lecture 7 post translational processing
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ost-Translational Processing (7.1)
Lecture 7
Post-Translational Processing
Protein synthesis consists of several steps: from
the translation of the information from mRNA
to the folded and fully processed, active protein
in its proper compartment of action.
The mRNA sequence predicts a specific length
polypeptide chain made up of the primary 20amino acids.
Fully processed protein products are almostalways shorter than their mRNA would predict,
and globally contain about 200 different amino
acids.
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ost-Translational Processing (7.1)
(determined by sequencing, biochemistry, X-ray crystallography)
uring translation, about 30-40 polypeptide residues areelatively protected by the ribosome (tunnel T and exit sites End E2 in the large subunit). Once the polypeptide chainmerges from the ribosome it starts to fold and can be subjeco post-translational modifications.
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ost-Translational Processing (7.1)
o after translation several additional steps must beonsidered as part of the complete protein biosyntheticrocess:
Covalent modification of
a: peptide bonds
b: the N-terminusc: the C-terminus
d: amino acid residues (side chains).
Noncovalent modifications: folding, addition of co-factors.
Translocation: compartment selection and transport (Trafficking/Targeting).
Involvement of molecular chaperones in 1, 2, and 3.
Why post-translational processing?
q adds functionality
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ost-Translational Processing (7.1)
q effects targeting
q regulates activity
q increases mechanical strength
q changes recognition
Next: Covalent Modifications
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ovalent Modifications (7.2)
Covalent Modifications
odifications involving the peptide bond (peptide bond cleavageomited proteolysis):
q usually carried out by enzymes calledpeptidases orproteases:
r activation of proenzymes (digestive enzymes, blood clotting cascade, complement
activation etc.) and prohormones (insulin)
r production of active neuropeptides and peptide hormones from high molecular we
precursors
r macromolecular assembly in virus particles (e.g. HIV protease)
r removal of signal sequences
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ovalent Modifications (7.2)
These reactions are often exquisitely specific for onlyone or a few peptide bonds.
Modifications involving the amino terminus:
q trimming of formyl group from formyl-Met
q proteolytic removal of N-terminal Met by aminopeptidases
q acetylation
q lipidation (myristoylation)
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ovalent Modifications (7.2)
odifications involving the carboxy terminus:
q amidation of C-terminal glycine
q attachment of membrane anchors
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ovalent Modifications (7.2)
Next: Side Chain Modifications
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ide Chain Modifications (7.3)
Side Chain Modifications
odifications involving amino acid side chains:
q disulfide cross-linking
q lysinonorleucine cross-linking (collagen)
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ide Chain Modifications (7.3)
q Phosphorylation of hydroxyls by kinases (serine, threonine, tyrosine):
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ide Chain Modifications (7.3)
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ide Chain Modifications (7.3)
lycogen phosphorylase is the ultimate enzyme in a cascade which catalyzes the degradation o
ycogen to glucose-1-phosphate. Phosphorylation of phosphorylase occurs on serine-14 and
nverts the inactivephosphorylase b to the activephosphorylase a.
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ide Chain Modifications (7.3)
Phosphorylation is reversibleand is used in manypathways to control activity. Enzymes that adda
phosphate to a hydroxyl side chain are commonly calledkinases. Enzymes that removea phosphate from aphosphorylated side chain are called phosphatases.
Glycosylation
q There are two basic types of glycosylation which occur on:
r asparagines (N-linked, see (a) below) and
r serines and threonines (O-linked, see (b) below)
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ide Chain Modifications (7.3)
(b)N-Acetylgalactosamine
q Covalently attached to the polypeptide as oligosaccharide chains containing 4 to 15 suga
q Sugars frequently comprise 50% or more of the total molecular weight of a glycoprotein
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ide Chain Modifications (7.3)
q Most glycosylated proteins are eithersecretedorremain membrane-bound
q Glycosylation is the most abundant form of post-translational modification
q Glycosylation confers resistance to protease digestion by steric protection
q Important incell-cell recognition
-linked glycosylation on asparagine (Asn) side chains:
q an alkali-stable bond between the amide nitrogen of asparagine and the C-1 of an amino
sugar residue
q occurs co-translationally in the endoplasmic reticulum (ER) during synthesis
q lipid-linked oligosaccharide complex is transferred to polypeptide byoligosaccharyl
transferase
q target sequence orconsensus site on protein is Asn-X-Ser/Thr
q further processing in Golgi apparatus
q Examples:
r Heavy chain of immunoglobulin G (IgG)
r Hen ovalbumin
r Ribonuclease B
-linked glycosylation on serine (Ser) or threonine (Thr) side chains
q an alkali-labile bond between the hydroxyl group of serine or threonine and an amino sug
q carried out by a class of membrane-bound enzymes calledglycosyl transferases which
reside in the endoplasmic reticulum (ER) or the Golgi apparatus
q nucleotide-linked monosaccharides added to protein side chain one at a time
q Example: Blood group antigens on erythrocyte surface:
r TheA antigen andB antigen are pentasaccharides which differ in composition of
5th sugar residue
r The O substance is a tetrasaccharide which is missing the 5th residue and does not
elecit an antibody response (non-antigenic).
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ide Chain Modifications (7.3)
,
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ide Chain Modifications (7.3)
Blood
Type
(Antigen)
Glycolsyl transferase Antibodies Against
Can Safely
Receive Blood
from
Can Safely
Donate Blood
O neither A & B O O, A, B & A
A UDP-GalNAc B O & A A & AB
B UDP-Gal A O & B B & AB
AB both neither O, A, B, & AB AB
Examples of monosaccharides used in glycosylation
q Prosthetic group attachment (heme, retinal etc.)
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ide Chain Modifications (7.3)
Heme group, attached to histidine side chain via Fe.
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ide Chain Modifications (7.3)
Retinal attached to lysine side chain via covalent Schiff base linkage.
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ide Chain Modifications (7.3)
Processing of pre-pro-insulin to active insulin
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ide Chain Modifications (7.3)
q Pre-pro-insulin is synthesized as a random coil on membrane-associated
ribosomes
q After membrane-transport the leader sequence (yellow) is cleaved off by a
protease and the resulting pro-insulin folds into a stable conformation.
q Disulfide bonds form between cysteine side chains.
q The connecting sequence (red) is cleaved off to form the mature and activ
insulin molecule.
Next: Noncovalent Modifications
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Noncovalent Modifications (7.4)
Noncovalent Modifications
ddition of metal ions and co-factors:
Nearly 50% of all proteins contain metal ions
Metal ions play regulatory as well as structural roles
q Calcium (Ca++): very important intra-cellular messenger, i.e. calmodulin
q Magnesium (Mg++): ATP enzymes
q Copper (Cu++), Nickel (Ni+), Iron (Fe++)
q Zinc (Zn++): Zinc finger domains are used for DNA recognition:
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Noncovalent Modifications (7.4)
zinc finger domain: Zn++ is bound by two cysteine and two histidine residues. Zinc finger
omains interact in the major groove with three consecutive bases from one strand of duplex B
rm DNA.
odifications involving tertiary structure (protein fold)
nzymes calledmolecular chaperones are responsible for detecting mis-folded proteins.
haperones only bind mis-folded proteins that exhibit large hydrophobic patches on their
rfaces.
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Noncovalent Modifications (7.4)
ubunit multimerization
any enzymes are only functional as multimeric units, either as homo- or hetero-oligomers.
xample: ribosomes!
Next: Chaperone-Assisted Protein Folding
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Chaperone-Assisted Protein Folding (7.5)
Chaperone-Assisted Protein Folding
Quote: "The traditional role of the human chaperone described in biochemical terms is
revent improper interactions between potentially complementary surfaces, and to disrany improper liaisons which occur."
haperones:
q Mediate folding and assembly.
q Do not convey steric information.
q Do not form part of the final structure.
q Suppress non-productive interactions by binding to transiently exposed portions o
the polypeptide chain.
q First identified asheat shock proteins (Hsp).
q Hsp expression is elevated when cells are grown at higher-than-normal temperatu
q Stabilize proteins during synthesis.q Assist in protein folding by binding and releasing unfolded/mis-folded proteins.
q Use an ATP-dependent mechanism.
Major types of chaperones:
q Hsp70 (cytoplasm, ER, chloroplasts, mitochondria):
r thought to bind and stabilize the nascent polypeptide chain as it is being
extruded from the ribosome.r also involved in "pulling" newly synthesized polypeptide into ER lumen.
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Chaperone-Assisted Protein Folding (7.5)
q Hsp60 (mitochondria, chloroplasts):
r forms large 28-subunit complexes called GroEL
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Chaperone-Assisted Protein Folding (7.5)
Next: Selenocysteine
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elenocysteine (7.6)
Selenocysteine: the 21st Amino Acid?
Although the element selenium was discovered in 1817 by Berzeliu
it was only shown over 100 years later to be an essential micronutri
in all three lines of decent. Subsequent analysis of several enzymes
that catalyze oxidation-reduction reactions showed that selenium
occurs in the form of the unusual amino acidselenocysteine. How
this amino acid is incorporated into the protein was unclear in these
days.
Today it is well established that the incorporation of selenocysteine
co-translational. Interestingly, the base triplet encoding this amino
acid is UGA, a codon that normally functions as a STOP signalin
translation. Since this codon has still retained its "normal" function
all organisms known to synthesize selenocysteine-containing protei
the incorporation of this amino acid requires a specific pathway. Th
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elenocysteine (7.6)
pathway has been elucidated for the bacteriumE. coli by August B
and coworkers.
General pathway
There are one cis-acting element and four trans-acting factors
involved in this incorporation. The cis-acting element is a specific
stem-loop structure of the mRNA directly 3' of the UGAcodon. Th
four trans-acting factors are:
q a specific tRNASec which is charged with serine by serinyl-tRNA
synthetase
q the enzyme selenophosphate synthetase which generates inorganic
selenophosphate
q the enzyme selenocysteine synthetase which uses selenophosphate t
convert seryl-tRNASec to selenocysteyl-tRNASecq a specific translation factor (SelB) that substitutes for EF-Tuand
recognizes the cis-acting element
In the first step the specific tRNASec is charged by the normal seryl-tRNA synthetase with serine, and that serine is subsequently
converted to selenocysteine by the enzyme selenocysteine synthetas
The low molecular weight selenium donor - selenophosphate - is
provided by the action of the selenophosphate synthetase. Finally, t
selenocysteyl-tRNASec is recognized by a specific translation factor
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elenocysteine (7.6)
SelB that delivers it to a UGAcodon at the ribosomal A-siteonly in
the presence of the stem-loop structure downstream of the codon on
the mRNA.
tRNASec
tRNASec differs in several aspects from the consensus for canonical
tRNAs. In particular it includes an acceptor stem elongated by extr
one base pair, an elongated D-arm with only four nucleotides in the
loop, and a UCAanticodon. These differences ensure that tRNASec
not recognized by the normal translation factor EF-Tu thus preventi
mis-incorporation.
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elenocysteine (7.6)
The two structures shown here are (a) tRNASec and (b) tRNASer ofE. coli. Residues which
normally conserved in tRNA are shown circled. Identity nucleotides in tRNASer and their
counterparts in tRNASec are shown boxed. tRNASec is the longest tRNA species known
consisting of 95 nucleotides.
Selenophosphate synthetase
This enzyme catalyses the synthesis of the low molecular weight
donor selenophosphate using ATP as the phosphate donor.
Interestingly, the gamma-phosphate is transferred to selenium
whereas the beta-phosphate is released, leaving AMP.
Selenocysteine synthetase
This enzyme possesses a prosthetic group (pyridoxal phosphate). T
active enzyme is composed of ten identical subunits arranged as a
stack of two five-membered rings. It converts the serine attached totRNASec to selenocysteine.
Translation factor SelB
This translation factor substitutes for elongation factor EF-Tu in the
specific incorporation of selenocysteine. This protein is considerab
longer than its regular counterpart. The N-terminal half of the prote
resembles EF-Tu in structure and function - GTP and tRNA binding
whereas the C-terminal half is responsible for the recognition of the
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elenocysteine (7.6)
specific stem-loop structure adjacent to the UGAcodon. A comple
between SelB, GTP, selenocysteyl-tRNASec and the stem-loop
structure of the mRNA has been detected and shown to be
functionally necessary.
Next: Summary
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ummary (7.7)
Summary
q Post-translational processing controlsfolding, targeting,activation
andstability of proteins
q Co- and post-translational modifications increasediversity and
functionality of proteins
q Common forms of co- or post-translational modifications are:
r proteolysis
r phosphorylation
r glycosylationr metal binding
q Chaperones mediate folding and assembly of newly synthesized
proteins
q Selenocysteine is sometimes called the 21st amino acid
r uses one of the three STOP codonsr has its own tRNASec
r requires other factors, including a cis-acting element on the
mRNA
Next Lecture: Protein Trafficking/Targeting
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