Posttranslational Modification and Targeting of Proteins

Transcription

1 Posttranslational Modification and Targeting of Proteins Graduate Biochemistry Term 2/2016 Assist. Prof. Dr. Panida Khunkaewla School of Chemistry, Institute of Science Suranaree University of Technology 1

2 What is posttranslational Modification? Modification of nascent protein by adding groups or cleavage of some parts to get mature protein Key wards: Adding Deleting 2

3 Diversity of Posttranslational modification of proteins Cleavage of signal peptides Phosphorylation Amidation Glycosylation Hydroxylation Ubiquitination Addition of prosthetic groups Iodination Prenylation Myristoylation Acylation Acetylation Methylation Oxidative crosslinking N-Glutamyl cyclization Carboxylation Adenylation Sulfonation 3

4 Location Nucleus Lysosome Mitochondria Golgi ER Cytosol Ribosome Plasma membrane Extracelullar fluid Extracellular matrix Modification acetylation, phosphorylation mannose-6-phosphate labelled N-linked sugar N-formyl acylation N- and O-linked ologosaccharide, sulfation, palmitoylation N-linked oligosaccharide, GPI-anchor acetylation, methylation, phosphorylation, myristoylation N- and O-glycosylation, GPI-anchor N- and O-glycosylation, acetylation, phosphorylation N- and O-glycosylation, phosphorylation, hydroxylation 4

5 Proteolytic cleavage Covalent cleavage of one or more peptide bonds in protein substrates by protease. Schematic processing of preproinsulin to proinsulin by signal peptidase in the ER and of proinsulin to insulin by proprotein convertases in the trans Golgi network. Christopher T. W. Posttranslational Modification of Proteins, 2006, Robert and Company Publisher. 5

6 Covalent modifications Covalent addition of a substrate fragment to protein side chain catalyzed by a posttranslational modification enzyme. Examples shown are generic phosphorylation, acetylation, and O-glycosylation. 6 Christopher T. W. Posttranslational Modification of Proteins, 2006, Robert and Company Publisher.

7 Irreversible Modification Irreversible changes include the proteolysis of peptide bonds, installation of phopsphopantertheinyl groups in fatty acids synthase, and N-methylation of lysine residues in histones. 7 Christopher T. W. Posttranslational Modification of Proteins, 2006, Robert and Company Publisher.

8 Reversible Modifications Reversible covalnt modifications including protein phosphorylations (due to balance of kinase and phosphatase), histone acetylation (due to the balance of histone acetyltransferase and histone deacetylase), and protein 8 ubiquitylations (due to the balance of ligases and deubiquitylating hydrolase Christopher T. W. Posttranslational Modification of Proteins, 2006, Robert and Company Publisher.

9 Purpose of posttranslational modification. Targeting of protein Stability of protein Function of protein Control protein activity 9

10 Targeting of proteins How are synthesized proteins directed to their final cellular destination? Secreted protein, membrane protein, inclusion protein in lysosomes Protein destines to mitochondria Protein destines to chloroplast Protein destines to nucleus Cytoplasmic proteins 10

11 Proteins can move between compartments in different ways Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; Cycles of membrane budding and fusion permit the lumen of any of these organelles to communicate with any other and with the cell exterior by means of transport vesicles. Blue arrows indicate the extensive network of outbound and inbound traffic route. Some organelles, most notably mitochondria and (in plant cells) plastids do not take part in this communication and are isolated from the traffic between organelles shown here. 11

12 Three major ways of protein moving between different compartments 1. In gated transport, the protein traffic between the cytosol and nucleus occurs through the nuclear pore complexes. The nuclear pore complexes function as selective gates that actively transport specific macromolecules and macromolecular assemblies, although they also allow free diffuse of smaller molecules. 2. Intransmembrane transport, membrane-bound protein translocators directly transport specific proteins across a membrane from the cytosol into a space that is topologically distinct. The transported protein molecules usually must unfold to snake through the translocator. The initial transport of selected proteins from the cytosol into the ER lumen or from the cytosol into mitochondria, for example, occurs in this way. 3. In vesicular transport, membrane-enclosed transport intermediates which may be small, spherical transport vesicles or larger, irregularly shaped organelle fragments-ferry protein from one compartment to another. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science;

13 Signal sequence Signal sequence is a short sequence of amino acids that directed a protein to it appropriate location in the cell and, for many protein, is removed during transport or after the protein has reached to its final destination

14 The Nobel Prize in Physiology or Medicine 1999 "for the discovery that proteins have intrinsic signals that govern their transport and localization in the cell PROTEIN ZIP CODES Albert B. et. al. Molecular Biology of the Cell, 4 th ed,

15 Signal sequence directed to ER The carboxyl terminus of the signal sequence is defined by a cleavage site, where protease action removes the sequence after the protein is imported into the ER. Signal sequences vary in length from 13 to 36 amino acid residues. All signal sequences have the following features: 1) about 10 to 15 hydrophobic amino acid residues 2) one or more positively charged residues, usually near the amino terminus, preceding the hydrophobic sequence 3) a short sequence at the carboxyl terminus (near the cleavage site) that is relatively polar, typically having amino acid residues with short side chains (especially Ala) at the positions closest to the cleavage site. 15

16 Examples of Signal sequences for protein translocation into ER Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed.,

17 Targeting of nuclear proteins 17 Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., 2005

18 Targeting of mitochondrial proteins Albert B. et. al. Molecular Biology of the Cell, 4 th ed,

19 Three protein translocators in the mitochondria membrane 19 Albert B. et. al. Molecular Biology of the Cell, 4 th ed, 2000

20 Protein import by mitochondria Albert B. et. al. Molecular Biology of the Cell, 4 th ed, 2000 The N-terminal signal sequence of the precursor protein is recognized by receptors of the TOM complex. The protein is thought to be translocated across both mitochondrial membranes at or near special contact sites. The signal sequence is cleaved off by a signal peptidase in the matrix to form the mature protein. The free signal sequence is then rapidly degraded (not shown) 20

21 The role of energy in protein import into the mitochondrial matrix Albert B. et. al. Molecular Biology of the Cell, 4 th ed, Albert B. et. al. Molecular Biology of the Cell, 4 th ed, 2000

22 Translocation of precursor proteins into chloroplast Albert B. et. al. Molecular Biology of the Cell, 4 th ed, 2000 A) The chloroplast signal sequence initiates translocation into the stroma through a membrane contact site by a mechanism similar to that used for translocation into the mitochondrial matrix. The signal sequence is then cleaved off, which initiates translocation across the thylakoid membrane. B) Translocation into the thylakoid space or thylakoid membrane can occur by any one of at least four routes: (1) Sec pathway, uses components that are homologs of Sec proteins, which mediate protein translocation across the bacterial plasma membrane. (2) SRP-like pathway, uses a chloroplast homolog of the signal recognition particle, or SRP (3) ΔpH pathway, it is driven by the H + gradient across the thylakoid membrane, and (4) spontaneous insertion pathway that seems to require no protein trarnslocator for membrane integration. 22

23 The signal hypothesis of protein targeting to ER Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed.,

24 How does a single-pass transmembrane protein become inserted into the ER Albert B. et. al. Molecular Biology of the Cell, 4 th ed, 2000 In addition to the start-transfer sequence as soluble proteins, however, the protein also contains a stoptransfer sequence (orange). When the stop-transfer sequence enters the translocator and interacts with a binding site, the translocator changes its conformation and discharges the protein laterally into the lipid bilayer. 24

25 Integrating of a single-pass membrane protein with an internal signal sequence into the ER (A) (B) Albert B. et. al. Molecular Biology of the Cell, 4 th ed, 2000 If there are more positively charged amino acids immediately preceding the hydrophobic core of the start-transfer sequence than there are following it, the start-transfer sequence is inserted into the translocator in the orientation. The part of the C-terminal to the start-transfer sequence will therefore be passed across the membrane. If there are more positively charged amino acids immediately following the hydrophobic core of the start-transfer sequence than there are preceding it, the start-transfer sequence is inserted into the translocator in the orientation shown here. The part of the N-terminal to the start-transfer sequence will therefore be passed across the membrane. Because translocation cannot start before a start-transfer sequence appears outside the ribosome, translocation of the N-terminal portion of the protein shown in (B) can occur only after this portion has been fully synthesized. 25

26 Integrating of a double-pass membrane protein with an internal signal sequence into the ER Albert B. et. al. Molecular Biology of the Cell, 4 th ed, 2000 An internal ER signal sequence acts as a start-transfer signal and initiates the transfer of the C-terminal part of the protein. At some point after a stop-transfer sequence has entered the translocator, the translocator discharges the sequence laterally into the membrane. 26

27 The attachment of a GPI anchor to a protein in the ER Albert B. et. al. Molecular Biology of the Cell, 4 th ed, ) Immediately after the completion of protein synthesis, the precursor protein remains anchored in the ER membrane by a hydrophobic C-terminal sequence of amino acids; the rest of the protein is in the ER lumen. 2) Within less than a minute, an enzyme in the ER cuts the protein free from its membrane-bound C terminus and simultaneously attaches the new C terminus to an amino group on a preassembled GPI intermediate. 3) The signal that specifies this modification is contained within the hydrophobic C-terminal sequence and a few amino acids adjacent to it on the lumenal side of the ER membrane; if this signal is added to other proteins, they too become modified in this way. Because of the covalently linked lipid anchor, the protein remains membrane-bound, with all of its amino acids exposed initially on the lumenal side of the ER and eventually on the cell exterior. 27

28 What happen to the newly synthesized in the ER lumen? Removing of signal sequences Folding of polypeptides Forming of disulfide bond Glycoprotein formation Play a key role in protein targeting 28

29 Glycosylation Adding of carbohydrate part into protein and there are 2 types of glycosylation 1. O-linked GalNac 2. N-linked GlcNac (Ser, Thr) (Asn) 29

30 30 Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., 2005

31 B. A. 31 Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., 2005

32 Process of N-glycosylation ER ER & Pre-Golgi Intermediates Pre-Golgi Intermediates & Golgi apparatus Transfer of lipid-liked Glc 3 Man 9 GlcNAc 2 to Asn in nascent polypeptides Removal of all three glucose residues by glucosidase I & II Transient reglucosylation by UDP-Glc: glycoprotein glucosyltransferase Removal of mannose residues by ER mannosidases Trimming of glucosylated inermediated by endomannosidase Golgi apparatus Removal of mannose residues by Golgi manosidase Elongation reaction by glycosyltransferase Adapted from Roth J. Chem. Rev. 2002, 102,

33 The role of N-linked glycosylation in ER protein folding Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; ) The ER-membrane-bound chaperone protein calnexin binds to incompletely folded proteins containing one terminal glucose on N-linked oligosaccharides, trapping the protein in the ER. 2) Removal of the terminal glucose by a glucosidase releases the protein from calnexin. 3) A glucosyl transferase is the crucial enzyme that determines whether the protein is folded properly or not: if the protein is still incompletely folded, the enzyme transfers a new glucose from UDP-glucose to the N-linked oligosaccharide, renewing the protein's affinity for calnexin and retaining it in the ER. 4) The cycle repeats until the protein has folded completely. Calreticulin functions similarly, except that it is a soluble ER resident protein. Another ER chaperone, ERp57 (not shown), collaborates with calnexin and calreticulin in retaining an incompletely folded protein in the ER. 33

34 The export and degradation of misfolded ER proteins Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; ) Misfolded soluble proteins in the ER lumen are translocated back into the cytosol, where they are deglycosylated, ubiquitylated, and degraded in proteasomes. 2) Misfolded membrane proteins follow a similar pathway. 3) Misfolded proteins are exported through the same type of translocator that mediated their import; accessory proteins that are associated with the translocator allow it to operate in the export direction. 34

35 O-glycosylation No consensus sequence for Ser and Thr Consensus for Hyl Gly X Hyl Y Arg Begins with GalNac transferase (Nacetylgalactosamine) Mannose common addition to core 35

36 Notes for glycosylation Usually be extracellular proteins or located at cell surface High structural information content molecular recognition Occurs along the secretory pathway Often stabilizes structure Difficult to get crystal structure for more than one or two carbohydrate residues 36

37 Utilization of different coats in vesicular traffic 37

38 The postulated role of SNAREs in guiding vesicular transport There are at least 20 different SNAREs in an animal cell, each associated with a particular membrane-enclosed organelles involved in the biosynthetic-secretory or endocytic pathway. These transmembrane proteins exist as complementary sets vesicle membrane SNAREs, called v-snares target membrane SNAREs, called t-snares, v-snaress and t-snares have characteristic helical domains. When a v-snares interacts with a t-snare, the helical domains of one wrap around the helical domains of the other to form stable trans-snare complexes, which lock the two membranes together. The specificity with which SNAREs interact determines the specificity of vesicle docking and fusion. SNAREs specify compartment identity and govern the orderly transfer of material during vesicle transport. 38

39 Transport from the ER to the Golgi apparatus is mediated by vesicular tubular cluster 1) Vesicular tubular clusters move along microtubules to carry proteins from the ER to the Golgi apparatus. 2) COPI coats mediate the budding of vesicles that return to the ER from these clusters. 3) The coats quickly disassemble after the vesicles have formed. 39

40 The retrieval pathway to the ER uses sorting signals (A) (B) The KDEL receptor present in vesicular tubular clusters and the Golgi apparatus, captures the soluble ER resident proteins and carries them in COPI-coated transport vesicles back to the ER. Upon binding its ligands in this low-ph environment, the KDEL receptor may change conformation, so as to facilitate its recruitment into budding COPIcoated vesicles. The retrieval of ER proteins begins in vesicular tubular clusters and continues from all parts of the Golgi apparatus. In the neutral-ph environment of the ER, the ER proteins dissociate from the KDEL receptor, which is then returned to the Golgi for reuse. 40

41 41

42 Oligosaccharide processing in the ER and the Golgi apparatus Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; Processing begins in the ER with the removal of the glucoses from the oligosaccharide initially transferred to the protein. A mannosidase in the ER membrane removes a specific mannose. Golgi mannosidase I removes three more mannoses and N-acetylglucosamine transferase I then adds an N-acetylglucosamine, which enables mannosidase II to remove two additional mannoses. This yields the final core of three mannoses that is present in a complex oligosaccharide. At this stage, the bond between the two N-acetylglucosamines in the core becomes resistant to attack by a highly specific endoglycosidase (Endo H). Since all later structures in the pathway are also Endo H-resistant, treatment with this enzyme is widely used to distinguish complex from high-mannose oligosaccharides. Additional N-acetylglucosamines, galactoses, and sialic acids are added. These final steps in the synthesis of a complex oligosaccharide occur in the cisternal compartments of the Golgi apparatus. Three types of glycosyl transferase enzymes act sequentially, using sugar substrates that have been activated by linkage to the indicated nucleotide. The membranes of the Golgi cisternae contain specific carrier proteins that allow each sugar nucleotide to enter in exchange for the nucleotide phosphates that are released after the sugar is attached to the protein on the lumenal face. 42

43 Albert B. et. al. Molecular Biology of the Cell, 4 th ed,