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Montioli R.,University of VeronaVerona | Oppici E.,University of VeronaVerona | Dindo M.,University of VeronaVerona | Roncador A.,University of VeronaVerona | And 4 more authors.
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2015

Abstract Liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5′-phosphate (PLP) enzyme, exists as two polymorphic forms, the major (AGT-Ma) and the minor (AGT-Mi) haplotype. Deficit of AGT causes Primary Hyperoxaluria Type 1 (PH1), an autosomal recessive rare disease. Although ∼ one-third of the 79 disease-causing missense mutations segregates on AGT-Mi, only few of them are well characterized. Here for the first time the molecular and cellular defects of G47R-Mi are reported. When expressed in Escherichia coli, the recombinant purified G47R-Mi variant exhibits only a 2.5-fold reduction of its kcat, and its apo form displays a remarkably decreased PLP binding affinity, increased dimer-monomer equilibrium dissociation constant value, susceptibility to thermal denaturation and to N-terminal region proteolytic cleavage, and aggregation propensity. When stably expressed in a mammalian cell line, we found ∼ 95% of the intact form of the variant in the insoluble fraction, and proteolyzed (within the N-terminal region) and aggregated forms both in the soluble and insoluble fractions. Moreover, the intact and nicked forms have a peroxisomal and a mitochondrial localization, respectively. Unlike what already seen for G41R-Mi, exposure of G47R-Mi expressing cells to pyridoxine (PN) remarkably increases the expression level and the specific activity in a dose-dependent manner, reroutes all the protein to peroxisomes, and rescues its functionality. Although the mechanism of the different effect of PN on the variants G47R-Mi and G41R-Mi remains elusive, the chaperoning activity of PN may be of value in the therapy of patients bearing the G47R mutation. © 2015 The Authors. Source


Zucal C.,University of TrentoTrento | D'Agostino V.,University of TrentoTrento | Loffredo R.,University of TrentoTrento | Mantelli B.,University of TrentoTrento | And 5 more authors.
Current Drug Targets | Year: 2015

The RNA-binding protein (RBP) HuR is one of the most widely studied regulators of the eukaryotic posttranscriptional gene expression and it plays a physiological role in mediating the cellular response to apoptotic, proliferating and survival stimuli. Following physiological or stress stimuli, HuR protein binds to Adenylate-Urydinilate rich elements (AREs) generally contained in the 3’UTR of transcripts, then it shuttles from the nucleus to the cytoplasm and regulates the half-life and/or translation of cargo mRNAs. Derangements in sub-cellular localization and expression of HuR have been associated with the pathophysiology of many diseases and this protein has been proposed as a potential drug target. Recent findings also re-evaluated HuR as a splicing and polyadenylation factor, expanding its spectrum of functional activity up to the maturation of pre-mRNAs. In this review, we generate a comprehensive picture of HuR functionality to discuss the implications of considering HuR as pharmacological target and the detrimental or positive impact that can be expected upon its modulation. Firstly, we focus on the recent findings about the mechanistic role of HuR in the nucleus and in the regulation of long non coding RNAs; then we describe the animal models and the clinical association and significance in cancer; finally, we have reviewed the pharmacological tools that influence HuR’s post-transcriptional control and the efforts made to identify specific HuR inhibitors. © 2015 Bentham Science Publishers Source

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