1. Molecular mechanisms regulating
protein expression
Aleš Hampl
Proteins
„Proteios“ – the first place (in geek)
• in most cell types, minimum 50% of their dry mass is represented
by proteins
• proteins play a key role in a vast majority of biological processes

2. Key role of proteins stems from their multiple functions
Enzymatic
Proteins - enzymes, which selectively modulate chemical reactions
Structural
Structural (building, supportive) proteins – colagen, elastin, keratin …
Signalling
Proteins mediating transfer of information – hormones, cytokines, receptors
Locomotive
Proteins that are responsible for movement – myosin, actin …
Transport
Proteins that transport various substances – haemoglobin, transferrin, …
Defensive
Proteins that prevent against unwanted substances – immunoglobulins …

3. Molecules of proteins are synthesized from individual
aminoacids by covalently binding their amino-
and carboxy-groups via peptidic bond
R1 R2
H O H O
N C C + N C C
H OH H OH
H H
amino carboxy
aminoacid 1 aminoacid 2
-H2O
„Alfa“ carbon
R1 O R2
H O
Growing
N C C N C C
peptide
chain H OH
H H H
N-terminus C-terminus
peptidic bond

4. Multiple functions of proteins stem from unique
features of individual proteins
DNA PROTEINS
The same
Sequence of Sequence of
principle
nucleotides aminoacids
(4 different nucleotides) (20 different aminoacids)
The same features
+ Different features
One function
Storing and transfer of
X +
Different functions
information
?
Higher structure of Sequence of
DNA is not influenced
by sequence of
nucleotides
X aminoacids
determines the higher
structure of proteins
!

5. Higher organization of molecule of protein is determined
by a sequence of aminoacids and by their side chains (R)
R1 O R2 O R3
H O
N C C N C C N C C
H OH
H H H H H
Primary Secondary Tertiary Qaurternary
structure structure structure structure
It is determined by
Linear sequence of It is determined by It is given by
interactions between
aminoacids in interactions between association of more
the components of
polypeptide chain. side chains (hydrogen then one of polypeptide
polypeptide backbone bonds, disulphidic subunits.
(alfa helix, beta sheat). bridges, ion
interactions, hydrofobic
interactions).
Higher organization of molecule of protein
Denaturation
• loss of a higher organization of molecule of protein produced by a change of physical and/or chemical
conditions of the environment, which is accompanied by a loss of function of protein,
and which can be reversible (e.g. damage to proteins caused by a fever).

6. What does higher organization of
protein do to its function?
Higher organization of protein decides
about its function.
&
Protein function typically depends on its ability
to recognize/bind other molecules.
Molecule of CDK10 interacting with ATP

7. DNA determines expression/metabolism
of proteins essentially by two mechanisms
At the level At the level
of transcripts of proteins
Synthesis of mRNA
Translation YES/NO
of given protein Primary sequence of aminoacids
of given protein
(determines features - stability
+ of given protein)
Stability of mRNA
of given protein

8. Synthesis of polypeptides according to mRNA sequence
realizes by the process called „translation“
Messenger RNA
(mRNA)
Ribosomes Key molecular components
of translational machinery
Transfer RNA
(tRNA)

9. Ribosomes – general features
They create environment for reading of mRNA codons
and for synthesis of polypeptide chain
Composition of ribosomes
Proteins - 1/3
Due to the number of ribosomes in cell,
Ribosomal RNA
rRNA is the most abundant type of RNA
rRNA – 2/3
(synthetized in nucleoli by RNA PolI)
Differences between ribosomes of eukaryotes
and prokaryotes are of medical significance
Eukaryotes Prokaryotes
Ribosomes are
X Ribosomes are
insensitive to sensitive to
certain antibiotics certain antibiotics

10. Ribosomes - structure
Large subunit
A – binding site for Aminoacyl-tRNA
E P A P – binding site for Peptidyl-tRNA
E – tRNA Exit site
Small subunit
Binding site for mRNA

11. Transfer RNA - tRNA
Ensures:
• transport of aminoacids to the place of synthesis of polypeptide chain
• interpretation (reading) of codons of mRNA
Length of tRNA – only about 80 nucleotides
Aminoacid binding
site 3` 5`
Aminoacyl-tRNA synthetase
• catalyses covalent bond between 3`
aminoacid and relevant tRNA
• requires ATP
• produces aminoacyl tRNA
(= „activated aminoacid“)
Hydrogen
bonds
5`
Anticodon

12. Translation Beginning of translation
Met-tRNA
3`UAC 5`
Growing polypeptide mRNA 5` AUG 3`
chain Aminoacyl tRNA START kodon
End of translation
UAG
3` mRNA 5`
UAA
3`
free tRNA mRNA 5` 3`
mRNA 5` UGA 3`
STOP kodony
E P A bind „release factor“
POLYRIBOSOME
(cluster of ribosomes translating
codons certain segment of mRNA)
ribosomes
reading of mRNA mRNA
5` =
movement of ribosomes on mRNA
100 nm

13. Regulation of translation
Occurs mostly at the level of initiation of translation
Blocking of mRNA by regulatory proteins
• binding of proteins to structures/sequences
located at 5`untranslated region of mRNA,
usually prevents binding of ribosomes
Shortening of poly-A tail of mRNA
• at 3`end of mRNA
• mechanism that is typical for storage
of dormant mRNA in developing/developed egg
Inactivation of factors (proteins) that
are required for initiation of translation
• global inhibition of translation
• also typical for developing/developed egg

14. Regulation of protein function takes
place also after their synthesis
Posttranslational modification of protein
• proteolytic digest of pro-protein (inactive
form) that produces active protein
(e.g. conversion of pro-insulin to insulin)
• addition of modifying chemical groups
(phosphorylation, glycosylation, acetylation,
methylation - and reversed processes)
Transport of protein to the site of its
function
• transport from cytoplasm to nucleus
(e.g. transcription factors)
• transport from cytoplasm to cell surface
(e.g. receptors)
Regulation of protein halflife
• halflife of proteins widely varies (from
seconds/minutes to days)

15. Regulation of protein halflife
Halflife of proteins decides about their functioning in cell
Degradation of proteins must be accomplished
by the mechanism that allows for precise regulation
Which one
?
Degradation of proteins
Hydrolytic cleavage of
by „ubiquitin-proteasome“
proteins in lysosomes
pathway

16. Nobel price for chemistry 2004
„for the discovery of ubiquitin-based mechanims
of degradation of proteins“
Aaron Ciechanover Avram Hershko Irwin Rose
*1947 *1937 *1926
Israel Israel USA
Technion - Israel Institute Technion - Israel Institute University of California
of Technology, Haifa of Technology, Haifa Irvine, CA, USA

17. „ubiquitin-proteasome“ pathway of protein degradation
KEY FACTS
At least 80% of • regulation of the level/function
types of proteins in of many proteins (e.g. cyclins,
cells is degraded by transcription factors, signalling proteins,…)
this pathway • elimination of denatured,
It is responsible for: abnormally synthesized,
abnormally posttranslationally
modified, and/or somehow else
Takes place both
damaged proteins
in cytoplasm and (in eukaryotes about 30% of newly synthesized
in nucleus proteins is degraded in several minutes after their
synthesis)
Its key players are:
Ubiquitin – evolutionary conserved protein, 76 aminoacids
Proteasome – proteolytic complex, function of which is dependent on
ATP, and which consists of three subunits:
• one central 20S proteasom (responsible for degradation of proteins)
• two 19S complexes (play regulatory role, substrate specificity)

18. Degradation of proteins
by „ubiquitin-proteasome“ pathway
ubiquitin-conjugating
enzyme
Target protein Target protein
ubiquitin
ligase
26S
Target protein
Proteasome
(~60 subunits)
ubiquitin-activating
enzyme
Step 1 Step 2 Step 3
Ubiquitin Recycled
(8,5 kDa)
Peptides
ubiquitine
Modified from Wang & Maldonado, Cellular & Molecular Immunology, 2006

19. Molecular machineries that are responsible for
translation and protein degradation as a cause
and/or participant in human diseases
YES or NO ???
Y E S

20. Abnormal function of ribosomes?
Diamond Blackfan anemia
• serious hypoplastic anemia
• develops in the first year of life Clinical
• accompanied by serious developmental abnormalities heterogeneity
• ¼ of pacients carries mutation in gene coding for Rsp19 and tissue
(component of 40S subunit of ribosome) nonspecific
• the only disease with the direct link to the mutation in the gene coding effects.
for ribosomal protein
Is it typical
for diseases
given by the
abnormal function
Other diseases that are linked to the factors of ribosomes?
involved in ribosome synthesis:
• Congenital X-linked diskeratosis
• Treacher Collins syndrome
• Shwachman Diamond syndrome
The question
to be answered

21. Abnormal translation as
a cause of cancer?
Supportive facts: Possible mechanisms:
Sensitivity to cancer
is linked to genes, which
control proteosynthesis
(e.g. TCS1/2, PTEN) and/or biogenesis
of ribosomes (e.g. DKC1, S19)
Experiments using transgenic
animals show that deregulated
expression of regulators of
translation has oncogenic
effects (e.g. mice with mutated gene
Dkc-1 tend to develop various tumors)
Some highly effective
anticancer drugs target
key regulators
of proteosynthesis
(e.g. Rapamycin targets mTOR kinase)

22. Abnormalities in degradation of proteins as a cause
of neurodegenerative diseases?
Proteinopathies
Neurodegenerative diseases accuring in late age,
which are typical by accumulation of aggregates of toxic proteins
Examples of diseases: Some abnormalities:
Cytosolic accumulation Levels and activities of 20/26S
• Parkinson`s disease proteasomes are lowered
• Late age Huntington disease in relevant loci of brain
in pacients with sporadic
Nuclear accumulation Parkinson`s disease
• Spinocerebelar ataxia type 1
Autosomal recessive loss of function
Extracellular accumulation mutation in gene coding for
• Alzheimer disease (beta amyloid) E3 ligase (parkin) causes
Parkinson`s disease.

23. Thank you for your attention
Questions and comments at:
ahampl@med.muni.cz