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Protein Synthesis and Protein Processing

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					   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
Peptide bond formation                  P-site          A-site
                                       NH2                N
                                                          NH2
                         CH3-S-CH2-CH2-CH             CH3-CH
                                       C
                                     O=C                O=C
                                       O                  O

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

                               cell makes interferon
                             in response to viral RNA

                                                              interferon binds to
                                                        receptors on neighboring cells
virus                                                       and activates the cells
                                 cell cannot
                                protect itself
        virus invades cell
                                                             cell synthesizes
                                                             antiviral proteins
                                                              in response to
                              virus replicates             interferon activation

           cell succumbs
                                  virus invades
                                   neighboring cell




                                                    cell protected from viral
                                                 infection by antiviral proteins
             Functions of two antiviral proteins

                                                           inactive
                                                        endonuclease
                 viral dsRNA
      ATP                         oligo 2-5 adenylate (2-5A)
                2-5A synthase
                                   [-A-2’-p-5’-A-2’-p-5’A-] N


                                                             active
                                                         endonuclease:
interferon
                                                     viral mRNA degraded
  induces




                  eIF2 kinase                    P
     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
 Translation of a secreted protein                  insertion through a pore

 2. the signal recognition particlea (SRP)                                    ER lumen c
    binds the signal peptideb and
    halts translation
                            SRP                               SRP receptor      cytosol




      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
      6. the SRP is released                      ER membrane; the rough ER
         and is recycled                          is ER studded with polysomes
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
             ER lumen                                                 protein is
                                                                     processed and
                                                                    secreted
   cytosol




         5’                                         UGA
• 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)
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)


                             P        Dolichol phosphate (polyprenol lipid carrier)
                  (2) UDP-
                                                                    ER lumen
              (1) UMP, (1) UDP

                                 (5) GDP-

                                          (5) GDP                      reorientation

             Cytosol

                                                        N-acetylglucosamine (GlcNAc) =
    Monosaccharides are transferred
   by specific glycosyltransferases                                  Mannose =
        from nucleotide sugars
Biosynthesis of N-linked oligosaccharides (second 7 steps)



           PP                                       ER lumen

       P        (4)                          Dolicol-phosphates are the
                                           sugar donors in the ER lumen;
                                         they are synthesized in the cytosol
                                      prior to being translocated to the lumen


           PP
                                                Dolicol-P-mannose =        P
           P          (3)
                                                 Dolicol-P-glucose =      P




                       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
Cytosol                 to the Golgi complex, where trimming and further
                             building of the oligosaccharides occurs
                  Formation of complex type oligosaccharides


                      Asn
                        I
                       X
                        I
                    Ser (Thr)
                                           Trimming by glycosidases;
                                        Building by glycosyltransferases

                                                      = common core structure




                              Asn
                                I
    Golgi lumen                 X
                                I
                            Ser (Thr)      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
Targeting of proteins to lysosomes (I-cell disease)
                                              • Proteins containing
                                                 mannose-6-phosphate
            Asn                                  are targeted to lysosomes

                                              • These proteins include the
                        UDP-                     lysosomal hydrolases

                                P             • 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
                                P                in the enzyme that transfers
                                                 GlcNAc phosphate to mannose
                                    P            residues in the Golgi

                                              • As a result, the hydrolases cannot
                  Asn                            be targeted to the lysosomes

                                              • The resulting deficiency in
                                    P            lysosomal hydrolases results in
                                                 an accumulation (inclusions)
                                                 of material in the lysosomes
 Proteolytic processing
         Processing of insulin (synthesized in the ER of pancreatic b-cells)

              Signal peptide
                                 N                       N
                                                             S       S               Disulfide bond
                                                             I       I                 formation
                                                             S       S
cleavage of                                                            C
signal peptide                                               Proinsulin
by signal                        C
peptidase            Preproinsulin


                                                                             B-chain
          Insulin                                                N
                         Further trimming by a                           S      S
     N                    carboxypeptidase B-like                        I       I
         S       S                                                       S      S
          I
                            enzyme removes two
                 I
                                                                         A-chain       C
         S       S            basic residues from
     N                  C C each of the new ends                             C-chain

                                                             Cleavage by trypsin-like enzymes
                                                                  releases the C-peptide
                                      The C-chain is
                       C-chain       packaged in the secretory
                                 vesicle and is secreted along with active insulin
            Preproopiomelanocortin
               • multiple functional polypeptides from a single precursor
               • processed in a cell-specific manner



     26aa        48aa      12aa     40aa     14aa   21aa      40aa      18aa 5aa    26aa
N                                                                                          C
    Signal                          Proopiomelanocortin
    peptide


                          g-MSH              Corticotropin           b-Lipotropin
                                               (ACTH)
                                                                                    31aa

                                            a-MSH                       b-MSH Endorphin


                                                              g-Lipotropin   Enkephalin (5aa)
   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
Peptide bond formation                  P-site          A-site
                                       NH2                N
                                                          NH2
                         CH3-S-CH2-CH2-CH             CH3-CH
                                       C
                                     O=C                O=C
                                       O                  O

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

                               cell makes interferon
                             in response to viral RNA

                                                              interferon binds to
                                                        receptors on neighboring cells
virus                                                       and activates the cells
                                 cell cannot
                                protect itself
        virus invades cell
                                                             cell synthesizes
                                                             antiviral proteins
                                                              in response to
                              virus replicates             interferon activation

           cell succumbs
                                  virus invades
                                   neighboring cell




                                                    cell protected from viral
                                                 infection by antiviral proteins
             Functions of two antiviral proteins

                                                           inactive
                                                        endonuclease
                 viral dsRNA
      ATP                         oligo 2-5 adenylate (2-5A)
                2-5A synthase
                                   [-A-2’-p-5’-A-2’-p-5’A-] N


                                                             active
                                                         endonuclease:
interferon
                                                     viral mRNA degraded
  induces




                  eIF2 kinase                    P
     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
 Translation of a secreted protein                  insertion through a pore

 2. the signal recognition particlea (SRP)                                    ER lumen c
    binds the signal peptideb and
    halts translation
                            SRP                               SRP receptor      cytosol




      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
      6. the SRP is released                      ER membrane; the rough ER
         and is recycled                          is ER studded with polysomes
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
             ER lumen                                                 protein is
                                                                     processed and
                                                                    secreted
   cytosol




         5’                                         UGA
• 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)
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)


                             P        Dolichol phosphate (polyprenol lipid carrier)
                  (2) UDP-
                                                                    ER lumen
              (1) UMP, (1) UDP

                                 (5) GDP-

                                          (5) GDP                      reorientation

             Cytosol

                                                        N-acetylglucosamine (GlcNAc) =
    Monosaccharides are transferred
   by specific glycosyltransferases                                  Mannose =
        from nucleotide sugars
Biosynthesis of N-linked oligosaccharides (second 7 steps)



           PP                                       ER lumen

       P        (4)                          Dolicol-phosphates are the
                                           sugar donors in the ER lumen;
                                         they are synthesized in the cytosol
                                      prior to being translocated to the lumen


           PP
                                                Dolicol-P-mannose =        P
           P          (3)
                                                 Dolicol-P-glucose =      P




                       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
Cytosol                 to the Golgi complex, where trimming and further
                             building of the oligosaccharides occurs
                  Formation of complex type oligosaccharides


                      Asn
                        I
                       X
                        I
                    Ser (Thr)
                                           Trimming by glycosidases;
                                        Building by glycosyltransferases

                                                      = common core structure




                              Asn
                                I
    Golgi lumen                 X
                                I
                            Ser (Thr)      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
Targeting of proteins to lysosomes (I-cell disease)
                                              • Proteins containing
                                                 mannose-6-phosphate
            Asn                                  are targeted to lysosomes

                                              • These proteins include the
                        UDP-                     lysosomal hydrolases

                                P             • 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
                                P                in the enzyme that transfers
                                                 GlcNAc phosphate to mannose
                                    P            residues in the Golgi

                                              • As a result, the hydrolases cannot
                  Asn                            be targeted to the lysosomes

                                              • The resulting deficiency in
                                    P            lysosomal hydrolases results in
                                                 an accumulation (inclusions)
                                                 of material in the lysosomes
 Proteolytic processing
         Processing of insulin (synthesized in the ER of pancreatic b-cells)

              Signal peptide
                                 N                       N
                                                             S       S               Disulfide bond
                                                             I       I                 formation
                                                             S       S
cleavage of                                                            C
signal peptide                                               Proinsulin
by signal                        C
peptidase            Preproinsulin


                                                                             B-chain
          Insulin                                                N
                         Further trimming by a                           S      S
     N                    carboxypeptidase B-like                        I       I
         S       S                                                       S      S
          I
                            enzyme removes two
                 I
                                                                         A-chain       C
         S       S            basic residues from
     N                  C C each of the new ends                             C-chain

                                                             Cleavage by trypsin-like enzymes
                                                                  releases the C-peptide
                                      The C-chain is
                       C-chain       packaged in the secretory
                                 vesicle and is secreted along with active insulin
            Preproopiomelanocortin
               • multiple functional polypeptides from a single precursor
               • processed in a cell-specific manner



     26aa        48aa      12aa     40aa     14aa   21aa      40aa      18aa 5aa    26aa
N                                                                                          C
    Signal                          Proopiomelanocortin
    peptide


                          g-MSH              Corticotropin           b-Lipotropin
                                               (ACTH)
                                                                                    31aa

                                            a-MSH                       b-MSH Endorphin


                                                              g-Lipotropin   Enkephalin (5aa)

				
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posted:12/4/2011
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