A cystatin C similar protein from Musa acuminata that inhibits cathepsin B involved in rheumatoid arthritis using in silico approach and in vitro cathepsin B inhibition by protein extract.

Rheumatoid arthritis (RA) is an auto-immune disease that affects the synovial lining of the joints, causes synovitis and culminates to joint destruction. Cathepsin B is responsible for digesting unwanted proteins in extracellular matrix but its hyper expression could implicate in pathological diseases like RA. Available treatments for RA are classified into non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs), and steroids, but the severe side effects associated with these drugs is one of concerns and cannot be ignored. Thus, any alternative therapy with minimum or no side effects would be a cornerstone. In our in silico studies a cystatin C similar protein (CCSP) has been identified from Musa acuminata that could effectively inhibit the cathepsin B activity. In silico and molecular dynamics studies showed that the identified CCSP and cathepsin B complex has binding energy -66.89 kcal/mol as compared to cystatin C - cathepsin B complex with binding energy of -23.38 kcal/mol. These results indicate that CCSP from Musa acuminata has better affinity towards cathepsin B as compared to its natural inhibitor cystatin C. Hence, CCSP may be suggested as an alternative therapeutic in combating RA by inhibiting its one of the key proteases cathepsin B. Further, in vitro experiments with fractionated protein extracts from Musa sp. peel inhibited cathepsin B to 98.30% at 300 µg protein concentration and its IC50 was found to be 45.92 µg indicating the presence of cathepsin B inhibitor(s) in protein extract of peel which was further confirmed by reverse zymography.Communicated by Ramaswamy H. Sarma.

Isolation and identification of a TetR family protein that regulates the biodesulfurization operon.

Biodesulfurization helps in removal of sulfur from organosulfur present in petroleum fractions. All microorganisms isolated to date harbor a desulfurization operon consisting of three genes dszA, -B and -C which encode for monooxygenases (DszA & C) and desulfinase (DszB). Most of the studies have been carried out using dibenzothiophene as the model organosulfur compound, which is converted into 2 hydroxybiphenyl by a 4S pathway which maintains the calorific value of fuel. There are few studies reported on the regulation of this operon. However, there are no reports on the proteins which can enhance the activity of the operon. In the present study, we used in vitro and in vivo methods to identify a novel TetR family transcriptional regulator from Gordonia sp. IITR100 which functions as an activator of the dsz operon. Activation by TetR family regulator resulted in enhanced levels of desulfurization enzymes in Gordonia sp. IITR100. Activation was observed only when the 385 bp full length promoter was used. Upstream sequences between - 385 and - 315 were found to be responsible for activation. We provide evidence that the TetR family transcription regulator serves as an activator in other biodesulfurizing microorganisms such as Rhodococcus erythropolis IGTS8 and heterologous host Escherichia coli. This is the first report on the isolation of a possible transcriptional regulator that activates the desulfurization operon resulting in improved biodesulfurization.

Improved conversion of Dibenzothiophene into sulfone by surface display of Dibenzothiophene monooxygenase (DszC) in recombinant Escherichia coli.

Biodesulfurization is an eco-friendly process for removing sulfur from petroleum fractions. The process could not be commercialized because of the inability of microorganisms to desulfurize a wide range of heterocyclic poly aromatic sulfur compounds like dibenzothiophene (DBT), 4, 6-Dimethyl DBT present in fuel and low desulfurization activity. In the present study, to improve the rates of conversion of dibenzothiophene to dibenzothiophene sulfone, the responsible enzyme dibenzothiophene monooxygenase DszC, is displayed on the surface of Escherichia coli. This helped in overcoming the mass transfer limitation and resulted in approximately 3 times faster conversion with respect to control (which contained intracellular enzyme). This is the first report demonstrating display of a biodesulfurization enzyme on bacterial cell surface.