Currently approximately 34 % of all approved clinical drugs in the US are targeting over a hundred different GPCRs[18]while ten years earlier the number was 27 % [43]. GPCR drug discovery is the most intensively studied area in drug research with significant progress constantly made in order to identify new targets.
It is worth noting that proteins can function in more ways than their designated group indicates. For instance, recent studies have shown that many proteins of the GPCR family have secondary functions as lipid scramblases – they actively induce lipid translocation across the bilayer, thus scrambling the lipid composition of the two opposing leaflets[12, 44]. One such GPCR is rhodopsin, the protein mainly in- volved in visual transduction. The transmembrane alpha-helices provide a favorable environment for the hydrophilic lipid head groups giving them easier entry to the membrane interior. There are also indications that the peripheral myelin protein P2, a member of the fatty acid binding (FABP) family, acts as a lipid transporter as is typical for fatty acid binding proteins, but also has an important role in holding the myelin sheath – a membrane complex vital for the nervous system – together [6]. These secondary functions of both rhodopsin and P2 are further explored in the following chapters of this Thesis.
create proteins. The genetic information is transported from the DNA chain to ribosomes via messenger RNA (mRNA), which makes a copy of a certain part of the DNA chain into its single-stranded structure. With the help of transfer RNA (tRNA), which acts as a transporter for the required amino acids, and ribosomal RNA (rRNA) located at the ribosomes, protein synthesis is able to take place.
During the RNA translation process, the ribosomes read the genetic chain en- coded in the nucleotides of the mRNA chain three nucleotides at a time. These nucleotide triplets are referred to as codons, which specify a certain amino acid, or the beginning and the end of the translation process. Since there are 64 possible nucleotide triplets, but only 20 different amino acids, a specific amino acid can be obtained from multiple nucleotide combinations.
Protein synthesis is initiated at the N-terminus of the amino acid chain, always containing methionine, from a specific start codon found in the mRNA. The ribo- some then reads the following nucleotide triplet and the tRNA attaches the corre- sponding amino acid to the previous one by a peptide bond. This continues until the ribosome reads a nucleotide triplet containing a stop codon, upon which the finished protein is released from the ribosome. The proteins are then folded and transported to their final destinations. Misfolded proteins are either captured by molecular chap- erones which attempt to refold them, or degraded back to amino acids in order to prevent them from causing harm.
Mutations
Mutations in protein structure occur mainly due to altered nucleotide base sequences in the DNA. The DNA replication process involves unwinding the double helix un- til new nucleotide pairs are generated for both divided halves. In rare cases, wrong types of nucleotides or additional nucleotide pairs are inserted into the newly gen- erated DNA, which modifies the genetic code. Enzymes performing verification of these new chains are able to correct most of these errors, but some mismatched nu- cleotide base pairs inevitably remain, resulting in a mutated DNA chain after the next cell division. If the mutation is located in the part of the DNA encoding the structure of a certain protein, the protein synthesis process produces a mutated pro- tein if the codons read from the mRNA make sense. Errors may also occur in a similar fashion when reading a correct mRNA sequence. The ribosomes operate in
a rapid fashion, so the tRNA molecules may occasionally attach wrong amino acids into the polypeptide chain. Additional proofreading mechanisms along the transla- tion process, so most mutated proteins are rapidly identified and discarded.
Several different types of mutations of the polypeptide chain may occur as a re- sult. Most common mutation types are substitutions – mutations of the nucleotide sequence where one nucleotide base is replaced with another. A silent mutation oc- curs when a change in one nucleotide base in a codon does not affect the generated amino acid. This is possible because certain amino acids can be obtained from several different nucleotide triplet combinations. Silent mutations are generally harmless as they do not affect the protein structure, but they can slow down the protein synthe- sis process.
Another common substitution is a missense mutation. This occurs when a nu- cleotide base of a codon is replaced with another in such a way that the resulting amino acid differs from the protein wild type. This is the main cause of point mu- tations found in proteins and can have significant effects in the protein function. As the initial effect of the mutation is quite small – the protein essentially retains its size and often folds properly – point mutated proteins are the most likely ones to pass post-translational inspection and find their way into the target cells.
The third type of substitution is called a nonsense mutation. It occurs when re- placing a nucleotide base in a codon changes the meaning of the codon from inserting a new amino acid to signal termination of translation. There are three different stop codons identified by the ribosome that signal the end of the protein. Missense mu- tations usually result in a truncated protein structure easily recognized by enzymes and dissolved back into amino acids.
Another common way the nucleotide base sequence can be altered is via frameshift mutation. It happens when additional nucleotide base pairs are inserted (or some are removed) into the replicated DNA. This in turn causes the nucleotides to be shifted in either direction. As the nucleotides in the mRNA are read in triplets, each triplet beginning from the insertion point is different from the wild type, resulting in ev- ery codon changing its original meaning. If three additional nucleotide base pairs are inserted, it merely corresponds to the addition of a single amino acid. Due to its nature, frameshift mutations usually involve the change of multiple amino acids and an early stop codon, resulting in an unusable protein.
The effects of mutations
While protein mutations generally have negative effects on the function of the cell, there are also many examples of beneficial mutations with positive effects. One well- known example is the ability to digest lactose contained in milk[45]. Human infants generate the enzyme lactase which breaks down the lactose sugar chains contained in milk. In most mammals, the activity of the enzyme greatly subsides with time when other food replaces milk as the main source of nutrition. However, many human populations have a point mutation in the gene encoding the enzyme, keeping the enzyme active throughout their lives. The lack of this mutation is known as lactose intolerance.
One example of a well-known mutation with both beneficial and negative effects is sickle-cell anemia[46]. A single point mutation in the haemoglobin gene changes the shape of the red blood cells from their typical fluid round shape into a rigid sickle shape. The sickle shaped cells are prone to clumping together at junctions of the blood vessels, thus blocking blood flow and resulting in many health problems such as swelling and stroke. On the other hand, the unnatural shape of the red blood cells makes them more resistant to several blood diseases such as malaria[47]. Geo- graphically, high representation of the sickle-cell gene in the population is generally found in areas where malaria has a high transmission intensity[48].
Most of the occurring mutations do not have such highly visible effects, but in- stead are seen only at a cellular level, facilitating or hindering cellular functions. For instance, a point mutation at a ligand-binding site of a protein may either accelerate the binding process or prevent the binding altogether[49]. A GPCR with the ability to facilitate lipid flip–flop across the lipid bilayer could become even more effective if additional polar amino acids appear along the lipid translocation path[50]. A fatty- acid binding protein found in the myelin sheath of the nervous system becomes more flexible when a point mutation occurs near the lid region of its barrel-like structure, which in turn increases its lipid stacking ability[51].
With recent advances in gene therapy, point mutated proteins can also be arti- ficially generated in laboratory conditions. Generating point mutated proteins can be an interesting alleyway into further studying the structure and function of many proteins and protein complexes. Many signaling proteins, among others, naturally form dimer structures – two identical proteins attached to each other by an interface
in the side of the protein[52]. One often asked question regarding protein func- tion is whether these types of proteins are able to function alone or if they need to dimerize in order to perform in their designated role. This is usually the case when the functional site is located at the dimerization interface. A way to separate two normally dimerized proteins is to generate point mutations at the dimerization in- terface which in turn reduces the ability of the two proteins to attach to each other.
These point mutated proteins can then be reconstituted into vesicles as monomers and their function studied. If the protein still works as intended in its monomeric form, it can be deduced that dimerization or the dimerization interface is not essen- tial for its function.
One such example can be found in opsin, a GPCR acting as a phospholipid scram- blase transporting lipids from one membrane leaflet to the other. Several different lipid translocation pathways have been suggested, one of them at the dimerization in- terface of two opsin proteins. A recent study conducted by using the aforementioned method showed that the point mutated dimerization interface (transmembrane he- lix IV) is not the main location of the scrambling activity and monomeric opsin was able to facilitate lipid flip–flop at a sufficient level[53]. In a nutshell, performing point mutations on specific amino acids at putative functional interfaces can gener- ally be used as a method of researching whether the interface in question is relevant for the studied function of the protein.
Artificially created proteins and targeting of mutated genes can also be used to treat certain protein-altering diseases. For instance, proteins facilitating programmed cell death are often disabled upon the onset of cancer. Gene therapy applied to pro- teins involved in the apoptotic pathway, such as p53, are used to heal the lost ability of the protein to aid in the controlled degradation of the sick cancer cell[54].
3 MYELIN SHEATH AND THE P2 PROTEIN
In this part of the Thesis we focus on a central part of the nervous system. The myelin sheath is the membrane layer that surrounds the axons transmitting neural signals across the body. A major component of this specific type of membrane is the peripheral membrane protein P2, which is a member of the fatty-acid binding protein (FABP) group. The Papers I-III presented in this Thesis, conducted in col- laboration with an experimental group, have provided new insight on the dynamics of the P2 protein, its function in the myelin sheath and how certain point mutations can be the root of many nervous diseases. In this chapter, we present an overview on the structure and function of the nervous system and the myelin sheath surrounding its axons, introduce the P2 protein, and discuss the many different diseases affecting the nervous system.