Day 10, Lecture 2: Proteins II: Post-translational modification, protein trafficking and clearance

Question 1

Difference in Translation from Free cytoplasmic Ribosomes and ER bound ribosomes

Answer 1

• Free Cytoplasmic ribosomes
  
  • Proteins of cytoplasm, nucleus, and mitochondria
• Endoplasmic Reticulum Bound ribosomes
  
  • Secreted, plasma membrane, ER, Golgi apparatus, and lysosomal proteins

Question 2

Does modification of proteins significantly increase the functional diversity of genes

Answer 2

yes

Question 3

Proteolytic cleavage

Answer 3

• Occurs in most proteins
  
  • simplest form
    
    • removal of the initition methionine present in most proteins
    • proteins secreted form the cell undergo cleavage
• Many proteins are synthesized as inactive precursors
  
  • activation occurs by limited proteolysis under proper conditions
• Preproprotein→Proprotein→Protein
• 2 major modes:
  
  • ​Removal of a short peptide form the N- or C-terminal region of a polypeptide, leaving a shortened molecule that folds into the active protein
  • Cleavage of polypeptides into segments generatign more than one active protein

Question 4

Trypsinogen

Answer 4

• Produced in the pancreas
• secreted into the intestinal lumen
• Fxn:
  
  • digestion of proteins in food

Question 5

Biomedical Implications of Proteolytic cleavage

Answer 5

• activation in the correct location:
  
  • Trypsin needs to be active in the early part of the small intestine for effective digestion
  • Glucagon is required in the systemic circulation (regulation of glucose metabolism)
• Hereditary Pancreatitis
  
  • Alteration in the gene coding for trypsin can lead to activation within the cell and organ injury
  • Pancreatic inflammation (Pancreatitis) due to the destructive nature of activated trypsin within the pancreas

Question 6

Chemical modification of proteins

Answer 6

• Addition of small chemical groups
  
  • phosphorylation
• addition of sugar side chains
  
  • N-linked glycosylation
  • O-linked glycosylation
• addition of fatty acid side chains
• Addition of peptide groups
  
  • Ubiquitination
• Modification of amino acids within the protein

Question 7

Question 8

Phosphorylation of proteins

Answer 8

• Common protein modification
  
  • Serine>Threonine>Tyrosine (1000:100:1)
• Serves to regulate the biologic activity of a protein (transient, reversible) or to target a location within the cell
• controls activity, structure, and cellular localization of both enzymes and many other types of proteins.
• Kinases
  
  • enzymes that phosphorylate proteins
• Phosphatases
  
  • Enzymes that remove phosphates

Question 9

What types of effects on protein confirmation can phosphorylation cause?

Answer 9

• Phosphorylation event can affect the protein that is modified in three important ways
  
  1. because each phosphate group carries two negative charges, the enzyme-catalyzed addition of a phosphate group to a protein can cause a major conformational change in the protein by, for example, attracting a cluster of positively charged amino acid side chains. This can, in turn, affect the binding of ligands elsewhere on the protein surface, dramtically changing the proteins activity. When the seond enzyme removes the phosphate group, the protein returns to its original conformation and restores its initial activity
  2. an attached phosphate group can form part of a strucutre that the binding sites of other proteins recognize. For example, The SH2 domain binds to a short peptide sequence containing a phosphorylated tyrosine side chain.
  3. the addition of a phosphate group can mask a binding site that otherwise holds two proteins together, and thereby disrupt protein-protein interactions. As a result, protein phosphorylation and dephosphorlyation very often drive the regulated assembly and disassembly of protein complexes

Question 10

What are the two major intracellular trafficing routes

Answer 10

Question 11

Secretory route

Answer 11

Question 12

Protein Signal Sequence

Answer 12

• Determines the site of protein synthesis
  
  • Free-cytoplasmic or ER-bound ribosomes
• 20-25 amino acids (mainly hydrophobic)
• Amino (N-terminal) end of polypeptide chain (first amino acids synthesized in a polypeptide)

Question 13

Protein Translocator

Answer 13

• Donut-shaped transmembrane protein of the RER, forms a pore in the ER memrbane; pore allows the growing polypeptide chain to enter the ER

Question 14

Signal peptidase

Answer 14

• Signal sequence is cleaved prior to completion of translation by the signal peptidase (located on the inner surface of the ER membrane)

Question 15

What is a preprotein

Answer 15

Protein with a secretory signal attached

Question 16

Cellular membrane proteins

Answer 16

• 20-residue stop-transfer sequences
  
  • arrest passage across the ER, become embedded in the membrane

Question 17

How a single pass transmembrane protein with a cleaved ER signal sequence is integrated into the ER membrane

Answer 17

Question 18

How integration of a double-pass transmembrane protein with an internal signal seqeunce into the ER membrane works

Answer 18

Question 19

insertion of the multipass membrane protein into ER membrane

Answer 19

Question 20

Post-translation processing in the secretory pathway

Answer 20

• Removal of signal sequence
• Partial proteolytic cleavage
• folding (and disulfide bond formation)
• Glycosylation
• Amino acid modification

Question 21

Glycosylation

Answer 21

• Common chemical modification of secreted and membrane bound proteins
• 2 general types
  
  • N-linked
    
    • Attachment through the amino (-N) group of the side chain of asparagine
  • O-linked
    
    • Attachment via the hydroxyl (-OH) group of serine or threonine
• Simple or complex with branching networks of 1000 sugars
• N-linked glycosalation promotes protein folding in two ways. First, it has a direct role in making folding intermediates more soluble, thereby preventing their aggregation. Second, the sequential modifications of the N-linked oligosaccaride establish a “glyco-code” that marks the progression of protein folding and mediates the binding of protein to chaperones and lectins

Question 22

What proteins are frequently modified by glycosylation and what are not

Answer 22

Question 23

Question 24

Question 25

Question 26

Glycoprotein Synthesis

Answer 26

• N-linked
  
  • Occurs in the lumen of the ER
  • Involves a lipid of the ER membrane (dolichol pyrophosphate)
  • Oligosaccharide is constructed on the lipid
  • transferred to the protein by a protein-oligosaccharide transferase in the ER
• O-linked
  
  • Occurs in the lumen of the Golgi
  • Oligosaccharide is constructed directly on the protein

Question 27

Answer 27

Question 28

Glycoprotein Transportation: Lysosomal Enzymes (acid Hydrolases)

Answer 28

Question 29

I-cell disease (lysosomal storage disease)

Answer 29

• Cuased by a deficiency of the ability to phosphorylate mannose
• Characterized by:
  
  • Course features
  • Hirsutism
  • small and delayed development
  • Flexion contractures of fingers, hips and knees
  • Facial features like Hurler’s, but clear corneas
  • Kyphotic posture
• death usually occurs by age 8
• Enzyme defect: deficiency
  
  • Metabolic defect: No N-linked oligosaccharides iwth Man-6-P are assembled on any lysosomal enzymes
  • Consequences:
    
    • No degradative enzymes packaged in lysosomes and all lysosomal enzymes are secreted

Question 30

What phenotype would be predicted for a mutation of an Asparagine on a lysosomal protein that is normally N-glycosylated?

Question 31

Localization (trafficking) of cytoplasmic proteins

Answer 31

• Proteins produced on free cytoplasmic ribosomes
• Contain a sorting signal
• Recognized by sorting signal receptor at target location
• Examples: import into nucleus, mitochondria, and peroxisome

Question 32

Nuclear localization of proteins

Answer 32

• Proteins produced on free cytoplasmic ribosomes
• Nuclear Localization Signal
  
  • Patch of Cationic amino acids (lysine and arginine) on the surface of the folded protein- not cleaved
• Recognized by nuclear import factor: importin-alpha
• Importin-beta transports NLS-importin-alpha into the nucleus through the nuclear pore
  
  • energy-dependent process

Question 33

Protein entry into cells

Answer 33

• 3 processes by which proteins enter cells
  
  • Phagocytosis
    
    • Performed by specialized cells
    • plasma membrane engulfs extracellular substances
    • internalized and fuses with lysosomes
  • pinocytosis
    
    • Nonselective uptake of fluid
    • Recycle the plasma membrane
  • Receptor-Mediated Endocytosis
    
    • Selective uptake of extracellular substances
    • Requires binding to a cell surface receptor

Question 34

Important functions of Receptor-mediated endocytosis

Answer 34

• Uptake of nutritive substances
• Waste disposal
• Mucosal transfer

Question 35

Question 36

Question 37

Protein degradation: what are the two major organelles involved in the degradation of proteins within the cell

Answer 37

• Lysosome
  
  • Extracellular proteins
  • Intracellular proteins within organelles
• Proteosome
  
  • Intracellular proteins

Question 38

Question 39

Question 40

Question 41

Question 42

Question 43

Question 44

Question 45

Question 46

Question 47