CadC-mediated activation of the cadBA promoter in Escherichia coli

The transcriptional activator CadC in Escherichia coli, a member of the ToxR-like proteins, activates transcription of the cadBA operon encoding the lysine decarboxylase CadA and the lysine-cadaverine antiporter CadB. cadBA is induced under conditions of acidic external pH and exogenous lysine; anoxic conditions raise the expression level up to 10 times. To characterize the binding mechanism of CadC, procedures for the purification of this membrane-integrated protein and its reconstitution into proteoliposomes were established. The binding sites of CadC upstream of the cadBA promoter region were determined by in vitro DNaseI protection analysis. Two regions were protected during DNaseI digestion, one from - 144 to - 112 bp, designated Cad1, and another one from - 89 to - 59 bp, designated Cad2. Binding of purified CadC to Cad1 and Cad2 was further characterized by DNA-binding assays, indicating that CadC was able to bind to both DNA fragments. Genetic analysis with promoter-lacZ fusions confirmed that both sites, Cad1 and Cad2, are essential for activation of cadBA transcription. Moreover, these experiments revealed that binding of H-NS upstream of the CadC-binding sites is necessary for repression of cadBA expression at neutral pH and under aerobic conditions. Based on these results, a model for transcriptional regulation of the cadBA operon is proposed, according to which H-NS is involved in the formation of a repression complex under non-inducing conditions. This complex is dissolved by binding of CadC to Cad1 under inducing conditions. Upon binding of CadC to Cad2 cadBA expression is activated. Copyright (C) 2005 S. Karger AG, Basel

Form Invariance of the Neutrino Mass Matrix

Consider the most general $3 \times 3$ Majorana neutrino mass matrix $\cal M$. Motivated by present neutrino-oscillation data, much theoretical effort is directed at reducing it to a specific texture in terms of a small number of parameters. This procedure is often {\it ad hoc}. I propose instead that for any $\cal M$ one may choose, it should satisfy the condition $U {\cal M} U^T = {\cal M}$, where $U \neq 1$ is a specific unitary matrix such that $U^N$ represents a well-defined discrete symmetry in the $\nu_{e,\mu,\tau}$ basis, $N$ being a particular integer not necessarily equal to one. I illustrate this idea with a number of examples, including the realistic case of an inverted hierarchy of neutrino masses.Comment: Version to appear in PR

Regulatory post-translational modifications and protein-protein interactions involved in function and proteostasis of aromatic amino acid hydroxylases

The non-heme iron and (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) dependent aromatic amino acid hydroxylases (AAAHs) family of enzymes include phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), and tryptophan hydroxylase 1 and 2 (TPH1 and TPH2). PAH catalyses the rate-limiting step in the catabolism of phenylalanine (L-Phe) that mainly takes place in the liver. TH catalyses the first and rate-limiting step in the biosynthesis of catecholamine neurotransmitters and hormones dopamine, norepinephrine and epinephrine in the brain and periphery. TPHs catalyse the first and rate-limiting step in the biosynthesis of serotonin in the peripheral (TPH1) and the central (TPH2) nervous systems. The AAAHs are of physiological and clinical importance. Dysfunctional PAH results in phenylketonuria (PKU), characterised by elevated levels of L-Phe in the blood, which can lead to brain damage. Catecholamine deficiency, due to dysfunctional TH, leads to motor dysfunction and neuropsychiatric disorders, such as TH deficiency (THD) and Parkinson’s disease. Reduced level of serotonin has been linked to anxiety disorder, depression, posttraumatic stress disorder and attention deficit hyperactivity disorder. Hence, the reactions catalysed by the AAAHs are important and tightly regulated. The aim of this thesis was to study the regulation of the AAAHs PAH and TH both in physiological and pathological states. We focused on regulatory mechanisms by selected post-translational modifications and protein-protein interactions and phosphorylation, investigating their role in the function, localisation and proteostasis of these enzymes using cellular and animal models. We investigated the role of DNAJC12, a type III member of the HSP40/DNAJ family, in the folding and degradation of wild-type (Wt) and mutant PAH. We observed a positive correlation between DNAJC12 and Wt and mutant PAH protein levels in the soluble cellular fractions. Detailed characterisations in liver lysates of the hyperphenylalaninemic Enu1 mouse (p.V106A-PAH mutation) revealed increased ubiquitination, instability, and aggregation of mutant PAH compared with Wt PAH. Furthermore, we showed that in the liver lysates, DNAJC12 interacts with both Wt and mono-ubiquitinated PAH; also, PAH mutation did not alter mRNA expression of DNAJC12. Our results support the role of DNAJC12 not only in proper folding but also in the processing of misfolded ubiquitinated PAH. We characterised a new custom-made Pah-R261Q knock-in mouse carrying mutation c.782G>A in the Pah gene. The homozygous Pah-R261Q mice exhibited reduced PAH activity and BH4 responsive hyperphenylalaninemia. Moreover, the mutant mice presented a reduced BH4 content in the liver, altered lipid metabolism, and increased oxidative stress, including increased mRNA expression of DNAJC12. Furthermore, the Pah-R261Q mice displayed large amyloid-like ubiquitinated PAH aggregates. The colocalisation of mutant PAH with selective autophagy markers indicated the involvement of the autophagic pathway in the clearance of mutant aggregates. These findings indicate a paradigm shift from a loss-of-function disorder to a toxic gain-of-function in PKU pathology. We next investigated the functional role of Ser31 phosphorylation in the regulation of TH in the cellular models. We observed that the perinuclear distribution of THpSer31 was concomitant with Golgi complex and synaptic vesicle marker in rat and human dopaminergic cells. The co-distribution of THpSer31 with vesicular monoamine transporter 2 (VMAT2) and α-synuclein (α-syn) in cells and their detection as co-immunoprecipitant in mouse brain lysate indicated an association of TH with vesicles. Furthermore, disruption of the microtubules caused accumulation of TH in the cell soma. Our study revealed that Ser31 phosphorylation regulates the subcellular localisation of TH by facilitating protein-protein interaction with VMAT2 and α-syn and enabling its transport toward axon terminals along microtubules. Finally, using SH-SY5Y cells, we sought to investigate the relationship between phosphorylation at different phosphosites and the nuclear distribution of TH, which was earlier proposed to be associated with Ser19 phosphorylation. We indeed observed that THpSer19 was predominantly nuclear, yet the phospho-null mutant of Ser19 (V5-TH-S19A) surprisingly accumulated significantly higher in the nuclear fraction when compared to Wt. Moreover, other phosphosites (Ser31 and Ser40) did not seem to influence the nuclear distribution of TH. When the phospho-null mutant of Thr8 (V5-TH-T8A) was expressed in SH-SY5Y cells, recombinant TH in the nuclear fraction was significantly reduced compared to Wt and the phospho-mimicking mutant V5-THT8E, indicating the potential role of Thr8 phosphorylation in the nuclear distribution of TH. In addition, inhibition of importin-β also reduced the amount of recombinant TH in the nucleus suggesting the involvement of the importin-β/RanGTP system in the nuclear localisation of TH in SH-SY5Y cells. To conclude, this study has brought new insights on the short-term regulation of AAAHs (PAH and TH) in physiological and pathological conditions by interacting with partners and by post-translational modifications, such as ubiquitination and phosphorylation (for TH), which ultimately affect their abundance, function and availability in different compartments of cells. Thus, this study has shed light on some of the molecular mechanisms involved in the proteostasis of AAAHs. Together, these findings open new research avenues to better understand disorders associated with the AAAHs.Doktorgradsavhandlin

Scanning tunneling spectroscopic studies of the pairing state of cuprate superconductors

Quasiparticle tunneling spectra of both hole-doped (p-type) and electron-doped (n-type) cuprates are studied using a low-temperature scanning tunneling microscope. The results reveal that neither the pairing symmetry nor the pseudogap phenomenon is universal among all cuprates, and that the response of n-type cuprates to quantum impurities is drastically different from that of the p-type cuprates. The only ubiquitous features among all cuprates appear to be the strong electronic correlation and the nearest-neighbor antiferromagnetic Cu2+-Cu2+ coupling in the CuO2 planes

Absolute differential cross sections for electron-impact excitation of CO near threshold: II. The Rydberg states of CO

Absolute differential cross sections for electron-impact excitation of Rydberg states of CO have been measured from threshold to 3.7 eV above threshold and for scattering angles between 20° and 140°. Measured excitation functions for the b 3Σ+, B 1Σ+ and E 1π states are compared with cross sections calculated by the Schwinger multichannel method. The behaviour of the excitation functions for these states and for the j 3Σ+ and C 1Σ+ states is analysed in terms of negative-ion states. One of these resonances has not been previously reported