This study aimed to enhance the expression level of a novel trypsin gene from Streptomyces fradiae ATCC14544 in Komagataella phaffii GS115 through the combinational use of propeptide engineering and ...self-degradation residues modification strategies. An artificial propeptide consisted of thioredoxin TrxA, the bovine propeptide DDDDK and the hydrophobic peptide FVEF was introduced to replace the original propeptide while the self-degradation residue sites were predicted and analyzed through alanine screening. The results showed that the quantity and enzymatic activity of asft with engineered propeptide reached 47.02 mg/mL and 33.9 U/mL, which were 9.6 % and 59.29 % higher than those of wild-type (42.9 mg/mL and 13.8 U/mL). Moreover, the introduction of R295A/R315A mutation further enhanced the enzymatic activity (58.86 U/mL) and obviously alleviated the phenomena of self-degradation. The tolerance of trypsin towards alkaline environment was also improved since the optimal pH was shifted from pH 9.0 to pH 9.5 and the half-life value at pH 10 was significantly extended. Finally, the fermentation media composition and condition were optimized and trypsin activity in optimal condition reached 160.58 U/mL, which was 2.73-fold and 11.64-fold of that before optimization or before engineering. The results obtained in this study indicated that the combinational use of propeptide engineering and self-degradation sites modification might have great potential application in production of active trypsins.
•Stress and antidepressants modulate brain neuroplasticity and BDNF levels.•BDNF production is tied to previous proBDNF making.•Binary sorting hypothesis proposes BDNF/proBDNF balance as crucial to ...neuroremodeling.•Neuronal remodeling is necessary for antidepressant effects on anxiety and depression.
Depression and posttraumatic stress disorder are assumed to be maladaptive responses to stress and antidepressants are thought to counteract such responses by increasing BDNF (brain-derived neurotrophic factor) levels. BDNF acts through TrkB (tropomyosin-related receptor kinase B) and plays a central role in neuroplasticity. In contrast, both precursor proBDNF and BDNF propeptide (another metabolic product from proBDNF cleavage) have a high affinity to p75 receptor (p75R) and usually convey apoptosis and neuronal shrinkage. Although BDNF and proBDNF/propeptide apparently act in opposite ways, neuronal turnover and remodeling might be a final common way that both act to promote more effective neuronal networking, avoiding neuronal redundancy and the misleading effects of environmental contingencies. This review aims to provide a brief overview about the BDNF functional role in antidepressant action and about p75R and TrkB signaling to introduce the “continuum-sorting hypothesis.” The resulting hypothesis suggests that both BDNF/proBDNF and BDNF/propeptide act as protagonists to fine-tune antidepressant-dependent neuroplasticity in crucial brain structures to modulate behavioral responses to stress.
A prospective, single-center, observational study.
To explore the association between serum levels of bone turnover markers and ossification of the posterior longitudinal ligament (OPLL) in the ...thoracic spine.
The relationship between bone turnover markers, such as N-terminal propeptide of type I procollagen (PINP) or tartrate-resistant acid phosphate 5b (TRACP-5b), and OPLL has previously been examined. However, the correlation between these markers and thoracic OPLL, which is more severe than cervical-only OPLL, remains unclear.
This prospective study included 212 patients from a single institution with compressive spinal myelopathy and divided them into those without OPLL (Non-OPLL group, 73 patients) and those with OPLL (OPLL group, 139 patients). The OPLL group was further subdivided into cervical OPLL (C-OPLL, 92 patients) and thoracic OPLL (T-OPLL, 47 patients) groups. Patients' characteristics and biomarkers related to bone metabolism, such as calcium, inorganic phosphate (Pi), 25-hydroxyvitamin D, 1α,25 dihydroxyvitamin D, PINP, and TRACP-5b, were compared between the Non-OPLL and OPLL groups, as well as the C-OPLL and T-OPLL groups. Bone metabolism biomarkers were also compared after adjusting for age, sex, body mass index, and the presence of renal impairment using propensity score-matched analysis.
The OPLL group had significantly lower serum levels of Pi and higher levels of PINP versus the Non-OPLL group as determined by propensity score-matched analysis. The comparison results between the C-OPLL and T-OPLL groups using a propensity score-matched analysis showed that T-OPLL patients had significantly higher concentrations of bone turnover markers, such as PINP and TRACP-5b, compared with C-OPLL patients.
Increased systemic bone turnover may be associated with the presence of OPLL in the thoracic spine, and bone turnover markers such as PINP and TRACP-5b can help screen for thoracic OPLL.
ABSTRACT
The type I procollagen carboxyterminal(C‐)propeptides are crucial in directing correct assembly of the procollagen heterotrimers. Defects in these domains have anecdotally been reported in ...patients with Osteogenesis Imperfecta (OI) and few genotype–phenotype correlations have been described. To gain insight in the functional consequences of C‐propeptide defects, we performed a systematic review of clinical, molecular, and biochemical findings in all patients in whom we identified a type I procollagen C‐propeptide defect, and compared this with literature data. We report 30 unique type I procollagen C‐propeptide variants, 24 of which are novel. The outcome of COL1A1 nonsense and frameshift variants depends on the location of the premature termination codon. Those located prior to 50–55 nucleotides upstream of the most 3’ exon–exon junction lead to nonsense‐mediated mRNA decay (NMD) and cause mild OI. Those located beyond this boundary escape NMD, generally lead to production of stable, overmodified procollagen chains, which may partly be retained intracellularly, and are usually associated with severe‐to‐lethal OI. Proα1(I)‐C‐propeptide defects that permit chain association result in more severe phenotypes than those inhibiting chain association. We demonstrate that the crystal structure of the proα1(III)‐C‐propeptide is a reliable tool to predict phenotypic severity for most COL1A1‐C‐propeptide missense variants, whereas for COL1A2‐C‐propeptide variants, the phenotypic outcome is milder than predicted.
The type I procollagen carboxyterminal (C‐) propeptides are crucial for correct assembly of the procollagen heterotrimers. Defects in these domains have anecdotally been reported in patients with osteogenesis imperfecta. Here we give a comprehensive overview of clinical, molecular and biochemical characteristics of C‐propeptide variants, thereby providing insights in their functional consequences and genotype‐phenotype correlations. We demonstrate that the crystal structure of the proα1(III)‐C‐propeptide is a reliable tool to predict phenotypic severity for most COL1A1‐C‐propeptide missense variants, while for COL1A2 the outcome is milder than predicted.
α-L-arabinofuranosidase (ARA) with enhanced specific activity and in large amounts, is needed for a variety of industrial applications. To improve ARA production with engineered methylotrophic yeast
..., a genetically modified
gene from
ND-1 was investigated. Through codon optimization and rational replacement of α-factor signal peptide with the native propeptide (MFSRRNLVALGLAATVSA), ARA production was improved from 2.61 ± 0.13 U/mL to 14.37 ± 0.22 U/mL in shaking flask culture (a 5.5-fold increase). Results of N-terminal sequencing showed that secreted active ARA of recombinant strain p-oARA had theoretical initial five amino acids (GPCDI) comparable to the mature sequences of α-oARA (EAEAG) and αp-oARA (NLVAL). The kinetic values have been determined for ARA of recombinant strain p-oARA (
= 747.55 μmol/min/mg,
= 5.36 mmol/L), optimal activity temperature 60°C and optimal pH 4.0. Scaling up of ARA production by p-oARA in a 7.5-L fermentor resulted in remarkably high extracellular ARA specific activity (479.50 ± 12.83 U/mg) at 168 h, and maximal production rate 164.47 ± 4.40 U/mL. In studies of corn stover degradation activity, degree of synergism for ARA and xylanase was 32.4% and enzymatic hydrolysis yield for ARA + xylanase addition was 15.9% higher than that of commercial cellulase, indicating significant potential of ARA for catalytic conversion of corn stover to fermentable sugars for biofuel production.
•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.
CTHRC1 is transiently expressed by activated fibroblasts during tissue repair and in certain cancers, and CTHRC1 derived from osteocytes is detectable in circulation. Because its biological activity ...is poorly understood, we investigated whether the N terminus of CTHRC1 encodes a propeptide requiring cleavage to become activated. The effects of full-length versus cleaved recombinant CTHRC1 on endothelial cell metabolism and gene expression were examined in vitro. Respirometry was performed on Cthrc1 null and wildtype mice to obtain evidence for biological activity of CTHRC1 in vivo. Cleavage of the propeptide observed in vitro was attenuated in the presence of protease inhibitors, and cleaved CTHRC1 significantly promoted glycolysis whereas full-length CTHRC1 was less effective. The respiratory exchange ratio was significantly higher in wildtype mice compared to Cthrc1 null mice, supporting the findings of CTHRC1 promoting glycolysis in vivo. Key enzymes involved in glycolysis were significantly upregulated in endothelial cells in response to treatment with CTHRC1. In healthy human subjects, 58% of the cohort had detectable levels of circulating full-length CTHRC1, whereas all subjects with undetectable levels of full-length CTHRC1 (with one exception) had measurable levels of truncated CTHRC1 (88 pg/ml to >400 ng/ml). Our findings support a concept where CTHRC1 induction in activated fibroblasts at sites of ischemia such as tissue injury or cancer functions to increase glycolysis for ATP production under hypoxic conditions, thereby promoting cell survival and tissue repair. By promoting glycolysis under normoxic conditions, CTHRC1 may also be a contributor to the Warburg effect characteristically observed in many cancers.
Abstract Biochemical markers of bone turnover provide insight into ongoing rates of skeletal metabolism and tumor–bone interactions in patients with malignant bone disease. This article reviews the ...available recent evidence assessing the potential of bone markers for detecting and monitoring malignant bone lesions in patients with advanced cancers, and for assessing overall skeletal health and response to antiresorptive therapies in patients at all stages of cancer progression. Most data thus far are for urinary N-terminal cross-linked telopeptide of type I collagen (NTX) in predicting risks of skeletal morbidity and death and monitoring response to zoledronic acid in patients with bone metastases. Ongoing studies are evaluating such correlations for other markers and therapies. Emerging evidence suggests that bone markers may help identify patients at high risk for bone metastasis or bone lesion progression, thereby allowing improved follow-up. Results from ongoing clinical trials evaluating such potential applications of bone markers are awaited.
There exists a marked circadian variation for several bone markers (BM), which is influenced by endogenous as well as exogenous factors including hormones, physical activity, and fasting. ...Consequently, was the aim of this review to provide an overview of the knowledge of the circadian variation of BM and which factors influence this rhythmicity. A systematic search of PubMed was performed for studies evaluating the circadian variation of BM and which factors influence this rhythmicity. The studies were screened for eligibility by a set of predetermined criteria including a list of relevant BM and a minimum study duration of 24 h with at least 3 blood samples of which two should be at least 6 h apart. In total were 29 papers included. There exists a marked circadian variation for most BM including Carboxy-terminal Cross-Linked Telopeptide of Type I Collagen (CTX) and osteocalcin (OC) with nighttime or early morning peak. Pro-collagen Type I N-terminal Propeptide (PINP) and PTH also showed circadian rhythm but with less amplitude. The inter-osteoblast-osteoclast regulatory markers such as OPG, RANKL, FGF23, and sclerostin showed no circadian rhythm. The markers were differently affected by exogenous factors like fasting, which greatly reduced the circadian variation of CTX but did not affect PINP or OC. The marked circadian variation and the factors which influence the rhythmicity, e.g., fasting are of great consequence when measuring BM. To reduce variation and heighten validity should circadian variation and fasting be kept in mind when measuring BM.
Most GH11 family endo-β-1,4-xylanases contain a propeptide region linked to the N-terminal region. The mechanistic basis of this region harboring key regulation information for enzyme function, ...however, remains poorly understood. We reported an investigation on the allosteric regulation mechanism of the propeptide based on biochemical characterization, molecular dynamics simulations, and evolutionary analysis. We discovered that the mutant of truncated propeptide shows a remarkably increased thermal stability (melting temperature increased by 11.5 °C) and catalytic efficiency (1.7-fold k cat/K m value of wild type). Molecular dynamics simulations reveal that long-range fluctuations in the propeptide lead to a conformational perturbation in the catalytic pocket and the thumb region. The probability of sampling the active conformation during the glycosylation step is reduced (i.e., catalytic efficiency). In-depth sequence analysis indicates that the propeptide has a strong plasticity and degeneration trend, and propeptide truncation experiments of the homologous enzyme XynB validated the feasibility of the truncation strategy. This work reveals the role of GH11 family propeptides in functional regulation and provides a straightforward and practical method to increase the robustness of GH11 family xylanases.
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