1212569_21823227.jpg logo_mu_cerne.gif 1212570_28446780.jpg logo_mu_cerne.gif Luděk Bláha, PřF MU, RECETOX www.recetox.cz BIOMARKERS AND TOXICITY MECHANISMS 03 – Mechanisms @proteins OPVK_MU_stred_2 1212569_21823227.jpg logo_mu_cerne.gif Major mechanisms (modes of action) to be discussed in detail •Proteins and inhibition of enzymatic activities •Mitotic poisons & microtubule toxicity •Ligand competition – receptor mediated toxicity • •Membrane nonspecific toxicity (narcosis) •Toxicity to membrane gradients • •DNA toxicity (genotoxicity) • •Complex mechanisms –Oxidative stress – redox toxicity –Defence processes as toxicity mechanisms and biomarkers - detoxification and stress protein induction –Toxicity to signal transduction – 1212569_21823227.jpg logo_mu_cerne.gif Proteins and enzyme inhibitions à toxicity mechanisms 1212569_21823227.jpg logo_mu_cerne.gif •Structure • – primary (sequence of aminoacids, AA), • - secondary, tertiary, quarternary (folding – important for functions) • •Proteins - large/long – key target for number of toxicants! • = polypeptides - tens to thousands of AA •Peptides (small, “πεπτός, "digested“, 2x AA to e.g. 20x AA) • may have various functions (e.g. protective - glutathione) • •Key functions of proteins •STRUCTURE and PROTECTION •CATALYSIS (enzymes) •TRANSFER (information and mass) • - receptors, channels, transporters •... student should know examples.. • Proteins as targets to toxicants https://www.mun.ca/biology/scarr/iGen3_06-04_Figure-L.jpg 1212569_21823227.jpg logo_mu_cerne.gif http://www.nc3rs.org.uk/downloaddoc.asp?id=744 Overview - interactions of small molecules with proteins 1212569_21823227.jpg logo_mu_cerne.gif CATALYTICAL PROTEINS = Enzymes •Catalysis - what is it? ... student should know • •Thousands of enzymes (vs. millions of compounds) –present in body fluids, membranes, cytoplasm, organelles.. –... student should know key examples • •Enzymology – science of enzymes –includes also interactions of enzymes with small molecules (xenobiotics) 1212569_21823227.jpg logo_mu_cerne.gif Enzymes vs toxicants •Interactions that make a chemical compound an enzyme (or protein) inhibitor • –Competitive vs. non-competitive • active site vs. side domains – –Specific vs nonspecific •affinity of the inhibition .... is determined by the effective concentration (lower the effective concentrations à higher the affinity) –Nonspecific inhibitions •Most of the chemical toxicants (!) •Compound interacts with functional groups on the surface of the protein (reactive toxicity) or affects the environment (high osmomolarity, changing pH) • • 1212569_21823227.jpg logo_mu_cerne.gif Most common interactions (and some examples) Hydrogen bond disruption alcohols, amines Ion bonds acids (COOH), alkalic compounds (amines) toxic metals Hg+2, Pb+2, Cd+2 , Ag+1 Tl+1, carbonyls S-S bonds toxic metals See also http://www.elmhurst.edu/~chm/vchembook/568denaturation.html http://www.elmhurst.edu/%7Echm/vchembook/images/568denathbond.gif http://www.elmhurst.edu/%7Echm/vchembook/images/568denatdisul.gif Non-specific interactions & denaturation 1212569_21823227.jpg logo_mu_cerne.gif 1 Kinetics of the enzyme reaction (Michaelis Menten) 1212569_21823227.jpg logo_mu_cerne.gif Michaelis Menten INHIBITIONS http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Michaelis_Menten2.gif http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/L/Lineweaver_Burk2.gif The kinetics informs about the nature of the interaction ! 1212569_21823227.jpg logo_mu_cerne.gif Enzyme inhibitions by toxicants – overview of key examples •Acetylcholinesterase (organophosphate pesticides) •Microsomal Ca2+-ATPase (DDE) •Inhibition of hemes – respiratory chains (cyanides) •d-Aminolevulinic Acid Dehydratase (ALAD) inhibition (lead - Pb) •Inhibition of proteinphosphatases (microcystins) •Glyphosate (roundup) action • •Enzyme inhibitions are beyond many other mechanisms à see e.g. CELL REGULATIONS etc. • 1212569_21823227.jpg logo_mu_cerne.gif 1 Acetylcholinesterase inhibition by organophosphates http://s2.hubimg.com/u/4316027_f520.jpg 1212569_21823227.jpg logo_mu_cerne.gif Acetylcholinesterase inhibition by organophosphates (and carbamates) http://1.bp.blogspot.com/-vJjcfxZOpBY/UghViEI7QXI/AAAAAAAADdw/0EuZdLLJHRw/s1600/nerve.tif http://www.scielo.br/img/revistas/jbchs/v15n6/22667f1.gif Nerve gases http://www.darkgovernment.com/news/wp-content/uploads/2012/12/sarin.jpg Insecticides - OPs http://www.ung.si/public/lpseminar/Image45.gif http://www2.mcdaniel.edu/Biology/eh01/pesticides/carbamates.gif Insecticides - Carbamates 1212569_21823227.jpg logo_mu_cerne.gif •Ca2+ in cells •* general signalling molecule (see later) •* stored in (endo-/sarcoplasmatic reticulum) •* assures contractility of muscles •* concentrations regulated by Ca2+-ATPase • •DDE à calcium metabolism in bird eggs à egg shell thinning • Inhibition of Ca2+-ATPase by DDE http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DDT.gif File:Elimination DDT DDE.svg DDE 1212569_21823227.jpg logo_mu_cerne.gif 1 1 Inhibition of hemes – e.g. Haemoglobin, Mitchochondria, CYP450 etc. (cyanide HCN, carbon monooxide – CO) http://uvahealth.com/Plone/ebsco_images/7351.jpg 1212569_21823227.jpg logo_mu_cerne.gif ALAD inhibition by lead (Pb) http://www.americanvintagehome.com/plumbing_heating_air_conditioning_information/wp-content/uploads /2014/05/lead-exposure.jpg Problem mostly in the USA Ban of Pb-containg petrols 1212569_21823227.jpg logo_mu_cerne.gif 1864 ALAD inhibition by lead (Pb) – inhibition of HAEM (!) synthesis 1212569_21823227.jpg logo_mu_cerne.gif 1 1b Inhibitions of PROTEINPHOSPHATASES by microcystins •Microcystins (7x AA – heptapeptides) •Cyanobacterial toxins produced in eutrophied waters (water blooms) up to tons/reservoir PPases – signalling enzymes (see further) 1212569_21823227.jpg logo_mu_cerne.gif •N-(phosphonomethyl)glycine •Broad-spectrum herbicide („RoundUp“) •Selective inhibition of ESPs 5-enolpyruvylshikimate-3-phosphate synthase; •(synthesis of aromatic AAs – Tyr, Trp, Phe) •Uptake via leafs - only to growing plants •„Non-toxic“ to other organisms (no ESPs in animals, AA-like chemical - rapid degradation) Glyphosate action 1212569_21823227.jpg logo_mu_cerne.gif Structural proteins (CYTOSKELETON) as target for toxicants 1212569_21823227.jpg logo_mu_cerne.gif Structures of microtubules – dynamic de/polymerization C:\Documents and Settings\Ludek Blaha\Obrázky\1.jpg http://www.primitive-streak.org/gfx/celldivision2.jpg Visualization of microtubules during cell division – separation of chromosomes 1212569_21823227.jpg logo_mu_cerne.gif Structure of actin-myosin system C:\Documents and Settings\Ludek Blaha\Obrázky\1.jpg 1212569_21823227.jpg logo_mu_cerne.gif Cytoskeleton – functions C:\Documents and Settings\Ludek Blaha\Obrázky\1.jpg - intracellular transport - cell replication and division (mitosis:chromosomes) - muscle movement - membrane (vesicles) fusion 1212569_21823227.jpg logo_mu_cerne.gif ACTIN – toxin effects on (DE)POLYMERIZATION C:\Documents and Settings\Ludek Blaha\Obrázky\1.jpg C:\Documents and Settings\Ludek Blaha\Obrázky\1.gif cytochalasin D (fungal toxin) C:\Documents and Settings\Ludek Blaha\Obrázky\1.gif Phalloidin (death cap - Amanita phalloides) 1212569_21823227.jpg logo_mu_cerne.gif Colchicine taxol C:\Documents and Settings\Ludek Blaha\Obrázky\1.jpg C:\Documents and Settings\Ludek Blaha\Obrázky\1.gif C:\Documents and Settings\Ludek Blaha\Obrázky\1.gif TUBULIN – toxin effects on (DE)POLYMERIZATION 1212569_21823227.jpg logo_mu_cerne.gif Toxicity to RECEPTORs (key-lock) discussed later during the lectures: see „signalling“ 1212569_21823227.jpg logo_mu_cerne.gif Various signalling types … now focus on nuclear receptors http://www.uic.edu/classes/bios/bios100/lectures/hormone_types.jpg