Inherited protein C deficiency (PCD) caused by mutations in protein C (PC) gene (PROC) increases the risk of thrombosis. Missense mutations in PC’s signal peptide and propeptide have been reported in ...patients with PCD, but their pathogenic mechanisms, except mutations in R42 residue, remain unclear.
To investigate the pathogenic mechanisms of inherited PCD caused by 11 naturally occurring missense mutations in PC’s signal peptide and propeptide.
Using cell-based assays, we evaluated the impact of these mutations on various aspects such as activities and antigens of secreted PC, intracellular PC expression, subcellular localization of a reporter protein, and propeptide cleavage. Additionally, we investigated their effect on pre-messenger RNA (pre-mRNA) splicing using a minigene splicing assay.
Our data revealed that certain missense mutations (L9P, R32C, R40C, R38W, and R42C) disrupted PC secretion by impeding cotranslational translocation to the endoplasmic reticulum or causing endoplasmic reticulum retention. Additionally, some mutations (R38W and R42L/H/S) resulted in abnormal propeptide cleavage. However, a few missense mutations (Q3P, W14G, and V26M) did not account for PCD. Using a minigene splicing assay, we observed that several variations (c.8A>C, c.76G>A, c.94C>T, and c.112C>T) increased the incidence of aberrant pre-mRNA splicing.
Our findings suggest that variations in PC’s signal peptide and propeptide have varying effects on the biological process of PC, including posttranscriptional pre-mRNA splicing, translation, and posttranslational processing. Additionally, a variation could affect the biological process of PC at multiple levels. Except for W14G, our results provide a clear understanding of the relationship between PROC genotype and inherited PCD.
•The mechanisms of protein C (PC) deficiency caused by mutations in PC’s signal peptide and propeptide remain unclear.•We systemically studied the pathogenic mechanisms of these mutations using cell-based assays.•These variations have varying effects on the biological process of PC to cause PC deficiency.•Understanding the mechanisms helps to evaluate pathogenic risk and inheritance patterns.
•IOF and International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) consider that Procollagen type I N-propeptide (PINP) and C-terminal telopeptide of type I collagen (β-CTX) are ...the reference bone turnover markers in osteoporosis.•Harmonization/standardization of PINP and β-CTX is mandatory for their clinical use.•Commutable reference materials and reference methods are needed to allow these steps.
Procollagen type I N-propeptide (PINP) and the C-terminal telopeptide of type I collagen (β-CTX) in blood have been designated as reference bone turnover markers in osteoporosis by the International Osteoporosis Foundation (IOF) and International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). The IFCC Committee on Bone Metabolism (C-BM) has examined current commercial assays and performed a multicentre study to examine the agreement between assays for PINP and β-CTX in serum and plasma. The results of these studies will inform our work towards the harmonization of PINP assays and the standardization of β-CTX assays in blood, with the development of common calibrators and reference measurement procedures in collaboration with the reagent manufacturing industry. Successful achievement of these goals will help develop universally acceptable practice guidelines for the management of osteoporosis with the inclusion of common reference intervals and treatment targets for PINP and β-CTX.
•The mediators of extracellular matrix deregulation downstream of Insulin-like growth factor II (IGF-II) were investigated.•Levels of pro-Lysyl Oxidase (ProLOX), its cleavage products LOX, and Lysyl ...oxidase propeptide (LOX-PP) as well as the enzymes that cleave ProLOX, Tolloid-like 1 (TLL1) isoforms and bone morphogenetic protein 1 (BMP1), increased in response to IGF-II.•LOX-PP levels were increased in human systemic sclerosis lung tissues and experimental murine lung fibrosis, and LOX-PP increased levels of markers of fibrosis by primary pulmonary fibroblasts.•The class E basic helix-loop-helix protein 40 (BHLHE40) regulates IGF-II-induced LOX-PP and TLL1 isoforms, and its deficiency increases matrix metalloprotease (MMP) 1, and restores MMP3 and cathepsin K levels repressed in response to IGF-II.•Our findings suggest that LOX-PP and BHLHE40 are new potential therapeutic targets in SSc-associated pulmonary fibrosis.
Pulmonary fibrosis (PF) is a clinically severe and commonly fatal complication of Systemic Sclerosis (SSc). Our group has previously reported profibrotic roles for Insulin-like Growth Factor II (IGF-II) and Lysyl Oxidase (LOX) in SSc-PF. We sought to identify downstream regulatory mediators of IGF-II. In the present work, we show that SSc lung tissues have higher baseline levels of the total (N-glycosylated/unglycosylated) LOX-Propeptide (LOX-PP) than control lung tissues. LOX-PP-mediated changes were consistent with the extracellular matrix (ECM) deregulation implicated in SSc-PF progression. Furthermore, Tolloid-like 1 (TLL1) and Bone Morphogenetic Protein 1 (BMP1), enzymes that can cleave ProLOX to release LOX-PP, were increased in SSc lung fibrosis and the bleomycin (BLM)-induced murine lung fibrosis model, respectively. In addition, IGF-II regulated the levels of ProLOX, active LOX, LOX-PP, BMP1, and isoforms of TLL1. The Class E Basic Helix-Loop-Helix protein 40 (BHLHE40) transcription factor localized to the nucleus in response to IGF-II. BHLHE40 silencing downregulated TLL1 isoforms and LOX-PP, and restored features of ECM deregulation triggered by IGF-II. Our findings indicate that IGF-II, BHLHE40, and LOX-PP may serve as targets of therapeutic intervention to halt SSc-PF progression.
Philadelphia chromosome-negative chronic myeloproliferative neoplasms (MPNs) arise due to acquired somatic driver mutations in stem cells and develop over 10–30 years from the earliest cancer stages ...(essential thrombocythemia, polycythemia vera) towards the advanced myelofibrosis stage with bone marrow failure. The JAK2V617F mutation is the most prevalent driver mutation. Chronic inflammation is considered to be a major pathogenetic player, both as a trigger of MPN development and as a driver of disease progression. Chronic inflammation in MPNs is characterized by persistent connective tissue remodeling, which leads to organ dysfunction and ultimately, organ failure, due to excessive accumulation of extracellular matrix (ECM). Considering that MPNs are acquired clonal stem cell diseases developing in an inflammatory microenvironment in which the hematopoietic cell populations are progressively replaced by stromal proliferation—“a wound that never heals”—we herein aim to provide a comprehensive review of previous promising research in the field of circulating ECM fragments in the diagnosis, treatment and monitoring of MPNs. We address the rationales and highlight new perspectives for the use of circulating ECM protein fragments as biologically plausible, noninvasive disease markers in the management of MPNs.
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•AO-proLapA and AO-mLapA are novel fungal M28 aminopeptidase structures.•AO-LapA propeptide acts as intramolecular chaperone.•Structures reveal molecular mechanism of AO-LapA ...catalytic domain autoinhibition.•Polar contacts between propeptide and catalytic domain mimic inhibitor interactions.
Leucyl aminopeptidase A from Aspergillus oryzae RIB40 (AO-LapA) is an exo-acting peptidase, widely utilised in food debittering applications. AO-LapA is secreted as a zymogen by the host and requires enzymatic cleavage of the autoinhibitory propeptide to reveal its full activity. Scarcity of structural data of zymogen aminopeptidases hampers a better understanding of the details of their molecular action of autoinhibition and how this might be utilised to improve the properties of such enzymes by recombinant methods for more effective bioprocessing. To address this gap in the literature, herein we report high-resolution crystal structures of recombinantly expressed AO-LapA precursor (AO-proLapA), mature LapA (AO-mLapA) and AO-mLapA complexed with reaction product l-leucine (AO-mLapA-Leu), all purified from Pichia pastoris culture supernatant. Our structures reveal a plausible molecular mechanism of LapA catalytic domain autoinhibition by propeptide and highlights the role of intramolecular chaperone (IMC). Our data suggest an absolute requirement for IMC in the maturation of cognate catalytic domain of AO-LapA. This observation is reinforced by our expression and refolding data of catalytic domain only (AO-refLapA) from Escherichia coli inclusion bodies, revealing a limited active conformation. Our work supports the notion that known synthetic aminopeptidase inhibitors and substrates mimic key polar contacts between propeptide and corresponding catalytic domain, demonstrated in our AO-proLapA zymogen crystal structure. Furthermore, understanding the atomic details of the autoinhibitory mechanism of cognate catalytic domains by native propeptides has wider reaching implications toward synthetic production of more effective inhibitors of bimetallic aminopeptidases and other dizinc enzymes that share an analogous reaction mechanism.
In patients with osteoporosis treated with antiresorptive agents, reduction in bone turnover explains much of the observed fracture risk reduction. Lower levels of bone turnover markers (BTMs) appear ...to be associated with a lower risk of fracture in bisphosphonate‐treated patients. BTMs were measured in a subset of subjects in the HORIZON Pivotal Fracture Trial. Annual infusions of zoledronic acid 5 mg significantly reduced BTMs: median decrease of 50% for β‐C‐terminal telopeptides of type 1 collagen (β‐CTX), 30% for bone alkaline phosphatase (ALP), and 56% for procollagen type 1 amino‐terminal propeptide (PINP). The mean level of BTMs decreased in treated patients but remained within the premenopausal range before the next injection. The percentage of zoledronic acid–treated patients with values below the premenopausal reference range at all time points was 1.7%, 17.8%, and 19% for bone ALP, CTX, and PINP, respectively. The third injection of zoledronic acid resulted in 60% reduction of β‐CTX within 9–11 days, followed by a gradual increase, indicating the persistence of osteoclastic bone resorption. The association between changes in BTMs and fracture incidence was assessed in 1132 patients who had PINP measurements at baseline and 1 yr. There was no association between low PINP levels at 1 yr and increased fracture incidence. In summary, (1) annual injections of zoledronic acid reduced BTMs in the premenopausal range, with a significant response persisting after the third infusion; and (2) low levels of PINP were not associated with increased fracture risk.
Growth and meat quality are concerned issues in aquaculture. The inhibition of MSTN can lead to significant muscle proliferation in mammals, as well as a decrease in muscle fat content. In fish, the ...effect of MSTN inhibition on muscle fat content is ambiguous. In this study, we constructed transgenic red carp with Larimichthys crocea MSTN1 propeptide (LcMSTN1 propeptide) to study how the inhibition of MSTN1 affects the growth and muscle fat content in fish. Southern Blotting showed that LcMSTN1 propeptide was integrated into the red carp genome. In muscle tissue, the expression of MSTN1 propeptide in transgenic red carp was significantly higher than that of non-transgenic red carp (P < 0.001). The mRNA levels of growth-related genes, MyoD, MyoG and MyHC, in the muscle tissue of transgenic red carp were also significantly higher than those in non-transgenic red carp (P < 0.01). However, there was no significant difference in both muscle fat content and the mRNA levels of lipid metabolism-related genes FAS and LPL between transgenic and non-transgenic red carps. Microscopical observation showed that the muscle fiber diameter in transgenic red carp was significantly larger than that of non-transgenic red carp, implying hypertrophy of muscle fibers. These results indicate that LcMSTN1 propeptide can promote the muscle growth, and does not affect muscle fat content in transgenic red carp, which is different from mammals.
•We constructed transgenic red carp with Larimichthys crocea MSTN1 Propeptide.•Muscle growth-related parameters were significantly larger in transgenic red carp.•Muscle fat contents and lipid metabolism didn't change significantly.
•No effect of liraglutide was seen on bone turnover markers in patients with prediabetes and schizophrenia treated with olanzapine and/or clozapine.•Liraglutide induced weight loss, but weight loss ...was not associated with increased bone metabolism.
Schizophrenia is associated with a lowered bone mineral density. The antidiabetic and body weight lowering glucagon-like peptide-1 receptor agonist liraglutide has shown to mitigate overweight and impaired glucose tolerance associated with olanzapine and clozapine. As liraglutide has been proposed to affect bone metabolism, we evaluated the effect of liraglutide on bone turnover markers (BTM) in patients with prediabetes and schizophrenia treated with olanzapine or clozapine. Patients diagnosed with a schizophrenia spectrum disorder treated with the antipsychotic compounds clozapine and/or olanzapine, having prediabetes and a BMI above 27 kg/m2 were randomized to 16 weeks of treatment with liraglutide or placebo. Fasting state serum sampled in the morning from patients (n=78) were analysed for the BTM collagen type 1 C-telopeptide (CTX) and procollagen type 1 N-terminal propeptide (P1NP). After 16 weeks of treatment, no significant changes of neither P1NP nor CTX were observed when comparing liraglutide to placebo. No association between changes of bone turnover markers and change of body weight were found in the group treated with liraglutide. In conclusion, no treatment effect on CTX nor P1NP was observed, and thus, this study does not raise any concerns in patients with schizophrenia and prediabetes treated with liraglutide regarding bone-related adverse effects.
Summary
Objective
Hyperandrogenism, hyperinsulinaemia and obesity, known characteristics of polycystic ovary syndrome (PCOS), may influence bone mineral density and biochemical markers of bone ...turnover (BTMs) can provide a noninvasive assessment of bone turnover. To this end, the serum concentrations of BTMs and 25‐hydroxyvitamin D (25OHD) were analysed in women with PCOS, and their possible associations with metabolic parameters of PCOS were determined.
Subjects and methods
Bone formation markers procollagen type I amino‐terminal propeptide (PINP) and osteocalcin (OC), and bone resorption marker carboxy‐terminal cross‐linking telopeptide of type I collagen (CTX), along with 25OHD, were measured in 298 women with PCOS and 194 healthy controls.
Results
Serum levels of PINP (47.0 ± 20.2 vs 58.1 ± 28.6 μg/L, P < .001) and OC (18.2 ± 7.5 vs 20.6 ± 9.8 μg/L, P < .001) were decreased in women with PCOS compared with controls, whereas no significant differences were found in CTX and 25OHD levels. Age‐stratified analyses suggested that PINP (50.5 ± 21.7 vs 68.2 ± 26.6 μg/L, P < .001) and OC levels (20.4 ± 7.6 vs 25.5 ± 9.6 μg/L, P < .001) were decreased only in the younger age group (≤30 years) women with PCOS compared with controls. The formation markers and resorption marker decreased with age in both study groups.
Conclusions
Bone formation markers were decreased in younger women with PCOS when compared with healthy women, which may affect bone mass in these women.
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