Learning Objectives :

1. Name 3 different types of post-translational modification of proteins

   2. Be able to describe what exactly occurs during this modification

   3. Be able to describe how it affects the protein's function

Hydroxylation

1. Definition : Adds hydroxyl ( -OH ) groups , primarily to proline or lysine residues.

2. What Occurs :

   • Example: Hypoxia-Inducible Factor ( HIF-α )

     • Under normal oxygen levels ( normoxia ) :

       • Prolyl hydroxylase enzymes hydroxylate HIF-α.

       • Hydroxylation enables binding to von Hippel-Lindau ( VHL ) protein

       • HIF-α is ubiquitinated and degraded by the proteasome.

     • Under low oxygen ( hypoxia ) :

       • Hydroxylation does not occur.

       • HIF-α stabilizes , binds with HIF-β , and activates gene transcription for hypoxia response.

3. Effect on Protein Function :

   • Regulates protein stability.

   • Hydroxylation under normoxia ensures degradation of HIF-α , preventing hypoxia response.

   • Lack of hydroxylation under hypoxia allows HIF-α to activate survival genes.

• Normoxia = HIF gets hydroxylated ➡️ becomes ubiquinated ➡️ proteosomal degradation

• Hypoxia = HIF doesn't get hydroxylated ➡️ becomes nuclear transcription factor ➡️ hypoxia response elements ( HREs ) are transcribed

Ubiquitination

1. Definition : Attaches ubiquitin proteins to lysine residues on target proteins.

2. What Occurs :

   • Marks proteins for degradation by the 26S proteasome.

   • Example : HIF-α ubiquitination under normoxia.

     • Ubiquitination is triggered by hydroxylation and VHL protein binding.

3. Effect on Protein Function :

   • Regulates protein turnover by targeting them for degradation.

   • Maintains cellular homeostasis by removing damaged or unnecessary proteins.

Lipidation

1. Definition : Adds lipid groups ( e.g., palmitate, myristate ) to cysteine residues.

2. What Occurs :

   • Anchors proteins to membranes, altering localization and function.

   • Examples :

     • AMPA Receptor ( GluA1-4 ) :

       • Lipidation at TM2 ➡️ Golgi retention.

       • Lipidation at TM4 ➡️ Membrane insertion; enables PKC phosphorylation.

     • Potassium Channel ( Kcnma1 ) :

       • Lipidation at S0-S1 loop ➡️ ER and Golgi exit.

       • Lipidation at STREX site ➡️ Regulates kinase activity ( e.g., PKA inhibition )

3. Effect on Protein Function :

   • Alters membrane localization , which impacts protein interactions and signaling.

   • Specific lipidation sites determine whether proteins are retained in organelles or inserted into membranes , influencing downstream activity.

Phosphorylation

1. Definition : Adds phosphate groups to serine, threonine, or tyrosine residues.

2. What Occurs :

   • Carried out by kinases and reversed by phosphatases.

   • Example: AMP-Activated Protein Kinase ( AMPK ) :

     • Activated under stress ( e.g., low ATP/high AMP )

     • Phosphorylates glucose transporters to increase glucose uptake

   • Energy regulation :

     • AMP rises during low ATP ➡️ Activates AMPK.

3. Effect on Protein Function :

   • Alters protein activity , stability , and interactions.

   • Phosphorylation of glucose transporters enhances glucose uptake , helping maintain energy balance under stress.

• Low ATP / High AMP ➡️ Activates AMPK ➡️ phosphorylates serine and threonine residues on glucose transporters ➡️ increases glucose import

Glycosylation

1. Definition : Addition of sugar groups ( glycans ) to proteins , primarily on extracellular domains.

2. What Occurs :

   • Facilitates cell-cell adhesion and protein-protein interactions.

   • Example - Sialic acid :

     • Negative charges repel but allow connections with positively charged glycans.

     • Example in podocytes: Glycosylation maintains podocyte-endothelial cell linkages.

3. Effect on Protein Function :

   • Enhances structural integrity and adhesion in cellular networks.

   • Modulates interactions with other proteins or cells , crucial for processes like immune responses and tissue stability.

Disulfide Bond Formation

1. Definition : Covalent linkage between two cysteine residues , forming disulfide bonds ( S-S )

2. What Occurs :

   • Stabilizes protein conformation by maintaining structural integrity.

   • Influenced by cellular redox states , which are regulated by reactive oxygen species ( ROS ) and antioxidants like glutathione.

   • Complex I and III of the mitochondrial electron transport chain can leak ROS , altering the redox environment and influencing disulfide bond formation.

     • This can lead to oxidative stress and misfolding if the redox balance is disrupted.

3. Effect on Protein Function :

   • Ensures correct protein folding and structural stability.

   • Misregulation can lead to misfolded proteins, resulting in dysfunction and disease.

   • Role of Glutathione : Glutathione maintains the proper redox environment for forming and breaking disulfide bonds.

     • It ensures that disulfide bonds are formed correctly by reducing incorrect linkages and preventing oxidative damage to cysteine residues.

SUMOylation

1. Definition : Addition of SUMO ( Small Ubiquitin-like Modifier ) proteins to target proteins.

2. What Occurs :

   • Modulates nuclear transport , transcriptional regulation , and DNA repair.

   • SUMO is attached to lysine residues , similar to ubiquitin.

3. Effect on Protein Function :

   • Alters protein localization and interactions.

   • Enhances or suppresses transcriptional activity , depending on the target.

Methylation

1. Definition: Adds methyl groups , primarily to lysine or arginine residues on histones.

2. What Occurs :

   • Regulates chromatin structure and gene expression.

   • Example: Histone methylation :

     • Specific methylation patterns can activate or repress gene transcription.

3. Effect on Protein Function :

   • Modulates gene expression by altering chromatin accessibility.

   • Plays a role in epigenetic regulation , influencing long-term cellular behavior.