Protein ActivityControl

Functional Proteins

Active vs inactive proteins

Steps in the creation of a functional protein

Cell 6.79

More than 100 different types ofcovalent modifications are known

Some ways in which the activity of gene regulatory proteins is regulated in eucaryotic cells

Each of these mechanisms is typically controlled by extracellular signals which are communicatedacross the plasma membrane to the gene regulatory proteins in the cell- SIGNAL TRANSDUCTION

Mechanisms are readily reversibleand therefore also provide the means to selectively inactivate gene regulatory proteins

Schematic representation of the four types of post-translational 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 qualityafter protein synthesis

Cell 6.85

ex. amiloid

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

Genomes 11.26

Structure of GroEL/GroES chaperonin(Hsp60)

Hsp70 chaperones bind to hydrophobic regionsin unfolded polypeptides, including those that are stillbeing translated, and hold protein in an open conformationuntil it is ready to be folded

Hps70 (dnaK)Hps40 (dnaJ)GrpE

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 CleavageChemical Modification

Intein splicing

Protein processing by proteolytic cleavage

Genomes 11.27

Ex. proteolytic cleavage : the pro-opiomelanocortinpolyprotein

Genomes 11.29

Ex. proteolytic cleavage: melitin and insulin

Genomes 11.28

Promelittin Melitin

24 aa

22 aa

Extracellular protease

Signal peptide- hydrophobic aa sequence thatattachs preproinsulin to the membrane, beforeexporting the protein through the membrane tothe extracellular environment

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

Genomes 9.22

Regulation the amount ofprotein that is beingsynthesized or changethe nature of the proteinin some way, for exampleby chemical modification

Common protein chemical modifications(after protein synthesis)

In red:Various chemicalgroups added to theaa side chains

Ex. of post-translational chemical modification ofcalf 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

Addition of lipid side chains

Acylation Serine, threonine, cysteine Many membrane proteins

N-myristoylation (miristic acid) N-terminal glycine Some protein kinases involved in signal transduction

Addition of biotin

Biotinylation Lysine Various carboxylase enzymes

150 different aa already described

Protein Degradation

Cell 6.82

Protein degradation... when?

Relation between N-terminal amino acid and half-life of E. coliβ-galactosidase proteins with modified N-terminal amino acids

N-terminal Amino Acid Half-life

Met, Ser, Ala, Thr, Val, Gly

Ile, Glu

Tyr, Gln

Pro

Phe, Leu, Asp, Lys

Arg

PestProGlnSerThr

Half-life less then 2 hours

more than 20 h

30 min

10 min

7 min

3 min

2 min

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

Lodish 3.13

Ubiquitin and the marking of protein withmultiubiquitin chains

Ubiquitin-mediatedproteolytic pathway

(7-8 residues)

The export and degradation of misfolded ER proteins

Cell 12.55

Protein Sorting

A simplified “roadmap” of proteintraffic

Cell 12.6

Through nuclear pores

Through translocator proteins

Through vesicules

Vesicle budding and fusion duringvesicular transport

Cell 12.7

Lodish 17.3

Overview of the secretoryand endocytic pathwaysof protein sorting

Lodish 17.1

Overview of major protein-sorting pathways in eukaryotic cells

Citosol

Non-secretory pathway

Free and membranes-bound ribosomes

Cell 12.37

Two ways in which a sorting signal (orsignal sequence) can be built into a protein

Cell 12.8

Some Typical Signal sequences

Hidrophylic aa

Hidrophylic aa/ hydrophobic aa

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 Gluor Asp residues

Chloroplast N-terminal (+) No common sequence motifs; generally rich in Ser, Thr, and small hydrophobic amino acid residues and poor in Gluand Asp residues

Peroxisome(matrix)

C-terminal (most proteins)N-terminal (few proteins)

( ) Usually Ser-Lys-Leu at extreme C-terminus

Nucleus Internal ( ) One cluster of 5 basic amino acids, or two smaller clusters of basic residues separated

Uptake-targeting Sequences that direct Proteins from the Cytosol to Organelles

The signal hypothesis

Cell 12.40

How ER signal sequences and SRP directribosomes to the ER membrane

Cell 12.42SRP- signal-recognition particle (citosol ribonucleoprotein)

Three ways in which protein translocation can bedriven trough structurally similar translocators

Cell 12.45

A model for how a soluble protein istranslocated across the ER membrane

Cell 12.46

Integration of a single-pass membrane proteinwith an internal sequence into the ER membrane

Cell 12.48

Integration of a double-pass membrane proteinwith 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 renaturationof 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