Protein detoxification

Protein detoxification is the process by which proteins containing methylated arginine are broken down and removed from the body.

Arginine (Arg) is a non-essential amino acid and one of the most commonly occuring natural amino acids. Methylated arginine is a modified version of arginine that is commonly formed from protein arginine (arginine incorporated in protein). Asymmetrically-methylated forms of arginine are toxic when released during protein turnover.

The protein detoxification pathway eliminates free methylated-arginine derivatives from the cell. Symmetrically-methylated forms are not toxic and are excreted unchanged by the kidney. Asymmetrically-methylated forms however are toxic and must first be broken down. This step requires the enzyme dimethylarginine dimethylaminohydrolase (DDAH). Impairment of DDAH function slows breakdown and increases the level of toxic asymmetrically-methylated arginine forms ^[1].

Long term exposure to these toxic amino acids is associated with endothelial dysfunction, arterial stiffness, insulin resistance, chronic kidney disease, heart disease, dementia and ageing^[2][3][4]. Therapeutic strategies that target the protein detoxification pathway aim to:

• reduce intracellular levels of free asymmetrically-methylated arginine derivatives,
• slow the ageing process
• delay the development of disorders associated with ageing.

In 1970, it was demonstrated that protein turnover releases the free methylated arginine derivatives:

1. asymmetric dimethylarginine (ADMA),
2. methylarginine,(also refered to as N-methyl-L-arginine, N-monomethylargine or L-NMMA) and
3. symmetrical dimethylarginine (SDMA)^[5].

The potential toxic role of the two asymmetrically methylated amino acids however was not fully appreciated until 1992 when Patrick Vallance and his London co-workers at the Wellcome Research Laboratories demonstrated that ADMA is a potent inhibitor of nitric oxide synthase (NOS)^[6]. Then, in 1996 MacAllister at St George's Hospital Medical School in London, recognised that inhibiting the enzyme DDAH increases intracellular concentrations of ADMA^[7]. To describe the process of protein turnover, the elimination of free methylated arginine derivatives and the catabolism of the two asymmetrically methylated arginine derivatives ADMA and L-NMMA, the Australian physician Trevor Tingate coined the term protein detoxification in 2010.

Protein arginine methylation occurs posttranslationally and is catalysed by protein arginine methyltransferase (PRMT). No direct synthesis of methylated arginine derivatives occurs from the free amino acid. The methylation of protein arginine plays an important role in the regulation of many cell processes including gene transcription, cell signal transduction, DNA repair and RNA processing^[8]. During protein turnover three arginine methylated derivatives are released: L-NMMA, SDMA and ADMA. SDMA is not directly toxic and is eliminated unchanged by renal excretion. Both L-NMMA and SDMA however, are potent inhibitors of NOS^[9]. Around 60mg of ADMA is produced per day^[10]. Unlike SDMA, 80% of ADMA and NMMA is catabolised by the enzyme DDAH. The activity of DDAH is therefore an important determinant of ADMA and NMMA levels, and thus NOS activity^[11].

• Enzymes, arginine and the free asymmetrically methylated arginine derivatives
• 
  Protein arginine N-methyltransferase 1 (PRMT1)
• 
  Dimethylarginine dimethylaminohydrolase (DDAH)
• 
  Asymmetric dimethylarginine (ADMA)
• 
  N-methyl-L-arginine (L-NMMA)

PRMTs are activated by shear stress^[12] and LDL cholesterol^[13]. Two types of PMRTs have been characterised. Type 1 PMRTs are found mainly in endothelial and smooth muscle cells and produce methylated proteins containing ADMA and L-NMMA. Type 2 PRMTs produce proteins that contain SDMA and L-NMMA^[14].

DDAH activity is inhibited by NO, reactive oxygen species (ROS) and L-arginine^[15]. Two isoforms of DDAH have been identified. DDAH-1 is found in tissues expressing neuronal NOS (nNOS) and in the liver, kidney and lung. Expression is increased by IL-1β and inhibited by oxLDL and TNF. Plasma levels of ADMA reflect DDAH-1 activity. DDAH-2 is found in tissues expressing endothelial NOS (eNOS) and inducible NOS (iNOS). Expression is increased by NADPHox, all trans retinoic acid, pioglitazone and estradiol and inhibited by hypoxia, hyperglycaemia and LPS^[16].

Nitric oxide (NO), also known as 'endothelium-derived relaxing factor', or 'EDRF', should be considered the fountain of youth. It protects blood vessels by mediating vasodilation, inhibiting platelet activation, smooth muscle proliferation and endothelial cell activation, reducing the stiffness of the arterial tree and protecting the heart. Free methylarginines by contrast inhibit NOS, reduce NO, increase central arterial pressure and reduce vessel protection^[17]. These changes markedly accelerate arterial stiffness leading to heart failure, kidney failure and dementia; the leading causes of death in later years. Protein detoxification removes free methylarginines and prevents the inhibition of NO generation that occurs when the process is inhibited.

Vascular endothelium

Shear stress in micro-circulation as a result of arterial stiffness

Endothelial dysfunction associated inhibtion of eNOS, reduced NO and vasoconstriction is associated with arterial stiffness. Increased pulse pressure drives arterial pulsations further down the arterial tree, increasing shear stress.

When William Osler stated that “man is as old as his arteries” he referred to arterial stiffening, a condition now acknowledged as a integrated bio-marker of ageing^[18]. Preservation of the arterial tree in a relaxed and elastic state is core doctrum of anti-ageing medicine. The recognition of free methylarginine derivatives as toxins that accelerate ageing by inhibiting the production of nitric oxide focuses on the importance of maintaining the protein detoxification pathway. This can be achieved by a combination of dietary, behavioural and therapeutic interventions.

• ADMA