Miguel Angel Vargas

Plasmodium falciparum heterochromatin protein 1 binds to tri-methylated histone 3 lysine 9 and is linked to mutually exclusive expression of var genes

Increasing experimental evidence shows a prominent role of histone modifications in the coordinated control of gene expression in the human malaria parasite Plasmodium falciparum. The search for the histone-mark-reading machinery that translates histone modifications into biological processes, such as formation of heterochromatin and antigenic variation is of foremost importance. In this work, we identified the first member of a histone modification specific recognition protein, an orthologue of heterochromatin protein 1 (PfHP1). Analysis of the PfHP1 amino-acid sequence revealed the presence of the two characteristic HP1 domains: a chromodomain (CD) and a chromo shadow domain (CSD). Recombinant CD binds to di- and tri-methylated lysine 9 from histone H3, but not to unmodified or methylated histone H3 in lysine 4. PfHP1 is able to interact with itself to form dimers, underlying its potential role in aggregating nucleosomes to form heterochromatin. Antibodies raised against PfHP1 detect this molecule in foci at the perinuclear region. ChIP analysis using anti-PfHP1 shows that this protein is linked to heterochromatin of subtelomeric non-coding repeat regions and monoallelic expression of the major virulence var gene family. This is the first report implicating an HP1 protein in the control of antigenic variation of a protozoan parasite.

Differential association of Orc1 and Sir2 proteins to telomeric domains in Plasmodium falciparum

Telomeres have the capacity to recruit proteins that facilitate the spreading of heterochromatin into subtelomeric DNA regions. In the human protozoan pathogen Plasmodium falciparum, the telomere-associated protein Sir2 has been shown to control the silencing of members of virulence genes at some, but not all, chromosome-end loci, indicating that additional proteins are involved in telomere position effect. Here, we identified, in P. falciparum, a novel telomere-associated protein that displays homology with the origin-of-recognition-complex 1 protein Orc1. Antibodies raised against this P. falciparum protein localized to telomeric clusters in the nuclear periphery and the nucleolus. It was found that, prior to DNA replication, P. falciparum Orc1 and Sir2 undergo drastic subcellular reorganization, such as dissociation from the telomere cluster and spreading into the nucleus and parasite cytoplasm. Relocation of Orc1 and Sir2 was also linked to the partial dissociation of telomere clusters. Super gel-shift and chromatin-immunoprecipitation experiments showed the physical association of Orc1 with telomere repeats but revealed a differential association with adjacent non-coding repeat DNA elements. Our data suggest that Plasmodium telomeres might fold back and that Orc1 cooperates with Sir2 in telomeric silencing.

Tanycyte pyroglutamyl peptidase II contributes to regulation of the hypothalamic-pituitary-thyroid axis through glial-axonal associations in the median eminence.

Pyroglutamyl peptidase II (PPII), a highly specific membrane-bound metallopeptidase that inactivates TRH in the extracellular space, is tightly regulated by thyroid hormone in cells of the anterior pituitary. Whether PPII has any role in the region where axons containing hypophysiotropic TRH terminate, the median eminence, is unknown. For this purpose, we analyzed the cellular localization and regulation of PPII mRNA in the mediobasal hypothalamus in adult, male rats. PPII mRNA was localized in cells lining the floor and infralateral walls of the third ventricle and coexpressed with vimentin, establishing these cells as tanycytes. PPII mRNA extended in a linear fashion from the tanycyte cell bodies in the base of the third ventricle to its cytoplasmic and end-feet processes in the external zone of the median eminence in close apposition to pro-TRH-containing axon terminals. Compared with vehicle-treated, euthyroid controls, animals made thyrotoxic by the i.p. administration of 10 microg L-T(4) daily for 1-3 d, showed dramatically increased accumulation of silver grains in the mediobasal hypothalamus and an approximately 80% increase in enzymatic activity. PPII inhibition in mediobasal hypothalamic explants increased TRH secretion, whereas i.p. injection of a specific PPII inhibitor increased cold stress- and TRH-induced TSH levels in plasma. We propose that an increase in circulating thyroid hormone up-regulates PPII activity in tanycytes and enhances degradation of extracellular TRH in the median eminence through glial-axonal associations, contributing to the feedback regulation of thyroid hormone on anterior pituitary TSH secretion.

Regional distribution of the membrane-bound pyroglutamate amino peptidase-degrading thyrotropin-releasing hormone in rat brain☆

The brain regional distribution of membrane-bound pyroglutamate aminopeptidase-degrading thyrotropin-releasing hormone (TRH) in rat was studied using a specific radiometric assay. The distribution was not homogeneous: a 10-fold difference was observed between regions. The highest activity was detected in olfactory bulb while the lowest was in the cervical part of spinal cord. There was no correlation with the regional distribution of enzyme activity vs TRH levels, previously reported TRH receptors or in vitro TRH release. The differential distribution of this enzyme is consistent with the hypothesis that it is responsible for extracellular degradation of neuroactive peptides.

Pyroglutamyl peptidase II inhibition specifically increases recovery of TRH released from rat brain slices

Pyroglutamyl peptidase II (EC 3.4.19-) is a highly specific membrane-bound thyrotropin releasing hormone (TRH) degrading enzyme. To study the functional significance of pyroglutamyl peptidase II in TRH degradation, we synthesized the reversible inhibitor N-1-carboxy-2-phenylethyl (Nimbenzyl)-histidyl-beta-naphthylamide (CPHNA). CPHNA inhibited the enzyme with a Ki of 8 microM, but had no effect no TRH receptors or no prolyl endopeptidase (EC 3.4.21.26). It weakly inhibited cytosolic pyroglutamyl peptidase I (EC 3.4.19.3). CPHNA at a concentration of 10(-4) M increased both the basal and potassium stimulated recovery of TRH released from hypothalamic slices by approximately two-fold. An even higher recovery was observed in slices from brain regions with relatively high levels of pyroglutamyl peptidase II. CPHNA had no effect on the basal recovery of gamma-aminobutyric acid or Met-enkephalin released from brain slices but decreased the potassium stimulated recovery of both Metenkephalin and gamma-aminobutyric acid. These data further support the involvement of pyroglutamyl peptidase II in the extracellular inactivation of brain TRH.

Glutamate-dependent transcriptional regulation of GLAST/EAAT1 : a role for YY1

Glutamate is the major excitatory transmitter in the vertebrate brain and its extracellular levels are tightly regulated to prevent excitotoxic effects. The Na+‐dependent glutamate/aspartate transporter GLAST/EAAT1 is regulated in the short and in the long term by glutamate. A receptors‐independent change in its membrane translocation rate, accounts for an acute modulation in GLAST/EAAT1 transport. In contrast, activation of the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate subtype of glutamate receptors represses the transcription of the chick glast gene. A glutamate responsive element has been mapped to the promoter region of this gene containing a bonafide binding site for the transcription factor Ying‐Yang 1. Using cultured chick cerebellar Bergmann glia cells, glutamate elicited a time and dose‐dependent increase in Ying‐Yang 1 DNA binding consistent with the negative response generated in a reporter gene construct controlled for Ying‐Yang 1. Over‐expression of this transcription factor leads to a substantial reduction in GLAST/EAAT1 transporter uptake and an important decrease in mRNA levels, all associated with the transcriptional repression of the chick glast promoter activity. These results provide evidence for an involvement of Ying‐Yang 1 in the transcriptional response to glutamate in glial cells and favor the notion of a relevant role of this factor in GLAST/EAAT1 transcriptional control.

Multifactorial Modulation of TRH Metabolism

Abstract1. Thyrotropin releasing hormone (TRH), synthesized in the paraventricular nucleus of the hypothalamus (PVN), is released in response to physiological stimuli through medianeminence nerve terminals to control thyrotropin or prolactin secretion from the pituitary.2. Several events participate in the metabolism of this neuropeptide: regulation of TRH biosynthesis and release as well as modulation of its inactivation by the target cell.3. Upon a physiological stimulus such as cold stress or suckling, TRH is released and levels of TRH mRNA increase in a fast and transient manner in the PVN; a concomitant increase in cfos is observed only with cold exposure.4. Hypothalamic cell cultures incubated with cAMP or phorbol esters show a rise in TRH mRNA levels; dexamethasone produces a further increase at short incubation times.TRH mRNA are thus controlled by transsynaptic and hormonal influences.5. Once TRH is released, it is inactivated by a narrow specificity ectoenzyme, pyroglu-tamyl peptidase II (PPII).6. In adenohypophysis, PPII is subject to stringent control: positive by thyroid hormones and negative by TRH; other hypothalamic factors such as dopamine and somatostatin also influence its activity.7. These combined approaches suggest that TRH action is modulated in a coordinate fashion.

Neuronal localization of pyroglutamate aminopeptidase II in primary cultures of fetal mouse brain.

Abstract: Pyroglutamate aminopeptidase II is a highly specific membrane‐bound ectopeptidase proposed to inactivate thyrotropin releasing hormone (TRH) in brain extracellular space. Its activity was measured in primary cell cultures of fetal brain in an attempt to define its cellular localization. Enzyme activity was detected in hypothalamic or cortical cell membrane fractions from 4‐ to 12‐day‐old cultures. When proliferation of nonneuronal cells was abolished by cytosine arabinoside treatment, pyroglutamate aminopeptidase II specific activity was increased as compared to untreated cultures, the opposite was observed for pyroglutamate aminopeptidase I activity. Treatment of cortical cells with the neu‐rotoxic agent glutamate reduced simultaneously pyroglutamate aminopeptidase II and glutamate decarboxylase activities. Glial cell cultures expressed pyroglutamate aminopeptidase I or glutamate synthase activities but not pyroglutamate aminopeptidase II. The data suggest that pyroglutamate aminopeptidase II is predominantly localized in neu‐ronal cells. This is consistent with a role for pyroglutamate aminopeptidase II in TRH‐ergic synaptic transmission. Key Words: Pyroglutamate aminopeptidase II—Thyrotropin releasing hormone—Peptidase—Neuronal cultures—Pyroglutamate aminopeptidase I—Synapsis. Cruz C. et al. Neuronal localization of pyroglutamate aminopeptidase II in primary cultures of fetal mouse brain. J. Neurochem. 56, 1594–1601 (1991).

Subcellular distribution of the enzymes degrading thyrotropin releasing hormone and metabolites in rat brain

In order to further understand the role of enzymes degrading Thyrotropin Releasing Hormone (TRH, pglu-his-proNH(2)) and metabolites, we studied their subcellular distribution in rat brain. Brain tissue was homogenized in 0.32 M sucrose, tris-HCl 0.01 M pH 7.4 and fractionated by differential and discontinuous gradient centrifugation; [(3)H]pro-TRH was incubated with the various subcellular fractions and the extent of degradation of each metabolite was measured after separation by thin layer chromatography. Several markers were simultaneously measured (lactate dehydrogenase, 5?-nucleotidase and hexosaminidase) to determine the pattern of distribution of the subcellular organelles. The post-proline cleaving enzyme responsible for pglu-his-pro formation and pyroglutamate amino-peptidase (which requires sulphydryl compounds for maximal activity) were found in cytosol but were barely detectable in the soluble component of synaptosomes; pyroglutamate aminopeptidase (dependent on metals) and post-proline dipeptidyl amino peptidase were found on the membranes of synaptosomes; imido peptidase was not enriched in any particular fraction. These data are consistent with the hypothesis that membrane-bound pyroglutamate aminopeptidase is responsible for TRH degradation once released into the synaptic cleft and that the post-proline dipeptidylaminopeptidase may participate in the extracellular catabolism of his-proNH(2) before it cyclizes to his-pro-DKP. They also suggest that post-proline cleaving enzyme and soluble pyroglutamate aminopeptidase may not play an important role in the regulation of TRH levels in nerve endings.

Regional distribution of in vitro release of thyrotropin releasing hormone in rat brain

To increase our knowledge of the TRH functions in brain and the processes of TRH compartmentalization and release, we studied the in vitro release of endogenous TRH in different brain areas. We also determined the correlation between TRH levels and release under both basal and stimulated conditions. TRH concentration was measured in tissues and media by specific radioimmunoassay. TRH-like material detected in olfactory bulb and hypothalamic incubates (basal or K+ stimulated) were shown to be chromatographically identical to synthetic TRH. Different brain regions showed high variability in the basal release of TRH (1-20% of tissue content). This suggests the existence of different pools. The response to depolarizing stimulus (56 mM K+) was significant only in the following regions: median eminence, total hypothalamus, preoptic area, nucleus accumbens-lateral septum, amygdala, mesencephalon, medulla oblongata and the cervical region of the spinal cord. These regions have been shown to contain a high number of receptors, a high concentration of TRH nerve endings and are susceptible to TRH effects. These results support the hypothesis that TRH functions as neuromodulator in these areas.