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Modifications of Amino Acid Residues 2

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Modifications of Amino Acid Residues 2

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Modifications of Amino Acid Residues: Part 2

Overview

AMINO ACID MODIFICATIONS

  • Disulfide cross-linking (between two cysteine residues).
  • Hydroxylation (the addition of a hydroxyl group).
  • Carboxylation (the addition of a carboxyl group).
  • Phosphorylation (the addition of a phosphate group).
  • Methylation (the addition of methyl groups)
  • Acetylation (the addition of acetyl groups)
  • Glycosylation (the addition of sugars such as glucosamine or mannose)
  • ADP-ribosylation (the addition of ADP-ribose)
  • Ubiquitination (the addition of one or more ubiquitins)

METHYLATION

  • Addition of –CH3 groups to Lys or Arg residues (+ charged side chains)
  • Arg: 1-2 methyl groups
  • Lys: 1, 2, or 3 methyl groups.
  • Doesn't affect charge of amino acid
  • -CH3 has few steric interactions (small group_
  • Histone methylation: affects DNA transcription

ACETYLATION

  • Addition of acetyl groups to Lys residues or amino termini
  • Histone acetylation: Lys acetylation = turns genes on/off
  • Lys acetylation neutralizes + Lys charge: affects histone binding affinity for DNA

GLYCOSYLATION

  • Addition of sugars (single or branched) to Asn, Ser or Thr residues
  • N-linked glycosylation: sugars added to NH2 group of Asn side chain
  • O-linked glycosylation: sugars added to OH group of Ser or Thr side chain
  • Secreted proteins & cell surface: sugars make proteins more hydrophilic (soluble)
  • Immune system: glycosylation patterns in diff. organisms --> identify pathogens

ADP-RIBOSYLATION

  • Addition of ADP-ribose to Arg, Gln, Cys & diphthamide (modified His)
  • Ribose: sugar component of RNA
  • ADP: dephosphorylated form of ATP
  • ADP-ribose: derived from NAD+ (coenzyme in electron transfer)
  • Gene regulation, DNA repair, cell survival
  • Poly-ADP-ribose polymerases (PARPs) add poly ADP-ribose chains to proteins
  • Inhibition of PARP1: anti-cancer therapeutic.

UBIQUITINATION

  • Addition of one or more ubiquitins to Lys residues
  • Trio of proteins add Ub to Lys residue
  • Ubiquitin: small protein in almost all tissues; targets proteins for degradation
  • Polyubiquitination: many Ubs can add to single Lys residue; one at a time
  • Polyubiquitination creates large hydrophobic strip --> signals chaperones to move protein to proteasome
  • Rids cell of unwanted/unneeded/misfolded proteins

CLINICAL CORRELATIONS

p53 silencing in cancer

  • Small molecule cancer drugs: prevent deacetylation of p53 to keep it active (acetylated) to fight cancer

Cholera, pertussis & diphtheria

  • Use ADP-ribosylation to permanently alter protein function in infected cells
  • Cholera toxin: causes constant secretion of water in intestinal lumen--> dehydration & diarrhea

Neurodegenerative disorders (e.g. Alzheimer's & Parkinson's disease)

  • Mutation/inhibition of ubiquitination proteins
  • Abnormal accumulation of proteins in cytosol --> build up of plaque in brain

Full-Length Text

  • Here we will continue to learn about post-translational modifications of the peptide residues in a polypeptide and how they affect proteins.
    • In this tutorial we will we will further explore the effects of modification of amino acid residues by looking at the addition of three kinds of groups to amino acids: sugars, ubiquitin, and hydrocarbon groups.

We will start with our diagram from the previous tutorial, which has our polypeptide chain of twenty amino acids.

  • Denote that we will focus on five modifications:
    • Methylation (the addition of methyl groups)
    • Acetylation (the addition of acetyl groups)
    • Glycosylation (the addition of sugars such as glucosamine or mannose)
    • ADP-ribosylation (the addition of ADP-ribose)
    • Ubiquitination (the addition of one or more ubiquitins)

Let's begin with methylation.

  • Denote that methylation is the addition of methyl groups to lysine or arginine residues, both of which have positively charged side chains.
    • Arginine may have one or two methyl groups added to it, and lysine may have one, two, or three methyl groups added to it.
  • Label amino acid 13 as arginine and draw two CH3 groups attached to it.
  • Indicate the following regarding methylation:
    • It doesn't affect the charge of the amino acid.
    • Because the methyl group is so small, it has few steric interactions.
    • Methylation seems to be a relatively minor modification (it neither affects the charge nor has steric interactions); however, it has quite an important functional effect.
  • Denote that methylation is the most well-known modification of histones (the proteins around which DNA is wrapped), which affects the transcription of DNA.
    • Mono-, di-, and tri-methylation have been shown to have different meanings, and protein domains such as Tudor domains and PHD domains, can recognize and interpret these methylation states of lysine and arginine residues, giving a range of responses to them.

Similar to methylation, amino acid residues can also be acetylated.

  • Denote that acetylation is the addition of acetyl groups to lysine residues and amino termini.
    • Lysine acetylation is another common modification of histones.
  • Label amino acid 4 as lysine and draw an acetyl group attached to it.
  • Indicate that lysine acetylation neutralizes the positive charge of amino group on the side chain, which, in histones, has an effect on its binding affinity for DNA; and can be used to turn genes on or off.
  • As a clinical correlation, denote that acetylation is essential the function of p53, which is silenced in many cancers.
    • Thus, small molecule cancer drugs have been developed that prevent deacetylation of p53 to keep it active (acetylated) to fight the cancer.

Now, let's look at the addition of sugar moieties to amino acid residues, starting with glycosylation.

  • Denote that glycosylation is the addition of sugars to asparagine, serine or threonine residues.
    • Single sugars, such as N-acetylglucosamine can be added, or branched chains of sugars can be added to these residues.
  • Label amino acid 17 as asparagine and draw a branched chain attached to it.
  • Indicate the following:
    • In N-linked glycosylation, sugars are added to the side chain amino group on asparagine.
    • In O-linked glycosylation, sugars are added to the side chain hydroxyl group of serine or threonine.
  • Write that the addition of sugars is common on cell surface and secreted proteins because sugars make proteins more hydrophilic and therefore, more soluble.
    • Different glycosylation patterns found in different organisms aids the immune system's ability to identify foreign (and potentially pathogenic) entities.

Next we will look at ADP-ribosylation.

  • Denote that ADP-ribosylation is the addition of ADP-ribose to arginine, glutamine, cysteine and diphthamide (a modified histidine) residues.
    • Ribose is the sugar component of RNA, and ADP is the de-phosphorylated form of ATP.
  • Denote that ADP-ribose is derived from NAD+ or nicotinamide adenine dinucleotide, a coenzyme found in all living cells that is important for electron transfer in metabolic reactions.
  • Label amino acid 18 as arginine and draw a pentagon with an ADP molecule attached to it.
    • The pentagon represents the pentose sugar ribose, which has five carbon atoms.
  • Write that one or many ADP-ribose moieties can be added to a protein, and that ADP-ribosylation has important effects on gene regulation, DNA repair and cell survival.
    • Poly-ADP-ribose polymerases (PARPs) add poly ADP-ribose chains to proteins, and inhibition of PARP1 has been a potent anti-cancer therapeutic.
  • As a clinical correlation, write that bacterial toxins, such as those of cholera, pertussis, and diphtheria use ADP-ribosylation to permanently alter the function of proteins in infected cells.
    • Cholera toxin causes constant secretion of water to the lumen of the intestines, resulting in dehydration and diarrhea.

Finally, we will look at ubiquitination.

  • Denote that ubiquitination is the addition of one or more ubiquitins to lysine residues; it requires a trio of proteins that add the ubiquitin to the lysine residue.
  • Denote that ubiquitin is a small protein found in almost all tissues and its addition to a protein targets the protein for degradation.
  • Label amino acid 15 as lysine and draw a chain of three ubiquitin molecules attached to it.
    • Although many ubiquitins can be added to a single lysine residue, they are added one at a time.
  • Indicate that poly-ubiquitination is the addition of multiple ubiquitins to a single residue.
    • A single ubiquitin has a small hydrophobic patch on it and poly-ubiquitination creates a large hydrophobic strip that signals chaperones to move the protein to the proteasome.
  • Write that ubiquitination helps to rid the cell of unwanted, unneeded and misfolded proteins, which makes it a powerful tool for regulation of cellular processes.
  • As a clinical correlate, write that mutation or inhibition of ubiquitination proteins is implicated in neurodegenerative disorders such as Alzheimer's and Parkinson's disease, both of which involve abnormal accumulation of proteins in the cytosol (a build up of plaque in the brain).