We used domain-selective biotinylation/125I-streptavidin blotting (Sargiacomo, M., M. P. Lisanti, L. Graeve, A. Le Bivic, and E. Rodriguez-Boulan. 1989 J. Membr. Biol. 107:277-286), in combination with lectin precipitation, to analyze the apical and basolateral glycoprotein composition of Madin-Darby canine kidney (MDCK) cells and to explore the role of glycosylation in the targeting of membrane glycoproteins. All six lectins used recognized both apical and basolateral glycoproteins, indicating that none of the sugar moieties detected were characteristic of the particular epithelial cell surface. Pulse-chase experiments coupled with domain-selective glycoprotein recovery were designed to detect the initial appearance of newly synthesized glycoproteins at the apical or basolateral cell surface. After a short pulse with a radioactive precursor, glycoproteins reaching each surface were biotinylated, extracted, and recovered via precipitation with immobilized streptavidin. Several basolateral glycoproteins (including two sulfated proteins) and at least two apical glycoproteins (one of them the major sulfated protein of MDCK cells) appeared at the corresponding surface after 20-40 min of chase, but were not detected in the opposite surface, suggesting that they were sorted intracellularly and vectorially delivered to their target membrane. Several "peripheral" apical proteins were detected at maximal levels on the apical surface immediately after the 15-min pulse, suggesting a very fast intracellular transit. Finally, domain-selective labeling of surface carbohydrates with biotin hydrazide (after periodate oxidation) revealed strikingly different integral and peripheral glycoprotein patterns, resembling the Con A pattern, after labeling with sulfo-N-hydroxy-succinimido-biotin. The approaches described here should be useful in characterizing the steady-state distribution and biogenesis of endogenous cell surface components in a variety of epithelial cell lines.

Adult renal epithelial neoplasms (RENs) comprise several distinct clinicopathologic entities with potential prognostic and therapeutic differences. Individual cases can show overlapping morphologic features, necessitating the use of ancillary methods. The purpose of this study was to determine the diagnostic utility of cytokeratin (CK) subtype expression pattern in a wide range of adult RENs. RENs (including clear cell [conventional] renal cell carcinoma (RCC), papillary RCC, chromophobe RCC, renal oncocytoma, collecting duct carcinoma (CDC), renal medullary carcinoma (RMC), urothelial carcinoma, metanephric adenoma (MA), tubulocystic carcinoma (TC) (also known as low-grade collecting duct carcinoma), and mucinous tubular and spindle cell carcinoma) were immunostained for CK subtypes (CK5/CK6, 7, 8, 13, 14, 17, 18, 19, 20), high molecular weight CKs 1, 5, 10, 14 (HMWCK), and vimentin (Vim). The expression pattern of normal kidney was also examined and correlated with RENs. Although there is some overlap, subtypes of RENs show distinctive CK expression profiles that may be useful in several differential diagnostic settings. Clear cell RCCs typically showed a restricted expression pattern of CK8, CK18 and Vim. Papillary RCCs typically expressed CK7, CK8, CK18, CK19, and Vim and could be distinguished from MA (CK7-). Chromophobe RCCs were typically CK7+, CK8+, CK18+, and Vim-, and could be distinguished from oncocytomas (typically CK7-). In oncocytomas, nonspecific staining of unblocked endogenous biotin is a potentially significant diagnostic pitfall. CDC, RMC, and TC demonstrated similar CK expression profiles (with the exception of HMWCK expression limited to CDC), supporting a close relationship between these entities. A panel of CK5/CK6, CK17, and Vim may be helpful in distinguishing CDC (typically CK5/CK6-, CK17-, Vim+) and urothelial carcinoma (typically CK5/CK6+, CK17+, Vim-). In conclusion, CK expression patterns may be helpful in several differential diagnostic situations when dealing with adult RENs.

A trophoblast cell surface antigen has been characterised by a monoclonal antibody (mAb) 5T4, raised following immunisation with solubilised wheat germ agglutinin binding glycoproteins from human syncytiotrophoblast plasma membrane (StMPM). The expression of the 72 kDa glycoprotein was assessed on cryostat sections of a range of neoplastic and non-neoplastic tissues, using an avidin-biotin immunoperoxidase technique. In products of conception, intense reactions were noted with villous syncytiotrophoblast membrane in normal early and term placenta, with weaker positivity of placental site trophoblast. Most normal or non-neoplastic tissues were negative, including liver, kidney, spleen, small intestine, ovary and testis. Faint or moderate positive reactions were present in some specialised epithelia. Of 115 neoplasms examined, 76 showed reactions with tumour cells including carcinomas of the bladder, breast, cervix, endometrium, lung, oesophagus, ovary, pancreas, stomach and testicular non-seminomatous germ cell tumours. Choriocarcinomas and placental site trophoblastic tumours were also positive. Most adenocarcinomas of colon and seminomas were negative as were all malignant melanomas and malignant lymphomas. A radioimmunoassay did not detect the antigen in either normal or pregnancy serum. The relatively low level of expression in normal tissues and reactivity with a wide range of carcinomas suggested that the antibody may be useful in diagnostic or targeting studies.

BACKGROUND

Preclinical studies in animal models have demonstrated tumor regression following intratumoral administration of an adenovirus vector containing wild-type p53 complementary DNA (Ad-p53). Therefore, in a phase I clinical trial, we administered Ad-p53 to 28 patients with non-small-cell lung cancer (NSCLC) whose cancers had progressed on conventional treatments.

METHODS

Patients received up to six, monthly intratumoral injections of Ad-p53 by use of computed tomography-guided percutaneous fine-needle injection (23 patients) or bronchoscopy (five patients). The doses ranged from 10(6) plaque-forming units (PFU) to 10(11) PFU.

RESULTS

Polymerase chain reaction (PCR) analysis showed the presence of adenovirus vector DNA in 18 (86%) of 21 patients with evaluable posttreatment biopsy specimens; vector-specific p53 messenger RNA was detected by means of reverse transcription-PCR analysis in 12 (46%) of 26 patients. Apoptosis (programmed cell death) was demonstrated by increased terminal deoxynucleotide transferase-mediated biotin uridine triphosphate nick-end labeling (TUNEL) staining in posttreatment biopsy specimens from 11 patients. Vector-related toxicity was minimal (National Cancer Institute's Common Toxicity Criteria: grade 3 = one patient; grade 4 = no patients) in 84 courses of treatment, despite repeated injections (up to six) in 23 patients. Therapeutic activity in 25 evaluable patients included partial responses in two patients (8%) and disease stabilization (range, 2-14 months) in 16 patients (64%); the remaining seven patients (28%) exhibited disease progression.

CONCLUSIONS

Repeated intratumoral injections of Ad-p53 appear to be well tolerated, result in transgene expression of wild-type p53, and seem to mediate antitumor activity in a subset of patients with advanced NSCLC.

A population of estrogen receptor-alpha (ER alpha) proteins, located at the plasma membrane, is postulated to mediate the rapid, nongenomic responses of GH3/B6/F10 pituitary cells to estrogen. To demonstrate the presence of ER alpha at the plasma membrane and to distinguish this receptor population from that in the nucleus, GH3/B6/F10 cells were first prepared in 2% paraformaldehyde/0.1% glutaraldehyde in PBS (P/G) without detergent, then exposed to one of several antibodies (Abs) raised against nuclear ER alpha. Ab binding was visualized as a fluorescent/chromagenic reaction product catalyzed by avidin-biotin-complexed alkaline phosphatase. With P/G fixation, Abs could only access antigens at the cell surface, as evidenced by the inability of 70K mol wt dextrans to permeate cells and the absence of intracellular staining by Abs to cytoplasmic or nuclear antigens. ER alpha Abs generated membrane, but not nuclear, staining in P/G-fixed cells; nuclear receptor labeling could only be detected in detergent-treated cells. Specificity of staining for ER alpha was confirmed by three approaches: first, treatment with an antisense oligodeoxynucleotide to nuclear ER alpha mRNA reduced immunolabeling of both membrane and nuclear ER alpha; second, labeling by two Abs raised against different ER alpha oligopeptides was neutralized by competing peptide; third, six Abs (ER21, H226, R4, H222, MC20, and C542) that recognize unique epitopes on rodent ER alpha produced immunolabeling, but neither primate-specific ER alpha Ab nor Ab to ER beta caused staining. In addition to demonstrating the plasma membrane ER alpha in GH3/B6/F10 cells, this method should be applicable to other cell types that exhibit nongenomic responses to estrogen or other steroid hormones.

With the development of covalent modification strategies for viral capsids comes the ability to convert them into modular carrier systems for drug molecules and imaging agents. With this overall goal in mind, we have used two orthogonal modification strategies to decorate the exterior surface of genome-free MS2 capsids with PEG chains, while installing 50-70 copies of a fluorescent dye inside as a drug cargo mimic. Despite the very high levels of modification, the capsids remained in the assembled state, as determined by TEM, size-exclusion chromatography, and dynamic light scattering analysis. The ability of the polymer coating to block the access of polyclonal antibodies to the capsid surface was probed using a sandwich ELISA, which indicated a 90% reduction in binding. Further experiments indicated that biotin groups placed at the distal ends of the polymer chains were still capable of binding to streptavidin, despite their proximity to the PEG layer. Finally, a modular strategy was developed for the attachment of small-molecule targeting groups to the polymer chains through an efficient oxime formation reaction. As a result of these studies, a robust and versatile new platform has emerged for the potential delivery of therapeutic cargo.

Methods for site-specific modification of proteins should be quantitative and versatile with respect to the nature and size of the biological or chemical targets involved. They should require minimal modification of the target, and the underlying reactions should be completed in a reasonable amount of time under physiological conditions. Sortase-mediated transpeptidation reactions meet these criteria and are compatible with other labeling methods. Here we describe the expression and purification conditions for two sortase A enzymes that have different recognition sequences. We also provide a protocol that allows the functionalization of any given protein at its C terminus, or, for select proteins, at an internal site. The target protein is engineered with a sortase-recognition motif (LPXTG) at the place where modification is desired. Upon recognition, sortase cleaves the protein between the threonine and glycine residues, facilitating the attachment of an exogenously added oligoglycine peptide modified with the functional group of choice (e.g., fluorophore, biotin, protein or lipid). Expression and purification of sortase takes ∼3 d, and sortase-mediated reactions take only a few minutes, but reaction times can be extended to increase yields.

Nuclear receptors TR2 and TR4 (TR2/TR4) were previously shown to bind in vitro to direct repeat elements in the mouse and human embryonic and fetal β-type globin gene promoters and to play critical roles in the silencing of these genes. By chromatin immunoprecipitation (ChIP) we show that, in adult erythroid cells, TR2/TR4 bind to the embryonic β-type globin promoters but not to the adult β-globin promoter. We purified protein complexes containing biotin-tagged TR2/TR4 from adult erythroid cells and identified DNMT1, NuRD, and LSD1/CoREST repressor complexes, as well as HDAC3 and TIF1β, all known to confer epigenetic gene silencing, as potential corepressors of TR2/TR4. Coimmunoprecipitation assays of endogenous abundance proteins indicated that TR2/TR4 complexes consist of at least four distinct molecular species. In ChIP assays we found that, in undifferentiated murine adult erythroid cells, many of these corepressors associate with both the embryonic and the adult β-type globin promoters but, upon terminal differentiation, they specifically dissociate only from the adult β-globin promoter concomitant with its activation but remain bound to the silenced embryonic globin gene promoters. These data suggest that TR2/TR4 recruit an array of transcriptional corepressors to elicit adult stage-specific silencing of the embryonic β-type globin genes through coordinated epigenetic chromatin modifications.

A small RNA encoded within the nucleus is an essential subunit of a RNA processing endonuclease (RNase MRP) hypothesized to generate primers for mitochondrial DNA replication from the heavy strand origin of replication. Controversy has arisen, however, concerning the authenticity of an intramitochondrial pool of MRP RNA, and has called into question the existence of pathways for nucleo-mitochondrial transport of nucleic acids in animal cells. In an effort to resolve this controversy, we combined ultrastructural in situ hybridization and biochemical techniques to assess the subcellular partitioning of MRP RNA. Cryosections of mouse cardiomyocytes were hybridized with biotin-labeled RNA probes complementary to different regions of MRP RNA and varying in length from 115 to 230 nucleotides, followed by immunogold labeling. In addition, we transfected mouse C2C12 myogenic cells with constructs bearing mutated forms of the mouse MRP RNA gene and compared the relative abundance of the resulting transcripts to that of control RNAs within whole cell and mitochondrial fractions. In the former analysis we observed preferential localization of MRP RNA to nucleoli and mitochondria in comparison to the nucleoplasm and cytoplasm. In the latter series of studies we observed that wild-type MRP RNA partitions to the mitochondrial fraction by comparison to other RNA transcripts that are localized to the extramitochondrial cytoplasmic space (28S rRNA) or to the nucleoplasm (U1 snRNA). Deletions within 5' or 3' regions of the MRP RNA gene produced transcripts that remain competent for mitochondrial targeting. In contrast, deletion of the midportion of the coding region (nt 118 to 175) of the MRP RNA gene resulted in transcripts that fail to partition to the mitochondrial fraction. We conclude that an authentic intramitochondrial pool of MRP RNA is present in these actively respiring cells, and that specific structural determinants within the MRP RNA molecule permit it to be partitioned to mitochondria.

The three-dimensional structure of hen egg-white avidin, crystallized in a tetragonal crystal form, has been solved at 2.7 A resolution by molecular replacement methods. After refinement the crystallographic R-factor is 16.8%, for the 7255 reflections in the 10.0 to 2.7 A resolution range. The asymmetric unit contains two avidin polypeptide chains (M(r) 2 x 15,600), which build up the functional tetramer through a crystallographic 2-fold axis parallel to the c unit cell direction. The avidin tetramer has almost exact 222 molecular symmetry; the three possible dimers display quite distinct packing interfaces. Each protomer is organized in an eight-stranded antiparallel orthogonal beta-barrel, with extended loop regions. The avidin binding site within each promoter is located in a deep pocket, at the center of the barrel, displaying both hydrophobic and polar residues for recognition of the tightly bound vitamin. Two Trp residues, Trp70 and Trp97, and Phe79 are in close contact with biotin. Moreover, the binding pocket is partly closed in its outer rim by residue Trp110 of a neighboring subunit. Once bound, biotin is almost completely buried in the protein core, with the exception of the valeryl side-chain carboxylate group which is exposed to solvent, hydrogen bonds to residues Ala39, Thr40 and Ser75, and triggers the formation of a network of hydrogen bonded water molecules. Modeling of synthetic biotin analogues allows us to rationalize functional data available for the binding of these compounds, and to analyze them in terms of biotin recognition mechanism. Hen egg-white avidin shows clear structural homology to streptavidin, from Streptomyces avidinii, but significant deviations can be observed in some regions.

The free energy of binding between avidin and seven biotin analogues has been calculated with the molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) method. We have studied how the force field and the method to generate geometries affect the calculated binding free energies. Four different force fields were compared, but we saw no significant difference in the results. However, mixing the force fields used for the geometry generation and energy calculations is not recommended. In the molecular dynamics simulations, explicit water molecules must be used, but the size of the simulated system and the boundary conditions are less important. In fact, nonperiodic simulations with a fixed protein outside a relatively small simulated system (18 A) seem to be a proper approach. The mean absolute error was 9-19 kJ/mol, with a standard error of 5-15 kJ/mol, which arises mainly from the entropy term.

Telomeres are transcribed from the telomeric C-rich strand, giving rise to UUAGGG repeat-containing telomeric transcripts or TERRA, which are novel structural components of telomeres. TERRA abundance is highly dependent on developmental status (including nuclear reprogramming), telomere length, cellular stresses, tumour stage and chromatin structure. However, the molecular mechanisms and factors controlling TERRA levels are still largely unknown. In this study, we identify a set of RNA-binding proteins, which endogenously bind and regulate TERRA in the context of primary mouse embryonic fibroblasts. The identification was carried out by biotin pull-down assays followed by LC-MALDI TOF/TOF mass spectrometry. Different members of the heterogeneous nuclear ribonucleoprotein family are among the ribonucleoprotein family that bind more abundantly to TERRA. Downregulation of TERRA-bound RBPs by small interfering RNA further shows that they can impact on TERRA abundance, their location and telomere lengthening. These findings anticipate an impact of TERRA-associated RBPs on telomere biology and telomeres diseases, such as cancer and aging.

Methylselenol has been implicated as an active anticancer selenium (Se) metabolite. However, its in vivo efficacy against prostate cancer (PCa) has yet to be established. Here, we evaluated the growth inhibitory effects of two presumed methylselenol precursors methylseleninic acid (MSeA) and Se-methylselenocysteine (MSeC) in comparison with selenomethionine (SeMet) and selenite in DU145 and PC-3 human PCa xenografts in athymic nude mice. Each Se was given by a daily single oral dose regimen starting the day after the subcutaneous inoculation of cancer cells. We analyzed serum, liver and tumor Se content to confirm supplementation status and apoptosis indices and tumor microvessel density for association with antitumor efficacy. Furthermore, we analyzed lymphocyte DNA integrity to detect genotoxic effect of Se treatments. The data show that MSeA and MSeC exerted a dose-dependent inhibition of DU145 xenograft growth and both were more potent than SeMet and selenite, in spite of less tumor Se retention than in the SeMet-treated mice. Selenite treatment increased DNA single-strand breaks in peripheral lymphocytes, whereas the other Se forms did not. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and cleaved caspase-3 indices (apoptosis) from MSeC-treated tumors were higher than tumors from control mice or MSeA-treated mice, whereas the microvessel density index was lower in tumors from MSeA-treated mice. In the PC-3 xenograft model, only MSeA was growth inhibitory at a dose of 3 mg/kg body wt. In summary, our data demonstrated superior in vivo growth inhibitory efficacy of MSeA over SeMet and selenite, against two human PCa xenograft models without the genotoxic property of selenite.

The multidrug transporter ABCG2 (BCRP/MXR/ABCP) can actively extrude a broad range of endogenous and exogenous substrates across biological membranes. ABCG2 limits oral availability and mediates hepatobiliary and renal excretion of its substrates, and thus influences the pharmacokinetics of many drugs. Recent work, relying mainly on the use of Abcg2(-/-) mice, has revealed important contributions of ABCG2 to the blood-brain, blood-testis and blood-fetal barriers. Together, these functions indicate a primary biological role of ABCG2 in protecting the organism from a range of xenobiotics. In addition, several other physiological functions of ABCG2 have been observed, including extrusion of porphyrins and/or porphyrin conjugates from hematopoietic cells, liver and harderian gland, as well as secretion of vitamin B(2) (riboflavin) and possibly other vitamins (biotin, vitamin K) into breast milk. However, the physiological significance of these processes has been difficult to establish, indicating that there is still a lot to learn about this intriguing protein.

In the CNS, receptor recycling is critical for synaptic plasticity; however, the recycling of receptors has never been observed at peripheral synapses. Using a novel imaging technique, we show here that nicotinic acetylcholine receptors (AChRs) recycle into the postsynaptic membrane of the neuromuscular junction. By sequentially labeling AChRs with biotin-bungarotoxin and streptavidin-fluorophore conjugates, we were able to distinguish recycled, preexisting, and new receptor pools at synapses in living mice. Time-lapse imaging revealed that recycled AChRs were incorporated into the synapse within hours of initial labeling, and their numbers increased with time. At fully functional synapses, AChR recycling was robust and comparable in magnitude with the insertion of newly synthesized receptors, whereas chronic synaptic activity blockade nearly abolished receptor recycling. Finally, using the same sequential labeling method, we found that acetylcholinesterase, another synaptic component, does not recycle. These results identify an activity-dependent AChR-recycling mechanism that enables the regulation of receptor density, which could lead to rapid alterations in synaptic efficacy.

An efficient mechanism-based tumor-targeting drug delivery system, based on tumor-specific vitamin-receptor mediated endocytosis, has been developed. The tumor-targeting drug delivery system is a conjugate of a tumor-targeting molecule (biotin: vitamin H or vitamin B-7), a mechanism-based self-immolative linker and a second-generation taxoid (SB-T-1214) as the cytotoxic agent. This conjugate (1) is designed to be (i) specific to the vitamin receptors overexpressed on tumor cell surface and (ii) internalized efficiently through receptor-mediated endocytosis, followed by smooth drug release via glutathione-triggered self-immolation of the linker. In order to monitor and validate the sequence of events hypothesized, i.e., receptor-mediated endocytosis of the conjugate, drug release, and drug-binding to the target protein (microtubules), three fluorescent/fluorogenic molecular probes (2, 3, and 4) were designed and synthesized. The actual occurrence of these processes was unambiguously confirmed by means of confocal fluorescence microscopy (CFM) and flow cytometry using L1210FR leukemia cells, overexpressing biotin receptors. The molecular probe 4, bearing the taxoid linked to fluorescein, was also used to examine the cell specificity (i.e., efficacy of receptor-based cell targeting) for three cell lines, L1210FR (biotin receptors overexpressed), L1210 (biotin receptors not overexpressed), and WI38 (normal human lung fibroblast, biotin receptor negative). As anticipated, the molecular probe 4 exhibited high specificity only to L1210FR. To confirm the direct correlation between the cell-specific drug delivery and anticancer activity of the probe 4, its cytotoxicity against these three cell lines was also examined. The results clearly showed a good correlation between the two methods. In the same manner, excellent cell-specific cytotoxicity of the conjugate 1 (without fluorescein attachment to the taxoid) against the same three cell lines was confirmed. This mechanism-based tumor-targeting drug delivery system will find a range of applications.

OBJECTIVE

To describe the histological changes in acute enthesopathy in early spondyloarthropathies (SpA).

METHODS

Clinically evident acute enthesopathy was confirmed by magnetic resonance imaging and ultrasonography in four cases of plantar fasciitis and one case of patellar tendon enthesitis. Ultrasound guided biopsy of insertional points was carried out with a Jamshedi needle. Control tissue was obtained from two subjects undergoing spinal grafting surgery. Standard histochemistry and immunohistochemistry analysis using the avidin-biotin immunoperoxidase complex method employing markers against CD3, CD8, CD34, and CD68 was used to determine cellular infiltrates at the insertion point.

RESULTS

The enthesis architecture was abnormal in the SpA group, with increased vascularity and cellular infiltration compared with normal subjects. The predominant infiltrating cell at the enthesis fibrocartilage was the macrophage, but there was a paucity of lymphocytes at the insertion point.

CONCLUSIONS

These preliminary findings have implications for a better understanding of the pathology in early SpA.

Expression of functional, recombinant alpha7 nicotinic acetylcholine receptors in several mammalian cell types, including HEK293 cells, has been problematic. We have isolated the recently described human ric-3 cDNA and co-expressed it in Xenopus oocytes and HEK293 cells with the human nicotinic acetylcholine receptor alpha7 subunit. In addition to confirming the previously reported effect on alpha7 receptor expression in Xenopus oocytes we demonstrate that ric-3 promotes the formation of functional alpha7 receptors in mammalian cells, as determined by whole cell patch clamp recording and surface alpha-bungarotoxin binding. Upon application of 1 mm nicotine, currents were undetectable in HEK293 cells expressing only the alpha7 subunit. In contrast, co-expression of alpha7 and ric-3 cDNAs resulted in currents that averaged 42 pA/pF with kinetics similar to those observed in cells expressing endogenous alpha7 receptors. Immunoprecipitation studies demonstrate that alpha7 and ric-3 proteins co-associate. Additionally, cell surface labeling with biotin revealed the presence of alpha7 protein on the plasma membrane of cells lacking ric-3, but surface alpha-bungarotoxin staining was only observed in cells co-expressing ric-3. Thus, ric-3 appears to be necessary for proper folding and/or assembly of alpha7 receptors in HEK293 cells.

Mapping protein-protein interactions for chromatin-associated proteins remains challenging. Here we explore the use of BioID, a proximity biotinylation approach in which a mutated biotin ligase (BirA*) is fused to a bait of interest, allowing for the local activation of biotin and subsequent biotinylation of proteins in the bait vicinity. BioID allowed for successful interactome mapping of core histones and members of the mediator complex. We explored the background signal produced by the BioID approach and found that using distinct types of controls increased the stringency of our statistical analysis with SAINTexpress. A direct comparison of BioID with our AP-MS protocol optimized for chromatin-associated protein complexes revealed that the approaches identified few shared interaction partners and enriched for distinct biological processes; yet, both approaches permitted the recovery of biologically meaningful interactions. While no clear bias could be observed for either technique toward protein complexes of particular functions, BioID allowed for the purification of proteins of lower cellular abundance. Finally, we were able to identify a strong association of MED4 with the centrosome by BioID and validated this finding by immunofluorescence. In summary, BioID complements AP-MS for the study of chromatin-associated protein complexes.

UNASSIGNED

This manuscript describes the application of BioID, a proximity biotinylation approach, to chromatin-associated proteins, namely core histones and members of the mediator complex. We observed that BioID was successful at identifying known interaction partners for the baits tested, but also allowed novel putative interaction partners to be identified. By performing a detailed comparison of BioID versus a standard method for interactome mapping (affinity purification coupled to mass spectrometry, AP-MS), we show that the approaches were complementary, allowing for purification of different interaction partners. These interaction partners were different in the biological processes they are associated with, but also in their abundance. BioID represents a significant technical development in the field of chromatin research by expanding the search space for interactome mapping beyond what is possible with AP-MS. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.

Our previous studies have demonstrated that DNA injury occurs in the brain after intracerebral hemorrhage (ICH). DNA damage can result from at least two pathways, either endonuclease-mediated DNA fragmentation or oxidative injury. The present study investigated the occurrence of the latter after ICH and the role of iron in such injury. Male Sprague-Dawley rats received an infusion of autologous whole blood or ferrous iron into the right basal ganglia. Control rats just had a needle insertion (sham). The rats were sacrificed 1, 3, or 7 days later. 8-Hydroxyl-2'-deoxyguanosine (8-OHdG) was analyzed by immunohistochemistry while the number of apurnic/apyrimidinic abasic sites (AP sites) was also quantified. 8-OHdG and AP sites are two hallmarks of DNA oxidation. Dinitrophenyl (DNP) was measured by Western blotting to compare the time course of protein oxidative damage to that of DNA. DNA repair Ku proteins were measured by Western blot analysis. DNA damage was also examined using DNA polymerase I-mediated biotin-dATP nick translation (PANT) labeling. An increase of 8-OHdG, AP sites and DNP levels and a decrease of Ku levels were observed. Abundant PANT-positive cells were also observed in the perihematomal area 3 days after ICH. In addition, intracerebral infusion of iron increased brain DNP levels and resulted in DNA injury. These results suggest that oxidative stress contributes to DNA damage and brain injury after ICH. Reducing DNA oxidative damage (for example, through iron chelation) may be a therapeutic target for ICH.

Hydrogen sulfide (H2S) has emerged as a new member of the gaseous transmitter family of signaling molecules and appears to play a regulatory role in the cardiovascular and nervous systems. Recent studies suggest that protein cysteine S-sulfhydration may function as a mechanism for transforming the H2S signal into a biological response. However, selective detection of S-sulfhydryl modifications is challenging since the persulfide group (RSSH) exhibits reactivity akin to other sulfur species, especially thiols. A modification of the biotin switch technique, using S-methyl methanethiosulfonate (MMTS) as an alkylating reagent, was recently used to identify a large number of proteins that may undergo S-sulfhydration, but the underlying mechanism of chemical detection was not fully explored. To address this key issue, we have developed a protein persulfide model and analogue of MMTS, S-4-bromobenzyl methanethiosulfonate (BBMTS). Using these new reagents, we investigated the chemistry in the modified biotin switch method and examined the reactivity of protein persulfides toward different electrophile/nucleophile species. Together, our data affirm the nucleophilic properties of the persulfide sulfane sulfur and afford new insights into protein S-sulfhydryl chemistry, which may be exploited in future detection strategies.

There is increasing interest in the concept of using nanopores as the sensing elements in biosensors. The nanopore most often used is the alpha-hemolysin protein channel, and the sensor consists of a single channel embedded within a lipid bilayer membrane. An ionic current is passed through the channel, and analyte species are detected as transient blocks in this current associated with translocation of the analyte through the channel-stochastic sensing. While this is an extremely promising sensing paradigm, it would be advantageous to eliminate the very fragile lipid bilayer membrane and perhaps to replace the biological nanopore with an abiotic equivalent. We describe here a new family of protein biosensors that are based on conically shaped gold nanotubes embedded within a mechanical and chemically robust polymeric membrane. While these sensors also function by passing an ion current through the nanotube, the sensing paradigm is different from the previous devices in that a transient change in the current is not observed. Instead, the protein analyte binds to a biochemical molecular-recognition agent at the mouth of the conical nanotube, resulting in complete blockage of the ion current. Three different molecular-recognition agents, and correspondingly three different protein analytes, were investigated: (i) biotin/streptavidin, (ii) protein-G/immunoglobulin, and (iii) an antibody to the protein ricin with ricin as the analyte.

BACKGROUND

Candida species are the most common cause of opportunistic fungal infection worldwide. Recent sequencing efforts have provided a wealth of Candida genomic data. We have developed the Candida Gene Order Browser (CGOB), an online tool that aids comparative syntenic analyses of Candida species. CGOB incorporates all available Candida clade genome sequences including two Candida albicans isolates (SC5314 and WO-1) and 8 closely related species (Candida dubliniensis, Candida tropicalis, Candida parapsilosis, Lodderomyces elongisporus, Debaryomyces hansenii, Pichia stipitis, Candida guilliermondii and Candida lusitaniae). Saccharomyces cerevisiae is also included as a reference genome.

RESULTS

CGOB assignments of homology were manually curated based on sequence similarity and synteny. In total CGOB includes 65617 genes arranged into 13625 homology columns. We have also generated improved Candida gene sets by merging/removing partial genes in each genome. Interrogation of CGOB revealed that the majority of tandemly duplicated genes are under strong purifying selection in all Candida species. We identified clusters of adjacent genes involved in the same metabolic pathways (such as catabolism of biotin, galactose and N-acetyl glucosamine) and we showed that some clusters are species or lineage-specific. We also identified one example of intron gain in C. albicans.

CONCLUSIONS

Our analysis provides an important resource that is now available for the Candida community. CGOB is available at http://cgob.ucd.ie.

A recently discovered superfamily of enzymes function using chemically novel mechanisms, in which S-adenosylmethionine (SAM) serves as an oxidizing agent in DNA repair and the biosynthesis of vitamins, coenzymes and antibiotics. Members of this superfamily, the radical SAM enzymes, are related by the cysteine motif CxxxCxxC, which nucleates the [4Fe-4S] cluster found in each. A common thread in the novel chemistry of these proteins is the use of a strong reducing agent--a low-potential [4Fe-4S](1+) cluster--to generate a powerful oxidizing agent, the 5'-deoxyadenosyl radical, from SAM. Recent results are beginning to determine the unique biochemistry for some of the radical SAM enzymes, for example, lysine 2,3 aminomutase, pyruvate formate lyase activase and biotin synthase.