Protein Post-Translational Modifications

A Newly Synthesized Protein is not Necessarily a Finished Protein:

• Translation produces a polypeptide with a defined primary sequence—but function depends on much more than sequence

• Folding, proteolytic processing, covalent modification, trafficking, and degradation determine the protein's functional state

• PTMs are one of the major mechanisms by which cells create distinct molecular states from the same gene product.

• Think in terms of a protein lifecycle: synthesis → folding → localization → activation → regulation → turnover

Genome vs Proteome

• The human proteome is vastly more complex than the human genome

• 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

Over billions of years, organisms have acquired mechanisms:

• Alternative splicing

• Cotranslational modifications

• Posttranslational modifications (PTMs)

  • These mechanisms allow genomes containing 20,000–25,000 genes to be converted into proteomes of more than one million proteins

    • enhancing the complexity and functional diversity of proteins in response to complex external stimuli and internal changes

Post Translational Modifications

• More than 500 types of protein PTMs

• These modifications play essential roles in various biological processes, including deoxyribonucleic acid (DNA) repair, signaling pathways, cell apoptosis, subcellular localization, protein–protein interactions, etc

• Studies have shown that PTMs are closely associated with kinds of human diseases, such as neurological diseases (Alzheimer’s and Parkinson’s diseases, neuroblastomas and glioblastomas), breast diseases (breast cancer/tumors/carcinomas and mammary tumors/carcinomas) and blood diseases

• Research on PTMs can facilitate the development of epigenetics, cellular functions and medical technology

• Accurate detection is the primary task in PTM research

• Modified Proteins are identified through experimentally, diverse techniques, such as radioactive label method, chromatin immunoprecipitation (ChIP), mass spectrometry (MS) and liquid chromatography

• Verified PTM protein sequences were organized in known databases including UniProt, PLMD, dbPTM and PhosphoSitePlus

• There are 908 917 experimental sites of more than 130 types of PTMs recorded in the dbPTM database

• Phosphorylation contributes to the highest proportion of 62.83% (571 032), and acetylation and ubiquitination separately account for 15.12% and 13.04%.

• Proteomic techniques are expensive, time-consuming and labor-intensive

• The artificial intelligence industry is accelerating!

• Computational proteomics use AI to predict PTMs

• This new avenue greatly reduces the costs and presents reliable results, which has become a frontier topic in biology and bioinformatics

• However, researchers should exercise significant caution. While AI-driven PTM site prediction is highly scalable, relying on these computational results without rigorous laboratory verification introduces heavy risks

What can a PTM Change?

• Protein conformation

• Protein activity

• Protein charge and stability

• Protein-Protein interactions (within and between cells)

• Interactions with DNA and RNA

• Binding affinity for proteins, DNA, RNA, lipids, or small molecules

• Subcellular localization and membrane association

• Protein half-life and degradation

Types of PTMs Based on the Nature of the Modification:

• Complex molecules (irreversible/reversible)

  • Lipidation

  • Glycosylation

• Proteins/peptides (reversible)

  • Ubiquitylation

  • Sumoylation

• Chemical groups (reversible)

  • Methylation

  • Phosphorylation

  • Acetylation

• Cleavage (irreversible)

  • Proteolysis

Timing of Post Translational Modifications

• Immediately after (or during) translation, before folding is complete

  • Early PTMs affect protein folding efficiency and structural stability

  • E.g., proteolytic cleavage

• Immediately after folding is complete

  • PTMs often direct proteins to their proper subcellular locations

  • E.g., glycosylation

• After folding and subcellular localization

  • PTMs can activate or deactivate specific functions of the protein

  • E.g., phosphorylation

• End of the protein life cycle

  • PTMs tag proteins for degradation, marking them for removal from the cell

  • E.g., ubiquitination

Cleavage of Polypeptides

• Proteolytic cleavage is a PTM

• Proteases hydrolyze specific peptide bonds in a precursor protein

• Cleavage can activate an inactive precursor, remove a targeting sequence, expose a new terminus, or generate multiple products

• Unlike many reversible PTMs, peptide-bond cleavage is usually irreversible in the cellular context

• Specificity depends on protease recognition, substrate accessibility, and cellular compartment

Zymogen Activation

• A zymogen, also called a proenzyme, is an inactive precursor of an enzyme

• The pancreas initially synthesizes protease enzymes as zymogens to help prevent enzymes from inappropriately digesting proteins within the pancreatic cells

• Enzymes like Trypsin are synthesized as proenzymes

• For trypsin, trypsinogen is an inactive precursor translated by the rough endoplasmic reticulum and transported to the Golgi apparatus for sorting

• Trypsinogen is always co-synthesized and packaged with a pancreatic secretory trypsin inhibitor (PSTI), which inhibits premature activation

• Thus, there are two mechanisms in place to maintain the inactivity of the protease within the pancreas: (1) synthesis of the zymogen or proenzyme form, and (2) co-expression of a trypsin inhibitor protein that will bind and inhibit any prematurely cleaved trypsin until it has reached the small intestine

• During packaging in the Golgi apparatus, trypsinogen and other digestive enzymes condense into core particles and are packed into zymogen granules

• The condensed enzymes are stable, and minimal activation occurs within the zymogen granules

• Once pancreatic cells receive secretory stimuli, zymogen granules are released into the pancreatic duct lumen, carrying digestive enzymes into the duodenum

• Once in the duodenum, enteropeptidase activates trypsinogen by removing the trypsinogen activation peptide (TAP) from the N-terminal region

Proteolysis as Protein Maturation

Many hormones and neuropeptides are synthesized as inactive precursor polypeptides that require limited endoproteolysis to become biologically active

• Example: Insulin

Insulin

• A vital peptide hormone produced by the beta cells of the pancreas that is critical for glucose uptake for:

  • Metabolism, glycogen synthesis/storage, lipolysis regulation, and other processes related to cell growth

• Synthesized as a precursor molecule and processed through a series of non-enzymatic and enzymatic events

How is Insulin made?

1. Expression of the INS gene leads to the translation of a 110-a.a. long precursor polypeptide ⇨ Preproinsulin

2. The first 24 a.a. signal peptide is cleaved and the rest of the peptide folds and forms disulfide bonds ⇨ Proinsulin

3. Removal of the 31 a.a. C-peptide (res. 57-87) creates the mature protein composed of two separate peptide chains ⇨ Insulin

Insulin is a secreted protein:

its processing is coordinated in the ER and Golgi apparatus

Glycosylation

• A process in which sugar groups are transferred to proteins catalyzed by glycosyltransferases

  • Attached oligosaccharides are called glycans

• Predominantly occurs in the ER and Golgi apparatus

• One of the most commonly occurring PTMs: ~50% of all cellular proteins are glycosylated

• Most glycans exist on the surface of cells and secreted proteins, with intricate and varied structures

• The human genome contains approximately 700 genes related to glycosylation and deglycosylation, including enzymes, transporters, and chaperones

• Changes in glycosylation patterns are linked to diseases

• Occurs as proteins traverse through the secretory pathway

  • Step-wise processes add, remove, and trim sugar moieties

Functions of Glycosylation

• Aids in protein folding and stability

• Facilitates protein-protein/protein-ligand interactions

• Helps mediate cell-cell adhesion and immune responses

Glycosylation requires the action of multiple enzymes located in different cellular compartments:

• Glycosyltransferases → link sugars to proteins, forming glycan structures

• Glycosidases → remove sugars, modifying and refining glycan structur

Glycans are primarily attached to either nitrogen or oxygen atoms on proteins:

• N-linked glycosylation

• O-linked glycosylation

N-Linked Glycosylation

• The most common type of glycosylation in eukaryotes

  • ~90% of glycoproteins contain N-linked glycans

• Glycans are attached to the side chain nitrogen of an asparagine residue within a specific consensus sequence:

  • Asn-X-Ser/Thr

  • (X = any a.a. except Pro)

• N-linked glycans tend to be large, bulky and diverse, requiring the action of many independent enzymes

• Fully processed glycans will belong to one of the following three types:

1. High-mannose

2. Hybrid

3. Complex

O-Linked Glycosylation

• Modifies the hydroxyl group of serine or threonine residues

• Sugars are added one at a time as monosaccharides by specific enzymes

• O-linked glycans are generally less branched and less diverse compared to N-linked glycans

• O-linked glycosylation primarily occurs in the Golgi apparatus, but occasionally also in the cytoplasm and nucleus

Erythropoietin (EPO)

Protein hormone:

• Stimulates red blood cell production

• Produced in the kidney under hypoxic conditions

• Binds to the EPO receptor on red blood cell progenitors in the bone marrow

• Activates the JAK2 signaling cascade, which in turn initiates the STAT5, PI3K, and MAPK pathways

• ⇨ Differentiation, survival, and proliferation of red blood cells

• Heavily glycosylated:

  • ~40% of the total MW is from glycans

  • Three N-linked glycans and one O-linked glycan at Asn24, Asn38, Asn83, and Ser126

  • Glycosylation sites do not overlap with residues involved in receptor binding

  • The presence of N-glycosylations does not compromise the binding site to the erythropoietin receptor

• Biological activity of incompletely glycosylated EPO is over 500-fold lower

  • Plasma half-life is significantly shorter due to increased rate of clearance from systemic circulation

Therapeutic Erythropoietin (EPO)

• Can be used to treat anemia caused by Chronic Kidney Disease (CKD) and cancer

• The recombinant EPO used for clinical treatment can be divided into four types according to their different glycosylation levels

• It is also exploited as a performance-enhancing drug: To boost aerobic capacity (“blood doping”)

  • Lance Armstrong

Glycosylation is essential for the biological activity of EPO:

• Cannot be produced in simpler organisms due to the lack of necessary glycosylation machinery (e.g., insulin in E. coli cells)

• Produced in mammalian cells instead (e.g., CHO cells) to mimic the glycosylation pattern of endogenous EPO

  • Enables detection in anti-doping test

• Newer variants have additional glycosylation sites engineered to increase the plasma half-life (and by extension the potency)

Protein lipidation can attach up to seven different types of lipids to proteins:

• Fatty acids (FA)

• Lipoic acids

• Isoprenoids

• Sterols

• Phospholipids

• Glycosylphosphatidylinositol anchors

• Lipid-derived electrophiles

Protein lipidation can be divided into 2 categories:

1. Lipid modifications that occur in the cytoplasm or on the cytoplasmic side of membranes

• S-palmitoylation, N-myristoylation and S-prenylation

2. Lipid modifications that occur in the lumen of secretory organelles, such as glycosylphosphatidylinositol (GPI) anchor and cholesterylation.

• glycosylphosphatidylinositol (GPI) anchor and cholesterylation

Lipid attachment to proteins plays a crucial role in:

• Regulating protein trafficking

• Localization

• Stability

• Conformation

• Interactions

• Signal transduction by enhancing the hydrophobicity of proteins

An increasing number of studies have revealed that aberrant regulation of lipidation is implicated in the pathogenesis of various diseases:

• Cancer

• Neurological disorders

• Cardiovascular diseases

• Inflammatory diseases

• Metabolic disorders

• Infectious diseases

• and others

  • This suggest the role of lipidation regulation in disease progression is promising for the development of potential therapeutic targets

Addition of Chemical Groups

• Mostly reversible

• Provides regulatory mechanisms

• Many different types of modifications, e.g.,

  • Phosphorylation

  • Acetylation

  • Methylation

  • Glutathionylation

  • Sulfation

  • Formylation

Phosphorylation

• Formed by adding a phosphate group from ATP to the side chains of amino acids by kinases

• Usually turns hydrophobic nonpolar proteins into hydrophilic polar proteins

• Phosphorylation is a reversible PTM, and the reverse process of phosphorylation is called dephosphorylation catalyzed by phosphatases

Phosphorylation modifications occur most commonly on the following residues:

• Serine (86.4%)

• Threonine (11.8%)

• Tyrosine (1.8%)

  • It is important to note that kinases can also act on the side chains of other amino acids, such as cysteine, lysine, histidine, arginine, aspartic acid, and glutamic acid, although with reduced frequency

The protein phosphorylation system consists of:

• Kinases

• Phosphatases

• Phosphorylation substrates

• Phosphorylation‐binding proteins

Protein phosphorylation extensively affects:

• Cellular stress response

• Protein stability

• Protein-protein interaction

• Conformation

• Protein activity

• Localization

• Transcriptional regulation

• Signal transduction

Phosphorylation – Protein Kinases

• Widely distributed in cells throughout the nucleus, cytosol, mitochondria, and microsomes

• 518 protein kinases have been identified and verified

• Protein kinases are mainly divided into the following categories according to the type of amino acids on which protein phosphorylation occurs:

  • Serine/threonine protein kinases (STKs)

  • Protein tyrosine kinases (PTKs)

  • Dual‐specificity kinases (DSKs)

  • Histidine protein kinases (HPKs)

Phosphorylation in Aging

• Aging is a process characterized by declines in both organism and organ functions

• This process is characterized by cell cycle arrest, abnormal accumulation of senescent cells in tissues, and altered neurotransmission and response ability to external stimuli

• Some protein phosphorylation significantly changes with age and has crucial physiological functions

Phosphorylation in Immune Regulation

• Phosphorylation plays a crucial role in regulating innate and acquired immunity, a process that is coregulated by kinases and phosphatases

• Phosphorylation and other PTMs work together to regulate the signaling networks of the immune system

Phosphorylation in Metabolism

• Protein kinases transfer a phosphate group from ATP to specific amino acid residues (serine, threonine, or tyrosine) on metabolic enzymes, while protein phosphatases remove it

• This structural change can turn a pathway on or off within seconds

• For example, phosphorylation regulates key steps in:

  • Glycolysis

  • Citric Acid Cycle (Krebs)

  • Oxidative Phosphorylation (electron transport chain)

  • Glycogen synthesis

  • Lipid metabolism

Phosphorylation in Growth and Development

It is essential for the precise regulation of:

• Cell proliferation

• Cell cycle arrest

• Differentiation into various cell types during embryonic development

  • Ex. Erk signaling dynamics

Extracellular signal-regulated kinase (ERK)

A member of the mitogen-activated protein kinase (MAPK) family, which is found in all eukaryotes and the ERK pathway plays a widespread role in the development

Diseases linked to Erk Signaling Cascade:

Cancers:

• Lung Cancer: Driven by mutations in upstream components like RAS or RAF that lead to continuous ERK activation and uncontrolled cell proliferation

• Breast, Ovarian, Pancreatic, and Colorectal Cancers: Frequently exhibit aberrant or hyperactivated ERK signaling, which promotes tumor growth, invasion, and metastasis

• Thyroid and Brain Tumors: Associated with elevated phosphorylation and activation of the MAPK/ERK pathway

Neurodegenerative and Neurological Diseases:

• Alzheimer’s Disease: Involves dysregulated ERK signaling linked to tau phosphorylation and beta-amyloid toxicity

• Parkinson’s Disease: Linked to ERK involvement in glial neuroinflammation, L-DOPA-induced dyskinesia, and LRRK2 mutation-related apoptosis

• Amyotrophic Lateral Sclerosis (ALS): Involves compromised signaling and motor-neuron viability issues tied to SOD1 mutations

• Huntington’s Disease and Prion Diseases: Associated with pathway-driven neuroinflammation and cell stress responses

• Multiple Sclerosis: Linked to microglial malfunction and neuroinflammation driven by inappropriate ERK overactivity

Cancer & Phosphorylation-Associated Targeted Therapies

• Compared with traditional cytotoxic anticancer drugs, targeted anticancer drugs have the advantages of high efficiency, low toxicity, and strong specificity

• Given the important roles of protein kinases in tumor growth and metastasis, if the kinase signaling pathway is effectively blocked, the malignant progression of tumors may be prevented

• To date, 68 small molecule kinase inhibitors have been approved

  • EGFR: Erlotinib, Afatinib, Dacomitinib, Gefitinib, Osimertinib, Lapatinib, Veratinib

Lapatinib for Advanced or Metastatic Breast Cancer

• Registered in Europe and in the U.S. in combination with capecitabine for patients with advanced breast cancer or metastatic breast cancer (MBC) whose tumors overexpress human epidermal growth factor receptor (HER)-2 (ErbB-2) and are progressive after prior therapy, which must have included anthracyclines and taxanes, and therapy with trastuzumab in the advanced disease setting

• A potent reversible and selective inhibitor of the intracellular domains of the tyrosine kinases EGFR (HER-1) and HER-2 (HER: Human Epidermal Growth Factor Receptor)

• Competes with ATP for the ATP-binding pocket

• This leads to downstream blocking of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin dependent transduction pathways, resulting in growth arrest or apoptosis of tumor cells

Acetylation

• Covalent attachment of an acetyl group

• A process in which acetyl group donors, such as acetyl‐CoA and acetyl phosphate, covalently bind to the protein N‐terminus and lysine side chains, forming N‐terminal and internal acetylation

• Most commonly occurs at the side chain nitrogen atom of Lys

• It serves as a regulatory switch for protein conformation and activity changes

Protein acetylation is a dynamic equilibrium state regulated by two families of enzymes:

• Lysine acetyltransferases (KATs)

• Lysine deacetylases (KDACs)

Functional mechanisms of protein acetylation:

• Altering protein conformation

• Modulating protein-protein interactions

• Regulating enzymatic activity

• Altering protein-DNA interactions

• Controlling subcellular localization

• Influencing protein stability

Lysine Acetyltransferases and Histones

• Histones are positively charged proteins in eukaryotic cell nuclei that package, organize, and regulate DNA

• p300 are members of the KAT superfamily, primarily acetylating histones

• By acetylating histones, p300 switches on DNA accessibility, enabling the binding of transcription factors to DNA and concurrent interaction with p300

• Histone acetyltransferase activity of p300 leads to the opening of chromatin structure, allowing RNA polymerase II and transcription factors to bind, initiating gene transcription

Crystal structure of p300 Acetyltransferase Domain bound with Acetyl-CoA:

• Active site contains a negatively charged cavity that binds unmodified Lys residues

• Flexible activation loop has multiple Lys residues that can occupy this pocket, blocking access to substrate lysines and preventing enzyme activity

• Once the lysines in the activation loop are acetylated, they lose their positive charge and can no longer bind to the negatively charged substrate pocket, relieving autoinhibition and enabling enzyme activity

Conformational and Functional States of p300: From an Autoinhibited Resting State to an Active Acetyltransferase:

• Inactive: p300 adopts a compact, autoinhibited conformation in which the active site is occluded, resulting in poor engagement with DNA-bound transcription factors (TFs) and other co-regulators Nucleosomes remain compact, chromatin is in-accessible, histone acetylation is low, and gene transcription is repressed.

• Active: p300 undergoes a conformational extension that relieves autoinhibition and exposes the active site. Binding of transcription factors and/or co-activators stabilizes the active conformation and promotes recruitment.

  • Active p300 acetylates nucleosomal histones, opening chromatin and increasing accessibility, thereby promoting gene transcription

Mechanisms of Gene Activation by Histone Acetylation

By weakening the protein-DNA interaction:

• Positively charged N-term tails of histones bind tightly to negatively charged phosphate groups

• Condensed, transcriptionally inactive nucleosome structure

• Acetylation neutralizes the positive charge on histones, reducing their affinity for DNA

• Relaxed, transcriptionally active nucleosome structure

• Allows transcription factors to access the DNA

Ubiquitination

• Covalent attachment of ubiquitin

• Most commonly occurs at Lys

  • Side chain nitrogen is covalently linked to the C-term carboxyl group (Gly) of ubiquitin

Ubiquitination/SUMOylation

• Can form polyubiquitin chains

  • C-term carboxyl group of each subsequent ubiquitin is linked to a Lys residue on the preceding molecule

• Requires three types of enzymes:

1. E1 (activating enzymes)

2. E2 (conjugating enzymes)

3. E3 (ligases)

• Note: Reversible by the action of deubiquitinating enzymes

The Guardian of the Genome – p53

• Also known as:

  • Transformation-related protein 53

  • Tumor suppressor p53

• Undergoes numerous reversible PTMs for precise control

• Protects against cancer

• Repairs DNA damage by regulating cell cycle and apoptosis:

  • Activates DNA repair proteins when damage is detected

  • Arrests cell growth upon DNA damage recognition until damage is fixed

  • Initiates apoptosis if DNA has sustained irreparable damage

P53: PTM Crosstalk

• E.g., p53 ubiquitination and acetylation

• Mechanism:

• Unstressed Cells

  • p53 levels are kept low by ubiquitination of CTD Lys and continuous degradation

• Stressed Cells (Exposed to Cellular Stress):

  • Upon cellular stress, phosphorylation of the TAD facilitates acetylation of the CTD Lys

  • Acetylation prevents ubiquitination of CTD Lys, thereby protecting p53 from proteasome-mediated degradation

• ⇨ One of many mechanisms of p53 regulation