Targeting by AutophaGy proteins (TAG): Targeting of IFNG-inducible GTPases to membranes by the LC3 conjugation system of autophagy

LC3 has been used as a marker to locate autophagosomes. However, it is also well established that LC3 can localize on various membranous structures other than autophagosomes. We recently demonstrated that the LC3 conjugation system (ATG7, ATG3, and ATG12–ATG5-ATG16L1) is required to target LC3 and IFNG (interferon, gamma)-inducible GTPases to the parasitophorus vacuole membrane (PVM) of a protist parasite Toxoplasma gondii and consequently for IFNG to control T. gondii infection. Here we show that not only LC3, but also its homologs (GABARAP, GABARAPL1, and GABARAPL2) localize on the PVM of T. gondii in a conjugation-dependent manner. Knockout/knockdown of all LC3 homologs led to a significant reduction in targeting of the IFNG-inducible GTPases to the PVM of T. gondii and the IFNG-mediated control of T. gondii infection. Furthermore, when we relocated the ATG12–ATG5-ATG16L1 complex, which specifies the conjugation site of LC3 homologs, to alternative target membranes, the IFNG-inducible GTPases were targeted to the new target membranes rather than the PVM of T. gondii. These data suggest that the localization of LC3 homologs onto a membrane by the LC3 conjugation system is necessary and sufficient for targeting of the IFNG-inducible GTPases to the membrane, implying Targeting by AutophaGy proteins (TAG). Our data further suggest that the conjugation of ubiquitin-like LC3 homologs to the phospholipids of membranes may change the destiny of the membranes beyond degradation through lysosomal fusion, as the conjugation of ubiquitin to proteins changes the destiny of the proteins beyond proteasomal degradation.     

Bisphosphonate conjugation to proteins as a means to impart bone affinity

Growth factors are endogenous proteins capable of stimulating new bone formation, but their clinical benefit for systemic stimulation of bone mass has not been demonstrated. The critical challenge is to deliver a significant dose of the proteins to bone after intravenous injection. This challenge may be overcome by derivatizing proteins with ligands that exhibit a high bone affinity (e.g., bisphosphonates). To demonstrate the feasibility of this approach, 1-amino-1,1-diphosphonate methane (aminoBP) was conjugated to a model protein, albumin. The conjugation was performed by (1) converting the amino group of aminoBP to a thiol group using 2-iminothiolane, (2) derivatizing the albumin amino groups with a thiol-reactive sulfosuccinimidyl-4-(N-maleimidomethyl)-1-cyclohexane carboxylate, and (3) reacting the derivatized albumin with thiolated aminoBP. Typically, 1-4 aminoBP molecules per albumin were obtained. The conjugated albumin exhibited a high affinity to hydroxyapatite that was proportional to the extent of conjugation. The conjugates were shown to exhibit a high affinity to bone matrix in vitro in a serum-containing medium. Once bound to bone matrix, the conjugates were found to desorb more slowly than the unmodified albumin, especially from bone whose organic matrix was removed by ashing. In conclusion, conjugation of bisphosphonates to albumin was shown to impart a high bone affinity to the protein, and such conjugates can be potentially targeted to bone.     

Leptin enhances feeding suppression and neural activation produced by systemically administered bombesin

Leptin amplifies feeding inhibition and neural activation produced by either cholecystokinin or intragastric preloads, suggesting that leptin may increase the efficacy of gastrointestinal meal-related signals. To determine whether leptin would similarly potentiate the feeding inhibitory actions of another putative satiety peptide, we evaluated the effects of third ventricular leptin administration on food intake and c-Fos activation in response to systemically administered bombesin (BN). Leptin (3.5 microg) was administered 1 h before either 0.9% saline or BN (0.32 and 1.0 nmol/kg) followed by 30-min access to Ensure liquid diet. Although neither leptin nor 0.32 nmol/kg BN alone suppressed Ensure intake, the combination reduced intake by 28%. The higher BN dose (1.0 nmol/kg) produced a significant suppression by itself but was further enhanced in the presence of leptin. Consistent with the behavioral results, c-Fos activation in the nucleus of the solitary tract was increased by combined dosages of leptin and 0.32 nmol/kg BN beyond the individual response to either peptide. In the presence of leptin, BN produced a 3.4- to 5.2-fold increase in the number of c-Fos-positive cells in the nucleus of the solitary tract compared with when BN was given alone. These data provide further support for the hypothesis that the effect of leptin on food intake may be mediated, in part, by modulating meal-related satiety signals.     

Targeting of proteins to mitochondria

A clear picture has emerged over the past years on how a 'classic' mitochondrial protein, like subunit IV of cytochrome c oxidase, might be targeted to mitochondria. The targeting and subsequent import process involves the commitment of the TOM (translocase in the outer mitochondrial membrane) receptor complex on the mitochondrial surface, a TIM (translocase in the inner mitochondrial membrane) translocation complex in the mitochondrial inner membrane, and assorted chaperones and processing enzymes within the organelle. Recent work suggests that while very many mitochondrial precursor proteins might follow this basic targeting pathway, a large number have further requirements if they are to be successfully imported. These include ribosome-associated factors and soluble factors in the cytosol, soluble factors in the mitochondrial intermembrane space, an additional TIM translocase in the inner membrane and a range of narrow specificity assembly factors in the inner membrane. This review is focused on the targeting of proteins up to the stage at which they enter the TOM complex in the outer membrane.     

Cardiovascular and lung inflammatory effects induced by systemically administered diesel exhaust particles in rats

Pollution by particulates has consistently been associated with increased cardiorespiratory morbidity and mortality. It has been suggested that ultrafine particles, of which diesel exhaust particles (DEP) are significant contributors, are able to translocate from the airways into the bloodstream in vivo. In the present study, we assessed the effect of systemic administration of DEP on cardiovascular and respiratory parameters. DEP were administered into the tail vein of rats, and heart rate, blood pressure, blood platelet activation, and lung inflammation were studied 24 h later. Doses of 0.02, 0.1, or 0.5 mg DEP/kg (8, 42, or 212 microg DEP/rat) induced a significant decrease of heart rate and blood pressure compared with saline-treated rats. Although the number of platelets was not affected, all the doses of DEP caused a shortening of the bleeding time. Similarly, in addition to triggering lung edema, the bronchoalveolar lavage analysis revealed the presence of neutrophil influx in DEP-treated rats in a dose-dependent manner. We conclude that the presence of DEP in the systemic circulation leads not only to cardiovascular and haemostatic changes but it also triggers pulmonary inflammation.     

Preparation of bioconjugates by solid-phase conjugation to ion exchange matrix-adsorbed carrier proteins

A solid-phase conjugation method utilizing carrier protein bound to an ion exchange matrix was developed. Ovalbumin was adsorbed to an anion exchange matrix using a batch procedure, and the immobilized protein was then derivatized with iodoacetic acid N-hydroxysuccinimid ester. The activated protein was conjugated with glutathione, the conjugation ratio determined by acid hydrolysis, and amino acid analysis performed with quantification of carboxymethyl cysteine. Elution of conjugates from the resin by a salt gradient revealed considerable heterogeneity in the degree of derivatization, and immunization experiments with the eluted conjugates showed that the more substituted conjugates gave rise to the highest titers of glutathione antibodies. Direct immunization with the conjugates adsorbed to the ion exchange matrix was possible and gave rise to high titers of glutathione antibodies. Conjugates of ovalbumin and various peptides were prepared in a similar manner and used for production of peptide antisera by direct immunization with the conjugates bound to the ion exchanger. Advantages of the method are its solid-phase nature, allowing fast and efficient reactions and intermediate washings, and the ability to release conjugates from the solid phase under mild conditions.     

A tracer study with systemically and locally administered dinitrophenylated osteopontin.

Osteopontin (OPN), a major non-collagenous matrix protein of bone, is also found in tissue fluids and in the circulation. It is still not clear whether circulating OPN contributes to bone formation. To elucidate this question, rat OPN was tagged with dinitrophenol groups and administered to rats either intravenously or by infusion with an osmotic minipump through a "surgical window" in the bone of the hemimandible. Dinitrophenylated rat albumin (ALB) was used as a control. The presence and distribution of tagged proteins were revealed by immunogold labeling on sections of tibia and alveolar bone. Tagged molecules of OPN were found in mineralization foci, surfaces and interfaces, and matrix accumulations among calcified collagen fibrils. Even though dinitrophenylated ALB was administered at several-fold higher concentrations, it did not accumulate in these sites. These results show that circulating OPN can be incorporated into specific compartments of forming bone and suggest that such molecules may play a more important role than previously suspected.     

Targeting malaria parasite proteins to the erythrocyte

The intraerythrocytic stages of the protozoan parasite Plasmodium falciparum reside within a parasitophorous vacuole (PV) and set up unique "extraparasite, intraerythrocyte" protein-trafficking pathways that target parasite-encoded proteins to the erythrocyte cytoplasm and cell surface. Two recent articles report the identification of trafficking motifs that regulate the transport of parasite-encoded proteins across the PV. These articles greatly aid the annotation of the parasite "secretome" catalog of proteins that are targeted to the erythrocyte cytoplasm or cell membrane.     

Targeting of proteins to the Golgi apparatus

 The proteins that reside in the Golgi carry out functions associated with post-translational modifications, including glycosylation and proteolytic processing, membrane transport, recycling of endoplasmic reticulum proteins and maintenance of the structural organisation of the organelle itself. The latter includes Golgi stacking, interconnections between stacks and the microtubule-dependent positioning of the organelle within the cell. There are a number of distinct groups of Golgi membrane proteins, including glycosyltransferases, recycling trans-Golgi network (TGN) proteins, peripheral membrane proteins and receptors. Considerable effort has been directed at understanding the basis of the localisation of Golgi glycosyltransferases and recycling TGN proteins; in both cases there is increasing evidence that multiple signals may be involved in their specific localisation. A number of models for the Golgi retention of glycosyltransferases have been proposed including oligomerisation, lipid-mediated sorting and intra-Golgi retrograde transport. More information is required to determine the contribution of each of these potential mechanisms in the targeting of different glycosyltransferases. Future work is also likely to focus on the relationship between the localisation of resident Golgi proteins and the maintenance of Golgi structure. Accepted: 15 October 1997     

Targeting of proteins to the Golgi apparatus

The Golgi apparatus maintains a highly organized structure in spite of the intense membrane traffic which flows into and out of this organelle. Resident Golgi proteins must have localization signals to ensure that they are targeted to the correct Golgi compartment and not swept further along the secretory pathway. There are a number of distinct groups of Golgi membrane proteins, including glycosyltransferases, recyclingtrans-Golgi network proteins, peripheral membrane proteins, receptors and viral glycoproteins. Recent studies indicate that there are a number of different Golgi localization signals and mechanisms for retaining proteins to the Golgi apparatus. This review focuses on the current knowledge in this field.     

Targeting proteomics to investigate metastasis-associated mitochondrial proteins

Mitochondria are essential organelles in eukaryotic cells and are responsible for regulating energy metabolism, ROS production, and cell survival. Recently, various cellular pathogeneses, including tumorigenesis and metastasis, have been reported to be associated with mitochondrial homeostasis. Consequently, exploiting the correlation between dysfunctional mitochondria and tumor progression has been implicated in the understanding of tumorigenesis, tumor metastasis, and chemoresistance, along with novel strategies to develop cancer therapeutics. To comprehensively understand the role of the mitochondria in cancer metastasis, it is necessary to resolve thousands of mitochondrial proteins and their post-translational modifications with high-throughput global assessments. We introduce mitochondrial proteomic strategies in this review and a discussion on their recent findings related to cancer metastasis. Additionally, the mitochondrial respiratory chain is believed to be a major site for ROS production, and elevated ROS is likely a key source to trigger dysfunctional mitochondria and impaired mitochondrial metabolism that subsequently contribute to the development of cancer progression. Equipment-based metabolomic analysis now allows the monitoring of disease progression and diagnosis. These newly emerging techniques, including proteomics, redox-proteomics, and metabolomics, are described in this review.     

Evidence that systemically administered salbutamol reduces food intake in rats by acting on central beta-adrenergic sites

Salbutamol was found to reduce food intake in a dose-related manner. The effect was prevented by the beta-adrenolytic drugs d,1-propranolol and d,1-alprenolol. In contrast, phentolamine, penfluridol and metergoline, which block alpha-adrenergic, dopamine and serotonin receptors respectively, or bilateral lesions of the ventral noradrenergic bundles were not able to counteract salbutamol's effect. The reduction of food intake induced by salbutamol was prevented by intracerebroventricular administration of d,1-propranolol. In addition, salbutamol was found to effectively reduce the apparent motivation for food as revealed by its effect on food-rewarded runaway behaviour. This effect was also counteracted by d,1-propranolol pretreatment. The findings indicate that food intake may be reduced by peripheral administration of salbutamol and that the effect is mediated by stimulation of central beta-adrenergic sites.     

The behavioural effects of systemically administered kainic acid: A pharmacological analysis

Systemic injection of kainic acid (KA), a powerful neuroexcitant and structural analogue of glutamate, induced a complex behaviour in the rat characterized by early “wet-dog-shakes” (WDS and delayed convulsions. 1) The WDS syndrome was antagonized by serotonin blockers (mianserin and cyproheptadine) and by GABAmimetic agents, which decrease serotonergic transmission; in contrast, WDS were potentiated by compounds which enhance serotonin-mediated events (fluoxetine, fenfluramine, imipramine and tranylcypromine) as well as by blockade of GABA receptors (bicuculline). In addition, WDS were antagonized by haloperidol (which possesses some anti-serotonin properties) whereas KA potentiated haloperidol-induced catalepsy, an effect which was blocked by cyproheptadine. This suggests that KA induces WDS and potentiates catalepsy via an increase in serotoninergic function. 2) KA induced convulsions were antagonized by GABAmimetic agents, in agreement with their broad anticonvulsant spectrum; in contrast, mianserine and cyproheptadine did not affect or even potentiated seizures. Thus KA seizures respond differently to pharmacological treatment than do WDS, and may me related to the nwuro-excitatory action of KA.     

The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent

1. A new method is described for labelling proteins to high specific radioactivities with (125)I. The protein is treated with a (125)I-labelled acylating agent, iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester, which reacts with free amino groups in the protein molecule to attach the (125)I-labelled groups by amide bonds. 2. Three protein hormones have been labelled by this method, human growth hormone, human thyroid-stimulating hormone and human luteinizing hormone. Specific radioactivities of up to 170, 120 and 55muCi/mug respectively have been obtained for these hormones. 3. The immunoreactivity of these labelled hormones has been investigated by using a radioimmunoassay system specific for each hormone. These preparations have also been compared with and found to be equal or superior to labelled hormones prepared by chemical substitution of (125)I into tyrosine residues of the proteins by using the chloramine-t-oxidation procedure. 4. With some antisera the immunoreactivity of the antigen was diminished by the introduction of a single I atom into the tyrosyl groups, whereas antigen containing a single (125)I-labelled 3-(4-hydroxyphenyl)propionamide group showed the same immunoreactivity as the unmodified antigen.     

Differential conjugation of polyamines to calf nuclear and nucleolar proteins

Nuclei and nucleoli isolated from calf liver contain acid-precipitable putrescine, spermidine and spermine conjugates. The polyamines are released upon peptide bond hydrolysis. All of the nuclear putrescine conjugate and a major portion of the polyamine conjugates are localized within the nucleolus. Nuclei and nucleoli also contain, in proportions consistent with the nucleolar/nuclear protein ratio, the putative conjugating enzyme, transglutaminase, as well as amine acceptor substrates to which radiolabeled putrescine can be conjugated by endogenous enzyme. Extraction of the isolated organelles with saline solutions of increasing ionic strength showed a differential distribution of the polyamine derivatives: all the covalently linked putrescine was associated with the less soluble components of the chromatin residue, while the spermidine and spermine conjugates were associated with several salt-extractable protein fractions as well as tightly bound to the chromatin pellet. Mono-gamma-glutamyl putrescine was detected after proteolytic digestion of the 600 mM NaCl fraction, further suggesting the enzymatic action of transglutaminase(s) in the conjugation process.     

Intein-mediated site-specific conjugation of Quantum Dots to proteins in vivo

We describe an intein based method to site-specifically conjugate Quantum Dots (QDs) to target proteins in vivo. This approach allows the covalent conjugation of any nanostructure and/or nanodevice to any protein and thus the targeting of such material to any intracellular compartment or signalling complex within the cells of the developing embryo. We genetically fused a pleckstrin-homology (PH) domain with the N-terminus half of a split intein (I_N). The C-terminus half (I_C) of the intein was conjugated to QDs in vitro. I_C-QD's and RNA encoding PH-I_N were microinjected into Xenopus embryos. In vivo intein-splicing resulted in fully functional QD-PH conjugates that could be monitored in real time within live embryos. Use of Near Infra Red (NIR)-emitting QDs allowed monitoring of QD-conjugates within the embryo at depths where EGFP is undetectable demonstrating the advantages of QD's for this type of experiment. In conclusion, we have developed a novel in vivo methodology for the site-specific conjugation of QD's and other artificial structures to target proteins in different intracellular compartments and signaling complexes.     

Targeting of membrane proteins to endosomes and lysosomes

The pathways involved in targeting membrane proteins to lysosomes are extraordinarily complex. Newly synthesized proteins in the ER are transported to the Golgi complex, and upon arrival at the trans Golgi network (TGN) are targeted either directly to endosomes, or first to the cell surface from where they can be rapidly internalized into the endocytic pathway for delivery to lysosomes. The routes to endosomes are specified by sorting motifs in the cytoplasmic tails of the proteins that are recognized at the TGN or plasma membrane. The molecular details of these processes are just emerging.