Arginine Residues Are More Effective than Lysine Residues in Eliciting the Cellular Uptake of Onconase

Sundlass, Nadia K.; Raines, Ronald T.

doi:10.1021/bi200979k

Cited by 23 publications

(25 citation statements)

References 46 publications

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“…Electrostatic interactions between basic amino acids in CPPs and negatively charged molecules at the surface of cell membranes have been recognized as important for the uptake of positively charged CPPs. Furthermore, arginine residues seem to favor the internalization of peptides compared to lysine residues . CTX has three lysine (K15, 23, and 27) and three arginine residues (R14, 25, and 36) in its sequence.…”

Section: Resultsmentioning

confidence: 99%

“…In this study, CTX and two analogs (CTX_M1 and CTX_M2) were synthesized and used to examine the effect of increasing the number of arginine and tryptophan residues on cellular uptake. These mutations, although conservative in nature, have been shown to affect, and in some cases, substantially improve cellular uptake of peptides . For example, we recently showed that mutation of both K9 and K10 to arginine residues resulted in a 2‐fold enhancement in cellular uptake of MCoTI‐II .…”

Section: Introductionmentioning

confidence: 99%

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Lysine to arginine mutagenesis of chlorotoxin enhances its cellular uptake

et al. 2017

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Chlorotoxin (CTX), a disulfide-rich peptide from the scorpion Leiurus quinquestriatus, has several promising biopharmaceutical properties, including preferential affinity for certain cancer cells, high serum stability, and cell penetration. These properties underpin its potential for use as a drug design scaffold, especially for the treatment of cancer; indeed, several analogs of CTX have reached clinical trials. Here, we focus on its ability to internalize into cells-a trait associated with a privileged subclass of peptides called cell-penetrating peptides-and whether it can be improved through conservative substitutions. Mutants of CTX were made using solid-phase peptide synthesis and internalization into human cervical carcinoma (HeLa) cells was monitored by fluorescence and confocal microscopy. CTX_M1 (ie, [K15R/K23R]CTX) and CTX_M2 (ie, [K15R/K23R/Y29W]CTX) mutants showed at least a twofold improvement in uptake compared to CTX. We further showed that these mutants internalize into HeLa cells largely via an energy-dependent mechanism. Importantly, the mutants have high stability, remaining intact in serum for over 24 h; thus, retaining the characteristic stability of their parent peptide. Overall, we have shown that simple conservative substitutions can enhance the cellular uptake of CTX, suggesting that such type of mutations might be useful for improving uptake of other peptide toxins.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Lysine to arginine mutagenesis of chlorotoxin enhances its cellular uptake

et al. 2017

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“…Although both Arg and Lys in CPP sequences play principal roles in cellular uptake, the two residues contribute differently (Wender et al 2008; Rothbard et al 2005; Futaki et al 2001; Wender et al 2000; Sundlass and Raines 2011; Takechi et al 2011). Polyarginine (Arg) n , (n=7 or 9), a synthetic CPP with n-consecutive Arg, was reported to enter cells more efficiently than polylysine (Lys) n (Mitchell et al 2000).…”

Section: Peptide-lipid and Peptide-water Interactionsmentioning

confidence: 99%

Cationic membrane peptides: atomic-level insight of structure–activity relationships from solid-state NMR

Hong

2012

Amino Acids

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Many membrane-active peptides, such as cationic cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs), conduct their biological functions by interacting with the cell membrane. The interactions of charged residues with lipids and water facilitate membrane insertion, translocation or disruption of these highly hydrophobic species. In this mini-review we will summarize high-resolution structural and dynamic findings towards the understanding of the structure-activity relationship of lipid membrane-bound CPPs and AMPs, as examples of the current development of solid-state NMR (SSNMR) techniques for studying membrane peptides. We will present the most recent atomic-resolution structure of the guanidinium-phosphate complex, as constrained from experimentally measured site-specific distances. These SSNMR results will be valuable specifically for understanding the intracellular translocation pathway of CPPs and antimicrobial mechanism of AMPs, and more generally broaden our insight into how cationic macromolecules interact with and cross the lipid membrane.

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“…Except for the mutations within the hydrophobic core (around Phe28 and Phe36) and the deletion of disulfide bonds, the effect of the amino acid substitutions on the thermal and thermodynamic stability is rather small. The triple mutant R73A/K76A/K78A–ONC is destabilized by only 10 °C and even more impressively, the replacement of 10 lysine residues with arginine results in a loss of only 9 kJ·mol −1 . Moreover, two circularly permuted ONC variants were found to possess high thermal stability ( T m = 81 °C at pH 6.0) .…”

Section: Stability and Folding Of Amphibian Rnase A Homologuesmentioning

confidence: 99%

Stability and folding of amphibian ribonuclease A superfamily members in comparison with mammalian homologues

Arnold

2014

The FEBS Journal

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Comparative studies on homologous proteins can provide knowledge on how limited changes in the primary structure find their expression in large effects on catalytic activity, stability or the folding behavior. For more than half a century, members of the ribonuclease A superfamily have been the subject of a myriad of studies on protein folding and stability. Both the unfolding and refolding kinetics as well as the structure of several folding intermediates of ribonuclease A have been characterized in detail. Moreover, the RNA-degrading activity of these enzymes provides a basis for their cytotoxicity, which renders them potential tumor therapeutics. Because amphibian ribonuclease A homologues evade the human ribonuclease inhibitor, they emerged as particularly promising candidates. Interestingly, the amphibian ribonuclease A homologues investigated to date are more stable than the mammalian homologues. Nevertheless, despite the generation of numerous genetically engineered variants, knowledge of the folding of amphibian ribonuclease A homologues remains rather limited. An exception is onconase, a ribonuclease A homologue from Rana pipiens, which has been characterized in detail. This review summarizes the data on the unfolding and refolding kinetics and pathways, as well on the stability of amphibian ribonuclease A homologues compared with those of ribonuclease A, the best known member of this superfamily.

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Arginine Residues Are More Effective than Lysine Residues in Eliciting the Cellular Uptake of Onconase

Cited by 23 publications

References 46 publications

Lysine to arginine mutagenesis of chlorotoxin enhances its cellular uptake

Lysine to arginine mutagenesis of chlorotoxin enhances its cellular uptake

Cationic membrane peptides: atomic-level insight of structure–activity relationships from solid-state NMR

Stability and folding of amphibian ribonuclease A superfamily members in comparison with mammalian homologues

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