Deamidation and protein repair studies of bio-catalysts from different organisms

Proteins undergo certain posttranslational modifications like acetylation, phosphorylation, methylation, deamidation etc as a step in protein biosynthesis. Protein post-translational modifications play a key role in many cellular processes such as cellular differentiation [G. Grotenbreg, H. Ploegh (2007)], protein degradation [R.Geiss-Friedlander, F.Melchior (2007)], signaling and regulatory processes [Morrison et al (2002)], regulation of gene expression, and protein-protein interactions. Deamidation is a type of posttranslational modification which changes the chemical nature of amide containing amino acids like asparagines (Asn). The reaction is initiated by the favorable nucleophilic attack on the side chain carbonyl by the peptide backbone nitrogen of the following amino acid residue. This leads to the formation of an unstable succinimidyl intermediate that non-enzymatically hydrolyzes into either L-aspartyl (normal) or L-isoaspartyl (abnormal) residue, resulting in a cumulative gain of net negative charge at their specific sites of chemical reaction [Q. Hasan et al (2005)] The metastable succinimide has a typical half life of 4 hours at pH-7.4, 37°C. The rapid hydrolysis of this intermediate mixture generates Asp (~30%) and predominantly isoAsp (~70%) residues. Each amide has a specific deamidation rate that is genetically determined by the sequence of residues immediately adjacent in the peptide chain and by secondary, tertiary and quarternary structure. The influence of local sequence and solution environment on the rates of deamidation has been extensively studied by researchers using synthetic /natual peptides. Isoaspartate forms most readily at sequences in which the side chain of the C flanking amino acid is relatively small and hydrophilic, and is less likely to be formed where bulky or hydrophobic residues are in this position. The most favorable C-flanking amino acids are Gly, Ser and His, leading to so called isoAsp hot spots at Asn-Gly, Asn-Ser, Asn-His and Asp-Gly sequences[A.Cimmino et al (2004)]. Protein deamidation occurs at flexible regions of proteins especially on those sites which are more exposed. Robinson and Robinson have recently designed a computational method which could estimate the probability of deamidation within a folded protein with known crystal structures [N. E.Robinson & A. B. Robinson (2001)]

S-adenosyl-L-methionine, trehalose and oleanolic acid in few plants

O jornal i distribui hoje uma edição especial durante o congresso da World Association of Newspapers, a decorrer em Hyderabad, Índia (fonte: Meios e Publicidade). A edição é um “best of com as melhores histórias do i e algumas páginas premiadas na Society for News Design” e coincide com a apresentação de Martim Avillez Figueiredo, director do diário, sobre o tema The Power of Print

Studies on Substrate Specificity and Activity Regulating Factors of Trehalose-6-Phosphate Synthase of Saccharomyces Cerevisiae

Purified trehalose-6-phosphate synthase (TPS) of Saccharomyces cerevisiae was effective over a wide range of substrates, although differing with regard to their relative activity. Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity, particularly when a pyrimidine glucose nucleotide like UDPG was used, rather than a purine glucose nucleotide like GDPG. A high Vmax and a low Km value of UDPG show its greater affinity with TPS than GDPG or TDPG. Among the glucosyl acceptors TPS showed maximum activity with G-6-P which was followed by M-6-P and F-6-P. Effect of heparin was also extended to the purification of TPS activity, as it helped to retain both stability and activity of the final purified enzyme. Metal co-factors, specifically MnCl2 and ZnCl2 acted as stimulators, while enzyme inhibitors had very little effect on TPS activity. Metal chelators like CDTA, EGTA stimulated enzyme activity by chelation of metal inhibitors. Temperature and pH optima of the purified enzyme were determined to be 40 °C and pH 8.5 respectively. Enzyme activity was stable at 0–40 °C and at alkaline pH

Purification and Characterization of a Trehalase–Invertase Enzyme with Dual Activity from Candida Utilis

Trehalose and sucrose, two important anti-stress non-reducing natural disaccharides, are catabolized by two enzymes, namely trehalase and invertase respectively. In this study, a 175 kDa enzyme protein active against both substrates was purified from wild type Candida utilis and characterized in detail. Substrate specificity assay and activity staining revealed the enzyme to be specific for both sucrose and trehalose. The ratio between trehalase and invertase activity was found to be constant at 1:3.5 throughout the entire study. Almost 40-fold purification and 30% yield for both activities were achieved at the final step of purification. The presence of common enzyme inhibitors, thermal and pH stress had analogous effects on its trehalase and invertase activity. Km values for two activities were similar while Vmax and Kcat also differed by a factor of 3.5. Competition plot for both substrates revealed the two activities to be occurring at the single active site. N-terminal sequencing and MALDI-TOF data analysis revealed higher similarity of the purified protein to previously known neutral trehalases. While earlier workers mentioned independent purification of neutral trehalase or invertase from different sources, the present study reports the purification of a single protein showing dual activity

Core-Shell Nanofibrous Scaffold Based on Polycaprolactone-Silk Fibroin Emulsion Electrospinning for Tissue Engineering Applications

The vast domain of regenerative medicine comprises complex interactions between specific cells’ extracellular matrix (ECM) towards intracellular matrix formation, its secretion, and modulation of tissue as a whole. In this domain, engineering scaffold utilizing biomaterials along with cells towards formation of living tissues is of immense importance especially for bridging the existing gap of late; nanostructures are offering promising capability of mechano-biological response needed for tissue regeneration. Materials are selected for scaffold fabrication by considering both the mechanical integrity and bioactivity cues they offer. Herein, polycaprolactone (PCL) (biodegradable polyester) and ‘nature’s wonder’ biopolymer silk fibroin (SF) are explored in judicious combinations of emulsion electrospinning rather than conventional electrospinning of polymer blends. The water in oil (W/O) emulsions’ stability is found to be dependent upon the concentration of SF (aqueous phase) dispersed in the PCL solution (organic continuous phase). The spinnability of the emulsions is more dependent upon the viscosity of the solution, dominated by the molecular weight of PCL and its concentration than the conductivity. The nanofibers exhibited distinct core-shell structure with better cytocompatibility and cellular growth with the incorporation of the silk fibroin biopolymer

Biochemical and computational approach of selected phytocompounds from Tinospora crispa in the management of COVID-19

A pandemic caused by the novel coronavirus (SARS-CoV-2 or COVID-19) began in December 2019 in Wuhan, China, and the number of newly reported cases continues to increase. More than 19.7 million cases have been reported globally and about 728,000 have died as of this writing (10 August 2020). Recently, it has been confirmed that the SARS-CoV-2 main protease (Mpro) enzyme is responsible not only for viral reproduction but also impedes host immune responses. The Mpro provides a highly favorable pharmacological target for the discovery and design of inhibitors. Currently, no specific therapies are available, and investigations into the treatment of COVID-19 are lacking. Therefore, herein, we analyzed the bioactive phytocompounds isolated by gas chromatography–mass spectroscopy (GC-MS) from Tinospora crispa as potential COVID-19 Mpro inhibitors, using molecular docking study. Our analyses unveiled that the top nine hits might serve as potential anti-SARS-CoV-2 lead molecules, with three of them exerting biological activity and warranting further optimization and drug development to combat COVID-19

Biochemical and Computational Approach of Selected Phytocompounds from Tinospora crispa in the Management of COVID-19

A pandemic caused by the novel coronavirus (SARS-CoV-2 or COVID-19) began in December 2019 in Wuhan, China, and the number of newly reported cases continues to increase. More than 19.7 million cases have been reported globally and about 728,000 have died as of this writing (10 August 2020). Recently, it has been confirmed that the SARS-CoV-2 main protease (Mpro) enzyme is responsible not only for viral reproduction but also impedes host immune responses. The Mpro provides a highly favorable pharmacological target for the discovery and design of inhibitors. Currently, no specific therapies are available, and investigations into the treatment of COVID-19 are lacking. Therefore, herein, we analyzed the bioactive phytocompounds isolated by gas chromatography–mass spectroscopy (GC-MS) from Tinospora crispa as potential COVID-19 Mpro inhibitors, using molecular docking study. Our analyses unveiled that the top nine hits might serve as potential anti-SARS-CoV-2 lead molecules, with three of them exerting biological activity and warranting further optimization and drug development to combat COVID-19