Depletion of plasma membrane-associated phosphoinositides mimics inhibition of TRPM7 channels by cytosolic Mg2+, spermine, and pH.

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is an ion channel/protein kinase belonging to the TRP melastatin and eEF2 kinase families. Under physiological conditions, most native TRPM7 channels are inhibited by cytoplasmic Mg2+, protons, and polyamines. Currents through these channels (ITRPM7) are robustly potentiated when the cell interior is exchanged with low Mg2+-containing buffers. ITRPM7 is also potentiated by phosphatidyl inositol bisphosphate (PI(4,5)P2) and suppressed by its hydrolysis. Here we characterized internal Mg2+- and pH-mediated inhibition of TRPM7 channels in HEK293 cells overexpressing WT voltage-sensing phospholipid phosphatase (VSP) or its catalytically inactive variant VSP-C363S. VSP-mediated depletion of membrane phosphoinositides significantly increased channel sensitivity to Mg2+ and pH. Proton concentrations that were too low to inhibit ITRPM7 when the VSP-C363S variant was expressed (pH 8.2) became inhibitory in WT VSP-expressing cells. At pH 6.5, protons inhibited ITRPM7 both in WT and VSP C363S-expressing cells but with a faster time course in the WT VSP-expressing cells. Inhibition by 150 µm Mg2+ was also significantly faster in the WT VSP-expressing cells. Cellular PI(4,5)P2 depletion increased the sensitivity of TRPM7 channels to the inhibitor 2-aminoethyl diphenyl borinate, which acidifies the cytosol. Single substitutions at Ser-1107 of TRPM7, reducing its sensitivity to Mg2+, also decreased its inhibition by spermine and acidic pH. Furthermore, these channel variants were markedly less sensitive to VSP-mediated PI(4,5)P2 depletion than the WT. We conclude that the internal Mg2+-, polyamine-, and pH-mediated inhibition of TRPM7 channels is not direct but, rather, reflects electrostatic screening and resultant disruption of PI(4,5)P2-channel interactions.

Hydroxyurea Induces Cytokinesis Arrest in Cells Expressing a Mutated Sterol-14α-Demethylase in the Ergosterol Biosynthesis Pathway.

Hydroxyurea (HU) has been used for the treatment of multiple diseases, such as cancer. The therapeutic effect is generally believed to be due to the suppression of ribonucleotide reductase (RNR), which slows DNA polymerase movement at replication forks and induces an S phase cell cycle arrest in proliferating cells. Although aberrant mitosis and DNA damage generated at collapsed forks are the likely causes of cell death in the mutants with defects in replication stress response, the mechanism underlying the cytotoxicity of HU in wild-type cells remains poorly understood. While screening for new fission yeast mutants that are sensitive to replication stress, we identified a novel mutation in the erg11 gene encoding the enzyme sterol-14α-demethylase in the ergosterol biosynthesis pathway that dramatically sensitizes the cells to chronic HU treatment. Surprisingly, HU mainly arrests the erg11 mutant cells in cytokinesis, not in S phase. Unlike the reversible S phase arrest in wild-type cells, the cytokinesis arrest induced by HU is relatively stable and occurs at low doses of the drug, which likely explains the remarkable sensitivity of the mutant to HU. We also show that the mutation causes sterol deficiency, which may predispose the cells to the cytokinesis arrest and lead to cell death. We hypothesize that in addition to the RNR, HU may have a secondary unknown target(s) inside cells. Identification of such a target(s) may greatly improve the chemotherapies that employ HU or help to expand the clinical usage of this drug for additional pathological conditions.

Mechanism of enzymatic reaction and protein-protein interactions of PLD from a 3D structural model.

The phospholipase D (PLD) superfamily catalyzes the hydrolysis of cell membrane phospholipids generating the key intracellular lipid second messenger phosphatidic acid. However, there is not yet any resolved structure either from a crystallized protein or from NMR of any mammalian PLDs. We propose here a 3D model of the PLD2 by combining homology and ab initio 3 dimensional structural modeling methods, and docking conformation. This model is in agreement with the biochemical and physiological behavior of PLD in cells. For the lipase activity, the N- and C-terminal histidines of the HKD motifs (His 442/His 756) form a catalytic pocket, which accommodates phosphatidylcholine head group (but not phosphatidylethanolamine or phosphatidyl serine). The model explains the mechanism of the reaction catalysis, with nucleophilic attacks of His 442 and water, the latter aided by His 756. Further, the secondary structure regions superimposed with bacterial PLD crystal structure, which indicated an agreement with the model. It also explains protein-protein interactions, such as PLD2-Rac2 transmodulation (with a 1:2 stoichiometry) and PLD2 GEF activity both relevant for cell migration, as well as the existence of binding sites for phosphoinositides such as PIP2. These consist of R236/W238 and R557/W563 and a novel PIP2 binding site in the PH domain of PLD2, specifically R210/R212/W233. In each of these, the polar inositol ring is oriented towards the basic amino acid Arginine. Since tumor-aggravating properties have been found in mice overexpressing PLD2 enzyme, the 3D model of PLD2 will be also useful, to a large extent, in developing pharmaceuticals to modulate its in vivo activity.

Two sites of action for PLD2 inhibitors: The enzyme catalytic center and an allosteric, phosphoinositide biding pocket.

Phospholipase D (PLD) has been implicated in many physiological functions, such as chemotaxis and phagocytosis, as well as pathological functions, such as cancer cell invasion and metastasis. New inhibitors have been described that hamper the role of PLD in those pathologies but their site of action is not known. We have characterized the biochemical and biological behavior of the PLD1/2 dual inhibitor 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), and the specific PLD2 inhibitor, N-[2-[1-(3-Fluorophenyl)-4-oxo-1,3,-8-triazaspiro[4.5]dec-8-yl]ethyl]-2-naphthalenecarboxamide (NFOT), and found that both FIPI and NFOT are mixed-kinetics inhibitors. Mutagenesis studies indicate that FIPI binds at S757 of PLD2, which is within the HKD2 catalytic site of the enzyme, whereas NFOT binds to PLD2 at two different sites, one being at S757/S648 and another to an allosteric site that is a natural site occupied by PIP2 (R210/R212). This latter site, along with F244/L245/L246, forms a hydrophobic pocket in the PH domain. The mechanism of action of FIPI is a direct effect on the catalytic site (and as such inhibits both PLD1 and PLD2 isoforms), whereas PLD2 affects both the catalytic site (orthosteric) and blocks PIP2 binding to PLD2 (allosteric), which negates the natural enhancing role of PIP2. Moreover, NFOT prevents cell invasion of cancer cells, which does not occur in cells overexpressing PLD2-F244A/L245A/L246A, or PLD2-R210A/R212A, or PLD2-S757/S648 mutants. This study provides new specific knowledge of enzyme regulation and mechanisms of activation and inhibition of PLD2 that are necessary to understand its role in cell signaling and to develop new inhibitors for cancer cell invasion and metastasis.

A single amino acid change humanizes long-chain fatty acid binding and activation of mouse peroxisome proliferator-activated receptor α.

Peroxisome proliferator-activated receptor α (PPARα) is an important regulator of hepatic lipid metabolism which functions through ligand binding. Despite high amino acid sequence identity (>90%), marked differences in PPARα ligand binding, activation and gene regulation have been noted across species. Similar to previous observations with synthetic agonists, we have recently reported differences in ligand affinities and extent of activation between human PPARα (hPPARα) and mouse PPARα (mPPARα) in response to long chain fatty acids (LCFA). The present study was aimed to determine if structural alterations could account for these differences. The binding of PPARα to LCFA was examined through in silico molecular modeling and docking simulations. Modeling suggested that variances at amino acid position 272 are likely to be responsible for differences in saturated LCFA binding to hPPARα and mPPARα. To confirm these results experimentally, LCFA binding, circular dichroism, and transactivation studies were performed using a F272I mutant form of mPPARα. Experimental data correlated with in silico docking simulations, further confirming the importance of amino acid 272 in LCFA binding. Although the driving force for evolution of species differences at this position are yet unidentified, this study enhances our understanding of ligand-induced regulation by PPARα and demonstrates the efficacy of molecular modeling and docking simulations.

Canonical Bcl-2 motifs of the Na+/K+ pump revealed by the BH3 mimetic chelerythrine: early signal transducers of apoptosis?

BACKGROUND/AIMS: Chelerythrine [CET], a protein kinase C [PKC] inhibitor, is a prop-apoptotic BH3-mimetic binding to BH1-like motifs of Bcl-2 proteins. CET action was examined on PKC phosphorylation-dependent membrane transporters (Na+/K+ pump/ATPase [NKP, NKA], Na+-K+-2Cl+ [NKCC] and K+-Cl- [KCC] cotransporters, and channel-supported K+ loss) in human lens epithelial cells [LECs]. METHODS: K+ loss and K+ uptake, using Rb+ as congener, were measured by atomic absorption/emission spectrophotometry with NKP and NKCC inhibitors, and Cl- replacement by NO3ˉ to determine KCC. 3H-Ouabain binding was performed on a pig renal NKA in the presence and absence of CET. Bcl-2 protein and NKA sequences were aligned and motifs identified and mapped using PROSITE in conjunction with BLAST alignments and analysis of conservation and structural similarity based on prediction of secondary and crystal structures. RESULTS: CET inhibited NKP and NKCC by >90% (IC50 values ~35 and ~15 µM, respectively) without significant KCC activity change, and stimulated K+ loss by ~35% at 10-30 µM. Neither ATP levels nor phosphorylation of the NKA α1 subunit changed. 3H-ouabain was displaced from pig renal NKA only at 100 fold higher CET concentrations than the ligand. Sequence alignments of NKA with BH1- and BH3-like motifs containing pro-survival Bcl-2 and BclXl proteins showed more than one BH1-like motif within NKA for interaction with CET or with BH3 motifs. One NKA BH1-like motif (ARAAEILARDGPN) was also found in all P-type ATPases. Also, NKA possessed a second motif similar to that near the BH3 region of Bcl-2. CONCLUSION: Findings support the hypothesis that CET inhibits NKP by binding to BH1-like motifs and disrupting the α1 subunit catalytic activity through conformational changes. By interacting with Bcl-2 proteins through their complementary BH1- or BH3-like-motifs, NKP proteins may be sensors of normal and pathological cell functions, becoming important yet unrecognized signal transducers in the initial phases of apoptosis. CET action on NKCC1 and K+ channels may involve PKC-regulated mechanisms; however, limited sequence homologies to BH1-like motifs cannot exclude direct effects.

Identification of the catalytic site of phospholipase D2 (PLD2) newly described guanine nucleotide exchange factor activity.

We have demonstrated that phospholipase D2 (PLD2) is a guanine nucleotide exchange factor (GEF) for Rac2 and determined the PLD2 domains and amino acid site(s) responsible for its GEF activity. Experiments using GST fusion proteins or GST-free counterparts, purified proteins revealed that the PX domain is sufficient to exert GEF activity similar to full-length PLD2. The PLD2-GEF catalytic site is formed by a hydrophobic pocket of residues Phe-107, Phe-129, Leu-166, and Leu-173, all of which are in the PX domain. A nearby Arg-172 is also important in the overall activity. PX mutants altering any of those five amino acids fail to have GEF activity but still bind to Rac2, while their lipase activity was mostly unaffected. In addition to the PX domain, a region in the pleckstrin homology domain (Ile-306-Ala-310) aids in the PX-mediated GEF activity by providing a docking site to hold Rac2 in place during catalysis. We conclude that PLD2 is a unique GEF, with the PX being the major catalytic domain for its GEF activity, whereas the pleckstrin homology domain assists in the PX-mediated activity. The physiological relevance of this novel GEF in cell biology is demonstrated here in chemotaxis and phagocytosis of leukocytes, as the specific PX and PH mutants abolished cell function. Thus, this study reveals for the first time the catalytic site that forms the basis for the mechanism behind the GEF activity of PLD2.

Prediction of and experimental support for the three-dimensional structure of replication protein A.

Replication protein A (RPA) is a heterotrimeric, multidomain, single-stranded DNA binding protein that is essential for DNA replication, repair, and recombination. Crystallographic and NMR studies on RPA protein fragments have provided structures for all domains; however, intact heterotrimeric RPA has resisted crystallization, and a complete protein structure has not yet been described. In this study, computational methods and experimental reactivity information (MRAN) were used to model the complete structure of RPA. To accomplish this, models of RPA's globular domains and its domain-linking regions were docked in various orders. We also determined rates of proteolytic cleavage and amino acid side chain chemical modifications in native, solution state RPA. These experimental data were used to select alternate modeling intermediates and final structural models, leading to a single model most consistent with our results. Using molecular dynamics simulations and multiple rounds of simulated annealing, we then relaxed this structural model and examined its flexibility. The family of resultant models is consistent with other, previously published, critical lines of evidence and with experimental reactivity data presented herein.

Reactivity-based analysis of domain structures in native replication protein A.

Replication protein A (RPA) is an essential heterotrimeric ssDNA binding protein that participates in DNA repair, replication, and recombination. Though X-ray and NMR experiments have been used to determine three-dimensional structure models of the protein's domain fragments, a complete RPA structural model has not been reported. To test whether the fragment structures faithfully represent the same portions in the native solution-state protein, we have examined the structure of RPA under biologically relevant conditions. We have probed the location of multiple amino acids within the native RPA three-dimensional structure using reactivity of these amino acids toward proteolytic and chemical modification reagents. In turn, we evaluated different structural models by comparing the observed native RPA reactivities with anticipated reactivities based on candidate structural models. Our results show that our reactivity analysis approach is capable of critically assessing structure models and can be a basis for selecting the most relevant from among alternate models of a protein structure. Using this analytical approach, we verified the relevance of RPA fragment models to the native protein structure. Our results further indicate several important features of native RPA's structure in solution, such as flexibility at specific locations in RPA, particularly in the C-terminal region of RPA70. Our findings are consistent with reported DNA-free structural models and support the role of conformational change in the ssDNA binding mechanism of RPA.

DNA damage induced hyperphosphorylation of replication protein A. 1. Identification of novel sites of phosphorylation in response to DNA damage.

Replication protein A (RPA) is the predominant eukaryotic single-stranded DNA binding protein composed of 70, 34, and 14 kDa subunits. RPA plays central roles in the processes of DNA replication, repair, and recombination, and the p34 subunit of RPA is phosphorylated in a cell-cycle-dependent fashion and is hyperphosphorylated in response to DNA damage. We have developed an in vitro procedure for the preparation of hyperphosphorylated RPA and characterized a series of novel sites of phosphorylation using a combination of in gel tryptic digestion, SDS-PAGE and HPLC, MALDI-TOF MS analysis, 2D gel electrophoresis, and phosphospecific antibodies. We have mapped five phosphorylation sites on the RPA p34 subunit and five sites of phosphorylation on the RPA p70 subunit. No modification of the 14 kDa subunit was observed. Using the procedures developed with in vitro phosphorylated RPA, we confirmed a series of phosphorylation events on RPA from HeLa cells that was hyperphosphorylated in vivo in response to the DNA damaging agents, aphidicolin and hydroxyurea.

Denaturation of replication protein A reveals an alternative conformation with intact domain structure and oligonucleotide binding activity.

Replication protein A (RPA) is a heterotrimeric, multidomain, single-stranded DNA-binding protein. Using spectroscopic methods and methylene carbene-based chemical modification methods, we have identified conformational intermediates in the denaturation pathway of RPA. Intrinsic protein fluorescence studies reveal unfolding profiles composed of multiple transitions, with midpoints at 1.5, 2.7, 4.2, and 5.3 M urea. CD profiles of RPA unfolding are characterized by a single transition. RPA is stabilized with respect to the CD-monitored transition when bound to a dA15 oligonucleotide. However, oligonucleotide binding appears to exert little, if any, effect on the first fluorescence transition. Methylene carbene chemical modification, coupled with MALDI-TOF mass spectrometry analysis, was also used to monitor unfolding of several specific RPA folds of the protein. The unfolding profiles of the individual structures are characterized by single transitions similar to the CD-monitored transition. Each fold, however, unravels with different individual characteristics, suggesting significant autonomy. Based on results from chemical modification and spectroscopic analyses, we conclude the initial transition observed in fluorescence experiments represents a change in the juxtaposition of binding folds with little unraveling of the domain structures. The second transition represents the unfolding of the majority of fold structure, and the third transition observed by fluorescence correlates with the dissociation of the 70- and 32-kD subunits.

The reverse transcriptase sequence of human immunodeficiency virus type 1 is under positive evolutionary selection within the central nervous system.

The human immunodeficiency virus type 1 (HIV-1) enters the central nervous system (CNS) during the acute phase of infection and causes AIDS-related encephalitis and dementia in 30% of individuals. Previous studies show that HIV-1 sequences derived from the CNS of infected patients, including the sequence encoding reverse transcriptase (RT), are genetically distinct from sequences in other tissues. The hypothesis of the current study is that the RT sequence of HIV-1 is under positive selection within the CNS. Multiple alignments of non-CNS-derived and CNS-derived HIV-1 RT sequences were constructed using the ClustalW 1.8 program. The multiple alignments were analyzed with the Synonymous/Nonsynonymous Analysis Program. Codon positions 122-125, 135-149, and 166-212 of the CNS-derived RT sequences underwent a greater accumulation of nonsynonymous than synonymous substitutions, which was markedly different from the analysis results of the non-CNS-derived RT sequences. These residues are located in the finger and palm subdomains of the RT protein structure, which encodes the polymerase active site. The analysis of CNS-derived partial-length RT sequences that encompass these regions yielded similar results. A comparison of CNS-derived RT sequences to a non-CNS-derived RT consensus sequence revealed that a majority of the nonsynonymous substitutions resulted in a specific amino acid replacement. These results indicate that reverse transcriptase is under positive selection within the CNS. The amino acid replacements were visualized on a three-dimensional structure of HIV-1 RT using the Sybyl software suite. The protein structure analysis revealed that the amino acid replacements observed among the CNS-derived sequences occurred in areas of known structural and functional significance.