Case 10 - BBS2042

1. Case 10 - Biotechnological Approaches

1.1 Learning Goals

  • Understand various biotechnological methods in researching:

    • Cardiac hypertrophy and changes in Renin-Angiotensin System (RAS) signalling.

    • Acute lymphocytic leukemia (ALL) and changes in Notch signalling.

1.2 Methods of Research

  • MaMTH: A method for studying protein interactions in cardiac hypertrophy and ALL.

  • FRET: A method for investigating molecular interactions in both cardiac hypertrophy and ALL.

  • Affinity purification mass spectrometry (AP-MS): Employed in both fields for studying protein interactions.

  • Phosphoproteomics by LC-MSMS: Used to explore phosphorylation changes in related signalling pathways.

2. Cardiac Hypertrophy

2.1 What is Cardiac Hypertrophy?

  • Definition: Enlargement of the heart muscle (myocardium) due to an increase in cardiomyocyte size, not the number of cells.

  • Triggers:

    • Hypertension (high blood pressure)

    • Valve disease (e.g., aortic stenosis)

    • Recovery from myocardial infarction

    • Genetic cardiomyopathies

    • Intense athletic training

2.2 Structural and Cellular Changes in Cardiac Hypertrophy

  • Changes observed in hypertrophic hearts include:

    • Increased size of cardiomyocytes.

    • Addition of sarcomeres, indicating increased muscle fiber structure.

    • Re-expression of fetal genes, notably:

    • ANP (Atrial natriuretic peptide)

    • BNP (Brain natriuretic peptide)

    • β-myosin heavy chain

2.3 RAS Signalling in Healthy Heart Tissue

  • RAS maintains cardiovascular homeostasis with low levels of angiotensin II under normal conditions.

  • Activation of the AT1 receptor leads to:

    • Mild vasoconstriction

    • Regulation of blood pressure

    • Short-term adaptations to increased workload

  • The AT2 receptor counteracts AT1 signalling by promoting:

    • Vasodilation

    • Anti-proliferative effects

    • Anti-fibrotic effects

2.4 Changes in the RAS System During Cardiac Hypertrophy

  • In pathological conditions, chronic activation of RAS occurs, leading to:

    • Increased angiotensin II production.

    • Prolonged stimulation of AT1 receptors in cardiac cells.

  • Local production of RAS components by cardiomyocytes, cardiac fibroblasts, and endothelial cells continues even with normal circulating hormones, which drives hypertrophic signalling.

  • Changes include:

    • Increased expression of Angiotensin-Converting Enzyme (ACE).

    • Enhanced activation of downstream growth pathways.

    • Shift from regulatory to pro-growth signalling role of RAS.

2.5 Angiotensin II Signalling in Cardiomyocytes

  • Angiotensin II binding to the AT1 receptor activates multiple intracellular signalling cascades important for hypertrophic growth:

  • Mechanism overview:

    • AT1 receptor engagement leads to activation of Gq proteins.

    • This stimulates enzymes such as phospholipase C (PLC), resulting in increased intracellular calcium levels.

    • Key pathways activated by Angiotensin II include:

    • MAPK signalling (includes MAPK1 and MAPK3).

    • Overall outcomes include:

      • Increased cardiomyocyte growth.

      • Enhanced protein synthesis.

      • Structural remodeling of the heart muscle.

3. Acute Lymphocytic Leukemia (ALL)

3.1 What is Acute Lymphocytic Leukemia?

  • Definition: A malignant cancer affecting lymphoid progenitor cells in the bone marrow leading to rapid proliferation of lymphoblasts.

  • Key features:

    • Rapid progression.

    • Accumulation of immature lymphoblasts.

    • Most common cancer in children, also found in adults.

  • Subtypes include:

    • B-cell ALL (B-ALL): ~80–85% of cases.

    • T-cell ALL (T-ALL): ~15–20% of cases.

3.2 The Role of NOTCH Signalling in Normal Immune Development

  • The Notch signalling pathway is critical for cell fate determination, differentiation, proliferation, and apoptosis.

  • In T lymphocyte development, Notch signalling requires four receptors:

    • NOTCH1, NOTCH2, NOTCH3, NOTCH4.

  • Activation occurs through interaction with neighbouring cells expressing:

    • Delta-like ligands (e.g., DLL4).

  • Notch signalling must involve direct cell contact.

3.3 NOTCH Signalling in Healthy T-cell Development

  • Regulated spatially and temporally to ensure proper T-cell development:

    1. Commit to the T-cell lineage.

    2. Proliferation of early thymocytes.

    3. Differentiation into mature T cells.

  • Excess Notch signalling may lead to harmful outcomes, requiring mechanisms for degradation of NICD (Notch intracellular domain).

3.4 NOTCH Signalling in Acute Lymphocytic Leukemia

  • Constitutive activation of NOTCH1 due to mutations, prevalent in 50–60% of T-ALL cases:

    1. HD domain mutations allowing ligand-independent activation.

    2. PEST domain mutations resulting in prolonged NICD stability due to loss of degradation targeting.

  • Both mutations perpetuate aberrant NOTCH signalling.

3.5 Consequences of Aberrant NOTCH Signalling in Leukemia

  • Chronic activation alters cellular processes leading to:

    • Upregulation of MYC, stimulating cell cycle progression.

    • Blocked differentiation into functional immune cells.

    • Enhanced metabolic activity, increasing glucose uptake to support rapid growth.

    • Interactions with oncogenic pathways including:

    • PI3K–AKT–mTOR pathway.

    • NF-kappa B pathway.

4. MaMTH

4.1 What is MaMTH?

  • Mammalian Membrane Two-Hybrid (MaMTH) is a method for detecting protein–protein interactions (PPIs) for membrane proteins in living cells.

  • Membrane proteins are challenging to analyze due to their cellular context, but MaMTH is tailored to overcome these difficulties.

  • Typical targets include membrane receptors, transporters, enzymes, and signalling complexes.

4.2 Importance of Studying Membrane Protein Interactions

  • Many major signalling pathways depend on membrane receptors.

  • Changes in interaction networks can lead to various diseases like cancer and cardiomyopathies.

4.3 Components of the MaMTH System

  • Bait Protein: Membrane protein fused to a transcription factor fragment and TEV protease site. Example include NOTCH1.

  • Prey Protein: Potential interacting partner fused to TEV protease.

  • Reporter Gene: Placed downstream of the transcription factor binding site, with common examples being luciferase or GFP.

4.4 Mechanism of MaMTH

  1. Bait receptor expression in the cell membrane, integrating transcription factor fragments and cleavage sites.

  2. Prey proteins expressed with TEV protease.

  3. Interaction between bait and prey brings TEV protease to the cleavage site.

  4. Proteolytic cleavage occurs releasing the transcription factor.

  5. Nuclear translocation of the transcription factor.

  6. Reporter activation and measurable signal generation.

4.5 Advantages of MaMTH

  • Works specifically with membrane proteins, overcoming limitations of other interaction assays.

  • Maintains mammalian cell context for realistic interactions and modifications.

  • Supports high-throughput screening of interaction partners.

  • Can detect dynamic receptor interactions during cellular signalling changes.

4.6 Limitations of MaMTH

  • Risk of false positives due to protein overexpression leading to artificial interactions.

  • False negatives can occur if the protein interaction is inhibited by other factors.

  • MaMTH shows only proximity, not the functional outcomes of interactions.

4.7 MaMTH in Cardiac Hypertrophy Research

  • Potentially useful for studying signalling through receptors like AGTR1, EGFR, and IGF1R.

  • Identifying interactions and studying changes during hypertrophy stimulates discovery in drug targets.

4.8 MaMTH in Acute Lymphocytic Leukemia Research

  • Applicable specifically to T-cell ALL for analyzing NOTCH1 interactions.

  • Identifying new partners can elucidate leukemic signalling and improve therapy resistance understanding.

5. FRET

5.1 What is FRET?

  • Förster Resonance Energy Transfer (FRET): A biophysical technique for studying molecular interactions and distances within living cells.

  • Effective in detecting:

    • Protein-protein interactions

    • Conformational changes

    • Enzyme activity

    • Signalling dynamics in real time.

5.2 Basic Principle of FRET

  • Involves two fluorescent molecules:

    • Donor fluorophore (excites upon light absorption).

    • Acceptor fluorophore (emits fluorescence after energy transfer).

  • Energy transfer is efficient only at close proximity (≤10 nm) and requires spectral overlap and proper orientation.

5.3 Key Components of a FRET Experiment

  • Donor Fluorophore: Commonly Cyan Fluorescent Protein (CFP) or Green Fluorescent Protein (GFP).

  • Acceptor Fluorophore: Examples include Yellow Fluorescent Protein (YFP) or mCherry.

  • Fusion Proteins: Proteins of interest fused with fluorescent tags to detect interactions.

5.4 Mechanism of FRET

  1. Excitation of the donor fluorophore.

  2. Energy transfer occurring if the acceptor is within a certain distance.

  3. Acceptor fluorescence emission.

  4. Signal measurement through changes in donor and acceptor fluorescence indicating interaction proximity.

5.5 Applications of FRET

  • Used to measure:

    • Protein-protein interactions

    • Conformational changes in proteins

    • Enzyme activity via cleavage or phosphorylation detection.

    • Real-time monitoring of signalling dynamics.

5.6 Advantages of FRET

  • High spatial resolution allows detection at 1-10 nm.

  • Can be performed in live cells enabling dynamic studies.

  • Non-invasive methodology minimizes disruption during observation.

5.7 Limitations of FRET

  • Requires meticulous experimental design to avoid disrupting protein functionality.

  • Signal monitoring might need precise optical equipment due to potential weak signals.

  • Orientation dependence makes interpretation complex.

5.8 FRET in Cardiac Hypertrophy Research

  • Significant in investigating signalling pathways involved, such as AGTR1 and MAPK pathways.

  • Useful for monitoring receptor conformational changes and calcium signalling associated with hypertrophy.

5.9 FRET in Acute Lymphocytic Leukemia Research

  • Beneficial in understanding signalling dynamics and protein interactions within leukemic cells.

  • Can evaluate changes in NOTCH1 receptor structure and interaction crucial for leukemic progression.

6. Affinity Purification Mass Spectrometry (AP-MS)

6.1 What is AP-MS?

  • A proteomics technique that identifies protein–protein interactions by combining affinity purification with mass spectrometry.

  • Aims to map interaction networks and is widely used in:

    • Signalling pathway research

    • Cancer biology

    • Drug discovery.

6.2 Basic Principle of AP-MS

  • Isolating proteins of interest along with their binding partners, followed by identification through mass spectrometry of these complexes.

6.3 Key Components of AP-MS

  • Bait Protein: The protein of interest, e.g., NOTCH1 for leukemia or AGTR1 for cardiac hypertrophy, typically tagged with an affinity tag.

  • Prey Proteins: The interacting proteins that bind to the bait through co-purification.

6.4 Affinity Purification Process

  • Cell lysates are treated with beads coated with antibodies targeting the affinity tag, capturing the bait and bound proteins.

6.5 Mass Spectrometry Identification

  1. Purified proteins are digested into peptides.

  2. Peptides are analyzed via liquid chromatography and mass spectrometry.

  • This method reveals identity and interactions of proteins in complexes.

6.6 Step-by-Step Workflow of AP-MS

  1. Bait protein expression in cells.

  2. Cell lysis to free proteins.

  3. Affinity purification with antibody-coated beads.

  4. Non-specific binding removal through washing.

  5. Protein digestion into peptides for analysis.

  6. Mass spectrometry to identify proteins from spectra data.

6.7 What AP-MS Can Measure

  • Enables detection of:

    • Protein–protein interactions

    • Elucidating complex formation

    • Signalling networks mapping

    • Comparing phenomena in different experimental conditions to observe dynamic changes.

6.8 Advantages of AP-MS

  • High-throughput capability allows detection of numerous interacting proteins simultaneously.

  • Conducted within physiological relevance through mammalian cells.

  • Facilitates complex-level analysis rather than straightforward pairwise assays.

  • Quantitative proteomics options for interaction strength measurement.

6.9 Limitations of AP-MS

  • Transient interactions may be lost during purification processes.

  • Background noise from non-specific binding may interfere with results.

  • Detected interactions require validation by alternative methods (e.g., Co-immunoprecipitation, FRET).

6.10 AP-MS in Cardiac Hypertrophy Research

  • Valuable in studying signalling complexes involving AGTR1 and MAPK1 driving hypertrophic changes.

  • Allows identification of hypertrophic-specific interactions in comparative studies of pathological versus normal tissues.

6.11 AP-MS in Acute Lymphocytic Leukemia Research

  • Critical for mapping NOTCH1 interactions in T-ALL research, potentially revealing mutation impacts and drug resistance mechanisms.

7. Phosphoproteomics by LC-MS/MS

7.1 What is Phosphoproteomics by LC-MS/MS?

  • A technique identifying and quantifying phosphorylation patterns across the proteome using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

  • Key role in signaling regulation related to diseases.

7.2 Basic Principle of Phosphoproteomics

  • Detects and quantifies phosphorylated proteins, providing insights into specific phosphorylation sites and their functions in signalling pathways. The workflow typically includes:

    1. Protein extraction.

    2. Protein digestion into peptides.

    3. Enrichment of phosphorylated peptides.

    4. Peptide separation via LC.

    5. Identification and quantification through MS/MS.

7.3 Key Components of the Method

  • Protein Extraction: Requires cell lysis and includes phosphatase inhibitors to retain phosphorylation during isolation.

  • Peptide Separation: Peptides must be enriched due to the low abundance of phosphorylated forms relative to non-phosphorylated.

7.4 Step-by-Step Workflow

  1. Sample preparation by lysing cells for protein extraction.

  2. Proteolytic digestion generating peptide fragments.

  3. Phosphopeptide enrichment using techniques like IMAC or TiO₂.

  4. Separation through liquid chromatography.

  5. MS/MS enables identification and quantification of associated phosphorylation sites.

7.5 What Phosphoproteomics Measures

  • Detects phosphorylation sites to:

    • Evaluate kinase activity via phosphorylation state changes.

    • Map signalling pathways by evaluating activated pathways through phosphorylation.

7.6 Advantages of Phosphoproteomics

  • Capable of simultaneous analysis of thousands of phosphorylation sites.

  • High sensitivity and quantitative techniques like SILAC support comprehensive profiling.

  • Useful for discovering new signalling pathways.

7.7 Limitations

  • Very complex samples complicate data analysis.

  • Transient phosphorylation requires precise timing for effective results.

  • Enrichment processes are crucial for detecting phosphorylated peptides.

7.8 Phosphoproteomics in Cardiac Hypertrophy Research

  • Signalling pathways like MAPK1 and AKT1 are critical for cardiomyocyte growth.

  • Helps identify changes in kinase signalling in response to hypertrophic stimuli.

7.9 Phosphoproteomics in Acute Lymphocytic Leukemia Research

  • Useful for decoding oncogenic signalling alterations in leukemia cells.

  • It enables tracking NOTCH signalling interactive mechanisms and identifies hyperactive kinases for potential drug targeting.