Transcript The Cell, 7e

9
Protein Synthesis,
Processing, and Regulation
9 Protein Synthesis, Processing, and Regulation
• Translation of mRNA
• Protein Folding and Processing
• Regulation of Protein Function
• Protein Degradation
Introduction
Translation is the synthesis of proteins
as directed by mRNA templates, the
first step in the formation of functional
proteins.
Polypeptide chains must fold into
appropriate conformations and often
undergo various processing steps,
sorting, and transport.
Introduction
Gene expression is regulated at the level
of translation in both prokaryotic and
eukaryotic cells.
There are also multiple controls on
amount and activities of proteins, which
ultimately regulate all aspects of cell
behavior.
Translation of mRNA
Proteins are synthesized from mRNA
templates by a process that has been
highly conserved throughout evolution.
All mRNAs are read in the 5′ to 3′
direction, and polypeptide chains are
synthesized from the amino to the
carboxy terminus.
Translation of mRNA
Each amino acid is specified by three
bases (a codon) in the mRNA.
Translation is carried out on ribosomes,
with tRNAs serving as adaptors.
Protein synthesis involves interactions
between the three types of RNA
(mRNA, tRNA, rRNA), plus other
proteins.
Translation of mRNA
tRNAs align amino acids with
corresponding codons on the mRNA
template.
They are 70–80 nucleotides long and
have characteristic cloverleaf structures
resulting from base pairing between
different regions.
Figure 9.1 Structure of tRNAs (Part 1)
Figure 9.1 Structure of tRNAs (Part 2)
Figure 9.1 Structure of tRNAs (Part 3)
Translation of mRNA
All tRNAs fold into compact L shapes, to
fit onto ribosomes during translation.
They have the sequence CCA at the 3′
end, and amino acids are covalently
attached to the ribose of the terminal
adenosine.
The anticodon loop binds to the
appropriate codon by complementary
base pairing.
Translation of mRNA
Attachment of amino acids to specific
tRNAs is mediated by enzymes called
aminoacyl tRNA synthetases.
Each of these 20 enzymes recognizes a
single amino acid, as well as the
correct tRNA to which it should attach.
Translation of mRNA
Attachment occurs in two steps:
1. The amino acid is joined to AMP,
forming aminoacyl AMP.
2. The amino acid is transferred to the 3′
CCA end of the tRNA and AMP is
released.
Figure 9.2 Attachment of amino acids to tRNAs
Translation of mRNA
The amino acid is then aligned on the
mRNA template by complementary
base pairing.
Most amino acids are specified by more
than one codon.
Cells have about 40 different tRNAs for
the 20 different amino acids.
Translation of mRNA
Some tRNAs can recognize more than
one mRNA codon, as a result of
nonstandard base pairing (wobble) at
the third codon position.
This allows G to pair with U, and inosine
(I) to pair with U, C, or A.
(Guanosine is modified to inosine in the
anticodons of some tRNAs.)
Figure 9.3 Nonstandard codon–anticodon base pairing (Part 1)
Figure 9.3 Nonstandard codon–anticodon base pairing (Part 2)
Translation of mRNA
Ribosomes are named according to
their sedimentation rates in ultracentrifugation: 70S for bacterial and
80S for eukaryotic.
Cells have many ribosomes, illustrating
the importance of protein synthesis.
E. coli has about 20,000; growing
mammalian cells can have 10 million.
Translation of mRNA
All ribosomes have two subunits.
Each subunit contains rRNA and
characteristic proteins.
The subunits of eukaryotic ribosomes
are larger and have more proteins than
prokaryotic ribosomes.
Figure 9.4 Ribosome structure (Part 1)
Figure 9.4 Ribosome structure (Part 2)
Translation of mRNA
Ribosomes can be formed in vitro by
self-assembly from purified ribosomal
proteins and rRNAs.
This provides an important experimental
tool, allowing analysis of the roles of
individual proteins and rRNAs.
Translation of mRNA
rRNAs form characteristic secondary
structures by complementary base
pairing.
Subsequent folding results in distinct 3-D
structures.
Figure 9.5 Structure of 16S rRNA
Translation of mRNA
It was first thought that rRNAs played
only a structural role in ribosomes.
It was later shown that rRNA has
catalytic activity.
Noller and colleagues in 1992 showed
that the large ribosomal subunit can
catalyze formation of peptide bonds
even after 90% of ribosomal proteins
have been removed.
Translation of mRNA
In 2000, unambiguous evidence for
rRNA catalysis came from highresolution structural analysis of the 50S
ribosomal subunit.
Ribosomal proteins are absent from the
site of the peptidyl transferase reaction,
showing that rRNA is responsible for
catalyzing peptide bond formation.
Figure 9.6 Structure of the 50S ribosomal subunit
Translation of mRNA
It is now thought that ribosomal proteins
play a largely structural role, and the
large ribosomal subunit functions as a
ribozyme.
This has evolutionary implications: RNAs
are thought to have been the first selfreplicating macromolecules.
Translation of mRNA
The role of rRNA in the formation of
peptide bonds extends the catalytic
activities of RNA beyond selfreplication to direct involvement in
protein synthesis.
This may provide an important link for
understanding the early evolution of
cells.
Translation of mRNA
mRNAs have noncoding untranslated
regions (UTRs) at the ends.
Most eukaryote mRNAs are monocistronic, encoding a single protein.
Prokaryotic mRNAs are often polycistronic, encoding multiple proteins,
each of which is translated from an
independent start site.
Figure 9.7 Prokaryotic and eukaryotic mRNAs
Translation of mRNA
In both prokaryotes and eukaryotes,
translation always starts with
methionine, usually encoded by AUG.
The signals that identify initiation codons
are different in prokaryotic and
eukaryotic cells.
Translation of mRNA
Initiation codons in bacterial mRNAs are
preceded by a Shine-Dalgarno
sequence, that aligns the mRNA on
the ribosome.
They can initiate translation at the 5′ end
of an mRNA and at internal initiation
sites of polycistronic mRNAs.
Translation of mRNA
Eukaryotic mRNAs are recognized by
the 7-methylguanosine cap at the 5′
end.
The ribosomes then scan downstream of
this cap until they encounter the
initiation codon.
Figure 9.8 Signals for translation initiation
Translation of mRNA
Translation occurs in three stages:
initiation, elongation, and termination.
A specific initiator, methionyl tRNA, and
the mRNA bind to the small ribosomal
subunit.
The large ribosomal unit then joins,
forming a functional ribosome.
Figure 9.9 Overview of translation
Translation of mRNA
Many nonribosomal proteins are also
required for various stages of
translation.
Table 9.1 Translation Factors
Translation of mRNA
In bacteria, initiation starts with a 30S
ribosomal subunit bound to initiation
factors IF1 and IF3.
Then the mRNA, initiator N-formylmethionyl
(fMet) tRNA, and IF2 (bound to GTP) join
the complex.
IF1 and IF3 are released, a 50S subunit
binds to the complex, and IF2 is released.
Figure 9.10 Initiation of translation in bacteria
Translation of mRNA
In eukaryotes initiation is more complex,
and requires at least 12 proteins,
designated eIFs (eukaryotic initiation
factors).
Figure 9.11 Initiation of translation in eukaryotic cells (Part 1)
Figure 9.11 Initiation of translation in eukaryotic cells (Part 2)
Translation of mRNA
Some viral and cellular eukaryotic
mRNAs have internal ribosome entry
sites (IRESs) at which translation can
initiate independently of the 5′ cap.
For viral mRNAs, IRES sequences bind
directly to eIF4G complexed to eIF4A,
or to 40S ribosomal subunits.
Figure 9.12 Initiation of translation at internal ribosome entry sites
Translation of mRNA
The mechanism of elongation in
prokaryotic and eukaryotic cells is
similar.
Ribosomes have three binding sites: P
(peptidyl), A (aminoacyl), and E (exit)
sites.
The initiator methionyl tRNA binds to the
P site.
Translation of mRNA
The next aminoacyl tRNA binds to the A
site by pairing with the second codon of
the mRNA.
An elongation factor (EF-Tu in
prokaryotes, eEF1α in eukaryotes)
complexed to GTP brings the aminoacyl
tRNA to the ribosome.
Figure 9.13 Elongation stage of translation (Part 1)
Figure 9.13 Elongation stage of translation (Part 2)
Figure 9.13 Elongation stage of translation (Part 3)
Translation of mRNA
Selection of the correct aminoacyl tRNA
determines the accuracy of protein
synthesis.
Base pairing alone can’t account for the
accuracy of protein synthesis.
A “decoding center” in the small
ribosomal subunit recognizes correct
codon-anticodon base pairs and
discriminates against mismatches.
Translation of mRNA
Insertion of the correct aminoacyl tRNA at
A triggers a conformational change that
induces hydrolysis of GTP/eEF1α and
release of the elongation factor.
The peptide bond is then formed,
catalyzed by the large ribosomal
subunit.
The initiator tRNA (uncharged) is now at
the P site.
Translation of mRNA
Translocation: the ribosome then moves
three nucleotides along the mRNA,
positioning the next codon in the A site.
This step translocates the peptidyl tRNA
from A to P, and the uncharged tRNA
from P to E.
Translation of mRNA
A new aminoacyl tRNA binds to the A
site and induces release of the
uncharged tRNA from the E site.
Translocation requires another
elongation factor (EF-G in prokaryotes,
eEF2 in eukaryotes) and is coupled to
GTP hydrolysis.
Translation of mRNA
As elongation continues, the eEF1α (or
EF-Tu) released from the ribosome
bound to GDP must be reconverted to
its GTP form.
This requires another elongation factor,
eEF1βγ (EF-Ts in prokaryotes).
Regulation of eEF1α by GTP binding
and hydrolysis is a common method of
protein regulation.
Figure 9.14 Regeneration of eEF1a/GTP
Translation of mRNA
Elongation continues until a stop codon
(UAA, UAG, or UGA) is translocated into
the A site.
Release factors recognize these signals
and terminate protein synthesis.
In prokaryotic cells RF1 recognizes UAA
or UAG, RF2 recognizes UAA or UGA.
In eukaryotic cells eRF1 recognizes all
three stop codons.
Figure 9.15 Termination of translation (Part 1)
Figure 9.15 Termination of translation (Part 2)
Figure 9.15 Termination of translation (Part 3)
Translation of mRNA
mRNAs can be translated
simultaneously by several ribosomes.
Once a ribosome has moved away from
the initiation site, another can bind to
the mRNA and begin synthesis.
A group of ribosomes bound to an
mRNA molecule is called a
polyribosome, or polysome.
Figure 9.16 Polysomes (Part 1)
Figure 9.16 Polysomes (Part 2)
Translation of mRNA
Regulation of translation plays a key role
in gene expression.
Regulation includes translational
repressor proteins and noncoding
microRNAs.
Global translational activity is modulated
in response to stress, nutrient
availability, and growth factor
stimulation.
Translation of mRNA
Regulation of ferritin translation (a
protein that stores iron) by repressor
proteins:
When iron is absent, iron regulatory
protein (IRP) binds to a the iron
response element (IRE) in the 5′ UTR,
blocking translation.
Figure 9.17 Translational regulation of ferritin
Translation of mRNA
Some translational repressors bind to
specific sequences in the 3′ UTR.
Some bind to initiation factor eIF4E,
interfering with its interaction with
eIF4G and inhibiting initiation of
translation.
Figure 9.18 Translational repressor binding to 3' untranslated sequences
Translation of mRNA
Proteins that bind to 3′ UTRs are also
responsible for localizing mRNAs to
specific regions of cells.
Localization to specific regions of eggs
or embryos is important in
development, allowing proteins to be
synthesized at appropriate sites.
Figure 9.19 Localization of mRNA in Xenopus oocytes
Translation of mRNA
Translational regulation is very important
during early development.
Many mRNAs with short poly-A tails are
stored in oocytes; translation is
activated at fertilization or later stages.
Lengthening the poly-A tails allows
binding of poly-A binding protein
(PABP), which stimulates translation.
Translation of mRNA
RNA interference (RNAi), mediated by
short double-stranded RNAs, is used
as an experimental tool to block gene
expression at the level of translation.
In cells, it is an important mechanism of
translational regulation.
Translation of mRNA
RNA interference is mediated by:
Small interfering RNAs (siRNAs)—
produced from double-stranded RNAs
by the nuclease Dicer.
MicroRNAs (miRNAs)—transcribed by
RNA polymerase II, then cleaved by
nucleases Drosha and Dicer.
Translation of mRNA
One strand of miRNA or siRNA is
incorporated into an RNA-induced
silencing complex (RISC).
siRNAs generally pair with their targets
and induce cleavage of the mRNA.
Figure 4.38 RNA interference
Figure 6.8 miRNAs (Part 1)
Figure 6.8 miRNAs (Part 2)
Translation of mRNA
Most miRNAs form mismatches in the 3′
UTRs that repress translation.
The miRNA/RISC complex represses
translation and targets the mRNA for
degradation by stimulating
deadenylation.
Figure 9.20 Regulation of translation by miRNAs
Translation of mRNA
As many as 1000 miRNAs are encoded
in mammals; each can target up to 100
different mRNAs.
Up to one-half of protein-coding genes
may be regulated by miRNAs.
They are important in embryonic
development, and may play a role to
cancer and other diseases.
Translation of mRNA
Translation can also be regulated by
modification of initiation factors.
This results in global effects on overall
translational activity rather than
translation of specific mRNAs.
Translation of mRNA
Phosphorylation of eIF2 and eIF2B by
regulatory protein kinases blocks the
exchange of bound GDP for GTP,
inhibiting initiation of translation.
Figure 9.21 Regulation of translation by phosphorylation of eIF2 and eIF2B (Part 1)
Figure 9.21 Regulation of translation by phosphorylation of eIF2 and eIF2B (Part 2)
Translation of mRNA
Regulation of eIF4E:
Growth factors activate protein kinases
that phosphorylate regulatory proteins
(eIF4E binding proteins, or 4E-BPs).
In the absence of growth factors, the
nonphosphorylated 4E-BPs bind to
eIF4E and inhibit translation.
Figure 9.22 Regulation of eIF4E
Protein Folding and Processing
Polypeptide chains must undergo folding
and other modifications to become
functional proteins.
3-D protein conformation results from
interactions between the side chains of
amino acids.
All information for the correct
conformation is provided by the amino
acid sequence.
Protein Folding and Processing
Chaperones are proteins that facilitate
folding of other proteins.
They act as catalysts that assist the selfassembly process without becoming
part of the folded protein.
They bind to and stabilize unfolded or
partially folded polypeptides.
Protein Folding and Processing
Chaperones bind to polypeptide chains
that are still being translated on
ribosomes.
The chain must be protected from
aberrant folding or aggregation with
other proteins until synthesis of an
entire domain is complete.
Figure 9.23 Action of chaperones during translation
Protein Folding and Processing
Chaperones also stabilize unfolded
polypeptide chains during transport into
organelles.
Example: Partially unfolded proteins
stabilized by chaperones are
transported across the mitochondrial
membrane.
Chaperones in the mitochondrion then
facilitate folding.
Figure 9.24 Action of chaperones during protein transport
Protein Folding and Processing
Many chaperones were initially identified
as heat-shock proteins (Hsp),
expressed in cells subjected to high
temperatures.
Hsp stabilize and facilitate refolding of
proteins that have been partially
denatured.
Protein Folding and Processing
Hsp70 chaperones and chaperonins are
found in both prokaryotic and
eukaryotic cells.
Hsp70 proteins stabilize polypeptide
chains during translation and transport
by binding to short hydrophobic
segments.
Protein Folding and Processing
The polypeptide is then transferred to a
chaperonin, where folding takes place.
Chaperonins consist of subunits
arranged in two stacked rings to form a
double-chambered structure.
This isolates the protein from the
cytosol and other unfolded proteins.
Figure 9.25 Sequential actions of chaperones
Protein Folding and Processing
Defects in protein folding are responsible
for protein misfolding diseases.
Cystic fibrosis is caused by a mutation
that results in one amino acid deletion
that leads to improper folding of protein
CFTR.
CFTR transports Cl‒ ions across
epithelial cell membranes.
Protein Folding and Processing
Alzheimer’s disease, Parkinson’s
disease, and type 2 diabetes are
associated with aggregation of
misfolded proteins.
The misfolded proteins form fibrous
aggregates called amyloids,
characterized by β-sheet structures.
Figure 9.26 Protein aggregation and amyloid formation (Part 1)
Figure 9.26 Protein aggregation and amyloid formation (Part 2)
Table 9.2 Representative Diseases Associated with Protein Aggregation
Protein Folding and Processing
Alzheimer’s disease is characterized by
two aggregate types in brain tissue:
• Neurofibrillary tangles (misfolded tau
proteins)
• Amyloid plaques (aggregates of
misfolded amyloid-β protein [Aβ])
Molecular Medicine, Ch. 9, p. 342
Protein Folding and Processing
Prions are misfolded proteins that can
self-replicate.
Diseases caused by prions include
scrapie in sheep, mad cow disease,
Creutzfeldt-Jakob disease, and kuru.
Protein Folding and Processing
Infection by prions is based on amyloid
formation of the protein PrP.
In mammalian cells the normal α-helical
form is PrPC.
In the infectious form, PrP forms a
misfolded amyloid structure, PrPSc.
Protein Folding and Processing
PrPSc can propagate by inducing
misfolding of PrPC proteins to the
amyloid state.
PrPSc can “replicate” by inducing
autocatalytic amyloid formation of
endogenous PrPC—a novel form of
propagation that does not require any
nucleic acid.
Figure 9.27 Prion propagation
Protein Folding and Processing
Two enzymes act as chaperones by
catalyzing protein folding:
• Protein disulfide isomerase (PDI)
catalyzes disulfide bond formation.
PDI is abundant in the ER, where an
oxidizing environment allows (S—S)
linkages.
Figure 9.28 The action of protein disulfide isomerase
Protein Folding and Processing
• Peptidyl prolyl isomerase
catalyzes isomerization of peptide
bonds that involve proline residues.
Isomerization between the cis and
trans configurations of prolyl-peptide
bonds could otherwise be a ratelimiting step in protein folding.
Figure 9.29 The action of peptidyl prolyl isomerase
Protein Folding and Processing
Proteolysis: cleavage of a 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.
Protein Folding and Processing
The signal sequence is inserted into a
membrane channel as it emerges from
the ribosome and the polypeptide chain
passes through as translation
proceeds.
The signal sequence is then cleaved by
a membrane protease (signal
peptidase).
Figure 9.30 The role of signal sequences in membrane translocation
Protein Folding and Processing
Proteolytic processing includes
formation of active enzymes or
hormones by cleavage of larger
precursors.
Example: Insulin is synthesized as a
precursor polypeptide that goes
through two cleavages to produce the
mature insulin.
Figure 9.31 Proteolytic processing of insulin
Protein Folding and Processing
In replication of HIV, a virus-encoded
protease cleaves precursor
polypeptides to form the viral structural
proteins.
The HIV protease is an important target
in drug development for treating AIDS
(in addition to reverse transcriptase) .
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 nitrogen atom in the
side chain of asparagine.
O-linked glycoproteins: the carbohydrate
is attached to the oxygen atom in the
side chain of serine or threonine.
Figure 9.32 Linkage of carbohydrate side chains to glycoproteins
Protein Folding and Processing
Glycosylation starts in the ER before
translation is complete.
An oligosaccharide is assembled on a
lipid carrier (dolichol phosphate) in
the ER membrane, then transferred to
an asparagine residue.
Further modifications result in many
different N-linked oligosaccharides.
Figure 9.33 Synthesis of N-linked glycoproteins
Protein Folding and Processing
O-linked oligosaccharides are added
within the Golgi apparatus.
They are formed by addition of one
sugar at a time.
Many cytoplasmic and nuclear proteins,
including transcription factors, are also
modified by addition of one O-linked Nacetylglucosamine residue.
Figure 9.34 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.
Protein Folding and Processing
Four types of lipid additions:
1. N-myristoylation: myristic acid (a
fatty acid) is attached to an N-terminal
glycine.
These proteins are associated with the
inner face of the plasma membrane.
Figure 9.35 Addition of a fatty acid by N-myristoylation
Protein Folding and Processing
2. Prenylation: prenyl groups are
attached to sulfur in the side chains of
cysteine near the C terminus.
Many of these proteins are involved in
control of cell growth and
differentiation, including the Ras
oncogene proteins, responsible for
many human cancers.
Figure 9.36 Prenylation of a C-terminal cysteine residue
Protein Folding and Processing
3. Palmitoylation: palmitic acid (a fatty
acid) is added to sulfur in the side
chains of internal cysteine residues.
This is also important in association of
some proteins with the cytosolic face of
the plasma membrane.
Figure 9.37 Palmitoylation
Protein Folding and Processing
4. Glycolipids (lipids linked to
oligosaccharides) are added to Cterminal carboxyl groups.
They anchor the proteins to the external
plasma membrane.
The glycolipids have phosphatidylinositol:
glycosylphosphatidylinositol (GPI)
anchors.
Figure 9.38 Structure of a GPI anchor
Regulation of Protein Function
Cells can regulate the amounts and the
activities of their proteins.
Three mechanisms:
• Regulation by small molecules
• Phosphorylation and other
modifications
• Protein-protein interactions
Regulation of Protein Function
Regulation by small molecules
Most enzymes are controlled by
changes in conformation, often as a
result of binding small molecules.
This type of regulation is common in
controlling metabolic pathways by
feedback inhibition.
Regulation of Protein Function
Feedback inhibition is an example of
allosteric regulation:
A regulatory molecule binds to an
enzyme site that is distinct from the
catalytic site.
Figure 9.39 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 9.40 Conformational differences between active and inactive Ras proteins
Regulation of Protein Function
The 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 25%
of human cancers. Ras proteins are
altered to be locked in the active GTPbound conformation and continually
signal cell division.
Regulation of Protein Function
Phosphorylation and other modifications
Phosphorylation is reversible; it can
activate or inhibit proteins in response
to environmental signals.
Protein kinases transfer phosphate
groups from ATP to the hydroxyl
groups of side chains of serine,
threonine, or tyrosine.
Regulation of Protein Function
Phosphorylation is reversed by protein
phosphatases, which catalyze
hydrolysis of phosphorylated amino
acids.
Figure 9.41 Protein kinases and phosphatases
Regulation of Protein Function
Protein kinases are often components of
signal transduction pathways.
Sequential action of a series of protein
kinases can transmit a signal from the
cell surface to target proteins in the
cell, resulting in changes in cell
behavior in response to environmental
stimuli.
Regulation of Protein Function
Example: In muscle cells, epinephrine
signals the 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 9.42 Regulation of glycogen breakdown by protein phosphorylation
Regulation of Protein Function
The signaling pathway is initiated by
allosteric regulation—epinephrine binds
to a cell surface receptor, and cAMP
binds to cAMP-dependent kinase.
The signal is then transmitted to its
target by the sequential action of
protein kinases.
Regulation of Protein Function
Aberrations in signaling pathways,
especially in protein-tyrosine kinases, are
responsible for some cancers.
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, Ch. 9, p. 353 (Part 3)
Regulation of Protein Function
Other covalent modifications include:
• Acetylation of lysine
• Methylation of lysine and arginine
• Nitrosylation (addition of NO groups)
to cysteine
• Glycosylation of serine and threonine
Figure 9.43 Modification of proteins by small molecules (Part 1)
Figure 9.43 Modification of proteins by small molecules (Part 2)
Figure 9.43 Modification of proteins by small molecules (Part 3)
Figure 9.43 Modification of proteins by small molecules (Part 4)
Regulation of Protein Function
Some proteins are regulated by covalent
attachment of polypeptides.
Addition of ubiquitin and other ubiquitinlike proteins, such as SUMO, affect a
variety of functions.
Addition of ubiquitin (ubiquitylation) is a
multistep process.
Figure 9.44 Modification of proteins by ubiquitin
Regulation of Protein Function
Histone modification by ubiquitin and
SUMO is one mechanism for regulating
transcriptional activity of chromatin.
Ubiquitylation is also important in
regulation of protein kinases, proteins
involved in DNA repair, and in the
control of endocytosis and vesicle
trafficking.
Regulation of Protein Function
Many of the proteins modified by SUMO
are transcription factors and other
nuclear proteins, whose localization is
affected by sumoylation.
Protein Degradation
A protein modified by SUMO is Ran
GTPase-activating protein (Ran GAP).
Ran GAP is associated with nuclear pore
complexes and is required for import of
proteins.
Addition of SUMO to Ran GAP is thus
necessary for all protein traffic between
the cytoplasm and nucleus.
Regulation of Protein Function
Protein-protein interactions
Many proteins consist of multiple
subunits; interactions between them
can regulate protein activity.
Example: cAMP-dependent protein
kinase has two regulatory and two
catalytic subunits in 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 9.45 Regulation of cAMP-dependent protein kinase
Protein Degradation
Protein levels in cells are determined by
rates of synthesis and rates of
degradation.
Half-lives of proteins vary greatly;
differential rates of degradation are
important in 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 eukaryotes is the
ubiquitin-proteasome pathway.
Ubiquitin is highly conserved in all
eukaryotes.
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 9.46 The ubiquitin-proteasome pathway
Protein Degradation
Many proteins that control fundamental
cellular processes are targets for
regulated ubiquitylation and proteolysis.
Example: Cyclins that regulate
progression through the division cycle
of eukaryotic cells.
Protein Degradation
Entry of cells into mitosis is controlled in
part by cyclin B, a regulatory subunit of
Cdk1 protein kinase.
The active cyclin B–Cdk1 complex
induces entry into mitosis.
Degradation of cyclin B by the
proteasome then leads to inactivation
of the Cdk1 kinase, allowing the cell to
exit mitosis and return to interphase.
Figure 9.47 Cyclin degradation during the cell cycle
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 cell contents.
Proteins move into lysosomes by
autophagy: vesicles (autophagosomes)
enclose small areas of cytoplasm or
organelles and then fuse with
lysosomes.
Figure 9.48 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.