Secretory ribonucleases are internalized by a dynamin-independent endocytic pathway

Haigis, Marcia C.; Raines, Ronald T.

doi:10.1242/jcs.00214

Cited by 88 publications

(136 citation statements)

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“…RNase A is a cationic protein that is internalized by mammalian cells via endocytosis (49). This internalization is critical to the action of cytotoxic RNase A variants and homologs (50).…”

Section: Bioconjugationmentioning

confidence: 99%

Fluorogenic Label for Biomolecular Imaging

Lavis¹,

2006

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Traditional small-molecule fluorophores are always fluorescent. That attribute can obscure valuable information in biological experiments. Here, we report on a versatile "latent" fluorophore that overcomes this limitation. At the core of the latent fluorophore is a derivative of rhodamine in which one nitrogen is modified as a urea. That modification enables rhodamine to retain half of its fluorescence while facilitating conjugation to a target molecule. The other nitrogen of rhodamine is modified with a "trimethyl lock", which enables fluorescence to be unmasked fully by a single userdesignated chemical reaction. An esterase-reactive latent fluorophore was synthesized in high yield and attached covalently to a cationic protein. The resulting conjugate was not fluorescent in the absence of esterases. The enzymatic activity of esterases in endocytic vesicles and the cytosol educed fluorescence, enabling the time-lapse imaging of endocytosis into live human cells and thus providing unprecedented spatiotemporal resolution of this process. The modular design of this "fluorogenic label" enables the facile synthesis of an ensemble of small-molecule probes for the illumination of numerous biochemical and cell biological processes.

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“…RNase A is a cationic protein that is internalized by mammalian cells via endocytosis (49). This internalization is critical to the action of cytotoxic RNase A variants and homologs (50).…”

Section: Bioconjugationmentioning

confidence: 99%

Fluorogenic Label for Biomolecular Imaging

Lavis¹,

2006

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“…By virtue of their basic pI values, RNases of the RNase A superfamily bind to the negatively charged surface of cells (23). As cells should be anxious to prevent foreign RNases from entering the cell, no RNase receptor is expected to be found but RNases enter cells by endocytosis (23).…”

Section: Contradictions In the Reason For The Cytotoxicity Of Rnases-mentioning

confidence: 99%

“…As cells should be anxious to prevent foreign RNases from entering the cell, no RNase receptor is expected to be found but RNases enter cells by endocytosis (23). To exert their cytotoxicity, the RNases have to be released from the endosomes into the cytosol.…”

Section: Contradictions In the Reason For The Cytotoxicity Of Rnases-mentioning

confidence: 99%

“…However, as elaborated above, other studies have pointed to a limitation of the cytotoxicity by the efficiency of internalization (11,15,40,45). Interestingly, for the cytotoxic variants G88R-RNase A and Onconase an enhanced binding to the cell surface can be seen (23); an improved interaction with the cell surface as reason for the cytotoxicity has also been proposed for domain-swapped RNase A multimers (25) and RNase A tandem enzymes (22). Our quantitative results unambiguously show that the cytotoxic variant G88R-RNase A accumulates in the cytosol (Fig.…”

Section: Subcellular Quantification Of Rnase a Variants Suggests Endomentioning

confidence: 99%

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Endocytotic Internalization as a Crucial Factor for the Cytotoxicity of Ribonucleases

Leich

Stöhr

Rietz

et al. 2007

Journal of Biological Chemistry

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The cytotoxic action of ribonucleases (RNases) requires the interaction of the enzyme with the cellular membrane, its internalization, translocation to the cytosol, and the degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the evasion from the intracellular ribonuclease inhibitor (RI) has not yet been fully elucidated. As cytosolic internalization is indispensable for the cytotoxicity of extracellular ribonucleases, we investigated the extent of cytosolic internalization of a cytotoxic, RI-evasive RNase A variant (G88R-RNase A) and of various similarly cytotoxic but RI-sensitive RNase A tandem enzyme variants in comparison to the internalization of the non-cytotoxic and RI-sensitive RNase A. After incubation of K-562 cells with the RNase A variants for 36 h, the internalized amount of RNases was analyzed by rapid cell disruption followed by subcellular fractionation and semiquantitative immunoblotting. The data indicate that an enhanced cellular uptake and an increased entry of the RNases into the cytosol can outweigh the abolishment of catalytic activity by RI. As all RNase A variants proved to be resistant to the proteases present in the different subcellular fractions for more than 100 h, our results suggest that the cytotoxic potency of RNases is determined by an efficient internalization into the cytosol.The ribonucleolytic activity of ribonucleases (RNases) 2 provides the potential to use these enzymes as therapeutics for tumor treatment. Particularly, members of the RNase A and RNase T1 superfamilies have shown promising cytotoxicity to cancer cells (1-3). Among these enzymes, Onconase TM (Alfacell Corp., Bloomfield, NJ), an RNase from the Northern Leopard frog, is farthest along the clinical trials (4). To overcome the disadvantage of the renal toxicity of Onconase and a possible immunogenicity of non-mammalian RNases, RNase A or human pancreatic RNase 1 evolved as targets for the development of antitumor agents (5). Because RNases from mammalian sources are silenced due to the tight binding by the intracellular inhibitor protein (RI) and, thus, are not cytotoxic (6), various strategies have been mapped out to accomplish cytotoxicity. Inspired by the cytotoxicity of the amphibian Onconase (5,7,8) and the dimeric bovine seminal RNase (9), tremendous efforts have been made to create RNase derivatives that evade RI binding. While the generation of chimeras (10 -13), the use of chemical modifications (14 -17), or site-directed mutagenesis (5, 6, 18 -21) proved to be strategies of rather varying success, we developed RNase A tandem enzymes (22) in which two RNase A molecules are coupled covalently by a peptide linker. Due to steric hindrance, a complete binding by RI should be prevented and in contrast to bovine seminal RNase, the RNase A entities of the tandem constructs cannot dissociate, which would result in a subsequent binding by RI. In fact, the RNase A tandem constructs have been found to...

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“…However, the selectivity of the action of RNAses on cells cannot be mediated only by their electrostatic properties 14 . In the work 15 , it was shown that the internalization of certain cytotoxic RNAses occurs via clathrin-and dynaminindependent endocytosis, after which they were translocated through the membrane of endosomes and enter to the cytosol, where the realization of their cytotoxicity is occurred. In the process of translocation through the membrane, the protein is forced to interact with a hydrophobic region of a lipid bilayer.…”

mentioning

confidence: 99%

Surface-Dependent Differences in the Adsorption of Pancreatic and Microbial Ribonucleases Visualized by Atomic Force Microscopy

Коновалова¹,

Kalacheva²,

Черепнев³

et al. 2016

Biosci., Biotech. Res. Asia

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A comparative study of the physical adsorption of RNAse A and RNAse Bacillus pumilis onto a negatively charged surface of mica, a hydrophobic surface of pyrolytic graphite and a surface of lipid layers of dipalmitoylphosphatidylcholine (DPPC) was performed by atomic force microscopy. It was found that microbial RNAse, unlike RNAse A, 1) is adsorbed onto the negatively charged surface of mica in the form of monomers and dimers; 2) exhibits enhanced tropism to the hydrophobic surface of pyrolytic graphite; 3) modifies morphotopography and thickness of the lipid bilayer of DPPC. The detected surface-dependent differences in adsorption of RNAses are consistent with the features of their structure and cytotoxic properties.

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Secretory ribonucleases are internalized by a dynamin-independent endocytic pathway

Cited by 88 publications

References 72 publications

Fluorogenic Label for Biomolecular Imaging

Fluorogenic Label for Biomolecular Imaging

Endocytotic Internalization as a Crucial Factor for the Cytotoxicity of Ribonucleases

Surface-Dependent Differences in the Adsorption of Pancreatic and Microbial Ribonucleases Visualized by Atomic Force Microscopy

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