The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication.

Rotavirus (RV) replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core shell, followed by the addition of the second layer of the virion. The viroplasms are composed of several viral proteins, including NSP5, which serves as the main building block. Microtubules, lipid droplets, and miRNA-7 are among the host components recruited in viroplasms. We investigated the interaction between RV proteins and host components of the viroplasms by performing a pull-down assay of lysates from RV-infected cells expressing NSP5-BiolD2. Subsequent tandem mass spectrometry identified all eight subunits of the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for folding at least 10% of the cytosolic proteins. Our confirmed findings reveal that TRiC is brought into viroplasms and wraps around newly formed double-layered particles. Chemical inhibition of TRiC and silencing of its subunits drastically reduced virus progeny production. Through direct RNA sequencing, we show that TRiC is critical for RV replication by controlling dsRNA genome segment synthesis, particularly negative-sense single-stranded RNA. Importantly, cryo-electron microscopy analysis shows that TRiC inhibition results in defective virus particles lacking genome segments and polymerase complex (VP1/VP3). Moreover, TRiC associates with VP2 and NSP5 but not with VP1. Also, VP2 is shown to be essential for recruiting TRiC in viroplasms and preserving their globular morphology. This study highlights the essential role of TRiC in viroplasm formation and in facilitating virion assembly during the RV life cycle. IMPORTANCE: The replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the distinct 11 double-stranded RNA genome segments are co-packaged to complete a genome in newly generated virus particles. In this study, we show for the first time that the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for the folding of at least 10% of the cytosolic proteins, is a component of viroplasms and is required for the synthesis of the viral negative-sense single-stranded RNA. Specifically, TRiC associates with NSP5 and VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited to viroplasms to assist replication.

In vitro methods to assess 11ß-hydroxysteroid dehydrogenase type 1 activity.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) converts inactive 11-keto-glucocorticoids to their active 11ß-hydroxylated forms. It also catalyzes the oxoreduction of other endogenous and exogenous substrates. The ubiquitously expressed 11ß-HSD1 shows high levels in liver and other metabolically active tissues such as brain and adipose tissue. Pharmacological inhibition of 11ß-HSD1 was found to ameliorate adverse metabolic effects of elevated glucocorticoids in rodents and humans, improve wound healing and delay skin aging, and enhance memory and cognition in rodent Alzheimer's disease models. Thus, there is an interest to develop 11ß-HSD1 inhibitors for therapeutic purposes. This chapter describes in vitro methods to assess 11ß-HSD1 enzyme activity for different purposes, be it in disease models, for the assessment of the kinetics of novel substrates or for the screening and characterization of inhibitors. 11ß-HSD1 protein expression and preparations of the different biological samples are discussed first, followed by a description of a well-established and easily adaptable 11ß-HSD1 enzyme activity assay. Finally, different readout methods are shortly described. This chapter should provide the reader with a toolbox of methods to assess 11ß-HSD1 activity with instructions in the form of a decision tree for the choice and implementation of an appropriate enzyme activity assay.

In vitro methods to assess 11ß-hydroxysteroid dehydrogenase type 2 activity.

11ß-Hydroxysteroid dehydrogenase type 2 (11ß-HSD2) converts active 11ß-hydroxyglucocorticoids to their inactive 11-keto forms, fine-tuning the activation of mineralocorticoid and glucocorticoid receptors. 11ß-HSD2 is expressed in mineralocorticoid target tissues such as renal distal tubules and cortical collecting ducts, and distal colon, but also in placenta where it acts as a barrier to reduce the amount of maternal glucocorticoids that reach the fetus. Disruption of 11ß-HSD2 activity by genetic defects or inhibitors causes the syndrome of apparent mineralocorticoid excess (AME), characterized by hypernatremia, hypokalemia and hypertension. Secondary hypertension due to 11ß-HSD2 inhibition has been observed upon consumption of excessive amounts of licorice and in patients treated with the azole fungicides posaconazole and itraconazole. Furthermore, inhibition of 11ß-HSD2 during pregnancy with elevated exposure of the fetus to cortisol can cause neurological complications with a lower intelligence quotient, higher odds of attention deficit and hyperactivity disorder as well as metabolic reprogramming with an increased risk of cardio-metabolic disease in adulthood. This chapter describes in vitro methods for the determination of 11ß-HSD2 activity that can be applied to identify inhibitors that may cause secondary hypertension and characterize the enzyme's activity in disease models. The included decision tree and the list of methods with their advantages and disadvantages aim to enable the reader to select and apply an in vitro method suitable for the scientific question and the equipment available in the respective laboratory.

Molecular mechanisms underlying the defects of two novel mutations in the HSD17B3 gene found in the Tunisian population.

17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3) converts Δ4-androstene-3,17-dione (androstenedione) to testosterone. It is expressed almost exclusively in the testes and is essential for appropriate male sexual development. More than 70 mutations in the HSD17B3 gene that cause 17ß-HSD3 deficiency and result in 46,XY Disorders of Sex Development (46,XY DSD) have been reported. This study describes three novel Tunisian cases with mutations in HSD17B3. The first patient is homozygous for the previously reported mutation p.C206X. The inheritance of this mutation seemed to be independent of consanguineous marriage, which can be explained by its high frequency in the Tunisian population. The second patient has a novel splice site mutation in intron 6 at position c.490 -6 T > C. A splicing assay revealed a complete omission of exon 7 in the resulting HSD17B3 mRNA transcript. Skipping of exon 7 in HSD17B3 is predicted to cause a frame shift in exon 8 that affects the catalytic site and results in a truncation in exon 9, leading to an inactive enzyme. The third patient is homozygous for the novel missense mutation p.K202M, representing the first mutation identified in the catalytic tetrad of 17ß-HSD3. Site-directed mutagenesis and enzyme activity measurements revealed a completely abolished 17ß-HSD3 activity of the p.K202M mutant, despite unaffected protein expression, compared to the wild-type enzyme. Furthermore, the present study emphasizes the importance of genetic counselling, detabooization of 46,XY DSD, and a sensitization of the Tunisian population for the risks of consanguineous marriage.

Activation of retinoic acid-related orphan receptor γ(t) by parabens and benzophenone UV-filters.

Retinoic acid-related orphan receptor γt (RORγt) regulates immune responses and its impaired function contributes to inflammatory and autoimmune diseases and may promote skin cancer. Synthetic inverse RORγt agonists block the production of Th17-associated cytokines including interleukin (IL)-17A and IL-22 and are under investigation for treatment of such pathologies. Unintentional RORγt activation in skin, following exposure to environmental chemicals, may promote inflammatory skin disease. Parabens and UV-filters, frequently used as additives in cosmetics and body care products, are intensively inspected for endocrine disrupting properties. This study assessed whether such compounds can interfere with RORγ activity using a previously established tetracycline-inducible reporter gene assay in CHO cells. These transactivation experiments revealed hexylparaben, benzylparaben and benzophenone-10 as RORγ agonists (EC50 values: 144 ± 97 nM, 3.39 ± 1.74 µM and 1.67 ± 1.04 µM, respectively), and they could restore RORγ activity after suppression by an inverse agonist. Furthermore, they enhanced RORγt-dependent transcription of the pro-inflammatory IL-17A and/or IL-22 genes in the murine T-cell model EL4. Virtual screening of a cosmetics database for structurally similar chemicals and in vitro testing of the most promising hits revealed benzylbenzoate, benzylsalicylate and 4-methylphenylbenzoate as RORγ agonists (low micromolar EC50 values). Moreover, an analysis of mixtures of the newly identified RORγ agonists suggested additive effects. This study presents novel RORγ(t) agonistic structural scaffolds. By activating RORγ(t) the identified parabens and UV-filters may potentially aggravate pathophysiological conditions, especially skin diseases where highest exposure of such chemicals can be expected. Follow-up studies should assess whether such compounds, either alone or as mixtures, can reach relevant concentrations in tissues and target cells to activate RORγ(t) in vivo.

Species-specific differences in the inhibition of 11ß-hydroxysteroid dehydrogenase 2 by itraconazole and posaconazole.

11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) converts active 11ß-hydroxyglucocorticoids to their inactive 11-keto forms, thereby preventing inappropriate mineralocorticoid receptor activation by glucocorticoids. Disruption of 11ß-HSD2 activity by genetic defects or inhibitors causes the syndrome of apparent mineralocorticoid excess (AME), characterized by hypokalemia, hypernatremia and hypertension. Recently, the azole antifungals itraconazole and posaconazole were identified to potently inhibit human 11ß-HSD2, and several case studies described patients with acquired AME. To begin to understand why this adverse drug effect was missed during preclinical investigations, the inhibitory potential of itraconazole, its main metabolite hydroxyitraconazole (OHI) and posaconazole against 11ß-HSD2 from human and three commonly used experimental animals was assessed. Whilst human 11ß-HSD2 was potently inhibited by all three compounds (IC50 values in the nanomolar range), the rat enzyme was moderately inhibited (1.5- to 6-fold higher IC50 values compared to human), and mouse and zebrafish 11ß-HSD2 were very weakly inhibited (IC50 values above 7 µM). Sequence alignment and application of newly generated homology models for human and mouse 11ß-HSD2 revealed significant differences in the C-terminal region and the substrate binding pocket. Exchange of the C-terminus and substitution of residues Leu170,Ile172 in mouse 11ß-HSD2 by the corresponding residues His170,Glu172 of the human enzyme resulted in a gain of sensitivity to itraconazole and posaconazole, resembling human 11ß-HSD2. The results provide an explanation for the observed species-specific 11ß-HSD2 inhibition by the studied azole antifungals. The obtained structure-activity relationship information should facilitate future assessments of 11ß-HSD2 inhibitors and aid choosing adequate animal models for efficacy and safety studies.