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.

Combining Stimulus-Triggered Release and Active Targeting Strategies Improves Cytotoxicity of Cytochrome c Nanoparticles in Tumor Cells.

Proteins often possess highly specific biological activities that make them potential therapeutics, but their physical and chemical instabilities during formulation, storage, and delivery have limited their medical use. Therefore, engineering of nanosized vehicles to stabilize protein therapeutics and to allow for targeted treatment of complex diseases, such as cancer, is of considerable interest. A micelle-like nanoparticle (NP) was designed for both, tumor targeting and stimulus-triggered release of the apoptotic protein cytochrome c (Cyt c). This system is composed of a Cyt c NP stabilized by a folate-receptor targeting amphiphilic copolymer (FA-PEG-PLGA) attached to Cyt c through a redox-sensitive bond. FA-PEG-PLGA-S-S-Cyt c NPs exhibited excellent stability under extracellular physiological conditions, whereas once in the intracellular reducing environment, Cyt c was released from the conjugate. Under the same conditions, the folate-decorated NP reduced folate receptor positive HeLa cell viability to 20%, while the same complex without FA only reduced it to 80%. Confocal microscopy showed that the FA-PEG-PLGA-S-S-Cyt c NPs were internalized by HeLa cells and were capable of endosomal escape. The specificity of the folate receptor-mediated internalization was confirmed by the lack of uptake by two folate receptor deficient cell lines: A549 and NIH-3T3. Finally, the potential as antitumor therapy of our folate-decorated Cyt c-based NPs was confirmed with an in vivo brain tumor model. In conclusion, we were able to create a stable, selective, and smart nanosized Cyt c delivery system.

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.

Graphene oxide as a protein matrix: influence on protein biophysical properties.

BACKGROUND: This study provides fundamental information on the influence of graphene oxide (GO) nanosheets and glycans on protein catalytic activity, dynamics, and thermal stability. We provide evidence of protein stabilization by glycans and how this strategy could be implemented when GO nanosheets is used as protein immobilization matrix. A series of bioconjugates was constructed using two different strategies: adsorbing or covalently attaching native and glycosylated bilirubin oxidase (BOD) to GO. RESULTS: Bioconjugate formation was followed by FT-IR, zeta-potential, and X-ray photoelectron spectroscopy measurements. Enzyme kinetic parameters (k(m) and k(cat)) revealed that the substrate binding affinity was not affected by glycosylation and immobilization on GO, but the rate of enzyme catalysis was reduced. Structural analysis by circular dichroism showed that glycosylation did not affect the tertiary or the secondary structure of BOD. However, GO produced slight changes in the secondary structure. To shed light into the biophysical consequence of protein glycosylation and protein immobilization on GO nanosheets, we studied structural protein dynamical changes by FT-IR H/D exchange and thermal inactivation. CONCLUSIONS: It was found that glycosylation caused a reduction in structural dynamics that resulted in an increase in thermostability and a decrease in the catalytic activity for both, glycoconjugate and immobilized enzyme. These results establish the usefulness of chemical glycosylation to modulate protein structural dynamics and stability to develop a more stable GO-protein matrix.

Chemical glycosylation of cytochrome c improves physical and chemical protein stability.

BACKGROUND: Cytochrome c (Cyt c) is an apoptosis-initiating protein when released into the cytoplasm of eukaryotic cells and therefore a possible cancer drug candidate. Although proteins have been increasingly important as pharmaceutical agents, their chemical and physical instability during production, storage, and delivery remains a problem. Chemical glycosylation has been devised as a method to increase protein stability and thus enhance their long-lasting bioavailability. RESULTS: Three different molecular weight glycans (lactose and two dextrans with 1 kD and 10 kD) were chemically coupled to surface exposed Cyt c lysine (Lys) residues using succinimidyl chemistry via amide bonds. Five neo-glycoconjugates were synthesized, Lac4-Cyt-c, Lac9-Cyt-c, Dex5(10kD)-Cyt-c, Dex8(10kD)-Cyt-c, and Dex3(1kD)-Cyt-c. Subsequently, we investigated glycoconjugate structure, activity, and stability. Circular dichroism (CD) spectra demonstrated that Cyt c glycosylation did not cause significant changes to the secondary structure, while high glycosylation levels caused some minor tertiary structure perturbations. Functionality of the Cyt c glycoconjugates was determined by performing cell-free caspase 3 and caspase 9 induction assays and by measuring the peroxidase-like pseudo enzyme activity. The glycoconjugates showed ≥94% residual enzyme activity and 86 ± 3 to 95 ± 1% relative caspase 3 activation compared to non-modified Cyt c. Caspase 9 activation by the glycoconjugates was with 92 ± 7% to 96 ± 4% within the error the same as the caspase 3 activation. There were no major changes in Cyt c activity upon glycosylation. Incubation of Dex3(1 kD)-Cyt c with mercaptoethanol caused significant loss in the tertiary structure and a drop in caspase 3 and 9 activation to only 24 ± 8% and 26 ± 6%, respectively. This demonstrates that tertiary structure intactness of Cyt c was essential for apoptosis induction. Furthermore, glycosylation protected Cyt c from detrimental effects by some stresses (i.e., elevated temperature and humidity) and from proteolytic degradation. In addition, non-modified Cyt c was more susceptible to denaturation by a water-organic solvent interface than its glycoconjugates, important for the formulation in polymers. CONCLUSION: The results demonstrate that chemical glycosylation is a potentially valuable method to increase Cyt c stability during formulation and storage and potentially during its application after administration.

Delivery of chemically glycosylated cytochrome c immobilized in mesoporous silica nanoparticles induces apoptosis in HeLa cancer cells.

Cytochrome c (Cyt c) is a small mitochondrial heme protein involved in the intrinsic apoptotic pathway. Once Cyt c is released into the cytosol, the caspase mediated apoptosis cascade is activated resulting in programmed cell death. Herein, we explore the covalent immobilization of Cyt c into mesoporous silica nanoparticles (MSN) to generate a smart delivery system for intracellular drug delivery to cancer cells aiming at affording subsequent cell death. Cyt c was modified with sulfosuccinimidyl-6-[3'-(2-pyridyldithio)-propionamido] hexanoate (SPDP) and incorporated into SH-functionalized MSN by thiol-disulfide interchange. Unfortunately, the delivery of Cyt c from the MSN was not efficient in inducing apoptosis in human cervical cancer HeLa cells. We tested whether chemical Cyt c glycosylation could be useful in overcoming the efficacy problems by potentially improving Cyt c thermodynamic stability and reducing proteolytic degradation. Cyt c lysine residues were modified with lactose at a lactose-to-protein molar ratio of 3.7 ± 0.9 using mono(lactosylamido)-mono(succinimidyl) suberate linker chemistry. Circular dichroism (CD) spectra demonstrated that part of the activity loss of Cyt c was due to conformational changes upon its modification with the SPDP linker. These conformational changes were prevented in the glycoconjugate. In agreement with the unfolding of Cyt c by the linker, a proteolytic assay demonstrated that the Cyt c-SPDP conjugate was more susceptible to proteolysis than Cyt c. Attachment of the four lactose molecules reversed this increased susceptibility and protected Cyt c from proteolytic degradation. Furthermore, a cell-free caspase-3 assay revealed 47% and 87% of relative caspase activation by Cyt c-SPDP and the Cyt c-lactose bioconjugate, respectively, when compared to Cyt c. This again demonstrates the efficiency of the glycosylation to improve maintaining Cyt c structure and thus function. To test for cytotoxicity, HeLa cells were incubated with Cyt c loaded MSN at different Cyt c concentrations (12.5, 25.0, and 37.5 µg/mL) for 24-72 h and cellular metabolic activity determined by a cell proliferation assay. While MSN-SPDP-Cyt c did not induced cell death, the Cyt c-lactose bioconjugate induced significant cell death after 72 h, reducing HeLa cell viability to 67% and 45% at the 25 µg/mL and 37.5 µg/mL concentrations, respectively. Confocal microscopy confirmed that the MSN immobilized Cyt c-lactose bioconjugate was internalized by HeLa cells and that the bioconjugate was capable of endosomal escape. The results clearly demonstrate that chemical glycosylation stabilized Cyt c upon formulation of a smart drug delivery system and upon delivery into cancer cells and highlight the general potential of chemical protein glycosylation to improve the stability of protein drugs.

Activation of caspase-dependent apoptosis by intracellular delivery of Cytochrome c-based nanoparticles.

BACKGROUND: Cytochrome c is an essential mediator of apoptosis when it is released from the mitochondria to the cytoplasm. This process normally takes place in response to DNA damage, but in many cancer cells (i.e., cancer stem cells) it is disabled due to various mechanisms. However, it has been demonstrated that the targeted delivery of Cytochrome c directly to the cytoplasm of cancer cells selective initiates apoptosis in many cancer cells. In this work we designed a novel nano-sized smart Cytochrome c drug delivery system to induce apoptosis in cancer cells upon delivery. RESULTS: Cytochrome c was precipitated with a solvent-displacement method to obtain protein nanoparticles. The size of the Cytochrome c nanoparticles obtained was 100-300 nm in diameter depending on the conditions used, indicating good potential to passively target tumors by the Enhanced Permeability and Retention effect. The surface of Cytochrome c nanoparticles was decorated with poly (lactic-co-glycolic) acid-SH via the linker succinimidyl 3-(2-pyridyldithio) propionate to prevent premature dissolution during delivery. The linker connecting the polymer to the protein nanoparticle contained a disulfide bond thus allowing polymer shedding and subsequent Cytochrome c release under intracellular reducing conditions. A cell-free caspase-3 assay revealed more than 80% of relative caspase activation by Cytochrome c after nanoprecipitation and polymer modification when compared to native Cytochrome c. Incubation of HeLa cells with the Cytochrome c based-nanoparticles showed significant reduction in cell viability after 6 hours while native Cytochrome c showed none. Confocal microscopy confirmed the induction of apoptosis in HeLa cells when they were stained with 4',6-diamidino-2-phenylindole and propidium iodide after incubation with the Cytochrome c-based nanoparticles. CONCLUSIONS: Our results demonstrate that the coating with a hydrophobic polymer stabilizes Cytochrome c nanoparticles allowing for their delivery to the cytoplasm of target cells. After smart release of Cytochrome c into the cytoplasm, it induced programmed cell death.

A Synergistic pH-Responsive Serum Albumin-Based Drug Delivery System Loaded with Doxorubicin and Pentacyclic Triterpene Betulinic Acid for Potential Treatment of NSCLC.

Nanosized drug delivery systems (DDS) have been studied as a novel strategy against cancer due to their potential to simultaneously decrease drug inactivation and systemic toxicity and increase passive and/or active drug accumulation within the tumor(s). Triterpenes are plant-derived compounds with interesting therapeutic properties. Betulinic acid (BeA) is a pentacyclic triterpene that has great cytotoxic activity against different cancer types. Herein, we developed a nanosized protein-based DDS of bovine serum albumin (BSA) as the drug carrier combining two compounds, doxorubicin (Dox) and the triterpene BeA, using an oil-water-like micro-emulsion method. We used spectrophotometric assays to determine protein and drug concentrations in the DDS. The biophysical properties of these DDS were characterized using dynamic light scattering (DLS) and circular dichroism (CD) spectroscopy, confirming nanoparticle (NP) formation and drug loading into the protein structure, respectively. The encapsulation efficiency was 77% for Dox and 18% for BeA. More than 50% of both drugs were released within 24 h at pH 6.8, while less drug was released at pH 7.4 in this period. Co-incubation viability assays of Dox and BeA alone for 24 h demonstrated synergistic cytotoxic activity in the low µM range against non-small-cell lung carcinoma (NSCLC) A549 cells. Viability assays of the BSA-(Dox+BeA) DDS demonstrated a higher synergistic cytotoxic activity than the two drugs with no carrier. Moreover, confocal microscopy analysis confirmed the cellular internalization of the DDS and the accumulation of the Dox in the nucleus. We determined the mechanism of action of the BSA-(Dox+BeA) DDS, confirming S-phase cell cycle arrest, DNA damage, caspase cascade activation, and downregulation of epidermal growth factor receptor (EGFR) expression. This DDS has the potential to synergistically maximize the therapeutic effect of Dox and diminish chemoresistance induced by EGFR expression using a natural triterpene against NSCLC.

Stimulus-responsive controlled release system by covalent immobilization of an enzyme into mesoporous silica nanoparticles.

Mesoporous silica nanoparticles (MSN) have emerged as an attractive class of drug delivery carriers for therapeutic agents. Herein, we explored the covalent immobilization of proteins into MSN to generate a stimulus-responsive controlled release system. First, MSN were functionalized with thiol groups using (mercaptopropyl)-trimethoxysilane (MPTMS). Functionalization was verified by X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared (FTIR) spectroscopy, and dynamic light scattering. The model enzyme carbonic anhydrase (CA) was coupled to sulfosuccinimidyl 6-[3'(2-pyridyldithio)-propionamido]hexanoate (Sulfo-LC-SPDP) at a low ratio of 1:1 to prevent enzyme inactivation and subsequently covalently immobilized into MSN via thiol-disulfide interchange. The enzyme could be released from MSN with 10 mM glutathione, which represents intracellular redox conditions, while it remained bound to the MSN at extracellular redox conditions represented by 1 µM glutathione. The activity of the released enzyme was >80% demonstrating that the enzyme was still largely functional and active after immobilization and release. Human cervical cancer (HeLa) cells were incubated with the MSN-CA bioconjugates at various concentrations for 24 h and the data show good biocompatibility. In summary, we demonstrate the potential of MSN as drug delivery systems for proteins.

Folate-Decorated Cross-Linked Cytochrome c Nanoparticles for Active Targeting of Non-Small Cell Lung Carcinoma (NSCLC).

The folate receptor alpha (FR), which is overexpressed in solid tumors including NSCLC, can be utilized for active tumor targeting to afford more effective cancer therapies. In this context, cytochrome c (Cyt c) has drawn attention to cancer research because it is non-toxic, yet, when delivered to the cytoplasm of cancer cells, can kill them by inducing apoptosis. Cyt c nanoparticles (NPs, 169 ± 9 nm) were obtained by solvent precipitation with acetonitrile, and stabilized by reversible homo-bifunctional crosslinking to accomplish a Cyt-c-based drug delivery system that combines stimulus-responsive release and active targeting. Cyt c was released under intracellular redox conditions, due to an S-S bond in the NPs linker, while NPs remained intact without any release under extracellular conditions. The NP surface was decorated with a hydrophilic folic acid-polyethylene glycol (FA-PEG) polymer for active targeting. The FA-decorated NPs specifically recognized and killed cancer cells (IC50 = 47.46 µg/mL) that overexpressed FR, but showed no toxicity against FR-negative cells. Confocal microscopy confirmed the preferential uptake and apoptosis induction of our NPs by FR-positive cancer cells. In vivo experiments using a Lewis lung carcinoma (LLC) mouse model showed visible NP accumulation within the tumor and inhibited the growth of LLC tumors.

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.

Key genes and drug delivery systems to improve the efficiency of chemotherapy.

Cancer cells can develop resistance to anticancer drugs, thereby becoming tolerant to treatment through different mechanisms. The biological mechanisms leading to the generation of anticancer treatment resistance include alterations in transmembrane proteins, DNA damage and repair mechanisms, alterations in target molecules, and genetic responses, among others. The most common anti-cancer drugs reported to develop resistance to cancer cells include cisplatin, doxorubicin, paclitaxel, and fluorouracil. These anticancer drugs have different mechanisms of action, and specific cancer types can be affected by different genes. The development of drug resistance is a cellular response which uses differential gene expression, to enable adaptation and survival of the cell to diverse threatening environmental agents. In this review, we briefly look at the key regulatory genes, their expression, as well as the responses and regulation of cancer cells when exposed to anticancer drugs, along with the incorporation of alternative nanocarriers as treatments to overcome anticancer drug resistance.

Cytochrome c: Using Biological Insight toward Engineering an Optimized Anticancer Biodrug.

The heme protein cytochrome c (Cyt c) plays pivotal roles in cellular life and death processes. In the respiratory chain of mitochondria, it serves as an electron transfer protein, contributing to the proliferation of healthy cells. In the cell cytoplasm, it activates intrinsic apoptosis to terminate damaged cells. Insight into these mechanisms and the associated physicochemical properties and biomolecular interactions of Cyt c informs on the anticancer therapeutic potential of the protein, especially in its ability to subvert the current limitations of small molecule-based chemotherapy. In this review, we explore the development of Cyt c as an anticancer drug by identifying cancer types that would be receptive to the cytotoxicity of the protein and factors that can be finetuned to enhance its apoptotic potency. To this end, some information is obtained by characterizing known drugs that operate, in part, by triggering Cyt c induced apoptosis. The application of different smart drug delivery systems is surveyed to highlight important features for maintaining Cyt c stability and activity and improving its specificity for cancer cells and high drug payload release while recognizing the continuing limitations. This work serves to elucidate on the optimization of the strategies to translate Cyt c to the clinical market.

Moisture-induced solid state instabilities in alpha-chymotrypsin and their reduction through chemical glycosylation.

BACKGROUND: Protein instability remains the main factor limiting the development of protein therapeutics. The fragile nature (structurally and chemically) of proteins makes them susceptible to detrimental events during processing, storage, and delivery. To overcome this, proteins are often formulated in the solid-state which combines superior stability properties with reduced operational costs. Nevertheless, solid protein pharmaceuticals can also suffer from instability problems due to moisture sorption. Chemical protein glycosylation has evolved into an important tool to overcome several instability issues associated with proteins. Herein, we employed chemical glycosylation to stabilize a solid-state protein formulation against moisture-induced deterioration in the lyophilized state. RESULTS: First, we investigated the consequences of moisture sorption on the stability and structural conformation of the model enzyme alpha-chymotrypsin (alpha-CT) under controlled humidity conditions. Results showed that alpha-CT aggregates and inactivates as a function of increased relative humidity (RH). Furthermore, alpha-CT loses its native secondary and tertiary structure rapidly at increasing RH. In addition, H/D exchange studies revealed that alpha-CT structural dynamics increased at increasing RH. The magnitude of the structural changes in tendency parallels the solid-state instability data (i.e., formation of buffer-insoluble aggregates, inactivation, and loss of native conformation upon reconstitution). To determine if these moisture-induced instability issues could be ameliorated by chemical glycosylation we proceeded to modify our model protein with chemically activated glycans of differing lengths (lactose and dextran (10 kDa)). The various glycoconjugates showed a marked decrease in aggregation and an increase in residual activity after incubation. These stabilization effects were found to be independent of the glycan size. CONCLUSION: Water sorption leads to aggregation, inactivation, and structural changes of alpha-CT as has been similarly shown to occur for many other proteins. These instabilities correlate with an increase in protein structural dynamics as a result of moisture exposure. In this work, we present a novel methodology to stabilize proteins against structural perturbations in the solid-state since chemical glycosylation was effective in decreasing and/or preventing the traditionally observed moisture-induced aggregation and inactivation. It is suggested that the stabilization provided by these chemically attached glycans comes from the steric hindrance that the sugars conveys on the protein surface therefore preventing the interaction of the protein internal electrostatics with that of the water molecules and thus reducing the protein structural dynamics upon moisture exposure.

Enantioselective Transesterification Catalysis by Nanosized Serine Protease Subtilisin Carlsberg Particles in Tetrahydrofuran.

Enzyme catalysis in organic solvents is a powerful tool for stereo-selective synthesis but the enantioselectivity is still hard to predict. To overcome this obstacle, we employed a nanoparticulate formulation of subtilisin Carlsberg (SC) and designed a series of 14 structurally related racemic alcohols. They were employed in the model transesterification reaction with vinyl butyrate and the enantioselectivities were determined. In general, short alcohol side chains led to low enantioselectivties, while larger and bulky side chains caused better discrimination of the enantiomers by the enzyme. With several bulky substrates high enantioselectivities with E>100 were obtained. Computational modeling highlighted that key to high enantioselectivity is the discrimination of the R and S substrates by the sole hydrophobic binding pocket based on their size and bulkiness. While bulky S enantiomer side chains could be accommodated within the binding pocket, bulky R enantiomer side chains could not. However, when also the S enantiomer side chain becomes too large and does not fit into the binding pocket anymore, enantioselectivity accordingly drops.

Effect of prolonged exposure to organic solvents on the active site environment of subtilisin Carlsberg.

The potential of enzyme catalysis as a tool for organic synthesis is nowadays indisputable, as is the fact that organic solvents affect an enzyme's activity, selectivity and stability. Moreover, it was recently realized that an enzyme's initial activity is substantially decreased after prolonged exposure to organic media, an effect that further hampers their potential as catalysts for organic synthesis. Regrettably, the mechanistic reasons for these effects are still debatable. In the present study we have made an attempt to explain the reasons behind the partial loss of enzyme activity on prolonged exposure to organic solvents. Fluorescence spectroscopic studies of the serine protease subtilisin Carlsberg chemically modified with polyethylene glycol (PEG-SC) and inhibited with a Dancyl fluorophore, and dissolved in two organic solvents (acetonitrile and 1,4-dioxane) indicate that when the enzyme is initially introduced into these solvents, the active site environment is similar to that in water; however prolonged exposure to the organic medium causes this environment to resemble that of the solvent in which the enzyme is dissolved. Furthermore, kinetic studies show a reduction on both V(max) and K(M) as a result of prolonged exposure to the solvents. One interpretation of these results is that during this prolonged exposure to organic solvents the active-site fluorescent label inhibitor adopts a different binding conformation. Extrapolating this to an enzymatic reaction we argue that substrates bind in a less catalytically favorable conformation after the enzyme has been exposed to organic media for several hours.

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.

Optimization and Characterization of Protein Nanoparticles for the Targeted and Smart Delivery of Cytochrome c to Non-Small Cell Lung Carcinoma.

The delivery of Cytochrome c (Cyt c) to the cytosol stimulates apoptosis in cells where its release from mitochondria and apoptotic induction is inhibited. We developed a drug delivery system consisting of Cyt c nanoparticles decorated with folate-poly(ethylene glycol)-poly(lactic-co-glycolic acid)-thiol (FA-PEG-PLGA-SH) to deliver Cyt c into cancer cells and tested their targeting in the Lewis Lung Carcinoma (LLC) mouse model. Cyt c-PLGA-PEG-FA nanoparticles (NPs) of 253 ± 55 and 354 ± 11 nm were obtained by Cyt c nanoprecipitation, followed by surface decoration with the co-polymer SH-PLGA-PEG-FA. The internalization of Cyt c-PLGA-PEG-FA nanoparticles (NPs) in LLC cells was confirmed by confocal microscopy. NP caspase activation was more efficient than the NP-free formulation. Caspase activity assays showed NPs retained 88-96% Cyt c activity. The NP formulations were more effective in decreasing LLC cell viability than NP-free formulation, with IC50 49.2 to 70.1 µg/mL versus 129.5 µg/mL, respectively. Our NP system proved to be thrice as selective towards cancerous than normal cells. In vivo studies using near infrared-tagged nanoparticles show accumulation in mouse LLC tumor 5 min post-injection. In conclusion, our NP delivery system for Cyt c shows superiority over the NP-free formulation and reaches a folic acid-overexpressing tumor in an immune-competent animal model.

On the role of protein structural dynamics in the catalytic activity and thermostability of serine protease subtilisin Carlsberg.

The effect of structural dynamics on enzyme activity and thermostability has thus far only been investigated in detail for the serine protease alpha-chymotrypsin (for a recent review see Solá et al., Cell Mol Life Sci 2007, 64(16): 2133-2152). Herein, we extend this type of study to a structurally unrelated serine protease, specifically, subtilisin Carlsberg. The protease was incrementally glycosylated with chemically activated lactose to obtain various subtilisin glycoconjugates which were biophysically characterized. Near UV-CD spectroscopy revealed that the tertiary structure was unaffected by the glycosylation procedure. H/D exchange FT-IR spectroscopy was performed to assess the changes in structural dynamics of the enzyme. It was found that increasing the level of glycosylation caused a linearly dependent reduction in structural dynamics. This led to an increase in thermostability and a decrease in the catalytic turnover rate for both, the enzyme acylation and deacylation steps. These results highlight the possibility that a structural dynamics-activity relationship might be a phenomenon generally found in serine proteases.