An 11-residue peptide with the sequence DSLEFIASKLA was identified from a genomic library of Bacillus subtilis by phage display as an efficient substrate for Sfp phosphopantetheinyl transferasecatalyzed protein labeling by small molecule-CoA conjugates. We name this peptide the ''ybbR tag,'' because part of its sequence is derived from the ybbR ORF in the B. subtilis genome. The site of Sfp-catalyzed ybbR tag labeling was mapped to the underlined Ser residue, and the ybbR tag was found to have a strong tendency for adopting an ␣-helical conformation in solution. Here we demonstrate that the ybbR tag can be fused to the N or C termini of target proteins or inserted in a flexible loop in the middle of a target protein for site-specific protein labeling by Sfp. The short size of the ybbR tag and its compatibility with various target proteins, the broad substrate specificity of Sfp for labeling the ybbR tag with small-molecule probes of diverse structures, and the high specificity and efficiency of the labeling reaction make Sfp-catalyzed ybbR tag labeling an attractive tool for expanding protein structural and functional diversities by posttranslational modification.coenzyme A ͉ posttranslational modification ͉ Bacillus subtillis ͉ phage display

Sfp phosphopantetheinyl transferase covalently attaches small-molecule probes including biotin and various organic fluorophores to a specific serine residue in the peptidyl carrier protein (PCP) or a short 11-residue peptide tag ybbR through a phosphopantetheinyl linker. We describe here a protocol for site-specific protein labeling by Sfp-catalyzed protein post-translational modification that includes (i) expression and purification of Sfp, (ii) synthesis of small-molecule probe-CoA conjugates, (iii) construction of target protein fusions with PCP or the ybbR tag, (iv) labeling PCP- or ybbR-tagged target protein fusions in cell lysates and on live cell surfaces and (v) imaging fluorophore-labeled cell surface receptors by fluorescence microscopy. To follow this protocol, we advise that you allow 3 d for the expression and purification of Sfp phosphopantetheinyl transferase, 1 d for the synthesis and purification of the small-molecule probe-CoA conjugates as the substrates of Sfp, 3 d for the cloning of target protein genes as fusions to the PCP or the ybbR tag in the appropriate plasmids and another 3 d for transfecting cell lines with the plasmids and the expression of PCP- or ybbR-tagged proteins. Labeling of the PCP- or the ybbR-tagged proteins in cell lysates or on cell surfaces should require only 15-30 min.

Cationic liposomes (CLs) are used worldwide as gene vectors (carriers) in nonviral clinical applications of gene delivery, albeit with unacceptably low transfection efficiencies (TE). We present three-dimensional laser scanning confocal microscopy studies revealing distinct interactions between CL-DNA complexes, for both lamellar L(alpha)(C) and inverted hexagonal H(II)(C) nanostructures, and mouse fibroblast cells. Confocal images of L(alpha)(C) complexes in cells identified two regimes. For low membrane charge density (sigma(M)), DNA remained trapped in CL-vectors. By contrast, for high sigma(M), released DNA was observed in the cytoplasm, indicative of escape from endosomes through fusion. Remarkably, firefly luciferase reporter gene studies in the highly complex L(alpha)(C)-mammalian cell system revealed an unexpected simplicity where, at a constant cationic to anionic charge ratio, TE data for univalent and multivalent cationic lipids merged into a single curve as a function of sigma(M), identifying it as a key universal parameter. The universal curve for transfection by L(alpha)(C) complexes climbs exponentially over approximately four decades with increasing sigma(M) below an optimal charge density (sigma(M)(*)), and saturates for at a value rivaling the high transfection efficiency of H(II)(C) complexes. In contrast, the transfection efficiency of H(II)(C) complexes is independent of sigma(M). The exponential dependence of TE on sigma(M) for L(alpha)(C) complexes, suggests the existence of a kinetic barrier against endosomal fusion, where an increase in sigma(M) lowers the barrier. In the saturated TE regime, for both L(alpha)(C) complexes and H(II)(C), confocal microscopy reveals the dissociation of lipid and DNA. However, the lipid-released DNA is observed to be in a condensed state, most likely with oppositely charged macro-ion condensing agents from the cytoplasm, which remain to be identified. Much of the observed bulk of condensed DNA may be transcriptionally inactive and may determine the current limiting factor to transfection by cationic lipid gene vectors.

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser22). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser22 is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle.

Short peptide tags S6 and A1, each 12 residues in length, were identified from a phage-displayed peptide library as efficient substrates for site-specific protein labeling catalyzed by Sfp and AcpS phosphopantetheinyl transferases (PPTases), respectively. S6 and A1 tags were selected for useful levels of orthogonality in reactivities with the PPTases: the catalytic efficiency, kcat/Km of Sfp-catalyzed S6 serine phosphopantetheinylation was 442-fold greater than that for AcpS. Conversely, the kcat/Km of AcpS-catalyzed A1 labeling was 30-fold higher than that for Sfp-catalyzed A1 labeling. S6 and A1 peptide tags can be fused to N- or C-termini of proteins for orthogonal labeling of target proteins in cell lysates or on live cell surfaces. The development of the orthogonal S6 and A1 tags represents a significant enhancement of PPTase-catalyzed protein labeling, allowing tandem or iterative covalent attachment of small molecules of diverse structures to the target proteins with high efficiency and specificity.

Cationic liposomes (CLs) are used as gene vectors (carriers) in worldwide human clinical trials of non-viral gene therapy. These lipid-gene complexes have the potential of transferring large pieces of DNA of up to 1 million base-pairs into cells. As our understanding of the mechanisms of action of CL-DNA complexes remains poor, transfection efficiencies are still low when compared to gene delivery with viral vectors. We describe recent studies with a combination of techniques (synchrotron x-ray diffraction for structure determination, laser-scanning confocal microscopy to probe the interactions of CL-DNA particles with cells, and luciferase reporter-gene expression assays to measure transfection efficiencies in mammalian cells), which collectively are beginning to unravel the relationship between the distinctly structured CL-DNA complexes and their transfection efficiency. The work described here is applicable to transfection optimization in ex vivo cell transfection, where cells are removed and returned to patients after transfection. CL-DNA complexes primarily form a multilayered sandwich structure with DNA layered between the cationic lipids (labeled L,~). On rare occasions, an inverted hexagonal structure with DNA encapsulated in lipid tubules (labeled H~~~) is observed. A major recent insight is that for L ,~ complexes the membrane charge density OM of the CL-vector, rather than the charge of the cationic lipid alone, is a key universal parameter that governs the transfection efficiency of L, ' complexes in cells. The parameter aM is a measure of the average charge per unit area of the membrane, thus taking into account the amount of neutral lipids. In contrast to LaC complexes, HITC complexes containing the lipid 1,2-dioleoyl-sn-glycerophosphatidylethanolamine (DOPE) exhibit no dependence on OM. The currenl limiting factor to transfection by cationic lipid vectors appears to be the tight association of a fraction of the delivered exogenous DNA with cationic cellular molecules, which may prevent optimal transcriptional activity. Future directions are outlined, which make use of surface-functionalized CL-DNA complexes suitable for transfection in vivo.

Pharyngocutaneous fistula remains a significant problem following salvage laryngectomy. Use of nonirradiated, vascularized flaps reduced the incidence and duration of fistula and should be considered during salvage laryngectomy.

The endothelial isoform of nitric-oxide synthase (eNOS) is regulated by a complex pattern of post-translational modifications. In these studies, we show that eNOS is dynamically regulated by S-nitrosylation, the covalent adduction of nitric oxide (NO)-derived nitrosyl groups to the cysteine thiols of proteins. We report that eNOS is tonically S-nitrosylated in resting bovine aortic endothelial cells and that the enzyme undergoes rapid transient denitrosylation after addition of the eNOS agonist, vascular endothelial growth factor. eNOS is thereafter progressively renitrosylated to basal levels. The receptor-mediated decrease in eNOS S-nitrosylation is inversely related to enzyme phosphorylation at Ser 1179 , a site associated with eNOS activation. We also document that targeting of eNOS to the cell membrane is required for eNOS S-nitrosylation. Acylation-deficient mutant eNOS, which is targeted to the cytosol, does not undergo S-nitrosylation. Using purified eNOS, we show that eNOS S-nitrosylation by exogenous NO donors inhibits enzyme activity and that eNOS inhibition is reversed by denitrosylation. We determine that the cysteines of the zinc-tetrathiolate that comprise the eNOS dimer interface are the targets of S-nitrosylation. Mutation of the zinc-tetrathiolate cysteines eliminates eNOS S-nitrosylation but does not eliminate NO synthase activity, arguing strongly that disruption of the zinc-tetrathiolate does not necessarily lead to eNOS monomerization in vivo. Taken together, these studies suggest that eNOS S-nitrosylation may represent an important mechanism for regulation of NO signaling pathways in the vascular wall.