT) and chemotherapy. In vitro studies demonstrate that the combination indexes against HeLa cells are 0.56 and 0.48 for 1 and 2, respectively, confirming their synergistic anticancer effect. More importantly, these SCCs also exhibit superior anticancer efficacy toward cisplatin-resistant A2780cis cell line by combining PDT and chemotherapy, showing promise in overcoming drug resistance. This study exploits a multicomponent approach to self-assembled metallacages that enables design of effective theranostic agents wherein the platinum acceptors are toxic chemotherapeutics and the BODIPY donors are imaging probes and photosensitizers. Since each piece may be independently tuned, i.e., Pt(II) polypyridyl fragment swapped for Pt(II) phosphine, the activity may be optimized without a total redesign of the system.

Tuning the Activity of Heterogeneous Cofacial Cobalt Porphyrins for Oxygen Reduction Electrocatalysis through Self-Assembly.

We report a suite of coordination-driven self-assembled prisms for heterogeneous electrocatalytic oxygen reduction (ORR) differing in the molecular clips linking two porphyrin faces in a cofacial arrangement. ORR activities and selectivities of monomeric CoTPyP along with cofacial prisms Ox-Co, Oxa-Co, and Benzo-Co were probed using cyclic voltammetry and rotating ring-disk techniques. All species were immobilized as heterogeneous catalysts on glassy carbon electrodes using a Nafion ink method. The selectivities of Ox-Co, Oxa-Co, and Benzo-Co prisms towards H2 O as determined by RRDE were 87, 97, and 75 %, respectively. The current density of the Oxa-Co plateaus at five times that of Pt/C when normalized per Co/Pt. The high synthetic yield (79 %), competitive overpotential (η ≈800 mV) and high selectivity (%H2 O ≈97 %) of the Oxa-Co highlights how self-assembly can be used to address multi-electron multi-proton transformations using polynuclear catalysts.

Synthesis, Characterization, and Catalytic Studies of Dinuclear d8 Metal-Phosphazane Complexes.

Herein we report the synthesis and characterization of two diphosphazane (P-N-P) ligands, along with their corresponding novel palladium and platinum complexes. Compounds were characterized by FTIR and NMR spectroscopy, single crystal X-ray diffraction (SC-XRD), and powder X-ray diffraction (PXRD)), as well as mass spectrometry. The Pd(II) complex of the p-phenyldiphosphazane was shown to effectively and efficiently catalyze Suzuki-Miyaura cross-coupling reactions between a variety of substrates. Yields were as high as 96%, with reaction times as short as 15 min at room temperature and open to air. No additional supporting ligands, such as triphenylphosphine, were needed. The work reported here expands the use of phosphazane ligands to support catalytic centers and provides an understanding of phosphazane metal-ligand bonding interactions (specifically diphosphazanes).

Coordination-Driven Self-Assembly of Ruthenium Polypyridyl Nodes Resulting in Emergent Photophysical and Electrochemical Properties.

Ruthenium polypyridyl complexes are among the most studied molecular species for photochemical applications such as light-harvesting and photocatalysis, with [Ru(bpy)3]2+ (bpy = 2,2'-bipyridine) serving as an iconic example. We report the use of the [Ru(bpy)2]2+ fragment as a 90° acceptor tecton (M) in coordination-driven self-assembly to synthesize a M4L4 metallacycle (L = 4,4'-bipyridine) and a M6L4 truncated tetrahedral cage [L = 2,4,6-tris(4-pyridyl)-1,3,5-triazine]. The M6L4 cage possesses emergent properties attributed to its unique electronic structure, which results in increased visible-light absorption and an emission band that decays biexponentially with times of 3 and 790 ns. The presence of multiple ruthenium centers in the cage results in multiple RuIII/II reduction events, with a cathodic shift of the first reduction relative to that of [Ru(bpy)3]Cl2 (0.56 V vs 1.05 V). The ligand-centered reduction shifts anodically (-1.29 vs -1.64 V) versus the first bpy reduction observed in the parent [Ru(bpy)3]Cl2. The photophysical properties are explained by the existence of two localized charge-transfer states in the cage molecule: one that draws upon the bipyridine π* orbitals and the other upon the 2,4,6-tris(4-pyridyl)-1,3,5-triazine π* orbitals.

A Self-Assembled Cofacial Cobalt Porphyrin Prism for Oxygen Reduction Catalysis.

Herein we report the first study of the oxygen reduction reaction (ORR) catalyzed by a cofacial porphyrin scaffold accessed in high yield (overall 53%) using coordination-driven self-assembly with no chromatographic purification steps. The ORR activity was investigated using chemical and electrochemical techniques on monomeric cobalt(II) tetra(meso-4-pyridyl)porphyrinate (CoTPyP) and its cofacial analogue [Ru8(η6-iPrC6H4Me)8(dhbq)4(CoTPyP)2][OTf]8 (Co Prism) (dhbq = 2,5-dihydroxy-1,4-benzoquinato, OTf = triflate) as homogeneous oxygen reduction catalysts. Co Prism is obtained in one self-assembly step that organizes six total building blocks, two CoTPyP units and four arene-Ru clips, into a cofacial motif previously demonstrated with free-base, Zn(II), and Ni(II) porphyrins. Turnover frequencies (TOFs) from chemical reduction (66 vs 6 h-1) and rate constants of overall homogeneous catalysis (kobs) determined from rotating ring-disk experiments (1.1 vs 0.05 h-1) establish a cofacial enhancement upon comparison of the activities of Co Prism and CoTPyP, respectively. Cyclic voltammetry was used to initially probe the electrochemical catalytic behavior. Rotating ring-disk electrode studies were completed to probe the Faradaic efficiency and obtain an estimate of the rate constant associated with the ORR.

Photophysical Enhancement of Triplet Emitters by Coordination-Driven Self-Assembly.

The quantum yields of organic fluorophores used as donors in coordination-driven self-assembly often suffer from the heavy atom effect of nearby metal sites. Here, the role of intersystem crossing from a deactivating process to one that delivers emissive triplet states was reversed. A phosphorescent trans bis-N-heterocyclic carbene platinum(II) compound, Pt(dhim)2 (C≡C-4-py)2 (D1; dhim=1,3-dihexyl-2-H-imidazol-2-ylidene), was used along with other linear donors 4,4'-bipyridine (D2) and 1,4-bis(4-pyridyl ethynyl)benzene (D3) in self-assembly reactions with Pt(dtbpy)X2 acceptors (dtbpy=4,4'-di-tert-butyl-2,2'-bipyridine) to afford three metallacycles. Photophysical investigations revealed that, although the building blocks used to construct M1 have relatively low quantum yields (Φ=1.2 and <1 % for D1 and 2, respectively), the metallacycle has a quantum yield of 14 %. This increase reflects a change in radiative rate constant from 3.6×104  s-1 for D1 to 2.1×105  s-1 for M1.

Exploring the Role of Carbonate in the Formation of an Organomanganese Tetramer.

The formation of metal-oxygen clusters is an important chemical transformation in biology and catalysis. For example, the biosynthesis of the oxygen-evolving complex in the enzyme photosystem II is a complicated stepwise process that assembles a catalytically active cluster. Herein we describe the role that carbonato ligands have in the formation of the known tetrameric complex [Mn(CO)3(µ3-OH)]4 (1). Complex 1 is synthesized in one step via the treatment of Mn2(CO)10 with excess Me3NO·2H2O. Alternatively, when anhydrous Me3NO is used, an OH-free synthetic intermediate (2) with carbonato ligands is produced. Complex 2 produces carbon dioxide, Me3NO·2H2O, and 1 when treated with water. Labeling studies reveal that the µ3-OH ligands in 1 are derived from the water and possibly the carbonato ligands in 2.

Phosphorescent Decanuclear Bimetallic Pt6M4 (M = Zn, Fe) Tetrahedral Cages.

Coordination-driven self-assembly delivers discrete, nanoscopic architectures that may preserve or enhance the physicochemical properties of their parent building blocks. Herein, we report the syntheses, characterization, and photophysical properties of two tetrahedral cages, [ZnII4L6](PF6)8 (C1) and [FeII4L6](OTf)8 (C2), where L = PtII(PEt3)2(C≡C-bpy)2 (PEt3 = triethylphosphine; C≡C-bpy = 5-ethynyl-2,2'-bipyridine) and OTf = trifluoromethanesulfonate. C1 and C2 were assembled in isolated yields of 72% and 81% by treating 2 equiv of Zn(NO3)2·6H2O or Fe(OTf)2 with 3 equiv of L, respectively. Both cages were fully characterized by NMR, electrospray ionization mass spectrometry, and single-crystal X-ray diffraction (SCXRD). The local D3 symmetry at each polypyridyl metal node raises the possibility of a number of isomeric cages; however, only the homochiral enantiomers (ΔΔΔΔ and ΛΛΛΛ) are formed based on 1H NMR and SCXRD. C1 exhibits phosphorescence centered at 545 nm with a quantum yield of 10% in N2-degassed acetonitrile at 25 °C. The quantum yield of C2 is significantly lower due to a nonradiative relaxation from 5MC (MC = metal-centered) states introduced by the FeII nodes.

RNA binding to APOBEC3G induces the disassembly of functional deaminase complexes by displacing single-stranded DNA substrates.

APOBEC3G (A3G) DNA deaminase activity requires a holoenzyme complex whose assembly on nascent viral reverse transcripts initiates with A3G dimers binding to ssDNA followed by formation of higher-order A3G homo oligomers. Catalytic activity is inhibited when A3G binds to RNA. Our prior studies suggested that RNA inhibited A3G binding to ssDNA. In this report, near equilibrium binding and gel shift analyses showed that A3G assembly and disassembly on ssDNA was an ordered process involving A3G dimers and multimers thereof. Although, fluorescence anisotropy showed that A3G had similar nanomolar affinity for RNA and ssDNA, RNA stochastically dissociated A3G dimers and higher-order oligomers from ssDNA, suggesting a different modality for RNA binding. Mass spectrometry mapping of A3G peptides cross-linked to nucleic acid suggested ssDNA only bound to three peptides, amino acids (aa) 181-194 in the N-terminus and aa 314-320 and 345-374 in the C-terminus that were part of a continuous exposed surface. RNA bound to these peptides and uniquely associated with three additional peptides in the N- terminus, aa 15-29, 41-52 and 83-99, that formed a continuous surface area adjacent to the ssDNA binding surface. The data predict a mechanistic model of RNA inhibition of ssDNA binding to A3G in which competitive and allosteric interactions determine RNA-bound versus ssDNA-bound conformational states.

Cigarette smoke induces distinct histone modifications in lung cells: implications for the pathogenesis of COPD and lung cancer.

Cigarette smoke (CS)-mediated oxidative stress induces several signaling cascades, including kinases, which results in chromatin modifications (histone acetylation/deacetylation and histone methylation/demethylation). We have previously reported that CS induces chromatin remodeling in pro-inflammatory gene promoters; however, the underlying site-specific histone marks formed in histones H3 and H4 during CS exposure in lungs in vivo and in lung cells in vitro, which can either drive gene expression or repression, are not known. We hypothesize that CS exposure in mouse and human bronchial epithelial cells (H292) can cause site-specific posttranslational histone modifications (PTMs) that may play an important role in the pathogenesis of CS-induced chronic lung diseases. We used a bottom-up mass spectrometry approach to identify some potentially novel histone marks, including acetylation, monomethylation, and dimethylation, in specific lysine and arginine residues of histones H3 and H4 in mouse lungs and H292 cells. We found that CS-induced distinct posttranslational histone modification patterns in histone H3 and histone H4 in lung cells, which may be considered as usable biomarkers for CS-induced chronic lung diseases. These identified histone marks (histone H3 and histone H4) may play an important role in the epigenetic state during the pathogenesis of smoking-induced chronic lung diseases, such as chronic obstructive pulmonary disease and lung cancer.

SIRT1 protects against cigarette smoke-induced lung oxidative stress via a FOXO3-dependent mechanism.

Oxidative and carbonyl stress is increased in lungs of smokers and patients with chronic obstructive pulmonary disease (COPD), as well as in cigarette smoke (CS)-exposed rodent lungs. We previously showed that sirtuin1 (SIRT1), an antiaging protein, is reduced in lungs of CS-exposed mice and patients with COPD and that SIRT1 attenuates CS-induced lung inflammation and injury. It is not clear whether SIRT1 protects against CS-induced lung oxidative stress. Therefore, we determined the effect of SIRT1 on lung oxidative stress and antioxidants in response to CS exposure using loss- and gain-of-function approaches, as well as a pharmacological SIRT1 activation by SRT1720. We found that CS exposure increased protein oxidation and lipid peroxidation in lungs of wild-type (WT) mice, which was further augmented in SIRT1-deficient mice. Furthermore, both SIRT1 genetic overexpression and SRT1720 treatment significantly decreased oxidative stress induced by CS exposure. FOXO3 deletion augmented lipid peroxidation products but reduced antioxidants in response to CS exposure, which was not affected by SRT1720. Interestingly, SRT1720 treatment exhibited a similar effect on lipid peroxidation and antioxidants (i.e., manganese superoxide dismutase, heme oxygenase-1, and NADPH quinone oxidoreductase-1) in WT and nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-deficient mice in response to CS exposure. This indicates that SIRT1 protects against CS-induced oxidative stress, which is mediated by FOXO3, but is independent of Nrf2. Overall, these findings reveal a novel function of SIRT1, which is to reduce CS-induced oxidative stress, and this may contribute to its protective effects against lung inflammation and subsequent development of COPD.

Proteomic and functional analyses of protein-DNA complexes during gene transfer.

One of the barriers to successful nonviral gene delivery is the crowded cytoplasm, which plasmids need to actively traverse for gene expression. Relatively little is known about how this process occurs, but our lab and others have shown that the microtubule network and motors are required for plasmid movement to the nucleus. To further investigate how plasmids exploit normal physiological processes to transfect cells, we have taken a proteomics approach to identify the proteins that comprise the plasmid-trafficking complex. We have developed a live cell DNA-protein pull-down assay to isolate complexes at certain time points post-transfection (15 minutes to 4 hours) for analysis by mass spectrometry (MS). Plasmids containing promoter sequences bound hundreds of unique proteins as early as 15 minutes post-electroporation, while a plasmid lacking any eukaryotic sequences failed to bind many of the proteins. Specific proteins included microtubule-based motor proteins (e.g., kinesin and dynein), proteins involved in protein nuclear import (e.g., importin 1, 2, 4, and 7, Crm1, RAN, and several RAN-binding proteins), a number of heterogeneous nuclear ribonucleoprotein (hnRNP)- and mRNA-binding proteins, and transcription factors. The significance of several of the proteins involved in protein nuclear localization and plasmid trafficking was determined by monitoring movement of microinjected fluorescently labeled plasmids via live cell particle tracking in cells following protein knockdown by small-interfering RNA (siRNA) or through the use of specific inhibitors. While importin ß1 was required for plasmid trafficking and subsequent nuclear import, importin α1 played no role in microtubule trafficking but was required for optimal plasmid nuclear import. Surprisingly, the nuclear export protein Crm1 also was found to complex with the transfected plasmids and was necessary for plasmid trafficking along microtubules and nuclear import. Our results show that various proteins involved in nuclear import and export influence intracellular trafficking of plasmids and subsequent nuclear accumulation.

ß-adrenergic receptor stimulation transactivates protease-activated receptor 1 via matrix metalloproteinase 13 in cardiac cells.

BACKGROUND: Chronic ß-adrenergic receptor (ß-AR) overstimulation, a hallmark of heart failure, is associated with increased cardiac expression of matrix metalloproteinases (MMPs). MMP-1 has been shown to cleave and activate the protease-activated receptor 1 (PAR1) in noncardiac cells. In the present study, we hypothesized that ß-AR stimulation would result in MMP-dependent PAR1 transactivation in cardiac cells. METHODS AND RESULTS: ß-AR stimulation of neonatal rat ventricular myocytes (NRVMs) or cardiac fibroblasts with isoproterenol transduced with an alkaline phosphatase-tagged PAR1 elicited a significant increase in alkaline phosphatase-PAR1 cleavage. This isoproterenol-dependent cleavage was significantly reduced by the broad-spectrum MMP inhibitor GM6001. Importantly, specific MMP-13 inhibitors also decreased alkaline phosphatase-PAR1 cleavage in isoproterenol-stimulated NRVMs, as well as in NRVMs stimulated with conditioned medium from isoproterenol-stimulated cardiac fibroblasts. Moreover, we found that recombinant MMP-13 stimulation cleaved alkaline phosphatase-PAR1 in NRVMs at DPRS(42)↓(43)FLLRN. This also led to the activation of the ERK1/2 pathway through Gαq in NRVMs and via the Gαq/ErbB receptor pathways in cardiac fibroblasts. MMP-13 elicited similar levels of ERK1/2 activation but lower levels of generation of inositol phosphates in comparison to thrombin. Finally, we demonstrated that either PAR1 genetic ablation or pharmacological inhibition of MMP-13 prevented isoproterenol-dependent cardiac dysfunction in mice. CONCLUSIONS: In this study, we demonstrate that ß-AR stimulation leads to MMP-13 transactivation of PAR1 in both cardiac fibroblasts and cardiomyocytes and that this likely contributes to pathological activation of Gαq and ErbB receptor-dependent pathways in the heart. We propose that this mechanism may underlie the development of ß-AR overstimulation-dependent cardiac dysfunction.

SIRT1 redresses the imbalance of tissue inhibitor of matrix metalloproteinase-1 and matrix metalloproteinase-9 in the development of mouse emphysema and human COPD.

Sirtuin1 (SIRT1), a protein/histone deacetylase, protects against the development of pulmonary emphysema. However, the molecular mechanisms underlying this observation remain elusive. The imbalance of tissue inhibitor of matrix metalloproteinases (TIMPs)/matrix metalloproteinases (MMPs) plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD)/emphysema. We hypothesized that SIRT1 protects against emphysema by redressing the imbalance between MMPs and TIMPs. To test this hypothesis, SIRT1-deficient and overexpressing/transgenic mice were exposed to cigarette smoke (CS). The protein level and activity of MMP-9 were increased in lungs of SIRT1-deficient mice exposed to CS compared with wild-type (WT) littermates, and these effects were attenuated by SIRT1 overexpression. SIRT1 deficiency decreased the level of TIMP-1, which was augmented in SIRT1 transgenic mice compared with WT littermates by CS. However, the level of MMP-2, MMP-12, TIMP-2, TIMP-3, or TIMP-4 was not altered by SIRT1 in response to CS exposure. SIRT1 reduction was associated with imbalance of TIMP-1 and MMP-9 in lungs of smokers and COPD patients. Mass spectrometry and immunoprecipitation analyses revealed that TIMP-1 acetylation on specific lysine residues was increased, whereas its interaction with SIRT1 and MMP-9 was reduced in mouse lungs with emphysema, as well as in lungs of smokers and COPD patients. SIRT1 deficiency increased CS-induced TIMP-1 acetylation, and these effects were attenuated by SIRT1 overexpression. These results suggest that SIRT1 protects against COPD/emphysema, in part, via redressing the TIMP-1/MMP-9 imbalance involving TIMP-1 deacetylation. Thus redressing the TIMP-1/MMP-9 imbalance by pharmacological activation of SIRT1 is an attractive approach in the intervention of COPD.

Cardiolipin biosynthesis in Streptococcus mutans is regulated in response to external pH.

Streptococcus mutans, a causative agent of dental caries in humans, adapts to changing environmental conditions, such as pH, in order to survive and cause disease in the oral cavity. Previously, we have shown that S. mutans increases the proportion of monounsaturated membrane fatty acids as part of its acid-adaptive strategy. Membrane lipids function as carriers of membrane fatty acids and therefore it was hypothesized that lipid backbones themselves could participate in the acid adaptation process. Lipids have been shown to protect other bacterial species from rapid changes in their environment, such as shifts in osmolality and the need for long-term survival. In the present study, we have determined the contribution of cardiolipin (CL) to acid resistance in S. mutans. Two ORFs have been identified in the S. mutans genome that encode presumptive synthetic enzymes for the acidic phospholipids: phosphatidylglycerol (PG) synthase (pgsA, SMU.2151c) and CL synthase (cls, SMU.988), which is responsible for condensing two molecules of PG to create CL. A deletion mutant of the presumptive cls gene was created using PCR-mediated cloning; however, attempts to delete pgsA were unsuccessful, indicating that pgsA may be essential. Loss of the presumptive cls gene resulted in the inability of the mutant strain to produce CL, indicating that SMU.988 encodes CL synthase. The defect in cls rendered the mutant acid sensitive, indicating that CL is required for acid adaptation in S. mutans. Addition of exogenous CL to the mutant strain alleviated acid sensitivity. MS indicated that S. mutans could assimilate exogenous CL into the membrane, halting endogenous CL incorporation. This phenomenon was not due to repression, as a cls gene transcriptional reporter fusion exhibited elevated activity when cells were supplemented with exogenous CL. Lipid analysis, via MS, indicated that CL is a reservoir for monounsaturated fatty acids in S. mutans. We demonstrated that the cls mutant exhibits elevated F-ATPase activity but it is nevertheless unable to maintain the normal membrane proton gradient, indicating cytoplasmic acidification. We conclude that the control of lipid backbone synthesis is part of the acid-adaptive repertoire of S. mutans.

Mass spectrometry detection of histidine phosphorylation on NM23-H1.

Phosphorylation is a ubiquitous protein post-translational modification that is intimately involved in most aspects of cellular regulation. Currently, most proteomic analyses are performed with phosphorylation searches for serine, threonine, and tyrosine modifications, as the phosphorylated residues of histidine and aspartic acid are acid labile and thus undetectable with most proteomic methodologies. Here, we present a novel buffer system to show histidine phosphorylation of NM23-H1, the product of the first identified putative human metastasis suppressor gene (NME1), which catalyzes the transfer of the γ-phosphate from nucleoside triphosphates to nucleoside diphosphates. On the basis of a pH titration of LC elution buffers and MS/MS identification, recombinant NM23-H1 subjected to autophosphorylation was shown to contain phosphorylated histidine at residue 118 at pH 5 and 6, with each level giving over 75% peptide coverage for identification. The solvent system presented permits the detection of all five possible phosphorylation moieties. Application of histidine and aspartic acid phosphorylation modifications to proteomic analyses will significantly advance the understanding of phosphorylation relay signaling in cellular regulation, including elucidation of the role of NM23-H1 in metastasis.

SIRT1 is a redox-sensitive deacetylase that is post-translationally modified by oxidants and carbonyl stress.

Sirtuin1 (SIRT1) deacetylase levels are decreased in chronic inflammatory conditions and aging where oxidative stress occurs. We determined the mechanism of SIRT1 redox post-translational modifications leading to its degradation. Human lung epithelial cells exposed to hydrogen peroxide (150-250 microM), aldehyde-acrolein (10-30 microM), and cigarette smoke extract (CSE; 0.1-1.5%) in the presence of intracellular glutathione-modulating agents at 1-24 h, and oxidative post-translational modifications were assayed in cells, as well as in lungs of mice lacking and overexpressing glutaredoxin-1 (Glrx1), and wild-type (WT) mice in response to cigarette smoke (CS). CSE and aldehydes dose and time dependently decreased SIRT1 protein levels, with EC(50) of 1% for CSE and 30 microM for acrolein at 6 h, and >80% inhibition at 24 h with CSE, which was regulated by modulation of intracellular thiol status of the cells. CS decreased the lung levels of SIRT1 in WT mice, which was enhanced by deficiency of Glrx1 and prevented by overexpression of Glrx1. Oxidants, aldehydes, and CS induced carbonyl modifications on SIRT1 on cysteine residues concomitant with decreased SIRT1 activity. Proteomics studies revealed alkylation of cysteine residue on SIRT1. Our data suggest that oxidants/aldehydes covalently modify SIRT1, decreasing enzymatic activity and marking the protein for proteasomal degradation, which has implications in inflammatory conditions.