Exploring the Mitochondrial Degradome by the TAILS Proteomics Approach in a Cellular Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most frequent neurodegenerative disease worldwide and the availability of early biomarkers and novel biotargets represents an urgent medical need. The main pathogenetic hallmark of PD is the specific loss of nigral dopaminergic neurons, in which mitochondrial dysfunction plays a crucial role. Mitochondrial proteases are central to the maintenance of healthy mitochondria and they have recently emerged as drug targets. However, an exhaustive characterization of these enzymes and their targets is still lacking, due to difficulties in analyzing proteolytic fragments by bottom-up proteomics approaches. Here, we propose the "mitochondrial dimethylation-TAILS" strategy, which combines the isolation of mitochondria with the enrichment of N-terminal peptides to analyze the mitochondrial N-terminome. We applied this method in a cellular model of altered dopamine homeostasis in neuroblastoma SH-SY5Y cells, which recapitulates early steps of PD pathogenesis. The main aim was to identify candidate mitochondrial proteases aberrantly activated by dopamine dysregulation and their cleaved targets. The proposed degradomics workflow was able to improve the identification of mitochondrial proteins if compared to classical shotgun analysis. In detail, 40% coverage of the mitochondrial proteome was obtained, the sequences of the transit peptides of two mitochondrial proteins were unveiled, and a consensus cleavage sequence for proteases involved in the processing of mitochondrial proteins was depicted. Mass spectrometry proteomics data have been submitted to ProteomeXchange with the identifier PXD013900. Moreover, sixty-one N-terminal peptides whose levels were affected by dopamine treatment were identified. By an in-depth analysis of the proteolytic peptides included in this list, eleven mitochondrial proteins showed altered proteolytic processing. One of these proteins (i.e., the 39S ribosomal protein L49 - MRPL49) was cleaved by the neprilysin protease, already exploited in clinics as a biotarget. We eventually demonstrated a mitochondrial subcellular localization of neprilysin in human cells for the first time. Collectively, these results shed new light on mitochondrial dysfunction linked to dopamine imbalance in PD and opened up the possibility to explore the mitochondrial targets of neprilysin as candidate biomarkers.

Mitochondrial alterations in Parkinson's disease human samples and cellular models.

Mitochondrial impairment is one of the most important hallmarks of Parkinson's disease (PD) pathogenesis. In this work, we wanted to verify the molecular basis of altered mitochondrial dynamics and disposal in Substantia nigra specimens of sporadic PD patients, by the comparison with two cellular models of PD. Indeed, SH-SY5Y cells were treated with either dopamine or 1-methyl-4-phenylpyridinium (MPP+) in order to highlight the effect of altered dopamine homeostasis and of complex I inhibition, respectively. As a result, we found that fusion impairment of the inner mitochondrial membrane is a common feature of both PD human samples and cellular models. However, the effects of dopamine and MPP+ treatments resulted to be different in terms of the mitochondrial damage induced. Opposite changes in the levels of two mitochondrial protein markers (voltage-dependent anion channels (VDACs) and cytochrome c oxidase subunit 5ß (COX5ß)) were observed. In this case, dopamine treatment better recapitulated the molecular picture of patients' samples. Moreover, the accumulation of PTEN-induced putative kinase 1 (PINK1), a mitophagy marker, was not observed in both PD patients samples and cellular models. Eventually, in transmission electron microscopy images, small electron dense deposits were observed in mitochondria of PD subjects, which are uniquely reproduced in dopamine-treated cells. In conclusion, our study suggests that the mitochondrial molecular landscape of Substantia nigra specimens of PD patients can be mirrored by the impaired dopamine homeostasis cellular model, thus supporting the hypothesis that alterations in this process could be a crucial pathogenetic event in PD.

RNASET2 silencing affects miRNAs and target gene expression pattern in a human ovarian cancer cell model.

Ribonucleases (RNases) are hydrolytic enzymes endowed with the ability to either process or degrade ribonucleic acids. Among the many biological functions assigned to RNases, a growing attention has been recently devoted to the control of cancer growth, in the attempt to bring novel therapeutic approaches to clinical oncology. Indeed, several enzymes belonging to different ribonuclease families have been reported in the last decade to display a marked oncosuppressive activity in a wide range of experimental models. The human RNASET2 gene, the only member of the highly conserved T2/Rh/S family of endoribonucleolytic enzymes described in our species, has been shown to display oncosuppressive roles in both in vitro and in vivo models representing several human malignancies. In the present study, we extend previous findings obtained in ovarian cancer models to shed further light on the cell-autonomous roles played by this gene in the context of its oncosuppresive role and to show that RNASET2 silencing can significantly affect the transcriptional output in one of the most thoroughly investigated human ovarian cancer cell lines. Moreover, we report for the first time that RNASET2-mediated changes in the cell transcriptome are in part mediated by its apparent ability to affect the cell's microRNA expression pattern.

Pleiotropic modes of action in tumor cells of RNASET2, an evolutionary highly conserved extracellular RNase.

As widely recognized, tumor growth entails a close and complex cross-talk among cancer cells and the surrounding tumor microenvironment. We recently described the human RNASET2 gene as one key player of such microenvironmental cross-talk. Indeed, the protein encoded by this gene is an extracellular RNase which is able to control cancer growth in a non-cell autonomous mode by inducing a sustained recruitment of immune-competent cells belonging to the monocyte/macrophage lineage within a growing tumor mass. Here, we asked whether this oncosuppressor gene is sensitive to stress challenges and whether it can trigger cell-intrinsic processes as well. Indeed, RNASET2 expression levels were consistently found to increase following stress induction. Moreover, changes in RNASET2 expression levels turned out to affect several cancer-related parameters in vitro in an ovarian cancer cell line model. Of note, a remarkable rearrangement of the actin cytoskeleton organization, together with changes in cell adhesion and motility, emerged as putative mechanisms by which such cell-autonomous role could occur. Altogether, these biological features allow to put forward the hypothesis that the RNASET2 protein can act as a molecular barrier for limiting the damages and tissue remodeling events occurring during the earlier step of cell transformation.

New Strategies for Expression and Purification of Recombinant Human RNASET2 Protein in Pichia pastoris.

Ribonucleases form a large family of enzymes involved in RNA metabolism and are endowed with a broad range of biological functions. Among the different RNase proteins described in the last decades, those belonging to the Rh/T2/S subfamily show the highest degree of evolutionary conservation, suggesting the occurrence of a key critical ancestral role for this protein family. We have recently defined the human RNASET2 gene as a novel member of a group of oncosuppressors called "tumor antagonizing genes," whose activity in the control of cancer growth is carried out mainly in vivo. However, to better define the molecular pathways underlying the oncosuppressive properties of this protein, further structural and functional investigations are necessary, and availability of high-quality recombinant RNASET2 is of paramount importance. Here, we describe a multi-step strategy that allows production of highly pure, catalytically competent recombinant RNASET2 in both wild-type and mutant forms. The recombinant proteins that were produced with our purification strategy will be instrumental to perform a wide range of functional assays aimed at dissecting the molecular mechanisms of RNASET2-mediated tumor suppression.

Loss of function of Ribonuclease T2, an ancient and phylogenetically conserved RNase, plays a crucial role in ovarian tumorigenesis.

In recent years, the role played by the stromal microenvironment has been given growing attention in order to achieve a full understanding of cancer initiation and progression. Because cancer is a tissue-based disease, the integrity of tissue architecture is a major constraint toward cancer growth. Indeed, a large contribution of the natural resistance to cancer stems from stromal microenvironment components, the dysregulation of which can facilitate cancer occurrence. For instance, recent experimental evidence has highlighted the involvement of stromal cells in ovarian carcinogenesis, as epitomized by ovarian xenografts obtained by a double KO of the murine Dicer and Pten genes. Likewise, we reported the role of an ancient extracellular RNase, called Ribonuclease T2 (RNASET2), within the ovarian stromal microenvironment. Indeed, hyperexpression of RNASET2 is able to control tumorigenesis by recruiting macrophages (mostly of the anticancer M1 subtype) at the tumor sites. We present biological data obtained by RNASET2 silencing in the poorly tumorigenetic and highly RNASET2-expressing human OVCAR3 cell line. RNASET2 knockdown was shown to stimulate in vivo tumor growth early after microinjection of OVCAR3 cells in nude mice. Moreover, we have investigated by molecular profiling the in vivo expression signature of human and mouse cell xenografts and disclosed the activation of pathways related to activation of the innate immune response and modulation of ECM components. Finally, we provide evidence for a role of RNASET2 in triggering an in vitro chemotactic response in macrophages. These results further highlight the critical role played by the microenvironment in RNASET2-mediated ovarian tumor suppression, which could eventually contribute to better clarify the pathogenesis of this disease.

Molecular signature induced by RNASET2, a tumor antagonizing gene, in ovarian cancer cells.

Using the Hey3Met2 human ovarian cancer cell line, we previously found the RNASET2 gene to possess a remarkable in vivo tumor suppressor activity, although no in vitro features such as inhibition of cell proliferation, clonogenic potential, impaired growth in soft agar and increase in apoptotic rate could be detected. This is reminiscent of the behavior of genes belonging to the class of tumor antagonizing genes (TAG) which act mainly within the context of the microenvironment. Here we present transcriptional profiles analysis which indicates that investigations of the mechanisms of TAG biological functions require a comparison between the in vitro and in vivo expression patterns. Indeed several genes displaying a biological function potentially related to tumor suppression could not be validated by subsequent in vivo expression analysis. On the other hand the fact that we could find congruency for three genes both in vivo and in vitro adds a warning to a too much stringent categorization of this class of genes which relies on the sensitivity of the methodological approaches.

Microenvironmental control of malignancy exerted by RNASET2, a widely conserved extracellular RNase.

A recent body of evidence indicates an active role for stromal (mis)-regulation in the progression of neoplasias. Within this conceptual framework, genes belonging to the growing but still poorly characterized class of tumor antagonizing/malignancy suppressor genes (TAG/MSG) seem to play a crucial role in the regulation of the cross-talk between stromal and epithelial cells by controlling malignant growth in vivo without affecting any cancer-related phenotype in vitro. Here, we have functionally characterized the human RNASET2 gene, which encodes the first human member of the widespread Rh/T2/S family of extracellular RNases and was recently found to be down-regulated at the transcript level in several primary ovarian tumors or cell lines and in melanoma cell lines. Although we could not detect any activity for RNASET2 in several functional in vitro assays, a remarkable control of ovarian tumorigenesis could be detected in vivo. Moreover, the control of ovarian tumorigenesis mediated by this unique tumor suppressor gene occurs through modification of the cellular microenvironment and the induction of immunocompetent cells of the monocyte/macrophage lineage. Taken together, the data presented in this work strongly indicate RNASET2 as a previously unexplored member of the growing family of tumor-antagonizing genes.