Posttranslational modifications covalent processing

The maturation of proteins into their final structural state often involves the cleavage or formation (or both) of covalent bonds, a process termed posttranslational modification. Many polypeptides are initially synthesized as larger precursors, called proproteins. The extra polypeptide segments in these proproteins often serve as leader sequences that target a polypeptide... 

Biosynthesis of the polypeptide chain is realised by a complicated process called translation. The basic polypeptide chain is subsequently chemically modified by the so-called posttranslational modifications. During this sequence of events the peptide chain can be cleaved by directed proteolysis, some of the amino acids can be covalently modified (hydroxylated, dehydrogenated, amidated, etc.) or different so-called prosthetic groups such as haem (haemoproteins), phosphate residues (phosphoproteins), metal ions (metal-loproteins) or (oligo)saccharide chains (glycoproteins) can be attached to the molecule by covalent bonds. Naturally, one protein molecule can be modified by more means. 

Posttranslational modifications include many covalent alterations Polypeptide processing, attachment of carbohydrate or lipid groups to specific side chains, and addition of many other low-molecular-weight ligands to side chains. 

Protein synthesis also involves a set of posttranslational modifications that prepare the molecule for its functional role, assist in folding, or target it to a specific destination. These covalent alterations include proteolytic processing, modification of certain amino acid side chains, and insertion of cofactors. 

Polypeptide chains are synthesized in the cytoplasm of a cell by the process known as translation. The polypeptide may be ready for use immediately after translation or it may require further maturation steps, such as facilitated folding, complex formation, transport to another cell compartment, or covalent modification of chemical groups on the protein. The term posttranslational modifications is used for the last of these processes. Some of the modifications take place in the cytoplasm or nucleus, particularly phosphorylation and acetylation, and others take place in the endoplasmic reticulum or Golgi apparatus, particularly the addition of sugar or polysaccharide residues. 

Studies of proteins present in a cell revealed a rich repertoire of expressed proteins way beyond what is expected from direct translation of the messages produced by a genome. Proteins can be modified posttranslationally by covalent attachment of one or more of several classes of molecules, by the formation of intramolecular or intermolecular linkages, by proteolytic processing of the newly synthesized polypeptide chain, or by any combination of these events. These modifications endow a protein with various properties that may be specifically required under a particular condition. 

Posttranslational modification (PTM) with functional groups is a universal mechanism for diversifying the activities of proteins. PTMs can affect many properties of proteins, such as localization, activity status, interaction networks, solubility, folding, turnover, or stabUity. It is therefore of vital importance to accurately determine the identities of modified proteins, the modified amino acid residues, and the covalently attached group. This chapter describes the process of PTM identification using the adenylylation (i.e., the covalent transfer of an adenosine monophosphate (AMP)) of rat sarcoma related in brain (Rab) proteins hy Legionella pneumophila enzymes as an example. It also deals with the development of PTM-specific antibodies from synthetic peptides. This account underlines the importance of chemical biology in the elucidation of PTMs. 

Hundreds of modifications to the protein backbone have been reported and reviewed." ° For many biopharmaceutical proteins, however, only a handful of modifications are usually detected. These include both posttranslational modifications that are results of intracellular enzymatic processes, and covalent modifications that occur during or after the manufacturing process, either induced by process conditions or resulting from degradation (discussed in Section II.A.4). 

Prenylated proteins have characteristic C-terminal sequences. For example, the three allelic Ras proteins (H-Ras, K-Ras, and N-Ras) expressed in mammalian tissues contain a C-terminal tetrapeptide which begins with cysteine, and ends with either methionine or serine. This part of the molecule is referred to as the CaaX box where C = cysteine, a = an aliphatic amino acid, and X = a prenylation specificity residue. The first step in the posttranslational processing of Ras proteins utilizes FTase and farnesyl diphosphate (FPP) to covalently attach a farnesyl group to the cysteine thiol of the CaaX box. While subsequent processing events involve proteolytic removal of the aaX tripeptide and methylation of the resulting C-termi-nal carboxylate group, only the farnesyl modification is required for mutant Ras proteins to associate with the cell membrane and transform a cell.2-6... 

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