Dataset Information

Rational design and evaluation of mammalian ribonuclease cytotoxins.

ABSTRACT: Mammalian pancreatic-type ribonucleases (ptRNases) comprise an enzyme family that is remarkably well suited for therapeutic exploitation. ptRNases are robust and prodigious catalysts of RNA cleavage that can naturally access the cytosol. Instilling cytotoxic activity requires endowing them with the ability to evade a cytosolic inhibitor protein while retaining other key attributes. These efforts have informed our understanding of ptRNase-based cytotoxins, as well as the action of protein-based drugs with cytosolic targets. Here, we address the most pressing problems encountered in the design of cytotoxic ptRNases, along with potential solutions. In addition, we describe assays that can be used to evaluate a successful design in vitro, in cellulo, and in vivo. The emerging information validates the continuing development of ptRNases as chemotherapeutic agents.

SUBMITTER: Lomax JE

PROVIDER: S-EPMC3304445 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Rational design and evaluation of mammalian ribonuclease cytotoxins.

Lomax Jo E JE Eller Chelcie H CH Raines Ronald T RT

Methods in enzymology 20120101

Mammalian pancreatic-type ribonucleases (ptRNases) comprise an enzyme family that is remarkably well suited for therapeutic exploitation. ptRNases are robust and prodigious catalysts of RNA cleavage that can naturally access the cytosol. Instilling cytotoxic activity requires endowing them with the ability to evade a cytosolic inhibitor protein while retaining other key attributes. These efforts have informed our understanding of ptRNase-based cytotoxins, as well as the action of protein-based d ...[more]

PMID: 22208989

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Project description:Background¹³C-Metabolic flux analysis (¹³C-MFA) is a standard technique to probe cellular metabolism and elucidate in vivo metabolic fluxes. 13C-Tracer selection is an important step in conducting ¹³C-MFA, however, current methods are restricted to trial-and-error approaches, which commonly focus on an arbitrary subset of the tracer design space. To systematically probe the complete tracer design space, especially for complex systems such as mammalian cells, there is a pressing need for new rational approaches to identify optimal tracers.ResultsRecently, we introduced a new framework for optimal ¹³C-tracer design based on elementary metabolite units (EMU) decomposition, in which a measured metabolite is decomposed into a linear combination of so-called EMU basis vectors. In this contribution, we applied the EMU method to a realistic network model of mammalian metabolism with lactate as the measured metabolite. The method was used to select optimal tracers for two free fluxes in the system, the oxidative pentose phosphate pathway (oxPPP) flux and anaplerosis by pyruvate carboxylase (PC). Our approach was based on sensitivity analysis of EMU basis vector coefficients with respect to free fluxes. Through efficient grouping of coefficient sensitivities, simple tracer selection rules were derived for high-resolution quantification of the fluxes in the mammalian network model. The approach resulted in a significant reduction of the number of possible tracers and the feasible tracers were evaluated using numerical simulations. Two optimal, novel tracers were identified that have not been previously considered for ¹³C-MFA of mammalian cells, specifically [2,3,4,5,6-¹³C]glucose for elucidating oxPPP flux and [3,4-¹³C]glucose for elucidating PC flux. We demonstrate that ¹³C-glutamine tracers perform poorly in this system in comparison to the optimal glucose tracers.ConclusionsIn this work, we have demonstrated that optimal tracer design does not need to be a pure simulation-based trial-and-error process; rather, rational insights into tracer design can be gained through the application of the EMU basis vector methodology. Using this approach, rational labeling rules can be established a priori to guide the selection of optimal ¹³C-tracers for high-resolution flux elucidation in complex metabolic network models.

Dataset Information

Rational design and evaluation of mammalian ribonuclease cytotoxins.

Publications

Rational design and evaluation of mammalian ribonuclease cytotoxins.

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