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 (GlcNAc 2 Man 3 )

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 posttranslational modifications 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)