Proteinfaltung und post-translationale Prozessierung Jonathan Howard Institute for Genetics

Der Aufbau einer Polypeptidkette

Eigenschaften der Seitenketten

Umdrehungspotential bei Polypeptidketten

Ramachandran plot: Umdrehungspotential ist begrenzt

Wechselwirkungen zwischen Proteinsträngen

Faltung, Entfaltung und Wiederfaltung eines Proteins

Wiederholte “Sekundarstrukturen”: Die a-Helix

Wiederholte “Sekundarstrukturen”: b-Stränge und b-Faltblätter

Proteine aus verschiedenen Sekundarstrukturelementen aufgebaut

Co-translationale Proteinfaltung

Chaperone helfen bei der Proteinfaltung Heatshock Protein 70 (hsp70, Mitglied einer Familie von hsp70 Proteinen) bindet an kurze hydrophobe Sequenzabschnitte (7 AS). Hsp70 besitzt eine ATPase Domäne, die anhaftendes ATP zu ADP spaltet, und so eine enge Anlagerung bewirkt, die die Faltung verunmöglicht. Nukleotid-Austausch-Faktoren setzen ADP frei und erlauben erneute ATP Anlagerung, so bekommt das Protein wieder Spielraum und kann seine Faltung fortsetzen. Figure 6-83. The hsp70 family of molecular chaperones. These proteins act early, recognizing a small stretch of hydrophobic amino acids on a protein's surface. Aided by a set of smaller hsp40 proteins, an hsp70 monomer binds to its target protein and then hydrolyzes a molecule of ATP to ADP, undergoing a conformational change that causes the hsp70 to clamp down very tightly on the target. After the hsp40 dissociates, the dissociation of the hsp70 protein is induced by the rapid re-binding of ATP after ADP release. Repeated cycles of hsp protein binding and release help the target protein to refold, as schematically illustrated in Figure 6-82. Figure 3-15. Chaperone-mediated protein folding. (a) Many proteins 1 fold into their proper three-dimensional structure with the assistance of Hsp70, a molecular chaperone that transiently binds to a nascent polypeptide as it emerges from a ribosome. Proper folding of some proteins 2 also depends on the chaperonin TCiP, a large barrel-shaped complex of Hsp60 units. (b) GroEL, the bacterial homolog of TCiP, is a barrel-shaped complex of 14 identical 60,000-MW subunits arranged in two stacked rings. In the absence of ATP or presence of ADP, GroEL exists in a “tight” conformational state (left) that binds partially folded or misfolded proteins. Binding of ATP shifts GroEL to a more open, “relaxed” state (right), which releases the folded protein. [Part (b) from A. Roseman et al., 1996, Cell 87:241. Figure 17-27. Folding of the hemagglutinin (HA) precursor polypeptide HA0 and formation of an HA0 trimer within the ER. While the nascent chain is still growing, two protein-folding catalysts, calnexin and calreticulin, associate with it, and three disulfide bonds form in the globular head domain. Following completion of translation, three additional disulfide bonds form and possibly rearrange in the monomer. Three HA0 chains then interact with each other, initially via their transmembrane α helices; this association apparently triggers the formation of a long stem containing one α helix (dark rod) from the luminal part of each HA0 polypeptide. Finally, interactions between the three globular heads occur, generating the mature trimeric spike. Calnexin and calreticulin bind to N-linked oligosaccharides with a single glucose residue on unfolded protein segments, thereby promoting the proper folding and assembly of newly synthesized glycoproteins such as HA (see Figure 17-36). [Adapted from sketch by Dan Hebert and Ari Helenius.See M-J. Gething et al., 1986, Cell 46:939; U. Tatu et al., 1995, EMBO J. 14:1340; and D. Hebert et al., 1997, J. Cell. Biol. 139:613.]

Chaperone helfen bei der Proteinfaltung Heatshock Protein 60 (hsp60, Mitglied einer Familie von hsp60 Proteinen, heisst GroEL in Bakterien und TCP-1 in Vetebraten) hat eine faßförmige Struktur, in deren Pore wiederholte Faltungsversuche durch- geführt werden können - ein Zyklus dauert 15 sec. Hsp60 arbeitet mit der vollständigen Proteinkette, also später in der Biosynthese als hsp70, das sich bereits an wachsende Proteinketten anlagert. Auch Hsp60 ist eine ATPase. Figure 6-84. The structure and function of the hsp60 family of molecular chaperones. (A) The catalysis of protein refolding. As indicated, a misfolded protein is initially captured by hydrophobic interactions along one rim of the barrel. The subsequent binding of ATP plus a protein cap increases the diameter of the barrel rim, which may transiently stretch (partly unfold) the client protein. This also confines the protein in an enclosed space, where it has a new opportunity to fold. After about 15 seconds, ATP hydrolysis ejects the protein, whether folded or not, and the cycle repeats. This type of molecular chaperone is also known as a chaperonin; it is designated as hsp60 in mitochondria, TCP-1 in the cytosol of vertebrate cells, and GroEL in bacteria. As indicated, only half of the symmetrical barrel operates on a client protein at any one time. (B) The structure of GroEL bound to its GroES cap, as determined by x-ray crystallography. On the left is shown the outside of the barrel-like structure and on the right a cross section through its center. (B, adapted from B. Bukace and A.L. Horwich, Cell 92:351–366, 1998.)

Abbau von nicht richtig gefaltenen Proteinen Bis zu einem Drittel aller neu synthetisierten Proteinketten werden gleich wieder abgebaut, weil sie in den verschiedenen Qualitätskontrollen scheitern. Aggregatbildung

Aggregatbildung Unlösliche Proteinaggregate sind ein besonderes Problem für langlebige Zellen wie z.B. Nervenzellen: Verschiedene neurodegenerative Erkrankungen lassen sich kausal auf die fortlaufende Ablagerung von unlöslichen Protein-Aggregaten zurückführen: Alzheimersche Krankheit - ß Amyloid Rinderwahnsinn - Prionen Veitstanz - Huntingtin mit Polyglutaminsequenzen Figure 6-85. The cellular mechanisms that monitor protein quality after protein synthesis. As indicated, a newly synthesized protein sometimes folds correctly and assembles with its partners on its own, in which case it is left alone. Incompletely folded proteins are helped to refold by molecular chaparones: first by a family of hsp70 proteins, and if this fails, then by hsp60-like proteins. In both cases the client proteins are recognized by an abnormally exposed patch of hydrophobic amino acids on their surface. These processes compete with a different system that recognizes an abnormally exposed patch and transfers the protein that contains it to a proteasome for complete destruction. The combination of all of these processes is needed to prevent massive protein aggregation in a cell, which can occur when many hydrophobic regions on proteins clump together and precipitate the entire mass out of solution. Cells quickly remove the failures of their translation processes. Recent experiments suggest that as many as one-third of the newly made polypeptide chains are selected for rapid degradation as a result of the protein quality control mechanisms just described. The final disposal apparatus in eucaryotes is the proteasome, an abundant ATP-dependent protease that constitutes nearly 1% of cellular protein. Present in many copies dispersed throughout the cytosol and the nucleus, the proteasome also targets proteins of the endoplasmic reticulum (ER): those proteins that fail either to fold or to be assembled properly after they enter the ER are detected by an ER-based surveillance system that retrotranslocate them back to the cytosol for degradation (discussed in Chapter 12).

Wo in der Zelle werden Proteine gefaltet? Figure 17-1. Overview of sorting of nuclear-encoded proteins in eukaryotic cells. All nuclear-encoded mRNAs are translated on cytosolic ribosomes. Ribosomes synthesizing nascent proteins in the secretory pathway 1 are directed to the rough endoplasmic reticulum (ER) by an ER signal sequence 2 . After translation is completed in the ER, these proteins move via transport vesicles to the Golgi complex dlccirc3; from whence they are further sorted to several destinations 4a, 4b, 4c . After synthesis of proteins lacking an ER signal sequence is completed on free ribosomes 1 , the proteins are released into the cytosol 2 . Those with an organelle- specific uptake-targeting sequence are imported into the mitochondrion 3a , chloroplast 3b , peroxisome 3c , or nucleus 3d . Mitochondrial and chloroplast proteins typically pass through the outer and inner membranes to enter the matrix or stromal space, respectively. Some remain there, and some 4a are sorted to other organellar compartments. Unlike mitochondrial and chloroplast proteins, which are imported in a partially unfolded form, most peroxisomal proteins cross the peroxisome membrane as fully folded proteins 4b . Folded nuclear proteins, often in the form of ribonucleoprotein particles, enter through visible nuclear pores by processes discussed in Chapter 11 4c.

Signalsequenzen leiten Proteine in verschiedene Kompartimente Figure 17-1. Overview of sorting of nuclear-encoded proteins in eukaryotic cells. All nuclear-encoded mRNAs are translated on cytosolic ribosomes. Ribosomes synthesizing nascent proteins in the secretory pathway 1 are directed to the rough endoplasmic reticulum (ER) by an ER signal sequence 2 . After translation is completed in the ER, these proteins move via transport vesicles to the Golgi complex dlccirc3; from whence they are further sorted to several destinations 4a, 4b, 4c . After synthesis of proteins lacking an ER signal sequence is completed on free ribosomes 1 , the proteins are released into the cytosol 2 . Those with an organelle- specific uptake-targeting sequence are imported into the mitochondrion 3a , chloroplast 3b , peroxisome 3c , or nucleus 3d . Mitochondrial and chloroplast proteins typically pass through the outer and inner membranes to enter the matrix or stromal space, respectively. Some remain there, and some 4a are sorted to other organellar compartments. Unlike mitochondrial and chloroplast proteins, which are imported in a partially unfolded form, most peroxisomal proteins cross the peroxisome membrane as fully folded proteins 4b . Folded nuclear proteins, often in the form of ribonucleoprotein particles, enter through visible nuclear pores by processes discussed in Chapter 11 4c.

Signalsequenzen leiten in verschiedene Kompartimente Figure 17-1. Overview of sorting of nuclear-encoded proteins in eukaryotic cells. All nuclear-encoded mRNAs are translated on cytosolic ribosomes. Ribosomes synthesizing nascent proteins in the secretory pathway 1 are directed to the rough endoplasmic reticulum (ER) by an ER signal sequence 2 . After translation is completed in the ER, these proteins move via transport vesicles to the Golgi complex dlccirc3; from whence they are further sorted to several destinations 4a, 4b, 4c . After synthesis of proteins lacking an ER signal sequence is completed on free ribosomes 1 , the proteins are released into the cytosol 2 . Those with an organelle- specific uptake-targeting sequence are imported into the mitochondrion 3a , chloroplast 3b , peroxisome 3c , or nucleus 3d . Mitochondrial and chloroplast proteins typically pass through the outer and inner membranes to enter the matrix or stromal space, respectively. Some remain there, and some 4a are sorted to other organellar compartments. Unlike mitochondrial and chloroplast proteins, which are imported in a partially unfolded form, most peroxisomal proteins cross the peroxisome membrane as fully folded proteins 4b . Folded nuclear proteins, often in the form of ribonucleoprotein particles, enter through visible nuclear pores by processes discussed in Chapter 11 4c.

Proteintranslokation in das endoplasmatische Reticulum Biogenese der sekretorischen Proteine und Membranprotein

Proteintranslokation in das endoplasmatische Reticulum

Proteintranslokation in das endoplasmatische Reticulum Signal recognition particle

Proteintranslokation in das endoplasmatische Reticulum The SRP cycle

Translokation in das Mitochondrion

Translokation in das Mitochondrion

Translokation in das Mitochondrion

Translokation in das Mitochondrion

Ende der Vorlesung 31. Mai 2011-3. Juni 2011