Physiology, Anatomy and

Biochemistry

Dr. Simone Cardaci, PhD

(IRCCS San Raffaele Hospital - Milan)
DIBIT1 - 4A1 – 8A
[Link]@[Link]
 Biochemistry Module

Skeletal, Cardiac and Smooth Muscle Metabolism

Structural and functional differences among skeletal, cardiac and smooth muscle types.
Structure of the sarcomere and the sliding filament model of muscle contraction.
Role of Ca2+ and ion channels in muscle contraction.
Bioenergetics of muscle

Brain Metabolism
Mechanisms of transport of metabolites across the blood brain barrier
Brain metabolism
Bioenergetic and redox cooperation between neurons and astrocytes
Metabolic cooperation in Glutamatergic and GABAergic synapses
Lactate and ketone bodies metabolism

Liver Metabolism
Overview the key properties and molecular specialization of the liver
Role of liver in homeostasis of glucose, lipids and amino acid metabolism
Detoxifying functions of the liver: catabolic function & disposal
Alcohol metabolism

Bone Metabolism
Anatomy and structure of bone
The chemical composition of bone and the biochemical bases of bone mineralization
Bone remodelling cycle
Regulation of serum calcium concentration and disorders of calcium metabolism
.
 Biochemistry Module

The Exocrine System

Overview of the structures of the exocrine system and the major acinar and ductular
secretions
Cellular physiology of exocrine secretion
Biochemistry of digestion

The Cytoskeleton and Cell Motors

Microtubules, microfilaments, and intermediate filaments
Motor proteins
Mechanisms of cargo transport

Extracellular matrix (ECM)

Composition, structure and function of the ECM
Biosynthesis and post-translational modification of collagens and elastin
Biosynthesis and turnover of proteoglycans.
Structure and function of integrins as receptors for ECM components.
 CYTOSKELETON
&
MOTOR PROTEINS
 The Cytoskeleton

• The cytoskeleton is a network of fibers

extending throughout the cytoplasm

• It organizes the cell’s structures and

activities

• It is composed of three types of

molecular structures:
 microtubules
 microfilaments
 intermediate filaments
 Functions of the Cytoskeleton

Tissue level: Subcellular level:

• muscle movement • anchors organelles
• organization of organelles
Cellular level:
• provides tensile strength
• determines shape of the
cell • movement of chromosomes

• motility of the cells • organizing cell polarity

• cell adhesion • intracellular movement of vesicles

• mitosis, meiosis • endocytosis (clathrin-mediated) and

phagocytosis

Stable, Strong, Dynamic, Adaptable

Microtubules
 Microfilaments

• Microfilaments are solid rods about 7

nm in diameter, built as a twisted
double chain of actin subunits.

• The structural role of microfilaments is

to bear tension, resisting pulling forces
within the cell.

• They form a 3-D network called the

cortex just inside the plasma
membrane to help support the cell’s
shape.

• Bundles of microfilaments make up the

core of microvilli of intestinal epithelial
cells.
 Microfilaments

Muscle contraction Cell Movement

Intermediate Filaments
 The Nuclear Lamina

• The nuclear envelope consists of an outer (ONM) and an inner

nuclear membrane (INM), which are fused at nuclear pore
complexes (NPC).

• The ONM is continuous with the endoplasmic reticulum (ER) and

associated with ribosomes as well as nesprins and other
proteins linking the nucleus to the cytoskeleton.

• The nuclear lamina is a proteinaceous network of intermediate

filaments underneath the INM that forms extensive contacts
with chromatin and INM proteins.

• Such interactions are critical for many cellular processes, such

as nuclear positioning, perception of mechanical stimuli from
the cell surface, nuclear stability, 3- dimensional
organization of chromatin and regulation of chromatin-binding
proteins, including transcription factors.

• The nuclear lamina consists of two components, LAMINS and

nuclear lamin-associated membrane proteins. The lamins are
type V intermediate filaments which can be categorized as
either A-type (lamin A, C) or B-type(lamin B1, B2)
 Nuclear Lamina-related diseases

Broad spectrum of disease manifestations in humans with mutations in NL-related genes, ranging
from muscle dystrophies to neurological disorders, lipodystrophies and progeria syndromes.
 Cytoskeleton is a dynamic structure

• Cytoskeleton subunits are small, so they can diffuse rapidly within cytoplasm,
whereas the assembled filaments cannot. In this way, cells can undergo rapid
structural reorganizations, disassembling filaments at one site and reassembling them
at another site far away.

• Intermediate filaments are made up of smaller subunits that are themselves

elongated and fibrous, whereas actin filaments and microtubules are made of
subunits that are compact and globular.

• The three types of cytoskeletal “polymers” are held together by weak noncovalent
interactions, which means that their assembly and disassembly can occur rapidly,
without covalent bonds being formed or broken.

• Hundreds of different cytoskeleton-associated accessory proteins regulate the spatial

distribution and the dynamic assembly of new filaments.

• In these processes, the accessory proteins bring cytoskeletal structure under the
control of extracellular and intracellular signals, including those that trigger the
dramatic transformations of the cytoskeleton that occur during the cell cycle. Acting
together, the accessory proteins enable a eucaryotic cell to maintain a highly organized
but flexible internal structure and, in many cases, to move.
 Formation of a Cytoskeletal Polymer

• For a new large filament to form, subunits must assemble into an initial
aggregate, or nucleus, that is stabilized by many subunit-subunit
contacts and can then elongate rapidly by addition of more subunits. The
initial process of nucleus assembly is called filament nucleation (slow lag
phase)

• The lag phase is followed by a phase of rapid filament elongation, during

which subunits add quickly onto the ends of the nucleated filaments.

• Finally, the system approaches a steady state at which the rate of

addition of new subunits to the filament ends is exactly balanced by the
rate of subunit dissociation from the ends.

• Cells use special proteins to catalyze filament nucleation at specific

sites, thereby determining the location where new cytoskeletal
filaments are assembled. Indeed, this type of regulation of filament
nucleation is a primary way that cells control their shape and movement.
 Cytoskeleton Filaments Polarity

• A microtubule is a stiff, hollow cylindrical structure built from 13

parallel protofilaments, each composed of alternating α-tubulin
and β-tubulin molecules, associated together by noncovalent
bonds.

• Each α or β monomer has a binding site for one molecule of

GTP. The GTP that is bound to the α-tubulin monomer is
physically trapped at the dimer interface and is never hydrolyzed
or exchanged. The nucleotide on the β-tubulin, in contrast, may
be in either the GTP or the GDP form, and it is exchangeable. The
hydrolysis of GTP at this site produces GDP and has an
important effect on microtubule dynamics.

• Each protofilament in a microtubule is assembled from subunits

that all point in the same direction, and the protofilaments
themselves are aligned in parallel. Therefore, the microtubule
itself has a distinct structural polarity, with α-tubulins exposed
at one end and β-tubulins exposed at the other end.
 Cytoskeleton Filaments Polarity

• The actin subunit is a single globular polypeptide

chain and is thus a monomer rather than a dimer. Like
tubulin, each actin subunit has a binding site for a
nucleotide, but for actin the nucleotide is ATP (or
ADP).

• As for tubulin, the actin subunits assemble head-to-tail

to generate filaments with a distinct structural polarity.
The actin filament can be considered to consist of two
parallel protofilaments that twist around each other
in a right-handed helix.
 Cytoskeleton Filaments Polarity

• The structural polarity of actin filaments and microtubules makes the two
ends of each polymer have a different kinetic rate constants for
association and dissociation.

• The more dynamic of the two ends of a filament, where both growth and
shrinkage are fast, is called the plus end, and the other end is called the
minus end.

• On microtubules, α subunits are exposed at the minus end, and β subunits

are exposed at the plus end. On actin filaments, the ATP-binding cleft on
the monomer points toward the minus end.

• Polar filaments can act as directional tracks for motor proteins

• The free energy released during spontaneous filament polymerization or

depolymerization can be used to do mechanical work (for example,
elongating microtubules can help push out membranes, and shrinking
microtubules can help pull mitotic chromosomes away from their sisters
during anaphase)
 Cytoskeleton Filaments Polarity

• The actin and tubulin subunits are both enzymes that can catalyze the
hydrolysis of a nucleoside triphosphate, ATP or GTP, respectively. For the free
subunits, this hydrolysis proceeds very slowly; however, it is accelerated when
the subunits are incorporated into filaments.

• The nucleoside diphosphate remains trapped in the filament structure. Thus,

two different types of filament structures can exist, one with the “T form” of the
nucleotide bound (ATP for actin, GTP for tubulin), and one with the “D form”
bound (ADP for actin, GDP for tubulin).

• In living cells, most of the free subunits are in the T form (as the free
concentration of both ATP and GTP is much higher than that of ADP and GDP).

• The T form are more prone to assembly, whereas the D form are more prone to
disassembly.

• Such properties allow filament polymerization described in a model called

dynamic instability.
 Dynamic Instability

• Dynamic instability refers to the coexistence of assembly and

disassembly at the ends of a microtubule.

• During polymerization, the tubulin dimers are in the GTP-bound state.

The GTP bound to α-tubulin is stable and it plays a structural function in
this bound state. However, the GTP bound to β-tubulin may be
hydrolyzed to GDP shortly after assembly, becoming more prone to
depolymerization.

• A GDP-bound tubulin subunit at the tip of a microtubule will tend to fall

off, although a GDP-bound tubulin in the middle of a microtubule cannot
spontaneously pop out of the polymer.

• Since tubulin adds onto the end of the microtubule in the GTP-bound
state, a cap of GTP-bound tubulin (T-cap) is proposed to exist at the tip
of the microtubule, protecting it from disassembly.

• When hydrolysis occurs to the tip of the microtubule, it begins a rapid

depolymerization and shrinkage. This switch from growth to shrinking is
called a catastrophe. GTP-bound tubulin can begin adding to the tip of
the microtubule again, providing a new cap and protecting the
microtubule from shrinking. This is referred to as "rescue".
 Treadmilling of Actin Filaments

• Actin filaments may undergo treadmilling, in which filament

length remains approximately constant, while actin monomers
_ +
add at the (+) end in the T form and dissociate from the (-)
end in the D form.

• The ATP or GTP hydrolysis that occurs along the way gives rise
to the difference in the free energy of the
association/dissociation reactions at the plus and minus ends
of the actin filament or microtubule and thereby makes
treadmilling possible.

• It happens in vitro; however its relevance in vivo is still

uncertain
 Drugs affecting Actin and Microtubules

The cancer-fighting taxane class of drugs block dynamic instability by stabilizing GDP-bound tubulin in the
microtubule. Thus, even when hydrolysis of GTP reaches the tip of the microtubule, there is no depolymerization and
the microtubule does not shrink back.
 Cytoskeleton Nucleation

• Microtubules are nucleated and organized by microtubule organizing

centers (MTOCs), such as the centrosome found in the center of
many animal cells, or the basal bodies found in cilia and flagella, or
the spindle pole bodies found in most fungi.

• In animal cells, the centrosome has a pair of centrioles, each with

nine triplets of microtubule filaments arranged in a ring (cartwheel
structure).

• The two centrioles are arranged at right angles to each other, and
surrounded by a dense, highly structured mass of protein termed the
pericentriolar material (PCM). The PCM contains proteins responsible
for microtubule nucleation and anchoring — including γ-tubulin rings
(from where microtubules extend).

• Microtubules are nucleated at their minus end, with the plus

end growing outward from each MTOC toward the cell
periphery

• In many cell types, the centrosome is replaced by a cilium during

cellular differentiation. However, once the cell starts to divide, the
cilium is replaced again by the centrosome.
 Actin Binding Proteins (ABPs)

3 groups:

• regulatory proteins:
polymerization/depolymerization,
Organisation of
capping and severing proteins
actin filaments
• banding and cross-linking proteins

• motor proteins Sliding and transport

 Regulatory Proteins

Tropomodulin CAP39
Severin
Gelsolin
- + Villin
CapZ


Prevents dissociation Inhibit polymerization
of the filament into of monomer subunits
monomer subunits into filament
Severin
Gelsolin
Cofilin

Profilin: controls turnover of the G actin pool

Thymosin: regulates actin polymerization by sequestering G actin subunits
Formin: assists actin polymerization in cooperation with profilin
 Actin-Banding and Cross-Linking proteins

• Organize actin filaments into bundles or

networks.

• Some actin-binding proteins such as α-actinin,

villin and fimbrin bind actin filaments into
parallel bundles.

• Depending on the length of actin cross-linking

protein, or the distance between actin-binding
domains, actin filaments in parallel bundles
may be held close, or may be far enough apart
to allow interaction with other proteins, e.g.,
myosin.
 Actin-Banding and Cross-Linking proteins

• Filamins dimerize to form V-shaped cross-linking proteins that are flexible due to hinge regions.

• Filamins organize actin filaments into loose networks that give some areas of the cytosol a gel-like consistency.

• Filamins may also have scaffolding roles relating to their ability to bind constituents of signal pathways such as plasma
membrane receptors, calmodulin, caveolin, protein kinase C, transcription factors, etc.
 Cell structures involving Actin

In contrast to microtubule nucleation, which occurs primarily deep within the cytoplasm near the nucleus, actin filament
nucleation most frequently occurs at the plasma membrane (consequently, the highest density of actin filaments in most
cells is at the cell periphery).

These actin filaments in the layer underlying the plasma membrane, called the cell cortex, determine the shape and
movement of the cell surface. For example, depending on their attachments to one another and to the plasma membrane,
actin structures can form many strikingly different types of cell surface projections. These include spiky bundles such as
microvilli or filopodia, flat protrusive veils called lamellipodia that help move cells over solid substrates, and the phagocytic
cups in macrophages.
.
MOLECULAR MOTORS
 General features of Motor Proteins

• Motor proteins are molecular motors that bind to a polarized cytoskeletal filament and use the energy derived from
repeated cycles of ATP hydrolysis to move steadily along it.

• All of them generate motion by coupling nucleoside triphosphate (e.g., ATP) hydrolysis to a large-scale conformational
change in a protein.

• The cytoskeletal motor proteins associate with their filament tracks through a “head” region, or motor domain, that
binds and hydrolyzes ATP. The identity of the track and the direction of movement along it are determined by the motor
domain (head), while the identity of the cargo (and therefore the biological function of the individual motor protein) is
determined by the tail of the motor protein.

• Through a mechanochemical cycle of filament binding, conformational change, filament release, conformational
relaxation, and filament rebinding, the motor protein and its associated cargo move one step at a time along the filament
(typically a distance of a few nanometers).

• Dozens of different motor proteins coexist in every eukaryotic cell. They differ in the type of filament they bind to (either
actin or microtubules), the direction in which they move along the filament, and the “cargo” they carry. Many motor
proteins carry membrane-enclosed organelles—such as mitochondria, Golgi stacks, or secretory vesicles—to their
appropriate locations in the cell. Other motor proteins cause cytoskeletal filaments to slide against each other, generating
the force that drives such phenomena as muscle contraction, ciliary beating, and cell division.
Actin-based Motor Proteins Are Members of the Myosin Superfamily
• Skeletal muscle myosin was the first motor protein identified. This myosin, called myosin II, is responsible for generating the force for
muscle contraction. It is an elongated protein that is formed from two heavy chains and two copies of each of two light chains. Each
of the heavy chains has a globular head domain at its N-terminus that contains the force-generating machinery, followed by a very
long amino acid sequence that forms an extended coiled-coil that mediates heavy chain dimerization.
• The two light chains bind close to the N-terminal head domain, while the long coiled-coil tail bundles itself with the tails of other
myosin molecules. These tail-tail interactions result in the formation of large bipolar “thick filaments” that have several hundred
myosin heads, oriented in opposite directions at the two ends of the thick filament.
• The human genome includes about 40 myosin genes (superfamily). The heavy chains generally start with a recognizable myosin motor
domain at the N-terminus, and then diverge widely with a variety of C-terminal tail domains.
• Each myosin head binds and hydrolyses ATP, using the energy of ATP hydrolysis to walk toward the plus end of an actin filament
(the exception is myosin VI, which moves toward the minus end). Movement speed varies widely, from about 0.2 to 60 μm/sec.
Conformational changes of Myosin sliding on Actin Filament

In the myosin cycle, the head remains bound to the actin filament for only about 5% of the
entire cycle time, allowing many myosins to work together to move a single actin filament.
 Phosphorylation regulates Myosin conformation

• Myosin activity can also be regulated by phosphorylation.

In non-muscle cells, myosin II can be phosphorylated on a
variety of sites on both heavy and light chains, affecting
both motor activity and thick filament assembly.

• The myosin II can exist in two different conformational

states in such cells, an extended state that is capable of
forming bipolar filaments, and a bent state in which the
tail domain apparently interacts with the motor head.

• Phosphorylation of the regulatory light chain by the

calcium-dependent myosin light-chain kinase (MLCK)
causes the myosin II to preferentially assume the
extended state, which promotes its assembly into a
bipolar filament and leads to cell contraction.
 Kinesin and kinesin-related proteins

• Kinesin is a motor protein that moves along

microtubules.

• Kinesin is similar structurally to myosin II in

having two heavy chains and two light chains
per active motor, two globular head motor
domains, and an elongated coiled-coil
responsible for heavy chain dimerization.

• There are at least ten families of kinesin-

related proteins, or KRPs, in the kinesin
superfamily.

• Most of them have the motor domain at the

N-terminus of the heavy chain and walk
toward the plus end of the microtubule.
 The mechanochemical cycles of kinesin

• At the start of the cycle, one of the two kinesin heads,

the front or leading head (dark green) is bound to the
microtubule, with the rear or trailing head (light green)
detached.

• Binding of ATP to the front head causes the rear head to

be thrown forward, past the binding site of the attached
head, to another binding site further toward the plus end
of the microtubule.

• Release of ADP from the second head (now in the front)

and hydrolysis of ATP on the first head (now in the rear)
brings the dimer back to the original state, but the two
heads have switched their relative positions, and the
motor protein has moved one step along the
microtubule protofilament.

• In this cycle, each head spends about 50% of its time

attached to the microtubule and 50% of its time
detached.
 Dyneins

• The dyneins are a family of minus-end-directed microtubule motors, but they are
unrelated to the kinesin superfamily.

• They are composed of two or three heavy chains (that include the motor
domain) and a large and variable number of associated light chains.
• Dyneins are the largest of the known molecular motors, and they are also among
the fastest: axonemal dyneins can move microtubules in a test tube at the
remarkable rate of 14 μm/sec. In comparison, the fastest kinesins can move their
microtubules at about 2–3 μm/sec.
• The dynein family has two major branches. The most ancient branch contains the
cytoplasmic dyneins, which are typically heavy-chain homodimers, with two large
motor domains as heads. Cytoplasmic dyneins are probably found in all eukaryotic
cells, and they are important for vesicle trafficking, as well as for localization of the
Golgi apparatus near the center of the cell.
• Axonemal (ciliary) dyneins, the other large branch, include heterodimers and
heterotrimers, with two or three motor- domain heads, respectively. They are
highly specialized for the rapid and efficient sliding movements of microtubules
that drive the beating of cilia and flagella.
 Dyneins: attachment to membranes

• For dynein, attachment to membranes is known to be mediated by a

large macromolecular assembly.

• Cytoplasmic dynein is itself a huge protein complex, and it requires

association with a second large protein complex called dynactin to
translocate organelles effectively.

• The dynactin complex includes a short actin-like filament that is made of

the actin-related protein Arp1 (distinct from Arp2 and Arp3, the
components of the ARP complex involved in the nucleation of
conventional actin filaments).

• Membranes of the Golgi apparatus are coated with the proteins ankyrin
and spectrin, which have been proposed to associate with the Arp1
filament in the dynactin complex to form a planar cytoskeletal array
reminiscent of the erythrocyte membrane cytoskeleton.
 The mechanochemical cycle of a cytoplasmic Dynein

• The dynein motor domain moves towards the minus end of the
microtubule.
• With no nucleotide bound at the AAA1 ATPase site, the dynein
motor domain is tightly bound to the microtubule (1).

• ATP binding induces rapid dissociation from the microtubule (2). A

slower ATP-driven change (~200 s–1) is the remodelling of the linker
domain, which is displaced across the AAA+ ring (3).

• After hydrolysis of ATP to ADP and inorganic phosphate (Pi), the

motor domain is thought to engage a new binding site on the
microtubule, initially via a weak interaction (4). Strong binding to
the microtubule accelerates the release of Pi from AAA1, inducing
the linker to revert to its straight form.

• This transition is speculated to represent the powerstroke: the

main step in which force (indicated by the light grey arrow) is
transmitted to the attached object (5). Finally, ADP is released from
AAA1 and the cycle restarts.
 Roles of kinesins and dyneins in organizing ER and Golgi

• Kinesins can tether ER-derived membranes to

preformed microtubule tracks, and walk toward
the microtubule plus ends, dragging the ER
membranes out into tubular protrusions and
forming a membranous web very much like the ER
in cells. Likewise, the outward movement of ER
tubules toward the cell periphery is associated with
microtubule growth in living cells.

• Conversely, dyneins are required for positioning

the Golgi apparatus near the cell center, moving
Golgi vesicles along microtubule tracks toward
minus ends at the centrosome.
 Cilia and flagella
• Cilia and flagella are highly specialized and efficient motility structures

• They share a common ultrastructure:

 an axoneme, a core of microtubules sheathed by the plasma membrane
 a basal body (equivalent to centrioles) that anchors the cilium or
flagellum
 dynein, which drives the bending movements of a cilium or flagellum

• The axoneme is composed of microtubules and their associated proteins,

arranged in a distinctive and regular pattern. Nine special doublet
microtubules (comprising one complete and one partial microtubule fused
together so that they share a common tubule wall) are arranged in a ring
around a pair of single microtubules.

• Molecules of ciliary dynein form bridges between the neighbouring doublet

microtubules around the circumference of the axoneme. When the motor
domain of this dynein is activated, the dynein molecules attached to one
microtubule doublet attempt to walk along the adjacent microtubule doublet,
tending to force the adjacent doublets to slide relative to one another, much
as actin thin filaments slide during muscle contraction. However, the presence
of other links between the microtubule doublets prevents this sliding, and the
dynein force is instead converted into a bending motion.
THE EXTRACELLULAR MATRIX
 The Extracellular Matrix (ECM)

• ECM is a complex network of secreted macromolecules located in the

extracellular space

• The macromolecular network comprises:

 organic fibrillar matrix (fibril-forming collagens, elastin, fibrillin,
laminin)
 organic non-fibrillar matrix (lattice-forming collagens, fibronectin,
proteoglycans)
 ions (Na+, K+, Ca2+, Mg2+; bound by the negatively charged
glycosaminoglycans)
 water (drained into ECM by osmotic forces)

• The ECM macromolecules are synthesized by a number of cell types of

mesenchymal origin:
 fibroblasts
 smooth muscle cells
 chondroblasts
 osteoblasts
 epithelial cells
 ECM Functions

• Provides support and anchoring to cells.

• Regulates basic cellular processes, including:

 polarity
 differentiation
 adhesion
 migration
 proliferation
• Acts as a 3D framework for the organization of tissues and organs in:
 tissue growth
 regenerative and healing processes
 determination and maintenance of tissue structure

• Is a “marketplace” for the inter-cellular exchange of metabolites, ions,

water.

• Relative abundance, distribution, and molecular organization of the ECM

vary enormously, depending upon tissue type, developmental stage and
pathological status (arthritis, cancer, fibrosis).
 ECM Dysfunctions

• The ECM has been found to be involved in many normal and pathologic
processes

• It plays important roles in development, in inflammatory states, and in

the spread of cancer cells.

• Involvement of certain components of the ECM has been documented in

both rheumatoid arthritis and osteoarthritis.

• Several diseases (eg, osteogenesis imperfecta and a number of types of

the Ehlers-Danlos syndrome) are due to genetic disturbances of the
synthesis of collagen.

• Specific components of proteoglycans (the glycosaminoglycans; GAGs)

are affected in the group of genetic disorders known as the
mucopolysaccharidoses.

• Changes occur in the ECM during the aging process.

 Collagens

• Collagen, the major component of most connective tissues,

constitutes approximately 25% of the protein of mammals. It
provides an extracellular framework for all metazoan animals
and exists in virtually every animal tissue.

• At least 28 types of collagen made up of over 30 distinct

polypeptide chains (each encoded by a separate gene) have
been identified in human tissues

• The elementary unit of collagens is tropocollagen, a rigid right-

handed triple helix or "super helix“ formed by 3 polypeptide
chains ( chains) enriched in glycine (33%) and
proline/hydroxyproline (27%).

• Each αchain comprises ~1000 aa (with a molecular weight up to

100 kDa) and has a left-handed helical structure.
 Collagens
• The characteristic repeating sequence of collagens is Gly-X-Y, where X and Y are most often proline and hydroxyproline, respectively. Due
to their restricted rotation and bulk structure, proline and hydroxyproline confer rigidity to the helix.
• The triple helices are then assembled into a quarter-staggered alignment to form fibrils. This arrangement leads to areas where there is
complete overlap of the molecules alternating with areas where there is a gap, giving the fibrils a regular banded appearance. Fibrils, in
turn, associate into thicker fibers (1-20 μm in diameter).
• Collagen fibers are further stabilized by the formation of covalent cross-links, both within and between the triple helical units. The main
fibril-forming collagens in skin and bone and in cartilage, respectively, are types I and II, although other collagens also adopt this structure.
However, there are many nonfibril-forming collagens and their structures and functions will be described briefly in the next slides
 Some Collagen Types Do Not Form Fibrils

• Fibrillar or fibril-forming collagens (types I, II, III, V, XI)

• Non-fibrillar collagens: several collagen types do not form fibrils in

tissues. They are characterized by interruptions of the triple helix
with stretches of protein lacking Gly-X-Y repeat sequences. Thus,
areas of globular structure are interspersed in the triple helical
structure.
 Network-like collagens such as type IV form networks in
basement membranes;
 fibril-associated collagens with interrupted triple helices
(FACITs) have interruptions in the triple helical domains;
 beaded filaments consist of long chains of collagen
molecules which have a regular beaded appearance;
 collagen VII forms the main part of anchoring fibrils in
epithelial tissues; transmembrane collagens have short
intracellular N-terminal domains and extracellular domains
with long interrupted triple helices; multiplexins are
collagens with multiple triple helix domains and
interruptions.
 Collagens types

The five most common types are:

• type I: skin, tendon, heart, bone (major
component of the organic fraction of the
bone)
• type II: cartilage (predominant
collagenous component of the cartilage)
• type III: reticular connective tissue
(major component of reticular fibers),
commonly found in association with
type I.
• type IV: forms basal lamina, the
epithelium-secreted layer of the
basement membrane.
• type V: cell surfaces, liver, cornea,
mucosa.
 Synthesis and post-translational modification of collagens

Newly synthesized collagen undergoes extensive posttranslational

modification before becoming part of a mature
extracellular collagen fiber.

1. Like most secreted proteins, collagen is synthesized on ribosomes in

a precursor form, pre-procollagen, which contains a leader or signal
sequence that directs the polypeptide chain into the lumen of the
endoplasmic reticulum (ER).

2. As it enters the ER, this leader sequence is removed

enzymatically. Hydroxylation of proline and lysine residues
and glycosylation of hydroxylysines in the procollagen molecule also
take place at this site and in the Golgi.

• The hydroxylation reactions are catalysed by lysyl-5-

hydroxylase, prolyl-3-hydroxylase, and prolyl-4-hydroxylase.
These reactions require α-ketoglutarate, O2 and ascorbic acid.
Ascorbic acid is required to reduce Fe3+ to fe2+, metal cofactor
of the hydroxylases.

• The glycosylation reactions involves the addition of galactose

and glucose to some hydroxylysine residues (galactosyl
transferase and glucosyl transferase)
 Synthesis and post-translational modification of collagens

3. Assembly of α-chains to form procollagen. This involves formation of disulfide bonds between
regions of the polypeptide chains known as registration or extention peptides, which are present
at both ends of procollagen. Formation of these disulfide bonds assists in the registration of the
three collagen molecules to form the triple helix.

4. Golgi apparatus modification: In the Golgi apparatus, the procollagen goes through one last
post-translational modification before being secreted out of the cell. In this step,
oligosaccharides (not monosaccharides as in step 3) are added, and then the procollagen is
packaged into a secretory vesicle destined for the extracellular space.

5. Secretion of procollagen molecules by exocytosis into the extracellular space.

6. Cleavage of the registration peptides catalysed by procollagen peptidases. The resulting

molecule is called tropocollagen.

7. Oxidative deamination of the hydroxylysine residues, as catalyzed by lysine oxidase, which

generates allysine residues. Allysine pairs can react via aldol condensation. Also, allysine and
lysine residues can form a Schiff base. In both cases, adjacent tropocollagen molecules will
become covalently cross-linked.

8. Self-assembly or polymerization of tropocollagen molecules to form collagen fibrils. Additional

covalent crosslinks between adjacent tropocollagen molecules stabilizes the fibrils.
 Diseases associated with collagens

• Collagen genes (> 30) have the prefix

COL followed by the type in arabic
numerals, then an A and the number of
the proα chain they encode. Thus,
COL1A1 and COL1A2 are the genes for
the proα1 and 2 chains of type I collagen,
COL2A1 is the gene for the proα1 chain
of type II collagen, and so on.

• The pathway of collagen biosynthesis is

complex, involving at least eight enzyme-
catalyzed posttranslational steps. Thus, it
is not surprising that a number of
diseases are due to mutations in
collagen genes or in genes encoding
some of the enzymes involved in these
posttranslational modifications.
 Osteogenesis Imperfecta

• Osteogenesis imperfecta (OI), also called brittle bone disease, is a congenital

disease caused by multiple genetic defects in the synthesis of type I collagen.

• It is characterized by fragile bones, thin skin, abnormal teeth and weak tendons.
The majority of individuals with this disease have mutations in genes encoding
α1(I) or α2(I) collagen chains (COL1A1 or COL1A2 genes). The Inheritance is often
autosomal dominant.

• Most of these mutations are single-base substitutions that convert glycine in the
Gly-X-Y repeat to bulky amino acids, preventing the correct folding of the collagen
chains into a triple helix and their assembly to form collagen fibrils. The
dominance of type 1 collagen in bone explains why bones are predominantly
affected.

• However, there is remarkable clinical variability characterized by bone fragility,

osteopenia, variable degrees of short stature, and progressive skeletal
deformities. The most common form of OI, with a presentation that is sometimes
mistaken for child abuse, has a good prognosis, with fractures decreasing after
puberty, though the general reduction in bone mass means that lifetime risk
remains high. Patients frequently develop deafness due to osteosclerosis, partly
from recurrent fractures of the stapes.

• Bisphosphonate drugs, which inhibit osteoclast activity and thereby inhibit normal
bone turnover, have reduced the incidence of fractures.
 Ehlers–Danlos Syndrome

• Group of 10 different collagen deficiency diseases. For many of them a genetic

cause exist and mutations were found in genes encoding for:

 fibrous proteins: COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, and

TNXB (tenascin X)

 enzymes: ADAMTS2 (disintegrin and metalloproteinase with

thrombospondin motifs 2, i.e. procollagen I N-proteinase), PLOD1 (a
lysyl hydroxylases, alpha-ketoglutarate- dependent), B4GALT7 (Beta-1,4-
galactosyltransferase 7, or galactosyltransferase I), DSE (Dermatan-sulfate
epimerase), and D4ST1/CHST14 (carbohydrate sulfotransferase 14)

• These mutations alter the structure, production, or processing of collagen or

proteins that interact with collagen. A defect in collagen can weaken
connective tissue in the skin, bones, blood vessels, and organs, resulting in the
features of the disorder. Therefore, symptoms may include loose joints, stretchy
skin, and abnormal scar formation. Complications may include aortic
dissection, joint dislocations, scoliosis, chronic pain, or early osteoarthritis.

• Most forms ofEDSs are inherited in an autosomal dominant pattern, a few are
inherited in an autosomal recessive pattern. It can also be sporadic mutation.
 Scurvy
Scurvy is a disease resulting from a lack of vitamin C (ascorbic acid). It results in defective collagen hydroxylation

Typical diseases affecting sailors and explorers in the past (no fresh fruits available on ships for long period)

Sir James Lind, a surgeon in the Royal Navy, conducted clinical tests that proved that oranges and lemons would cure and
prevent scurvy, the disease that killed 1 million seamen between 1600 and 1800.
 ELASTIC FIBERS

The ability of a tissue to expand and recoil (compliance = change of volume) depends on the elastic fibers.

• Elastin

• Fibrillin
 ELASTIN

• Elastin is a major protein component of tissues that require

elasticity such as arteries, lungs, bladder, skin and elastic ligaments
and cartilage.
• There is only one gene for elastin.

• It comprises a ~65 kDa soluble protein (tropoelastin) that

contains, primarily, glycine and valine (one in seven of its amino
acids is valine), and modified alanine and proline residues.
• Tropoelastin does not contain hydroxylysine residues and is not
glycosylated.
• Its primary sequence consists of alternating hydrophilic and
hydrophobic lysine and valine-rich domains, respectively.
• The lysines are involved in intermolecular crosslinking, while the
week interactions between valine residues in the hydrophobic
domains impart elasticity to the molecule.
• Tropoelastin is highly cross-linked to form insoluble complexes.
 Elastin cross-linking

• The most common interchain cross-link in elastin is the result of *

the conversion of the amine groups of lysine to reactive *
aldehydes by lysyl oxidase.
*
• This results in the spontaneous formation three-dimensional
elastic polymer, crosslinked by desmosine (a amino acid
derivative found uniquely in elastin) *

* Allysine
* Lysine
Major Differences between Collagen and Elastin
 Diseases associated with Elastin

• Deletions in the elastin gene (located at 7q11.23) have been found in approximately 90% of
subjects with the Williams-Beuren syndrome, a developmental disorder affecting connective
tissue and the central nervous system.

• The mutations, by affecting synthesis of elastin, probably play a causative role in the
supravalvular aortic stenosis often found in this condition.

• Fragmentation or, alternatively, a decrease of elastin is found in conditions such as pulmonary

emphysema , cutis laxa, and aging of the skin.
 Fibrillin

• Fibrillins are large glycoproteins (about 350 kDa) that are

major structural component of microfibrils.

• Microfibrils are fine fiber-like strands 10 to 12 nm in

diameter which provide a scaffold for the deposition of
elastin in the ECM, thus contributing to the stability of the
elastic fibers.

(stability = fibrillin; elasticity = elastin)

• Fibrillin-1 is the main fibrillin present, but fibrillins-2

and 3 have also been identified, and fibrillin-2 is
thought to be important in deposition of microfibrils
early in development
 The Marfan Syndrome

• Marfan syndrome is a relatively rare genetic disease of

connective tissues caused by mutations in the fibrillin gene
(frequency: 1 in 10,000 births).

• People with this disease have typically tall stature, long

arms and legs, and arachnodactyly (long, ‘spidery’ fingers).

• The disease in a mild form causes loose joints, deformed

spine, floppy mitral valves (leading to cardiac regurgitation),
and eye problems such as lens dislocation. In severely
affected individuals, the aortic wall is prone to rupture
because of defects in elastic fiber formation.

Abraham Lincoln
 Groups of Macromolecules in ECM

 Fibrous structural proteins (Collagen and Elastin)

 Glycoconjugates (proteins or lipids that contain covalently linked sugar (glycan) chains)

• Adhesive glycoproteins, which connect the matrix elements to one another and to cells (Fibronectin and Laminin)

• Glycolipids (lipid + oligosaccharides). Membrane lipids whose polar head is modified with oligosaccharides.

• Proteoglycans (protein + glycosaminoglycans). The basic proteoglycan unit consists of a "core protein" linked to one
or more glycosaminoglycan chain(s) (heavily glycosylated).
 Fibronectin (FN)

• Fibronectin is a high molecular weight glycoprotein (~440 kDa)

• It binds to integrins (membrane-spanning receptors)

• It also interacts with ECM components such as collagen, fibrin, heparin and
heparan sulfate

• At least 20 different tissue-specific isoforms of fibronectin have been

identified, all produced by alternative splicing of a single precursor mRNA

• Cellular fibronectin, mainly produced by fibroblasts, is assembled into

insoluble fibrils

• Plasma fibronectin, mostly secreted by hepatocytes, circulates in a soluble

form
 Fibronectin (FN)

• Fibronectin is involved in cell adhesion, differentiation, growth, migration

• Anchoring basal lamina to other ECM components

• Along with fibrin, plasma fibronectin is deposited at sites of injury, forming a

clot that stops bleeding and protects the underlying tissue

• Fibronectin is necessary for embryogenesis (guiding cell attachment and

migration during embryonic development)

• The loss of fibronectin from the surface of many tumor cells may contribute
to their release into the circulation and penetration through the ECM
(metastasis)
 Laminin (LN)

• LN is a high molecular weight glycoprotein (~850 kDa)

• About 50 oligosaccharides are linked to the LN N-terminus, which makes LN one

of the most complicated glycoproteins ever

• It is heterotrimeric (, , and  chains), and folds into a cross-shaped molecule.

LN trimers undergo reversible self-association to form polymers in the presence
of calcium

• Five , four , and three  chains have been identified so far, which can associate
to form at least 15 LN variants

• LN interacts with cell integrins via multiple binding sites

• LN generates a scaffold for anchoring of cells and ECM molecules [including

heparan sulfate (directly), and collagen IV (mostly indirectly via
nidogen/entactin)] in the basement membrane.
 Cell Adhesion Proteins (CAMs)
Cell Adhesion Proteins (CAMs, Cell Adhesion Molecules) can be classified into 4 main families

• Immunoglobulin family CAMs

• Caderins
• Integrins
• Selectins

Ca2+ bindings proteins

 Integrins

• Integrins enable interactions between the cell and the ECM

• The heterodimerization of 19 α-integrin and 8 β-integrin subunits is thought

to yield 25 integrin αβ heterodimers.

• The α and β chains span the cell membrane, interacting with the ECM
outside the cell and the cytoskeleton and signaling molecules inside.

• In such a manner, integrins can transduce signals from the ECM into
biochemical and mechanical events in the cytoplasm that ultimately lead
to alterations in cell morphology and function.

• Upon ligand binding, a conformational change occurs that renders the

cytosolic tail of integrins accessible for intracellular interactors leading to
the recruitment of diverse proteins such as talin, vinculin and α-actinin
that link integrins to the actin cytoskeleton which in turn allows the
generation of traction forces through actomyosin contraction (focal
adhesion).

• Signaling and adaptor proteins (e.g., the Src tyrosine kinase, focal adhesion
kinase [FAK], paxillin, and the integrin-linked kinase [ILK]) are also recruited
that mediate a multiplicity of biological effects (gene expression,
differentiation, proliferation).
 Selectins

• Selectins are single-chain transmembrane glycoproteins that bind to sugar moieties

(lectin = carbohydrate-binding protein)

• There are three subsets of selectins:

 E-selectin (in endothelial cells)

 L-selectin (in leukocytes)
 P-selectin (in platelets and endothelial cells)

• They share a similar cassette structure: an N-terminal, calcium-dependent lectin

domain, an epidermal growth factor (EGF)-like domain, a variable number of consensus
repeat units (2, 6, and 9 for L-, E-, and P-selectin, respectively), a transmembrane
domain (TM) and an intracellular cytoplasmic tail (cyto).

• Selectins are involved in constitutive lymphocyte homing, and in chronic and acute
inflammation processes, including post-ischemic inflammation in muscle, kidney and
heart, skin inflammation, atherosclerosis, glomerulonephritis and lupus erythematosus
and cancer metastasis.
 Cadherins

• Selectins are single-chain transmembrane glycoproteins that bind to sugar moieties

(lectin = carbohydrate-binding protein)

• There are three subsets of selectins:

 E-selectin (in endothelial cells)

 L-selectin (in leukocytes)
 P-selectin (in platelets and endothelial cells)

• They share a similar cassette structure: an N-terminal, calcium-dependent lectin

domain, an epidermal growth factor (EGF)-like domain, a variable number of consensus
repeat units (2, 6, and 9 for L-, E-, and P-selectin, respectively), a transmembrane
domain (TM) and an intracellular cytoplasmic tail (cyto).

• Selectins are involved in constitutive lymphocyte homing, and in chronic and acute
inflammation processes, including post-ischemic inflammation in muscle, kidney and
heart, skin inflammation, atherosclerosis, glomerulonephritis and lupus erythematosus
and cancer metastasis.
 Proteoglycans

• Proteoglycan are gel-forming components of the ECM

• A proteoglycan consists of a core protein bound

covalently to glycosaminoglycans (GAGs).

• At least 30 proteoglycans have been characterized, for

example, syndecan, betaglycan, serglycin, perlecan,
aggrecan, versican, decorin, biglycan, and fibromodulin.

• They are very large complexes with an overall structure

resembling that of a bottlebrush.

• Proteoglycans vary in tissue distribution, nature of the

core protein, attached GAGs, and their function. The
amount of carbohydrate in a proteoglycan is usually
much greater than that found in a glycoprotein, and
may comprise up to 95% of its weight.
 Glycosaminoglycans

• GAGs are long, linear carbohydrate polymers made of repeating

disaccharide units, one component of which is always an amino
sugar, either D-glucosamine or D-galactosamine.

• The other component of the repeating disaccharide (except in the

case of keratan sulfate) is a uronic acid, either D-glucuronic acid
(GlcUA) or its 5′-epimer, L-iduronic acid (IdUA).

• There are at least seven GAGs: hyaluronic acid (hyaluronan),

chondroitin sulfate, keratan sulfates I and II, heparin, heparan sulfate,
and dermatan sulfate.

• With the exception of hyaluronic acid, all the GAGs contain sulfate
groups, either as O-esters or as N-sulfate (in heparin and heparan
sulfate).

• Hyaluronic acid is also exceptional because it appears to exist as a

polysaccharide in the ECM, with no covalent attachment to protein.

• Under physiological conditions they are negatively charged (due to the

presence of sulfate and uronic acid groups).
 Biosynthesis of GAGs

• With the exception of hyaluronic acid (HA), all GAGs are attached
to a Ser/Trh residue of the core protein via the trisaccharide Gal-
Gal-Xyl

• In particular, an O-glycosidic bond forms between xylose (Xyl) and

Ser (bond that is unique to proteoglycans). This linkage is formed
by transfer of a Xyl residue to Ser from UDP-xylose. Two residues
of Gal are then added to the Xyl residue, forming a link
trisaccharide, Gal-Gal-Xyl-Ser.

• The link trisaccharide Gal-Gal-Xyl is attached to a Ser/Thr side

chain on the core protein while this is still in the rER, and acts as a
primer for polysaccharide growth.

• Further glycosylation occurs in the Golgi apparatus in multiple

enzymatic steps. These involve position- and linkage-specific
glycosyltransferases, epimerases and sulfotransferares.
 Degradation of GAGs and Mucopolysaccharidosis
• GAGs are degraded in the lysosome. The protein component is
hydrolyzed by proteases, while the GAG chains are degraded by the
sequential action of acid hydrolases (these include endoglycosidases,
exoglycosidases, and sulfatases, generally acting in sequence)

• If one of the enzymes involved in the stepwise pathway is missing,

the entire degradation process is halted at that point and the
undegraded molecules accumulate in the lysosome, leading to
some forms (more than a dozen) of lysosomal storage diseases
(mucopolysaccharidosis).

• These diseases can be diagnosed by the identification of specific GAG

chains in the urine, followed by assay of the specific hydrolases in
leukocytes and fibroblasts as well as test for specific genes. Prenatal
diagnosis is now sometimes possible using amniotic fluid cells or
chorionic villus biopsy samples

• The diseases are usually inherited in an autosomal recessive

manner. In general, these conditions are chronic and progressive and
affect multiple organs. Many patients exhibit organomegaly (eg,
hepato- and splenomegaly); severe abnormalities in the
development of cartilage and bone; abnormal facial appearance; and
mental retardation. In addition, defects in hearing, vision, and the
cardiovascular system may be present.
 Function of proteoglycans

• Occupy space between cells and collagen

• Organize water molecules
 compressibility
 flexibility/elasticity
 a certain degree of rigidity (due to their highly negative charge)
 silly putty properties (bounce + resilience)
• Highly viscous
 lubricating fluid in the joints
• Specific binding to other macromolecules
• Associate with collagen fibers
 network
 in the bone, combine with calcium salts (calcium carbonate,
hydroxyapatite)
• Anchoring of cells to matrix fibers
• Cell migration and adhesion
 passageways between cells
Biomedical applications of hyaluronic acid
 Matrix Remodelling

• The ECM is in a constant state of synthesis and degradation, repair and remodeling: for example, during cell migration,
morphogenesis, angiogenesis, and in response to inflammation and injury.

• ECM turnover is mediated by a family of matrix metalloproteinases (MMPs), about 30 zinc endoproteinases with
specificity for different components of the matrix. The MMP family includes collagenases, stromelysins, matrilysins
and elastases; these enzymes, with broad substrate specificities, catalyze degradation of collagen, aggrecan and
accessory ECM proteins, such as fibronectin and laminin.

• MMPs may be integral plasma membrane proteins, may be bound to the plasma membrane by a
glycosylphosphatidylinositol (GPI) glycan anchor, or secreted into the extracellular space.

• MMPs exist as zymogens until activated locally by proteolytic cleavage in response to cellular signals or extracellular
enzymes.

• Tissue inhibitors of MMPs, known as TIMPs are a family of proteins that inactivate MMPs and limit their activity in
tissues. The balance between the activation and inhibition of MMPs is critical to the integrity and function of the ECM;
alterations in MMP activity are associated with skeletal dysplasias, coronary artery disease, arthritis and metastasis
[Link]