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Protein Activity Control Functional Proteins Active vs inactive proteins Steps in the creation of a functional protein More than 100 different types of covalent modifications are known Cell 6.79 Some ways in which the activity of gene regulatory proteins is regulated in eucaryotic cells Mechanisms are readily reversible and therefore also provide the means to selectively inactivate gene regulatory proteins Each of these mechanisms is typically controlled by extracellular signals which are communicated across the plasma membrane to the gene regulatory proteins in the cell- SIGNAL TRANSDUCTION Schematic representation of the four types of posttranslational processing events Genomes 11.23 Protein Folding The aminoacid sequence contains all the information needed to fold the polypeptide into its correct tertiary structure The cellular mechanisms that monitor protein quality after protein synthesis ex. amiloid Cell 6.85 How a protein folds into a compact conformation Cell 3.6 The co-translational folding of a protein Cell 6.81 Molecular chaperones of Escherichia coli Hsp70 chaperones bind to hydrophobic regions in unfolded polypeptides, including those that are still being translated, and hold protein in an open conformation until it is ready to be folded Hps70 (dnaK) Hps40 (dnaJ) GrpE Structure of GroEL/GroES chaperonin (Hsp60) Genomes 11.26 Chaperone-and chaperonin- mediated protein folding Lodish 3.11 Chaperons • Ligam-se às proteínas em estádios precoces da sua síntese • Impedem enrolamentos não produtivos • Permitem que a proteína se enrole na forma termodinamicamente mais estável (estado de menor energia) • A sequência de aminoácidos do polipéptido é que determina a estrutura final Protein processing Proteolytic Cleavage Chemical Modification Intein splicing Protein processing by proteolytic cleavage Genomes 11.27 Ex. proteolytic cleavage : the pro-opiomelanocortin polyprotein Genomes 11.29 Ex. proteolytic cleavage: melitin and insulin Promelittin Melitin 22 aa Extracellular protease 24 aa Signal peptide- hydrophobic aa sequence that attachs preproinsulin to the membrane, before exporting the protein through the membrane to the extracellular environment Genomes 11.28 Ex. of protein processing by proteolytic cleavage • Botulism (from the latim botulus “sausage”)- is a rare but serious paralytic illness caused by the botulinum neurotoxin (BoNT) produced by Clostridium botulinum. • This disease is characterized by descending flaccid paralysis as a result of inhibition of acetylcholine release at the neuromuscular junction (BoNT belongs to the group of zinc-metalloproteases) • It is synthesized as a single-chain polypeptide of approx. 150 kDa, subsequently cleaved to form a di-chain molecules, in which a single disulfide bond links the light (50 kDa) and heavy chains (100 kDa) Chemical Modification Primary and secondary levels of gene regulation Regulation the amount of protein that is being synthesized or change the nature of the protein in some way, for example by chemical modification Genomes 9.22 Common protein chemical modifications (after protein synthesis) In red: Various chemical groups added to the aa side chains Ex. of post-translational chemical modification of calf histone H3 Genomes 11.30 Modification Amino acids that are modified Examples of proteins Addition of small chemical groups Acetylation (CH3CooH) Lysine Histones Methylation (CH3) Lysine Histones Phosphorylation (P) Serine, threonine, tyrosine Some proteins involved in signal transduction Hydroxylation (OH) Proline, lysine Collagen N-formylation (COH) N-terminal glycine Melittin Addition of sugar side chains O-linked glycosylation (hydroxil groups) Serine, threonine Many membrane proteins and secreted proteins N-linked glycosylation (amino group) Asparagine Many membrane proteins and secreted proteins Acylation Serine, threonine, cysteine Many membrane proteins N-myristoylation (miristic acid) N-terminal glycine Some protein kinases involved in signal transduction Lysine Various carboxylase enzymes Addition of lipid side chains Addition of biotin Biotinylation 150 different aa already described Protein Degradation Protein degradation... when? Cell 6.82 Relation between N-terminal amino acid and half-life of E. coli β-galactosidase proteins with modified N-terminal amino acids N-terminal Amino Acid Half-life Met, Ser, Ala, Thr, Val, Gly more than 20 h Ile, Glu 30 min Tyr, Gln 10 min Pro 7 min Phe, Leu, Asp, Lys 3 min Arg 2 min Pest Pro Gln Ser Thr Half-life less then 2 hours Acerca da degradação de proteínas • Várias vias de degradação – Lisossomas: contêm uma série de hidrolases e enzimas proteolíticos, degradando essencialmente proteínas transmembranares e do lúmen dos organitos – Proteossoma: complexo multiproteico que degrada proteínas ubiquitinadas, localizadas sobretudo no núcleo e no citosol. Ex. Factores de transcrição, proteínas da regulação do ciclo celular como as cinases e fosfatases etc. • Processos altamente selectivos e rápidos • Eucariotas- descrito o Proteossoma Ubiquitin and the marking of protein with multiubiquitin chains Ubiquitin-mediated proteolytic pathway (7-8 residues) Lodish 3.13 The export and degradation of misfolded ER proteins Cell 12.55 Protein Sorting A simplified “roadmap” of protein traffic Through nuclear pores Through translocator proteins Through vesicules Cell 12.6 Vesicle budding and fusion during vesicular transport Cell 12.7 Overview of the secretory and endocytic pathways of protein sorting Lodish 17.3 Overview of major protein-sorting pathways in eukaryotic cells Citosol Non-secretory pathway Lodish 17.1 Free and membranes-bound ribosomes Cell 12.37 Two ways in which a sorting signal (or signal sequence) can be built into a protein Cell 12.8 Some Typical Signal sequences Hidrophylic aa Hidrophylic aa/ hydrophobic aa Uptake-targeting Sequences that direct Proteins from the Cytosol to Organelles Target Organelle Usual Signal Location within Protein Signal Removal* Nature of Signal Endoplasmic reticulum N-terminal (+) "Core" of 6 - 12 mostly hydrophobic amino acids, often preceded by one or more basic amino acids Mitochondrion N-terminal (+) 20 to 50 nonconsecutive Arg or Lys residues, often with Ser and Thr; no Glu or Asp residues Chloroplast N-terminal (+) No common sequence motifs; generally rich in Ser, Thr, and small hydrophobic amino acid residues and poor in Glu and Asp residues Peroxisome (matrix) C-terminal (most proteins) N-terminal (few proteins) ( ) Usually Ser-Lys-Leu at extreme C-terminus Internal ( ) One cluster of 5 basic amino acids, or two smaller clusters of basic residues separated Nucleus The signal hypothesis Cell 12.40 How ER signal sequences and SRP direct ribosomes to the ER membrane SRP- signal-recognition particle (citosol ribonucleoprotein) Cell 12.42 Three ways in which protein translocation can be driven trough structurally similar translocators Cell 12.45 A model for how a soluble protein is translocated across the ER membrane Cell 12.46 Integration of a single-pass membrane protein with an internal sequence into the ER membrane Cell 12.48 Integration of a double-pass membrane protein with an internal signal sequence into the ER membrane Cell 12.49 Lodish 17.21 Lodish 17.25 Lodish 17.16 Cell 12.57 Steps at which eukaryotic gene expression can be controlled Cell 7.5 Protein denaturation agents Denaturation and spontaneous renaturation of a small protein Genomes 11.24 The structure and function of the Hsp60 family of molecular chaperones Cell 6.84 Intein splicing Intein splicing- self-splicing of proteins -150 conserved aa peptide - cut, generally occurs downstream a Ser,Thr or Cys aa - present in bacteria, Archae and eucaryotes A polypeptide might have several inteins Some of them after excision, are proteins themselves