Question

What are post-translational protein modifications? How do they affect protein structures and functions? How can they be measured (name at least two ways)? Why should we care about them (hint: there are likely a few reasons; tell me about all of them)?

Answer 1

Post-translational modifications are changes to proteins that are made after translation has occurred. post-translational modification consist of cleaving peptide bonds, as in processing a propeptide to a mature form or removing the initiator methionine residue. The formation of disulfide bonds from cysteine residues may also be referred to as a post-translational modification

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.

2.

Structural changes

Disulfide bridges, the covalent linkage of two cysteine amino acids

Proteolytic cleavage, cleavage of a protein at a peptide bond Isoaspartate formation, via the cyclisation of asparagine or aspartic acid amino-acid residues racemisation of serine by protein-serine epimerase of alanine in dermorphin, a frog opioid peptide of methionine in deltorphin, also a frog opioid peptide protein splicing, self-catalytic removal of inteins analogous to mRNA processing.

Posttranslational modifications play a fundamental role in regulating the folding of proteins, their targeting to specific subcellular compartments, their interaction with ligands or other proteins, and their functional state, such as catalytic activity in the case of enzymes or the signaling function of proteins involved in signal transduction pathways. Some posttranslational modifications (e.g., phosphorylation) are readily reversible by the action of specific deconjugating enzymes. The interplay between modifying and demodifying enzymes allows for rapid and economical control of protein function. A similar control by protein degradation and de novo synthesis would take much longer time and cost much more bioenergy. A very powerful way to study posttranslational modifications is by ‘proteomics,’ an extremely rapid and sensitive methodology for the systematic identification of proteins from cells or tissues. This involves separation of proteins and their isoforms by size and/or charge heterogeneity by two-dimensional gel electrophoresis, recovery of individual spots from the gel followed by mass spectrometry. The technique not only yields sequence information to identify the protein.

3.

Phosphorylation

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 9.6) and p15Ink4b (pI 5.5) served as negative controls for nonspecific binding of non-phosphorylated proteins. FT = flow-through fraction, W = pooled wash fractions, E = pooled elution fractions and L = non-enriched total cell extract.

Glycosylation

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. Carbohydrates in the form of aspargine-linked (N-linked) or serine/threonine-linked (O-linked) oligosaccharides are major structural components of many cell surface and secreted proteins.

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.

Ubiquitination

Ubiquitin is an 8-kDa polypeptide consisting of 76 amino acids that is appended to the ε-NH2 of lysine in target proteins via the C-terminal glycine of ubiquitin. 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. 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. After epoxomicin-treatment, HeLa cells lysates (150 µg) were processed by four different methods. 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. Compared to Supplier C’s kit and an antibody-based method, the Thermo Scientific Pierce Ubiquitin Enrichment Kit, yielded more ubiquitinated protein in the elution fraction (and less protein in the flow-through fraction), indicating significantly better enrichment of ubiquitinated proteins. GSH Resin is a negative control for comparison.

4. Post-translational modifications (PTMs) such as glycosylation and phosphorylation play an important role on the function of haemostatic proteins and are critical in the setting of disease. Such secondary level changes to haemostatic proteins have wide ranging effects on their ability to interact with other proteins. This review aimed to summarize the knowledge of the common PTMs associated with haemostatic proteins and the implications of such modifications on protein function. Haemostatic proteins that represent the main focus for studies specific to PTMs are von Willebrand factor, tissue factor, factor VIII, antithrombin and fibrinogen. These proteins are susceptible to PTMs by glycosylation, phosphorylation, sulphation, citrullination and nitration, respectively, with a significant impact on their function. During synthesis, vWF must undergo extensive PTMs, with N-linked glycosylation being the most common. Increased phosphorylation of tissue factor results in increased affinity for platelets to the vessel endothelium. Citrullination of antithrombin leads to an increased anticoagulant function of this protein and therefore an anticoagulant state that inhibits clot formation. On the contrary, nitration of fibrinogen has been shown to result in a prothrombotic state, whilst sulphation is required for the normal function of Factor VIII. From this review, it is evident that PTMs of haemostatic proteins as a change in protein structure at a secondary level greatly influences the behaviour of the protein at a tertiary level.