A novel binding protein for a member of CyP40-type Cyclophilins: N.crassa CyPBP37, a growth and thiamine regulated protein homolog to yeast Thi4p

Cyclophilins belong to the family of peptidyl-prolyl cis/trans isomerases (PPIases), which are ubiquitous and highly conserved enzymes capable of cis/trans isomerizing Xaa-Pro peptide bonds. Members of the CyP40-type cyclophilins have originally been described as components of hormone receptor complexes. Here, we describe NcCyP41, a CyP40 ortholog from Neurospora crassa, its expression in Escherichia coli and subsequent purification. Characterization of NcCyP41 reveals that it is a heat shock protein, which is active as a cyclosporin A-sensitive PPIase. Affinity chromatography using immobilized recombinant NcCyP41 yielded two major NcCyP41-binding proteins: Hsp80 (a Hsp90 ortholog from N.crassa) and CyPBP37. CyPBP37 has not been described. In addition, this is the first record describing an interaction between a member of Cyp40-type cyclophilins and of CyPBP37-type proteins, respectively. CyPBP37 expression is repressed by thiamine and in the stationary phase in N.crassa. CyPBP37 is present in different isoforms. The expression of a CyPBP37 ortholog in yeast, Thi4p, is diminished in a mutant lacking one of the two CyP40 orthologs (Cpr7p). In addition, the DeltaCpr7p deletion mutant shows a thiamine-dependent growth defect. We conclude that, in yeast, Cpr7p and Thi4p interact functionally.     

Immunoreactive-beta-endorphin, -adrenocorticotropin and -calcitonin in extracts of anaplastic or differentiated (rat) medullary thyroid carcinoma.

Fifteen analogs of luliberin (2, LRH) were synthesized by the solid phase method and examined for their ability to block ovulation in the rat. Two analogs, [Ac-DAla¹,DPhe²,DTrp^3,6]-LRH and [Ac-DPhe¹,DPhe²,DTrp^3,6]-LRH, each blocked ovulation at a single injection dose of 250 μg administered at noon on the day of proestrus; three peptides, [Ac-DPro¹,DPhe²,DTrp^3,6]-LRH, [Ac-DThi¹,DPhe²,DTrp^3,6]-LRH and [Ac-DTrp¹,DPhe²,DTrp^3,6]-LRH, were effective at doses of 500 μg each; and four others, [Ac-DTrp¹,DPhe²,DTrp³,DTrp(Nps)⁶]-LRH, [Chlorambucil-DPhe¹, DPhe², DTrp^3,6]-LRH, [1,DThi²,DTrp^3,6]-LRH and [(Thi-DTrp-Ser-Tyr)₂-DLys⁶]-LRH, gave partial inhibition at doses tested.     

An investigation of position 3 in arginine vasopressin with aliphatic,aromatic, conformationally‐restricted,polar and charged amino acids

We report the solid‐phase synthesis and some pharmacological properties of 23 new analogs of arginine vasopressin (AVP) which have the Phe³ residue replaced by a broad variety of amino acids. Peptides 1–9 have at position 3: (1) the mixed aromatic/aliphatic amino acid thienylalanine (Thi) and the aliphatic amino acids; (2) cyclohexylalanine (Cha); (3) norleucine (Nle); (4) Leu; (5) norvaline (Nva); (6) Val; (7) alpha‐aminobutyric acid (Abu); (8) Ala; (9) Gly. Peptides 10–23 have at position 3: the aromatic amino acids, (10) homophenylalanine (Hphe); (11) Tyr; (12) Trp; (13) 2‐naphthylalanine (2‐Nal); the conformationally‐restricted amino acids (14) Pro; (15) 2‐aminotetraline‐2‐carboxylic acid (Atc); the polar amino acids (16) Ser; (17) Thr; (18) Gln; and the charged amino acids (19) Asp; (20) Glu; (21) Arg; (22) Lys; (23) Orn. All 23 new peptides were evaluated for agonistic and, where appropriate, antagonistic activities in in vivo antidiuretic (V₂‐receptor) and vasopressor (V_1a‐receptor) assays and in in vitro (no Mg²⁺) oxytocic assays. The corresponding potencies (units/mg) in these assays for AVP are: 323±16; 369±6 and 13.9±0.5. Peptides 1–9 exhibit the following potencies (units/mg) in these three assays: (1) 379±14; 360±9; 36.2±1.9; (2) 294±21; 73.4±2.7; 0.33±0.02; (3) 249±28; 84.6±4.3; 4.72±0.16; (4) 229±19; 21.4±0.6; 2.1±0.2; (5) 134±5; 31.2±0.9; 28.4±0.2; (6) 114±9; 45.3±2.3; 11.3±1.6; (7) 86.7±2.5; 4.29±0.13; 0.45±0.03; (8) 15.5±1.5; 0.16±0.01; ∼0.02; (9) 3.76±0.03; <0.02; in vitro oxytocic agonism was not detected. These data show that the aliphatic amino acids Cha, Nle, Leu, Nva and Val are well‐tolerated at position 3 in AVP with retention of surprisingly high levels of antidiuretic activity. Peptides 2–9 exhibit significant gains in both antidiuretic/vasopressor (A/P) and antidiuretic/oxytocic (A/O) selectivities relative to AVP. [Thi³]AVP appears to be a more potent antidiuretic and oxytocic agonist than AVP and is equipotent with AVP as a vasopressor agonist. The antidiuretic potencies of peptides 10–23 exhibit drastic losses relative to AVP. They range from a low of 0.018±0.001 units/mg for the Lys³ analog (peptide 22) to a high of 24.6±4.6 units/mg for the Hphe³ analog (peptide 10). Their vasopressor potencies are also drastically reduced. These range from a low of <0.002 units/mg for peptide 22 to a high of 8.99±0.44 units/mg for the Atc³ analog (peptide 15). Peptides 10–23 exhibit negligible or undetectable in vitro oxytocic agonism. The findings on peptides 10–23 show that position 3 in AVP is highly intolerant of changes with aromatic, conformationally‐restricted, polar and charged amino acids. Furthermore, these findings are in striking contrast to our recent discovery that position 3 in the potent V₂/V_1a/OT antagonist d(CH₂)₅d ‐Tyr(Et)²VAVP tolerates a broad latitude of structural change at position 3 with many of the same amino acids, to give excellent retention of antagonistic potencies. The data on peptides 1–4 offer promising clues to the design of more potent and selective AVP V₂ agonists. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.