MOLECULAR STRUCTURE AND METABOLISM

This course will introduce students to fundamental concepts of molecular structure and function; these will serve as a basis for understanding both the biochemical basis for topics such as metabolism as well as aspects to be covered in the second semester such as membrane transport phenomena and second messenger signaling. The basic concepts of prokaryotic and eukaryotic DNA replication, transcription and translation, as well as protein processing and trafficking will be discussed. The information learned during this course will be the basis for understanding many of basic concepts in cell biology.

Fundamental Concepts

• Water as a biological solvent
• Acid-base chemistry (pH, pK, Henderson-Hasselbalch equation)
• Equilibrium (Gibbs free energy, coupled reactions, K)
• Kinetics (activation energy, catalysts)

Macromolecules – Proteins

• Amino acid structure, classifications, abbreviations
• Acid-base properties of amino acids
• Structure (with forces that stabilize) and function of proteins: primary, secondary, tertiary, and quaternary structure (with emphasis on peptide bond formation and on protein folding)
• Interaction of proteins with metals, lipids, sugars, and nucleic acids; denaturation/renaturation
• Hemoglobin as an example of protein structure/function: how the oxygen-carrying ability of hemoglobin results from its primary, secondary, tertiary, and quaternary structures

Protein Interactions

• Protein interactions and networks on and in cells
• Fundamentals of self-recognition and protein assembly
• Cooperative mechanisms of interaction
• Protein interactions and function in cell receptors and ribosomes
• Methods for measuring and characterizing protein interactions
• Antagonist design in drug discovery using HIV-1 envelope protein interaction machine case study

Enzymes

• General properties
• Function as catalysts
• Types of catalysis (acid, base, covalent) using chymotrypsin as example
• Coenzymes
• Kinetics (Michaelis-Menton equation, Lineweaver-Burk plots Km and Vmax)
• Inhibition (competitive and non-competitive)
• Allosteric enzymes
• Regulation of enzyme activity
• Nomenclature

Macromolecules – Carbohydrates

• Definition
• Classification as mono, di, and polysaccharides
• Isomers
• Redox reactions
• Linkage to proteins and lipids
• Proteoglycans and glycosaminoglycans

Membranes I

• Membrane structure
• Transport across membranes (passive and facilitated diffusion and active transport)

Nucleic Acid Structure

• DNA and RNA structures and their relevance to cellular function and to widely used molecular biology methods

Nucleic Acid Analysis

• Specific methods, including nucleic acid purification, hybridization, sequencing, sequence analysis, gene expression analysis and PCR
• DNA cloning (including creation and screening of DNA libraries)
• Expression of recombinant proteins and site-directed mutagenesis (including selection of target sites, design principles)

DNA Replication

• Basics of DNA replication, semiconservative replication, replication forks, origin of replication, semidiscontinuous synthesis, primers, proof-reading, proteins of DNA replication and the replication of a bacterial chromosome
• Detailed look at elongation and initiation of DNA replication, and replication of eukaryotic chromosomes including the replication of the ends of linear chromosomes

DNA Mutation and Repair

• An overview of types of DNA damage, their causes, and the cell pathways that sense and repair this damage. DNA damage results both from internal and external assaults on the cell. During the normal process of DNA replication, polymerase errors not corrected lead to permanent changes in the nucleotide sequence that can have dire consequences on cell viability. We are exposed every day to environmental factors such as alkylating agents, toxic hydrocarbons and pesticides, which target and modify DNA in harmful ways.

Prokaryotic transcription

• RNA structure and transcription in prokaryotic cells, elements of bacterial promoters, structure and function of bacterial RNA polymerase, processivity and transcription termination
• Regulation of transcription, binding of transcription regulators to DNA, characteristics and function of DNA binding proteins, role of different sigma factors, negative and positive regulation, two component regulatory systems and global gene regulation

Eukaryotic transcription I & II

• Transcription in eukaryotic cells, the basic transcription unit including promoters, terminators, up- and downstream regulatory sequences, transcription factors, cis and trans elements
• Inhibitors of transcription and post-transcriptional modifications to RNA

RNA processing

• RNA processing in eukaryotic cells, types and structure of RNA
• Processing of rRNA, tRNA, and mRNA
• RNA transport
• RNA stability
• RNA interference

Prokaryotic & Eukaryotic Translation

• Protein synthesis, the genetic code, the mechanism of protein synthesis
• Comparison of the key features of prokaryotic and eukaryotic translation machinery
• Regulation and inhibition of protein synthesis

Translation Regulation

• Regulatory aspects of translation
• Translational block of maternal mRNA
• Subcellular localization of mRNAs destined for different parts of the cell (using examples in the yeast and neurons)

Regulatory RNA

• Regulation of gene expression in prokaryotes and eukaryotes by small RNAs, with emphasis on the role of small interfering RNAs in transcriptional and post-transcriptional gene regulation

Protein Processing

• Protein processing overview
• Protein folding
• Protein modification
• Nuclear targeting

Epigenetics

• This lecture will focus on how changes in DNA methylation, the modifications of histones (methylation, acetylation) and the structure of chromatin contribute to the regulation of gene expression. These can be heritable changes in gene activity that are independent of changes in DNA sequence. Epigenetic modifications in disease states as well as therapeutic applications will be discussed.

Protein Trafficking: ER and Mitochondria

• The general strategies by which proteins are transported into various organelles will be discussed. The specific mechanisms and players involved in transport into the nucleus, endoplasmic reticulum and mitochondria will then be described in detail, including unique experimental approaches used to uncover the transport processes.

Protein Trafficking: ER to Golgi and Beyond

• Protein trafficking from the ER through the Golgi Complex via the secretory pathway will be described. In particular, the dynamic nature of the Golgi will be discussed, including vesicle formation, vesicle targeting, vesicle fusion, vesicle vs. cisternal maturation, and sorting at the Trans-Golgi Network.

Protein Degradation and Ubiquitin-Like Modifications

• The lecture will focus on how proteins are targeted for degradation by ubiquitin-mediated proteolysis. The role of protein ubiquitin and ubiquitin-like modifications in the cellular processes will be discussed.

Membranes II

• Membrane receptors and their role in signal transduction

Introduction to Metabolism

• Anabolism and catabolism
• Basal metabolic rate
• Basic thermodynamics and thermodynamic coupling
• High energy bonds
• Biological redox reactions
• Intermediary metabolism (overview)
• Glycolysis as an example of an oxidative
• Catabolic pathway

Purine & Pyrimidine Synthesis & Catabolism

• Structure, nomenclature and functions
• De novo synthesis and salvage pathways and their regulation
• Degradation of purines degradation of pyrimidines
• Emphasis on the discussion of the biochemical mechanisms of the reactions involved in biosynthesis and its regulation

Glycogen Metabolism; Regulation of Glycolysis; Gluconeogensis; Pentose Pathway

• Structure and function of glycogen, storage sites, pathways of synthesis and degradation, regulation of the pathways by covalent modification and allosteric effectors
• Cellular uptake and utilization of glucose, and regulation of glycolysis (with emphasis on gluco/hexokinase, phosphofructokinase, pyruvate kinase)
• Metabolism of fructose and galactose
• Pentose phosphate pathway
• Gluconeogenesis reactions, regulation, compartmentalization

Acetyl CoA Generation & Utilization, Mitochondrial Electron Transport, Mechanisms of ATP Generation; Ion Transport

• Overview of catabolic pathways and central role of acetyl CoA in cellular formation of ATP energy source in cells
• Molecular organization, cellular localization, and regulation of enzyme systems that convert pyruvate to ATP
• Enzyme components, structural organization and regulation of the pyruvate dehydrogenase multienzyme complex that forms acetyl CoA from pyruvate
• Tricarboxylic acid cycle, its multienzyme molecular organization and reactions leading from acetyl CoA to respiration and phosphorylation
• Biological oxidation and reduction
• Mitochondrial electron transport (respiratory chain), its organization in membranes and the function of ETC protein complexes to reduce oxygen and drive and the formation of proton gradients in mitochondria
• The ATP synthase protein machine and its mechanism of function and regulation in the oxidative phosphorylation that leads to ATP

Lipids, Triglycerides, Eicosanoids, Cholesterol Metabolism

• Glycerol- and sphingosine-based polar lipids with emphasis on TG, PL and sphingoglycolipid synthesis
• Eicosanoid metabolism and biological activities (emphasis on PGI2, TXA2, LTC4)
• Cholesterol synthesis and regulation
• Overview of digestion
• Function, classification, structure and function of lipoproteins

Fatty Acid Synthesis, Fatty Acid Oxidation; Ketones

• Significance and overview, fatty acid synthesis, modification of endogenous and dietary fatty acids, fatty acid oxidation
• Ketogenesis, peroxisomal degradation

Steroid Hormones & Nuclear Receptors

• This lecture will cover the synthetic pathway that leads from cholesterol to the generation of steroid hormones. The hormone-receptor interactions will also be discussed as well as the general family of nuclear receptors.

Amino Acid Metabolism

• Overview of amino acid metabolism with emphasis on amino acid pool
• Nitrogen catabolism
• Urea cycle
• Use of alpha-keto acid skeletons in energy metabolism (gluco- and ketogenic amino acids)
• Metabolism of branched - chain amino acids with emphasis on the production and roles of gln and ala

Review & Integration of Metabolism

• A refocus on the "big picture"
• Role of liver, muscle, adipose and brain in the fed and short-term fasted states
• Review (overview) of the key pathways of the fed and fasted states with emphasis on regulation
• Tissue interrelationships
• Key molecules
• Adaptation to long-term fasting with a focus on tissue inter-relationships
• Aerobic-anaerobic transitions
• Resting muscle-contracting muscle transitions

 
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