Expression and localization of augmenter of liver regeneration in human muscle tissue

The contractile activity of skeletal muscular fibres depends, predominantly, on the energy supplied by mitochondrial oxidative phosphorylation, a biological process that involves several enzymes and proteins, 13 of which are encoded by mitochondrial DNA (mt-DNA), and all the rest by nuclear DNA (n-DNA). Mitochondrial phosphorylation-associated biochemical events require then an efficient cross-talk between mt- and n-genome that is facilitated by several nuclear encoded proteins (Garstka et al. 1994). Among them, mitochondrial transcription factor A (mt-TFA) is one of the most important (Parisi & Clayton 1991; Shadel & Clayton 1993; Larsson et al. 1994, 1996). Our group identified and isolated a protein from rat liver that is able to augment, in vivo in rat, hepatocyte proliferative spur consequent to 40% partial hepatectomy. This factor, termed augmenter of liver regeneration (ALR) (Francavilla et al. 1994; Hagiya et al. 1994), belongs to the ancestral and well-conserved ERV1/ALR protein family (Polimeno et al. 1999) and it is present in all eukaryotic cells (Francavilla et al. 1994; Giorda et al. 1996), in yeast (Lisowsky 1994), fruit fly (Klebes et al. 2005) and even viruses (Senkevich et al. 2000). These proteins, highly divergent in their amino terminal domain, show a strong homology in their well-conserved, carboxy terminal domain (Hofhaus et al. 1999), where a CXXC motif indispensable for the sulphydryl oxidase enzyme activity of this protein family is present (Lisowsky et al. 2001). Of particular interest is the 40% homology of ALR protein (Alrp) with saccharomyces cerevisiae essential for respiration and viability 1 (scErv1) (Lisowsky et al. 1995; Polimeno et al. 1999), a factor present in yeast and known to be important for the biogenesis of mitochondria and the viability of the cell (Lisowsky 1994). It is already well known that Alrp activity and mitochondrial metabolism are strictly related: (i) Alrp is predominantly localized in the mitochondrial inter-membrane space (Hofhaus et al. 1999; Lange et al. 2001), (ii) Alrp and its homologue scErv1 are proteins necessary for mitochondrial biogenesis (Hofhaus et al. 1999), characterized by direct interaction with cytochrome c (Allen et al. 2005; Dabir et al. 2007; Koehler & Tienson 2008), and an essential function for the maturation of cytosolic Fe/S proteins (Lange et al. 2001), and (iii) complementation studies have demonstrated that the C-terminal part of rat Alrp and scErv1 can functionally be interchanged, even if the two organisms are phylogenetically distant (Lisowsky et al. 1995). Alrp mRNA in rat has been identified in all tissues in relatively low quantities with the exception of testis, muscle, nervous system and liver (Hagiya et al. 1994; Giorda et al.1996), where it has been detected in large quantity, suggesting that Alrp may play a fundamental role for the proper functioning of these organs. Alrp, when injected in intact rats, produces a burst of mitochondrial metabolism and a significant increase of the expression of mitochondrial genes and of nuclear-encoded mt-TFA. This latter event is associated with an enhanced cytochrome content and oxidative phosphorylation capacity of liver mitochondria, exhibiting a significantly higher rate of oxygen consumption and ATP production (Polimeno et al. 2000). An increased ALR mRNA and serum protein expression have been detected in regenerating liver after 70% partial hepatectomy in rats (Yang et al. 1997; Gandhi et al. 1999), when an increased energetic demand is required for the regenerating tissue (Onoue et al. 1996; Guerrieri et al. 1999). Despite this knowledge, few reports investigate the presence of Alrp in mammalian tissues and no data are available on human tissues. Tury et al. (2005) report in their study the presence of Alrp in rat brain and Thasler et al. (2005) documented the presence of Alrp in human hepatocellular carcinoma. We therefore surmised that it would be of interest to study ALR expression in human skeletal muscular tissue, particularly rich in mitochondria. In this article, we report the first results on the characterization and localization of ALR in human muscular tissue samples taken from patients hospitalized in the Orthopedic Unit of the Policlinico of Bari, evaluating: type I and type II muscle fibres, identified according to the presence of mitochondrial enzymes (ATPase and NADH-tetrazolium reductase), detected by enzymatic techniques; Alrp presence, by immunohistochemistry, using a specific polyclonal antibody; ALR mRNA expression, by molecular biology technique; Alrp subcellular localization by electron microscopy.