Pro‐Thyrotropin‐Releasing Hormone Processing by Recombinant PC1

Abstract

Pro-thyrotropin-releasing hormone (proTRH) is the precursor to thyrotropin-releasing hormone (TRH ; pGlu-His-Pro-NH2 ), the hypothalamic releasing factor that stimulates synthesis and release of thyrotropin from the pituitary gland . Five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven cryptic peptides are formed following posttranslational proteolytic cleavage of the 26-kDa rat proTRH precursor. The endopeptidase(s) responsible for the physiological conversion of proTRH to the TRH progenitor form is currently unknown. We examined the in vitro processing of [3H]leucine-labeled or unlabeled proTRH by partially purified recombinant PC1 . Recombinant PC1 processed the 26-kDa TRH precursor by initially cleaving the prohormone after the basic amino acid at either position 153 or 159 . Based on the use of our well-established antibodies, we propose that the initial cleavage gave rise to the formation of a 15-kDa N-terminal peptide (preproTRH 25 -152 or pre-proTRH 25 -15a) and a 10-kDa C-terminal peptide . Some initial cleavage occurred after amino acid 108 to generate a 16.5-kDa Cterminal peptide. The 15-kDa N-terminal intermediate was further processed to a 6-kDa peptide (prepro-TRH25-,6 or preproTRH25-82) and a 3.8-kDa peptide (pre-proTRH 83 -108), whereas the 10-kDa C-terminal intermediate was processed to a 5.4-kDa peptide . The optimal pH for these cleavages was 5.5 . ZnCl 2 , EDTA, EGTA, and the omission of Ca t ' inhibited the formation of pYE2, (preproTRH25-50), one of the proTRH N-terminal products, by 48, 82, 72, and 45%, respectively . This study provides evidence, for the first time, that recombinant PC 1 enzyme can process proTRH to its predicted peptide intermediates.

Regulation of hypothalamic prohormone convertases 1 and 2 and effects on processing of prothyrotropin-releasing hormone

“…The assays used for proTRH-derived peptides were developed in our laboratory using commercially synthesized or standard available peptides and primary antibodies (24). The trace was iodinated using the chloramine T oxidation-reduction method followed by HPLC purification (24).…”

“…The assays used for proTRH-derived peptides were developed in our laboratory using commercially synthesized or standard available peptides and primary antibodies (24). The trace was iodinated using the chloramine T oxidation-reduction method followed by HPLC purification (24). The N-terminal antiserum anti-pEH24, which was used to study pro-TRH processing (6,82), and TRH (7,24) RIAs were performed as standard in our laboratory.…”

“…The trace was iodinated using the chloramine T oxidation-reduction method followed by HPLC purification (24). The N-terminal antiserum anti-pEH24, which was used to study pro-TRH processing (6,82), and TRH (7,24) RIAs were performed as standard in our laboratory. All RIAs were performed on the same volume of material, in duplicate.…”

“…In the hypothalamus, these enzymes displayed an extensive overlapping pattern of expression. Both convertases are effectively targeted to the regulated secretory pathway, where they process many neuropeptide precursors in the brain and prohormones in the pituitary, including pro-TRH (7,24), POMC (9), prosomatostatin (25), provasopressin (26), pro-neuropeptide Y (proNPY) (27), proneurotensin (28), proenkephalin (29), and pro-cocaine amphetamine-regulated transcript (proCART) (30). The critical role of PC1 and PC2 in prohormone processing is underscored by studies of animals that lack the genes encoding PC1 (31) and PC2 (32), as well as 7B2 (33,34), a neuropeptide essential for the maturation of PC2.…”

Differential Adaptations of the Hypothalamus-Pituitary-Thyroid Axis Between Food Restriction and Anorexia

“…Those processes are conducted by two prohormone convertases (PCs), PC1/3 and PC2, which cleave proTRH at basic residues flanking TRH sequence (Nillni et al, 1995(Nillni et al, , 1996Schaner et al, 1997). PCs activity may also be regulated by TH (Espinosa et al, 2007;Perello et al, 2006).…”