Molecular Mechanism of Disease Lecture-21 PDF

Course HSS 2305 A Molecular Mechanism of Disease Session Fall 2023 Lecture-21 Protein-Ligand, Protein-Protein Interaction Chapter 2 Ajoy Basak, Ph. D. Adjunct and Part-time Professor, Pathology and Laboratory Medicine,...

Course HSS 2305 A Molecular Mechanism of Disease Session Fall 2023 Lecture-21 Protein-Ligand, Protein-Protein Interaction Chapter 2 Ajoy Basak, Ph. D. Adjunct and Part-time Professor, Pathology and Laboratory Medicine, Faculty of Medicine, U Ottawa, Roger Guindon Building 451 Smyth Road Ottawa, ON K1H 8M5 Tel 613-878-7043 (Cell) E-mail: [email protected] Alternate: [email protected] Affiliate Investigator, Chronic Disease Program, Ottawa Hospital Research Institute Web: https://med.uottawa.ca/pathology/people/basak-ajoy 1 2  Interaction Between Proteins and Ligands (Non-Receptor Mediated) A ligand in biology is a molecule that binds/interacts or forms a complex with a protein molecule leading to change in conformation/geometry and activity of the ligand to initiate or alter cellular responses  For receptor mediated event the ligand is called signaling molecule  Types of Protein-Ligand Interactions  Protein : Protein/Enzyme (Most common interaction event in physiological systems)  Protein : Peptide  Protein : Lipid  Protein : Carbohydrate  Protein : Nucleic acid (DNA/RNA)  Protein : Small Organic Molecule Nature of Protein Interaction Non-covalent. Hydrophobic. Ionic Covalent. Cross linking. Disulphide bond 5 Protein : Protein / Enzyme Interaction Protein : Protein interactions regulate the majority of cellular processes & are significant points of intervention for development of therapeutics and Drugs for disease intervention 6  Protein:Protein Interactions  All known protein interactions can be found in the website “STRING” (Search Tool for the Retrieval of Interacting Genes/Proteins)  “STRING” data is based on experimental data, Computational prediction methods and Public Text collections.  The last version out in 2023 is v11.0. The v9.0 contains 5.2 million proteins from 1133 species. Web site: http://string-db.org/newstring_cgi/show_network_section.pl 7 Tryptophan Pathway of A Typical Example of Protein:Protein Interactions Genes in Bacteria: TrpA: Tryptophan Synthase or Synthetase alpha subunit TrpB: Tryptophan Synthase beta subunit TrpC, TrpD, TrpE, TrpS FolC: Dihydro Folate Synthase PabA/B: Para Amino Benzoate A/B TnaA: Tryptophanase AccD: Acetyl-CoA carboxylase beta subunit  Protein-Protein interaction network visualized by STRING.  In this view, the color saturation of the edges represents the confidence score of a functional association 8  Thicker the line means stronger the interaction. How Protein Interaction Can Be Detected And Affinity Potential or Binding Strength Be Measured? 9 Detection & Measurement of Protein Binding Efficiency 1. Fluorescence Method 2. Co-immunoprecipitation Method 3. Labeled Protein Method 4. Native (Non denatured) Gel Electrophoresis Method 5. Immobilization Method 6. Surface Plasmon Resonance (SPR) method (Label free method) Detection & Measurement of Protein Binding Efficiency 1. Fluorescence method  One protein (P1) is labeled with a fluorescence group (such as Fluorescein molecule shown below)  The second protein (P2) is unlabeled  Upon binding the fluorescence intensity of labeled P1 protein is normally suppressed and / or peak position shifted. 578 nm (Peak) Fluorescein molecule  Emission spectra of Fluorescein labeled-P1 Protein upon interaction with increasing concentrations of unlabeled P2 protein  The spectra were recorded at at a fixed Excitation wavelength (514 nm) Detection & Measurement of Protein Binding Efficiency 2. Co-immuno-precipitation (Co-IP) method:  The two interacting proteins are incubated to allow binding  The mixture was treated separately with Antibody (Ab) specific for either protein  In either case one can notice the presence of both proteins by gel electrophoresis and western blot. Two Interacting proteins A & B Both proteins are precipitated by either antibody (Ab against Antibody (Ab) against protein A or Ab against either protein A or B protein B) Detection & Measurement of Protein Binding Efficiency 3. Radiolabeled Method:  One protein is labeled with radioactivity (eg I131, H3 or C14)  Second protein not labeled  Upon interaction radioactivity could be seen in the complex. Protein-1 Protein 2 Molecular Weight = M1 Molecular Weight = M2 (Radioactive Labeled Protein) (Non-radioactive Protein) Protein-1 Protein 2 Radioactivity Found in M1+M2 complex Detection & Measurement of Protein Binding Efficiency 4. Native (Non denatured) Gel Electrophoresis Method  Here Gel Electrophoresis is performed in absence of SDS (a detergent) to detect formation of complex between two proteins  Under SDS condition non-covalent interaction between two proteins cannot be observed since SDS disrupts such binding MW : Molecular Weight Band (Lane C) MW (kDa) Showing the 50 formation of 35 Complex Between 15 the Two Proteins A and B Detection & Measurement of Protein Binding Efficiency 5. Immobilization Method  Here one protein is attached covalently on a resin.  The second protein (with a Fluorescent or Radioactive Tag or Label) solution was added to the above resin  The resin was washed extensively  The Protein-2 will remain attached which could later be released from resin by buffer with high ionic strength like high NaCl (e.g. ~ 500 mM) Resin or Bead 15  6. Surface Plasmon Resonance Method  Here the light beam of single wavelength is projected in a multi-angle fashion using a prism on a surface of gold particle on which one ligand (eg DEXTRAN) is immobilized.  Surface Plasmon Resonance Angle provides information about binding of DEXTRAN with any other ligand added to it. It also measures the binding constant of each interaction.  Human Diseases and Protein Interaction  Many human diseases are the result of abnormal Protein–Protein interactions involving endogenous proteins, proteins from pathogens or both.  Studies have identified and/or characterized specific interactions from various disease systems, including Cervical Cancer, Bacterial Infection, Leukemia and Neurodegenerative Diseases.  Strategies have been developed to generate inhibitors or blockers of Protein–Protein interactions that may lead to useful therapeutic use.  Thus the intervention of these aberrant associations is of great and obvious clinical significance.  Because of the diverse nature of Protein–Protein interactions, the successful design of therapeutics requires detailed knowledge of each system at a molecular and atomic level.  Example-1: Human Papilloma Virus (HPV) Infection 18  Human Papilloma Virus (HPV) Infection  Most common sexually transmitted infection (Skin to Skin)  Many types (>100 varieties) are known, some lead to cancer, others lead to skin lesion  One type is responsible for 70% Cervical Cancer and another type for 70-90% anogenital warts (also called Condylomata)  No Specific symptoms but appearance of anogenital warts considered as indicator of sexual abuse.  Sometimes warts appear inside the body or inside skin  Vaccine (Immunization) is available as preventative measure  HPV DNA test Condylomata: Reddish Cauliflower type bulges in the skin 19 Pathway showing the role of E1 and E2 proteins of HPV-I DNA duplication 20 Initiation of HPV DNA replication. (A) Schematic representation of viral proteins E1 and E2 required for replication of HPV genome. E1 and E2 are ~650 and 370 amino acids in length, respectively. OBD: Origin Binding Domain; TAD: TransActivation Domain; H: Hinge region; DBD: DNA-Binding Domain. (B) (I) Replication is initiated by the recruitment of E1 (blue), by E2 (yellow), to the viral origin. This recruitment step involves an essential protein-protein interaction between the TAD of E2 and the helicase domain of E1 that can be antagonized by the Indandione or Repaglinide class of small molecule inhibitors. (II) E2 recruits additional E1 molecules and promotes their assembly into a replication- competent double hexameric helicase. ATP also stimulates the oligomerization of E1 and is further needed to power the helicase activity of E1. Biphenylsulfonacetic acid inhibitors abrogate the ATPase and helicase activities of E1. (III) Finally, E1 interacts with host cell replication factors such as polymerase α primase (pol α; orange) to promote bidirectional replication of the viral genome. 21  Protein-Protein interaction in HPV infection: Potential targets  DNA Replication of HPV is mediated by E1 and E2 proteins  E2 has a DNA binding domain that forms a dimer  Dimerization inhibitors are considered for viral inhibition  The E1 enzyme inhibitors are also considered for blocking HPV infection  E2-mediated transcription of HPV chromatin GCN5 is a Histone Acetyl Transferase Potential targets for papilloma virus E2 inhibition. Arrows indicate potential targets for inhibition of papilloma virus infection. The E1 helicase (cyan) and E2 (magenta). The DNA- binding domain of E2 forms a dimer. Dimerization inhibitors are considered for viral inhibition. The structure of bromodomain (blue) of GCN5 bound to the acetylated histone H4 peptide (yellow) is shown in duplicate to represent the tandem bromodomains of Brd4. Relevant domains of unknown structure are shown as thick lines.  Example of Protein interaction as target The structure of a papilloma virus E2–small-molecule inhibitor complex (a) The E2 transactivation domain (magenta) bound to an inhibitor (orange) (Arrow). Inhibitor binding induces the formation of a hydrophobic pocket in the protein domain to prevent E1 binding. (b) Chemical structure of the inhibitor. EFFECTS OF PROTEIN:PROTEIN INTERACTION IN BIOLOGY 1. Modify the kinetic properties of enzymes 2. Act as a general mechanism to allow for substrate channeling 3. Construct a new binding site for small effector molecules 4. Inactivate or suppress a protein’s functional activity 5. Change the specificity of a protein for its substrate through interaction with different binding partners 6. Serve a regulatory role in either upstream or downstream level 25 Rao VS, Srinivas K, Sujini GN, Kumar GN. Protein-protein interaction detection: methods and analysis. Int J Proteomics. 2014;2014:147648.  Example-2: Hypercholesterolemia 26  Interaction of h(human)PCSK9 with hLDL-R protein: It leads to LDL-R degradation and High LDL-Cholesterol in Blood b: Crystal Structure of hPCSK9, showing its various characteristic domains and the binding interaction of its cleaved prodomain segment (shown by green arrow) with its catalytic domain (shown by yellow arrow). Blue arrow shows the C- terminal domain Human Proprotein Convertase Subtilisin Kexin 9 (PCSK9) binds with Low Density Lipoprotein Receptor (LDL-R) which leads to LDL-R’s degradation in the lysosome. This results in accumulation of cholesterol in the blood 692 Prodomain Catalytic Domain 152 422 Pro-PCSK9 Protein C-Terminal Domain 31 (Endoplasmic Autocatalytic cleavage Reticulum) 692 152 31 Prodomain 422 bound PCSK9 153 Prodomain bound (Golgi) Binding of hPCSK9 prodomain of PCSK9 with its catalytic domain hLDL-R (Human Low Density Lipoprotein) : hPCSK9 Complex EGF-A domain of LDL-R involved in binding with PCSK9 via latter’s prodomain Binding of prodomain of hPCSK9 with its catalytic domain 152 31 422 1 153 Binding of hLDL-R protein with the catalytic domain of Prodomain bound hPCSK9 hLDL-R hLDL-R forms complex with - hLDL-R Degradation prodomsin bound PCSK9 - Cholesterol Accumulation Disruption of hLDL-R : PCSK9 Complex in presence of a PCSK9 peptide derived from its catalytic domain, leading to LDL-R enhancement and cholesterol clearance 152 692 365 384 31 153 422 Prodomain 365 384 bound hPCSK9 1 hLDL-R hLDL-R Degradation is Blocked More Cholesterol Clearance by LDL-R  In hLDLR : hPCSK9 interaction  EGFA domain is involved  Catalytic domain of hPCSK9 is involved  Addition of a synthetically prepared EGFA peptide can block the above hLDLR:hPCSK9 interaction  Also Peptide derived from PCSK9 catalytic domain can block hLDLR:hPCSK9 interaction  Either peptide is expected act as competitive inhibitor and therefore lower hLDL- cholesterol level in blood hLDL-R: hPCSK9 hLDL-R complex crystal hLDL-R structure Interaction area hLDL-R 32 Example-3: Cancer 33  Protein-Protein Interactions and Angiogenesis  Tumor growth depends on the development of new blood vessels, a process that is known as Angiogenesis  This provides oxygen, nutrients and blood supply to the cancer cells for their survival and growth  The interaction of two proteins Matrix Metallo Proteinase 2 (MMP2) and the Membrane Bound Integrin αvβ3 Receptor is crucial to this process.  Angiogenesis begins when MMP2 interacts with the alphav subunit of αvβ3, which then degrades the collagen matrix that surrounds cells, making room for new blood vessels to proliferate.  Small-molecule inhibitors that block the ability of MMP2 to interact with αvβ3 would disrupt angiogenesis, and provide a powerful novel therapy for cancer. 34  Protein-protein interactions and angiogenesis. The interaction of MMP2 and αvβ3 (a membrane-based receptor of the integrin family) 35  Angiogenesis Inhibitors Based Drugs for Cancer  Bevacizumab to be used alone for glioblastoma that has not improved with other treatments and to be used in combination with other drugs to treat metastatic colorectal cancer, some non-small cell lung cancers, and metastatic renal cell cancer.  Bevacizumab was the first angiogenesis inhibitor that was shown to slow tumor growth and, more important, to extend the lives of patients with some cancers.  Other approved Antiangiogenic Inhibitor Based Cancer Drugs are Sorafenib, Sunitinib, Pazopanib and Everolimus.  Sorafenib is approved for hepatocellular carcinoma and kidney cancer  Sunitinib and Everolimus for both kidney cancer and neuroendocrine tumors  Pazopanib for kidney cancer 36  Multiprotein Complex  Many Examples are known in which different proteins each with separate function becomes associated to form a much larger “Multiprotein Complex”  First example: Pyruvate Dehydrogenase isolated from E. Coli  It is a complex consisting of 3 enzymes based on 60 polypeptide chains  Catalyze a series of reactions in Glycolysis and TCA Cycle  Lack of activity of this enzyme complex lead to Neurological diseases 37 Protein : Lipid Interaction 38  Protein – Lipid Interaction and Alzheimer’s Disease  Lipid rafts (Membrane Domains) promote interaction of the Amyloid Precursor Protein (APP) with the Beta Secretase Enzyme (BACE-1)  This leads to increased generation of the Amyloid β peptide  It promotes Alzheimer’s Disease  Protein : Lipid interaction also plays role in GPCR-mediated pathway signaling 39 Protein : Carbohydrate Interaction 40  Lectins  Important Class of Carbohydrate Binding Proteins  Many involve carbohydrates found on cell surface as a membrane glycoprotein or glycolipid.  Such Interactions play key role in Cellular adhesion, Signal transduction, Host-Pathogen recognition events and Inflammatory response  Possess carbohydrate binding domain 41 Mannose Binding Lectin recognizes bacterial surfaces by their particular spacing of carbohydrate residues and they promote bacterial infection 42