Chapter 39: Proteolysis Flashcards
What is the breakdown of proteins or peptides into amino acids by the action of enzymes? What enzymes do this and what type of rxn is it?
- Proteolysis
- Proteases —> Peptide bond hydrolysis to cleave proteins
**use H2O to break it up
**polypeptides can be cleaved enzymatically OR chemically
Importance of proteolysis
- Elimination of misfolded and damaged proteins (Sickle cell, prion dx)
- Regulation of cellular metabolism
- The generation of active proteins: cleavage of a precursor to an enzyme (proteases, lysosome) or proteasome/ubiquitin pathway thus activating them
- Recycling of amino acids (purine/pyrimidine salvage pathways)
If a damaged protein IS NOT REPAIRED, it is degraded in specialized organelles like _________ or the ___________ pathway
- lysosomes
- ubiquitin/proteasome pathway
How are proteases categorized?
By their active sites
**What type of proteases include plasmin, tissue plasminogen activator (TPA), urokinase (thrombolytic activity) and
trypsin, chymotrypsin (digestion)
Serine proteases: have serine residue at the active site
What type of proteases needs a zinc ion at active site? Example enzymes: carboxypeptidase A, collagenases
Metalloproteases (Zinc proteases)
What type of protease is cathepsin B? What residue does it have in its active site?
Cysteine proteases (Thiol proteases): cystine residue at the active site
***What type of proteases are pepsin, HIV protease, renin? What residue do they have at their active site?
Aspartic proteases (Aspartyl proteases): have aspartic acid residue at the active site
**What is the catalytic triad of the serine protease?
aspartate, histidine, and serine
Different serine proteases have different substrate specificities
Functions: digestion, blood clotting, complement system
Serine protease steps:
1. _________ attracts the H+ from the serine hydroxyl, allowing the Ser-O- to attack the peptide bond.
2. This attachment to H+ is stabilized by binding to ___________
First acylation reaction
3. The nucleophilic ____________ attacks the substrate and forms a tetrahedral intermediate
4. Next, a covalent acyl-enzyme releases the _____________ fragment
Second deacylation reaction
5. A _________ molecule attacks the acyl-enzyme forming a second tetrahedral intermediate. This is followed by the release of the ___________ fragment.
- Histidine
- Aspartate: stabilizes His-H+
- Serine
- C-terminal
- water
- N-terminal
**Overall
1. Histidine-H+ allows Ser-O- to attack —> 1st tetrahedral intermediate —> C-terminal fragment released by acyl-enzyme
2. Water attacks acyl-enzyme —> 2nd tetrahedral int and release of N-terminal fragment
__________ and _________ proteases use histidine to act as a proton-withdrawing group (H+) so they can attack the peptide bond (nucleophilic attack via their functional group)
________ and __________ proteases use water as the nucleophile rather than a functional group of the enzyme itself
- Serine and cysteine
- Aspartyl and metalloproteases
Serine proteases used in thrombolytic therapy: Coagulation (Secondary hemostasis)
Circulating coagulation factors undergo a cascade of proteolytic reactions to generate __________, which forms a mesh that stabilizes platelets in the clot.
- fibrin
Blood clotting factors are activated BY proteolysis
Serine proteases used in thrombolytic therapy: Coagulation (Secondary hemostasis)
Fibrinolysis occurs AFTER primary and secondary
_____________ to help the clot to contract/degrade. To dissolve fibrin, ____________ MUST be activated which in turn activates _____________.
- Hemostasis
- Plasminogen
- Plasmin (can also activate this)
***Activators of plasminogen?
Activators: activated plasminogen activates plasmin to degrade fibrin
1. Tissue Plasminogen Activator (TPA) → activated by fibrin binding
2. Urokinase → found in urine and cells lining renal tubes to lyse fibrin deposits there
3. Factor XIa, XIIa, Kallikrein
***Inhibitors of plasmin? Inhibitor of fibrin degradation?
Inhibitors of plasmin: stop plasmin from degrading fibrin
1. Alpha2-antiplasmin
2. Alpha2-macroglobulin
Inhibitor of fibrin degradation:
1. Thrombin-activated fibrinolysis inhibitor
**What is the use of drugs to break up or dissolve blood clots called?
Thrombolytic therapy
What are Alteplase, Urokinase, Streptokinase (not a serine protease) used for?
Thrombolytic agents used for tx of acute pulmonary embolisms: break up the clot
Streptokinase: activates plasminogen to break up the clot
**THESE ARE NOT anti-coagulants. The clot MUST be present for them to work on it
**What are inactive substances (usually proteins) that are activated by a protease cleaving off a repressing subunit? Where are they most commonly found?
- Zygmogens
- Stomach and pancreas: used for digestion
IMPT Zymogen activation in the pancreas:
- Formation of ______________ is the MOST IMPORTANT initiating step. This is activated by the enzyme ______________ that is secreted in the duodenum.
- TRYPSIN (serine protease)
- Entropeptidase (trypsinogen —> trypsin)
IMPT Zymogen activation in the pancreas:
Once trypsin is formed, it activates what 4 enzymes via protease activity?
- Chymotrypsin
- Elastase
- Carboxypeptidase
- Lipase
I TRYPted over ELASTic and broke my CHYna, LIP and CAR
What is the protease of choice for mass spectrometry (MS)-based proteomics to identify unknown proteins in a sample?
Trypsin: cleaves C-terminal of Arg and Lys resulting in a + charge on the C-terminus
IMPT Zymogen activation in the stomach:
- Exposure to WHAT causes plasminogen to activate and cleave itself to form active pepsin? This breaks the salt bridges between what residues?
- LOW pH OF THE STOMACH
- The Low pH breaks salt bridges between positively charged Lys and Arg and negatively charged Glu and Asp residues
**Attachment of what (a small protein found in all species) marks a protein for degradation via proteasomes?
ubiquitin
What are protein complexes that degrade unneeded or damaged proteins by proteolysis?
Proteasomes: present in cytoplasm and nuclei of eukaryotic cells, associate with centrosomes, cytoskeletal networks and the outer surface of the ER
The process of adding ubiquitin to the target protein:
- Ubiquitin undergoes an ATP requiring reaction to form a thioester bond with WHAT enzyme?
- Ubiquitin is then transferred to a reactive cysteine on WHAT enzyme?
- What enzyme then transfers ubiquitin to a lysine residue on the target protein?
- Several ubiquitin molecules are added to lysine residues and the polyubiquinated protein is transported to what?
- E1: ubiquitin activating enzyme**
- E2: ubiquitin conjugating enzyme**
- E3: ubiquitin-ligase enzyme**
- 26S proteosome
Degradation of target protein in the proteosome:
- In the proteasome, the polyubiquitinated protein is lysed into what (2 things)?
- What are the chymotrypsin-like, Trypsin‐like (TL) and Peptidylglutamyl peptide hydrolyzing (PGPH) parts of the proteosome?
- amino acids and small peptides
small peptides are further lysed by cytosolic proteases - The proteolytic actives AKA the active parts of the proteosome
***The target proteins for ubiquitin are rich is WHAT 4 amino acids? These proteins are rapidly degraded because they contain sites for what rxn?
- Proline (P), Glutamate (E), Serine (S), Threonine (T)
PEST - Phosphorylation: method to terminate activated proteins
What is a proteins half life determined by?
Amino terminal residues
***In cancer, _____________ is necessary for metastasis and angiogenesis. The use of _____________ inhibitors can reduce the invasive and metastatic capabilities of tumor cells.
**1. proteolysis
**2. protease inhibitors
**the imbalance of proteases and anti-proteases leads to many diseases like cancer
**HIV/AIDS
1. Why are proteases important in HIV/AIDS? What do they release? 2. What do protease inhibitors block the action of? Effect?
3. In AIDS, proteolysis of polyproteins is necessary for AIDS infected cells to do WHAT?
**1. They allow for HIV replication and AIDS development: HIV protease releases enzymes and proteins for viral RNA packaging.
- HIV protease enzymes: stops HIV virus from multiplying/stops HIV life cycle —> DOES NOT cure HIV, it just lowers the viral count and slows progression of HIV
-competitive inhibitors of HIV proteases are used in cocktail therapy and must be taken for life
**3. Spread virions
Review of cell parts/functions
What is the method in which proteins reach their target organelles? Why is location of translation important?
- Protein sorting
- Proteins translated in cytosol are transported to DIFFERENT sites than those translated on surface of ER
**Protein sorting methods:
- How do proteins that are made in the cytosol enter the nucleus?
- How are proteins transported across an organelles membrane?
- How are proteins that are translated in the ER redistributed to other organelles?
- Nuclear pore complexes: selective access
- Translocators: primarily used for chloroplasts, mitochondria, peroxisomes and ER
- Transport vesicles
Purpose of signal sequences? How can proteins end up in DIFFERENT cell locations?
- Signal sequences: an AA sequence that determines where a protein is transported to
- By “swapping” signal sequences —> allows for RELOCATION of a protein to end up in a different cell location (knock sideways)
Nuclear transport: NPCs
- Where are nuclear pore complexes (NPC) located? ***Why are they important for transport into the nucleus?
- Located in the nuclear envelope (lipid bilayer around nucleus)
***2. THEY ARE THE ONLY WAY MOLECULES CAN ENTER OR EXIT THE NUCLEUS
Nuclear transport: NPCs
**1. What type of molecules need signal sequences to enter/exit the cell?
**2. Protein import requires WHAT type of signal sequence?
3. Vs export?
- Macromolecules
small, water-soluble molecules freely cross the nuclear envelope and DONT req NPCs - Import: Nuclear localization signals (NLS): most common is pro-lys-lys-lys-arg-lys
- Export: Nuclear export sequence (NES)
What is the most abundant GTPase out of the 5 in the superfamily?
Superfamily of 5: Ras, Rho, Rab, Arf, **Ran (most abundant)
GTPases: affinity for GDP & GTP
1. What is bound to GTPase when it is active (ON)?
**2. When on, what stimulates GTP hydrolysis by GTPases to release a phosphate (P transfer)?
- What is bound to GTPase when it is inactive (OFF)?
**4. What removes GDP from the GTPase and replaces it with GTP?
- GTP = ON —> will interact with GAP
**2. GAP (GTPase activating proteins): causes GTP —> GDP + P via hydrolysis - GDP = OFF —> NO signal generated
**4. GEF (guanosine nucleotide exchange factors): GDP —> GTP (active GTPase)
IMPT
1. Where is RanGAP localized to?
2. What is bound to Ran in the cytoplasm?
3.. Where is RanGEF localized to?
3. What is bound to Ran in the nucleus?
- RanGAP: Cytoplasmic side of NPC, stimulates hydrolysis GTP —> GDP + P
- Ran-GDP
- RanGEF: nucleoplasm side of NPC, GDP —> GTP
- Ran-GTP
Nuclear transport: nuclear export sequence (NES)
1. What binds to the NES of the cargo protein?
2. What stabilizes the interaction above?
**2. Once through the NPC, what causes the hydrolysis of RanGTP in the cytoplasm and what is released?
**3. RanGDP wither adds to the concentration of the cytoplasm or is transported back to the nucleus by what?
- Exportin
- RanGTP: stabilizes the exportin-cargo protein interaction
- RanGAP: hydrolyzes RanGTP —> RanGDP to release exportin and cargo protein
- Ntf2: Once in nucleus, RanGEF dissociates Ntf2 and convert RanGDP —> RanGTP
Nuclear transport: Nuclear localization sequence (NLS)
**1. What binds to the NLS of the cargo protein?
**2. What flexible filamentous proteins does it jump between to travel through the NPC channel to the nucleoplasm?
**3. Once through the NPC, what releases the cargo protein in the nucleoplasm?
- Importins (Karyopherins)
- FG (phegly) Nups
- RanGTP: releases the cargo protein from karyopherin (importin)
**The remaining RanGTP-importin can travel back through the NPC and be hydrolyzed by RanGAP on cytoplasmic side (RanGTP —> RanGDP + importin)
Post-translational import into organelles: mitochondria
- What TWO protein translocators are used for import into the mitochondria?
- Aside from protein translocators, mitochondrial transport requires what (2 things)?
**3. What unfolds the protein for transport into the matrix of the mitochondria? - Once in the matrix, what cleaves the signal sequence and what refolds the protein
- TOM20/22 —> TOM40 —> TIM —> mitochondrial matrix
a. TOM: translocase of outer membrane
b. TIM: translocase of inner membrane - ATP, a cleavable N-terminal signal sequence
- Heat shock protein 70 (HSP70)
- Protease: cleaves, **chaperones: REFOLD
**mitochondria can make their own proteins but most come via import from the cytosol
Protein import into the ER: usually an entry pt for proteins with other destinations
- What are the 2 types of proteins that enter the ER?
- Proteins generally enter the ER WHILE they are being synthesized, what is this called?
- Water-soluble proteins (end up secreted or on the inside of an organelle), transmembrane proteins (end up in different membranes)
- Co-translational transport
Co-translational transport: soluble proteins
**1. What cytosolic protein binds to the ER signal sequence and directs the protein/ribosome complex to the ER membrane?
**2. Once at the ER membrane, where does the complex bind?
3. When the signal sequence (bound to receptor) reaches the translocator, what is the result? What is displaced?
**4. At the protein translocator, a signal peptidase cleaves WHAT end of the protein?
**5. What end of the protein is captured by chaperones to fold it **6. What side of the ER does the protein end up in?
- Signal recognition particle (SRP)
- SRP receptor
- The sequence causes the protein translocator to OPEN and attach to it, displacing the SRP
- N-terminal signal sequence
- C-terminus
- ER lumen
co-translational import of a SINGLE TRANSMEMBRANE region (simple membrane protein):
- What contains the start and stop sequences for insertion into ER membrane?
- Where is the signal sequence for ER localization?
**3. What HALTS translocation into the ER lumen?
IMPT4. Where do the regions of the SIMPLE transmembrane protein reside
- Membrane protein mRNA: the start and stop sequences signal the protein translocator
- N-terminal signal sequence (similar to soluble proteins)
- Stop-transfer sequence
- N-terminus: ER lumeN; C-terminus: Cytosol (C=C)
co-translational import of a MULTIPLE TRANSMEMBRANE region (protein):
- What is used to initiate transcription that is NOT CLEAVED OFF (forms an alpha helix)?
- When does translocation stop?
- Internal Start-transfer signal
- Once a stop-transfer signal is reached —> can occur multiple times to “stitch” a protein into the membrane
***Importance of having the proper sequences/signals in protein translocation?
ONCE translocation initiates IT CANNOT BE REVERSED —> the protein MUST enter the organelles correctly by having the proper start-transfer signals and signal sequences OR IT CANNOT BE REVERSED
IMPT Where do most proteins in the ER transport to?
Golgi apparatus
Soluble proteins that are retained in the ER lumen contain WHAT on their C-terminus? If they do not have this, where are they transported?
- ER retention signal: Lys-Asp-Glu-Leu (KDEL)
- Other parts of the endomembrane system (consists of ER, golgi, lysosomes, vacuoles)
What is responsible for molecular traffic (transporting proteins across the membrane) between the endomembrane system (membrane-enclosed compartments)?
Vesicles
**What are coated vesicles?
Vesicles that BUD from membranes & are coated with specific proteins
What buds from the Golgi apparatus on their way to the cell membrane OR from the plasma membrane inward? **BEST studied vesicles
Clathrin coated vesicles (CCV)
For protein transport from ER —> golgi:
Cis side of golgi vs trans side
Cis side: receives proteins from ER
Trans side: sends proteins in vesicles to the cell membrane
Vesicle formation:
1. What captures cargo receptor-ligand complex?
2. What binds to this to initiate budding?
3. What binds and causes the neck of the bud to constrict and pinch off? What does this require?
**4. Newly formed vesicles lack WHAT?
**5. What mediates the fusion of the vesicle and organelle membrane to deliver the protein?
- Adaptin
- Clathrin: binds to the adaptin
- Dynamin proteins: GTP hydrolysis required
- they are NAKED because adaptin and clathirin is removed —> free to be carried by motor proteins to the target organelle
- SNARES
