Week 11 Flashcards
How does the lac operon work?
There is a repressor that sits upstream of the Lac genes that prevents RNA polymerase transcription. The presence of lactose will remove this repressor, leading to the downstream transcription of the genes needed for lactose catabolism. Thus, nutrient sensing is coupled to gene expression.
What is the difference between “push systems” and “pull systems” when it comes to considering cellular metabolism?
Most people tend to think of metabolism as a “pull” system, wherein the cell decides what it ‘wants’, and takes what it needs to make it. A “push” system considers the current metabolic environment of the cell as instructive towards the cell’s available metabolic options.
What is one of the large differences between cellular metabolism of multicellular and single celled organisms?
Multicellular organisms need to centrally coordinate growth so that cells only proliferate when it is advantageous for the orgaism as a whole. Cells should not all immediately enter the cell cycle in a multicellular organism when nutrients are available (for obvious reasons), although single cellular organisms such as yeast do just that.
In non-transformed cells, what regulates nutrient uptake?
Growth factors (not nutrient availability). Diabetes and insulin is a good example.
What are the two requirements for tissue-specific, growth-factor dependent proliferation?
1) A progenitor cell with proliferative capacity
2) A local, tissue-specific growth factor
Why is growth-factor stimulation/proliferation coupled with differentiation?
As a mechanism to limit the extent of growth in the presence of growth factor stimulation.
What is the Warburg effect?
Otto Warburg found that tumor cells consumed glucose and excreted lactate at high rates. This was later shown to be a product of proliferation - anaerobic glycolysis favors lipid formation, which is needed for cell division.
How does metabolism activate cancer cells?
Through mutations of the PI3K pathway. PI3K activates AKT, and AKT activation is important for GLUT1 translocation to the cell membrane. AKT also activates and phosphorylates a number of proximal enzymes in the glycolytic pathway. This enables the glycolytic pathway and cells that have constitutively activated PI3K tend to have lots of glucose uptake.
How do PET scans work?
A Fluoride-tagged (radioactive) glucose is given i.v.. This is incorporated into cells via GLUT transporters and its phosphorylation retains it in the cell. However, it cannot be further catabolized, and thus it can be visualized. Cells (tumors) can be visualized based on their glucose uptake.
What is the role of CD28 in immunometabolism?
CD28 activation allows the activated T cell to quickly take up massive amounts of glucose. (Byproduct of AKT activation). Facilitates pre-translated GLUT1 from endosomes to cell surface. This shows that the Warburg effect is not exclusive to cancer.
How is immunometabolism related to T cell differentiation?
Activation of T cells leads to immediate increased uptake of glucose due to GLUT1 upregulation on the cell surface. This extra glucose is shunted into pathways that produce acetyl-CoA, which is required for the chromatin modifications needed to execute T cell differentiation transcriptional programs.
How does checkpoint signaling affect immunometabolism in T cells?
CTLA-4 signaling blocks AKT activation, and PD-1 signaling blocks PI3K modifications of lipids into signaling PIP3. Both lead to the downregulation of GLUT transporters on the cell surface and decreased glucose uptake.
What is the largest effect of checkpoint inhibitor on T cells?
It is mostly metabolic, not transcriptional. Chromatin modifications are largely superimposable, but glycolysis is extremely different.
Structure of MHC class I
B2 microglobulin and three alpha subunits. a1 and a2 provide peptide-binding specificity, while a3 provides structural support and includes a transmembrane portion. Peptide binding cleft has charged regions that recognize the N-terminus and C-terminus of the peptide (so it is ‘closed’)
Structure of MHC Class II
Homodimer of alpha and Beta chains, both of which have a variable and constant region, and both of which have transmembrane portions. Peptide binding cleft is ‘open’, allowing for the inclusion of longer peptides that can stick out over the peptide binding cleft area. Recognizes bound peptide throughout the groove.
Contents of entire TCR/CD3 complex.
TCR to recognize pMHC complex (often a:B). Two CD3 heterodimers - one CD3e:CD3d and one CD3gCD3e and two Zeta chains. Each CD3 molecule has a single ITAM, and each Zeta chain has two ITAMs, for a total of 10 ITAMs per signaling complex.
What is the first intracellular event downstream of TCR signaling?
ITAMs on CD3 and Zeta chains are phosphorylated by Src-family kinases such as Lck. This then recruits Syk-family kinases such as Zap70.
What do SH2 domains recognize and bind to?
Phosphotyrosines.
What is the general archetecture of Syk Kinases?
They have a kinase domain and at the N-terminal they have 2 SH2 domains spaced in stereotypical way. They can recognize and bind phosphotyrosine sites of ITAMs.
What generally recognizes phosphorylated ITAMs?
Syk-family kinases. They contain two SH2 domains that are specifically spaced apart to recognize and bind to the two phosphokinases on activated ITAMs.
How is the TCR/CD3 signaling complex assembled in the plasma membrane?
TCRa and B chains actually have positively charged arginines and lysines along their transmembrane helicies. The CD3 chains have negatively charged residues along their transmembrane helicies. This is remarkable because the plasma membrane is disfavorable to charges.
The assembly of the TCR complex is partially conferred by the neutralization of these charged residues by one another. This establishes a very specific spatial relationship between all components.
Differences in structure and binding affinities for CD4 and CD8 coreceptors.
CD8 is a homodimer, whereas CD4 is a monomer. CD8 binds to its cognate MHC I with higher affinity than does CD4 to its own (by an order of magnitude).
How do CD4 and CD8 potentiate TCR signaling?
Both enhance the strength of the TCR:pMHC interaction by binding to the side of the MHC complex.
Additionally, both assocaite with Lck kinase through a cysteine motif in the cytoplasmic domain, and thus assist with the recruitment of Lck kinase to the TCR signaling complex. Both CD4 and CD8 are constitutively assocaited with Lck in the cytoplasmic domain. Bringing Lck into proximity of the ITAMs of TCR complex allows for phosphorylation of tyrosine residues in the CD3 cytoplasmic domains by Lck, and the subsequent ZAP70 recruitment.
What happens upon ZAP70 recruitment to the TCR signaling complex?
ZAP70 phosphorylates LAT (Linker for the Activation of T cells), which leads to the phosphotyrosine-mediated assembly of a scaffolding complex called the LAT-SLP76 complex.
What is LAT?
Linker for the Activation of T cells is a signaling protein involved in TCR complex signaling that is phosphorylated by ZAP70 and recruits Gads and SLP76 to form a scaffolding complex called the LAT-SLP76 complex. Both LAT and SLP76 are basically just proteins with lots of binding sites, so they can pull lots of different signaling enzymes from the cytosol.
What (among others) enzymes and messenger proteins does LAT/SLP76 scaffolding protein recruit to the activated TCR signaling complex?
SOS (Ras, MAPK), VAV (Rac, Cdc42 activation), PLC-g (IP3 and DAG)
What is the role of Phospholipase C gamma in TCR signaling?
PLCg takes phosphatidylinositol biphosphate (PIP2) in the membrane and cleaves the PIP headgroup (IP3) off. IP3 is soluble and diffuses through the cytoplasm and induces calcium responses. The lipid remnant is diacylglycerol (DAG) acts as a signaling molecule in the membrane and recruits protein kinase Cs and Ras exchange factors.
