Deciphering the Transcriptional Regulation of Cholesterol Catabolic Pathway in Mycobacteria: IDENTIFICATION OF THE INDUCER OF KstR REPRESSOR*

Cholesterol degradation plays a prominent role in Mycobacterium tuberculosis infection; therefore, to develop new tools to combat this disease, we need to decipher the components comprising and regulating the corresponding pathway. A TetR-like repressor (KstR) regulates the upper part of this complex catabolic pathway, but the induction mechanism remains unknown. Using a biophysical approach, we have discovered that the inducer molecule of KstR in M. smegmatis mc²155 is not cholesterol but 3-oxo-4-cholestenoic acid, one of the first metabolic intermediates. Binding this compound induces dramatic conformational changes in KstR that promote the KstR-DNA interaction to be released from the operator, retaining its dimeric state. Our findings suggest a regulatory model common to all cholesterol degrading bacteria in which the first steps of the pathway are critical to its mineralization and explain the high redundancy of the enzymes involved in these initial steps.     

Pro-apoptotic Role of Cdc25A: ACTIVATION OF CYCLIN B1/Cdc2 BY THE Cdc25A C-TERMINAL DOMAIN*

Cdc25A is a dual specificity protein phosphatase that activates cyclin/cyclin-dependent protein kinase (Cdk) complexes by removing inhibitory phosphates from conserved threonine and tyrosine in Cdks. To address how Cdc25A promotes apoptosis, Jurkat cells were treated with staurosporine, an apoptosis inducer. Upon staurosporine treatment, a Cdc25A C-terminal 37-kDa fragment, designated C37, was generated by caspase cleavage at Asp-223. Thr-507 in C37 became dephosphorylated, which prevented 14-3-3 binding, as shown previously. C37 exhibited higher phosphatase activity than full-length Cdc25A. C37 with alanine substitution for Thr-507 (C37/T507A) that imitated the cleavage product during staurosporine treatment interacted with Cdc2, Cdk2, cyclin A, and cyclin B1 and markedly activated cyclin B1/Cdc2. The dephosphorylation of Thr-507 might expose the Cdc2/Cdk2-docking site in C37. C37/T507A also induced apoptosis in Jurkat and K562 cells, resulting from activating cyclin B1/Cdc2 but not Cdk2. Thus, this study reveals that Cdc25A is a pro-apoptotic protein that amplifies staurosporine-induced apoptosis through the activation of cyclin B1/Cdc2 by its C-terminal domain.     

Neuroendocrine Regulation and Metabolism of Glucose and Lipids in Primary Chronic Insomnia: A Prospective Case-Control Study

Objectives

To investigate the relation between primary chronic insomnia and insulin sensitivity, visceral adiposity, non alcoholic fatty liver disease and neuroendocrine hormones.

Materials and Methods

In a case-controlled, prospective clinical trial 13 women with primary chronic insomnia according to DSM-IV criteria were compared to 12 healthy controls matched for age, sex, BMI, body composition and menopausal status. All participants had a sleep assessment including polysomnographic studies and neuropsychiatric evaluation. Insulin sensitivity was evaluated using the euglycaemic hyperinsulinemic clamp. Hepatic fat content, visceral adipose tissue and intramyocellular lipid accumulation were assessed using magnetic resonance imaging and spectroscopy. The hormonal stress axis was evaluated by measurements of midnight and early morning salivary cortisol, urinary catecholamines and plasma metanephrines. Body composition was determined using body impedance analysis and indirect calorimetry.

Results

Although the diagnosis of primary chronic insomnia was made by established clinical criteria, standard polysomongraphic studies failed to identify altered sleep continuity and architecture when compared to matched controls. However, women with primary chronic insomnia showed significantly higher midnight salivary cortisol concentrations (1.46 vs. 0.76 nmol/l, p = 0.02), indicating dysregulation of the hypothalamo-pituitary-adrenal (HPA) axis. Plasma glucose and lipid concentrations, insulin sensitivity, hepatic and intramyocellular fat content, visceral adipose tissue mass and body composition did not differ between the two groups.

Conclusion

Healthy women with clinically diagnosed primary chronic insomnia demonstrate a dysregulation of circadian cortisol secretion despite normal sleep continuity and architecture. Increased midnight cortisol levels, however, were not associated with impaired metabolism of glucose and lipids.     

Three-dimensional Structure of Saccharomyces Invertase: ROLE OF A NON-CATALYTIC DOMAIN IN OLIGOMERIZATION AND SUBSTRATE SPECIFICITY*

Invertase is an enzyme that is widely distributed among plants and microorganisms and that catalyzes the hydrolysis of the disaccharide sucrose into glucose and fructose. Despite the important physiological role of Saccharomyces invertase (SInv) and the historical relevance of this enzyme as a model in early biochemical studies, its structure had not yet been solved. We report here the crystal structure of recombinant SInv at 3.3 Å resolution showing that the enzyme folds into the catalytic β-propeller and β-sandwich domains characteristic of GH32 enzymes. However, SInv displays an unusual quaternary structure. Monomers associate in two different kinds of dimers, which are in turn assembled into an octamer, best described as a tetramer of dimers. Dimerization plays a determinant role in substrate specificity because this assembly sets steric constraints that limit the access to the active site of oligosaccharides of more than four units. Comparative analysis of GH32 enzymes showed that formation of the SInv octamer occurs through a β-sheet extension that seems unique to this enzyme. Interaction between dimers is determined by a short amino acid sequence at the beginning of the β-sandwich domain. Our results highlight the role of the non-catalytic domain in fine-tuning substrate specificity and thus supplement our knowledge of the activity of this important family of enzymes. In turn, this gives a deeper insight into the structural features that rule modularity and protein-carbohydrate recognition.     

Glutamine Synthetase in Rice: A COMPARATIVE STUDY OF THE ENZYMES FROM ROOTS AND LEAVES

Chromatographic, kinetic, and regulatory properties of glutamine synthetase in rice were investigated. By DEAE-Sephacel column chromatography, two forms (glutamine synthetase 1 and glutamine synthetase 2) were identified in leaves and one form (glutamine synthetase R) was identified in roots. Purification on hydroxyapatite and gel electrophoresis showed that glutamine synthetase R was distinct from the leaf enzymes. The three isoforms were purified to similar specific activities and their properties were studied. Heat lability, pH optimum about 8, K(m) for l-glutamate of 20 millimolar, and inhibition by glucosamine 6-phosphate were the main characteristics of glutamine synthetase 2. Heat stability, pH optimum about 7.5, K(m) for l-glutamate of 2 millimolar, and no effect of glucosamine 6-phosphate differentiated glutamine synthetase 1 from glutamine synthetase 2. Glutamine synthetase R was also a labile protein but its kinetic and regulatory properties were quite similar to those of glutamine synthetase 1. These results clearly demonstrate the existence of three isoforms of glutamine synthetase in rice, two of which are located in the leaves and the third in the roots.     

DREPANOTREMA NORDESTENSE (LUCENA, 1953) (PULMONATA: PLANORBIDAE): A NEW RECORD FOR THE BOLIVIAN ALTIPLANO AND ITS DIFFERENTIATION BY MORPHOLOGY AND ENZYMES

In 1995, a small planorbid snail new for the Northern Bolivian Altiplanowas discovered in several temporary ditches. Conchological andanatomical characteristics of this snail are similar to thoseof Drepanotrema nordestense (Lucena 1953), a tropical planorbid snailreported from the northeastern region of Brazil. A comparative studywas carried out with the closely related species D. aeruginosumfrom Puerto Rico and Guadeloupe using morphological (shell andanatomy of the reproductive system) and genetic (allozyme) markers.This study confirmed a clear distinction between the two species.Several malacological surveys have been carried out in the NorthernBolivian Altiplano since the first field trip by D'Orbigny in1835, the last one in 1975. That D. nordestense and relatedspecies were not reported in these earlier surveys may indicatea relatively recent introduction. (Received 6 March 2000; accepted 1 June 2000)     

Rewired Metabolism in Drug-resistant Leukemia Cells: A METABOLIC SWITCH HALLMARKED BY REDUCED DEPENDENCE ON EXOGENOUS GLUTAMINE*

Cancer cells that escape induction therapy are a major cause of relapse. Understanding metabolic alterations associated with drug resistance opens up unexplored opportunities for the development of new therapeutic strategies. Here, we applied a broad spectrum of technologies including RNA sequencing, global untargeted metabolomics, and stable isotope labeling mass spectrometry to identify metabolic changes in P-glycoprotein overexpressing T-cell acute lymphoblastic leukemia (ALL) cells, which escaped a therapeutically relevant daunorubicin treatment. We show that compared with sensitive ALL cells, resistant leukemia cells possess a fundamentally rewired central metabolism characterized by reduced dependence on glutamine despite a lack of expression of glutamate-ammonia ligase (GLUL), a higher demand for glucose and an altered rate of fatty acid β-oxidation, accompanied by a decreased pantothenic acid uptake capacity. We experimentally validate our findings by selectively targeting components of this metabolic switch, using approved drugs and starvation approaches followed by cell viability analyses in both the ALL cells and in an acute myeloid leukemia (AML) sensitive/resistant cell line pair. We demonstrate how comparative metabolomics and RNA expression profiling of drug-sensitive and -resistant cells expose targetable metabolic changes and potential resistance markers. Our results show that drug resistance is associated with significant metabolic costs in cancer cells, which could be exploited using new therapeutic strategies.     

Glucuronyltransferase Activity of KfiC from Escherichia coli Strain K5 Requires Association of KfiA: KfiC AND KfiA ARE ESSENTIAL ENZYMES FOR PRODUCTION OF K5 POLYSACCHARIDE, N-ACETYLHEPAROSAN*

Heparan sulfate is a ubiquitous glycosaminoglycan in the extracellular matrix of most animals. It interacts with various molecules and exhibits important biological functions. K5 antigen produced by Escherichia coli strain K5 is a linear polysaccharide N-acetylheparosan consisting of GlcUA β1–4 and GlcNAc α1–4 repeating disaccharide, which forms the backbone of heparan sulfate. Region 2, located in the center of the K5-specific gene cluster, encodes four proteins, KfiA, KfiB, KfiC, and KfiD, for the biosynthesis of the K5 polysaccharide. Here, we expressed and purified the recombinant KfiA and KfiC proteins and then characterized these enzymes. Whereas the recombinant KfiC alone exhibited no GlcUA transferase activity, it did exhibit GlcUA transferase and polymerization activities in the presence of KfiA. In contrast, KfiA had GlcNAc transferase activity itself, which was unaffected by the presence of KfiC. The GlcNAc and GlcUA transferase activities were analyzed with various truncated and point mutants of KfiA and KfiC. The point mutants replacing aspartic acid of a DXD motif and lysine and glutamic acid of an ionic amino acid cluster, and the truncated mutants deleting the C-terminal and N-terminal sites, revealed the essential regions for GlcNAc and GlcUA transferase activity of KfiC and KfiA, respectively. The interaction of KfiC with KfiA is necessary for the GlcUA transferase activity of KfiC but not for the enzyme activity of KfiA. Together, these results indicate that the complex of KfiA and KfiC has polymerase activity to synthesize N-acetylheparosan, providing a useful tool toward bioengineering of defined heparan sulfate chains.     

Identification of Novel in Vivo Phosphorylation Sites of the Human Proapoptotic Protein BAD: PORE-FORMING ACTIVITY OF BAD IS REGULATED BY PHOSPHORYLATION*

BAD is a proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Although much attention has been devoted to the identification of phosphorylation sites in murine BAD, little data are available with respect to phosphorylation of human BAD protein. Using mass spectrometry, we identified here besides the established phosphorylation sites at serines 75, 99, and 118 several novel in vivo phosphorylation sites within human BAD (serines 25, 32/34, 97, and 124). Furthermore, we investigated the quantitative contribution of BAD targeting kinases in phosphorylating serine residues 75, 99, and 118. Our results indicate that RAF kinases represent, besides protein kinase A, PAK, and Akt/protein kinase B, in vivo BAD-phosphorylating kinases. RAF-induced phosphorylation of BAD was reduced to control levels using the RAF inhibitor BAY 43-9006. This phosphorylation was not prevented by MEK inhibitors. Consistently, expression of constitutively active RAF suppressed apoptosis induced by BAD and the inhibition of colony formation caused by BAD could be prevented by RAF. In addition, using the surface plasmon resonance technique, we analyzed the direct consequences of BAD phosphorylation by RAF with respect to association with 14-3-3 and Bcl-2/Bcl-X_L proteins. Phosphorylation of BAD by active RAF promotes 14-3-3 protein association, in which the phosphoserine 99 represented the major binding site. Finally, we show here that BAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation.Apoptosis is a genetically programmed, morphologically distinct form of cell death that can be triggered by a variety of physiological and pathological stimuli (1–3). This form of cellular suicide is widely observed in nature and is not only essential for embryogenesis, immune responses, and tissue homeostasis but is also involved in diseases such as tumor development and progression. Bcl-2 family proteins play a pivotal role in controlling programmed cell death. The major function of these proteins is to directly modulate outer mitochondrial membrane permeability and thereby regulate the release of apoptogenic factors from the intermembrane space into the cytoplasm (for a recent review, see Ref. 4). On the basis of various structural and functional characteristics, the Bcl-2 family of proteins is divided into three subfamilies, including proteins that either inhibit (e.g. Bcl-2, Bcl-X_L, or Bcl-w) or promote programmed cell death (e.g. Bax, Bak, or Bok) (5, 6). A second subclass of proapoptotic Bcl-2 family members, the BH3²-only proteins, comprises BAD, Bik, Bmf, Hrk, Noxa, truncated Bid, Bim, and Puma (4). BH3-only proteins share sequence homology only at the BH3 domain. The amphipathic helix formed by the BH3 domain (and neighboring residues) associates with a hydrophobic groove of the antiapoptotic Bcl-2 family members (7, 8). Originally, truncated Bid has been reported to interact with Bax and Bak (9), suggesting that some BH3-only proteins promote apoptosis via at least two different mechanisms: inactivating Bcl-2-like proteins by direct binding and/or by inducing modification of Bax-like molecules. BAD (Bcl-2-associated death promoter, Bcl-2 antagonist of cell death) was described to promote apoptosis by forming heterodimers with the prosurvival proteins Bcl-2 and Bcl-X_L, thus preventing them from binding with Bax (10). More recently, two major models have been suggested for how BH3-only proteins may induce apoptosis. In the direct model, all BH3-only proteins promote cell death by directly binding and inactivating their specific anti-death Bcl-2 protein partner (11, 12). In this model, the relative killing potency of different BH3-only proteins is based on their affinities for antiapoptotic proteins. Thus, the activation of Bax/Bak would be mediated through their release from antiapoptotic counterparts. Contrary to this model, Kim et al. (13) provided support for an alternative hierarchy model, in which BH3-only proteins are divided into two distinct subsets. According to this model, the inactivator BH3-only proteins, like BAD, Noxa, and some others, respond directly to survival factors, resulting in phosphorylation, 14-3-3 binding, and suppression of the proapoptotic function. In the absence of growth factors, these proteins engage specifically their preferred antiapoptotic Bcl-2 proteins. The targeted Bcl-2 proteins then release the other subset of BH3-only proteins designated the activators (truncated Bid, Bim, and Puma) that in turn bind to and activate Bax and Bak.Non-phosphorylated BAD associated with Bcl-2/Bcl-X_L is found at the outer mitochondrial membrane. Phosphorylation of specific serine residues, Ser-112 and Ser-136 of mouse BAD (mBAD) or the corresponding phosphorylation sites Ser-75 and Ser-99 of human BAD (hBAD), results in association with 14-3-3 proteins and subsequent relocation of BAD (14, 15). Phosphorylation of mBAD at Ser-155 (Ser-118 of hBAD) within its BH3 domain disrupts the association with Bcl-2 or Bcl-X_L, promoting cell survival (16). Therefore, the phosphorylation status of BAD at these serine residues reflects a checkpoint for cell death or survival. Although the C-RAF kinase was the first reported BAD kinase (17), its target sites were not clearly defined. However, there is a growing body of evidence for direct participation of RAF in regulation of apoptosis via BAD (18, 19). In addition, Kebache et al. (20) reported recently that the interaction between adaptor protein Grb10 and C-RAF is essential for BAD-mediated cell survival. On the other hand, numerous reports suggest that PKA (21), Akt/PKB (22), PAK (18, 23, 24), Cdc2 (25), RSK (26, 27), CK2 (28), and PIM kinases (29) are involved in BAD phosphorylation as well. The involvement of c-Jun N-terminal kinase in BAD phosphorylation is controversially discussed. Whereas Donovan et al. (30) reported that c-Jun N-terminal kinase phosphorylates mBAD at serine 128, Zhang et al. (31) claimed that c-Jun N-terminal kinase is not a BAD-serine 128 kinase. On the other hand, it has been shown that c-Jun N-terminal kinase is able to suppress IL-3 withdrawal-induced apoptosis via phosphorylation of mBAD at threonine 201 (32). Thus, taken together, with respect to regulation of mBAD by phosphorylation, five serine phosphorylation sites (at positions 112, 128, 136, 155, and 170) and two threonines (117 and 201) have been identified so far. Intriguingly, only little data are available regarding the role of phosphorylation in regulation of hBAD protein, although significant structural differences between these two BAD proteins exist.During apoptosis, some members of the Bcl-2 family of proteins, such as Bax or Bak, have been shown to induce permeabilization of the outer mitochondrial membrane, allowing proteins in the mitochondrial intermembrane space to escape into the cytosol, where they can initiate caspase activation and cell death (for a review, see Refs. 33 and 34). Despite intensive investigation, the mechanism whereby Bax and Bak induce outer membrane permeability remains controversial (34). Based on crystal structure (35), it became evident that Bcl-X_L has a pronounced similarity to the translocation domain of diphtheria toxin (36), a domain that can form pores in artificial lipid bilayers. This discovery provoked the predominant view that upon commitment to apoptosis, the proapoptotic proteins Bax and Bak also form pores in the outer mitochondrial membrane (37). As expected from the structural considerations, Bcl-X_L was found to form channels in synthetic lipid membranes (38). Since then, other Bcl-2 family members like Bcl-2, Bax, and the BH3-only protein Bid have been reported to have channel-forming ability. These pores can be divided into two different types: proteinaceous channels of defined size and ion specificity (38–42) and large lipidic pores that allow free diffusion of 2-megadalton macromolecules (43, 44). With respect to the BH3-only protein BAD, no pore-forming abilities have been reported so far, although human BAD has been found to possess per se high affinity for negatively charged phospholipids and liposomes, mimicking mitochondrial membranes (14).The RAF kinases (A-, B-, and C-RAF) play a central role in the conserved Ras-RAF-MEK-ERK signaling cascade and mediate cellular responses induced by growth factors (45–47). Direct involvement of C-RAF in inhibition of proapoptotic properties of BAD established a link between signal transduction and apoptosis control (48, 49). However, the early works did not identify the exact RAF phosphorylation sites on BAD (17). Here we demonstrate that hBAD serves as a substrate of RAF isoforms. With respect to hBAD phosphorylation by PKA, Akt/PKB, and PAK1 in vivo, we observed different specificity compared with RAF kinases. hBAD phosphorylation by RAF was accompanied by reduced apoptosis in HEK293 cells (transformed human embryonic kidney cells) and NIH 3T3 cells (a mouse embryonic fibroblast cell line). Furthermore, we show that in vitro phosphorylation of hBAD by RAF at serines 75, 99, and 118 regulates the binding of 14-3-3 proteins and association with Bcl-2 and Bcl-X_L. By use of mass spectrometry, we detected several novel in vivo phosphorylation sites of hBAD in addition to the established phosphorylation sites, serines 75, 99, and 118. Finally, we show here that hBAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins.     

Cyclic Di-AMP Homeostasis in Bacillus subtilis: BOTH LACK AND HIGH LEVEL ACCUMULATION OF THE NUCLEOTIDE ARE DETRIMENTAL FOR CELL GROWTH*

The genome of the Gram-positive soil bacterium Bacillus subtilis encodes three potential diadenylate cyclases that may synthesize the signaling nucleotide cyclic di-AMP (c-di-AMP). These enzymes are expressed under different conditions in different cell compartments, and they localize to distinct positions in the cell. Here we demonstrate the diadenylate cyclase activity of the so far uncharacterized enzymes CdaA (previously known as YbbP) and CdaS (YojJ). Our work confirms that c-di-AMP is essential for the growth of B. subtilis and shows that an excess of the molecule is also harmful for the bacteria. Several lines of evidence suggest that the diadenylate cyclase CdaA is part of the conserved essential cda-glm module involved in cell wall metabolism. In contrast, the CdaS enzyme seems to provide c-di-AMP for spores. Accumulation of large amounts of c-di-AMP impairs the growth of B. subtilis and results in the formation of aberrant curly cells. This phenotype can be partially suppressed by elevated concentrations of magnesium. These observations suggest that c-di-AMP interferes with the peptidoglycan synthesis machinery. The activity of the diadenylate cyclases is controlled by distinct molecular mechanisms. CdaA is stimulated by a regulatory interaction with the CdaR (YbbR) protein. In contrast, the activity of CdaS seems to be intrinsically restricted, and a single amino acid substitution is sufficient to drastically increase the activity of the enzyme. Taken together, our results support the idea of an important role for c-di-AMP in B. subtilis and suggest that the levels of the nucleotide have to be tightly controlled.     

Multimodal Mechanism of Action for the Cdc34 Acidic Loop: A CASE STUDY FOR WHY UBIQUITIN-CONJUGATING ENZYMES HAVE LOOPS AND TAILS*

Together with ubiquitin ligases (E3), ubiquitin-conjugating enzymes (E2) are charged with the essential task of synthesizing ubiquitin chains onto protein substrates. Some 75% of the known E2s in the human proteome contain unique insertions in their primary sequences, yet it is largely unclear what effect these insertions impart on the ubiquitination reaction. Cdc34 is an important E2 with prominent roles in cell cycle regulation and signal transduction. The amino acid sequence of Cdc34 contains an insertion distal to the active site that is absent in most other E2s, yet this acidic loop (named for its four invariably conserved acidic residues) is critical for Cdc34 function both in vitro and in vivo. Here we have investigated how the acidic loop in human Cdc34 promotes ubiquitination, identifying two key molecular events during which the acidic loop exerts its influence. First, the acidic loop promotes the interaction between Cdc34 and its ubiquitin ligase partner, SCF. Second, two glutamic acid residues located on the distal side of the loop collaborate with an invariably conserved histidine on the proximal side of the loop to suppress the pK_a of an ionizing species on ubiquitin or Cdc34 which greatly contributes to Cdc34 catalysis. These results demonstrate that insertions can guide E2s to their physiologically relevant ubiquitin ligases as well as provide essential modalities that promote catalysis.     

Studies in the Metabolism of Mould Fungi: I. A PRELIMINARY STUDY OF THE METABOLISM OF CARBON, NITROGEN, AND SULPHUR BY ASPERGILLUS NIDULANS

The growth and metabolism of a normal strain (A 69) of Aspergillusnidulans upon a simple chemically defined medium has been followedanalytically. The main metabolic products were mycelial material,which reached its maximum after about 8 days, and CO² Negligiblequantities of small-molecule carbon-compounds were detected.A minute amount of volatile acid which was maximal at the timeof maximal felt-weight was found. Nitrate, the sole nitrogen-source,was all utilized. The felt took up most of the nitrogen andretained it in spite of the decrease in mycelial matter dueto senescence. Negligible amounts of volatile-base nitrogenwere formed. Sulphate was partly assimilated by the mould. Nonon-sulphate-sulphur compounds could be detected in the liquor.Like the mycelial nitrogen, once taken up the sulphur remainedin the felt throughout the period of the experiments (20 days).     

Proteome of Acidic Phospholipid-binding Proteins: SPATIAL AND TEMPORAL REGULATION OF CORONIN 1A BY PHOSPHOINOSITIDES*

Reversible interactions between acidic phospholipids in the cellular membrane and proteins in the cytosol play fundamental roles in a wide variety of physiological events. Here, we present a novel approach to the identification of acidic phospholipid-binding proteins using nano-liquid chromatography-tandem mass spectrometry. We found more than 400 proteins, including proteins with previously known acidic phospholipid-binding properties, and confirmed that several candidates, such as Coronin 1A, mDia1 (Diaphanous-related formin-1), PIR121/CYFIP2, EB2 (end plus binding protein-2), KIF21A (kinesin family member 21A), eEF1A1 (translation elongation factor 1α1), and TRIM2, directly bind to acidic phospholipids. Among such novel proteins, we provide evidence that Coronin 1A activity, which disassembles Arp2/3-containing actin filament branches, is spatially and temporally regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). Whereas Coronin 1A co-localizes with PI(4,5)P₂ at the plasma membrane in resting cells, it is dissociated from the plasma membrane during lamellipodia formation where the PI(4,5)P₂ signal is significantly reduced. Our in vitro experiments show that Coronin 1A preferentially binds to PI(4,5)P₂-containing liposomes and that PI(4,5)P₂ antagonizes the ability of Coronin 1A to disassemble actin filament branches, indicating a spatiotemporal regulation of Coronin 1A via a direct interaction with the plasma membrane lipid. Collectively, our proteomics data provide a list of potential acidic phospholipid-binding protein candidates ranging from the actin regulatory proteins to translational regulators.     

Folding of a Cyclin Box: LINKING MULTITARGET BINDING TO MARGINAL STABILITY,OLIGOMERIZATION, AND AGGREGATION OF THE RETINOBLASTOMA TUMOR SUPPRESSOR AB POCKET DOMAIN*

The retinoblastoma tumor suppressor (Rb) controls the proliferation, differentiation, and survival of cells in most eukaryotes with a role in the fate of stem cells. Its inactivation by mutation or oncogenic viruses is required for cellular transformation and eventually carcinogenesis. The high conservation of the Rb cyclin fold prompted us to investigate the link between conformational stability and ligand binding properties of the RbAB pocket domain. RbAB unfolding presents a three-state transition involving cooperative secondary and tertiary structure changes and a partially folded intermediate that can oligomerize. The first transition corresponds to unfolding of the metastable B subdomain containing the binding site for the LXCXE motif present in cellular and viral targets, and the second transition corresponds to the stable A subdomain. The low thermodynamic stability of RbAB translates into a propensity to rapidly oligomerize and aggregate at 37 °C (T₅₀ = 28 min) that is suppressed by human papillomavirus E7 and E2F peptide ligands, suggesting that Rb is likely stabilized in vivo through binding to target proteins. We propose that marginal stability and associated oligomerization may be conserved for function as a “hub” protein, allowing the formation of multiprotein complexes, which could constitute a robust mechanism to retain its cell cycle regulatory role throughout evolution. Decreased stability and oligomerization are shared with the p53 tumor suppressor, suggesting a link between folding and function in these two essential cell regulators that are inactivated in most cancers and operate within multitarget signaling pathways.     

Trp2313-His2315 of Factor VIII C2 Domain Is Involved in Membrane Binding: STRUCTURE OF A COMPLEX BETWEEN THE C2 DOMAIN AND AN INHIBITOR OF MEMBRANE BINDING*

Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 Å) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed β-strand of the C2 domain, Trp²³¹³-His²³¹⁵. This result indicates that the Trp²³¹³-His²³¹⁵ segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.     

myo-Inositol Metabolism in 41 A3 Neuroblastoma Cells: Effects of High Glucose and Sorbitol Levels

Neuroblastoma cells were used to determine the effect of high carbohydrate and polyol levels on myo-inositol metabolism. The presence of elevated concentrations of glucose or sorbitol caused a significant decrease in both inositol accumulation and incorporation into phospholipid. These conditions, however, did not alter the accumulation of the other phospholipid head groups or the growth rate and water content of the cells. Two weeks of growth in either of the modified conditions was necessary to obtain a maximal effect on inositol incorporation. In contrast, growth in elevated concentrations of fructose, mannitol, or dulcitol had no effect on inositol metabolism. The reduced inositol accumulation and incorporation into lipids seen with glucose or sorbitol supplementation resulted in a decrease in the total phosphatidylinositol content of the cell without changing the levels of the other phospholipids. Kinetic analysis of cells grown in the presence of elevated glucose indicated that V'max for inositol uptake was significantly decreased with little change in the K'm. These data suggest that glucose decreases myo-inositol uptake in this system by noncompetitive inhibition. Cells grown in the presence of increased glucose also had elevated levels of intracellular sorbitol and decreased levels of myo-inositol. These results suggest that the high levels of glucose and sorbitol which exist in poorly regulated diabetes may be at least partially responsible for diabetic neuropathy via a reduction in the cellular content of myo-inositol and phosphatidylinositol. This system may be a useful model to determine the effect of reduced inositol phospholipid levels on neural cell function.