Functions & Dysfunctions of Protein Processing

Question 1

What are the start and stop codons?

Answer 1

AUG (Methionine); UAG,UAA, UGA

Question 2

What are the ribosomal and eukaryotic subunits in translation?

Answer 2

Prokaryotes:
30S and 50S

Eukaryotes:
40S and 60S

Question 3

What are the prokaryotic translation inhibitors?

Answer 3

1) Streptomycin - binds to 30s, impairs initiation
2) Clindamycin/Erythromycin: binds to 50s, blocks ribosome translocation
3) Tetracycline - binds to 30S, impairs elongation
4) Chloramphenicol - inhibits peptide transferase activity, impairs peptide bond formation

Question 4

Eukaryotic Translation Inhibitors

Answer 4

1) Shiga toxin & Ricin - bind to 60s, blocks aminoacyl-tRNA
2) Diptheria toxin: no ribosomal translocation - inactivates GTP-bound EF-2
3) Cycloheximide - impairs peptide bond formation

Question 5

What is an elongation Inhibitor?

Answer 5

Puromycin - premature chain termination

-Stops ribosome from functioning

Question 6

What are the types of mutations (w/ clinical example)?

Answer 6

1) Silent - no AA change
2) Missense - changes AA in a protein with no impact on function (Sickle cell)
3) Nonsense - codon becomes stop codon - chain termination
4) Frameshift - one or more nucleotides deleted or inserted —- HUGE change in sequence (Duchenne Muscular Dystrophy)

Question 7

Sickle Cell Anemia

Answer 7

• Missense mutation
• Glutamic acid changes to Valine
• HbA conformational change, RBC become rigid, rod-shaped
• RBCs = poor 02 capacity, clog capillaries

Question 8

Duchenne Muscular Dystrophy

Answer 8

• Frameshift mutation on in-frame and out-of-frame Dystropin gene (Dystropin…think “dystrophy”)
• No expression of dystrophin
• In males, muscle wasting, death within 10 years
• Mild form = Becker muscular dystrophy

Question 9

Protein Sorting Pathways

Answer 9

1) Cytoplasmic - cytosol, mitochondria, nucleus, peroxisomes. Starts and ends on free ribosomes
2) Secretory - ER, lysosomes, plasma membranes, secretion. Translation starts on free ribosomes, ends on ribosomes sent to the ER

Question 10

Cytoplasmic Pathway destinations and translocation signals

Answer 10

1) Cytoplasm - None
2) Mitochondria - N-terminal hydrophobic alpha helix
3) Nucleus - KKKRK sequence
4) Peroxisomes - C-terminal SKL sequence

Question 11

Secretory Pathway destinations and translocation signals

Answer 11

1) ER lumen - C-terminal KDEL
2) Lysosomes - Mannose 6 Phosphate (you know this)
3) Secretion - Tryptophan-rich domain
4) Membranes - N-terminal apolar region

Question 12

Explain the Mitochondrial Protein Import process

Answer 12

1) Translation sequence recognized by TIM and TOM — inner and outer membrane proteins, respectively
2) Proteins moved across membrane

Question 13

Explain the Nuclear Protein Import Process

Answer 13

• Proteins move through nuclear pores
• Large proteins need NUCLEAR LOCALIZATION SIGNALS
• Four basic residues (Lysine & Arginine)

Question 14

What are the steps in the Secretory Pathway?

Answer 14

• Proteins have:
  
  • ER signal peptide
  • 15-60 AA at N-terminus

1) SRP (signal recognition particle) binds to ER signal and ribosome and then wraps around ribosome-mRNA complex
2) This halts translation and SRP guides/docks to translocon (protein receptor)
3) Once in ER lumen, translation resumes, enzymes cleave signal to release protein
4) Protein undergoes PTMs

Question 15

What is I-Cell Disease?

Answer 15

• Deficiency in Mannose-6-Phosphate
• Proteins not target to lysosomes
• Results in:
  1) Failure to Thrive
  2) Developmental delays
  3) Abnormal skeletal development, hepatomegaly
  4) Death usual by age 7

Question 16

Explain protein folding

Answer 16

• The idea that small proteins can spontaneously fold into tertiary conformations
• Large proteins can’t, need help from CHAPERONES

Question 17

What are chaperones and chaperonins?

Answer 17

• Molecules that help large proteins fold into their tertiary structure without risk of:
  1) aggregation
  2) proteolysis

-Chaparonins are barrel-structure molecules that use ATP to fold large, unfolded proteins

Question 18

Name a chaperone and chaperoning protein

Answer 18

Chaperone - HSP70 (heat shock protein)

Chaperonin - HSP60

Question 19

Type of PTMs

Answer 19

1) Protein folding
2) Proteolytic cleavage
3) Covalent modifications

Question 20

Explain Proteolytic cleavage

Answer 20

-Reveals active site of enzymes, making them active (i.e. trypsinogen -> trypsin)

Question 21

What are some types of covalent modifications?

Answer 21

1) Glycosylation
2) Phosphorylation
3) Disulfide bond formation
4) Acetylation

Question 22

Glycosylation (detail, functional group, residue affected) —– CLINICAL?????

Answer 22

1) 0-glycosylation; OH; Ser, Thr
2) N-glycosylation; Acid-Amide; Asn, Glu

-Cataract formation, similar to galacticol

Question 23

Acetylation (detail, functional group, residue affected, impacts)

Answer 23

• Covalent amine linkage
• Amine
• Lysine
• Catalyzed by HAT and HDAC, important for GENE REGULATION
• Impacts are heritable (epigenetics)

Question 24

Disulfide Bond formation (detail, functional group, residue affected, impacts)

Answer 24

• oxidation leading to cysteine residue linkage
• SH (sulfahydryl)
• Cys

-Stabilize proteins, formation occurs in ER lumen

Question 25

Phosphorylation (detail, functional group, residue affected, impacts)

Answer 25

• Phosphate added via ester bond
• OH
• Ser, Try, Thr, Asp, His

-Important for enzyme activity, protein function, cell growth, cancer???

Question 26

Explain the PTMs of collagen

Answer 26

• Collagen biggest structural protein
• Modification necessary for collagen
• Vitamin C = important for LYSYL and PROLYL HYDROXYLASES
  
  • Defects = skin, bone, join problems —- Ehlers-Danlos

Question 27

Ehlers-Danlos Syndrome

Answer 27

• From defect in Prolyl hydroxylases In collagen formation
• overly flexible joints
• blood vessel walls, intestines or uterus may rupture

Question 28

Alzheimer’s Disease (AD)

Answer 28

• Memory loss, cognitive dysfunction as a result of:
  1) amyloid precursor protein (APP) breakdown to amyloid beta peptide
  2) Hyperphosphorylation of Tau

Brain aging = sporadic form

Question 29

Parkinson’s disease (PD)

Answer 29

1) a-synuclein (AS) protein deposit as Lewy bodies in neurons (familial form)
2) Cause reduced availability of dopamine (from tyrosine precursor)

Brain aging = sporadic form

Question 30

Huntington’s Disease (HD)

Answer 30

• Mutation in Huntington gene = CAG repeats
• Leads to abnormal Huntington protein, which misfiles and aggregates
• Cell death in basal ganglia

Question 31

Creutzfeldt-Jakob Disease

Answer 31

• “Prion Protein” misfolding
• TRANSMISSIBLE, like catching a cold
• Transmissable Spongiform Encephalopathies (holes in brain like a sponge)