BIOCHEM 2.4 - PROTEIN TARGETING & PROTEOLYSIS

Protein/Enzyme Regulation

• Most enzymes are proteins subject to regulation principles.
• Regulation occurs at the levels of expression/availability (transcriptional regulation) and translational regulation.
• Regulation can involve binding, sequestering, modifying, or degrading proteins.
• Binding can form complexes or prevent associations.
• Sequestering involves placing proteins in the wrong compartment or separating them from cofactors or coenzymes.
• Modification involves adding or removing functional groups to control activity.
• Degradation involves proteolysis or protein breakdown.

Basic Protein Concepts

• Structure relates to function.
• Similar structures often have similar functions.
• Motifs are super secondary structures that are structurally important.
• Domains represent tertiary structural portions with functional importance.
• Protein half-life is tied to activity and cellular needs.
• Continuous synthesis and degradation balance cellular content.

Protein Half-Life

• Proteins exhibit variable half-lives linked to their cellular functions.
• N-terminal residues can promote rapid protein turnover.
• N-terminal arginine correlates to a shorter half-life.
• N-terminal methionine correlates to a longer half-life.
• Half-life is controlled via post-translational modification.
• Modifications can stabilize or target proteins for degradation, such as ubiquitylation.

Protein Degradation

• Protein degradation is a mechanism for cellular maintenance that manages protein availability.
• Damaged proteins are degraded.
• General degradation triggers include environmental stress, inappropriate modification/damage (ROS damage), and when a functional need has been met (e.g., rapid signaling molecule removal).
• Mechanisms include modification to target and proteolytic cleavage of proteins into amino acid components.

Proteolysis

• Proteolysis degrades proteins, and consists of specialized mechanisms that operate in cell compartments.
• Proteolysis mechanisms include lysosomes, ubiquitin-proteasome activity, calpains, mitochondrial proteinases, and membrane proteinases.
• Lysosomes break down intracellular proteins and those entering via endocytosis.
• Ubiquitin-proteasomes clear most intracellular proteins and remove damaged ones.
• Calpains degrade cytoskeletal proteins.
• Mitochondrial proteinases act on oxidatively modified mitochondrial proteins.
• Membrane proteinases degrade membrane-associated, secretory, endocytosed, and newly synthesized but faulty proteins.

Processing Proteins

• Dietary proteins are processed through digestive organs.
• Endogenous proteins may have short lives or transient roles, or be misfolded.
• Endogenous proteins are usually ubiquitinated for proteasome processing.

Ubiquitin/Ubiquitylation

• Ubiquitin is a highly conserved small protein with 76 amino acids.
• It covalently binds to the ε-amino group of lysine residues.
• A 3-enzyme complex adds ubiquitin.
• Ubiquitin enzymes are an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3).
• Ubiquitin can create long chains/tails in tandem, or alone at multiple sites.

Autophagy/Ubiquitin

• Autophagy and ubiquitin can function independently or dependently for cellular waste management.
• It can also function in tandem with the proteasome.

Proteolysis via Proteasome

• The proteasome is a barrel-shaped complex with a core particle and regulatory particle(s).
• Degradation requires recognition of the molecule to be destroyed with ubiquitylation.
• Targeted proteins go to the proteasome by ubiquitin recognition or shuttle proteins.
• Deubiquitinating enzyme removes ubiquitin so the rest of the protein undergoes denaturing and shunting.

Proteasome Proteolysis

• Proteasome recycling or destruction is occasionally necessary.
• It can be triggered by inhibition or cellular starvation, with reliance on Autophagy.
• Autophagy acts to replace altered or inhibited proteolysis.

Protein Degradation to Evaluate Protein Composition

• The Edman Degradation Method removes one amino acid to sequence acids in proteins.
• Phenyl isothiocyanate (PITC) converts the N-terminal amino acid to a phenyl thiocarbamoyl derivative in alkaline solution.
• Acid hydrolysis removes the amino acid as a PTH derivative.
• Amino acid is identified by HPLC; using chromatographic evaluation (charge, size, and/or hydrophobicity).

Implications of Altered Proteolysis

• Protein targeting and degradation play a key role in homeostasis; targeting and degradation processes must to remain intact.