Hina Younus

Alda-1 is an agonist and chemical chaperone for the common human aldehyde dehydrogenase 2 variant

In approximately one billion people, a point mutation inactivates a key detoxifying enzyme, aldehyde dehydrogenase (ALDH2). This mitochondrial enzyme metabolizes toxic biogenic and environmental aldehydes, including the endogenously produced 4-hydroxynonenal (4HNE) and the environmental pollutant acrolein, and also bioactivates nitroglycerin. ALDH2 is best known, however, for its role in ethanol metabolism. The accumulation of acetaldehyde following the consumption of even a single alcoholic beverage leads to the Asian alcohol-induced flushing syndrome in ALDH2*2 homozygotes. The ALDH2*2 allele is semidominant, and heterozygotic individuals show a similar but less severe phenotype. We recently identified a small molecule, Alda-1, that activates wild-type ALDH2 and restores near-wild-type activity to ALDH2*2. The structures of Alda-1 bound to ALDH2 and ALDH2*2 reveal how Alda-1 activates the wild-type enzyme and how it restores the activity of ALDH2*2 by acting as a structural chaperone.

Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequences of Fistulina hepatica and Cylindrobasidium torrendii

Wood decay mechanisms in Agaricomycotina have been traditionally separated in two categories termed white and brown rot. Recently the accuracy of such a dichotomy has been questioned. Here, we present the genome sequences of the white-rot fungus Cylindrobasidium torrendii and the brown-rot fungus Fistulina hepatica both members of Agaricales, combining comparative genomics and wood decay experiments. C. torrendii is closely related to the white-rot root pathogen Armillaria mellea, while F. hepatica is related to Schizophyllum commune, which has been reported to cause white rot. Our results suggest that C. torrendii and S. commune are intermediate between white-rot and brown-rot fungi, but at the same time they show characteristics of decay that resembles soft rot. Both species cause weak wood decay and degrade all wood components but leave the middle lamella intact. Their gene content related to lignin degradation is reduced, similar to brown-rot fungi, but both have maintained a rich array of genes related to carbohydrate degradation, similar to white-rot fungi. These characteristics appear to have evolved from white-rot ancestors with stronger ligninolytic ability. F. hepatica shows characteristics of brown rot both in terms of wood decay genes found in its genome and the decay that it causes. However, genes related to cellulose degradation are still present, which is a plesiomorphic characteristic shared with its white-rot ancestors. Four wood degradation-related genes, homologs of which are frequently lost in brown-rot fungi, show signs of pseudogenization in the genome of F. hepatica. These results suggest that transition toward a brown-rot lifestyle could be an ongoing process in F. hepatica. Our results reinforce the idea that wood decay mechanisms are more diverse than initially thought and that the dichotomous separation of wood decay mechanisms in Agaricomycotina into white rot and brown rot should be revisited.

Protective effect of thymoquinone on glucose or methylglyoxal-induced glycation of superoxide dismutase

Glycation plays an important role in various oxidative stress related diseases. Superoxide dismutase (SOD) constitutes an essential defense against oxidative stress. The damage caused by oxidative stress is exacerbated if the antioxidant enzymes themselves are inactivated by glycation. Thymoquinone (TQ) has been reported to have various pharmacological activities. Therefore, the glycation of SOD by glucose or methylglyoxal (MG) and its protection by TQ has been investigated. Incubation of SOD with glucose, MG or both at 37 °C resulted in a progressive decrease in the activity of the enzyme, and a parallel decrease in the amount of protein on SDS-PAGE gels for glucose incubated SOD and formation of high molecular weight aggregates for MG or both glucose and MG incubated enzyme. TQ offered protection against glucose or MG induced loss in SOD activity and fragmentation/cross-linking. The antiglycating activity of TQ appears to be better for mild glycating agents. It is also effective in protecting against strong glycating agents, more when the exposure time to the glycating agent is short. TQ has also earlier been reported to have anti-diabetic effects, and this along with the observed antiglycating effect makes it an effective compound against diabetes and its complications.

Stabilization of pancreatic ribonuclease A by immobilization on Sepharose-linked antibodies that recognize the labile region of the enzyme

The stabilizing potential of the antibodies recognizing the labile region of pancreatic ribonuclease A (RNase) has been investigated. The dodecapeptide SRNLTKDRAKPV corresponding to the labile region 32--43 on RNase was synthesized by the solid-phase method. Antiserum raised against the dodecapeptide-bovine serum albumin conjugate showed good cross-reactivity with the peptide and native RNase. RNase immobilized on Sepharose support precoupled either with the antipeptide immunoglobulin (IgG) or anti-RNase IgG proved to be more resistant to thermal inactivation than the soluble enzyme. Besides, stability against inactivation by trypsin at 55 degrees C was markedly high when enzyme was immobilized on the antipeptide IgG support, as compared to the soluble and other immobilized preparations. These results suggest that matrices bearing antibodies recognizing specific labile regions of enzyme may be useful in selectively improving their stability against specific forms of inactivation.

Cross-linked stem bromelain : A more stabilized active preparation

Cross-linking of the protease stem bromelain (bromelain) with 0.25 and 1.25% glutaraldehyde (GTA) results in the formation of a large molecular mass, multimeric and soluble aggregate having comparable activity to the unmodified bromelain. Both 0.25 and 1.25% GTA cross-linked (CL) bromelain preparations were more stable against urea, guanidine hydrochloride (GdnHCl) and temperature-induced inactivation, and exhibited slightly better storage stability compared to the unmodified protease. Such a high molecular weight, soluble, active and stable preparation may be useful in industry, i.e. in the textile industry for improving the properties of a fabric without loss of fabric strength and shape.

A structural study on the protection of glycation of superoxide dismutase by thymoquinone

Accumulation of advanced glycation end products (AGEs) in tissues and serum plays important roles in diabetes-associated complications. Therefore, the identification of antiglycating compounds is attracting considerable interest. In this study, the structural changes associated with the glycation of superoxide dismutase (SOD) and its protection by thymoquinone (TQ) have been investigated by biophysical techniques. Incubation of SOD with glucose, methylglyoxal (MG) or both at 37̊C resulted in progressive hyperchromicity at 280nm, intrinsic fluorescence quenching at 310nm, decrease in negative ellipticity at 208nm, AGE-specific fluorescence enhancement in the wavelength range 400-480nm and Thioflavin T (ThT) fluorescence enhancement at 480nm (fibrillar state enhancement). Therefore, glycation by glucose or MG induced both tertiary and secondary structural changes in SOD and formation of AGEs and fibrils. The changes were more and faster with MG than with glucose since MG is a stronger glycating agent than glucose. TQ offered protection against glucose or MG-induced glycation of SOD as observed by a reduction in the structural changes, formation of AGEs and fibrils. Thus, TQ can be used for reducing diabetic complications many of which are due to protein glycation.

Enzyme immobilization and molecular modeling studies on an organic–inorganic polypyrrole–titanium(IV)phosphate nanocomposite

In this work we report the synthesis of an electrically conductive polypyrrole–titanium(IV)phosphate (PPy–TiP) nanocomposite using simple, facile in situ chemical oxidative polymerization of polypyrrole (PPy) in the presence of titanium(IV)phosphate (TiP) for immobilization of the yeast alcohol dehydrogenase enzyme. FTIR, FE-SEM and TGA were employed for the characterization of PPy, TiP and the PPy–TiP nanocomposite. YADH was successfully immobilized on the PPy–TiP nanocomposite with a loading efficiency of 69%. The immobilized YADH showed no change in the pH optima (pH 8.0) and there was a broadening of the peak both at acidic as well as in alkaline pH. The optimum temperature of immobilized YADH was increased by 5 °C with almost the same residual activity. Immobilized YADH showed improved thermal stability at 60 °C and retained about 71% activity after 5 h of incubation. Also, the immobilized YADH showed greater reusability and retained 75% activity after 10th successive use. All the results were compared to those of free YADH, immobilized YADH on PPy and immobilized YADH on TiP. The affinity of immobilized YADH for ethanol was decreased as evident from the Km value (223.71 mM) and also there was a decrease in the maximum velocity (201.53 μM min−1) as compared to soluble YADH. The improved residual activity, stability and reusability of YADH immobilized on the PPy–TiP nanocomposite make the enzyme more suitable for industry-based applications. Molecular docking was used to query the protein – the newly synthesized chemical entity interactions that help in understanding the affinity in enzyme activity.

Study on immobilization of yeast alcohol dehydrogenase on nanocrystalline Ni-Co ferrites as magnetic support.

The covalent binding of yeast alcohol dehydrogenase (YADH) enzyme complex in a series of magnetic crystalline Ni-Co nanoferrites, synthesized via sol-gel auto combustion technique was investigated. The structural analysis, morphology and magnetic properties of Ni-Co nanoferrites were determined by X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), vibrating-sample magnetometer (VSM), high resolution transmission electron microscopy (HRTEM) and Fourier transform infrared spectroscopy (FTIR). The comparative analysis of the HRTEM micrographs of bare magnetic nanoferrite particles and particles immobilized with enzyme revealed an uniform distribution of the particles in both the cases without undergoing change in the size which was found to be in the range 20-30 nm. The binding of YADH to Ni-Co nanoferrites and the possible binding mechanism have been suggested by comparing the FTIR results. The binding properties of the immobilized YADH enzyme were also studied by kinetic parameters, optimum operational pH, temperature, thermal stability and reusability. The immobilized YADH exhibits enhanced thermal stability as compared to the free enzyme over a wide range of temperature and pH, and showed good durability after recovery by magnetic separation for repeated use.

Activation of Human Salivary Aldehyde Dehydrogenase by Sulforaphane: Mechanism and Significance.

Cruciferous vegetables contain the bio-active compound sulforaphane (SF) which has been reported to protect individuals against various diseases by a number of mechanisms, including activation of the phase II detoxification enzymes. In this study, we show that the extracts of five cruciferous vegetables that we commonly consume and SF activate human salivary aldehyde dehydrogenase (hsALDH), which is a very important detoxifying enzyme in the mouth. Maximum activation was observed at 1 μg/ml of cabbage extract with 2.6 fold increase in the activity. There was a ~1.9 fold increase in the activity of hsALDH at SF concentration of ≥ 100 nM. The concentration of SF at half the maximum response (EC50 value) was determined to be 52 ± 2 nM. There was an increase in the Vmax and a decrease in the Km of the enzyme in the presence of SF. Hence, SF interacts with the enzyme and increases its affinity for the substrate. UV absorbance, fluorescence and CD studies revealed that SF binds to hsALDH and does not disrupt its native structure. SF binds with the enzyme with a binding constant of 1.23 x 107 M-1. There is one binding site on hsALDH for SF, and the thermodynamic parameters indicate the formation of a spontaneous strong complex between the two. Molecular docking analysis depicted that SF fits into the active site of ALDH3A1, and facilitates the catalytic mechanism of the enzyme. SF being an antioxidant, is very likely to protect the catalytic Cys 243 residue from oxidation, which leads to the increase in the catalytic efficiency and hence the activation of the enzyme. Further, hsALDH which is virtually inactive towards acetaldehyde exhibited significant activity towards it in the presence of SF. It is therefore very likely that consumption of large quantities of cruciferous vegetables or SF supplements, through their activating effect on hsALDH can protect individuals who are alcohol intolerant against acetaldehyde toxicity and also lower the risk of oral cancer development.

Molecular hybridization approach of bio-potent CuII/ZnII complexes derived from N, O donor bidentate imine scaffolds: Synthesis, spectral, human serum albumin binding, antioxidant and antibacterial studies

Novel bio-relevant monometallic Schiff base complexes of the type, [Cu(L1)2] (1), [Zn(L1)2]·2H2O (2), [Cu(L2)2]·2H2O (3) and [Zn(L2)2]·H2O (4) [L1(E)-3-(((3-chloro-4-hydroxyphenyl)imino)methyl)naphthalen-2-ol and L2(E)-2-chloro-4-((1-(5-chloro-2-hydroxyphenyl)ethylidene)amino)phenol] were synthesized and characterized. A comparative account of analytical, spectroscopic (FT-IR, 1H and 13C NMR, Mass, UV-vis and EPR), thermal (TGA/DTA), XRD and SEM studies revealed a correlation between the structure and function of these biologically active molecular entities. HSA (Human serum albumin) binding profiles of the metal complexes (1-4) were monitored using biophysical techniques viz., absorbance, fluorescence, circular dichromism (CD) and foster resonance energy transfer (FRET). The intrinsic binding constant (Kb) demonstrated substantial binding propensity of L1 linked complexes (1 and 2) in comparison to L2 complexes (3 and 4) suggesting L1 to be more bio-active pharmacophore due to higher planarity and conjugation as compared to L2 ligand. The outcome of fluorescence study revealed static quenching mechanism on the basis of the quenching of HSA by the complexes (1-4). However, modifications in the secondary structure of HSA by complexes (1-4) inferred via CD measurements which revealed the enhancement of α-helicity (67.47% to 69.20%) with the preference order of 1>2>3>4. Furthermore, in-vitro antibacterial study against different bacteria and antioxidant activities against DPPH and superoxide radical (O2-) at variable concentrations outspread discernible bio-potencies of the metal complexes as compared to free ligand scaffolds due to the chelation effect.