Download Document

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 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)