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YS-121 [2-(4-chloro-6-(2,3-dimethylphenylamino)pyrimidin-2-ylthio)octanoic acid] is the result of target-oriented structural derivatization of pirinixic acid. It is a potent dual PPARα/γ-agonist, as well as a potent dual 5-LO/mPGES-1-inhibitor. Additionally, recent studies showed an anti-inflammatory efficacy in vivo. Because of its interference with many targets, YS-121 is a promising drug candidate for the treatment of inflammatory diseases. Ongoing preclinical studies will thus necessitate huge amounts of YS-121. To cope with those requirements, we have optimized the synthesis of YS-121. Surprisingly, we isolated and characterized byproducts during the resulting from nucleophilic aromatic substitution reactions by different tertiary alkylamines at a heteroaromatic halide. These amines should actually serve as assisting bases, because of their low nucleophilicity. This astonishing fact was not described in former publications concerning that type of reaction and, therefore, might be useful for further reaction improvement in general. Furthermore, we could develop a proposal for the mechanism of that byproduct formation.
Tectonin β-propeller containing protein 2 (TECPR2) was first identified in a mass- spectrometric approach as an interactor of GABARAP, an ATG8-family protein playing a role in autophagy. The mammalian ATG8 protein family consists of seven members, namely MAP1LC3A (LC3A), MAP1LC3B (LC3B), MAP1LC3C (LC3C), GABARAP, GABARAPL1 and GABARAPL2. All share an ubiquitin-like core and possess two additional N-terminal α-helices, which are important for the distinct functions of the proteins. First determined in various organelles the ATG8 proteins are shown to be involved in autophagy, supporting the formation and cargo recruitment of autophagosomes, the vesicles transporting cargo for autophagic degradation.
Autophagy is the process of recycling cytoplasmic contents by degradation of misfolded proteins or damaged organelles in order to supply nutrients. Also clearance of pathogens can be achieved via autophagy. Importantly, LC3B is incorporated into the autophagosomal membrane and is therefore used as the main marker for autophagosomes. Previous studies exhibited that depletion of TECPR2 leads to a loss of LC3B-positive structures in cells, which suggests TECPR2 to positively regulate autophagic processes.
A frame shift deletion in the gene encoding for TECPR2 causes the generation of a premature stop codon and subsequent an unstable version of the protein, which is then degraded. Mutation in the TECPR2 gene triggers a neurodegenerative disorder termed hereditary spastic paraparesis (HSP). HSPs are a diverse group of neurodegenerative diseases that are characterized by spasticity in prevalent lower extremities and were mediated by a loss of axonal integrity of the corticospinal motor neurons. In the context of HSP more than 50 gene loci were identified by now. While TECPR2 is a human ATG8 binding protein and positive regulator of autophagy causing a form of HSP, the exact function of TECPR2 is unknown.
This study primarily focused on the determination of TECPR2’s binding mode to ATG8 proteins in vitro and in cells. The association of TECPR2 to all ATG8-family proteins was confirmed in in vitro pulldown experiments. Following fragment-based binding and peptide array experiments, the LC3-interacting region (LIR) of TECPR2 could be verified with mutants of TECPR2 lacking the LIR motif. Nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) were conducted to gain deeper insights into the binding preference to the different ATG8-family members. Moreover, the crystal structure of TECPR2-LIR was solved. In cells colocalization studies with overexpressed ATG8 proteins unraveled a preferential binding to the LC3-subfamily.
Further, mass spectrometric analysis revealed novel association partners of TECPR2: SEC24D, HOPS and BLOC-1, all of those participating in different endomembrane trafficking pathways. Interaction and colocalization of TECPR2 with these components was validated with several immunoprecipitation experiments and the N-terminal part of the protein comprising the WD40-domain could be defined as the binding site for all three of the association partners. In further approaches, the requirement of the LIR-motif and the necessity of the availability of LC3 protein for the particular interactions were determined. Interestingly, in the absence of LC3C the binding of TECPR2 to SEC24D was completely disrupted whereas a loss of LC3B only resulted in a decreased association. Notably, the binding proteins were not subjected to autophagosomal degradation, indicating that TECPR2 may operate as a multifunctional scaffold protein. While depletion of TECPR2 destabilized HOPS and BLOC-1, the autophagy defect observed in TECRP2-deficient cells could not be attributed to functional impairment of these two complexes.
Moreover, loss of TECPR2 led to a decline in protein levels of SEC24D and of its heterodimer partner SEC23A. Thus, TECPR2 is required to regulate the protein levels of SEC23A and SEC24D and subsequently the formation of the heterodimers. Together, SEC24D and SEC23A form the inner coat of COPII vesicles. These vesicles are responsible for the anterograde transport of cargo from the ER toward the Golgi compartment. COPII-coated vesicles are secreted form ER at distinct sites, termed ER exit sites (ERES). The small GTPase SAR1A maintains the vesicle budding, coating and secretion at the ERES. Together with SEC13, SEC31 forms the outer coat of the COPII vesicles and therefore serves as a general ERES marker.
Consistent with a defect in COPII coat assembly, the number of ERES diminished in the absence of TECPR2. These phenotypes could be rescued by the wildtype TECPR2 protein but not by the LIR-mutant. Intriguingly, these results were mimicked by depletion of LC3C, which localized to ERES. By monitoring the release of various cargos from ER in dependency of TECPR2 or LC3C, a role of both proteins in ER export was determined. These facts indicated that TECPR2 cooperates with LC3C to facilitate COPII assembly, ERES maintenance and ER export. Notably, fibroblast derived from a HSP patient carrying mutated TECPR2 showed diminished SEC24D protein levels and delayed ER export.
Concurrent with emerging evidence for a role of ERES in autophagosome formation, depletion of TECPR2 or LC3C or overexpression of a constitutive inactive SAR1 mutant reduced puncta formation of the early autophagosomal protein WIPI2.
In summary, this study uncovered a role for TECPR2 in ER export at ERES through interaction and stabilization of SEC24D, a COPII coat protein. This process also depended on ATG8-family protein LC3C, which is localized at ERES. Both proteins are required for correct COPII-mediated secretion. Moreover, the presence of TECPR2 and LC3C on ER allows development of omegasomes, membranous structures budding ER to form autophagosomes, by stabilization of WIPI2 and therefore contribute to autophagosome formation.
Autophagy is a highly conserved catabolic process through which defective or otherwise harmful cellular components are targeted for degradation via the lysosomal route. Regulatory pathways, involving post-translational modifications such as phosphorylation, play a critical role in controlling this tightly orchestrated process. Here, we demonstrate that TBK1 regulates autophagy by phosphorylating autophagy modifiers LC3C and GABARAP-L2 on surface-exposed serine residues (LC3C S93 and S96; GABARAP-L2 S87 and S88). This phosphorylation event impedes their binding to the processing enzyme ATG4 by destabilizing the complex. Phosphorylated LC3C/GABARAP-L2 cannot be removed from liposomes by ATG4 and are thus protected from ATG4-mediated premature removal from nascent autoph-agosomes. This ensures a steady coat of lipidated LC3C/GABARAP-L2 throughout the early steps in autophagosome formation and aids in maintaining a unidirectional flow of the autophagosome to the lysosome. Taken together, we present a new regulatory mechanism of autophagy, which influences the conjugation and de-conjugation of LC3C and GABARAP-L2 to autophagosomes by TBK1-mediated phosphorylation.
Modification of SMN2 exon 7 (E7) splicing is a validated therapeutic strategy against spinal muscular atrophy (SMA). However, a target-based approach to identify small-molecule E7 splicing modifiers has not been attempted, which could reveal novel therapies with improved mechanistic insight. Here, we chose as a target the stem-loop RNA structure TSL2, which overlaps with the 5′ splicing site of E7. A small-molecule TSL2-binding compound, homocarbonyltopsentin (PK4C9), was identified that increases E7 splicing to therapeutic levels and rescues downstream molecular alterations in SMA cells. High-resolution NMR combined with molecular modelling revealed that PK4C9 binds to pentaloop conformations of TSL2 and promotes a shift to triloop conformations that display enhanced E7 splicing. Collectively, our study validates TSL2 as a target for small-molecule drug discovery in SMA, identifies a novel mechanism of action for an E7 splicing modifier, and sets a precedent for other splicing-mediated diseases where RNA structure could be similarly targeted.
Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins.
Nanopores are key in portable sequencing and research given their ability to transport elongated DNA or small bioactive molecules through narrow transmembrane channels. Transport of folded proteins could lead to similar scientific and technological benefits. Yet this has not been realised due to the shortage of wide and structurally defined natural pores. Here we report that a synthetic nanopore designed via DNA nanotechnology can accommodate folded proteins. Transport of fluorescent proteins through single pores is kinetically analysed using massively parallel optical readout with transparent silicon-on-insulator cavity chips vs. electrical recordings to reveal an at least 20-fold higher speed for the electrically driven movement. Pores nevertheless allow a high diffusive flux of more than 66 molecules per second that can also be directed beyond equillibria. The pores may be exploited to sense diagnostically relevant proteins with portable analysis technology, to create molecular gates for drug delivery, or to build synthetic cells.
Synthesis, crystal structure and structure–property relations of strontium orthocarbonate, Sr2CO4
(2021)
Carbonates containing CO4 groups as building blocks have recently been discovered. A new orthocarbonate, Sr2CO4 is synthesized at 92 GPa and at a temperature of 2500 K. Its crystal structure was determined by in situ synchrotron single-crystal X-ray diffraction, selecting a grain from a polycrystalline sample. Strontium orthocarbonate crystallizes in the orthorhombic crystal system (space group Pnma) with CO4, SrO9 and SrO11 polyhedra as the main building blocks. It is isostructural to Ca2CO4. DFT calculations reproduce the experimental findings very well and have, therefore, been used to predict the equation of state, Raman and IR spectra, and to assist in the discussion of bonding in this compound.
In this study, we describe the synthesis of 1,4-disustituted-1,2,3-triazolo-quinazoline ribonucleosides or acyclonucleosides by means of 1,3-dipolar cycloaddition between various O or N-alkylated propargyl-quinazoline and 1'-azido-2',3',5'-tri-O-benzoylribose or activated alkylating agents under microwave conditions. None of the compounds selected showed significant anti-HCV activity in vitro.
A series of novel mono-1,2,3-triazole and bis-1,2,3-triazole acyclonucleoside analogues of 9-(4-hydroxybutyl)guanine was prepared via copper(I)-catalyzed 1,3-dipolar cycloaddition of N-9 propargylpurine, N-1-propargylpyrimidines/as-triazine with the azido-pseudo-sugar 4-azidobutylacetate under solvent-free microwave conditions, followed by treatment with K2CO3/MeOH, or NH3/MeOH. All compounds studied in this work were screened for their antiviral activities [against human rhinovirus (HRV) and hepatitis C virus (HCV)] and antibacterial activities against a series of Gram positive and negative bacteria.
Die Beteiligung an Schlüsselfunktionen in zellulären Signalwegen macht Kinasen zu einem vielversprechenden Ansatzpunkt in der Wirkstoffentwicklung bei verschiedenen menschlichen Erkrankungen wie z.B. Krebs oder auch Autoimmun- und Entzündungskrankheiten. Die Prävention von post-translationalen Modifikationen durch Phosphorylierung und somit die Regulierung der nachgeschalteten Signalwege ist das Ziel von Kinaseinhibitoren. Die katalytische Aktivität von Kinasen ist abhängig von ATP, welches im hochkonservierten aktiven Zentrum bindet. Bedingt durch diese kinomweite hohe Konservierung stellt die Entwicklung von hoch selektiven ATP-mimetischen Inhibitoren eine Herausforderung dar. Typische ATP-Mimetika sind flach und die oft hydrophoben Moleküle weisen meist eine große Zahl an frei rotierbaren Bindungen auf. Um das aus dieser Flexibilität hervorgehende Problem der teils mangelnden Selektivität zu umgehen, kann eine bioaktive Konformation des Inhibitors durch Makrozyklisierung fixiert werden. Als Konsequenz dieser konformationellen Einschränkung können die entropischen Kosten während des Bindens reduziert werden und folglich zu einer gesteigerten Affinität gegenüber der Kinase führen.
Der Grundstein dieser Arbeit war der makrozyklische Pyrazolo[1,5-a]pyrimidin basierte FLT3 Kinaseinhibitor ODS2004070 (37). Im Rahmen eines kinomweiten Screenings konnten hohe Affinitäten zu verschiedensten Kinasen detektiert werden, was 37 zu einer guten Leitstruktur für das Design von potenten und selektiven Kinaseinhibitoren machte. Im Rahmen dieser Arbeit blieb das literaturbekannte Pyrazolo[1,5-a]pyrimidin basierte ATP-mimetische Bindemotiv sowie das makrozyklische Grundgerüst 37 bis auf einige wenige Variation unverändert.
Strukturelle Optimierungen zur Fokussierung der Selektivität wurden am sekundären Amin zwischen Bindemotiv und Linker als auch über die freie Carbonsäure durchgeführt. Mit einer Anzahl von mehr als 430 identifizierten Phosphorylierungsstellen ist die pleiotropisch und konstitutiv aktive Casein Kinase 2 (CK2) an verschiedensten zellulären Prozessen wie dem Verlauf des Zellzyklus, der Apoptose oder der Transkription regulatorisch beteiligt. Die Fehlregulation von CK2 wird häufig mit der Pathologie von Krankheiten wie zum Beispiel Krebs assoziiert, was CK2 zu einem vielversprechenden Ziel klinischer Untersuchungen macht.
Im Rahmen des CK2-Projekts war es möglich, durch spezifische Modifikationen an 37, die hoch selektiven und potenten CK2-Inhibitoren 47 und 60 zu entwickeln. Ebenfalls gezeigt wurde, dass kleine strukturelle Veränderungen, wie z.B. Makrozyklisierung, einen signifikanten Effekt auf Selektivität und Potenz des Inhibitors haben kann.
Weiter Untersuchungen der Verbindungen lenkten den Fokus weiterer Arbeiten u.a. auf die Serin/Threonin Kinase 17A (STK17A) oder auch death-associated protein kinase-related apoptosis-inducing protein kinase 1 (DRAK1) genannt. Sie ist Teil der DAPK Familie und gehört zusammen mit anderen Kinasen zu den weniger erforschten Kinasen. Bis heute ist nicht viel über ihre zellulären Funktionen und die Beteiligung an pathophysiologischen Prozessen bekannt. Berichtet wurde jedoch eine Überexpression in verschiedenen Formen von Hirntumoren des zentralen Nervensystems (Gliom). Strukturelle Modifikationen, unter Erhalt des makrozyklischen Grundgerüsts 37, führten zu dem hoch selektiven und potenten DRAK1 Inhibitor 121, der alle Kriterien für eine chemical probe Verbindung erfüllt.
Ein weiteres Ziel dieser Arbeit war die AP-2-assoziierte Protein Kinase 1 (AAK1) aus der NAK Familie, bestehend aus AAK1, BIKE und GAK. Sie ist als potenzielles therapeutisches Ziel für viele verschieden Krankheiten wie z.B. neuropathische Schmerzen, Schizophrenie und Parkinson identifiziert. Durch die Regulierung der Clathrin-mediierten Endozytose ist AAK1 an intrazellulären Bewegungen verschiedener nicht zusammenhängenden RNS- und DNSViren, wie beispielsweise HCV, DENV oder EBOV, beteiligt. Ebenfalls berichtet wurde eine mögliche Assoziation mit dem SARS-CoV-2 Virus, was das Interesse an neuen selektiven AAK1 Inhibitoren verstärkte. Die Entwicklung der hochpotenten und selektiven AAK1 Inhibitoren 61 und 63 basierte ebenfalls auf dem makrozyklischen Grundgerüst 37, das bereits im CK2- und DRAK1-Projekt verwendet wurde.
Zusammenfassend lässt sich sagen, dass es im Rahmen dieser Arbeit gelungen ist, ausgehend von einem höchst unselektiven makrozyklischen Grundgerüst, hochpotente und selektive Kinaseinhibitoren für CK2, DRAK1 und AAK1 zu entwickeln und zu charakterisieren. Im Zuge von Untersuchungen verschiedener Struktur-Wirkungsbeziehungen wurde gezeigt, dass es durch geringfügige strukturelle Modifikationen möglich ist, die kinomweite Selektivität zu variieren und auf eine Kinase zu fokussieren. Diese Arbeit brachte nicht nur die erwähnten Inhibitoren hervor, sondern bildet auch die Grundlage für weitere Projekte zur Entwicklung von hoch potenten und selektiven Verbindungen als potenzielle chemische Werkzeuge für den Einsatz in der Forschung.
Perchlorinated polysilanes were synthesized by polymerization of tetrachlorosilane under cold plasma conditions with hydrogen as a reducing agent. Subsequent selective cleavage of the resulting polymer yielded oligochlorosilanes SinCl2n+2 (n = 2, 3) from which the octachlorotrisilane (n = 3, Cl8Si3, OCTS) was used as a novel precursor for the synthesis of single-crystalline Si nanowires (NW) by the well-established vapor–liquid–solid (VLS) mechanism. By adding doping agents, specifically BBr3 and PCl3, we achieved highly p- and n-type doped Si-NWs by means of atmospheric-pressure chemical vapor deposition (APCVD). These as grown NWs were investigated by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM), as well as electrical measurements of the NWs integrated in four-terminal and back-gated MOSFET modules. The intrinsic NWs appeared to be highly crystalline, with a preferred growth direction of [111] and a specific resistivity of ρ = 6 kΩ·cm. The doped NWs appeared to be [112] oriented with a specific resistivity of ρ = 198 mΩ·cm for p-type Si-NWs and ρ = 2.7 mΩ·cm for n-doped Si-NWs, revealing excellent dopant activation.
Synthesis and crystal structure of 2-(2-hydroxyphenyl)-1,3-bis(4-methoxybenzyl)-1,3-diazinan-5-ol
(2022)
The redetermined structure of 2-(2-hydroxyphenyl)-1,3-bis(4-methoxybenzyl)-1,3-diazinan-5-ol, C26H30N2O4, at 173 K has orthorhombic (Pbca) symmetry. It was previously described by Bolte et al. [ Private Communication (refcode EWICEV). CCDC, Cambridge, England]. The title compound resulted from the condensation reaction between 1,3-bis{[(4-methoxyphenyl)methyl]amino}propan-2-ol and 2-hydroxybenzaldehyde in CH3OH. The structure exhibits disorder. One of the 4-methoxybenzyl groups, the hydroxy group bonded to the 1,3-diazinan ring, and the methyl group of the methoxy residue are disordered over two orientations, with occupancies of 0.807 (3)/0.193 (3), 0.642 (5)/0.358 (5), and 0.82 (4)/0.18 (4), respectively. The dihedral angles between the mean planes of the central 1,3-diazinan-5-ol and the 4-methoxyphenyl rings (both occupancy components of the disordered ring) are 88.65 (13), 85.79 (14) and 83.4 (7)°. The crystal packing is sustained by C—H...O and O—H...π interactions, giving rise to infinite chains running along the b-axis direction.
The title thiourea was synthesized by reaction of 3,4,5-trimethoxybenzoyl isothiocyante with 3-fluoroaniline. The 3,4,5-trimethoxybenzoyl isothiocyante was produced in situ by reaction of 3,4,5-trimethoxybenzoyl chloride with ammonium thiocyanate in dry acetonitrile. The structure was confirmed by the spectroscopic, elemental analysis and single crystal X-ray diffraction data. It crystallizes in the monoclinic space group P21/c with unit cell dimensions a = 13.0966(9), b = 16.6460(13), c = 7.8448(5), β = 106.721(5)°, V 1637.9(2) ų, Z = 4.
The respiratory chain of Escherichia coli contains two different types of terminal oxidase that are differentially regulated as a response to changing environmental conditions. These oxidoreductases catalyze the reduction of molecular oxygen to water and contribute to the proton motive force. The cytochrome bo3 oxidase (cyt bo3) acts as the primary terminal oxidase under atmospheric oxygen levels, whereas the bd‐type oxidase is most abundant under microaerobic conditions. In E. coli, both types of respiratory terminal oxidase (HCO and bd‐type) use ubiquinol‐8 as electron donor. Here, we assess the inhibitory potential of newly designed and synthesized 3‐alkylated Lawson derivatives through L‐proline‐catalyzed three‐component reductive alkylation (TCRA). The inhibitory effects of these Lawson derivatives on the terminal oxidases of E. coli (cyt bo3 and cyt bd‐I) were tested potentiometrically. Four compounds were able to reduce the oxidoreductase activity of cyt bo3 by more than 50 % without affecting the cyt bd‐I activity. Moreover, two inhibitors for both cyt bo3 and cyt bd‐I oxidase could be identified. Based on molecular‐docking simulations, we propose binding modes of the new Lawson inhibitors. The molecular fragment benzyl enhances the inhibitory potential and selectivity for cyt bo3, whereas heterocycles reduce this effect. This work extends the library of 3‐alkylated Lawson derivatives as selective inhibitors for respiratory oxidases and provides molecular probes for detailed investigations of the mechanisms of respiratory‐chain enzymes of E. coli.
Supersilylated tetrachlorodigermane (tBu3Si)Cl2GeGeCl2(SitBu3) and trigermoxetane (tBu3Si)3Ge3Cl3O
(2004)
In contrast to the tetrachlorodigermane (tBu3Si)Cl2Ge-GeCl2(SitBu3), the cis,transcyclotrigermane (tBu3SiGeCl)3 is sensitive to oxygen. Its treatment with O2 at ambient temperature leads to the trigermoxetane (tBu3Si)3Ge3Cl3O. According to an X-ray structure analysis of single crystals consisting of cocrystallized (tBu3Si)3Ge3Cl3O and (tBu3Si)Cl2Ge-GeCl2(SitBu3) the trigermaoxetane contains an almost planar Ge3O-ring while the tetrachlorodigermane (tBu3Si)Cl2Ge- GeCl2(SitBu3) possesses a Si-Ge-Ge-Si chain which is exactly all trans,
Stimulated emission from chemically formed excited iodine molecules has been observed. The emission originates from the vibrational state ν′ = 55 of I2(B 3 II). The excited molecules are produced by a three body recombination reaction.
The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread . Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse.
Post-translational modification of proteins with ubiquitin-like SUMO modifiers is a tightly regulated and highly dynamic process. The SENP family of SUMO-specific isopeptidases comprises six cysteine proteases. They are instrumental in counterbalancing SUMO conjugation, but their regulation is not well understood. We demonstrate that in hypoxic cell extracts, the catalytic activity of SENP family members, in particular SENP1 and SENP3, is inhibited in a rapid and fully reversible process. Comparative mass spectrometry from normoxic and hypoxic cells defines a subset of hypoxia-induced SUMO1 targets, including SUMO ligases RanBP2 and PIAS2, glucose transporter 1, and transcriptional regulators. Among the most strongly induced targets, we identified the transcriptional co-repressor BHLHE40, which controls hypoxic gene expression programs. We provide evidence that SUMOylation of BHLHE40 is reversed by SENP1 and contributes to transcriptional repression of the metabolic master regulator gene PGC-1α. We propose a pathway that connects oxygen-controlled SENP activity to hypoxic reprogramming of metabolism.
Sucrose- and H+-dependent charge movements associated with the gating of sucrose transporter ZmSUT1
(2010)
Background: In contrast to man the majority of higher plants use sucrose as mobile carbohydrate. Accordingly proton-driven sucrose transporters are crucial for cell-to-cell and long-distance distribution within the plant body. Generally very negative plant membrane potentials and the ability to accumulate sucrose quantities of more than 1 M document that plants must have evolved transporters with unique structural and functional features.
Methodology/Principal Findings: To unravel the functional properties of one specific high capacity plasma membrane sucrose transporter in detail, we expressed the sucrose/H+ co-transporter from maize ZmSUT1 in Xenopus oocytes. Application of sucrose in an acidic pH environment elicited inward proton currents. Interestingly the sucrose-dependent H+ transport was associated with a decrease in membrane capacitance (Cm). In addition to sucrose Cm was modulated by the membrane potential and external protons. In order to explore the molecular mechanism underlying these Cm changes, presteady-state currents (Ipre) of ZmSUT1 transport were analyzed. Decay of Ipre could be best fitted by double exponentials. When plotted against the voltage the charge Q, associated to Ipre, was dependent on sucrose and protons. The mathematical derivative of the charge Q versus voltage was well in line with the observed Cm changes. Based on these parameters a turnover rate of 500 molecules sucrose/s was calculated. In contrast to gating currents of voltage dependent-potassium channels the analysis of ZmSUT1-derived presteady-state currents in the absence of sucrose (I = Q/τ) was sufficient to predict ZmSUT1 transport-associated currents.
Conclusions: Taken together our results indicate that in the absence of sucrose, ‘trapped’ protons move back and forth between an outer and an inner site within the transmembrane domains of ZmSUT1. This movement of protons in the electric field of the membrane gives rise to the presteady-state currents and in turn to Cm changes. Upon application of external sucrose, protons can pass the membrane turning presteady-state into transport currents.
Tsetse flies are the transmitting vector of trypanosomes causing human sleeping sickness and animal trypanosomiasis in sub-saharan Africa. 3-alkylphenols are used as attractants in tsetse fly traps to reduce the spread of the disease. Here we present an inexpensive production method for 3-ethylphenol (3-EP) and 3-propylphenol (3-PP) by microbial fermentation of sugars. Heterologous expression in the yeast Saccharomyces cerevisiae of phosphopantetheinyltransferase-activated 6-methylsalicylic acid (6-MSA) synthase (MSAS) and 6-MSA decarboxylase converted acetyl-CoA as a priming unit via 6-MSA into 3-methylphenol (3-MP). We exploited the substrate promiscuity of MSAS to utilize propionyl-CoA and butyryl-CoA as alternative priming units and the substrate promiscuity of 6-MSA decarboxylase to produce 3-EP and 3-PP in yeast fermentations. Increasing the formation of propionyl-CoA by expression of a bacterial propionyl-CoA synthetase, feeding of propionate and blocking propionyl-CoA degradation led to the production of up to 12.5 mg/L 3-EP. Introduction of a heterologous ‘reverse ß-oxidation’ pathway provided enough butyryl-CoA for the production of 3-PP, reaching titers of up to 2.6 mg/L. As the concentrations of 3-alkylphenols are close to the range of the concentrations deployed in tsetse fly traps, the yeast broths might become promising and inexpensive sources for attractants, producible on site by rural communities in Africa.