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As noted above, the large number of different PTMs precludes a thorough review of all possible protein modifications. Therefore, this overview only touches on a small number of the most common types of PTMs studied in protein research today. Furthermore, greater focus is placed on phosphorylation, glycosylation and ubiquitination, and therefore these PTMs are described in greater detail on pages dedicated to the respective PTM. Reversible protein phosphorylation, principally on serine, threonine or tyrosine residues, is one of the most important and well-studied post-translational modifications.

Phosphorylation plays critical roles in the regulation of many cellular processes, including cell cycle, growth, apoptosis and signal transduction pathways. In the following example, western blot analysis was used to evaluate phosphoprotein specificity in lysates obtained from serum-starved HeLa and NIH 3T3 cancer cell lines stimulated with epidermal growth factor EGF and platelet derived growth factor PDGF , respectively. Highly pure phosphoprotein enrichment from complex biological samples. Western blot analysis was performed with the Thermo Scientific Pierce Phosphoprotein Enrichment Kit , and cell lysates were prepared according to the kit instructions to enrich for phosphoproteins.

Protein detection was achieved using phospho-specific antibodies that recognize key regulatory proteins involved in growth factor signaling. Cytochrome C pI 9. Protein glycosylation is acknowledged as one of the major post-translational modifications, with significant effects on protein folding, conformation, distribution, stability and activity. Glycosylation encompasses a diverse selection of sugar-moiety additions to proteins that ranges from simple monosaccharide modifications of nuclear transcription factors to highly complex branched polysaccharide changes of cell surface receptors.

Types of glycosylation. Glycopeptide bonds can be categorized into specific groups based on the nature of the sugar—peptide bond and the oligosaccharide attached, including N-, O- and C-linked glycosylation, glypiation and phosphoglycosylation. Following an initial monoubiquitination event, the formation of a ubiquitin polymer may occur, and polyubiquitinated proteins are then recognized by the 26S proteasome that catalyzes the degradation of the ubiquitinated protein and the recycling of ubiquitin.

Co And Posttranslational Proteolysis Of Proteins

The following experiment provides an example of methods used to detect ubiquitinated proteins. Detection of ubiquitin in HeLa cell lysates. Western blot analysis was performed to compare four methods for detecting ubiquitin protein in HeLa cell lysates. The resulting flow-through F and elution E fractions were volume-normalized to the original unprocessed lysate H and identical volumes electophoresed for western blot detection.

Protein modifications (video) | DNA | Khan Academy

GSH Resin is a negative control for comparison. Nitric oxide NO is produced by three isoforms of nitric oxide synthase NOS , and it is a chemical messenger that reacts with free cysteine residues to form S-nitrothiols SNOs. S-nitrosylation is a critical PTM used by cells to stabilize proteins, regulate gene expression and provide NO donors, and the generation, localization, activation and catabolism of SNOs are tightly regulated.

S-nitrosylation is a reversible reaction, and SNOs have a short half-life in the cytoplasm because of the host of reducing enzymes, including glutathione GSH and thioredoxin, that denitrosylate proteins. Therefore, SNOs are often stored in membranes, vesicles, the interstitial space and lipophilic protein folds to protect them from denitrosylation.

For example, caspases, which mediate apoptosis, are stored in the mitochondrial intermembrane space as SNOs. In response to extra- or intracellular cues, the caspases are released into the cytoplasm, and the highly reducing environment rapidly denitrosylates the proteins, resulting in caspase activation and the induction of apoptosis. S-nitrosylation is not a random event, and only specific cysteine residues are S-nitrosylated.

Because proteins may contain multiple cysteines and due to the labile nature of SNOs, S-nitrosylated cysteines can be difficult to detect and distinguish from non-S-nitrosylated amino acids. The biotin switch assay, developed by Jaffrey et al. The transfer of one-carbon methyl groups to nitrogen or oxygen N- and O-methylation, respectively to amino acid side chains increases the hydrophobicity of the protein and can neutralize a negative amino acid charge when bound to carboxylic acids. Methylation is mediated by methyltransferases, and S-adenosyl methionine SAM is the primary methyl group donor.

Methylation occurs so often that SAM has been suggested to be the most used substrate in enzymatic reactions after ATP. Additionally, while N-methylation is irreversible, O-methylation is potentially reversible. Methylation is a well-known mechanism of epigenetic regulation, as histone methylation and demethylation influences the availability of DNA for transcription.

Amino acid residues can be conjugated to a single methyl group or multiple methyl groups to increase the effects of modification. The figure below provides an illustration of PMTs associated with nucleosome core particles. Representation showing post-translational modifications associated with histone particles.

Nucleosomes are represented by red spheres wrapped by DNA shown in gray. X , H2B, H3, and H4. These PTMs impact gene expression by altering chromatin structure and recruiting histone modifiers. PTM events mediate diverse biological functions such as transcriptional activation and inactivation, chromosome packaging, and DNA damage and repair processes. N-acetylation, or the transfer of an acetyl group to nitrogen, occurs in almost all eukaryotic proteins through both irreversible and reversible mechanisms.

N-terminal acetylation requires the cleavage of the N-terminal methionine by methionine aminopeptidase MAP before replacing the amino acid with an acetyl group from acetyl-CoA by N-acetyltransferase NAT enzymes. This type of acetylation is co-translational, in that N-terminus is acetylated on growing polypeptide chains that are still attached to the ribosome.

Histone acetylation is a reversible event that reduces chromosomal condensation to promote transcription, and the acetylation of these lysine residues is regulated by transcription factors that contain histone acetyletransferase HAT activity. While transcription factors with HAT activity act as transcription co-activators, histone deacetylase HDAC enzymes are co-repressors that reverse the effects of acetylation by reducing the level of lysine acetylation and increasing chromosomal condensation.

Sirtuins silent information regulator are a group of NAD-dependent deacetylases that target histones. As their name implies, they maintain gene silencing by hypoacetylating histones and have been reported to aid in maintaining genomic stability. While acetylation was first detected in histones, cytoplasmic proteins have been reported to also be acetylated, and therefore acetylation seems to play a greater role in cell biology than simply transcriptional regulation.

Enter your information below:. Email Address. First Name. Last Name. ZIP Code. Yemen Zambia Zimbabwe. Login to automatically fill out the form with your account information. Login using another user account. You have entered an invalid username or password. Send request. Email address. And so, let's see how phosphorylation regulates this protein. And again, the proteins that you're looking at, they really all represent one protein.

It's just, we're going through the motions of the changes that happen to this one protein. It's not like we're looking at six different proteins in the membrane. So here's our first step. And when you look at this enzyme, you can see that there are three receptor sites for a particular ion, this is the sodium ion.

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Represented by these dark blue circles. And then there are two receptor sites that look kind of more squarish, and those are receptor sites for the potassium ions, and those are represented by these light blue squares. So those are the potassium ions. And so, back to the first step, and what's happening here. The sodium ions will attach to the receptor sites on the enzyme.

And just to clarify, this is the intracellular space, so this is basically the cytoplasm.

POST TRANSLATIONAL MODIFICATIONS of protein explained in simple way

And so these sodium ions are coming from the cytoplasm. And then out here is the extracellular space, the outside of the cell. Back to our first step.


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So the sodium ions attach to the receptor site. When the receptor sites are full, it's going to cause something to happen. It's going to cause an ATP molecule, adenosine triphosphate, to break down into ADP, adenosine diphosphate, plus phosphate. And then this phosphate will attach itself to the protein, and that is phosphorylation.

And so when this protein gets phosphorylated, when the phosphate group is attached to it, it causes there to be some sort of change in the conformation of the protein. And that change in conformation causes the protein to turn itself around by degrees and face the outside of the cell.

That's what you're looking at right over here. So our protein is still phosphorylated. I know I drew it in a different place, but just keep in mind that there's still a phosphate group attached to that protein.

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And then the protein releases the sodium ions into the extracellular space. So the next step is number four. And again, we are still phosphorylated, there's still a phosphate group attached to our protein. And so in the next step, the potassium ions on the outside of the cells will attach themselves to the protein on the receptor sites. Take note, there are three receptor sites for sodium but only two for potassium.

When the potassium receptor sites are full, it's going to cause a different change to happen. This phosphate group is going to be removed. And that is called dephosphorylation. That phosphate group ends up in the inside of the cell, and it gets recycled in some other way. And when this protein is dephosphorylated, when that protein is removed, it's going to cause a different change in the conformation of the protein.

And that change in conformation causes the protein now to turn around again by degrees and it faces the inside of the cell. So that's our fifth step, you can see the protein is facing the inside of the cell. And in the last step, the potassium ions are released into the inside part of the cell.

And then we're back to our first step. And so you can see that the phosphorylation and dephosphorylation basically regulates the activity of this protein.


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  • And the end result that we're trying to get to is that we want there to be, on the outside of the cell, a rather high concentration of sodium, and a rather low concentration of potassium. And on the inside of the cell we want there to be a rather low concentration of sodium, and a relatively high concentration of potassium. That's accomplished by the fact that for every three sodiums that are pumped out, two potassiums are pumped in.

    And so here's an example, a very common example of how phosphorylation regulates a protein. And this doesn't happen just in the sodium-potassium pump, it happens in many, many enzymes and proteins in our bodies and in cells. Let's move on to two other protein modifications that also have to do with regulating an enzyme or something similar to that.

    So the next protein modification I want to talk about is methylation. And in particular, the methylation of certain proteins called histones. And those are these green circles that you're looking at. Histones are these proteins around which DNA wraps itself. So they're found in the chromosomes, and they help to package DNA in a very tight and organized manner. And sometimes histones are methylated, so let's put some methyl groups on our histones.

    Methylating and demethylating histones helps to turn certain genes on and off.