Refine
Year of publication
Document Type
- Article (179)
- Part of a Book (1)
- Contribution to a Periodical (1)
Has Fulltext
- yes (181)
Is part of the Bibliography
- no (181)
Keywords
- Cell biology (7)
- autophagy (5)
- Biochemistry (4)
- RNA (4)
- aging (4)
- mitochondria (4)
- rna (4)
- Angiogenesis (3)
- E. coli (3)
- Phosphorylation (3)
Institute
- Exzellenzcluster Makromolekulare Komplexe (181) (remove)
The 3′ untranslated regions (3′ UTRs) of transcripts serve as important hubs for posttranscriptional gene expression regulation. Here, we find that the exonisation of intergenic Alu elements introduced new terminal exons and polyadenylation sites during human genome evolution. While Alu exonisation from introns has been described previously, we shed light on a novel mechanism to create alternative 3′ UTRs, thereby opening opportunities for differential posttranscriptional regulation. On the mechanistic level, we show that intergenic Alu exonisation can compete both with alternative splicing and polyadenylation in the upstream gene. Notably, the Alu-derived isoforms are often expressed in a tissue-specific manner, and the Alu-derived 3′ UTRs can alter mRNA stability. In summary, we demonstrate that intergenic elements can affect processing of preceding genes, and elucidate how intergenic Alu exonisation can contribute to tissue-specific posttranscriptional regulation by expanding the repertoire of 3′ UTRs.
Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s−1). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.
Proton-pumping complex I of the mitochondrial respiratory chain is among the largest and most complex membrane protein complexes. The enzyme contributes substantially to oxidative energy-conversion in eukaryotic cells. Its malfunctions are implicated in many hereditary and degenerative disorders. Here, we report the X-ray structure of mitochondrial complex I at 3.6- 3.9 Å resolution describing in detail the central subunits that execute the bioenergetic function. A continuous axis of basic and acidic residues running centrally through the membrane arm connects the ubiquinone reduction site in the hydrophilic arm to four putative proton-pumping units. The binding position for a substrate analogous inhibitor and blockage of the predicted ubiquinone binding site provide a model for the ‘deactive’ form of the enzyme. The proposed transition into the active form is based on a concerted structural rearrangement at the ubiquinone reduction site rendering support for a two-state stabilization-change mechanism of protonpumping.
Filamentous, heterocyst-forming cyanobacteria exchange nutrients and regulators between cells for diazotrophic growth. Two alternative modes of exchange have been discussed involving transport either through the periplasm or through septal junctions linking adjacent cells. Septal junctions and channels in the septal peptidoglycan are likely filled with septal junction complexes. While possible proteinaceous factors involved in septal junction formation, SepJ (FraG), FraC, and FraD, have been identified, little is known about peptidoglycan channel formation and septal junction complex anchoring to the peptidoglycan. We describe a factor, SjcF1, involved in regulation of septal junction channel formation in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. SjcF1 interacts with the peptidoglycan layer through two peptidoglycan-binding domains and is localized throughout the cell periphery but at higher levels in the intercellular septa. A strain with an insertion in sjcF1 was not affected in peptidoglycan synthesis but showed an altered morphology of the septal peptidoglycan channels, which were significantly wider in the mutant than in the wild type. The mutant was impaired in intercellular exchange of a fluorescent probe to a similar extent as a sepJ deletion mutant. SjcF1 additionally bears an SH3 domain for protein-protein interactions. SH3 binding domains were identified in SepJ and FraC, and evidence for interaction of SjcF1 with both SepJ and FraC was obtained. SjcF1 represents a novel protein involved in structuring the peptidoglycan layer, which links peptidoglycan channel formation to septal junction complex function in multicellular cyanobacteria. Nonetheless, based on its subcellular distribution, this might not be the only function of SjcF1.
Maintenance of mitochondria is achieved by several mechanisms, including the regulation of mitochondrial proteostasis. The matrix protease CLPXP, involved in protein quality control, has been implicated in ageing and disease. However, particularly due to the lack of knowledge of CLPXP's substrate spectrum, only little is known about the pathways and mechanisms controlled by this protease. Here we report the first comprehensive identification of potential mitochondrial CLPXP in vivo interaction partners and substrates using a combination of tandem affinity purification and differential proteomics. This analysis reveals that CLPXP in the fungal ageing model Podospora anserina is mainly associated with metabolic pathways in mitochondria, e.g. components of the pyruvate dehydrogenase complex and the tricarboxylic acid cycle as well as subunits of electron transport chain complex I. These data suggest a possible function of mitochondrial CLPXP in the control and/or maintenance of energy metabolism. Since bioenergetic alterations are a common feature of neurodegenerative diseases, cancer, and ageing, our data comprise an important resource for specific studies addressing the role of CLPXP in these adverse processes.
Cardiac arrhythmias are often associated with mutations in ion channels or other proteins. To enable drug development for distinct arrhythmias, model systems are required that allow implementing patient-specific mutations. We assessed a muscular pump in Caenorhabditis elegans. The pharynx utilizes homologues of most of the ion channels, pumps and transporters defining human cardiac physiology. To yield precise rhythmicity, we optically paced the pharynx using channelrhodopsin-2. We assessed pharynx pumping by extracellular recordings (electropharyngeograms--EPGs), and by a novel video-microscopy based method we developed, which allows analyzing multiple animals simultaneously. Mutations in the L-type VGCC (voltage-gated Ca(2+)-channel) EGL-19 caused prolonged pump duration, as found for analogous mutations in the Cav1.2 channel, associated with long QT syndrome. egl-19 mutations affected ability to pump at high frequency and induced arrhythmicity. The pharyngeal neurons did not influence these effects. We tested whether drugs could ameliorate arrhythmia in the optogenetically paced pharynx. The dihydropyridine analog Nemadipine A prolonged pump duration in wild type, and reduced or prolonged pump duration of distinct egl-19 alleles, thus indicating allele-specific effects. In sum, our model may allow screening of drug candidates affecting specific VGCCs mutations, and permit to better understand the effects of distinct mutations on a macroscopic level.
Crystallization and X-ray diffraction studies of a complete bacterial fatty-acid synthase type I
(2015)
While a deep understanding of the fungal and mammalian multi-enzyme type I fatty-acid synthases (FAS I) has been achieved in recent years, the bacterial FAS I family, which is narrowly distributed within the Actinomycetales genera Mycobacterium, Corynebacterium and Nocardia, is still poorly understood. This is of particular relevance for two reasons: (i) although homologous to fungal FAS I, cryo-electron microscopic studies have shown that bacterial FAS I has unique structural and functional properties, and (ii) M. tuberculosis FAS I is a drug target for the therapeutic treatment of tuberculosis (TB) and therefore is of extraordinary importance as a drug target. Crystals of FAS I from C. efficiens, a homologue of M. tuberculosis FAS I, were produced and diffracted X-rays to about 4.5 Å resolution.
Mechanisms regulating protein degradation ensure the correct and timely expression of transcription factors such as hypoxia inducible factor (HIF). Under normal O2 tension, HIFα subunits are targeted for proteasomal degradation, mainly through vHL-dependent ubiquitylation. Deubiquitylases are responsible for reversing this process. Although the mechanism and regulation of HIFα by ubiquitin-dependent proteasomal degradation has been the object of many studies, little is known about the role of deubiquitylases. Here, we show that expression of HIF2α (encoded by EPAS1) is regulated by the deubiquitylase Cezanne (also known as OTUD7B) in an E2F1-dependent manner. Knockdown of Cezanne downregulates HIF2α mRNA, protein and activity independently of hypoxia and proteasomal degradation. Mechanistically, expression of the HIF2α gene is controlled directly by E2F1, and Cezanne regulates the stability of E2F1. Exogenous E2F1 can rescue HIF2α transcript and protein expression when Cezanne is depleted. Taken together, these data reveal a novel mechanism for the regulation of the expression of HIF2α, demonstrating that the HIF2α promoter is regulated by E2F1 directly and that Cezanne regulates HIF2α expression through control of E2F1 levels. Our results thus suggest that HIF2α is controlled transcriptionally in a cell-cycle-dependent manner and in response to oncogenic signalling.
Shrew-1, also called AJAP1, is a transmembrane protein associated with E-cadherin-mediated adherence junctions and a putative tumor suppressor. Apart from its interaction with β-catenin and involvement in E-cadherin internalization, little structure or function information exists. Here we explored shrew-1 expression during postnatal differentiation of mammary gland as a model system. Immunohistological analyses with antibodies against either the extracellular or the cytoplasmic domains of shrew-1 consistently revealed the expression of full-length shrew-1 in myoepithelial cells, but only part of it in luminal cells. While shrew-1 localization remained unaltered in myoepithelial cells, nuclear localization occurred in luminal cells during lactation. Based on these observations, we identified two unknown shrew-1 transcript variants encoding N-terminally truncated proteins. The smallest shrew-1 protein lacks the extracellular domain and is most likely the only variant present in luminal cells. RNA analyses of human tissues confirmed that the novel transcript variants of shrew-1 exist in vivo and exhibit a differential tissue expression profile. We conclude that our findings are essential for the understanding and interpretation of future functional and interactome analyses of shrew-1 variants.
We investigate complexes of two paramagnetic metal ions Gd3+ and Mn2+ to serve as polarizing agents for solid-state dynamic nuclear polarization (DNP) of 1H, 13C, and 15N at magnetic fields of 5, 9.4, and 14.1 T. Both ions are half-integer high-spin systems with a zero-field splitting and therefore exhibit a broadening of the mS = −1/2 ↔ +1/2 central transition which scales inversely with the external field strength. We investigate experimentally the influence of the chelator molecule, strong hyperfine coupling to the metal nucleus, and deuteration of the bulk matrix on DNP properties. At small Gd-DOTA concentrations the narrow central transition allows us to polarize nuclei with small gyromagnetic ratio such as 13C and even 15N via the solid effect. We demonstrate that enhancements observed are limited by the available microwave power and that large enhancement factors of >100 (for 1H) and on the order of 1000 (for 13C) can be achieved in the saturation limit even at 80 K. At larger Gd(III) concentrations (≥10 mM) where dipolar couplings between two neighboring Gd3+ complexes become substantial a transition towards cross effect as dominating DNP mechanism is observed. Furthermore, the slow spin-diffusion between 13C and 15N, respectively, allows for temporally resolved observation of enhanced polarization spreading from nuclei close to the paramagnetic ion towards nuclei further removed. Subsequently, we present preliminary DNP experiments on ubiquitin by site-directed spin-labeling with Gd3+ chelator tags. The results hold promise towards applications of such paramagnetically labeled proteins for DNP applications in biophysical chemistry and/or structural biology.
ANGIOGENES : knowledge database for protein-coding and noncoding RNA genes in endothelial cells
(2016)
Increasing evidence indicates the presence of long noncoding RNAs (lncRNAs) is specific to various cell types. Although lncRNAs are speculated to be more numerous than protein-coding genes, the annotations of lncRNAs remain primitive due to the lack of well-structured schemes for their identification and description. Here, we introduce a new knowledge database “ANGIOGENES” (http://angiogenes.uni-frankfurt.de) to allow for in silico screening of protein-coding genes and lncRNAs expressed in various types of endothelial cells, which are present in all tissues. Using the latest annotations of protein-coding genes and lncRNAs, publicly-available RNA-seq data was analyzed to identify transcripts that are expressed in endothelial cells of human, mouse and zebrafish. The analyzed data were incorporated into ANGIOGENES to provide a one-stop-shop for transcriptomics data to facilitate further biological validation. ANGIOGENES is an intuitive and easy-to-use database to allow in silico screening of expressed, enriched and/or specific endothelial transcripts under various conditions. We anticipate that ANGIOGENES serves as a starting point for functional studies to elucidate the roles of protein-coding genes and lncRNAs in angiogenesis.
The ubiquitin-binding zinc finger (UBZ) is a type of zinc-coordinating β-β-α fold domain found mainly in proteins involved in DNA repair and transcriptional regulation. Here, we report the crystal structure of the UBZ domain of Y-family DNA polymerase (pol) η and the crystal structure of the complex between the UBZ domain of Werner helicase-interacting protein 1 (WRNIP1) and ubiquitin, crystallized using the GFP fusion technique. In contrast to the pol η UBZ, which has been proposed to bind ubiquitin via its C-terminal α-helix, ubiquitin binds to a novel surface of WRNIP1 UBZ composed of the first β-strand and the C-terminal α-helix. In addition, we report the structure of the tandem UBZ domains of Tax1-binding protein 1 (TAX1BP1) and show that the second UBZ of TAX1BP1 binds ubiquitin, presumably in a manner similar to that of WRNIP1 UBZ. We propose that UBZ domains can be divided into at least two different types in terms of the ubiquitin-binding surfaces: the pol η type and the WRNIP1 type.
The discovery of antibiotics represented a key milestone in the history of medicine. However, with the rise of these life-saving drugs came the awareness that bacteria deploy defense mechanisms to resist these antibiotics, and they are good at it. Today, we appear at a crossroads between discovery of new potent drugs and omni-resistant superbugs. Moreover, the misuse of antibiotics in different industries has increased the rate of resistance development by providing permanent selective pressure and, subsequently, enrichment of multidrug resistant pathogens. As a result, antimicrobial resistance has now become an urgent threat to public health worldwide (http:// www.who.int/drugresistance/documents/surveillancereport/en/). The development of multidrug resistance (MDR) in an increasing number of pathogens, including Pseudomonas, Acinetobacter, Klebsiella, Salmonella, Burkholderia, and other Gram-negative bacteria is a serious issue. Membrane efflux pump complexes of the Resistance-Nodulation-Division (RND) superfamily play a key role in the development of MDR in these bacteria. These pumps, together with other transporters, contribute to intrinsic and acquired resistance of bacteria toward most, if not all, of the compounds available in our antimicrobial arsenal. Given the enormous drug polyspecificity of MDR efflux pumps, studies on their mechanism of action are extremely challenging, and this has negatively impacted both on the development of new antibiotics that are able to evade these efflux pumps and on the design of pump inhibitors. The collection of articles in this eBook, published as a Research Topic in Frontiers in Microbiology, section of Antimicrobials, Resistance, and Chemotherapy, aims to update the reader about the latest advances on the structure and function of RND efflux transporters, their roles in the overall multidrug resistance phenotype of Gram-negative pathogens, and on the strategies to inhibit their activities. ...
Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (∼1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy.
Biallelic mutations in TMEM126B cause severe complex i deficiency with a variable clinical phenotype
(2016)
Complex I deficiency is the most common biochemical phenotype observed in individuals with mitochondrial disease. With 44 structural subunits and over 10 assembly factors, it is unsurprising that complex I deficiency is associated with clinical and genetic heterogeneity. Massively parallel sequencing (MPS) technologies including custom, targeted gene panels or unbiased whole-exome sequencing (WES) are hugely powerful in identifying the underlying genetic defect in a clinical diagnostic setting, yet many individuals remain without a genetic diagnosis. These individuals might harbor mutations in poorly understood or uncharacterized genes, and their diagnosis relies upon characterization of these orphan genes. Complexome profiling recently identified TMEM126B as a component of the mitochondrial complex I assembly complex alongside proteins ACAD9, ECSIT, NDUFAF1, and TIMMDC1. Here, we describe the clinical, biochemical, and molecular findings in six cases of mitochondrial disease from four unrelated families affected by biallelic (c.635G>T [p.Gly212Val] and/or c.401delA [p.Asn134Ilefs∗2]) TMEM126B variants. We provide functional evidence to support the pathogenicity of these TMEM126B variants, including evidence of founder effects for both variants, and establish defects within this gene as a cause of complex I deficiency in association with either pure myopathy in adulthood or, in one individual, a severe multisystem presentation (chronic renal failure and cardiomyopathy) in infancy. Functional experimentation including viral rescue and complexome profiling of subject cell lines has confirmed TMEM126B as the tenth complex I assembly factor associated with human disease and validates the importance of both genome-wide sequencing and proteomic approaches in characterizing disease-associated genes whose physiological roles have been previously undetermined.
Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction.
The endoplasmic reticulum–mitochondria encounter structure (ERMES) connects the mitochondrial outer membrane with the ER. Multiple functions have been linked to ERMES, including maintenance of mitochondrial morphology, protein assembly and phospholipid homeostasis. Since the mitochondrial distribution and morphology protein Mdm10 is present in both ERMES and the mitochondrial sorting and assembly machinery (SAM), it is unknown how the ERMES functions are connected on a molecular level. Here we report that conserved surface areas on opposite sides of the Mdm10 β-barrel interact with SAM and ERMES, respectively. We generated point mutants to separate protein assembly (SAM) from morphology and phospholipid homeostasis (ERMES). Our study reveals that the β-barrel channel of Mdm10 serves different functions. Mdm10 promotes the biogenesis of α-helical and β-barrel proteins at SAM and functions as integral membrane anchor of ERMES, demonstrating that SAM-mediated protein assembly is distinct from ER-mitochondria contact sites.
50 years of amino acid hydrophobicity scales : revisiting the capacity for peptide classification
(2016)
Background: Physicochemical properties are frequently analyzed to characterize protein-sequences of known and unknown function. Especially the hydrophobicity of amino acids is often used for structural prediction or for the detection of membrane associated or embedded β-sheets and α-helices. For this purpose many scales classifying amino acids according to their physicochemical properties have been defined over the past decades. In parallel, several hydrophobicity parameters have been defined for calculation of peptide properties. We analyzed the performance of separating sequence pools using 98 hydrophobicity scales and five different hydrophobicity parameters, namely the overall hydrophobicity, the hydrophobic moment for detection of the α-helical and β-sheet membrane segments, the alternating hydrophobicity and the exact ß-strand score.
Results: Most of the scales are capable of discriminating between transmembrane α-helices and transmembrane β-sheets, but assignment of peptides to pools of soluble peptides of different secondary structures is not achieved at the same quality. The separation capacity as measure of the discrimination between different structural elements is best by using the five different hydrophobicity parameters, but addition of the alternating hydrophobicity does not provide a large benefit. An in silico evolutionary approach shows that scales have limitation in separation capacity with a maximal threshold of 0.6 in general. We observed that scales derived from the evolutionary approach performed best in separating the different peptide pools when values for arginine and tyrosine were largely distinct from the value of glutamate. Finally, the separation of secondary structure pools via hydrophobicity can be supported by specific detectable patterns of four amino acids.
Conclusion: It could be assumed that the quality of separation capacity of a certain scale depends on the spacing of the hydrophobicity value of certain amino acids. Irrespective of the wealth of hydrophobicity scales a scale separating all different kinds of secondary structures or between soluble and transmembrane peptides does not exist reflecting that properties other than hydrophobicity affect secondary structure formation as well. Nevertheless, application of hydrophobicity scales allows distinguishing between peptides with transmembrane α-helices and β-sheets. Furthermore, the overall separation capacity score of 0.6 using different hydrophobicity parameters could be assisted by pattern search on the protein sequence level for specific peptides with a length of four amino acids.
Plant-released flavonoids induce the transcription of symbiotic genes in rhizobia and one of the first bacterial responses is the synthesis of so called Nod factors. They are responsible for the initial root hair curling during onset of root nodule development. This signal exchange is believed to be essential for initiating the plant symbiosis with rhizobia affiliated with the Alphaproteobacteria. Here, we provide evidence that in the broad host range strain Sinorhizobium fredii NGR234 the complete lack of quorum sensing molecules results in an elevated copy number of its symbiotic plasmid (pNGR234a). This in turn triggers the expression of symbiotic genes and the production of Nod factors in the absence of plant signals. Therefore, increasing the copy number of specific plasmids could be a widespread mechanism of specialized bacterial populations to bridge gaps in signaling cascades.
Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin(-/-)mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.