Regulation and function of H3K36 di-methylation by the trithorax-group protein complex AMC.

The Drosophila Ash1 protein is a trithorax-group (trxG) regulator that antagonizes Polycomb repression at HOX genes. Ash1 di-methylates lysine 36 in histone H3 (H3K36me2) but how this activity is controlled and at which genes it functions is not well understood. We show that Ash1 protein purified from Drosophila exists in a complex with MRG15 and Caf1 that we named AMC. In Drosophila and human AMC, MRG15 binds a conserved FxLP motif near the Ash1 SET domain and stimulates H3K36 di-methylation on nucleosomes. Drosophila MRG15-null and ash1 catalytic mutants show remarkably specific trxG phenotypes: stochastic loss of HOX gene expression and homeotic transformations in adults. In mutants lacking AMC, H3K36me2 bulk levels appear undiminished but H3K36me2 is reduced in the chromatin of HOX and other AMC-regulated genes. AMC therefore appears to act on top of the H3K36me2/me3 landscape generated by the major H3K36 methyltransferases NSD and Set2. Our analyses suggest that H3K36 di-methylation at HOX genes is the crucial physiological function of AMC and the mechanism by which the complex antagonizes Polycomb repression at these genes.

Short-term inhibition of DPP-4 enhances endothelial regeneration after acute arterial injury via enhanced recruitment of circulating progenitor cells.

BACKGROUND: Endothelial injuries regularly occur in atherosclerosis and during interventional therapies of the arterial occlusive disease. Disturbances in the endothelial integrity can lead to insufficient blood supply and bear the risk of thrombus formation and acute vascular occlusion. At present, effective therapeutics to restore endothelial integrity are barely available. We analyzed the effect of pharmacological DPP-4-inhibition by Sitagliptin on endogenous progenitor cell-based endothelial regeneration via the SDF-1α/CXCR4-axis after acute endothelial damage in a mouse model of carotid injury. METHODS AND RESULTS: Induction of a defined endothelial injury was performed in the carotid artery of C57Bl/6 mice which led to a local upregulation of SDF-1α expression. Animals were treated with placebo, Sitagliptin or Sitagliptin+AMD3100. Using mass spectrometry we could prove that Sitagliptin prevented cleavage of the chemokine SDF-1α. Accordingly, increased SDF-1α concentrations enhanced recruitment of systemically applied and endogenous circulating CXCR4+ progenitor cells to the site of vascular injury followed by a significantly accelerated reendothelialization as compared to placebo-treated animals. Improved endothelial recovery, as well as recruitment of circulating CXCR4+ progenitor cells (CD133+, Flk1+), was reversed by CXCR4-antagonization through AMD3100. In addition, short-term Sitagliptin treatment did not significantly promote neointimal or medial hyperplasia. CONCLUSION: Sitagliptin can accelerate endothelial regeneration after acute endothelial injury. DPP-4 inhibitors prevent degradation of the chemokine SDF-1α and thus improve the recruitment of regenerative circulating CXCR4+ progenitor cells which mediate local endothelial cell proliferation without adversely affecting vessel wall architecture.

In vitro reconstitution of an mRNA-transport complex reveals mechanisms of assembly and motor activation.

The assembly and composition of ribonucleic acid (RNA)-transporting particles for asymmetric messenger RNA (mRNA) localization is not well understood. During mitosis of budding yeast, the Swi5p-dependent HO expression (SHE) complex transports a set of mRNAs into the daughter cell. We recombinantly reconstituted the core SHE complex and assessed its properties. The cytoplasmic precomplex contains only one motor and is unable to support continuous transport. However, a defined interaction with a second, RNA-bound precomplex after its nuclear export dimerizes the motor and activates processive RNA transport. The run length observed in vitro is compatible with long-distance transport in vivo. Surprisingly, SHE complexes that either contain or lack RNA cargo show similar motility properties, demonstrating that the RNA-binding protein and not its cargo activates motility. We further show that SHE complexes have a defined size but multimerize into variable particles upon binding of RNAs with multiple localization elements. Based on these findings, we provide an estimate of number, size, and composition of such multimeric SHE particles in the cell.

Mouse cytomegalovirus egress protein pM50 interacts with cellular endophilin-A2.

The herpesvirus replication cycle comprises maturation processes in the nucleus and cytoplasm of the infected cells. After their nuclear assembly viral capsids translocate via primary envelopment towards the cytoplasm. This event is mediated by the nuclear envelopment complex, which is composed by two conserved viral proteins belonging to the UL34 and UL31 protein families. Here, we generated recombinant viruses, which express affinity-tagged pM50 and/or pM53, the pUL34 and pUL31 homologues of the murine cytomegalovirus. We extracted pM50- and pM53-associated protein complexes from infected cells and analysed their composition after affinity purification by mass spectrometry. We observed reported interaction partners and identified new putative protein-protein interactions for both proteins. Endophilin-A2 was observed as the most prominent cellular partner of pM50. We found that endophilin-A2 binds to pM50 directly, and this interaction seems to be conserved in the pUL34 family.

Characterization of the insertase for ß-barrel proteins of the outer mitochondrial membrane.

The TOB-SAM complex is an essential component of the mitochondrial outer membrane that mediates the insertion of ß-barrel precursor proteins into the membrane. We report here its isolation and determine its size, composition, and structural organization. The complex from Neurospora crassa was composed of Tob55-Sam50, Tob38-Sam35, and Tob37-Sam37 in a stoichiometry of 1:1:1 and had a molecular mass of 140 kD. A very minor fraction of the purified complex was associated with one Mdm10 protein. Using molecular homology modeling for Tob55 and cryoelectron microscopy reconstructions of the TOB complex, we present a model of the TOB-SAM complex that integrates biochemical and structural data. We discuss our results and the structural model in the context of a possible mechanism of the TOB insertase.

DOT1A-dependent H3K76 methylation is required for replication regulation in Trypanosoma brucei.

Cell-cycle progression requires careful regulation to ensure accurate propagation of genetic material to the daughter cells. Although many cell-cycle regulators are evolutionarily conserved in the protozoan parasite Trypanosoma brucei, novel regulatory mechanisms seem to have evolved. Here, we analyse the function of the histone methyltransferase DOT1A during cell-cycle progression. Over-expression of DOT1A generates a population of cells with aneuploid nuclei as well as enucleated cells. Detailed analysis shows that DOT1A over-expression causes continuous replication of the nuclear DNA. In contrast, depletion of DOT1A by RNAi abolishes replication but does not prevent karyokinesis. As histone H3K76 methylation has never been associated with replication control in eukaryotes before, we have discovered a novel function of DOT1 enzymes, which might not be unique to trypanosomes.

Cbp3-Cbp6 interacts with the yeast mitochondrial ribosomal tunnel exit and promotes cytochrome b synthesis and assembly.

Mitochondria contain their own genetic system to express a small number of hydrophobic polypeptides, including cytochrome b, an essential subunit of the bc(1) complex of the respiratory chain. In this paper, we show in yeast that Cbp3, a bc(1) complex assembly factor, and Cbp6, a regulator of cytochrome b translation, form a complex that associates with the polypeptide tunnel exit of mitochondrial ribosomes and that exhibits two important functions in the biogenesis of cytochrome b. On the one hand, the interaction of Cbp3 and Cbp6 with mitochondrial ribosomes is necessary for efficient translation of cytochrome b transcript [corrected]. On the other hand, the Cbp3-Cbp6 complex interacts directly with newly synthesized cytochrome b in an assembly intermediate that is not ribosome bound and that contains the assembly factor Cbp4. Our results suggest that synthesis of cytochrome b occurs preferentially on those ribosomes that have the Cbp3-Cbp6 complex bound to their tunnel exit, an arrangement that may ensure tight coordination of cytochrome b synthesis and assembly.

The RSC chromatin remodelling enzyme has a unique role in directing the accurate positioning of nucleosomes.

Nucleosomes impede access to DNA. Therefore, nucleosome positioning is fundamental to genome regulation. Nevertheless, the molecular nucleosome positioning mechanisms are poorly understood. This is partly because in vitro reconstitution of in vivo-like nucleosome positions from purified components is mostly lacking, barring biochemical studies. Using a yeast extract in vitro reconstitution system that generates in vivo-like nucleosome patterns at S. cerevisiae loci, we find that the RSC chromatin remodelling enzyme is necessary for nucleosome positioning. This was previously suggested by genome-wide in vivo studies and is confirmed here in vivo for individual loci. Beyond the limitations of conditional mutants, we show biochemically that RSC functions directly, can be sufficient, but mostly relies on other factors to properly position nucleosomes. Strikingly, RSC could not be replaced by either the closely related SWI/SNF or the Isw2 remodelling enzyme. Thus, we pinpoint that nucleosome positioning specifically depends on the unique properties of the RSC complex.

Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y.

Nucleosomal incorporation of specialized histone variants is an important mechanism to generate different functional chromatin states. Here, we describe the identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y. Their messenger RNAs are found in certain human cell lines, in addition to several normal and malignant human tissues. In keeping with their primate specificity, H3.X and H3.Y are detected in different brain regions. Transgenic H3.X and H3.Y proteins are stably incorporated into chromatin in a similar fashion to the known H3 variants. Importantly, we demonstrate biochemically and by mass spectrometry that endogenous H3.Y protein exists in vivo, and that stress stimuli, such as starvation and cellular density, increase the abundance of H3.Y-expressing cells. Global transcriptome analysis revealed that knockdown of H3.Y affects cell growth and leads to changes in the expression of many genes involved in cell cycle control. Thus, H3.Y is a novel histone variant involved in the regulation of cellular responses to outside stimuli.

Proteins at the polypeptide tunnel exit of the yeast mitochondrial ribosome.

Oxidative phosphorylation in mitochondria requires the synthesis of proteins encoded in the mitochondrial DNA. The mitochondrial translation machinery differs significantly from that of the bacterial ancestor of the organelle. This is especially evident from many mitochondria-specific ribosomal proteins. An important site of the ribosome is the polypeptide tunnel exit. Here, nascent chains are exposed to an aqueous environment for the first time. Many biogenesis factors interact with the tunnel exit of pro- and eukaryotic ribosomes to help the newly synthesized proteins to mature. To date, nothing is known about the organization of the tunnel exit of mitochondrial ribosomes. We therefore undertook a comprehensive approach to determine the composition of the yeast mitochondrial ribosomal tunnel exit. Mitochondria contain homologues of the ribosomal proteins located at this site in bacterial ribosomes. Here, we identified proteins located in their proximity by chemical cross-linking and mass spectrometry. Our analysis revealed a complex network of interacting proteins including proteins and protein domains specific to mitochondrial ribosomes. This network includes Mba1, the membrane-bound ribosome receptor of the inner membrane, as well as Mrpl3, Mrpl13, and Mrpl27, which constitute ribosomal proteins exclusively found in mitochondria. This unique architecture of the tunnel exit is presumably an adaptation of the translation system to the specific requirements of the organelle.

Characterisation of the CipC-like protein AFUA_5G09330 of the opportunistic human pathogenic mould Aspergillus fumigatus.

Aspergillus fumigatus is currently the major airborne fungal pathogen that menaces immunocompromised individuals. Germination of inhaled conidia is a hallmark of the early infection process, but little is known about the underlying mechanisms. The intention of our ongoing studies is the identification of A. fumigatus proteins that are differentially expressed during germination and may provide insights in the germination process. Using a proteomic approach, we identified AFUA_5G09330 as a major hyphal-specific protein. This result was confirmed using monoclonal antibodies generated in this study. AFUA_5G09330 belongs to a fungal-specific protein family. The eponymous CipC protein of A. nidulans has been shown to be induced by concanamycin A, and transcriptional data from Cryptococcus neoformans demonstrate a strong up-regulation of the expression of a homologous gene during infection. Our data provide evidence that AFUA_5G09330 is a monomeric, cytoplasmic protein. We found no evidence for an overexpression of AFUA_5G09330 induced by concanamycin A or other stress conditions. AFUA_5G09330 is exclusively found in the hyphal morphotype that enables an invasive growth of A. fumigatus during infection. Further studies are required to define the biological function of this hyphae-specific protein and its potential relevance for the pathogenicity of A. fumigatus.

Synergy between CD26/DPP-IV inhibition and G-CSF improves cardiac function after acute myocardial infarction.

Ischemic cardiomyopathy is one of the main causes of death, which may be prevented by stem cell-based therapies. SDF-1alpha is the major chemokine attracting stem cells to the heart. Since SDF-1alpha is cleaved and inactivated by CD26/dipeptidylpeptidase IV (DPP-IV), we established a therapeutic concept--applicable to ischemic disorders in general--by combining genetic and pharmacologic inhibition of DPP-IV with G-CSF-mediated stem cell mobilization after myocardial infarction in mice. This approach leads to (1) decreased myocardial DPP-IV activity, (2) increased myocardial homing of circulating CXCR-4+ stem cells, (3) reduced cardiac remodeling, and (4) improved heart function and survival. Indeed, CD26 depletion promoted posttranslational stabilization of active SDF-1alpha in heart lysates and preserved the cardiac SDF-1-CXCR4 homing axis. Therefore, we propose pharmacological DPP-IV inhibition and G-CSF-based stem cell mobilization as a therapeutic concept for future stem cell trials after myocardial infarction.

Cross-talk between type three secretion system and metabolism in Yersinia.

Pathogenic yersiniae utilize a type three secretion system (T3SS) to inject Yop proteins into host cells in order to undermine their immune response. YscM1 and YscM2 proteins have been reported to be functionally equivalent regulators of the T3SS in Yersinia enterocolitica. Here, we show by affinity purification, native gel electrophoresis and small angle x-ray scattering that both YscM1 and YscM2 bind to phosphoenolpyruvate carboxylase (PEPC) of Y. enterocolitica. Under in vitro conditions, YscM1, but not YscM2, was found to inhibit PEPC with an apparent IC(50) of 4 mum (K(i) = 1 mum). To analyze the functional roles of PEPC, YscM1, and YscM2 in Yop-producing bacteria, cultures of Y. enterocolitica wild type and mutants defective in the formation of PEPC, YscM1, or YscM2, respectively, were grown under low calcium conditions in the presence of [U-(13)C(6)]glucose. The isotope compositions of secreted Yop proteins and nine amino acids from cellular proteins were analyzed by mass spectrometry. The data indicate that a considerable fraction of oxaloacetate used as precursor for amino acids was derived from [(13)C(3)]phosphoenolpyruvate by the catalytic action of PEPC in the wild-type strain but not in the PEPC(-) mutant. The data imply that PEPC is critically involved in replenishing the oxaloacetate pool in the citrate cycle under virulence conditions. In the YscM1(-) and YscM2(-) mutants, increased rates of pyruvate formation via glycolysis or the Entner-Doudoroff pathway, of oxaloacetate formation via the citrate cycle, and of amino acid biosynthesis suggest that both regulators trigger the central metabolism of Y. enterocolitica. We propose a "load-and-shoot cycle" model to account for the cross-talk between T3SS and metabolism in pathogenic yersiniae.

Increased alpha-synuclein aggregation following limited cleavage by certain matrix metalloproteinases.

Recent evidence indicates that protein aggregation and in particular the formation of toxic protein oligomers is a key mechanism in synucleinopathies such as Parkinson's disease (PD). Post mortem brain tissue studies as well as animal studies furthermore suggest that matrix metalloproteinases (MMPs) are also involved in the pathogenesis of PD. We used confocal single molecule spectroscopy to characterize the influence of MMPs and other proteases on the aggregation of alpha-synuclein. These studies were complemented by the characterization of alpha-synuclein fragment patterns generated by these proteases using gel electrophoresis and mass spectrometry. Limited digestion by MMP-1 and MMP-3, but not by MMP-9, increased the tendency of alpha-synuclein to aggregate. Proteinase K and Trypsin did not increase the level of de novo aggregation of alpha-synuclein. SDS-PAGE as well as MALDI-ToF analysis of limitedly digested alpha-synuclein demonstrate that all proteases generate different fragments of alpha-synuclein. We provide mass spectrometry data of proteolytic alpha-synuclein fragments and propose specific cleavage sites for MMP-1 and MMP-9 in alpha-synuclein. We furthermore found four additional cleavage sites of MMP-3 that had not been described previously. In order to increase aggregation of alpha-synuclein, specific cleavage between the highly charged C-terminal domain and the aggregation-prone NAC domain of alpha-synuclein seems to be crucial. Our findings obtained in vitro in a well-characterized model of pathological alpha-synuclein aggregation indicate that MMP-1 and MMP-3 may also influence pathogenesis of PD in vivo by generation of specific aggregation-enhancing alpha-synuclein fragments resulting from limited proteolysis.

Analysis of histone modifications by mass spectrometry.

Histone N-termini undergo diverse post-translational modifications that significantly extend the information potential of the genetic code. Moreover, they appear to mark specific chromatin regions, modulating epigenetic control, lineage commitment, and overall function of chromosomes. It is widely accepted that histone modifications affect chromatin function, but the exact mechanisms of how modifications on histone tails and specific combinations of modifications are generated, and how they cross-talk with one another, is still enigmatic. Mass spectrometry is ideal for the analysis of histone modifications and is becoming the gold standard for histone post-translational modification analysis since it allows the quantification of modifications and combinations. This unit describes how high-resolution mass spectrometry can be used to study histone post-translational modifications.

Functional characterization of Mia40p, the central component of the disulfide relay system of the mitochondrial intermembrane space.

Mia40p and Erv1p are components of a translocation pathway for the import of cysteine-rich proteins into the intermembrane space of mitochondria. We have characterized the redox behavior of Mia40p and reconstituted the disulfide transfer system of Mia40p by using recombinant functional C-terminal fragment of Mia40p, Mia40C, and Erv1p. Oxidized Mia40p contains three intramolecular disulfide bonds. One disulfide bond connects the first two cysteine residues in the CPC motif. The second and the third bonds belong to the twin CX(9)C motif and bridge the cysteine residues of two CX(9)C segments. In contrast to the stabilizing disulfide bonds of the twin CX(9)C motif, the first disulfide bond was easily accessible to reducing agents. Partially reduced Mia40C generated by opening of this bond as well as fully reduced Mia40C were oxidized by Erv1p in vitro. In the course of this reaction, mixed disulfides of Mia40C and Erv1p were formed. Reoxidation of fully reduced Mia40C required the presence of the first two cysteine residues in Mia40C. However, efficient reoxidation of a Mia40C variant containing only the cysteine residues of the twin CX(9)C motif was observed when in addition to Erv1p low amounts of wild type Mia40C were present. In the reconstituted system the thiol oxidase Erv1p was sufficient to transfer disulfide bonds to Mia40C, which then could oxidize the variant of Mia40C. In summary, we reconstituted a disulfide relay system consisting of Mia40C and Erv1p.

OPA1 processing reconstituted in yeast depends on the subunit composition of the m-AAA protease in mitochondria.

The morphology of mitochondria in mammalian cells is regulated by proteolytic cleavage of OPA1, a dynamin-like GTPase of the mitochondrial inner membrane. The mitochondrial rhomboid protease PARL, and paraplegin, a subunit of the ATP-dependent m-AAA protease, were proposed to be involved in this process. Here, we characterized individual OPA1 isoforms by mass spectrometry, and we reconstituted their processing in yeast to identify proteases involved in OPA1 cleavage. The yeast homologue of OPA1, Mgm1, was processed both by PARL and its yeast homologue Pcp1. Neither of these rhomboid proteases cleaved OPA1. The formation of small OPA1 isoforms was impaired in yeast cells lacking the m-AAA protease subunits Yta10 and Yta12 and was restored upon expression of murine or human m-AAA proteases. OPA1 processing depended on the subunit composition of mammalian m-AAA proteases. Homo-oligomeric m-AAA protease complexes composed of murine Afg3l1, Afg3l2, or human AFG3L2 subunits cleaved OPA1 with higher efficiency than paraplegin-containing m-AAA proteases. OPA1 processing proceeded normally in murine cell lines lacking paraplegin or PARL. Our results provide evidence for different substrate specificities of m-AAA proteases composed of different subunits and reveal a striking evolutionary switch of proteases involved in the proteolytic processing of dynamin-like GTPases in mitochondria.

Proteomic approach for characterization of hop-inducible proteins in Lactobacillus brevis.

Resistance to hops is a prerequisite for the capability of lactic acid bacteria to grow in beer and thus cause beer spoilage. Bactericidal hop compounds, mainly iso-alpha-acids, are described as ionophores which exchange H+ for cellular divalent cations, e.g., Mn2+, and thus dissipate ion gradients across the cytoplasmic membrane. The acid stress response of Lactobacillus brevis TMW 1.465 and hop adaptation in its variant L. brevis TMW 1.465A caused changes at the level of metabolism, membrane physiology, and cell wall composition. To identify the basis for these changes, a proteomic approach was taken. The experimental design allowed the discrimination of acid stress and hop stress. A strategy for improved protein identification enabled the identification of 84% of the proteins investigated despite the lack of genome sequence data for this strain. Hop resistance in L. brevis TMW 1.465A implies mechanisms to cope with intracellular acidification, mechanisms for energy generation and economy, genetic information fidelity, and enzyme functionality. Interestingly, the majority of hop-regulated enzymes are described as manganese or divalent cation dependent. Regulation of the manganese level allows fine-tuning of the metabolism, which enables a rapid response to environmental (stress) conditions. The hop stress response indicates adaptations shifting the metabolism into an energy-saving mode by effective substrate conversion and prevention of exhaustive protein de novo synthesis. The findings further demonstrate that hop stress in bacteria not only is associated with proton motive force depletion but obviously implies divalent cation limitation.

Yersinia protein kinase YopO is activated by a novel G-actin binding process.

Pathogenic bacteria of the genus Yersinia employ a type III secretion system to inject effector proteins (Yops) into host cells. The Yops down-regulate host cell functions through unique biochemical activities. YopO, a serine/threonine kinase required for Yersinia virulence, is activated by host cell actin via an unknown process. Here we show that YopO kinase is activated by formation of a 1:1 complex with monomeric (G) actin but is unresponsive to filamentous (F) actin. Two separate G-actin binding sites, one in the N-terminal kinase region (amino acids 89-440) and one in the C-terminal guanine nucleotide dissociation inhibitor-like region (amino acids 441-729) of YopO, were identified. Actin binding to both of these sites was necessary for effective autophosphorylation of YopO on amino acids Ser-90 and Ser-95. A S90A/S95A YopO mutant was strongly reduced in substrate phosphorylation, suggesting that autophosphorylation activates YopO kinase activity. In cells the kinase activity of YopO regulated rounding/arborization and was specifically required for inhibition of Yersinia YadA-dependent phagocytosis. Thus, YopO kinase is activated by a novel G-actin binding process, and this appears to be crucial for its anti-host cell functions.

A gamma-secretase-like intramembrane cleavage of TNFalpha by the GxGD aspartyl protease SPPL2b.

Gamma-secretase and signal peptide peptidase (SPP) are unusual GxGD aspartyl proteases, which mediate intramembrane proteolysis. In addition to SPP, a family of SPP-like proteins (SPPLs) of unknown function has been identified. We demonstrate that SPPL2b utilizes multiple intramembrane cleavages to liberate the intracellular domain of tumor necrosis factor alpha (TNFalpha) into the cytosol and the carboxy-terminal counterpart into the extracellular space. These findings suggest common principles for regulated intramembrane proteolysis by GxGD aspartyl proteases.