The following points highlight the eight important steps in maturation of proteins. The steps are: 1. Protein folding and Chaperones 2. Enzymes in Protein Folding 3. Protein Cleavage 4. Glycosylation 5. Modification by Lipids 6. Protein Degradation 7. The Ubiquitin-Proteasome Pathway 8. Lysosome Mediated Proteolysis.
Step # 1. Protein Folding and Chaperones:
The experiments of Anfinsen and collaborators half a century ago, demonstrated that information for protein folding into a three-dimensional conformation is provided by its amino acid sequence. Working with the enzyme ribonuclease, they found that denatured proteins could spontaneously refold into an active conformation.
This implied that the information required to specify the three- dimensional conformation of a protein is contained in its primary amino acid sequence. Thus, the three-dimensional structure of a protein corresponds to its thermodynamically most stable conformation, determined by interactions between its constituent amino acids. Protein folding thus appeared to be a self-assembly process that did not require additional cellular factors.
Since the order of nucleotides in DNA specifies the sequence of amino acids in the polypeptide chain, it follows that the nucleotide sequence of a gene contains all the information needed to determine the three-dimensional structure of its protein product. Recent studies have however, shown that protein folding is a complex process and requires mediation by additional cellular proteins.
Proteins which act as catalysts that facilitate folding of other proteins are called molecular chaperones. Chaperones catalyse protein folding by assisting the self-assembly process. Chaperones do not convey information required for folding, and the folded conformation is determined solely by the amino acid sequence of the protein.
They appear to function by binding to unfolded or partially folded polypeptides that are intermediates along the pathway to the final folded state. In the absence of chaperones, unfolded or partially folded polypeptide chains remain unstable within the cell, would either fold incorrectly or aggregate into insoluble complexes.
In some cases, chaperones bind to nascent polypeptide chains that are still being translated on ribosomes, thus preventing incorrect folding. In the case of cytosolic proteins that are transported into mitochondria, chaperones within the mitochondrion facilitate transfer of the polypeptide chain across the membrane and its subsequent folding within the organelle.
In addition, chaperones are involved in the assembly of proteins that comprise multiple polypeptide chains, in the assembly of macromolecular structures such as nucleoplasmin, and in the regulation of protein degradation.
Many of the molecular chaperones have been identified to be heat-shock proteins (Hsp), that are expressed in cells subjected to high temperatures or other forms of environmental stress. Heat-shock proteins are thought to stabilise the refolding of proteins that have been partially denatured in response to elevated temperature.
Many of the heat-shock proteins are also expressed in cells under normal conditions and are believed to be essential for cellular functions. These proteins act as molecular chaperones both under normal conditions and in cells under environmental stress.
Step # 2. Enzymes in Protein Folding:
At least two types of enzymes are known to catalyse protein folding by breaking and reforming covalent bonds. Protein disulphide isomerase catalyses the breakage and reformation of disulphide bonds between cysteine residues in many proteins.
Disulphide bonds are generally restricted to secreted proteins and some membrane proteins that are synthesised in ribosomes bound to endoplasmic reticulum, because the cytosol contains reducing agents that maintain cysteine residues in their reduced, -SH form, thereby preventing the formation of disulphide, S-S linkages.
Peptidyl prolyl isomerase is the second enzyme that plays a role in protein folding by catalysing the isomerisation of peptide bonds that involve proline residues.
In contrast with peptide bonds between most amino acids that are almost always in the transform, proline is unusual in that the equilibrium between the cis and trans conformations of peptide bonds that precede proline residues is only slightly in favour of the trans form. Isomerisation between the cis and trans configurations of prolyl peptide bonds is catalysed by peptidyl prolyl isomerase.
Step # 3. Protein Cleavage:
Cleavage, also called proteolysis, is an important step in maturation of many proteins. During translation the initiator methionine from the amino acid terminal of the newly formed chain is removed by cleavage, soon after the amino terminus of the polypeptide chain emerges from the ribosome. After cleavage, chemical groups such as acetyl groups or fatty acid chains are added to the amino terminal residues.
The amino terminus of secreted proteins undergoes proteolytic transformation in order to facilitate translocation of these proteins across membranes in both prokaryotes and eukaryotes. The same is true of proteins destined for incorporation into the plasma membrane, lysosomes, mitochondria and chloroplasts.
These proteins contain signal sequences about 20 amino acids long at their amino terminus that direct proteins to their destinations, and are removed by proteolytic cleavage after crossing the membrane. The signal sequence contains mostly hydrophobic amino acids which are able to cross the hydrophobic interior of the membrane.
The remainder of the polypeptide chain passes through a channel in the membrane. After traversing the membrane, the signal sequence is cleaved by a specific membrane protease (signal peptidase) and the mature protein is released.
The formation of active enzymes and hormones also takes place by cleavage of larger precursors. The precursor of insulin is a longer polypeptide from which insulin is derived by two cleavages. The initial precursor (preproinsulin) contains an amino terminal signal sequence that directs the polypeptide chain to the endoplasmic reticulum. Removal of the signal sequence yields the second precursor, pro-insulin which is converted to insulin.
The two chains of insulin are held together by disulphide bonds following proteolytic removal of an internal peptide bond. The proteins of many animal viruses are derived from larger precursors.
In replication of HIV, a virus-encoded protease cleaves precursor polypeptides to form the virus structural proteins. Since the HIV protease (as also reverse transcriptase) has a central role in virus multiplication, it is an important target for development of drugs (protease inhibitors) for treatment of AIDS.
Step # 4. Glycosylation:
The addition of carbohydrates to proteins to form glycoproteins is called glycosylation. Most glycoproteins are secreted or incorporated into the plasma membrane. Some nuclear and cytosolic proteins are also glycosylated. Glycosylation is either N-linked or O-linked.
In N-linked glycoproteins, the carbohydrate is attached to the nitrogen atom in the side chain of asparagines. In O-linked glycoproteins, the oxygen atom in the side chain of serine or threonine is the site of carbohydrate attachment. Glycosylation of proteins takes place while they are traversing the endoplasmic reticulum-Golgi network.
Step # 5. Modification by Lipids:
The attachment of lipids to some proteins in eukaryotic cells facilitates their incorporation into the plasma membrane. A fatty acid, myristic acid (a 14 carbon fatty acid) is attached to the amino terminus (N-terminal glycine residue) of the polypeptide chain during translation, called N-myristoylation.
The initiator methionine is first removed by proteolysis before fatty acid addition. Proteins modified by N-myristoylation are usually associated with the inner face of the plasma membrane. When lipids are attached to the side chains of cysteine, serine or threonine it is called prenylation. The prenyl groups of lipids are attached to the sulfur atoms in the side chains of cysteine.
Importantly, many plasma membrane-associated proteins that are involved in cell growth and differentiation are prenylated. For example, the ras oncogene proteins responsible for uncontrolled growth of many human cancers. In another type of fatty acid modification, palmitic acid (a 16-carbon fatty acid) is added to sulphur atoms of the side chains of internal cysteine residues, called palmitoylation.
Proteins modified by palmitoylation associate with the cytosolic face of the plasma membrane. Lipids linked to oligosaccharides, that is glycolipids are added to the C-terminal carboxyl groups of some proteins, where they serve as anchors for attachment of proteins to the external face of the plasma membrane. The glycolipids attached to these proteins contain phosphatidylinositol, hence they are referred to as glycosyl-phosphatidylinositol or GPI anchors.
Step # 6. Protein Degradation:
Besides synthesis, degradation of proteins takes place that maintains appropriate levels of protein in cells. The half-life of proteins within cells could vary from minutes to several days. Some of the rapidly degrading proteins include the transcription factors whose rapid turnover allows their levels to change quickly in response to external stimuli, and to function as regulatory molecules.
Some proteins are rapidly degraded in response to specific signals and play a role in regulation of intracellular enzyme activity. Undesirable proteins are also degraded. Eukaryotic cells have two major pathways for degradation of proteins, the ubiquitin-proteasome pathway and the lysosomal proteolytic pathway.
Step # 7. The Ubiquitin-Proteasome Pathway:
The ubiquitin polypeptide contains 76 amino acids. Nuclear and cytosolic proteins destined for proteolyses bind to ubiquitin to the amino group of the side chain of a lysine residue.
The addition of further ubiquitins forms a multi-ubiquitin chain which is recognised and degraded by a large protease complex called the proteasome. Ubiquitin-mediated proteolysis is called ubiquitination, the process takes place in several steps and requires energy in the form of ATP. Ubiquitin is then released and can be reused in another cycle.
Step # 8. Lysosome Mediated Proteolysis:
Lysosomes are single membrane-bound organelles in eukaryotic cells that contain several digestive enzymes, all of which are active at low pH not higher than 4.5. The acidic environment within lysosomes is maintained by proton pumps in the lysosomal membrane. Lysosomes play a significant role in degrading both externally acquired proteins (by endocytosis) as well as in turnover of organelles and cytosolic proteins.
Proteins targeted for lysosomes become enclosed in autophagic vesicles derived from membranes of the endoplasmic reticulum. The vesicles fuse with the membrane of the lysosome and release the protein that is acted upon by the digestive enzymes and degraded.