MCB Paper Discussion

Phosphorylation on Tyrosine vs. Serine/Threonine

Tyrosine phosphorylation is crucial in signal transduction and regulating enzymatic activity. In contrast, phosphorylation on serine or threonine is more involved in controlling protein function by altering their conformation or localization. Tyrosine's unique side chain structure leads to different cellular signaling pathways.

Molecular Consequence of Phosphorylating Insulin Receptor Tyrosines

Phosphorylation of the insulin receptor's three tyrosine residues initiates insulin signaling. This creates docking sites for substrates and triggers a cascade leading to glucose uptake and metabolic regulation. It's a critical step in insulin-mediated effects.

Single-chain Variable Fragment (scFv)

An scFv is a fusion of the variable regions of an antibody's heavy and light chains, linked by a short peptide. It retains antigen-binding ability in a smaller, versatile format, useful for therapeutic and diagnostic applications.

scFv45 Target Recognition

scFv45 in the Haque/Tonks paper specifically recognizes the oxidized, inactive form of PTP1B. It distinguishes between active and inactive PTP1B forms, aiding in understanding protein regulation and therapeutic targeting.

Localization of scFv45 in Insulin/H2O2 Treated Cells

Morphological evaluation of scFv45 localization in insulin or H2O2 treated cells shows it binds to oxidized PTP1B. This implies PTP1B is oxidized in response to these treatments, affecting cellular signaling.

Biochemical Effect of scFv45 Expression with Insulin

Biochemically, scFv45 binding to oxidized PTP1B inhibits its activity in insulin-treated cells. This enhances insulin signaling by preventing dephosphorylation of the insulin receptor, illustrating a regulatory mechanism.

Latent vs. Non-latent Transcription Factors

Latent transcription factors are inactive under normal conditions, becoming active in response to specific signals. Non-latent ones are usually active and directly influence gene expression. NF-κB is latent; AP-1 is typically non-latent.

TLR4 and Gram-Negative Bacteria

Defective TLR4 in mice increases susceptibility to Gram-negative bacteria due to the inability to recognize lipopolysaccharides (LPS). TLR4 is crucial for sensing LPS, triggering immune responses against these bacteria.

LPS: Protective and Harmful

LPS helps defend wild-type mice against bacterial infections but can cause fatal septic shock. The overactive immune response to LPS in wild-type mice can be detrimental, leading to death.

NF-κB Forms Failing to Stimulate Transcription

Some NF-κB forms fail to stimulate transcription if lacking transactivation domains or if forming complexes with inhibitors. This prevents DNA-binding or transactivating functions, hindering transcription.

NF-κB Diminishing Transcription

NF-κB forms might diminish transcription by competing with active forms for binding sites or forming inactive complexes with coactivators, impeding transcriptional machinery.

Ankyrin Repeats in I-κB Proteins

Ankyrin repeats in I-κB proteins bind to NF-κB, inhibiting its activity. They mask NF-κB's nuclear localization signals, preventing its nuclear translocation and activation of transcription.

NEMO in NF-κB Pathway

NEMO (NF-κB Essential Modulator) is a regulatory subunit of the IκB kinase complex, crucial for NF-κB activation. It facilitates I-κB phosphorylation and degradation, releasing NF-κB for nuclear translocation.

Positive Feedback in NF-κB Pathway

Positive feedback is contributed by molecules like IKKs that enhance NF-κB activation by phosphorylating I-κB, leading to its degradation and NF-κB release.

Recognizing Feedback Molecules in Pathways

Identifying feedback molecules involves recognizing proteins that amplify (positive feedback) or reduce (negative feedback) signaling. Look for enhancers of activation or inhibitors of key pathway components.

Northern Blot vs. RT-qPCR

Northern blot visualizes and quantifies RNA, showing RNA size and integrity, useful for detecting alternatively spliced forms. RT-qPCR quantifies specific RNA sequences but might miss alternative splicing or modifications. In context like Figure 3B, Northern blot provides more comprehensive RNA profile information.

Difference in Phosphorylation

Tyrosine phosphorylation differs from serine/threonine due to its side chain structure and role in signal transduction and enzymatic regulation, while serine/threonine phosphorylation alters protein conformation/localization.

Phosphorylation in Insulin Receptor

Phosphorylating the three tyrosine residues in the insulin receptor initiates insulin signal transduction, creating docking sites for substrates and triggering downstream metabolic effects.

Single-chain Variable Fragment

A single-chain variable fragment (scFv) is a fusion of antibody heavy and light chain variable regions, maintaining antigen-binding ability in a smaller format for therapeutic/diagnostic use.

scFv45 in Haque/Tonks Paper

scFv45 specifically recognizes the oxidized, inactive form of PTP1B, distinguishing between active and inactive forms for therapeutic targeting.

Localization of scFv45

Morphologically, scFv45 binds to oxidized PTP1B in insulin/H2O2 treated cells, indicating PTP1B oxidation as a regulatory mechanism in cellular signaling.

Biochemical Effect of scFv45

scFv45 inhibits oxidized PTP1B activity in insulin-treated cells, enhancing insulin signaling by preventing insulin receptor dephosphorylation.

Latent Transcription Factor

A latent transcription factor is inactive under normal conditions but activates in response to specific signals, unlike non-latent transcription factors like AP-1 which are continually active.

TLR4 and Gram-negative Bacteria

Defective TLR4 makes mice susceptible to Gram-negative bacteria by failing to recognize lipopolysaccharides (LPS), crucial for initiating immune response.

Ligand Dual Role in Wild-Type Mice

The same ligand, LPS, can defend wild-type mice against bacterial infections and cause fatal septic shock due to excessive immune response.

NF-κB Failure to Stimulate Transcription

Some NF-κB forms fail to stimulate transcription due to lack of transactivation domains or complexes with inhibitors, impeding DNA-binding/transactivation functions.

Diminished Transcription by NF-κB

These NF-κB forms may diminish transcription by competing with active forms for binding sites or forming inactive complexes with coactivators.

Ankyrin Repeats in I-κB

Ankyrin repeats in I-κB bind to NF-κB, inhibiting its activity by masking nuclear localization signals and preventing nuclear translocation.

NEMO Description

NEMO (NF-κB Essential Modulator) is a regulatory subunit in the IKK complex, crucial for NF-κB activation via I-κB phosphorylation/degradation.

Positive Feedback in NF-κB Pathway

Positive feedback in the NF-κB pathway is contributed by molecules like IKKs, which enhance NF-κB activation by phosphorylating I-κB.

Negative Feedback in NF-κB Pathway

Negative feedback in the NF-κB pathway is contributed by molecules like I-κB and A20, inhibiting NF-κB or the IKK complex.

Recognizing Feedback Molecules

Recognize feedback molecules by identifying proteins that amplify (positive feedback) or attenuate (negative feedback) the signaling cascade.

Northern Blot vs RT-qPCR

Northern blot is better than RT-qPCR for visualizing RNA transcripts' size and integrity, useful in complex RNA populations or when transcript size matters, as shown in Figure 3B.

TLR4 RNA Feedback in Poltorak Paper

Figure 4 shows either positive or negative feedback for TLR4 RNA; the absence of this process could disrupt immune response balance, affecting organism health.

EMSA Basic Steps

EMSA identifies DNA-binding proteins by mixing radioactive DNA with protein, running gel electrophoresis, and detecting shifted DNA-protein complexes; radioactive DNA and protein are key entities.

EMSA Results in Sen and Baltimore Paper

Figure 7B's EMSA results show specific DNA-protein interactions, termed "winner winner chicken dinner" for their clear, conclusive evidence of binding specificity.

Methylation Interference Assay

Methylation interference assay reveals protein-DNA binding sites by methylating DNA, binding protein, and identifying obstructed methylation sites indicating binding regions.