Design and Characterization of an HIV-Specific Ribonuclease Zymogen

Turcotte, Rebecca F.; Raines, Ronald T.

doi:10.1089/aid.2008.0146

Cited by 25 publications

(30 citation statements)

References 52 publications

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“…For example, the circular Str2 zymogen of RNase 1 has T m = 48°C and gains 11,000‐fold in catalytic efficiency upon activation with HIV‐1 protease (). In contrast, the circular permutation of RNase A led to a zymogen with T m = 46°C and only a 48‐fold gain in catalytic efficiency . (Here, T m refers to the temperature at the midpoint of the thermal transition from the native state to denatured states.…”

Section: Discussionmentioning

confidence: 99%

“…The first zymogen to be created de novo was a circularly permuted variant of ribonuclease (RNase) A in which the native N‐ and C‐termini were connected with a linker containing the sequence recognized by the plasmepsin II protease . Iterations of this strategy produced RNase A zymogens activated by the NS3 protease and HIV‐1 protease . In these RNase A zymogens, the linker occludes the active site, leading to the suppression of enzymatic activity by up to 10 3 ‐fold, depending on the design parameters and nature of the activating protease.…”

Section: Introductionmentioning

confidence: 99%

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Circular zymogens of human ribonuclease 1

2019

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The endogenous production of enzymes as zymogens provides a means to control catalytic activities. Here, we describe the heterologous production of ribonuclease 1 (RNase 1), which is the most prevalent secretory ribonuclease in humans, as a zymogen. In folded RNase 1, the N and C termini flank the enzymic active site. By using intein‐mediated cis‐splicing, we created circular proteins in which access to the active site of RNase 1 is obstructed by an amino‐acid sequence that is recognized by the HIV‐1 protease. Installing a sequence that does not perturb the RNase 1 fold led to only modest inactivation. In contrast, the ancillary truncation of residues from each terminus led to a substantial decrease in the catalytic activity of the zymogen with the maintenance of thermostability. For optimized zymogens, activation by HIV‐1 protease led to a > 104‐fold increase in ribonucleolytic activity at a rate comparable to that for the cleavage of endogenous viral substrates. Molecular modeling indicated that these zymogens are inactivated by conformational distortion in addition to substrate occlusion. Because protease levels are elevated in many disease states and ribonucleolytic activity can be cytotoxic, RNase 1 zymogens have potential as generalizable prodrugs.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Circular zymogens of human ribonuclease 1

2019

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“…The potential therapeutic value of ptRNases has been extended beyond cancer with the creation of zymogens that can be engineered to be disease-specific. To date, protease-activatable ptRNase zymogens have been developed to combat malaria, hepatitis C, and HIV (Johnson et al , 2006; Plainkum et al , 2003; Turcotte and Raines, 2008). Another member of the ptRNase family, angiogenin, has been designed as a hyperactive variant capable of enhanced neovascularization (Dickson et al , 2009).…”

Section: Prospectusmentioning

confidence: 99%

Rational Design and Evaluation of Mammalian Ribonuclease Cytotoxins

Lomax

Eller

Raines

2012

Methods in Enzymology

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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.

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“…This mechanism is based on the control of enzyme activation rather than inhibition [1]. Inspired by protease zymogens, Raines and co-workers extended this strategy to bovine pancreatic ribonuclease (RNase A) by connecting the amino and carboxyl termini with a peptide segment that caps the active site and thus acts like a pro-segment of a natural zymogen and inhibits the native ribonucleolytic activity [2], [3], [4]. Upon cleavage of this linker by a specific protease, near-wild type activity of RNase A is reconstituted.…”

Section: Introductionmentioning

confidence: 99%

Towards Tricking a Pathogen’s Protease into Fighting Infection: The 3D Structure of a Stable Circularly Permuted Onconase Variant Cleavedby HIV-1 Protease

et al. 2013

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Onconase® is a highly cytotoxic amphibian homolog of Ribonuclease A. Here, we describe the construction of circularly permuted Onconase® variants by connecting the N- and C-termini of this enzyme with amino acid residues that are recognized and cleaved by the human immunodeficiency virus protease. Uncleaved circularly permuted Onconase® variants are unusually stable, non-cytotoxic and can internalize in human T-lymphocyte Jurkat cells. The structure, stability and dynamics of an intact and a cleaved circularly permuted Onconase® variant were determined by Nuclear Magnetic Resonance spectroscopy and provide valuable insight into the changes in catalytic efficiency caused by the cleavage. The understanding of the structural environment and the dynamics of the activation process represents a first step toward the development of more effective drugs for the treatment of diseases related to pathogens expressing a specific protease. By taking advantage of the protease’s activity to initiate a cytotoxic cascade, this approach is thought to be less susceptible to known resistance mechanisms.

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Design and Characterization of an HIV-Specific Ribonuclease Zymogen

Cited by 25 publications

References 52 publications

Circular zymogens of human ribonuclease 1

Circular zymogens of human ribonuclease 1

Rational Design and Evaluation of Mammalian Ribonuclease Cytotoxins

Towards Tricking a Pathogen’s Protease into Fighting Infection: The 3D Structure of a Stable Circularly Permuted Onconase Variant Cleavedby HIV-1 Protease

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