302 Hypothesis Accelerated removal of deamidated proteins and endogenous electric felds: possible implications Isabella Panfoli, Silvia Ravera, Daniela Calzia and Alessandro Morelli Department of Biology, University of Genoa, V.le Benedetto XV 3, 16132 Genova, Italy Abstract. Changes in protein structure through the spontaneous deamidation of asparaginyl (Asn) and glutaminyl (Gln) residues have been observed in many proteins. Amide residues were supposed to serve as clocks for development and aging. Deamidated proteins are rapidly degraded by as yet unclear molecular mechanisms. Deamidation leads to elevation of the ratio of charged versus polar residues (CH-PO) of a protein and to a decrease in its pI value. We had reported that those enzymes, characterized by a high CH-PO, are prone to inactivation and loss of ordered structure by exposure to direct current from low voltage in solution. Nano-local endogenous electric felds arise within cells. Endogenous currents may cause the unfolding of the products of deamidation at Asn. In turn, these unfolded proteins would be removed, likely by proteolysis. Key words: Aging — Asparagine — CH-PO — Deamidation — Direct electric current — Glutamine — Proteasome Abbreviations: CH-PO, ratio of charged versus polar residues of a protein; dc, direct electric current; Asn, asparagine; Gln, glutamine. Correspondence to: Isabella Panfoli, Department of Biology, Uni- versity of Genova, V.le Benedetto XV 3, 16132 Genova, Italy E-mail: Isabella.Panfoli@unige.it Protein deamidation Many biological peptides and proteins possess labile as- paraginyl (Asn) and glutaminyl (Gln) residues. Deamida- tion of Asn and Gln residues is now recognized as one of the major pathways of spontaneous protein degradation, together with protein aggregation, oxidation, and glyca- tion (Robinson 2002; Reissner and Aswad 2003; Li et al. 2006). Te frst isolation and characterization of the amidated and deamidated forms of a naturally occurring peptide was accomplished by counter-current distribution of insulin in 1952 (reviewed in Robinson 2004a). However, only about 40 years ago, it was discovered that deamidation occurs in vivo under genetic control, over a very wide, physiologically relevant deamidation rate range (Robinson et al. 1970). Subsequently, much has been learned, especially about Asn, whose deamidation rate is faster than Gln. Between 1971 and 1973, Robinson and co-workers examined the deamidation of 65 synthetic peptides labeled with C 14 and measured the deamidation rates at pH 7, directly measuring the sequence- determined deamidation rates of model peptides (reviewed in Robinson 2004a). It is now established that a large per- centage of proteins deamidate to a substantial extent during their biological lifetimes. Te deamidation of Gln proceeds both enzymatically and non-enzymatically. By contrast, only the nonenzy- matic deamidation of Asn has been reported. Deamidation rates seem to depend upon the sequence and higher-order structure of the protein and to afect protein structure and function in natural and engineered mutant sequences (Clarke 2003). In fact, since the side chains of Asn and Gln are not charged, deamidation introduces a negative charge at the site of occurrence, afecting the structure of the peptides and proteins in which they are incorporated. Te occurrence of Asn and Gln in proteins was determined by Robinsons (2004b) from a set of 4835 representative proteins from the January 2003 PDB databank. Authors found that about 72% of proteins possessed more Asn and 63% have more Gln than expected by chance. Te research literature on the history deamidation was reviewed and summarized (Robinson 2004a). Gen. Physiol. Biophys. (2010), 29, 302–308 doi:10.4149/gpb_2010_03_302