Mingming Pu

Chestnut Hill College, Boston, Massachusetts, United States

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Publications (6)30.4 Total impact

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    ABSTRACT: Despite the profound physiological consequences associated with peripheral membrane protein localization, only a rudimentary understanding of the interactions of proteins with membrane surfaces exists because these questions are inaccessible by commonly used structural techniques. Here, we combine high resolution field-cycling (31)P NMR relaxation methods with spin-labeled proteins to delineate specific interactions of a bacterial phospholipase C with phospholipid vesicles. Unexpectedly, discrete binding sites for both a substrate analogue and a different phospholipid (phosphatidylcholine) known to activate the enzyme are observed. The lifetimes for the occupation of these sites (when the protein is anchored transiently to the membrane) are >1-2 micros (but <1 ms), which represents the first estimate of an off-rate for a lipid dissociating from a specific site on the protein and returning to the bilayer. Furthermore, analyses of the spin-label induced NMR relaxation corroborates the presence of a discrete tyrosine-rich phosphatidylcholine binding site whose location is consistent with that suggested by modeling studies. The methodology illustrated here may be extended to a wide range of peripheral membrane proteins.
    Journal of Biological Chemistry 08/2010; 285(35):26916-22. · 4.65 Impact Factor
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    ABSTRACT: 31P NMR relaxation studies from 0.005 to 11.7 T are used to monitor water-soluble inositol 1,2-(cyclic) phosphate (cIP) binding to phosphatidylinositol-specific phospholipase C spin-labeled at H82C, a position near the active site of the enzyme, and to determine how activating phosphatidylcholine (PC) molecules affect this interaction. We show that, in the absence of an interface, cIP binding to the protein is not rate-limiting, and that lower activation by PC vesicles as opposed to micelles is likely due to hindered product release. The methodology is general and could be used for determining distances in other weakly binding small molecule ligand-protein interactions.
    Biochemistry 08/2009; 48(35):8282-4. · 3.38 Impact Factor
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    ABSTRACT: Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes simultaneously interact with the substrate, PI, and with nonsubstrate lipids such as phosphatidylcholine (PC). For Bacillus thuringiensis PI-PLC these interactions are synergistic with maximal catalytic activity observed at low to moderate mole fractions of PC (X(PC)) and maximal binding occurring at low mole fractions of anionic lipids. It has been proposed that residues in alpha-helix B help to modulate membrane binding and that dimerization on the membrane surface both increases affinity for PC and activates PI-PLC, yielding the observed PI/PC synergy. Vesicle binding and activity measurements using a variety of PI-PLC mutants support many aspects of this model and reveal that while single mutations can disrupt anionic lipid binding and the anionic lipid/PC synergy, the residues important for PC binding are less localized. Interestingly, at high X(PC) mutations can both decrease membrane affinity and increase activity, supporting a model where reductions in wild-type activity at X(PC) > 0.6 result from both dilution of the substrate and tight membrane binding of PI-PLC, limiting enzyme hopping or scooting to the next substrate molecule. These results provide a direct analysis of vesicle binding and catalytic activity and shed light on how occupation of the activator site enhances enzymatic activity.
    Biochemistry 06/2009; 48(29):6835-45. · 3.38 Impact Factor
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    ABSTRACT: The enzymatic activity of the peripheral membrane protein, phosphatidylinositol-specific phospholipase C (PI-PLC), is increased by nonsubstrate phospholipids with the extent of enhancement tuned by the membrane lipid composition. For Bacillus thuringiensis PI-PLC, a small amount of phosphatidylcholine (PC) activates the enzyme toward its substrate PI; above 0.5 mol fraction PC (XPC), enzyme activity decreases substantially. To provide a molecular basis for this PC-dependent behavior, we used fluorescence correlation spectroscopy to explore enzyme binding to multicomponent lipid vesicles composed of PC and anionic phospholipids (that bind to the active site as substrate analogues) and high resolution field cycling 31P NMR methods to estimate internal correlation times (tauc) of phospholipid headgroup motions. PI-PLC binds poorly to pure anionic phospholipid vesicles, but 0.1 XPC significantly enhances binding, increases PI-PLC activity, and slows nanosecond rotational/wobbling motions of both phospholipid headgroups, as indicated by increased tauc. PI-PLC activity and phospholipid tauc are constant between 0.1 and 0.5 XPC. Above this PC content, PI-PLC has little additional effect on the substrate analogue but further slows the PC tauc, a motional change that correlates with the onset of reduced enzyme activity. For PC-rich bilayers, these changes, together with the reduced order parameter and enhanced lateral diffusion of the substrate analogue in the presence of PI-PLC, imply that at high XPC, kinetic inhibition of PI-PLC results from intravesicle sequestration of the enzyme from the bulk of the substrate. Both methodologies provide a detailed view of protein-lipid interactions and can be readily adapted for other peripheral membrane proteins.
    Journal of Biological Chemistry 04/2009; 284(24):16099-107. · 4.65 Impact Factor
  • Biophysical Journal 01/2009; 96(3). · 3.67 Impact Factor
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    ABSTRACT: D-3-deoxyphosphatidylinositol (D-3-deoxy-PI) derivatives have cytotoxic activity against various human cancer cell lines. These phosphatidylinositols have a potentially wide array of targets in the phosphatidylinositol-3-kinase (PI3K)/Akt signaling network. To explore the specificity of these types of molecules, we have synthesized D-3-deoxydioctanoylphosphatidylinositol (D-3-deoxy-diC8PI), D-3,5-dideoxy-diC8PI, and D-3-deoxy-diC8PI-5-phosphate and their enantiomers, characterized their aggregate formation by novel high-resolution field cycling (31)P NMR, and examined their susceptibility to phospholipase C (PLC), their effects on the catalytic activities of PI3K and PTEN against diC8PI and diC8PI-3-phosphate substrates, respectively, and their ability to induce the death of U937 human leukemic monocyte lymphoma cells. Of these molecules, only D-3-deoxy-diC8PI was able to promote cell death; it did so with a median inhibitory concentration of 40 microM, which is much less than the critical micelle concentration of 0.4 mM. Under these conditions, little inhibition of PI3K or PTEN was observed in assays of recombinant enzymes, although the complete series of deoxy-PI compounds did provide insights into ligand binding by PTEN. D-3-deoxy-diC8PI was a poor substrate and not an inhibitor of the PLC enzymes. The in vivo results are consistent with the current thought that the PI analogue acts on Akt1, since the transcription initiation factor eIF4e, which is a downstream signaling target of the PI3K/Akt pathway, exhibited reduced phosphorylation on Ser209. Phosphorylation of Akt1 on Ser473 but not Thr308 was reduced. Since the potent cytotoxicity for U937 cells was completely lost when L-3-deoxy-diC8PI was used as well as when the hydroxyl group at the inositol C5 in D-3-deoxy-diC8PI was modified (by either replacing this group with a hydrogen or phosphorylating it), both the chirality of the phosphatidylinositol moiety and the hydroxyl group at C5 are major determinants of the binding of 3-deoxy-PI to its target in cells.
    Journal of the American Chemical Society 07/2008; 130(24):7746-55. · 10.68 Impact Factor

Publication Stats

44 Citations
30.40 Total Impact Points

Institutions

  • 2008–2010
    • Chestnut Hill College
      Boston, Massachusetts, United States
  • 2009
    • Brandeis University
      • Department of Biochemistry
      Waltham, Massachusetts, United States
  • 2008–2009
    • Boston College, USA
      • Chemistry Department
      Boston, MA, United States