2. Questions
1. Who is Dalton
2. Footnotes about regulation of the RAG-complex
3. Mechanism of PKCα in cytoskeletal organization
4. Mechanism of SGK in cell survival/metabolism
5. Define autophagy
6. Steps in micro-autophagy
7. Define Ubiquitin
8. Steps in Ubiquitination
9. Region, band and sub-band of PTEN on Chromosomal 10
3. John Dalton
• He was an English chemist, physicist and meteorologist. He was born on
September 6, 1766, in Eaglesfield, United Kingdom and died on July 27,
1844, in Manchester, United Kingdom.
• He is best known for developing Dalton's atomic theory points.
• Which are;
Elements are made of extremely small particles called atoms.
Atoms of a given element are identical in size, mass and other properties;
atoms of different elements differ in size, mass and other properties.
Atoms cannot be subdivided, created or destroyed.
Atoms of different elements combine in simple whole-number ratios to form
chemical compounds.
4. Regulation of the RAG complex
Rag Genes
There are four Rag genes, RagA was thought to be functionally
redundant with RagB, and RagC was thought to be functionally
redundant with RagD.
Rags are in their ‘active’ form when GTP-bound RagA/B forms a
complex with GDP-bound RagC/D. In contrast, in the ‘inactive form’,
GDP-bound RagA/B complexes with GTP-bound RagC/D. The Rag
GTPases ‘active’ and ‘inactive’ complex is controlled by the guanine
nucleotide exchange factors (GEFs) and GTPase-activating proteins
(GAPs). GAP toward Rags (GATOR) complexes acts upstream of Rag
GTPases and regulates mTORC1 signaling
5. GATOR complexes
GAP toward Rags (GATOR) is made up of two subcomplexes called
GATOR1 and GATOR2. During amino acid deprivation, GATOR1 displays
GAP activity towards RagA/B, acting as a negative regulator of mTORC1 by
promoting GTP hydrolysis of RagA/B. In contrast, during amino acid
abundance, GATOR2 inhibits GATOR1 and positively regulates mTORC1.
Leucine sensor Sestrin2, and arginine sensor CASTOR1 binds GATOR2 and
releases GATOR1
In amino acid-rich conditions, the ‘active’ Rag GTPase heterodimer (GTP-
bound RagA/B and GDP-bound RagC/D) recruit mTORC1 from an
unknown location to the lysosome. Once at the lysosome mTORC1
encounters the small GTPase Ras homolog enriched in brain (Rheb). Rheb
increases the catalytic activity of mTORC1 and results in mTORC1
activation
6. CASTOR1
Cellular arginine sensor for mTORC1 (CASTOR1) regulates arginine
sensing to positively regulate mTORC1. When arginine is limiting
CASTOR1 binds to and inhibits GATOR2 resulting in mTORC1 inhibition.
Arginine directly binds to CASTOR1 which leads to disruption of the
CASTOR1–GATOR2 increasing mTORC1 activity
7. Sestrin 2
• This is a stress inducible proteins that regulate metabolism by sensing
nutrient level and redox status in cells. It has been identified as a
leucine sensor. In the absence of leucine, Sestin2 binds to GATOR2
resulting in mTORC1 inhibition. Leucine directly binds to Sestrin2
promoting the dissociation of Sestrin2–GATOR2 increasing mTORC1
activity. GATOR2 inhibits GATOR1 decreasing GATOR1's GAP activity
toward RagA/B and subsequently promoting mTORC1 activity
8. Ragulator
The Ragulator complex, also known as late endosomal/lysosomal
adaptor, MAPK, and mTOR activator (LAMTOR) complex. It consists of
five LAMTOR components and functions as a scaffolding protein that
tethers the Rags and mTORC1 to the lysosomal surface and in presence
of amino acids, it promotes ‘active’ Rag GTPase state thus facilitating
mTORC1 lysosomal localization and activation
9. v-ATPase
Vacuole-ATPase is a highly conserved proton pump that hydrolyzes ATP
by pumping protons across membranes acidifying organelles (such as
the lysosome) and maintaining cytosolic pH. It interacts with Ragulator
and promotes its Guanine Nucleotide Exchange Factor (GEF) activity
for RagA/B resulting in mTORC1 activation
SLC38A9
Solute carrier family 38 member 9 (SLC38A9 ) facilitates the efflux of
essential amino acids from the lysosome in an arginine-dependent
fashion. Arginine stimulates the interaction of the SLC38A9-Ragulator-
Rag GTPases complex promoting mTORC1 signaling
10. Mechanism of PKCα in cytoskeletal organization
• Protien Kinase C alpha (PKCα) a specific member of the protein
kinase family that can be activated by calcium and the second
messenger diacylglycerol.
• Its binding to cytoskeletal filaments helps the kinase to navigate
through the crowed cytoplasm.
• It Phosphorylates myosin II which is able to bind actin, thus activating
the intrinsic ATPase activity of myosin II, leading to contraction of the
cytoskeleton
• PKC phosphorylates vinculin, a cytoskeletal protein localized at
adhesion plaques, thus controlling cell shape and adhesion
11. Mechanism of SGK in cell survival/metabolism
Serum and glucocorticoid-regulated protein kinase (SGK) is a member
of protein kinase family that regulates diverse effects of extracellular
agonists by phosphorylating regulatory proteins that control cellular
processes such as ion transport and growth.
SGK also negatively regulates cell death through inhibiting forkhead
transcription factor 3a (FOXO3a) activity. Anti-apoptotic function of
SGK1 is also supported that FOXO3a is a critical regulator of apoptotic
genes.
12. Autophagy
Autophagy is a process in which a cell eats its own contents
• It involves the delivery of cytoplasmic materials to the lysosome for
degradation
Types of Autophagy
• Macro autophagy is a type of autophagic process in which the substrates
are sequestered within cytosolic double-membrane vesicles termed
autophagosomes
• Micro autophagy refers to a process by which cytoplasmic contents enter
the lysosome through an invagination or deformation of the lysosomal
membrane
• Chaperone-mediated autophagy (CMA) is a lysosomal pathway of
proteolysis that is responsible for the degradation of specific cytosolic
proteins under conditions of prolonged nutrient deprivation.
13. Mechanism of Autophagy
Initiation: This involves Nucleation and formation of an isolation
membrane, also called a phagophore. The isolation membrane is derived
from the Endoplasmic Reticulum, though other membrane
Elongation: This involves Formation of a vesicle, called the
autophagosome, from the isolation membrane, inside which intracellular
organelles and cytosolic structures are sequestered.
Elongation and closure of the initiation membrane require the coordinated
action of two ubiquitin-like conjugation systems that result in the covalent
linkage of the lipid phosphatidylethanolamine (PE) to microtubule-
associated protein light chain 3 (LC3). PE-lipidated LC3 is increased during
autophagy, and it is therefore a useful marker for identifying cells in which
autophagy is occurring.
Maturation of the autophagosome by fusion with lysosomes, to deliver
digestive enzymes that degrade the contents of the autophagosome
14.
15. Steps in micro autophagy
Membrane invagination and autophagic tubes formation:
• This involves Formation of the autophagic tubes by the lysosomal membrane
Vesicle formation
• This involves lipid enrichment in the autophagic tubes due to removal of
transmembrane proteins leading to spontaneous vesicle formation via phase
separation mechanism
Vesicle expansion and scission
• Enlargement of vesicle is mediated by binding enzymes inside of unclosed vesicle.
Vesicle degradation and recycling
• Vesicle moves freely in the lumen and are degraded by hydrolases to release
nutrients back into the cystosol.
16. Define Ubiquitin
Ubiqutin is a small protein of 76 amino acids with a molecular mass
of about 8.6kDa that exists in all eukaryotic cells.
Ubiquitination : An enzymatic Post Transitional Modification (PTM) in
that Ubiquitin protein is attached to a substrate protein.
Monoubiquitination: The addition of a single ubiquitin molecule to
one substrate protein residue.
Polyubiquitination: Refers to the formation of a ubiquitin chain on a
single lysine residue on the substrate, further ubiquitin molecules are
added to the first ubiquitin molecule
17. Steps in Ubiquitination
Activation
• The activation step is catalyzed by Ubiquitin activating enzymes(E1) upon
ATP hydrolysis
• During the activation the ubiquitin molecule carboxyl glycine residue is
linked to the E2 enzyme cysteine residue via a thio-ester bond (cysteine
residue being the active site of the enzyme)
Cojugation
• This step is catalyzed by Ubiquitin Conjugating Enzyme (E2)
• The E2 enzyme catalyzes the transfer of Ubiquitin from E1 to the active
cysteine site of E2 via a thio-esterification reaction
• The E2 enzyme binds both the activated Ubiquitin and the E1 enzyme.
18. Ligation
• Catalyzed by ubiquitin ligases (E3)
• This enzyme shuttles the ubiquitin to the target protein thus the
substrate recognition Module of the system
• It’s the enzyme that catalyzes the formation of an isopeptide bond
between the lysine of the target protein and the c-terminal glycine
residue of Ubiquitin Protein
• E4 enzymes (Ubiquitin Chain Elongation Factors ) are capable of
adding preformed polyubiquitin chains to substrate proteins
19. Chromosomal location of PTEN
• Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)
is a tumor suppressor gene located at chromosome 10q23. 31,
encoding for a 403-amino acid protein that possesses both lipid and
protein phosphatase activities
