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

The Pah-R261Q mouse reveals oxidative stress associated with amyloid-like hepatic aggregation of mutant phenylalanine hydroxylase

Phenylketonuria (PKU) is caused by autosomal recessive variants in phenylalanine hydroxylase (PAH), leading to systemic accumulation of L-phenylalanine (L-Phe) that may reach neurotoxic levels. A homozygous Pah-R261Q mouse, with a highly prevalent misfolding variant in humans, reveals the expected hepatic PAH activity decrease, systemic L-Phe increase, L-tyrosine and L-tryptophan decrease, and tetrahydrobiopterin-responsive hyperphenylalaninemia. Pah-R261Q mice also present unexpected traits, including altered lipid metabolism, reduction of liver tetrahydrobiopterin content, and a metabolic profile indicative of oxidative stress. Pah-R261Q hepatic tissue exhibits large ubiquitin-positive, amyloid-like oligomeric aggregates of mutant PAH that colocalize with selective autophagy markers. Together, these findings reveal that PKU, customarily considered a loss-of-function disorder, can also have toxic gain-of-function contribution from protein misfolding and aggregation. The proteostasis defect and concomitant oxidative stress may explain the prevalence of comorbid conditions in adult PKU patients, placing this mouse model in an advantageous position for the discovery of mutation-specific biomarkers and therapies.publishedVersio

Hydrogen and Carbon Nanotubes from Pyrolysis-Catalysis of Waste Plastics: A Review

More than 27 million tonnes of waste plastics are generated in Europe each year representing a considerable potential resource. There has been extensive research into the production of liquid fuels and aromatic chemicals from pyrolysis-catalysis of waste plastics. However, there is less work on the production of hydrogen from waste plastics via pyrolysis coupled with catalytic steam reforming. In this paper, the different reactor designs used for hydrogen production from waste plastics are considered and the influence of different catalysts and process parameters on the yield of hydrogen from different types of waste plastics are reviewed. Waste plastics have also been investigated as a source of hydrocarbons for the generation of carbon nanotubes via the chemical vapour deposition route. The influences on the yield and quality of carbon nanotubes derived from waste plastics are reviewed in relation to the reactor designs used for production, catalyst type used for carbon nanotube growth and the influence of operational parameters

Obesity-Related Oxidative Stress: the Impact of Physical Activity and Diet Manipulation

Obesity-related oxidative stress, the imbalance between pro-oxidants and antioxidants (e.g., nitric oxide), has been linked to metabolic and cardiovascular disease, including endothelial dysfunction and atherosclerosis. Reactive oxygen species (ROS) are essential for physiological functions including gene expression, cellular growth, infection defense, and modulating endothelial function. However, elevated ROS and/or diminished antioxidant capacity leading to oxidative stress can lead to dysfunction. Physical activity also results in an acute state of oxidative stress. However, it is likely that chronic physical activity provides a stimulus for favorable oxidative adaptations and enhanced physiological performance and physical health, although distinct responses between aerobic and anaerobic activities warrant further investigation. Studies support the benefits of dietary modification as well as exercise interventions in alleviating oxidative stress susceptibility. Since obese individuals tend to demonstrate elevated markers of oxidative stress, the implications for this population are significant. Therefore, in this review our aim is to discuss (i) the role of oxidative stress and inflammation as associated with obesity-related diseases, (ii) the potential concerns and benefits of exercise-mediated oxidative stress, and (iii) the advantageous role of dietary modification, including acute or chronic caloric restriction and vitamin D supplementation

Multimessenger NuEM Alerts with AMON

The Astrophysical Multimessenger Observatory Network (AMON), has developed a real-time multi-messenger alert system. The system performs coincidence analyses of datasets from gamma-ray and neutrino detectors, making the Neutrino-Electromagnetic (NuEM) alert channel. For these analyses, AMON takes advantage of sub-threshold events, i.e., events that by themselves are not significant in the individual detectors. The main purpose of this channel is to search for gamma-ray counterparts of neutrino events. We will describe the different analyses that make-up this channel and present a selection of recent results

Combined dark matter searches towards dwarf spheroidal galaxies with Fermi-LAT, HAWC, H.E.S.S., MAGIC, and VERITAS

Cosmological and astrophysical observations suggest that 85% of the total matter of the Universe is made of Dark Matter (DM). However, its nature remains one of the most challenging and fundamental open questions of particle physics. Assuming particle DM, this exotic form of matter cannot consist of Standard Model (SM) particles. Many models have been developed to attempt unraveling the nature of DM such as Weakly Interacting Massive Particles (WIMPs), the most favored particle candidates. WIMP annihilations and decay could produce SM particles which in turn hadronize and decay to give SM secondaries such as high energy \u1d6fe rays. In the framework of indirect DM search, observations of promising targets are used to search for signatures of DM annihilation. Among these, the dwarf spheroidal galaxies (dSphs) are commonly favored owing to their expected high DM content and negligible astrophysical background. In this work, we present the very first combination of 20 dSph observations, performed by the Fermi-LAT, HAWC, H.E.S.S., MAGIC, and VERITAS collaborations in order to maximize the sensitivity of DM searches and improve the current results. We use a joint maximum likelihood approach combining each experiment’s individual analysis to derive more constraining upper limits on the WIMP DM self-annihilation cross-section as a function of DM particle mass. We present new DM constraints over the widest mass range ever reported, extending from 5 GeV to 100 TeV thanks to the combination of these five different \u1d6fe-ray instruments

Southern African Large Telescope Spectroscopy of BL Lacs for the CTA project

In the last two decades, very-high-energy gamma-ray astronomy has reached maturity: over 200 sources have been detected, both Galactic and extragalactic, by ground-based experiments. At present, Active Galactic Nuclei (AGN) make up about 40% of the more than 200 sources detected at very high energies with ground-based telescopes, the majority of which are blazars, i.e. their jets are closely aligned with the line of sight to Earth and three quarters of which are classified as high-frequency peaked BL Lac objects. One challenge to studies of the cosmological evolution of BL Lacs is the difficulty of obtaining redshifts from their nearly featureless, continuum-dominated spectra. It is expected that a significant fraction of the AGN to be detected with the future Cherenkov Telescope Array (CTA) observatory will have no spectroscopic redshifts, compromising the reliability of BL Lac population studies, particularly of their cosmic evolution. We started an effort in 2019 to measure the redshifts of a large fraction of the AGN that are likely to be detected with CTA, using the Southern African Large Telescope (SALT). In this contribution, we present two results from an on-going SALT program focused on the determination of BL Lac object redshifts that will be relevant for the CTA observatory

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

Insights into the cellular role of Serine19 phosphorylation in tyrosine hydroxylase

Tyrosine hydroxylase (TH) is the rate limiting enzyme that catalyzes the first step in the biosynthesis of catecholamines. The posttranslational modification of its regulatory domain controls the regulation of TH enzyme activity. Different kinases are responsible for the phosphorylation of the enzyme at Thr8, Ser19, Ser31 and Ser40 N terminal residues. It is well established that phosphorylation at Ser31 and Ser40 (THpSer31 and THpSer40, respectively) leads to an increase of TH activity. However, phosphorylation of Ser19 does not directly increase TH activity but induces its binding to the 14-3-3 protein, leading to the enhancement of TH activity through a mechanism yet not fully understood. The main goal of this work was to investigate the cellular role of Ser19 phosphorylation in TH. Site-directed mutagenesis was used to substitute Ser residue with Ala and Glu at position 19 of recombinant V5-tagged TH to create a phospho-null (V5-TH-S19A) and phospho-mimic mutant (V5-TH-S19E), respectively. Native-PAGE analysis showed that there is no significant difference in the oligomeric state of TH in nuclear and cytosolic fractions of neuroblastoma cells, regardless of the phosphorylation state of Ser19. Immunofluorescence (IF) analysis of the subcellular distribution of endogenous THpSer19 showed that it was predominantly distributed in the nucleus of neuroblastoma cells. However, Western blot results showed that the band intensity for THpSer19 in the cytosolic fraction was stronger than the band found in the nuclear fraction. Proximity ligation assay (PLA) suggested that the amount of TH present in the nucleus is phosphorylated at Ser19. Western blot and immunofluorescence analysis showed that the phospho-site mutants did not accumulate preferentially in the nucleus or in the cytosol. Both IF and PLA analysis showed that 14-3-3 proteins are distributed in the cytoplasm and nucleus of neuroblastoma cells. PLA analysis indicated that also in the nucleus THpSer19 interacts with 14-3-3 protein. These results, thus, have an impact on fundamental understanding of the distribution of THpSer19 and one of the binding partners in nucleus, such as 14-3-3γ.Master i BiomedisinMAMD-MEDBIBMED39

Phosphorylation at serine 31 targets tyrosine hydroxylase tovesicles for transport along microtubules

Tyrosine hydroxylase (TH) catalyzes the conversion of L-tyrosine into L-DOPA, which is the rate-limiting step in the synthesis of catecholamines, such as dopamine, in dopaminergergic neurons. Low dopamine levels and death of the dopaminergic neurons are hallmarks of Parkinson's disease (PD), where α-synuclein is also a key player. TH is highly regulated, notably by phosphorylation of several Ser/Thr residues in the N-terminal tail. However, the functional role of TH phosphorylation at the Ser-31 site (THSer(P)-31) remains unclear. Here, we report that THSer(P)-31 co-distributes with the Golgi complex and synaptic-like vesicles in rat and human dopaminergic cells. We also found that the TH microsomal fraction content decreases after inhibition of cyclin-dependent kinase 5 (Cdk5) and ERK1/2. The cellular distribution of an overexpressed phospho-null mutant, TH1-S31A, was restricted to the soma of neuroblastoma cells, with decreased association with the microsomal fraction, whereas a phospho-mimic mutant, TH1-S31E, was distributed throughout the soma and neurites. TH1-S31E associated with vesicular monoamine transporter 2 (VMAT2) and α-synuclein in neuroblastoma cells, and endogenous THSer(P)-31 was detected in VMAT2– and α-synuclein–immunoprecipitated mouse brain samples. Microtubule disruption or co-transfection with α-synuclein A53T, a PD-associated mutation, caused TH1-S31E accumulation in the cell soma. Our results indicate that Ser-31 phosphorylation may regulate TH subcellular localization by enabling its transport along microtubules, notably toward the projection terminals. These findings disclose a new mechanism of TH regulation by phosphorylation and reveal its interaction with key players in PD, opening up new research avenues for better understanding dopamine synthesis in physiological and pathological states