SNX17 protects integrins from degradation by sorting between lysosomal and recycling pathways.

The FERM-like domain-containing sorting nexins of the SNX17/SNX27/SNX31 family have been proposed to mediate retrieval of transmembrane proteins from the lysosomal pathway. In this paper, we describe a stable isotope labeling with amino acids in culture-based quantitative proteomic approach that allows an unbiased, global identification of transmembrane cargoes that are rescued from lysosomal degradation by SNX17. This screen revealed that several integrins required SNX17 for their stability, as depletion of SNX17 led to a loss of β1 and β5 integrins and associated a subunits from HeLa cells as a result of increased lysosomal degradation. SNX17 bound to the membrane distal NPXY motif in β integrin cytoplasmic tails, thereby preventing lysosomal degradation of β integrins and their associated a subunits. Furthermore, SNX17-dependent retrieval of integrins did not depend on the retromer complex. Consistent with an effect on integrin recycling, depletion of SNX17 also caused alterations in cell migration. Our data provide mechanistic insight into the retrieval of internalized integrins from the lysosomal degradation pathway, a prerequisite for subsequent recycling of these matrix receptors

Atypical parkinsonism-associated retromer mutant alters endosomal sorting of specific cargo proteins

The retromer complex acts as a scaffold for endosomal protein complexes that sort integral membrane proteins to various cellular destinations. The retromer complex is a heterotrimer of VPS29, VPS35, and VPS26. Two of these paralogues, VPS26A and VPS26B, are expressed in humans. Retromer dysfunction is associated with neurodegenerative disease, and recently, three VPS26A mutations (p.K93E, p.M112V, and p.K297X) were discovered to be associated with atypical parkinsonism. Here, we apply quantitative proteomics to provide a detailed description of the retromer interactome. By establishing a comparative proteomic methodology, we identify how this interactome is perturbed in atypical parkinsonism-associated VPS26A mutants. In particular, we describe a selective defect in the association of VPS26A (p.K297X) with the SNX27 cargo adaptor. By showing how a retromer mutant leads to altered endosomal sorting of specific PDZ ligand–containing cargo proteins, we reveal a new mechanism for perturbed endosomal cargo sorting in atypical parkinsonism

Identification of a Novel CYP11B2 Variant in a Family with Varying Degrees of Aldosterone Synthase Deficiency

Isolated aldosterone synthase deficiency is a rare autosomal recessive disorder caused by pathogenic variants in CYP11B2, resulting in impaired aldosterone synthesis. We report on a neonate with isolated aldosterone synthase deficiency caused by a novel homozygous CYP11B2 variant Chr8: NM_000498.3: c.400G>A p.(Gly134Arg). The patient presented shortly after birth with severe signs of aldosterone deficiency. Interestingly, segregation analysis revealed that the patient's asymptomatic father was also homozygous for the CYP11B2 variant. Biochemical evaluation of the father indicated subclinical enzyme impairment, characterized by elevated aldosterone precursors. Apparently, this homozygous variant led to different clinical phenotypes in two affected relatives. In this manuscript we elaborate on the biochemical and genetic work-up performed and describe potential pitfalls in CYP11B2 sequencing due to its homology to CYP11B1

Racial differences in systemic sclerosis disease presentation: a European Scleroderma Trials and Research group study

Objectives. Racial factors play a significant role in SSc. We evaluated differences in SSc presentations between white patients (WP), Asian patients (AP) and black patients (BP) and analysed the effects of geographical locations.Methods. SSc characteristics of patients from the EUSTAR cohort were cross-sectionally compared across racial groups using survival and multiple logistic regression analyses.Results. The study included 9162 WP, 341 AP and 181 BP. AP developed the first non-RP feature faster than WP but slower than BP. AP were less frequently anti-centromere (ACA; odds ratio (OR) = 0.4, P < 0.001) and more frequently anti-topoisomerase-I autoantibodies (ATA) positive (OR = 1.2, P = 0.068), while BP were less likely to be ACA and ATA positive than were WP [OR(ACA) = 0.3, P < 0.001; OR(ATA) = 0.5, P = 0.020]. AP had less often (OR = 0.7, P = 0.06) and BP more often (OR = 2.7, P < 0.001) diffuse skin involvement than had WP.AP and BP were more likely to have pulmonary hypertension [OR(AP) = 2.6, P < 0.001; OR(BP) = 2.7, P = 0.03 vs WP] and a reduced forced vital capacity [OR(AP) = 2.5, P < 0.001; OR(BP) = 2.4, P < 0.004] than were WP. AP more often had an impaired diffusing capacity of the lung than had BP and WP [OR(AP vs BP) = 1.9, P = 0.038; OR(AP vs WP) = 2.4, P < 0.001]. After RP onset, AP and BP had a higher hazard to die than had WP [hazard ratio (HR) (AP) = 1.6, P = 0.011; HR(BP) = 2.1, P < 0.001].Conclusion. Compared with WP, and mostly independent of geographical location, AP have a faster and earlier disease onset with high prevalences of ATA, pulmonary hypertension and forced vital capacity impairment and higher mortality. BP had the fastest disease onset, a high prevalence of diffuse skin involvement and nominally the highest mortality

Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling

Following endocytosis into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are retrieved and recycled back to the cell surface. Here we describe the discovery of an ancient and conserved multiprotein complex that orchestrates cargo retrieval and recycling and, importantly, is biochemically and functionally distinct from the established retromer pathway. We have called this complex 'retriever'; it is a heterotrimer composed of DSCR3, C16orf62 and VPS29, and bears striking similarity to retromer. We establish that retriever associates with the cargo adaptor sorting nexin 17 (SNX17) and couples to CCC (CCDC93, CCDC22, COMMD) and WASH complexes to prevent lysosomal degradation and promote cell surface recycling of α5β1 integrin. Through quantitative proteomic analysis, we identify over 120 cell surface proteins, including numerous integrins, signalling receptors and solute transporters, that require SNX17-retriever to maintain their surface levels. Our\ua0identification of retriever establishes a major endosomal retrieval and recycling pathway

Scanning tunneling microscopy and spectroscopy study of carbon nanotubes and epitaxial graphene

Die Dissertation beschäftigt sich mit zwei sp2 hybridisierten Kohlenstoffsystemen, nämlich funktionalisierten Kohlenstoffnanoröhren sowie epitaktischem Graphen. Trotz der großen Ähnlichkeit ergeben sich grundverschiedene experimentelle Herausforderungen bei der Untersuchung mittels Rastertunnelmikroskopie (STM). Epitaktisches Graphen liegt als wohldefinierte, makroskopisch homogene Probe vor, die sich ideal für STM eignet. Kohlenstoffnanoröhren hingegen liegen als Feststoff vor, der zunächst mit Lösungsmitteln dispergiert wird. Anschließend muss die Suspension zu einer für STM zugänglichen dünnen Schicht verarbeitet werden. Der beste Weg zum Erreichen einer reproduzierbaren Bedeckung mit Nanoröhren besteht aus einem Trockentransfer-Verfahren. Zu diesem Zweck wird die Nanoröhren-Suspension durch eine mikroporöse Membran gefiltert. Das Filtrat, das sogenannte bucky paper, wird dann unter UHV Bedingungen in Kontakt mit der Au(111) Oberfläche gebracht. Sachtes Bewegen schält einzelne Nanoröhren aus dem Verband heraus auf die Substratoberfläche. Die Löslichkeit der Nanoröhren kann durch chemische Funktionalisierung verbessert werden. Aromatische Moleküle (z.B. 9-Amino-Anthrazen) wurden vom kooperierenden Lehrstuhl für Organische Chemie (Prof. Dr. A. Hirsch) nass-chemisch mittels pi-stacking Funktionalisierung an die Nanoröhren angeheftet. Die Dekorierung der Seitenwand der Nanoröhre mit Funktionalmolekülen konnte im STM lokal gezeigt werden. Weitere beeindruckende Auflösung der Funktionalmoleküle wurde mittels Spektroskopiebildern erzielt. Während der Trockentransfer-Präparation unterliegen die Nanoröhren einer gewissen Defunktionalisierung. Eine potentielle Anwendung der pi-stacking Funktionalisierung wäre daher die Präparation großer und thermisch instabiler organischer Moleküle. Die eindimensionale Struktur der Nanoröhre kann dabei als Templat fungieren und führt unter bestimmten Voraussetzungen zur Erzeugung von molekularen "Nanodrähten". Zur Unterstützung der Identifizierung von Funktionalmolekülen wurde 9-Amino-Anthrazen auf Au(111) sublimiert. Bevorzugte Dekorierung der Stufenkanten und der fcc-Bereiche der rekonstruierten Au(111) Oberfläche wurde beobachtet. Unter dem Aspekt der Identifizierung geeigneter sind STM Aufnahmen einzelner Moleküle. Bei positiven Tunnelspannungen, bei denen unbesetzte elektronische Zustände zum Tunnelstrom beitragen, ähnelt die Abbildung stark der berechneten Ladungsdichte des ersten unbesetzten Zustands (LUMO) des freien Moleküls. Aufnahmen, die besetzten Zuständen entsprechen, ähneln jedoch nicht dem höchsten besetzten Zustand (HOMO), sondern aus bisher unbekannten Gründen dem HOMO-16. Das zweite untersuchte Kohlenstoff-basierte System ist epitaktisches Graphen auf Siliziumkarbid. Eine wichtige Frage auf dem Weg zu Graphen-basierten Bauelementen betrifft die Einstellung der Ladung der Graphenschicht. Ladungstransfer aufgrund der Adsorption des organischen Moleküls PTCDA wurde untersucht, was in gewisser Analogie zur pi-stacking Funktionalisierung der Nanoröhren gesehen werden kann. Zuerst war es jedoch notwendig, die intrinsischen elektronischen Eigenschaften des Graphens verschiedener Lagenanzahlen mit Rastertunnelspektroskopie (STS) zu charakterisieren. Ein ausgeprägtes Minimum in der elektronischen Zustandsdichte konnte dabei mit dem Dirac-Punkt identifiziert werden. Bei höheren Tunnelspannungen konnten Defekte erzeugt und manipuliert sowie Stark-verschobene Bildpotential-Zustände nachgewiesen werden. Die Adsorptionsgeometrie der PTCDA Moleküle auf epitaktischem Graphen unterscheidet sich bemerkenswert von der auf Graphit. Obwohl die kristallographische Struktur des Graphen der des Graphit sehr stark ähnelt, wurde nicht die bekannte Herringbone-Struktur planar adsorbierter PTCDA Moleküle beobachtet. Vielmehr kann auf eine nicht-planare Adsorptionsgeometrie geschlossen werden. Anhand der mit ortsaufgelöster STS beobachten Verschiebung des Dirac-Minimums konnte eine zusätzliche Dotierung der Graphenschicht mit Elektronen nachgewiesen werden. Auch die PTCDA Schicht selbst wurde mit STS untersucht. An zwei verschiedenen PTCDA Konfigurationen wurden zwei verschiedene Klassen von Tunnelspektren beobachtet, die beide jeweils gut mit Messungen aus der Literatur übereinstimmen. Während dieser STS Experimente wurden Veränderungen in der Anordnung der PTCDA Moleküle beobachtet, die der schwachen, nicht-planaren Adsorption der PTCDA Moleküle auf der Graphenoberfläche zugeschrieben werden.This dissertation deals with two sp2 hybridized carbon-based systems, namely functionalized carbon nanotubes and epitaxial graphene. Despite their similarity, investigation via scanning tunneling microscopy (STM) is subject to entirely different experimental challenges. Epitaxial graphene is available as well-defined macroscopically homogeneous samples which are extremely well suited for STM. On the contrary carbon nanotubes are available initially in the form of powder and subsequently dispersed with solvents. Hence, these suspensions have to be processed onto a substrate to form a thin layer suitable for STM. The best way to achieve reproducible coverage of nanotubes consists of a dry transfer process. For this purpose the nanotube suspension is filtrated through a microporous membrane. The obtained bucky paper then is brought into contact with a Au(111) surface under UHV conditions. Gentle motion peels individual nanotubes onto the substrate surface. Solubility of carbon nanotubes can be enhanced by chemical functionalization. Aromatic molecules (e.g. 9-amino-anthracene) were attached wet-chemically via pi-stacking functionalization by coworkers from the Chair of Organic Chemistry (Prof. A. Hirsch). These moieties could be detected locally on the nanotube sidewall with STM. Further impressive resolution of the functional molecules was obtained with spectroscopy images. Upon the dry transfer process, the nanotubes are subject to certain defunctionalization. This leads to one possible application of pi-stacking functionalization in preparation of large and thermally instable aromatic molecules via carbon nanotubes onto surfaces. The one-dimensional structure can act as a template and leads to the generation of molecular "nanowires" on certain conditions. To maintain the identification of functional molecules, 9-amino-anthracene was evaporated onto Au(111). Preferential decoration of step edges and fcc sites of the reconstructed Au(111) surface was observed. More suitable for the purpose of identification are STM pictures of individual molecules. Images taken with bias voltages corresponding to unoccupied sample states reveal strong resemblance with the calculated charge density of the lowest unoccupied molecular orbital (LUMO) of the free molecule. Images obtained by tunneling out of occupied sample states do not resemble the highest occupied molecular orbital (HOMO) but the HOMO-16 for reasons that are not clear at present. The second carbon-based system studied here is epitaxial graphene on silicon carbide. An important question on the way to graphene-based future electronic components concerns the adjustment of the graphene sheet charge. Charge transfer upon adsorption of the organic molecule PTCDA was investigated which can be seen as a certain analog to pi-stacking functionalization of carbon nanotubes. First of all, it was necessary to analyse the intrinsic electronic properties of different graphene thicknesses by means of scanning tunneling spectroscopy (STS). The Dirac point could be identified by a distinct minimum in the electronic density of states. At higher bias voltages, defects have been created and manipulated as well as Stark-shifted image potential states were observed. The adsorption geometry of PTCDA molecules on epitaxial graphene differs remarkably from that on graphite. Despite the fact that the crystal structure of graphene resembles that of graphite, the well-known herringbone structure of PTCDA molecules adsorbed in planar geometry was not observed. Instead, a non-planar adsorption geometry is infered. Additional electron doping of the graphene sheet could be proved by means of the Dirac minimum shift revealed by spatially resolved STS. Also the PTCDA layer itself was investigated with STS. Two archetypes of tunneling spectra were observed at two different PTCDA configurations whereas both the tunneling spectra nicely agree with measurements from literature. During these STS experiments an alternation of the PTCDA assembly has been observed which is attributed to the weak non-planar adsorption of PTCDA molecules on the graphene surface

Betrachtungen und Befunde zur medialen Kinderkultur aus einem Forschungsprojekt

Treumann KP. Betrachtungen und Befunde zur medialen Kinderkultur aus einem Forschungsprojekt. In: Lauffer J, Volkmer I, eds. Kommunikative Kompetenz in einer sich verändernden Medienwelt. Opladen: Leske + Budrich; 1995: 270-283