Mass Spectrometry Profiling of Pituitary Glands.

Many chronic diseases are associated with hypothalamic-pituitary-adrenal axis dysfunction. Therefore, proteomic profiling of the pituitary gland has potential to uncover new information on the underlying pathways affected in these conditions. This could lead to identification of new biomarkers or drug targets for development of novel therapeutics.

Development of an Assay for Measuring Proprotein-Conversion Activity on a Multiplex Magnetic Bead-Based Array Platform.

This chapter describes a protocol for measuring prolyl oligopeptidase (POP) activity using a biotinylated peptide substrate coupled to magnetic microspheres. The complex is incubated in the presence of a pituitary extract and activity can be detected by loss of the biotin label. The assay can be multiplexed for measuring multiple proprotein-cleaving enzymes simultaneously and can be used in analyses of neuropsychiatric diseases in which proteolytic cleavage of biologically active peptides is known to play a role.

Proteomic Profiling of the Pituitary Gland in Studies of Psychiatric Disorders.

Psychiatric disorders have been associated with perturbations of the hypothalamic-pituitary-adrenal axis. Therefore, proteomic studies of the pituitary gland have the potential to provide new insights into the underlying pathways affected in these conditions as well as identify new biomarkers or targets for use in developing improved medications. This chapter describes a protocol for preparation of pituitary protein extracts followed by characterization of the pituitary proteome by label-free liquid chromatography-tandem mass spectrometry in expression mode (LC-MS).

pCAP-based peptide substrates: the new tool in the box of tyrosine phosphatase assays.

Robust, facile high throughput assays based on non-peptidic probes are available to detect the enzyme activity of protein tyrosine phosphatases. However, these assays cannot replace the use of peptide-based probes in many applications; for example when a closer mimic of the physiological target is desired or in substrate profiling expeditions. Phosphotyrosine peptides are often used in these assays, but their use is complicated by either poor sensitivity or the need for indirect detection methods, among other pitfalls.

Thiuram disulfides as pseudo-irreversible inhibitors of lymphoid tyrosine phosphatase.

We screened a small library of thiuram disulfides for inhibition of lymphoid tyrosine phosphatase (LYP) activity. The parent thiuram disulfide, disulfiram, inhibited LYP activity in vitro and in Jurkat T cells, whereas diethyldithiocarbamate failed to inhibit LYP at the concentrations tested. Compound 13, an N-(2-thioxothiazolidin-4-one) analogue, was found to be the most potent LYP inhibitor in this series, with an IC50 value of 3 μM.

Schizophrenia: metabolic aspects of aetiology, diagnosis and future treatment strategies.

Despite decades of research, the pathophysiology and aetiology of schizophrenia remains incompletely understood. The disorder is frequently accompanied by metabolic symptoms including dyslipidaemia, hyperinsulinaemia, type 2 diabetes and obesity. These symptoms are a common side effect of currently available antipsychotic medications.

Pharmacology of iron transport.

Elucidating the molecular basis for the regulation of iron uptake, storage, and distribution is necessary to understand iron homeostasis. Pharmacological tools are emerging to identify and distinguish among different iron transport pathways. Stimulatory or inhibitory small molecules with effects on iron uptake can help characterize the mechanistic elements of iron transport and the roles of the transporters involved in these processes.

Metabolic, hormonal and stress-related molecular changes in post-mortem pituitary glands from schizophrenia subjects.

Objectives: To identify a molecular profile for schizophrenia using post-mortem pituitaries from schizophrenia and control subjects.

Methods: Molecular profiling analysis of pituitaries from schizophrenia (n = 14) and control (n = 15) subjects was carried out using a combination of liquid chromatography tandem mass spectrometry (LC-MS(E)), multiplex analyte profiling (MAP), two-dimensional difference gel electrophoresis (2D-DIGE) and Western blot analysis.

Results: This led to identification of differentially expressed molecules in schizophrenia patients including hypothalamic-pituitary-adrenal axis-associated constituents such as cortisol, pro-adrenocorticotropic hormone, arginine vasopressin precursor, agouti-related protein, growth hormone, prolactin and secretagogin, as well as molecules associated with lipid transport and metabolism such as apolipoproteins A1, A2, C3 and H.

Discovery of a novel series of inhibitors of lymphoid tyrosine phosphatase with activity in human T cells.

The lymphoid tyrosine phosphatase LYP, encoded by the PTPN22 gene, is a critical regulator of signaling in T cells and recently emerged as a candidate target for therapy of autoimmune diseases. Here, by library screening, we identified a series of noncompetitive inhibitors of LYP that showed activity in primary T cells. Kinetic analysis confirmed that binding of the compounds to the phosphatase is nonmutually exclusive with respect to a known bidentate competitive inhibitor.

Analysis of the human pituitary proteome by data independent label-free liquid chromatography tandem mass spectrometry.

Studies of pituitary-related disorders would be facilitated by enhanced knowledge of the pituitary proteome. To construct a data set of human pituitary proteins, separate protein extracts were prepared from 15 post-mortem pituitaries and analyzed by data independent label-free nanoflow liquid chromatography mass spectrometry (nLC-MS(E) ). The detected mass/time features were aligned and quantified using the Rosetta Elucidator(®) system and annotated using results from ProteinLynx Global Server.

Altered levels of circulating insulin and other neuroendocrine hormones associated with the onset of schizophrenia.

Recently, we showed that the circulating levels of insulin-related peptides and the secretory granule protein chromogranin A were increased in small cohorts of first onset schizophrenia patients. Assuming that this effect was associated with impaired insulin signalling, we investigated the possibility that secretion of other hormones is also affected in schizophrenia. Multiplex immunoassay analysis of 21 hormones and hormone-related molecules was carried out using sera from 236 first and recent onset schizophrenia patients and 230 matched controls.

Gold(I) phosphine mediated selective inhibition of lymphoid tyrosine phosphatase.

Selective protein tyrosine phosphatase (PTP) inhibition is often difficult to achieve owing to the high degree of similarity of the catalytic domains of this family of enzymes. Selective inhibitors of the lymphoid specific tyrosine phosphatase, LYP, are of great interest due to the involvement of LYP in several autoimmune disorders. This manuscript describes a study into the mechanistic details of selective LYP inhibition by a Au(I)-phosphine complex.

Identifying potent, selective protein tyrosine phosphatase inhibitors from a library of Au(I) complexes.

Therapeutic inhibition of protein tyrosine phosphatase activity is a compelling yet challenging approach to the treatment of human disease. Toward this end, a library of 40 gold complexes with the general formula R(3)P-Au-Cl was screened to identify novel inhibitors of PTP activity. The most promising inhibitor obtained for the lymphoid tyrosine phosphatase LYP, (2-pyridine)(Ph(2))P-Au-Cl, is one of the most potent and selective LYP inhibitors identified to date with an IC(50) of 1.

Profiling protein tyrosine phosphatase activity with mechanistic probes.

The protein tyrosine phosphatases are a family of enzymes that play critical roles in regulating physiological processes including cellular signaling, growth, differentiation, and the immune response. As new roles emerge for these enzymes in human disease, interest in understanding their mechanism of regulation and function has increased correspondingly. The recent development of mechanism-based probes for phosphatase activity has paved the way for detailed studies of tyrosine phosphatase activity and regulation in both physiological and pathological cellular signaling.

Gold(I)-mediated inhibition of protein tyrosine phosphatases: a detailed in vitro and cellular study.

Gold(I) complexes containing N-heterocyclic carbene ligands were synthesized, characterized, and along with the antiarthritic drug, auranofin, tested as inhibitors of the cysteine-dependent protein tyrosine phosphatases, which are implicated in several disease states. These compounds exhibit potencies in the low micromolar range against the enzymes in vitro. At therapeutically relevant concentrations, all compounds inhibit PTP activity in Jurkat T leukemia cells with some selectivity.

A low-spin alkylperoxo-iron(III) complex with weak Fe-O and O-O bonds: implications for the mechanism of superoxide reductase.

The synthesis of a mononuclear, five-coordinate ferrous complex [([15]aneN4)FeII(SPh)](BF4) (1) is reported. This complex is a new model of the reduced active site of the enzyme superoxide reductase (SOR), which is comprised of a [(NHis)4(Scys)FeII] center. Complex 1 reacts with alkylhydroperoxides (tBuOOH, cumenylOOH) at low temperature to give a metastable, dark red intermediate (2a: R = tBu; 2b: R = cumenyl) that has been characterized by UV-vis, EPR, and resonance Raman spectroscopy.

Mononuclear, dinuclear, and pentanuclear [[N,S(thiolate)]iron(II)] complexes: nuclearity control, incorporation of hydroxide bridging ligands, and magnetic behavior.

The mixed N3S(thiolate) ligand 1-[bis[2-(pyridin-2-yl)ethyl]amino]-2-methylpropane-2-thiol (Py2SH) was used in the synthesis of four iron(II) complexes: [(Py2S)FeCl] (1), [(Py2S)FeBr] (2), [(Py2S)4Fe5II(mu-OH)2](BF4)4 (3), and [(Py2S)2Fe2II(mu-OH)]BF4 (4). The X-ray structures of 1 and 2 revealed monomeric iron(II)-alkylthiolate complexes with distorted trigonal-bipyramidal geometries. The paramagnetic 1H NMR spectra of 1 and 2 display resonances from delta = -25 ppm to +100 ppm, consistent with a high-spin iron(II) ion (S = 2).

A new bis(imidazolyl)(alkylthiolate) tripodal ligand and the spontaneous formation of a disulfide-linked, hydroxo-bridged dinuclear zinc complex.

The new sterically encumbered, tripodal N2S(alkylthiolate) ligand, LIm2SH, has been synthesized and used to prepare [(LIm2S)ZnCH3], which upon protonolysis under acidic conditions leads to the synthesis of a novel dinucleating ligand and a zinc dimer with an unusual structure.