Structural mechanism for tyrosine hydroxylase inhibition by dopamine and reactivation by Ser40 phosphorylation

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of dopamine (DA) and other catecholamines, and its dysfunction leads to DA deficiency and parkinsonisms. Inhibition by catecholamines and reactivation by S40 phosphorylation are key regulatory mechanisms of TH activity and conformational stability. We used Cryo-EM to determine the structures of full-length human TH without and with DA, and the structure of S40 phosphorylated TH, complemented with biophysical and biochemical characterizations and molecular dynamics simulations. TH presents a tetrameric structure with dimerized regulatory domains that are separated 15 Å from the catalytic domains. Upon DA binding, a 20-residue α-helix in the flexible N-terminal tail of the regulatory domain is fixed in the active site, blocking it, while S40-phosphorylation forces its egress. The structures reveal the molecular basis of the inhibitory and stabilizing effects of DA and its counteraction by S40-phosphorylation, key regulatory mechanisms for homeostasis of DA and TH.

A Glance into MTHFR Deficiency at a Molecular Level

MTHFR deficiency still deserves an investigation to associate the phenotype to protein structure variations. To this aim, considering the MTHFR wild type protein structure, with a catalytic and a regulatory domain and taking advantage of state-of-the-art computational tools, we explore the properties of 72 missense variations known to be disease associated. By computing the thermodynamic ΔΔG change according to a consensus method that we recently introduced, we find that 61% of the disease-related variations destabilize the protein, are present both in the catalytic and regulatory domain and correspond to known biochemical deficiencies. The propensity of solvent accessible residues to be involved in protein-protein interaction sites indicates that most of the interacting residues are located in the regulatory domain, and that only three of them, located at the interface of the functional protein homodimer, are both disease-related and destabilizing. Finally, we compute the protein architecture with Hidden Markov Models, one from Pfam for the catalytic domain and the second computed in house for the regulatory domain. We show that patterns of disease-associated, physicochemical variation types, both in the catalytic and regulatory domains, are unique for the MTHFR deficiency when mapped into the protein architecture.

5-Hydroxymethylcytosine-mediated active demethylation is required for mammalian neuronal differentiation and function

Although high levels of 5-hydroxymethylcytosine (5hmC) accumulate in mammalian neurons, our knowledge of its roles in terminal differentiation or as an intermediate in active DNA demethylation is incomplete. We report high-resolution mapping of DNA methylation and hydroxymethylation, chromatin accessibility, and histone marks in developing postmitotic Purkinje cells (PCs) in Mus musculus. Our data reveal new relationships between PC transcriptional and epigenetic programs, and identify a class of genes that lose both 5-methylcytosine (5mC) and 5hmC during terminal differentiation. Deletion of the 5hmC writers Tet1, Tet2, and Tet3 from postmitotic PCs prevents loss of 5mC and 5hmC in regulatory domains and gene bodies, and hinders transcriptional and epigenetic developmental transitions. Our data demonstrate that Tet-mediated active DNA demethylation occurs in vivo, and that acquisition of the precise molecular properties of adult PCs require continued oxidation of 5mC to 5hmC during the final phases of differentiation.

Indications of Goal Displacement in Regulatory Enforcement Agencies: An Empirical Exploration

This paper describes indications of goal displacement in regulatory enforcement agencies as reported by enforcement professionals from a range of regulatory domains. The findings suggest that the occurrence of this phenomenon in these agencies may be more prevalent and multifaceted than expected. Among the goal-displacement types reported as most impactful were goal narrowing, induced by calamities, goal diversion through ongoing organizational reform, and goal diversion brought upon by strict regimes of output management. A systematic exploration of these various goal-displacement types as conducted here sheds light onto the intricate nature of goal alignment of these agencies.

Identifying stress responsive genes using overlapping communities in co-expression networks

Background This paper proposes a workflow to identify genes that respond to specific treatments in plants. The workflow takes as input the RNA sequencing read counts and phenotypical data of different genotypes, measured under control and treatment conditions. It outputs a reduced group of genes marked as relevant for treatment response. Technically, the proposed approach is both a generalization and an extension of WGCNA. It aims to identify specific modules of overlapping communities underlying the co-expression network of genes. Module detection is achieved by using Hierarchical Link Clustering. The overlapping nature of the systems’ regulatory domains that generate co-expression can be identified by such modules. LASSO regression is employed to analyze phenotypic responses of modules to treatment. Results The workflow is applied to rice (Oryza sativa), a major food source known to be highly sensitive to salt stress. The workflow identifies 19 rice genes that seem relevant in the response to salt stress. They are distributed across 6 modules: 3 modules, each grouping together 3 genes, are associated to shoot K content; 2 modules of 3 genes are associated to shoot biomass; and 1 module of 4 genes is associated to root biomass. These genes represent target genes for the improvement of salinity tolerance in rice. Conclusions A more effective framework to reduce the search-space for target genes that respond to a specific treatment is introduced. It facilitates experimental validation by restraining efforts to a smaller subset of genes of high potential relevance.

Chemical Approaches for Studying the Biology and Pharmacology of Membrane Transporters: The Histidine/Large Amino Acid Transporter SLC7A5 as a Benchmark

The localization of membrane transporters at the forefront of natural barriers makes these proteins very interesting due to their involvement in the absorption and distribution of nutrients and xenobiotics, including drugs. Over the years, structure/function relationship studies have been performed employing several strategies, including chemical modification of exposed amino acid residues. These approaches are very meaningful when applied to membrane transporters, given that these proteins are characterized by both hydrophobic and hydrophilic domains with a different degree of accessibility to employed chemicals. Besides basic features, the chemical targeting approaches can disclose information useful for pharmacological applications as well. An eminent example of this picture is the histidine/large amino acid transporter SLC7A5, known as LAT1 (Large Amino Acid Transporter 1). This protein is crucial in cell life because it is responsible for mediating the absorption and distribution of essential amino acids in peculiar body districts, such as the blood brain barrier and placenta. Furthermore, LAT1 can recognize a large variety of molecules of pharmacological interest and is also considered a hot target for drugs due to its over-expression in virtually all human cancers. Therefore, it is not surprising that the chemical targeting approach, coupled with bioinformatics, site-directed mutagenesis and transport assays, proved fundamental in describing features of LAT1 such as the substrate binding site, regulatory domains and interactions with drugs that will be discussed in this review. The results on LAT1 can be considered to have general applicability to other transporters linked with human diseases.

Leveraging interindividual variability of regulatory activity refines genetic regulation of gene expression in schizophrenia

Schizophrenia is a polygenic psychiatric disorder with limited understanding about the mechanistic changes in gene expression regulation. To elucidate on this, we integrate interindividual variability of regulatory activity with gene expression and genotype data captured from the prefrontal cortex of 272 cases and controls. We show that regulatory element activity is structured into 10,936 and 10,376 cis-regulatory domains in cases and controls, respectively, which display distinct regulatory element coordination structures in both states. By studying the interplay among genetic variants, gene expression and cis-regulatory domains, we ascertain that changes in coordinated regulatory activity tag alterations in gene expression levels (p=8.62e-06, OR=1.60), unveil case-specific QTL effects, and identify regulatory machinery changes for genes affecting synaptic function and dendritic spine morphology in schizophrenia. Altogether, we show that accounting for coordinated regulatory activity provides a novel mechanistic approach to reduce the search space for unveiling genetically perturbed regulation of gene expression in schizophrenia.

Structure-Based Understanding of ABCA3 Variants

ABCA3 is a crucial protein of pulmonary surfactant biosynthesis, associated with recessive pulmonary disorders such as neonatal respiratory distress and interstitial lung disease. Mutations are mostly private, and accurate interpretation of variants is mandatory for genetic counseling and patient care. We used 3D structure information to complete the set of available bioinformatics tools dedicated to medical decision. Using the experimental structure of human ABCA4, we modeled at atomic resolution the human ABCA3 3D structure including transmembrane domains (TMDs), nucleotide-binding domains (NBDs), and regulatory domains (RDs) in an ATP-bound conformation. We focused and mapped known pathogenic missense variants on this model. We pinpointed amino-acids within the NBDs, the RDs and within the interfaces between the NBDs and TMDs intracellular helices (IHs), which are predicted to play key roles in the structure and/or the function of the ABCA3 transporter. This theoretical study also highlighted the possible impact of ABCA3 variants in the cytosolic part of the protein, such as the well-known p.Glu292Val and p.Arg288Lys variants.

Redundant enhancers in the iab-5 domain cooperatively activate Abd-B in the A5 and A6 abdominal segments of Drosophila

The Abdominal-B (Abd-B) gene belongs to Bithorax complex and its expression is controlled by four regulatory domains, iab-5, iab-6, iab-7 and iab-8, each of which is thought to be responsible for directing the expression of Abd-B in one of the abdominal segments from A5 to A8. A variety of experiments have supported the idea that BX-C regulatory domains are functionally autonomous and that each domain is both necessary and sufficient to orchestrate the development of the segment they specify. Unexpectedly, we discovered that this model does not always hold. Instead, we find that tissue-specific enhancers located in the iab-5 domain are required for the proper activation of Abd-B not only in A5 but also in A6. Our findings indicate that the functioning of the iab-5 and iab-6 domains in development of the adult cuticle A5 and A6 in males fit better with an additive model much like that first envisioned by Ed Lewis.