Protein Folding and Processing

Proteolysis—cleavage of the polypeptide chain

removes portions such as the initiator methionine
from the amino terminus.
Many proteins have amino-terminal signal
sequences that target the protein for transport to a
specific destination.
The signal sequence is inserted into a membrane
channel as it emerges from the ribosome and the
rest of the polypeptide chain passes through as
translation proceeds.
The signal sequence is then cleaved by a membrane
protease (signal peptidase).
Figure 8.27 The role of signal sequences in membrane translocation
Protein Folding and Processing
Proteolytic processing also includes formation of active enzymes or
hormones by cleavage of larger precursors.
Example: Insulin is synthesized as a precursor polypeptide that goes
through 2 cleavages to produce the mature insulin.
Protein Folding and Processing

In the replication of HIV, a virus-encoded

protease cleaves precursor
polypeptides to form the viral structural
proteins.
The HIV protease (in addition to reverse
transcriptase) is an important target for
the development of drugs used for
treating AIDS.
Protein Folding and Processing

Glycosylation adds carbohydrate

chains to proteins to form
glycoproteins.
The carbohydrate moieties play
important roles in protein folding in the
ER, in targeting proteins for transport,
and as recognition sites in cell-cell
interactions.
Protein Folding and Processing

N-linked glycoproteins: the carbohydrate

is attached to the N atom in the side
chain of asparagine.
O-linked glycoproteins: the carbohydrate
is attached to the O atom in the side
chain of serine or threonine.
Figure 8.29 Linkage of carbohydrate side chains to glycoproteins
Protein Folding and Processing

Glycosylation starts in the ER before

translation is complete.
A 14-sugar oligosaccharide is
transferred to an asparagine residue of
the growing polypeptide chain.
The oligosaccharide is assembled on a
lipid carrier (dolichol phosphate) in
the ER membrane.
Figure 8.30 Synthesis of N-linked glycoproteins
Protein Folding and Processing

The N-linked oligosaccharide is modified

by removal of three glucose residues,
and further modifications occur in the
Golgi apparatus.
Figure 8.31 Processing of N-linked oligosaccharides
Protein Folding and Processing

O-linked oligosaccharides are also

added within the Golgi apparatus.
They are formed by the addition of one
sugar at a time.
Figure 8.32 Examples of O-linked oligosaccharides
Protein Folding and Processing

Some eukaryotic proteins are modified

with lipids, which often serve to anchor
them to the plasma membrane.
There are four types of lipid additions.
Protein Folding and Processing

1. N-myristoylation: myristic acid (a 14-

carbon fatty acid) is attached to an N-
terminal glycine.
 These proteins are associated with
the inner face of the plasma
membrane.
Figure 8.33 Addition of a fatty acid by N-myristoylation
Protein Folding and Processing

2. Prenylation: prenyl groups are

attached to sulfur atoms in the side
chains of cysteine located near the C
terminus.
 Many of these proteins are involved
in control of cell growth and
differentiation, including the Ras
oncogene proteins, which are
responsible for many human cancers.
Figure 8.34 Prenylation of a C-terminal cysteine residue
Protein Folding and Processing

3. Palmitoylation: palmitic acid (a 16-

carbon fatty acid) is added to sulfur
atoms of the side chains of internal
cysteine residues.
Figure 8.35 Palmitoylation
Protein Folding and Processing

4. Glycolipids (lipids linked to

oligosaccharides) are added to C-
terminal carboxyl groups.
 They anchor the proteins to the
external face of the plasma
membrane.
 The glycolipids contain
phosphatidylinositol, and are called
glycosylphosphatidylinositol (GPI)
anchors.
Figure 8.36 Structure of a GPI anchor
Regulation of Protein Function

Regulation of protein function allows the

cell to regulate not only the amounts
but also the activities of its proteins.
There are three general mechanisms of
control of cellular proteins:
• regulation by small molecules
• phosphorylation
• protein-protein interactions
Regulation of Protein Function

Most enzymes are controlled by

changes in conformation, often as a
result of binding small molecules.
End products of many biosynthetic
pathways inhibit the enzymes that
catalyze the first step in their synthesis.
Regulation of Protein Function

Feedback inhibition is an example of

allosteric regulation, in which a
regulatory molecule binds to an
enzyme site that is distinct from the
catalytic site (allo = “other”; steric =
“site”).
Figure 8.37 Feedback inhibition
Regulation of Protein Function

Many cellular proteins are regulated by

GTP or GDP binding, including the Ras
oncogene proteins.
X-ray crystallography has revealed
subtle conformational differences
between the inactive GDP-bound and
active GTP-bound forms.
Figure 8.38 Conformational differences between active and inactive Ras proteins
Regulation of Protein Function

This small difference in protein

conformation determines whether Ras
can interact with its target molecule,
which signals the cell to divide.
Mutations in ras genes contribute to about
20% of human cancers, by altering
structure of Ras proteins so they are
locked in the active GTP-bound
conformation and continually signal cell
division.
Regulation of Protein Function

Protein phosphorylation is a reversible

covalent modification process that can
activate or inhibit a wide variety of
proteins in response to environmental
signals.
It is catalyzed by protein kinases, which
transfer phosphate groups from ATP to
the hydroxyl groups of side chains of
serine, threonine, or tyrosine.
Figure 8.39 Protein kinases and phosphatases
Regulation of Protein Function

Protein phosphorylation is reversed by

protein phosphatases, which catalyze
hydrolysis of phosphorylated amino
acids.
Regulation of Protein Function

Protein kinases are often components of

signal transduction pathways.
The sequential action of a series of
protein kinases can transmit a signal
received at the cell surface to target
proteins within the cell, resulting in
changes in cell behavior in response to
environmental stimuli.
Regulation of Protein Function

Example of protein kinase action: In

muscle cells epinephrine signals
breakdown of glycogen to glucose-1-
phosphate, providing energy for
increased muscular activity.
This is catalyzed by glycogen
phosphorylase, which is regulated by a
protein kinase.
Figure 8.40 Regulation of glycogen breakdown by protein phosphorylation
Regulation of Protein Function

The signaling pathway is initiated by

allosteric regulation due to binding of
small molecules to targets –
epinephrine to its cell surface receptor,
and cAMP to cAMP-dependent kinase.
The signal is then transmitted to its
intracellular target by the sequential
action of protein kinases.
Regulation of Protein Function

Aberrations in signaling pathways,

especially involving protein-tyrosine
kinases are responsible for some kinds of
cancer.
The first protein-tyrosine kinase was
discovered in 1980 in studies of Rous
sarcoma virus.
Small molecule inhibitors of these enzymes
are promising drugs for cancer treatment.
Key Experiment 8.2 The Discovery of Protein-Tyrosine Kinases: Identification of phosphotyrosine
in immunoglobulin phosphorylated by Src
Regulation of Protein Function

Other covalent modifications include

methylation and acetylation of lysine
and arginine residues, and
nitrosylation—addition of NO groups
to side chains of cysteine.
O-linked glycosylation of proteins may
also play a regulatory role.
Figure 8.41 Nitrosylation
Regulation of Protein Function

Many proteins are regulated by protein-

protein interactions.
Example: cAMP-dependent protein
kinase is composed of two regulatory
and two catalytic subunits; this is the
inactive form.
Regulation of Protein Function

cAMP binds to the regulatory subunits,

which induces conformational change
and dissociation of the complex.
The free catalytic subunits are then
enzymatically active protein kinases.
cAMP acts as an allosteric regulator by
altering protein-protein interactions.
Figure 8.42 Regulation of cAMP-dependent protein kinase
Protein Degradation

Proteins levels in cells are determined

by rates of synthesis and rates of
degradation.
Differential rates of protein degradation
are an important aspect of cell
regulation.
Protein Degradation

Many regulatory proteins have short half

lives; this allows levels to change
quickly in response to external stimuli.
Faulty or damaged proteins are
recognized and rapidly degraded.
Protein Degradation

The major pathway of protein

degradation in eukaryotic cells is the
ubiquitin-proteasome pathway.
Ubiquitin is highly conserved in all
eukaryotes; it is a marker that targets
proteins for rapid proteolysis.
Protein Degradation

Ubiquitin is attached to the amino group

of the side chain of a lysine residue,
then more are added to form a chain.
Polyubiquinated proteins are recognized
and degraded by a large protease
complex, the proteasome.
Figure 8.43 The ubiquitin-proteasome pathway
Protein Degradation

Ubiquitination is a multistep process

involving several enzymes—E1, E2,
and E3.
The specificity of E3 enzymes selectively
targets proteins for degradation.
Protein Degradation

Example: Controlled degradation of

cyclins, proteins that regulate
progression through the division cycle
of eukaryotic cells.
Entry into mitosis is regulated by cyclin B,
a regulatory subunit of protein kinase
Cdk1.
Protein Degradation

Cdk1 also activates a ubiquitin ligase

that targets cyclin B for degradation at
the end of mitosis.
This inactivates Cdk1 and the cell enters
interphase.
Ubiquitination of cyclin B is highly
selective, targeted by a cyclin B
sequence called the destruction box.
Figure 8.44 Cyclin degradation during the cell cycle
Protein Degradation

Ubiquitin can have other functions:

 Addition of one ubiquitin to some
proteins is involved in regulation of
DNA repair, transcription, and
endocytosis.
 SUMO (small ubiquitin-related
modifier) and other ubiquitin-like
proteins serve as markers for protein
localization and regulators of protein
activity.
Protein Degradation

Protein degradation can also take place

in lysosomes—membrane-enclosed
organelles that contain digestive
enzymes, including proteases.
Lysosomes digest extracellular proteins
taken up by endocytosis; and take part
in turnover of organelles and proteins.
Protein Degradation

Containment of digestive enzymes in

lysosomes prevents uncontrolled
degradation of the cell contents.
Movement of proteins into lysosomes is
accomplished by autophagy: vesicles
(autophagosomes) enclose small areas
of cytoplasm or organelles. The
vesicles then fuse with lysosomes.
Figure 8.45 Autophagy
Protein Degradation

Autophagy is activated in nutrient

starvation, allowing cells to degrade
nonessential proteins and organelles
and reutilize the components.
Autophagy also plays a role in many
developmental processes, such as
insect metamorphosis, which involve
extensive tissue remodeling.