Chapter 1: The cell
There are about 20K protein-encoding genes (1.5%) of human genome. Variously function as enzymes, structural components, signaling molecules and are used to assemble and maintain all of the cells in the body. This is a low estimate: many genes produce different RNA transcripts to encode protein isoforms.
Many of same 20K genes are homologs in worms. Noncoding DNA is expected to play a large role in differentiating humans and worms.
80% of the genome: binds proteins (regulation of gene expression), has a functional activity (related to regulation of gene expression often in cell-type specific fashion.)
Noncoding regions provide “architectural planning.” Many of the polymorphisms associated with disease are located in non-protein-coding regions of genome.
Classes of non-protein-coding sequences:
Promoter/enhancer—regions providing binding sites for transcription factors
Chromatin structures—binding sites for factors that maintain higher order structure
Noncoding regulatory RNAs—$>$60% of of genome is transcribed into RNA that is never translated (to protein) but regulate gene expression (e.g. micro-RNA and long noncoding RNA)
Mobile genetic elements—Make up a 3rd of the genome. Called “jumping genes”: segments can move around genome vary widely in number and position even one related species. Implicated in regulation and chromatin organization. Function is not well understood. (e.g. transposons)
Special structural regions—Particularly, telomeres: chromosome ends & centromeres: chromosome “tethers”
Person-to-person variation is encoded in about 15 million base-pairs (0.5%). The two most common forms of variation:
SNPs vary at a single nucleotide and almost always biallelic (only to choices: A or T). > 6million identified. Occur across genome—exons, introns, intergenetc regions, and coding regions. 1% occur in coding regions about by change (1.5% of genome is a coding region). May have direct effect on disease susceptibility when fall in non-coding region or can be “neutral” and have no effect on gene function or carrier phenotype. “Neutral” SNPs may be useful markers to be co-inherited with a disease-associated gene.
May prove useful in assessing genetic risk of multigenic complex diseases e.g type II diabetes and hypertension. However, effect of SNPs on disease susceptibility is weak and yet to be seen if they can be used to develop strategies for disease prevention.
Linkage disequilibrium:
Variation of large (1K to millions of base-pair) contiguous DNA. Sometimes loci are biallelic (duplicated or deleted) other times CNVs are complex rearrangements with multiple alleles. CNVs are estimated to make up for 5-24 million base pair variation between individuals. About half involve coding sequences—responsible for the portion of human phenotype variation.
Not all genetic variation can be explained by alterations to DNA sequence. Epigenetics plays an important role.
Type-specific variation in DNA transcription and translation among terminally differentiated can depend on the following epigenetic factors:
Histones and histone modifying factors—Histones are highly conserved low molecular weight proteins. Nucleosomes are 147 base pair sequences wrapped around histones. Chromatin: DNA-histone complexes separated by short DNA linkers. DNA is not uniformly and compactly wound.
Two types of chromatin: (1) heterochomatin: cytochemically dense and transcriptionally inactive, (2) euchromatin: dispersed and active.
Histones are regulated by nuclear proteins and chemical modifications. chromatin remodeling complexes can reposition nucleosomes thereby allowing/preventing proteins to bind to regulatory elements e.g. promoters.
Chromatin writers, erasers, and reader complexes: Writers “mark” or modify histones via 70 different covalent modifications e.g. methylation, acetylation, phosporylation of amino acid residues on histones. Marks may make DNA more accessible to RNA polymerase inducing transcription.
Erasers reverse marks.
Readers bind to histones with particular marks to regulate gene expression.
Histone methylation—Methylation of lysines and arginines can either active or repress transcription depending on which histone residue is marked.
Histone acetylation—histone acetyl transferase (HAT) and histone deactylase (HDAC) acetylate and deacetylate lysine residues causing chromatin to open and close, respectively.
Histone phosphorylation—Phosphorylation of serine residues will open or condense chromatin.
DNA methylation—Typically results in silencing of transcription. Regulated by methyltransferase, demethylating enzymes e.g. methylated-DNA-binding proteins.
Chromatin organizing factors—Believed to bind to noncoding regions and control long-rage looping (important for regulating spatial relationships between gene enhancers and promoters).
Dysregulation of the “epigenome” can has been shown to play a central role in malignancy. Unlike other genetic modifications, epigenetic alterations such as marking is reversible and amenable to therapeutic interventions.
Noncoding RNA is transcribed from genes but not translated. Two of many are discussed.
### miRNA
Function primarily to modulate translation of targeted mRNA. Posttranscriptional silencing of gene expression by miRNA is important to all eukaryotes. The human genome codes about 1K miRNA genes and regulate multiple protein-coding genes and even entire programs of gene expression.
Primary micro-RNA (pri-miRNA) (multiple connected hair-pin loops or RNA) is transcribed from the miRNA gene and processed to pre-miRNA in the nucleus, which is then exported by specific transport proteins and it is trimmed by the Dicer enzyme to become mature double stranded miRNA. The strands unpair and form a multiprotein aggregate called RNA-induced silencing complex (RISC). The RISC then induces cleavage or translational repression of the targeted mRNA after binding by base-pair matching. The specificity of miRNA binding and silencing is determined by seed sequence in the 3′ untranslated region (UTR).
Small interfering RNA (siRNA) are analogous to miRNA and can be introduced experimentally to test gene function and silence pathogenic genes.
The number of lncRNA may exceed coding mRNA by 10-20 times.
Means of gene expression regulation:
Gene activation: aides in binding of ribonucleoprotein transcription complex (RNPTC) to gene
Gene suppression: decoy lncRNA occupies active site of RNPTC
Promotion of chromatin modification: e.g. marking
Assembly of protein complexes: act as scaffolding for multi-subunit protein complexes that influence protein architecture or gene activity
One example of lncRNA is XIST, which is transcribed from the X chromosome forms a repressive “cloak” of the same X chromosome.
Normal housekeeping functions are compartmentalized within membrane-bounded intracellular organelles. This allows for:
Storing functionally important yet potentially harmful degradative enzymes or reactive metabolites.
Creating unique chemical environments (e.g. low pH, high calcium) that regulate enzyme function or metabolic pathways.
Rough endoplasmic reticulum—Site where new proteins that are destined for the plasma membrane are made. (Proteins destined for the cytosol are made on free ribosomes.)
Golgi apparatus—Site where proteins are assembled
Smooth endoplasmic reticulum—Used to make steroid hormones, lipoproteins, and make hydrophobic compounds water-soluble. (Likely abundant in cells in gonads and liver.)
Endosomal vesicles—Shuttle materials different place within the cell, the cell surface, or other organelles
Sites of catabolism:
Lysosomes—digest macromolecules (proteins, polysaccharides, lipids, and nucleic acids)
Proteasomes—“Grind” denatured proteins into shorter peptides into histocompatability molecules. They can also be used to degrade regulatory proteins to affect transcription.
Peroxisomes—Break down fatty acids and make hydrogen peroxide in process
Cytoskeleton—Necessary for movement of cell and organelles and proteins within the cell. It is also necessary for establishing cell polarity i.e. the top (apical) and bottom/side (basolateral)
Mitochondria—Major source of ATP but also source of metaboic intermediates that are needed for anabolic metabolism, the site of synthesis of macromolecules e.g. heme and contain regulators of programmed cell death
Asymetric partition of phospholipids affects other cellular processes:
Phosphatidylinositol—on the inner membrane can be phosphorylated to serve as electrostatic scaffold or hydrolyzed to generate intracellular second signals e.g. diacylglycerol and inositol triphosphate
Phosphatidylserine—Normally on inner membrane and involved in negative electrostatic protein interaction. When on outer membrane, it signals apoptosis (cell death) and serves as blood clotting cofactor.
Glycolipids & sphingomyelin—important for cell-to-cell interactions including inflammatory cell recruitment and sperm-egg interactions
Lipid rafts—Important for cell-cell, cell-matrix interactions, and specialized endocytic pathways.
Role of glycoproteins:
Ion and metabolite transport
Fluid-phase and receptor mediated uptake of macromolecules
Cell-ligand, cell-matrix, and cell-cell interaction
These proteins can be integrated into the membrane via 4 general ways, which defines function:
Integral/transmembrane have at least one hydrophobic α-helical segment that traverse membrane. Integral proteins usually have positively charged amino acids in the cytoplasm anchored to negatively charged head groups of membrane phospholipids.
Proteins can be attached to prenyl groups (related to cholesterol) or fatty acids that insert to the cytosolic side of the membrane
Extracellular insertion into membrane may occur through glycosylphosphatidylinositol (GPI) anchors
Peripheral membrane proteins may associate (non-covalent bonding) with true transmembrane proteins
Plasma membrane protein complexes can be aggregated by chaperone molecules in the RER or by lateral diffusion followed by formation in situ. The latter method is common among many protein receptors.
Small non-polor molecules (e.g. O2 and CO2), small polar molecules (≤75 daltons) (e.g. water, ethanol, urea), and hydrophobic moleucles (e.g. steroid-based molecules, estradiol, vitamin D) can all cross the plasma membrane with relative ease.
In tissues that transport water in high volumes (e.g. renal tubular epithelium), specialized transmembrane proteis, aquaporins, augment passive water transport.
Carrier proteins and Channel proteins are used for transport of ions and large molecules. Movement is passive in most cases; driven mostly by electrical or chemical concentration gradient. Transport is active when carrier molecules use energy (ATP hydrolysis or coupled ion gradient). An infamous example is the multidrug resistant (MDR) protein, which pumps polar compounds (drugs) out of the cell.
Channel proteins—Act as hydrophilic pores that allow rapid movement of solutes (usually restricted by size or charge)
Carrier proteins—Proteins that bind to ligand and move proteins by conformational changes; transport is slow.
The cytosol often has a high concentration of charged metabolites that tend to increase intra-cellular osmolarity (hypertonic) leading to flow of water into cell. The cells has to constantly pump small inorganic ions (e.g. Na+, Cl−) to prevent overhydration, osmotic swelling, and eventual rupture (lysis).
There are two types of endocytosis
Caveolae-mediated endocytosis—Caveolin is the major structural protein that form Caveola, noncoated plasma membranes invaginations that fold into the cell. They likely are involved in transmembrane delivery of molecules but also are involved in transmembrane signaling or cellular adhesion. Internalization of caveolae is sometimes called potocytosis.
Caveolae are associated with:
GPI-linked molecules
cyclic adenosine monophosphate (cAMP) binding proteins
SRC-family kinases
Folate receptor
Pinocytosis—Begins at clathrin-coated pit, specialized portions of plasma membrane. It then rapidly invaginates and pinches off to form a clathrin-coated vesicle containing, extracelullar milieu and sometimes receptor bound macromolecules. The vesicles uncoat and fuse with the early endosome where they are discharge contents for digestion or passed to lysosome.
Receptor-mediated endocytosis—Similar to pinocytosis, macromolecules bind to receptors in clathrin-coated pits before they are endocytosed in vesicles that fuse with lysosomes. Molecules such transferrin and low-density lipoprotein release iron and cholesterol.
Defects in receptor-mediated transport of LDL are responsible for familial hypercholesterolemia.
The cytoskeleton is essential for cell shape, polarity, organelle relationship, and movement. There are three main classes of cytoskeletal proteins:
Actin microfilaments—Globular protein actin (G-actin) monomers bind together (noncovalently) to crate long double-stranded helical filaments (F-actin). F-actin has a positive and negative end per se and G-actin is added/removed at the positive end.
In muscle cells, myosin binds to actin and moves along it via ATP hydrolysis: the basis of muscle contraction.
Intermediate filaments—These characterize a large family of filaments and have tissue specific patterns of expression.
Lamin A, B, and C: nuclear lamina of all cells
Vimetin: mesenchymal cells (fibroblasts, endothelium)
Desmin: scaffolding for actin and myosin contract in muscle cells
Neurofilaments: provide strength and rigidity for axons of neurons
Glial fibrillary acidic protein: glial cells around neurons
Cytokeratins: Impart tensile strenght and allow cells to bear mechanical stress. Important for nuclear morphology and regulating transcription.
Microtubules: Hollow tubes of α and β-tubulin dimers. Have positive and negative ends. The negative end is usually embedded in the microtubule organizing center (MTOC) or centrosome). Kinesin and dynein proteins are responsible for anterograde (negative to positive) and retrograde (positive to negative) transport, respectively.
Microtubules are adapted to form motile cilia and flagella.
Cell junctions—provide mechanical links and enable receptors to recognize ligands on other cells—are organized into 3 basic types.
Occluding (tight) junctions—Prevent paracellular (between cell) movement and help restrict ion flows and maintain cell polarity. Composed of multiple transmembrane proteins: occludin, claudin, zonulin, and catenin.
Anchoring junctions (desmosomes)—Desomosomes that adhere individual cells are called spot desmosomes or macula adherens. Those that attach cells to the extracellular matrix are called hemidesmosomes. Adhesion between broad bands of cells are called belt desomosomes. Anchoring junctions are formed by cadherins, E-cadherins, and integrins, respectively.
Communicating (gap) junctions—Consist of dense array of pores called connexons (hexamer transmembrane porteins). Permit the passage of ions, nucleotides, sugars, amino acids, vitamins, and small molecules. Permeability is increased and reduced intracellular Calcium and lowered intracellular pH, respectively. Very important in cardiac cells.
The endoplasmic reticulum (ER) is the site of synthesis of all transmembrane proteins, organelles, lipids for plasma membrane, and the ER itself. It has two domains, smooth and rough, distinguished by absence/presence of ribosomes. Membrane-bound ribosomes translate mRNA into proteins into the ER lumen or become integrated into the ER membrane. Proteins can oligomerize—form polypeptide complexes—form disulfide bonds, and N-linked oligosaccharides (attached to asparagine). Chaperone molecules keep proteins in the ER until processing is complete. If processing fails, it can be kept and degraded in ER but most often is destroyed my a proteaosome. Many failed folding can cause ER stress response (unfolded protein response) that triggers cell death.
The Golgi apparatus consists of stacked cisternae that modify proteins from cis to trans as it moves from the ER to the plasma membrane. It also shuttles macromolecules between cisternae. N-linked oligosaccharides are added are processed and O-linked oligosaccharides are added. Some of the glycosylation is important for transport to lysosomes. The cis Golgi network can recycle proteins to ER and trans Golgi network sends proteins and lipids to organelles or secretory vesicles.
The process of waste disposal depends on the activity of lysosomes and proteasomes
Lysomose are very acidic (≤5 pH) membrane bound organelles that contain a variety of hydrolases (e.g. proteases, nucleases, lipases, glycosidases, phosphatases, and sulfatases) that were synthesized in the ER lumen and tagged with a mannose-6-phosphate (M6P) residue in the Golgi apparatus. Proteins are delivered to the lysosomes through trans-Golgi vesicles with M6P receptors. Other molecules are delivered through three other pathways:
Material take in by fluid-phase pinocytosis or receptor mediated endocytosis pass into the cytosol as an early endosome before maturing into a lysosome
During autophagy a double membrane from the ER that encircles obsolete organelles. It then fuses with a lysosome to digest contents.
Phagocytosis usually occurs by macrophages or neutrophils. These cells engulf large fragments of ECM or debris with a phagosome, which fuses with lysosome.
Misfolded and misbehaving proteins are identified and small peptides, ubiquitin are attached to the protein. These poly-ubiquitinated proteins are then unfolded and then passed into a proteasome to be spliced into short peptide sequences that are then degraded and recycled.
The mitochondria has two membranes. The enzymes involved the respiratory chain are on the inner membrane which is folded into cristae. This membrane encloses the core matrix space where the enzymes of the Citric Acid Cycle reside. ATP synthesis occurs in the inner membrane space (between inner and outer membranes). Hydrogen ions pump H+ from the core matrix space to the inner membrane space. The flow of the ions back into the core matrix space is used to generate ATP. However, proteins such as thermogenin on the inner membrane can also be used to generate heat in place of ATP.
Glucose and glutamine are important sources of carbon moieties that can be spun off from the TCA cycle to create lipids, nucleic acids, and proteins as governed by different growth factors, nutrient supplies, oxygen availability, and cellular signals.
The mitochondria regulate two primary pathways of cell death.
Necrosis—External cell injury can damage the mitochondria causing it to dissipate the electric potential generated by the ion gradient ending production of ATP and causing cell death.
Apoptosis—When the mitochondria are damaged by irreversible cell injury or stress; or the cell cannot create sufficient amounts of survival proteins, the mitochondria releases Cytochrome C that binds with proteins to activate caspases that induce apoptosis.
Most cell signals can be classified to several groups:
Damage
Contact with neighboring cell
Flow of signals between gap junctions
Contact with ECM
Mediation through integrins in connection with luekocyte attraction
Secreted molecules
Hormones, cytokines, growth factors, etc.
Signaling Pathways are classified into 4 different types
Paracrine—signaling to cells in a given neighborhood; this implies that the signal must be taken up quickly, has minimal diffusion, or is trapped in the ECM
Autocrine—signaling to the same cell that secreted a given signal; such a signal might affect differentiation or feedback regulator
Synaptic—signaling of neurotransmitters by neurons into specialized junctions (synapses)
Endocrine—signal is released into blood stream to act on distal cells
Types of receptors
Intracellular—lipid soluable ligands that can easliy cross the plasma membrane
Cell-surface—trans membrane proteins that can
Open ion channels (e.g. at synapses)
Active G-proteins
Active other endogenous regulatory enzymes (e.g. tyrosine kinanse)
Change a proteins binding or stability to activate a transcription factor
Items 2 and 3 are associated with cell proliferation while 4 is a common regulatory pathway for normal development. Mutations in these types of receptors are often implicated implicated in developmental disorders and cancers.
Signal transduction and cell receptors are grouped based on on signaling mechanisms and biochemical pathways they use and activate.
- Kinase activity— (De)phosphorylation of tyrosine, serine/threonine, or lipids by a kinase or phosphotase play in important role in regulation. (Phophotases usually play an inhibitory role in signal transduction).
- *Receptor tyrosine kinase*—Ligand-induced cross-linking
activates the intracellular tyrosine kinase domains for the
transmembrane recetor
- *Non-receptor tyrosine kinase*—A separate intracellular protein
phosphoylates a tyrosine on the receptor (or associated protein)
to activate the pathway.G-protein coupled receptors—This receptor traverses the membrane 7 times and is connected to a large G-protein. Binding of the ligand catalyzes the exchange of GDP for GTP and subsequently transduction.
Nuclear receptors—Lipid-soluble ligand diffuse through the membrane, form a receptor-ligand complex that regulates gene expression
Others
Notch
Wnt
Receptor activation should not be seen as an isolated and linear sequence of events but is interconnected with many different and diverging effects. As such an Adaptor proteins (transmembrane or cytosolic) play a key role in mediating and organizing transduction pathways by acting as physical links between enzymes, promoting assembly of complexes, mediate protein-protein interactions, or influencing recruitment of proteins to signaling complexes.
Transduction pathways that affect transcription factors (e.g. phophorylation) can allow for translocation into nucleus, expose genes, or binding motifs. Transcription factors are needed for growth (e.g. MYC, JUN) and other induce cell-cycle arrest (e.g. p53). They are modular per se containing domains that bind to DNA or the RNA polymerase complex, for example.
DNA binding transcription factors can bind (or dissociate) from regions close to a given gene (promoters), or longer range regulatory elements in the “neighborbood” of a gene, on an enhancer, or even on a different chromosome.
For transcription factors to induce transcription, they must have protein-protein domains that affect histone modification enzymes, chromatin remodeling complexes, and RNA polymerase.
Growth factors and promote cell growth and proliferation during normal states as well as after cell injury by: promoting entry into the cell cycle, relieve blocks of the cell cycle, prevent apoptosis, enhance biosynthesis of macromolecules required for division—as well as promote a number of non-growth cellular activities such as migration, differentiation, and synthetic activities. Many genes that code for proteins involved in growth factors pathways are called proto-oncogenes as a gain-of-function mutation causing constitutive activity can result in uncontrolled proliferation and tumor formation.
One should note that, although all the discussed receptor pathways involved kinase activity per se, there are variety of other pathways.
EGF and TGF-α are secreted by macrophages and some epithelial cells. These growth factors are mitogenic for hepatocytes, fibroblasts, and many epithelial cells. The EGR receptor family includes 4 membrane bound receptors with tyrosine kinase activity. EGFR1 (or ERB-B1) is mutated or amplified in lung, head/neck, breast, and brain cancers while ERBB2 (or HER2) is overexpressed in a subset of breast cancers. Targeted antibodies and small molecule antagonists have been successfully implemented as treatments thereof.
HGF have mitogenic effects on hepatocytes and most epithelial cells (biliary, pulmonary, renal, mammary, and epidermal). It acts as a morphogen (i.e. it induces differentiation) and promotes migration during embryonic development. It is sometimes called a scatter factor in the case of the latter. It is produced by fibroblasts, mesenchymal (connective tissue or stromal stem cells), endothelial, and non-hepatocyte liver liver cells. HGF is created as and inactive pro-HGF and activated at the sites of injury. Overexpression/mutation in the receptor for HGF, MET, is commong in renal and thyroid papillary carcinomas.
PDGF induce proliferation of fibroblasts, endothelial, and smooth muscle cell proliferation; extracellular matrix synthesis; and is chemotactic for the aforementioned cells, which recruits cells into the areas of inflammation and tissue injury. Family of growth factors is designated by two protein chains. PDGF (AA, AB, and BB) are constitutively active while PDGF-CC and PDGF-DD must be cleaved first. They are produced by platelets, macrophages, endothelium, and smooth muscle cells in addition to tumors. They bind to PDGFR α and β.
VEGF and placental growth factor (PIGF) are a homodimeric proteins. VEGF-A is the major angiogenic factor released after injury or by tumors. VEGF-B and PIGF are implicated in embryonic vessel development. VEGF-C and D are involved in angiogenesis and lymphogenesis. In general, VEGFs are involved formation of vascular lumen as well as adult endothelium maintenance with the most expression in fenestrated epithelium (e.g. pigment epithelium in the retina, choroid plexus in the brain, and podocytes in the kidney). VEGFs also promote endothelial cell migration, proliferation, vascular dilation, increased permeability. VEGF activity is induced by hypoxia (HIF-1) and PDGF, and TGF-α.
The cell cycle is divided in to 4 primary phases: G1 (presynthetic growth), S (DNA synthesis), G2 (premitotic growth), and M (mitotic). Cells that are not actively replicating are in G0. The cell cycle is driven by cyclin proteins and cyclin-dependent kinases (CDKs). There are 4 types of cyclins that act in sequence to activate CDKs.
There are checkpoints at the G1-S juncture that checks the integrity of the DNA before replication and at the G2-M juncture that checks that the DNA was properly replicated before division occurs. If satisfactory conditions are not met, the cell undergoes apoptosis or enter a non-replicative state called senescence. These checkpoints are controlled by CDK inhibitors, which modulate CDK-cyclin activity. Defects in these checkpoints are often implicated in malignant tumors.