CELL

The human genome contains some 3.2 billion DNA base pairs.

Within the genome there are only about 20,000 protein-encoding genes, constituting just 1.5% of the
genome

Nearly 80% is devoted to regulation of gene expression

There are five major classes of functional non–protein coding sequences in the human genome
• Promoter and enhancer regions that provide binding sites for transcription factors.
• Binding sites for factors that organize and maintain higher order chromatin structures.
• Noncoding regulatory RNAs. Over 60% of the genome is transcribed into RNAs that are never
translated but regulate gene expression through a variety of mechanisms. The two best-studied
varieties—
• micro-RNAs (miRNAs) and
• long noncoding RNAs (lncRNAs)
• Mobile genetic elements (e.g., transposons) make up more than a third of the human genome.
The two most common forms of DNA variation

SNPs are variants at single nucleotide positions and are almost always biallelic.

SNPs occur across the genome—within exons, introns, intergenic regions, and coding regions.

Roughly 1% of SNPs occur in coding regions.

SNPs located in noncoding regions can occur within genomic regulatory elements, thereby altering gene
expression; in such instances, SNPs influence disease susceptibility directly.

Some SNPs, termed “neutral” variants, are thought to have no effect on gene function or individual phenotype.

Even “neutral” SNPs may be useful markers if they happen to be coinherited with a disease-associated
polymorphism.
CNVs are a form of genetic variation consisting of different numbers of large contiguous stretches of
DNA; these can range from 1000 base pairs to millions of base pairs.

CNVs can be biallelic and simply duplicated or, alternatively, deleted in some individual.

CNVs are responsible for between 5 million and 24 million base pairs of sequence difference
between any two individuals.

Approximately 50% of CNVs involve gene-coding sequences; thus CNVs may underlie a large
portion of human phenotypic diversity.
Nucleosomes consist of DNA segments 147 bp long that are wrapped around a central core
structure of highly conserved low molecular weight proteins called histones.

At the light microscopic level, nuclear chromatin is recognizable as cytochemically dense and
transcriptionally inactive heterochromatin and disperse, transcriptionally active euchromatin.

Histone methylation- Both lysines and arginines ; methylation of histone lysine residues can lead
to transcriptional activation or repression, depending on which histone residue is marked.
Histone acetylation- Lysine residues are acetylated by histone acetyltransferases (HATs), whose
modifications tend to open the chromatin and increase transcription. In turn, these changes can be
reversed by histone deacetylases (HDACs), leading to chromatin condensation.
Histone phosphorylation- Serine residues can be modified by phosphorylation; depending on the
specific residue, the DNA may be opened for transcription or condensed and inactive.
DNA methylation- High levels of DNA methylation in gene regulatory elements typically result in
transcriptional silencing.
Chromatin organizing factors- Much less is known about these proteins, which are believed to
bind to noncoding regions and control long-range looping of DNA, thus regulating the spatial
relationships between enhancers and promoters 
 
Genes expression regulated by noncoding RNAs.

These genomic sequences are transcribed but not translated.

• small RNA molecules called microRNAs (miRNAs) and

• long noncoding RNAs (lncRNAs) (>200 nucleotides in length).

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated
genes (Cas), such as the Cas9 nuclease.
Bacteria use the system to sample the DNA of infecting agents and integrate portions into their
genomes as CRISPRs. These CRISPR segments are subsequently transcribed and processed into
guide RNA sequences that bind and direct the Cas9 nuclease to specific sites so that it can be
cleaved to disable infecting agent.

Application
Repair of inherited genetic diseases and the creation of pathogenic mutations in inducible
pluripotent stem cells.
• By isolating certain cellular functions within distinct compartments, potentially injurious degradative
enzymes or toxic metabolites can be kept at usefully high concentrations without risking damage to
more delicate intracellular constituents.

• Compartmentalization also allows the creation of unique intracellular environments (e.g., low pH or
high calcium) that permit more efficient functioning of certain enzymes or metabolic pathways.
• Proteins destined for the plasma membrane or secretion are physically assembled in the rough
endoplasmic reticulum (RER) and Golgi apparatus.

 Membrane-bound ribosomes on the cytosolic face of RER translate mRNA into proteins that are
extruded into the ER lumen or become integrated into the ER membrane. 
 From the RER, proteins and lipids destined for other organelles or extracellular export are shuttled into
the Golgi apparatus. This consists of stacked cisternae that progressively modify proteins in an orderly
fashion from cis (near the ER) to trans (near the plasma membrane).
 As cisternae mature, the N-linked oligosaccharides originally added in the ER are pruned and
extended in a stepwise fashion; O-linked oligosaccharides (sugar moieties linked to serine or
threonine) are also appended.

• Proteins intended for the cytosol are synthesized on free ribosomes.

Proteins lacking a signal sequence, translation remains on free ribosomes in the cytosol, forming
polyribosomes as multiple ribosomes attach to the mRNA; such transcribed proteins remain within the
cytoplasm.

• Smooth endoplasmic reticulum (SER) is used for steroid hormone and lipoprotein synthesis
and modification of hydrophobic compounds into water-soluble molecules
Chaperone molecules assist in folding and retaining proteins in the ER until the modifications
are complete and the proper conformation is achieved

Most common mutation of the CFTR protein in cystic fibrosis. In mutant CFTR, a codon deletion
leads to the absence of a single amino acid (Phe508) which results in its misfolding, ER
retention and catabolism and therefore reduced surface expression.
 Lysosomes are membrane-bound organelles containing roughly 40 different acid hydrolases (i.e., that
function best at pH ≤5)
 Include proteases, nucleases, lipases, glycosidases, phosphatases, and sulfatases.
 Enzymes are initially synthesized in the ER lumen and then tagged with mannose-6-phosphate
(M6P) within the Golgi apparatus. These M6P-modified proteins are subsequently delivered to lysosomes
through trans Golgi vesicles that express M6P receptors.
 3 Pathway

• The material internalized by fluid-phase or receptor-mediated endocytosis

• Senescent organelles and/or large, denatured protein complexes can be ferried into lysosomes by a
process called autophagy 
• Phagocytosis of microorganisms or large fragments of matrix or debris occurs primarily in professional
phagocytes (macrophages or neutrophils). The material is engulfed to form a phagosome that
subsequently fuses with lysosomes.
• Peroxisomes contain catalase, peroxidase, and other oxidative enzymes; they play
a specialized role in the breakdown of very long-chain fatty acids, generating
hydrogen peroxide in the process.

• Proteasomes are “disposal” complexes that degrade denatured or otherwise

“tagged” cytosolic proteins
The plasma membrane is liberally studded with a variety of proteins and glycoproteins involved in

(1) ion and metabolite transport;

(2) fluid-phase and receptor mediated uptake of macromolecules; and
(3) cell-ligand, cell-matrix, and cell-cell interactions.

Multiple transmembrane-spanning proteins are often pores or molecular transporters, while proteins
that are superficially attached to the membrane via labile linkages are more likely to participate in
signaling.
In general, proteins associate with the lipid bilayer by one of four mechanisms

• Most proteins are integral or transmembrane proteins, having one or more relatively hydrophobic α-helical
segments that traverse the lipid bilayer.

• Proteins synthesized on free ribosomes in the cytosol may be modified posttranslationally by addition of
prenyl groups (e.g., farnesyl, related to cholesterol) or fatty acids (e.g., palmitic or myristic acid) that insert into
the cytosolic side of the plasma membrane.

• Proteins on the extracellular face of the membrane may be anchored by glycosylphosphatidylinositol (GPI)
tails that are added posttranslationally.

• Peripheral membrane proteins may noncovalently associate with true transmembrane proteins.
Passive Diffusion-
• Small, nonpolar molecules like O2 and CO2 readily dissolve in lipid bilayers and therefore rapidly
diffuse across them.
• Larger hydrophobic molecules, (e.g., steroid-based molecules like estradiol or vitamin D) can also
cross lipid bilayers with relative impunity.
• While small polar molecules such as water (18 Da) can also diffuse across membranes at low rates,
in tissues responsible for significant water movement (e.g., renal tubular epithelium), special integral
membrane proteins called aquaporins form transmembrane channels for water
• In contrast, the lipid bilayer is an effective barrier to the passage of larger polar molecules (>75 Da);
at 180 Da, for example, glucose is effectively excluded.

Channel proteins create hydrophilic pores, which, when open, permit rapid movement of solutes
(usually restricted by size and charge).

Carrier proteins bind their specific solute and undergo a series of conformational changes to transfer
the ligand across the membrane; their transport is relatively slow
Caveolae-mediated endocytosis

• Caveolae are noncoated plasma membrane invaginations associated with GPI-

linked molecules, cyclic adenosine monophosphate (cAMP) binding proteins, src-
family kinases, and the folate receptor

• Caveolin is the major structural protein of caveolae, are enriched in

glycosphingolipids and cholesterol.

• Internalization of caveolae along with bound molecules and associated extracellular

fluid is called potocytosis—literally “cellular sipping.”

• Delivery of some molecules (e.g., folate), caveolae regulate transmembrane

signaling and cellular adhesion via internalization of receptors and integrins.
Receptor-mediated endocytosis

• Macromolecules bound to membrane receptors (such as transferrin or low-density lipoprotein [LDL]

receptors) are taken up at specialized regions of the plasma membrane called clathrin-coated pits.

• The receptors are efficiently internalized by membrane invaginations driven by the associated clathrin
matrix, eventually pinching off to form clathrin-coated vesicles.

• The vesicles then rapidly lose their clathrin coating and fuse with an acidic intracellular structure called
the early endosome; the endosomal vesicles undergo progressive maturation to late endosomes,
ultimately fusing with lysosomes.

• In the acidic environment of the endosomes, LDL and transferrin receptors release their cargo
(cholesterol and iron, respectively), which is then transported into the cytosol.
• Mitochondria evolved from ancestral prokaryotes that were engulfed by primitive
eukaryotes about 1.5 billion years ago. Their origin explains why mitochondria contain
their own DNA that encodes about 1% of total cellular protein

• Ovum contributes the vast majority of cytoplasmic organelles in the fertilized zygote,
mitochondrial DNA is almost entirely maternally inherited. 

• Mitochondria are short-lived—being degraded through autophagy (a process called

mitophagy)—with estimated half-lives of 1 to 10 days, depending on the tissue, nutritional
status, metabolic demands, and intercurrent injury.
Energy generation

Has got two separate membranes with distinct functions:

• The inner membrane contains the enzymes of the respiratory chain folded into cristae.
• The outer membrane is studded with porin proteins that form voltage-dependent anion channels that
are permeable to small (<5000 Da) molecules
• Matrix space that harbors the bulk of the enzymes of the glycolytic and tricarboxylic acid cycles,
intermembrane space, which is the site of nucleotide phosphorylation

Mitochondria oxidize substrates to CO2, transferring the high-energy electrons from the original molecule
(e.g., sugar) to molecular oxygen. Oxidation of various metabolites drives proton pumps that transfer H+
from the core matrix into the intermembrane space. As the H+ ions flow down their electrochemical
gradient and out of the intermembrane space, the energy released is used to generate ATP.

Nonshivering thermogenesis
An inner membrane protein enriched in brown fat called thermogenin (also called uncoupling protein-1
[UCP-1]) is a hydrogen ion transporter that can dissipate the proton gradient, uncoupling it from oxidative
phosphorylation. 
Intermediate metabolism

• Oxidative phosphorylation efficiently generates 36 to 38 ATP molecules per glucose molecule, but also
“burns” substrates to their core CO2 and H2O, leaving no carbon moieties to use for building lipids and
proteins

• Both benign and malignant lesions to aerobic glycolysis, a phenomenon called the Warburg effect.
Whereby each glucose molecule is catabolized to lactic acid (even in the presence of adequate oxygen),
generating only two net ATP molecules but “spinning off” intermediates that can be converted into new
lipids, amino acids and proteins, and nucleic acids. 

Cell death
Apoptosis 

• Mitochondria integrate intracellular proapoptotic and antiapoptotic effector signals to generate a final “go” or
“no go” signal for apoptosis.

• A net proapoptotic signal results in mitochondrial outer membrane permeabilization (MOMP) that releases
cytochrome C (and other proteins) into the cytoplasm. In turn, these proteins activate intracellular
programmed cell death pathways
Intracellular receptors

• Include transcription factors that are activated by lipid-soluble ligands that can easily transit plasma
membranes; eg, vitamin D and steroid hormones that activate nuclear hormone receptors.

• Small and/or nonpolar signaling ligand produced by one cell type can influence the activity of adjacent
cells.

Cell-surface receptors

Are generally transmembrane proteins with extracellular domains that bind activating ligands. Depending
on the receptor, ligand binding can:

• Open ion channels, typically at the synapse between electrically excitable cells.
• Activate an associated GTP-binding regulatory protein (G protein).
• Activate an endogenous or associated enzyme (often a tyrosine kinase).
• Trigger a proteolytic event or change protein binding or stability to activate a latent transcription factor.
• Actin microfilaments are 5- to 9-nm diameter fibrils formed from the globular protein actin
(G-actin).

• G-actin monomers noncovalently polymerize into long filaments (F-actin) that intertwine
to form double-stranded helices with a defined polarity

• Polymerization, bundling, and branching to form networks that control cell shape and
movement.

• ATP hydrolysis by myosin slides the actin filaments relative to one another to cause
muscle contraction. 
Intermediate filaments are 10-nm diameter fibrils that comprise a large and heterogeneous family that
includes keratin proteins and nuclear lamins.

• Vimentin, in mesenchymal cells (fibroblasts, endothelium).

• Desmin in muscle cells forms the scaffold on which actin and myosin contract.
• Neurofilaments are critical for neuronal axon structure and confer both strength and rigidity.
• Glial fibrillary acidic protein is expressed in glial cells.
• Cytokeratins are expressed in epithelial cells.
• There are at least 30 distinct different cytokeratins that are expressed in different cell lineages (e.g., lung
vs. gastrointestinal epithelium).
• Lamins are intermediate filament proteins that form the nuclear lamina, define nuclear shape, and can
regulate transcription.
• Microtubules are 25-nm-thick fibrils composed of noncovalently polymerized α- and
β-tubulin dimers organized into hollow tubes.

• These fibrils are extremely dynamic and polarized, with “+” and “−” ends.

• There are two varieties of these motor proteins, kinesins and dyneins, that typically
(but not exclusively) transport cargo in anterograde (− to +) or retrograde (+ to −)
directions, respectively.

• Mediate sister chromatid segregation during mitosis.

• Form the core of primary cilia, single
• Can be adapted to form the core of motile cilia (e.g., in bronchial epithelium) or
flagella (in sperm).
Cells connect and communicate with each other via junctional complexes that form mechanical
links and facilitate receptor-ligand interactions, mediate interaction with the ECM

Occluding junctions (tight junctions)

• Seal adjacent epithelial cells together to create a continuous barrier that restricts the
paracellular (between cells) movement of ions and other molecules.

• Complexes that mediate the cell-cell interactions are composed of transmembrane proteins
including the tetraspanning claudin and tight junction– associated MARVEL protein (TAMP)
families.
These connect to a host of intracellular adaptor and scaffolding proteins, including the three
members of the zonula occludens protein family (ZO-1, ZO-2, ZO-3) and cingulin

• Junctions are dynamic structures that can be modified to facilitate epithelial healing and
inflammatory cell migration across epithelial lined mucosal surfaces.
Desmosomes

• Anchoring junctions mechanically attach cells—and their cytoskeletons—to other cells or the ECM.

• Adherens junctions are often closely associated with and beneath tight junctions, are more basal compared
to tight junctions.

• Formed by homotypic extracellular interactions between transmembrane glycoproteins called Cadherins.

• Linked with intracellular actin microfilaments through which they can also influence cell shape and/or motility

• Linked to intracellular intermediate filaments, allowing extracellular forces to be mechanically dissipated

Hemidesmosomes

• When desmosomes attach the cell to the extracellular matrix (ECM) they are referred to as
hemidesmosomes.

• Transmembrane connector proteins are called integrins

• Attach to intermediate filaments and link the cytoskeleton

• Their component proteins can generate intracellular signals when cells are subjected to shear stress
(e.g., endothelium in the bloodstream or cardiac myocytes in a failing heart).
Communicating junctions (gap junctions)

• Permit the diffusion of chemical or electrical signals from one cell to another.

• Consists of a dense planar array of 1.5- to 2-nm pores (called connexons) formed by a pair of hexamers
of transmembrane connexin proteins.

• Permeability of the junction is rapidly reduced by lowered intracellular pH or increased intracellular

calcium

• Gap junctions in cardiac myocytes allow cell-to-cell calcium fluxes that allow the many cells of the
myocardium to behave as a functional syncytium with coordinated waves of contraction
• The ECM is a protein network that constitutes a significant proportion of any tissue.

• Cell interactions with the ECM are critical for development, healing, and maintenance of normal tissue architecture

• Mechanical support for cell anchorage, cell migration, and maintenance of cell polarity.
• Regulator of cell proliferation by binding and displaying growth factors and by signaling via cellular integrin family
receptors
• Scaffolding for tissue renewal.
• Foundation for establishment of tissue microenvironments.

The ECM occurs in two basic forms:

 Interstitial matrix- occupies the spaces between stromal cells within connective tissue and between parenchymal
epithelium and the underlying supportive vascular and smooth muscle structures in some organs. Interstitial matrix
is synthesized by mesenchymal cells

 Basement membrane- interstitial matrix within connective tissues becomes highly organized around epithelial
cells, endothelial cells, and smooth muscle cells, where it forms basement membranes,
Collagens

Collagens are composed of three separate polypeptide chains braided into a ropelike triple helix.

Fibrillar collagens:

• Collagen types (e.g., types I, II, III, and V) form linear fibrils stabilized by interchain hydrogen bonding;

• Comprise a major proportion of the connective tissue in bone, tendon, cartilage, blood vessels, and skin,
as well as in healing wounds and scars.

Nonfibrillar collagens (e.g., type IV collagen)

• Contribute to structures of planar basement membranes;

• Provide anchoring fibrils that maintain structure of stratified squamous epithelium (e.g., type VII collagen;
mutations lead to blistering skin diseases
Elastin

• Morphologically, elastic fibers consist of a central core of elastin with an associated meshlike network of
fibrillin glycoprotein.
• Elasticity is especially important in cardiac valves and large blood vessels

Proteoglycans and Hyaluronan

• Proteoglycans form highly hydrated compressible gels that confer resistance to compressive forces; in
joint cartilage, proteoglycans also provide a layer of lubrication between adjacent bony surfaces.
• Serve as reservoirs for growth factors secreted into the ECM (e.g., FGF and HGF).
• Some are integral cell membrane proteins that have roles in cell proliferation, migration, and adhesion 

Fibronectin

• Large (450-kDa), disulfide-linked heterodimer that exists in tissue and plasma forms; it is synthesized by a
variety of cells including fibroblasts, monocytes, and endothelium.
• Has specific domains that can bind to distinct ECM components (e.g., collagen, fibrin, heparin, and
proteoglycans), as well as attach to cell integrins.
• In healing wounds, tissue and plasma fibronectin provide the scaffolding for subsequent ECM deposition,
angiogenesis, and reepithelialization
Laminin

• Laminin is the most abundant glycoprotein in basement membranes.

• 820-kDa cross-shaped heterotrimer that connects cells to underlying ECM components such as type IV
collagen and heparan sulfate.
• Besides mediating cell attachment to basement membrane, laminin can also modulate cell proliferation,
differentiation, and motility.

Integrins

• Integrins are a large family of transmembrane heterodimeric glycoproteins composed of α- and β-subunits;
Allow cells to attach to ECM constituents such as laminin and fibronectin, thus functionally and structurally
linking the intracellular cytoskeleton with the outside world.
• Integrins also facilitate cell-cell adhesive interactions;
• On leukocytes, they mediate the firm adhesion to and migration across endothelium and epithelium at sites
of Inflammation, they also play a critical role in platelet aggregation.
• DNA replication is confined to a discrete Synthesis or S-phase, and chromosome segregation occurs at
Mitosis or M-phase.

• Two Gap phases separate S phase and mitosis, known as G1 and G2. These are not periods of
inactivity, but rather periods where cells obtain mass, integrate growth signals, organize a replicated
genome, and prepare for chromosome segregation.

• Central machines that drive cell cycle progression are the cyclin-dependent kinases (CDKs). These are
serine/threonine protein kinases that phosphorylate key substrates to promote DNA synthesis and
mitotic progression.

• Cyclin-binding allows inactive CDKs to adopt an active configuration

• CDK activity can also be negatively regulated by the binding of small inhibitory proteins, the CKIs, or by
inhibitory tyrosine phosphorylation

Ubiquitin-dependent proteolysis .. cyclins

Cell cycle control by RB

• RB is a central regulator of the cell cycle. Functionally RB represents a transcriptional corepressor. It forms
complexes with the E2F family of transcription factors. Importantly, the resulting RB-E2F complexes switch E2F
promoter sites from activator to repressor sites.

• RB binds preferentially E2F1, E2F2, and E2F3, but can also attach to E2F4 and E2F5. These factors form complexes
with the DP dimerization partners DP1 or DP2 to form the E2F component of RB-E2F complexes. The E2F component
of these complexes contacts the DNA via E2F transcription factor binding sites in the gene promoters. RB-E2F
complexes downregulate transcription of the genes.

• Classical RB-E2F target genes often control the cell cycle by contributing to DNA replication and the transition from
the G1 to S phase. Specifically, genes such as DNA polymerase α (POLA1), cyclin A (CCNA2), thymidine kinase
(TK1), dihydrofolate reductase (DHFR), cyclin-dependent kinase 1 CDC2/CDK1 (CDK1), and minichromosome
maintenance complex component 3 and 5 (MCM3/5, DNA replication licensing factors) are considered bona fide RB-
E2F targets.
Cell cycle control by P53

• Its main functions are the induction of apoptosis and cell cycle arrest.

• p53 is involved in DNA repair, control of metabolic pathways, embryo implantation, and driving cells into
senescence.

• The CDKN1A gene, coding for the cyclin-dependent kinase inhibitor p21/WAF1/CIP1/CDKN1A, was the
first discovered transcriptional target of p53. Its binding spectrum is wide, as p21 forms complexes with
CDK1 (also named CDC2), CDK2, CDK3, CDK4, and CDK6 together with specific cyclins forming
complexes with these kinases

•  p21 is a target of p53, it depends on p53 activity. p53 levels are induced by cellular stress, for example
following DNA damage or viral infection. Elevated p53 concentrations yield increased p53 transcriptional
activity triggering cell cycle arrest and apoptosis.

• p53 binds to elements in the p21/CDNKN1A promoter and activates its transcription. The p21 protein
then inhibits all cyclin-CDK pairs that are involved in hyperphosphorylation of RB.

• Hypophosphorylated RB successively forms complexes with E2F transcription factors. RB-E2F

complexes downregulate transcription via binding to E2F binding sites in the promoters of target genes.
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