Proteolytic Cleavage of Proteins
Glycoproteins: Synthesis and Clinical Consequences
Acylation
Methylation
Phosphorylation
Sulfation
Prenylation
Vitamin C-Dependent Modifications
Vitamin K-Dependent Modifications
Selenoproteins
Ubiquitin and Targeted Protein Degradation
Return to The Medical Biochemistry Page
Secreted and Membrane-Associated Proteins
Proteins that are membrane bound or are destined for excretion are synthesized by ribosomes associated with the membranes of the endoplasmic reticulum (ER). The ER associated with ribosomes is termed rough ER (RER). This class of proteins all contain an N-terminus termed a signal sequence or signal peptide. The signal peptide is usually 13-36 predominantly hydrophobic residues. The signal peptide is recognized by a multi-protein complex termed the signal recognition particle (SRP). This signal peptide is removed following passage through the endoplasmic reticulum membrane. The removal of the signal peptide is catalyzed by signal peptidase. Proteins that contain a signal peptide are called preproteins to distinguish them from proproteins. However, some proteins that are destined for secretion are also further proteolyzed following secretion and, therefore contain pro sequences. This class of proteins is termed preproproteins.
Mechanism of synthesis of membrane bound or secreted proteins. Ribosomes engage the ER membrane through interaction of the signal recognition particle, SRP in the ribosome with the SRP receptor in the ER membrane. As the protein is synthesized the signal sequence is passed through the ER membrane into the lumen of the ER. After sufficient synthesis the signal peptide is removed by the action of signal peptidase. Synthesis will continue and if the protein is secreted it will end up completely in the lumen of the ER. If the protein is membrane associated a stop transfer motif in the protein will stop the transfer of the protein through the ER membrane. This will become the membrane spanning domain of the protein.
back to the top
Proteolytic Cleavage
Most proteins undergo proteolytic cleavage following translation. The simplest form of this is the removal of the initiation methionine. Many proteins are synthesized as inactive precursors that are activated under proper physiological conditions by limited proteolysis. Pancreatic enzymes and enzymes involved in clotting are examples of the latter. Inactive precursor proteins that are activated by removal of polypeptides are termed proproteins.
A complex example of post-translational processing of a preproprotein is the cleavage of prepro-opiomelanocortin (POMC) synthesized in the pituitary (see the Peptide Hormones page for discussion of POMC). This preproprotein undergoes complex cleavages, the pathway of which differs depending upon the cellular location of POMC synthesis.
Another is example of a preproprotein is insulin. Since insulin is secreted from the pancreas it has a prepeptide. Following cleavage of the 24 amino acid signal peptide the protein folds into proinsulin. Proinsulin is further cleaved yielding active insulin which is composed of two peptide chains linked togehter through disulfide bonds.
Still other proteins (of the enzyme class) are synthesized as inactive precursors called zymogens. Zymogens are activated by proteolytic cleavage such as is the situation for several proteins of the blood clotting cascade.
back to the topAcylation
Many proteins are modified at their N-termini following synthesis. In most cases the initiator methionine is hydrolyzed and an acetyl group is added to the new N-terminal amino acid. Acetyl-CoA is the acetyl donor for these reactions. Some proteins have the 14 carbon myristoyl group added to their N-termini. The donor for this modification is myristoyl-CoA. This latter modification allows association of the modified protein with membranes. The catalytic subunit of cyclicAMP-dependent protein kinase (PKA) is myristoylated.
back to the topMethylation
Post-translational methylation of proteins occurs on nitrogens and oxygens. The activated methyl donor is S-adenosylmethionine (SAM). The most common methylations are on the ε-amine of lysine residues. Additional nitrogen methylations are found on the imidazole ring of histidine, the guanidino moiety of arginine and the R-group amides of glutamate and aspartate. N-methylation is a permanent modification and there are no known mammalian enzymes that can remove the methyl group. Methylation of the oxygen of the R-group carboxylates of lgutamate and aspartate also takes place and forms methyl esters. Proteins can also be methylated on the thiol R-group of cysteine. Methylation of histones in DNA is an important regulator of chromatin structure and consequently of transcriptional activity.
As indicated below, many proteins are modified at their C-terminus by prenylation near a cysteine residue in the consensus CAAX. Following the prenylation reaction the protein is cleaved at the peptide bond of the cysteine and the carboxylate residue is methylated by a prenylated protein methyltransferase. One such protein that undergoes this type of modification is the proto-oncogene RAS.
back to the topPhosphorylation
Post-translational phosphorylation is one of the most common protein modifications that occurs in animal cells. The vast majority of phosphorylations occur as a mechanism to regulate the biological activity of a protein and as such are transient. In other words a phosphate (or more than one in many cases) is added and later removed.
Physiologically relevant examples are the phosphorylations that occur in glycogen synthase and glycogen phosphorylase in hepatocytes in response to glucagon release from the pancreas. Phosphorylation of synthase inhibits its activity, whereas, the activity of phosphorylase is increased. These two events lead to increased hepatic glucose delivery to the blood.
The enzymes that phosphorylate proteins are termed kinases and those that remove phosphates are termed phosphatases. Protein kinases catalyze reactions of the following type:
ATP + protein <——> phosphoprotein + ADP
In animal cells serine, threonine and tyrosine are the amino acids subject to phosphorylation. The largest group of kinases are those that phsophorylate either serines or threonines and as such are termed serine/threonine kinases. The ratio of phosphorylation of the three different amino acids is approximately 1000/100/1 for serine/threonine/tyrosine.
Although the level of tyrosine phosphorylation is minor, the importance of phosphorylation of this amino acid is profound. As an example, the activity of numerous growth factor receptors is controlled by tyrosine phosphorylation.
back to the topSulfation
Sulfate modification of proteins occurs at tyrosine residues such as in fibrinogen and in some secreted proteins (eg gastrin). The universal sulfate donor is 3'-phosphoadenosyl-5'-phosphosulphate (PAPS).
Since sulfate is added permanently it is necessary for the biological activity and not used as a regulatory modification like that of tyrosine phosphorylation.
back to the topPrenylation
Prenylation refers to the addition of the 15 carbon farnesyl group or the 20 carbon geranylgeranyl group to acceptor proteins, both of which are isoprenoid compounds derived from the cholesterol biosynthetic pathway. The isoprenoid groups are attached to cysteine residues at the carboxy terminus of proteins in a thioether linkage (C-S-C). A common consensus sequence at the C-terminus of prenylated proteins has been identified and is composed of CAAX, where C is cysteine, A is any aliphatic amino acid (except alanine) and X is the C-terminal amino acid. In order for the prenylation reaction to occur the three C-terminal amino acids (AAX) are first removed. Following attachment of the prenyl group the carboxylate of the cysteine is methylated in a reaction utilizing S-adenosylmethionine as the methyl donor.
In addition to numerous prenylated proteins that contain the CAAX consensus, prenylation is known to occur on proteins of the RAB family of RAS-related G-proteins. There are at least 60 proteins in this family that are prenylated at either a CC or CXC element in their C-termini. The RAB family of proteins are involved in signaling pathways that control intracellular membrane trafficking.
Some of the most important proteins whose functions depend upon prenylation are those that modulate immune responses. These include proteins involved in leukocyte motility, activation, and proliferation and endothelial cell immune functions. It is these immune modulatory roles of many prenylated proteins that are the basis for a portion of the anti-inflammatory actions of the statin class of cholesterol synthesis-inhibiting drugs due to a reduction in the synthesis of farnesylpyrophosphate and geranylpyrophosphate and thus reduced extent of inflammatory events. Other important examples of prenylated proteins include the oncogenic GTP-binding and hydrolyzing protein RAS and the γ-subunit of the visual protein transducin, both of which are farnesylated. In addition, numerous GTP-binding and hydrolyzing proteins (termed G-proteins) of signal transduction cascades have γ-subunits modified by geranylgeranylation.
back to the topVitamin C-Dependent Modifications
Modifications of proteins that depend upon vitamin C as a cofactor include proline and lysine hydroxylations and carboxy terminal amidation. The hydroxylating enzymes are identified as prolyl hydroxylase and lysyl hydroxylase. The donor of the amide for C-terminal amidation is glycine. The most important hydroxylated proteins are the collagens. Several peptide hormones such as oxytocin and vasopressin have C-terminal amidation.
back to the topVitamin K-Dependent Modifications
Vitamin K is a cofactor in the carboxylation of glutamic acid residues. The result of this type of reaction is the formation of a γ-carboxyglutamate (gamma-carboxyglutamate), referred to as a gla residue.
Structure of a gla Residue
The formation of gla residues within several proteins of the blood clotting cascade is critical for their normal function. The presence of gla residues allows the protein to chelate calcium ions and thereby render an altered conformation and biological activity to the protein. The coumarin-based anticoagulants, warfarin and dicumarol function by inhibiting the carboxylation reaction.
back to the topSelenoproteins
Selenium is a trace element and is found as a component of several prokaryotic and eukaryotic enzymes that are involved in redox reactions. The selenium in these selenoproteins is incorporated as a unique amino acid, selenocysteine, during translation. A particularly important eukaryotic selenoenzyme is glutathione peroxidase. This enzyme is required during the oxidation of glutathione by hydrogen peroxide (H2O2) and organic hydroperoxides.
Structure of the Selenocysteine Residue
Incorporation of selenocysteine by the translational machinery occurs via an interesting and unique mechanism. The tRNA for selenocysteine is charged with serine and then enzymatically selenylated to produce the selenocysteinyl-tRNA. The anticodon of selenocysteinyl-tRNA interacts with a stop codon in the mRNA (UGA) instead of a serine codon. The selenocysteinyl-tRNA has a unique structure that is not recognized by the termination machinery and is brought into the ribosome by a dedicated specific elongation factor. An element in the 3' non-translated region (UTR) of selenoprotein mRNAs determines whether UGA is read as a stop codon or as a selenocysteine codon.
back to the topUbiquitin and Targeted Protein Degradation
Proteins are in a continual state of flux, being synthesized and degraded. In addition, when proteins become damaged they must be degraded to prevent aberrant activities of the defective proteins and/or other proteins associated with those that have been damaged. One of the major mechanisms for the destruction of cellular proteins involves a complex structure referred to as the proteosome. In eukayotic cells the proteosome is found in the cytosol and the nucleus and has a large mass such that it has a sedimentation coefficient of 26S. The 26S proteosome comprises a 20S barrel-shaped catalytic core as well as 19S regulatory complexes at both ends. Degradation of proteins in the proteosome occurs via an ATP-dependent mechanism.
Proteins that are to be degraded by the proteosome are first tagged by attachment of multimers of the 76 amino acid polypeptide ubiquitin. Many proteins involved in cell cycle regulation, control of proliferation and differentiation, programmed cell death (apoptosis), DNA repair, immune and inflammatory processes and organelle biogenesis have been discovered to undergo regulated degradation via the 26S proteosome. Of clinical significance are the recent findings that deregulation of the functions of the proteosome can contribute to the pathogenesis of various human diseases such as cancer, myeloproliferative diseases, and neurodegenerative diseases.
The degradation of proteins via the 26S proteosome involves a two-step process starting with ubiquitination of the protein followed by entry into and degradation by the proteosome complex with release of ubiquitin monomers that can be re-used to tag additional proteins. The process of ubiquitin addition to the substrate protein involves multiple ubiquitin additions such that the targeted protein is polyubiquitinated.
Attachment of ubiquitin involves a series of three enzyme activities. The first, identified as E1 (also called ubiquitin-activating enzymes), activates ubiquitin in an ATP-dependent manner such that the ubiquitin is bound to the E1 enzyme via a high-energy thiol ester. The next class of enzyme, referred to as E2 (also called ubiquitin-conjugating enzymes or ubiquitin-carrier proteins), transfers the ubiquitin via an E2 thiol ester intermediate to the substrate protein. The substrate proteins are recognizable by E2 because they are bound by the third class of enzyme called E3 or ubiquitin-protein ligases. The E3 enzymes carry out the final step in the process which is the covalent attachment of ubiquitin to the substrate protein. The ubiquitin is generally transferred to the ε-amino group of an internal lysine residue in the substrate protein. There are however, examples where the ubiquitin is attached to the N-terminal amino group in a substrate protein. Whereas, ubiquitination targets proteins for degradation in the proteosome there needs to be a mechanism to ensure that inappropriately tagged proteins, i.e. those that are not destined for degradation, can be untagged. There are a family of enzymes called isopeptidases that carry out this vital function of removing ubiquitin from from proteins to which it is mistakenly attached.
Process of ubiquitination and proteosome-mediated protein degradation
Inactivation of a critical activity such as that catalyzed by the E1 enzymes results in lethality. However, there are numerous pathological states that can be attributed, in part, to mutations in recognition motifs in ubiquitination substrates and enzymes in the ubiquitination process. Disease states associated with the ubiquitin modification system can be classified into two groups. One group results from a loss of function mutation in a ubiquitin system enzyme or target protein that results in stabilization of the protein that should normally be degraded. The other group results from gain of function mutations that result in abnormal or accelerated degradation of target proteins. The most obvious disease state that could be expected to arise as a result of defective ubiquitination processes is cancer. In fact, many malignancies are known to result from defective ubiquitin-mediated degradation of growth promoting proteins such as FOS, MYC, and SRC. Likewise, inappropriate degradation of key regulators of cell cycle progression such as the tumor suppressor p53 and p27KIP1 (CDKN1B), which is an inhibitor of cyclin-dependent kinases (CDKs which control progression through the cell cycle) is also associated with various types of cancer. In addition to cancers, defective ubiquitination is found associated with neurodegenerative diseases such as Parkinson disease, Alzheimer disease, and amyotrophic lateral sclerosis.
back to the topReturn to The Medical Biochemistry Page
Michael W. King, Ph.D / IU School of Medicine / miking at iupui.edu
