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Chapter 5 Protein Synthesis, Processing, and Trafficking 47

mature products is irreversible. Here are some features of the most Protein γ-Carboxylation: A Rare Posttranslational
common protein modifications: BOX 5.1 Modification Crucial for Life
• Acetylation: ribosome-associated Met-aminopeptidases and acety- γ-Carboxylation of glutamic acid residues in the Gla domain serves to
lases cotranslationally cleave off the N-terminal methionine and coordinate calcium ions and is essential for the proper biologic activity
acetylate the second residue if it is small (Gly, Ala, Ser, Thr, Cys. of factors involved in blood coagulation. These factors are prothrombin,
Pro or Val). Approximately 90% of human proteins are factors VII, IX and X which are involved in the coagulant response, and
N-acetylated (acetyl group: COCH 3). In general, the N-terminal protein C and S which play roles in an antithrombotic pathway that
residue affects the lifetime of a protein. limits coagulation. Bone proteins osteocalcin and matrix Gla protein
also require processing by γ-carboxylation for full activity.
This posttranslational modification is catalyzed by γ-glutamyl carbox-
Another type of acetylation is posttranslational and occurs on the ylase, an endoplasmic reticulum (ER) membrane protein. Its obligate
α-amino group of the side chain of lysine. This modification abolishes cofactor, reduced vitamin K, is produced by the action of vitamin
the positive charge of this amino acid. The N-terminal tails of his- K-epoxide reductase (VKOR) which converts oxidized vitamin K to the
tones, are targets of acetylation that is carried out by histone acetyl reduced form. The activity of VKOR is inhibited by warfarin, a potent
transferases (HATs) and is reversed by histone deacetylases (HDACs). anticoagulant compound. γ-Carboxylase homozygous null mutants
The degree of histone acetylation increases transcriptional activity by manifested dramatic effects on development with partial midembryonic
relaxing chromatin structure. loss and postnatal hemorrhage. Similar effects were observed in pro-
thrombin or factor V-deficient mice. Thus, the results of these studies
• Methylation: is the reversible addition of a methyl group (CH 3) to have suggested that the functionally critical substrates for γ-carboxylation
lysine or arginine residues. This modification abolishes the posi- are primarily restricted to components of the blood coagulation
cascade. These results highlight the importance of a rare protein
tive charge and increases the hydrophobicity of the protein. modification for blood coagulation.
Histone methylation by histone methyltransferases affects tran-
scriptional activity by modifying the accessibility to DNA either
positively or negatively.
unusual nucleotide, a 3′-phosphoadenosine 5′-phosphosulfate
Other processing reactions add anchors to the proteins for membrane (PAPS), is the universal sulfate donor for TPST-catalyzed reac-
association. Among the most important of these modifications are: tions. Addition of sulfate occurs almost exclusively on secreted or
membrane proteins and is believed to play a role in the modula-
• Myristoylation: is the attachment of a 14-carbon myristoyl group tion of protein-protein interactions. This protein modification is
to the N-terminal glycine residue of a protein. This modification critical in the processes of blood coagulation, various immune
is co- or posttranslational, irreversible and allows the association functions, intracellular trafficking, and ligand recognition by
of the target protein with membranes or with other proteins or several G protein-coupled receptors. Notably, coagulation factors
lipids. V and VIII, the gut peptides gastrin and cholecystokinin are
• Prenylation: involves the attachment of the 15 carbon farnesyl modified by sulfation.
group or 20 carbon geranyl group to acceptor proteins that harbor • γ-Carboxylation: this modification converts glutamate into
at the C-terminus the CAAX consensus sequence (C = cysteine, γ-carboxyglutamate that is found in several components of the
AA = any aliphatic amino acid except alanine, X = any amino coagulation pathway, as well as in a number of other proteins.
acid). AAX is first removed by a CAAX protease and the prenyl This modification is carried out by the enzyme γ-glutamyl carbox-
group is attached to the side chain of cysteine that is finally also ylase (GGCX) that requires vitamin K as an essential cofactor. The
methylated by a prenylcysteine methyltransferase that uses formation of modified glutamate residues is crucial for the process
S-adenosylmethionine. This complex modification occurs on RAS of blood coagulation and is described in more detail in Box 5.1.
protein family members and variants involving CC or CXC ele- • Ubiquitination: this posttranslational modification results from
ments at the C-terminus take place on the RAB protein family the sequential activity of three enzymes, the first activates a small
that is involved in signaling pathways that control intracellular protein of 76 amino acids named ubiquitin, the second transfers
membrane traffic (see later). Lack of prenylation on these other- ubiquitin to ubiquitin ligase that links ubiquitin to lysine residues
wise soluble proteins leads to lack of membrane association and present in target proteins and then other ubiquitin molecules to
generates severe phenotypes. ubiquitin lysine residues (polyubiquitination). Seven lysine resi-
dues in ubiquitin may serve as sites of linkage in formation of a
Other modifications are crucial to regulate protein function: polyubiquitin chain. Initially discovered as a mechanism to regu-
late protein degradation via the proteasome, in recent years,
• Phosphorylation: is the transfer of a phosphate group from ATP to ubiquitination was described to regulate different cellular processes
target amino acids in a protein (serine, threonine or tyrosine with including DNA repair, vesicle trafficking, and inflammation.
a ratio of occurrence of 1000:100:1). It can be reversed by the According to the pattern of modification of the target proteins
action of protein phosphatases that remove the phosphate group. (monoubiquitinated, polyubiquitinated) different processes can
It has been estimated that the human genome encodes about 500 be triggered (see next section).
protein kinases and 100 protein phosphatases. The catalytic or • Sumoylation: this modification belongs to a group of ubiquitin-
biologic activity of many enzymes is transiently regulated by like proteins (Ubl) that are added to proteins via posttranslational
reversible phosphorylation. Another role of phosphorylation reactions. The small peptide SUMO (small ubiquitin-related
emerged from studies on intracellular signaling pathways. Phos- modifier) is among the best characterized Ubl and over 50 proteins
phorylation of a protein substrate can create an interaction site are modified by SUMO addition.
(or docking site) to bind another protein. Interestingly, many pairs • Proteolysis: this modification consists in the cleavage of the poly-
of docking sites that bind specific small protein domains to peptide chain at a specific site during its maturation (proinsulin
promote protein–protein interaction have been identified. An to insulin), or occurs upon secretion of its inactive form (zymogen)
example is the SH2 domain of the c-Src protein kinase and other to prevent potential toxic effects of the processed protein in the
signal-transducing proteins that recognizes specific phosphotyro- cell (digestive proteases) or because proteolysis is regulated as
sine in other proteins. About 111 SH2 domains are found in occurs for coagulation factors that are secreted into the blood in
proteins encoded by the human genome. an inactive form and are activated by proteolysis at sites of
• Sulfation: this modification occurs at tyrosine residues and is cata- damaged blood vessels. Proteolysis can take place in more than
lyzed by tyrosyl-protein sulfotransferases (TPST) which are one site to generate smaller products as for instance in the matura-
membrane-associated enzymes of the trans-Golgi network. An tion of hormones starting from a larger precursor. Proteolytic