Animals ; Autophagy/genetics* ; Cholesterol/metabolism* ; Gene Expression Regulation ; Integrases/metabolism* ; Leydig Cells/metabolism* ; Male ; Mice, Knockout ; Multienzyme Complexes/metabolism* ; Phosphoproteins/metabolism* ; Primary Cell Culture ; Progesterone Reductase/metabolism* ; RNA Splicing Factors/metabolism* ; Scavenger Receptors, Class B/metabolism* ; Sequestosome-1 Protein/metabolism* ; Signal Transduction ; Sirtuin 1/genetics* ; Sodium-Hydrogen Exchangers/metabolism* ; Steroid 17-alpha-Hydroxylase/metabolism* ; Steroid Isomerases/metabolism* ; Testosterone/genetics*

In vitro multi-enzyme molecular machines that follow the designed multi-enzyme pathways, require the rational optimization and adaptation of several purified or partially purified enzyme components, in order to convert certain substrates into target compounds in vitro in an efficient manner. This type of molecular machine is component-based and modularized, so that its design, assembly, and regulation processes are highly flexible. Recently, the advantages of in vitro multi-enzyme molecular machines on the precise control of reaction process and the enhancement of product yield have suggested their great application potential in biomanufacturing. Studies on in vitro multi-enzyme molecular machines have become an important branch of synthetic biology, and are gaining increasing attentions. This article systematically reviews the enzyme component-/module-based construction strategy of in vitro multi-enzyme molecular machines, as well as the research progress on the improvement of compatibility among enzyme components/modules. The current challenges and future prospects of in vitro multi-enzyme molecular machines are also discussed.
Biotechnology ; Enzymes ; chemistry ; metabolism ; Multienzyme Complexes ; chemistry ; metabolism ; Synthetic Biology

OBJECTIVETo study the association between endoplasmic reticulum stress (ERS) pathway mediated by inositol-requiring kinase 1 (IRE1) and the apoptosis of type II alveolar epithelial cells (AECIIs) exposed to hyperoxia.

METHODSThe primarily cultured AECIIs from preterm rats were devided into an air group and a hyperoxia group. The model of hyperoxia-induced cell injury was established. The cells were harvested at 24, 48, and 72 hours after hyperoxia exposure. An inverted phase-contrast microscope was used to observe morphological changes of the cells. Annexin V/PI double staining flow cytometry was performed to measure cell apoptosis. RT-PCR and Western blot were used to measure the mRNA and protein expression of glucose-regulated protein 78 (GRP78), IRE1, X-box binding protein-1 (XBP-1), and C/EBP homologous protein (CHOP). An immunofluorescence assay was performed to measure the expression of CHOP.

RESULTSOver the time of hyperoxia exposure, the hyperoxia group showed irregular spreading and vacuolization of AECIIs. Compared with the air group, the hyperoxia group showed a significantly increased apoptosis rate of AECIIs and significantly increased mRNA and protein expression of GRP78, IRE1, XBP1, and CHOP compared at all time points (P<0.05). The hyperoxia group had significantly greater fluorescence intensity of CHOP than the air group at all time points. In the hyperoxia group, the protein expression of CHOP was positively correlated with the apoptosis rate of AECIIs and the protein expression of IRE1 and XBP1 (r=0.97, 0.85, and 0.88 respectively; P<0.05).

CONCLUSIONSHyperoxia induces apoptosis of AECIIs possibly through activating the IRE1-XBP1-CHOP pathway.

Animals ; Apoptosis ; Cells, Cultured ; Endoplasmic Reticulum Stress ; physiology ; Endoribonucleases ; physiology ; Epithelial Cells ; physiology ; Female ; Hyperoxia ; metabolism ; pathology ; Multienzyme Complexes ; physiology ; Protein-Serine-Threonine Kinases ; physiology ; Pulmonary Alveoli ; pathology ; Rats ; Rats, Sprague-Dawley ; Transcription Factor CHOP ; physiology ; X-Box Binding Protein 1 ; physiology

The mitochondrial respiratory chain consists of 5 enzyme complexes that are responsible for ATP generation. The paradigm of the electron transport chain as discrete enzymes diffused in the inner mitochondrial membrane has been replaced by the solid state supercomplex model wherein the respiratory complexes associate with each other to form supramolecular complexes. Defects in these supercomplexes, which have been shown to be functionally active and required for forming stable respiratory complexes, have been associated with many genetic and neurodegenerative disorders demonstrating their biomedical significance. In this review, we will summarize the functional and structural significance of supercomplexes and provide a comprehensive review of their assembly and the assembly factors currently known to play a role in this process.
Adenosine Triphosphate ; metabolism ; Arylamine N-Acetyltransferase ; metabolism ; Cardiolipins ; metabolism ; Electron Transport ; Humans ; Mitochondria ; enzymology ; metabolism ; Multienzyme Complexes ; chemistry ; metabolism

OBJECTIVETo study the protection of Zibu Piyin Recipe (ZBPYR) on the insulin signal pathway in the hippocampus of Pi-yin deficiency diabetic encephalopathy rats and to explore its possible mechanisms.

METHODSThe type 2 diabetic model was established using high fat diet and intraperitoneal injection of small dose streptozotocin (STZ). The Pi-yin deficiency model was established referring to classic compound factors. The learning and memory capabilities were tested in rats by the behavioral changes. The protein expressions of hippocampal IRE1alpha, JNK, and IRS-1 were detected using Western blot.

RESULTSThere was statistical difference in the learning and memory capabilities of Pi-yin deficiency rats when compared with the blank control group (P<0.05). The learning and memory capabilities could be improved by ZBPYR. The protein expressions of hippocampal phospho-IRS-1, phospho-JNK, and total IRE1alpha were enhanced (P<0.05). But they were weakened after treatment of ZBPYR.

CONCLUSIONSZBPYR could significantly improve the learning and memory capabilities of Pi-yin deficiency diabetic rats. Its functions might be correlated with improving the endoplasmic reticulum stress to regulate the insulin signaling pathway.

Animals ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Endoplasmic Reticulum Stress ; Endoribonucleases ; metabolism ; Hippocampus ; drug effects ; metabolism ; Insulin ; metabolism ; Insulin Receptor Substrate Proteins ; metabolism ; Insulin Resistance ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Male ; Multienzyme Complexes ; metabolism ; Protein-Serine-Threonine Kinases ; metabolism ; Rats ; Rats, Sprague-Dawley

Farnesoid X receptor (FXR) belongs to the nuclear receptor superfamily. It is highly related to the formation of metabolic syndrome and the glucose homeostasis, and therefore represents an important drug target against metabolic diseases and diabetes. In recent years, great progress has been made in the agonists, antagonists, and crystal structures of FXR. The diverse FXR ligands and their structure-activity relationship are reviewed in this article. The advances in the crystal structures of FXR in complex with different ligands are also introduced.
Animals ; Anticholesteremic Agents ; chemical synthesis ; chemistry ; pharmacology ; Azepines ; chemical synthesis ; chemistry ; pharmacology ; Benzene Derivatives ; chemical synthesis ; chemistry ; pharmacology ; Chenodeoxycholic Acid ; analogs & derivatives ; chemical synthesis ; chemistry ; pharmacology ; Crystallization ; Humans ; Indoles ; chemical synthesis ; chemistry ; pharmacology ; Isoxazoles ; chemical synthesis ; chemistry ; pharmacology ; Ligands ; Molecular Structure ; Multienzyme Complexes ; chemical synthesis ; chemistry ; pharmacology ; Pregnenediones ; chemical synthesis ; chemistry ; pharmacology ; Receptors, Cytoplasmic and Nuclear ; agonists ; antagonists & inhibitors ; metabolism ; Structure-Activity Relationship

The experiment was conducted by directed evolution strategy (error-prone PCR) to improve the activity of aflatoxin detoxifzyme with the high-throughput horse radish peroxidas and recessive brilliant green (HRP-RBG) screening system. We built up a mutant library to the order of 10(4). Two rounds of EP-PCR and HRP-RBG screening were used to obtain three optimum mutant strains A1773, A1476 and A2863. We found that mutant A1773 had upper temperature tolerance of 70 degrees C and that its enzyme activity was 6.5 times higher than that of the parent strain. Mutant strains A1476 worked well at pH 4.0 and its enzyme activity was 21 times higher than that of the parent strain. Mutant A2863 worked well at pH 4.0 and pH 7.5, and its enzyme activity was 12.6 times higher than that of the parent strain. With DNA sequencing we found that mutant A1773 revealed two amino acid substitutions, Glu127Lys and Gln613Arg. Mutant A1476 revealed four amino acid substitutions: Ser46Pro, Lys221Gln, Ile307Leu and Asn471lle. Mutant A2863 revealed four amino acid substitutions: Gly73Ser, Ile307Leu, Va1596Ala and Gln613Arg. The results provided a useful illustration for the deep understanding of the relationship between the function and structure of aflatoxin detoxifzyme.
Aflatoxin B1 ; antagonists & inhibitors ; chemistry ; Amino Acid Substitution ; Directed Molecular Evolution ; Enzyme Activation ; Enzyme Stability ; Multienzyme Complexes ; genetics ; metabolism ; Mutant Proteins ; genetics ; metabolism ; Point Mutation ; Polymerase Chain Reaction ; methods ; Protein Engineering

Besides the catalytic domain, some xylanases contained a non-catalytic domain which is named as carbohydrate binding module (CBM). CBM can be used to improve their binding-ability to insoluble substrates. We illustrated the importance of CBM by reviewing the source of CBMs, type of families, features of binding to insoluble substrates, specific amino acids involved in substrate-binding, linker peptides connecting the catalytic domain, and the effect of CBMs on xylanase thermostability. CBM is important for xylanase to break down complicate carbohydrates. Perspectives on engineering xylanase activity according to the characteristics of CBMs were given.
Binding Sites ; Carbohydrate Metabolism ; Catalysis ; Endo-1,4-beta Xylanases ; metabolism ; Multienzyme Complexes ; chemistry ; Substrate Specificity

OBJECTIVESTo investigate the relationships between proteasome active center LMP7 subunit and the occurrence and development of alcoholic liver disease.

METHODSEighty male Wistar rats, 170 to 190 g, were randomly divided into two groups: a model group (60 rats) and a control group (20 rats). The model group was given alcoholic intragastric administration plus an olive oil diet. Gavage, twice a day, was used to administer ethanol (30%) in a dose of 4 g/kg/d to the model group rats in the first 4 weeks. In the next 4 weeks, 40% ethanol in a dose of 5 g/kg/d was used, and then in the last 4 weeks, 50% ethanol in a dose of 6 g/kg/d was used. After infusion for 12 weeks, 15 rats (fatty liver group) were sacrificed. Others were divided into two groups; one was the hepatitis group with continued alcohol intragastric administration, the other was the hepatitis control group, receiving equal amounts of normal saline. Both groups were sacrificed after 4 weeks. By HE staining, histological pathology of the rat livers was analyzed. The expression of proteasome LMP7 subunit mRNA was examined by reverse transcription and real-time PCR. The content of LMP7 subunit protein was determined by Western blot.

RESULTSThe LMP7 mRNA level of the fatty liver group was 36% of the control group. The level of the hepatitis control group was 51% of the control group. The level of the hepatitis group was the lowest, which was only 26% of the control group. Western blot results showed that the level of the LMP7 protein content of the control group was 0.50+/-0.01; the level was 0.39+/-0.02 of the fatty liver group; 0.30+/-0.04 of the hepatitis group, and 0.38+/-0.02 of the hepatitis control group. The differences of the LMP7 protein content and mRNA expression correlated with the severity of the pathological alterations of the livers.

CONCLUSIONSThe proteasome LMP7 mRNA expression and protein content decreased in the alcoholic liver group. It may be one of the factors responsible for the decreased activity of proteasome and may play an important role in the pathogenesis of alcoholic liver disease.

Animals ; Liver ; pathology ; Liver Diseases, Alcoholic ; metabolism ; pathology ; Male ; Multienzyme Complexes ; metabolism ; Proteasome Endopeptidase Complex ; Rats ; Rats, Wistar

AMP-Activated Protein Kinases ; Animals ; Antioxidants ; pharmacology ; Apoptosis ; drug effects ; Humans ; Infant, Newborn ; Infection ; pathology ; Ischemia ; pathology ; Leukomalacia, Periventricular ; etiology ; Multienzyme Complexes ; physiology ; Nitric Oxide Synthase Type II ; antagonists & inhibitors ; Oligodendroglia ; cytology ; Protein-Serine-Threonine Kinases ; physiology ; Reactive Oxygen Species ; metabolism ; Stem Cells ; cytology