Protective Effects of Recombinant Human Angiogenin in Keratinocytes: New Insights on Oxidative Stress Response Mediated by RNases

Abstract

Human angiogenin (ANG) is a 14-kDa ribonuclease involved in different pathophysiological processes including tumorigenesis, neuroprotection, inflammation, innate immunity, reproduction, the regeneration of damaged tissues and stress cell response, depending on its intracellular localization. Under physiological conditions, ANG moves to the cell nucleus where it enhances rRNA transcription; conversely, recent reports indicate that under stress conditions, ANG accumulates in the cytoplasmic compartment and modulates the production of tiRNAs, a novel class of small RNAs that contribute to the translational inhibition and recruitment of stress granules (SGs). To date, there is still limited and controversial experimental evidence relating to a hypothetical role of ANG in the epidermis, the outermost layer of human skin, which is continually exposed to external stressors. The present study collects compelling evidence that endogenous ANG is able to modify its subcellular localization on HaCaT cells, depending on different cellular stresses. Furthermore, the use of recombinant ANG allowed to determine as this special enzyme is effectively able to counter at various levels the alterations of cellular homeostasis in HaCaT cells, actually opening a new vision on the possible functions that this special enzyme can support also in the stress response of human skin.

Tailoring the stress response of human skin cells by substantially limiting the nuclear localization of angiogenin

“…4 (panel B), following this ailment, R31Q/R32Q exhibited dynamic re-localization effectively gathering at the level of cytosolic aggregates attributable to SGs. This behaviour, fully in line with what concerns the unmodified protein [ 17 ], clearly suggests that the introduced mutations force the variant protein to localize in the cytosol without however affecting its recruitment on the granules during the stress response. However, the main objective of this work was to verify whether a purely cytosolic localization conferred to R31Q/R32Q a noteworthy contribution to the recovery operations that the cell implements in response to stress.…”

“…S1 - panel B, by incubating the cells for 60 min in the presence of 2 μM R31Q/R32Q, no significant alteration of cell morphology was observed as well as no appearance of SGs was detected by following the distribution of a well-known SGs marker as PABP (Poly-Adenine Binding Protein). Overall, in the experimental conditions used, both tests allowed us to assert that R31Q/R32Q did not present significant toxicity, in an entirely similar way to what has already been verified and described for the parental protein ANG and ANG WT [ 17 ].…”

“…tRNA molecules provide a reservoir of RNA species whose function extends beyond translation, indeed, as widely reported by increasingly findings, even tRNA fragments (tRFs) can affect various cellular processes [ 10 , 11 ] and are involved in several human diseases [ 12 , 13 ]. Among these tRFs, a prominent role in cellular stress response, increasingly documented, is undoubtedly covered by angiogenin-cleaved tRNA halves (tiRNAs), some of which [ 14 , 15 ] are able to directly inhibit protein synthesis by displacing the translation initiation factor eIF4G from mRNA on ribosomes [ 16 , 17 ]. These active small noncoding RNAs are also involved in the regulation of mRNA stability, interaction with cytochrome C to modulate apoptosis and the SGs assembly [ 18 ].…”

“…This emphasizes even more the meaningfulness of dynamic subcellular localization of ANG in the cell and above all how cytoplasmic localization is the essential requirement for handling altered cellular homeostasis. In this regard, it is worth noting our recent contribution to the study of the stress-induced role of ANG [ 17 ] where we highlighted the ability of human keratinocytes to intercept recombinant ANG from the culture medium and the benefits that this uptake guaranteed to the cells subjected to oxidative stress. These indications are in line with other reports that support the idea concerning the key role of ANG in aiding the cell, namely its ability to flow into the cytoplasm when cellular homeostasis is compromised and to ensure the release of tiRNAs [ 32 , 33 ].…”

“…The goal behind this strategy is to draw a solid connection between the cytoplasmic localization of ANG in its active form and the ability of the protein to contribute effectively to the cell stress response. To do this, on the wake of our previous paper [ 17 ], we chose human keratinocytes as model cell line and designed a variant of ANG in which the substitution of two arginine residues (R 31 /R 32 ) with two glutamine residues resulted in an almost total suppression of the net positive charge of the NLS. This double variant, to date never studied and named R31Q/R32Q, was produced in recombinant form and extensively studied for its rescue properties in stressed HaCaT cells in comparison with the properties previously reported for unmodified recombinant ANG.…”