Deglycosylation of glucoamylase from Aspergillus niger: Effects on structure,activity and stability

A comparative structure–function study was performed to establish possible roles of carbohydrates in stabilization of glycoproteins, using glucoamylase (GA) as a model system. In addition to kinetic properties, stability toward elevated temperatures, extremes of pH, high salt concentrations together with circular dichroism, intrinsic/extrinsic fluorescence studies, proteolysis and affinity for interaction with hydrophobic ligands were investigated. Related to all the main properties examined, with one exception, glycosylation provided improvement in functional characteristics of the enzyme, especially in relation to its thermostability. Results are explained in terms of provision of stabilizing intermolecular interactions by the sugar molecules. The improvement in protein rigidity together with reduction of surface hydrophobicity appear to be especially important in relation to prevention of aggregation, an important mechanism of irreversible thermoinactivation, occurring at elevated temperatures.     

Acid-Induced Formation of Molten Globule States in the Wild Type <Emphasis Type="Italic">Escherichia coli</Emphasis> 5-Enolpyruvylshikimate 3-Phosphate Synthase and its Three Mutated Forms: G96A,A183T and G96A/A183T

Recent advances in protein chemistry have led to progress in the understanding of protein folding and properties of possible intermediates during the folding of proteins. The molten globule (MG) state, a major intermediate of protein folding, has a denatured state with native-like secondary structure. In the present work, the acid-induced unfolding of wild type Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) and its three different variants (G96A, A183T and G96A/A183T) were studied by far- and near-UV circular dichroism (CD), intrinsic fluorescent emission spectroscopy and 1-anilino naphthalene-8-sulfonate (ANS) binding. At pH < 3.0, these EPSPS variants acquire partially folded state, which show the characteristics of the MG state, e.g., a drastic reduction of defined tertiary structure and almost no change in the secondary structure. ANS binding experiments show that hydrophobic surface of these variants is exposed to a greater extent in comparison to the native form, at acidic pH. Wild type, G96A, A183T and G96A/A183T acquire MG states at pH 2.0, 1.5, 3.0 and 3.0, respectively, which show that pH stability of MG state of G96A has increased in comparison to wild type; and pH stability of MG states of two other mutants is lower than that of the wild type. The results suggest that there is a direct relationship between stability of protein and pH stability of its folding intermediates.     

The effect of surface charge balance on thermodynamic stability and kinetics of refolding of firefly luciferase

Thermodynamic stability and refolding kinetics of firefly luciferase and three representative mutants with depletion of negative charge on a flexible loop via substitution of Glu by Arg (ER mutant) or Lys (EK mutant) as well as insertion of another Arg in ER mutants (ERR mutant) was investigated. According to thermodynamic studies, structural stability of ERR and ER mutants are enhanced compared to WT protein, whereas, these mutants become prone to aggregation at higher temperatures. Accordingly, it was concluded that enhanced structural stability of mutants depends on more compactness of folded state, whereas aggregation at higher temperatures in mutants is due to weakening of intermolecular repulsive electrostatic interactions and increase of intermolecular hydrophobic interactions. Kinetic results indicate that early events of protein folding are accelerated in mutants.     

Rubredoxin reductase from Alcanivorax borkumensis: expression and characterization

Oil pollution is an environmental problem of increasing importance. Alcanivorax borkumensis, with a high potential for biotechnological applications, is a key marine hydrocarbonoclastic bacterium and plays a critical role in the bioremediation of oil-polluted marine systems. In oil degrading bacteria, the first step of alkane degradation is catalyzed by a monooxygenase. The reducing electrons are tunneled from NAD(P)H via rubredoxin, one of the most primitive metalloproteins, to the hydroxylase. Rubredoxin reductase is a flavoprotein catalyzing the reduction of rubredoxin. There are two rubredoxin genes, alkG and rubA, in A. borkumensis genome. In this work, the genes encoding rubredoxin reductase (ABO_0162, rubB) and AlkG(ABO_2708, alkG) were cloned and functionally overexpressed in E. coli. Our results demonstrate that RubB could reduce AlkG, therefore compensating for the absence of AlkT, also a rubredoxin reductase, missing in A. borkumensis SK2 genome. These results will increase our knowledge concerning biological alkane degradation and will lead us to design more efficient biotransformation and bioremediation systems.     

Inhibitory Phenotype of HBV-Specific CD4+ T-Cells Is Characterized by High PD-1 Expression but Absent Coregulation of Multiple Inhibitory Molecules

Background

T-cell exhaustion seems to play a critical role in CD8⁺ T-cell dysfunction during chronic viral infections. However, up to now little is known about the mechanisms underlying CD4⁺ T-cell dysfunction during chronic hepatitis B virus (CHB) infection and the role of inhibitory molecules such as programmed death 1 (PD-1) for CD4⁺ T-cell failure.

Methods

The expression of multiple inhibitory molecules such as PD-1, CTLA-4, TIM-3, CD244, KLRG1 and markers defining the grade of T-cell differentiation as CCR7, CD45RA, CD57 and CD127 were analyzed on virus-specific CD4⁺ T-cells from peripheral blood using a newly established DRB1*01-restricted MHC class II Tetramer. Effects of in vitro PD-L1/2 blockade were defined by investigating changes in CD4⁺ T-cell proliferation and cytokine production.

Results

CD4⁺ T-cell responses during chronic HBV infection was characterized by reduced Tetramer⁺CD4⁺ T-cell frequencies, effector memory phenotype, sustained PD-1 but low levels of CTLA-4, TIM-3, KLRG1 and CD244 expression. PD-1 blockade revealed individualized patterns of in vitro responsiveness with partly increased IFN-γ, IL-2 and TNF-α secretion as well as enhanced CD4⁺ T-cell expansion almost in treated patients with viral control.

Conclusion

HBV-specific CD4⁺ T-cells are reliably detectable during different courses of HBV infection by MHC class II Tetramer technology. CD4⁺ T-cell dysfunction during chronic HBV is basically linked to strong PD-1 upregulation but absent coregulation of multiple inhibitory receptors. PD-L1/2 neutralization partly leads to enhanced CD4⁺ T-cell functionality with heterogeneous patterns of CD4⁺ T-cell rejunivation.     

Isolation and characterization of a protease-producing novel haloalkaliphilic bacterium <Emphasis Type="Italic">Halobiforma</Emphasis> sp. strain BNMIITR from Sambhar lake in Rajasthan,India

A novel haloalkaliphilic bacterium designated as strain BNMIITR was isolated from a soil sample collected from Sambhar lake, Rajasthan, in northern India. Colonies of the isolated strain were dark orange and comprised Gram-negative bacilli; there was a slight pleomorphism towards the stationary phase of growth. Experiments revealed that the isolate can grow in the range of 2–5 M NaCl, pH 6–11 and 18–55 °C, with optimum growth observed at 3 M NaCl, pH 8–8.5 and 45 °C. No growth was observed in culture medium without NaCl. The isolate showed no requirement for magnesium sulphate heptahydrate (MgSO₄ ^.7H₂O) for growth. Major cellular fatty acids were C _14:0, C _15:0 iso, C _15:0 anteiso, C _16:0, C _17:0 iso, C _17:0 anteiso and C _20:2 w6, 9c. The result of 16S rRNA gene sequence analysis showed 98 % sequence similarity with Halobiforma lacisalsi and Hbf. haloterrestris. Halobiforma sp. strain BNMIITR showed resistance towards several antibiotics and produced an extracellular alkaline protease. The crude enzyme was found to be active in broad range of alkaline pH and temperature (30–80 °C).     

Designing a Novel Multi-epitope T Vaccine for “Targeting Protein for Xklp-2” (TPX2) in Hepatocellular Carcinoma Based on Immunoinformatics Approach

International Journal of Peptide Research and Therapeutics - Hepatocellular carcinoma (HCC) is one of the leading cancer-related deaths worldwide. Recently, studies for HCC treatment are focused on...     

Comparison of the molten globule states of thermophilic and mesophilic alpha-amylases

In recent years great interest has been generated in the process of protein folding, and the formation of intermediates during the folding process has been proven with new experimental strategies. In the present work, we have examined the molten globule state of Bacillus licheniformis alpha-amylase (BLA) by intrinsic fluorescence and circular dichroism spectra, 1-anilino naphthalene-8-sulfonate (ANS) binding and proteolytic digestion by pepsin, for comparison to its mesophilic counterpart, Bacillus amyloliquefaciens alpha-amylase (BAA). At pH 4.0, both enzymes acquire partially folded state which show characteristics of molten globule state. They unfold in such a way that their hydrophobic surfaces are exposed to a greater extent compared to the native forms. Chemical denaturation studies by guanidine hydrochloride and proteolytic digestion with pepsin show that molten globule state of BLA is more stable than from BAA. Results from gel filtration indicate that BAA has the same compactness at pH 4.0 and 7.5. However, molten globule state of BLA is less compact than its native state. The effects of polyols such as trehalose, sorbitol and glycerol on refolding of enzymes from molten globule to native state were also studied. These polyols are effective on refolding of mesophilic alpha-amylase but only slightly effect on BLA refolding. In addition, the folding pathway and stability of intermediate state of the thermophilic and the mesophilic alpha-amylases are discussed.     

Dorsal premotor neurons encode the relative position of the hand, eye, and goal during reach planning

When reaching to grasp an object, we often move our arm and orient our gaze together. How are these movements coordinated? To investigate this question, we studied neuronal activity in the dorsal premotor area (PMd) and the medial intraparietal area (area MIP) of two monkeys while systematically varying the starting position of the hand and eye during reaching. PMd neurons encoded the relative position of the target, hand, and eye. MIP neurons encoded target location with respect to the eye only. These results indicate that whereas MIP encodes target locations in an eye-centered reference frame, PMd uses a relative position code that specifies the differences in locations between all three variables. Such a relative position code may play an important role in coordinating hand and eye movements by computing their relative position.     

Differential interactions of a biological photosensitizer with liposome membranes having varying surface charges

The present work demonstrates the interaction of promising cancer cell photosensitizer, harmane (HM), with liposome membranes of varying surface charges, dimyristoyl-l-α-phosphatidylcholine (DMPC) and dimyristoyl-l-α-phosphatidylglycerol (DMPG). Electrostatic interaction of the cationic probe (HM) with the surface charges of the lipids is responsible for differential modulation of the spectral properties of the drug in different lipid environments. Estimation of partition coefficient (K(p) (±10%) = 5.58 × 10(4) in DMPC and 3.28 × 10(5) in DMPG) of HM between aqueous buffer and lipid phases reflect strong binding interaction of the drug with both the lipids. Evidence for greater degree of partitioning of HM into DMPG membrane compared to DMPC membrane has been deduced and further substantiated from experimental studies such as steady-state fluorescence anisotropy, micropolarity determination. The molecular modeling investigation by docking simulation coupled with fluorescence quenching experiment has been exploited to substantiate the location of drug at the lipid head-group region. Modulation of the dynamical properties of the drug within the lipid environments has also been addressed. Rotational relaxation dynamics studies unravel the impartation of a significant degree of motional restriction on the probe molecule within the lipids and reinforce the differential interactions of HM with the two lipid systems along the lines of other findings. Fluorescence kinetics studies reveal a faster association (in terms of apparent rate constants describing the process of interaction) of the drug with DMPG membrane compared to DMPC. This result is argued in connection with the electrostatic interaction between the drug and the liposome surface charges.