Post-translational Modifications (PTMs)

Post-Translational Modifications (PTMs)

• Most proteins undergo chemical modifications before becoming functional.
• Post-translational modifications (PTMs) is the collective term for these modifications
• PTMs are crucial for generating protein heterogeneity
• Help utilize identical proteins for different cellular functions in different cell types.
• Genome has approximately 20-25,000 genes
• Transcriptome has approximately 100,000 transcripts
• Proteome has over 1,000,000 proteins
• Alternative promoters, alternative splicing and mRNA editing contribute to proteome complexity.

Enzymes Mediating PTMs

• PTMs occur at distinct amino acid side chains, mediated by enzymatic activity.
• Enzymes comprise 5% of the proteome and perform more than 200 types of PTMs.
• Enzymes include kinases, phosphatases, transferases, ligases, and proteases.
• These enzymes add or remove functional groups, proteins, lipids, or sugars to/from amino acid side chains.
• Many proteins modify themselves using autocatalytic domains like autokinase and autoprolytic domains.

Purposes of PTMs

• Targeting (e.g., membrane targeting)
• Regulation of stability (e.g., secreted glycoproteins and ubiquitination)
• Structural role in proteins (e.g., surface glycoproteins)
• Control of activity (e.g., phosphorylation and caspases)

Types of PTMs

• Most PTMs are reversible.
• Kinases phosphorylate proteins at specific side chains
• Phosphatases hydrolyze the phosphate group to remove it.
• Proteolytic cleavage is irreversible, cleaving peptide bonds permanently.
• Types of post-translational modifications:
• Proteolytic processing: removal of the peptide sequences or domains.
• Phosphorylation: addition of a phosphate.
• Glycosylation: addition of carbohydrates.
• Acetylation: addition of an acetyl group.
• Alkylation: addition of an alkyl group (like methyl or ethyl).
• Hydroxylation, carboxylation, and oxidation of side chains.
• Biotinylation: acylation of lysine residues with a biotin appendage.
• Glutamylation: covalent linkage of glutamic acid to tubulin and other proteins.
• Glycylation: covalent linkage of 1 or more than 40 glycine residues to the C-terminal tail of tubulin.
• Isoprenlyation: addition of an isoprenoid group.
• Sulfation: addition of a sulfate group to a tyrosine.
• Selenation: selenocysteine/selenomethionine modification.
• Amidation: usually at the C terminus

Phosphorylation

• Reversible protein phosphorylation on serine, threonine, tyrosine, or histidine residues is the most important and well-studied post-translational modification.
• Phosphorylation plays a critical role in regulating many cellular processes:
• Cell cycle
• Growth
• Apoptosis
• Signal transduction pathways

Abundance and Mechanism

• A most common mechanism for regulating protein function and transmitting signals.
• More pervasive in eukaryotic cells, though observed for bacterial proteins.
• Estimated that 1/3 of proteins in the human proteome are substrates for phosphorylation.
• Phosphoproteomics: branch of proteomics identifying and characterizing phosphorylated proteins.
• Occurs at the side chains of 3 amino acids: serine, threonine, and tyrosine in eukaryotic cells.
• These above amino acids have hydroxyl (-OH) groups attack the terminal phosphate group (γ-PO32-) on adenosine triphosphate (ATP).
• The phosphate group transfers to the amino acid side chain
• This reaction is unidirectional due to the large amount of free energy released.

Kinases and Phosphatases

• Protein phosphorylation is a reversible PTM mediated by kinases and phosphatases that phosphorylate and dephosphorylate substrates, respectively.
• Kinases and phosphatases facilitate the dynamic nature of phosphorylation.
• The extent of the phosphoproteome in a given cell depends on the spatial and temporal balance of kinase and phosphatase concentrations.
• Over 500 kinases have been predicted in the human proteome, which is comprised of the kinome.
• Kinase activity substrates are diverse.
• Substrates include proteins, carbohydrates, nucleotides, and lipids.
• ATP is co-substrate for almost all protein kinases, but guanosine triphosphate is used by a small number of kinases.
• The ATP-binding site is generally conserved, although substrate specificity of kinases varies.
• Kinase specificity is shown in protein kinase subfamilies which include serine/threonine kinases or tyrosine kinases.
• Approximately 80% of mammalian kinome comprises serine/threonine kinases, and more than 90% of the phosphoproteome consists of pS and pT.
• The relative abundance ratio of pS:pT:pY in a cell is approximate 1800:200:1.
• Tyrosine phosphorylation, though not as prevalent as pS and pT, is critical for biomedical research because of its connection to human disease (e.g., dysregulation of tyrosine kinases receptors (RTKs).
• Kinase substrate specificity relies not only on the target amino acid, but also consensus sequences flanking it.
• Kinases can phosphorylate single or multiple amino acids on an individual protein,
• Kinase single protein consensus sequences allow some kinases to phosphorylate proteins.
• Other kinase consensus sequence variants allow multiple substrates (>300).
• Kinases have regulatory subunits for activating or inhibiting signals
• Basal state/dephosphorylated signals for protein Kinases are inactive
• Activity is controlled by phosphorylation
• Small quantities of Kinases are always active
• They deactivate when phosphorylized.
• Kinases such as Src require phosphorylation and dephosphorylation to activate.

Protein Activity Regulation

• Protein activity can be affected by phosphorylation in 2 ways
• Catalytic activity directly regulated by phosphorylation
• Recruitment of proteins containing conservative domains
• Domains show a specificity in bonding
• SH2 domains show a specificity for phosphotyrosine
• WW domains recognize phosphoserine
• FHA domains recognize phosphothreonine
• Protein activity regulation is essential for signal transduction
• Effector proteins which are downstream, are recruited to signals of phosphorylated proteins

Protein Phosphatases

• Phosphorylation dependent signaling regulated by 3 methods
• ligand removal
• Kinase or subsrate proteolysis
• Phosphatase-dependent dephosphorylation
• Human proteome contains approximately 150 protein phosphatases
• Phosphatases show a specificity of pS/pT or pY residues
• Dephosphorylation achieved by the 2 groups but uses different pathways.

Signal Transduction Cascades

• PTM achieved through reversible protein phosphorylation is perfect for signal transduction
• Cellular responsiveness increased.
• Signal Transduction Cascades are shown via:
• Proteins sensing signals and relaying them-to secondary messengers + signaling enzymes
• Activation of downstream kinases by phosphorylation
• Achieving specific responses through the kinases activation of downstream substrates
• Signal Trasnduction cades can be linear
• Signalling pathaways also amplify
• Kinase actvation results in activation for additional kinases
• Growth factor is able to activate these global programs

Methylation

• Hydrophobicity of protein increased due to the methyl groups transferred to nitrogen/oxygen
• Amino acids negative charges can be neutralized
• SAM is the main methyl donor and is mediated by Methyltransferases
• Substrates in enzymatic reactions heavily use SAM
• Mulyiple or a single group can be added
• Histone methylation plays an important role as it influences transcription via the ability of DNA

N-acetylation

• Common gene transcription regulation via acetylation at e-NH2 of lysine on histones
• Histone acetyletransferase regulates lysine residued acetylation through transcription factors that contain HAT
• Histone deacetylase is a reverse reaction achieved via HDAC enzymes

Chromatin and PTMs

• Nucleosomes that are condensed also improve replication, transcription and DNA repair.
• These histone-post translaational modification informations are transferred
• Modifications acheived through enzymes reversibly modify nucleosomes as nucleosome-modelling complexes
• Kinases, acetlyases, methylases and histone deacetylases are present
• There are 2 mechanisms
• 1. Changing the chromatinstructure with an activated or suppressed transcriptional state
• 2. Docking site achieved for regulators

Proteolysis

• Cells use this mechanism to break the bonds as stable peptide bonds are under conditions that are physiological.
• Over 11,000 varying protreases that all cleave proteins
• They all differ in:
• Specificity of substate
• Length of Activity
• Mechanism of pepetide cleavage
• Location

Degradative Proteolysis

• Proteins must be maintained at consistent homeostatic concentration
• Unassembled proteins + misfolded proteins must be degraded.
• Peptides and single amino acids must also be degraded
• Proteases also achieve enzyme activity by cleaving zygomens and signal peptides. These act like molecualr signals to regular enzyme pathways such as with temporol and/or spatial control mechansisms

Degradation

• Protein degradation done via 2 methods
• Lysosomal via lysonomal protein degragation by enzymes
• Cytosolic achieved via a ubiquitin-proteasome pathway

Ubiquitin

• ATP dependent, pathways are selective for degradation by ubiquitin-mediated means
• Eukaryotes have this highly-convered 76 residue protein
• Degradation through conjugation with ubiquitin
• Protein structure contains stable globular folding
• Ubiquitin shows around 96% sequence identity in humans and yeast
• Contributes in many cellular processes
• E1 enzyme forming a thioester bond achieved via combining with the ubiquitin C-terminal GLy
• Ubiquitin E2 carrier with a ubiquitin-conjugating portion
• Ubiquitin Ligase 3 selects with the E2 S ubiquitin for degradation
• Selected porteins achieved here
• Multiple ubiquitins are available here
• Ubiquitination cascade uses UBI for all functions: ATP, substrate and functions

E1 Activating Enzyme

• Catalysis in 2 steps
• 1. C-terminus activated and an intermedaite is formed
• 2. Second catalytic Cys residue then forms a thioester bond
• Activation then transferred

E2 Ubiquitin Conjugating Enzyme:

• Activates UBi and carries to the substrate
• E1 is also achieved from a Thioester Bond
• Greater E2 available
• Paired with a number of E3's

E3 Ubiquitin Ligases:

• Lys and substrate both selection with specificity
• Location activated UB near the lys and substate
• Ubquitin has a tranfer promoting function from E2 to the lys residue
• Differing ligases available via distinctive functions

The Proteasome

• Proteins degrades thanks to protein degrading machines that contribute 1% of the proteins
• Present in the nucleus and fluid
• Central core acts a catalyst with the cylinder
• 19S cap is present here
• Function achived through unfolding and removal
• Proteasomal degradtaion achieved through a number of means: cell division, apoptosis
• Cell arest caused by inhibitors
• Cancer treamtent is acheived via a number of method

Explore post-translational modifications (PTMs) and their roles in cells, including enzymes that mediate PTMs and the impact of modifications like phosphorylation, acetylation, and glycosylation on protein activity and function. Scenarios where proteolytic processing is crucial. Histone modification.