7 Flashcards
What attracts Phagocytes?
ATP leakage = “find me Signal”
what is the “eat me” signal that is recognized by phagocytes ?
Phosphatidylserine flips to outside of cell, a key “eat me” signal recognized by phagocytes
what is a PEST sequence?
A PEST sequence is a protein degradation signal rich in proline (P), glutamic acid (E), serine (S), and threonine (T).
These sequences target proteins for rapid degradation by the ubiquitin-proteasome system or calpain-mediated proteolysis.
what proteins PEST ssequences?
Cyclins (e.g., Cyclin D, Cyclin E)
p53
IκB (Inhibitor of NF-κB)
c-Fos
c-Myc
FOS and JUN (AP-1 Transcription Factors)
β-Catenin
Notch Intracellular Domain (NICD)
Lysosomal Hydrolases work at —- pH?
Low (~4.7)
what are endosomes?
They play a crucial role in endocytosis, directing cargo to lysosomes.
How do Lysosomes become acidic?
Depends on V-class ATPase Proton Pumps and Anion Channels (Cl-)
26S Proteasome and the subunits?
The 26S proteasome is a multi-subunit complex that consists of two main parts:
20S core particle – The catalytic part, responsible for breaking down proteins.
19S regulatory particles – These help in recognizing substrates, unfolding them, and feeding them into the core particle.
** 2- 19S caps and central 20S core
What is E1, E2, and E3?
-E1 Ub Activating Enzyme
-E2 Ub Conjugating Enzyme
-E3 Ub Ligase (~1000 in humans)
What is a RING protein?
A RING protein (Really Interesting New Gene) is a type of E3 ubiquitin ligase that plays a crucial role in ubiquitin-mediated protein degradation.
- These proteins contain a specific RING domain—a zinc finger-like motif that is key to their function in the ubiquitination process
why is Lysine so important for degradation?
lysine residues on target proteins are essential for the attachment of ubiquitin molecules, which tag proteins for degradation by the proteasome
What are the STEPS in the ubiquitin-mediated protein degradation pathway?
- ATP activates proteasome
-E1 becomes active and transfers Ubiquitin (8.5 KDa) to E2
- E2 binds E3 and transfers Ub to target protein (e.g. Hh or Wnt)
- process repeats to get polyUbiquitination
-polyUb-protein is recognized by cap (PTM)
- Deubiquitination recycles the Ub
What family does JNK belong to & what is relationship between p-Jun and p-JNK?
JNK belongs to the MAPK family (stress-activated protein kinases).
p-JNK phosphorylates and activates c-Jun, leading to p-c-Jun.
p-c-Jun (activated c-Jun) is a transcription factor that regulates genes involved in cell stress responses, apoptosis, and other cellular processes.
Components of the Shh Pathway (ligand, receptor…)
Ligand:
- Sonic Hedgehog (Shh)
Receptors:
- Patched1 (PTCH1= transmembrane protein)
- Smoothened (SMO = GPCR)
Transducer:
- Gli
Effectors:
Target genes (e.g., Cyclins, Bcl2)
Regulators:
Primary cilia,
Ubiquitin ligases; Proteasome, Su(fu).
Hh signal ABSENT Pathway (STEPS)
Shh ligand is absent.
Patched1 (PTCH1) receptor binds to Shh and inhibits Smoothened (SMO).
Smoothened (SMO) is inactive and localized in the cytoplasm.
Gli3 is cleaved (by GSK-3) into a REPRESSOR form and inhibits gene expression.
Primary cilia do not facilitate SMO signaling; ciliary localization of SMO is inhibited.
No downstream gene activation of target genes (e.g., Cyclins, Bcl2).
Hh signal PRESENT Pathway (STEPS)
Shh ligand binds to Patched1 (PTCH1) receptor on the cell surface. (PTCH1 inhibited)
Smoothened (SMO) becomes active, accumulates in primary cilia, and activates downstream signaling.
Gli Active form goes to nucleus
Gli transcription factors bind to target gene promoters (e.g., Cyclins, Bcl2) to activate transcription.
PKA in the Shh pathway?
PKA primarily acts as a negative regulator of Shh signaling, keeping it off until Shh binding to PTCH1 releases the inhibition.
shh off:
PKA phosphorylates Gli3, leading to its cleavage into a repressor form.
What is Gli called in Drosophila?
In Drosophila, Gli is called Cubitus interruptus (Ci).
What determines Gli behaviour?
Presence of phosphates at specific residues in Gli determines its behaviour! (activator or repressor)
(via PKA)
what are the 3 types of wnt signalling pathways?
Canonical:
- b-catenin and the proteasome
Non cononical:
- Planar Cell Polarity (PCP)
- Ca2+ dependent
what does LRP serve as?
a Wnt Co-receptor
- is is a low-density lipoprotein receptor-related protein)
what is the receptor in Wnt signalling pathway?
- Frizzled (GPCR)
What does active Groucho do?
Groucho (Gro) ensures repression
- no wnt = no target gene activity
what forms the destruction box in wnt signalling?
- Axin
- GSK-3
- APC
- CK1
What is the phenotype when APC is KO?
APC KO leads to increased β-catenin activity which leads to uncontrolled cell proliferation = cancer
what is Dickkopf to Wnt ?
Dkk is a wnt antagonist
When wnt/active is present you get ?
transcription
- activation turns on markers such as:
Lgr5
c-Jun & c-Myc
Cyclin D1
MMPs & Connexins
RARs & EGF receptor
Dickkopf (Dkk) – Wnt antagonist
Canonical Wnt Pathway (STEPS)
-Wnt binds Frizzled
-LRP is Phosphorylated
-Dishevelled binds Fzd & Axin
-Axin is recruited (to p-LRP) & destruction box is broken
-b-catenin moves to nucleus
-translocation displaces Groucho (Gro)
-TCF (LEF) transcription factors are active
