Biomedical subjects

Thomas Köcher

Publications and source records attributed to Thomas Köcher.

At least 19 recordsLinked to original sources

PubMed2006

HURP is part of a Ran-dependent complex involved in spindle formation.

BACKGROUND: GTP-loaded Ran induces the assembly of microtubules into aster-like and spindle-like structures in Xenopus egg extract. The microtubule-associated protein (MAP), TPX2, can mediate Ran's role in aster formation, but factors responsible for the transition from aster-like to spindle-like structures have not been described. RESULTS: Here we identify a complex that is required for the conversion of aster-like to spindle-like structures. The complex consists of two characterized MAPs (TPX2, XMAP215), a plus end-directed motor (Eg5), a mitotic kinase (Aurora A), and HURP, a protein associated with hepatocellular carcinoma. Formation and function of the complex is dependent on Aurora A activity. HURP protein was further characterized and shown to bind microtubules and affect their organization both in vitro and in vivo. In egg extract, anti-HURP antibodies disrupt the formation of both Ran-dependent and chromatin and centrosome-induced spindles. HURP is also required for the proper formation and function of mitotic spindles in HeLa cells. CONCLUSIONS: HURP is a new and essential component of the mitotic apparatus. HURP acts as part of a multicomponent complex that affects the growth or stability of spindle MTs and is required for spindle MT organization.

Animals↗

PubMed2006

A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysine-binding activities.

Polycomb response elements (PREs) are specific cis-regulatory sequences needed for transcriptional repression of HOX and other target genes by Polycomb group (PcG) proteins. Among the many PcG proteins known in Drosophila, Pho is the only sequence-specific DNA-binding protein. To gain insight into the function of Pho, we purified Pho protein complexes from Drosophila embryos and found that Pho exists in two distinct protein assemblies: a Pho-dINO80 complex containing the Drosophila INO80 nucleosome-remodeling complex, and a Pho-repressive complex (PhoRC) containing the uncharacterized gene product dSfmbt. Analysis of PhoRC reveals that dSfmbt is a novel PcG protein that is essential for HOX gene repression in Drosophila. PhoRC is bound at HOX gene PREs in vivo, and this targeting strictly depends on Pho-binding sites. Characterization of dSfmbt protein shows that its MBT repeats have unique discriminatory binding activity for methylated lysine residues in histones H3 and H4; the MBT repeats bind mono- and di-methylated H3-K9 and H4-K20 but fail to interact with these residues if they are unmodified or tri-methylated. Our results establish PhoRC as a novel Drosophila PcG protein complex that combines DNA-targeting activity (Pho) with a unique modified histone-binding activity (dSfmbt). We propose that PRE-tethered PhoRC selectively interacts with methylated histones in the chromatin flanking PREs to maintain a Polycomb-repressed chromatin state.

Amino Acid Sequence↗

PubMed2006

NuSAP, a mitotic RanGTP target that stabilizes and cross-links microtubules.

Nucleolar and spindle-associated protein (NuSAP) was recently identified as a microtubule- and chromatin-binding protein in vertebrates that is nuclear during interphase. Small interfering RNA-mediated depletion of NuSAP resulted in aberrant spindle formation, missegregation of chromosomes, and ultimately blocked cell proliferation. We show here that NuSAP is enriched on chromatin-proximal microtubules at meiotic spindles in Xenopus oocytes. When added at higher than physiological levels to Xenopus egg extract, NuSAP induces extensive bundling of spindle microtubules and causes bundled microtubules within spindle-like structures to become longer. In vitro reconstitution experiments reveal two direct effects of NuSAP on microtubules: first, it can efficiently stabilize microtubules against depolymerization, and second, it can cross-link large numbers of microtubules into aster-like structures, thick fibers, and networks. With defined components we show that the activity of NuSAP is differentially regulated by Importin (Imp) alpha, Impbeta, and Imp7. While Impalpha and Imp7 appear to block the microtubule-stabilizing activity of NuSAP, Impbeta specifically suppresses aspects of the cross-linking activity of NuSAP. We propose that to achieve full NuSAP functionality at the spindle, all three importins must be dissociated by RanGTP. Once activated, NuSAP may aid to maintain spindle integrity by stabilizing and cross-linking microtubules around chromatin.

Animals↗

PubMed2006

A general precursor ion-like scanning mode on quadrupole-TOF instruments compatible with chromatographic separation.

MS protein identification and quantitation are key proteomic techniques in biological research. Besides identification of proteins, MS is used increasingly to characterize secondary protein modifications. This often requires trimming the analytical strategy to a specific type of modification. Direct analysis of protein modifications in proteomic samples is often hampered by the limited dynamic range of current analytical tools. Here we present a fast, sensitive, multiplexed precursor ion scanning mode--implemented on a quadrupole-TOF instrument--that allows the specific detection of any modified peptide or molecule that reveals itself by a specific fragment ion or pattern of fragment ions within a complex proteomic sample. The high mass accuracy of the TOF mass spectrometer is available for the marker ion specificity and the precursor ion mass determination. The method is compatible with chromatographic separation. Fragment ions and intact molecular ions are acquired quasi-simultaneously by continuously switching the collision energy between elevated and low levels. Using this technique many secondary modifications can be analyzed in parallel; however, the number of peptides carrying a specific modification that can be analyzed successfully is limited by the chromatographic resolution or, more generally, by the depth of the resolved time domain.

Acetylation↗

PubMed2006

PhosTShunter: a fast and reliable tool to detect phosphorylated peptides in liquid chromatography Fourier transform tandem mass spectrometry data sets.

A database independent search algorithm for the detection of phosphopeptides is described. The program interrogates the tandem mass spectra of LC-MS/MS data sets regarding the presence of phosphorylation specific signatures. To achieve maximum informational content, the complementary fragmentation techniques electron capture dissociation (ECD) and collisionally activated dissociation (CAD) are used independently for peptide fragmentation. Several criteria characteristic for peptides phosphorylated on either serine or threonine residues were evaluated. The final algorithm searches for product ions generated by either the neutral loss of phosphoric acid or the combined neutral loss of phosphoric acid and water. Various peptide mixtures were used to evaluate the program. False positive results were not observed because the program utilizes the parts-per-million mass accuracy of Fourier transform ion cyclotron resonance mass spectrometry. Additionally, false negative results were not generated owing to the high sensitivity of the chosen criteria. The limitations of database dependent data interpretation tools are discussed and the potential of the novel algorithm to overcome these limitations is illustrated.

Algorithms↗

PubMed2005

Elucidation of the stator organization in the V-ATPase of Neurospora crassa.

V-ATPases are membrane protein complexes that pump protons in the lumen of various subcellular compartments at the expense of ATP. Proton pumping is done by a rotary mechanism that requires a static connection between the membrane pumping domain (V(0)) and the extrinsic catalytic head (V(1)). This static connection is composed of several known subunits of the V-ATPase, but their location and topological relationships are still a matter of controversy. Here, we propose a model for the V-ATPase of Neurospora crassa on the basis of single-particle analysis by electron microscopy. Comparison of the resulting map to that of the A-ATPase from Thermus thermophilus allows the positioning of two subunits in the static connecting region that are unique to eukaryotic V-ATPases (C and H). These two subunits seem to be located on opposite sides of a semicircular arrangement of the peripheral connecting elements, suggesting a role in stabilizing the stator in V-ATPases.

Intracellular Membranes↗

PubMed2005

Fragmentation of peptides in MALDI in-source decay mediated by hydrogen radicals.

In-source decay (ISD) in matrix-assisted laser desorption/ionization (MALDI) shares some similarities with the novel fragmentation technique electron capture dissociation (ECD). In both reactions, the otherwise strong N-C(alpha) bond is cleaved, forming fragment ions of the c and z types, while labile posttranslational modifications are preserved. Therefore, it is tempting to assume that ISD and ECD have some mechanistic aspects in common. Because electrons are present in the MALDI plume, we investigated the previously suggested possibility that ISD is a variation of ECD. However, experiments with peptides with only one site for efficient protonation revealed that ISD is not caused by electron capture. Instead, ICD seems to be induced by hydrogen atoms generated by a photochemical reaction of the matrix. We provide evidence for this reaction by hydrogen/deuterium exchange experiments with peptides containing a minimal number of exchangeable hydrogen atoms. The hydrogen atom model in ECD is indirectly supported by the proposed fragmentation mechanism for ISD, because our data suggest that hydrogen radicals can induce fragmentation by cleavage of the N-C(alpha) bond, independent from their origin.

Electrons↗

PubMed2005

Identification of 40LoVe, a Xenopus hnRNP D family protein involved in localizing a TGF-beta-related mRNA during oogenesis.

Asymmetric distribution of cellular components underlies many biological processes, and the localization of mRNAs within domains of the cytoplasm is one important mechanism of establishing and maintaining cellular asymmetry. mRNA localization often involves assembly of large ribonucleoproteins (RNPs) in the cytoplasm. Using an RNA affinity chromatography approach, we investigated localization RNP formation on the vegetal localization element (VLE) of the mRNA encoding Vg1, a Xenopus TGF-beta family member. We identified 40LoVe, an hnRNP D family protein, as a specific VLE binding protein from Xenopus oocytes. Interaction of 40LoVe with the VLE strictly correlates with the ability of the RNA to localize, and antibodies against 40LoVe inhibit vegetal localization in vivo in oocytes. Our results associate an hnRNP D protein with mRNA localization and have implications for several functions mediated by this important protein family.

Amino Acid Sequence↗

PubMed2004

Genome-wide analysis of mRNAs regulated by the THO complex in Drosophila melanogaster.

In yeast cells, the THO complex has been implicated in mitotic recombination, transcription elongation and mRNA nuclear export. The stable core of THO consists of Tho2p, Hpr1p, Mft1p and Thp2p. Whether a complex with similar functions assembles in metazoa has not yet been established. Here we report that Drosophila melanogaster THO consists of THO2, HPR1 and three proteins, THOC5-THOC7, which have no orthologs in budding yeast. Gene expression profiling in cells depleted of THO components revealed that <20% of the transcriptome was regulated by THO. Nonetheless, export of heat-shock mRNAs under heat stress was strictly dependent on THO function. Notably, 8% of upregulated genes encode proteins involved in DNA repair. Thus, although THO function seems to be conserved, the vast majority of mRNAs are transcribed and exported independently of THO in D. melanogaster.

Active Transport, Cell Nucleus↗

PubMed2004

Importin alpha associates with membranes and participates in nuclear envelope assembly in vitro.

Importin alpha is well known as an adaptor that functions with Importin beta in the nuclear import of proteins containing specific nuclear localization signals (NLSs). We show here that either an excess or a lack of Importin alpha blocks nuclear envelope (NE) assembly in vitro, and our data suggest that soluble Importin alpha functions in NE assembly in conjunction with NLS-containing partner proteins. Surprisingly, a significant proportion of Importin alpha is found to fractionate with Xenopus egg membranes. We demonstrate that membrane association of Importin alpha is regulated by phosphorylation. Using mutant forms of Importin alpha that either do not bind membranes or are not released from them by phosphorylation, we provide evidence that membrane-associated Importin alpha is required for NE formation. Unlike other functions of Importin alpha, this membrane-associated activity does not require interaction with NLS proteins.

Active Transport, Cell Nucleus↗

PubMed2004

RanBP2/Nup358 provides a major binding site for NXF1-p15 dimers at the nuclear pore complex and functions in nuclear mRNA export.

Metazoan NXF1-p15 heterodimers promote the nuclear export of bulk mRNA across nuclear pore complexes (NPCs). In vitro, NXF1-p15 forms a stable complex with the nucleoporin RanBP2/Nup358, a component of the cytoplasmic filaments of the NPC, suggesting a role for this nucleoporin in mRNA export. We show that depletion of RanBP2 from Drosophila cells inhibits proliferation and mRNA export. Concomitantly, the localization of NXF1 at the NPC is strongly reduced and a significant fraction of this normally nuclear protein is detected in the cytoplasm. Under the same conditions, the steady-state subcellular localization of other nuclear or cytoplasmic proteins and CRM1-mediated protein export are not detectably affected, indicating that the release of NXF1 into the cytoplasm and the inhibition of mRNA export are not due to a general defect in NPC function. The specific role of RanBP2 in the recruitment of NXF1 to the NPC is highlighted by the observation that depletion of CAN/Nup214 also inhibits cell proliferation and mRNA export but does not affect NXF1 localization. Our results indicate that RanBP2 provides a major binding site for NXF1 at the cytoplasmic filaments of the NPC, thereby restricting its diffusion in the cytoplasm after NPC translocation. In RanBP2-depleted cells, NXF1 diffuses freely through the cytoplasm. Consequently, the nuclear levels of the protein decrease and export of bulk mRNA is impaired.

Animals↗

PubMed2003

Complexes between the nonsense-mediated mRNA decay pathway factor human upf1 (up-frameshift protein 1) and essential nonsense-mediated mRNA decay factors in HeLa cells.

mRNAs harbouring premature translation-termination codons are usually degraded by the nonsense-mediated mRNA decay (NMD) pathway. Human up-frameshift protein 1 (Hupf1) is an NMD factor that is conserved between yeast and mammals. To isolate cellular complexes that are formed with Hupf1 and to explore the role of cellular proteins in NMD, we generated a HeLa cell line that stably expresses Hupf1 bearing a double-affinity tag (termed Hupf1-2tag). Hupf1-2tag is localized in the cytoplasm similar to the endogenous Hupf1 protein, and the Hupf1-2tag cell line is fully NMD-competent. Using affinity chromatography, Hupf1-2tag-associated proteins were isolated. MS and immunoblotting identified the NMD factors Hupf2 and Hupf3a/b as interaction partners of Hupf1. Size-exclusion chromatography indicates that the NMD factors Hupf1, Hupf2 and the large isoform of Hupf3a might exist in a stable, high-molecular-mass complex of approx. 1.3 MDa. Interestingly, the poly(A)-binding protein was also identified by MS to be associated specifically with Hupf1-2tag. In contrast with the interaction with Hupf2 and Hupf3a/b, the association of poly(A)-binding protein with Hupf1 is highly sensitive to treatment of the isolated complexes with RNase. Components of the exon-exon junction complex or the translational eukaryotic release factor (eRF) 3 were not identified in complexes associated with Hupf1-2tag. We discuss these findings in the context of current models of NMD.

Base Sequence↗

PubMed2003

The conserved Nup107-160 complex is critical for nuclear pore complex assembly.

Nuclear pore complexes (NPCs) are large multiprotein assemblies that allow traffic between the cytoplasm and the nucleus. During mitosis in higher eukaryotes, the Nuclear Envelope (NE) breaks down and NPCs disassemble. How NPCs reassemble and incorporate into the NE upon mitotic exit is poorly understood. We demonstrate a function for the conserved Nup107-160 complex in this process. Partial in vivo depletion of Nup133 or Nup107 via RNAi in HeLa cells resulted in reduced levels of multiple nucleoporins and decreased NPC density in the NE. Immunodepletion of the entire Nup107-160 complex from in vitro nuclear assembly reactions produced nuclei with a continuous NE but no NPCs. This phenotype was reversible only if Nup107-160 complex was readded before closed NE formation. Depletion also prevented association of FG-repeat nucleoporins with chromatin. We propose a stepwise model in which postmitotic NPC assembly initiates on chromatin via early recruitment of the Nup107-160 complex.

Animals↗

PubMed2003

The conformational state of Tes regulates its zyxin-dependent recruitment to focal adhesions.

The function of the human Tes protein, which has extensive similarity to zyxin in both sequence and domain organization, is currently unknown. We now show that Tes is a component of focal adhesions that, when expressed, negatively regulates proliferation of T47D breast carcinoma cells. Coimmunoprecipitations demonstrate that in vivo Tes is complexed with actin, Mena, and vasodilator-stimulated phosphoprotein (VASP). Interestingly, the isolated NH2-terminal half of Tes pulls out alpha-actinin and paxillin from cell extracts in addition to actin. The COOH-terminal half recruits zyxin as well as Mena and VASP from cell extracts. These differences suggest that the ability of Tes to associate with alpha-actinin, paxillin, and zyxin is dependent on the conformational state of the molecule. Consistent with this hypothesis, we demonstrate that the two halves of Tes interact with each other in vitro and in vivo. Using fibroblasts lacking Mena and VASP, we show that these proteins are not required to recruit Tes to focal adhesions. However, using RNAi ablation, we demonstrate that zyxin is required to recruit Tes, as well as Mena and VASP, but not vinculin or paxillin, to focal adhesions.

Actinin↗

PubMed2003

Nanoelectrospray-based detection and sequencing of substoichiometric amounts of phosphopeptides in complex mixtures.

A novel immobilized metal affinity chromatography (IMAC)-based protocol was successfully used for sample preparation prior to nanoelectrospray-based sequencing of phosphopeptides. In a first step, phosphorylated peptides are detected in an unseparated peptide mixture using precursor ion scanning in the negative ion mode on a triple-quadrupole mass spectrometer. Then the phosphorylated peptides are enriched by passing the sample over an IMAC column and sequenced in the positive ion mode. The IMAC-based enrichment allows the sequencing of phosphorylated peptides even if other, much more abundant, peptides are present at the same m/z value in the original mixture. Using this two-step approach, we are able to combine the simplicity, sensitivity and specificity of precursor ion scanning-based detection of phosphopeptides using the nanoelectrospray ion source with the ability to sequence phosphorylated peptides even if they are present in substoichiometric amounts.

Animals↗

PubMed2003

Preprocessing of tandem mass spectrometric data to support automatic protein identification.

Liquid chromatography tandem mass spectrometry is a major tool for identifying proteins. The fragment spectra of peptides can be interpreted automatically in conjunction with a sequence database search. With the development of powerful automatic search engines, research now focuses on optimizing the result returned from database searches. We present a series of preprocessing steps for fragment spectra to increase the accuracy and specificity of automatic database searches. After processing, the correct amino acid sequences from the database can be related better to the fragment spectra. This increases the sensitivity and reliability of protein identifications, especially with very large genomic databanks, and can be important for the systematic characterization of post-translational modifications.

Algorithms↗

PubMed2003

MOF-regulated acetylation of MSL-3 in the Drosophila dosage compensation complex.

Dosage compensation ensures equal expression of X-linked genes in males and females. In Drosophila, equalization is achieved by hypertranscription of the male X chromosome. This process requires an RNA/protein containing dosage compensation complex (DCC). Here we use RNA interference of individual DCC components to define the order of complex assembly in Schneider cells. We show that interaction of MOF with MSL-3 leads to specific acetylation of MSL-3 at a single lysine residue adjacent to one of its chromodomains. We observe that localization of MSL-3 to the X chromosome is RNA dependent and acetylation sensitive. We find that the acetylation status of MSL-3 determines its interaction with roX2 RNA. Furthermore, we find that RPD3 interacts with MSL-3 and that MSL-3 can be deacetylated by the RPD3 complex. We propose that regulated acetylation of MSL-3 may provide a mechanistic explanation for spreading of the dosage compensation complex along the male X chromosome.

Acetylation↗

PubMed2002

An efficient protein complex purification method for functional proteomics in higher eukaryotes.

The ensemble of expressed proteins in a given cell is organized in multiprotein complexes. The identification of the individual components of these complexes is essential for their functional characterization. The introduction of the 'tandem affinity purification' (TAP) methodology substantially improved the purification and systematic genome-wide characterization of protein complexes in yeast. The use of this approach in higher eukaryotic cells has lagged behind its use in yeast because the tagged proteins are normally expressed in the presence of the untagged endogenous version, which may compete for incorporation into multiprotein complexes. Here we describe a strategy in which the TAP approach is combined with double-stranded RNA interference (RNAi) to avoid competition from corresponding endogenous proteins while isolating and characterizing protein complexes from higher eukaryotic cells. This strategy allows the determination of the functionality of the tagged protein and increases the specificity and the efficiency of the purification.

Animals↗