Michael B. Morris

Measuring Protein Self-Association Using Pulsed-Field-Gradient Nmr-Spectroscopy - Application to Myosin Light-Chain-2

SummaryAt the millimolar concentrations required for structural studies, NMR spectra of the calcium-binding protein myosin light chain 2 (MLC2) showed resonance line widths indicative of extensive self-association. Pulsed-field-gradient (PFG) NMR spectroscopy was used to examine whether MLC2 aggregation could be prevented by the zwitterionic bile salt derivative 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). PFG NMR measurements indicated that CHAPS was capable of preventing MLC2 self-association, but only at concentrations well above the critical micelle concentration of ∼7.5 mM. CHAPS was most effective at a concentration of 22.5 mM, where the apparent molecular mass of MLC2 correponded to a protein monomer plus seven molecules of bound detergent. The resolution and sensitivity of 2D 15N-1H HSQC spectra of MLC2 were markedly improved by the addition of 25 mM CHAPS, consistent with a reduction in aggregation following addition of the detergent. The average amide nitrogen T2 value for MLC2 increased from ∼30 ms in the absence of CHAPS to ∼56 ms in the presence of 25 mM CHAPS. The results of this study lead us to propose that PFG NMR spectroscopy can be used as a facile alternative to conventional techniques such as analytical ultracentrifugation for examining the self-association of biological macromolecules.

L-Proline induces differentiation of ES cells: a novel role for an amino acid in the regulation of pluripotent cells in culture

The development of cell therapeutics from embryonic stem (ES) cells will require technologies that direct cell differentiation to specific somatic cell lineages in response to defined factors. The initial step in formation of the somatic lineages from ES cells, differentiation to an intermediate, pluripotent primitive ectoderm-like cell, can be achieved in vitro by formation of early primitive ectoderm-like (EPL) cells in response to a biological activity contained within the conditioned medium MEDII. Fractionation of MEDII has identified two activities required for EPL cell formation, an activity with a molecular mass of <3 kDa and a second, much larger species. Here, we have identified the low-molecular-weight activity as l-proline. An inhibitor of l-proline uptake, glycine, prevented the differentiation of ES cells in response to MEDII. Supplementation of the culture medium of ES cells with >100 M l-proline and some l-proline-containing peptides resulted in changes in colony morphology, cell proliferation, gene expression, and differentiation kinetics consistent with differentiation toward a primitive ectoderm-like cell. This activity appeared to be associated with l-proline since other amino acids and analogs of proline did not exhibit an equivalent activity. Activation of the mammalian target of rapamycin (mTOR) signaling pathway was found to be necessary but not sufficient for l-proline activity; addition of other activators of the mTOR signaling pathway failed to alter the ES cell phenotype. This is the first report describing a role for amino acids in the regulation of pluripotency and cell differentiation and identifies a novel role for the imino acid l-proline.

Pharmacological characterisation of the P2Y11 receptor in stably transfected haematological cell lines.

The recently cloned P2Y11 receptor is unique amongst P2Y receptors with its coupling to the adenylyl cyclase pathway. P2Y11 has previously been shown to be expressed in human acute promyelocytic leukemia (APL) HL-60 and NB4 cell lines, and both cell types elevate cyclic AMP (cAMP) levels upon stimulation with extracellular ATP. Acute erythroleukemic K562 cells and acute monocytic leukemia U937 cells did not elevate cAMP levels upon exposure to 1 mM extracellular ATP. However, K562 and U937 cells stably transfected with P2Y11 (K11 and U11 cells, respectively) were responsive to extracellular ATP, with an EC50 of 31 and 21 μM, respectively. The most potent agonists in both K11 and U11 cells were ATPγS (adenosine 5′-O-[3-thiotriphosphate]), ATPαS (adenosine 5′-O-[1-thiotriphosphate]), dATP and ADPβS (adenosine 5′-O-[2-thiobisphosphate]), which were of similar or greater potency compared to ATP itself. ADP and α,β-methylene ATP were less potent compared to ATP. The order of potency for ATP breakdown products was ATP > ADP > AMP ≥ Ado. UTP, a known activator of P2Y2 and P2Y4, was largely ineffective. In the transfected cells, ATP-induced cAMP elevation was inhibited by suramin (0.5 mM), but not XAC (20 μM) nor PPADS (100 μM). AMPS inhibited ATP-induced cAMP elevation in both K11 and U11 cells (EC50 ~ 3 mM) and may be a P2Y11-selective inhibitor. These results are similar to those observed for HL-60 cells and NB4 cells implicating P2Y11 as the receptor responsible for the ATP-induced cAMP elevations in these cells.

Selective modulation of P-glycoprotein-mediated drug resistance.

Multidrug resistance associated with the overexpression of the multidrug transporter P-glycoprotein is a serious impediment to successful cancer treatment. We found that verapamil reversed resistance of CEM/VLB100 cells to vinblastine and fluorescein-colchicine, but not to colchicine. Chlorpromazine reversed resistance to vinblastine but not to fluorescein-colchicine, and it increased resistance to colchicine. Initial influx rates of fluorescein-colchicine were similar in resistant and parental cells, whereas vinblastine uptake was about 10-fold lower in the resistant cells. These results provide indirect evidence that fluorescein-colchicine is transported from the inner leaflet of the membrane and vinblastine from the outer membrane leaflet. Verapamil inhibited fluorescein-colchicine transport in inside-out vesicles made from resistant cells, whilst chlorpromazine was found to activate the transport of fluorescein-colchicine. The chlorpromazine-induced activation of fluorescein-colchicine transport was temperature-dependent and may reflect its interaction with phospholipids localised in the same bilayer leaflet. Conversely, chlorpromazine localisation in this leaflet may be responsible for its allosteric inhibition of vinblastine transport from the opposing membrane leaflet. The proposed relationship between the selectivity of modulation of P-glycoprotein and the membrane localisation of the cytotoxic drug substrates and modulators may have important implications in the rational design of regimes for the circumvention of multidrug resistance clinically.

Pharmacological profile of a novel cyclic AMP‐linked P2 receptor on undifferentiated HL‐60 leukemia cells

1 Extracellular ATP (EC50 = 146±57 μm) and various ATP analogues activated cyclic AMP production in undifferentiated HL‐60 cells. 2 The order of agonist potency was: ATPγS (adenosine 5′‐O‐[3‐thiotriphosphate])BzATP (2′&3′O‐(4‐benzoylbenzoyl)‐adenosine‐5′‐triphosphate)dATP>ATP. The following agonists (in order of effectiveness at 1 mm) were all less effective than ATP at concentrations up to 1 mm: β,γ methylene ATP2‐methylthioATP>ADPAp4A (P1, P4‐di(adenosine‐5′) tetraphosphate)Adenosine>UTP. The poor response to UTP indicates that P2Y2 receptors are not responsible for ATP‐dependent activation of adenylyl cyclase. 3 Several thiophosphorylated analogs of ATP were more potent activators of cyclic AMP production than ATP. Of these, ATPγS (EC50 = 30.4±6.9 μm) was a full agonist. However, adenosine 5′‐O‐[1‐thiotriphosphate] (ATPαS; EC50 = 45±15 μm) and adenosine 5′‐O‐[2‐thiodiphosphate] (ADPβS; EC50 = 33.3±5.0 μm) were partial agonists. 4 ADPβS (IC50 = 146±32 μm) and adenosine 5′‐O‐thiomonophosphate (AMPS; IC50 = 343±142 μm) inhibited cyclic AMP production by a submaximal concentration of ATP (100 μm). Consistent with its partial agonist activity, ADPβS was estimated to maximally suppress ATP‐induced cyclic AMP production by about 65%. AMPS has not been previously reported to inhibit P2 receptors. 5 The broad spectrum P2 receptor antagonist, suramin (500 μm), abolished ATP‐stimulated cyclic AMP production by HL‐60 cells but the adenosine receptor antagonists xanthine amine congener (XAC; 20 μm) and 8‐sulpho‐phenyltheophylline (8‐SPT; 100 μm) were without effect. 6 Extracellular ATP also activated protein kinase A (PK‐A) consistent with previous findings that PK‐A activation is involved in ATP‐induced differentiation of HL‐60 cells ( Jiang et al., 1997 ). 7 Taken together, the data indicate the presence of a novel cyclic AMP‐linked P2 receptor on undifferentiated HL‐60 cells.

Signal transduction and white cell maturation via extracellular ATP and the P2Y11 receptor.

Extracellular ATP promotes a wide range of physiological effects in many tissues. Of particular interest is the effect of ATP on leukaemia‐derived HL‐60 and NB4 cell lines, which are induced to mature to neutrophil‐like cells. The differentiation process appears to be mediated by ATP binding to a cell‐surface purinergic P2Y receptor, resulting in the stimulation of adenylyl cyclase, elevation of cAMP levels and activation of protein kinase A. In 1997, a novel ATP‐selective P2Y receptor, P2Y11, was cloned and shown to be linked to both cAMP and Ca2+ signalling pathways. The pharmacological profile of ATP analogues used by P2Y11 for cAMP production in transfected cells is reviewed in the present paper and shown to be closely similar to the profiles for cAMP production and differentiation of myeloblastic HL‐60 cells and promyelocytic NB4 cells, both of which express P2Y11. Additional data are provided showing that HL‐60 mature to neutrophil‐like cells in response to extracellular ATP, as measured by upregulation of the N‐formyl peptide receptor, N‐formyl peptide‐mediated actin polymerization and superoxide production. It is proposed that P2Y11 is responsible for the ATP‐mediated differentiation of these cells lines and that this receptor may play a role in the maturation of granulocytic progenitors in the bone marrow.

The amino acid transporter SNAT2 mediates L-proline-induced differentiation of ES cells.

There is an increasing appreciation that amino acids can act as signaling molecules in the regulation of cellular processes through modulation of intracellular cell signaling pathways. In culture, embryonic stem (ES) cells can be differentiated to a second, pluripotent cell population, early primitive ectoderm-like cells in response to biological activities within the conditioned medium MEDII. The amino acid l-proline has been identified as a component of MEDII required for ES cell differentiation. Here, we define the primary l-proline transporter on ES and early primitive ectoderm-like cells as sodium-coupled neutral amino acid transporter 2 (SNAT2). SNAT2 uptake of l-proline can be inhibited by the addition of millimolar concentrations of other substrates. The addition of excess amino acids was used to regulate the uptake of l-proline by ES cells, and the effect on differentiation was analyzed. The ability of SNAT2 substrates, but not other amino acids, to prevent changes in morphology, gene expression, and differentiation kinetics suggested that l-proline uptake through SNAT2 was required for ES cell differentiation. These data reveal an unexpected role for amino acid uptake and the amino acid transporter SNAT2 in regulation of pluripotent cells in culture and provides a number of specific, inexpensive, and nontoxic culture additives with the potential to improve the quality of ES cell culture.

Structural modelling and dynamics of proteins for insights into drug interactions.

Proteins are the workhorses of biomolecules and their function is affected by their structure and their structural rearrangements during ligand entry, ligand binding and protein-protein interactions. Hence, the knowledge of protein structure and, importantly, the dynamic behaviour of the structure are critical for understanding how the protein performs its function. The predictions of the structure and the dynamic behaviour can be performed by combinations of structure modelling and molecular dynamics simulations. The simulations also need to be sensitive to the constraints of the environment in which the protein resides. Standard computational methods now exist in this field to support the experimental effort of solving protein structures. This review presents a comprehensive overview of the basis of the calculations and the well-established computational methods used to generate and understand protein structure and function and the study of their dynamic behaviour with the reference to lung-related targets.

Extracellular ATP couples to cAMP generation and granulocytic differentiation in human NB4 promyelocytic leukaemia cells

Priming of NB4 promyelocytic cells with all‐trans retinoic acid, followed by extracellular ATP in the presence of a phosphodiesterase inhibitor, elevated cAMP and activated protein kinase A. The order of potency for cAMP production was ATP (EC50 = 95 ± 13 μmol/L) > ADP > AMP = adenosine. The order of potency of ATP analogues was 2′‐ and 3′‐O‐(4‐benzoylbenzoyl)‐ATP (EC50 = 54 ± 15 μmol/L) = adenosine 5′‐O‐(3‐thio) triphosphate (EC50 = 66 ± 4 μmol/L) > ATP > β,γ‐methylene ATP (EC50 = 200 ± 55 μmol/L). Adenosine 5′‐O‐thiomonophosphate and adenosine 5′‐O‐(2‐thio) diphosphate inhibited ATP‐induced cAMP production. Differentiation also occurred as measured by increased expression of CD11b and N‐formyl peptide receptor and changes in cell morphology. UTP did not elevate cAMP or induce differentiation, indicating that P2Y2, P2Y4, and P2Y6 receptors were not involved. The P2Y11 receptor, a cAMP‐linked receptor on promyelocytic HL‐60 cells, was detected in NB4 cells by reverse transcription–polymerase chain reaction and northern blotting. This receptor has the same order of potency with respect to cAMP production as that observed in HL‐60 cells.

Determination of the parameters of self-association by direct fitting of the omega function

Nonlinear regression is used to fit the omega function vs. protein concentration curves (first described by B.K. Milthorpe, P.D. Jeffrey and L.W. Nichol, Biophys. Chem. 3 (1975) 169) obtained from sedimentation equilibrium experiments on self-associating macromolecules. Nonlinear regression allows the direct fit of these curves with discrete or indefinite self-association reaction models in order to obtain values for the equilibrium constants and second virial coefficient. The method is independent of the choice of reference concentration and avoids the original method of extrapolating an omega function curve to zero concentration and then using the extrapolated value to construct a monomer activity curve used for analysis. This extrapolation can become very difficult for mild to strong self-associations where incorrectly extrapolated values lead to systematic error in the monomer activity curves. The method is applied to results from a mild, indefinite self-association, exemplified by the self-association of human spectrin, and to computer-simulated data of weak, mild and strong, indefinite self-associations.