New horizons in specific hormone proteolysis
Authors: Jakob Triebel, Juan Pablo Robles, Magdalena Zamora, Carmen Clapp, Thomas Bertsch
Journal: Trends in Endocrinology and Metabolism (2022)

Dysfunction of proteolytic processing of protein hormones is associated with endocrine and metabolic diseases.Point mutations in protein cleavage sites modify protein hormone maturation and the generation of cleaved protein hormones, impacting metabolic and vascular homeostasis and disease.Clinical trials have focused on modifying the levels of cleaved protein hormones, such as vasoinhibin, for the treatment of diabetic retinopathy and peripartum cardiomyopathy.Clinical trials have mainly investigated substrate depletion or supplementation therapies, such as established treatments in metabolic disease.The cleavage sites in the protein hormone at which the proteolysis occurs and the proteases with their determinants define the hormone–metabolism junction. Proteolysis of protein hormones is primarily acknowledged in the context of breakdown and metabolic clearance by hepatorenal elimination. However, less explored is the specific proteolytic processing of large protein hormones, for which canonical signaling pathways were already established [e.g., prolactin (PRL)], to generate unique messengers that impact cellular functions via pathways unrelated to the receptors of their precursor molecules. Yet, the proteolysis of PRL to generate new messengers evolved under positive selection, and cleaved protein hormones regulate essential functions to maintain homeostasis at the organismal, tissue, or organ levels. The cleavage sites at which proteolysis occurs and the proteases with their determinants define a hormone–metabolism junction at which specific proteolytic cleavage, pathological alteration, and hepatorenal elimination occur. Proteolysis of protein hormones is primarily acknowledged in the context of breakdown and metabolic clearance by hepatorenal elimination. However, less explored is the specific proteolytic processing of large protein hormones, for which canonical signaling pathways were already established [e.g., prolactin (PRL)], to generate unique messengers that impact cellular functions via pathways unrelated to the receptors of their precursor molecules. Yet, the proteolysis of PRL to generate new messengers evolved under positive selection, and cleaved protein hormones regulate essential functions to maintain homeostasis at the organismal, tissue, or organ levels. The cleavage sites at which proteolysis occurs and the proteases with their determinants define a hormone–metabolism junction at which specific proteolytic cleavage, pathological alteration, and hepatorenal elimination occur. The physiology of protein hormones is conventionally described by a sequence of events through which hormones are synthesized, are released into the circulation, act on primary target cell(s), and are then subjected to proteolytical breakdown and hepatorenal elimination. The plasma half-time of protein hormones is relatively short. For example, the protein hormone PRL (see Glossary), best known for its fundamental role in lactation, has a half-time of disappearance from the circulation of ~25–50 min [1.Gardner D.G. Shoback D. Greenspan's Basic & Clinical Endocrinology. McGraw-Hill Education, 2018Google Scholar], and its metabolic fate is proteolysis and clearance by the liver and kidney. A similar plasma half-time has also been determined for human growth hormone (GH), essential for somatic growth [1.Gardner D.G. Shoback D. Greenspan's Basic & Clinical Endocrinology. McGraw-Hill Education, 2018Google Scholar,2.Haffner D. et al.Metabolic clearance of recombinant human growth hormone in health and chronic renal failure.J. Clin. Invest. 1994; 93: 1163-1171Crossref PubMed Scopus (131) Google Scholar], and for placental lactogen [PL; also known as chorionic somatomammotropin hormone (CSH1/2)], as determined by studies in animals [3.Byatt J.C. et al.Serum half-life and in-vivo actions of recombinant bovine placental lactogen in the dairy cow.J. Endocrinol. 1992; 132: 185-193Crossref PubMed Scopus (32) Google Scholar,4.Reddy S. Watkins W.B. Uptake of 125-I-labelled human placental lactogen and human placental lactogen by the tissues of normal and lactating rats.J. Endocrinol. 1975; 65: 183-194Crossref Scopus (10) Google Scholar]. Renal elimination is a cornerstone of the metabolic clearance rate (MCR) of these three protein hormones, as indicated by the direct correlation between their MCR and glomerular filtration rate (GFR) and the reduction in their MCR in patients with chronic renal failure. Clearing by glomerular filtration and urine excretion is further exemplified by the facts that filtered GH associates with low peritubular uptake and secretion, and only degradation products of GH return to the circulation [5.Johnson V. Maack T. Renal extraction, filtration, absorption, and catabolism of growth hormone.Am. J. Phys. 1977; 233: F185-F196PubMed Google Scholar,6.Rabkin R. et al.Removal and excretion of immunoreactive rat growth hormone by the isolated kidney.Am. J. Phys. 1981; 240: F282-F287PubMed Google Scholar]. In addition, di- or multimeric molecular complexes, formed by the binding of GH to GH-binding proteins or of PRL to immunoglobulin complexes, and post-translational modifications, such as glycosylation, significantly alter the renal MCR of these two hormones [7.Baumann G. et al.The metabolic clearance, distribution, and degradation of dimeric and monomeric growth hormone (GH): implications for the pattern of circulating GH forms.Endocrinology. 1986; 119: 1497-1501Crossref PubMed Scopus (58) Google Scholar, 8.Hattori N. Inagaki C. Anti-prolactin (PRL) autoantibodies cause asymptomatic hyperprolactinemia: bioassay and clearance studies of PRL-immunoglobulin G complex.J. Clin. Endocrinol. Metab. 1997; 82: 3107-3110Crossref PubMed Scopus (111) Google Scholar, 9.Leanos-Miranda A. et al.Antiprolactin autoantibodies in systemic lupus erythematosus: frequency and correlation with prolactinemia and disease activity.J. Rheumatol. 2001; 28: 1546-1553PubMed Google Scholar, 10.Hoffmann T. et al.Glycosylation of human prolactin regulates hormone bioactivity and metabolic clearance.J. Endocrinol. Investig. 1993; 16: 807-816Crossref PubMed Scopus (46) Google Scholar]. The main site of extrarenal disposal of all protein hormones is the liver, where cellular uptake by receptor-mediated endocytosis is followed by degradation in endocytotic vesicles via lysosomal interaction and ubiquitin–proteasome-associated pathways [11.Bullier-Picard F. et al.In-vivo uptake of human growth hormone in male rat liver.J. Endocrinol. 1989; 121: 19-25Crossref PubMed Scopus (16) Google Scholar]. Inside vesicles and lysosomes, protein hormones are exposed to an acidic pH, whereupon they uncoil, and are cleaved by proteases, such as cathepsins. Moreover, proteolysis determines the removal of signal peptides from prehormones and the processing of inactive precursor molecules or prohormones. One of the best-known examples of the latter is that of proopiomelanocortin (POMC), so named because it encodes opioid, melanotropic, and corticotropic activities. POMC, a 226-amino acid precursor, is cleaved by prohormone-converting enzymes type 1 and 2 (PSK1 and PSK2) and carboxypeptidase E to generate adrenocorticotropic hormone; α-, β-, and γ-melanocyte-stimulating hormone (MSH); and β-endorphin, and other protein hormones, respectively. The processing of POMC by proteolytic enzymes is tissue specific, depending on the differential distribution of responsible enzymes in the various tissues exposed to POMC. Another relevant example is insulin produced by pancreatic β cells, which is generated by the enzymatic cleavage of proinsulin and the subsequent proteolytic removal of the C peptide [1.Gardner D.G. Shoback D. Greenspan's Basic & Clinical Endocrinology. McGraw-Hill Education, 2018Google Scholar]. Preproinsulin and proinsulin are also cleaved by PSK1, PSK2, and carboxypeptidase E and secretory granules contain insulin and C peptide in equimolar amounts and only minute quantities of proinsulin and partially cleaved intermediates [1.Gardner D.G. Shoback D. Greenspan's Basic & Clinical Endocrinology. McGraw-Hill Education, 2018Google Scholar]. The circulatory half-life of endogenous insulin is ~3–5 min, and it is degraded mainly by insulinases in the liver and kidney. Similar proteolytic processing occurs during the maturation of numerous other protein hormones, such as glucagon and somatostatin. Based on these findings, a rigid understanding of protein hormones developed, in which the cellular synthesis, release, endocrine, paracrine, and/or autocrine effects via a specific receptor in the target tissue, and the subsequent metabolic clearance by hepatorenal elimination, represent their essential physiology, with few or no exceptions. The role of proteolysis was largely confined to prohormone processing and degradation. This long-held intellectual framework prevented the exploration of other avenues of proteolysis with functional significance, such as of the specific proteolytic processing of protein hormones for which canonical signaling pathways were already established, such as PRL and GH, not for the purpose of metabolic clearance, but instead to generate additional unique messengers that impact cellular functions via pathways unrelated to the receptors of their precursor molecules. Interest focused on the detailed characterization of the canonical pathways and effects of PRL, GH, and PL, while the role of their cleaved products as relevant functional messengers was underestimated. Protein hormones signal through seven-transmembrane domain (TMD) and single-TMD receptors, which are further subdivided into growth factor receptors, cytokine receptors, and guanylyl cyclase-linked receptors. The seven-TMD receptors depend on G protein transducers, and single-TMD receptors mostly harbor intrinsic tyrosine kinase activity. The transforming growth factor (TGF)-β family signals through serine/threonine kinase domains and guanylyl cyclase-linked receptors to induce the synthesis of cGMP. The critical components of PRL and GH signaling are single-TMD receptors, which, after ligand binding, activate Janus kinase tyrosine kinases, which phosphorylate cytoplasmatic signal transducer and activator of transcription proteins (the JAK/STAT signaling pathway). The different modes of canonical protein hormone signaling result in effects in which activation depends on systemic (endocrine), local (autocrine or paracrine), or intracellular (intracrine) routes of communication. These modes of operation are subject to regulation at the hypothalamic, pituitary, and/or the peripheral level, which thereby define three tiers of control in endocrine axes. However, proteolytically cleaved fragments of protein hormones activate non-canonical signaling pathways, which are not activated by their uncleaved precursor molecules. A prime example is vasoinhibin, a molecular fragment generated by the proteolytic processing of PRL, GH, and PL by proteases such as cathepsin D [12.Baldocchi R.A. et al.Mass spectrometric analysis of the fragments produced by cleavage and reduction of rat prolactin: evidence that the cleaving enzyme is cathepsin D.Endocrinology. 1993; 133: 935-938Crossref PubMed Scopus (72) Google Scholar,13.Piwnica D. et al.Cathepsin D processes human prolactin into multiple 16K-like N-terminal fragments: study of their antiangiogenic properties and physiological relevance.Mol. Endocrinol. 2004; 18: 2522-2542Crossref PubMed Scopus (81) Google Scholar], matrix metalloproteases (MMPs) [14.Macotela Y. et al.Matrix metalloproteases from chondrocytes generate an antiangiogenic 16 kDa prolactin.J. Cell Sci. 2006; 119: 1790-1800Crossref PubMed Scopus (91) Google Scholar], bone morphogenetic protein 1 [15.Ge G. et al.Bone morphogenetic protein 1 processes prolactin to a 17-kDa antiangiogenic factor.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 10010-10015Crossref PubMed Scopus (72) Google Scholar], thrombin [16.Zamora M. et al.Thrombin cleaves prolactin into a potent 5.6-kDa vasoinhibin: implication for tissue repair.Endocrinology. 2021; 162: bqab177Crossref Scopus (7) Google Scholar], and plasmin [17.Friedrich C. et al.Plasmin generates vasoinhibin-like peptides by cleaving prolactin and placental lactogen.Mol. Cell. Endocrinol. 2021; 538111471Crossref Scopus (3) Google Scholar]. In contrast to its parent molecule PRL, vasoinhibin is antiangiogenic and inhibits vasodilation and vasopermeability. It does not activate the PRL receptor, but instead interacts with a multicomponent complex formed by plasminogen activator inhibitor-1, urokinase plasminogen activator, and the urokinase plasminogen activator receptor [18.Bajou K. et al.PAI-1 mediates the antiangiogenic and profibrinolytic effects of 16K prolactin.Nat. Med. 2014; 20: 741-747Crossref PubMed Scopus (65) Google Scholar] to block the activation of the Ras-Raf-MAPK pathway, the Ras-Tiam1-Rac1-Pak1 pathway, and the Ca2+/calmodulin and PI3K/Akt activation of endothelial nitric oxide synthase induced by several proangiogenic and vasopermeability factors [vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin (IL)-1β, and bradykinin] [19.Clapp C. et al.Peptide hormone regulation of angiogenesis.Physiol. Rev. 2009; 89: 1177-1215Crossref PubMed Scopus (132) Google Scholar,20.Clapp C. et al.Regulation of blood vessels by prolactin and vasoinhibins.Adv. Exp. Med. Biol. 2015; 846: 83-95Crossref Scopus (42) Google Scholar]. The bioactive site of vasoinhibin is an HGR motif, a short linear motif at residues 74–76 (or residues 46–48 without the signal peptide) [21.Robles J.P. et al.The HGR motif is the antiangiogenic determinant of vasoinhibin: implications for a therapeutic orally active oligopeptide.Angiogenesis. 2022; 25: 57-70Crossref Scopus (5) Google Scholar]. The generation of vasoinhibin, its bioactive domain, and specific signaling pathways exemplify how cleaved protein hormones convert into messengers with new functionality that act beyond the canonical signaling pathways of their uncleaved precursors (Figure 1 and Boxes 1 and 2).Box 1VasoinhibinVasoinhibin is generated by the proteolytic cleavage of PRL, GH, and PL.Vasoinhibin isoforms vary in size, depending on the cleavage site and cleaving enzyme, ranging from 5.6 to 18 kDa.Vasoinhibin interacts with plasminogen activator inhibitor-1 and blocks the activation of the Ras-Raf-MAPK pathway, the Ras-Tiam1-Rac1-Pak1 pathway, and the Ca2+/calmodulin and PI3K/Akt activation of endothelial nitric oxide synthase induced by several proangiogenic and vasopermeability factors.The bioactive site of vasoinhibin derived from PRL is a short motif of three residues (HGR).Vasoinhibin inhibits angiogenesis, vasopermeability, and vasodilation.The vasoinhibin receptor remains poorly defined.Box 2Cleaved protein hormonesCleaved protein hormones are generated by proteolytic cleavage of protein hormones for which canonical signaling pathways may already exist.Cleaved protein hormones are subject to evolutionary conservation and positive selection.Various isoforms exist, depending on the cleaving enzyme and tissue.Cleaved protein hormones contain folded structures and intrinsically disordered regions.Cleaved protein hormones can signal via short linear motifs and through more than one signaling pathway.Targets include metabolic and organ system homeostasis. Vasoinhibin is generated by the proteolytic cleavage of PRL, GH, and PL. Vasoinhibin isoforms vary in size, depending on the cleavage site and cleaving enzyme, ranging from 5.6 to 18 kDa. Vasoinhibin interacts with plasminogen activator inhibitor-1 and blocks the activation of the Ras-Raf-MAPK pathway, the Ras-Tiam1-Rac1-Pak1 pathway, and the Ca2+/calmodulin and PI3K/Akt activation of endothelial nitric oxide synthase induced by several proangiogenic and vasopermeability factors. The bioactive site of vasoinhibin derived from PRL is a short motif of three residues (HGR). Vasoinhibin inhibits angiogenesis, vasopermeability, and vasodilation. The vasoinhibin receptor remains poorly defined. Cleaved protein hormones are generated by proteolytic cleavage of protein hormones for which canonical signaling pathways may already exist. Cleaved protein hormones are subject to evolutionary conservation and positive selection. Various isoforms exist, depending on the cleaving enzyme and tissue. Cleaved protein hormones contain folded structures and intrinsically disordered regions. Cleaved protein hormones can signal via short linear motifs and through more than one signaling pathway. Targets include metabolic and organ system homeostasis. If proteolytic processing of protein hormones is considered as the consecutive breakdown of a longer chain of amino acids by one or more proteases, the enzymatic cleavage sites appear as arbitrary regions that are already exposed at either the solvent-accessible surface area of the folded intact protein precursor, regions that become exposed when the protein reorganizes its 3D structure after a first cleavage, or sites that become solvent accessible dependent on pH-induced structural changes. The cleavage sites of the substrates would then have features resembling natural substrates and act as active site intruders. Enzymes involved in protein catabolism would act catalytically promiscuously and generate fragments that are physiologically irrelevant. Along this line, the biological activity of a cleaved protein hormone fragment, such as vasoinhibin, would be an accident of birth. However, the contrary is suggested by recent evidence. Bioactive sites of protein hormone fragments and cleavage sites have evolved and are conserved; in addition, predicted proteolysis sites in large-scale protein analyses coincide with transcribed exon junctions [22.Campbell K.L. et al.Protein hormone fragmentation in intercellular signaling: hormones as nested information systems.Biol. Reprod. 2021; 104: 887-901Crossref Scopus (4) Google Scholar]. For example, the HGR motif determining the bioactive site of vasoinhibin is conserved in mammals, birds, reptiles, amphibians, and fish [21.Robles J.P. et al.The HGR motif is the antiangiogenic determinant of vasoinhibin: implications for a therapeutic orally active oligopeptide.Angiogenesis. 2022; 25: 57-70Crossref Scopus (5) Google Scholar]. In addition, the cleavage site responsible for the generation of a 15-kDa vasoinhibin isoform evolved in higher primates and has remained conserved [23.Leuchs A. et al.A comparative phylogenetic analysis of prolactin cleavage sites for the generation of vasoinhibin in vertebrates.Gen. Comp. Endocrinol. 2022; 320114011Crossref Scopus (1) Google Scholar]. Cleavage sites are usually defined as eight residues relevant for the recognition of the substrate, the formation of the transition state, and the subsequent disruption of the peptide bond between the residues. The eight residues within a given protein sequence are numbered P4, P3, P2, P1, P1', P2', P3', and P4', with cleavage occurring between P1 and P1' [24.Schechter I. Berger A. On the size of the active site in proteases. I. Papain.Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4697) Google Scholar] (Figure 1). Proteases usually require a specific sequence of residues at the eight positions of the cleavage site (consensus sequence), and alterations in this sequence, such as point mutations, can either inhibit or facilitate the cleavage. It appears that there can be more than one cleavage site per protease, such that isoforms of various sizes are generated. For example, β-endorphin, a cleavage product of POMC, and an endogenous μ-opioid receptor agonist with pain-relieving effects may comprise 26, 27, or 31 amino acids, depending on where cleavage occurs. The variability of vasoinhibin isoforms is even larger and comprises isoforms between 5.6 and 18 kDa, which reflects cleavage by more than one protease at various cleavage sites and is linked not only to tissue-specific cleavage, but also to differential effects in various organic systems [25.Yun B.Y. et al.Differential activity of 16K rat prolactin in different organic systems.Anim. Cells Syst. (Seoul). 2019; 23: 135-142Crossref Scopus (3) Google Scholar] (Boxes 1 and 2). Dysfunction of the proteolytic processing of protein hormones is usually a disease modifier in multi-etiological endocrine and metabolic entities and syndromes, which is likely to have contributed to the systematic undervaluation of cleaved protein hormones and their physiological and pathological significance. However, it is now acknowledged that the whole chain of instances in which proteolytic processing of protein hormones occurs is susceptible to dysfunction. Such dysfunction may affect the proteolytic removal of signal peptides, prepro- and prohormone processing, as well as the generation of cleaved protein hormones from mature protein hormones with established canonical signaling, whereby 'dysfunction' relates to sequence aberrations in the substrate or cleaving enzyme, such as mutations, changes in enzyme activity or microenvironmental factors determining their activity, or alterations in the enzyme–substrate ratio. Consequences may affect the secretory pathway, protein conformation, and secreted or circulating hormone levels. For example, an Ala2Thr mutation in preproinsulin is projected to lead to a conformational change from an α-helix to β-sheet, severely compromising the efficiency of signal peptide cleavage of preproinsulin. Ala2Thr mutation carriers secrete insulin at only 50% of the levels of their unaffected relatives and the mutation co-segregates with diabetes mellitus whereas the unaffected relatives remain healthy. Therefore, the mutation appears as a pathogenic cause of maturity-onset diabetes of the young (MODY) with a defect in insulin biosynthesis, classified as INS/MODY 10 [26.Zhang J. et al.Identification of Ala2Thr mutation in insulin gene from a Chinese MODY10 family.Mol. Cell. Biochem. 2020; 470: 77-86Crossref Scopus (2) Google Scholar,27.Nkonge K.M. et al.The epidemiology, molecular pathogenesis, diagnosis, and treatment of maturity-onset diabetes of the young (MODY).Clin. Diabetes Endocrinol. 2020; 6: 20Crossref PubMed Google Scholar]. In addition, cleaving or converting enzymes may be affected. For example, mutations in the gene encoding carboxypeptidase E (CPE) were responsible for the manifestation of a syndrome characterized by obesity, intellectual disability, and hypogonadotropic hypogonadism in three siblings. Whole-exome sequencing revealed a homozygous nonsense c.405>A (p.Y135*) mutation in CPE that was present in the index case and their affected siblings. Their parents were phenotypically normal heterozygous mutation carriers [28.Durmaz A. et al.A new cause of obesity syndrome associated with a mutation in the carboxypeptidase gene detected in three siblings with obesity, intellectual disability and hypogonadotropic hypogonadism.J. Clin. Res. Pediatr. Endocrinol. 2021; 13: 52-60Crossref Scopus (8) Google Scholar]. Mutations in PRL, CSH1/2, or GH1 or the cleaving enzymes responsible for the generation of vasoinhibin are possible but have not yet been investigated in conjunction with associated phenotypes or diseased conditions, such as preeclampsia, diabetic retinopathy, or peripartum cardiomyopathy [29.Triebel J. et al.Human prolactin point mutations and their projected effect on vasoinhibin generation and vasoinhibin-related diseases.Front. Endocrinol. 2017; 8: 294Crossref Scopus (6) Google Scholar]. However, for the latter two, clinical studies were conducted or are underway, in which the generation of vasoinhibin is pharmacologically stimulated or inhibited. For the treatment of diabetic retinopathy and diabetic macular edema, an ongoing clinical study is investigating the effect of levosulpiride, a selective dopamine D2 receptor antagonist, on retinal alterations [30.Robles-Osorio M.L. et al.Basis and design of a randomized clinical trial to evaluate the effect of levosulpiride on retinal alterations in patients with diabetic retinopathy and diabetic macular edema.Front. Endocrinol. 2018; 9: 242Crossref PubMed Scopus (14) Google Scholar]. The rationale behind this trial is that the levosulpiride-induced hyperprolactinemia promotes MMP-mediated conversion to vasoinhibin, which could lead to beneficiary outcomes in terms of vasoinhibin-mediated antagonization of diabetes-induced retinal alterations [31.Nunez-Amaro C.D. et al.Levosulpiride increases the levels of prolactin and antiangiogenic vasoinhibin in the vitreous of patients with proliferative diabetic retinopathy.Transl. Vis. Sci. Technol. 2020; 9: 27Crossref Scopus (7) Google Scholar] (NCT03161652i). For the treatment of peripartum cardiomyopathy, a trial investigated the effect of bromocriptine, a dopamine D2 receptor agonistii. The rationale of treatment with bromocriptine is the inhibition of vasoinhibin generation due to enhanced activity of cathepsin D by substrate depletion to prevent detrimental effects on myocardial microvascularization. The trial demonstrated that bromocriptine treatment was associated with a high rate of left ventricular recovery [32.Hilfiker-Kleiner D. et al.Bromocriptine for the treatment of peripartum cardiomyopathy: a multicentre randomized study.Eur. Heart J. 2017; 38: 2671-2679Crossref PubMed Scopus (166) Google Scholar]; thus, adding bromocriptine to standard heart failure therapy has been included as a consideration in the guidelines for the management of cardiovascular diseases during pregnancy [33.Regitz-Zagrosek V. et al.2018 ESC Guidelines for the management of cardiovascular diseases during pregnancy.Eur. Heart J. 2018; 39: 3165-3241Crossref PubMed Scopus (762) Google Scholar]. Both treatment rationales resemble concepts in the treatment of metabolic diseases, where substrate depletion or supplementation therapies are established. The generation of vasoinhibin depends on PRL levels, and hyperprolactinemia promotes the conversion of PRL to vasoinhibin by providing more substrate for cleaving proteases. Hyperprolactinemic mice overexpressing PRL in the liver have enhanced levels of circulating vasoinhibin associated with increased blood pressure [34.Chang A.S. et al.Prolactin alters blood pressure by modulating the activity of endothelial nitric oxide synthase.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 12538-12543Crossref Scopus (21) Google Scholar]. Dopamine D2 receptor antagonist-induced hyperprolactinemia results in higher levels of vasoinhibin in ocular tissues and fluids of rats [35.Adan-Castro E. et al.Sulpiride-induced hyperprolactinaemia increases retinal vasoinhibin and protects against diabetic retinopathy in rats.J. Neuroendocrinol. 2022; (Published online January 14, 2022. https://doi.org/10.1111/jne.13091)Crossref Scopus (3) Google Scholar] and humans [31.Nunez-Amaro C.D. et al.Levosulpiride increases the levels of prolactin and antiangiogenic vasoinhibin in the vitreous of patients with proliferative diabetic retinopathy.Transl. Vis. Sci. Technol. 2020; 9: 27Crossref Scopus (7) Google Scholar] and can protect against diabetic retinopathy and diabetic macular edema. These observations unravel interactions at the level of the PRL–vasoinhibin axis influencing the pathophysiology of vascular diseases and the development of new treatments. Endocrine disease usually comprises a dysregulation of effector hormone levels, which is related to gland dysfunction due to autoimmunity and genetic causes, or cancer. Metabolic diseases are classified as disorders that give rise to intoxication (group 1), disorders involving energy metabolism (group 2), and disorders involving complex molecules (group 3) [36.Saudubray J.M. et al.Inborn Metabolic Diseases: Diagnosis and Treatment. Springer, 2016Crossref Google Scholar]. Disorders that give rise to intoxication include inborn errors of intermediary metabolism that lead to acute or progressive intoxication from the accumulation of small molecules proximal to the metabolic block, including errors of amino acid catabolism, such as phenylketonuria, tyrosinemia, and maple syrup urine disease. However, these entities affect the metabolism of single amino acids, such as phenylalanine (phenylketonuria) or tyrosine (tyrosinemia), or the branched chain amino acids leucine, isoleucine, and valine (maple syrup urine disease), and not the protein level. Nevertheless, the pathobiochemical basis of a dysregulation in specific hormone proteolysis resembles that of inborn errors of metabolism and of endocrine disease, because quantitative or qualitative alterations of the substrate or the cleaving enzymes can result in effector hormone levels that are too low or too high relative to what is required to maintain homeostasis. This demonstrates that disorders related to cleaved protein hormones fit neither into classic endocrine diseases nor smoothly into the established classification for metabolic diseases. Rather, the cleavage sites in the protein hormone at which proteolysis occurs and the proteases with their determinants define a hormone–metabolism junction at which specific proteolytic cleavage, pathological alteration, and hepatorenal elimination can occur (Figure 1). The stronger consideration of the various paths that can be taken at this junction leads to a deeper understanding of the associated pathologies. Birds of a feather flock together and cleaved protein hormones share common features with respect to their evolution, generation, structure–function relationships, signal transduction, and effects (see Outstanding questions). Cleaved protein hormones are subject to evolutionary conservation and positive selection and are usually generated by more than one enzyme. Various isoforms exist depending on where the cleavage occurs, and the processing is tissue specific and depends on the expression of the cleaving enzyme, as well as on factors in the microenvironment. Cleaved protein hormones may not only be folded, but may also comprise intrinsically disordered regions and their bioactive site may consist of short linear motifs as small as three amino acids. Cleaved protein hormones signal through more than one receptor or signaling pathway, and their main target is metabolic and organ system homeostasis. In general, physiological relevance is to be anticipated for any product of hormone proteolysis, as suggested by the evolution and conservation of fragment-generating cleavage sites and the ever-expanding list of functions ascribed to protein hormone fragments, which were considered physiologically irrelevant or only resulting from proteolytic breakdown and metabolic clearance. Acknowledging this trend, combined with the emergence of new technologies to study protease–substrate relationships [37.Uliana F. et al.Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen.Nat. Commun. 2021; 12: 1693Crossref Scopus (3) Google Scholar], yields new horizons in specific hormone proteolysis, the exploration of which will lead to a deeper understanding of diseases related to hormone metabolism.Outstanding questionsWhich phenotypes are associated with genetic alterations in protein hormone cleavage sites and cleaving enzymes?Which evolutionary forces drive the emergence of cleaved protein hormones and their cleavage sites?Which factors control the generation of cleaved protein hormones in target tissues?What are the principles determining the interplay between local and systemic effects of cleaved protein hormones?What are the best tools to study the generation and effects of cleaved protein hormones? Which phenotypes are associated with genetic alterations in protein hormone cleavage sites and cleaving enzymes? Which evolutionary forces drive the emergence of cleaved protein hormones and their cleavage sites? Which factors control the generation of cleaved protein hormones in target tissues? What are the principles determining the interplay between local and systemic effects of cleaved protein hormones? What are the best tools to study the generation and effects of cleaved protein hormones? M.Z. is supported by a fellowship from the German Academic Exchange Service for Bi-nationally Supervised Doctoral Degrees/Cotutelle and is a doctoral student from 'Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM)' and received fellowship 768182 from CONACYT. The authors declare the following competing interests: J.T., J.P.R., M.Z., C.C., and T.B. are inventors of submitted Mexican (MX/E/2019/079075) and multinational (PCT/ EP2020/069154) patent applications. The Universidad Nacional Autónoma de México (UNAM) and J.T. and T.B. are owners of pending patents. ihttps://clinicaltrials.gov/ct2/show/NCT03161652. iihttps://clinicaltrials.gov/ct2/show/NCT00998556. signaling via established, specific cell surface receptors. site at which a protein hormone is enzymatically cleaved; usually comprises eight amino acids. sequence of amino acids at which the most efficient proteolytic cleavage occurs. pathological alterations in the retina of patients with diabetes mellitus. hormone levels and effects controlled by hypothalamic, pituitary, and peripheral signals. junction between two exons. volume of circulating blood plasma that is filtered by the kidney per time unit. protein hormone essential for somatic growth. molecules with the same effect but different structure (size). volume of circulating blood plasma that is cleared of a protein hormone per time unit. signaling unrelated to specific cell surface receptors. syndrome in pregnant women with cardiac insufficiency. protein hormone secreted by the placenta. syndrome in pregnant women with elevated blood pressure and proteinuria. inactive insulin precursor molecules. protein hormone essential for lactation. protein generated by the proteolytic cleavage of PRL, GH, and PL, with antiangiogenic effects.