Giuseppina Pitari

Pantetheinase activity of membrane‐bound Vanin‐1: lack of free cysteamine in tissues of Vanin‐1 deficient mice

Pantetheinase (EC 3.5.1.‐) is an ubiquitous enzyme which in vitro has been shown to recycle pantothenic acid (vitamin B5) and to produce cysteamine, a potent anti‐oxidant. We show that the Vanin‐1 gene encodes pantetheinase widely expressed in mouse tissues: (1) a pantetheinase activity is specifically expressed by Vanin‐1 transfectants and is immunodepleted by specific antibodies; (2) Vanin‐1 is a GPI‐anchored pantetheinase, and consequently an ectoenzyme; (3) Vanin‐1 null mice are deficient in membrane‐bound pantetheinase activity in kidney and liver; (4) in these organs, a major metabolic consequence is the absence of detectable free cysteamine; this demonstrates that membrane‐bound pantetheinase is the main source of cysteamine in tissues under physiological conditions. Since the Vanin‐1 molecule was previously shown to be involved in the control of thymus reconstitution following sublethal irradiation in vivo, this raises the possibility that Vanin/pantetheinase might be involved in the regulation of some immune functions maybe in the context of the response to oxidative stress.

Vanin-1-/- mice exhibit a glutathione-mediated tissue resistance to oxidative stress

ABSTRACT Vanin-1 is an epithelial ectoenzyme with pantetheinase activity and generating the amino-thiol cysteamine through the metabolism of pantothenic acid (vitamin B5). Here we show that Vanin-1−/− mice, which lack cysteamine in tissues, exhibit resistance to oxidative injury induced by whole-body γ-irradiation or paraquat. This protection is correlated with reduced apoptosis and inflammation and is reversed by treating mutant animals with cystamine. The better tolerance of the Vanin-1−/− mice is associated with an enhanced gamma-glutamylcysteine synthetase activity in liver, probably due to the absence of cysteamine and leading to elevated stores of glutathione (GSH), the most potent cellular antioxidant. Consequently, Vanin-1−/− mice maintain a more reducing environment in tissue after exposure to irradiation. In normal mice, we found a stress-induced biphasic expression of Vanin-1 regulated via antioxidant response elements in its promoter region. This process should finely tune the redox environment and thus change an early inflammatory process into a late tissue repair process. We propose Vanin-1 as a key molecule to regulate the GSH-dependent response to oxidative injury in tissue at the epithelial level. Therefore, Vanin/pantetheinase inhibitors could be useful for treatment of damage due to irradiation and pro-oxidant inducers.

Is pantetheinase the actual identity of mouse and human vanin-1 proteins?

Pantetheinase is an amidohydrolase involved in the dissimilative pathway of CoA, allowing the turnover of the pantothenate moiety. We have determined the N‐terminal sequence as well as the sequences of a number of tryptic and chymotryptic peptides of the protein isolated from pig kidney. These sequence stretches were used as probes to search in the SwissProt database and significant similarities were found with a GPI‐anchored protein (mouse vanin‐1, with a suggested role in lymphocyte migration), with two putative proteins encoded by human cDNAs (VNN1 and VNN2) and with human biotinidase. On the basis of sequence similarity, we propose that vanin‐1 and VNN1 should be identified as pantetheinase.

Vanin genes are clustered (human 6q22-24 and mouse 10A2B1) and encode isoforms of pantetheinase ectoenzymes.

Abstract. The mouse Vanin-1 molecule plays a role in thymic reconstitution following damage by irradiation. We recently demonstrated that it is a membrane pantetheinase (EC 3.56.1.–). This molecule is the prototypic member of a larger Vanin family encoded by at least two mouse (Vanin-1 and Vanin-3) and three human (VNN1, VNN2, VNN3) orthologous genes. We now report (1) the structural characterization of the human and mouse Vanin genes and their organization in clusters on the 6q22–24 and 10A2B1 chromosomes, respectively; (2) identification of the human VNN3 gene and the demonstration that the mouse Vanin-3 molecule is secreted by cells, and (3) that the Vanin genes encode different isoforms of the mammalian pantetheinase activity. Thus, the Vanin family represents a novel class of secreted or membrane-associated ectoenzymes. We discuss here their possible role in processes pertaining to tissue repair in the context of oxidative stress.

Physiological roles of ovotransferrin.

BACKGROUND Ovotransferrin is an iron-binding glycoprotein, found in avian egg white and in avian serum, belonging to the family of transferrin iron-binding glycoproteins. All transferrins show high sequence homology. In mammals are presents two different soluble glycoproteins with different functions: i) serum transferrin that is present in plasma and committed to iron transport and iron delivery to cells and ii) lactoferrin that is present in extracellular fluids and in specific granules of polymorphonuclear lymphocytes and committed to the so-called natural immunity. To the contrary, in birds, ovotransferrin remained the only soluble glycoprotein of the transferrin family present both in plasma and egg white. SCOPE OF REVIEW Substantial experimental evidences are summarized, illustrating the multiple physiological roles of ovotransferrin in an attempt to overcome the common belief that ovotransferrin is a protein dedicated only to iron transport and to iron withholding antibacterial activity. MAJOR CONCLUSIONS Similarly to the better known family member protein lactoferrin, ovotransferrin appears to be a multi-functional protein with a major role in avian natural immunity. GENERAL SIGNIFICANCE Biotechnological applications of ovotransferrin and ovotransferrin-related peptides could be considered in the near future, stimulating further research on this remarkable protein. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.

Immunology and Homeopathy. 5. The Rationale of the ‘Simile’

The foundation of homeopathic medicine is the ‘Similia Principle’, also known as the ‘Principle of Similarity’ or also as the ‘Simile’, which reflects the inversion of pharmacological effects in healthy subjects as compared with sick ones. This article describes the inversion of effects, a widespread medical phenomenon, through three possible mechanisms: non-linearity of dose–response relationship, different initial pathophysiological states of the organism, and pharmacodynamics of body response to the medicine. Based on the systemic networks which play an important role in response to stress, a unitary and general model is designed: homeopathic medicines could interact with sensitive (primed) regulation systems through complex information, which simulate the disorders of natural disease. Reorganization of regulation systems, through a coherent response to the medicine, could pave the way to the healing of the cellular, tissue and neuro-immuno-endocrine homeodynamics. Preliminary evidence is suggesting that even ultra-low doses and high-dilutions of drugs may incorporate structural or frequency information and interact with chaotic dynamics and physical-electromagnetic levels of regulation. From the clinical standpoint, the ‘simile’ can be regarded as a heuristic principle, according to which the detailed knowledge of pathogenic effects of drugs, associated with careful analysis of signs and symptoms of the ill subject, could assist in identifying homeopathic remedies with high grade of specificity for the individual case.

PPARalpha regulates the production of serum Vanin-1 by liver.

The membrane‐bound Vanin‐1 pantetheinase regulates tissue adaptation to stress. We investigated Vnn1 expression and its regulation in liver. Vnn1 is expressed by centrolobular hepatocytes. Using novel tools, we identify a soluble form of Vnn1 in mouse and human serum and show the contribution of a cysteine to its catalytic activity. We show that liver contributes to Vanin‐1 secretion in serum and that PPARalpha is a limiting factor in serum Vnn1 production. Functional PPRE sites are identified in the Vnn1 promoter. These results indicate that serum Vnn1 might be a reliable reporter of PPARalpha activity in liver.

Cystamine restores GSTA3 levels in Vanin-1 null mice.

Free cysteamine levels in mouse tissues have been strictly correlated to the presence of membrane-bound pantetheinase activity encoded by Vanin-1. Vanin-1 is involved in many biological processes in mouse, from thymus homing to sexual development. Vanin-1 -/- mice are fertile and grow and develop normally; they better control inflammation and most of the knockout effects were rescued by cystamine treatment. Gene structure analysis showed the presence of an oxidative stimuli-responsive ARE-like sequence in the promoter. In this paper we investigate antioxidant-detoxifying enzymatic activities at the tissue level, comparing Vanin-1 -/- and wild-type mice. In Vanin-1 null animals we pointed out a decrease in the Se-independent glutathione peroxidase activity. The decrease in enzymatic activity appeared to be correlated to an impairment of GST isoenzyme levels. In particular a significant drop in GSTA3 together with a minor decrement in GSTM1 and an increase in GSTP1 levels was detected in Vanin-1 -/- livers. Cystamine administration to Vanin-1 -/- mice restored specifically GSTA3 levels and the corresponding enzymatic activity without influencing protein expression. A possible role of cystamine on protein stability/folding can be postulated.

Enzymatic synthesis of S-aminoethyl-L-cysteine from pantetheine.

The recently characterized compound S-aminoethylcysteine ketimine can be synthesized from purified S-aminoethylcysteine by enzymatic systems (transaminases or L-amino acid oxidase) present in mammalian tissues. S-Aminoethylcysteine, which could be considered as the natural precursor of the ketimine, is produced from L-serine and cysteamine by the action of the enzyme cystathionine-beta-synthase. We demonstrate in this paper that pantetheine, a normal cellular component, is an efficient cysteamine donor for the synthesis of S-aminoethylcysteine and of S-aminoethylcysteine ketimine in the place of free cysteamine, and we describe the enzymatic system, composed of partially purified enzymes, for the in vitro synthesis of S-aminoethylcysteine ketimine from pantetheine. This seems to indicate a new biological role for pantetheine.

Neuroprotective effects of PrxI over-expression in an in vitro human Alzheimer's disease model.

Peroxiredoxins are ubiquitous proteins that recently attracted major interests in view of the strict correlation observed in several cell lines and/or tissues between different levels of their expression and the increased capacity of cells to survive in different pathophysiological conditions. They are recently considered as the most important enzymes regulating the concentration of hydroperoxides inside the cells. Most of neurodisorders such as Parkinson, Huntington, Alzheimers diseases, and ischemic injury are characterized by conditions of oxidative stress inside cells. In these pathophysiological conditions, a strict correlation between cell survival and Prx expression has been found. In CNS all the Prx isoforms are present though with different expression pattern depending on cell phenotype. Interestingly, neurons treated with amyloid beta peptide (Aβ), showed an overexpression of PrxI. In this study, the neuroprotective effect of PrxI after Aβ exposure and the underlying mechanisms by which PrxI expression counteracts cell death was investigated in a well established human AD in vitro model. Taking advantage on cells transfected by a construct where human PrxI is fused with a Green fluorescent protein (GFP) at the C‐terminus, we report some events at the basis of cell survival after Aβ injury, suggesting possible new signal cascades dealing with the antiapoptotic effect of PrxI. The results obtained indicated a protective role for PrxI in counteracting Aβ injury by increasing cell viability, preserving neurites, and decreasing cell death. J. Cell. Biochem. 114: 708–715, 2013.