1,720,957 research outputs found
AMP-activated Protein Kinase (AMPK): New molecular insights and novel downstream targets
Get PDFAMP-activated Protein Kinase (AMPK) is a central regulator of energy homeostasis and a promising drug target for metabolic disorders. It exists as complexes of three subunits, a catalytic alpha, and two regulatory beta and gamma subunits. The regulation of AMPK involves reversible phosphorylation and allosteric regulation by adenine nucleotides. It is activated by phosphorylation of Thr172 on the catalytic alpha subunit as a consequence of various energy-depleting conditions. Once activated, AMPK regulates a plethora of metabolic processes through the phosphorylation of target proteins to maintain energy homeostasis. The beta subunit has a vital role as a structural scaffold stabilising the AMPK heterotrimeric complex. It is also known to regulate AMPK activity through different posttranslational modifications (i.e., phosphorylation and myristoylation). Although myristoylation of Glycine-2 (Gly2) of the beta subunit has been shown to be required for sensing stress signals and achieving maximum AMPK activity in vitro, its physiological relevance at the cellular and organismal levels remains unknown. Critically, the underlying molecular mechanism by which beta subunit myristoylation controls AMPK activity is elusive.
The primary aim of this thesis was to investigate the molecular basis of AMPK regulation by the beta subunit myristoyl switch. I showed that mouse embryonic fibroblasts (MEFs) isolated from knock-in (KI) mice carrying Gly2 to Ala point mutation of beta1 and beta2 isoforms (beta1/2 G2A double knock-in (DKI)) displayed increased activity and phosphorylation of Thr172 in the beta subunit. Using proximity ligation assay, I found that the loss of beta1 myristoylation impedes the interaction/proximity of the phosphatases PPM1A/B with AMPK in cells. In vivo, beta1 G2A KI mice showed increased AMPK activity in the liver and were protected from high-fat diet-induced obesity, hepatic lipid accumulation, and insulin resistance.
The second aim of the thesis focused on the identification of novel AMPK substrates to expand our understanding of the AMPK system/signalling in the control of metabolic and also in non-metabolic processes. We performed an unbiased phosphoproteomics analysis which revealed that AMPK phosphorylates several proteins involved in regulating Golgi structure and function. I observed that pharmacological activation of AMPK induces Golgi fragmentation in wild-type but not in AMPK-deficient human U2OS cells and MEFs. We identified AMPK-dependent phosphorylation of three Golgi-related proteins and focused on the Oxysterol-binding protein like 9 (OSBPL9), a novel AMPK substrate phosphorylated on a threonine residue (Thr335). Interestingly, knockdown of OSBPL9 in cells induced Golgi fragmentation, linking the AMPK-OSBPL9 pathway to Golgi regulation.
Collectively, this study expands our understanding of the regulation and novel biological roles of AMPK. This will advance future studies to elucidate the significance of AMPK in the treatment of metabolic as well as non-metabolic disorders.SSV-EN
Targeted protein degradation through cytosolic delivery of monobody binders using bacterial toxins
Get PDFMonobodies are small engineered binding proteins that, upon expression in cells, can inhibit signaling of cytosolic oncoproteins with outstanding selectivity. Efficacy may be further increased by inducing degradation of monobody targets through fusion to the von Hippel-Lindau (VHL) substrate receptor of the Cullin2-E3 ubiquitin ligase complex. However, potential therapeutic use is currently limited, because of the inability of monobody proteins to cross cellular membranes. Here, we use a chimeric bacterial toxin, composed of the Shiga-like toxin B (Stx2B) subunit and the translocation domain of Pseudomonas aeruginosa exotoxin A (ETA-II) for delivery of VHL-monobody protein fusions to target endogenous tyrosine kinases in cancer cells. Depending on the expression of the Stx2B receptor Gb3 on the cell surface, we show that monobodies are taken up by an endocytic route, but are not degraded in lysosomes. Delivery of monobodies fused to a nuclear localization signal resulted in accumulation in the nucleus, thereby indirectly, but unequivocally, demonstrating cytosolic delivery. Delivery of VHL fused to monobodies targeting the Lck tyrosine kinase in T-cells resulted in reduced Lck protein levels, which was dependent on the expression of Gb3. This led to the inhibition of proximal signaling events downstream of the T-cell receptor complex. This work provides a prime example of the delivery of a stoichiometric protein inhibitor of an endogenous target protein to cells and inducing its degradation without the need of genetic manipulation of target cells. It lays the foundation for further in vivo exploitation of this delivery system.UPHANEpub ahead of print
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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