Eukaryotic evolution, transcription, exons and introns

• During evolution, a eukaryotic organism engulfed a prokaryotic organism, leading to a mutually beneficial relationship.
• Mitochondria originated from internalized aerobic prokaryotes that performed oxidative phosphorylation.
• Chloroplasts derived from prokaryotes that performed photosynthesis.
• Peroxisomes have a similar origin to mitochondria and chloroplasts.
• The central dogma describes the unidirectional flow of genetic information: DNA → RNA → protein → property (phenotype).

Transcription

• Transcription involves copying DNA into a single-stranded RNA molecule with a complementary base sequence.
• RNA polymerase facilitates transcription.
• Transcription serves as is the first step in gene expression.

Exons and Introns

• Exons are gene sequences coding for a gene product.
• In eukaryotes, exons are separated by non-coding sequences called introns.
• During transcription in eukaryotes, both exons and introns get transcribed into messenger RNAs. Importantly, introns get removed from the transcript before translation.

RNA Maturation

• RNA maturation involves post-transcriptional modifications in eukaryotic cells to convert primary transcript RNA into mature RNA.
• A 5' end cap is added to protect mRNA from degradation by RNases.
• A 3' polyadenylation tail is added for nuclear export, translation, and mRNA stability.
• Introns get removed, and exons get joined together through RNA splicing.

RNA Splicing

• RNA splicing is a post-transcriptional process where introns are removed, and exons are joined to generate mature mRNA.
• Multi-exon genes can have exons joined in different combinations, leading to alternative matured mRNA strands/ isoforms.
• Alternative splicing allows a single gene to code for multiple proteins.
• Proteins translated from alternatively spliced mRNAs differ in their amino acid sequence and biological functions.

Translation and Genetic Code

• Translation is the process in the cytoplasm where a polypeptide chain is synthesized from an mRNA molecule.
• Ribosomes read the mRNA base sequence as triplets.
• The genetic code describes the relationship between base triplets and corresponding amino acids in the polypeptide.

Heterotrophs

• Heterotrophs synthesize ATP by degrading organic material produced by other organisms.
• ATP is used to produce the heterotroph's own organic compounds and carry out cellular processes.
• Examples of heterotrophs: all animals, protozoans, fungi, and most bacteria.

Autotrophs

• Autotrophs either use sunlight as an energy source (photosynthetic) or produce ATP through oxidative processes (chemosynthetic).
• Autotrophs use ATP to create their own organic compounds or carry out other cellular processes.
• Examples of autotrophs: plants and certain bacteria.

Prokaryotes

• Prokaryotic cells lack a nucleus separated from the cytosol by a nuclear membrane.
• Genetic material resides in the cell plasma.
• Prokaryotes lack membrane-bound organelles or a cytoskeleton.
• Examples of prokaryotes: bacteria and cyanobacteria.

Eukaryotes

• Eukaryotic cells have a nucleus separated from the cell plasma by a membrane.
• Eukaryotes have a structured internal membrane system (nuclear membrane, endoplasmic reticulum, mitochondria) and cytoskeleton.
• Most organisms are eukaryotes, including unicellular eukaryotes, plants, animals, and fungi.

Primary Biogenic Elements

• Primary biogenic elements: carbon (C), hydrogen (H), oxygen (O), nitrogen (N), and phosphorus (P).
• They constitute 98% of cells.
• These elements form the bulk of organic matter in living organisms, and H and O form/ are a large component of water.

Secondary Biogenic Elements

• Secondary biogenic elements constitute approximately 2% of cells.
• Examples: sulfur (S), iron (Fe), chlorine (Cl), sodium (Na), potassium (K), and calcium (Ca).

Organic Substances

• Organic substances are compounds formed by joining several carbon atoms.
• High-energy organic substances formed through metabolism include carbohydrates, fats, and proteins.

Amino Acids

• Amino acids are molecules containing amino and carboxyl groups.
• α-amino acids have the amino group attached to the α-carbon of the carboxylic acid chain.
• Proteins in most living cells are built from 20 different α-amino acids.

Deoxyribonucleic Acid (DNA)

• DNA serves as the hereditary material of cells constructed of two polynucleotide chains forming a double helix.
• Nucleotides in DNA contain deoxyribose sugar and nitrogenous bases: adenine (A), cytosine (C), guanine (G), and thymine (T).
• The two chains in the helix are bound by hydrogen bonds between complementary bases (A with T, two bonds; G with C, three bonds).
• DNA molecules differ in the base sequence of the polynucleotide chains.
• DNA stores information, transmits it to daughter cells, and indirectly controls protein synthesis.

Ribonucleic Acid (RNA)

• RNA is a polymer composed of ribonucleotide units.
• RNA nucleotides contain ribose sugar and nitrogenous bases: adenine (A), cytosine (C), guanine (G), and uracil (U).
• Major types of RNA molecules: messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and small nuclear RNA (snRNA).
• mRNAs transmit genetic information from DNA to proteins and are produced during transcription.
• rRNAs, along with ribosomal proteins, build ribosomes.
• tRNAs are involved in translation, transporting amino acids to ribosomes and decoding genetic information from mRNA to proteins.
• snRNAs participate in mRNA splicing.
• Other RNA types like small interfering RNA (siRNA) and microRNA (miRNA) regulate gene expression.
• RNA viruses have genomes composed of RNA.

Cytoplasm (Cell Plasma)

• Cytoplasm is a gelatinous material filling cells where metabolic processes take place.

Lipids

• Lipids are organic compounds found in living organisms with varying composition and structure, soluble in apolar solvents.
• Hydrolyzable lipids: neutral fats (glycerol and three fatty acids) and phospholipids (polar part formed by phosphoric acid and an alcohol molecule).
• Nonhydrolyzable lipids: steroids and poly-isoprenoids (e.g., carotenoids).

Amphipathic Molecule

• Amphipathic molecules (ions) contain apolar and polar (or ionic) parts.
• Phospholipids are examples of amphipathic molecules.
• In water, amphipathic molecules interact via their apolar (hydrophobic) hydrocarbon chains, forming a micelle.

Polar and Apolar Molecules

• Polar molecules or parts of molecules have unequal charge distribution (dipoles).
• Polar particles interact readily with water (hydrophilic).
• Apolar molecules or parts of molecules do not undergo charge separation and do not significantly interact with water.

Cell Cortex

• The cell cortex is a three-dimensional protein network on the intracellular side of the cell membrane.
• Primarily composed of actin filaments and actin-binding proteins; spectrin dimers are also essential in many cells.
• The net-like structure is anchored to transmembrane proteins by anchoring proteins.
• They maintain plasma membrane integrity, determine cell shape, cell motility, and regulate membrane protein function.

Glycocalyx

• Glycocalyx is a viscous coating layer on the extracellular side of the cell membrane.
• It is made of carbohydrates (glycoproteins, proteoglycans, and glycolipids), mainly associated with proteins.
• The glycocalyx protects the cell surface, mediates cell adhesion and signal transduction.
• The glycocalyx's composition helps immune cells differentiate between normal (self) and abnormal (non-self) cells.

Asymmetry of the Lipid Bilayer

• The lipid composition of the outer and inner layers of the cell membrane differs due to active translocation by flippase and floppase enzymes.
• Phosphatidylserine is only in the inner layer of the plasma membrane in intact cells. In apoptotic cells, it appears in the outer layer as an "eat-me" signal.
• Geometrical differences contribute to spontaneous membrane curvature.

Scramblase, Flippase, Floppase

• These are enzyme proteins involved in the vertical asymmetry of lipid bilayers by assisting in the translocation of phospholipids between the two layers of the membrane.
• Scramblases facilitate random lipid shuffling between layers without energy investment and are non-specific.
• Flippases translocate lipids inwards, while floppases translocate lipids outwards, both using ATP hydrolysis and being specific for their substrate.

Lipid Rafts

• Lipid rafts are cholesterol-, glyco-, and sphingolipid-rich domains in the cell membrane.
• They are held together mostly by lipid-lipid and protein-lipid interactions.
• Lipid rafts have lower fluidity, increased thickness, rigidity, and packing density compared to other membrane regions.
• They assemble or segregate various proteins, dynamically regulating the quality and quantity of interacting molecules, thus enhancing signal transduction.

Signalosome

• A signalosome presents as a supramolecular protein complex of various signaling elements, with association and activities primarily regulated by protein-protein interactions.
• Composition and assembly dynamically change in space and time, ensuring specificity and speed of signal transduction.

Endoplasmic Reticulum (ER)

• The endoplasmic reticulum, is a membrane system in eukaryotic cells near the nucleus.
• Rough ER (RER) binds ribosomes, which synthesize proteins destined for the membrane or ER lumen.
• RER proteins achieve final structure in the lumen, are modified, and packaged into vesicles for transport to the Golgi apparatus.
• Smooth ER (SER) lacks ribosomes and participates in metabolic reactions, including phospholipid and fatty acid synthesis, and detoxification.

Golgi Complex

• The Golgi complex consists of stacked, flattened membrane disks (cisternae).
• It has a polarized organization with compartments: cis-Golgi network, cis-, medial-, and trans- cisternae, and trans-Golgi network.
• Its function involves posttranslational modification of proteins from the endoplasmic reticulum, sorting, and shipping proteins to various destinations, including lysosomes, the cell membrane, or secretion outside the cell.

Lysosome

• A lysosome is a membrane-bound organelle where the degradation of intracellular and extracellular materials occurs.
• Lysosomes contain acid hydrolases that function at an acidic pH generated by H+-ATPases in their membrane.
• Primary lysosomes contain only enzymes, while secondary lysosomes contain enzymes and materials to be digested.

Peroxisome

• A peroxisome is a membrane-bound organelle with a possible endosymbiotic origin, abundant in liver and kidney cells.
• Peroxisome enzymes (e.g., peroxidases) are used in oxidative decomposition processes, mainly of fatty acids, alcohol, and amino acids.
• Catalase, another peroxisomal enzyme, neutralizes hydrogen peroxide by converting it to H2O.

Storage Disease

• Storage diseases are characterized by lysosomal dysfunction caused by inherited mutation of lysosomal enzymes.
• Lack of enzyme activity leads to the accumulation of undigested substrates in lysosomes.
• Examples: Tay-Sachs disease, Fabry disease, I-cell disease.

Lipid-Water Partitioning Coefficient

• It characterizes hydrophobic character, expressed as the ratio of a molecule's equilibrium concentrations in contiguous lipid- and water phases: R= CL/CV.
• Lipid-soluble/more hydrophobic molecules enter the cell faster and reach higher intracellular concentrations.

Passive Transport

• Passive transport is material flow through biological membranes that does not require cellular energy.
• The concentration difference between the two sides of the membrane and the electric potential difference provide the driving force.
• Transport occurs through the membrane lipid bilayer or membrane proteins (e.g., ion channels).

Facilitated Diffusion

• Facilitated diffusion involves specific proteins that help transfer substances (e.g., ions, glucose, some medicines) through biological membranes without direct energy input.
• This process allows for the transfer of materials that would otherwise not pass through the membrane.

Glucose Uniport

• Glucose uniport is a type of facilitated transport that does not require ATP hydrolysis; glucose gets transported down its concentration gradient.
• The transporter molecule oscillates between two conformations, exposing the glucose-binding site to either the exterior or the interior of the cell.
• Examples: GLUT-1 (glucose uptake in most cells), GLUT-2 (glucose release on the basolateral surface of intestinal epithelium), and GLUT-4 (insulin-dependent glucose uptake in muscle and fat tissues).

Ion Channel Gating

• A suitable trigger causes a conformational change in the protein resulting in the transition among different conducting and non-conducting states (closed, open, inactivated) of the channels.
• Ion channels are classified into voltage-gated, ligand-gated, intracellular signal-gated, membrane stretch-gated channels, and G-protein gated based on the trigger.

Ion Channel Selectivity

• Specific ion species are allowed passage through the pore of an open ion channel depending on the pore size and charge.
• Highly selective channels (e.g., K+, Na+, Ca2+, Cl-) are usually formed by four subunits.
• Mildly selective (e.g., acetylcholine receptor)channels are usually formed by five subunits.
• Non-selective channels (e.g., gap junction channel) are usually formed by six subunits.

Voltage Sensor Mechanism

• A voltage is a domain in voltage-gated ion channels, composed of alpha-helical segments and containing positively charged amino acid side chains.
• Structural rearrangement of voltage-sensors in response to changes in the membrane potential leads to conformational changes in the pore.

Gap Junction

• Gap junctions are non-selective channels connecting neighboring cells, permeable to small molecules (up to 2000 Da).
• Six connexin molecules form a connexon, and two connexons couple to form a gap junction or channel.
• Permeability is regulated by pH and Ca2+ concentration.

Active Transport

• Active transport transports ions against their electrochemical potential gradients or uncharged molecules against their concentration gradients using energy directly from ATP hydrolysis (primary active transport) or indirectly by ion flow provided by an existing ion gradient (secondary active transport).
• Examples of primary: Na+/K+-pump, Ca2+-pump, P-glycoprotein, and lysosomes V-types proton ATPase.

Secondary Active Transporters

• Secondary active transporters do not couple directly to ATP hydrolysis.
• The electrochemical potential difference of an ion is harnessed.
• An example is glucose-Na+ symport, which takes up glucose from the small intestine into the intestinal epithelia using a sodium gradient.
• The sodium electrochemical gradient is maintained by Na+/K+ ATPase using ATP hydrolysis.

Na+/Glucose Cotransport

• An example of secondary active transport in kidney and small intestine.
• The Na+/glucose symporter transports 2 Na+ ions and one glucose molecule into cells simultaneously at the apical surface of epithelial cells.
• The electrochemical potential of Na+ provides the energy for glucose transport against its concentration gradient.
• The Na+/K+-ATPase pump maintains the Na+ electrochemical gradient for this process.

V-Type Transporters

• Vacuolar-type proton transporters transport protons into membrane enclosed organelles.
• They are responsible for the low pH of lysosomes and synaptic vesicles.
• They are also present in cells that acidify their environment, like osteoclasts, tumor cells, macrophages, and sperm.
• They use ATP hydrolysis, but they are not transiently phosphorylated like the P-type transporters.

P-Type Transporters

• P-type transporters involve transient phosphorylation during their operation, which leads to the transport of the ion.
• The Na+/K+-ATPase and plasma membrane Ca2+-ATPase create ion gradients required for essential cell operations and are found in all cell types.

ABC Proteins

• ABC (ATP-binding cassette) proteins have two ATP-binding sites (NBDs) and two transmembrane domains (TMDs).
• NBDs bind and hydrolyze ATP.
• TMDs form the substrate-binding sites.
• ABC proteins are categorized into channel-type proteins, channel regulators, and active pumps based on their function.

Multidrug Resistance

• Multidrug resistance is the resistance of cancer cells to numerous anticancer agents.
• Cancer cells express ABC transporters that extrude drugs from the cells.
• ABC transporters contribute to drug resistance in tumor cells, including P-glycoprotein (Pgp=ABCB1=MDR1), multidrug resistance proteins (MRP1=ABCC1), and BCRP (=ABCG2).

P-Glycoprotein

• Pgp is an active transporter (active pump) type human ABC protein.
• It protects the body from toxic compounds, external and internal.
• Pgp is also often expressed in stem cells, tumor stem cells, and cancer cells.
• Pgp is involved in the chemotherapy resistance of tumors .

ABCG2 (Breast Cancer Resistance Protein, BCRP)

• ABCG2 is an active transporter type ABC protein with a wide substrate spectrum.
• It is expressed in barrier regions of the body, stem cells, and tumor cells.
• Its physiological substrate is uric acid, and it is involved in eliminating uric acid from the body.

Cystic Fibrosis Transmembrane Conductance Regulator

• The cystic fibrosis transmembrane conductance regulator serves as a channel type ABC protein where inactivation mutations of the CFTR Cl--ion channel cause cystic fibrosis (CF).
• CF is a multiorgan hereditary disease caused by inactivating mutations of this Cl- channel.
• High viscosity of secreted mucus causes symptoms affecting the lungs, the gastro-intestinal system and the reproductive system as well.

TAP1/TAP2 Oligopeptide Transporter

• TAP1/TAP2 forms a heterodimeric transporter found in the endoplasmic reticulum (ER) membrane.
• It pumps oligopeptides into the ER lumen, where they bind to the MHC I protein.
• The MHC I complex is transported to the plasma membrane to be presented to cytotoxic T-cells.

Sulfonylurea Receptor 1 (SUR1), KATP Channel

• SUR1, together with Kir6.2 subunits, forms an ATP-sensitive KATP potassium channel.
• It is involved in the regulation of insulin secretion in pancreatic β cells.

SLC (Solute Carrier) Proteins

• SLC proteins include secondary active transporters (coupled transporters) and passive transporters.
• They transport inorganic ions or water-soluble small molecules through the plasma membrane or the membrane of intracellular organelles.

Glucose and Amino Acid Uptake through the Intestinal Epithelium

• Directional transport of glucose through the intestinal epithelium is mediated by the segregation of transporter proteins in the plasma membrane.
• Na+-glucose symporter in the apical membrane actively uptakes glucose, while GLUT2 in the basal membrane facilitates glucose diffusion to the extracellular fluid to blood according to its concentration gradient.
• The Na+ gradient driving glucose uptake is maintained by the Na+/K+-pump, also expressed in the basolateral membrane of enterocytes.

Plasma Membrane Na+/Ca2+ Antiport

• Electrogenic, secondary active transporter located in the cytoplasm membrane.
• It transports 3 Na+ ions into the cell and 1 Ca2+ ion out of the cytosol.
• The transporter is powered by the electrochemical gradient of Na+, and NCX is significant in cardiac myocytes as it restores the resting Ca2+ concentration.

Plasma Membrane Ca2+ ATPase

• Primary active transporter, P-type ATPase, located in the cytoplasm membrane.
• It transports Ca2+ from the cytosol to the extracellular space.
• 2 H+ ions are transported into the cytosol.

SERCA

• SERCA serves as a primary active, P-type ATPase transporter
• Functioning occurs in the sarcoplasmic and endoplasmic reticulum membrane.
• Transports Ca2+ from the cytosol into the lumen of the ER/SR at the cost of ATP hydrolysis.

Ryanodine Receptor

• An intracellular ligand-gated channel found on the sarcoplasmic and endoplasmic reticulum membrane.
• The ligand activating the channel in skeletal muscle cells is a part of the DHP receptor, whereas in the cardiac myocytes and neurons the ligand is Ca2+.
• Releases Ca2+ from the ER/SR store into the cytosol

IP3 Receptor

• An intracellular ligand-gated Ca2+ channel on the endoplasmic reticulum membrane.
• The ligand activating the channel is IP3 upon receptor-ligand interaction.

Calmodulin

• Functions as a cytosolic Ca2+-binding protein with 4 Ca2+ binding pockets.
• Depending on the Ca2+-saturation of the binding sites the conformation of calmodulin changes dramatically thereby enabling calmodulin to interact with and activate target proteins.

Pump-Leak Model of Osmo- and Volume Regulation

• Operates for homeostatic regulation of the cell volume in isotonic medium
• The tendency of inorganic ions to reach thermodynamic equilibrium results in a net influx of ions (Donnan effect) is counterbalanced by the Na+/K+ -pump.

RVD

• This cell volume regulatory mechanism is induced by cell swelling in hypotonic medium and leads to the reduction of the cell volume and loss of water even if the hypotonic condition is maintained.
• Short-term RVD: net loss of inorganic ions.
• Long-term RVD: reduction of the cytosolic osmolality by reducing the concentration of metabolites either via efflux through transporters (e.g. taurine transporter) or by favoring anabolic processes.

RVI

• This cell volume regulatory mechanism is induced by cell shrinkage in hypertonic medium and leads to the increase in the cell volume by gaining water even if the hypertonic condition is maintained.
• Short-term RVI: net accumulation of inorganic ions.
• Long-term RVI: increase of the cytosolic osmolality by increasing the concentration of metabolites either via metabolite influx through transporters (e.g. taurine transporter) or by favoring catabolic processes.

Steady-State pH of the Cytosol

• The pH refers to when the efflux (e.g. the Cl-/HCO3- antiport) from the cells equals to the rate of the acid efflux , and thus the cytosolic pH remains constant.
• Graph of the pH-dependence of the acid efflux rate intercepts the pH-dependence of the base efflux rate.

Na+/H+ Antiport

• Electroneutral exchanger in the cytoplasm membrane that mediates the influx of Na+ and efflux of H+ from the cytosol.
• Regulation of cytosolic pH running at high speed at acidic cytosolic pH removing excess H+ whereas reduced at alkaline cytosolic pH.

Cl-/HCO3- Antiport

• An electroneutral exchanger in the cytoplasm membrane that mediates the influx of 1 Cl- into, and the efflux of 1 HCO3- from the cytosol during one duty cycle.
• Regulation of cytosolic pH running at highs speed at alkaline pH removing excess base whereas transport rate reduces when the cytosolic pH decreases.