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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.
Alu elements are retrotransposons that frequently form new exons during primate evolution. Here, we assess the interplay of splicing repression by hnRNPC and nonsense-mediated mRNA decay (NMD) in the quality control and evolution of new Alu-exons. We identify 3100 new Alu-exons and show that NMD more efficiently recognises transcripts with Alu-exons compared to other exons with premature termination codons. However, some Alu-exons escape NMD, especially when an adjacent intron is retained, highlighting the importance of concerted repression by splicing and NMD. We show that evolutionary progression of 3' splice sites is coupled with longer repressive uridine tracts. Once the 3' splice site at ancient Alu-exons reaches a stable phase, splicing repression by hnRNPC decreases, but the exons generally remain sensitive to NMD. We conclude that repressive motifs are strongest next to cryptic exons and that gradual weakening of these motifs contributes to the evolutionary emergence of new alternative exons.
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.
Relative orientation of POTRA domains from cyanobacterial Omp85 studied by pulsed EPR spectroscopy
(2016)
Many proteins of the outer membrane of Gram-negative bacteria and of the outer envelope of the endosymbiotically derived organelles mitochondria and plastids have a β-barrel fold. Their insertion is assisted by membrane proteins of the Omp85-TpsB superfamily. These proteins are composed of a C-terminal β-barrel and a different number of N-terminal POTRA domains, three in the case of cyanobacterial Omp85. Based on structural studies of Omp85 proteins, including the five POTRA-domain-containing BamA protein of Escherichia coli, it is predicted that anaP2 and anaP3 bear a fixed orientation, whereas anaP1 and anaP2 are connected via a flexible hinge. We challenged this proposal by investigating the conformational space of the N-terminal POTRA domains of Omp85 from the cyanobacterium Anabaena sp. PCC 7120 using pulsed electron-electron double resonance (PELDOR, or DEER) spectroscopy. The pronounced dipolar oscillations observed for most of the double spin-labeled positions indicate a rather rigid orientation of the POTRA domains in frozen liquid solution. Based on the PELDOR distance data, structure refinement of the POTRA domains was performed taking two different approaches: 1) treating the individual POTRA domains as rigid bodies; and 2) using an all-atom refinement of the structure. Both refinement approaches yielded ensembles of model structures that are more restricted compared to the conformational ensemble obtained by molecular dynamics simulations, with only a slightly different orientation of N-terminal POTRA domains anaP1 and anaP2 compared with the x-ray structure. The results are discussed in the context of the native environment of the POTRA domains in the periplasm.
Diffuse invasion of the surrounding brain parenchyma is a major obstacle in the treatment of gliomas with various therapeutics, including anti-angiogenic agents. Here we identify the epi-/genetic and microenvironmental downregulation of ephrinB2 as a crucial step that promotes tumour invasion by abrogation of repulsive signals. We demonstrate that ephrinB2 is downregulated in human gliomas as a consequence of promoter hypermethylation and gene deletion. Consistently, genetic deletion of ephrinB2 in a murine high-grade glioma model increases invasion. Importantly, ephrinB2 gene silencing is complemented by a hypoxia-induced transcriptional repression. Mechanistically, hypoxia-inducible factor (HIF)-1α induces the EMT repressor ZEB2, which directly downregulates ephrinB2 through promoter binding to enhance tumour invasiveness. This mechanism is activated following anti-angiogenic treatment of gliomas and is efficiently blocked by disrupting ZEB2 activity. Taken together, our results identify ZEB2 as an attractive therapeutic target to inhibit tumour invasion and counteract tumour resistance mechanisms induced by anti-angiogenic treatment strategies.
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.
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.
Recently, the conserved intracellular digestion mechanism ‘autophagy’ has been considered to be involved in early tumorigenesis and its blockade proposed as an alternative treatment approach. However, there is an ongoing debate about whether blocking autophagy has positive or negative effects in tumor cells. Since there is only poor data about the clinico-pathological relevance of autophagy in gliomas in vivo, we first established a cell culture based platform for the in vivo detection of the autophago-lysosomal components. We then investigated key autophagosomal (LC3B, p62, BAG3, Beclin1) and lysosomal (CTSB, LAMP2) molecules in 350 gliomas using immunohistochemistry, immunofluorescence, immunoblotting and qPCR. Autophagy was induced pharmacologically or by altering oxygen and nutrient levels. Our results show that autophagy is enhanced in astrocytomas as compared to normal CNS tissue, but largely independent from the WHO grade and patient survival. A strong upregulation of LC3B, p62, LAMP2 and CTSB was detected in perinecrotic areas in glioblastomas suggesting micro-environmental changes as a driver of autophagy induction in gliomas. Furthermore, glucose restriction induced autophagy in a concentration-dependent manner while hypoxia or amino acid starvation had considerably lesser effects. Apoptosis and autophagy were separately induced in glioma cells both in vitro and in vivo. In conclusion, our findings indicate that autophagy in gliomas is rather driven by micro-environmental changes than by primary glioma-intrinsic features thus challenging the concept of exploitation of the autophago-lysosomal network (ALN) as a treatment approach in gliomas.
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.
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.
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.
Abstract: The hallmarks of Alzheimer’s disease (AD) are characterized by cognitive decline and behavioral changes. The most prominent brain region affected by the progression of AD is the hippocampal formation. The pathogenesis involves a successive loss of hippocampal neurons accompanied by a decline in learning and memory consolidation mainly attributed to an accumulation of senile plaques. The amyloid precursor protein (APP) has been identified as precursor of Aβ-peptides, the main constituents of senile plaques. Until now, little is known about the physiological function of APP within the central nervous system. The allocation of APP to the proteome of the highly dynamic presynaptic active zone (PAZ) highlights APP as a yet unknown player in neuronal communication and signaling. In this study, we analyze the impact of APP deletion on the hippocampal PAZ proteome. The native hippocampal PAZ derived from APP mouse mutants (APP-KOs and NexCreAPP/APLP2-cDKOs) was isolated by subcellular fractionation and immunopurification. Subsequently, an isobaric labeling was performed using TMT6 for protein identification and quantification by high-resolution mass spectrometry. We combine bioinformatics tools and biochemical approaches to address the proteomics dataset and to understand the role of individual proteins. The impact of APP deletion on the hippocampal PAZ proteome was visualized by creating protein-protein interaction (PPI) networks that incorporated APP into the synaptic vesicle cycle, cytoskeletal organization, and calcium-homeostasis. The combination of subcellular fractionation, immunopurification, proteomic analysis, and bioinformatics allowed us to identify APP as structural and functional regulator in a context-sensitive manner within the hippocampal active zone network.
Author Summary: More than 20 years ago, the amyloid precursor protein (APP) was identified as the precursor protein of the Aβ peptide, the main component of senile plaques in brains affected by Alzheimer’s disease. However, little is known about the physiological function of amyloid precursor protein. Allocating APP to the proteome of the structurally and functionally dynamic presynaptic active zone highlights APP as a hitherto unknown player within the presynaptic network. The hippocampus is the most prominent brain region for learning and memory consolidation, and a vulnerable target for neurodegenerative disease, e. g. Alzheimer’s disease. Therefore, our experimental design is focused on the hippocampal neurotransmitter release site. Currently, the underlying mechanism of how APP acts within presynaptic networks is still elusive. Within the scope of this research article, we constructed a network of APP within the presynaptic active zone and how deletion of APP affects these individual networks. We combine bioinformatics tools and biochemical approaches to address the dataset provided by proteomics. Furthermore, we could unravel that APP executes regulatory functions within the synaptic vesicle cycle, cytoskeletal rearrangements and Ca2+-homeostasis. Taken together, our findings offer a new perspective on the physiological function of APP in the central nervous system and may provide a molecular link to the pathogenesis of Alzheimer’s disease.
Ruijs-Aalfs syndrome is a segmental progeroid syndrome resulting from mutations in the SPRTN gene. Cells derived from patients with SPRTN mutations elicit genomic instability and people afflicted with this syndrome developed hepatocellular carcinoma. Here we describe the molecular mechanism by which SPRTN contributes to genome stability and normal cellular homeostasis. We show that SPRTN is a DNA-dependent mammalian protease required for resolving cytotoxic DNA-protein crosslinks (DPCs)— a function that had only been attributed to the metalloprotease Wss1 in budding yeast. We provide genetic evidence that SPRTN and Wss1 function distinctly in vivo to resolve DPCs. Upon DNA and ubiquitin binding, SPRTN can elicit proteolytic activity; cleaving DPC substrates and itself. SPRTN null cells or cells derived from patients with Ruijs-Aalfs syndrome are impaired in the resolution of covalent DPCs in vivo. Collectively, SPRTN is a mammalian protease required for resolving DNA-protein crosslinks in vivo whose function is compromised in Ruijs-Aalfs syndrome patients.
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.
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.
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.
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. ...