PubMed · 5771574
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1969-05-24. Asparaginase.. https://pubmed.ncbi.nlm.nih.gov/5771574/
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Enzymes capable of converting L-asparagine to L-aspartate can be classified as bacterial-type or plant-type L-asparaginases. Bacterial-type L-asparaginases are further divided into subtypes I and II, defined by their intra-/extra-cellular localization, substrate affinity, and oligomeric form. Plant-type L-asparaginases are evolutionarily and structurally distinct from the bacterial-type enzymes. They function as potassium-dependent or -independent Ntn-hydrolases, similar to the well characterized aspartylglucosaminidases with (alphabeta)2 oligomeric structure. The review discusses the structural aspects of both types of L-asparaginases and highlights some peculiarities of their catalytic mechanisms. The bacterial-type enzymes are believed to have a disordered active site which gets properly organized on substrate binding. The plant-type enzymes, which are more active as isoaspartyl aminopeptidases, pose a chemical challenge common to other Ntn-hydrolases, which is how an N-terminal nucleophile can activate itself or cleave its own alpha-amide bond before the activation is even possible. The K+ -independent plant-type L-asparaginases show an unusual sodium coordination by main-chain carbonyl groups and have a key arginine residue which by sensing the arrangement at the oligomeric (alphabeta)-(alphabeta) interface is able to discriminate among substrates presented for hydrolysis.
RNA interference (RNAi) is an evolutionarily conserved cellular defense mechanism that protects cells from hostile genes and regulates the function of normal genes during growth and development. In this study, we established proof of principle of small interfering RNA (siRNA) silencing in hard-to-transfect human T cell lines and primary human CD4 T cells. We used public and in-house programs to design four siRNAs each for GFP, for our novel cellular gene HALP, and for their corresponding scrambled siRNA controls. We generated siRNA expression cassettes (SECs) by PCR and directly transfected the PCR products into T cells using amaxa Nucleofector technology. The most effective SECs were selected and cloned into a TA cloning vector and titered with their respective controls to increase transfection efficiency. Flow cytometry and fluorescence microscopy analyses were performed for GFP siRNAs, and Northern blot analysis was done to assess the HALP silencing effect. These experiments demonstrate that SECs are an excellent screening tool to identify siRNA sequences effective in silencing expression of genes of interest. The vector expressing the most effective siRNA robustly inhibited GFP expression (up to 92%) in the context of co-transfection in human T cell lines and primary CD4 T cells. The optimized siRNA for our endogenous cellular gene HALP also silenced its target RNA expression by more than 90%. These studies demonstrate that the combination of SEC, siRNA expression vectors and Nucleofector technology can be successfully applied to hard-to-transfect human T cell lines and primary T cells to effectively silence genes.
Thermal unfolding monitored by spectroscopy or calorimetry is widely used to determine protein stability. Equilibrium thermodynamic analysis of such unfolding is often hampered by its irreversibility, which usually results from aggregation of thermally denatured protein. In addition, heat-induced protein misfolding and aggregation often lead to formation of amyloid-like structures. We propose a convenient method to monitor in real time protein aggregation during thermal folding/ unfolding transition by recording turbidity or 90 degrees light scattering data in circular dichroism (CD) spectroscopic experiments. Since the measurements of turbidity and 90 degrees light scattering can be done simultaneously with far- or near-UV CD data collection, they require no additional time or sample and can be directly correlated with the protein conformational changes monitored by CD. The results can provide useful insights into the origins of irreversible conformational changes and test the linkage between protein unfolding or misfolding and aggregation in various macromolecular systems, including globular proteins and protein-lipid complexes described in this study, as well as a wide range of amyloid-forming proteins and peptides.