L4 Post-Translational Modifications in Biomolecules

Post-Translational Modifications

PTMs are chemical changes made to a protein after it has been synthesised by ribosomes.

Locations of PTMs

Usually occur in the endoplasmic reticulum, Golgi apparatus, or cytoplasm.

Post-Translational Modifications (PTMs)

Chemical modifications that fine-tune a protein's properties, activity, location, and stability.

Phosphorylation

- Addition of a phosphate group (PO4^3) to a protein after it is made; one of the most common and important PTMs in cells.

- Protein-OH + ATP → Protein-O-PO3; -Kinase transfers the phosphate from ATP to the hydroxyl group on the protein.

Glycosylation

Attachment of sugar chains to proteins.

Ubiquitination

Tags proteins for degradation.

Acetylation

Addition of an acetyl group to proteins.

Methylation

Addition of a methyl group to proteins.

Prenylation

Attachment of a lipid group to proteins.

Myristoylation

Attachment of myristoyl fatty acid to proteins.

Palmitoylation

Attachment of palmitoyl fatty acid to proteins.

Phosphate donor

ATP (adenosine triphosphate) is the phosphate donor in phosphorylation.

Serine (Ser)

Most common phosphorylation site; small side chain, abundant in proteins.

Threonine (Thr)

Similar chemistry to serine but bulkier and less common.

Tyrosine (Tyr)

Less abundant but critical in signalling; aromatic ring affects local structure.

Receptor Kinases

Membrane-bound receptors that detect extracellular signals and convert them into intracellular phosphorylation events.

Ligand Binding

Triggers autophosphorylation of the receptor, activating downstream signalling proteins.

Epidermal Growth Factor (EGF)

Binding to EGFR causes a conformational change in the dimerised receptor, activating signalling pathways.

Receptor Tyrosine Kinases (RTKs)

Most receptor kinases; involved in cell growth, differentiation, and metabolism.

Downregulation of Receptor Kinases

Occurs through endocytosis and degradation, phosphatases removing phosphate groups.

Autophosphorylation

Phosphorylation of a receptor by itself after ligand binding.

Signalling Cascade

A series of biochemical events triggered by receptor activation that leads to multiple signalling pathways being activated.

Phosphorylation Motifs

Short amino acid sequences around the phosphorylation site that kinases recognise.

Function of Phosphorylation Motifs

It acts like a 'postal address' that kinases use to recognise where to dock and transfer the phosphate.

Phosphorylated Residue Location

The phosphorylated residue is usually in the centre of the motif.

Kinase Specificity

Kinases can't just phosphorylate any Ser/Thr/Tyr - they have specificity based on shape and charge.

Example of Phosphorylation Motif

[R/K]-X-[S/T], where R/K arginine/lysine, X = any amino acid, S/T = serine/threonine.

Another Example of Phosphorylation Motif

[S/T]-X-X-[E/D].

Phosphorylation Cascades

Sequential activation of multiple kinases in a chain where one kinase phosphorylates the next, amplifying the signal.

Example of Phosphorylation Cascade

Ras → Raf → MEK → ERK.

What Can Be Phosphorylated?

Enzymes, receptors, transcription factors, structural proteins, transport proteins, and scaffold proteins.

Prevalence of Glycosylation

Affects >50% of all proteins in humans.

Functions of Glycosylation

1. Aids protein folding and stability

2. Mediates cell-cell recognition

3. Signalling

4. Modulates immune recognition.

Catalysts of Glycosylation

Catalysed by glycosyltransferases, reversed partially by glycosidases.

General Reaction of Glycosylation

Protein + Sugar-Nucleotide → Glycoprotein + Nucleotide.

Where is found Mannose and what are its functions?

Often found in the core structure of N-linked glycans and essential for forming the initial core sugar structure.

Role of Mannose

Important for protein quality control in the ER, signaling whether a glycoprotein is correctly folded.

Sialic Acid

Typical terminal sugar in glycans that adds a negative charge to the polypeptide.

Influence of Sialic Acid

Influences circulation half-life of glycoproteins in blood and protects glycoproteins from clearance.

Where is N-acetylglucosamine found?

Found in core linkages of N-linked glycans and O-linked glycosylation initiation.

O-GlcNAcylation

A dynamic PTM performed inside the cytoplasm and nucleus that regulates several cellular processes.

O-linked Glycosylation

Attachment of a sugar(s) to the hydroxyl oxygen of serine.

N-linked Glycosylation

Glycan is added to asparagine residues.

Characteristics of O-glycans

Generally smaller but more diverse than N-glycans.

What Can Be Glycosylated?

Proteins like secreted proteins, membrane-bound receptors, extracellular matrix proteins, and lipids such as glycolipids.

Function of Ubiquitination

Regulates protein fate through degradation by the proteosome and influences protein trafficking, DNA repair, and signalling.

Ubiquitination Reaction

Involves an enzyme cascade that attaches ubiquitin to lysine residues on substrate proteins.

Activation

Ubiquitin is activated by an E1 enzyme (ubiquitin-activating enzymes) using ATP.

Conjugation

Activated ubiquitin is transferred to an E2 enzyme (ubiquitin-conjugating enzyme).

Ligation

An E3 ligase facilitates transfer of ubiquitin from E2 to the substrate protein.

E1 (Ubiquitin-activating enzyme)

Activates ubiquitin by forming a high energy bond with its C-terminal glycine.

E2 (Ubiquitin-conjugating enzyme)

Receives activated ubiquitin from E1 and carries ubiquitin to the substrate.

E3 (Ubiquitin ligase)

Recognises specific substrate proteins and catalyses the transfer of ubiquitin from E2 to substrate lysines.

What Can Be Ubiquitinated

Proteins with exposed lysine residues can be ubiquitinated.

Substrates for Ubiquitination

Includes misfolded/damaged proteins for degradation, regulatory proteins controlling cell cycle, DNA repair, signal transduction, and histones involved in chromatin remodelling.

Where Does Ubiquitination Take Place

Ubiquitination occurs primarily in the cytoplasm and nucleus.

Target Proteins

Can be soluble, membrane-bound, or nuclear in nature.

What is the 26S Proteosome?

The 26S Proteosome is a large protein complex responsible for degrading ubiquitinated proteins.

Recognition by Proteosome

Recognises proteins tagged with polyubiquitin chains, especially those linked via lysine.

Deubiquitinating Enzymes (DUBs)

Remove attached ubiquitin molecules before degradation, freeing ubiquitin for reuse.

Protein Unfolding

Removal of the ubiquitin allows the proteins to be unfolded and fed into the proteosome core.

Proteolytic Sites

Cleaves the protein into small peptides (~3-25 amino acids) within the proteosome core.

Protease Activities

The proteosome has multiple protease activities (chymotrypsin-like, trypsin-like, etc.) for broad substrate cleavage.

Why are Release of Small Peptides

The small peptides are released into the cytoplasm or nucleus for further degradation by peptidases.

Immune System Priming

Small peptides may be up-taken by immune cells and used to prime the immune system to recognise unknown antigens.

Informing the Immunoproteosome

Viral/bacterial proteins, abnormal tumour proteins, or normal turnover proteins can inform the immunoproteosome to protect the cell/host.