Reverse Zymography Alone does not Confirm Presence of a Protease Inhibitor

Reverse zymography is applied for identification and semi-quantification of protease inhibitors that are of protein in nature. However, a protein that shows band in reverse zymography against a protease used for digestion of the gel need not be an inhibitor; it might be resistant to degradation by the protease. We demonstrate that in reverse zymography, avidin, streptavidin and the leaf extract of Catharanthus roseus behave like inhibitors of proteases like papain, ficin, bromelain extracts from pineapple leaf, stem and fruit and trypsin. Still, they do not act as inhibitors of those proteases when enzyme assays were done in solution. In reverse zymography, the extract of pineapple crown leaf shows two major inhibitor bands against its own proteases. Identification of these proteins from sequences derived from MALDI TOF MS analysis indicated that they are fruit and stem bromelains. Avidin, streptavidin and bromelains are ‘kinetically stable proteins’ that are usually resistant to proteolysis. Thus, it is recommended that identification of an inhibitor of a protease by reverse zymography should be supported by independent assay methods for confirmation.     

Metastable states of HYPK-UBA domain's seeds drive the dynamics of its own aggregation

Protein aggregation is a multi-step process that requires sequential structural transitions of monomers during their incorporation into oligomers. Such process involves the formation of various intermediate stages in protein structures. Seed-nucleation mediated oligomerization is observed in many aggregation-prone proteins. Understanding of the protein seed's structural features and mechanisms of its transition-state formation are important for knowing the details of post-nucleation aggregation process. We have identified the metastable states in the seeds of the Ubiquitin associated (UBA) domain of Huntingtin Interacting Protein K (HYPK). This is studied by monitoring the events of dynamic transitions of metastable seeds to aggregates or monomers through microscopy, biophysical and computational techniques. HYPK-UBA seeds can exist in specific metastable state(s) that show transition from closed to open conformations, thereby reorienting the helix associated hydrophobic patches to cause its self-aggregation. Metastable seeds show inter-seed exchange of monomers through simultaneous dissociation-association phenomenon. Monomer release from metastable seeds can cause the dissolution of the aggregates. Like metastable monomers, metastable seeds also show reduction in their secondary structure by altering the molecular contacts and solvent accessible hydrophobic surfaces. Induction of metastable seeds from the ground-state is a slow thermodynamic process and it results from excitable perturbations. Conclusively, we propose the concept that the thermodynamic induction of metastable states in HYPK-UBA seed potentiates the molecule to switch its conformations that increases the protein's self-aggregation by the mechanism of hydrophobic patch collapse, while also releasing the monomers from oligomeric seeds due to structural instability.     

Genetic Evidence for Preferential Strand Transfer during Meiotic Recombination in Yeast

During meiotic recombination in the yeast Saccharomyces cerevisiae, heteroduplexes are formed as an intermediate in the exchange process. In the formation of an asymmetric heteroduplex, one chromosome acts as a donor of a single DNA strand and the other acts as a recipient. We present genetic evidence that the nontranscribed strand is donated more frequently than the transcribed strand in spores that have an unrepaired mismatch at the HIS4 locus.