8. Protein Synthesis and Protein Processing

8. Protein Synthesis and Protein Processing a). Ribosome structure b). Protein synthesis i). Initiation of protein synthesis ii). Peptide bond formation; peptidyl transferase iii). Elongation and termination iv). Inhibitors of protein synthesis Antiviral action of interferon Induction of 2-5A synthase Induction of eIF2 kinase Antibiotics c). Protein processing i). Synthesis of secreted and integral membrane proteins ii). Glycosylation and protein targeting iii). Proteolytic processing

P-site A-site • Peptide bond formation • peptide bond formation is • catalyzed by peptidyl transferase • peptidyl transferase is contained within • a sequence of 23S rRNA in the • prokaryotic large ribosomal subunit; • therefore, it is probably within • the 28S rRNA in eukaryotes • the energy for peptide bond formation • comes from the ATP used in tRNA charging • peptide bond formation results in a shift • of the nascent peptide from the P-site • to the A-site N NH2 CH3-S-CH2-CH2-CH O=C O tRNA NH2 CH3-CH O=C O tRNA C NH2 CH3-S-CH2-CH2-CH O=C OH tRNA NH CH3-CH O=C O tRNA

Induction and action of interferon cell makes interferon in response to viral RNA interferon binds to receptors on neighboring cells and activates the cells virus cell cannot protect itself virus invades cell cell synthesizes antiviral proteins in response to interferon activation virus replicates cell succumbs virus invades neighboring cell cell protected from viral infection by antiviral proteins

Functions of two antiviral proteins inactive endonuclease viral dsRNA oligo 2-5 adenylate (2-5A) ATP 2-5A synthase [-A-2’-p-5’-A-2’-p-5’A-] N active endonuclease: viral mRNA degraded interferon induces P eIF2 kinase eIF2 eIF2 viral dsRNA active inactive: viral protein synthesis cannot initiate

Inhibitors of protein synthesis Inhibitor Process Affected Site of Action Kasugamycin initiator tRNA binding 30S subunit Streptomycin initiation, elongation 30S subunit Tetracycline aminoacyl tRNA binding A-site Erythromycin peptidyl transferase 50S subunit Lincomycin peptidyl transferase 50S subunit Clindamycin peptidyl transferase 50S subunit Chloramphenicol peptidyl transferase 50S subunit Staphylococcus resistance to erythromycin • certain strains of Staphylococcus can carry a plasmid that encodes • an RNA methylase • this RNA methylase converts a single adenosine residue in 23S rRNA • to N6-dimethyladenosine • this is the site of action of erythromycin, lincomycin, and clindamycin • N6-dimethyladenosine blocks the action of these antibiotics • the organism that produces erythromycin has its own RNA methylase • and thus is resistent to the antibiotic it makes

Protein maturation: modification, secretion, targeting 3. the SRP docks with the SRP receptor on the cytosolic side of the ER membrane and positions the signal peptide for insertion through a pore Translation of a secreted protein 2. the signal recognition particlea (SRP) binds the signal peptideb and halts translation ERlumen c cytosol SRP SRP receptor 5’ AUG polysome for secreted protein 1. translation initiates as usual on a cytosolic mRNA athe signal recognition particle (SRP) consists of protein and RNA (7SL RNA); it binds to the signal peptide, to the ribosome, and to the SRP receptor on the ER membrane bthe signal peptide is a polypeptide extension of 10-40 residues, usually at the N-terminus of a protein, that consists mostly of hydrophobic amino acids cER = endoplasmic reticulum

4. translation resumes and the nascent polypeptide moves into the ER lumen 5. signal peptidase, which is in the ER lumen, cleaves off the signal peptide ER lumen cytosol 5’ 7. the ribosomes dock onto the ER membrane; the rough ER is ER studded with polysomes 6. the SRP is released and is recycled

8. translation continues with the nascent polypeptide emerging into the ER lumen 9. at termination of translation, the completed protein is within the ER and is further processed prior to secretion completed protein is processed and secreted ER lumen cytosol UGA 5’

Examples of secreted proteins: • polypeptide hormones (e.g., insulin) • albumin • collagen • immunoglobulins • Integral membrane proteins are also synthesized by the same mechanisms; • they may be considered “partially secreted” • Examples of integral membrane proteins: • polypeptide hormone receptors (e.g., insulin receptor) • transport proteins • ion channels • cytoskeletal anchoring proteins (e.g., band 3)

P P P P P • Glycosylation of proteins • most integral membrane proteins and secreted proteins are glycosylated • during translation on the ER membrane the protein begins to be glycosylated • various oligosaccharide modifications occur in the ER and in the Golgi complex • O-linked (Ser, Thr linked) oligosaccharides (linked to hydroxyl group) • N-linked (Asn linked) oligosaccharides (linked to amide group) Biosynthesis of N-linked oligosaccharides (first 7 steps) Dolichol phosphate (polyprenol lipid carrier) (2) UDP- ER lumen (1) UMP, (1) UDP (5) GDP- (5) GDP reorientation Cytosol N-acetylglucosamine (GlcNAc) = Mannose = Monosaccharides are transferred by specific glycosyltransferases from nucleotide sugars

PP P P PP P P Biosynthesis of N-linked oligosaccharides (second 7 steps) ER lumen Dolicol-phosphates are the sugar donors in the ER lumen; they are synthesized in the cytosol prior to being translocated to the lumen (4) Dolicol-P-mannose = Dolicol-P-glucose = (3) PP Cytosol

Transfer of oligosaccharide to protein PP Transfer of oligosaccharide chain to the growing polypeptide ER lumen Asn I X I Ser (Thr) Linkage is to the amide group of an asparagine followed by any (X) amino acid (except proline) followed by serine or threonine Following synthesis, the protein is transferred to the Golgi complex, where trimming and further building of the oligosaccharides occurs Cytosol

Formation of complex type oligosaccharides Asn I X I Ser (Thr) Trimming by glycosidases; Building by glycosyltransferases = common core structure Asn I X I Ser (Thr) Golgi lumen A complex type oligosaccharide fucose = galactose = sialic acid = come from nucleotide sugars translocated across the Golgi membrane Cytosol The type of carbohydrate determines whether the protein is targeted to the membrane, to a vesicle, or is secreted

P P Targeting of proteins to lysosomes (I-cell disease) • Proteins containing • mannose-6-phosphate • are targeted to lysosomes Asn • These proteins include the • lysosomal hydrolases UDP- • Phosphate groups are added to • mannose by transfer of GlcNAc • phosphate from UDP-GlcNAc Asn • Patients with I-cell (for inclusion • body) disease have a deficiency • in the enzyme that transfers • GlcNAc phosphate to mannose • residues in the Golgi P • As a result, the hydrolases cannot • be targeted to the lysosomes Asn • The resulting deficiency in • lysosomal hydrolases results in • an accumulation (inclusions) • of material in the lysosomes P

Proteolytic processing Processing of insulin (synthesized in the ER of pancreatic b-cells) Signal peptide N N Disulfide bond formation S I S S I S cleavage of signal peptide by signal peptidase C Proinsulin C Preproinsulin B-chain Insulin N S I S S I S Further trimming by a carboxypeptidase B-like enzyme removes two basic residues from each of the new ends N S I S S I S C A-chain N C C C-chain Cleavage by trypsin-like enzymes releases the C-peptide The C-chain is packaged in the secretory vesicle and is secreted along with active insulin C-chain

Preproopiomelanocortin • multiple functional polypeptides from a single precursor • processed in a cell-specific manner 5aa 26aa 48aa 12aa 40aa 14aa 21aa 40aa 18aa 26aa C N Signal peptide Proopiomelanocortin g-MSH Corticotropin (ACTH) b-Lipotropin 31aa a-MSH b-MSH Endorphin g-Lipotropin Enkephalin (5aa)