Native RNase Research Articles

PH-responsive organic/inorganic hybrid nanocolloids for transcellular delivery of ribonucleolytic payloads toward targeted anti-glioma therapy

Proteins have been appreciated to be a superlative modality of therapeutics in view of their direct roles in regulating diverse sets of biological events, nonetheless, the clinical applications of the proteinic therapeutics have been strictly limited to act on the cell surface receptors owing to their inherent cell-impermeable character of the proteins. To this obstacle, we contrived carboxylation reaction upon the proteins (RNase A) into the overall negatively charged pro-RNase, followed by elaboration of intelligent pH-responsive pro-RNase delivery nanocolloids based on co-precipitation of pro-RNase and Arg-Gly-Asp (RGD)-functionalized poly(ethylene glycol) (PEG)-block-polyanion with aids of inorganic calcium phosphate (CaP). The resulting nanocolloids appeared to actively accumulate into glioma due to the specific binding affinities of RGD and glioma-enriched αVβ3 and αVβ5 integrins. Furthermore, the pH responsiveness to the acidic endolysosomal microenvironment of all compositions of nanocolloids (including: decarboxylation of pro-RNase composition to restore the native RNase A, ionization of CaP composition to elicit osmotic pressure, and charge reversal of PEG-block-polyanion into membrane-disruptive polycation) could stimulate not only efficient endolysosomal escape for translocation into the cytosol but also structural disassembly for ready liberation of the RNase A payloads, eventually exerting non-specific RNA degradation for apoptosis of the affected cells. Systemic dosage of the proposed nanocolloids demonstrated potent anti-tumor efficacies towards xenograft glioma due to massive RNA degradation. Therefore, our proposed RNase A prodrug nanocolloids could represent as a versatile platform for engineering transcellular protein delivery systems, which are expected to spur thriving emergence of a spectrum of proteins in precision intervention of intractable diseases.

Slow Evolution toward “Super-Aggregation” of the Oligomers Formed through the Swapping of RNase A N-Termini: A Wish for Amyloidosis?

Natively monomeric RNase A can oligomerize upon lyophilization from 40% acetic acid solutions or when it is heated at high concentrations in various solvents. In this way, it produces many dimeric or oligomeric conformers through the three-dimensional domain swapping (3D-DS) mechanism involving both RNase A N- or/and C-termini. Here, we found many of these oligomers evolving toward not negligible amounts of large derivatives after being stored for up to 15 months at 4 °C in phosphate buffer. We call these species super-aggregates (SAs). Notably, SAs do not originate from native RNase A monomer or from oligomers characterized by the exclusive presence of the C-terminus swapping of the enzyme subunits as well. Instead, the swapping of at least two subunits’ N-termini is mandatory to produce them. Through immunoblotting, SAs are confirmed to derive from RNase A even if they retain only low ribonucleolytic activity. Then, their interaction registered with Thioflavin-T (ThT), in addition to TEM analyses, indicate SAs are large and circular but not “amyloid-like” derivatives. This confirms that RNase A acts as an “auto-chaperone”, although it displays many amyloid-prone short segments, including the 16–22 loop included in its N-terminus. Therefore, we hypothesize the opening of RNase A N-terminus, and hence its oligomerization through 3D-DS, may represent a preliminary step favoring massive RNase A aggregation. Interestingly, this process is slow and requires low temperatures to limit the concomitant oligomers’ dissociation to the native monomer. These data and the hypothesis proposed are discussed in the light of protein aggregation in general, and of possible future applications to contrast amyloidosis.

A stimulus-responsive hexahedron DNA framework facilitates targeted and direct delivery of native anticancer proteins into cancer cells.

The targeted and direct intracellular delivery of proteins plays critical roles in biological research and disease treatments, yet remains highly challenging. Current solutions to such a challenge are limited by the modification of proteins that may potentially alter protein functions inside cells or the lack of targeting capability. Herein, we develop a stimulus-responsive and bivalent aptamer hexahedron DNA framework (HDF) for the targeted and direct delivery of native therapeutic proteins into cancer cells. The unmodified proteins are caged inside the HDF nanostructures assembled from six programmable single stranded DNAs to protect the proteins from degradation by cathepsins and enhance their targeting capability and delivery efficiency with the nanostructure-integrated aptamers. In addition, the protein drugs can be selectively released from the HDF nanostructures by the intracellular ATP molecules to induce tumor cell apoptosis, highlighting their promising application potential for cell biology and precise protein medicines.

E. coli RNase I exhibits a strong Ca2+-dependent inherent double-stranded RNase activity.

Since its initial characterization, Escherichia coli RNase I has been described as a single-strand specific RNA endonuclease that cleaves its substrate in a largely sequence independent manner. Here, we describe a strong calcium (Ca2+)-dependent activity of RNase I on double-stranded RNA (dsRNA), and a Ca2+-dependent novel hybridase activity, digesting the RNA strand in a DNA:RNA hybrid. Surprisingly, Ca2+ does not affect the activity of RNase I on single stranded RNA (ssRNA), suggesting a specific role for Ca2+ in the modulation of RNase I activity. Mutation of a previously overlooked Ca2+ binding site on RNase I resulted in a gain-of-function enzyme that is highly active on dsRNA and could no longer be stimulated by the metal. In summary, our data imply that native RNase I contains a bound Ca2+, allowing it to target both single- and double-stranded RNAs, thus having a broader substrate specificity than originally proposed for this traditional enzyme. In addition, the finding that the dsRNase activity, and not the ssRNase activity, is associated with the Ca2+-dependency of RNase I may be useful as a tool in applied molecular biology.

Structure and Dynamics of Ribonuclease A during Thermal Unfolding: The Failure of the Zimm Model.

Disordered regions as found in intrinsically disordered proteins (IDP) or during protein folding define response time to stimuli and protein folding times. Neutron spin-echo spectroscopy is a powerful tool to directly access the collective motions of the unfolded chain to enlighten the physical origin of basic conformational relaxation. During the thermal unfolding of native ribonuclease A, we examine the structure and dynamics of the disordered state within a two-state transition model using polymer models, including internal friction, to describe the chain dynamics. The presence of four disulfide bonds alters the disordered configuration to a more compact configuration compared to a Gaussian chain that is defined by the additional links, as demonstrated by coarse-grained simulation. The dynamics of the disordered chain is described by Zimm dynamics with internal friction (ZIF) between neighboring amino acids. Relaxation times are dominated by mode-independent internal friction. Internal friction relaxation times show an Arrhenius-like behavior with an activation energy of 33 kJ/mol. The Zimm dynamics is dominated by internal friction and suggest that the characteristic motions correspond to overdamped elastic modes similar to the motions observed for folded proteins but within a pool of disordered configurations spanning the configurational space. For IDP, internal friction dominates while solvent friction and hydrodynamic interactions are smaller corrections.

Cholesteryl-Conjugated Ribonuclease A Exhibits Enzyme Activity in Aqueous Solution and Resistance to Dimethyl Sulfoxide.

Using bovine pancreatic ribonuclease A (RNase A) and cholesterol, we synthesized cholesteryl-conjugated ribonuclease A (CHRNase A) to evaluate the influence of a conjugated hydrophobic moiety on protein function. Nuclear magnetic resonance and matrix-assisted laser desorption/ionization time-of-flight spectrometry suggested that one cholesteryl group was conjugated to RNase A. Differential scanning calorimetry indicated that CHRNase A was denatured in the solid state but was folded in phosphate buffer (0.05 mol/L, pH 6.5). CHRNase A resembled RNase A in its secondary structure, but circular dichroism (CD) spectra revealed that the helical content of CHRNase A was decreased and the tertiary structure of CHRNase A differed from that of RNase A. Furthermore, fluorescence measurements, CD spectra, an 8-anilino-1-naphthalenesulfonic acid ammonium salt-based assay, and surface tension measurements suggested that cholesterol was conjugated to a tyrosine residue on the protein surface. The relative activity of CHRNase A to RNase A was 79 ± 7%, and the enzyme activity of CHRNase A by adding β-cyclodextrin (β-CyD) increased to 129 ± 7%. Therefore, we considered that the cholesteryl group interacted with substrate (cytidine 2′3′-cyclic monophosphate monosodium salt) to inhibit the enzyme reaction. Finally, the environment around tyrosine residues in CHRNase A in dimethyl sulfoxide was similar to that of native RNase A in phosphate buffer (0.05 mol/L, pH 6.5). These results suggest that cholesterol conjugation to RNase A altered RNase A functionality, including improvement of RNase A resistance to dimethyl sulfoxide and modulation of the ability of β-CyD to control RNase A enzymatic activity.

C-Mannosylation Enhances the Structural Stability of Human RNase 2.

SummaryC-Mannosylation is a relatively rare form of protein glycosylation involving the attachment of an α-mannopyranosyl residue to C-2 of the indole moiety of the amino acid tryptophan. This type of linkage was initially discovered in RNase 2 from human urine but later confirmed to be present in many other important proteins. Based on NMR experiments and extensive molecular dynamics simulations on the hundred microsecond timescale we demonstrate that, for isolated glycopeptides and denatured RNase 2, the C-linked mannopyranosyl residue exists as an ensemble of conformations, among which 1C4 is the most abundant. However, for native RNase 2, molecular dynamics and NMR studies revealed that the mannopyranosyl residue favors a specific conformation, which optimally stabilizes the protein fold through a network of hydrogen bonds and which leads to a significant reduction of the protein dynamics on the microsecond timescale. Our findings contribute to the understanding of the biological role of C-mannosylation.

RNase 7 downregulates TH2 cytokine production by activated human T cells.

The antimicrobial peptide (AMP) RNase 7 is constitutively expressed in the epidermis of healthy human skin and has been found to be upregulated in chronic inflammatory skin diseases such as atopic dermatitis and psoriasis. Activated T cells in lesional skin of patients with atopic dermatitis (AD) and psoriasis (PSO) might be directly exposed to RNase 7. In addition to their antimicrobial activity, immunoregulatory functions have been published for several AMPs. In this study, we investigated immunoregulatory effects of the antimicrobial peptide RNase 7 on activated T cells. Isolated human CD3+T cells were stimulated with RNase 7 and screened for possible effects by mRNA microarray analysis. The results of the mRNA microarray were confirmed in isolated CD4+T cells and in polarized TH2 cells using skin-derived native RNase 7 and a recombinant ribonuclease-inactive RNase 7 mutant. Activation of GATA3 was analysed by electrophoretic mobility shift assay. Treatment of activated human CD4+T cells and TH2 cells with RNase 7 selectively reduced the expression of TH2 cytokines (IL-13, IL-4 and IL-5). Experiments with a ribonuclease-inactive recombinant RNase 7 mutant showed that RNase 7 ribonuclease activity is dispensable for the observed regulatory effect. We further demonstrate that CD4+T cells from AD patients revealed a significantly less pronounced downregulation of IL-13 in response to RNase 7 compared to healthy control. Finally, we show that GATA3 activation was diminished upon cultivation of T cells with RNase 7. Our data indicate that RNase 7 has immunomodulatory functions on TH2 cells and decreases the production of TH2 cytokines in the skin.

Modelling of electrokinetic phenomena for capture of PEGylated ribonuclease A in a microdevice with insulating structures.

Synthesis of PEGylated proteins results in a mixture of protein-polyethylene glycol (PEG) conjugates and the unreacted native protein. From a ribonuclease A (RNase A) PEGylation reaction, mono-PEGylated RNase A (mono-PEG RNase A) has proven therapeutic effects against cancer, reason for which there is an interest in isolating it from the rest of the reaction products. Experimental trapping of PEGylated RNase A inside an electrokinetically driven microfluidic device has been previously demonstrated. Now, from a theoretical point of view, we have studied the electrokinetic phenomena involved in the dielectrophoretic streaming of the native RNase A protein and the trapping of the mono-PEG RNase A inside a microfluidic channel. To accomplish this, we used two 3D computational models, a sphere and an ellipse, adapted to each protein. The effect of temperature on parameters related to trapping was also studied. A temperature increase showed to rise the electric and thermal conductivities of the suspending solution, hindering dielectrophoretic trapping. In contrast, the dynamic viscosity of the suspending solution decreased as the temperature rose, favoring the dielectrophoretic manipulation of the proteins. Also, our models were able to predict the magnitude and direction of the velocity of both proteins indicating trapping for the PEGylated conjugate or no trapping for the native protein. In addition, a parametric sweep study revealed the effect of the protein zeta potential on the electrokinetic response of the protein. We believe this work will serve as a tool to improve the design of electrokinetically driven microfluidic channels for the separation and recovery of PEGylated proteins in one single step.

Glycation of Ribonuclease A affects its enzymatic activity and DNA binding ability.

Prolonged non-enzymatic glycation of proteins results in the formation of advanced glycation end products (AGEs) that cause several diseases. The glycation of Ribonuclease A (RNase A) at pH 7.4 and 37°C with ribose, glucose and fructose has been monitored by UV-vis, fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption ionization spectroscopy-time of flight (MALDI-TOF) methods. The enzymatic activity and DNA binding ability of glycated RNase A was also investigated by an agarose gel-based assay. A precipitation assay examined the ribonucleolytic activity of the glycated enzyme. An increase in incubation time resulted in the formation of high molecular weight AGEs with a decrease in ribonucleolytic activity. Ribose exhibits the highest potency as a glycating agent and showed the greatest reduction in the ribonucleolytic activity of the enzyme. Interestingly, glycated RNase A was unable to bind with the ribonuclease inhibitor (RI) and DNA. The glycated form of the protein was also found to be ineffective in DNA melting unlike native RNase A.

Chemical grafting of Sepharose 6B and its use in the purification of PEGylated RNase A

PEGylation is the covalent binding of at least one chain of polyethylene glycol (PEG) to a protein, often resulting in a heterogeneous population of conjugated species and unmodified protein. Despite some recent developments, one of the major challenges in the area of PEGylated proteins is related to the purification. Sequential chromatographic steps have been extensively used for the resolution of the different PEGylated species. This study, presents the use of a chemically modified support as an alternative for the separation of PEGylated ribonuclease (RNase) species from its unmodified counterpart as well as the resolution of the different PEGylated species in a single chromatographic step using hydrophobic interaction chromatography (HIC) media. Sepharose-6B was subjected to chemical modification (covalent addition of PEG 550, PEG 2000 and PEG 5000g/mol) to increase the selectivity of this media for PEGylated RNase A. The effect of ammonium sulfate concentration as well as the length of the PEG molecule on the chromatographic behavior of purified monoPEGylated and diPEGylated RNase was evaluated. The complete separation of native RNase from its PEGylated conjugates was achieved using a linear gradient elution of 1.5M ammonium sulfate in 25mM potassium phosphate pH7.0. With step gradient optimization, the resolution of PEGylated conjugates (mono and di-PEG RNase) was also achieved injecting an actual PEGylation reaction mixture as sample, using the same elution buffers and Sepharose 6B-PEG5000 as stationary phase. The purity obtained for monoPEGylated RNase A was 85% with a yield of 96% comparable to what has been reported previously for other HIC/SEC media. The use of PEGylated Sepharose 6B was demonstrated as an attractive alternative for the separation and resolution of PEGylated proteins in a single chromatographic step.

Switching the enzymatic activity of ribonuclease a based on enzyme/polymer complex formation

Context: Enzymes are ideal for various applications in both medicine and biotechnology. Switching on/off the enzymatic activity of enzymes by polymeric modification would expand the applications of enzymes in a wide range of research fields. Objectives: On/off switching the enzymatic activity of RNase A and the confirmation of the enzyme/polymer complex formation which leads to improve the heat resistance of RNase A. Materials and Methods: ?-Methoxy-?-methacryloyl poly (ethylene glycol) (PEG-MA) macromonomer and PEAMA-g-PEG were synthesized. Bovine RNase A, cytidine 2?,3?-cyclic monophosphate sodium salt (cCMP), and 3-(N-morpholino)propanesulfonic acid (MOPS) were obtained from Sigma Chemical Co. (St. Louis, USA). PAAc (Mn = 5,000 g/mol) were purchased from Wako (Osaka, Japan). Sodium dihydrogen phosphate dihydrate (NaH2PO4.2H2O) was obtained from Nacalai Tesque Inc. (Kyoto, Japan). The enzymatic activity of RNase A was estimated as follows. The RNase A concentration was determined by measuring the absorbance at 280 nm with an appropritate blank, using an extinction coefficient of 7.10 mL mg-1 cm-1. A total of 1.5 mL of 0.1 mg/mL cCMP solution prepared in 0.1 M MOPS (pH 7.0) was mixed with 10 ?L of the RNase A solution in 50 mM sodium phosphate buffer (pH 7.0). The increase in light scattering intensity of the solution was monitored by measuring the absorbance at 284 nm for 60 s in a UV-vis spectrophotometer at room temperature. After heat treatment, the enzymatic activities were measured. Far-UV and near-UV CD spectra were monitored using a spectropolarimeter (model J-720W; Jasco, Tokyo, Japan). Results: We have found that poly (acrylic acid) (PAAc) suppressed the enzymatic activity of RNase A completely and the recovery of enzymatic activity were observed (94%) by the external addition of PEAMA-g-PEG to the RNase A/PAAc complex. Our present findings suggest that the complexation between PEAMA-g-PEG and RNase A has occurred which improve the heat resistance (64%) of RNase A. Heat treatment has been carried out at 98°C for 10 minutes where the native RNase A lost all of its enzymatic activity. CD spectral analysis also indicates that the conformation of the enzyme was not altered due to the complexation. Conclusion: Poly (N,N-diethylaminoethyl methacrylate)-graft-poly (ethylene glycol) (PEAMA-g-PEG) restored the enzymatic activity of RNase A completely which was inactivated upon the addition of poly(acrylic acid) (PAAc) to RNase A. Complexation of RNase A with PEAMA-g-PEG induced the improvement of heat resistance of RNase A . Circular dichroism (CD) spectral analysis clearly indicated that the complexation of enzyme with polymer has almost no influence on the conformation of enzyme. DOI: http://dx.doi.org/10.3329/jbs.v20i0.17650 J. bio-sci. 20: 33-39, 2012

Effects of chemical modifications in the partition behavior of proteins in aqueous two-phase systems: A case study with RNase A

Chemical modification of proteins is gaining importance due to the improvement in properties and the broader range of applications that these protein conjugates have. Once modified, several purification strategies need to be applied to isolate the conjugates of interest. Aqueous two-phase systems (ATPS) are an attractive alternative for the primary recovery of proteins and their conjugates. However, to better understand which biochemical parameters affect in greater degree the partition behavior of these modified proteins in ATPS, it becomes necessary to characterize the partition behavior of different species. In this work, ribonuclease A (RNase A) was selected as a model protein to address the partition behavior of chemically modified proteins in ATPS. Native, mono-PEGylated, Uniblue A, Dabsyl Chloride, and Direct Red 83 chemically modified RNase A's were partitioned in 16 different polyethylene glycol (PEG)-potassium phosphate ATPS. Results suggest that while the effects of system design parameters govern the partition of native RNase A, the behavior of the chemically modified species is more influenced by the physicochemical characteristics of the modifying molecules, that in most cases promote partition toward the top polymer-rich phase with recovery percentages as high as 86%. It has been found that both, the hydrophobicity and molecular weight of the modifying species play a preponderant role in conjugate partition behavior since as hydrophobicity increases partition is promoted towards the PEG-rich phase balancing the effect of the molecular weight of the modifying molecules that tends to shift partition towards the salt rich phase.

Protein prosthesis: β‐peptides as reverse‐turn surrogates

The introduction of non-natural modules could provide unprecedented control over folding/unfolding behavior, conformational stability, and biological function of proteins. Success requires the interrogation of candidate modules in natural contexts. Here, expressed protein ligation is used to replace a reverse turn in bovine pancreatic ribonuclease (RNase A) with a synthetic β-dipeptide: β²-homoalanine-β³-homoalanine. This segment is known to adopt an unnatural reverse-turn conformation that contains a 10-membered ring hydrogen bond, but one with a donor-acceptor pattern opposite to that in the 10-membered rings of natural reverse turns. The RNase A variant has intact enzymatic activity, but unfolds more quickly and has diminished conformational stability relative to native RNase A. These data indicate that hydrogen-bonding pattern merits careful consideration in the selection of beneficial reverse-turn surrogates.

Superfolder GFP reporters validate diverse new mRNA targets of the classic porin regulator, MicF RNA

MicF is a textbook example of a small regulatory RNA (sRNA) that acts on a trans-encoded target mRNA through imperfect base pairing. Discovery of MicF as a post-transcriptional repressor of the major Escherichia coli porin OmpF established the paradigm for a meanwhile common mechanism of translational inhibition, through antisense sequestration of a ribosome binding site. However, whether MicF regulates additional genes has remained unknown for almost three decades. Here, we have harnessed the new superfolder variant of GFP for reporter-gene fusions to validate newly predicted targets of MicF in Salmonella. We show that the conserved 5' end of MicF acts by seed pairing to repress the mRNAs of global transcriptional regulator Lrp, and periplasmic protein YahO, while a second targeting region is also required to regulate the mRNA of the lipid A-modifying enzyme LpxR. Interestingly, MicF targets lpxR at both the ribosome binding site and deep within the coding sequence. MicF binding in the coding sequence of lpxR decreases mRNA stability through exacerbating the use of a native RNase E site proximal to the short MicF-lpxR duplex. Altogether, this study assigns the classic MicF sRNA to the growing class of Hfq-associated regulators that use diverse mechanisms to impact multiple loci.

Kinetic and thermodynamic analysis of the conformational folding process of SS-reduced bovine pancreatic ribonuclease A using a selenoxide reagent with high oxidizing ability

Redox-coupled folding pathways of bovine pancreatic ribonuclease A (RNase A) with four intramolecular disulfide (SS) bonds comprise three phases: (I) SS formation to generate partially oxidized intermediate ensembles with no rigid folded structure; (II) SS rearrangement from the three SS intermediate ensemble (3S) to the des intermediates having three native SS linkages; (III) final oxidation of the last native SS linkage to generate native RNase A. We previously demonstrated that DHSox, a water-soluble selenoxide reagent for rapid and quantitative SS formation, allows clear separation of the three folding phases. In this study, the main conformational folding phase (phase II) has been extensively analyzed at pH 8.0 under a wide range of temperatures (5–45°C), and thermodynamic and kinetic parameters for the four des intermediates were determined. The free-energy differences (ΔG) as a function of temperature suggested that the each SS linkage has different thermodynamic and kinetic roles in stability of the native structure. On the other hand, comparison of the rate constants and the activation energies for 3S→des with those reported for the conformational folding of SS-intact RNase A suggested that unfolded des species (desU) having three native SS linkages but not yet being folded are involved in very small amounts (<1%) in the 3S intermediate ensemble and the desU species would gain the native-like structures via X-Pro isomerization like SS-intact RNase A. It was revealed that DHSox is useful for kinetic and thermodynamic analysis of the conformational folding process on the oxidative folding pathways of SS-reduced proteins.

SiRNA-Aptamer Chimeras on Nanoparticles: Preserving Targeting Functionality for Effective Gene Silencing

SiRNA-aptamer chimeras are emerging as a highly promising approach for cell-type specific delivery of siRNA due to the outstanding targeting capability of aptamers and the compatibility of chimeras with native ribonuclease (Dicer) processing. For efficient RNA interference (RNAi), however, additional challenges must be addressed, in particular how to get siRNA out of the endosome after cell entry and how to preserve aptamer targeting specificity when chimeras are combined with delivery carriers. Here, we report a rationally designed nanoparticle vector that simultaneously displays large surface area for high siRNA payload, exposed aptamer for specific targeting, proton sponge effect for endosome escape, and fluorescence for imaging and quantification. A key concept of this work is to graft chimeras onto nanoparticle surface via a two-step process: first immobilizing siRNA onto nanoparticle via noncovalent interactions to facilitate intracellular unpackaging and reduce nanoparticle surface charge (avoiding nonspecific electrostatic interactions between aptamers and nanoparticles) and then coupling siRNA and aptamer with retained conformation and high accessibility. Compared with conventional one-step adsorption of siRNA-aptamer chimeras onto nanoparticles with random orientations and conformations, which does not elicit much improved RNAi effect than nontargeted nanoparticle-siRNA complexes (∼6-8% improvement of the total cell population), under the same RNA concentration our approach shows selective gene silencing and enables 34% more silenced cells of the total cell population over nontargeted nanoparticle-siRNA complexes. This remarkable difference in RNAi efficiency using nanoparticle-chimera complexes is directly related to cell uptake discrepancy resulting from aptamer conformation on the nanoparticle surface (intact vs random).

RNase A oligomerization through 3D domain swapping is favoured by a residue located far from the swapping domains

Bovine pancreatic ribonuclease A forms 3D domain-swapped oligomers by lyophilization from 40% acetic acid solutions or if subjected to various thermally-induced denaturation procedures.Considering that the intrinsic swapping propensity of bovine seminal RNase, the only member of the pancreatic-type RNase super-family that is dimeric in nature, is decreased from 70 to 30% if Arg80 is substituted by Ser (the corresponding residue in native RNase A), we introduced the opposite mutation in position 80 of the pancreatic enzyme. Our aim was to detect if the RNase A tendency to aggregate through domain swapping could increase.Aggregation of the S80R-RNase A mutant was induced either through the ‘classic’ acetic acid lyophilization, or through a thermally-induced method. The results indicate that the S80R mutant aggregates to a higher extent than the native protein, and that the increase occurs especially through N-terminal swapping.Additional investigations on the dimeric and multimeric species formed indicate that the S80R mutation increases their stability against regression to monomer, and does not significantly change their structural and functional features.

Monitoring the Glycosylation Status of Proteins Using Raman Spectroscopy

Protein-based biopharmaceuticals are becoming increasingly widely used as therapeutic agents, and the characterization of these biopharmaceuticals poses a significant analytical challenge. In particular, monitoring posttranslational modifications (PTMs), such as glycosylation, is an important aspect of this characterization because these glycans can strongly affect the stability, immunogenicity, and pharmacokinetics of these biotherapeutic drugs. Raman spectroscopy is a powerful tool, with many emerging applications in the bioprocessing arena. Although the technique has a relatively rich history in protein science, only recently has Raman spectroscopy been investigated for assessing posttranslational modifications, including phosphorylation, acetylation, trimethylation, and ubiquitination. In this investigation, we develop for the first time Raman spectroscopy combined with multivariate data analyses, including principal components analysis and partial least-squares regression, for the determination of the glycosylation status of proteins and quantifying the relative concentrations of the native ribonuclease (RNase) A protein and RNase B glycoprotein within mixtures.

Abstract 2573: Combinations of QBI-139, a clinical stage ribonuclease drug

Abstract RNA has been recognized as a drug target for cancer therapy, as evidenced by the ongoing clinical trials of RNAi and antisense therapies. An alternative approach that circumvents the delivery and stability issues of RNAi and antisense is to harness the activity of naturally occurring enzymes that degrade RNA. The ribonuclease approach is partially validated by objective clinical responses seen in clinical trials of ribonuclease (RNase) discovered in frog eggs. Our research has focused on creating variants of human RNases with the ability of frog RNase to evade the human RNase inhibitor. Diminished binding of mammalian RNases to the inhibitor have been shown to endow the RNase with the ability to kill cancer cells. One of the RNase variants, called QBI-139, demonstrated particularly strong anti-cancer activity against a variety of tumor types in in vivo models. QBI-139 is differentiated from the frog RNase because QBI-139 is 95 % identical to a native human RNase, while the frog RNase is only 20% similar. This difference may be what has led to a significantly improved tolerability profile for QBI-139 across species. QBI-139 was selected for clinical development and is now in a Phase I trial of solid tumors. In charting the clinical development path for QBI-139, the ability of the RNase to work in combination with a variety of standard of care agents had to be evaluated. Based in the single agent activity of QBI-139 in colon, non-small cell lung, ovarian and pancreatic cancers, the potency of QBI-139 in combination with the standard of care agents for these indications was explored. In in vitro screening, QBI-139 has demonstrated additive and synergistic activity in combination with cisplatin, 5-FU and docetaxel. The treatments were evaluated using fixed ratios of QBI-139 and the combination drug based on their EC50 values. The Combination Index (CI) was determined using median effect analysis. A CI of 1 indicates additive effects, whereas a CI &amp;lt; 1 indicates synergy. Synergy was demonstrated with QBI-139 in combination with cisplatin against solid tumors of non-small cell lung cancer (A549 and H1975) and an additive effect in ovarian (Ovcar-3) cancer. 5-FU and docetaxel also were synergistic with QBI-139 against solid tumors of colon (HCT-116) and ovarian (SK-OV-3) cancer, respectively.The goal of this project is development of an innovative, differentiated therapy, QBI-139, to provide clinical benefit to patients with colon, non-small cell lung and ovarian cancer, which are among the seven deadliest forms of cancer in the United States. In addition to being serious diseases, effective treatment of these cancers also represent unmet medical needs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2573. doi:10.1158/1538-7445.AM2011-2573