Endocytosis and Protein Degradation Flashcards
- Describe two major routes for small volume endocytosis. How are these endocytic pathways related to viral infections and to bacterial ingestion by the macrophage?
1) Phagocytosis:
-In multicellular organisms is normally carried out by specialized cells in the blood, e.g.,
macrophages and neutrophils.
-These cells recognize foreign organisms like bacteria, engulf them, and deliver them to lysosomes for degradation.
-Macrophages and neutrophils also recognize apoptotic cells (one signal is
negatively charged phosphatidylserine that moves from the inner to the outer leaflet) and aged cells (roughly
10^11 of our red blood cells are phagocytosed
every day)
2)Pinocytosis:
-Of vesicles involves
small volumes, and usually is associated with specific uptake of ligands and receptors.
-Vesicles are typically formed by two mechanisms, either clathrin coat proteins or caveolae. Much more is known about the
clathrin coat pathway than that using caveolae
(EX for clathrin)
- Cholesterol and Control.
- When you eat cholesterol, you form LDL particles in the bloodstream.
- LDL particle binds to LDL receptor on cell surface; LDLRs are clustered in membrane pits where adaptor protein AP2 binds them + clathrin.
- Clathrin covers the nascent vesicle and dynamin pinches it off.
- This creates the vesicle.
- Soon after, clathrin coat dissembles and vesicle (early endosome) moves to late endosome and gets acidified –> pH drop. This exposes LDL particle to lysosomal lumen.
- LDL dissociates from LDLR at low pH and LDLR recycled to plasma membrane as LDL gets broken down.
- LDLR goes through several 10 minute cycles, with a lifetime of ~20 hours. This cycle happens even if the receptor is empty.
(EX Caveolae formation)
- They are little caves that form vesicles with caveolin protein (as opposed to coats) in the membrane. Important for lipid rafts.
- Encoded for by genes cav-1, cav-2, and cav-3.
- Cav-3 is predominantly found in skeletal and cardiac muscle, and mutations can lead to disease (like Limb-Girdle and Rippling Muscle Disease).
- Look different from clathrin-coated pits.
- Some viruses can actually be endocytosed via clathrin coated pits (like cholera).
- So cells can take up viruses by:
1) clathrin-mediated entry:
e. g. vesicular stomatitis virus – a problem for horses and cows in Colorado – sores and blisters on the mouth and in the throat – transmitted by flies – especially bad in 2014
2) fusion-entry:
e. g. HIV).
3) macropinocytosis-mediated entry:
(e. g. vaccinia virus and Ebola virus)
4) phagocytosis-like-mediated entry:
(e. g. herpes simplex virus)
5) phagocytosis mediated entry:
(e. g. bacteria)
6) caveolin-mediated entry:
(e. g. simian virus 40).
- Explain how quality control of protein synthesis ensured in the ER
- In the ER, quality control includes providing an optimized oxidizing environment, providing folding enzymes, like ERp57, providing chaperones, like BiP (from the Hsp70 family), and providing folding sensors (like UDP glucose: glycoprotein glucosyltransferase).
- ERp57 = thiol oxidoreductase that allows formation of disulfide bonds –>breaks improper ones and reforms proper ones
- BiP is a member of the Hsp70 family and uses ATP to help proteins fold properly
*Folding sensor pathway:
-monitor unfolded proteins and hold them in the ER until they fold properly or get shuttled to the degradation pathway.
-Calnexin (CNX) and calreticulin are lectins that bind the oligosaccharide chain on a protein if there is a glucose.
-While protein is bound, it can get fixed (like by ERp57) and try to fold properly.
Then glucose is removed by a glucosidase and protein is released.
-If it is folded properly, it can leave ER; if it’s not, glucosyltransferase puts another glucose on the protein so it gets bound by CNX/calreticulin again.
-Can go through several cycles of this, but eventually the protein ‘times out’ and is retrotranslocated out of ER for degradation.
- Describe two types of molecular chaperones.
So first of all whats the problem with having misfolded proteins? They are like that one bad influence in a friend group, not only are they fucking up but their hydrophobic parts can fuck up other proteins that were gonna be something one day and make a difference. #nevertrump anyways yeah they can make more aggregates happen.
But then chaperones show up and are like a role model that guides the protein into folding into a productive member of society ( i mean cell)
Hsp70:
-helps protein fold by binding to exposed hydrophobic patches and preventing aggregation
Hsp60:
- Complex of proteins; 2 barrels with cavity inside. This structure exists in bacteria –>sooo its old AF.
- Misfold will bind to regions at top of entrance of chamber (hydropbic interactions)
- Then moves inside and then a cap comes on (GroES = cap)
- ATP causes barrel to expand, pulls protein and then releases trying to change conformation
- The lid comes off, protein comes out whether its if right or not.
- The other chamber is empty but when the other one c comes out, it can also put some work in.
- If still fucked it will probs go back in.
-an isolation chamber into which misfolded proteins are fed to prevent their aggregation and to help them refold. (GroES = cap).
Chaperone proteins are a necessary part of quality control in the cytosol.
- Describe the proteasome, protein degradation, and the role of ubiquitin.
Protein Degredation General Info:
-Three major protein degradation pathways exist in eukaryotic cells:
1)the ubiquitin-proteasome system (UPS),
2)the lysosome (mentioned above as part of the endocytosis pathway),
3) and autophagy.
-The UPS is responsible for the rapid degradation of proteins when fast adaptation is needed, and UPS protein makes up about 1% of the protein
in a cell.
-Autophagy is mainly involved in the degradation of long-lived proteins and entire organelles; it plays
an important role during development and is required for the adaptation to environmental stresses such as starvation and involve direct transport into the lysosomal lumen from cytoplasm.
-Lysosomes are on another card in this deck
Proteasome:
- Huge complex of proteins that unwind the misfolded protein and feed it into a compartment where it gets cut into short peptides. (1% of cellular protein).
- Requires ATP for the denaturing of the peptide, and then the 19S cap recognizes proteins tagged with ubiquitin to be degraded.
- Proteolysis occurs in central chamber.
- Only beta subunits are proteolytically active; alpha subunit just regulates entry into the chamber.
- B1 = caspase-like –> cleaves after acidic AAs
- B2 = trypsin-like –> cleaves after basic AAs
- B5 = chymotrypsin-like –> cleaves after hydrophobic AAs
- Proteins that get retrotranslocated are polyubiquitinated so as to get targeted to proteasome.
Ubiquitin:
- 76 AA protein (very conserved).
- E1 = activation –> gets ubiquitin ready.
- E2 transfers the ubiquitin to E3.
- E3 is most prevalent –> attaches a string of at least 4 ubiquitins to the peptide.
- Describe the functions of the lysosome. What storage diseases occur due to mutations in lysosomal proteins such as degradative enzymes and transporters?
Function:
- Degrades ALL cellular components
- Targeted via the endocytic pathway
- Monoubiquitinated transmembrane proteins
- Regulated at the Multivesicular Body
Lysosomal storage diseases anyone?
Tay-Sachs:
-beta-hexosaminidase, breaks down gangliosides in neurons
Gaucher’s disease:
-beta-glucosidase, breaks down glucosylceramide in monocytes and leukocytes
Niemann-Pick:
(1) sphingomyelinase, breaks down sphingomyelin in macrophages
and (2) cholesterol transporter, moves cholesterol from the lysosome to the cytosol.
can be useful for ebola virus
