Show Yourself, Asparaginase: An Enzymatic Reaction Explained through a Hands-On Interactive Activity.

Determining the catalytic activity of an enzyme can be the perfect method for its identification, for example during purification procedures or for isolation purposes. Herein, we used a pharmaceutically relevant protein to bring the concept of enzymatic activity to the classroom. We designed a hands-on interactive activity in which a medically relevant enzyme, asparaginase, was distinguished from a nonenzymatic protein based on its specific enzymatic activity. The experiment was carried out in the classroom, designed to impact different educational levels from elementary to high school. Our main purposes were to promote the emerging field of protein-based drugs as a source of scientific careers in bionanotechnology and to show the students an image of a "scientist" as that of a common and educated person working in an exciting profession. In addition of being inexpensive, this activity proved to be adaptable for various educational levels and can be easily implemented in different scenarios, for example, scientific fairs, some schools, and so forth.

Purification and characterization of a cytochrome c with novel caspase-3 activation activity from the pathogenic fungus Rhizopus arrhizus.

BACKGROUND: Members of Rhizopus species are the most common cause of mucormycosis, a rare but often fatal fungal infection. Host induced pathogen apoptosis and pathogen induced host cell apoptosis are often involved in fungal infections. In many organisms, the release of mitochondrial cytochrome c can trigger apoptosis by activating caspase proteases, but the role of fungal cytochrome c in apoptosis remains unknown. RESULTS: DNA sequence encoding Rhizopus arrhizus cytochrome c was cloned and expressed in E. coli. Both native and recombinant cytochrome c were purified using ion exchange followed by gel filtration chromatography. The identities of purified proteins were confirmed by MALDI-MS and UV-Visible spectroscopy. For the first time, we demonstrated that Rhizopus arrhizus cytochrome c could activate human capspase-3 in HeLa cell extracts. We also found that Rhizopus arrhizus cytochrome c has redox potential, peroxidase activity, and spectral properties similar to human and horse cytochrome c proteins. CONCLUSIONS: Rhizopus arrhizus cytochrome c can activate human caspase-3 in HeLa cell extracts and it possesses similar physical and spectral properties as human and horse cytochrome c. This protein was found to have a previously unknown potential to activate human caspase-3, an important step in the apoptosis cascade.

Oxidative Stress- and Autophagy-Inducing Effects of PSI-LHCI from Botryococcus braunii in Breast Cancer Cells.

Botryococcus braunii (B. braunii) is a green microalga primarily found in freshwater, reservoirs, and ponds. Photosynthetic pigments from algae have shown many bioactive molecules with therapeutic potential. Herein, we report the purification, characterization, and anticancer properties of photosystem I light-harvesting complex I (PSI-LHCI) from the green microalga B. braunii UTEX2441. The pigment-protein complex was purified by sucrose density gradient and characterized by its distinctive peaks using absorption, low-temperature (77 K) fluorescence, and circular dichroism (CD) spectroscopic analyses. Protein complexes were resolved by blue native-PAGE and two-dimensional SDS-PAGE. Triple-negative breast cancer MDA-MB-231 cells were incubated with PSI-LHCI for all of our experiments. Cell viability was assessed, revealing a significant reduction in a time- and concentration-dependent manner. We confirmed the internalization of PSI-LHCI within the cytoplasm and nucleus after 12 h of incubation. Cell death mechanism by oxidative stress was confirmed by the production of reactive oxygen species (ROS) and specifically superoxide. Furthermore, we monitored autophagic flux, apoptotic and necrotic features after treatment with PSI-LHCI. Treated MDA-MB-231 cells showed positive autophagy signals in the cytoplasm and nucleus, and necrotic morphology by the permeabilization of the cell membrane. Our findings demonstrated for the first time the cytotoxic properties of B. braunii PSI-LHCI by the induction of ROS and autophagy in breast cancer cells.

A Cytochrome c-Chlorotoxin Hybrid Protein as a Possible Antiglioma Drug.

Malignant gliomas are the most lethal form of primary brain tumors. Despite advances in cancer therapy, the prognosis of glioma patients has remained poor. Cytochrome c (Cytc), an endogenous heme-based protein, holds tremendous potential to treat gliomas because of its innate capacity to trigger apoptosis. To this end, a hybrid cytochrome c-chlorotoxin (Cytc-CTX) protein was biosynthesized to enable cellular uptake of the cell impenetrable Cytc using CTX transporters. A nucleotide sequence containing 1 : 1 Cytc and CTX was constructed and separated by a hexahistidine-tag and an enterokinase cleavage site. The sequence was cloned into a pBTR1 plasmid, expressed in Escherichia coli, purified via 2-dimensional chromatography. The identity and size of the protein were determined by Western blot and mass spectrometry. Cytc in this soluble hybrid protein has similar structure and stability as human Cytc and the hybrid protein is endocytosed into a glioma cell line, while displaying potent cytotoxicity and a favorable therapeutic index. Its facile, low-cost, and high yield synthesis, biocompatibility, and robustness suggest that the hybrid protein is a promising candidate for antiglioma drug evaluation.

Thiol-maleimide poly(ethylene glycol) crosslinking of L-asparaginase subunits at recombinant cysteine residues introduced by mutagenesis.

L-Asparaginase is an enzyme successfully being used in the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, and non-Hodgkin's lymphoma. However, some disadvantages still limit its full application potential, e.g., allergic reactions, pancreatitis, and blood clotting impairment. Therefore, much effort has been directed at improving its performance. A popular strategy is to randomly conjugate L-asparaginase with mono-methoxy polyethylene glycol, which became a commercial FDA approved formulation widely used in recent years. To improve this formulation by PEGylation, herein we performed cysteine-directed conjugation of the L-asparaginase subunits to prevent dissociation-induced loss of activity. The recombinant cysteine conjugation sites were introduced by mutagenesis at surface-exposed positions on the protein to avoid affecting the catalytic activity. Three conjugates were obtained using different linear PEGs of 1000, 2000, and 5000 g/mol, with physical properties ranging from a semi-solid gel to a fully soluble state. The soluble-conjugate exhibited higher catalytic activity than the non-conjugated mutant, and the same activity than the native enzyme. The cysteine-directed crosslinking of the L-asparaginase subunits produced a higher molecular weight conjugate compared to the native tetrameric enzyme. This strategy might improve L-asparaginase efficiency for leukemia treatment by reducing glomerular filtration due to the increase in hydrodynamic size thus extending half-live, while at the same time retaining full catalytic activity.