Term
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Definition
| A member of the chaperonin family of molecular chaperones that is required for the proper folding of many proteins. To function properly, GroEL requires the lid-like cochaperonin protein complex GroES. |
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Term
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Definition
| A chaperonin which usually works in conjunction with GroEL. |
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Term
| By which mechanisms does the GroEL-GroES folding machine work? |
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Definition
The GroEL-GroES machine seems to work by a combination of the following mechanisms:
1) Provides the protein a region of open space within the packed cell to fold.
2) "Iterative annealing" - the GroEL double donut recognizes unfolded/misfolder proteins. How? The inside of the cavity is lined with mostly hydrophobic residues so it tends to have an affinity for nonpolar molecules, such as the ones exposed (the marker of a misfolded or unfolded protein). Then the GroES cap binds, changing the chemical environment of the inside of the chaperonin (similar to the lid of a jar being screwed on) by rotating the charged, polar groups in place of the nonpolar groups. This creates a sort of aqueous environment and forces the protein to want to "hide" its hydrophobic residues, thus "stretching" and further unfolding the protein. ATP hydrolysis provided by the cap allows ~13 seconds for the protein to fold. After 13 seconds, GroES dissociates, and the protein is released to try and refold again (either in the cytosol or another GroEL).
*Ironically, proteins have the tendency to become unfolded due to this process rather than become folded.
3) Proteins can fold faster inside the box than in solution. Cavity size and charge can affect folding rates. |
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Term
| What is the structure of GroEL? |
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Definition
| GroEL is a dual-ringed tetradecamer, with both the cis and trans rings consisting of seven subunits each. The conformational changes that occur within the central cavity of GroEL cause for the inside of GroEL to become hydrophillic, rather than hydrophobic, and is likely what facilitates protein folding. |
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Term
| Describe the process of "iterative annealing". |
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Definition
The GroEL double donut recognizes unfolded/misfolder proteins. How? The inside of the cavity is lined with mostly hydrophobic residues so it tends to have an affinity for nonpolar molecules, such as the ones exposed (the marker of a misfolded or unfolded protein). Then the GroES cap binds, changing the chemical environment of the inside of the chaperonin (similar to the lid of a jar being screwed on) by rotating the charged, polar groups in place of the nonpolar groups. This creates a sort of aqueous environment and forces the protein to want to "hide" its hydrophobic residues, thus "stretching" and further unfolding the protein. ATP hydrolysis provided by the cap allows ~13 seconds for the protein to fold. After 13 seconds, GroES dissociates, and the protein is released to try and refold again (either in the cytosol or another GroEL).
*Ironically, proteins have the tendency to become unfolded due to this process rather than become folded. |
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Term
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Definition
| Very large protein complexes designed to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that carry out such reactions are called proteases. |
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Term
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Definition
| Any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein. |
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Term
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Definition
| A small regulatory protein that has been found in almost all tissues (ubiquitously) of eukaryotic organisms. Among other functions, it directs protein recycling. Ubiquitin can be attached to proteins and label them for destruction. The ubiquitin tag directs proteins to the proteasome, which is a large protein complex in the cell that degrades and recycles unneeded proteins. |
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Term
| Ubiquitin-proteasome pathway |
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Definition
| Plays a role in the degradation of the bulk of proteins in the cytoplasmic and nuclear compartments. In this pathway proteins are targeted for degradation by covalent ligation (linking) with ubiquitin, a reaction that requires ATP. Following the binding of the first ubiquitin molecule with the epsilon-amino group of a lysine residue of the substrate protein, a polyubiquitin chain is usually formed, in which the C-terminus of each ubiquitin unit is linked to a specific Lys residue of the previous ubiquitin. Central to this pathway is the 26S proteasome, a high molecular mass multifunctional protease which requires ATP for its catalytic activity. Substrates of the 26S proteasome are not only old or damaged proteins, but also short lived proteins functioning as regulatory factors in a large array of cellular processes, such as cell cycle progression, cell growth and gene expression, inflammatory response and immune surveillance. A number of inhibitors of the catalytic activity of proteasomes have been developed and successfully employed in the study of their functional and structural properties, as well as of their involvement in different cellular processes. |
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Term
| How are proteins targeted for destruction? |
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Definition
| By the actions of three enzymes. The E1 enzyme activates ubiquitin in an ATP driven reaction that creates a high energy, covalent, thioester E1-ubiquitin bond. One of several different E2 enzymes then transfer the activated ubituitin to the target protein bound to a specific E3 enzyme, again via a thioester E2-ubiquitin intermediate. E3 then catalyzes the final transfer to the epsilon amino group of one or more specific lysine residues on the target protein. This step is usually repeated to generate polyubiquitin chains of various lengths. |
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Term
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Definition
| Activates ubiquitin in an ATP driven reaction that creates a high energy, covalent, thioester E1-ubiquitin bond. |
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Term
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Definition
| Transfer the activated ubituitin to the target protein bound to a specific E3 enzyme, again via a thioester E2-ubiquitin intermediate. |
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Term
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Definition
| Catalyzes the final transfer to the epsilon amino group of one or more specific lysine residues on the target protein. This step is usually repeated to generate polyubiquitin chains of various lengths. |
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Term
| What are the steps in protein degradation? |
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Definition
1) Ubiquitination
2) Degradation by the proteasome |
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Term
| How does the proteasome degrade proteins? |
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Definition
| Polyubiquitinated proteins bind to the 19S cap. At least four ubiquitins are required for efficient binding. ATP is then used to unfold the bound protein. The mechanism is not fully understood but it at least superficially resembles the action of some chaperones. The unfolded protein is threaded into the small channel of the 20S cylinder. Deubiquitinating enzymes cleave away the intact ubiquitin so that it can be reused. The 20S particle cleaves the protein into peptides from ~3-30 amino acids in length. Where the proteasome decides to cleave is an important but unresolved question. There are some inherent sequence preferences, but also a strong context dependence (i.e. on the structure of the protein and the pattern of amino acids both next to and relatively distant from the cleaved residue). Also the 19S cap influences the cleavage pattern. Peptides can then be transported through the ER for antigen presentation by MHC class I, or simply recycled to build new proteins. The latter process involves further degradation by free endo- and exopeptidases in the cell. Many other proteins not mentioned here participate in the ubiquitin-proteasome pathway. Identifying them and their mechanisms of action is a highly active area of research, since the ubiquitin-proteasome pathway is critical in numerous cell regulatory responses. |
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Term
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Definition
| Another name for the intact proteasome. (26S refers to how it sediments on denisty gradients). It is made up of the 20S cylinder and the 19S “cap”. |
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Term
| Where do polyubiquitinated proteins bind? What is required for effecient binding? |
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Definition
| The proteins bind to the 19S cap. At least four ubiquitins are required for effective binding. |
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Term
| What happens to the unfolded protein during degradation? |
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Definition
| It is threaded into the small channel of the 20S cylinder. The 20S particle cleaves the protein into peptides from ~3-30 amino acids in length. Where the proteasome decides to cleave is an important but unresolved question. There are some inherent sequence preferences, but also a strong context dependence (i.e. on the structure of the protein and the pattern of amino acids both next to and relatively distant from the cleaved residue). Also the 19S cap influences the cleavage pattern. |
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Term
| What happens to the ubiquitin during protein degradation? |
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Definition
| De-ubiquitinating enzymes cleave away the intact ubiquitin so that it can be reused. |
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Term
| What happens to the peptides following cleavage by the 20S particle? |
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Definition
| Peptides can then be transported through the ER for antigen presentation by MHC class I, or simply recycled to build new proteins. The latter process involves further degradation by free endo- and exopeptidases in the cell. |
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Term
| What are some diseases of the ubiquitin-proteasome pathway? |
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Definition
1) Cancer
- decreased degradation of oncoproteins
- increased degradation of tumor suppressors (p53, p27)
2) Neurodegenerative diseases
- Alzheimer's, Parkinson's, Huntington's
- observe accumulation of ubiquitinated proteins in plaques, Lewy bodies, etc.
- not clear whether cause or by-product, byt some disease-causing mutations have been identified
3) Cystic fibrosis
- clears misfolded deltaF508 CFTR
4) Autoimmune disease
- improper processing of peptide antigens |
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Term
| What are the two broad mechanisms by which defective proteins bring about disease? |
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Definition
1) Loss of protein function
2) Formation of alternate conformations which poison nearby cells, tissues, and even organs.
*Aggregation of protein molecules plays a central role in both of these mechanisms, especially in promoting ‘toxic folds’. |
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Term
| What are three ways in which protein structure and/or function can be perturbed? |
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Definition
1) Direct knockout - Mutation of a residue essential for function. Structure and stability of protein are unchanged, but can't function because a critical side chain has been altered.
2) Destabilization - Pushes the equilibrium towards the unfolded state as a result of a side chain in a tightly packed, hydrophobic core being changed to one of substantially different size, shape or charge or deletion of a stretch of amino acids.
3) Toxic conformation - A mutation shifts the conformational equilibrium not to the unfolded state, but to an incorrectly folded state. It could be as simple as mutating a surface charged residue to a hydrophobic one, which causes the protein to aggregate. |
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Term
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Definition
| In a protein, mutation of a residue that is essential for function, for example, one that is involved in substrate binding or catalysis. The structure and stability of the protein are essentially unchanged; it simply cannot function because a critical side chain has been altered. |
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Term
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Definition
| A type of protein mutation that pushes the equilibrium (shown by the upper left arrows) toward the unfolded state. The protein is so traumatized by this mutation that it cannot muster enough energy to fold. An example would be side chain in the tightly-packed, hydrophobic core being changed to one of a substantially different size, shape or charge. Another example is deletion of a stretch of amino acids. |
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Term
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Definition
| Occurs when a mutation shifts the conformational equilibrium not to the unfolded state, but to an incorrectly folded state. It could be as simple as mutating a surface charged residue to a hydrophobic one, which causes the protein to aggregate (e.g. the Glu to Val mutation responsible for sickle cell anemia). Mutations can also cause the conformation of the protein to change more substantially, as in the amyloid diseases. Aggregation is a common manifestation of misfolding, although the molecular mechanisms by which aggregation leads to cell death and disease are not known. |
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Term
| What are three general ways in which defective proteins cause disease? |
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Definition
1) Lack of a functional protein.
- Alteration of critical residue
- Destabilization (won't fold)
- Folds slowly (degraded or mislocalized in a cell)
2) Lethal conformation
- Large, highly stable, fibrous aggregates
- Pre-fibrillar misfolded species
3) Too much function - "always on" mutations |
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Term
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Definition
A tumor suppressor protein in humans that is crucial in multicellular organisms, where it regulates the cell cycle. >50% of all tumors have point mutation(s) in p53. It is the most frequently mutated protein in cancer.
p53 acts as a transcription factor when activated by DNA damage or other insult by binding specific target sites on the chromosome and activating expression of select proteins that result in cell-cycle arrest or apoptosis. |
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Term
| T or F: Most cancer cells express zero p53. |
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Definition
| False! Many cancerous cells express p53 but at least one of the p53 alleles contains a mutation that compromises its function. p53 mutations tend to be dominant negative. The physical picture of many dominant negative phenotypes is that the mutant protein interacts with the WT protein, either in a physiologically relevant fashion (e.g. mult-subunit complex) or in a non-functional manner (aggregation). p53 is the latter. |
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Term
| Dominant negative mutations |
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Definition
| Commonly the type of mutation seen in p53, have an altered gene product that acts antagonistically to the wild-type allele. These mutations usually result in an altered molecular function (often inactive) and are characterised by a dominant or semi-dominant phenotype. Marfan syndrome is another example of this type of mutation. |
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Term
| Where do most of the tumorigenic p53 mutations exist? |
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Definition
[image]
In the DNA binding domain. |
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Term
| What is the difference in effect of contact mutations and stability mutants? |
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Definition
DNA contact mutations - alter side chains that directly bind to DNA. They act by simply reducing DNA binding w/out changing overall protein structure or stability.
Stability mutants - often very distant from binding site and do not change DNA binding residues. They decrease protein stability by disrupting hydrophobic, electrostatic, H-bonding, or van der Waals interactions. |
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Term
| What happens to "less stable" p53? |
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Definition
| Less stable p53 leads to faster degradation by the ubiquitin/proteasome pathway—not enough p53 around to do the job. |
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Term
| What treatment strategy would you devise for mutations in the DNA contact region of p53? In the stability region? |
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Definition
Unfortunately, there is little that can be done for DNA binding mutants, as the mutation would require the swapping of a single amino acid in the polypeptide chain.
However, for mutations in the stability region, you can introduce a small molecule that binds to a nook unique to the p53 protein (other than the binding site) and, by Le Chatlier's principle, there would be a shift towards the native state. Or, you can introduce small molecules that disrupt interaction of p53 with HDM2 (an E3 ubiquitin ligase, which tags p53 for destruction). |
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Term
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Definition
Fatal disease with 30 year average life span characterized by thick, sticky mucus in lung, pancreas, and intestines. Related to an inability to absorb nutrients (high infant mortality) and buildup of fluids in lungs leading to infection and lung degradation.
70% of cases are caused by a deletion of Phe508 in cystic fibrosis transmembrane conductance regulator (CFTR). |
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Term
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Definition
[image]
CFTR is a member of a family of membrane proteins called ABC transporters (ABC stands for ATP binding cassette). These proteins, of which about 50 are known, pump various solutes in and out of the cell. They are involved in multidrug resistance. The complete CFTR structure has not yet been solved, although those of a few other ABC transporters have. Only the nucleotide binding domain 1 (NBD1) structure is known (see reference above). Mutations of the CFTR gene affect functioning of the chloride ion channels in these cell membranes, leading to CF. |
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Term
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Definition
[image]
(Green structure above)
An intracellular domain of CFTR and a mutational hotsot for CF. The delta(F508) mutation as well as many other disease-causing mutations are located in NBD1. Removal of Phe508, a surface aminio acid that interacts with one of the cytoplasmic loops of TMD1, results in improper trafficking and assembly of delta-F CFTR, ultimately leading to its degradation by the ubiquitin-proteasome pathway. |
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Term
| What is the effect of a F508 mutation on CFTR? |
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Definition
| Deletion of F508 has little effect on the functional properties of native CFTR. Mutant can bind nucleotide and function just as well as wild-type. However, the folding pathway is changed. Takes much longer to fold, increasing chance of misfolding (aggregation in this case) via off- pathway side reactions. Nearly all of F508 CFTR never makes it out of the endoplasmic reticulum- gets processed and degraded by ubiquitin/proteasome machinery. Much less CFTR makes it to the native state- not enough to do the job. |
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Term
| T or F: F508 mutations make it impossible for CFTR to bind nucleotides. |
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Definition
| False. F508 mutations affect the folding pathways. The mutations make the protein take much longer to fold, increasing the chance of misfolding and aggregation via off-pathway side reactions. Thus, much less CFTR makes it to the native state, resulting in not enough to do the job. |
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Term
| What are some of the treatment options for F508 CFTR? |
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Definition
1) (Totally conceptual) - Folding is much better at 25C.
2) Small organic molecules (glycerol, myoinositol, benzoflavones) have been shown to improve the yield of functional CFTR molecules (maybe by stabilizing the protein? it's not well understood).
3) Overexpressing chaperones
4) Inhibiting degradation of the ubiquitin/proteasome pathway. |
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Term
| α1-antitrypsin (α1-AT) deficiency |
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Definition
| Characterized by lung disease (emphysema) and liver disease (cirrhosis, cancer). α1-AT is a member of the serpin family (serine protease inhibitors). Serine proteases are enzymes that bind and cleave the polypeptide chain at specific locations. Neutrophil elastase is its main target and is from activated neutrophils at sites of inflammation, digests connective tissues. This is thought to facilitate movement of inflammatory cells through the extracellular stroma. If neutrophil elastase activity is unchecked by α1-AT, excessive connective tissue damage occurs which leads to emphysema. Serpins bind to target protease and prevent it from binding substrate. Serpin is then cleaved but does not readily dissociate from enzyme. If released, cleaved serpin cannot re-bind; it has lost inhibitory activity (suicide substrate). |
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Term
| What is the difference between S-type and Z-type α1-AT deficiency? |
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Definition
S-type α1-AT deficiency results from a Glu264 --> Val mutation.
Z-type α1-AT deficiency results from a Glu342 --> Lys mutation. |
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Term
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Definition
| Enzymes that bind and cleave the polypeptide chain in which one of the amino acids in the active site of the enzyme is serine. |
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Term
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Definition
| (α1-AT) A group of proteins with similar structures that were first identified as a set of proteins able to inhibit proteases. |
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Term
| How does α1-AT lead to disease? |
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Definition
| Neutrophil elastase is from activated neutrophils at sites of inflammation, digests connective tissues. This is thought to facilitate movement of inflammatory cells through the extracellular stroma. If neutrophil elastase activity is unchecked by α1-AT, excessive connective tissue damage occurs which leads to emphysema. |
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Term
| Where is α1-AT found in the body? |
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Definition
| α1-AT is found circulating in the plasma. |
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Term
| What is the main target of α1-AT? |
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Definition
| Neutrophil elastase-- α1-AT clears away scar tissue. |
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Term
| What is the mechanism of uncleaved α1-AT? |
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Definition
| The "molecular mousetrap". It uses "bait" (the reactive center loop (RCL)) to attract neutrophil elastase. Target protease binds the reactive center loop (RCL) of α1-AT and cleaves it at residue P1. The RCL is a frustrated beta strand- it would prefer to be in the middle of the α1-AT beta-sheet. Once cleaved, it can do so. The RCL inserts into the beta-sheet and drags the protease along with it. It then makes the protease less unstable by "stretching it" or something. In order for the loop to insert, the sheet has to split in the middle and open up. The enzyme is then chopped by by soluble proteases. |
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Term
| What are some inherent problems with the unique α1-AT inhibitory mechanism? |
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Definition
| The central beta sheet must be flexible enough to accept the RCL as part of the normal inhibitory process. If the beta sheet is structurally weakened, then it may be unusually prone to strand insertion, at premature or inopportune times. |
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Term
| T or F: α1-AT can regenerate after binding to proteases. |
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Definition
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Term
| What is the inherent problem with the S-type α1-AT mutation? |
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Definition
| Glu264 is hydrogen bonded to Tyr38 in wild-type α1-AT. The Glu264-Tyr38 pair is located underneath the central beta- sheet in a location that has become known as the “shutter region”. Removal of that H-bond may weaken the central beta sheet. If the beta sheet is structurally weakened, then it may be unusually prone to strand insertion, at premature or inopportune times. |
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Term
| How does mutant α1-AT affect the liver? |
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Definition
| Because α1-AT is synthesized in the liver, there is an over abundance and the molecules are at an increased risk of bumping into each other. Polymerization occurs when the central beta sheet (blue protein) aberrantly opens and allows part of the reactive loop of a second protein to insert into the lower portion of the sheet. This causes the neighbor to insert into its neighbor, and so on, leading to a "beads on a string" polymer effect. These are very stable, and it becomes difficult for the lungs to clear these aggregates. |
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Term
| What therapies exist to treat α1-AT serpinopathies? |
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Definition
[image]
Polymerization can be blocked and even reversed by various peptides that correspond to portions of the RCL. P1-P14 indicate the amino acid numbers of the RCL. P2-P8 peptides are especially effective (shown in cyan at left). They bind in the pocket of the beta sheet where part of the RCL from a second antitrypsin molecule would occupy (in the polymer). Peptides as short as four amino acids inhibit polymerization in vitro. A somewhat unexpected finding is that the most effective peptide found so far, Trp-Met-Asp-Phe, is not part of the RCL sequence. The X-ray structure showed that it binds by inserting the hydrophobic side chains into the beta sheet. The P3-P8 peptide from antithrombin (TAVVIA) and the P2-P7 peptide from antitrypsin (FLEAIG) can partially reverse polymerization of WT antithrombin and Z-type antitrypsin, respectively, as demonstrated by the native polyacrylamide gel at right. |
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