In liver, macroautophagy is the principal mechanism for degrading intracellular protein and is a major source of endogenous amino acids for gluconeogenesis and other critical pathways early in starvation. The fact that autophagy is an ongoing process and subject to immediate feedback control by specific regulatory amino acids attests to its importance in cellular homeostasis. Although the effects of amino acids are strongly modulated by insulin and glucagon in the hepatocytes, amino acids are believed to represent prime regulators since the full range of deprivation-induced autophagy can be elicited by these agents in the absence of hormones. Of the 20 amino acids that turn over in protein, only 8 as a group (Leu, Tyr, Phe, Gln, Pro, Met, His, and Trp) possess direct inhibitory activity at physiological concentrations. Leucine is the dominant regulator since it is the most potent and is always required for maximal effectiveness. Although actively sought, a molecular mechanism of amino acid inhibition of autophagy has not been established. The hypothesis that leucine has its own signal transduction mechanism, characterized by a recognition site at the cell surface, originated from a series of evidences obtained with leucine and structurally related analogues. These studies have clearly pointed out a close relationship between autophagic response and extracellular, but not intracellular, amino acid concentration. This hypothesis has been enforced by the result obtained with the Leu8-MAP, a large, not transportable, branched leucine-mimetic peptide. It was synthesized by attaching 8 residues of leucine to the 4α + 4ε amino termini of a branched polylysinic core (termed MAP for Multiple Antigen Peptide), thus creating a compact molecule of about 1900Da with leucine moieties arranged peripherally. When compared on a molar basis Leu8-MAP was as effective in suppressing autophagy as leucine and had the same apparent Km (0.1 mM). Inhibition was specific for leucine since Ile8-MAP and Val8-MAP evoked no response. The branched structure of MAP, together with the redundancy of leucine residues, has made possible the synthesis of a biologically active, biotin conjugated, azide derivative of the peptide [Leu7-(ASA)MAP- Biotin]. This provided a useful photo-probe to test the presence of the putative leucine receptor and to attempt its isolation and purification. An approximately 103 kD protein, whose labeling was prevented > 90% by 5 mM Leucine, was found in plasma membrane enriched fractions of hepatocytes; valine and isoleucine did not compete, indicating that the photo-labeling was leucine specific. Biotin based affinity purification yielded a highly purified protein suitable for sequence determination by mass spectrometry. The protein was identified, with high degree of confidence (Mowse score > 350), as a multifunctional glycoprotein located on the hepatic plasma membrane, where it forms functional dimers. The functional link of this protein with autophagy is supported by the effects exerted by two distinct monoclonal antibodies. When added to isolated hepatocytes one antibody induced the inhibition of autophagy similarly to the amino acid; the second instead was able to modulate the responsiveness of the cells, since significantly enhanced the anti-proteolytic effect of the leucine, without exerting autophagy inhibition “per se”. The antibodies results, together with the high leucine specificity of the photo-labeling, strongly suggest that the identified protein has the function of leucine receptor in liver. However, important aspects, as the site of leucine interaction and the mechanism of signal transduction, need further investigation.

Does leucine regulate autophagy through a plasma membrane receptor? Identification of a hepatic glycoprotein supporting this hypothesis

MIOTTO, GIOVANNI
2005

Abstract

In liver, macroautophagy is the principal mechanism for degrading intracellular protein and is a major source of endogenous amino acids for gluconeogenesis and other critical pathways early in starvation. The fact that autophagy is an ongoing process and subject to immediate feedback control by specific regulatory amino acids attests to its importance in cellular homeostasis. Although the effects of amino acids are strongly modulated by insulin and glucagon in the hepatocytes, amino acids are believed to represent prime regulators since the full range of deprivation-induced autophagy can be elicited by these agents in the absence of hormones. Of the 20 amino acids that turn over in protein, only 8 as a group (Leu, Tyr, Phe, Gln, Pro, Met, His, and Trp) possess direct inhibitory activity at physiological concentrations. Leucine is the dominant regulator since it is the most potent and is always required for maximal effectiveness. Although actively sought, a molecular mechanism of amino acid inhibition of autophagy has not been established. The hypothesis that leucine has its own signal transduction mechanism, characterized by a recognition site at the cell surface, originated from a series of evidences obtained with leucine and structurally related analogues. These studies have clearly pointed out a close relationship between autophagic response and extracellular, but not intracellular, amino acid concentration. This hypothesis has been enforced by the result obtained with the Leu8-MAP, a large, not transportable, branched leucine-mimetic peptide. It was synthesized by attaching 8 residues of leucine to the 4α + 4ε amino termini of a branched polylysinic core (termed MAP for Multiple Antigen Peptide), thus creating a compact molecule of about 1900Da with leucine moieties arranged peripherally. When compared on a molar basis Leu8-MAP was as effective in suppressing autophagy as leucine and had the same apparent Km (0.1 mM). Inhibition was specific for leucine since Ile8-MAP and Val8-MAP evoked no response. The branched structure of MAP, together with the redundancy of leucine residues, has made possible the synthesis of a biologically active, biotin conjugated, azide derivative of the peptide [Leu7-(ASA)MAP- Biotin]. This provided a useful photo-probe to test the presence of the putative leucine receptor and to attempt its isolation and purification. An approximately 103 kD protein, whose labeling was prevented > 90% by 5 mM Leucine, was found in plasma membrane enriched fractions of hepatocytes; valine and isoleucine did not compete, indicating that the photo-labeling was leucine specific. Biotin based affinity purification yielded a highly purified protein suitable for sequence determination by mass spectrometry. The protein was identified, with high degree of confidence (Mowse score > 350), as a multifunctional glycoprotein located on the hepatic plasma membrane, where it forms functional dimers. The functional link of this protein with autophagy is supported by the effects exerted by two distinct monoclonal antibodies. When added to isolated hepatocytes one antibody induced the inhibition of autophagy similarly to the amino acid; the second instead was able to modulate the responsiveness of the cells, since significantly enhanced the anti-proteolytic effect of the leucine, without exerting autophagy inhibition “per se”. The antibodies results, together with the high leucine specificity of the photo-labeling, strongly suggest that the identified protein has the function of leucine receptor in liver. However, important aspects, as the site of leucine interaction and the mechanism of signal transduction, need further investigation.
2005
Giornale di gerontologia volume 53
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/2484929
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