l08 protein metabolism
TRANSCRIPT
Molecular mechanisms regulatingprotein expression
Aleš Hampl
• in most cell types, minimum 50% of their dry mass is representedby proteins
• proteins play a key role in a vast majority of biological processes
Proteins„Proteios“ – the first place (in geek)
Enzymatic Proteins - enzymes, which selectively modulate chemical reactions
Structural Structural (building, supportive) proteins – colagen, elastin, keratin …
SignallingProteins mediating transfer of information – hormones, cytokines, receptors
Key role of proteins stems from their multiple functions
Locomotive Proteins that are responsible for movement – myosin, actin …
Defensive Proteins that prevent against unwanted substances – immunoglobulins …
Transport Proteins that transport various substances – haemoglobin, transferrin, …
Molecules of proteins are synthesized from individual aminoacids by covalently binding their amino-
and carboxy-groups via peptidic bond
C
H
R1
CN
H
H OH
O
C
H
R2
CN
H
H OH
O
amino carboxy
+
aminoacid 1 aminoacid 2
-H2O
C
H
R1
CN
H
H
O
C
H
R2
CN
HOH
O
peptidic bond
N-terminus C-terminus
Growingpeptide chain
„Alfa“ carbon
Multiple functions of proteins stem from unique features of individual proteins
DNA
Sequence ofnucleotides
(4 different nucleotides)
PROTEINS
Sequence ofaminoacids
(20 different aminoacids)
X
The same features+
One functionStoring and transfer of
information
Different features+
Different functions
The same principle
X
Higher structure ofDNA is not influenced
by sequence ofnucleotides
Sequence ofaminoacids
determines the higherstructure of proteins
?
!
Higher organization of molecule of protein is determinedby a sequence of aminoacids and by their side chains (R)
C
H
R1
CN
H
H
O
C
H
R2
N
H
C
H
R3
CN
HOH
O
C
O
Primarystructure
Linear sequence ofaminoacids in
polypeptide chain.
Secondary structure
It is determined by interactions betweenthe components of
polypeptide backbone(alfa helix, beta sheat).
Tertiary structure
It is determined by interactions betweenside chains (hydrogen
bonds, disulphidicbridges, ion
interactions, hydrofobicinteractions).
Qaurternary structureIt is given by
association of more then one of polypeptide
subunits.
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).
What does higher organization ofprotein do to its function?
Higher organization of protein decides about its function.
Protein function typically depends on its abilityto recognize/bind other molecules.
&
Molecule of CDK10 interacting with ATP
DNA determines expression/metabolism of proteins essentially by two mechanisms
Synthesis of mRNA of given protein
Stability of mRNA of given protein
Primary sequence of aminoacids of given protein
(determines features - stability of given protein)
At the levelof transcripts
At the level of proteins
+
Tran
slat
ion
YES/
NO
Synthesis of polypeptides according to mRNA sequence realizes by the process called „translation“
Key molecular components of translational machinery
Messenger RNA (mRNA)
Ribosomes
Transfer RNA(tRNA)
Ribosomes – general features
They create environment for reading of mRNA codons and for synthesis of polypeptide chain
Proteins - 1/3
Ribosomal RNA rRNA – 2/3
Composition of ribosomes
Due to the number of ribosomes in cell,rRNA is the most abundant type of RNA(synthetized in nucleoli by RNA PolI)
Eukaryotes
Ribosomes are insensitive to
certain antibiotics
Prokaryotes
Ribosomes are sensitive to
certain antibiotics
Differences between ribosomes of eukaryotes and prokaryotes are of medical significance
X
Ribosomes - structure
Large subunit
Small subunit
E P AA – binding site for Aminoacyl-tRNAP – binding site for Peptidyl-tRNAE – tRNA Exit site
Binding site for mRNA
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 bindingsite
Hydrogenbonds
Anticodon
Aminoacyl-tRNA synthetase
• catalyses covalent bond betweenaminoacid and relevant tRNA
• requires ATP• produces aminoacyl tRNA
(= „activated aminoacid“)
3` 5`
5`
3`
Translation
ribosomes
mRNA
100 nm
POLYRIBOSOME(cluster of ribosomes translating
certain segment of mRNA)
mRNA 5` AUG 3`START kodon
3`UAC 5`
Met-tRNA
Beginning of translation
mRNA 5` UAG 3`
End of translation
mRNA 5` UAA 3`mRNA 5` UGA 3`
STOP kodony
bind „release factor“E P A
5`
3`
reading of mRNA=
movement of ribosomes on mRNA
codons
Aminoacyl tRNA
free tRNA
Growing polypeptidechain
Regulation of translation
Occurs mostly at the level of initiation of translation
Blocking of mRNA by regulatory proteins• binding of proteins to structures/sequenceslocated 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 storageof dormant mRNA in developing/developed egg
Inactivation of factors (proteins) thatare required for initiation of translation• global inhibition of translation• also typical for developing/developed egg
Regulation of protein function takesplace also after their synthesis
Posttranslational modification of protein• proteolytic digest of pro-protein (inactiveform) 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 itsfunction• 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 (fromseconds/minutes to days)
Regulation of protein halflife
Halflife of proteins decides about their functioning in cell
Degradation of proteins must be accomplishedby the mechanism that allows for precise regulation
Which one?
Hydrolytic cleavage ofproteins in lysosomes
Degradation of proteinsby „ubiquitin-proteasome“
pathway
Nobel price for chemistry 2004„for the discovery of ubiquitin-based mechanims
of degradation of proteins“
Aaron Ciechanover*1947
Israel
Technion - Israel Institute of Technology, Haifa
Avram Hershko*1937
Israel
Technion - Israel Institute of Technology, Haifa
Irwin Rose*1926
USA
University of California Irvine, CA, USA
„ubiquitin-proteasome“ pathway of protein degradation
KEY FACTS
At least 80% oftypes of proteins in cells is degraded by
this pathway
• regulation of the level/functionof many proteins (e.g. cyclins,
transcription factors, signalling proteins,…)
• elimination of denatured, abnormally synthesized, abnormally posttranslationallymodified, and/or somehow elsedamaged proteins(in eukaryotes about 30% of newly synthesized
proteins is degraded in several minutes after theirsynthesis)
It is responsible for:
Takes place bothin cytoplasm and
in nucleus
Its key players are:Ubiquitin – evolutionary conserved protein, 76 aminoacidsProteasome – 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)
Degradation of proteinsby „ubiquitin-proteasome“ pathway
Target protein
Target protein Target protein
Step 1 Step 2 Step 3
26SProteasome
(~60 subunits)
PeptidesRecycledubiquitine
Ubiquitin(8,5 kDa)
ubiquitin-activatingenzyme
ubiquitin-conjugatingenzyme
ubiquitinligase
Modified from Wang & Maldonado, Cellular & Molecular Immunology, 2006
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
Abnormal function of ribosomes?
Diamond Blackfan anemia• serious hypoplastic anemia• develops in the first year of life• accompanied by serious developmental abnormalities• ¼ of pacients carries mutation in gene coding for Rsp19
(component of 40S subunit of ribosome)• the only disease with the direct link to the mutation in the gene codingfor ribosomal protein
Other diseases that are linked to the factors involved in ribosome synthesis:• Congenital X-linked diskeratosis• Treacher Collins syndrome• Shwachman Diamond syndrome
Clinical heterogeneityand tissuenonspecificeffects.
Is it typicalfor diseases given by the
abnormal function of ribosomes?
The questionto be answered
Abnormal translation as a cause of cancer?
Possible mechanisms:Supportive facts:
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)
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
Cytosolic accumulation• Parkinson`s disease• Late age Huntington disease
Nuclear accumulation• Spinocerebelar ataxia type 1
Extracellular accumulation• Alzheimer disease (beta amyloid)
Examples of diseases:Levels and activities of 20/26S
proteasomes are loweredin relevant loci of brain
in pacients with sporadicParkinson`s disease
Some abnormalities:
Autosomal recessive loss of function mutation in gene coding for
E3 ligase (parkin) causesParkinson`s disease.
Thank you for your attention
Questions and comments at:[email protected]