Conjugation of the photoluminescent silicon nanoparticles to streptavidin

We have covalently attached multiple photoluminescent silicon nanoparticles (SNs) to streptavidin molecules. Conjugation of SNs to a target protein is achieved using the multistage photoassisted procedure. In a first step, the terminal hydrogen in the freshly prepared SNs is substituted with an alkane monolayer that serves as a platform for chemical linkage to a heterobifunctional cross-linker: 4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester. A resulting surface coating stabilizes nanoparticles against oxidation and aggregation. Next, an open end of bifunctional cross-linker-diazirine succinimidyl ester is reacted with carboxyl moieties of streptavidin and forms an amide bond. Gel and capillary electrophoresis of the SN-streptavidin complex demonstrated separate elution of the conjugation product and unreacted protein. Then, the number of SNs per protein molecule was determined by measuring complex charge variation by capillary electrophoresis. Conjugate functionality was tested by allowing it to interact with biotinylated polystyrene microbeads. Intense photoluminescence at carefully washed microbeads demonstrated selective binding of silicon nanoparticle bearing streptavidin to biotinylated microbeads. The high quantum yield of streptavidin-SN conjugate in combination with the small size and biocompatibility of silicon nanoparticles presents an attractive platform for the fluorescence labeling in diverse bioassays.     

A Golgi Lipid Signaling Pathway Controls Apical Golgi Distribution and Cell Polarity during Neurogenesis

1. Download high-res image (156KB)
2. Download full-size image

     

Genetic ablation of phosphatidylinositol transfer protein function in murine embryonic stem cells

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between signal transduction, membrane-trafficking, and lipid metabolic pathways in eukaryotic cells. The best characterized mammalian PITPs are PITP alpha and PITP beta, two highly homologous proteins that are encoded by distinct genes. Insights into PITP alpha and PITP beta function in mammalian systems have been gleaned exclusively from cell-free or permeabilized cell reconstitution and resolution studies. Herein, we report for the first time the use of genetic approaches to directly address the physiological functions of PITP alpha and PITP beta in murine cells. Contrary to expectations, we find that ablation of PITP alpha function in murine cells fails to compromise growth and has no significant consequence for bulk phospholipid metabolism. Moreover, the data show that PITP alpha does not play an obvious role in any of the cellular activities where it has been reconstituted as an essential stimulatory factor. These activities include protein trafficking through the constitutive secretory pathway, endocytic pathway function, biogenesis of mast cell dense core secretory granules, and the agonist-induced fusion of dense core secretory granules to the mast cell plasma membrane. Finally, the data demonstrate that PITP alpha-deficient cells not only retain their responsiveness to bulk growth factor stimulation but also retain their pluripotency. In contrast, we were unable to evict both PITP beta alleles from murine cells and show that PITP beta deficiency results in catastrophic failure early in murine embryonic development. We suggest that PITP beta is an essential housekeeping PITP in murine cells, whereas PITP alpha plays a far more specialized function in mammals than that indicated by in vitro systems that show PITP dependence.     

Penicillin acylase-catalyzed synthesis of beta-lactam antibiotics in highly condensed aqueous systems: beneficial impact of kinetic substrate supersaturation

Advantages of performing penicillin acylase-catalyzed synthesis of new penicillins and cephalosporins by enzymatic acyl transfer to the beta-lactam antibiotic nuclei in the supersaturated solutions of substrates have been demonstrated. It has been shown that the effective nucleophile reactivity of 6-aminopenicillanic (6-APA) and 7-aminodesacetoxycephalosporanic (7-ADCA) acids in their supersaturated solutions continue to grow proportionally to the nucleophile concentration. As a result, synthesis/hydrolysis ratio in the enzymatic synthesis can be significantly (up to three times) increased due to the nucleophile supersaturation. In the antibiotic nuclei conversion to the target antibiotic the remarkable improvement (up to 14%) has been gained. Methods of obtaining relatively stable supersaturated solutions of 6-APA, 7-ADCA, and D-p-hydroxyphenylglycine amide (D-HPGA) have been developed and syntheses of ampicillin, amoxicillin, and cephalexin starting from the supersaturated homogeneous solutions of substrates were performed. Higher synthetic efficiency and increased productivity of these reactions compared to the heterogeneous "aqueous solution-precipitate" systems were observed. The suggested approach seems to be an effective solution for the aqueous synthesis of the most widely requested beta-lactam antibiotics (i.e., amoxicillin, cephalexin, cephadroxil, cephaclor, etc.).     

The Cirque du Soleil of Golgi membrane dynamics

The role of lipid metabolic enzymes in Golgi membrane remodeling is a subject of intense interest. Now, in this issue, Schmidt and Brown (2009. J. Cell Biol. doi:10.1083/jcb.200904147) report that lysophosphatidic acid–specific acyltransferase, LPAAT3, contributes to Golgi membrane dynamics by suppressing tubule formation.The idea that active remodeling of glycerolipid acyl chains contributes to the membrane transformations required for membrane trafficking is not new (Kozlov et al., 1989; Chernomordik et al., 1995). However, identification of specific enzymes that execute such functions in living cells has proven elusive. Now, an interesting study by Schmidt and Brown (see p. 211 of this issue) demonstrates that a lysophosphatidic acid acyltransferase (LPAAT) is directly involved in regulating mammalian Golgi trafficking functions. A variety of experimental approaches converge on a coherent model where LPAAT3 quenches the formation of Golgi-derived tubules. In doing so, LPAAT3 opposes what is most likely a phospholipase A₂–mediated tubulation pathway. This balance of PLA₂ and LPAAT3 activities has functional consequences for membrane trafficking from the mammalian Golgi complex.Glycerolipids, such as phosphatidic acid (PtdOH), consist of a glycerol backbone to which three additional constituents are esterified. Fatty acyl chains are attached at the sn-1 and sn-2 positions, and these lend glycerolipids their hydrophobic character. The headgroup at the sn-3 position can be very simple (an −OH group to generate diacylglycerol; DAG) or complex (i.e., another glycerolipid molecule). In the case of phospholipids, the headgroup is linked to the backbone by a phosphoester bond (PtdOH representing the simplest case). The three-dimensional shape of a phospholipid molecule (cone, inverted cone, cylinder) is governed by the ratio of the axial area of the headgroup to that of the acyl chain region. Because the sn-2 acyl chain is often unsaturated, and therefore kinked, a suitably bulky headgroup is required to match the axial area of the acyl chain region and generate a cylindrical molecule that packs into orderly membrane bilayers. The basic principle is lipid shape can be regulated at the level of either the headgroup or the acyl chains, and enrichment of non-cylindrical lipid molecules will physically deform membranes in predictable ways (Burger, 2000; Kooijman et al., 2005).Phospholipase A₂ (PLA₂) hydrolyzes the acyl chain from the sn-2 position of a glycerolipid molecule and, in doing so, generates a molecule with a glycerol backbone esterified to a fatty acid at sn-1 and to the headgroup at sn-3. This lyso-lipid exhibits a small axial area for the acyl chain region (and is shaped as an inverted cone that promotes positive membrane curvature). What LPAATs do is re-acylate the sn-2 position with a second fatty acid (or more accurately, a fatty acyl-CoA with release of CoA as product), often an unsaturated one in higher eukaryotes, so that the axial area of the acyl chain region is much increased. When the headgroup of the glycerolipid is small, as is the case with PtdOH and DAG, the renovated glycerolipid molecule now assumes a cone shape that promotes negative membrane curvature. The general deacylation/reacylation cycle driven by sequential PLA₂/LPAAT actions of this sort is termed the Lands cycle (Fig. 1; Lands and Hart, 1965). Although originally discovered as a metabolic pathway for phospholipid acyl chain remodeling in liver, the Lands cycle now resurfaces as a mechanism for controlling mammalian Golgi membrane dynamics.Open in a separate windowFigure 1.The Lands cycle. PLA₂ hydrolyzes the acyl-chain from a glycerophospholipid to generate a free fatty acid and a lysophospholipid product. Reacylation of lysophospholipid back to a glycerophospholipid (often with a different acyl chain at sn-2) is catalyzed by an LPAAT and involves consumption of a fatty acyl-CoA. This figure was adapted from Figure 5 in Shimizu (2009).LPAATs have been studied previously from the perspective of the enzymology of lipid metabolism, but their functions from the cell biological point of view remain poorly understood. The human genome sequence database identifies nine potential LPAATs (Leung, 2001; Shindou and Shimizu, 2009). A functional involvement of the Lands cycle (and LPAATs) with the Golgi complex was initially forecast by pharmacological studies with PLA₂ and LPAAT inhibitors—the former insults interfering with various membrane trafficking pathways and the latter promoting others (de Figueiredo et al., 1998, 2000; Drecktrah et al., 2003; Chambers et al., 2005). Unfortunately, inhibitor studies of this sort are difficult to interpret. For instance, do the pleiotropic effects of the drugs report inhibition of multiple enzyme isoforms with various execution points, or are these reflections of “off-target” effects?Schmidt and Brown (2009) now report the integral membrane protein LPAAT3 localizes to ER/Golgi membranes and exhibits lyso-PtdOH acyltransferase activity. Modulation of LPAAT3 expression has significant consequences for Golgi organization and function. siRNA-mediated silencing of LPAAT3 expression resulted in Golgi fragmentation into mini-stacks, an exquisite sensitivity of Golgi integrity to brefeldin A (BFA), and elevated mis-localization of Golgi resident proteins to the ER. Reciprocally, elevated LPAAT3 expression retards Golgi collapse into the ER upon BFA challenge. These various effects correlate with enhanced formation of Golgi-derived tubules in the face of LPAAT3 inhibitors (lyso-PtdOH formation favored) and depressed tubule biogenesis when LPAAT3 activity is increased (conversion of lyso-PtdOH to PtdOH favored). Tubulation is clearly relevant to membrane transport, as enhancement can (in specific cases) accelerate rates of cargo trafficking. Interference with tubule biogenesis, or maintenance, retards trafficking from the Golgi complex, and both anterograde and retrograde trafficking pathways are affected (Schmidt and Brown, 2009).The simple physical principle that connects the Lands cycle to tubulation is that production of inverted cone lyso-PtdOH by a PLA₂ strongly promotes positive membrane curvature and tubulation, whereas LPAAT3-mediated reacylation of lyso-PtdOH to PtdOH has the opposite effect (Fig. 2). Curvature parameters have been measured for lyso-PtdOH and PtdOH at physiological salt and pH concentrations, and the respective spontaneous radii of curvatures are +20Å and −46Å, respectively (for oleoyl molecular species; Kooijman et al., 2005). Interestingly, the measurements for lyso-PtdOH yield among the highest positive curvature values recorded to date. One testable question for future investigation is whether the PtdOH molecular species generated in Golgi membranes by LPAAT3 differ from those of bulk Golgi membrane PtdOH; that is, whether reacylation generates PtdOH molecular species with distinct properties such as unsaturated acyl chains at sn-2. Such a result would forecast an acyl-chain preference for LPAAT3 and the resultant molecular species would assume more extreme cone shapes that may contribute to the membrane transformations that accompany fission processes (Burger, 2000; Kooijman et al., 2005). It is also possible that newly remodeled PtdOH is a precursor for DAG, which may be the operative fission-ogenic glycerolipid. DAG assumes even more extreme negative curvatures than does PtdOH, and it is not subject to electrostatic penalties associated with packing the highly negatively charged PtdOH headgroup. Critical roles for DAG in Golgi membrane trafficking are well established (Kearns et al., 1997; Baron and Malhotra, 2002; Fernandez-Ulibarri et al., 2007; Asp et al., 2009).Open in a separate windowFigure 2.Protein domains consolidate the positive membrane curvature generated by lyso-PtdOH. PLA₂ hydrolyzes the acyl-chain from a phosphatidylcholine (PtdCho: cylindrical lipid) to generate a free fatty acid (FFA) and a lyso-PtdCho product (positive curvature). That lyso-PtdCho (LPC) species is further metabolized to lyso-PtdOH (LPA; greater positive curvature) by phospholipases with concomitant release of the choline (Cho) headgroup. The LPA is bound by proteins that “sense” curvature or bend membranes (e.g., BAR domain), leading to further sorting of LPA to the site of deformation (in this case a budding profile). LPAAT3 antagonizes this pathway by consuming lyso-PtdOH into PtdOH synthesis. The figure was adapted from one generously provided to V.A. Bankaitis by Wonhwa Cho (University of Illinois-Chicago, Chicago, IL).How may proteins interface with the Lands cycle in the Golgi system? Do proteins provide the primary driving force for membrane deformation, or is lipid metabolism the major factor? It is unlikely to be solely the latter—at least in this case. Simple activity of PLA₂ in generating lyso-PtdOH (or other lyso-lipids) is insufficient to impose significant positive curvature to membranes. The liberated fatty acid product will promote negative curvature—thereby countering the effects of the lyso-lipid. Enrichment of lyso-lipid into domains is a prerequisite for membrane deformation, and such enrichment can be reinforced by proteins in several ways. First, protein domains that bend membranes (e.g., BAR-domains; Frost et al., 2009) could bind lyso-lipids by virtue of their shape characteristics and thereby consolidate them into positively curved domains (Fig. 2). Second, coat or motor proteins that mechanically generate tubules could drive a physical rearrangement of membrane lipids to structures that best fit their shape (Roux et al., 2005; Krauss et al., 2008; Sorre et al., 2009). In this scenario, lyso-lipids re-sort preferentially to tubules and generate a positive feedback loop for membrane deformations with positive curvature.Reconstituted systems for membrane deformation use metabolically inert membranes, and are deprived of the active interface between lipid metabolism, proteins, and membrane dynamics. What blind spots are inherent in such protein-driven membrane deformation assays remains to be seen, but it will prove increasingly true that diverse pathways of lipid metabolism, including the Lands cycle, lubricate the actions of proteins in productive membrane deformation pathways. Which is more important—proteins or lipids—in this arena? Let’s call it an equal-opportunity collaboration.Given the intense interest concerning interfaces of lipid metabolism and Golgi function, it is difficult to believe that the concept of lipid metabolism as an active participant in membrane trafficking was ignored during the halcyon days when proteins involved in vesicle biogenesis were being discovered. Since the first demonstrations that specific lipid metabolic pathways are central to these processes (Bankaitis et al, 1990; Cleves et al., 1991), work from numerous laboratories has greatly expanded the lipid–Golgi interface. The report of Schmidt and Brown (2009) adds new sets of activities to the ever-growing roster of lipid metabolic enzymes whose actions contribute to the remarkable Cirque du Soleil of Golgi membrane dynamics. Indeed, we may soon wonder whether there is any such thing as a simple “housekeeping” lipid metabolic pathway in eukaryotic cells.     

Specific and nonspecific membrane-binding determinants cooperate in targeting phosphatidylinositol transfer protein beta-isoform to the mammalian trans-Golgi network

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and specific steps in membrane trafficking through the secretory pathway in eukaryotes. Herein, we describe the cis-acting information that controls PITPbeta localization in mammalian cells. We demonstrate PITPbeta localizes predominantly to the trans-Golgi network (TGN) and that this localization is independent of the phospholipid-bound state of PITPbeta. Domain mapping analyses show the targeting information within PITPbeta consists of three short C-terminal specificity elements and a nonspecific membrane-binding element defined by a small motif consisting of adjacent tryptophan residues (the W(202)W(203) motif). Combination of the specificity elements with the W(202)W(203) motif is necessary and sufficient to generate an efficient TGN-targeting module. Finally, we demonstrate that PITPbeta association with the TGN is tolerant to a range of missense mutations at residue serine 262, we describe the TGN localization of a novel PITPbeta isoform with a naturally occurring S262Q polymorphism, and we find no other genetic or pharmacological evidence to support the concept that PITPbeta localization to the TGN is obligately regulated by conventional protein kinase C (PKC) or the Golgi-localized PKC isoforms delta or epsilon. These latter findings are at odds with a previous report that conventional PKC-mediated phosphorylation of residue Ser262 is required for PITPbeta targeting to Golgi membranes.     

Mutational alterations affecting the export competence of a truncated but fully functional maltose-binding protein signal peptide.

The wild-type maltose-binding protein (MBP) signal peptide is 26 amino acids in length. A mutationally altered MBP signal peptide has been previously described that is missing one of the basic residues from the hydrophilic segment and seven residues from the hydrophobic core; however, it still facilitates MBP secretion to the periplasm at a rate and efficiency comparable to those of the wild-type structure. Thus, this truncated signal peptide (designated the R2 signal peptide) must retain all of the essential features required for proper export function. In this study, alterations were obtained in the R2 signal peptide that resulted in an export-defective MBP. For the first time, signal sequence mutations were obtained that resulted in the synthesis of a totally export-defective MBP. As was previously the case for the wild-type signal peptide, the introduction of either charged residues or helix-breaking proline residues adversely affected export function. Despite these similarities, the position of these alterations within the R2 signal peptide, their relative effects on MBP secretion and processing, and an analysis of the ability of various extragenic prl mutations to suppress the secretion defects provide additional insight into the minimal requirements for a functional MBP signal peptide.     

Kinetics of the enzymatic synthesis of benzylpenicillin

The kinetics of the enzymatic synthesis of benzylpenicillin catalysed by penicillin amidase (EC 3.5.1.11) from Escherichia coli have been studied. Both free phenylacetic acid (PAA) and its activated derivative, phenylacetylglycine (PAG), were used in the synthesis as acylating agents for 6-aminopenicillanic acid (6-APA). The catalytic rate constants for synthesis carried out at pH 6.0 were 11.2 and 25.2 s⁻¹, respectively, i.e. they are close and have high absolute values. The main feature of the enzymatic synthesis of benzylpenicillin from phenylacetylglycine, compared with the synthesis from phenylacetic acid, is the shape of the progress curve of antibiotic accumulation. In the former case, benzylpenicillin gradually accumulates until equilibrium is reached. Thus, if the reaction is carried out at the thermodynamically optimum pH of synthesis (low pH), penicillin can be obtained in high yield. In the case of phenylacetylglycine, the kinetic curves are more complex and are characterized by a clear-cut maximum. The presence of the maximum, its value and position on the time axis depend on reagent concentration and on the pH used. A kinetic scheme is proposed which describes well the experimental dependencies. The possibility of using activated acid derivatives in synthesis and the advantages of using computer calculations for process optimization are discussed.     

Phosphatidylinositol transfer proteins and functional specification of lipid signaling pools

The diversity of lipid species in biological membranes testifies to the multiple roles of these molecules as structural units, precursors to second messengers, as scaffolding units that impose spatial and temporal regulation on assembly of proteins, and as regulators of the catalytic activities of proteins. Such diverse lipid functions must be appropriately coordinated so that these can be specifically and appropriately coupled to dedicated biological processes. Evidence from multiple sources is building towards a concept where Sec14-like PITPs are specific components of lipid metabolic nanoreactors and, in this capacity, help impose a functional specification of lipid signaling pools.