119 research outputs found
Molekulare Ursachen des Mohr-Tranebjaerg-Syndromes
The molecular basis of the Mohr-Tranebjaerg syndrome:
A structural and functional analysis of the proteins DDP1 and Tim13
Mohr-Tranebjaerg syndrome is a mitochondrial disorder caused by a defects in the biogenesis of the human TIM23 translocase
Tim8 and Tim13 of yeast belong to a family of evolutionary conserved zinc finger proteins that are organised in hetero-oligomeric complexes in the mitochondrial intermembrane space (IMS). The TIM8-13 complex assists the import of Tim23, the major component of the translocase for matrix-targeted proteins. Mutations in DDP1/TIMM8A, the gene encoding the human homolog of Tim8, cause the Mohr-Tranebjaerg syndrome (MTS), a progressive neurodegenerative disorder. This work shows that DDP1 and human Tim13 are zinc binding proteins which together form a 70 kDa complex in the intermembrane space of human mitochondria. Similar to yeast, the human DDP1-hTim13 complex facilitates import of yeast and human Tim23. It has been additionally analysed the structural and functional consequences of a MTS-missense mutation (C66W) directly affecting the conserved Cys4 metal binding motif. In this connection the C66W mutation impairs the ability to bind zinc. As a consequence, the mutated DDP1 loses its ability to assemble into a hetero-oligomeric complex with its partner protein human Tim13. Thus, it was suggested that an assembly defect of DDP1 is the molecular basis of Mohr-Tranebjaerg syndrome in patients carrying the C66W mutation.Molekulare Ursachen des Mohr-Tranebjaerg-Syndromes:
Untersuchungen zur Struktur und Funktion von DDP1 und Tim13
Mutationen im TIMM8A/DDP1-Gen verursachen das Mohr-Tranebjaerg-Syndrom (MTS), eine X-chromosomal vererbte neurodegenerative Erkrankung, die durch eine progressive sensorineurale Taubheit, Dystonie, mentale Retardierung und kortikale Blindheit gekennzeichnet ist. Das TIMM8A/DDP1-Gen kodiert fĂŒr ein kleines mitochondriales Protein, das "deafness dystonia peptide 1" (DDP1), welches strukturelle Ăhnlichkeiten zu einer Familie kleiner Zinkfinger-Proteine aufweist, die in der Hefe S.cerevisiae am Import mitochondrialer Innenmembranproteine beteiligt sind. In der Hefe werden fĂŒnf Mitglieder dieser Proteinfamilie exprimiert: yTim9, yTim10, yTim12, yTim8 und yTim13. Allen gemeinsam ist ein konserviertes Cys4-Metallbindungs-Motiv, das vermutlich zur Formation eines Zinkfingers beitrĂ€gt. Der Mensch kodiert dagegen fĂŒr sechs kleine Tim-Proteine: ein Tim9-, zwei Tim10- (Tim10a, Tim10b), zwei Tim8- (DDP1, DDP2) und ein Tim13-Homolog. Schwerpunkt dieser Arbeit war die strukturelle und funktionelle Charakterisierung des krankheitsassoziierten DDP1-Proteins.
Dabei konnten folgende Ergebnisse erzielt werden:
DDP1, wie auch alle anderen im Rahmen dieser Arbeit identifizierten menschlichen kleinen Tim-Proteine, ist als lösliches Protein im mitochondrialen Intermembranraum lokalisiert. In Àhnlicher Weise wie die Hefe-Komponenten agieren die menschlichen kleinen Tim-Proteine in Form hetero-oligomerer Komplexe. Das MTS-assoziierte DDP1 assembliert zusammen mit seinem Partnerprotein Tim13 zu einem hetero-hexameren Komplex von 70 kDa. Die Funktion des DDP1/Tim13-Komplexes scheint evolutionÀr konserviert zu sein, da der in Hefe rekonstituierte menschliche Komplex in der Lage ist, den kÀltesensitiven PhÀnotyp einer Dtim8/Dtim13-Deletionsmutante zu komplementieren.
In gleicher Weise wie der yTim8/yTim13-Komplex der Hefe unterstĂŒtzt der menschliche DDP1/Tim13-Komplex den mitochondrialen Import von Tim23-Vorstufenproteinen. Es gibt jedoch Unterschiede in den energetischen Erfordernissen zwischen höheren und niederen Eukaryonten: WĂ€hrend in der Hefe S.cerevisiae der Import von Tim23-Vorstufenproteinen nur bei einem erniedrigten Membranpotential ĂŒber der Innenmembran durch den yTim8/yTim13-Komplex unterstĂŒtzt wird (Paschen et al., 2000) ist das humane Tim23-Vorstufenprotein auch unter gĂŒnstigen energetischen Bedingungen auf die UnterstĂŒtzung durch den DDP1/Tim13-Komplex angewiesen. Diese Unterschiede können erklĂ€ren, warum eine in Hefe nicht-essentielle Komponente im Menschen mit einer progressiven neurodegenerativen Erkrankung assoziiert ist.
Im Rahmen dieser Arbeit wurden zudem die funktionellen und strukturellen Auswirkungen einer missense Mutation im TIMM8A/DDP1-Gen aufgeklÀrt. Durch die Mutation wird das dem C-terminus am nÀchsten gelegene Cystein der vier konservierten Cysteinreste (Cys4-Motiv) des DDP1-Proteins gegen ein Tryptophan ausgetauscht (C66W-Mutation) (Tranebjaerg et al., 2000).
Die vier konservierten Cysteinreste vermitteln die koordinierte Bindung von Zinkionen in einem molaren VerhĂ€ltnis von 1:1, was vermutlich zur Ausbildung einer Zinkfinger-Struktur fĂŒhrt. Die C66W-Mutation hat den vollstĂ€ndigen Verlust der ZinkbindungskapazitĂ€t des DDP1-Proteins zur Folge. Das DDP1C66W-Protein ist in der Hefe nicht funktionell und kann weder den kĂ€ltesensitiven PhĂ€notyp noch den Tim23-Importdefekt der Dtim8/Dtim13-Deletionsmutante komplementieren. Der Funktionsverlust scheint auf eine erhöhte InstabilitĂ€t des mutierten Proteins zurĂŒckzugehen. Vermutlich kommt es durch die fehlende Ausbildung einer Zinkfingerstruktur zu einer Miss- bzw. Fehlfaltung des DDP1-Proteins und in Folge zur raschen Degradation im mitochondrialen Intermembranraum. DarĂŒber hinaus hat die Zinkfingerstruktur eine wichtige Funktion fĂŒr die Interaktion von DDP1 mit seinem Partnerprotein. DDP1C66W ist nicht in der Lage mit Tim13 zu höhermolekularen Komplexen zu assemblieren. Dies kann erklĂ€ren, warum die in einem MTS-Patienten beschriebene missense Mutation in ihrer Konsequenz - dem Fehlen von DDP1 - nicht von loss-of-function Mutationen, wie Stopmutationen, Insertionen/Deletionen zu unterscheiden ist (Tranebjaerg et al., 2000)
Differential recruitment of DNA Ligase I and III to DNA repair sites
DNA ligation is an essential step in DNA replication, repair and recombination. Mammalian cells contain three DNA Ligases that are not interchangeable although they use the same catalytic reaction mechanism. To compare the recruitment of the three eukaryotic DNA Ligases to repair sites in vivo we introduced DNA lesions in human cells by laser microirradiation. Time lapse microscopy of fluorescently tagged proteins showed that DNA Ligase III accumulated at microirradiated sites before DNA Ligase I, whereas we could detect only a faint accumulation of DNA Ligase IV. Recruitment of DNA Ligase I and III to repair sites was cell cycle independent. Mutational analysis and binding studies revealed that DNA Ligase I was recruited to DNA repair sites by interaction with PCNA while DNA Ligase III was recruited via its BRCT domain mediated interaction with XRCC1. Selective recruitment of specialized DNA Ligases may have evolved to accommodate the particular requirements of different repair pathways and may thus enhance efficiency of DNA repair
Direct and dynamic detection of HIV-1 in living cells.
In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA) at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research
An Intracellular Nanotrap Redirects Proteins and Organelles in Live Bacteria
Owing to their small size and enhanced stability, nanobodies derived from camelids have previously been used for the construction of intracellular "nanotraps," which enable redirection and manipulation of green fluorescent protein (GFP)-tagged targets within living plant and animal cells. By taking advantage of intracellular compartmentalization in the magnetic bacterium Magnetospirillum gryphiswaldense, we demonstrate that proteins and even entire organelles can be retargeted also within prokaryotic cells by versatile nanotrap technology. Expression of multivalent GFP-binding nanobodies on magnetosomes ectopically recruited the chemotaxis protein CheW(1)-GFP from polar chemoreceptor clusters to the midcell, resulting in a gradual knockdown of aerotaxis. Conversely, entire magnetosome chains could be redirected from the midcell and tethered to one of the cell poles. Similar approaches could potentially be used for building synthetic cellular structures and targeted protein knockdowns in other bacteria. IMPORTANCE Intrabodies are commonly used in eukaryotic systems for intracellular analysis and manipulation of proteins within distinct subcellular compartments. In particular, so-called nanobodies have great potential for synthetic biology approaches because they can be expressed easily in heterologous hosts and actively interact with intracellular targets, for instance, by the construction of intracellular "nanotraps" in living animal and plant cells. Although prokaryotic cells also exhibit a considerable degree of intracellular organization, there are few tools available equivalent to the well-established methods used in eukaryotes. Here, we demonstrate the ectopic retargeting and depletion of polar membrane proteins and entire organelles to distinct compartments in a magnetotactic bacterium, resulting in a gradual knockdown of magneto-aerotaxis. This intracellular nanotrap approach has the potential to be applied in other bacteria for building synthetic cellular structures, manipulating protein function, and creating gradual targeted knockdowns. Our findings provide a proof of principle for the universal use of fluorescently tagged proteins as targets for nanotraps to fulfill these tasks
DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells
DNA methylation plays a central role in the epigenetic regulation of gene expression in vertebrates. Genetic and biochemical data indicated that DNA methyltransferase 1 (Dnmt1) is indispensable for the maintenance of DNA methylation patterns in mice, but targeting of the DNMT1 locus in human HCT116 tumor cells had only minor effects on genomic methylation and cell viability. In this study, we identified an alternative splicing in these cells that bypasses the disrupting selective marker and results in a catalytically active DNMT1 protein lacking the proliferating cell nuclear antigenâbinding domain required for association with the replication machinery. Using a mechanism-based trapping assay, we show that this truncated DNMT1 protein displays only twofold reduced postreplicative DNA methylation maintenance activity in vivo. RNA interferenceâmediated knockdown of this truncated DNMT1 results in global genomic hypomethylation and cell death. These results indicate that DNMT1 is essential in mouse and human cells, but direct coupling of the replication of genetic and epigenetic information is not strictly required
Np95 interacts with de novo DNA methyltransferases, Dnmt3a and Dnmt3b, and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells
Recent studies have indicated that nuclear protein of 95 kDa (Np95) is essential for maintaining genomic methylation by recruiting DNA methyltransferase (Dnmt) 1 to hemi-methylated sites. Here, we show that Np95 interacts more strongly with regulatory domains of the de novo methyltransferases Dnmt3a and Dnmt3b. To investigate possible functions, we developed an epigenetic silencing assay using fluorescent reporters in embryonic stem cells (ESCs). Interestingly, silencing of the cytomegalovirus promoter in ESCs preceded DNA methylation and was strictly dependent on the presence of either Np95, histone H3 methyltransferase G9a or Dnmt3a and Dnmt3b. Our results indicate a regulatory role for Np95, Dnmt3a and Dnmt3b in mediating epigenetic silencing through histone modification followed by DNA methylation
Four-color single-molecule imaging with engineered tags resolves the molecular architecture of signaling complexes in the plasma membrane
Development of a PNGase Rc column for online deglycosylation of complex glycoproteins during HDX-MS
Protein glycosylation is one of the most common PTMs and many cell surface receptors, extracellular proteins, and biopharmaceuticals are glycosylated. However, HDX-MS analysis of such important glycoproteins has so far been limited by difficulties in determining the HDX of the protein segments that contain glycans. We have developed a column containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup to allow improved analysis of glycoproteins. We show that HDX-MS with the PNGase Rc column enables efficient online removal of N-linked glycans and the determination of the HDX of glycosylated regions in several complex glycoproteins. Additionally, we use the PNGase Rc column to perform a comprehensive HDX-MS mapping of the binding epitope of a mAb to c-Met, a complex glycoprotein drug target. Importantly, the column retains high activity in the presence of common quench-buffer additives like TCEP and urea and performed consistent across 114 days of extensive use. Overall, our work shows that HDX-MS with the integrated PNGase Rc column can enable fast and efficient online deglycosylation at harsh quench conditions to provide comprehensive analysis of complex glycoproteins
Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes
The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments
Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells
Signal-regulatory protein α (SIRPα) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRPα and the âdonât eat meâ ligand CD47 and as a marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRPα (hSIRPα)âspecific nanobodies (Nbs). We identified high-affinity Nbs targeting the hSIRPα/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis. For non-invasive in vivo imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography in novel hSIRPα/hCD47 knock-in mice, we demonstrated the applicability of 64Cu-hSIRPα-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRPα-Nbs presented in this study have potential as versatile theranostic probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for in vivo stratification and monitoring of individual responses during cancer immunotherapies
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