The Host-Pathogen interaction of human cyclophilin A and HIV-1 Vpr requires specific N-terminal and novel C-terminal domains

<p>Abstract</p> <p>Background</p> <p>Cyclophilin A (CypA) represents a potential key molecule in future antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication. CypA interacts with the virus proteins Capsid (CA) and Vpr, however, the mechanism through which CypA influences HIV-1 infectivity still remains unclear.</p> <p>Results</p> <p>Here the interaction of full-length HIV-1 Vpr with the host cellular factor CypA has been characterized and quantified by surface plasmon resonance spectroscopy. A C-terminal region of Vpr, comprising the 16 residues ⁷⁵GCRHSRIGVTRQRRAR⁹⁰, with high binding affinity for CypA has been identified. This region of Vpr does not contain any proline residues but binds much more strongly to CypA than the previously characterized N-terminal binding domain of Vpr, and is thus the first protein binding domain to CypA described involving no proline residues. The fact that the mutant peptide Vpr^75-90R80A binds more weakly to CypA than the wild-type peptide confirms that Arg-80 is a key residue in the C-terminal binding domain. The N- and C-terminal binding regions of full-length Vpr bind cooperatively to CypA and have allowed a model of the complex to be created. The dissociation constant of full-length Vpr to CypA was determined to be approximately 320 nM, indicating that the binding may be stronger than that of the well characterized interaction of HIV-1 CA with CypA.</p> <p>Conclusions</p> <p>For the first time the interaction of full-length Vpr and CypA has been characterized and quantified. A non-proline-containing 16-residue region of C-terminal Vpr which binds specifically to CypA with similar high affinity as full-length Vpr has been identified. The fact that this is the first non-proline containing binding motif of any protein found to bind to CypA, changes the view on how CypA is able to interact with other proteins. It is interesting to note that several previously reported key functions of HIV-1 Vpr are associated with the identified N- and C-terminal binding domains of the protein to CypA.</p

Brain catecholamine depletion and motor impairment in a Th knock-in mouse with type B tyrosine hydroxylase deficiency

Tyrosine hydroxylase catalyses the hydroxylation of L-tyrosine to l-DOPA, the rate- limiting step in the synthesis of catecholamines. Mutations in the TH gene encoding tyrosine hydroxylase are associated with the autosomal recessive disorder tyrosine hydroxylase deficiency, which manifests phenotypes varying from infantile parkinsonism and DOPA-responsive dystonia, also termed type A, to complex encephalopathy with perinatal onset, termed type B. We generated homozygous Th knock-in mice with the mutation Th-p.R203H, equivalent to the most recurrent human mutation associated with type B tyrosine hydroxylase deficiency (TH-p.R233H), often unresponsive to l-DOPA treatment. The Th knock-in mice showed normal survival and food intake, but hypotension, hypokinesia, reduced motor coordination, wide-based gate and catalepsy. This phenotype was associated with a gradual loss of central catecholamines and the serious manifestations of motor impairment presented diurnal fluctuation but did not improve with standard l-DOPA treatment. The mutant tyrosine hydroxylase enzyme was unstable and exhibited deficient stabilization by catecholamines, leading to decline of brain tyrosine hydroxylase-immunoreactivity in the Th knock-in mice. In fact the substantia nigra presented an almost normal level of mutant tyrosine hydroxylase protein but distinct absence of the enzyme was observed in the striatum, indicating a mutation-associated mislocalization of tyrosine hydroxylase in the nigrostriatal pathway. This hypomorphic mouse model thus provides understanding on pathomechanisms in type B tyrosine hydroxylase deficiency and a platform for the evaluation of novel therapeutics for movement disorders with loss of dopaminergic input to the striatum

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

16 pags, 7 figs . -- The online version contains supplementary movie1: https://static-content.springer.com/esm/art%3A10.1038%2Fs41467-021-27657-y/MediaObjects/41467_2021_27657_MOESM3_ESM.mp4Tyrosine 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.This research was supported by the grant PID2019-105872GB-I00/AEI/10.13039/ 501100011033 from the Spanish Ministry of Science and Innovation to J.M.V. and J.C. as well as FRIMEDBIO (261826) from the Research Council of Norway to A.M.; the Western Norway Regional Health Authorities (912246 to A.M. and 912264 to R.K.), the K.G.Peer reviewe

Personalized medicine to improve treatment of dopa-responsive dystonia—a focus on tyrosine hydroxylase deficiency

Dopa-responsive dystonia (DRD) is a rare movement disorder associated with defective dopamine synthesis. This impairment may be due to the fact of a deficiency in GTP cyclohydrolase I (GTPCHI, GCH1 gene), sepiapterin reductase (SR), tyrosine hydroxylase (TH), or 6-pyruvoyl tetrahydrobiopterin synthase (PTPS) enzyme functions. Mutations in GCH1 are most frequent, whereas fewer cases have been reported for individual SR-, PTP synthase-, and TH deficiencies. Although termed DRD, a subset of patients responds poorly to L-DOPA. As this is regularly observed in severe cases of TH deficiency (THD), there is an urgent demand for more adequate or personalized treatment options. TH is a key enzyme that catalyzes the rate-limiting step in catecholamine biosynthesis, and THD patients often present with complex and variable phenotypes, which results in frequent misdiagnosis and lack of appropriate treatment. In this expert opinion review, we focus on THD pathophysiology and ongoing efforts to develop novel therapeutics for this rare disorder. We also describe how different modeling approaches can be used to improve genotype to phenotype predictions and to develop in silico testing of treatment strategies. We further discuss the current status of mathematical modeling of catecholamine synthesis and how such models can be used together with biochemical data to improve treatment of DRD patients

Phenylalanine Hydroxylase from <em>Legionella pneumophila</em> Is a Thermostable Enzyme with a Major Functional Role in Pyomelanin Synthesis

<div><h3>Background</h3><p><em>Legionella pneumophila</em> is a pathogenic bacterium that can cause Legionnaires’ disease and other non-pneumonic infections in humans. This bacterium produces a pyomelanin pigment, a potential virulence factor with ferric reductase activity. In this work, we have investigated the role of phenylalanine hydroxylase from <em>L. pneumophila</em> (lpPAH), the product of the <em>phhA</em> gene, in the synthesis of the pyomelanin pigment and the growth of the bacterium in defined compositions.</p> <h3>Methodology/Principal Findings</h3><p>Comparative studies of wild-type and <em>phhA</em> mutant corroborate that lpPAH provides the excess tyrosine for pigment synthesis. <em>phhA</em> and <em>letA</em> (<em>gacA</em>) appear transcriptionally linked when bacteria were grown in buffered yeast extract medium at 37°C. <em>phhA</em> is expressed in <em>L. pneumophila</em> growing in macrophages. We also cloned and characterized lpPAH, which showed many characteristics of other PAHs studied so far, including Fe(II) requirement for activity. However, it also showed many particular properties such as dimerization, a high conformational thermal stability, with a midpoint denaturation temperature (<em>T</em>_m) = 79±0.5°C, a high specific activity at 37°C (10.2±0.3 µmol L-Tyr/mg/min) and low affinity for the substrate (<em>K</em>_m (L-Phe) = 735±50 µM.</p> <h3>Conclusions/Significance</h3><p>lpPAH has a major functional role in the synthesis of pyomelanin and promotes growth in low-tyrosine media. The high thermal stability of lpPAH might reflect the adaptation of the enzyme to withstand relatively high survival temperatures.</p> </div

Conformational stability of lpPAH.

<p>(A) Far-UV CD spectrum of lpPAH (6 µM in 50 mM Na-phosphate buffer, pH 6.5) at 37°C (<b>^____</b>), at 85°C (––) and at 37°C after heating the sample to 100°C (⋅⋅⋅⋅⋅). [θ], mean residual ellipticity. (B) CD-monitored (at 222 nm) thermal denaturation lpPAH (6 µM in 20 mM Na-Hepes, 200 mM NaCl, pH 7.0) without (•) or with (○) 6 µM Fe(II) (added as ferrous ammonium sulphate) and 6 µM Fe(II) and 5 mM L-Phe (▾). The lines show a fitting of the data to a two-state unfolding equation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046209#pone.0046209-Swint1" target="_blank">[79]</a> and points are averaged over ten data points after conversion to fraction unfolded <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046209#pone.0046209-Agashe1" target="_blank">[80]</a>. (C) DSC-monitored thermal denaturation of lpPAH (30 µM) in 20 mM Na-Hepes, pH 7.0. The scan rate was 1°C/min.</p

Intracellular expression of <i>phhA</i> and growth of <i>L. pneumophila</i> in U937 cell macrophages.

<p>Intracellular expression of <i>phhA</i> and <i>letA</i> transcripts in macrophages, which were infected with wild-type (WT) 130b for 24 h, 48 and 72 h and then RT-PCR was done using primers that amplify the specific transcripts. That the PCR products obtained resulted from mRNA templates was confirmed by the lack of product obtained when the PCR did not incorporate reverse transcriptase (- RT). The results are representative of two independent experiments.</p

Transcriptional linkage between <i>phhA</i> and <i>letA</i>.

<p>(A) The gray horizontal arrows denote the <i>phhA</i> and <i>letA</i> genes. The thin horizontal lines below the genes signify the approximate size and location of transcripts identified by RT-PCR analysis, including an intergenic transcript. (B) Wild-type strain 130b was grown in BYE at 37°C, and then RNA was analyzed by RT-PCR utilizing primer pairs specific to either <i>phhA, letA,</i> or the intergenic region spanning <i>phhA</i> and <i>letA</i>. That the PCR products obtained resulted from mRNA templates was confirmed by the lack of product obtained when the PCR did not incorporate RT (-RT). PCR products obtained from genomic DNA appear in the left-most lanes, indicating that the mRNAs observed are full-length. The results presented are representative of at least three independent experiments.</p