Unraveling the Mechanistic Complexity of the Glomerulocystic Phenotype in <i>Dicer</i> Conditional KO Mice by 2D Gel Electrophoresis Coupled Mass Spectrometry

Fiumara, Claudia Vincenza; Scumaci, Domenica; Iervolino, Anna; Perri, Angela Mena; Concolino, Antonio; Tammè, Laura; Petrillo, Federica; Capasso, Giovambattista; Cuda, Giovanni

doi:10.1002/prca.201700006

Cited by 3 publications

(3 citation statements)

References 37 publications

(47 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Procedures were performed according to GE Healthcare Ettan protocol book [ 16 , 17 ]. The second dimension was carried out on 10% SDS polyacrylamide gels, until the bromophenol blue reached the bottom of the gels [ 18 ]. The gels were fixed and stained using silver staining method, which is compatible with mass spectrometry analysis [ 19 ].…”

Section: Methodsmentioning

confidence: 99%

DJ-1 Proteoforms in Breast Cancer Cells: The Escape of Metabolic Epigenetic Misregulation

Scumaci

Olivo

Fiumara

et al. 2020

Cells

Self Cite

View full text Add to dashboard Cite

Enhanced glycolysis is a hallmark of breast cancer. In cancer cells, the high glycolytic flux induces carbonyl stress, a damaging condition in which the increase of reactive carbonyl species makes DNA, proteins, and lipids more susceptible to glycation. Together with glucose, methylglyoxal (MGO), a byproduct of glycolysis, is considered the main glycating agent. MGO is highly diffusible, enters the nucleus, and can react with easily accessible lysine- and arginine-rich tails of histones. Glycation adducts on histones undergo oxidization and further rearrange to form stable species known as advanced glycation end-products (AGEs). This modification alters nucleosomes stability and chromatin architecture deconstructing the histone code. Formation of AGEs has been associated with cancer, diabetes, and several age-related diseases. Recently, DJ-1, a cancer-associated protein that protects cells from oxidative stress, has been described as a deglycase enzyme. Although its role in cell survival results still controversial, in several human tumors, its expression, localization, oxidation, and phosphorylation were found altered. This work aimed to explore the molecular mechanism that triggers the peculiar cellular compartmentalization and the specific post-translational modifications (PTM) that, occurring in breast cancer cells, influences the DJ-1 dual role. Using a proteomic approach, we identified on DJ-1 a novel threonine phosphorylation (T125) that was found, by the in-silico tool scansite 4, as part of a putative Akt consensus. Notably, this threonine is in addition to histidine 126, a key residue involved in the formation of catalytic triade (glu18-Cys106-His126) inside the glioxalase active site of DJ. Interestingly, we found that pharmacological modulation of Akt pathway induces a functional tuning of DJ-1 proteoforms, as well as their shuttle from cytosol to nucleus, pointing out that pathway as critical in the development of DJ-1 pro-tumorigenic abilities. Deglycase activity of DJ-1 on histones proteins, investigated by coupling 2D tau gel with LC-MS/MS and 2D-TAU (Triton-Acid-Urea)-Western blot, was found correlated with its phosphorylation status that, in turn, depends from Akt activation. In normal conditions, DJ-1 acts as a redox-sensitive chaperone and as an oxidative stress sensor. In cancer cells, glycolytic rewiring, inducing increased reactive oxygen species (ROS) levels, enhances AGEs products. Alongside, the moderate increase of ROS enhances Akt signaling that induces DJ-1-phosphorylation. When phosphorylated DJ-1 increases its glyoxalase activity, the level of AGEs on histones decreases. Therefore, phospho-DJ-1 prevents glycation-induced histones misregulation and its Akt-related hyperactivity represents a way to preserve the epigenome landscape sustaining proliferation of cancer cells. Together, these results shed light on an interesting mechanism that cancer cells might execute to escape the metabolic induced epigenetic misregulation that otherwise could impair their malignant proliferative potential.

show abstract

Section: Methodsmentioning

confidence: 99%

DJ-1 Proteoforms in Breast Cancer Cells: The Escape of Metabolic Epigenetic Misregulation

Scumaci

Olivo

Fiumara

et al. 2020

Cells

Self Cite

View full text Add to dashboard Cite

show abstract

“…The protein spots were analyzed in terms of volume using Image Master 2D-Platinum software, version 6.0 (GE Healthcare BioSciences) [53], [54], [55]. The protein spots showing a differential expression profile were manually excised from the silver-stained gel using OneTouch Plus Spotpicker (Gel company), destained, and in-gel digested as previously reported [56], [57], [58]. The resulting tryptic peptides were purified using Pierce C18 Spin Columns (Thermo Fisher Scientific Inc.) according to the manufacturer's recommended procedure, eluted with 40 μl of 70% acetonitrile, and dehydrated in a vacuum evaporator 23 …”

Section: Methodsmentioning

confidence: 99%

In Preclinical Model of Ovarian Cancer, the SGK1 Inhibitor SI113 Counteracts the Development of Paclitaxel Resistance and Restores Drug Sensitivity

D’Antona

Dattilo

Catalogna

et al. 2019

Translational Oncology

Self Cite

View full text Add to dashboard Cite

Ovarian cancer is the second most common gynecological malignancy worldwide. Paclitaxel is particularly important in the therapy of ovarian carcinomas, but the treatment efficacy is counteracted by the development of resistance to chemotherapy. The identification of target molecules that can prevent or control the development of chemoresistance might provide important tools for the management of patients affected by ovarian cancer. Serum- and glucocorticoid-regulated kinase 1 (SGK1) appears to be a key determinant of resistance to chemo- and radiotherapy. Specifically, SGK1 affects paclitaxel sensitivity in RKO colon carcinoma cells by modulating the specificity protein 1 (SP1)–dependent expression of Ran-specific GTPase-activating protein (RANBP1), a member of the GTP-binding nuclear protein Ran (RAN) network that is required for the organization and function of the mitotic spindle. SGK1 inhibition might thus be useful for counteracting the development of paclitaxel resistance. Here, we present in vitro data obtained using ovarian carcinoma cell lines that indicate that the SGK1 inhibitor SI113 inhibits cancer cell proliferation, potentiates the effects of paclitaxel-based chemotherapy, counteracts the development of paclitaxel resistance, and restores paclitaxel sensitivity in paclitaxel-resistant A2780 ovarian cancer cells. The results were corroborated by preclinical studies of xenografts generated in nude mice through the implantation of paclitaxel-resistant human ovarian cancer cells. The SGK1 inhibitor SI113 synergizes with paclitaxel in the treatment of xenografted ovarian cancer cells. Taken together, these data suggest that SGK1 inhibition should be investigated in clinical trials for the treatment of paclitaxel-resistant ovarian cancer.

show abstract

“…twice a day for 5 days) during chronic ethanol intake using two-bottle choice self-administration j control and cocaine HCl (15 mg/kg i.p. once a day for 7 days) k italics for the samples analyzed by 2-DE l after recovery from peripheral Bacille Calmette-Guerin challenge [193] glomerulus S collagen alpha-3(IV) chain (Col4a3) or Alport mouse, Q9QZS0 [194] mpkCCD C11 cells f C cAMP-dependent protein kinase catalytic subunit alpha (Prkaca) and beta (Prkcb) P05132 and P68404 [195] g bladder S large-conductance, voltagedependent and Ca 2+ -dependent K + channel, or calciumactivated potassium channel subunit alpha-1 (Kcnma1) Q08460 [196] urethra S estrogen receptor beta (Esr2) O08537 [197] h…”

Section: Accepted Manuscript Sciatic Nerve In-frame Deletion Ubiquiti...mentioning

confidence: 99%

What if? Mouse proteomics after gene inactivation

Gianazza

Miller

Guerrini

et al. 2019

Journal of Proteomics

View full text Add to dashboard Cite

Unraveling the Mechanistic Complexity of the Glomerulocystic Phenotype in Dicer Conditional KO Mice by 2D Gel Electrophoresis Coupled Mass Spectrometry

Abstract: Our results contribute to gain new insights into molecular mechanisms through which Dicer endonuclease controls kidney development and physiological functions.

Cited by 3 publications

References 37 publications

DJ-1 Proteoforms in Breast Cancer Cells: The Escape of Metabolic Epigenetic Misregulation

DJ-1 Proteoforms in Breast Cancer Cells: The Escape of Metabolic Epigenetic Misregulation

In Preclinical Model of Ovarian Cancer, the SGK1 Inhibitor SI113 Counteracts the Development of Paclitaxel Resistance and Restores Drug Sensitivity

What if? Mouse proteomics after gene inactivation

Contact Info

Product

Resources

About