Biomedical subjects

Barbara Imperiali

Publications and source records attributed to Barbara Imperiali.

At least 19 recordsLinked to original sources

PubMed2006

In vitro biosynthesis of UDP-N,N'-diacetylbacillosamine by enzymes of the Campylobacter jejuni general protein glycosylation system.

In Campylobacter jejuni 2,4-diacetamido-2,4,6-trideoxy-alpha-d-glucopyranose, termed N,N'-diacetylbacillosamine (Bac2,4diNAc), is the first carbohydrate in the glycoprotein N-linked heptasaccharide. With uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) as a starting point, two enzymes of the general protein glycosylation (Pgl) pathway in C. jejuni (PglF and PglE) have recently been shown to modify this sugar nucleotide to form UDP-2-acetamido-4-amino-2,4,6-trideoxy-alpha-d-glycopyranose (UDP-4-amino-sugar) [Schoenhofen, I. C., et al. (2006) J. Biol. Chem. 281, 723-732]. PglD has been proposed to catalyze the final step in N,N'-diacetylbacillosamine synthesis by N-acetylation of the UDP-4-amino-sugar at the C4 position. We have cloned, overexpressed, and purified PglD from the pgl locus of C. jejuni NCTC 11168 and identified it as the acetyltransferase that modifies the UDP-4-amino-sugar to form UDP-N,N'-diacetylbacillosamine, utilizing acetyl-coenzyme A as the acetyl group donor. The UDP-N,N'-diacetylbacillosamine product was purified from the reaction by reverse phase C18 HPLC and the structure determined by NMR analysis. Additionally, the full-length PglF was overexpressed and purified in the presence of detergent as a GST fusion protein, allowing for derivation of kinetic parameters. We found that the UDP-4-amino-sugar was readily synthesized from UDP-GlcNAc in a coupled reaction using PglF and PglE. We also demonstrate the in vitro biosynthesis of the complete heptasaccharide lipid-linked donor by coupling the action of eight enzymes (PglF, PglE, PglD, PglC, PglA, PglJ, PglH, and PglI) in the Pgl pathway in a single reaction vessel.

Acetylglucosamine↗

PubMed2006

In vitro evidence for the dual function of Alg2 and Alg11: essential mannosyltransferases in N-linked glycoprotein biosynthesis.

The biosynthesis of asparagine-linked glycoproteins utilizes a dolichylpyrophosphate-linked glycosyl donor (Dol-PP-GlcNAc(2)Man(9)Glc(3)), which is assembled by the series of membrane-bound glycosyltransferases that comprise the dolichol pathway. This biosynthetic pathway is highly conserved throughout eukaryotic evolution. While complementary genetic and bioinformatic approaches have enabled identification of most of the dolichol pathway enzymes in Saccharomyces cerevisiae, the roles of two of the mannosyltransferases in the pathway, Alg2 and Alg11, have remained ambiguous because these enzymes appear to catalyze only two of the remaining four unannotated transformations. To address this issue, a biochemical approach was taken using recombinant Alg2 and Alg11 from S. cerevisiae and defined dolichylpyrophosphate-linked substrates. A cell-membrane fraction isolated from Escherichia coli overexpressing thioredoxin-tagged Alg2 was used to demonstrate that this enzyme actually carries out an alpha1,3-mannosylation, followed by an alpha1,6-mannosylation, to form the first branched pentasaccharide intermediate of the pathway. Then, using thioredoxin-tagged Alg2 for the chemoenzymatic synthesis of the dolichylpyrophosphate pentasaccharide, it was thus possible to define the biochemical function of Alg11, which is to catalyze the next two sequential alpha1,2-mannosylations. The elucidation of the dual function of each of these enzymes thus completes the identification of the entire ensemble of glycosyltransferases that comprise the dolichol pathway.

Asparagine↗

PubMed2006

Lanthanide-binding tags as luminescent probes for studying protein interactions.

Herein, we report a method for studying protein-peptide interactions which exploits the luminescence properties of Tb(III). Lanthanide-binding tags (LBTs) are short peptide sequences comprising 15-20 naturally occurring amino acids that bind Tb(III) with high affinity. These genetically encodable luminescent tags are smaller in size than the Aequorea victoria fluorescent proteins (AFPs) and benefit from the long-lived luminescence lifetime of lanthanides. In this study, luminescence resonance energy transfer (LRET) was used to monitor the interaction between SH2 domains and different phosphopeptides. For the study, the SH2 domains of Src and Crk kinase were each coexpressed with an LBT, and phosphorylated and nonphosphorylated peptides were chemically synthesized with organic fluorophores. The LRET between the protein-bound Tb(III) and the peptide-based organic fluorophore was shown to be specific for the recognition of the SH2 domain and the peptide binding partner. This method can detect differences in binding affinity and can be used to measure the dissociation constant for the protein-peptide interaction. In addition, decay experiments can be used to calculate the distance between a site in the bound peptide and the protein using Förster theory. In all of these experiments, the millisecond luminescence lifetime of Tb(III) can be exploited using time-resolved detection to eliminate background fluorescence from organic fluorophores.

Amino Acid Sequence↗

PubMed2006

Direct biochemical evidence for the utilization of UDP-bacillosamine by PglC, an essential glycosyl-1-phosphate transferase in the Campylobacter jejuni N-linked glycosylation pathway.

Campylobacter jejuni has a general N-linked glycosylation pathway, encoded by the pgl gene cluster. In C. jejuni, a heptasaccharide is transferred from an undecaprenyl pyrophosphate donor [GalNAc-alpha1,4-GalNAc-alpha1,4-(Glcbeta1,3)-GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac-alpha1-PP-undecaprenyl, where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose)] to the asparagine side chain of target proteins at the Asn-X-Ser/Thr motif. In this study, we have cloned, overexpressed in Escherichia coli, and purified PglC, the glycosyl-1-phosphate transferase responsible for the first step in the biosynthesis of the undecaprenyl-linked heptasaccharide donor. In addition, we report the first synthetic route to uridine 5'-diphosphobacillosamine. Using the uridine 5'-diphosphobacillosamine and undecaprenyl phosphate, we demonstrate the ability of PglC to produce undecaprenyl pyrophosphate bacillosamine using radiolabeled HPLC and mass spectral analysis. In addition, we revealed that PglC does not accept uridine 5'-diphospho-N-acetylglucosamine or uridine 5'-diphospho-N-acetylgalactosamine as substrates but will accept uridine 5'-diphospho-6-hydroxybacillosamine, an analogue of bacillosamine that retains the C-6 hydroxyl functionality from the biosynthetic precursor. The in vitro characterization of PglC as a bacillosamine 1-phosphoryl transferase provides direct evidence for the early steps in the C. jejuni N-linked glycosylation pathway, and the coupling of PglC with the latter glycosyltransferases (PglA, PglJ, PglH, and PglI) allows for the "one-pot" chemoenzymatic synthesis of the undecaprenyl pyrophosphate heptasaccharide donor.

Campylobacter jejuni↗

PubMed2006

Expression of N-terminal Cys-protein fragments using an intein refolding strategy.

The inclusion body expression and refolding of a pH-sensitive intein fusion protein (Ssp DnaB intein) delivered sufficient quantities of an N-terminal Cys-polypeptide for native chemical ligations. This strategy circumvents premature intein cleavage under expression conditions and allows the expression and purification of proteins with uncertain solubility properties. The expressed protein resembles the C-terminal portion of the amphiphilic immunity protein Im7, which can be ligated to synthetic thioesters to yield synthetic protein analogues for protein folding studies.

Bacterial Proteins↗

PubMed2006

Optimal Sox-based fluorescent chemosensor design for serine/threonine protein kinases.

Fluorescent chemosensors of protein kinase activity provide a continuous, high-throughput sensing format for the study of the roles of these enzymes, which are crucial for regulating cellular function. Specifically, chemosensors using the nonnatural amino acid, Sox, and physiological Mg(2+) levels report phosphorylation with dramatic fluorescence changes that are amenable to real-time and high-throughput analysis. In this article, we report 15 probes for a total of six distinct serine/threonine kinases with large fluorescence increases and good reactivity toward the target kinase. The sensing mechanism is detailed, and the optimal sensing motif is determined. These versatile and powerful sensors provide tools for researchers studying the roles of the targeted kinases in signal transduction, and the design principles provide guidelines for the generation of future fluorescent chemosensors for any serine/threonine kinase.

Enzyme Activation↗

PubMed2006

Asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems.

Asparagine-linked protein glycosylation is a prevalent protein modification reaction in eukaryotic systems. This process involves the co-translational transfer of a pre-assembled tetradecasaccharide from a dolichyl-pyrophosphate donor to the asparagine side chain of nascent proteins at the endoplasmic reticulum (ER) membrane. Recently, the first such system of N-linked glycosylation was discovered in the Gram-negative bacterium, Campylobacter jejuni. Glycosylation in this organism involves the transfer of a heptasaccharide from an undecaprenyl-pyrophosphate donor to the asparagine side chain of proteins at the bacterial periplasmic membrane. Here we provide a detailed comparison of the machinery involved in the N-linked glycosylation systems of eukaryotic organisms, exemplified by the yeast Saccharomyces cerevisiae, with that of the bacterial system in C. jejuni. The two systems display significant similarities and the relative simplicity of the bacterial glycosylation process could provide a model system that can be used to decipher the complex eukaryotic glycosylation machinery.

Asparagine↗

PubMed2005

Investigating bacterial N-linked glycosylation: synthesis and glycosyl acceptor activity of the undecaprenyl pyrophosphate-linked bacillosamine.

The chemical synthesis and biological activity of undecaprenyl pyrophosphate bacillosamine (Und-PP-Bac), an obligatory intermediate in the asparagine-linked glycosylation pathway of Campylobacter jejuni, are reported. The key transformation involves the coupling of bacillosamine phosphate and undecaprenyl phosphate. The synthetic Und-PP-Bac can be used to investigate the activity of the enzyme PglA, which catalyzes the first glycosyl transfer in substrate biosynthesis for N-linked protein glycosylation in the pathogenic gram-negative bacterium. The availability of this synthetic substrate makes it possible to access polyprenyl-linked oligosaccharides, such as the GalNAc-alpha-1,3-bacillosamine-alpha-1-PP-Und intermediate, that will enable exploration of the remaining enzymes in the prokaryotic glycosylation pathway. Study of the bacterial glycosylation system will provide insight into the corresponding eukaryotic process, which is currently poorly understood.

Campylobacter jejuni↗

PubMed2005

In vitro assembly of the undecaprenylpyrophosphate-linked heptasaccharide for prokaryotic N-linked glycosylation.

Campylobacter jejuni has a general N-linked glycosylation pathway (encoded by the pgl gene cluster), which culminates in the transfer of a heptasaccharide: GalNAc-alpha1,4-GalNAc-alpha1,4-(Glcbeta1,3)-GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac [where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose)] from a membrane-anchored undecaprenylpyrophosphate (Und-PP)-linked donor to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Herein we report, the cloning, overexpression, and purification of four of the glycosyltransferases (PglA, PglH, PglI, and PglJ) responsible for the biosynthesis of the Und-PP-linked heptasaccharide. Starting with chemically synthesized Und-PP-linked Bac and various combinations of enzymes, we have deduced the precise functions of these glycosyltransferases. PglA and PglJ add the first two GalNAc residues on to the isoprenoid-linked Bac carrier, respectively. Elongation of the trisaccharide with PglH results in a hexasaccharide revealing the polymerase activity of PglH. The final branching glucose is then added by PglI, which prefers native lipids for optimal activity. The sequential activities of the glycosyl transferases in the pathway can be reconstituted in vitro. This pathway represents an ideal venue for investigating the integrated functions of a series of enzymatic processes that occur at a membrane interface.

Amino Acid Motifs↗

PubMed2005

Semisynthesis of a glycosylated Im7 analogue for protein folding studies.

To establish a system to address questions concerning the influence of glycosylation on protein folding pathways, we have developed a semisynthetic route toward the immunity protein Im7. This fourhelix protein has been used extensively as model protein for folding studies. Native chemical ligation (NCL) affords an N-linked chitobiose glycoprotein analogue of Im7 with an Ala29Cys mutation. The semisynthetic approach relies on the solid-phase peptide synthesis (SPPS) of N-terminal thioesters (including helix I), in glycosylated or unglycosylated form, in combination with the expression of the C-terminal fragment of Im7 (containing helices II-IV). Detailed kinetic and thermodynamic analysis of the protein folding behavior reveals that semisynthetic Im7 analogues are well suited for protein folding studies and that the folding mechanism of the glycoprotein of this Im7 variant is not significantly altered over the unglycosylated analogue.

Amino Acid Sequence↗

PubMed2005

Protein oligomerization: how and why.

A large fraction of cellular proteins are oligomeric. Protein oligomerization may often be an advantageous feature from the perspective of protein evolution and has probably evolved by a variety of mechanisms. The study of protein oligomerization may provide insights into the early protein environment and the evolution of modern proteins. Oligomeric mini-proteins, short peptides with discrete protein-like structures, may serve as valuable models for understanding features of protein oligomerization.

Biopolymers↗

PubMed2005

The engineering of membrane-permeable peptides.

Reversible lipid attachment was investigated as a means to deliver small peptides into cells. Two labile straight chain alkyl motifs were developed: a cysteine dodecane disulfide (Cdd) building block and a tyrosine- or serine-myristate ester. Both moieties are cleaved on cell internalization and are compatible with Fmoc solid phase peptide synthesis. A series of fluorophore-labeled peptides that varied in lipophilic content, net charge, and charge distribution were synthesized. The peptides were screened for cellular uptake efficiency as monitored by fluorescence microscopy. Effective peptide transport is based on a distributed net positive charge introduced as lysine residues at the C and/or N terminus of the peptide and the presence of a hydrophobic domain exhibiting an estimated log P4.0. The incorporation of labile lipid motifs into peptides enhances lipophilic character of the peptides and contributes to cellular uptake with minimal alteration to the native sequence.

Amino Acid Sequence↗

PubMed2005

Improving glycopeptide synthesis: a convenient protocol for the preparation of beta-glycosylamines and the synthesis of glycopeptides.

[reaction: see text] Herein we apply a recently introduced protocol using ammonium carbamate in methanol to the amination of crude chitobiose leading to 1,beta-aminochitobiose. This simple, one-step procedure allows a facile preparation of unstable glycosylamines in contrast to the commonly implemented ammonium bicarbonate based amination of water-soluble carbohydrates. The new amination protocol leads to an improved synthesis of the key chitobiosyl-asparagine building block for the SPPS of glycopeptides. The utility of the method is demonstrated with the synthesis of a 39-amino acid glycoprotein.

Amination↗

PubMed2005

In situ photoactivation of a caged phosphotyrosine peptide derived from focal adhesion kinase temporarily halts lamellar extension of single migrating tumor cells.

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, mediates integrin-based cell signaling by transferring signals regulating cell migration, adhesion, and survival from the extracellular matrix to the cytoplasm. Following autophosphorylation at tyrosine 397, FAK binds the Src homology 2 domains of Src and phosphoinositide 3-kinase, among several other possible binding partners. To further investigate the role of phosphorylated FAK in cell migration in situ, peptides comprising residues 391-406 of human FAK with caged phosphotyrosine 397 were synthesized. Although the caged phosphopeptides were stable to phosphatase activity, the free phosphopeptides showed a half-life of approximately 10-15 min in cell lysates. Migrating NBT-II cells (a rat bladder tumor cell line) were microinjected with the caged FAK peptide and locally photoactivated using a focused laser beam. The photoactivation of caged FAK peptide in 8-microm diameter spots over the cell body led to the temporary arrest of the leading edge migration within approximately 1 min of irradiation. In contrast, cell body migration was not inhibited. Microinjection of a non-caged phosphorylated tyrosine 397 FAK peptide into migrating NBT-II cells also led to lamellar arrest; however, this approach lacks the temporal control afforded by the caged phosphopeptides. Photoactivation of related phosphotyrosine peptides with altered sequences did not result in transient lamellar arrest. We hypothesize that the phosphorylated FAK peptide competes with the endogenous FAK for binding to FAK effectors including, but not limited to, Src and phosphoinositide 3-kinase, causing spatiotemporal misregulation and subsequent lamellar arrest.

Animals↗

PubMed2005

A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates.

New methods to quantify protein kinase activities directly from complex cellular mixtures are critical for understanding biological regulatory pathways. Herein, a fluorescence-based chemosensor strategy for the direct measurement of kinase activities in crude mammalian cell lysates is described. We first designed a new fluorescent peptide reporter substrate for each target kinase. These kinase chemosensors were readily phosphorylated by recombinant target enzyme and underwent a several-fold fluorescence increase upon phosphorylation. Then, using unfractionated cell lysates, a homogeneous kinase assay was developed that was reproducible, linear and highly preferential for monitoring changes in cellular activity of the target kinase. The general protocol was developed for the kinase Akt and then easily extended to measure protein kinase A (PKA) and mitogen-activated protein kinase-associated protein kinase 2 (MK2) activities. This assay platform is immediately useful for studying protein kinase signaling in crude cellular extracts.

Biosensing Techniques↗

PubMed2005

Caged phosphoproteins.

We present the chemical and biological synthesis of caged phosphoproteins using the in vitro nonsense codon suppression methodology. Specifically, phosphoamino acid analogues of serine, threonine, and tyrosine with a single photocleavable o-nitrophenylethyl caging group were synthesized as the amino acyl tRNA adducts for insertion into full-length proteins. For this purpose, a novel phosphitylating agent was developed. The successful incorporation of these bulky and charged amino acids into the alpha-subunit of the nicotinic acetyl choline receptor (nAChR) and the vasodilator-stimulated phosphoprotein (VASP) using an in vitro translation system is reported.

Cyclic AMP-Dependent Protein Kinases↗

PubMed2005

Chemoenzymatic synthesis of glycopeptides with PglB, a bacterial oligosaccharyl transferase from Campylobacter jejuni.

The gram-negative bacterium Campylobacter jejuni has a general N-linked glycosylation pathway encoded by the pgl gene cluster. One of the proteins in this cluster, PgIB, is thought to be the oligosaccharyl transferase due to its significant homology to Stt3p, a subunit of the yeast oligosaccharyl transferase complex. PgIB has been shown to be involved in catalyzing the transfer of an undecaprenyl-linked heptasaccharide to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Using a synthetic disaccharide glycan donor (GaINAc-alpha1,3-bacillosamine-pyrophosphate-undecaprenyl) and a peptide acceptor substrate (KDFNVSKA), we can observe the oligosaccharyl transferase activity of PgIB in vitro. Furthermore, the preparation of additional undecaprenyl-linked glycan variants reveals the ability of PgIB to transfer a wide variety of saccharides. With the demonstration of PgIB activity in vitro, fundamental questions surrounding the mechanism of N-linked glycosylation can now be addressed.

Amino Acid Sequence↗

PubMed2005

Design of a heterospecific, tetrameric, 21-residue miniprotein with mixed alpha/beta structure.

The study of short, autonomously folding peptides, or "miniproteins," is important for advancing our understanding of protein stability and folding specificity. Although many examples of synthetic alpha-helical structures are known, relatively few mixed alpha/beta structures have been successfully designed. Only one mixed-secondary structure oligomer, an alpha/beta homotetramer, has been reported thus far. In this report, we use structural analysis and computational design to convert this homotetramer into the smallest known alpha/beta-heterotetramer. Computational screening of many possible sequence/structure combinations led efficiently to the design of short, 21-residue peptides that fold cooperatively and autonomously into a specific complex in solution. A 1.95 A crystal structure reveals how steric complementarity and charge patterning encode heterospecificity. The first- and second-generation heterotetrameric miniproteins described here will be useful as simple models for the analysis of protein-protein interaction specificity and as structural platforms for the further elaboration of folding and function.

Amino Acid Sequence↗