Identification of selenoprotein O substrates using a biotinylated ATP analog.

Selenoprotein O is one of 25 human selenoproteins that incorporate the 21st amino acid selenocysteine. Recent studies have revealed a previously undocumented mechanism of redox regulation by which SelO protects cells from oxidative damage. SelO catalyzes the covalent addition of AMP from ATP to the hydroxyl side chain of protein substrates in a post translational modification known as AMPylation. Although AMPylation was discovered over 50 years ago, methods to detect and enrich substrates are limited. Here, we describe protocols to clone, purify, and identify the substrates of bacterial SelO using a biotinylated ATP analog. Identification of SelO substrates and the functional consequences of AMPylation will illuminate the significance of this evolutionarily conserved selenoprotein.

Prion-derived tetrapeptide stabilizes thermolabile insulin via conformational trapping.

Unfolding followed by fibrillation of insulin even in the presence of various excipients grappled with restricted clinical application. Thus, there is an unmet need for better thermostable, nontoxic molecules to preserve bioactive insulin under varying physiochemical perturbations. In search of cross-amyloid inhibitors, prion-derived tetrapeptide library screening reveals a consensus V(X)YR motif for potential inhibition of insulin fibrillation. A tetrapeptide VYYR, isosequential to the ß2-strand of prion, effectively suppresses heat- and storage-induced insulin fibrillation and maintains insulin in a thermostable bioactive form conferring adequate glycemic control in mouse models of diabetes and impedes insulin amyloidoma formation. Besides elucidating the critical insulin-IS1 interaction (R4 of IS1 to the N24 insulin B-chain) by nuclear magnetic resonance spectroscopy, we further demonstrated non-canonical dimer-mediated conformational trapping mechanism for insulin stabilization. In this study, structural characterization and preclinical validation introduce a class of tetrapeptide toward developing thermostable therapeutically relevant insulin formulations.

De Novo designed 13 mer hairpin-peptide arrests insulin and inhibits its aggregation: role of OH-π interactions between water and hydrophobic amino acids.

Background: Protein aggregation in the cellular systems can be highly fatal causing a series of diseases including neurodegenerative diseases like ALS, Alzheimer, Prion Diseases, Parkinson's and other diseases like type II diabetes. To date, there is no crucial mechanism invented that shows how a protein molecule unfolds or misfolds. Insulin fibrillation in type II diabetes is an alarming event that brings every year deaths of millions of people around the globe. Pharmaceutical companies are still in the cultivation of finding newer therapeutic agents which halt/impede insulin aggregation to combat diabetes II and improve the patient's life expectancy. Methods and Results: Here in this report, we have engineered four short 13 mer peptides (N-term-DMYY- N-term-DITT- N-term-DIFF- N-term-KVYY-) which target monomeric insulin in its globular form. The de Novo designed peptides are found to be non-cytotoxic in human HEK293 cells. Among these four peptides, only DITT- showed complete inhibition of insulin fibrillation, whereas DIFF- and DIYY- and KVYY- lost their functionality to impede insulin aggregation to a great extent. High-resolution multi-dimensional NMR experiments portrayed the 13 mer sequences of peptides in the beta-hairpin forms. A series of biophysical techniques like CD, ThT assay, DLS, SEM, ITC, size-exclusion chromatography, and molecular dynamics simulation strongly evidenced inhibition of insulin fibrillation by N-term-DITT- compared to those by the other peptides. Conclusion and significance: Here we tried to unravel how DITT- could impede insulin fibrillation.

A Small Molecule Impedes Insulin Fibrillation: Another New Role of Phenothiazine Derivatives.

Protein misfolding is interrelated to several diseases, including neurodegenerative diseases and type II diabetes. Misfolded/unfolded proteins produce soluble oligomers that accumulate into "amyloid plaques". Inhibition of amyloid-plaque formation by those misfolded/unfolded proteins will lead to the invention of new therapeutic approaches for amyloid-related diseases. Herein, methylene blue (MB), a well-defined drug against multiple diseases and disorders, is used to impede insulin fibrillation. In this study, we perform an array of in vitro experiments to monitor the effects of MB on the fibrillation of bovine insulin. Our results confirm that MB distresses the kinetics of insulin fibrillation by interacting with insulin in its monomeric form. A thioflavin T assay indicates that insulin fibrillation is interrupted upon the addition of MB. The same results are confirmed by circular dichroism, dynamic light scattering (DLS), and size-exclusion chromatography (SEC). According to the DLS data, the insulin fibrils are 800 nm in diameter, and the addition of MB reduces the size of the fibrils, which remain 23 nm in size, and this indicates that no fibrillation of insulin occurs in the presence of MB. This data is also supported by SEC. Saturation transfer difference NMR spectroscopy and molecular dynamics simulations demonstrate the interactions between insulin and MB at the atomic level.

LINCRNA00273 promotes cancer metastasis and its G-Quadruplex promoter can serve as a novel target to inhibit cancer invasiveness.

Discovery of anti-metastatic drugs is of immense clinical significance as metastasis is responsible for 90% of all cancer deaths. Here we report the inhibitory effect of a bis schiff base (M2) on cancer cell migration and invasion in vitro and in vivo. M2 has shown good solubility and permeability across the intestinal cell wall and hence can be classified as BCS (Biopharmaceutical classification system) class I. Microarray studies identified a long non coding intergenic RNA, LINC00273 as a novel molecular target of M2. We report that LINC00273 harbors a unique (4n-1) parallel G-Quadruplex structure in its promoter as validated by DMS footprint. M2 is proposed to stabilize this G-quadruplex structure resulting in the down-regulation of LINC00273 expression. Dual Luciferase reporter assay also suggests inhibition of LINC00273 promoter activity by M2. Involvement of this linc in metastasis is proven by siRNA and shRNA mediated knock down of LINC00273 in vitro and in vivo in nude mice which significantly decelerates cancer cell migration and invasion and also makes the cells unresponsive to TGF-ß's pro-metastatic effects. Furthermore, the real time expression of LINC00273 in thirty seven human clinical samples is found to be positively correlated with the histopathological staging of metastasis.

Reverse Watson-Crick G-G base pair in G-quadruplex formation.

A stable intermediate dimeric G-rich form as a precursor of tetrameric G-quadruplex structures has been detected via MALDI-TOF spectrometry. Molecular dynamics simulation offered detailed insights at the atomic level, assigning reverse Watson-Crick G-G base pairing (not Hoogsteen) in the G-rich dimer. In support of this, cisplatin formed a stable adduct by binding to the dimeric G-rich structure, eliminating the possibility of G-G Hoogsteen hydrogen bond formation.