Concepts to Refresh-Biochem PDF

[Foundations: Biochemistry Summary Sheet] **[Genome: ]** **DNA packaging:** - DNA double helix wrapped around histones - Histones (basic) have a positive charge that binds to the negative phosphate group (acidic) of DNA - Histone modification help regulate DNA packing and...

[Foundations: Biochemistry Summary Sheet] **[Genome: ]** **DNA packaging:** - DNA double helix wrapped around histones - Histones (basic) have a positive charge that binds to the negative phosphate group (acidic) of DNA - Histone modification help regulate DNA packing and gene expression (epigenetics) - Tightly wrapped= decreased gene expression; needs to be loosened to interact with transcription factors - 4 histone types: - H2A, H2B, H3, and H4 - DNA wrapped around 8 histones= nucleosome - Nucleosomes wrapped tightly into a helical fiber - Helical fiber wraps into supercoil **DNA structure:** - Consists of two strands of nucleotide polymers - Nucleotide consists of a base, sugar, and phosphodiester linkage - Four bases: - Adenosine and Guanine= purines - Thymine and Cytosine= pyrimidines - **A and T pair with 2 hydrogen bonds** - **C and G pair with 3 hydrogen bonds** - **Chargaff's rule:** number of A=T and C=G in nuclear genome; not the case for mitochondrial genome or in prokaryotes**.** - Nucleoside Purine/pyrimidine base attached to the deoxyribose sugar - Nucleotide Purine/pyrimidine base attached to deoxyribose sugar with the phosphate attached. - Stabilized by: - Base stacking - Hydrophobic interactions - Van der Waals forces **DNA replication:** 1. Helicase unwinds the parental double helix 2. Single-stranded binding proteins stabilize the unwound parental DNA 3. Leading strand is synthesized continuously in the 5' to 3' direction by DNA polymerase a. Goes towards the replication fork b. DNA polymerase has polymerizing and proof-reading activity (3' to 5' exonuclease activity) 4. Lagging strand is synthesized discontinuously c. Primase synthesizes a short RNA primer d. Primer is extended by DNA polymerase to form Okazaki fragments e. Goes away from the replication fork 5. RNA primer is replaced by another DNA polymerase 6. DNA ligase joins the Okazaki fragment to the growing strand - **DNA gyrase** prevents supercoiling of proximal DNA strand - Replication is semi-conservative; with strands running anti-parallel **Genome Variability:** Germline mutation DNA changes that are passed on Somatic mutation DNA changes not passed on - Inversion - Deletion - Translocation - Transition: purine substituted for a purine or a pyrimidine for a pyrimidine - Transversion: purine substituted for a pyrimidine - Duplication Mutation uncommon alteration in DNA sequence SNP common inherited change in a single base pair that occurs in at least 1% of the population - Ex) p53 - Stabilizes to stop cell division to repair DNA - If cant be repaired then DNA is degraded - Tumour suppressor - SNP increases cancer susceptibility **DNA damage and repair mechanisms** +-------------+-------------+-------------+-------------+-------------+ | **Stressor* | Replication | Oxygen | Ionizing | UV Light | | * | stress: | radicals | radiation | | | | | | | Polycyclic | | | - Toxins | Ionizing | Chemo | aromatic | | | | radiation | | hydrocarbon | | | - Venom | | | s | | | | Chemo | | | | | - Chemo | | | | +=============+=============+=============+=============+=============+ | **Damage** | Base | Single-stra | Double-stra | Bulky | | | mismatch | nd | nd | adducts/int | | | | break | break | rastrand | | | - DNA | | | crosslinks | | | polymer | | Interstrand | | | | ase | | crosslinks | | | | can't | | | | | | proof | | | | | | read | | | | +-------------+-------------+-------------+-------------+-------------+ | **Repair** | Mismatch | Base | Homologous | Nucleotide | | | excision | excision | recombinati | Excision | | | repair | repair | on | Repair | | | | | & | | | | | | Non-homolog | | | | | | ous | | | | | | end-joining | | +-------------+-------------+-------------+-------------+-------------+ - Mitochondrial DNA is maternally inherited - Genome size isn't what makes us distinct - Sophistication of gene expression helps us adapt to change in stim - Bring risk associated with responding to mutations **[Gene Expression: ]** **RNA:** - 2' hydroxyl on sugar; Uracil replaces Thymine - Classifications: - RNA - Messenger RNA - Non-coding RNA - Housekeeping ncRNA - tRNA - rRNA - Regulatory ncRNA - Long non-coding RNA - Small non-coding RNA - Micro RNA - Small nuclear RNA - PIWI interacting RNA **Location:** - DNA: nucleus and mitochondria - RNA: cytoplasm **Protein-coding gene:** - Regulatory sequence controls when and where expression happens in protein-coding region - Promoter and enhancer regulate transcription into preMRNA which is modified - **mRNA 5' and 3' untranslated regions regulate transcription** **Transcription:** - Initiation, Elongation, Termination - RNA polymerase binds promoter; promoter itself isn't transcribed - Initiation of RNA synthesis by binding NTP to antisense strand of DNA and moves 5' to 3' - NTP loses 2 phosphates to generate energy (ATP) - Elongates until termination signal - Pre-mRNA detaches from the template strand and DNA rewinds; RNA polymerase detaches from the DNA **Post-transcriptional modifications:** - 5' cap prevents degradation and helps binding - Removal of introns and exons joining by differential splicing spliceosome - Catalyzed **small nuclear ribonucleoproteins** (snRNPs) of spliceosome - Spliceosome recognizes splicing signals to bring introns together - Have GU at 5' end and AG at 3' end - Branching point within the intron has Adenosine - Splice out as a lariat structure - Poly-A tail enables export to cytoplasm and prevents degradation **Prokaryotes:** - Single transcript - One step, fast - Cytoplasm - Single RNA polymerase synthesises all the three types of RNA - Several co-regulated genes (operon) - Corresponds directly to protein sequence (no introns) **Thalassemia** mutation G to A in exon-intron junction - snRNP doesn't recognize junction **Systemic Lupus Erythematosus** body produces antibodies against snRNPS - defective splicing **Translation:** - Ribosome: - A Aminoacyl site - P peptidyl transfer site - E exit site - P and A site has transfer of charges to cause to cause PP binding **Stop codons:** - UAG, UAA, UGA **Post-transcriptional modifications:** - Phosphorylation - Methylation in CG-rich promoter regions inhibits transcription - Polycomb Repressive Complex 2 (PRC2) using SAM to close chromatin and prevent RNAPII binding - Acetylation Histone acetyltransferases (HATs) with acetyl-CoA - Chromatin opens to allow RNAPII to bind promoter and induce expression **Euchromatin** transcriptionally able; lose and accessible by transcription factors **Heterochromatin** No transcription; tight and inaccessible by TF **[Proteins and Enzymes: ]** **Levels of structure:** 1. Primary structure a. AA sequence b. Determines higher order structure c. Convention: amino terminal to carboxyl terminal 2. Secondary structure d. Localized conformation of polypeptide backbone 3. Tertiary structure e. 3D structure of polypeptide, including all its side chain 4. Quaternary structure f. Spatial arrangement of polypeptide chain with multiple subunits **Amino acids:** - Free amino (positive charge) and carboxyl (negative charge) groups are ionized at physiological pH - 20 common AA classified by: - Polarity Non-polar vs polar - Ionizability basic vs acidic - Special chemical properties - Non-polar: - MCAT Methionine - Grades Glutamic Acid - Are Alanine (simplest AA) - Like Leucine - Fights Phenylalanine - I Isoleucine - Prove Proline (rigid structure) - Victorious Valine - Polar: - Santa's Serine - Team Threonine - Crafts Cysteine - New Asparagine - Quilts Glutamine - Acidic (donate H+): - Erectile Glutamate - Dysfunction Aspartate - Basic (accept H+) : - Knights Lysine - Riding Arginine - Horses Histidine **Acid-Base properties of AA:** - Henderson-Hasselbalch: relates ratio of conjugate base to acid at a given pH - Buffer: solution containing a mixture of weak acid and its conjugate base; resists changes when strong acids or bases are added; **resists near pKa value** - **Molecule is at isoelectric pH when its net charge is 0** **Peptide bond:** - Linked with dehydration reaction, forming an amide (peptide) bond - Planar and alpha-amino and carboxyl groups are not charged - Cleaved by proteases **Immunoglobulins:** - 2 heavy chains and 2 light chains; stabilized by intra- and inter-chain disulphide bonds - Organized into complement (Fc) and antigen-binding (Fab) regions consisting of globular-sheet domains - Almost all beta-sheets - Detection: - Antigen-binding region of Fab has residues of the heavy and light chain - Binding to epitope due to complementary surface, H-bonds, and electrostatic forces - Antibody can be used to detect and quantify proteins either: - Directly ELISA - Protein separation by electrophoresis Western Blot Elisa: - Wells pre-coated with capture antibody that binds to specific antigen - Primary antibody binds to immobilized analyte - 2^nd^ antibody binds and is linked with fluorescence - Detects protein presence with an idea of concentration - Can give false positives Western Blot: - Separate proteins by weight in polyacrylamide gel and use Ab to detect - Very specific - Used in Autoimmune disease detection **Proteins as biomarkers** - **Cardiac troponins in MI** - Released in acute cases - Released with death and damage of cardiac myocytes - Detected a few hours after event Secondary structure: - Alpha helix - Right-handed - Side chains sticking out - H-bonds - Beta-sheet - Parallel or anti-parallel - Ex) Alzheimer's Disease - Normally alpha helix proteolytic cleavage beta-amyloid fibril - Nucleated aggregates form stacks and compromise neurons **Post-translational modifications:** - Phosphorylation: - Transfer of phosphate group from ATP to Ser, Thr, or Tyr - Protein kinases, removed by phosphatases - Regulation of metab and signal transduction - Glycosylation: - Attachment of complex carb to Asn or Ser - Secreted, extracell or luminal proteins - Stability, targeting, recognition - Proteolysis: - Cleavage of chain at specific sites - Activation of other proteases - Synthesis of peptide hormones **Protein stability and denaturation:** - Native conformation due to primary structure but only marginally stable - Hydrophobic interactions, salt bridges, hydrogen bonds - Denaturation= loss of 2-4 structure and function, often irreversible - Proteins more tolerant to conservative mutations with polar surface residues than non-conservative replacements (leads to conformational change and functional change) **Hemoglobin A:** - Heterotetramer (a2b2) **Enzymes:** - Convert substrate to product without changing reaction equilibrium - Active site decreases activation energy of reaction by binding the transition state - Binding triggers conformational change that brings catalytic group into right position to facilitate the reaction - Kinetics: - Described by Michaelis-Menten kinetics; Vmax and Km - Vo depends on the substrate concentration when S is low, but not when S is high - Reversible inhibition: - Competitive: - Vmax remains the same but Km increases - Binds the active site preferentially therefore need increase in S - Non-competitive: - Km is unchanged but Vmax is decreased - Binds allosteric site therefore doesn't matter what S is because it stays bound **Mechanisms for regulation:** - Substrate availability - Allosteric control - Covalent modification - Localisation - Enzyme synthesis or degradation **[Cell Structure: ]** **Cell compartments: topological relationships:** - How do proteins know where to go? - Depends on what membranes it needs to cross - Proteins continuous with the cytosol can travel throughout the cell - Proteins can be retained or exported **Cytoskeletal networks:** - 3D structure that provides structure, strength, support, motility - Extends throughout the cytosol - Rapidly changes - Three main elements: - Microtubules - Hollow, heterodimers of tubulin - One end attached to microtubule-organizing center= centrosome - More rigid and rupture when stretched - Help position organelles - Actin filaments - Helical actin polymers; G-actin monomers that polymerize by positive and negative ends - Treadmilling: hydrolyze ATP and change cell shape - Muscle contraction and cell signalling - Flexible, organized in linear bundles - Concentrated in cortex - Intermediate filaments - Fibrous insoluble proteins nuclear lamina - Beneath the nuclear membrane - Flexible, helping cell to distribute stress and gain tensile strength - Mechanical strength and tensile strength - Cytoplasmic: - Keratin filaments (epithelium) - Vimentin and vimentin-related filaments (CT cells, muscle cells, and glial cells) - Neurofilaments (all animal cells) - Nuclear: - Nuclear lamins - Ex) Progeria - Rapid aging where lamin network is compromised **Imaging cytoskeletal units:** - Fluorescence microscopy **Cellular Junctions:** - Tight junction seals neighbours, prevents leakage, helps polarize cells - Adherens junction connects **actin filaments** - Desmosomes connects **intermediate filaments** - Gap junction cell communication with small, intracellular, water-solubel molecules - Hemidesmosome anchor intermediate filaments and nucleus to basement membrane **Lipid bilayer:** - Phospholipids and cholesterol= amphipathic - Membrane proteins: - Peripheral surface, based on membrane-association by electrostatic interactions - Integral span whole bilayer; based on hydrophobic interactions; hard ot access therefore need detergent - Lipids can rotate and diffuse within membrane plane **Membrane permeability:** - Permeable: - Small, non-polar molecules - Distribution critical to cell function - Not crazy permeable - Small, uncharged polar molecules - Larger uncharged polar molecules - Impermeable - Ions - Function of membrane to establish gradients for inorganic ions **Membrane transporters and channels** - Simple diffusion - Passive transport: - Channel-mediated - Active transport: - Ex) Na/K-ATPase pumps **[Cell Dynamics: ]** - Proteins need to be delivered to specific cellular location or exported out = protein targeting - Carried out based on info within coding sequence - Gated transport: - cytosol to nucleus - nuclear pore complexes; selective gates for active transport - Protein translocation: - into organelles passing membranes - protein usually unfolds and snakes through translocator - Vesicular transport: - endocytic - vesicles derived from lumen - discharge cargo by fusing membrane - Ex) Lysosome - If no sorting sequence, it ends up in the cytosol - Targeting sequences: - Cytosol no signal (default) - **Lysosomal targeting attachment of M6P** - **Proteasomal targeting PEST-rich sequences** - Nucleus internal basic sequence - Mitochondria N-terminal amphipathic helix - Peroxisomal import C-terminal SKL - ER N-terminal hydrophobic sequence - ER retention C-terminal KDEL **Sorting Roadmap:** - Secretory pathway: - ER Golgi PM or lysosome - Endocytic pathway: - Ingested in vesicles derived from PM early endosomelate endosomes lysosomes - All ATP-dependent **Lysosomal protein targeting:** - Proteins targeted here are meant to clear cellular waste through protein degradation 1. Lysosome hydrolase precursor enters golgi from ER 2. Phosphate is adding forming M6P a. Sorting signal b. Sugar added for solubility (glycosylation) 3. M6P binds M6Pr in golgi to prelysosomal endosome 4. Receptor-dependent transport of vesicle from prelysosomal endosome 5. Endosome is acidified by H+ pump 6. M6P dissociates 7. M6Pr organize within budding vesicle 8. Receptors recycled back to trans golgi c. Some leaked outside of cell to PM helpful with disease treatment **Niemann-Pick C Disease:** - Affects kids at school age; rapid neurological impairment - Death in teen years - NS mutation: **Gly992 Tyrp** - Affects ability of cell to deliver cholesterol from NPC1 MOA: - NPC1 delivers cholesterol to NPC2 on golgi - Cells loat cholesterol by LDL endocytosis - Increased unesterified cholesterol: - Decreased LDL receptor synthesis less intake - Decreased cholesterol synthesis less production - Decreased esterification and storage less storage and modification Therapies: - **Miglustat** decrease glycolipid synthesis; reduces lysosomal congestion - **Cyclodextrin**s synthetic cholesterol carriers; facilitate transport - **Histone deacetylation inhibitors (HDACi)** blocks the inhibition of transcription increase transcription of NPC1 that are functional **Fabray disease:** - Deficient alpha-galactosidase enzyme - Unable to degrade glycosphingolipids Therapy: - Enzyme replacement therapy: - After M6P and M6Pr are recycled there can be mistargeting - Health alpha-galactosidase taken up with M6Pr and can allow some processing - Very expensive and unethical because of that **[Introduction to Metabolic Biochemistry: ]** - Catabolic pathway: - Breakdown biomolecules to get complex macromolecules, ATP, and/or electron donors (NADH, FADH2) - Anabolic pathway - Use ATP and reduced co-factors to make complex macromolecules - Catabolic and Anabolic pathways must differ by at least one step to not be a futile cycle **Thermodynamics and free energy:** - [Energy intake equals energy expenditure plus weight gain or loss] - Exergonic reactions are favoured - Final product loses free energy and has negative delta G - Endergonic reactions are unfavourable - Product has a positive delta G - Can be made to proceed if coupled to a exergonic reaction **Redox reactions and ATP synthesis:** - Oxidoreductase enzymes transfer electrons to coenzymes NAD+ or FAD, reducing them to form NADH and FADH2 - NADH and FADH2 are subsequently re-oxidized to NAD+ and FAD in the ETC coupled to ATP synthesis by oxidative phosphorylation **Glycogen:** - Branched polymer of glucose - Glycogen to glucose glycogenolysis - Glucose to glycogen glycogenesis - Two most important storage organs: - Liver (control of blood glucose level) - Skeletal muscle (for its own energy needs) **Glycolysis:** - Catabolic pathway converting one molecule of glucose to two molecules of pyruvate in the cytosol - Glucose + 2 ADP + 2 NAD+ 2 Pyruvate + 2 ATP + 2 NADH - Requires molecular oxygen - In anaerobic conditions forms lactate through the Cori cycle **TCA cycle:** - Catabolism of carbs, AA, and FAs converge (mitochondria) - Carbon enters as Acetyl-CoA - Key intermediate and allosteric regulator of many enzymes - Provides maximum energy: up to 32 ATP per glucose oxidized **Gluconeogenesis:** - Used mainly by the liver to replenish BGL - Uses precursors like pyruvate, lactate, glycerol, and some AA - Can't use acetyl-CoA **[Signal Transduction and cell fate]** **General principles:** - Cell-surface receptors - Hydrophilic signal molecule binds surface receptor on the PM - Activates second messenger system to cause change in DNA leads to effect - Intracellular receptors - Carrier protein brings small hydrophobic signal molecule to the cell - Crosses through the membrane and interacts with intracellular receptor in the nucleus to effect change **Classification of extracellular signals:** - Classified chemically, by nature of transmission, or by system - Signal molecules are polar and need membrane receptors, or non-polar and can cross PM - Contact-dependent signal and receptor bound to membrane - Paracrine released and effect close by - Synaptic Neuro - Endocrine released to have effect far away **Important themes in signal transduction** 1. Extracellular signals (ligands) bind to specific cellular receptors a. Relay signals with small molecules and intracellular signalling proteins b. Membrane receptors: i. G-Protein coupled receptors ii. Enzyme-coupled receptors iii. Ion-channel-coupled receptors 2. GTP hydrolysis by G-proteins acts to regulate the activity and timing of many signalling pathways c. G-proteins turn pathways on and off: when bound to CTP they bind and activate downstream proteins d. Regulators of G protein (RGS) act as timers because of intrinsic GTPase,act as a subunit-specific GTPase activating proteins (GAPs) e. Further regulated by: iv. GEF v. GAP f. Two main classes of G-proteins vi. Heterotrimeric G-proteins directly activated by GPCRs vii. Monomeric G-proteins important role in cell processes (ex Ras) 3. Signals propagated within the cell by transient formation of second messengers g. Second messengers are kept at low intracellular levels until needed, then are transiently generated from abundant cellular precursors. h. Common examples: viii. cAMP ix. Cytosolic free Ca 2+ x. Hydrolysis of phospholipids (ex. diacylglycerol, inositol-3-PO4) i. Activated PKA induces gene transcription to induce transcription and translation for new effector protein. 4. Signals are amplified and regulated using protein phosphorylation cascades j. Protein kinase transfer phosphate groups to specific AA (Ser, Thr, or Tyr) xi. Reversed by phosphatases k. Phosphorylation can modify target activity and/or provide docking sites for additional signalling proteins l. Targets can be protein kinases, amplifying signal cascade at each step xii. Highly conserved and regulated, but generally slower than Ca2+ signalling 5. Assembly of signalling complexes are facilitated using limited modular protein domains m. Intracellular signalling complexes enhances the speed, efficiency, and specificity of the response. xiii. Scaffolding protein speeds signalling complex; requires previous activation to pass signal xiv. Without: moment the ligand occupies receptor invites proteins to bind inactive receptor to assemble complex **GPCR activation of protein kinase A:** - Bound receptor changes shape and interacts with Gs protein to release GDP and bind GTP - Alpha Gs subunit dissociates and activates adenylyl cyclase - When ligand is no longer present, receptor reverts to resting state; GTP on alpha subunit is hydrolyzed to GDP and adenylyl cyclase is deactivated. - cAMP-dependent PKA becomes active when regulatory subunit comes off - Kinase phosphorylates protein for intracellular effects