You are studying a neurodegenerative disease whose genetic cause maps to a mutat

Question

You are studying a neurodegenerative disease whose genetic cause maps to a mutation in the p175 gene, which from BLAST searches may belong to the family of 7-transmembrane cell surface receptors. [See scheme of the protein below with each pre- dicted transmembrane segment shown with its most basic flanking region marked.] You quickly generate a polyclonal antibody to human p175, because you want to use Western blotting to show that the disease results from a lack of expression, or perhaps truncation, of the receptor. Unexpectedly though, the Westerns show that patient (“P”) and normal (“N”) nervous tissue express this protein at comparable levels and that the protein is apparently the same size whether the disease is present or not (see below, left 2 lanes). As a side experiment, you treated the intact, live cells with trypsin prior to Western analysis (below, right 2 lanes). This provided the only clue so far that anything was different about p175 in disease--p175 in normal cells was cleaved into several smaller fragments, whereas p175 in patient cells was trypsin insensitive.

(1) Why does the protein product get digested into four discreet products though there are seven membrane anchors? If you inserted an epitope tag at the amino terminus and expressed the protein in cultured cells and did the same experiment, would the labeled species get smaller after trypsin treatment, not change, or disappear altogether?

(2) Explain what this experiment tells you about the protein in the diseased nerve cells. Assuming that the protein in fact encodes some kind of hormone receptor normally present on the cell surface, what kind of mutation might cause the disease and how would it affect the proteins’ function as a receptor?

(3) Now suppose you took patient and normal cell extracts from the minus trypsin samples and digested them with and without endoglycosidase H prior to the Western analysis. What would you expect to see and how would that be consistent with your hypothesis about the disease in A? Is there a precedent for diseases like this?

Schematic of p175 with predicted membrane anchors TM1 TM3 TM4 TM2 Western on extracts of patient and normal cell samples patient preincubation with trypsin TM5 TM6 TM7

Explanation / Answer

(1).Possible reason why we get 4 bands is beacuse there are 4 trypsin digest sites in the entire sequence. Trypsin cleaves at the C terminal of Lysine and Arginine residues.

It's good to keep in mind that protein digestion is not as simple as eating an egg and magically getting amino acids. A large protein molecule breaks down via a few intermediate steps, in the stomach and in the small intestine, before it becomes the tiny amino acids. So, let's take a look at how proteins are broken down by your digestive system.

Protein digestion begins in the stomach with the action of an enzyme that we previously learned about called pepsin. Pepsin is the active protein-digesting enzyme of the stomach. When pepsin acts on the protein molecule, it breaks the bonds that hold the protein molecule together, called peptide bonds. So, you can think of pepsin as the enzyme that breaks peptide bonds. When these bonds are broken, you get chains of amino acids linked together called polypeptides. Since we know that the prefix 'poly' means 'many,' we can easily recall that a polypeptide is many amino acid units joined together. These polypeptides then move into your small intestine, where digestion will be completed by additional enzymes.

In the small intestine, pancreatic enzymes that we previously learned about, called trypsin, chymotrypsin, and carboxypeptidase, really go to work breaking down the polypeptides. These enzymes enter the duodenum via the pancreatic duct. These pancreatic enzymes are helped by the brush border enzymes. We previously learned that the brush border enzymes are special enzymes found on the microvilli of the small intestine that complete digestion.

The peptide bonds holding the polypeptides together continue to be hydrolyzed, or broken down, and result in smaller units called peptides. Peptides are simply defined as two or more amino acids linked together. Enzymes continue to break down polypeptides and peptides into amino acids. Because amino acids are very small, they are able to be absorbed through the small intestine lining and into your bloodstream.

(2).

The capability to form amyloidal protein structures that are considered to be genetic is from the findings that an increasing number of proteins show no signs of protein related diseases. It has been found that amyloidal proteins can be converted from its own protein that has a function rather than disease- related characteristics in living organisms.

In these protein mutations, different factors that affect the formation of amyloid fibril formation and different chains form amyloid fibrils at different speeds. In different polypeptide molecules, hydrophobicity, hydrophillicity, changes in charge, degree of exposure to solvent, the number of aromatic side chains, surface area, and dipole moment can affect the rate of aggregation of protein. It has been found that the concentration of protein, pH and ionic strength of the solution the protein is in as well as the amino acid sequence it is in determines the aggregation rate from the unstructured, non-homologous protein sequences.

As the hydrophobicity of the side chains increases or decreases can change the tendency for the protein to aggregate.

Charge in a protein can create aggregations through interaction of the polypeptide chain with other macromolecules around it. Also, the low tendency for -sheets to form along with the high tendency for -helixes to form contributes in facilitating amyloid formation.

It was found that the degree in which the protein sequence are exposed to solvent tend to affect the formation of amyloids. Proteins that are exposed to solvent seem to promote aggregation. Even though some other parts of the protein that had a high tendency to aggregate were not involved in the aggregation, they seem to at least be partially unexposed to the solvent but other regions that were exposed to solvent that were not involved in the aggregation had a low tendency to form amyloid fibrils.

It has even been raised that protein sequences have evolved over time to avoid forming clusters of hydrophobic residues by alternating the patterns of hydrophobic and hydrophillic regions to lower the tendency for protein aggregation to occur.

(3). Endoglycosidase H (Endo H, New England Biolabs) sample digestion was performed for 24 h at 37 °C with 5000 units of Endo H in G5 reaction buffer (New England Biolabs). For peptide-N-glycosidase F (PNGase F, New England Biolabs) cleavage, samples were incubated in G7 reaction buffer (New England Biolabs) with 2.5% Nonidet P-40 (ANew England Biolabs) and 500 units of PNGaseF for 24 h at 37 °C. The reactions were stopped with 100 l of 2 M Tris, and the fluorescence was recorded immediately.

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