Protein folding,maturation & targeting

Secretory pathway: signal peptide recognition

• Glycosylation is important– alters the properties of proteins– changing their stability– solubility– act as recognition signals– influence cell-cell interactions

• Glycosylation site– by the type of amino acid– its neighboring sequence in the protein– the availability of enzymes & substrates for the reactions.

Glycosyltransferases in Eukaryotic Cells

Biosynthesis of N-Iinked oligosaccharides

N-Iinked gIycosyIation

• Asn-X-Thr/Ser• (Man)5(GlcNAch-pyrophosphoryl-dolichol• Reorientation

• Cotranslational• Glycosidases • Classes of N-linked oligosaccharides

– High-mannose type– Complex type

• with a larger variety of sugars and linkages

• Common core region (GlcNAc2Man3)

O-linked glycosylation

• O-glycosylation is posttranslational • Only residues on the protein surface serve as

acceptors – (GalNAc-Ser/Thr)

• Stepwise addition of sugars• Heterogeneity in glycoproteins is common– the types and amounts of glycosyltransferases

MEMBRANE AND ORGANELLE TARGETING

• Protein transport uses carrier vesicles

Sorting signals

• Mannose 6-phosphate– I-cell disease

• C-terminal KDEL (Lys-Asp-Glu-Leu) sequence• Polypeptide-specific glycosylation and

sulfation • Polysialic acid modification

Targeting of enzymes to Iysosomes

• The secretory pathway to – Lysosomes– Plasma membrane– Secretion from the cell– Proteins of the ER and Golgi apparatus

• N-terminal signal sequence• Internal signal sequence• Hydrophobic anchoring sequences

Mitochondrial proteins

• N-terminal presequences • A positively charged α-helix

Nuclear Targeting

• Localization signals– Clusters of basic amino acids

• Peroxisome targeting – Carboxy-terminal tripeptide, Ser-Lys-Leu (SKL)– N-terminal nonapeptide

• dual location– Contain two targeting signals – Gene duplication and divergence – Alternative transctiption initiation sites

Targeting

• Alternative splicing • Alternative translation initiation

Maturation events (Posttranslational Modifications)

• Some are very common– Partial proteolysis• Either end or from within

– in the ER and Golgi » Insulin

• others are highly restricted • Reversible modifications– regulate protein activity

• familial hyperproinsulinemia • a common means of enzyme activation– Zymogen

Maturation of human proinsulin.

• Amino acids can be modified after incorporation into proteins– Permanent – Reversible

• Amino-termini– Removal – Acetylarion – Alteration

• Myristic or palmitic acid – G-proteins

– Pyroglutamyl formation – Elongation

• Disulfide bond formation– a means of localization – Cysteine modification

• S-palmitoylation

• Multiple sulfatase deficiency – Unmodified sulfatases are catalytically inactive

• Lysine ε-amino groups – Acetylation & methylation– Isopeptide linkage– amide linkages • Biotin

• Serine & threonine hydroxyl– Glycosylation – Phosphorylation

• Tyrosine residues – Growth factor receptors – Oncogenes

• Protein kinases & protein phosphatases• ADP-ribosylation on– Diphthamide– Arginine & cysteine

• Formation of y-carboxyglutamate – II,VII, IX, and X – Blocked by coumarin derivatives

Modified Amino Acids in Proteins

Collagen biosynthesis requires many posttranslationalmodifications

• Hydroxylation of proline and lysine residues – In the Gly-X-Y- sequence • at Y positions

– Lysine hydroxylation• Interchain cross-linking and for glycosylation

Collagen structure

Selected Disorders in Collagen Biosynthesis and Structure

Regulation of translation

• At the initiation stage– Phosphorylation of initiation factors– Global regulation • Phosphorylation of elF-2a.

– no eIF-2a-GTP is available for initiation

• Heme-regulated kinase • double-stranded RNA dependent kinase

– Interferon

• Initiation factor eIF-4e is activated by phosphorylation

Regulation of translation

• Regulation of translation of mRNAs

– iron response element (IRE)– 5'-IRE– 3'-IRE

• Polypyrimidine tract

RNA silencing and interference

• Small RNA molecules – Micro-RNAs• represses translation bur does not affect mRNA stability

– Small interfering RNA (siRNA) • Cleavage and inactivation of the target mRNA

PROTEIN DEGRADATION AND TURNOVER

Ubiquitin-dependent proteolysis

• Destabilizing PEST sequences (rich in Pro, Glu, Ser, & Thr)

• Ubiquitin-interacting motif • N-end rule

• Polyubiquitinylation is necessary to signal proteolysis

ATP and ubiquitin-dependent Protein degradation.

Model of the proteasome

• Lysosomes – from the extracellular environment– Some intracellular protein • Recognition of a specific peptide sequence

Other Proteolytic Systems

• Caspases (cysteine aspartyl proteases)– Stress-induced apoptosis

• thiol proteases(calpains)