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
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 …
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
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 ofDNA is not influenced by sequence of nucleotides X aminoacids determines the higher structure of proteins !
Higher organization of molecule of protein is determinedby 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).
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
DNA determines expression/metabolism of proteins essentially by two mechanisms At the level At the level of transcripts of proteins Synthesis of mRNATranslation YES/NO of given protein Primary sequence of aminoacids of given protein (determines features - stability + of given protein) Stability of mRNA of given protein
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)
Ribosomes – general featuresThey 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
Ribosomes - structureLarge subunit A – binding site for Aminoacyl-tRNAE P A P – binding site for Peptidyl-tRNA E – tRNA Exit siteSmall subunit 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 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
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 mRNA5` = movement of ribosomes on mRNA 100 nm
Regulation of translationOccurs 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
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)
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 proteinsHydrolytic cleavage of by „ubiquitin-proteasome“ proteins in lysosomes pathway
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 USATechnion - Israel Institute Technion - Israel Institute University of California of Technology, Haifa of Technology, Haifa Irvine, CA, USA
„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)
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 3Ubiquitin Recycled(8,5 kDa) Peptides ubiquitine Modified from Wang & Maldonado, Cellular & Molecular Immunology, 2006
Molecular machineries that are responsible fortranslation 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 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 functionOther 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
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 transgenicanimals 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 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.
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