Functions & Dysfunctions of Protein Processing Flashcards
How does streptomycin inhibit prokaryotic translation?
Streptomycin binds to the 30S subunit to disrupt INITIATION of translation. Interferes with the 30S subunit and 50S subunit interacting.
How does clindamycin inhibit prokaryotic translation?
Clindamycin (and erythromycin!) bind to the 50S subunit to disrupt TRANSLOCATION of the ribosome.
How does erythromycin inhibit prokaryotic translation?
Erythromycin binds to the 50S subunit to disrupt TRANSLOCATION of the ribosome.
How does tetracycline inhibit prokaryotic translation?
Tetracyclines bind to the 30S subunit to disrupt ELONGATION. It blocks entry of the aminoacyl-tRNA to ribosomal complex and impairs elongation. (the aminoacyl t-RNA would come in during the first step of elongation normally. This occurs higher up in the pathway before clindamycin and erythromycin bind.
How does chloramphenicol block translation?
Inhibits peptidyl transferase activity and impairs peptide fond formation. The peptide bond-forming with help of peptidyl transferase is the 2nd step of elongation.
why should we look at the differences in protein synthesis between prokaryotes and eukaryotes?
- To selectively inhibit prokaryotic protein synthesis (this is the molecular basis of abx)
- To understand the mechanism of human disease
What 2 subunits make up a prokaryotic ribosome
30S and 50S
what 2 subunits make up a eukaryotic ribosome
40S and 60S
Where is peptidyl transferase activity housed
In the large (bigger number!) subunits
How does GTP-bound EF-2 relate to ribosomal translocation?
The GTP bound EF will get hydrolyzed and help to translocate the ribosome. This is the 3rd step of elongation.
What does Diptheria toxin inactivate?
EF2-GTP. This will INHIBIT elongation.
How does puromycin cause premature chain termination?
Puromycin forms a puromycylated chain causing premature chain release. This premature chain is more resistant to hydrolysis. Stops ribosome from functioning.
Where does puromycin enter to inhibit elongation?
It enters the A site and adds to the growing chain.
What is a silent mutation?
A mutation that does not change the amino acid
What is a missense mutation?
Changes the amino acid in the protein with:
A. No effect OR
B. Vastly different function
What is a nonsense mutation?
Changes a codon into a STOP codon.
In a nonsense mutation, what does the stop codon cause?
Premature termination of the chain. The protein will either be degraded or formed as a truncated version.
What is a frameshift mutation?
One or more nucleotides are deleted or inserted into the open reading frame.
What does out of frame (frameshift mutation) cause?
OOF causes a change in the codon sequence and thus alters the amino acid sequence of the protein. (DMD)
How does the hydrophobic nature of Val relate to sickle cell anemia?
The hydrophobicity of Val from Glutamate (hydrophilic) changes the HbA conformation and causes it to aggregate and form rigid rod structures
This deforms the RBC into “sickle” shape. They have poor O2 capacity and clog capillaries.
What mutation causes sickle cell anemia?
A missense mutation of the 6th codon in the allele of the gene for human beta globin (HBB)
In relation to DMD, where do OOF and in-frame deletions come in?
OOF (out of frame): results in little to no expression of dystrophin = severe DMD
In-Frame: Expression of truncated forms of dystrophin - a milder form of DMD called “Becker muscular dystrophy”
How does DMD affect different body systems?
Leads to muscle wasting (musculoskeletal system, respiratory problems, cardiac myopathies)
Why do only males get DMD
It is a sex-linked gene
What’s the benefit of having a truncated protein in disease?
While it’s still bad, it will be a milder version of the disease because you do have SOME expression instead of NO expression. I.e. DMD
What are the 2 protein sorting pathways
- Cytoplasmic
2. Secretory
what kinds of proteins use the cytosolic sorting pathway
Proteins destined for the cytosol, mitochondria, nucleus, and peroxisomes
what kinds of proteins use the secretory sorting pathway
Proteins destined for the ER, lysosomes, plasma membranes, or for secretion
What happens if there is no sorting signal?
In the cytoplasmic pathway, if there is no translocation signal it will go to the cytoplasm
Translocation signal for the mitochondria
N-terminal hydrophobic alpha-helix signal peptide
MitochondriA has ALPHA-helix
Translocation signal for the nucleus
A KKKRK signal sequence. This is Lys and Arg rich.
Translocation signal for peroxisomes
C-terminal SKL signal sequence
Translocation signal for the ER lumen
C-terminal KDEL retention signal (Lys, Asp, Glu, Leu)
Translocation signal for lysosomes
Mannose 6-phosphate signal group (I-Cell disease)
Translocation signal for secretion
Trp-rich domain signal region. Absence of retention motifs.
“Sec-rich-on”
Translocation signal for membranes
N-terminal apolar region (stop transfer sequence)
*like the mitochondria signal (Alpha helix b/c of the A) but membranes aren’t as cool so they don’t have the alpha helix, they only have the APOLAR
what is special about proteins with a Rough ER sequence
They each of an ER-targeting signal peptide composed of 15-60 amino acids at N terminus of the protein
What does KDEL mean
It is the translocation signal for proteins destined for the ER lumen. K=Lysine D=Aspartate E=Glutamate L=Leucine
What organelle/(s) are involved in I-cell disease?
Lysosomes. Tagging of lysosomal proteins with mannose 6P is defective. There are high numbers of lysosomal enzymes in the blood.
Sx of I-Cell disease
- Failure to thrive
- Developmental delay
- Abnormal skeletal development
- Coarse facial features
- Claw hands
- Cornea clouding
- Hepatomegaly
- Splenomegaly
- CHF
- Defective heart valves
What does HSP70 do?
HSP70 (heat shock proteins 70) are chaperones that unfolded proteins bind to for protection. Nuclear-coded mitochondrial proteins bind to this and then go through TOM and TIM into the mitochondria.
To cross the nuclear membrane, some proteins need ____
a nuclear ionization signal if they are large (>40 kDa). If they are small, they can pass through specific pores.
What kinds of residues do proteins need for nuclear import?
4 continuous basic residues (Lys and Arg)
What pathway is SRP (signal recognition particle) needed?
Secretory pathway.
What does an SRP do?
- Bind to ER-targeting signal and ribosome during translation
- Wraps itself around the ribosome-mRNA-peptide complex, causes tethering, and halts translation (for now)
- Once directed to the ER lumen, translation begins again
- Luminal enzymes cleave the signal to release the protein
- Protein undergoes post-translational modifications in ER/Golgi
- Addition signal sequences guide protein to final destination
Explain trypsinogen and chymotrypsinogen to trypsin and chymotrypsin in relation to proteolytic cleavage
Proteolytic cleavage, a type of post-translational processing, converts inactive forms to active enzymes by unmasking the enzyme site. Trypsinogen and chymotrypsinogen have a “masking” sequence that is removed to reveal the active enzyme (trypsin and chymotrypsin) by proteolytic cleavage.
Is protein folding part of post-translational processing?
Yes. Small proteins can fold into native conformations spontaneously. Large proteins cannot and are at risk for aggregations and proteolysis.
What do large proteins require in post-translational processing?
Chaperones to protect the protein and help it fold into its proper tertiary form (ex. HSP70)
What do chaperonins do?
Admit unfolded proteins and catalyze folding in ATP dependent manner (ex. HSP60)
What are the 4 covalent post-translational modifications
- Glycosylation
- Phosphorylation
- Disulfide bond formation
- Acetylation
Acetylation modification
Definition: covalent linkage to amine
Functional group: AMine (-NH3)
Residue affected: Lys
The acetyl group donor is acetyl coA
Glycosylation modification
There is O and N glycosylation.
O: Hydroxyl (-OH), Ser&Thr
N: Acid amide (-CONH2), Asn&Gln
“O (oh sir) N that glyc sauce”
Phosphorylation modification
Phosphate linked via esterification
HYdroxyl (-OH)
Ser, Tyr, Thr, Asp, His
Disulfide bond modification
Oxidation to achieve covalent linkage of cysteine residues
Sulfhydryl (-SH)
Cysteine
What kinds of residues and kinases are present in phosphorylation?
Phosphorylation uses the activity of serine and threonine and tyrosine kinase (transfers a phosphate onto a protein from ATP). To inactivate the enzyme, use a protein phosphatase to remove the phosphate from the active enzyme.
What enzyme catalyzes disulfide bond formation
Protein disulfide isomerase
Where does the formation and reorganization of disulfide bonds occur
In the ER lumen
What is acetylation catalyzed by?
Histone acetyltransferase (HAT)
What is deacetylation catalyzed by?
Histone deacetylase (HDAC)
where are proteins typically acetylated?
Lysine residues
which vitamin is essential for the activity of lysyl and prolyl hydroxylases
Ascorbic Acid (Vit. C)
What are some post-translational modifications of collagen?
- Lysines modified to form 5-hydroxylysine
- Lysines deaminated to aldehydes
- Prolines hydroxylated to hydroxyprolines
what are some results of lysyl hydroxylase defects
skin, bone, and joint disorders (Ehlers-Danlos, Nevo, Bruck, Epidermolysis Bullosa Simplex)
Alzheimers Disease sx
Loss of memory, cognitive function, and language
Parkinsons disease sx
Fine motor control issues
Huntingtons disease sx
Loss of movement, cognitive issues, psych issues
Crutzfeldt-Jacob sx
Memory failure, behavior changes, lack of coordination, extremity weakness, coma
What happens to proteins in Alzheimers
Amyloid precursor protein (APP) breaks down to form Amyloid beta peptide –> forms extracellular plaques
What happens to Tau protein in Alzheimers
Tau (neurofibrillary tangles, intracellular) gets hyperphosphorylated
Mutations in APP and Tau cause what form of AD?
The familial form of Alzheimer’s (early onset)
What is the common denominator in sporadic form of AD?
Brain aging aka not genetic
What aggregates are in PD?
An aggregation os alpha-synuclein (AS) protein form insoluble fibers which lead to Lewy bodies
Where are Lewy Bodies located and what does this lead to
Located in the dopaminergic neurons in the substantia nigra. Leads to reduced availability of dopamine.
What does a mutation in the Huntington gene cause
CAG triplet repeats. Leads to polyglutamine repeats which form intramolecular H-bonds which misfold and aggregate. Selective death of cells in the basal ganglia cause sx.
what’s unique about Creutzfeldt-Jacob disease?
It is transmissible. Infection by misfolded proteins converts normal proteins to misfolded form. Key word=prion.
