lecture 7 post translational processing

7/29/2019 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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(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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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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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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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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odifications involving the carboxy terminus:

q amidation of C-terminal glycine

q attachment of membrane anchors



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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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q Phosphorylation of hydroxyls by kinases (serine, threonine, tyrosine):



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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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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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(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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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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,



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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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Heme group, attached to histidine side chain via Fe.



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Retinal attached to lysine side chain via covalent Schiff base linkage.



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Processing of pre-pro-insulin to active insulin



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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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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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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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q Hsp60 (mitochondria, chloroplasts):

r forms large 28-subunit complexes called GroEL



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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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lecture 7 post translational processing

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