A common East-Asian ALDH2 mutation causes metabolic disorders and the therapeutic effect of ALDH2 activators.

Obesity and type 2 diabetes have reached pandemic proportion. ALDH2 (acetaldehyde dehydrogenase 2, mitochondrial) is the key metabolizing enzyme of acetaldehyde and other toxic aldehydes, such as 4-hydroxynonenal. A missense Glu504Lys mutation of the ALDH2 gene is prevalent in 560 million East Asians, resulting in reduced ALDH2 enzymatic activity. We find that male Aldh2 knock-in mice mimicking human Glu504Lys mutation were prone to develop diet-induced obesity, glucose intolerance, insulin resistance, and fatty liver due to reduced adaptive thermogenesis and energy expenditure. We find reduced activity of ALDH2 of the brown adipose tissue from the male Aldh2 homozygous knock-in mice. Proteomic analyses of the brown adipose tissue from the male Aldh2 knock-in mice identifies increased 4-hydroxynonenal-adducted proteins involved in mitochondrial fatty acid oxidation and electron transport chain, leading to markedly decreased fatty acid oxidation rate and mitochondrial respiration of brown adipose tissue, which is essential for adaptive thermogenesis and energy expenditure. AD-9308 is a water-soluble, potent, and highly selective ALDH2 activator. AD-9308 treatment ameliorates diet-induced obesity and fatty liver, and improves glucose homeostasis in both male Aldh2 wild-type and knock-in mice. Our data highlight the therapeutic potential of reducing toxic aldehyde levels by activating ALDH2 for metabolic diseases.

Roux-en-Y and One-Anastomosis Gastric Bypass Surgery Are Superior to Sleeve Gastrectomy in Lowering Glucose and Cholesterol Levels Independent of Weight Loss: a Propensity-Score Weighting Analysis.

BACKGROUND: The superior effects of gastric bypass surgery in preventing cardiovascular diseases compared with sleeve gastrectomy are well-established. However, whether these effects are independent of weight loss is not known. METHODS: In this retrospective cohort study, we compared the change in cardiometabolic risks of 1073 diabetic patients undergoing Roux-en-Y gastric bypass (RYGB) (n = 265), one-anastomosis gastric bypass (OAGB) (n = 619), and sleeve gastrectomy (SG) (n = 189) with equivalent weight loss from the Min-Shen General Hospital. Propensity score-weighting, multivariate regression, and matching were performed to adjust for baseline differences. RESULTS: After 12 months, OAGB and, to a lesser extent, RYGB exhibited superior effects on glycemic control compared with SG in patients with equivalent weight loss. The effect was significant in patients with mild-to-modest BMI reduction but diminished in patients with severe BMI reduction. RYGB and OAGB had significantly greater effects in lowering total and low-density lipoprotein cholesterol than SG, regardless of weight loss. The results of matching patients with equivalent weight loss yielded similar results. The longer length of bypassed biliopancreatic (BP) limbs was correlated with a greater decrease in glycemic levels, insulin resistance index, lipids, C-reactive protein (CRP) levels, and creatinine levels in patients receiving RYBG. It was correlated with greater decreases in BMI, fasting insulin, insulin resistance index, and C-reactive protein levels in patients receiving OAGB. CONCLUSION: Diabetic patients receiving OAGB and RYGB had lower glucose and cholesterol levels compared with SG independent of weight loss. Our results suggest diabetic patients with cardiovascular risk factors such as hypercholesterolemia to receive bypass surgery.

Genome-Wide Association Studies for Albuminuria of Nondiabetic Taiwanese Population.

INTRODUCTION: Chronic kidney disease, which is defined by a reduced estimated glomerular filtration rate and albuminuria, imposes a large health burden worldwide. Ethnicity-specific associations are frequently observed in genome-wide association studies (GWAS). This study conducts a GWAS of albuminuria in the nondiabetic population of Taiwan. METHODS: Nondiabetic individuals aged 30-70 years without a history of cancer were enrolled from the Taiwan Biobank. A total of 6,768 subjects were subjected to a spot urine examination. After quality control using PLINK and imputation using SHAPEIT and IMPUTE2, a total of 3,638,350 single-nucleotide polymorphisms (SNPs) remained for testing. SNPs with a minor allele frequency of less than 0.1% were excluded. Linear regression was used to determine the relationship between SNPs and log urine albumin-to-creatinine ratio. RESULTS: Six suggestive loci are identified in or near the FCRL3 (p = 2.56 × 10-6), TMEM161 (p = 4.43 × 10-6), EFCAB1 (p = 2.03 × 10-6), ELMOD1 (p = 2.97 × 10-6), RYR3 (p = 1.34 × 10-6), and PIEZO2 (p = 2.19 × 10-7). Genetic variants in the FCRL3 gene that encode a secretory IgA receptor are found to be associated with IgA nephropathy, which can manifest as proteinuria. The PIEZO2 gene encodes a sensor for mechanical forces in mesangial cells and renin-producing cells. Five SNPs with a p-value between 5 × 10-6 and 5 × 10-5 are also identified in five genes that may have a biological role in the development of albuminuria. CONCLUSION: Five new loci and one known suggestive locus for albuminuria are identified in the nondiabetic Taiwanese population.

Anti-Diabetic Therapy and Heart Failure: Recent Advances in Clinical Evidence and Molecular Mechanism.

Diabetic patients have a two- to four-fold increase in the risk of heart failure (HF), and the co-existence of diabetes and HF is associated with poor prognosis. In randomized clinical trials (RCTs), compelling evidence has demonstrated the beneficial effects of sodium-glucose co-transporter-2 inhibitors on HF. The mechanism includes increased glucosuria, restored tubular glomerular feedback with attenuated renin-angiotensin II-aldosterone activation, improved energy utilization, decreased sympathetic tone, improved mitochondria calcium homeostasis, enhanced autophagy, and reduced cardiac inflammation, oxidative stress, and fibrosis. The RCTs demonstrated a neutral effect of the glucagon-like peptide receptor agonist on HF despite its weight-reducing effect, probably due to it possibly increasing the heart rate via increasing cyclic adenosine monophosphate (cAMP). Observational studies supported the markedly beneficial effects of bariatric and metabolic surgery on HF despite no current supporting evidence from RCTs. Bromocriptine can be used to treat peripartum cardiomyopathy by reducing the harmful cleaved prolactin fragments during late pregnancy. Preclinical studies suggest the possible beneficial effect of imeglimin on HF through improving mitochondrial function, but further clinical evidence is needed. Although abundant preclinical and observational studies support the beneficial effects of metformin on HF, there is limited evidence from RCTs. Thiazolidinediones increase the risk of hospitalized HF through increasing renal tubular sodium reabsorption mediated via both the genomic and non-genomic action of PPARγ. RCTs suggest that dipeptidyl peptidase-4 inhibitors, including saxagliptin and possibly alogliptin, may increase the risk of hospitalized HF, probably owing to increased circulating vasoactive peptides, which impair endothelial function, activate sympathetic tones, and cause cardiac remodeling. Observational studies and RCTs have demonstrated the neutral effects of insulin, sulfonylureas, an alpha-glucosidase inhibitor, and lifestyle interventions on HF in diabetic patients.

Emerging Therapy for Diabetic Cardiomyopathy: From Molecular Mechanism to Clinical Practice.

Diabetic cardiomyopathy is characterized by abnormal myocardial structure or performance in the absence of coronary artery disease or significant valvular heart disease in patients with diabetes mellitus. The spectrum of diabetic cardiomyopathy ranges from subtle myocardial changes to myocardial fibrosis and diastolic function and finally to symptomatic heart failure. Except for sodium-glucose transport protein 2 inhibitors and possibly bariatric and metabolic surgery, there is currently no specific treatment for this distinct disease entity in patients with diabetes. The molecular mechanism of diabetic cardiomyopathy includes impaired nutrient-sensing signaling, dysregulated autophagy, impaired mitochondrial energetics, altered fuel utilization, oxidative stress and lipid peroxidation, advanced glycation end-products, inflammation, impaired calcium homeostasis, abnormal endothelial function and nitric oxide production, aberrant epidermal growth factor receptor signaling, the activation of the renin-angiotensin-aldosterone system and sympathetic hyperactivity, and extracellular matrix accumulation and fibrosis. Here, we summarize several important emerging treatments for diabetic cardiomyopathy targeting specific molecular mechanisms, with evidence from preclinical studies and clinical trials.

ER ribosomal-binding protein 1 regulates blood pressure and potassium homeostasis by modulating intracellular renin trafficking.

BACKGROUND: Genome-wide association studies (GWASs) have linked RRBP1 (ribosomal-binding protein 1) genetic variants to atherosclerotic cardiovascular diseases and serum lipoprotein levels. However, how RRBP1 regulates blood pressure is unknown. METHODS: To identify genetic variants associated with blood pressure, we performed a genome-wide linkage analysis with regional fine mapping in the Stanford Asia-Pacific Program for Hypertension and Insulin Resistance (SAPPHIRe) cohort. We further investigated the role of the RRBP1 gene using a transgenic mouse model and a human cell model. RESULTS: In the SAPPHIRe cohort, we discovered that genetic variants of the RRBP1 gene were associated with blood pressure variation, which was confirmed by other GWASs for blood pressure. Rrbp1- knockout (KO) mice had lower blood pressure and were more likely to die suddenly from severe hyperkalemia caused by phenotypically hyporeninemic hypoaldosteronism than wild-type controls. The survival of Rrbp1-KO mice significantly decreased under high potassium intake due to lethal hyperkalemia-induced arrhythmia and persistent hypoaldosteronism, which could be rescued by fludrocortisone. An immunohistochemical study revealed renin accumulation in the juxtaglomerular cells of Rrbp1-KO mice. In the RRBP1-knockdown Calu-6 cells, a human renin-producing cell line, transmission electron and confocal microscopy revealed that renin was primarily retained in the endoplasmic reticulum and was unable to efficiently target the Golgi apparatus for secretion. CONCLUSIONS: RRBP1 deficiency in mice caused hyporeninemic hypoaldosteronism, resulting in lower blood pressure, severe hyperkalemia, and sudden cardiac death. In juxtaglomerular cells, deficiency of RRBP1 reduced renin intracellular trafficking from ER to Golgi apparatus. RRBP1 is a brand-new regulator of blood pressure and potassium homeostasis discovered in this study.

A genome-wide association study for melatonin secretion.

Melatonin exerts a wide range of effects among various tissues and organs. However, there is currently no study to investigate the genetic determinants of melatonin secretion. Here, we conducted a genome-wide association study (GWAS) for melatonin secretion using morning urine 6-hydroxymelatonin sulfate-to-creatinine ratio (UMCR). We initially enrolled 5000 participants from Taiwan Biobank in this study. After excluding individuals that did not have their urine collected in the morning, those who had history of neurological or psychiatric disorder, and those who failed to pass quality control, association of single nucleotide polymorphisms with log-transformed UMCR adjusted for age, sex and principal components of ancestry were analyzed. A second model additionally adjusted for estimated glomerular filtration rate (eGFR). A total of 2373 participants underwent the genome-wide analysis. Five candidate loci associated with log UMCR (P value ranging from 6.83 × 10-7 to 3.44 × 10-6) encompassing ZFHX3, GALNT15, GALNT13, LDLRAD3 and intergenic between SEPP1 and FLJ32255 were identified. Similar results were yielded with further adjustment for eGFR. Interestingly, the identified genes are associated with circadian behavior, neuronal differentiation, motor disorders, anxiety, and neurodegenerative diseases. We conducted the first GWAS for melatonin secretion and identified five candidate genetic loci associated with melatonin level. Replication and functional studies are needed in the future.

A Novel ALDH2 Activator AD-9308 Improves Diastolic and Systolic Myocardial Functions in Streptozotocin-Induced Diabetic Mice.

Diabetes mellitus has reached epidemic proportion worldwide. One of the diabetic complications is cardiomyopathy, characterized by early left ventricular (LV) diastolic dysfunction, followed by development of systolic dysfunction and ventricular dilation at a late stage. The pathogenesis is multifactorial, and there is no effective treatment yet. In recent years, 4-hydroxy-2-nonenal (4-HNE), a toxic aldehyde generated from lipid peroxidation, is implicated in the pathogenesis of cardiovascular diseases. Its high bioreactivity toward proteins results in cellular damage. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is the major enzyme that detoxifies 4-HNE. The development of small-molecule ALDH2 activator provides an opportunity for treating diabetic cardiomyopathy. This study found that AD-9308, a water-soluble andhighly selective ALDH2 activator, can improve LV diastolic and systolic functions, and wall remodeling in streptozotocin-induced diabetic mice. AD-9308 treatment dose-dependently lowered serum 4-HNE levels and 4-HNE protein adducts in cardiac tissue from diabetic mice, accompanied with ameliorated myocardial fibrosis, inflammation, and apoptosis. Improvements of mitochondrial functions, sarco/endoplasmic reticulumcalcium handling and autophagy regulation were also observed in diabetic mice with AD-9308 treatment. In conclusion, ADLH2 activation effectively ameliorated diabetic cardiomyopathy, which may be mediated through detoxification of 4-HNE. Our findings highlighted the therapeutic potential of ALDH2 activation for treating diabetic cardiomyopathy.

Genome-wide association study for circulating fibroblast growth factor 21 and 23.

Fibroblast growth factors (FGFs) 21 and 23 are recently identified hormones regulating metabolism of glucose, lipid, phosphate and vitamin D. Here we conducted a genome-wide association study (GWAS) for circulating FGF21 and FGF23 concentrations to identify their genetic determinants. We enrolled 5,000 participants from Taiwan Biobank for this GWAS. After excluding participants with diabetes mellitus and quality control, association of single nucleotide polymorphisms (SNPs) with log-transformed FGF21 and FGF23 serum concentrations adjusted for age, sex and principal components of ancestry were analyzed. A second model additionally adjusted for body mass index (BMI) and a third model additionally adjusted for BMI and estimated glomerular filtration rate (eGFR) were used. A total of 4,201 participants underwent GWAS analysis. rs67327215, located within RGS6 (a gene involved in fatty acid synthesis), and two other SNPs (rs12565114 and rs9520257, located between PHC2-ZSCAN20 and ARGLU1-FAM155A respectively) showed suggestive associations with serum FGF21 level (P = 6.66 × 10-7, 6.00 × 10-7 and 6.11 × 10-7 respectively). The SNPs rs17111495 and rs17843626 were significantly associated with FGF23 level, with the former near PCSK9 gene and the latter near HLA-DQA1 gene (P = 1.04 × 10-10 and 1.80 × 10-8 respectively). SNP rs2798631, located within the TGFB2 gene, was suggestively associated with serum FGF23 level (P = 4.97 × 10-7). Additional adjustment for BMI yielded similar results. For FGF23, further adjustment for eGFR had similar results. We conducted the first GWAS of circulating FGF21 levels to date. Novel candidate genetic loci associated with circulating FGF21 or FGF23 levels were found. Further replication and functional studies are needed to support our findings.

High TSH Level within Normal Range Is Associated with Obesity, Dyslipidemia, Hypertension, Inflammation, Hypercoagulability, and the Metabolic Syndrome: A Novel Cardiometabolic Marker.

(1) Background: Overt and subclinical hypothyroidism has been associated with increased cardiometabolic risks. Here we further explore whether thyroid function within normal range is associated with cardiometabolic risk factors in a large population-based study. (2) Methods: We screened 24,765 adults participating in health examinations in Taiwan. Participants were grouped according to high-sensitive thyroid-stimulating hormone (hsTSH) level as: <50th percentile (0.47-1.48 mIU/L, the reference group), 50-60th percentile (1.49-1.68 mIU/L), 60-70th percentile (1.69-1.94 mIU/L), 70-80th percentile (1.95-2.3 mIU/L), 80-90th percentile (2.31-2.93 mIU/L), and >90th percentile (>2.93 mIU/L). Cardiometabolic traits of each percentile were compared with the reference group. (3) Results: Elevated hsTSH levels within normal range were dose-dependently associated with increased body mass index, body fat percentage, waist circumferences, blood pressure, hemoglobin A1c (HbA1c), fasting insulin, homeostasis model assessment of insulin resistance (HOMA-IR), high homeostasis model of assessment of beta-cell (HOMA-ß), triglycerides, total cholesterols, fibrinogen, and uric acids (p-for-trend <0.001), but not with fasting glucose levels. The association remained significant after adjustment of age, sex, and lifestyle. As compared to the reference group, subjects with the highest hsTSH percentile had significantly increased risk of being overweight (adjusted odds ratio (adjOR): 1.35), increased body fat (adjOR: 1.29), central obesity (adjOR: 1.36), elevated blood pressure (adjOR: 1.26), high HbA1c (adjOR: 1.20), hyperinsulinemia (adjOR: 1.75), increased HOMA-IR (adjOR: 1.45), increased HOMA-ß (adjOR: 1.40), hypertriglyceridemia (adjOR: 1.60), hypercholesterolemia (adjOR: 1.25), elevated hsCRP (adjOR: 1.34), increased fibrinogen (adjOR: 1.45), hyperuricemia (adjOR: 1.47), and metabolic syndrome (adjOR: 1.42), but significant risk of low fasting glucose (adjOR: 0.89). Mediation analysis indicates that insulin resistance mediates the majority of the association between thyroid hormone status and the metabolic syndrome. (4) Conclusion: Elevated hsTSH within the normal range is a cardiometabolic risk marker associated with central obesity, insulin resistance, elevated blood pressure, dyslipidemia, hyperuricemia, inflammation, and hypercoagulability.

Active Transport of Membrane Components by Self-Organization of the Min Proteins.

Heterogeneous distribution of components in the biological membrane is critical in the process of cell polarization. However, little is known about the mechanisms that can generate and maintain the heterogeneous distribution of the membrane components. Here, we report that the propagating wave patterns of the bacterial Min proteins can impose steric pressure on the membrane, resulting in transport and directional accumulation of the component in the membrane. Therefore, the membrane component waves represent transport of the component in the membrane that is caused by the steric pressure gradient induced by the differential levels of binding and dissociation of the Min proteins in the propagating waves on the membrane surface. The diffusivity, majorly influenced by the membrane anchor of the component, and the repulsed ability, majorly influenced by the steric property of the membrane component, determine the differential spatial distribution of the membrane component. Thus, transportation of the membrane component by the Min proteins follows a simple physical principle, which resembles a linear peristaltic pumping process, to selectively segregate and maintain heterogeneous distribution of materials in the membrane. VIDEO ABSTRACT.

A flexible loop for mannan recognition and activity enhancement in a bifunctional glycoside hydrolase family 5.

BACKGROUND: An array of glycoside hydrolases with multiple substrate specificities are required to digest plant cell wall polysaccharides. Cel5E from Clostridium thermocellum and Cel5A from Thermotoga maritima are two glycoside hydrolase family 5 (GH5) enzymes with high sequence and structural similarity, but notably possess different substrate specificities; the former is a bifunctional cellulase/xylanase and the latter is a cellulase/mannanase. A specific loop in TmCel5A, Tmloop, is one of the most structurally divergent regions compared to CtCel5E and interacts with substrates, suggesting the importance for mannan recognition. METHOD: A Tmloop inserted CtCel5E and its related mutants were produced to investigate the role of Tmloop in catalysis. Crystal structure of CtCel5E-TmloopF267A followed by site-direct mutagenesis reveals the mechanism. RtCelB, a homolog with Tmloop was identified to have mannanase activity. RESULT: Tmloop incorporation enables CtCel5E to gain mannanase activity. Tyr270, His277, and Trp282 in the Tmloop are indispensable for CtCel5E-Tmloop catalysis, and weakening hydrophobic environment near the Tmloop enhances enzyme kcat. Using our newly identified loop motif to search for structurally conserved homologs in other subfamilies of GH5, we identified RtCelB. This homolog, originally annotated as a cellulase also possesses mannanase and xylanase activities. CONCLUSION: Our studies show that Tmloop enhances GH5 enzyme promiscuity and plays a role in catalysis. GENERAL SIGNIFICANCE: The study identified a loop of GH5 for mannan recognition and catalysis. Weakening the hydrophobic environment near the loop can also enhance the enzyme catalytic rate. Our findings provide a new insight on mannan recognition and activity enhancement of GH5.

Quantitative Proteomics Analysis Reveals the Min System of Escherichia coli Modulates Reversible Protein Association with the Inner Membrane.

The Min system of Escherichia coli mediates placement of the division septum at the midcell. It oscillates from pole to pole to establish a concentration gradient of the division inhibition that is high at the poles but low at the midcell; the cell middle thereby becomes the most favorable site for division. Although Min oscillation is well studied from molecular and biophysical perspectives, it is still an enigma as to whether such a continuous, energy-consuming, and organized movement of the Min proteins would affect cellular processes other than the division site selection. To tackle this question, we compared the inner membrane proteome of the wild-type and Δmin strains using a quantitative approach. Forty proteins that showed differential abundance on the inner membrane of the mutant cells were identified and defined as proteins of interest (POIs). More than half of the POIs were peripheral membrane proteins, suggesting that the Min system affects mainly reversible protein association with the inner membrane. In addition, 6 out of 10 selected POIs directly interacted with at least one of the Min proteins, confirming the correlation between POIs and the Min system.Further analysis revealed a functional relationship between metabolism and the Min system. Metabolic enzymes accounted for 45% of the POIs, and there was a change of metabolites in the related reactions. We hypothesize that the Min system could alter the membrane location of proteins to modulate their enzymatic activity. Thus, the metabolic modulation in the Δmin mutant is likely an adaptive phenotype in cells of abnormal size and chromosome number due to an imbalanced abundance of proteins on the inner membrane. Taken together, the current work reports novel interactions of the Min system and reveals a global physiological impact of the Min system in addition to the division site placement.

Structures of exoglucanase from Clostridium cellulovorans: cellotetraose binding and cleavage.

Exoglucanase/cellobiohydrolase (EC 3.2.1.176) hydrolyzes a ß-1,4-glycosidic bond from the reducing end of cellulose and releases cellobiose as the major product. Three complex crystal structures of the glycosyl hydrolase 48 (GH48) cellobiohydrolase S (ExgS) from Clostridium cellulovorans with cellobiose, cellotetraose and triethylene glycol molecules were solved. The product cellobiose occupies subsites +1 and +2 in the open active-site cleft of the enzyme-cellotetraose complex structure, indicating an enzymatic hydrolysis function. Moreover, three triethylene glycol molecules and one pentaethylene glycol molecule are located at active-site subsites -2 to -6 in the structure of the ExgS-triethylene glycol complex shown here. Modelling of glucose into subsite -1 in the active site of the ExgS-cellobiose structure revealed that Glu50 acts as a proton donor and Asp222 plays a nucleophilic role.

Biochemical characterization and structural analysis of a bifunctional cellulase/xylanase from Clostridium thermocellum.

We expressed an active form of CtCel5E (a bifunctional cellulase/xylanase from Clostridium thermocellum), performed biochemical characterization, and determined its apo- and ligand-bound crystal structures. From the structures, Asn-93, His-168, His-169, Asn-208, Trp-347, and Asn-349 were shown to provide hydrogen-bonding/hydrophobic interactions with both ligands. Compared with the structures of TmCel5A, a bifunctional cellulase/mannanase homolog from Thermotoga maritima, a flexible loop region in CtCel5E is the key for discriminating substrates. Moreover, site-directed mutagenesis data confirmed that His-168 is essential for xylanase activity, and His-169 is more important for xylanase activity, whereas Asn-93, Asn-208, Tyr-270, Trp-347, and Asn-349 are critical for both activities. In contrast, F267A improves enzyme activities.

Self-assembly of MinE on the membrane underlies formation of the MinE ring to sustain function of the Escherichia coli Min system.

The pole-to-pole oscillation of the Min proteins in Escherichia coli results in the inhibition of aberrant polar division, thus facilitating placement of the division septum at the midcell. MinE of the Min system forms a ring-like structure that plays a critical role in triggering the oscillation cycle. However, the mechanism underlying the formation of the MinE ring remains unclear. This study demonstrates that MinE self-assembles into fibrillar structures on the supported lipid bilayer. The MinD-interacting domain of MinE shows amyloidogenic properties, providing a possible mechanism for self-assembly of MinE. Supporting the idea, mutations in residues Ile-24 and Ile-25 of the MinD-interacting domain affect fibril formation, membrane binding ability of MinE and MinD, and subcellular localization of three Min proteins. Additional mutations in residues Ile-72 and Ile-74 suggest a role of the C-terminal domain of MinE in regulating the folding propensity of the MinD-interacting domain for different molecular interactions. The study suggests a self-assembly mechanism that may underlie the ring-like structure formed by MinE-GFP observed in vivo.

Mutations in the substrate entrance region of ß-glucosidase from Trichoderma reesei improve enzyme activity and thermostability.

ß-Glucosidase (EC 3.2.1.21) plays an essential role in biofuel production since it can cleave ß-1,4-glycosidic bond to convert cellobiose into fermentable glucose. Based on the structure of Trichoderma reesei ß-glucosidase 2 (TrBgl2) we solved, the amino acids in the outer channel of active site were mutated in this study. Mutants P172L and P172L/F250A showed the most enhanced k(cat)/K(m) and k(cat) values by 5.3- and 6.9-fold, respectively, compared to the wild type (WT) toward 4-nitrophenyl-ß-D-glucopyranoside (p-NPG) substrate at 40°C. L167W and P172L/F250A mutations resulted in shift of optimal temperature to 50°C, at which WT was almost inactive. However, thin-layer chromatography analysis revealed that mutant L167W had the best synergism with T. reesei cellulases on degrading cellulosic substrates into glucose. This study enhances our understanding on the roles of amino acids in the substrate entrance region away from the active site and provides engineered T. reesei ß-glucosidases with better activity and/or thermostability to hydrolyze cellobiose.

Structure of the catalytic domain of the Clostridium thermocellum cellulase CelT.

Cellulases hydrolyze cellulose, a major component of plant cell walls, to oligosaccharides and monosaccharides. Several Clostridium species secrete multi-enzyme complexes (cellulosomes) containing cellulases. C. thermocellum CelT, a family 9 cellulase, lacks the accessory module(s) necessary for activity, unlike most other family 9 cellulases. Therefore, characterization of the CelT structure is essential in order to understand its catalytic mechanism. Here, the crystal structure of free CelTΔdoc, the catalytic domain of CelT, is reported at 2.1 Šresolution. Its structure differs in several aspects from those of other family 9 cellulases. CelTΔdoc contains an additional α-helix, α-helices of increased length and two additional surface-exposed ß-strands. It also contains three calcium ions instead of one as found in C. cellulolyticum Cel9M. CelTΔdoc also has two flexible loops at the open end of its active-site cleft. Movement of these loops probably allows the substrate to access the active site. CelT is stable over a wide range of pH and temperature conditions, suggesting that CelT could be used to convert cellulose biomass into biofuel.

Construction and characterization of different fusion proteins between cellulases and ß-glucosidase to improve glucose production and thermostability.

A ß-glucosidase from Clostridium cellulovorans (CcBG) was fused with one of three different types of cellulases from Clostridium thermocellum, including a cellulosomal endoglucanase CelD (CtCD), a cellulosomal exoglucanase CBHA (CtCA) and a non-cellulosomal endoglucanase Cel9I (CtC9I). Six bifunctional enzymes were constructed with either ß-glucosidase or cellulase in the upstream. CtCD-CcBG showed the favorable specific activities on phosphoric acid swollen cellulose (PASC), an amorphous cellulose, with more glucose production (2 folds) and less cellobiose accumulation (3 folds) when compared with mixture of the single enzymes. Moreover, CtCD-CcBG had significantly improved thermal stability with a melting temperature (T(m)) of 10.9°C higher than that of CcBG (54.5°C) based on the CD unfolding experiments. This bifunctional enzyme is thus useful in industrial application to convert cellulose to glucose.

Calpain specificity and expression in chicken tissues.

We have compared ubiquitous calpains in chicken (Gallus gallus), turkey (Meleagris gallopavo) and mammals. In chicken, we studied their distribution in different tissues. The calpain activity was determined by casein zymography, a technique avoiding any prior sample purification, thus limiting any autolysis and denaturation reactions. Our results show that two ubiquitous calpains are present in chicken: (1) a mu-calpain having a greater calcium sensitivity and a lower electrophoretic mobility than the mammalian one, (2) a mu/m-calpain, named like this by Sorimachi et al. [Sorimachi, H., Tsukahara, T., Okada-Ban, M., Sugita, H., Ishiura, S., Suzuki, K., 1995. Identification of a third ubiquitous calpain species-chicken muscle expresses four distinct calpains. Biochim. Biophys. Acta, 1261, 381-93.], having a calcium sensitivity intermediate between that of the two mammalian mu-calpain and the m-calpain. Tissue distribution of the two chicken isozymes vary and mu/m-calpain predominates, whereas mu-calpain levels are very low in some tissues, unlike in mammalian tissues. The characteristics of mu/m-calpain and its preponderance in all organs suggest that it may play a different role in chicken than in mammals.