Hamburg Unit

Hamburg, Germany

Hamburg Unit

Hamburg, Germany
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Grill C.,University of Iceland | Bergsteinsdo ttir K.,University of Iceland | Ogmundsdo ttir M.H.,University of Iceland | Pogenberg V.,Hamburg Unit | And 4 more authors.
Human Molecular Genetics | Year: 2013

The basic-helix-loop-helix-leucine zipper (bHLHZip) protein MITF (microphthalmia-associated transcription factor) is a master regulator of melanocyte development. Mutations in the MITF have been found in patients with the dominantly inherited hypopigmentation and deafness syndromes Waardenburg syndrome type 2A (WS2A) and Tietz syndrome (TS). Additionally, both somatic and germline mutations have been found in MITF in melanoma patients. Here, we characterize the DNA-binding and transcription activation properties of 24 MITF mutations found in WS2A, TS and melanoma patients. We show that most of the WS2A and TS mutations fail to bindDNAand activate expression from melanocyte-specific promoters. Someof the mutations, especially R203K and S298P, exhibit normal activity and may represent neutral variants. Mutations found in melanomas showed normal DNA-binding and minor variations in transcription activation properties; some showed increased potential to form colonies. Our results provide molecular insights into how mutations in a single gene can lead to such different phenotypes. © The Author 2013. Published by Oxford University Press. All rights reserved.

Fodor K.,Hamburg Unit | Fodor K.,Eötvös Loránd University | Wolf J.,Ruhr University Bochum | Reglinski K.,Ruhr University Bochum | And 5 more authors.
Traffic | Year: 2015

Peroxisomes entirely rely on the import of their proteome across the peroxisomal membrane. Recognition efficiencies of peroxisomal proteins vary by more than 1000-fold, but the molecular rationale behind their subsequent differential import and sorting has remained enigmatic. Using the protein cargo alanine-glyoxylate aminotransferase as a model, an unexpected increase from 34 to 80% in peroxisomal import efficiency of a single-residue mutant has been discovered. By high-resolution structural analysis, we found that it is the recognition receptor PEX5 that adapts its conformation for high-affinity binding rather than the cargo protein signal motif as previously thought. During receptor recognition, the binding cavity of the receptor shrinks to one third of its original volume. This process is impeded in the wild-type protein cargo because of a bulky side chain within the recognition motif, which blocks contraction of the PEX5 binding cavity. Our data provide a new insight into direct protein import efficiency by removal rather than by addition of an apparent specific sequence signature that is generally applicable to peroxisomal matrix proteins and to other receptor recognition processes. The translocation efficiency of peroxisomal targets defining the peroxisomal proteome is not uniform. Using a low-efficiency peroxisomal target - alanine-glyoxylate aminotransferase - we have discovered an unexpected more than twofold increase in import efficiency of a single-residue mutant. We found that it is the recognition receptor PEX5 that adapts its conformation for high-affinity binding rather than the cargo protein signal motif as previously thought. During receptor recognition, the binding cavity of the receptor shrinks to one third of its original volume. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Hoffmann N.A.,Structural and Computational Biology Unit | Jakobi A.J.,Structural and Computational Biology Unit | Jakobi A.J.,Hamburg Unit | Moreno-Morcillo M.,Structural and Computational Biology Unit | And 7 more authors.
Nature | Year: 2015

Transcription of genes encoding small structured RNAs such as transfer RNAs, spliceosomal U6 small nuclear RNA and ribosomal 5S RNA is carried out by RNA polymerase III (Pol III), the largest yet structurally least characterized eukaryotic RNA polymerase. Here we present the cryo-electron microscopy structures of the Saccharomyces cerevisiae Pol III elongating complex at 3.9 Å resolution and the apo Pol III enzyme in two different conformations at 4.6 and 4.7 Å resolution, respectively, which allow the building of a 17-subunit atomic model of Pol III. The reconstructions reveal the precise orientation of the C82-C34-C31 heterotrimer in close proximity to the stalk. The C53-C37 heterodimer positions residues involved in transcription termination close to the non-template DNA strand. In the apo Pol III structures, the stalk adopts different orientations coupled with closed and open conformations of the clamp. Our results provide novel insights into Pol III-specific transcription and the adaptation of Pol III towards its small transcriptional targets. © 2015 Macmillan Publishers Limited. All rights reserved.

Posse V.,Gothenburg University | Shahzad S.,Karolinska Institutet | Falkenberg M.,Gothenburg University | Hallberg B.M.,Karolinska Institutet | And 2 more authors.
Nucleic Acids Research | Year: 2015

A single-subunit RNA polymerase, POLRMT, transcribes the mitochondrial genome in human cells. Recently, a factor termed as the mitochondrial transcription elongation factor, TEFM, was shown to stimulate transcription elongation in vivo, but its effect in vitro was relatively modest. In the current work, we have isolated active TEFM in recombinant form and used a reconstituted in vitrotranscription system to characterize its activities. We show that TEFM strongly promotes POLRMT processivity as it dramatically stimulates the formation of longer transcripts. TEFM also abolishes premature transcription termination at conserved sequence block II, an event that has been linked to primer formation during initiation of mtDNA synthesis. We show that POLRMT pauses at a wide range of sites in a given DNA sequence. In the absence of TEFM, this leads to termination; however, the presence of TEFM abolishes this effect and aids POLRMT in continuation of transcription. Further, we show that TEFM substantially increases the POLRMT affinity to an elongation-like DNA:RNA template. In combination with previously published in vivo observations, our data establish TEFM as an essential component of the mitochon-drial transcription machinery. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Bakiri L.,National Cancer Research Center | MacHo-Maschler S.,University of Veterinary Medicine Vienna | Custic I.,Research Institute of Molecular Pathology | Custic I.,Hamburg Unit | And 9 more authors.
Cell Death and Differentiation | Year: 2015

Epithelial-to-mesenchymal transition (EMT) is essential for embryonic morphogenesis and wound healing and critical for tumour cell invasion and dissemination. The AP-1 transcription factor Fra-1 has been implicated in tumorigenesis and in tumour-associated EMT in human breast cancer. We observed a significant inverse correlation between Fra-1 mRNA expression and distant-metastasis-free survival in a large cohort of breast cancer patients derived from multiple array data sets. This unique correlation among Fos genes prompted us to assess the evolutionary conservation between Fra-1 functions in EMT of human and mouse cells. Ectopic expression of Fra-1 in fully polarized, non-tumourigenic, mouse mammary epithelial EpH4 cells induced a mesenchymal phenotype, characterized by a loss of epithelial and gain of mesenchymal markers. Proliferation, motility and invasiveness were also increased in the resulting EpFra1 cells, and the cells were tumourigenic and efficiently colonized the lung upon transplantation. Molecular analyses revealed increased expression of Tgfβ1 and the EMT-inducing transcription factors Zeb1, Zeb2 and Slug. Mechanistically, Fra-1 binds to the tgfb1 and zeb2 promoters and to an evolutionarily conserved region in the first intron of zeb1. Furthermore, increased activity of a zeb2 promoter reporter was detected in EpFra1 cells and shown to depend on AP-1-binding sites. Inhibiting TGFβ signalling in EpFra1 cells moderately increased the expression of epithelial markers, whereas silencing of zeb1 or zeb2 restored the epithelial phenotype and decreased migration in vitro and tumorigenesis in vivo. Thus Fra-1 induces changes in the expression of genes encoding EMT-related transcription factors leading to the acquisition of mesenchymal, invasive and tumorigenic capacities by epithelial cells. This study defines a novel function of Fra-1/AP-1 in modulating tgfb1, zeb1 and zeb2 expression through direct binding to genomic regulatory regions, which establishes a basis for future in vivo genetic manipulations and preclinical studies using mouse models. © 2015 Macmillan Publishers Limited.

Reinhard L.,Karolinska Institutet | Sridhara S.,Karolinska Institutet | Hallberg B.M.,Karolinska Institutet | Hallberg B.M.,Hamburg Unit
Nucleic Acids Research | Year: 2015

Mitochondrial RNA polymerase produces long polycistronic precursors that contain the mRNAs, rRNAs and tRNAs needed for mitochondrial translation. Mitochondrial RNase P (mt-RNase P) initiates the maturation of the precursors by cleaving at the 5′ ends of the tRNAs. Human mt-RNase P is only active as a tripartite complex (mitochondrial RNase P proteins 1-3; MRPP1-3), whereas plant and trypanosomal RNase Ps (PRORPs) - albeit homologous to MRPP3 - are active as single proteins. The reason for this discrepancy has so far remained obscure. Here, we present the crystal structure of human MRPP3, which features a remarkably distorted and hence non-productive active site that we propose will switch to a fully productive state only upon association with MRPP1, MRPP2 and pre-tRNA substrate. We suggest a mechanism in which MRPP1 and MRPP2 both deliver the pre-tRNA substrate and activate MRPP3 through an induced-fit process. © 2015 The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Hallberg B.M.,Karolinska Institutet | Hallberg B.M.,Hamburg Unit | Larsson N.-G.,Max Planck Institute for Biology of Ageing | Larsson N.-G.,Karolinska Institutet
Cell Metabolism | Year: 2014

Understanding regulation of mitochondrial DNA (mtDNA) expression is of considerable interest given that mitochondrial dysfunction is important in human pathology and aging. Similar to the situation in bacteria, there is no compartmentalization between transcription and translation in mitochondria; hence, both processes are likely to have a direct molecular crosstalk. Accumulating evidence suggests that there are important mechanisms for regulation of mammalian mtDNA expression at the posttranscriptional level. Regulation of mRNA maturation, mRNA stability, translational coordination, ribosomal biogenesis, and translation itself all form the basis for controlling oxidative phosphorylation capacity. Consequently, a wide variety of inherited human mitochondrial diseases are caused by mutations of nuclear genes regulating various aspects of mitochondrial translation. Furthermore, mutations of mtDNA, associated with human disease and aging, often affect tRNA genes critical for mitochondrial translation. Recent advances in molecular understanding of mitochondrial translation regulation will most likely provide novel avenues for modulating mitochondrial function for treating human disease. © 2014 Elsevier Inc.

Parret A.H.,Hamburg Unit | Besir H.,European Molecular Biology Laboratory | Meijers R.,Hamburg Unit
Current Opinion in Structural Biology | Year: 2016

Gene synthesis enables the exploitation of the degeneracy of the genetic code to boost expression of recombinant protein targets for structural studies. This has created new opportunities to obtain structural information on proteins that are normally present in low abundance. Unfortunately, synthetic gene expression occasionally leads to insoluble or misfolded proteins. This could be remedied by recent insights in the effect of codon usage on translation initiation and elongation. In this review, we discuss the interplay between optimal gene and vector design to enhance expression in a particular host and highlight the benefits and potential pitfalls associated with protein expression from synthetic genes. © 2016 The Authors

Celie P.H.N.,Netherlands Cancer Institute | Parret A.H.A.,Hamburg Unit | Perrakis A.,Netherlands Cancer Institute
Current Opinion in Structural Biology | Year: 2016

A variety of methods to create specific constructs for protein expression, in a broad range of organisms, are available nowadays. Restriction enzyme-free, ligation-independent and recombinase-based cloning methods have enabled high-throughput protein expression for structural and functional studies. These methods are also instrumental for modification of target genes including gene truncations, site-specific mutagenesis and domain swapping. Here, we describe the most common cloning techniques that are currently at hand for recombinant protein expression studies, including a brief overview of techniques associated with co-expression experiments. We also provide an inventory of many of the available reagents for the various cloning methods, and an overview for some computational tools that can help with the design of expression constructs. © 2016 Elsevier Ltd.

Pogenberg V.,Hamburg Unit | Ogmundsdottir M.H.,University of Iceland | Bergsteinsdottir K.,University of Iceland | Schepsky A.,University of Iceland | And 6 more authors.
Genes and Development | Year: 2012

Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte development and an important oncogene in melanoma. MITF heterodimeric assembly with related basic helix-loop-helix leucine zipper transcription factors is highly restricted, and its binding profile to cognate DNA sequences is distinct. Here, we determined the crystal structure of MITF in its apo conformation and in the presence of two related DNA response elements, the E-box and M-box. In addition, we investigated mouse and human Mitf mutations to dissect the functional significance of structural features. Owing to an unusual three-residue shift in the leucine zipper register, the MITF homodimer shows a marked kink in one of the two zipper helices to allow an out-of-register assembly. Removal of this insertion relieves restricted heterodimerization by MITF and permits assembly with the transcription factor MAX. Binding of MITF to the M-box motif is mediated by an unusual nonpolar interaction by Ile212, a residue that is mutated in mice and humans with Waardenburg syndrome. As several related transcription factors have low affinity for the M-box sequence, our analysis unravels how these proteins discriminate between similar target sequences. Our data provide a rational basis for targeting MITF in the treatment of important hereditary diseases and cancer. © 2012 by Cold Spring Harbor Laboratory Press.

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