The nuclear transport capacity of a human-pancreatic ribonuclease variant is critical for its cytotoxicity

Abstract

We have previously described a human pancreatic-ribonuclease variant, named PE5, which carries a non-contiguous extended bipartite nuclear localization signal. This signal comprises residues from at least three regions of the protein. We postulated that the introduction of this signal in the ribonuclease provides it with cytotoxic activity because although the variant poorly evades the ribonuclease inhibitor in vitro, it is routed to the nucleus, which is devoid of the inhibitor. In this work, we have investigated the relationship between the cytotoxicity produced by PE5 and its ability to reach the nucleus. First, we show that this enzyme, when incubated with HeLa cells, specifically cleaves nuclear RNA while it leaves cytoplasmic RNA unaffected. On the other hand, we have created new variants in which the residues of the nuclear localization signal that are important for the nuclear transport have been replaced. As expected, the individual changes produce a significant decrease in the cytotoxicity of the resulting variants. We conclude that the nuclear transport of PE5 is critical for its cytotoxicity. Therefore, routing a ribonuclease to the nucleus is an alternative strategy to endow it with cytotoxic activity.

Transcriptional repression of RUNX2 is associated with aggressive clinicopathological outcomes, whereas nuclear location of the protein is related to metastasis in prostate cancer

“…RUNX1 is important for hematopoiesis and its ablation leads to leukemia, and RUNX3 is a critical regulator and its promoter hypermethylation is associated with several types of cancer. [3][4][5][6] These findings strongly suggest that RUNX1 and RUNX3 function as tumor suppressors. The RUNX2 gene, on the other hand, is a unique member of the RUNX family that was shown to have a dual function in several studies.…”

A human ribonuclease induces apoptosis associated with p21WAF1/CIP1induction and JNK inactivation

“…The different mechanisms are likely related to their mechanism of action. Onconase is driven into the cytoplasm and its natural substrates are t-RNA and most probably the non-coding RNA (microRNA) [53] whereas PE5 is located in the nucleolus [10] and targets nuclear RNA [12]. Interestingly, nucleolus and biogenesis of rRNA are important factors distinguishing cancer from normal cells and may have an active role in tumorigenesis [54].…”

“…Nuclear import assays showed that PE5 was efficiently transported to the nucleus where it was initially localized in the nucleolus. The variant carries a conformational bipartite nuclear localization signal [11] which targets the protein to the nucleus, specifically in the nucleolus [10], where RI is absent, therefore producing the degradation of nuclear RNA [12]. …”

“…Since PE5 is directed to the nucleus where it degrades nuclear RNA [12] while onconase displays its activity degrading cytosolic RNA, we sought to compare the cytotoxic properties of both RNases on this NCI/ADR-RES cell line. We also wanted to check whether this RNase could be a candidate to have its antitumor properties evaluated in vivo .…”

Rational Design and Evaluation of Mammalian Ribonuclease Cytotoxins

“…An alternative strategy to engender RI-evasion is to bypass RI contact altogether. The human ptRNase variant PE5 carries a non-contiguous nuclear localization signal and has been shown to possess potent cytotoxicity that is dependent upon its nuclear uptake (Tubert et al , 2011). …”