Добірка наукової літератури з теми "Tripartite GFP"

Various methods can provide a readout of the physical interaction between two biomolecules. A recently described tripartite split-GFP system has the potential to report by direct visualization via a fluorescence signal the intimate association of minimally tagged proteins expressed at their endogenous level in their native cellular milieu and can capture transient or weak interactions. Here we document the utility of this tripartite split-GFP system to assess in living cells protein–protein interactions in a dynamic cytoskeletal structure—the septin collar at the yeast bud neck. We show, first, that for septin–septin interactions, this method yields a robust signal whose strength reflects the known spacing between the subunits in septin filaments and thus serves as a “molecular ruler.” Second, the method yields little or no spurious signal even with highly abundant cytosolic proteins readily accessible to the bud neck (including molecular chaperone Hsp82 and glycolytic enzyme Pgk1). Third, using two proteins (Bni5 and Hsl1) that have been shown by other means to bind directly to septins at the bud neck in vivo, we validate that the tripartite split-GFP method yields the same conclusions and further insights about specificity. Finally, we demonstrate the capacity of this approach to uncover additional new information by examining whether three other proteins reported to localize to the bud neck (Nis1, Bud4, and Hof1) are able to interact physically with any of the subunits in the septin collar and, if so, with which ones.

ABSTRACT Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.

Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite » split-FP, that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein–protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology.

Bacterial micro-compartments (BMC) are complex macromolecular assemblies that participate in varied metabolic processes in about 20% of bacterial species. Most of these organisms carry BMC genetic information organized in operons that often include several paralog genes coding for components of the compartment shell. BMC shell constituents can be classified depending on their oligomerization state as hexamers (BMC-H), pentamers (BMC-P) or trimers (BMC-T). Formation of hetero-oligomers combining different protein homologs is theoretically feasible, something that could ultimately modify BMC shell rigidity or permeability, for instance. Despite that, it remains largely unknown whether hetero-oligomerization is a widespread phenomenon. Here, we demonstrated that the tripartite GFP (tGFP) reporter technology is an appropriate tool that might be exploited for such purposes. Thus, after optimizing parameters such as the size of linkers connecting investigated proteins to GFP10 or GFP11 peptides, the type and strength of promoters, or the impact of placing coding cassettes in the same or different plasmids, homo-oligomerization processes could be successfully monitored for any of the three BMC shell classes. Moreover, the screen perfectly reproduced published data on hetero-association between couples of CcmK homologues from Syn. sp. PCC6803, which were obtained following a different approach. This study paves the way for mid/high throughput screens to characterize the extent of hetero-oligomerization occurrence in BMC-possessing bacteria, and most especially in organisms endowed with several BMC types and carrying numerous shell paralogs. On the other hand, our study also unveiled technology limitations deriving from the low solubility of one of the components of this modified split-GFP approach, the GFP1-9.

ABSTRACT Here, the class I polyhydroxyalkanoate synthase (PhaC) from Ralstonia eutropha was investigated regarding the functionality of its conserved C-terminal region and its ability to tolerate translational fusions to its C terminus. MalE, the maltose binding protein, and green fluorescent protein (GFP) were considered reporter proteins to be translationally fused to the C terminus. Interestingly, PhaC remained active only when a linker was inserted between PhaC and MalE, whereas MalE was not functional. However, the extension of the PhaC N terminus by 458 amino acid residues was required to achieve a functionality of MalE. These data suggested a positive interaction of the extended N terminus with the C terminus. To assess whether a linker and/or N-terminal extension is generally required for a functional C-terminal fusion, GFP was fused to the C terminus of PhaC. Both fusion partners were active without the requirement of a linker and/or N-terminal extension. A further reporter protein, the immunoglobulin G binding ZZ domain of protein A, was translationally fused to the N terminus of the fusion protein PhaC-GFP and resulted in a tripartite fusion protein mediating the production of polyester granules displaying two functional protein domains.

Bladder cancer (BC) has a 70% telomerase reverse transcriptase (TERT or hTERT in humans) promoter mutation prevalence, commonly at −124 base pairs, and this is associated with increased hTERT expression and poor patient prognosis. We inserted a green fluorescent protein (GFP) tag in the mutant hTERT promoter allele to create BC cells expressing an hTERT-GFP fusion protein. These cells were used in a fluorescence-activated cell sorting–based pooled CRISPR-Cas9 Kinome knockout genetic screen to identify tripartite motif containing 28 (TRIM28) and TRIM24 as regulators of hTERT expression. TRIM28 activates, while TRIM24 suppresses, hTERT transcription from the mutated promoter allele. TRIM28 is recruited to the mutant promoter where it interacts with TRIM24, which inhibits its activity. Phosphorylation of TRIM28 through the mTOR complex 1 (mTORC1) releases it from TRIM24 and induces hTERT transcription. TRIM28 expression promotes in vitro and in vivo BC cell growth and stratifies BC patient outcome. mTORC1 inhibition with rapamycin analog Ridaforolimus suppresses TRIM28 phosphorylation, hTERT expression, and cell viability. This study may lead to hTERT-directed cancer therapies with reduced effects on normal progenitor cells.

We recently discovered that MG53, a muscle-specific tripartite motif (TRIM) family protein, functions as a sensor of oxidation to nucleate the assembly of cell membrane repair machinery. Our data showed that disulfide bond formation mediated by Cys242 is critical for MG53-mediated translocation of intracellular vesicles toward the injury sites. Here we test the hypothesis that leucine zipper motifs in the coiled-coil domain of MG53 constitute an additional mechanism that facilitates oligomerization of MG53 during cell membrane repair. Two leucine zipper motifs in the coiled-coil domain of MG53 (LZ1 - L176/L183/L190/V197 and LZ2 - L205/L212/L219/L226) are highly conserved across the different animal species. Chemical cross-linking studies show that LZ1 is critical for MG53 homodimerization, whereas LZ2 is not. Mutations of the conserved leucines into alanines in LZ1, not in LZ2, diminish the redox-dependent oligomerization of MG53. Live cell imaging studies demonstrate that the movement of green fluorescent protein (GFP)-tagged MG53 mutants (GFP-LA1 and GFP-LA2) is partially compromised in response to mechanical damage of the cell membrane, and the GFP-LA1/2 double mutant is completely ineffective in translocation toward the injury sites. In addition to the leucine zipper-mediated intermolecular interaction, redox-dependent cross talk between MG53 appears to be an obligatory step for cell membrane repair, since in vivo modification of cysteine residues with alkylating reagents can prevent the movement of MG53 toward the injury sites. Our data show that oxidation of the thiol group of Cys242 and leucine zipper-mediated interaction among the MG53 molecules both contribute to the nucleation process for MG53-mediated cell membrane repair.

RhoB est une petite GTPase rapidement activée par les facteurs de croissance et les stress cellulaires, qui régule des processus biologiques fondamentaux comme la migration, l'angiogenèse, la réparation de l'ADN, l'apoptose ainsi que la réponse à des thérapeutiques anticancéreuses. L'activité des petites GTPases est finement régulée par leur localisation subcellulaire. Cependant, l'activation de RhoB en cellules vivantes n'avait jamais été investiguée. Ce travail a permis d'adapter et de valider une méthode innovante d'analyse des interactions protéine-protéine par complémentation split-GFP tripartite, pour la détection sensible et spécifique de l'activation des petites GTPases en cellules vivantes. Nous avons ensuite développé un modèle cellulaire optimisé par la combinaison de la technologie split-GFP tripartite et d'un intracorps anti-GFP amplificateur de fluorescence, pour détecter la régulation de l'activation de RhoB avec une haute résolution spatiale. Ce biosenseur a mis en évidence la translocation de la forme active de RhoB en réponse au sérum à partir des endosomes pour s'accumuler au niveau de la membrane plasmique, révélant ainsi une nouvelle plateforme de signalisation membranaire de RhoB. Ce biosenseur permettra d'analyser le profil d'activation de RhoB et d'autres petites GTPases, sous d'autres stimulations ou dans différents contextes cellulaires, et d'identifier leurs partenaires et les modulateurs de leur activation
RhoB is a small GTPase that is rapidly activated in response to growth factors and cellular stress. It regulates fundamental biological processes such as cell migration, angiogenesis, DNA repair, apoptosis and response to anticancer therapies. Small GTPases activity is tightly regulated by their subcellular localization. However, RhoB activation had never been investigated in living cells. In this work, we have adapted and validated an innovative method of protein-protein interactions analysis using tripartite split-GFP complementation, for the sensitive and specific detection of small GTPases activation in living cells. Then, we developed an optimized cellular model by combining the tripartite split-GFP technology with an anti-GFP intrabody fluorescence-enhancer to detect the regulation of RhoB activation with high spatial resolution. This biosensor highlighted the translocation of active RhoB from endosomes to accumulate at the plasma membrane upon serum stimulation, revealing a novel membrane signaling platform of RhoB. Future studies based on this biosensor will enable the analysis of RhoB activation profile and other small GTPases upon various stimuli or in different cellular contexts, as well as the identification of the GTPases partners and activation modulators

Les microcompartiments bactériens (BMC) sont des structures protéiques naturellement présentes chez certaines bactéries dans lesquelles ils agissent comme des bioréacteurs et métabolisent des substrats spécifiques. Selon le type de BMC, le set d’enzymes encapsulé au sein du BMC peut fixer le CO2 atmosphérique ou cataboliser l’éthanolamine, le 1,2-propanediol ou la choline. La coque polyédrique des BMCs est composée de 3 sous-unités différentes dont les BMC-H, un protomère s’oligomérisant en hexamère, sous-unité principale de la coque des BMCs. Des études génomiques ont estimé une moyenne de 3,5 homologues BMC-H par opéron BMC, avec certains organismes tel que Clostridium saccharolyticum WM1 codant jusqu’à 15 BMC-H, répartis dans 3 opérons BMC différents.Bien qu’il fut longtemps considéré que seuls des homo-hexamères existaient, il fut démontré récemment que des hétéro-hexamères pouvaient également se former entre homologues BMC-H, dans 2 bactéries exprimant le β-carboxysome. En effet, les homologues BMC-H partagent généralement une forte identité de séquence, notamment sur leur interfaces intra-hexamère. Outre ouvrir à la possibilité que des hétéro-hexamères se forment dans des organismes dotés d’un autre type de BMC, ces études récentes ont soulevé la question de potentielles cross-interactions entre BMC-H venant de différents types de BMC, au sein d’un même organisme.Un objectif de ma thèse fut d’examiner la formation d’hétéro-hexamères dans la nature. Pour cela, la tripartite GFP a été adaptée à l’étude des interactions protéines-protéines entre BMC-H et appliquée au cas des BMC-H de Klebsiella pneumoniae 342. Cet organisme est en effet doté de 3 loci codant 3 BMCs de différents types, loci qui comptabilisent un total de 11 homologues BMC-H. Ainsi, en plus de nous permettre de déterminer si des hétéro-hexamères se forment en dehors des BMC-H compris dans le β-carboxysome, leur étude pourrait apporter de premiers éléments de réponse sur la possibilité de cross-interactions entre BMC-H provenant de différents types de BMC.Une nouvelle méthode pour améliorer l’efficacité catalytique d’une voie (autre que par l’ingénierie enzymatique) suscite de plus en plus d’intérêt de nos jours : l’organisation spatiale des enzymes. L’idée est qu’en plaçant à proximité ou de manière arrangée les enzymes d’une voie métabolique, il serait possible d’augmenter l’efficacité de la voie.La majorité des hexamères formés par les BMC-H ont la propriété intrinsèque de s'auto-assembler et de former des macrostructures (nanotubes, Swiss-rolls, feuillets 2D) lorsqu'ils sont exprimés seuls dans E. coli. Cette particularité a déjà été exploitée dans de multiples études pour créer un échafaudage protéique pour l’immobilisation d’enzymes. Dans ces preuves de concept, un seul BMC-H a été utilisé pour construire l'échafaudage, ce qui permettait uniquement d'immobiliser les enzymes de manière aléatoire.Nous proposons ici d'aller plus loin dans l'idée d'organisation spatiale et visons à élaborer une plateforme protéique à partir d'un hétéro-hexamère. Cet hétéro-hexamère serait composé de 2 à 6 BMC-H différents, chaque BMC-H constituant un point d'ancrage pour un futur domaine enzymatique. Avec une telle plateforme, l’organisation spatiale des enzymes serait contrôlée plus finement, ce qui améliorerait encore l’efficacité de la catalyse d’une voie métabolique.Pour atteindre cet objectif, des BMC-H ont été designés de novo par 2 équipes collaboratrices de design computationnel. Je les ai étudiés et ai recherché des couples BMC-H qui présenteraient des interfaces intra-hexamères orthogonales. En effet, pour pouvoir contrôler précisément l'organisation sur la plateforme, cela nécessiterait d'assurer un ordre spécifique des BMC-H au sein de l'hétéro-hexamère et de contrôler étroitement quel BMC-H est adjacent à quel autre et d'empêcher toute autre association
Bacterial microcompartments (BMC) are protein structures, naturally found in some bacteria in which they act as bioreactor and process specific substrates. For instance, depending on the BMC type, the enzymatic set they encapsulate can fixate atmospheric CO2 or catabolize the ethanolamine, 1,2-propanediol or the choline. The BMC shell is polyhedral and is composed of 3 different subunits, including the BMC-H, a protomer associating as an hexamer which are the main and the most diverse shell subunits, in terms of number of homologs within a single BMC operon. Indeed, genomic surveys indicate an average of 3,5 BMC-H homologs per operon, with some organisms like Clostridium saccharolyticum WM1 coding for up to 15 BMC-H split between 3 BMC types.Although it has long been thought that only homo-hexamers existed, it was recently evidenced that hetero-hexamer formation occurred between BMC-H homologs in 2 different β-carboxysome-expressing bacteria. Indeed, numerous BMC-H homologs share a high sequence identity, notably at the intra-hexamer interfaces. Besides paving the way for possible hetero-hexamer formation beyond the β-carboxysome, inside organisms equipped with one BMC type, these recent studies raise the question of possible cross-interactions between BMC-H coming from multiple BMC types.One objective during my PhD thesis was to examine the occurrence of hetero-hexamers in nature. To this end, the tripartite GFP was adapted to study protein-protein interactions among BMC-H and implemented on the case study of Klebsiella pneumonia 342 BMC-H. Of note, this organism is very interesting because it has in its genome 3 BMC loci, comprising a total of 11 BMC-H homologs. Then, besides allowing to determine whether hetero-hexamers do form aside from the β-CBX, in 3 other BMC types, their study would also bring some answer elements to the question of the cross-interactions between BMC-H arising from different BMC types.A novel method to enhance a pathway catalytic efficiency (other than by classical enzymatic engineering) is gaining more and more interests nowadays: enzyme spatial organization. The idea is that, by putting in close proximity or in an arranged fashion the enzymes from a metabolic pathway, one could increase the efficiency of the pathway, through substrate channelling between the different enzymes, for instance, or enzyme clusterisation.The majority of hexamers formed by the BMC-H have the intrinsic property to self-assemble and form higher-ordered macrostructures (nanotubes, Swiss-rolls, 2D sheets) when recombinantly expressed alone in E. coli. This peculiarity has already been exploited in multiple studies to create a protein scaffold for the immobilization of enzymes. In these proof-of-concepts, a sole BMC-H was used to build the scaffold, which would only permit to immobilized different enzymes in a random fashion.Here, we propose to go further with the idea of spatial organization and aimed to elaborate a protein platform starting from an hetero-hexamer. This hetero-hexamer would be composed by 2 up to 6 different BMC-H with each BMC-H constituting an anchoring point for a future enzymatic domain. With such platform, the spatial organization of the enzymes would be more finely controlled which would further enhance the catalysis efficiency of a metabolic pathway.To meet this goal, de novo designed BMC-H were created by 2 collaborator teams of computational design. I studied them and searched for BMC-H couples that would depict orthogonal intra-hexamer interfaces. Indeed, to be able to control precisely the organization onto the platform, this would require to ensure a specific BMC-H order within the hetero-hexamer and thus, tightly control which BMC-H is adjacent to which one and prevent any other association

O Vírus da Imunodeficiência Humana (HIV) é o agente etiológico da Síndrome da Imunodeficiência Adquirida (AIDS). A AIDS é uma doença de distribuição mundial, e estima-se que existam atualmente pelo menos 36,9 milhões de pessoas infectadas com o vírus. Durante o seu ciclo replicativo, o HIV promove diversas alterações na fisiologia da célula hospedeira a fim de promover sua sobrevivência e potencializar a replicação. A rápida progressão da infecção pelo HIV-1 em humanos e em modelos animais está intimamente ligada à função da proteína acessória Nef. Dentre as diversas ações de Nef está a regulação negativa de proteínas importantes na resposta imunológica, como o receptor CD4. Sabe-se que esta ação resulta da indução da degradação de CD4 em lisossomos, mas os mecanismos moleculares envolvidos ainda são totalmente elucidados. Nef forma um complexo tripartite com a cauda citosólica de CD4 e a proteína adaptadora 2 (AP-2), em vesículas revestidas por clatrina nascentes, induzindo a internalização e degradação lisossomal de CD4. Pesquisas anteriores demonstraram que o direcionamento de CD4 aos lisossomos por Nef envolve a entrada do receptor na via dos corpos multivesiculares (MVBs), por um mecanismo atípico, pois, embora não necessite da ubiquitinação de carga, depende da ação de proteínas que compõem os ESCRTs (Endosomal Sorting Complexes Required for Transport) e da ação de Alix, uma proteína acessória da maquinaria ESCRT. Já foi reportado que Nef interage com subunidades dos complexos AP-1, AP-2, AP-3 e Nef não parece interagir com subunidades de AP-4 e AP-5. Entretanto, o papel da interação de Nef com AP-1 e AP-3 na regulação negativa de CD4 ainda não está totalmente elucidado. Ademais, AP-1, AP-2 e AP-3 são potencialmente heterogêneos devido à existência de isoformas múltiplas das subunidades codificadas por diferentes genes. Todavia, existem poucos estudos para demonstrar se as diferentes combinações de isoformas dos APs são formadas e se possuem propriedades funcionais distintas. O presente trabalho procurou identificar e caracterizar fatores celulares envolvidos na regulação do tráfego intracelular de proteínas no processo de regulação negativa de CD4 induzido por Nef. Mais especificamente, este estudo buscou caracterizar a participação do complexo AP-1 na modulação negativa de CD4 por Nef de HIV-1, através do estudo funcional das duas isoformas de ?-adaptina, subunidades de AP-1. Utilizando a técnica de Pull-down demonstramos que Nef é capaz de interagir com ?2. Além disso, nossos dados de Imunoblot indicaram que a proteína ?2-adaptina, e não ?1-adaptina, é necessária no processo de degradação lisossomal de CD4 por Nef e que esta participação é conservada para degradação de CD4 por Nef de diferentes cepas virais. Ademais, por citometria de fluxo, o silenciamento de ?2, e não de ?1, compromete a diminuição dos níveis de CD4 por Nef da membrana plasmática. A análise por imunofluorêsncia indireta também revelou que a diminuição dos níveis de ?2 impede a redistribuição de CD4 por Nef para regiões perinucleares, acarretando no acúmulo de CD4, retirados por Nef da membrana plasmática, em endossomos primários. A depleção de ?1A, outra subunidade de AP-1, acarretou na diminuição dos níveis celulares de ?2 e ?1, bem como, no comprometimento da eficiente degradação de CD4 por Nef. Além disso, foi possível observar que, ao perturbar a maquinaria ESCRT via super-expressão de HRS (uma subunidade do complexo ESCRT-0), ocorreu um acumulo de ?2 em endossomos dilatados contendo HRS-GFP, nos quais também detectou-se CD4 que foi internalizado por Nef. Em conjunto, os resultados indicam que ?2-adaptina é uma importante molécula para o direcionamento de CD4 por Nef para a via ESCRT/MVB, mostrando ser uma proteína relevante no sistema endo-lisossomal. Ademais, os resultados indicaram que as isoformas ?-adaptinas não só possuem funções distintas, mas também parecem compor complexos AP-1 com diferentes funções celulares, já que apenas a variante AP-1 contendo ?2, mas não ?1, participa da regulação negativa de CD4 por Nef. Estes estudos contribuem para o melhor entendimento dos mecanismos moleculares envolvidos na atividade de Nef, que poderão também ajudar na melhor compreensão da patogênese do HIV e da síndrome relacionada. Em adição, este trabalho contribui para o entendimento de processos fundamentais da regulação do tráfego de proteínas transmembrana no sistema endo-lisossomal.
The Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). AIDS is a disease which has a global distribution, and it is estimated that there are currently at least 36.9 million people infected with the virus. During the replication cycle, HIV promotes several changes in the physiology of the host cell to promote their survival and enhance replication. The fast progression of HIV-1 in humans and animal models is closely linked to the function of an accessory protein Nef. Among several actions of Nef, one is the most important is the down-regulation of proteins from the immune response, such as the CD4 receptor. It is known that this action causes CD4 degradation in lysosome, but the molecular mechanisms are still incompletely understood. Nef forms a tripartite complex with the cytosolic tail of the CD4 and adapter protein 2 (AP-2) in clathrin-coated vesicles, inducing CD4 internalization and lysosome degradation. Previous research has demonstrated that CD4 target to lysosomes by Nef involves targeting of this receptor to multivesicular bodies (MVBs) pathway by an atypical mechanism because, although not need charging ubiquitination, depends on the proteins from ESCRTs (Endosomal Sorting Complexes Required for Transport) machinery and the action of Alix, an accessory protein ESCRT machinery. It has been reported that Nef interacts with subunits of AP- 1, AP-2, AP-3 complexes and Nef does not appear to interact with AP-4 and AP-5 subunits. However, the role of Nef interaction with AP-1 or AP-3 in CD4 down-regulation is poorly understood. Furthermore, AP-1, AP-2 and AP-3 are potentially heterogeneous due to the existence of multiple subunits isoforms encoded by different genes. However, there are few studies to demonstrate if the different combinations of APs isoforms are form and if they have distinct functional properties. This study aim to identify and characterize cellular factors involved on CD4 down-modulation induced by Nef from HIV-1. More specifically, this study aimed to characterize the involvement of AP-1 complex in the down-regulation of CD4 by Nef HIV-1 through the functional study of the two isoforms of ?-adaptins, AP-1 subunits. By pull-down technique, we showed that Nef is able to interact with ?2. In addition, our data from immunoblots indicated that ?2- adaptin, not ?1-adaptin, is required in Nef-mediated targeting of CD4 to lysosomes and the ?2 participation in this process is conserved by Nef from different viral strains. Furthermore, by flow cytometry assay, ?2 depletion, but not ?1 depletion, compromises the reduction of surface CD4 levels induced by Nef. Immunofluorescence microscopy analysis also revealed that ?2 depletion impairs the redistribution of CD4 by Nef to juxtanuclear region, resulting in CD4 accumulation in primary endosomes. Knockdown of ?1A, another subunit of AP-1, resulted in decreased cellular levels of ?1 and ?2 and, compromising the efficient CD4 degradation by Nef. Moreover, upon artificially stabilizing ESCRT-I in early endosomes, via overexpression of HRS, internalized CD4 accumulates in enlarged HRS-GFP positive endosomes, where co-localize with ?2. Together, the results indicate that ?2-adaptin is a molecule that is essential for CD4 targeting by Nef to ESCRT/MVB pathway, being an important protein in the endo-lysosomal system. Furthermore, the results indicate that ?-adaptins isoforms not only have different functions, but also seem to compose AP-1 complex with distinct cell functions, and only the AP-1 variant comprising ?2, but not ?1, acts in the CD4 down-regulation induced by Nef. These studies contribute to a better understanding on the molecular mechanisms involved in Nef activities, which may also help to improve the understanding of the HIV pathogenesis and the related syndrome. In addition, this work contributes with the understanding of primordial process regulation on intracellular trafficking of transmembrane proteins.

碩士
國立清華大學
生物資訊與結構生物研究所
106
ABSTRACT Tripartite split-GFP complementation assay is a new protein-protein interaction technique improved from bimolecular fluorescence complementation (BiFC). Tripartite split GFP system is composed of a larger fragment, GFP1-9 (residues 1–193), and two shorter β-strands : GFP10 (residues 194–212) and GFP11 (residues 213–233). There are some disadvantages of BiFC because BiFC is based on bulky fragments that may increase the difficulty of protein folding and interfere with protein function. To test whether tripartite split-GFP is a useful tool in planta, we first chose two proteins involved in the phosphate starvation reponse: the phosphate starvation response 1 (PHR1) and the SPX domain-containing protein 1 (SPX1) proteins. The PHR1 and SPX1 sandwitch proteins (GFP10-PHR1-GFP11 and GFP10-SPX1-GFP11) were used for transient expression in tobacco leaves and localized in the nucleus. Next, we confirmed the previous results that PHR1 interacts with SPX1 using tripartite split-GFP system. This assay showed that PHR1 and SPX1 can form homo-dimer/oligomers itself. We used the Arabidopsis nitrogen limitation adaptation (NLA/BAH1) to verify the specificity of the interaction between PHR1 and SPX1. Our results showed that NLA interacts with both PHR1 and SPX1. In addition, we chose the other PHR1 family protein PHR1-like 3 (PHL3) to test the interaction of PHL3 with PHR1 and SPX1 respectively. The signal strength between PHR1, SPX1 and PHL3 are different. From the strongest to the weakest is PHR1 and PHL3, PHR1 dimer, SPX1 and PHR1, and then the signal of SPX1 and PHL3 was the strongest. Furthermore, we generated and obtained the Arabidopsis transgenic liens overexpessing GFP10-PHR1, SPX1-GFP11 and GFP1–9. Confonal analysis of these lines showed few signals in the root hair of seedlings, indicating that the tripartite spli-GFP system used in transgenic plants of Arabidopsis needs to be improved.

Understanding the risk perception is essential to explaining people’s judgment and decisions during drug safety crises. In addition to affective and cognitive components, the experiential facet of risk perception captures “gut-level” reactions in heuristic-based risk judgments. However, few empirical studies have explicated the validity of the tripartite approach to analyzing risk perception or examined whether experiential risk perception is a conceptually sound construct distinct from the well-established dual-factor model. Building upon the tripartite model of risk perception, this study acknowledges the current research gap and compares three fundamental components of risk perception as well as their relative capabilities to predict individuals’ behavioral intention. Results of an online survey conducted shortly after a substandard vaccine crisis in China empirically support the discriminant validity of the tripartite model, which exhibits significantly better model fit than either single-factor or dual-factor models. A pretest-posttest analysis has further identified a highly controversial gap between experiential and affective risk perceptions: instructional risk message stimuli have provoked a significant change in participants’ experiential risk perception but not in the other two components. Moreover, three dimensions of risk perception reveal different patterns of association with behavioral intention. Implications for risk and crisis management are further discussed.

Abstract During 1961-63, ICT was involved in an almost constant round of negotiations with virtually every major computer company in America and Britain, as well as Machines Bull in France. The threads of this tapestry of negotiation are very difficult to untangle. For example, ICT and Machines Bull were both independently talking to RCA and General Electric in the United States, at the same time as they were talking to each other; and simultaneously ICT, English Electric, and RCA were considering some tripartite agreement. There were thus at least six separate, but interlocking, sets of negotiations involving just these companies. The course of these negotiations becomes much clearer when viewed in the context of ICT’s objective in seeking liaisons with other companies. ICT’s objective was to strengthen its position in computers in two ways. First, it needed to close the technology gap between its own computers and those available in the United States-this implied a close relationship with an American company. Second, it needed to increase its electronic production facilities-this implied either merging with, or acquiring, another British computer company.

Banco de la República is celebrating its 100th anniversary in 2023. This is a very significant anniversary and one that provides an opportunity to highlight the contribution the Bank has made to the country’s development. Its track record as guarantor of monetary stability has established it as the one independent state institution that generates the greatest confidence among Colombians due to its transparency, management capabilities, and effective compliance with the central banking and cultural responsibilities entrusted to it by the Constitution and the Law. On a date as important as this, the Board of Directors of Banco de la República (BDBR) pays tribute to the generations of governors and officers whose commitment and dedication have contributed to the growth of this institution.1 Banco de la República’s mandate was confirmed in the National Constitutional Assembly of 1991 where the citizens had the opportunity to elect the seventy people who would have the task of drafting a new constitution. The leaders of the three political movements with the most votes were elected as chairs to the Assembly, and this tripartite presidency reflected the plurality and the need for consensus among the different political groups to move the reform forward. Among the issues considered, the National Constitutional Assembly gave special importance to monetary stability. That is why they decided to include central banking and to provide Banco de la República with the necessary autonomy to use the instruments for which they are responsible without interference from other authorities. The constituent members understood that ensuring price stability is a state duty and that the entity responsible for this task must be enshrined in the Constitution and have the technical capability and institutional autonomy necessary to adopt the decisions they deem appropriate to achieve this fundamental objective in coordination with the general economic policy. In particular, Article 373 established that “the State, through Banco de la República, shall ensure the maintenance of the purchasing power of the currency,” a provision that coincided with the central banking system adopted by countries that have been successful in controlling inflation. In 1999, in Ruling 481, the Constitutional Court stated that “the duty to maintain the purchasing power of the currency applies to not only the monetary, credit, and exchange authority, i.e., the Board of Banco de la República, but also those who have responsibilities in the formulation and implementation of the general economic policy of the country” and that “the basic constitutional purpose of Banco de la República is the protection of a sound currency. However, this authority must take the other economic objectives of state intervention such as full employment into consideration in their decisions since these functions must be coordinated with the general economic policy.” The reforms to Banco de la República agreed upon in the Constitutional Assembly of 1991 and in Act 31/1992 can be summarized in the following aspects: i) the Bank was assigned a specific mandate: to maintain the purchasing power of the currency in coordination with the general economic policy; ii) the BDBR was designatedas the monetary, foreign exchange, and credit authority; iii) the Bank and its Board of Directors were granted a significant degree of independence from the government; iv) the Bank was prohibited from granting credit to the private sector except in the case of the financial sector; v) established that in order to grant credit to the government, the unanimous vote of its Board of Directors was required except in the case of open market transactions; vi) determined that the legislature may, in no case, order credit quotas in favor of the State or individuals; vii) Congress was appointed, on behalf of society, as the main addressee of the Bank’s reporting exercise; and viii) the responsibility for inspection, surveillance, and control over Banco de la República was delegated to the President of the Republic. The members of the National Constitutional Assembly clearly understood that the benefits of low and stable inflation extend to the whole of society and contribute mto the smooth functioning of the economic system. Among the most important of these is that low inflation promotes the efficient use of productive resources by allowing relative prices to better guide the allocation of resources since this promotes economic growth and increases the welfare of the population. Likewise, low inflation reduces uncertainty about the expected return on investment and future asset prices. This increases the confidence of economic agents, facilitates long-term financing, and stimulates investment. Since the low-income population is unable to protect itself from inflation by diversifying its assets, and a high proportion of its income is concentrated in the purchase of food and other basic goods that are generally the most affected by inflationary shocks, low inflation avoids arbitrary redistribution of income and wealth.2 Moreover, low inflation facilitates wage negotiations, creates a good labor climate, and reduces the volatility of employment levels. Finally, low inflation helps to make the tax system more transparent and equitable by avoiding the distortions that inflation introduces into the value of assets and income that make up the tax base. From the monetary authority’s point of view, one of the most relevant benefits of low inflation is the credibility that economic agents acquire in inflation targeting, which turns it into an effective nominal anchor on price levels. Upon receiving its mandate, and using its autonomy, Banco de la República began to announce specific annual inflation targets as of 1992. Although the proposed inflation targets were not met precisely during this first stage, a downward trend in inflation was achieved that took it from 32.4% in 1990 to 16.7% in 1998. At that time, the exchange rate was kept within a band. This limited the effectiveness of monetary policy, which simultaneously sought to meet an inflation target and an exchange rate target. The Asian crisis spread to emerging economies and significantly affected the Colombian economy. The exchange rate came under strong pressure to depreciate as access to foreign financing was cut off under conditions of a high foreign imbalance. This, together with the lack of exchange rate flexibility, prevented a countercyclical monetary policy and led to a 4.2% contraction in GDP that year. In this context of economic slowdown, annual inflation fell to 9.2% at the end of 1999, thus falling below the 15% target set for that year. This episode fully revealed how costly it could be, in terms of economic activity, to have inflation and exchange rate targets simultaneously. Towards the end of 1999, Banco de la República announced the adoption of a new monetary policy regime called the Inflation Targeting Plan. This regime, known internationally as ‘Inflation Targeting,’ has been gaining increasing acceptance in developed countries, having been adopted in 1991 by New Zealand, Canada, and England, among others, and has achieved significant advances in the management of inflation without incurring costs in terms of economic activity. In Latin America, Brazil and Chile also adopted it in 1999. In the case of Colombia, the last remaining requirement to be fulfilled in order to adopt said policy was exchange rate flexibility. This was realized around September 1999, when the BDBR decided to abandon the exchange-rate bands to allow the exchange rate to be freely determined in the market.Consistent with the constitutional mandate, the fundamental objective of this new policy approach was “the achievement of an inflation target that contributes to maintaining output growth around its potential.”3 This potential capacity was understood as the GDP growth that the economy can obtain if it fully utilizes its productive resources. To meet this objective, monetary policy must of necessity play a countercyclical role in the economy. This is because when economic activity is below its potential and there are idle resources, the monetary authority can reduce the interest rate in the absence of inflationary pressure to stimulate the economy and, when output exceeds its potential capacity, raise it. This policy principle, which is immersed in the models for guiding the monetary policy stance, makes the following two objectives fully compatible in the medium term: meeting the inflation target and achieving a level of economic activity that is consistent with its productive capacity. To achieve this purpose, the inflation targeting system uses the money market interest rate (at which the central bank supplies primary liquidity to commercial banks) as the primary policy instrument. This replaced the quantity of money as an intermediate monetary policy target that Banco de la República, like several other central banks, had used for a long time. In the case of Colombia, the objective of the new monetary policy approach implied, in practical terms, that the recovery of the economy after the 1999 contraction should be achieved while complying with the decreasing inflation targets established by the BDBR. The accomplishment of this purpose was remarkable. In the first half of the first decade of the 2000s, economic activity recovered significantly and reached a growth rate of 6.8% in 2006. Meanwhile, inflation gradually declined in line with inflation targets. That was how the inflation rate went from 9.2% in 1999 to 4.5% in 2006, thus meeting the inflation target established for that year while GDP reached its potential level. After this balance was achieved in 2006, inflation rebounded to 5.7% in 2007, above the 4.0% target for that year due to the fact that the 7.5% GDP growth exceeded the potential capacity of the economy.4 After proving the effectiveness of the inflation targeting system in its first years of operation, this policy regime continued to consolidate as the BDBR and the technical staff gained experience in its management and state-of-the-art economic models were incorporated to diagnose the present and future state of the economy and to assess the persistence of inflation deviations and expectations with respect to the inflation target. Beginning in 2010, the BDBR established the long-term 3.0% annual inflation target, which remains in effect today. Lower inflation has contributed to making the macroeconomic environment more stable, and this has favored sustained economic growth, financial stability, capital market development, and the functioning of payment systems. As a result, reductions in the inflationary risk premia and lower TES and credit interest rates were achieved. At the same time, the duration of public domestic debt increased significantly going from 2.27 years in December 2002 to 5.86 years in December 2022, and financial deepening, measured as the level of the portfolio as a percentage of GDP, went from around 20% in the mid-1990s to values above 45% in recent years in a healthy context for credit institutions.Having been granted autonomy by the Constitution to fulfill the mandate of preserving the purchasing power of the currency, the tangible achievements made by Banco de la República in managing inflation together with the significant benefits derived from the process of bringing inflation to its long-term target, make the BDBR’s current challenge to return inflation to the 3.0% target even more demanding and pressing. As is well known, starting in 2021, and especially in 2022, inflation in Colombia once again became a serious economic problem with high welfare costs. The inflationary phenomenon has not been exclusive to Colombia and many other developed and emerging countries have seen their inflation rates move away from the targets proposed by their central banks.5 The reasons for this phenomenon have been analyzed in recent Reports to Congress, and this new edition delves deeper into the subject with updated information. The solid institutional and technical base that supports the inflation targeting approach under which the monetary policy strategy operates gives the BDBR the necessary elements to face this difficult challenge with confidence. In this regard, the BDBR reiterated its commitment to the 3.0% inflation target in its November 25 communiqué and expects it to be reached by the end of 2024.6 Monetary policy will continue to focus on meeting this objective while ensuring the sustainability of economic activity, as mandated by the Constitution. Analyst surveys done in March showed a significant increase (from 32.3% in January to 48.5% in March) in the percentage of responses placing inflation expectations two years or more ahead in a range between 3.0% and 4.0%. This is a clear indication of the recovery of credibility in the medium-term inflation target and is consistent with the BDBR’s announcement made in November 2022. The moderation of the upward trend in inflation seen in January, and especially in February, will help to reinforce this revision of inflation expectations and will help to meet the proposed targets. After reaching 5.6% at the end of 2021, inflation maintained an upward trend throughout 2022 due to inflationary pressures from both external sources, associated with the aftermath of the pandemic and the consequences of the war in Ukraine, and domestic sources, resulting from: strengthening of local demand; price indexation processes stimulated by the increase in inflation expectations; the impact on food production caused by the mid-2021 strike; and the pass-through of depreciation to prices. The 10% increase in the minimum wage in 2021 and the 16% increase in 2022, both of which exceeded the actual inflation and the increase in productivity, accentuated the indexation processes by establishing a high nominal adjustment benchmark. Thus, total inflation went to 13.1% by the end of 2022. The annual change in food prices, which went from 17.2% to 27.8% between those two years, was the most influential factor in the surge in the Consumer Price Index (CPI). Another segment that contributed significantly to price increases was regulated products, which saw the annual change go from 7.1% in December 2021 to 11.8% by the end of 2022. The measure of core inflation excluding food and regulated items, in turn, went from 2.5% to 9.5% between the end of 2021 and the end of 2022. The substantial increase in core inflation shows that inflationary pressure has spread to most of the items in the household basket, which is characteristic of inflationary processes with generalized price indexation as is the case in Colombia. Monetary policy began to react early to this inflationary pressure. Thus, starting with its September 2021 session, the BDBR began a progressive change in the monetary policy stance moving away from the historical low of a 1.75% policy rate that had intended to stimulate the recovery of the economy. This adjustment process continued without interruption throughout 2022 and into the beginning of 2023 when the monetary policy rate reached 12.75% last January, thus accumulating an increase of 11 percentage points (pp). The public and the markets have been surprised that inflation continued to rise despite significant interest rate increases. However, as the BDBR has explained in its various communiqués, monetary policy works with a lag. Just as in 2022 economic activity recovered to a level above the pre-pandemic level, driven, along with other factors, by the monetary stimulus granted during the pandemic period and subsequent months, so too the effects of the current restrictive monetary policy will gradually take effect. This will allow us to expect the inflation rate to converge to 3.0% by the end of 2024 as is the BDBR’s purpose.Inflation results for January and February of this year showed declining marginal increases (13 bp and 3 bp respectively) compared to the change seen in December (59 bp). This suggests that a turning point in the inflation trend is approaching. In other Latin American countries such as Chile, Brazil, Perú, and Mexico, inflation has peaked and has begun to decline slowly, albeit with some ups and downs. It is to be expected that a similar process will take place in Colombia in the coming months. The expected decline in inflation in 2023 will be due, along with other factors, to lower cost pressure from abroad as a result of the gradual normalization of supply chains, the overcoming of supply shocks caused by the weather, and road blockades in previous years. This will be reflected in lower adjustments in food prices, as has already been seen in the first two months of the year and, of course, the lagged effect of monetary policy. The process of inflation convergence to the target will be gradual and will extend beyond 2023. This process will be facilitated if devaluation pressure is reversed. To this end, it is essential to continue consolidating fiscal sustainability and avoid messages on different public policy fronts that generate uncertainty and distrust. 1 This Report to Congress includes Box 1, which summarizes the trajectory of Banco de la República over the past 100 years. In addition, under the Bank’s auspices, several books that delve into various aspects of the history of this institution have been published in recent years. See, for example: Historia del Banco de la República 1923-2015; Tres banqueros centrales; Junta Directiva del Banco de la República: grandes episodios en 30 años de historia; Banco de la República: 90 años de la banca central en Colombia. 2 This is why lower inflation has been reflected in a reduction of income inequality as measured by the Gini coefficient that went from 58.7 in 1998 to 51.3 in the year prior to the pandemic. 3 See Gómez Javier, Uribe José Darío, Vargas Hernando (2002). “The Implementation of Inflation Targeting in Colombia”. Borradores de Economía, No. 202, March, available at: https://repositorio.banrep.gov.co/handle/20.500.12134/5220 4 See López-Enciso Enrique A.; Vargas-Herrera Hernando and Rodríguez-Niño Norberto (2016). “The inflation targeting strategy in Colombia. An historical view.” Borradores de Economía, No. 952. https://repositorio.banrep.gov.co/handle/20.500.12134/6263 5 According to the IMF, the percentage change in consumer prices between 2021 and 2022 went from 3.1% to 7.3% for advanced economies, and from 5.9% to 9.9% for emerging market and developing economies. 6 https://www.banrep.gov.co/es/noticias/junta-directiva-banco-republica-reitera-meta-inflacion-3