Protein Post-Translational Modification: The Precise Regulator of Life Activities

The human genome contains approximately 20,000 to 25,000 protein-coding genes. However, the number of protein species in the human body is far greater than the number of genes, estimated to be between 200,000 and 2,000,000. The generation of diversity from genes to mRNA and then to proteins is a multi-level and multi-mechanism process. These mechanisms include genetic-level variations (such as mutations, fusions, duplications), transcriptional-level regulation (such as alternative splicing, selection of transcription start and termination sites, regulation by non-coding RNAs), and post-translational modifications (PTMs) (such as phosphorylation, ubiquitination, acetylation, glycosylation, lipidation, oxidation). These mechanisms cooperate to greatly enrich the structural and functional diversity of proteins, thereby supporting the complex physiological functions and adaptability of organisms.

I. Definition and Overview of Protein Post-Translational Modification

Protein post-translational modification refers to the process of adding various chemical groups to amino acid residues through covalent bonding after protein synthesis, or performing processing such as cleavage and folding of proteins, thereby altering the structure, stability, activity, localization of proteins, and their ability to interact with other molecules. These modification processes usually occur in organelles such as the cytoplasm, endoplasmic reticulum, and Golgi apparatus, and are catalyzed by a series of highly specific enzymes. Currently, there are many known types of protein post-translational modifications, including phosphorylation, ubiquitination, acetylation, methylation, glycosylation, ubiquitin-like protein modification, lipidation, oxidation, etc. Each modification has its unique biological significance and mechanism of action. They cooperate to form a complex and precise regulatory network that accurately regulates protein functions and cellular physiological activities.

II. Common Types of Protein Post-Translational Modifications and Their Functions

(I) Phosphorylation

Phosphorylation is one of the most common post-translational modifications of proteins, mainly occurring on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues. Phosphorylation modification is usually catalyzed by protein kinases, which transfer the phosphate group from ATP molecules to specific amino acid residues, thereby changing the structure and function of proteins. Phosphorylation modification plays a central role in cellular signal transduction. It can regulate protein activity, stability, subcellular localization, and interaction with other proteins. For example, in processes such as cell growth, differentiation, apoptosis, and metabolic regulation, many key signaling pathways rely on protein phosphorylation modification to transmit and amplify signals. When extracellular signaling molecules (such as hormones, growth factors) bind to cell surface receptors, they activate a series of protein kinase cascades, transmitting signals from receptors to the interior of cells through phosphorylation modification, ultimately leading to changes in the activity of intracellular target proteins and triggering corresponding cellular responses. In addition, phosphorylation modification is also involved in the regulation of gene expression. Some transcription factors can bind to DNA after phosphorylation to regulate gene transcriptional activity. As one of the most widely studied types of post-translational modifications, antibodies for detecting phosphorylation are now relatively common.

(II) Ubiquitination

Ubiquitination modification is the process of covalently linking ubiquitin molecules to target proteins, mainly completed through the synergistic effect of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). Ubiquitination modification plays a key role in many biological processes such as protein degradation, cell cycle regulation, DNA repair, and signal transduction. Its most typical function is to mark target proteins for entry into the proteasomal degradation pathway through the formation of polyubiquitin chains, thereby regulating protein levels and intracellular homeostasis. For example, at various stages of the cell cycle, many cyclins are degraded by the proteasome through ubiquitination modification, ensuring the normal progression of the cell cycle. In addition, ubiquitination modification can also regulate protein subcellular localization, activity, and interaction with other proteins, and participate in the regulation of various intracellular signaling pathways, such as the NF-κB signaling pathway and Wnt signaling pathway.

(III) Acetylation

Acetylation modification mainly occurs on lysine residues, catalyzed by acetyltransferases, which transfer the acetyl group from acetyl-CoA to the amino group of lysine residues. Acetylation modification plays an important role in gene expression regulation, chromatin structure remodeling, cellular metabolism, and protein stability. In gene expression regulation, histone acetylation is an important epigenetic modification method. Acetylation modification can neutralize the positive charge of histones, weaken the binding between histones and DNA, loosen the chromatin structure, thereby promoting the binding of transcription-related factors such as transcription factors and RNA polymerase to DNA, and activating gene transcription. In addition to histones, many non-histone proteins also undergo acetylation modification. These modifications can regulate protein activity, stability, subcellular localization, and interaction with other proteins, and participate in various intracellular metabolic processes and signal transduction pathways.

(IV) Glycosylation

Glycosylation modification refers to the process of linking sugar groups to proteins, mainly divided into two types: N-glycosylation and O-glycosylation. N-glycosylation modification occurs on asparagine residues, while O-glycosylation modification mainly occurs on serine, threonine, or hydroxylysine residues. Glycosylation modification plays an important role in protein folding, stability, subcellular localization, intercellular recognition, and signal transduction. During protein folding, glycosylation modification can act as a chaperone to help proteins fold correctly and prevent protein aggregation. Glycosylation modification can also regulate protein stability and affect protein transport and subcellular localization in cells. In terms of intercellular recognition and signal transduction, glycoproteins on the cell surface interact with extracellular ligands, receptors, or extracellular matrix through their glycosylation modifications, participating in processes such as cell-cell adhesion, immune recognition, and cell migration. For example, in the immune system, glycosylation modification of antibodies can affect their binding affinity to antigens and immune effector functions.

III. Regulatory Mechanisms of Protein Post-Translational Modification

The regulation of protein post-translational modification is a complex process involving the interaction of multiple factors. Firstly, the activity of modifying enzymes and demodifying enzymes is a key factor regulating protein post-translational modification. The expression level, activity state of modifying enzymes and demodifying enzymes, and their binding affinity with substrates all affect the degree of protein post-translational modification. For example, the balance between the activity of protein kinases and phosphatases determines the phosphorylation level of proteins; the balance between the activity of acetyltransferases and deacetylases determines the acetylation level of proteins. Secondly, intracellular signal transduction pathways also regulate protein post-translational modification. Extracellular signaling molecules can regulate the activity of modifying enzymes and demodifying enzymes by activating specific signaling pathways, thereby changing the post-translational modification state of proteins. For example, the PI3K-Akt signaling pathway activated by growth factors can regulate the activity of protein kinases, thereby affecting protein phosphorylation modification. In addition, intracellular metabolic status, redox status, etc. also affect protein post-translational modification. For example, the intracellular energy metabolism status can affect the level of acetyl-CoA, thereby regulating the degree of acetylation modification; oxidative stress can induce oxidative modification of proteins, such as tyrosine nitration and cysteine oxidation.

IV. Research Methods and Technologies for Protein Post-Translational Modification

With the continuous development of biotechnology, the methods and technologies for studying protein post-translational modification have become increasingly diversified. Traditional research methods include immunoprecipitation (IP), Western Blot (WB), mass spectrometry (MS) analysis, etc. IP and WB technologies can be used to detect the post-translational modification status of specific proteins. Through specific antibodies that recognize and bind to modified proteins, qualitative and quantitative analysis of protein modifications can be achieved. In recent years, with the continuous development of proteomics technology, mass spectrometry-based proteomics technology has become a powerful tool for studying protein post-translational modification. Through high-throughput proteomics analysis, it is possible to simultaneously identify and quantitatively analyze the post-translational modification status of a large number of proteins in cells or tissues, revealing the global changes and regulatory networks of protein modifications.

WB Example:

WB result of Phospho-NF-kB p105/p50 (Ser337) Recombinant Rabbit mAb

Primary antibody: Phospho-NF-kB p105/p50 (Ser337) Recombinant Rabbit mAb at 1/5000 dilution

Lane 1: untreated NIH/3T3 whole cell lysate 20 µg

Lane 2: NIH/3T3 treated with 20 ng/mL TNF-alpha and 100 nM Calyculin A for 10 minutes whole cell lysate 20 µg

Secondary antibody: Goat Anti-Rabbit IgG, (H+L), HRP conjugated at 1/10000 dilution

Predicted MW: 105 kDa

Observed MW: 55 kDa

CoIP Example:

After enriching total proteins using the target protein antibody, use pan-modification antibodies to detect the post-translational modification of the enriched proteins; or use pan-modification antibodies to enrich all modified proteins, then use the target protein antibody to detect the post-translational modification of the protein.

Endogenous and exogenous immunoprecipitation experiments were performed in MDA-MB-231 and HEK293T cells. The results showed that specific lactylation bands were detected at the molecular weight of RCC2 in both endogenous and exogenous immunoprecipitates (Figures D, E) using abs9649 Immunoprecipitation (IP/CoIP) Kit (Magnetic Bead Method) [1]

References:

[1] Luo C, Xu H, Yu Z, Liu D, Zhong D, Zhou S, Zhang B, Zhan J, Sun F. Meiotic chromatin-associated HSF5 is indispensable for pachynema progression and male fertility. Nucleic Acids Res. 2024 Sep 23;52(17):10255-10275. doi: 10.1093/nar/gkae701. PMID: 39162221; PMCID: PMC11417359.

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