Question

Some proteins can function as enzymes. How does a protein become phosphorylated? What effect can this post-translational modification (PTM) have on a protein?

Answer 1

A.Protein phosphorylation is a reversible post-translational modification of proteins in which an amino acid residue is phosphorylated by a protein kinase by the addition of a covalently bound phosphate group. Phosphorylation alters the structural conformation of a protein, causing it to become activated, deactivated, or modifying its function.A 13000 human proteins have sites that are phosphorylated.

Protein phosphorylation was first reported in 1906 by Phoebus Levene at the Rockefeller Institute for Medical Research with the discovery of phosphorylated vitellin.However, it was nearly 50 years until the enzymatic phosphorylation of proteins by protein kinases was discovered.

While phosphorylation is a prevalent post-translational modification (PTM) for regulating protein function, it only occurs at the side chains of three amino acids, serine, threonine and tyrosine, in eukaryotic cells.

Figure 1:Protein phosphorylation. (a) A phosphate group is transferred from ATP to a hydroxyl group on the protein by a kinase and can be removed by a phosphatase. (b) Phosphorylation can occur on Ser, Thr or Tyr residues.

Phosphorylation introduces a charged and hydrophilic group in the side chain of amino acids, possibly changing a protein's structure by altering interactions with nearby amino acids. Some proteins such as p53 contain multiple phosphorylation sites, facilitating complex, multi-level regulation. Because of the ease with which proteins can be phosphorylated and dephosphorylated, this type of modification is a flexible mechanism for cells to respond to external signals and environmental conditions.

Reversible phosphorylation of proteins occurs in both prokaryotic and eukaryotic organisms.

B.Protein post-translational modifications (PTMs) increase the functional diversity of the proteome by the covalent addition of functional groups or proteins, proteolytic cleavage of regulatory subunits, or degradation of entire proteins.

These modifications include phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation and proteolysis and influence almost all aspects of normal cell biology and pathogenesis. Therefore, identifying and understanding PTMs is critical in the study of cell biology and disease treatment and prevention.

Within the last few decades, scientists have discovered that the human proteome is vastly more complex than the human genome. While it is estimated that the human genome comprises between 20,000 and 25,000 genes, the total number of proteins in the human proteome is estimated at over 1 million. These estimations demonstrate that single genes encode multiple proteins. Genomic recombination, transcription initiation at alternative promoters, differential transcription termination, and alternative splicing of the transcript are mechanisms that generate different mRNA transcripts from a single gene.

The increase in complexity from the level of the genome to the proteome is further facilitated by protein post-translational modifications (PTMs). PTMs are chemical modifications that play a key role in functional proteomic because they regulate activity, localization, and interaction with other cellular molecules such as proteins, nucleic acids, lipids and cofactors.

Post-translational modifications are key mechanisms to increase proteomic diversity.While the genome comprises 20,000 to 25,000 genes, the proteome is estimated to encompass over 1 million proteins. Changes at the transcriptional and mRNA levels increase the size of the transcriptome relative to the genome, and the myriad of different post-translational modifications exponentially increases the complexity of the proteome relative to both the transcriptome and genome.

Additionally, the human proteome is dynamic and changes in response to a legion of stimuli, and post-translational modifications are commonly employed to regulate cellular activity. PTMs occur at distinct amino acid side chains or peptide linkages, and they are most often mediated by enzymatic activity. Indeed, it is estimated that 5% of the proteome comprises enzymes that perform more than 200 types of post-translational modifications. These enzymes include kinases, phosphatases, transferases and ligases, which add or remove functional groups, proteins, lipids or sugars to or from amino acid side chains; and proteases, which cleave peptide bonds to remove specific sequences or regulatory subunits. Many proteins can also modify themselves using autocatalytic domains, such as autokinase and autoprotolytic domains.

Post-translational modification can occur at any step in the "life cycle" of a protein. For example, many proteins are modified shortly after translation is completed to mediate proper protein folding or stability or to direct the nascent protein to distinct cellular compartments (e.g., nucleus, membrane). Other modifications occur after folding and localization are completed to activate or inactivate catalytic activity or to otherwise influence the biological activity of the protein. Proteins are also covalently linked to tags that target a protein for degradation. Besides single modifications, proteins are often modified through a combination of post-translational cleavage and the addition of functional groups through a step-wise mechanism of protein maturation or activation.

Protein PTMs can also be reversible depending on the nature of the modification. For example, kinases phosphorylate proteins at specific amino acid side chains, which is a common method of catalytic activation or inactivation. Conversely, phosphatases hydrolyze the phosphate group to remove it from the protein and reverse the biological activity. Proteolytic cleavage of peptide bonds is a thermodynamically favorable reaction and therefore permanently removes peptide sequences or regulatory domains.

Consequently, the analysis of proteins and their post-translational modifications is particularly important for the study of heart disease, cancer, neurodegenerative diseases and diabetes. The characterization of PTMs, although challenging, provides invaluable insight into the cellular functions underlying etiological processes. Technically, the main challenges to studying post-translationally modified proteins are the development of specific detection and purification methods. Fortunately, these technical obstacles are being overcome with a variety of new and refined proteomics technologies.

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