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Structural and Biochemical Characterization of the GABA(A) Receptor Interacting Protein Muskelin
(2015)
In a study from 2011, the protein muskelin was described as a central coordinator of the retrograde transport of GABA(A) receptors in neurons. As muskelin governs the transport along actin filaments as well as microtubules, it might be the first representative of a novel class of regulators, which coordinate cargo transport across the borders of these two independent systems of transport paths and their associated motorproteins. To establish a basis for understanding the mode of operation of muskelin, the aim of this thesis was an in-depth biochemical and structural characterization of muskelin and its interaction with the GABA(A) receptor.
One focus of the work was the analysis of the oligomerization of muskelin. As could be demonstrated, the oligomerization is based on two independent interactions mediated by different domains of the protein: a known interaction of the N-terminal discoidin domain with the C-terminal portion, termed head-to-tail interaction, and a dimerization of the LisH motif in muskelin that was so far neglected in the literature. For the detailed studies of both binding events, the solution of a crystal structure of a fragment of muskelin, comprising the Discoidin domain and the LisH motif, was an important basis. The fragment crystallized as a dimer, with dimerization being mediated solely by the LisH motif. Biochemical analysis corroborated that the LisH motif in muskelin serves as a dimerization element, and, moreover, showed that the C-terminal domain of the protein substantially stabilizes this dimerization. In addition, the crystal structure revealed the molecular composition of the surface of the head in the head-to-tail interaction, namely the discoidin domain. This information enabled to map the amino acids contributing to binding, which showed that the binding site of the head-to-tail interaction coincides with the generic ligand binding site of the discoidin domain.
As part of the analyses, residues that are critical for LisH-dimerization and the head-to-tail binding, respectively, were identified, whose mutation specifically interfered with each of the interactions separately. These mutations allowed to investigate the interplay of these interactions during oligomerization. It could be shown that recombinant muskelin assembles into a tetramer to which both interactions, the LisH-dimerization and the head-to-tail binding, contribute independently. When one of the two interactions was disturbed, only a dimer mediated via the respective other interaction could be formed; when both interactions were disturbed, the protein was present as monomer. Furthermore, Frank Heisler in the group of Matthias Kneussel was able to show the drastic impact of an impaired LisH-dimerization on muskelin in cells using these mutations. Disturbing the LisH-dimerization led to a complete redistribution of the originally cytoplasmic muskelin to the nucleus which was accompanied by a severe impairment of its function during GABA(A) receptor transport. Following up on these results in an analysis of muskelin variants, for which alterations of the subcellular localization had been published earlier, the crucial influence of LisH-dimerization to the subcellular localization and thereby the role of muskelin in the cell was confirmed.
The biochemical studies of the interaction of muskelin and the alpha1 subunit of the GABA(A) receptor demonstrated a direct binding with an affinity in the low micromolar range, which is mediated primarily by the kelch repeat domain in muskelin. For the binding site on the GABA(A) receptor, it was confirmed that the thirteen most C-terminal residues of the intracellular domain are critical for the binding of muskelin. In accordance with the strong conservation of these residues among the alpha subunits of the GABA(A) receptor, it could be shown that an interaction with muskelin in vitro is also possible for the alpha2 and alpha5 subunits. Based on the comparison of the binding sites between the homologous subunits, tentative conclusions can be drawn about the details of the binding, which may serve as a starting point for follow-up studies.
This thesis thereby makes valuable contributions to the understanding of muskelin, in particular the significance of its oligomerization. It furthermore provides an experimental framework for future studies that address related topics, such as the characterization of other muskelin interaction partners, or the questions raised in this work.
A chromatographic procedure 1 is described by means of which cytochrome oxidase has been purified from a variety of organisms including the fungus N eurospora crassa,2,3 the unicellular alga Po/ytoma mirum, 4 the insect Locusta migratoria ,5 the frog Xenopus muel/eri,4 and the mammal Rattus norwegicus. 4 This procedure can be used to equal effect for large-scale preparations, starting from grams of mitochondrial protein, or for small-scale preparations starting from milligrams. The cytochrome oxidase preparations from the different organisms are enzymically active. They show similar subunit compositions.
The fungus Neurospora crassa represents a eukaryotic cell with high biosynthetic activities. Cell mass doubles in 2-4 hr during expone ntial growth , even in simple salt media with sucrose as the sole carbon source. The microorgani sm forms a mycelium of long hyphae durlng vegetative growth . The mitochondria can be isolated under relatively gentle condi tions since a few breaks in the threadlike hyphae are sufficient to cause the outflow of the organelles. This article describes two methods for the physical disruption of the hyphae : (I) The cell s are opened in a grind mill between two rotating corundum di sks. This is a continuous and fast procedure and allows large- and small-scale preparations of mitochondria. (2) Hyphae are ground with sand in a mortar and pestle. This procedure can be applied to microscale preparations of mitochondria starting with minute amounts of cells. Other procedures for the isolation of Neurospora mitochondria after the physical di sruption or the enzymatic degradation of the cell wall have been described elsewhere
The A TPase eomplex has been isolated from mitoehondria of N eurospora crassa by immunologieal teehniques. The protein ean be obtained rapidly and qua ntitatively in high purity by miero- or large-seale immunopreeipitation. Immunopreeipitation has been applied to labeled and doubly labeled mitoehondrial proteins in order to investigate the number and moleeular weights of subunit polypeptides , the site of synthesis of subunit polypeptides, and the dieycIohexyIcarbodiimide-binding protein . The A TPase complex obtained by large-seale immunopreeipitation has been used as starting ma terial for the isolation of hydrophobie polypeptides.
no abstract available
no abstract available
Background: Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, we enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors. Methods: For this study, the oncolytic vaccinia virus GLV-1h255, containing the mmp-9 gene, was constructed and used to treat PC-3 tumor-bearing mice, achieving an intra-tumoral over-expression of MMP-9. The intra-tumoral MMP-9 content was quantified by immunohistochemistry in tumor sections. Therapeutic efficacy of GLV-1h255 was evaluated by monitoring tumor growth kinetics and intra-tumoral virus titers. Microenvironmental changes mediated by the intra-tumoral MMP-9 over-expression were investigated by microscopic quantification of the collagen IV content, the blood vessel density (BVD) and the analysis of lymph node metastasis formation. Results: GLV-1h255-treatment of PC-3 tumors led to a significant over-expression of intra-tumoral MMP-9, accompanied by a marked decrease in collagen IV content in infected tumor areas, when compared to GLV-1h68-infected tumor areas. This led to considerably elevated virus titers in GLV-1h255 infected tumors, and to enhanced tumor regression. The analysis of the BVD, as well as the lumbar and renal lymph node volumes, revealed lower BVD and significantly smaller lymph nodes in both GLV-1h68- and GLV-1h255- injected mice compared to those injected with PBS, indicating that MMP-9 over-expression does not alter the metastasis-reducing effect of oncolytic VACV. Conclusions: Taken together, these results indicate that a GLV-1h255-mediated intra-tumoral over-expression of MMP-9 leads to a degradation of collagen IV, facilitating intra-tumoral viral dissemination, and resulting in accelerated tumor regression. We propose that approaches which enhance the oncolytic effect by increasing the intra-tumoral viral load, may be an effective way to improve therapeutic outcome.
Retinitis pigmentosa (RP) ist eine vererbte Form der Erblindung, die durch eine progressive Degeneration von Photorezeptorzellen in der Retina verursacht wird. Neben „klassischen“ RP-Krankheitsgenen, die direkt oder indirekt mit dem Sehprozess und der Aufrechterhaltung der Photorezeptoren in Verbindung stehen, können auch Mutationen in Genen für konstitutive Spleißfaktoren zur Photorezeptordegeneration führen. RP kann daher als Paradebeispiel einer Erkrankung mit paradoxer Gewebespezifität angesehen werden: Defekte in essentiellen und ubiquitär exprimierten Genen führen zu einem Phänotyp, der nur wenige Zelltypen betrifft. Um Einblicke in diesen außergewöhnlichen Pathomechanismus zu erhalten, wurde im Rahmen der vorliegenden Arbeit ein Tiermodell für Spleißfaktor-vermittelte RP im Zebrafisch Danio rerio etabliert. Zunächst wurde gezeigt, dass eine RP verursachende Punktmutation des Spleißfaktors Prpf31 auch in dessen Zebrafisch-Homolog zu einem Verlust der physiologischen Aktivität führt. Als Modell für die Prpf31-Mangelsituation diente dann die durch ein Antisense-Morpholino induzierte partielle Reduktion der Prpf31-Expression in Zebrafischlarven. Konsistent mit einem RP-Phänotyp zeigte sich in diesen Larven eine starke Beeinträchtigung des Sehvermögens. Sie wurde – ebenfalls analog zu RP – durch defekte Photorezeptoren verursacht, die bei ansonsten normal entwickelter Retina eine deutlich veränderte Morphologie aufwiesen. Daraufhin konnten in einer genomweiten Transkriptomanalyse der Augen von Prpf31-defizienten Larven erstmals in vivo photorezeptorspezifische Gene identifiziert werden, deren Expression durch den Mangel an Prpf31 beeinträchtigt war. Im zweiten Teil der Arbeit wurde untersucht, ob es neben den bereits bekannten RP-Krankheitsgenen weitere Spleißfaktoren gibt, deren Defekt die Degeneration von Photorezeptoren auslösen kann. Dazu wurde in Zebrafischlarven ein Mangel an Prpf4 erzeugt, einem Spleißfaktor, der bislang nicht mit RP in Verbindung gebracht worden war. Der Phänotyp dieser Fische war nicht von dem des Prpf31 RP-Modells zu unterscheiden. Dies lieferte einen Hinweis darauf, dass auch Defekte in Prpf4 in der Lage sein könnten, RP auszulösen. Tatsächlich konnte durch genetisches Screening ein RP-Patient mit einer Punktmutation in Prpf4 identifiziert werden (Kollaboration mit Hanno Bolz, Universität Köln). Die biochemische Analyse dieser Mutation zeigte, dass sie zu einem Defekt der Integration von Prpf4 in spleißosomale Untereinheiten und zu dessen Funktionsverlust in vivo führt. Mit dem in dieser Arbeit etablierten Tiermodell konnte zum ersten Mal in vivo ein von Spleißfaktor-Mutationen verursachter Pathomechanismus von Retinitis pigmentosa nachvollzogen werden. Die vom Prpf31-Mangel betroffenen Photorezeptortranskripte stellen vielversprechende Kandidaten für die Vermittlung der Gewebespezifität dar und unterstützen die Hypothese, dass ihre ineffiziente Prozessierung den RP-Phänotyp auslöst. Die Entdeckung eines weiteren Spleißfaktors, dessen Defizienz ebenfalls zu defekten Photorezeptoren führt, zeigt, dass offenbar der Funktionsverlust des Spleißosoms generell in der Lage ist, die Degeneration dieser Zellen zu verursachen. Dies ist nicht zuletzt auch von klinischer Relevanz, da vermutet werden kann, dass sich unter den vielen bisher nicht identifizierten RP-Krankheitsgenen weitere Spleißfaktoren befinden.
Upon synthesis, nascent polypeptide chains are subject to major rearrangements of their side chains to obtain an energetically more favorable conformation in a process called folding. About one third of all cellular proteins pass through the secretory pathway and undergo oxidative folding in the endoplasmic reticulum (ER). During oxidative folding, the conformational rearrangements are accompanied by the formation of disulfide bonds – covalent bonds between cysteine side chains that form upon oxidation. Protein disulfide isomerase (PDI) assists in the folding of substrates by catalyzing the oxidation of pairs of cysteine residues and the isomerization of disulfide bonds as well as by acting as chaperones. In addition to PDI itself, a family of related ER-resident proteins has formed. All PDI family members share the thioredoxin fold in at least one of their domains and exhibit a subset of the PDI activities. Despite many studies, the role of most PDI family members remains unclear. The project presented in this thesis was aimed to establish tools for the biochemical characterization of single members of the PDI family and their role in the folding process. A combination of fluorescence based assays was developed to selectively study single functions of PDI family members and relate their properties of either catalysis of oxidation or catalysis of isomerization or chaperone activity to the rest of the protein family. A binding assay using isothermal titration calorimetry (ITC) was established to complement the activity assays. Using ITC we could show for the first time that members of the PDI family can distinguish between folded and unfolded proteins selectively binding the latter. The unique information provided by this method also revealed a two-site binding of unfolded proteins by PDI itself. In addition to the functional characterization, experiments were conducted to further investigate the oligomeric state of PDI. We could show that the equilibrium between structurally different states of PDI is heavily influenced by the redox state of the protein and its environment. This new data could help to further our understanding of the interplay between oxidases like PDI and their regenerative enzymes like Ero1, which may be governed by structural changes in response to the change in redox status. Another structural approach was the screening of all investigated PDI family members for suitable crystallization conditions. As a result of this screening we could obtain protein crystals of human ERp27 and were able to solve the structure of this protein with X-ray crystallography. The structure gives insight into the mechanisms of substrate binding domains within the PDI family and helps to understand the interaction of ERp27 with the redox active ERp57. In collaboration with the group of Heike Hermanns we could further show the physiological importance of this interaction under oxidative stress. In conclusion, the project presented in this thesis provides novel tools for an extensive analysis of the activities of single PDI family members as well as a useful set of methods to characterize novel oxidoreductases and chaperones. The initial results obtained with the our novel methods are very promising. At the same time, the structural approach of this project could successfully solve the structure of a PDI family member and give information about the interplay within the PDI family.
Radioactive amino acids were incorporated into isolated mitochondria from Neurospora crassa. Then the mitochondrial ribosomes were isolated and submitted to density gradient centrifugation. A preferential labelling of polysomes was observed. However, when the mitochondrial suspension was treated with puromycin after amino acid incorporation, no radioactivity could be detected in either the monosomes or the polysomes. The conclusion is drawn that isolated mitochondria under these conditions do not incorporate significant amounts of amino acids into proteins of their ribosomes.
Radioactive amino acids were incorporated in vivo into N eurospora crassa cells, and the mitochondrial ribosomes were isolated. The incorporation of radioactivity into the proteins of these ribosomes was inhibited by cycloheximide, but not by chloramphenicol. It is therefore concluded that these proteins are synthesized on the cycloheximide sensitive and chloramphenicol insensitive cytoplasmic ribosomes.
Radioaetive leueine was ineorporated by N eurospora crassa mitoehondria in vivo in the presence of cyeloheximide. When the membrane protein of these mitochondria was ehromatographieally separated on oleyl polymethaerylie aeid resin, & nurober of fraetions were obtained whieh differ with respeet to their eontents of radioaetivity and eytoehromes. The highest speeifie radioaetivity was found in the fraction eontaining eytoehrome aa3• This fraetion proved to be a pure and enzymatically aetive cytoehrome oxidase. Its ratio of absorbanee at 280 nm (ox)/ 443 nm (red.) was 2.1. By means of sodium dodeeylsulfate gel-electrophoresis, this enzymewas separated into five polypeptides with molecular weights of 30000, 20000, 13000, 10000, and 8000. Only the polypeptide with the molecular weight 20000 displayed a high specific radioaetivity.
Cytochrome oxidasewas prepared from Neurospora crassa by chromatography on oleyl polymethacrylic acid resin and separated into seven polypeptides by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Incorporation oflabelled amino acids into the single polypeptideswas investigated after a pulse labelling in the absence and presence of chloramphenicol, and afterwashing out the inhibitor. Chloramphenicol (4 mg/ml) inhibited amino acid incorporation into all polypeptides 90-95%• while labeHing of the whole membrane protein was inhibited only 30%• Mter washing out the inhibitor and further growth of the cells. the four smaller polypeptides were highly labelled, whereas the other polypeptides showed only a. small increase in radioactivity. It is concluded that the four small-sized polypeptides of cytochrome oxidase are synthesized but not integrated into the functional enzyme under the action of chloramphenicol.
Different pool sizes of the precursor polypeptides of cytochrome oxidase from Neurospora crassa.
(1972)
Pulse-labelling experiments with growing Neurospora crassa revealed that the polypeptides composing the protein moiety of a cytochrome oxidase preparation are derived from at least four independent pools of precursor polypeptides. The pool sizes range from 2 ° f 0 to 25 °/0 of the amount of the corresponding polypeptide present in cytochrome oxidase. The smallest pool is assigned to a polypeptide of mitochondrial origm. Serial pools were found for one of the polypeptides.
A cytochrome b preparation from Neurospora crassa mitochondria is found to consist of three polypeptides (apparent molecular weight 10 000, 11 000 and 32 000), a cytochrome aa3 preparation of six to seven polypeptides (apparent molecular weight 8 000, 11 000, 13 000, 18 000, 28 000 and 36 000). Selective incorporation of radioactive amino acids by eilher mitochondrial protein synthesis when the cytoplasmic one is blocked or by the cytoplasmic protein synthesis, when the mitochondrial one is blocked, indicates that one cytochrome b polypeptide (mw 32 000) and one to three cytochrome aa3 polypeptides (mw 36 000, 28 000 and 18 000) are mitochondrial translation products, the other cytochrome b and cytochrome aa3 polypeptides cytoplasmic translation products. The delayed appearance of labeling in the cytochrome b and cytochrome aa3 polypeptides compared to the average cell protein after a pulse of <~H leueine revealed that these polypeptides are derived from separate pools of precursor polypeptides. The pool sizes range from 2 p. cent to 25 p. cent of the amount of the corresponding polypeptide present in the cytochromes. The 32 000 molecular weight polypeptide of cytochrome band at least the 18 000 molecular weight polypeptide of cytochrome aa\(_3\) are mitochondrial translation products as well in the fungus Neurospora crassa as in the insect Locusta migratoria. So, despite the fact that the size of mitochondrial DNA and mitochondrial ribosomes is reduced in insects, the products have maintained their characteristics.
Cytochrome oxidase isolated from N eurospora crassa was resolved into seven protein eomponents by eleetrophoresis in polyaerylamide gels eontaining sodium dodeeylsulfate. The apparent molecular weights were determined tobe 41000, 28500, 21000, 16000, 14000, 11500 and 10000 for the eomponents 1, 2, 3, 4, 5, 6, and 7, respectively. The components 1, 2 and 3 are synthesized on mitochondrial ribosomes as shown by the incorporation of radioactive amino aeids in the presenee of cyeloheximide. Amino-acidanalysis of the isolated components 1, 2 and 3 revealed a high content of apolar amino acids and a low eontent of basic amino aeids compared to an average amino-aeid eomposition of components 4-7. Components 1, 2 and 3 eontribute 27.9°/0, 18°/0 and 14.2°/0 to the whole eytoehrome oxidase protein. This was calculated from the contributions of the single eomponents to the totalleueine eontent of the enzyme and the leueine eontents (nmol leueine per mg protein) of the single eomponents as determined by amino-aeid analysis. Equimolar relations of the components 1, 2 and 3 are found by dividing the amounts of protein by their apparent molecular weights. A stoichiometry of 1:1:1 results assuming a minimal molecular weight of 150000 for the whole cytochrome oxidase protein. On the basis of the heme a content a molecular weight of about 70000 per heme group was determined, using an absorption coeffieient L1e605 (redueed minus oxidized) of 12 mM-1 cm-1• It is concluded that the smallest structural unit of eytochrome oxidase contains two heme groups.