Biochemistry Notes on Post-Translational Modifications

Final Exam Format

• 65 Questions (out of 60)

• Proteins (Lectures 1-8): 30%

• Carbohydrates (Lecture 9): 10%

• Lipids (Lecture 10 and 11): 10%

• Nucleic Acid (Lectures 12-25): 50%

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Objectives

• What are post-translational modifications?

• Give examples of post-translational modifications. Where have you seen such examples?

• What are some functions of post-translational modifications?

• USE READING MATERIAL PROVIDED FOR REFERENCE

Post-Translational Modifications

• Chemical modifications of proteins after translation.

• Most proteins undergo this process.

Why Post-Translational Modification?

• Adds functionality

• Effects targeting

• Regulates activity

• Increases mechanical strength

• Changes recognition

Steps After Translation

• After translation, several additional steps may be considered as part of the complete protein biosynthetic process:

  1. Covalent modification

     • a: the N-terminus e.g myristoylation

     • b: the C-terminus e.g GPI anchor

     • c: amino acid residues (side chains) e.g acetylation, phosphorylation

     • d. Glycosylation

  2. Noncovalent modifications: addition of co-factors.

  3. Folding using chaperones

  4. Ubiquitination

Modifications Involving Peptide Bonds

• Modifications involving the peptide bond (peptide bond cleavage or limited proteolysis): usually carried out by enzymes called peptidases or proteases:

  • activation of proenzymes (digestive enzymes, blood clotting cascade, complement activation etc.) and prohormones (insulin).

  • production of active neuropeptides and peptide hormones from high molecular weight precursors

  • removal of signal sequences

Modifications Involving Amino Acid Side Chains

• disulfide cross-linking

  • $2 <br>ewline SH + O=O \rightarrow S-S + H_2O$

Processing of Preproinsulin to Insulin: Proteolytic Cleavage

• Pre-pro-insulin is synthesized as a random coil on membrane-associated ribosomes

• After membrane-transport, the leader sequence (yellow) is cleaved off by a protease, and the resulting pro-insulin folds into a stable conformation.

• Disulfide bonds form between cysteine side chains.

• The connecting sequence (red) is cleaved off to form the mature and active insulin molecule.

• (signal sequence)

• Question: Does proinsulin or insulin run faster on a polyacrylamide gel?

Deformylation and Acetylation

• Deformylation of formyl methionyl proteins

• Acetylation of cytoplasmic proteins of eukaryotes (60-90%) Ex: acetylation of histones
  $R-CH-C-H<em>2N + N-Acetyltransferases \rightarrow R-CH-C-NH-COCH</em>3$

• Myristoylation of N-terminus
  $\text{Myristoyl CoA} + \text{Protein} \rightarrow N-\text{MyristoylProtein} + CoA$

Histone Tails are Post-Translationally Modified at a Specific Amino Acid: Acetylation

• Histone Acetylase (HAT) adds an acetyl group to lysine in the histone tail.

• Reversible process: Histone Deacteylase (HDAC) removes the acetyl group.

Attachment of Membrane Anchors & Amidation

• Attachment of membrane anchors

• Amidation, especially peptide hormones

• Usually removal of an N-terminal Gly

Lipidation

• palmitate + cysteine of the protein

• farnesyl group + cysteine

• GPI + carboxyl terminal of protein

• Modifications involving the carboxy terminus: LIPIDATION

Vitamin C-Dependent Modifications

• Proline + α-ketoglutarate + $O<em>2 \rightarrow$ 4-hydroxyproline + $CO</em>2$ + succinate

• Vitamin C (ascorbic acid) required for this reaction

• $Vit C Fe^{+3} \rightarrow Fe^{+2}$

• Prolyl Hydroxylase + $Fe^{+2}$

• Hydroxylation is a form of post-translational modification.

Phosphorylation

• Phosphorylation of hydroxyls by kinases (serine, threonine, tyrosine)

• Phosphorylation is reversible and is used in many pathways to control activity.

• Enzymes that add a phosphate to a hydroxyl side chain are commonly called kinases.

• Enzymes that remove a phosphate from a phosphorylated side chain are called phosphatases.

• Example: Phosphorylation of the CTD in RNA Polymerase II (eukaryotes) is a form of post-translational regulation and post-translational modification.

Glycosylation

• Covalently attached to the polypeptide as oligosaccharide chains containing 4 to 15 sugars

• Sugars frequently comprise 50% or more of the total molecular weight of a glycoprotein

• Most glycosylated proteins are either secreted or remain membrane-bound

• Glycosylation is the most abundant form of post- translational modification

• Glycosylation confers resistance to protease digestion by steric protection.

• Important in cell-cell recognition

Types of Glycosylation

• There are two basic types of glycosylation which occur on:

  • asparagines (N-linked)

  • serines and threonines (O-linked)

N-Linked Glycosylation

• N-linked glycosylation on asparagine (Asn) side chains:

  • an alkali-stable bond between the amide nitrogen of asparagine and the C-1 of an amino sugar residue

  • occurs co-translationally in the endoplasmic reticulum (ER) during synthesis (can occur post- translationally as well)

  • oligosaccharide complex is transferred to polypeptide by specific enzymes.

  • target sequence or consensus site on protein is Asn- X-Ser/Thr further processing in Golgi apparatus

  • Examples: Heavy chain of immunoglobulin G (IgG)

O-Linked Glycosylation

• O-linked glycosylation on serine (Ser) or threonine (Thr) side chains

  • a bond between the hydroxyl group of serine or threonine and an amino sugar

  • carried out by a class of membrane-bound enzymes called specific enzymes which reside in the endoplasmic reticulum (ER) or the Golgi apparatus

  • Example: Blood group antigens on erythrocyte surface: N-linkage O-linkage

Prosthetic Group Attachment

• Prosthetic group attachment (heme)

• Heme attached to histidine group via Fe

Non-Covalent Modifications

• Addition of metals and cofactors.

• 50% of proteins contain metals

• Metals have structural roles

• Examples:

  • Calcium (Ca++): very important intra-cellular messenger, i.e. calmodulin

  • Magnesium (Mg++): ATP enzymes

  • Copper (Cu++), Nickel (Ni+), Iron (Fe++)

  • Zinc (Zn++): Zinc finger domains are used for DNA recognition E.g Nuclear Hormone Receptors such as Estrogen Receptor.

Zinc Finger Domain

• A zinc finger domain: $Zn^{++}$ is bound by two cysteine and two histidine residues.

• Zinc finger domains interact in the major groove with three consecutive bases from one strand of duplex B-form DNA.

• Zinc Finger attached to nuclear receptors such as Estrogen Receptor

Chaperonin-Assisted Protein Folding

• Hydrolysis of several ATP molecules is required

• Unfolded polypeptide folds with the help of chaperones, using ATP

• $n ATP \rightarrow n ADP + n P_i$

Ubiquitination

• Ubiquitination- A type of Post-translational Modification

  • most highly conserved protein known.

  • 76 residues polypeptide that marks a protein for degradation.

  • Ubiquitination requires energy

  • ubiquitin is an 8-kDa polypeptide consisting of 76 amino acids that is appended to the $NH_2$ of lysine in target proteins via the C-terminal glycine of ubiquitin.

  • Following an initial monoubiquitination event, the formation of a ubiquitin polymer may occur, and polyubiquitinated proteins are then recognized by the proteasome that catalyzes the degradation of the ubiquitinated protein and the recycling of ubiquitin.

  • Controls a number of processes: DNA repair, gene transcription, cell cycle, quality Control of newly produced proteins.