ADP-Ribosylation, a Multifaceted Posttranslational Modification Involved in the Control of Cell Physiology in Health and Disease.

Posttranslational modifications (PTMs) regulate protein functions and interactions. ADP-ribosylation is a PTM, in which ADP-ribosyltransferases use nicotinamide adenine dinucleotide (NAD+) to modify target proteins with ADP-ribose. This modification can occur as mono- or poly-ADP-ribosylation. The latter involves the synthesis of long ADP-ribose chains that have specific properties due to the nature of the polymer. ADP-Ribosylation is reversed by hydrolases that cleave the glycosidic bonds either between ADP-ribose units or between the protein proximal ADP-ribose and a given amino acid side chain. Here we discuss the properties of the different enzymes associated with ADP-ribosylation and the consequences of this PTM on substrates. Furthermore, the different domains that interpret either mono- or poly-ADP-ribosylation and the implications for cellular processes are described.

Variants with increased negative electrostatic potential in the Cx50 gap junction pore increased unitary channel conductance and magnesium modulation.

Gap junction (GJ) channels are oligomers of connexins forming channels linking neighboring cells. GJs formed by different connexins show distinct unitary channel conductance (γj), transjunctional voltage-dependent gating (Vj-gating) properties, and modulation by intracellular magnesium ([Mg2+]i). The underlying molecular determinants are not fully clear. Previous experimental evidence indicates that residues in the amino terminal (NT) and initial segment of the first extracellular (E1) domain influence the γj, Vj-gating, and/or [Mg2+]i modulation in several GJs. Increasing negatively charged residues in Cx50 (connexin50) E1 (G46D or G46E) increased γj, while increasing positively charged residue (G46K) reduced the γj Sequence alignment of Cx50 and Cx37 in the NT and E1 domains revealed that in Cx50 G8 and V53, positions are negatively charged residues in Cx37 (E8 and E53, respectively). To evaluate these residues together, we generated a triple variant in Cx50, G8E, G46E, and V53E simultaneously to study its γj, Vj-gating properties, and modulation by [Mg2+]i Our data indicate that the triple variant and individual variants G8E, G46E, and V53E significantly increased Cx50 GJ γj without a significant change in the Vj gating. In addition, elevated [Mg2+]i reduced γj in Cx50 and all the variant GJs. These results and our homology structural models suggest that these NT/E1 residues are likely to be pore-lining and the variants increased the negative electrostatic potentials along the GJ pore to facilitate the γj of this cation-preferring GJ channel. Our results indicate that electrostatic properties of the Cx50 GJ pore are important for the γj and the [Mg2+]i modulation.

Genetic selection for mistranslation rescues a defective co-chaperone in yeast.

Despite the general requirement for translation fidelity, mistranslation can be an adaptive response. We selected spontaneous second site mutations that suppress the stress sensitivity caused by a Saccharomyces cerevisiae tti2 allele with a Leu to Pro mutation at residue 187, identifying a single nucleotide mutation at the same position (C70U) in four tRNAProUGG genes. Linkage analysis and suppression by SUF9G3:U70 expressed from a centromeric plasmid confirmed the causative nature of the suppressor mutation. Since the mutation incorporates the G3:U70 identity element for alanyl-tRNA synthetase into tRNAPro, we hypothesized that suppression results from mistranslation of Pro187 in Tti2L187P as Ala. A strain expressing Tti2L187A was not stress sensitive. In vitro, tRNAProUGG (C70U) was mis-aminoacylated with alanine by alanyl-tRNA synthetase, but was not a substrate for prolyl-tRNA synthetase. Mass spectrometry from protein expressed in vivo and a novel GFP reporter for mistranslation confirmed substitution of alanine for proline at a rate of ∼6%. Mistranslating cells expressing SUF9G3:U70 induce a partial heat shock response but grow nearly identically to wild-type. Introducing the same G3:U70 mutation in SUF2 (tRNAProAGG) suppressed a second tti2 allele (tti2L50P). We have thus identified a strategy that allows mistranslation to suppress deleterious missense Pro mutations in Tti2.

Pin1: Intimate involvement with the regulatory protein kinase networks in the global phosphorylation landscape.

BACKGROUND: Protein phosphorylation is a universal regulatory mechanism that involves an extensive network of protein kinases. The discovery of the phosphorylation-dependent peptidyl-prolyl isomerase Pin1 added an additional layer of complexity to these regulatory networks. SCOPE OF REVIEW: We have evaluated interactions between Pin1 and the regulatory kinome and proline-dependent phosphoproteome taking into consideration findings from targeted studies as well as data that has emerged from systematic phosphoproteomic workflows and from curated protein interaction databases. MAJOR CONCLUSIONS: The relationship between Pin1 and the regulatory protein kinase networks is not restricted simply to the recognition of proteins that are substrates for proline-directed kinases. In this respect, Pin1 itself is phosphorylated in cells by protein kinases that modulate its functional properties. Furthermore, the phosphorylation-dependent targets of Pin1 include a number of protein kinases as well as other enzymes such as phosphatases and regulatory subunits of kinases that modulate the actions of protein kinases. GENERAL SIGNIFICANCE: As a result of its interactions with numerous protein kinases and their substrates, as well as itself being a target for phosphorylation, Pin1 has an intricate relationship with the regulatory protein kinase and phosphoproteomic networks that orchestrate complex cellular processes and respond to environmental cues. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Peroxide-mediated oxidation and inhibition of the peptidyl-prolyl isomerase Pin1.

Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase that plays a critical role in mediating protein conformational changes involved in signaling processes related to cell cycle control. Pin1 has also been implicated as being neuroprotective in aging-related neurodegenerative disorders including Alzheimer's disease where Pin1 activity is diminished. Notably, recent proteomic analysis of brain samples from patients with mild cognitive impairment revealed that Pin1 is oxidized and also displays reduced activity. Since the Pin1 active site contains a functionally critical cysteine residue (Cys113) with a low predicted pK(a), we hypothesized that Cys113 is sensitive to oxidation. Consistent with this hypothesis, we observed that treatment of Pin1 with hydrogen peroxide results in a 32Da mass increase, likely resulting from the oxidation of Cys113 to sulfinic acid (Cys-SO(2)H). This modification results in loss of peptidyl-prolyl isomerase activity. Notably, Pin1 with Cys113 substituted by aspartic acid retains activity and is no longer sensitive to oxidation. Structural studies by X-ray crystallography revealed increased electron density surrounding Cys113 following hydrogen peroxide treatment. At lower concentrations of hydrogen peroxide, oxidative inhibition of Pin1 can be partially reversed by treatment with dithiothreitol, suggesting that oxidation could be a reversible modification with a regulatory role. We conclude that the loss of Pin1 activity upon oxidation results from oxidative modification of the Cys113 sulfhydryl to sulfenic (Cys-SOH) or sulfinic acid (Cys-SO(2)H). Given the involvement of Pin1 in pathological processes related to neurodegenerative diseases and to cancer, these findings could have implications for the prevention or treatment of disease.

Enhanced ubiquitination and proteasomal degradation of catalytically deficient human choline acetyltransferase mutants.

Choline acetyltransferase (ChAT) is essential for cholinergic neuron function as it mediates synthesis of the neurotransmitter acetylcholine. ChAT mutations have been linked to the neuromuscular disorder congenital myasthenic syndrome (CMS). One CMS-related ChAT mutation, V18M, reduces enzyme activity and cellular protein levels, and is positioned within a highly conserved proline-rich motif with the sequence 14 PKLPVPP20 . We demonstrate that N-terminal truncation that includes this proline-rich motif, as well as mutation of prolines-17/19 together to alanine (P17A/P19A), dramatically reduces ChAT steady-state protein levels and cellular activity when expressed in cholinergic SN56 neural cells. The in vitro activity of bacterially expressed recombinant P17A/P19A-ChAT is also reduced, although this is not caused by changes in protein secondary structure or thermal stability. Treatment of SN56 cells with the proteasome inhibitor MG132 increases cellular P17A/P19A-ChAT steady-state protein levels, and by immunoprecipitation we found that ChAT is ubiquitinated and that polyubiquitination of P17A/P19A-ChAT is increased compared to wild-type (WT) ChAT. Using a novel fluorescent-biorthogonal pulse-chase protocol in SN56 cells, we determined that the protein half-life of P17A/P19A-ChAT (2.2 h) is substantially reduced compared to WT-ChAT (19.7 h). Lastly, we show that two CMS-related ChAT mutants (V18M and A513T) have enhanced ubiquitination, and that treatment with MG132 can partially restore both the steady-state protein levels as well as cellular activity of some CMS-mutant ChAT. These results identify a novel mechanism for regulation of ChAT through the ubiquitin-proteasome system that is influenced by the conserved N-terminal proline-rich motif of ChAT and may be implicated in CMS pathology. Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons. In this study we find that steady-state protein levels of human 69-kDa ChAT are regulated by the ubiquitin-proteasome system. Mutation of a highly conserved N-terminal proline-rich motif in human 69-kDa ChAT reduces both cellular ChAT protein levels, through enhanced ubiquitination and proteasomal degradation, and enzyme activity. Ubiquitination of catalytically deficient congenital myasthenic syndrome (CMS)-mutant ChAT is increased in cells, and importantly proteasome inhibition partially restores steady-state protein levels as well as cellular activity of some CMS-mutant ChAT proteins.

Substrate-assisted catalysis by PARP10 limits its activity to mono-ADP-ribosylation.

ADP-ribosylation controls many processes, including transcription, DNA repair, and bacterial toxicity. ADP-ribosyltransferases and poly-ADP-ribose polymerases (PARPs) catalyze mono- and poly-ADP-ribosylation, respectively, and depend on a highly conserved glutamate residue in the active center for catalysis. However, there is an apparent absence of this glutamate for the recently described PARP6-PARP16, raising questions about how these enzymes function. We find that PARP10, in contrast to PARP1, lacks the catalytic glutamate and has transferase rather than polymerase activity. Despite this fundamental difference, PARP10 also modifies acidic residues. Consequently, we propose an alternative catalytic mechanism for PARP10 compared to PARP1 in which the acidic target residue of the substrate functionally substitutes for the catalytic glutamate by using substrate-assisted catalysis to transfer ADP-ribose. This mechanism explains why the novel PARPs are unable to function as polymerases. This discovery will help to illuminate the different biological functions of mono- versus poly-ADP-ribosylation in cells.

Active transporters as enzymes: an energetic framework applied to major facilitator superfamily and ABC importer systems.

Active membrane transporters are dynamic molecular machines that catalyse transport across a membrane by coupling solute movement to a source of energy such as ATP or a secondary ion gradient. A central question for many active transporters concerns the mechanism by which transport is coupled to a source of energy. The transport process and associated energetic coupling involve conformational changes in the transporter. For efficient transport, the conformational changes must be tightly regulated and they must link energy use to movement of the substrate across the membrane. The present review discusses active transport using the well-established energetic framework for enzyme-mediated catalysis. In particular, membrane transport systems can be viewed as ensembles consisting of low-energy and high-energy conformations. The transport process involves binding interactions that selectively stabilize the higher energy conformations, and in this way promote conformational changes in the system that are coupled to decreases in free energy and substrate translocation. The major facilitator superfamily of secondary active transporters is used to illustrate these ideas, which are then be expanded to primary active transport mediated by ABC (ATP-binding cassette) import systems, with a focus on the well-studied maltose transporter.

Quinone reductase 2 is an adventitious target of protein kinase CK2 inhibitors TBBz (TBI) and DMAT.

Quinone reductase 2 (NQO2) exhibits off-target interactions with two protein kinase CK2 inhibitors, 4,5,6,7-1H-tetrabromobenzimidazole (TBBz) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT). TBBz and DMAT induce apoptosis in cells expressing an inhibitor-resistant CK2, suggesting that the interaction with NQO2 may mediate some of their pharmacological effects. In this study, we have fully characterized the binding of TBBz and DMAT to NQO2. Fluorescence titrations showed that TBBz and DMAT bind oxidized NQO2 in the low nanomolar range; in the case of TBBz, the affinity for NQO2 was 40-fold greater than its affinity for CK2. A related CK2 inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB), which failed to cause apoptosis in cells expressing inhibitor-resistant CK2, binds NQO2 with an affinity 1000-fold lower than those of TBBz and DMAT. Kinetic analysis indicated that DMAT inhibits NQO2 by binding with similar affinities to the oxidized and reduced forms. Crystal structure analysis showed that DMAT binds reduced NQO2 in a manner different from that in the oxidized state. In oxidized NQO2, TBBz and DMAT are deeply buried in the active site and make direct hydrogen and halogen bonds to the enzyme. In reduced NQO2, DMAT occupies a more peripheral region and hydrogen and halogen bonds with the enzyme are mediated through three water molecules. Therefore, although TBB, TBBz, and DMAT are all potent inhibitors of CK2, they exhibit different activity profiles toward NQO2. We conclude that the active site of NQO2 is fundamentally different from the ATP binding site of CK2 and the inhibition of NQO2 by CK2 inhibitors is adventitious.

Binding of DNA-Intercalating Agents to Oxidized and Reduced Quinone Reductase 2.

Quinone reductase 2 (NQO2) is an enzyme that might have intracellular signaling functions. NQO2 can exist in either an oxidized state or a reduced state, and binding of compounds to one or both of these states inhibits enzymatic activity and could also affect intracellular signaling. A wide range of planar aromatic compounds bind NQO2, and we have identified three DNA-intercalating agents [ethidium bromide, acridine orange (AO), and doxorubicin] as novel nanomolar inhibitors of NQO2. Ethidium and AO, which carry a positive charge in their aromatic ring systems, bound reduced NQO2 with an affinity 50-fold higher than that of oxidized NQO2, while doxorubicin bound only oxidized NQO2. Crystallographic analyses of oxidized NQO2 in complex with the inhibitors indicated that the inhibitors were situated deep in the active site. The aromatic faces were sandwiched between the isoalloxazine ring of FAD and the phenyl ring of F178, with their edges making direct contact with residues lining the active site. In reduced NQO2, ethidium and AO occupied a more peripheral position in the active site, allowing several water molecules to interact with the polar end of the negatively charged isoalloxazine ring. We also showed that AO inhibited NQO2 at a nontoxic concentration in cells while ethidium was less effective at inhibiting NQO2 in cells. Together, this study shows that reduced NQO2 has structural and electrostatic properties that yield a preference for binding of planar, aromatic, and positively charged molecules that can also function as DNA-intercalating agents.

Crystal and solution structure analysis of FhuD2 from Staphylococcus aureus in multiple unliganded conformations and bound to ferrioxamine-B.

Iron acquisition is a central process for virtually all organisms. In Staphylococcus aureus, FhuD2 is a lipoprotein that is a high-affinity receptor for iron-bound hydroxamate siderophores. In this study, FhuD2 was crystallized bound to ferrioxamine-B (FXB), and also in its ligand-free state; the latter structures are the first for hydroxamate-binding receptors within this protein family. The structure of the FhuD2-FXB conformation shows that residues W197 and R199 from the C-terminal domain donate hydrogen bonds to the hydroxamate oxygens, and a ring of aromatic residues cradles the aliphatic arms connecting the hydroxamate moieties of the siderophore. The available ligand-bound structures of FhuD from Escherichia coli and YfiY from Bacillus cereus show that, despite a high degree of structural conservation, three protein families have evolved with critical siderophore binding residues on either the C-terminal domain (S. aureus), the N-terminal domain (E. coli), or both (B. cereus). Unliganded FhuD2 was crystallized in five conformations related by rigid body movements of the N- and C-terminal domains. Small-angle X-ray scattering (SAXS) indicates that the solution conformation of unliganded FhuD2 is more compact than the conformations observed in crystals. The ligand-induced conformational changes for FhuD2 in solution are relatively modest and depend on the identity of the siderophore. The crystallographic and SAXS results are used to discuss roles for the liganded and unliganded forms of FhuD2 in the siderophore transport mechanism.

Chloroquine binding reveals flavin redox switch function of quinone reductase 2.

Quinone reductase 2 (NQO2) is an FAD-linked enzyme and the only known human target of two antimalarial drugs, primaquine (PQ) and chloroquine (CQ). The structural differences between oxidized and reduced NQO2 and the structural basis for inhibition by PQ and CQ were investigated by x-ray crystallography. Structures of oxidized NQO2 in complex with PQ and CQ were solved at 1.4 Å resolution. CQ binds preferentially to reduced NQO2, and upon reduction of NQO2-CQ crystals, the space group changed from P2(1)2(1)2(1) to P2(1), with 1-Å decreases in all three unit cell dimensions. The change in crystal packing originated in the negative charge and 4-5º bend in the reduced isoalloxazine ring of FAD, which resulted in a new mode of CQ binding and closure of a flexible loop (Phe(126)-Leu(136)) over the active site. This first structure of a reduced quinone reductase shows that reduction of the FAD cofactor and binding of a specific inhibitor lead to global changes in NQO2 structure and is consistent with a functional role for NQO2 as a flavin redox switch.

The role of the master cancer regulator Pin1 in the development and treatment of cancer.

This review examines the complex role of Pin1 in the development and treatment of cancer. Pin1 is the only peptidyl-prolyl isomerase (PPIase) that can recognize and isomerize phosphorylated Ser/Thr-Pro peptide bonds. Pin1 catalyzes a structural change in phosphorylated Ser/Thr-Pro motifs that can modulate protein function and thereby impact cell cycle regulation and tumorigenesis. The molecular mechanisms by which Pin1 contributes to oncogenesis are reviewed, including Pin1 overexpression and its correlation with poor cancer prognosis, and the contribution of Pin1 to aggressive tumor phenotypes involved in therapeutic resistance is discussed, with an emphasis on cancer stem cells, the epithelial-to-mesenchymal transition (EMT), and immunosuppression. The therapeutic potential of Pin1 inhibition in cancer is discussed, along with the promise and the difficulties in identifying potent, drug-like, small-molecule Pin1 inhibitors. The available evidence supports the efficacy of targeting Pin1 as a novel cancer therapeutic by analyzing the role of Pin1 in a complex network of cancer-driving pathways and illustrating the potential of synergistic drug combinations with Pin1 inhibitors for treating aggressive and drug-resistant tumors.

Specific quinone reductase 2 inhibitors reduce metabolic burden and reverse Alzheimer's disease phenotype in mice.

Biological aging can be described as accumulative, prolonged metabolic stress and is the major risk factor for cognitive decline and Alzheimer's disease (AD). Recently, we identified and described a quinone reductase 2 (QR2) pathway in the brain, in which QR2 acts as a removable memory constraint and metabolic buffer within neurons. QR2 becomes overexpressed with age, and it is possibly a novel contributing factor to age-related metabolic stress and cognitive deficit. We found that, in human cells, genetic removal of QR2 produced a shift in the proteome opposing that found in AD brains while simultaneously reducing oxidative stress. We therefore created highly specific QR2 inhibitors (QR2is) to enable evaluation of chronic QR2 inhibition as a means to reduce biological age-related metabolic stress and cognitive decline. QR2is replicated results obtained by genetic removal of QR2, while local QR2i microinjection improved hippocampal and cortical-dependent learning in rats and mice. Continuous consumption of QR2is in drinking water improved cognition and reduced pathology in the brains of AD-model mice (5xFAD), with a noticeable between-sex effect on treatment duration. These results demonstrate the importance of QR2 activity and pathway function in the healthy and neurodegenerative brain and what we believe to be the great therapeutic potential of QR2is as first-in-class drugs.

Flavin adenine dinucleotide content of quinone reductase 2: analysis and optimization for structure-function studies.

Quinone reductase 2 (NQO2) is a broadly expressed enzyme implicated in responses to a number of compounds, including protein kinase inhibitors, resveratrol, and antimalarial drugs. NQO2 includes a flavin adenine dinucleotide (FAD) cofactor, but X-ray crystallographic analysis of human NQO2 expressed in Escherichia coli showed that electron density for the isoalloxazine ring of FAD was weak and there was no electron density for the adenine mononucleotide moiety. Reversed-phase high-performance liquid chromatography (HPLC) of the NQO2 preparation indicated that FAD was not present and only 38% of the protomers contained flavin mononucleotide (FMN), explaining the weak electron density for FAD in the crystallographic analysis. A method for purifying NQO2 and reconstituting with FAD such that the final content approaches 100% occupancy with FAD is presented here. The enzyme prepared in this manner has a high specific activity, and there is strong electron density for the FAD cofactor in the crystal structure. Analysis of NQO2 crystal structures present in the Protein Data Bank indicates that many may have sub-stoichiometric cofactor content and/or contain FMN rather than FAD. This method of purification and reconstitution will help to optimize structural and functional studies of NQO2 and possibly other flavoproteins.

The Unusual Cosubstrate Specificity of NQO2: Conservation Throughout the Amniotes and Implications for Cellular Function.

Human Quinone Reductase 2 (NQO2) is a pharmacological target and has appeared in numerous screening efforts as an off-target interactor with kinase-targeted drugs. However the cellular functions of NQO2 are not known. To gain insight into the potential cellular functions of NQO2, we have carried out a detailed evolutionary analysis. One of the most striking characteristics of NQO2 is that it uses conventional dihydronicotinamide cosubstrates, NADH and NADPH, extremely inefficiently, raising questions about an enzymatic function in cells. To characterize the ability of NQO2 to serve as an enzyme, the NQO2 gene was disrupted in HCT116 cells. These NQO2 knockouts along with the parental cells were used to demonstrate that cellular NQO2 is unable to catalyze the activation of the DNA cross-linking reagent, CB1954, without the addition of exogenous dihydronicotinamide riboside (NRH). To find whether the unusual cosubstrate specificity of NQO2 has been conserved in the amniotes, recombinant NQO2 from a reptile, Alligator mississippiensis, and a bird, Anas platyrhynchos, were cloned, purified, and their catalytic activity characterized. Like the mammalian enzymes, the reptile and bird NQO2 were efficient catalysts with the small and synthetic cosubstrate N-benzyl-1,4-dihydronicotinamide but were inefficient in their use of NADH and NADPH. Therefore, the unusual cosubstrate preference of NQO2 appears to be conserved throughout the amniotes; however, we found that NQO2 is not well-conserved in the amphibians. A phylogenetic analysis indicates that NQO1 and NQO2 diverged at the time, approximately 450 MYA, when tetrapods were beginning to evolve.

Studies of the maltose transport system reveal a mechanism for coupling ATP hydrolysis to substrate translocation without direct recognition of substrate.

The ATPase activity of the maltose transporter (MalFGK(2)) is dependent on interactions with the maltose-binding protein (MBP). To determine whether direct interactions between the translocated sugar and MalFGK(2) are important for the regulation of ATP hydrolysis, we used an MBP mutant (sMBP) that is able to bind either maltose or sucrose. We observed that maltose- and sucrose-bound sMBP stimulate equal levels of MalFGK(2) ATPase activity. Therefore, the ATPase activity of MalFGK(2) is coupled to translocation of maltose solely by interactions between MalFGK(2) and MBP. For both maltose and sucrose, the ability of sMBP to stimulate the MalFGK(2) ATPase was greatly reduced compared with wild-type MBP, indicating that the mutations in sMBP have interfered with important interactions between MBP and MalFGK(2). High resolution crystal structure analysis of sMBP shows that in the closed conformation with bound sucrose, three of four mutations are buried, and the fourth causes only a minor change in the accessible surface. In contrast, in the open form of sMBP, all of the mutations are accessible, and the main chain of Tyr(62)-Gly(69) is destabilized and occupies an alternative conformation due to the W62Y mutation. On this basis, the compromised ability of sMBP to stimulate ATP hydrolysis by MalFGK(2) is most likely due to a disruption of interactions between MalFGK(2) and the open, rather than the closed, conformation of sMBP. Modeling the open sMBP structure bound to MalFGK(2) in the transition state for ATP hydrolysis points to an important site of interaction and suggests a mechanism for coupling ATP hydrolysis to substrate translocation that is independent of the exact structure of the substrate.

Synthetic triterpenoids target the Arp2/3 complex and inhibit branched actin polymerization.

Synthetic triterpenoids are anti-tumor agents that affect numerous cellular functions including apoptosis and growth inhibition. Here, we used mass spectrometric and protein array approaches and uncovered that triterpenoids associate with proteins of the actin cytoskeleton, including actin-related protein 3 (Arp3). Arp3, a subunit of the Arp2/3 complex, is involved in branched actin polymerization and the formation of lamellipodia. 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)-Im and CDDO-Me were observed to 1) inhibit the localization of Arp3 and actin at the leading edge of cells, 2) abrogate cell polarity, and 3) inhibit Arp2/3-dependent branched actin polymerization. We confirmed our drug effects with siRNA targeting of Arp3 and observed a decrease in Rat2 cell migration. Taken together, our data suggest that synthetic triterpenoids target Arp3 and branched actin polymerization to inhibit cell migration.

Site-specific IGFBP-1 hyper-phosphorylation in fetal growth restriction: clinical and functional relevance.

Phosphorylation enhances IGFBP-1 binding to IGF-I, thereby limiting the bioavailability of IGF-I that may be important in fetal growth. Our goal in this study was to determine whether changes in site-specific IGFBP-1 phosphorylation were unique to fetal growth restriction. To establish a link, we compared IGFBP-1 phosphorylation (sites and degree) in amniotic fluid from FGR (N = 10) and controls (N = 12). The concentration of serine phosphorylated IGFBP-1 showed a negative correlation with birth weight in FGR (P = 0.049). LC-MS/MS analysis revealed all four previously identified phosphorylation sites (Ser98, Ser101, Ser119, and Ser169) to be common to FGR and control groups. Relative phosphopeptide intensities (LC-MS) between FGR and controls demonstrated 4-fold higher intensity for Ser101 (P = 0.026), 7-fold for Ser98/Ser101 (P = 0.02), and 23-fold for Ser169 (P = 0.002) in the FGR group. Preliminary BIAcore data revealed 4-fold higher association and 1.7-fold lower dissociation constants for IGFBP-1/IGF-I in FGR. A structural model of IGFBP-1 bound to IGF-I indicates that all the phosphorylation sites are on relatively mobile regions of the IGFBP-1 sequence. Residues Ser98, Ser101, and Ser169 are close to structured regions that are involved in IGF-I binding and, therefore, could potentially make direct contact with IGF-I. On the other hand, residue Ser119 is in the middle of the unstructured linker that connects the N- and C-terminal domains of IGFBP-1. The model is consistent with the assumption that residues Ser98, Ser101, and Ser169 could directly interact with IGF-I, and therefore phosphorylation at these sites could change IGF-I interactions. We suggest that site-specific increase in IGFBP-1 phosphorylation limits IGF-I bioavailability, which directly contributes to the development of FGR. This study delineates the potential role of higher phosphorylation of IGFBP-1 in FGR and provides the basis to substantiate these findings with larger sample size.

Stimulation of the maltose transporter ATPase by unliganded maltose binding protein.

ATP hydrolysis by the maltose transporter (MalFGK(2)) is regulated by maltose binding protein (MBP). Binding of maltose to MBP brings about a conformational change from open to closed that leads to a strong stimulation of the MalFGK(2) ATPase. In this study, we address the long-standing but enigmatic observation that unliganded MBP is also able to stimulate MalFGK(2). Although the mechanism of this stimulation is not understood, it is sometimes attributed to a small amount of closed (but unliganded) MBP that may exist in solution. To gain insight into how MBP regulates the MalFGK(2) ATPase, we have investigated whether the open or the closed conformation of MBP is responsible for MalFGK(2) stimulation in the absence of maltose. The effect of MBP concentration on the stimulation of MalFGK(2) was assessed: for unliganded MBP, the apparent K(M) for stimulation of MalFGK(2) was below 1 microM, while for maltose-bound MBP, the K(M) was approximately 15 microM. We show that engineered MBP molecules in which the open-closed equilibrium has been shifted toward the closed conformation have a decreased ability to stimulate MalFGK(2). These results indicate that stimulation of the MalFGK(2) ATPase by unliganded MBP does not proceed through a closed conformation and instead must operate through a different mechanism than stimulation by liganded MBP. One possible explanation is that the open conformation is able to activate the MalFGK(2) ATPase directly.