The Noc-Domain Containing C-Terminus of Noc4p Mediates Both Formation of the Noc4p-Nop14p Submodule and Its Incorporation into the SSU Processome

Noc1p, Noc3p and Noc4p are eukaryotic proteins which play essential roles in yeast ribosome biogenesis and contain a homologous stretch of about 45 aminoacids (Noc-domain) of unknown function. Yeast Noc4p is a component of the small ribosomal subunit (SSU) processome, can be isolated as a stable Noc4p-Nop14p SSU-processome submodule from yeast cells, and is required for nuclear steps of small ribosomal subunit rRNA maturation. We expressed a series of mutated alleles of NOC4 in yeast cells and analysed whether the corresponding protein variants support vegetative growth, interact with Nop14p, and are incorporated into the SSU-processome. The data reveal that the essential C-terminus of Noc4p which contains 237 aminoacids including the Noc-domain represents a protein-protein interaction module. It is required and sufficient for its association with Nop14p and several nuclear precursors of the small ribosomal subunit. The N-terminal Noc4-part seems to be targeted to pre-ribosomes via the C-terminus of Noc4p and plays there an essential role in SSU-processome function. Replacement of the Noc4p-Noc-domain by its homologues Noc1p-counterpart results in a hybrid Noc4p variant which fails to associate with Nop14p and pre-ribosomes. On the other hand, exchange of 6 amino acids in the Noc1-Noc-domain of this hybrid Noc4p protein is sufficient to restore its essential in vivo functions. These data suggest that Noc-domains of Noc1p and Noc4p share a common structural backbone in which diverging amino acids play crucial roles in mediating specific regulated interactions. Our analysis allows us to distinguish between different functions of certain domains within Noc4p and contribute to the understanding of how incorporation of Noc4p into ribosomal precursors is coupled to rRNA processing and maturation of the small ribosomal subunit

Collagen VI regulates motor circuit plasticity and motor performance by cannabinoid modulation

Collagen VI is a key component of muscle basement membranes, and genetic variants can cause monogenic muscular dystrophies. Conversely, human genetic studies recently implicated collagen VI in central nervous system function, with variants causing the movement disorder dystonia. To elucidate the neurophysiological role of collagen VI, we generated mice with a truncation of the dystonia-related collagen alpha 3 (VI) (COL6A3) C-terminal domain (CTD). These Col6a3(CTT) mice showed a recessive dystonia-like phenotype in both sexes. We found that COL6A3 interacts with the cannabinoid receptor 1 (CB1R) complex in a CTD-dependent manner. Col6a3(CTT) mice of both sexes have impaired homeostasis of excitatory input to the basal pontine nuclei (BPN), a motor control hub with dense COL6A3 expression, consistent with deficient endocannabinoid signaling. Aberrant synaptic input in the BPN was normalized by a CB1R agonist, and motor performance in Col6a3(CTT) mice of both sexes was improved by CB1R agonist treatment. Our findings identify a readily therapeutically addressable synaptic mechanism for motor control

Cross-talk between monocyte invasion and astrocyte proliferation regulates scarring in brain injury

Scar formation after brain injury is still poorly understood. To further elucidate such processes, here, we examine the interplay between astrocyte proliferation taking place predominantly at the vascular interface and monocyte invasion. Using genetic mouse models that decrease or increase reactive astrocyte proliferation, we demonstrate inverse effects on monocyte numbers in the injury site. Conversely, reducing monocyte invasion using CCR2-/- mice causes a strong increase in astrocyte proliferation, demonstrating an intriguing negative cross-regulation between these cell types at the vascular interface. CCR2-/- mice show reduced scar formation with less extracellular matrix deposition, smaller lesion site and increased neuronal coverage. Surprisingly, the GFAP+ scar area in these mice is also significantly decreased despite increased astrocyte proliferation. Proteomic analysis at the peak of increased astrocyte proliferation reveals a decrease in extracellular matrix synthesizing enzymes in the injury sites of CCR2-/- mice, highlighting how early key aspects of scar formation are initiated. Taken together, we provide novel insights into the cross-regulation of juxtavascular proliferating astrocytes and invading monocytes as a crucial mechanism of scar formation upon brain injury.Instituto de Biotecnologia y Biologia Molecula

Retinal regions shape human and murine Müller cell proteome profile and functionality

The human macula is a highly specialized retinal region with pit‐like morphology and rich in cones. How Müller cells, the principal glial cell type in the retina, are adapted to this environment is still poorly understood. We compared proteomic data from cone‐ and rod‐rich retinae from human and mice and identified different expression profiles of cone‐ and rod‐associated Müller cells that converged on pathways representing extracellular matrix and cell adhesion. In particular, epiplakin (EPPK1), which is thought to play a role in intermediate filament organization, was highly expressed in macular Müller cells. Furthermore, EPPK1 knockout in a human Müller cell‐derived cell line led to a decrease in traction forces as well as to changes in cell size, shape, and filopodia characteristics. We here identified EPPK1 as a central molecular player in the region‐specific architecture of the human retina, which likely enables specific functions under the immense mechanical loads in vivo

Adipocyte-derived extracellular vesicles increase insulin secretion through transport of insulinotropic protein cargo

Adipocyte-derived extracellular vesicles (AdEVs) are membranous nanoparticles that convey communication from adipose tissue to other organs. Here, to delineate their role as messengers with glucoregulatory nature, we paired fluorescence AdEV-tracing and SILAC-labeling with (phospho)proteomics, and revealed that AdEVs transfer functional insulinotropic protein cargo into pancreatic β-cells. Upon transfer, AdEV proteins were subjects for phosphorylation, augmented insulinotropic GPCR/cAMP/PKA signaling by increasing total protein abundances and phosphosite dynamics, and ultimately enhanced 1st-phase glucose-stimulated insulin secretion (GSIS) in murine islets. Notably, insulinotropic effects were restricted to AdEVs isolated from obese and insulin resistant, but not lean mice, which was consistent with differential protein loads and AdEV luminal morphologies. Likewise, in vivo pre-treatment with AdEVs from obese but not lean mice amplified insulin secretion and glucose tolerance in mice. This data suggests that secreted AdEVs can inform pancreatic β-cells about insulin resistance in adipose tissue in order to amplify GSIS in times of increased insulin demand

Analysis of ribosome biogenesis factor-modules in yeast cells depleted from pre-ribosomes

Formation of eukaryotic ribosomes requires more than 150 biogenesis factors which transiently interact with the nascent ribosomal subunits. Previously, many pre-ribosomal intermediates could be distinguished by their protein composition and rRNA precursor (pre-rRNA) content. We purified complexes of ribosome biogenesis factors from yeast cells in which de novo synthesis of rRNA precursors was down-regulated by genetic means. We compared the protein composition of these largely pre-rRNA free assemblies with the one of analogous pre-ribosomal preparations by semi-quantitative mass spectrometry. The experimental setup minimizes the possibility that the analysed pre-rRNA free protein modules were derived from (partially) disrupted pre-ribosomal particles and provides thereby strong evidence for their pre-ribosome independent existence. In support of the validity of this approach (i) the predicted composition of the analysed protein modules was in agreement with previously described rRNA-free complexes and (ii) in most of the cases we could identify new candidate members of reported protein modules. An unexpected outcome of these analyses was that free large ribosomal subunits are associated with a specific set of ribosome biogenesis factors in cells where neo-production of nascent ribosomes was blocked. The data presented strengthen the idea that assembly of eukaryotic pre-ribosomal particles can result from transient association of distinct building blocks

In-vivo- und In-vitro-Charakterisierung von Proteinkomplexen eukaryotischer Ribosomenbiogenesefaktoren

Zur Synthese eukaryotischer Ribosomen werden mehr als 150 Ribosomenbiogenesefaktoren benötigt, die vorübergehend bei der Bildung neusynthetisierter Untereinheiten mit präribosomalen Partikeln interagieren. Während der letzten Jahre konnten verschiedenste präribosomale Zwischenstufen identifiziert werden, die sich in ihrer Proteinzusammensetzung und ihrem jeweiligen (prä-) rRNA–Gehalt unterscheiden. Bei diesem hoch dynamischen und komplexen Vorgang stellt sich grundsätzlich die Frage, in welcher Form die zahlreichen beschriebenen Ribosomenbiogenesefaktoren an präribosomale Partikel assoziieren. Dabei besteht die Möglichkeit der Bindung einzelner Proteine an unabhängigen Bindungsstellen der (prä-) rRNA, der sequenziellen Assoziation einzelner Proteine an bereits gebundene ribosomale Proteine und Biogenesefaktoren oder auch der Assoziation vorgeformter prä-rRNA unabhängiger Proteinmodule. Weiterhin ist bisher auch unklar, ob diese Proteine als Einzelproteine oder im Komplex mit anderen Faktoren im Laufe der Ribosomenreifung von präribosomalen Partikeln abdissoziieren. In der vorliegenden Arbeit konnte eine systematische Methode etabliert werden, die es ermöglicht, die Existenz prä-rRNA freier Proteinkomplexe von Ribosomenbiogenesefaktoren im Modellorganismus S. cerevisiae in vivo zu analysieren. Dazu wurden die Biogenesefaktoren Noc1p, Nop7p, Rio2p, Enp1p, Rix1p und Arx1p in Abwesenheit von rRNA–Neusynthese affinitätsgereinigt. Die Zusammensetzung assoziierter, ohne prä-rRNA vorliegender Zusammenschlüsse wurde mittels semiquantitativer Massenspektrometrie mit dem jeweiligen Aufbau von präribosomalen Partikeln verglichen, die in Anwesenheit von rRNA–Synthese gereinigt wurden. Im Zuge dieser Untersuchung konnten die Zusammensetzungen bereits beschriebener rRNA freier Proteinkomplexe bestätigt werden. Dabei handelt es sich um den Rix1p-Ipi1p-Ipi3p-Komplex, den Nop7p-Erb1p-Ytm1p-Komlex und das potentielle Dimer der beiden Proteine Noc1p und Noc2p. Weiterhin konnten in fast allen Fällen neue Komponenten dieser Komplexe identifiziert werden. So scheint Rrp5p, eine Komponente des SSU-Prozessoms, prä-rRNA unabhängig mit dem Noc1p-Noc2p-Komplex zu interagieren und die DEXD/H Box RNA-Helikase Drs1p Teil des Nop7p-Erb1p-Ytm1p-Komplexes zu sein. Diese Komplexe aus LSU-Biogenesefaktoren könnten vermutlich funktionelle Blöcke in der Bildung von prä-60S Partikeln darstellen. Weiterhin scheint nach Abschalten der rRNA-Synthese in vivo ein Rio2p-Enp1p-Modul mit bis zu fünf weiteren Komponenten zu existieren. Dabei handelt es sich wahrscheinlich eher um einen Komplex, der im Laufe der Ribosomenreifung im Ganzen vom 40S Präribosom abdissoziiert und in Abwesenheit von rRNA-Synthese als reiner Proteinkomplex erhalten bleibt. Bei dem hier gewählten Ansatz war es im Gegensatz zu vorangegangenen Analysen unwahrscheinlich, dass sich diese Proteinmodule durch ein Zerfallen präribosomaler Partikel ausgebildet hatten. Vielmehr lassen die vorliegenden Ergebnisse vermuten, dass sie in vivo unabhängig von ablaufender Ribosomenbiosynthese existieren und als funktionelle Einheiten mit präribosomalen Partikeln interagieren können oder im Laufe der Ribosomenreifung aus präribosomalen Partikeln als Proteinkomplexe freigesetzt werden. Zusätzlich zu diesen prä-rRNA unabhängigen Proteinmodulen konnten mehrere Ribosomenbiogenesefaktoren identifiziert werden, die nicht nur an neusynthetisierte große ribosomale Untereinheiten gebunden vorliegen. Arx1p, Tif6p/eIF6, Nmd3p und möglicherweise zusätzlich Rei1p, Alb1p und Lsg1p scheinen auch Assoziation an reife 60S Untereinheiten zu zeigen, die möglicherweise durch Termination der Transkription aus Polysomen freigesetzt werden. Dies lässt vermuten, dass sie eine doppelte Funktion in der Biogenese der großen Untereinheit und deren Stabilität und Recycling während der Translation ausfüllen könnten. Durch anschließende heterologe Expression der jeweiligen Komponenten der Proteinkomplexe in Insektenzellen konnten die prä-rRNA unabhängigen Interaktionen innerhalb der Noc1p-Noc2p-Rrp5p Proteinkomplexe bestätigt und genauer charakterisiert werden. Auch innerhalb des Rio2p-Enp1p-Moduls konnten direkte Wechselwirkungen identifiziert werden, die jedoch noch genauer untersucht werden müssen. Prinzipiell scheinen Proteinmodule von Ribosomenbiogenesefaktoren mit dieser Methode in der nötigen Menge und Reinheit für anschließende funktionelle und strukturelle Untersuchungen exprimiert und gereinigt werden zu können. Anfängliche Bindungstests dieser in vitro rekonstituierten Proteinkomplexe an kurze definierte rRNA-Fragmente waren nicht erfolgreich. Somit konnte nicht ermittelt werden, ob und, wenn ja, an welcher Stelle die identifizierten Proteinmodule in der Hefezelle mit präribosomalen Partikeln als vorgeformte Komplexe interagieren können. Da in der Vergangenheit zumindest für Rrp5p bereits eine Bindung an definierte Abschnitte der prä-rRNA nachgewiesen werden konnte, müssen in diesem Zusammenhang gegebenenfalls die Bindungsbedingungen entsprechend angepasst werden. Grundsätzlich konnten im Rahmen der vorliegenden Arbeit Grundlagen zu weiterführenden Analysen der Architektur von Proteinkomplexen von Ribosomenbiogenesefaktoren und zur Untersuchung deren in vitro Bindung an präribosomale Partikel geschaffen werden, um langfristig einen Einblick in die funktionelle Architektur präribosomaler Partikel zu gewinnen

The Proteome of Native Adult Müller Glial Cells From Murine Retina

To date, the proteomic profiling of Muller cells, the dominant macroglia of the retina, has been hampered because of the absence of suitable enrichment methods. We established a novel protocol to isolate native, intact Muller cells from adult murine retinae at excellent purity which retain in situ morphology and are well suited for proteomic analyses. Two different strategies of sample preparation-an in StageTips (iST) and a subcellular fractionation approach including cell surface protein profiling were used for quantitative liquid chromatography-mass spectrometry (LC-MSMS) comparing Muller cell-enriched to depleted neuronal fractions. Pathway enrichment analyses on both data sets enabled us to identify Muller cell-specific functions which included focal adhesion kinase signaling, signal transduction mediated by calcium as second messenger, transmembrane neurotransmitter transport and antioxidant activity. Pathways associated with RNA processing, cellular respiration and phototransduction were enriched in the neuronal subpopulation. Proteomic results were validated for selected Muller cell genes by quantitative real time PCR, confirming the high expression levels of numerous members of the angiogenic and anti-inflammatory annexins and antioxidant enzymes (e. g. paraoxonase 2, peroxiredoxin 1, 4 and 6). Finally, the significant enrichment of antioxidant proteins in Muller cells was confirmed by measurements on vital retinal cells using the oxidative stress indicator CM-H2DCFDA. In contrast to photoreceptors or bipolar cells, Muller cells were most efficiently protected against H2O2-induced reactive oxygen species formation, which is in line with the protein repertoire identified in the proteomic profiling. Our novel approach to isolate intact glial cells from adult retina in combination with proteomic profiling enabled the identification of novel Muller glia specific proteins, which were validated as markers and for their functional impact in glial physiology. This provides the basis to allow the discovery of novel glial specializations and will enable us to elucidate the role of Muller cells in retinal pathologies-a topic still controversially discussed

Identification of Autoantigens in Body Fluids by Combining Pull-Downs and Organic Precipitations of Intact Immune Complexes with Quantitative Label-Free Mass Spectrometry

Most autoimmune diseases are multifactorial diseases and are caused by the immunological reaction against a number of autoantigens. Key for understanding autoimmune pathologies is the knowledge of the targeted autoantigens, both initially and during disease progression. We present an approach for autoantigen identification based on isolation of intact autoantibody–antigen complexes from body fluids. After organic precipitation of high molecular weight proteins and free immuno­globulins, released autoantigens were identified by quantitative label-free liquid chromatography mass spectrometry. We confirmed feasibility of target enrichment and identification from highly complex body fluid proteomes by spiking of a predefined antibody–antigen complex at low level of abundance. As a proof of principle, we studied the blinding disease autoimmune uveitis, which is caused by autoreactive T-cells attacking the inner eye and is accompanied by autoantibodies. We identified three novel autoantigens in the spontaneous animal model equine recurrent uveitis (secreted acidic phosphoprotein osteopontin, extracellular matrix protein 1, and metallo­proteinase inhibitor 2) and confirmed the presence of the corresponding autoantibodies in 15–25% of patient samples by enzyme-linked immuno­sorbent assay. Thus, this workflow led to the identification of novel autoantigens in autoimmune uveitis and may provide a versatile and useful tool to identify autoantigens in other autoimmune diseases in the future