Post Translational Modifications

Define post translational modifications and a brief overview of what they regulate and when they occur.

PTMs are chemical modifications of a protein after its translation. They regulate activity, localization, and inter and intramolecular interactions. They can occur anytime in aprotein life cycle (immediately after translation, after folding, after the interaction with another protein, or signal for destruction).

Define proteome.

The entire complement of proteins, including PTMS that is produced by an organism or cellular system at a particular time and under defined conditions.

What is the theoretical max for a protein isoform?

2ⁿ where n is the # of PTMs

What are the three major types of PTMs?

Covalent modification, proteolytic cleavage, degradation of entire proteins.

What amino acid residues contain a hydroxyl group?

Ser, Thr, Tyr

What amino acids contain an amine group?

Lys, Arg, His

What amino acid contains a thiolate ion?

Cysteine

What amino acids have carboxylates?

Asp, Glu

What amino acids contain amides?

Asn, Gln

Describe phosphorylation.

A reversible PTM in which a phosphate group is added via kinase. It is incredibly important in signal transduction, cell death, cell growth, and cell cycle. Roughly one third of proteins in the human proteome are substrates for phosphorylation.

List the two primary ways that phosphorylation regulates protein function and cell signaling in two primary ways.

Inducing conformation changes that activate or inactivate catalytic activity. Or serve as a recognition site for domains in other proteins that specifically interact with phospho amino acid.

In an inactive kinase what does the activation loop do?

The activation loop adopts a conformation where it distorts the active site (aka phos SH2 in Src).

Describe the SH2 domain.

First recognized in Src and Fps kinases, is present in many many many oncogenes and signaling proteins. It binds pTry residues. It provides specificity and selectivity in recruitment of signaling proteins.

What is a characteristic of the conserved tyrosine binding motif?

A basic pocket.

What is lipidation?

reversible. A method to target proteins to membranes in organelles or in the plasma membrane. Occurs through the enzymatic addition of hydrophobic groups to proteins.

What are the three main types of lipidation?

Prenylation, N-myristoylation, palmitoylation.

Which one of the three lipidation types are the most dynamic and used by the cell to alter the subcellular localization?

Palmitoylation

What chemical group does calcium bind to in the blood clotting cascade, after Vitamin K oxidation?

Dual COO^-

Is glycosylation typically on the cell surface or cell interior?

Cell surface

What are the major functions of glycosylation?

1. proper protein folding

2. effect the stability/solubility of a protein

3. cell to cell adhesion

4. trafficking

5. mask hydrophobic regions that may be binding sites

What are the types of protein glycosylation?

N-linked glycosylation, O-linked glycosylation, glycosaminoglycans on proteoglycans.

Describe O linked glycosylation

O-GlcNAc, single sugar addition, on sites of Ser/Thr phos, commonly on transcription factors and regulatory proteins.

O-mannose, O-fucose, O-glucose modifications for cell surface adhesion modifications

Describe N linked glycosylation

Begins with the co translational enzymatic addition of a 14 sugar precursor onto an asparagine side chain at NXT or NXS motifs in the E

Describe glycosaminoglycans on proteoglycans.

Sugar combinations attached to linkers on protein backbones to generate highly charged bottle brush like structures in the extracellular space.

Describe Asn-linked glycosylation.

N glycans co translationally linked to Asn in the ER then processed by glycosyltransferases and glycosidases in the ER and Golgi. Function in protein folding and structure stability, cell to cell adhesion, immune cell recognition and activation, protein trafficking, and receptor ligand recognition.

Describe how N glycosylation contributes to compartmentalization.

Oligosacharyltransferase recognizes a consensus sequence and transfers 14 14-sugar precursor glycan. Three terminal glucoses are trimmed and this functions as a quality control mechanism to monitor folding, as they are only removed upon proper folding in the golgi.

Describe what chaperones are and how they work.

When proteins are misfolded in the ER as part of the unfolded protein response. A glucose is then transferred to the oligomannose N glycans that signals a chaperone response to attempt to correct folding.

What are glycosaminoglycans?

Variably sulfated repeating disaccharide polymers attached to protein scaffold. Function as lubricants and mechanical support in cartilage, skin and cornea. Cell migration, adhesion, differentiation, signaling, anti coagulation, and wound healing and repair are all important functions of these.

Provide a basic summary of Ubiquitination

Reversible PTM that involves the attachment of the C-terminal glycine of the group to a lysine residue on the target protein.

List some of the uses for ubiquitination.

1. targets cellular proteins for proteasomal degradation

2. alters subcellular localization

3. modulates intermolecular interactions

4. alter enzyme activity and conformation

5. protein protein interaction

What removes ubiquitin?

Deubiquitinates

What can monoubiquitylation mean?

Histone regulation

What can multiubiquitylation mean?

Endocytosis

What can polyubiquitylation mean?

Lys48- degradation

Lys63- DNA repair