Proteins and peptides

Proteins are large biomolecules consisting of more than 50 amino acids connected by multiple peptide bonds, while peptides are small biomolecules consisting of less than 50 amino acids. Proteins fulfill a variety of functions, including regulating physiological activity and providing structure to cells, and their functions are closely tied to their conformation. After ingestion, dietary proteins are denatured by gastric acid and subsequently cleaved by pepsin and proteases into monopeptides, dipeptides, tripeptides, and tetrapeptides. These end products are absorbed in the small intestine via proton symporter and Na⁺-coupled carrier proteins. Intracellularly, endogenous proteins are degraded by the ubiquitin proteasome system, while endocytosed dietary proteins are degraded by the lysosome. Accumulation of damaged or misfolded proteins/peptides has been observed in many neurological diseases such as Alzheimer disease, Parkinson disease, Huntington disease, Creutzfeldt-Jakob disease, and myotonic muscular dystrophy.

Composition

Proteins consist of a chain of ≥ 50 amino acids (AAs) that are connected by multiple peptide bonds (polypeptide chain).

• Peptide: a chain of < 50 connected AAs
  • Dipeptide: two amino acids
  • Tripeptide: three amino acids
  • Oligopeptide: up to 10 amino acids
• Polypeptide: 10-100 amino acids

Peptide bond

• Formation
  • Formed in ribosomes during translation
  • Chemical reaction: A covalent bond (R1–CO–NH–R2) is formed when the carboxyl group (COOH) of an AA reacts with the amino group (NH₂) of another AA and causes the release of an H₂O molecule (i.e., a condensation reaction).
• Characteristics: planar, stable, partial double-bond character
• Polarity: polar with a dipole moment due to the difference in electronegativity between carbon and nitrogen, which affects the protein's overall structure and interactions
• Role: forms primary structure of proteins, establishing the peptide backbone , and providing both stability and directionality (from N-terminus to C-terminus)
• Cleavage of peptide bonds: occurs through hydrolysis, where H₂0 is added and is facilitated by enzymes such as proteases; critical in metabolism and digestion

The structure of proteins is crucial for their function!

Proper protein folding must occur for a protein to be functional (see article on translation and protein synthesis).

Creutzfeldt-Jakob disease and Alzheimer's disease
Misfolded proteins can lead to serious diseases. Creutzfeldt-Jakob disease (CJD) is caused by misfolded proteins called "prions." Prions are found in brain cells and exist in two forms: normal, which has mostly α-helices, and abnormal, which has mostly β-sheets. The abnormal prions can convert normal prions into their misfolded form, leading to the accumulation of insoluble proteins in the brain. This causes cell death and results in a sponge-like appearance in the brain, known as "spongiform encephalopathy." Alzheimer's disease is another condition caused by misfolded proteins, specifically the accumulation of β-amyloid fibrils in the brain, leading to cognitive decline.

Denaturation of proteins

Denaturation leads to loss of function; while the primary structure remains intact, the secondary and tertiary structures are altered. It is often caused by a change in external conditions.

• Types
  • Reversible denaturation: protein can refold if conditions are restored (= renaturation)
  • Irreversible denaturation: permanent structural loss due to chemical (e.g., oxidation, deamination, glycosylation) and/or physical changes
• Causes of denaturation
  • pH changes
  • Temperature shifts
  • Chemical agents: urea, detergents

References:^[1]

• Process
  1. Stomach
     • Gastric acid causes denaturation.
     • Cleavage via pepsin
  2. Duodenum: further cleavage from pancreatic and intestinal proteases
  3. Enterocytes
     • Absorption of di-, tri-, and tetrapeptides, likely via a proton symporter
     • Absorption of single amino acids: via Na⁺ coupled carrier proteins for specific AA groups (neutral, branched-chain, aromatic, acidic, basic)
  4. AAs enter the bloodstream and travel to the liver via the portal vein.
• Proteases: enzymes that split peptide bonds via hydrolysis
  • Examples of proteases
    • Serine proteases
      • Catalytic triad: consists of a specific arrangement of three amino acid residues: serine, histidine, and aspartate. These residues interact to activate one another, enabling the cleavage of peptide bonds.
      • Mechanism: The oxygen atom of the serine residue performs a nucleophilic attack on the positively polarized carbon atom of the peptide bond.
      • Examples: chymotrypsin, trypsin, elastase, plasmin, thrombin
    • Aspartate proteases
      • Characteristics: have two catalytically active aspartic acid residues in their active site
      • Examples: pepsin, renin
    • Threonine proteases
      • Characteristics: a catalytically active threonine residue is present in the active site
      • Mechanism: The two aspartate residues (Asp) work together. One is protonated (Asp-COOH) and one is deprotonated (Asp-COO⁻). They activate a water molecule to perform the nucleophilic attack.
      • Example: proteolytically active centers of proteasomes, where ubiquitin-tagged proteins are degraded
    • Metalloproteases
      • Characteristics: contain a catalytically crucial metal ion in their active site
      • Mechanism: The metal ion (usually Zn²⁺) acts as a Lewis acid. It coordinates with a water molecule, making it more acidic and a better nucleophile to attack the peptide bond.
      • Examples: angiotensin-converting enzyme, carboxypeptidase A
    • Cysteine proteases
      • Characteristics: use a cysteine residue as the nucleophile (specifically, its S-H group)
      • Example: caspases responsible for apoptosis
• Zymogens
  • Proteases that are first secreted in an inactive form to avoid damage to the immediate surrounding tissue
  • For example, pancreatic proteases are first secreted as inactive precursors (zymogens) before being activated in the duodenum.

Carboxypeptidases are exopeptidases (they cleave from the C-terminus), while trypsin/chymotrypsin are endopeptidases (they cleave in the middle of the chain).

Trypsinogen is first activated by enteropeptidase via proteolytic cleavage at the N-terminal. The resulting trypsin then activates other zymogens, including further trypsinogen (positive feedback loop).

The inactive zymogen pepsinogen is activated to pepsin by gastric acid.

References:^{[2][3][3][4][5][6][7]}

Protein degradation

Endogenous proteins (those synthesized in cells) are degraded by proteasomes. Exogenous proteins are degraded by lysosomes.

Ubiquitin proteasome system (UPS)

• Description
  • Via ubiquitination, proteins are targeted for degradation in proteasomes.
  • Proteasome: a barrel-like protein complex consisting of two units that breaks down marked or damaged proteins into peptides via ATP hydrolysis of peptide bonds
  • Not all ubiquitinated proteins are marked for degradation. In fact, ubiquitination may communicate changes to protein activity, location, or interactions.
  • Either a single ubiquitin molecule (monoubiquitylation) or a chain of ubiquitin (polyubiquitylation) can be added to the protein.
• Pathway
  1. Ubiquitination: addition of ubiquitin to the ε-amino group of lysine residues of a substrate protein; occurs in three stages
     • Activation: performed by ubiquitin-activating enzymes (E1s)
     • Conjugation: performed by ubiquitin-conjugating enzymes (E2s)
     • Ligation: performed by ubiquitin ligases (E3s)
  2. Degradation
     • Polyubiquitinated proteins are recognized by proteasomes.
     • Proteins are broken down into peptides via hydrolysis of peptide bonds.

Some cases of Parkinson disease have been linked to defects in the ubiquitin-proteasome system.

Lysosomes

• Foreign proteins are endocytosed into cells and form an endosome.
• Endosomes merge with lysosomes.
• Lysosomal hydrolases break down proteins into peptides via hydrolysis of peptide bonds.

Examples of diseases associated with aberrant proteolysis

There are many diseases associated with aberrant proteolysis; this list is not exhaustive.

• Conditions that lead to increased tissue protein breakdown
  • Chronic inflammatory diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus, Sjögren disease)
  • Diabetes mellitus
• Conditions caused by increased protein breakdown
  • Emphysema ; and α1-antitrypsin deficiency
  • Pancreatitis ; and possibly resulting exocrine pancreatic insufficiency
  • Malignancy induced cachexia
    • Pro-inflammatory cytokines (e.g., IL-1, IL-6, and TNF-α) released from tumor cells induce ubiquitination and proteasomal degradation of cellular proteins → degradation of myosin chains in skeletal muscle cells → increased muscle catabolism
    • TNF-α activates the extrinsic pathway of apoptosis
• Conditions caused by accumulation of damaged or misfolded proteins/peptides (see “Protein misfolding” in “Translation and protein synthesis” for more details)
  • Age-related neurological diseases/neurodegenerative diseases (e.g., Alzheimer disease, Parkinson disease, Huntington disease)
  • Prion-related conditions (e.g., Creutzfeldt-Jakob disease)
  • Amyloidosis
  • Myotonic muscular dystrophy
  • Cardiovascular diseases
  • Inflammatory responses and autoimmune diseases
  • Malignancy

References:^[8]