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:ahampl@med.muni.cz