1,720,957 research outputs found
Metamorphosis by ATG13 and ATG101 in human autophagy initiation
No full textMax Planck society 10.13039/501100004189DFG SFB119
Altered tRNA dynamics during translocation on slippery mRNA as determinant of spontaneous ribosome frameshifting
Full text linkWhen reading consecutive mRNA codons, ribosomes move by exactly one triplet at a time to synthesize a correct protein. Some mRNA tracks, called slippery sequences, are prone to ribosomal frameshifting, because the same tRNA can read both 0- and –1-frame codon. Using smFRET we show that during EF-G-catalyzed translocation on slippery sequences a fraction of ribosomes spontaneously switches from rapid, accurate translation to a slow, frameshifting-prone translocation mode where the movements of peptidyl- and deacylated tRNA become uncoupled. While deacylated tRNA translocates rapidly, pept-tRNA continues to fluctuate between chimeric and posttranslocation states, which slows down the re-locking of the small ribosomal subunit head domain. After rapid release of deacylated tRNA, pept-tRNA gains unconstrained access to the –1-frame triplet, resulting in slippage followed by recruitment of the –1-frame aa-tRNA into the A site. Our data show how altered choreography of tRNA and ribosome movements reduces the translation fidelity of ribosomes translocating in a slow mode
Rate-limiting steps of autophagy initiation
No full textDie Autophagieforschung hat seit der Identifizierung der für die Aufrechterhaltung des Prozesses verantwortlichen „ATG“-Gene und -Proteine enorme Fortschritte gemacht. Eine Analyse der allgemeinen Funktionen aller beteiligten Proteine war möglich. Die Regulierung der menschlichen Autophagie, insbesondere der Autophagie-Initiierung, bleibt jedoch unklar. Die Biogenese von Autophagosomen umfasst eine koordinierte Ansammlung vieler Proteine, die in Unterkomplexen organisiert sind, an einer Initiierungsstelle zwischen dem endoplasmatischen Retikulum (ER) und einer neu entstehenden Isolationsmembran (IM). Wie die Ansammlung molekular reguliert und koordiniert wird, ist noch nicht vollständig verstanden. Unsere vorherige Arbeit mit In-vitro-Rekonstitution und Reinigung vollständiger menschlicher Autophagie-Initiationsproteine in großem Maßstab war die erste, die eine spontane superkomplexe Ansammlung von Autophagie-Initiationsproteinen zeigte. Damit wurde die Notwendigkeit erkannt, die spontane Ansammlung nach Bedarf zu regulieren. Nur ein Kernkomplex von ATG9-13-101 ermöglichte die superkomplexe Ansammlung. Bei genauerer Betrachtung dieses Komplexes wurde ein seltenes, aufkommendes Konzept der Proteinmetamorphose identifiziert, das die Interaktionen von ATG13 und ATG101 mit ATG9 steuert. Dies führte zu der Hypothese, dass der kinetische Flaschenhals, der für die Hemmung oder Hochregulierung der Superkomplex-Assemblierung nach Bedarf verantwortlich ist, in diesen Interaktionen liegen könnte. ATG13 und ATG101 sind metamorphe Proteine der HORMA-Domänenfamilie. Metamorphe Proteine haben sich so entwickelt, dass sie zwei Konformere mit völlig unterschiedlichen Strukturfaltungen desselben Proteins aufweisen. Der Wechsel zwischen der Standardfaltung und der zweiten, ausgelösten Faltung ist oft geschwindigkeitsbegrenzend, da der Wechsel spontan und sehr langsam erfolgen kann. Ziel dieser Arbeit ist es, den geschwindigkeitsbegrenzenden Schritt der ATG9-13-101-Komplex-Assemblierung zu isolieren. Zu diesem Zweck wurde ein kinetischer Test auf Basis der Fluoreszenzpolarisation entwickelt und Interaktionen quantifiziert. Bei der Assoziation der beiden metamorphen Proteine ATG13 und ATG101 wurde eine unerwartete geschwindigkeitsbegrenzende Interaktion identifiziert. Eine detaillierte Charakterisierung von ATG101-Mutanten zur Identifizierung alternativer Konformere führte zur Untersuchung der ATG101-Homodimerisierung. Schließlich wird ein Modell zur Beschleunigung der obligatorischen, geschwindigkeitsbegrenzenden ATG13-ATG101-Assoziation diskutiert. Schließlich beinhalten Stoffwechselwege, die metamorphe Proteine enthalten, aufgrund der langsamen Faltungsumwandlungen häufig eine aktive Katalyse. Die Arbeit in dieser Dissertation, die eine detaillierte Charakterisierung aller Wechselwirkungen innerhalb dieses Komplexes liefert, bietet eine solide Grundlage für zukünftige Ziele zur Identifizierung von Katalysatoren der Autophagie-Initiierung.Autophagy research has progressed tremendously since the identification of ‘ATG’ genes and proteins responsible for maintaining the process. A dissection of general functions of all proteins involved was possible. However, the regulation of human autophagy, particularly autophagy initiation remains unclear. Autophagosome biogenesis involves a coordinated assembly of many proteins organized in subcomplexes, at an initiation site between the endoplasmic reticulum (ER) and a newly forming Isolation Membrane (IM). How the assembly is regulated and coordinated molecularly is poorly understood. Our previous work using in vitro reconstitution purifying full-length human autophagy initiation proteins on a large scale was the first to show a spontaneous super-complex assembly of autophagy initiation proteins. With this, a need to regulate spontaneous assembly in an on-demand manner was identified. Only a core complex of ATG9-13-101 enabled super-complex assembly. A closer look within this complex identified a rare, emerging concept of protein metamorphosis that governed interactions of ATG13 and ATG101 to ATG9. It led to a hypothesis that the kinetic bottleneck responsible for inhibiting or upregulating super-complex assembly on-demand could be within these interactions. ATG13 and ATG101 are metamorphic proteins of the HORMA domain family. Metamorphic proteins have evolved to exhibit two conformers with entirely different structural folds of the same protein Switching between the default fold and the second, triggered fold is often rate-limiting, because the switch can happen spontaneously at a very slow rate. This thesis aims to isolate the rate-limiting step of ATG9-13-101 complex assembly. For this, a kinetic assay based on fluorescence polarization was developed and interactions quantified. An unexpected rate-limiting interaction was identified in the association of the two metamorphic proteins ATG13 and ATG101. A detailed characterization of ATG101 mutants in order to identify alternate conformers led to investigation of ATG101 homodimerization. Eventually, a model of accelerating the obligatory, rate-limiting ATG13-ATG101 association is discussed. Finally, pathways which harbour metamorphic proteins often involve active catalysis because of the slow fold conversions. Work in this thesis, providing a detailed characterization of all interactions within this complex, provides a strong platform for future goals aiming to identify catalysts of autophagy initiation.2025-08-1
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe 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
Full text link“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
Full text linkWe 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
Full text linkWe 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
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