Thimet Oligopeptidase—A Classical Enzyme with New Function and New Form

Peptidases generate bioactive peptides that can regulate cell signaling and mediate intercellular communication. While the processing of peptide precursors is initiated intracellularly, some modifications by peptidases may be conducted extracellularly. Thimet oligopeptidase (TOP) is a peptidase that processes neuroendocrine peptides with roles in mood, metabolism, and immune responses, among other functions. TOP also hydrolyzes angiotensin I to angiotensin 1–7, which may be involved in the pathophysiology of COVID-19 infection. Although TOP is primarily cytosolic, it can also be associated with the cell plasma membrane or secreted to the extracellular space. Recent work indicates that membrane-associated TOP can be released with extracellular vesicles (EVs) to the extracellular space. Here we briefly summarize the enzyme’s classical function in extracellular processing of neuroendocrine peptides, as well as its more recently understood role in intracellular processing of various peptides that impact human diseases. Finally, we discuss new findings of EV-associated TOP in the extracellular space.

Alterations in Gene Expression of Renin-Angiotensin System Components and Related Proteins in Colorectal Cancer

Hypothesis/Introduction. Recent studies suggest involvement of the renin-angiotensin system (RAS) in cancers, including colorectal cancer (CRC). This study focuses on the association of genes encoding 17 proteins related to the RAS within a Japanese male CRC population. Materials and Methods. Quantitative expression of the RNA of these 17 genes in normal and cancerous tissues obtained using chip arrays from the public functional genomics data repository, Gene Expression Omnibus (GEO) application, was compared statistically. Results. Expression of four genes, AGT (angiotensinogen), ENPEP (aminopeptidase A) MME (neprilysin), and PREP (prolyl endopeptidase), was significantly upregulated in CRC specimens. Expression of REN (renin), THOP (thimet oligopeptidase), NLN (neurolysin), PRCP (prolyl carboxypeptidase), ANPEP (aminopeptidase N), and MAS1 (Mas receptor) was downregulated in CRC specimens. Conclusions. Presuming gene expression parallel protein expression, these results suggest that increased production of the angiotensinogen precursor of angiotensin (ANG) peptides, with the reduction of the enzymes that metabolize it to ANG II, can lead to accumulation of angiotensinogen in CRC tissues. Downregulation of THOP, NLN, PRCP, and MAS1 gene expression, whose proteins contribute to the ACE2/ANG 1-7/Mas axis, suggests that reduced activity of this RAS branch could be permissive for oncogenicity. Components of the RAS may be potential therapeutic targets for treatment of CRC.

Thimet Oligopeptidase Biochemical and Biological Significances: Past, Present, and Future Directions

Thimet oligopeptidase (EC 3.4.24.15; EP24.15, THOP1) is a metallopeptidase ubiquitously distributed in mammalian tissues. Beyond its previously well characterized role in major histocompatibility class I (MHC-I) antigen presentation, the recent characterization of the THOP1 C57BL6/N null mice (THOP1−/−) phenotype suggests new key functions for THOP1 in hyperlipidic diet-induced obesity, insulin resistance and non-alcoholic liver steatosis. Distinctive levels of specific intracellular peptides (InPeps), genes and microRNAs were observed when comparing wild type C57BL6/N to THOP1−/− fed either standard or hyperlipidic diets. A possible novel mechanism of action was suggested for InPeps processed by THOP1, which could be modulating protein-protein interactions and microRNA processing, thus affecting the phenotype. Together, research into the biochemical and biomedical significance of THOP1 suggests that degradation by the proteasome is a step in the processing of various proteins, not merely for ending their existence. This allows many functional peptides to be generated by proteasomal degradation in order to, for example, control mRNA translation and the formation of protein complexes.

Extracellular Thimet Oligopeptidase is Released with Extracellular Vesicles from Human Prostate Cancer Cells

ABSTRACTAndrogen signaling plays a central role in the development of prostate cancer. Androgen hormone synthesis is tightly governed by the hypothalamic–pituitary–gonadal (HPG) axis, including gonadotropin-releasing hormone (GnRH). Thimet oligopeptidase (TOP) is a biologically significant peptidase known to cleave GnRH and potentially regulate its activity. Thus, TOP can play an important role in the HPG axis through regulating the downstream production and release of gonadal steroid hormones, including androgens, which may further affect prostate cancer development. TOP is known to be secreted out to the extracellular space. Here, we report that extracellular TOP can be associated with extracellular vesicles (EVs). Western blot analysis of EVs isolated from PC3 or DU145 prostate cancer cells revealed that TOP protein is, indeed, carried by the EVs. Budding of EVs from stimulated PC3 prostate cancer cells can also be visualized by confocal microscopy. Significantly, the TOP enzyme carried by EVs is enzymatically active. The present study shows that EV-associated TOP is a novel form of this extracellular peptidase that may play a role in the disease progression of prostate cancer cells.

The Relevance of Thimet Oligopeptidase in the Regulation of Energy Metabolism and Diet-Induced Obesity

Thimet oligopeptidase (EC 3.4.24.15; EP24.15; THOP1) is a potential therapeutic target, as it plays key biological functions in processing biologically functional peptides. The structural conformation of THOP1 provides a unique restriction regarding substrate size, in that it only hydrolyzes peptides (optimally, those ranging from eight to 12 amino acids) and not proteins. The proteasome activity of hydrolyzing proteins releases a large number of intracellular peptides, providing THOP1 substrates within cells. The present study aimed to investigate the possible function of THOP1 in the development of diet-induced obesity (DIO) and insulin resistance by utilizing a murine model of hyperlipidic DIO with both C57BL6 wild-type (WT) and THOP1 null (THOP1−/−) mice. After 24 weeks of being fed a hyperlipidic diet (HD), THOP1−/− and WT mice ingested similar chow and calories; however, the THOP1−/− mice gained 75% less body weight and showed neither insulin resistance nor non-alcoholic fatty liver steatosis when compared to WT mice. THOP1−/− mice had increased adrenergic-stimulated adipose tissue lipolysis as well as a balanced level of expression of genes and microRNAs associated with energy metabolism, adipogenesis, or inflammation. Altogether, these differences converge to a healthy phenotype of THOP1−/− fed a HD. The molecular mechanism that links THOP1 to energy metabolism is suggested herein to involve intracellular peptides, of which the relative levels were identified to change in the adipose tissue of WT and THOP1−/− mice. Intracellular peptides were observed by molecular modeling to interact with both pre-miR-143 and pre-miR-222, suggesting a possible novel regulatory mechanism for gene expression. Therefore, we successfully demonstrated the previously anticipated relevance of THOP1 in energy metabolism regulation. It was suggested that intracellular peptides were responsible for mediating the phenotypic differences that are described herein by a yet unknown mechanism of action.