Protein structure, targeting and sortingPresentation Transcript
(a) The linear sequence of amino acids (10 structure)folds into helices or sheets (20 structure) which pack into a globular or fibrous domain (30 structure). Some individualproteins self-associate into complexes (40 structure). (b) Proteins display functions that arise from specific binding interactions and conformationalchanges in the structure of a properly folded protein.
Sometimes the primary sequence of amino acids is sufficient to spontaneously direct the folding of proteins into their proper shape. However, often newly-made proteins require the help of molecular chaperones to attain their final shape. Members of the heatshock protein family (Hsp70 and Hsp60) briefly bind to and stabilize hydrophobic regions of proteins (especially rich in Trp, Phe, Leu) allowing proper folding instead of aggregation with other immature proteins. Heat-denatured proteins can be renatured through the activity of molecular chaperones and heatshock proteins are made during times of stress. A number of diseases, including Alzheimers disease, may be considered to be protein-folding diseases. Prion diseases, such as "mad cow" disease, may "self- propagate" based upon a misfolded protein that can, in turn, misfold other versions of the same protein.
Amyloid Fibers-involved in Alzheimer’s Protein amyloid fibers are often found to have a β-pleated sheet structure regardless of their sequence, leading some to believe that it is the molecules misfolding that leads to aggregation.Enzymes act on the APP (Amyloid Precursor Protein) and cut it into fragments of protein, one of which iscalled beta-amyloid and is crucial in the formation of senile plaques in Alzheimer.
(a) Many proteins fold into their proper 3-D structures with the assistance of Hsp70-like proteins (top). These chaperones transiently bind to a nascent polypeptide as it emerges from a ribosome. Proper folding of other proteins (bottom) depends on chaperonins such as the prokaryotic GroEL, ahollow, barrel-shaped complex of 14 identical 60,000-MW subunits arranged in two stackedrings. One end of GroEL is transiently blocked by the co-chaperonin GroES, an assembly of10,000-MW subunits. (b) In the absence of ATP or presence of ADP, GroEL exists in a “tight” conformational state that binds partly folded or misfolded proteins. Binding of ATP shifts GroEL to a more open, “relaxed” state, which releases the folded protein.
The ER membrane-bound chaperone protein calnexin, or a resident chaperone calreticulin binds to incompletely folded proteins, trapping the protein in the ER. Glucosyl transferasedetermines whether the protein is folded properly or not: if the protein is still incompletely folded, the enzyme renews the proteins affinity for calnexin & retains it in the ER. The cycle repeats until the protein has folded completely.
Misfolded soluble proteins in the ER lumen or membraneproteins are translocated back into the cytosol, where they are deglycosylated, ubiquitylated, and degraded in proteasomes. Misfolded proteins are exported through the same type of translocator that mediated their import; accessory proteins allow it to operate in the export direction.
(a) Enzyme E1 is activated by attachment of an ubiquitin (Ub) molecule (1) and then transfers this Ub molecule to E2 (2). Ubiquitin ligase (E3) transfers the bound Ub molecule on E2 to the side-chain-NH2 of a lysineresidue in a target protein (3).Ub molecules are added to thetarget protein by repeating steps 1–3 , forming a polyubiquitin chain that directs the tagged protein to a proteasome (4).Within this complex, the protein is cleaved into small peptide fragments (5). (b) Computer-generated image reveals that a proteasome has a cylindrical structure with a cap at each end of a core region. Proteolysis of ubiquitin-tagged proteins occurs along the inner wall of the core.
After the amino chain is made, manyproteins undergo posttranslationalprocessing (including removal ofstretches of amino acids).1. In prokaryotes, the N-formyl group is always removed in the mature protein and often the methionine and, sometimes, a number of N-terminal amino acids are cleaved away from the final protein product. Example: Proinsulin is converted to the active hormone by the enzymatic removal of a long internal section of polypeptide. The two remaining chains continue to be covalently connected by disulfide bonds connecting cysteine residues in insulin.2. Recently discovered, the process of protein splicing (analagous to RNA splicing) removes inteins and splices the exteins together to make a mature protein.
PROTEIN TARGETING AND SORTING Free and bound populations of ribosomes are active participants in protein synthesis. Free ribosomes are suspended in the cytosol and synthesize proteins that reside in the cytosol. Bound ribosomes are attached to the cytosolic side of the endoplasmic reticulum. They synthesize proteins of the endomembrane system as well as proteins secreted from the cell. Secretory proteins are released entirely into the cisternal space, but membrane proteins remain partially embedded in the ER membrane. While bound and free ribosomes are identical in structure, their location depends on the signal peptidase of proteins that they are synthesizing.
Overview of major protein-sorting pathways in eukaryotic cells.
In cotranslational import, proteins to be targeted to the ER initially have an N-terminal peptide, the ER signal sequence, translated by a cytosolic ribosome. The ER signal sequence is bound by a signal-recognition particle (SRP), a ribonucleoprotein complex composed of 6 peptides and a 300 nucleotide RNA molecule. The SRP binds to the SRP receptor to dock the ribosome on the ER membrane. When the SRP receptor binds GTP, the nascent polypeptide enters the pore.
The SRP is released with hydrolysis of the GTP. The growing polypeptide translocates through a hydrophilic pore created by one or more membrane proteins called the translocon. The ribosome fits tightly across the cytoplasmic side of the pore and the ER-lumen side is somehow closed off until the polypeptide is about 70 amino acids long. When the polypepide is complete, the signal peptidase cleave the signal to release the protein into the ER lumen while retaining the signal peptide, for a time, in the membrane. Afterwards the ribosome is released and the pore closes completely.
Integral membrane proteins are inserted into the ERmembrane as they are made, rather than into the lumen.
Major topological classes of integral membrane proteins synthesized onthe rough ER. The hydrophobic segments of the protein chain form helices embedded in the membrane bilayer; the regions outside the membrane are hydrophilic and fold into various conformations. All type IV proteins have multiple transmembrane helices. The type IV topology depicted here corresponds to that of G protein–coupled receptors: seven helices, the N-terminus on the exoplasmic side of the membrane, and the C-terminus on thecytosolic side. Other type IV proteins may have a different number of helices and various orientations of the N-terminus and C-terminus.
Other kinds of signal peptides are used to target polypeptides to mitochondria, chloroplasts, the nucleus, and other organelles that are not part of the endomembrane system. In these cases, translation is completed in the cytosol before the polypeptide is imported into the organelle. Each of these polypeptides has a “postal” code that ensures its delivery to the correct cellular location. In principle, a signal could be required for either retention in, or exit from a compartment.
Posttranslational import allows some polypeptides to enter organelles after protein synthesis. Like cotranslational importinto the ER, posttranslational import into a mitochondrion (and chloroplast) involves a signal sequence (called a transit sequence), a membrane receptor, pore-forming membrane proteins, and a peptidase.
In the mitochondrion, the membrane receptor recognizes the signal sequence directly without the intervention of a cytosolic SRP. Furthermore, chaperone proteins play several crucial roles in the mitochondrial process: o Chaperones keep the polypeptide partially unfolded after synthesis in the cytosol so that binding of the transit sequence and translocation can occur. o Chaperones drive the translocation itself by binding to and releasing from the polypeptide within the matrix, an ATP-requiring process o Chaperones often help the polypeptide fold into its final conformation.
Protein import into the mitochondrial matrix. Precursor proteinssynthesized on cytosolic ribosomes are maintained in an unfolded or partially folded state by bound chaperones, such as Hsc70 (1). After a precursor protein binds to an import receptor near a site of contact with the innermembrane (2), it is transferred into the general import pore (3). The translocating protein then moves through this channel and an adjacent channel in the inner membrane (4-5). Note that translocation occurs at rare “contact sites” at which the inner and outer membranes appear to touch.Binding of the translocating protein by the matrix chaperone Hsc70 and subsequent ATP hydrolysis by Hsc70 helps drive import into the matrix.Once the uptake-targeting sequence is removed by a matrix protease and Hsc70 is released from the newly imported protein (6), it folds into themature, active conformation within the matrix (7). Folding of some proteins depends on matrix chaperonins.
Pathways for transporting proteins from the cytosol to the inner mitochondrial membrane.
In all three pathways, proteins cross the outer membrane via the Tom40 general import pore. Proteins delivered by pathways A and B contain an N- terminal matrix-targeting sequence that is recognized by the Tom20/22 import receptor in the outer membrane. Although both these pathways use the Tim23/17 inner- membrane channel, they differ in that the entire precursor protein enters the matrix and then is redirected to the inner membrane in pathway B. Matrix Hsc70 plays a role similar its role in the import of soluble matrix proteins. Proteins delivered by pathway C contain internal sequences that are recognized by the Tom70 import receptor. A different inner-membrane translocation channel (Tim22/54) is used in this pathway. Two intermembrane proteins (Tim9 and Tim10) facilitate transfer between the outer and inner channels.
The major difference is that the Two pathways for transportinginternal targeting sequence in proteins proteins from the cytosol to thesuch as cytochrome b2 destined for mitochondrial intermembranethe intermembrane space is space. Pathway A, the major onerecognized by an innermembrane for delivery to the inter-membraneprotease, which cleaves the protein on space, is similar to pathway A forthe inter-membrane-space delivery to the inner membrane.side of the membrane. Thereleased protein then foldsand binds to its hemecofactor within theintermembranespace. Pathway Binvolves directdelivery to theintermembranespace through theTom40 generalimport pore in theouter membrane.
Two of the four pathways for transportingproteins from the cytosol to the thylakoid lumen. In these pathways, unfoldedprecursors are delivered to the stroma via the same outer-membrane proteins that importstromal-localized proteins. Cleavage of the N- terminal stromal-import sequence by a stromal protease then reveals the thylakoid- targeting sequence. At this point the two pathways diverge. In the SRP dependent pathway (left), plastocyanin and similar proteins are kept unfolded in the stromalspace by a set of chaperones and, directed by the thylakoid targeting sequence, bind to proteins that are closely related to the bacterial SRP, SRP receptor, and SecYtranslocon, which mediate movement into thelumen. After the thylakoid-targeting sequence is removed in the thylakoid lumen by a separate endoprotease, the protein folds into its mature conformation. In the pH dependent pathway (right), metal-binding proteins fold in the stroma, and complex redox cofactors are added. Two arginine residues (RR) at the N-terminus of the thylakoid-targeting sequence and a pH gradient across the inner membrane are required for transport of the folded protein into the thylakoid lumen. The translocon in the thylakoid membrane is composed of at least four proteins related to proteins in the bacterial inner membrane.
(1) Catalase and most other peroxisomal matrix proteins contain a C-terminal Import of PTS1 uptake-targeting sequence (red)peroxisomal that binds to the cytosolic receptor Pex5. (2) Pex5 with the bound matrix protein matrix interacts with the Pex14 receptor located proteins on the peroxisome membrane. (3) Thedirected by matrix protein–Pex5 complex is then PTS1 transferred to a set of membrane targeting proteins (Pex10, Pex12, and Pex2) that sequence. are necessary for translocation into the peroxisomal matrix by an unknown mechanism. (4) At some point, either during translocation or in the lumen, Pex5 dissociates from the matrix protein and returns to the cytosol, a process that involves the Pex2/10/12 complex and additional membrane and cytosolic proteins. Note that folded proteins can be imported into peroxisomes and that the targeting sequence is not removed in the matrix.
Mutations are changes in the genetic material of a cell or virus. MUTATION AND DNA REPAIR MECHANISMS.pptx These include large-scale mutations in which long segments of DNA are affected (for example, translocations, duplications, and inversions). A chemical change in just one base pair of a gene causes a spontaneous or point mutation. If these occur in gametes or cells producing gametes, they may be transmitted to future generations.
For example, sickle-cell disease is caused by a mutation of a single base pair in the gene that codes for one of the polypeptides of hemoglobin. A change in a single nucleotide from T to A in the DNA template leads to an abnormal protein.