Similar metabolic pathways are affected in both Congenital Myasthenic Syndrome-22 and Prader-Willi Syndrome.

Loss of prolyl endopeptidase-like (PREPL) encoding a serine hydrolase with (thio)esterase activity leads to the recessive metabolic disorder Congenital Myasthenic Syndrome-22 (CMS22). It is characterized by severe neonatal hypotonia, feeding problems, growth retardation, and hyperphagia leading to rapid weight gain later in childhood. The phenotypic similarities with Prader-Willi syndrome (PWS) are striking, suggesting that similar pathways are affected. The aim of this study was to identify changes in the hypothalamic-pituitary axis in mouse models for both disorders and to examine mitochondrial function in skin fibroblasts of patients and knockout cell lines. We have demonstrated that Prepl is downregulated in the brains of neonatal PWS-IC-p/+m mice. In addition, the hypothalamic-pituitary axis is similarly affected in both Prepl-/- and PWS-IC-p/+m mice resulting in defective orexigenic signaling and growth retardation. Furthermore, we demonstrated that mitochondrial function is altered in PREPL knockout HEK293T cells and can be rescued with the supplementation of coenzyme Q10. Finally, PREPL-deficient and PWS patient skin fibroblasts display defective mitochondrial bioenergetics. The mitochondrial dysfunction in PWS fibroblasts can be rescued by overexpression of PREPL. In conclusion, we provide the first molecular parallels between CMS22 and PWS, raising the possibility that PREPL substrates might become therapeutic targets for treating both disorders.

The proprotein convertase FURIN is a novel aneurysm predisposition gene impairing TGF-ß signaling.

AIM: Aortic aneurysms (AA) frequently involve dysregulation of transforming growth factor ß (TGF-ß)-signaling in the aorta. Here, FURIN was tested as aneurysm predisposition gene given its role as proprotein convertase in pro-TGF-ß maturation. METHODS AND RESULTS: Rare FURIN variants were detected by whole-exome sequencing of 781 unrelated aortic aneurysm patients and affected relatives. Thirteen rare heterozygous FURIN variants occurred in 3.7% (29) unrelated index AA patients, of which 72% had multiple aneurysms or a dissection.FURIN maturation and activity of these variants were decreased in vitro. Patient-derived fibroblasts showed decreased pro-TGF-ß processing, phosphorylation of downstream effector SMAD2 and kinases ERK1/2, and steady-state mRNA levels of the TGF-ß-responsive ACTA2 gene. In aortic tissue, collagen and fibrillin fibers were affected. One variant (R745Q), observed in 10 unrelated cases, affected TGF-ß signaling variably, indicating effect modification by individual genetic backgrounds. CONCLUSION: FURIN is a novel, frequent genetic predisposition for abdominal-, thoracic-, and multiple aortic or middle sized artery aneurysms in older patients, by affecting intracellular TGF-ß signaling, depending on individual genetic backgrounds.

Epigenetic modulations of immune cells: from normal development to tumor progression.

The dysfunction of immune cell development often impairs immunological homeostasis, thus causing various human diseases. Accumulating evidence shows that the development of different immune cells from hematopoietic stem cells are highly fine-tuned by different epigenetic mechanisms including DNA methylation, histone modifications, chromatin remodeling and RNA-related regulations. Understanding how epigenetic regulators modulate normal development of immune cells contributes to the identification of new strategies for various diseases. Here, we review recent advances suggesting that epigenetic modulations can orchestrate immune cell development and functions through their impact on critical gene expression. We also discuss the aberrations of epigenetic modulations in immune cells that influence tumor progression, and the fact that underlying mechanisms affect how epigenetic drugs interfere with tumor progression in the clinic.

Bone-derived C-terminal FGF23 cleaved peptides increase iron availability in acute inflammation.

Inflammation leads to functional iron deficiency by increasing the expression of the hepatic iron regulatory peptide hepcidin. Inflammation also stimulates fibroblast growth factor 23 (FGF23) production by increasing both Fgf23 transcription and FGF23 cleavage, which paradoxically leads to excess in C-terminal FGF23 peptides (Cter-FGF23), rather than intact FGF23 (iFGF23) hormone. We determined that the major source of Cter-FGF23 is osteocytes and investigated whether Cter-FGF23 peptides play a direct role in the regulation of hepcidin and iron metabolism in response to acute inflammation. Mice harboring an osteocyte-specific deletion of Fgf23 showed a ∼90% reduction in Cter-FGF23 levels during acute inflammation. Reduction in Cter-FGF23 led to a further decrease in circulating iron in inflamed mice owing to excessive hepcidin production. We observed similar results in mice showing impaired FGF23 cleavage owing to osteocyte-specific deletion of Furin. We next showed that Cter-FGF23 peptides bind members of the bone morphogenetic protein (BMP) family, BMP2 and BMP9, which are established inducers of hepcidin. Coadministration of Cter-FGF23 and BMP2 or BMP9 prevented the increase in Hamp messenger RNA and circulating hepcidin levels induced by BMP2/9, resulting in normal serum iron levels. Finally, injection of Cter-FGF23 in inflamed Fgf23KO mice and genetic overexpression of Cter-Fgf23 in wild type mice also resulted in lower hepcidin and higher circulating iron levels. In conclusion, during inflammation, bone is the major source of Cter-FGF23 secretion, and independently of iFGF23, Cter-FGF23 reduces BMP-induced hepcidin secretion in the liver.

Loss of hypothalamic Furin affects POMC to proACTH cleavage and feeding behavior in high-fat diet-fed mice.

OBJECTIVE: The hypothalamus regulates feeding and glucose homeostasis through the balanced action of different neuropeptides, which are cleaved and activated by the proprotein convertases PC1/3 and PC2. However, the recent association of polymorphisms in the proprotein convertase FURIN with type 2 diabetes, metabolic syndrome, and obesity, prompted us to investigate the role of FURIN in hypothalamic neurons controlling glucose and feeding. METHODS: POMC-Cre+/- mice were bred with Furinfl/fl mice to generate conditional knockout mice with Furin-deletion in neurons expressing proopiomelanocortin (POMCFurKO), and Furinfl/fl mice were used as controls. POMCFurKO and controls were periodically monitored on both normal chow diet and high fat diet (HFD) for body weight and glucose tolerance by established in-vivo procedures. Food intake was measured in HFD-fed FurKO and controls. Hypothalamic Pomc mRNA was measured by RT-qPCR. ELISAs quantified POMC protein and resulting peptides in the hypothalamic extracts of POMCFurKO mice and controls. The in-vitro processing of POMC was studied by biochemical techniques in HEK293T and CHO cell lines lacking FURIN. RESULTS: In control mice, Furin mRNA levels were significantly upregulated on HFD feeding, suggesting an increased demand for FURIN activity in obesogenic conditions. Under these conditions, the POMCFurKO mice were hyperphagic and had increased body weight compared to Furinfl/fl mice. Moreover, protein levels of POMC were elevated and ACTH concentrations markedly reduced. Also, the ratio of α-MSH/POMC was decreased in POMCFurKO mice compared to controls. This indicates that POMC processing was significantly reduced in the hypothalami of POMCFurKO mice, highlighting for the first time the involvement of FURIN in the cleavage of POMC. Importantly, we found that in vitro, the first stage in processing where POMC is cleaved into proACTH was achieved by FURIN but not by PC1/3 or the other proprotein convertases in cell lines lacking a regulated secretory pathway. CONCLUSIONS: These results suggest that FURIN processes POMC into proACTH before sorting into the regulated secretory pathway, challenging the dogma that PC1/3 and PC2 are the only convertases responsible for POMC cleavage. Furthermore, its deletion affects feeding behaviors under obesogenic conditions.

The proprotein convertase furin regulates the development of thymic epithelial cells to ensure central immune tolerance.

The generation of mature T cells and establishment of central tolerance is predominantly orchestrated by thymic epithelial cells (TECs). Proprotein convertases are responsible for the proteolysis of proproteins into their mature bioactive counterparts. Here, we found that Furin, a member of the subtilisin/kexin-like PCs family, is highly expressed in TECs compared with other members of this family. TEC-specific deletion of Furin caused severe thymic atrophy and predominantly reduced the number of medullary TECs and thymic tuft cells, and to a less degree, cortical TECs. Furin deletion attenuated the proliferation of TECs, impaired thymopoiesis, and led to autoimmune disorders in mice. Furin promotes the development of TECs via cleavage of proIGF1 receptor and pro-Insulin receptor and the activation of downstream ERK/MAPK and Akt signaling pathways. Thus, this study uncovered the role of furin in TEC development and function and highlighted the importance of post-translational modification of immature proproteins in TEC biology.

Role of Furin in Colon Cancer Stem Cells Malignant Phenotype and Expression of LGR5 and NANOG in KRAS and BRAF-Mutated Colon Tumors.

Proprotein convertases or PCs are known to regulate the malignant phenotype of colon cancer cells by different mechanisms, but their effects on cancer stem cells (CSCs) have been less widely investigated. Here, we report that PCs expression is altered in colon CSCs, and the inhibition of their activity reduced colon CSCs growth, survival, and invasion in three-dimensional spheroid cultures. In vivo, repression of PCs activity by the general PC inhibitors α1-PDX, Spn4A, or decanoyl-RVKR-chloromethylketone (CMK) significantly reduced tumor expression levels of the stem cell markers LGR5 and NANOG that are associated with reduced tumor xenografts. Further analysis revealed that reduced tumor growth mediated by specific silencing of the convertase Furin in KRAS or BRAF mutated-induced colon tumors was associated with reduced expression of LGR5 and NANOG compared to wild-type KRAS and BRAF tumors. Analysis of various calcium regulator molecules revealed that while the calcium-transporting ATPase 4 (ATP2B4) is downregulated in all the Furin-silenced colon cancer cells, the Ca2+-mobilizing P2Y receptors, was specifically repressed in BRAF mutated cells and ORAI1 and CACNA1H in KRAS mutated cells. Taken together, our findings indicate that PCs play an important role in the malignant phenotype of colon CSCs and stem cell markers' expression and highlight PCs repression, particularly of Furin, to target colon tumors with KRAS or BRAF mutation.

The proprotein convertase furin in cancer: more than an oncogene.

Furin is the first discovered proprotein convertase member and is present in almost all mammalian cells. Therefore, by regulating the maturation of a wide range of proproteins, Furin expression and/or activity is involved in various physiological and pathophysiological processes ranging from embryonic development to carcinogenesis. Since many of these protein precursors are involved in initiating and maintaining the hallmarks of cancer, Furin has been proposed as a potential target for treating several human cancers. In contrast, other studies have revealed that some types of cancer do not benefit from Furin inhibition. Therefore, understanding the heterogeneous functions of Furin in cancer will provide important insights into the design of effective strategies targeting Furin in cancer treatment. Here, we present recent advances in understanding how Furin expression and activity are regulated in cancer cells and their influences on the activity of Furin substrates in carcinogenesis. Furthermore, we discuss how Furin represses tumorigenic properties of several cancer cells and why Furin inhibition leads to aggressive phenotypes in other tumors. Finally, we summarize the clinical applications of Furin inhibition in treating human cancers.

Prolyl endopeptidase-like is a (thio)esterase involved in mitochondrial respiratory chain function.

Deficiency of the serine hydrolase prolyl endopeptidase-like (PREPL) causes a recessive metabolic disorder characterized by neonatal hypotonia, feeding difficulties, and growth hormone deficiency. The pathophysiology of PREPL deficiency and the physiological substrates of PREPL remain largely unknown. In this study, we connect PREPL with mitochondrial gene expression and oxidative phosphorylation by analyzing its protein interactors. We demonstrate that the long PREPLL isoform localizes to mitochondria, whereas PREPLS remains cytosolic. Prepl KO mice showed reduced mitochondrial complex activities and disrupted mitochondrial gene expression. Furthermore, mitochondrial ultrastructure was abnormal in a PREPL-deficient patient and Prepl KO mice. In addition, we reveal that PREPL has (thio)esterase activity and inhibition of PREPL by Palmostatin M suggests a depalmitoylating function. We subsequently determined the crystal structure of PREPL, thereby providing insight into the mechanism of action. Taken together, PREPL is a (thio)esterase rather than a peptidase and PREPLL is involved in mitochondrial homeostasis.

Differential Effects of Furin Deficiency on Insulin Receptor Processing and Glucose Control in Liver and Pancreatic ß Cells of Mice.

The insulin receptor (IR) is critically involved in maintaining glucose homeostasis. It undergoes proteolytic cleavage by proprotein convertases, which is an essential step for its activation. The importance of the insulin receptor in liver is well established, but its role in pancreatic ß cells is still controversial. In this study, we investigated the cleavage of the IR by the proprotein convertase FURIN in ß cells and hepatocytes, and the contribution of the IR in pancreatic ß cells and liver to glucose homeostasis. ß-cell-specific Furin knockout (ßFurKO) mice were glucose intolerant, but liver-specific Furin knockout (LFurKO) mice were normoglycemic. Processing of the IR was blocked in ßFurKO cells, but unaffected in LFurKO mice. Most strikingly, glucose homeostasis in ß-cell-specific IR knockout (ßIRKO) mice was normal in younger mice (up to 20 weeks), and only mildly affected in older mice (24 weeks). In conclusion, FURIN cleaves the IR non-redundantly in ß cells, but redundantly in liver. Furthermore, we demonstrated that the IR in ß cells plays a limited role in glucose homeostasis.

In Vivo Analysis of the Contribution of Proprotein Convertases to the Processing of FGF23.

Fibroblast growth factor 23 (FGF23) is a hormone secreted from fully differentiated osteoblasts and osteocytes that inhibits phosphate reabsorption by kidney proximal tubules. The full-length (i.e., intact) protein mediates FGF23 endocrine functions, while endoproteolytic cleavage at a consensus cleavage sequence for the proprotein convertases (PCs) inactivates FGF23. Two PCs, furin and PC5, were shown to cleave FGF23 in vitro at RHTR179↓, but whether they are fulfilling this function in vivo is currently unknown. To address this question, we used here mice lacking either or both furin and PC5 in cell-specific manners and mice lacking the paired basic amino acid-cleaving enzyme 4 (PACE4) in all cells. Our analysis shows that furin inactivation in osteoblasts and osteocytes results in a 25% increase in circulating intact FGF23, without any significant impact on serum phosphate levels, whether mice are maintained on a normal or a low phosphate diet. Under conditions of iron deficiency, FGF23 is normally processed in control mice, but its processing is impaired in mice lacking furin in osteoblasts and osteocytes. In contrast, FGF23 is normally cleaved following erythropoietin or IL-1ß injections in mice lacking furin or both furin and PC5, and in PACE4-deficient mice. Altogether, these studies suggest that furin is only partially responsible for FGF23 cleavage under certain conditions in vivo. The processing of FGF23 may therefore involve the redundant action of multiple PCs or of other peptidases in osteoblasts, osteocytes and hematopoietic cells.

Loss of Furin in ß-Cells Induces an mTORC1-ATF4 Anabolic Pathway That Leads to ß-Cell Dysfunction.

FURIN is a proprotein convertase (PC) responsible for proteolytic activation of a wide array of precursor proteins within the secretory pathway. It maps to the PRC1 locus, a type 2 diabetes susceptibility locus, but its specific role in pancreatic ß-cells is largely unknown. The aim of this study was to determine the role of FURIN in glucose homeostasis. We show that FURIN is highly expressed in human islets, whereas PCs that potentially could provide redundancy are expressed at considerably lower levels. ß-cell-specific Furin knockout (ßFurKO) mice are glucose intolerant as a result of smaller islets with lower insulin content and abnormal dense-core secretory granule morphology. mRNA expression analysis and differential proteomics on ßFurKO islets revealed activation of activating transcription factor 4 (ATF4), which was mediated by mammalian target of rapamycin C1 (mTORC1). ßFurKO cells show impaired cleavage or shedding of vacuolar-type ATPase (V-ATPase) subunits Ac45 and prorenin receptor, respectively, and impaired lysosomal acidification. Blocking V-ATPase pharmacologically in ß-cells increased mTORC1 activity, suggesting involvement of the V-ATPase proton pump in the phenotype. Taken together, these results suggest a model of mTORC1-ATF4 hyperactivation and impaired lysosomal acidification in ß-cells lacking Furin, causing ß-cell dysfunction.

Loss of Proprotein Convertase Furin in Mammary Gland Impairs proIGF1R and proIR Processing and Suppresses Tumorigenesis in Triple Negative Breast Cancer.

In triple negative breast cancer (TNBC) cell lines, the proprotein convertase Furin cleaves and then activates several protein precursors involved in oncogenesis. However, the in vivo role of Furin in the mammary gland and how mammary gland-specific Furin knockout specifically influences tumor initiation and progression of TNBC is unknown. Here, we report that Furin is frequently overexpressed in TNBC tumors and this correlates with poor prognosis in patients with TNBC tumors. In a whey acidic protein (WAP)-induced mammary epithelial cell-specific Furin knockout mouse model, mice show normal mammary development. However, loss of Furin in mammary glands inhibits primary tumor growth and lung metastasis in an oncogene-induced TNBC mouse model. Further analysis of TNBC mice lacking Furin revealed repressed maturation of the Furin substrates proIGF1R and proIR that are associated with reduced expression and activation of their downstream effectors PI3K/AKT and MAPK/ERK1/2. In addition, these tissues showed enhanced apoptotic signaling. In conclusion, our findings reveal that upregulated Furin expression reflects the poor prognosis of TNBC patients and highlights the therapeutic potential of inhibiting Furin in TNBC tumors.

Loss of the proprotein convertase Furin in T cells represses mammary tumorigenesis in oncogene-driven triple negative breast cancer.

Immunotherapeutic interventions have become an important treatment for various cancer types including triple negative breast cancer (TNBC). Previous studies have shown that T cell-specific Furin deficient mice show regulatory CD4+ T cells (Tregs) malfunction phenotypes due to impaired cleavage of proTGF-ß1. However, it is unknown how this phenotype influences tumor initiation and progression in TNBC. Here, we first show that there is a higher level of Furin expression in different immune cells compared to other proprotein convertase members, and its expression is clearly upregulated once immune cells are activated. Moreover, Furin expression levels negatively correlated with an abundance of different infiltrating immune cells in TNBC tumor samples. In an oncogene-induced TNBC mouse model, we demonstrate that Furin inactivation in T cells inhibits primary tumor growth and lung metastasis. Disruption of Furin in T cells in these mice led to a decreased peripheral and tumor-infiltrating Tregs. As a consequence, tumor-infiltrating CD8+ T cells showed a strong proliferative capacity and upregulated expression of IFN-γ and TNF-α. In these mice the repressed tumor growth was associated with induced apoptosis, which led to reduced lung metastases formation. Taken together, these finding revealed the potential therapeutic benefit of targeting Furin in cancer, particularly for immunotherapeutic interventions to treat TNBC.

The proprotein convertase furin is a pro-oncogenic driver in KRAS and BRAF driven colorectal cancer.

Mutations in KRAS and/or BRAF that activate the ERK kinase are frequently found in colorectal cancer (CRC) and drive resistance to targeted therapies. Therefore, the identification of therapeutic targets that affect multiple signaling pathways simultaneously is crucial for improving the treatment of patients with KRAS or BRAF mutations. The proprotein convertase furin activates several oncogenic protein precursors involved in the ERK-MAPK pathway by endoproteolytic cleavage. Here we show that genetic inactivation of furin suppresses tumorigenic growth, proliferation, and migration in KRAS or BRAF mutant CRC cell lines but not in wild-type KRAS and BRAF cells. In a mouse xenograft model, these KRAS or BRAF mutant cells lacking furin displayed reduced growth and angiogenesis, and increased apoptosis. Mechanistically, furin inactivation prevents the processing of various protein pecursors including proIGF1R, proIR, proc-MET, proTGF-ß1 and NOTCH1 leading to potent and durable ERK-MAPK pathway suppression in KRAS or BRAF mutant cells. Furthermore, we identified genes involved in activating the ERK-MAPK pathway, such as PTGS2, which are downregulated in the KRAS or BRAF mutant cells after furin inactivation but upregulated in wild-type KRAS and BRAF cells. Analysis of human colorectal tumor samples reveals a positive correlation between enhanced furin expression and KRAS or BRAF expression. These results indicate that furin plays an important role in KRAS or BRAF-associated ERK-MAPK pathway activation and tumorigenesis, providing a potential target for personalized treatment.

Transgenic Artifacts Caused by Passenger Human Growth Hormone.

The minigene encoding human growth hormone (hGH) has been incorporated into over 300 transgenic mouse lines to improve transgene expression. However, unexpected and functional hGH expression can drastically alter physiology. We list here the mouse lines in which ectopic hGH has been confirmed, and we provide a wiki for lines awaiting analysis.

Prodomain of Furin Promotes Phospholipid Transfer Protein Proteasomal Degradation in Hepatocytes.

BACKGROUND: Phospholipid transfer protein (PLTP) is one of the major modulators of lipoprotein metabolism and atherosclerosis development; however, little is known about the regulation of PLTP. The effect of hepatic prodomain of furin (profurin) expression on PLTP processing and function is investigated. METHODS AND RESULTS: We used adenovirus expressing profurin in mouse liver to evaluate PLTP activity, mass, and plasma lipid levels. We coexpressed PLTP and profurin in human hepatoma cell line cells and studied their interaction. We found profurin expression significantly reduced plasma lipids, plasma PLTP activity, and mass in all tested mouse models, compared with controls. Moreover, the expression of profurin dramatically reduced liver PLTP activity and protein level. We further explored the mechanism using in vivo and ex vivo approaches. We found that profurin can interact with intracellular PLTP and promote its ubiquitination and proteasomal degradation, resulting in less PLTP secretion from the hepatocytes. Furin does not cleave PLTP; instead, it forms a complex with PLTP, likely through its prodomain. CONCLUSIONS: Our study reveals that hepatic PLTP protein is targeted for proteasomal degradation by profurin expression, which could be a novel posttranslational mechanism underlying PLTP regulation.

PREPL deficiency: delineation of the phenotype and development of a functional blood assay.

PurposePREPL deficiency causes neonatal hypotonia, ptosis, neonatal feeding difficulties, childhood obesity, xerostomia, and growth hormone deficiency. Different recessive contiguous gene deletion syndromes involving PREPL and a variable combination of SLC3A1 (hypotonia-cystinuria syndrome), CAMKMT (atypical hypotonia-cystinuria syndrome), and PPM1B (2p21 deletion syndrome) have been described. In isolated PREPL deficiency, previously described only once, the absence of cystinuria complicates the diagnosis. Therefore, we developed a PREPL blood assay and further delineated the phenotype.MethodsClinical features of new subjects with PREPL deficiency were recorded. The presence of PREPL in lymphocytes and its reactivity with an activity-based probe were evaluated by western blot.ResultsFive subjects with isolated PREPL deficiency, three with hypotonia-cystinuria syndrome, and two with atypical hypotonia-cystinuria syndrome had nine novel alleles. Their IQs ranged from 64 to 112. Adult neuromuscular signs included ptosis, nasal dysarthria, facial weakness, and variable proximal and neck flexor weakness. Autonomic features are prevalent. PREPL protein and reactivity were absent in lymphocytes from subjects with PREPL deficiency, but normal in the clinically similar Prader-Willi syndrome.ConclusionPREPL deficiency causes neuromuscular, autonomic, cognitive, endocrine, and dysmorphic clinical features. PREPL is not deficient in Prader-Willi syndrome. The novel blood test should facilitate the confirmation of PREPL deficiency.

Endosome to trans-Golgi network transport of Proprotein Convertase 7 is mediated by a cluster of basic amino acids and palmitoylated cysteines.

Proprotein Convertase 7 (PC7) is a Furin-like endoprotease that cleaves precursor proteins at basic amino acids. PC7 is concentrated in the trans-Golgi network (TGN) but it shuttles between the plasma membrane and the TGN depending on sequences in the cytoplasmic tail. A short region containing a three amino acids motif, P724-L725-C726, is essential and sufficient for internalization of PC7 but not for TGN localization, which requires the additional presence of the juxtamembrane region. In this study we have investigated the contribution of a cluster of basic amino acids and two reversibly palmitoylated cysteine residues to endocytic trafficking. Stable cell lines overexpressing chimeric proteins (CD25 and CD46) containing the cytoplasmic domain of PC7 in which the basic cluster alone or together with both palmitoylated cysteines are mutated showed enhanced surface expression as demonstrated by immunofluorescence experiments and surface biotinylation. The mutant proteins no longer recycled to the TGN in antibody uptake experiments and accumulated in an endosomal compartment. Recycling of wild type PC7 to the TGN is blocked by nocodazole, suggesting that PC7 shuttles to the TGN via late endosomes, similar to Furin. Unlike furin, however, PC7 was found to recycle to a region within the TGN, which is deficient in sialyltransferase, as shown by resialylation experiments. In conclusion, a novel motif, composed of a basic amino acid cluster and two palmitoylated cysteines are essential for TGN localization and endocytic trafficking.

Functional and clinical relevance of novel and known PCSK1 variants for childhood obesity and glucose metabolism.

OBJECTIVE: Variants in Proprotein Convertase Subtilisin/Kexin Type 1 (PCSK1) may be causative for obesity as suggested by monogenic cases and association studies. Here we assessed the functional relevance in experimental studies and the clinical relevance through detailed metabolic phenotyping of newly identified and known PCSK1 variants in children. RESULTS: In 52 obese children selected for elevated proinsulin levels and/or impaired glucose tolerance, we found eight known variants and two novel heterozygous variants (c.1095 + 1G > A and p.S24C) by sequencing the PCSK1 gene. Patients with the new variants presented with extreme obesity, impaired glucose tolerance, and PCOS. Functionally, c.1095 + 1G > A caused skipping of exon8 translation and a complete loss of enzymatic activity. The protein was retained within the endoplasmic reticulum (ER) causing ER stress. The p.S24C variant had no functional effect on protein size, cell trafficking, or enzymatic activity. The known variants rs6230, rs35753085, and rs725522 in the 5' end did not affect PCSK1 promoter activity. In clinical association studies in 1673 lean and obese children, we confirmed associations of rs6232 and rs6234 with BMI-SDS and of rs725522 with glucose stimulated insulin secretion and Matsuda index. We did not find the new variants in any other subjects. CONCLUSIONS: We identified and functionally characterized two rare novel PCSK1 variants of which c.1095 + 1G > A caused complete loss of protein function. In addition to confirming rs6232 and rs6234 in PCSK1 as polygenic risk variants for childhood obesity, we describe an association of rs725522 with insulin metabolism. Our results support the contribution of PCSK1 variants to obesity predisposition in children.