Cellular Signalling and Metabolic Regulation

Flashcards covering GPCR/RTK signalling, PKA/PKC/Ras pathways, insulin resistance, Rb protein, glycolytic/TCA/gluconeogenic regulation, and lipids/lipoproteins/bile acid synthesis.

What are the structural characteristics and activation mechanism of G-Protein Coupled Receptors (GPCRs)?

GPCRs are 7-transmembrane domain receptors that undergo a conformational change upon ligand binding, allowing the $\alpha$ subunit of a heterotrimeric G protein to exchange GDP for GTP.

How do different $\text{G}\alpha$ subunits influence adenylyl cyclase activity and [cAMP] levels?

$\text{G}\alpha_s$ stimulates adenylyl cyclase to increase [cAMP], while $\text{G}\alpha_i$ inhibits it to decrease [cAMP].

How is cAMP produced and subsequently degraded in the cell?

cAMP is produced from ATP by adenylyl cyclase and is hydrolysed to $5\text{'-AMP}$ by phosphodiesterases (PDEs) to ensure transient signalling.

Describe the structure and activation of Protein Kinase A (PKA).

PKA is a tetramer of 2 Regulatory (R) and 2 Catalytic (C) subunits. When 4 cAMP molecules bind to the R subunits (2 per subunit), the active C subunits are released to phosphorylate serine/threonine residues.

What is the function of A-Kinase Anchoring Proteins (AKAPs)?

AKAPs bind PKA regulatory subunits to localise PKA to specific subcellular compartments, such as organellar membranes or the cytoskeleton, ensuring precise and rapid responses.

Which second messengers are produced by the action of Phospholipase C $\beta$ (PLC$\beta$)?

PLC$\beta$ hydrolyses $PIP_2$ into Diacylglycerol (DAG), which stay in the membrane, and Inositol $1,4,5\text{-trisphosphate (IP}_3)$, which diffuses into the cytosol.

What are the three components required for the activation of Protein Kinase C (PKC)?

PKC activation requires elevated cytosolic $Ca^{2+}$, Diacylglycerol (DAG), and the negatively charged phospholipid phosphatidylserine.

How is the [$Ca^{2+}$] gradient maintained in a resting cell?

Resting cells maintain low cytosolic free calcium (around $100\,\text{nM}$) by pumping $Ca^{2+}$ into the ER/SR reservoir (millimolar range) or extruding it using proteins like the Na+-Ca2+ Exchanger (NCX), which moves $3\,\text{Na}^+$ in for $1\,\text{Ca}^{2+}$ out.

What is the role of Calmodulin (CaM) in calcium signalling?

Calmodulin is a conserved EF-hand protein with four EF-hand motifs. Upon binding four $Ca^{2+}$ ions, it undergoes a conformational change to activate protein kinases, phosphatases, and ion channels.

How does insulin activate the Insulin Receptor (IR)?

Insulin binding to the homodimeric Receptor Tyrosine Kinase (RTK) induces a conformational change leading to trans-autophosphorylation of tyrosine residues, creating docking sites for SH2-domain proteins like IRS.

What is the primary metabolic route for insulin signalling downstream of IRS?

The PI3K/AKT pathway, where PI3K converts $PIP_2$ to $PIP_3$, recruiting and activating AKT (Protein Kinase B) to drive glucose uptake and glycogen synthesis.

Describe the regulation of the Retinoblastoma (Rb) protein during the cell cycle.

Active (hypophosphorylated) Rb binds E2F to inhibit S-phase gene transcription. Hyperphosphorylation by cyclin-CDK complexes inactivates Rb, releasing E2F to promote cell division.

What mechanism does the EGF receptor use for activation?

Ligand binding induces the formation of an asymmetric dimer where one subunit (the activator) induces a conformational change in the other (the receiver) to activate its kinase domain.

How is the small GTPase Ras activated by RTKs?

RTK autophosphorylation allows the adapter GRB2 to bind via its SH2 domain. GRB2 then recruits SOS (a Ras-GEF) via its SH3 domains to catalyse the exchange of GDP for GTP on Ras.

What are the three irreversible steps of Glycolysis?

Reaction 1 (Hexokinase/Glucokinase), Reaction 3 (Phosphofructokinase-1), and Reaction 10 (Pyruvate Kinase).

How does Fructose-2,6-bisphosphate ($F\text{-2,6-BP}$) regulate glycolysis in the liver?

It is the most potent activator of PFK-1. Its levels are increased by insulin (activating PFK-2) and decreased by glucagon (activating FBPase-2 via PKA-mediated phosphorylation).

What is the role of the Pyruvate Dehydrogenase Complex (PDC) and its coenzymes?

The PDC converts pyruvate to acetyl-CoA (the link reaction). It requires five coenzymes: TPP (vitamin B1), Lipoamide, CoA (vitamin B5), FAD (vitamin B2), and $\text{NAD}^+$ (vitamin B3).

Identify the major rate-limiting enzyme of the TCA cycle.

Isocitrate Dehydrogenase, which is activated by ADP and inhibited by ATP and NADH.

Distinguish between Glycogen Phosphorylase and Glycogen Synthase regulation by PKA.

PKA activates glycogen breakdown by phosphorylating Phosphorylase Kinase (which activates Glycogen Phosphorylase) and inhibits glycogen synthesis by phosphorylating Glycogen Synthase (inactivating it).

What are the four enzyme bypasses used in Gluconeogenesis to overcome glycolytic barriers?

Pyruvate Carboxylase and PEP Carboxykinase (bypass Pyruvate Kinase), Fructose $1,6\text{-bisphosphatase}$ (bypasses PFK-1), and Glucose $6\text{-phosphatase}$ (bypasses Hexokinase).

What is the rate-limiting step of fatty acid synthesis (lipogenesis)?

The carboxylation of Acetyl CoA to form Malonyl CoA, catalysed by Acetyl CoA Carboxylase (ACC) and requiring ATP and biotin.

How does Malonyl-CoA prevent the simultaneous synthesis and degradation of fatty acids?

Malonyl-CoA (a synthesis intermediate) acts as a potent inhibitor of Carnitine Palmitoyltransferase I (CPTI), blocking the entry of fatty acids into the mitochondria for $\beta\text{-oxidation}$.

Describe the regulation of HMG-CoA Reductase via its Sterol-Sensing Domain (SSD).

High cholesterol causes the SSD (transmembrane domains 2-6) to bind Insig proteins. This recruits E3 ubiquitin ligases like gp78, leading to the polyubiquitination and proteasomal degradation of the enzyme.

What is the function of the Sterol Response Element (SRE) and SREBP-2?

The SRE is a DNA sequence in promoters. SREBP-2 is a transcription factor that, under low cholesterol conditions, is cleaved to release its N-terminal domain, which enters the nucleus to bind the SRE and activate cholesterol synthesis genes.

What are the three main classes of lipoproteins based on their major apoprotein?

Apo B-48 Family (chylomicrons), Apo B-100 Family (VLDL, IDL, LDL), and Apo A Family (HDL).

What is the role of the Farnesoid X Receptor (FXR) in bile acid metabolism?

FXR acts as a bile acid sensor (activated by CDCA). It reduces bile acid synthesis by inducing SHP to inhibit CYP7A1, increases excretion via BSEP, and decreases uptake via NTCP.

State the enzyme deficiency and accumulated metabolite in Tay-Sachs disease.

Hexosaminidase A deficiency leads to an excessive accumulation of ganglioside $GM2$.

How many protons are pumped across the inner mitochondrial membrane for two electrons passed from NADH to $O_2$?

A total of $10\text{H}^+$ are transported: 4 by Complex I, 4 by Complex III, and 2 by Complex IV.

What is the ATP yield (P/O ratio) for NADH and $FADH_2$ in oxidative phosphorylation?

The P/O ratio is 2.5 for NADH and 1.5 for $FADH_2$, based on a requirement of $4\text{H}^+$ per ATP synthesised and transported.

What are the structural components of the ATP Synthase (F1F0-ATPase) complex?

$F_0$ is the membrane-embedded proton channel and molecular rotor ($c\text{-complex}$ and $\gamma/ε\text{ stalk}$), while $F_1$ is the catalytic unit (3$\alpha$ and 3$\beta$ subunits) that synthesises ATP in the matrix.