Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone

Abstract

The biogenesis of rat thyrotropin releasing hormone (TRH) involves the processing of its precursor (proTRH) into five biologically active TRH peptides and several non-TRH peptides where two of them had been attributed potential biological functions. This process implicates 1) proper folding of proTRH in the endoplasmic reticulum after its biosynthesis and exit to the Golgi apparatus and beyond, 2) initial processing of proTRH in the trans Golgi network and, 3) sorting of proTRH-derived peptides to the regulated secretory pathway. Previous studies have focused on elucidating the processing and sorting determinants of proTRH. However, the role of protein folding in the sorting of proTRH remains unexplored. Here we have investigated the role in the secretion of proTRH of a sequence comprising 22 amino acid residues, located at the N-terminal region of proTRH, residues 31-52. Complete deletion of these 22 amino acids dramatically compromised the biosynthesis of proTRH, manifested as a severe reduction in the steady state level of pro-TRH in the endoplasmic reticulum. This effect was largely reproduced by the deletion of only three amino acid residues, 40 PGL 42 , within the proTRH 31-52 sequence. The decreased steady state level of the mutant ⌬PGL was due to enhanced endoplasmic reticulum-associated protein degradation. However, the remnant of ⌬PGL that escaped degradation was properly processed and sorted to secretory granules. Thus, these results suggest that the N-terminal domain within the prohormone sequence does not act as "sorting signal" in late secretion; instead, it seems to play a key role determining the proper folding pathway of the precursor and, thus, its stability.

The α-Helical Structure of Prodomains Promotes Translocation of Intrinsically Disordered Neuropeptide Hormones into the Endoplasmic Reticulum

“…In contrast, mutants lacking the hormone domain were present in the conditioned medium of transiently transfected cells. Notably, a similar finding was described previously for TRH constructs lacking the prodomain (75).…”

Regulation of the hypothalamic Thyrotropin Releasing Hormone (TRH) neuron by neuronal and peripheral inputs

“…At the TGN, proTRH products are sorted to and stored into specialized secretory granules (SGs) that undergo secretion only after appropriate stimuli. In these SGs the final processing steps take place, which involves endoproteolysis by specific prohormone convertases at pair of basic residues [82; 83; 84], removal of the basic residues by a carboxypeptidase [85; 86; 87], and amidation [5; 88]. If one of these regulated steps is compromised, the biosynthesis of the prohormone derived peptides and their secretion may be affected.…”

“…1). By expressing several deletions and point mutations cDNA constructs within the preproTRH 31-52 sequence, and by monitoring the steady state production of proTRH in the ER, we identified a single tripeptide Pro-Gly-Leu ( 40 PGL 42 ) sequence, which is conserved in mammals and it turn out to be important for the stability of proTRH, in terms of resistance to protein degradation [84]. These studies revealed that PGL is primarily involved in the stability of proTRH in the early secretory pathway.…”

“…Deletion of PGL destabilizes proTRH by targeting the protein to the proteasome for degradation. This was the first evidence showing that the structural role played by a short motif located in the N-terminal region of proTRH takes place early in the ER, and has important consequences on precursor stability rather than the sorting process to the SGs per se [84]. The processing modifications occur while proTRH is transported from the TGN to newly formed immature secretory granules [89].…”

Role of Lysyl Oxidase Propeptide in Secretion and Enzyme Activity

“…A key intracellular function of LOX‐PP is most likely the maintenance of LOX in an inactive state within the secretory pathway [Kagan and Li, 2003]. Propeptides may also function as intramolecular chaperones to facilitate correct folding and the eventual targeting of these proteins to their destinations [Yasuda et al, 2005; Romero et al, 2008]. While the extracellular processing and activation of secretory pro‐LOX upon release of the propeptide moiety have been elucidated [Cronshaw et al, 1995; Panchenko et al, 1996; Uzel et al, 2001] the intracellular role of LOX‐PP within the secretory pathway has not been definitively described.…”