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International Journal of Molecular... Feb 2024The employment of 2-deoxyribose-5-phosphate aldolase (DERA) stands as a prevalent biocatalytic route for synthesizing statin side chains. The main problem with this...
The employment of 2-deoxyribose-5-phosphate aldolase (DERA) stands as a prevalent biocatalytic route for synthesizing statin side chains. The main problem with this pathway is the low stability of the enzyme. In this study, mesocellular silica foam (MCF) with different pore sizes was used as a carrier for the covalent immobilization of DERA. Different functionalizing and activating agents were tested and kinetic modeling was subsequently performed. The use of succinic anhydride as an activating agent resulted in an enzyme hyperactivation of approx. 140%, and the stability almost doubled compared to that of the free enzyme. It was also shown that the pore size of MCF has a decisive influence on the stability of the DERA enzyme.
Topics: Hydroxymethylglutaryl-CoA Reductase Inhibitors; Silicon Dioxide; Aldehyde-Lyases; Fructose-Bisphosphate Aldolase; Biocatalysis
PubMed: 38396648
DOI: 10.3390/ijms25041971 -
Applied Microbiology and Biotechnology Feb 2024Steroid-based drugs are now mainly produced by the microbial transformation of phytosterol, and a two-step bioprocess is adopted to reach high space-time yields, but...
Steroid-based drugs are now mainly produced by the microbial transformation of phytosterol, and a two-step bioprocess is adopted to reach high space-time yields, but byproducts are frequently observed during the bioprocessing. In this study, the catabolic switch between the C19- and C22-steroidal subpathways was investigated in resting cells of Mycobacterium neoaurum NRRL B-3805, and a dose-dependent transcriptional response toward the induction of phytosterol with increased concentrations was found in the putative node enzymes including ChoM2, KstD1, OpccR, Sal, and Hsd4A. Aldolase Sal presented a dominant role in the C22 steroidal side-chain cleavage, and the byproduct was eliminated after sequential deletion of opccR and sal. Meanwhile, the molar yield of androst-1,4-diene-3,17-dione (ADD) was increased from 59.4 to 71.3%. With the regard of insufficient activity of rate-limiting enzymes may also cause byproduct accumulation, a chromosomal integration platform for target gene overexpression was established supported by a strong promoter L2 combined with site-specific recombination in the engineered cell. Rate-limiting steps of ADD bioconversion were further characterized and overcome. Overexpression of the kstD1 gene further strengthened the bioconversion from AD to ADD. After subsequential optimization of the bioconversion system, the directed biotransformation route was developed and allowed up to 82.0% molar yield with a space-time yield of 4.22 g·L·day. The catabolic diversion elements and the genetic overexpression tools as confirmed and developed in present study offer new ideas of M. neoaurum cell factory development for directed biotransformation for C19- and C22-steroidal drug intermediates from phytosterol. KEY POINTS: • Resting cells exhibited a catabolic switch between the C19- and C22-steroidal subpathways. • The C22-steroidal byproduct was eliminated after sequential deletion of opccR and sal. • Rate-limiting steps were overcome by promoter engineering and chromosomal integration.
Topics: Aldehyde-Lyases; Androstadienes; Cell Differentiation; Phytosterols; Polyenes
PubMed: 38300290
DOI: 10.1007/s00253-023-12847-z -
Cell Communication and Signaling : CCS Jan 2024Parkinson's disease (PD), a chronic and severe neurodegenerative disease, is pathologically characterized by the selective loss of nigrostriatal dopaminergic neurons....
BACKGROUND
Parkinson's disease (PD), a chronic and severe neurodegenerative disease, is pathologically characterized by the selective loss of nigrostriatal dopaminergic neurons. Dopamine (DA), the neurotransmitter produced by dopaminergic neurons, and its metabolites can covalently modify proteins, and dysregulation of this process has been implicated in neuronal loss in PD. However, much remains unknown about the protein targets.
METHODS
In the present work, we designed and synthesized a dopamine probe (DA-P) to screen and identify the potential protein targets of DA using activity-based protein profiling (ABPP) technology in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In situ pull-down assays, cellular thermal shift assays (CETSAs) and immunofluorescence were performed to confirm the DA modifications on these hits. To investigate the effects of DA modifications, we measured the enzymatic activities of these target proteins, evaluated glycolytic stress and mitochondrial respiration by Seahorse tests, and systematically analyzed the changes in metabolites with unbiased LC-MS/MS-based non-targeted metabolomics profiling.
RESULTS
We successfully identified three glycolytic proteins, aldolase A, α-enolase and pyruvate kinase M2 (PKM2), as the binding partners of DA. DA bound to Glu166 of α-enolase, Cys49 and Cys424 of PKM2, and Lys230 of aldolase A, inhibiting the enzymatic activities of α-enolase and PKM2 and thereby impairing ATP synthesis, resulting in mitochondrial dysfunction.
CONCLUSIONS
Recent research has revealed that enhancing glycolysis can offer protection against PD. The present study identified that the glycolytic pathway is vulnerable to disruption by DA, suggesting a promising avenue for potential therapeutic interventions. Safeguarding glycolysis against DA-related disruption could be a potential therapeutic intervention for PD.
Topics: Humans; Parkinson Disease; Dopamine; Fructose-Bisphosphate Aldolase; Chromatography, Liquid; Neurodegenerative Diseases; Tandem Mass Spectrometry; Proteins; Phosphopyruvate Hydratase
PubMed: 38287374
DOI: 10.1186/s12964-024-01478-0 -
The Turkish Journal of Pediatrics 2023Sphingosine phosphate lyase insufficiency syndrome (SPLIS) caused by inactivating mutations in the human SGPL1 gene results in congenital nephrotic syndrome, adrenal...
BACKGROUND
Sphingosine phosphate lyase insufficiency syndrome (SPLIS) caused by inactivating mutations in the human SGPL1 gene results in congenital nephrotic syndrome, adrenal insufficiency, ichthyosis, immunodeficiency, and a wide range of pathological neurological features. We present a novel mutation in the SGPL1 gene causing hypocalcemia, primary adrenal insufficiency (PAI), nephrotic syndrome, subclinical hypothyroidism, lymphopenia, ptosis, and pathologic neuroimaging findings.
CASE
A Turkish male infant presented with bruising at 2 months of age and was diagnosed with hypocalcemia, PAI, and subclinical hypothyroidism. At the age of 15 months, he was admitted to the hospital with ptosis. Other systemic manifestations included persistent lymphopenia and nephrotic syndrome. Magnetic resonance imaging (MRI) of the brain and orbit demonstrated asymmetric contrast enhancement in the left cavernosal sinus, orbital apex, and thinning at the bilateral optic nerve. Whole exome sequencing (WES) revealed a homozygous c.1432C > G (p.Gln478Glu) variant in the SGPL1 gene (NM_003901.4), which has not previously been reported in the literature.
CONCLUSIONS
Novel mutations in SGPL1 are still being identified. This case reminded us that SPLIS should not be considered for patients with nephrotic syndrome alone. Still, PAI may also include patients with neurological disorders, hypocalcemia, and pathological neuroimaging findings such as thinning at the bilateral optic nerve.
Topics: Infant; Humans; Male; Hypocalcemia; Nephrotic Syndrome; Hypothyroidism; Lymphopenia; Mutation; Aldehyde-Lyases
PubMed: 38204317
DOI: 10.24953/turkjped.2022.187 -
Nature Communications Dec 2023One-carbon (C1) substrates, such as methanol or formate, are attractive feedstocks for circular bioeconomy. These substrates are typically converted into formaldehyde,...
One-carbon (C1) substrates, such as methanol or formate, are attractive feedstocks for circular bioeconomy. These substrates are typically converted into formaldehyde, serving as the entry point into metabolism. Here, we design an erythrulose monophosphate (EuMP) cycle for formaldehyde assimilation, leveraging a promiscuous dihydroxyacetone phosphate dependent aldolase as key enzyme. In silico modeling reveals that the cycle is highly energy-efficient, holding the potential for high bioproduct yields. Dissecting the EuMP into four modules, we use a stepwise strategy to demonstrate in vivo feasibility of the modules in E. coli sensor strains with sarcosine as formaldehyde source. From adaptive laboratory evolution for module integration, we identify key mutations enabling the accommodation of the EuMP reactions with endogenous metabolism. Overall, our study demonstrates the proof-of-concept for a highly efficient, new-to-nature formaldehyde assimilation pathway, opening a way for the development of a methylotrophic platform for a C1-fueled bioeconomy in the future.
Topics: Escherichia coli; Methanol; Formaldehyde; Sarcosine; Fructose-Bisphosphate Aldolase; Metabolic Engineering
PubMed: 38123535
DOI: 10.1038/s41467-023-44247-2 -
Journal of Investigative Medicine High... 2023Dermatomyositis (DM) is a rare inflammatory myopathy with an incidence of 9.63 per 1 000 000 people and typically presents with skin rash and muscle weakness. We...
Dermatomyositis (DM) is a rare inflammatory myopathy with an incidence of 9.63 per 1 000 000 people and typically presents with skin rash and muscle weakness. We report a case of DM that presented with proximal muscle weakness, normal creatine phosphokinase (CPK), negative myositis antibody panel, and non-specific histopathological findings on muscle biopsy, without initial skin involvement. A 67-year-old male presented with subacute bilateral proximal lower-extremity weakness and weight loss of 20 pounds over 3 months. Laboratory investigation was significant for elevated erythrocyte sedimentation rate, C-reactive protein, CPK, and aldolase, with negative myositis-specific antibodies. Femur magnetic resonance imaging revealed subcutaneous, fascial, and muscle edema throughout quadriceps and gluteal muscles. Muscle biopsy showed myofiber atrophy with perivascular and endomysial T-lymphocytes and histiocytes, as well as scattered necrotic myofibers. He was diagnosed with inflammatory myositis and started on prednisone and monthly IVIG infusions. At 2-month follow-up, he reported new rashes on the extensor surfaces of the hands consistent with Gottron's papules, mechanic's hands, and livedo reticularis of feet and arms. Cases of DM that present with myopathy and later develop skin changes are rare. Our patient had several months of progressive proximal muscle weakness, and skin changes occurred after he was started on treatment. Laboratory findings include elevated CPK, aldolase, and myositis-specific auto-antibodies. Muscle biopsy helps in diagnosis; however, findings may be nonspecific-as was the case in our patient. Corticosteroids are first-line treatment. Long-term follow-up studies are necessary to better understand the incidence of late-onset development of typical skin findings.
Topics: Male; Humans; Aged; Dermatomyositis; Myositis; Skin; Muscle Weakness; Aldehyde-Lyases
PubMed: 38097369
DOI: 10.1177/23247096231217829 -
Cancer Biology & Therapy Dec 2023Radioresistance is the major obstacle that affects the efficacy of radiotherapy which is an important treatment for cervical cancer. By analyzing the databases, we found...
Radioresistance is the major obstacle that affects the efficacy of radiotherapy which is an important treatment for cervical cancer. By analyzing the databases, we found that aldolase A (ALDOA), which is a key enzyme in metabolic reprogramming, has a higher expression in cervical cancer patients and is associated with poor prognosis. We detected the expression of ALDOA in the constructed cervical cancer radioresistance (RR) cells by repetitive irradiation and found that it was upregulated compared to the control cells. Functional assays were conducted and the results showed that the knockdown of ALDOA in cervical cancer RR cells inhibited the proliferation, migration, and clonogenic abilities by regulating the cell glycolysis. In addition, downregulation of ALDOA enhanced radiation-induced apoptosis and DNA damage by causing G2/M phase arrest and further promoted radiosensitivity of cervical cancer cells. The functions of ALDOA in regulating tumor radiosensitivity were also verified by the mouse tumor transplantation model . Therefore, our study provides new insights into the functions of ALDOA in regulating the efficacy of radiotherapy and indicates that ALDOA might be a promising target for enhancing radiosensitivity in treating cervical cancer patients.
Topics: Animals; Female; Humans; Mice; Cell Line, Tumor; Cell Proliferation; DNA Damage; Fructose-Bisphosphate Aldolase; Gene Expression Regulation, Neoplastic; Glycolysis; Radiation Tolerance; Uterine Cervical Neoplasms
PubMed: 38010897
DOI: 10.1080/15384047.2023.2287128 -
Journal of Translational Medicine Nov 2023LIPH, a membrane-associated phosphatidic acid-selective phospholipase A1a, can produce LPA (Lysophosphatidic acid) from PA (Phosphatidic acid) on the outer leaflet of...
BACKGROUND
LIPH, a membrane-associated phosphatidic acid-selective phospholipase A1a, can produce LPA (Lysophosphatidic acid) from PA (Phosphatidic acid) on the outer leaflet of the plasma membrane. It is well known that LIPH dysfunction contributes to lipid metabolism disorder. Previous study shows that LIPH was found to be a potential gene related to poor prognosis with pancreatic ductal adenocarcinoma (PDAC). However, the biological functions of LIPH in PDAC remain unclear.
METHODS
Cell viability assays were used to evaluate whether LIPH affected cell proliferation. RNA sequencing and immunoprecipitation showed that LIPH participates in tumor glycolysis by stimulating LPA/LPAR axis and maintaining aldolase A (ALDOA) stability in the cytosol. Subcutaneous, orthotopic xenograft models and patient-derived xenograft PDAC model were used to evaluate a newly developed Gemcitabine-based therapy.
RESULTS
LIPH was significantly upregulated in PDAC and was related to later pathological stage and poor prognosis. LIPH downregulation in PDAC cells inhibited colony formation and proliferation. Mechanistically, LIPH triggered PI3K/AKT/HIF1A signaling via LPA/LPAR axis. LIPH also promoted glycolysis and de novo synthesis of glycerolipids by maintaining ALDOA stability in the cytosol. Xenograft models show that PDAC with high LIPH expression levels was sensitive to gemcitabine/ki16425/aldometanib therapy without causing discernible side effects.
CONCLUSION
LIPH directly bridges PDAC cells and tumor microenvironment to facilitate aberrant aerobic glycolysis via activating LPA/LPAR axis and maintaining ALDOA stability, which provides an actionable gemcitabine-based combination therapy with limited side effects.
Topics: Humans; Fructose-Bisphosphate Aldolase; Phosphatidylinositol 3-Kinases; Cell Line, Tumor; Carcinoma, Pancreatic Ductal; Pancreatic Neoplasms; Gemcitabine; Cell Proliferation; Glycolysis; Phenotype; Gene Expression Regulation, Neoplastic; Tumor Microenvironment
PubMed: 37990271
DOI: 10.1186/s12967-023-04702-6 -
International Journal of Molecular... Oct 2023Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is an inborn error of metabolism caused by inactivating mutations in , the gene encoding...
Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is an inborn error of metabolism caused by inactivating mutations in , the gene encoding sphingosine-1-phosphate lyase (SPL), an essential enzyme needed to degrade sphingolipids. SPLIS features include glomerulosclerosis, adrenal insufficiency, neurological defects, ichthyosis, and immune deficiency. Currently, there is no cure for SPLIS, and severely affected patients often die in the first years of life. We reported that adeno-associated virus (AAV) 9-mediated gene therapy (AAV-SPL) given to newborn knockout mice that model SPLIS and die in the first few weeks of life prolonged their survival to 4.5 months and prevented or delayed the onset of SPLIS phenotypes. In this study, we tested the efficacy of a modified AAV-SPL, which we call AAV-SPL 2.0, in which the original cytomegalovirus (CMV) promoter driving the transgene is replaced with the synthetic "CAG" promoter used in several clinically approved gene therapy agents. AAV-SPL 2.0 infection of human embryonic kidney (HEK) cells led to 30% higher SPL expression and enzyme activity compared to AAV-SPL. Newborn knockout mice receiving AAV-SPL 2.0 survived ≥ 5 months and showed normal neurodevelopment, 85% of normal weight gain over the first four months, and delayed onset of proteinuria. Over time, treated mice developed nephrosis and glomerulosclerosis, which likely resulted in their demise. Our overall findings show that AAV-SPL 2.0 performs equal to or better than AAV-SPL. However, improved kidney targeting may be necessary to achieve maximally optimized gene therapy as a potentially lifesaving SPLIS treatment.
Topics: Animals; Humans; Mice; Aldehyde-Lyases; Dependovirus; Lysophospholipids; Mice, Knockout; Parvovirinae; Phosphates; Sphingosine; Genetic Therapy
PubMed: 37958544
DOI: 10.3390/ijms242115560 -
Basic Research in Cardiology Nov 2023Cardiovascular disease (CVD) is a major threat to human health, accounting for 46% of non-communicable disease deaths. Glycolysis is a conserved and rigorous biological... (Review)
Review
Cardiovascular disease (CVD) is a major threat to human health, accounting for 46% of non-communicable disease deaths. Glycolysis is a conserved and rigorous biological process that breaks down glucose into pyruvate, and its primary function is to provide the body with the energy and intermediate products needed for life activities. The non-glycolytic actions of enzymes associated with the glycolytic pathway have long been found to be associated with the development of CVD, typically exemplified by metabolic remodeling in heart failure, which is a condition in which the heart exhibits a rapid adaptive response to hypoxic and hypoxic conditions, occurring early in the course of heart failure. It is mainly characterized by a decrease in oxidative phosphorylation and a rise in the glycolytic pathway, and the rise in glycolysis is considered a hallmark of metabolic remodeling. In addition to this, the glycolytic metabolic pathway is the main source of energy for cardiomyocytes during ischemia-reperfusion. Not only that, the auxiliary pathways of glycolysis, such as the polyol pathway, hexosamine pathway, and pentose phosphate pathway, are also closely related to CVD. Therefore, targeting glycolysis is very attractive for therapeutic intervention in CVD. However, the relationship between glycolytic pathway and CVD is very complex, and some preclinical studies have confirmed that targeting glycolysis does have a certain degree of efficacy, but its specific role in the development of CVD has yet to be explored. This article aims to summarize the current knowledge regarding the glycolytic pathway and its key enzymes (including hexokinase (HK), phosphoglucose isomerase (PGI), phosphofructokinase-1 (PFK1), aldolase (Aldolase), phosphoglycerate metatase (PGAM), enolase (ENO) pyruvate kinase (PKM) lactate dehydrogenase (LDH)) for their role in cardiovascular diseases (e.g., heart failure, myocardial infarction, atherosclerosis) and possible emerging therapeutic targets.
Topics: Humans; Cardiovascular Diseases; Heart Failure; Oxidative Phosphorylation; Aldehyde-Lyases; Metabolic Networks and Pathways
PubMed: 37938421
DOI: 10.1007/s00395-023-01018-w