Functions and Dysfunctions of Protein Processing

Question 1

Why is it important to study the difference in protein synthesis between prokaryotes and eukaryotes? (2)

Answer 1

1. to be able to selectively inhibit prokaryotic protein synthesis (clinical use - molecular basis for development of abx)
2. to be able to understand the mechanism of human diseases (research use - allow for the development of treatment and/or prevention)

Question 2

Streptomycin binds to ____ subunit to disrupt initiation of translation

• interferes w/ binding of fmet-tRNA and impairs initiation

Answer 2

30S (prok)

Question 3

Shiga toxin and Ricin binds to the ____ subunit to disrupt elongation

• blocks entry of aminoacyl-tRNA ribosomal complex

Answer 3

60S (euk)

Question 4

Clindamycin and erythyromycin bind to the ____ subunit to disrupt translocation of the ribosome

Answer 4

50S (prok)

Question 5

Tetracyclines bind to the ____ subunit to disrupt elongation

• blocks entry of aminoacyl-tRNA to ribosomal complex

Answer 5

30S (prok)

Question 6

peptidyl transferase activity is housed in the _____ subunits

Answer 6

large

Question 7

diphtheria toxin inactivates _______ and inhibits _______

• interferes with ribosomal translocation

Answer 7

• EF2-GTP
• elongation

Question 8

Chloramphenicol inhibits ______ ________ activity and impairs ______ bond formation

Answer 8

• peptidyl transferase
• peptide

Question 9

Cycloheximide inhibits ______ ________ (euk) and impairs _______ bond formation

Answer 9

• peptidyl transferase
• peptide

Question 10

• causes premature chain termination (prok/euk)
• resembles 3’ end of the aminoaceylated-tRNA
• enters the A site and adds to the growing chain
• forms a puromycylated chain, leading to premature chain release
• more resistant to hydrolysis
• stops the ribosome from functioning

Answer 10

puromycin

Question 11

What are the 4 types of genetic mutations?

Answer 11

1. silent: does not change amino acid
2. missense: changes amino acid in protein with either no effect on protein function or a protein with vastly different function
3. nonsense (null mutation): codon changes into a stop codon causing premature chain termination, protein either degraded or formed as a truncated version
4. frameshift: one or more nucleotides are deleted or added into ORF, out of frame causes change in the codon sequence and consequently alteration in the amino acid sequence of the protein (e.g. Duchenne muscular dystrophy, β-thalassemia)

Question 12

• arises from a missense mutation of 6th codon in the allele of gene for human β-globin (HBB), subunit of adult hemoglobin
• mutation changes GAG to GTG which changes glutamic acid (negatively charged, hydrophillic) to valine (hydrophobic)
• alters conformation of HbA which causes it to aggregate and form rigid, rod-like structures
• deforms the RBC’s into sickle-like shape
• deformed erythrocytes have poor oxygen capacity and tend to clog capillaries, further restricting blood supply to tissues

Answer 12

sickle cell anemia

Question 13

• large in frame and out of frame deletions to the dystrophin gene leads to partially or non-functioning dystrophin protein
• OOF deletions result in little/no expression of dystrophin protein, gives rise to the severe form of condition
• presented in 1:3,500 males
• leads to muscle wasting (wheelchair by age 12, death by respiratory failure within 10 years, sx onset by years 3-5)
• in frame deletions result in expression of truncated forms of dystrophin, giving rise to a milder for of the disease called Becker ______ _______

Answer 13

Duchenne muscular dystrophy

Question 14

What are the 2 types of protein sorting?

Answer 14

cytoplasmic and secretory

Question 15

• type of protein sorting for proteins destined for cytosol, mito, nucleus, and peroxisomes
• protein syn begins and ends on free ribosomes in cytoplasm
• absence or presence of certain translocation signals play role in final targeting

Answer 15

cytoplasmic pathway

Question 16

• type of protein sorting for proteins destined for ER, lysosomes, plasma membranes, or for secretion
• translation begins on free ribosomes but terminates on ribosomes sent to ER
• proteins have N-terminal hydrophobic α-helix ER targeting signal sequences present on the first 20 amino acid residues of the polypeptide

Answer 16

secretory pathway

Question 17

How does the cytoplasmic pathway know where to send mito, nucleus, and peroxisome proteins?

Answer 17

• mito: N-terminal hydrophobic α-helix signal peptide
• nucleus: KKKRK signal sequence (Lys, Arg rich)
• peroxisome: C-terminal SKL signal sequence (Ser, Lys, Leu)

Question 18

How does the secretory pathway know where to send ER lumen, lysosome, secretory, and membrane proteins?

Answer 18

• ER lumen: C-terminal KDEL retention signal (Lys, Asp, Glu, Leu)
• lysosome: mannose 6-phosphate signal group (I-cell disease)
• secretory: tryptophan-rich domain signal region, absence of retention motifs
• membrane: N-terminal apolar region (stop transfer sequence)

Question 19

How are mitochondrial proteins imported?

Answer 19

• unfolded proteins protected by binding to chaperones such as heat shock proteins 70 (HSP70)
• translocation sequences recognized by transporters present in the mito membrane
• proteins are passed across TOM and TIM

Question 20

How are nuclear proteins imported?

Answer 20

• via nuclear pores
• small proteins able to pass through specific pores
• large proteins (>40 kDa) require nuclear localization signals
• four continuous basic residues (Lys and Arg)

Question 21

What is the process of secretory pathway protein processing?

Answer 21

• each protein has ER-targeting signal peptide that is 15-60 amino acids at N terminus of protein
• signal peptide: 1 or 2 basic amino acids (Lys or Arg) near N terminus, extremely hydrophobic sequence (10-15 residues) on C terminus of basic residues
• signal recognition particle (SRP) binds to ER targeting signal and ribosome during translation
• SRP wraps itself around ribosome-mRNA-peptide complex, tethering it to ER membrane and halting translation temporarily
• translation resumes when protein is directed into ER lumen
• enzymes on luminal side cleave the signal to release the protein
• protein undergoes post translational mods in ER and/or golgi
• additional signal sequences serve to guide each protein to final location

Question 22

• disease caused by lysosomal proteins not tagged with M6P
• defective or missing GIcNAc phosphotransferase (enzyme that adds M6P to lysosomal hydrolases), enzymes are not phosphorylated and hence not sorted into vesicles and not delivered into lysosomes
• enzymes are carried to cell surface and secreted (into blood)
• high plasma levels of lysosonal enzymes
• by 6 months: failure to thrive, developmental delays, physical manifestations
• developmental delays more pronounced than cognitive delays
• hepatomegaly, splenomegaly, defective heart valves
• death by age 7, usually due to congestive heart failure or recurrent respiratory tract infections

Answer 22

inclusion cell disease (I cell disease)

Question 23

Describe the process of protein folding in terms of small and large proteins

Answer 23

• small proteins can fold into native conformations spontaneously
• large proteins cannot and are at risk for aggregation and proteolysis
• large proteins need auxiliary proteins, chaperones which protect the protein and help fold into proper tertiary structure
• other proteins, chaperonins, have barrel shaped compartments that admit unfolded proteins and catalyze their folding in an ATP-dependent manner

Question 24

• type of post-translational processing
• converts inactive forms of protein to active enzymes by unmasking active site (e.g. trypsinogen and chymotrypsinogen to trypsin and chymotrypsin)
• converts nascent precursor proteins to mature ones (e.g. proinsulin to insulin)

Answer 24

proteolytic cleavage

Question 25

What are the 4 types of post-translational mods?

Answer 25

• glycosylation
• phosphorylation
• disulfide bond formation
• acetylation

Question 26

• type of post-translational modification that involves a covalent linkage to an amine (NH3+)
• residue affected: Lys
• acetyl group donor is usually acetyl CoA
• histones are acetylated and deacetylated on their N terminal lysines, critical for gene regulation
• rxn catalyzed by histone acetyltransferase (HAT) and deacetylation by histone deacetylase (HDAC) enzymes
• regulates protein function
• also facilitates crosstalk w/ other types of modifications such as phospho, methylation, ubiquination, etc for dynamic control of cellular signaling
• pattern of histone mods are heritable (epigenetics)

Answer 26

acetylation

Question 27

• type of post-translational modification for proteins that are destined for cell surface and plasma
• covalently linked to sugar residues in the ER lumen
• functional group: (OH)
• residue affected: Ser and Thr for O-linkage, Asn and Gln for N-linkage
• O-links are formed with the hydroxyl groups of Ser or Thr residues
• N-links are always with Asn

Answer 27

glycosylation