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Protein aggregation of the p63 transcription factor underlies severe skin fragility in AEC syndrome
(2018)
The p63 gene encodes a master regulator of epidermal commitment, development, and differentiation. Heterozygous mutations in the C-terminal domain of the p63 gene can cause ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome, a life-threatening disorder characterized by skin fragility and severe, long-lasting skin erosions. Despite deep knowledge of p63 functions, little is known about mechanisms underlying disease pathology and possible treatments. Here, we show that multiple AEC-associated p63 mutations, but not those causative of other diseases, lead to thermodynamic protein destabilization, misfolding, and aggregation, similar to the known p53 gain-of-function mutants found in cancer. AEC mutant proteins exhibit impaired DNA binding and transcriptional activity, leading to dominant negative effects due to coaggregation with wild-type p63 and p73. Importantly, p63 aggregation occurs also in a conditional knock-in mouse model for the disorder, in which the misfolded p63 mutant protein leads to severe epidermal defects. Variants of p63 that abolish aggregation of the mutant proteins are able to rescue p63’s transcriptional function in reporter assays as well as in a human fibroblast-to-keratinocyte conversion assay. Our studies reveal that AEC syndrome is a protein aggregation disorder and opens avenues for therapeutic intervention.
Membrane proteins frequently assemble into higher order homo- or hetero-oligomers within their natural lipid environment. This complex formation can modulate their folding, activity as well as substrate selectivity. Non-disruptive methods avoiding critical steps, such as membrane disintegration, transfer into artificial environments or chemical modifications are therefore essential to analyze molecular mechanisms of native membrane protein assemblies. The combination of cell-free synthetic biology, nanodisc-technology and non-covalent mass spectrometry provides excellent synergies for the analysis of membrane protein oligomerization within defined membranes. We exemplify our strategy by oligomeric state characterization of various membrane proteins including ion channels, transporters and membrane-integrated enzymes assembling up to hexameric complexes. We further indicate a lipid-dependent dimer formation of MraY translocase correlating with the enzymatic activity. The detergent-free synthesis of membrane protein/nanodisc samples and the analysis by LILBID mass spectrometry provide a versatile platform for the analysis of membrane proteins in a native environment.
In optogenetics, rhodopsins were established as light-driven tools to manipulate neuronal activity. However, during long-term photostimulation using channelrhodopsin (ChR), desensitization can reduce effects. Furthermore, requirement for continuous presence of the chromophore all-trans retinal (ATR) in model systems lacking sufficient endogenous concentrations limits its applicability. We tested known, and engineered and characterized new variants of de- and hyperpolarizing rhodopsins in Caenorhabditis elegans. ChR2 variants combined previously described point mutations that may synergize to enable prolonged stimulation. Following brief light pulses ChR2(C128S;H134R) induced muscle activation for minutes or even for hours (‘Quint’: ChR2(C128S;L132C;H134R;D156A;T159C)), thus featuring longer open state lifetime than previously described variants. Furthermore, stability after ATR removal was increased compared to the step-function opsin ChR2(C128S). The double mutants C128S;H134R and H134R;D156C enabled increased effects during repetitive stimulation. We also tested new hyperpolarizers (ACR1, ACR2, ACR1(C102A), ZipACR). Particularly ACR1 and ACR2 showed strong effects in behavioral assays and very large currents with fast kinetics. In sum, we introduce highly light-sensitive optogenetic tools, bypassing previous shortcomings, and thus constituting new tools that feature high effectiveness and fast kinetics, allowing better repetitive stimulation or investigating prolonged neuronal activity states in C. elegans and, possibly, other systems.
Structural and functional dissection of the DH and PH domains of oncogenic Bcr-Abl tyrosine kinase
(2017)
The two isoforms of the Bcr-Abl tyrosine kinase, p210 and p190, are associated with different leukemias and have a dramatically different signaling network, despite similar kinase activity. To provide a molecular rationale for these observations, we study the Dbl-homology (DH) and Pleckstrin-homology (PH) domains of Bcr-Abl p210, which constitute the only structural differences to p190. Here we report high-resolution structures of the DH and PH domains and characterize conformations of the DH–PH unit in solution. Our structural and functional analyses show no evidence that the DH domain acts as a guanine nucleotide exchange factor, whereas the PH domain binds to various phosphatidylinositol-phosphates. PH-domain mutants alter subcellular localization and result in decreased interactions with p210-selective interaction partners. Hence, the PH domain, but not the DH domain, plays an important role in the formation of the differential p210 and p190 Bcr-Abl signaling networks.
Regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking in response to neuronal activity is critical for synaptic function and plasticity. Here, we show that neuronal activity induces the binding of ephrinB2 and ApoER2 receptors at the postsynapse to regulate de novo insertion of AMPA receptors. Mechanistically, the multi-PDZ adaptor glutamate-receptor-interacting protein 1 (GRIP1) binds ApoER2 and bridges a complex including ApoER2, ephrinB2, and AMPA receptors. Phosphorylation of ephrinB2 in a serine residue (Ser-9) is essential for the stability of such a complex. In vivo, a mutation on ephrinB2 Ser-9 in mice results in a complete disruption of the complex, absence of ApoER2 downstream signaling, and impaired activity-induced and ApoER2-mediated AMPA receptor insertion. Using compound genetics, we show the requirement of this complex for long-term potentiation (LTP). Together, our findings uncover a cooperative ephrinB2 and ApoER2 signaling at the synapse, which serves to modulate activity-dependent AMPA receptor dynamic changes during synaptic plasticity.
SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining individual-resolution cross-linking and immunoprecipitation (iCLIP) and mass spectrometry, we show that elevated arginine methylation of SRSF5 and lower phosphorylation levels of cobound SRSF2 enhance shuttling of SRSF5 in P19 cells by modulating protein-protein and protein-RNA interactions. Moreover, SRSF5 is bound to pluripotency-specific transcripts such as Lin28a and Pou5f1/Oct4 in the cytoplasm. SRSF5 depletion reduces and overexpression increases their cytoplasmic mRNA levels, suggesting that enhanced mRNA export by SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19 cells leads to dramatically reduced SRSF5 shuttling. Our findings indicate that posttranslational modification of SR proteins underlies the regulation of their mRNA export activities and distinguishes pluripotent from differentiated cells.
The yeast bc1 complex (complex III) and cytochrome oxidase (complex IV) are mosaics of core subunits encoded by the mitochondrial genome and additional nuclear-encoded proteins imported from the cytosol. Both complexes build in the mitochondrial inner membrane various supramolecular assemblies. The formation of the individual complexes and their supercomplexes depends on the activity of dedicated assembly factors. We identified a so far uncharacterized mitochondrial protein (open reading frame YDR381C-A) as an important assembly factor for complex III, complex IV, and their supercomplexes. Therefore, we named this protein Cox interacting (Coi) 1. Deletion of COI1 results in decreased respiratory growth, reduced membrane potential, and hampered respiration, as well as slow fermentative growth at low temperature. In addition, coi1Δ cells harbour reduced steady-state levels of subunits of complexes III and IV as well as of the assembled complexes and supercomplexes. Interaction of Coi1 with respiratory chain subunits seems transient, as it appears to be a stoichiometric subunit neither of complex III nor of complex IV. Collectively, this work identifies a novel protein that plays a role in the assembly of the mitochondrial respiratory chain.
Cells respond to protein misfolding and aggregation in the cytosol by adjusting gene transcription and a number of post-transcriptional processes. In parallel to functional reactions, cellular structure changes as well; however, the mechanisms underlying the early adaptation of cellular compartments to cytosolic protein misfolding are less clear. Here we show that the mammalian ubiquitin ligase C-terminal Hsp70-interacting protein (CHIP), if freed from chaperones during acute stress, can dock on cellular membranes thus performing a proteostasis sensor function. We reconstituted this process in vitro and found that mainly phosphatidic acid and phosphatidylinositol-4-phosphate enhance association of chaperone-free CHIP with liposomes. HSP70 and membranes compete for mutually exclusive binding to the tetratricopeptide repeat domain of CHIP. At new cellular locations, access to compartment-specific substrates would enable CHIP to participate in the reorganization of the respective organelles, as exemplified by the fragmentation of the Golgi apparatus (effector function).
The degradation of nonfunctional mitochondrial proteins is of fundamental relevance for maintenance of cellular homeostasis. The heteromeric CLPXP protein complex in the mitochondrial matrix is part of this process. In the fungal aging model Podospora anserina, ablation of CLPXP leads to an increase in healthy lifespan. Here, we report that this counterintuitive increase depends on a functional autophagy machinery. In PaClpXP mutants, autophagy is involved in energy conservation and the compensation of impairments in respiration. Strikingly, despite the impact on mitochondrial function, it is not mitophagy but general autophagy that is constitutively induced and required for longevity. In contrast, in another long-lived mutant ablated for the mitochondrial PaIAP protease, autophagy is neither induced nor required for lifespan extension. Our data provide novel mechanistic insights into the capacity of different forms of autophagy to compensate impairments of specific components of the complex mitochondrial quality control network and about the biological role of mitochondrial CLPXP in the control of cellular energy metabolism.
We demonstrate high-density labelling of cellular DNA and RNA using click chemistry and perform confocal and super-resolution microscopy. We visualize the crescent and ring-like structure of densely packed RNA in nucleoli. We further demonstrate click chemistry with unnatural amino acids for super-resolution imaging of outer-membrane proteins of E. coli.
The inner boundary and the cristae membrane are connected by pore-like structures termed crista junctions (CJs). The MICOS complex is required for CJ formation and enriched at CJs. Here, we address the roles of the MICOS subunits Mic27 and Mic10. We observe a positive genetic interaction between Mic27 and Mic60 and deletion of Mic27 results in impaired formation of CJs and altered cristae membrane curvature. Mic27 acts in an antagonistic manner to Mic60 as it promotes oligomerization of the F1FO-ATP synthase and partially restores CJ formation in cells lacking Mic60. Mic10 impairs oligomerization of the F1FO-ATP synthase similar to Mic60. Applying complexome profiling, we observed that deletion of Mic27 destabilizes the MICOS complex but does not impair formation of a high molecular weight Mic10 subcomplex. Moreover, this Mic10 subcomplex comigrates with the dimeric F1FO-ATP synthase in a Mic27-independent manner. Further, we observed a chemical crosslink of Mic10 to Mic27 and of Mic10 to the F1FO-ATP synthase subunit e. We corroborate the physical interaction of the MICOS complex and the F1FO-ATP synthase. We propose a model in which part of the F1FO-ATP synthase is linked to the MICOS complex via Mic10 and Mic27 and by that is regulating CJ formation.
Lieblingsbild
(2017)
Dieses Bild ist wichtig, weil wir daran verstanden haben, wie in der Zelle fehlerhaftes Spleißen verhindert wird. Dazu muss man wissen, dass unsere Gene sich aus Exons und dazwischenliegenden Introns zusammensetzen. Während des Spleißens werden die Introns entfernt und die Exons in ein reifes Transkript zusammengefügt, das dann für ein Protein kodiert. Allerdings gibt es innerhalb der Introns viele Bereiche, die einem Exon sehr ähnlich sehen. Werden diese sogenannten "PseudoExons" fälschlicherweise während des Spleißprozesses erkannt und in das reife Transkript eingebaut, kann das fatale Folgen für das kodierte Protein und oft die gesamte Zelle haben. ...
"Ästhetisch ist, was hilft"
(2017)
Lieblingsbild
(2017)
Das rechte Bild stellt die Elektronendichte eines menschlichen Proteins dar, gewonnen durch die Röntgenstrahl-Beugung an Kristallen dieses Proteins. Die Struktur hat Sagar Bhogaraju 2016 aufgeklärt. Das linke Bild stellt ein erstes Strukturmodell auf der Basis der gemessenen Elektronendichte dar. ...
Synaptic release sites are characterized by exocytosis-competent synaptic vesicles tightly anchored to the presynaptic active zone (PAZ) whose proteome orchestrates the fast signaling events involved in synaptic vesicle cycle and plasticity. Allocation of the amyloid precursor protein (APP) to the PAZ proteome implicated a functional impact of APP in neuronal communication. In this study, we combined state-of-the-art proteomics, electrophysiology and bioinformatics to address protein abundance and functional changes at the native hippocampal PAZ in young and old APP-KO mice. We evaluated if APP deletion has an impact on the metabolic activity of presynaptic mitochondria. Furthermore, we quantified differences in the phosphorylation status after long-term-potentiation (LTP) induction at the purified native PAZ. We observed an increase in the phosphorylation of the signaling enzyme calmodulin-dependent kinase II (CaMKII) only in old APP-KO mice. During aging APP deletion is accompanied by a severe decrease in metabolic activity and hyperphosphorylation of CaMKII. This attributes an essential functional role to APP at hippocampal PAZ and putative molecular mechanisms underlying the age-dependent impairments in learning and memory in APP-KO mice.
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.
The full-length translation-regulating add adenine riboswitch (Asw) from Vibrio vulnificus has a more complex conformational space than its isolated aptamer domain. In addition to the predicted apo (apoA) and holo conformation that feature the conserved three-way junctional purine riboswitch aptamer, it adopts a second apo (apoB) conformation with a fundamentally different secondary structure. Here, we characterized the ligand-dependent conformational dynamics of the full-length add Asw by NMR and by single-molecule FRET (smFRET) spectroscopy. Both methods revealed an adenine-induced secondary structure switch from the apoB-form to the apoA-form that involves no tertiary structural interactions between aptamer and expression platform. This strongly suggests that the add Asw triggers translation by capturing the apoA-form secondary structure in the holo state. Intriguingly, NMR indicated a homogenous, docked aptamer kissing loop fold for apoA and holo, while smFRET showed persistent aptamer kissing loop docking dynamics between comparably stable, undocked and docked substates of the apoA and the holo conformation. Unraveling the folding of large junctional riboswitches thus requires the integration of complementary solution structural techniques such as NMR and smFRET.
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.
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.
The turnover of endoplasmic reticulum (ER) ensures the correct biological activity of its distinct domains. In mammalian cells, the ER is degraded via a selective autophagy pathway (ER-phagy), mediated by two specific receptors: FAM134B, responsible for the turnover of ER sheets and SEC62 that regulates ER recovery following stress. Here, we identified reticulon 3 (RTN3) as a specific receptor for the degradation of ER tubules. Oligomerization of the long isoform of RTN3 is sufficient to trigger fragmentation of ER tubules. The long N-terminal region of RTN3 contains several newly identified LC3-interacting regions (LIR). Binding to LC3s/GABARAPs is essential for the fragmentation of ER tubules and their delivery to lysosomes. RTN3-mediated ER-phagy requires conventional autophagy components, but is independent of FAM134B. None of the other reticulon family members have the ability to induce fragmentation of ER tubules during starvation. Therefore, we assign a unique function to RTN3 during autophagy.