Post-translational Protein Processing

Post-translational protein processing (including post-translational modification) is the folding, sorting, cleavage, and modifications required to make a protein functional after it is translated. As the protein folds, it forms complex secondary, tertiary, and quaternary structures. In addition, new functional groups or molecules may be added to the polypeptide chain, including phosphoryl, methyl, or acetyl groups; carbohydrates; and lipids. Proteins also have to be sorted into the correct intracellular compartment to either carry out their function, be packaged for secretion, or be inserted into the appropriate cellular membrane.

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Amino Acid Overview

Amino acids are the building blocks of proteins. Understanding the basics of amino acids allows a more comprehensive understanding of protein folding and modification.

Amino acid structure

Amino acids that make up proteins are known as α-amino acids. Each of these amino acids has a central carbon known as the “alpha carbon,” which makes 4 bonds:

  • Hydrogen ion
  • Carboxyl group: made up of carboxylic acid (–COOH), creating the C-terminal end of the amino acid
  • Amine group: made up of an amine group (–NH2), creating the N-terminal end of the amino acid 
  • R side chain: the unique functional group of an amino acid

Properties of amino acids

Amino acids may be categorized by characteristics of their R groups, which may be:

  • Polar or nonpolar
  • Hydrophobic or hydrophilic
  • Charged or noncharged at physiologic pH
  • Acidic or basic

Classification of amino acids by R group

Image by Lecturio.

Protein Folding

Levels of protein structure

Protein structure, which is often referred to as protein folding, has 4 levels. These levels are:

  1. Primary
  2. Secondary
  3. Tertiary
  4. Quaternary

Primary structure

  • The primary structure is the linear sequence of the amino acids in the peptide chain.
  • “Beads on a string” joined by peptide bonds
  • This structure ultimately determines all the properties of the protein.

Example of primary structure of a protein

Image by Lecturio.

Secondary structure

  • Occurs between amino acids that are relatively close to each other (typically about 3–10 amino acids apart) 
  • 3 common motifs include:
    • α-helix: a coil with the R groups on the outside
    • β-strands and sheets: 
      • planar structure formed by zigzagging amino acid strands
      • R groups protrude from the top and bottom of the sheet.
    • Reverse turns: a short sequence, usually involving proline and/or glycine, occurring between α-helices and/or β-pleated sheets
  • Secondary structures are stabilized by hydrogen bonds between the carboxyl oxygen and the amine hydrogens.
  • Some simple fibrous proteins (e.g., keratin) have only primary and secondary structures.
  • Computer models are often able to predict secondary structures based on the amino acid sequence.

Examples of α-helices and β-pleated sheets

Image by Lecturio.

Tertiary structure

Tertiary structure is the complex looping and folding that occurs as a result of interactions and bonding between portions of the protein that are farther apart.  Examples of interactions that create tertiary structure include:

  • Hydrogen bonds: form between polar side chains
  • Disulfide bridges: strong covalent bonds that form between two cysteines
  • Ionic bonds: form between a positively charged/acidic R group (e.g., carboxyl group on aspartic acid) and a negatively charged/basic R group (e.g., amine group on lysine)
  • Metallic bonds: 2 regions of a protein bound to a metal (e.g., iron)
  • Hydrophobic interactions between nonpolar side chains: orient inward, away from water, to create spaces of hydrophobic exclusion.

Example of tertiary structure

Image by Lecturio.

Quaternary structure

In a quaternary structure, multiple subunits of a protein come together to form a single protein.

  • Each subunit has its own primary, secondary, and tertiary structures.
  • Subunits are held together by the same forces that generate tertiary structure:
    • Hydrogen bonds
    • Ionic bonds
    • Disulfide bridges (covalent bonds)
    • Metallic bonds
    • Hydrophobic interactions
  • Tertiary folding and quaternary folding produce several common motifs:
    • β–α–β
    • β-barrels (common in membrane channels)
    • Helix–turn–helix

Chaperone proteins

Chaperone proteins assist in protein folding.

  • Chaperones are barrel-like proteins that take in misfolded proteins and use ATP energy to refold them.
  • These proteins can bind to hydrophobic regions of unfolded proteins, allowing proper folding to take place.
  • Found in various cellular compartments such as:
    • Cytosol
    • Mitochondria
    • Lumen of the endoplasmic reticulum

Chaperone proteins assist in protein folding

Image by Lecturio.

Protein denaturing

A denatured protein is a protein that has been unfolded and is no longer functional. This unfolding occurs under certain conditions, which include changes in:

  • pH
  • Temperature
  • Ionic concentration

Proteins can become denatured (or unfolded) as a result of changes in pH, temperature, or ionic concentration.

Image by Lecturio.

Protein Sorting

Proteins need to be sorted and will end up remaining in the cell, being placed on the cell wall, or being exported/secreted.

Exported and surface proteins

Proteins destined for the cell surface and/or secretion from the cell are synthesized within the rough endoplasmic reticulum (RER):

  • During translation, a signal peptide sequence on the end of the growing polypeptide chain indicates that a protein is destined for secretion from the cell.
  • A signal recognition protein (SRP) binds to the signal peptide sequence, pausing elongation.
  • The SRP guides the entire ribosome to the RER and associates it with a pore.
  • When synthesis resumes, the growing polypeptide is deposited within the RER.
  • Proteins are then directed to the lysosomes or plasma membrane or packaged for exocytosis (secretion):
    • Secreted proteins follow the exocytic (secretory) pathway: RER → Golgi apparatus (GA) → plasma membrane (PM)
    • Proteins destined for the GA, PM, or secretion are carried in transport vesicles.

Docking a ribosome on the rough endoplasmic reticulum
SRP: signal recognition protein

Image by Lecturio.

Intracellular proteins

  • Proteins synthesized in the cytosol, unassociated with the RER, are kept inside the cell.
  • Other specific signal peptides may direct these proteins to their final location in the cell (e.g., the nucleus).

Protein Modification

After a polypeptide is synthesized, it undergoes further modification in order to form a functional protein. This modification may include cleaving off portions of the polypeptide chain or adding a functional group.

Protein cleavage

Protein cleavage is the process of removing certain polypeptides in order for the protein to become functional.

  • Many proteins are not functional immediately after translation; these proteins are called pro-proteins.
  • Polypeptides are cleaved by proteolytic enzymes.
  • Examples of amino acids or peptides that are typically cleaved off:
    • The first amino acid (methionine) matches the start codon in mRNA, signaling the start of translation.
    • Signal peptides assist the protein in getting to its proper location but are not part of the functional protein itself.
    • Enzymes and hormones are frequently translated as pro-proteins, which require cleavage in order to become functional/active (e.g., insulin precursor (proinsulin) C-peptide is cleaved in GA).

Addition of a functional group

Proteins are further modified by the covalent addition of functional groups and other molecules.

  • Common modifications include:
    • Phosphorylation
    • Acetylation
    • Methylation
    • Ubiquitylation
    • Glycosylation
    • Lipidation
  • Common sites that are modified:
    • Hydroxyl groups in serine, threonine, and tyrosine
    • Amine groups in lysine, arginine, and histidine
    • Carboxylate groups in aspartate and glutamate
    • The N- and C-terminals
  • General principles:
    • Hydrophobic groups may help a protein incorporate into a membrane.
    • Addition of cofactors can enhance enzymatic activity.

Phosphorylation

  • Addition of phosphoryl group (most common)
  • Common functions:
    • Regulates enzymatic activity
    • Cellular energy exchange: ATP, guanosine triphosphate (GTP), nicotinamide adenine dinucleotide phosphate (NADPH)

Acetylation and methylation

  • Addition of acetyl group or methyl group
  • Common functions:
    • Activates many pharmaceuticals
    • Regulates gene expression and protein synthesis
    • Histone modifications

Acetylation of a polypeptide
CoA: coenzyme A
NAT: N-terminal acetyltransferases

Image: “Protein-acetylation-nterminal” by Hbf878. License: CC0 1.0

Ubiquitylation

  • Addition of ubiquitin
  • Functions: 
    • Targets proteins for degradation in proteasome
    • Histone modifications

Glycosylation

  • Addition of carbohydrate to create a glycoprotein
  • Types of glycoproteins include:
    • N-glycoproteins: bound to nitrogen on the side chain of asparagine
    • O-glycoproteins: bound to oxygen on the side chain of serine or threonine
  • Glycosylated proteins are commonly associated with the cell membrane or are secreted; common examples include:
    • Hormones
    • Immune system functions (e.g., found in some antibodies)
    • Cellular identity (e.g., ABO blood types)
  • Glycosylation affects many different cellular processes and is implicated in: 
    • Cancer
    • Diabetes
    • Alzheimer’s disease

An N-linked versus an O-linked glycoprotein

Image by Lecturio.

Lipidation

  • Addition of a lipid molecule to create a proteolipid
  • Typically occurs on proteins associated with a phospholipid membrane
  • Examples of lipidation:
    • Addition of a glycosylphosphatidylinositol (GPI) “anchor”: commonly used to anchor cell surface proteins
    • N-myristoylation: addition of a myristoyl group to some proteins involved in signal transduction, oncogenesis, and host defense
    • Prenylation or palmitoylation: addition of a prenyl or a palmitic acid group to membrane proteins, making them more hydrophobic

Clinical Relevance

Abnormalities in post-translational modification and/or protein folding or sorting can lead to a number of clinically important medical conditions.

  • Protease inhibitors: HIV uses the process of proteolysis during its life cycle to create functional structural proteins from precursors. These proteases are a target of the anti-HIV drugs called protease inhibitors.
  • Alzheimer’s disease: neurodegenerative disease resulting in dementia: It is thought that misfolded and/or abnormally modified proteins, including the β-amyloid peptide and tau proteins, are associated with Alzheimer’s disease. Whether Alzheimer’s disease results in increased misfolded proteins or the misfolded proteins cause the disease is still being explored.
  • Parkinson’s disease: progressive neurodegenerative movement disorder that presents with tremors, stiffness, and slowing of movement: Parkinson’s disease is thought to be caused at least in part by accumulation of a protein called α-synuclein in the neurons of the nigrostriatal pathway, leading to the death of these neurons. Misfolding of α-synuclein leads to the formation of insoluble aggregates, which accumulate and disrupt signaling.
  • Cystic fibrosis (CF): autosomal recessive disorder caused by mutations in the CFTR gene. The mutations lead to dysfunction of chloride channels, which results in hyperviscous mucus and the accumulation of secretions. There are 5 classes of mutations. Class II is a group of mutations that cause abnormal post-translational processing; because of these abnormalities, the proteins are not brought to the correct cellular locations (and are often defective). This class includes the common mutation F508del. Common presentations of CF include chronic respiratory infections, failure to thrive, and pancreatic insufficiency.

References

  1. Barrett, K. E., Barman, S. M., Brooks, H. L., Yuan, J. X. (2019). General principles & energy production in medical physiology. Ganong’s review of medical physiology, 26th ed. New York: McGraw-Hill Education. Retrieved from https://accessmedicine.mhmedical.com/content.aspx?aid=1159051016
  2. Bender, D. A., Murray, R. K. (2018). Glycoproteins. In Rodwell, V. W., et al. (Eds.). Harper’s Illustrated Biochemistry, 31st ed. New York: McGraw-Hill Education. Retrieved from https://accessmedicine.mhmedical.com/content.aspx?aid=1160192722
  3. Botham, K. M., Murray, R. K. (2018). Intracellular traffic & sorting of proteins. In Rodwell, V. W., et al. (Eds.). Harper’s Illustrated Biochemistry, 31st ed. New York: McGraw-Hill Education. Retrieved from https://accessmedicine.mhmedical.com/content.aspx?aid=1160192994
  4. Cooper, G. M. (2000). Protein folding and processing. Sinauer Associates. Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK9843/
  5. Sweeney, P., et al. (2017). Protein misfolding in neurodegenerative diseases: implications and strategies. Translational Neurodegeneration 6(6). https://doi.org/10.1186/s40035-017-0077-5
  6. Weil, P. A. (2018). The diversity of the endocrine system. In Rodwell, V. W., et al. (Eds.). Harper’s Illustrated Biochemistry, 31st ed. New York: McGraw-Hill Education. Retrieved from https://accessmedicine.mhmedical.com/content.aspx?aid=1160191674

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