HSS 2305 A Lecture 21: Interactions Overview

Course Information

• Course: HSS 2305 A, Molecular Mechanism of Disease
• Session: Fall 2023
• Lecturer: Ajoy Basak, Ph.D.
• Contact Information: Provided

Lecture 21: Protein-Ligand, Protein-Protein Interactions

• A ligand is a molecule that binds to a protein, often leading to a change in conformation and activity of the ligand to initiate or alter cellular responses.
• In receptor-mediated events, the ligand is called a signaling molecule.
• Common types of protein-ligand interactions include: protein-protein/enzyme, protein-peptide, protein-lipid, protein-carbohydrate, protein-nucleic acid (DNA/RNA), and protein-small organic molecule.
• Protein-protein interactions are frequently non-covalent; covalent interactions include cross-linking and disulfide bonds; non-covalent interactions include hydrophobic and ionic interactions.
• Protein-protein interactions are critical regulators of many cellular processes.
• Tools like STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) are used to find known protein interactions, based on data and computational prediction. This database (STRING) contains 5.2 million proteins from 1133 species in version 9.0 and 11.0 (2023).
• Protein-protein interactions are essential for various biological functions, including the regulation of DNA replication in Human Papilloma Virus (HPV) infection.
• Certain proteins, like E1 and E2, mediate HPV DNA replication. E2 has a DNA-binding domain that dimerizes for viral inhibition. Inhibitors may target the E1 enzyme or E2 dimerization for HPV-infection blockage.

Methods for Detecting and Measuring Protein Binding

• Fluorescence Method: One protein is labeled with a fluorescent molecule (e.g., fluorescein). Upon binding, the fluorescence intensity changes.
• Co-immunoprecipitation (Co-IP) Method: Two interacting proteins are incubated together. An antibody specifically targeting one protein is added, precipitating both proteins. Gel electrophoresis and Western blots are used to confirm the presence of both proteins.
• Radiolabeled Method: One protein is labeled with radioactivity (e.g., I131, H3 or C14). When two proteins interact, the radioactivity is transferred to the complex.
• Native (Non-denatured) Gel Electrophoresis Method: This method runs proteins in the absence of SDS to detect complex formations.
• Immunolocalization: A method used to detect the presence of proteins by using antibodies specific for a certain protein.
• Immobilization Method: One protein is covalently attached to a resin and the second protein is added. The resin is washed, and the remaining proteins are released and analyzed.
• Surface Plasmon Resonance (SPR) Method: A label-free protein binding detection method.
• Specific methods for Protein-protein interactions depend on the target proteins and the type of interaction being analyzed.

Effects of Protein-Protein Interactions in Biology

• Modifying enzyme kinetic properties.
• Acting as a substrate channeling mechanism.
• Creating new binding sites for small effector molecules.
• Inactivating or suppressing protein function.
• Altering substrate specificity.
• Regulating biological processes at upstream or downstream levels

Examples of Protein Interactions in Disease

• Hypercholesterolemia: The interaction of PCSK9 with LDL-R leads to LDL-R degradation, resulting in elevated blood cholesterol.
• HPV Infection: Interactions between E1 and E2 proteins are involved in HPV DNA replication.
• Cancer (Angiogenesis): In cancers, the MMP2 and aβ3 integrin interaction is crucial for angiogenesis (new blood vessel growth).
• Multiprotein Complex (Pyruvate Dehydrogenase Complex): A complex of three enzymes crucial for glycolysis and the tricarboxylic acid cycle, whose dysfunction can lead to neurological diseases.
• Alzheimer's Disease: Lipid rafts are implicated in Alzheimer's progression, facilitating interaction between amyloid precursor protein (APP) and β-secretase enzyme, leading to amyloid-β peptide generation.

Additional Notes

• Many human diseases result from abnormal protein-protein interactions or pathogen-host interactions.
• Specific protein interactions are a significant target for therapeutic intervention due to their important role in many biological processes, including several diseases.
• Various small molecule inhibitors target protein-protein interactions and are under development for treatment of human diseases.

Explore the dynamics of protein-ligand and protein-protein interactions in this quiz based on HSS 2305 A. Understand the critical roles these interactions play in cellular processes and how they can be mediated through various molecular mechanisms.