Download Protein Activity Control

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