Lecture 3 pt 4

Covalent modifications overview

• Covalent modification = post-translational modification (PTM) of proteins; covalent conjugation of chemical groups onto a protein.
• In this course, these covalent modifications are directed by enzymes in most cases; an enzyme is required to make the modification.
• Many (if not all) covalent modifications are reversible and can be removed by another enzyme (erasers).
• Context dependence: effects of a given modification are highly context-specific; the same modification can have different effects on different proteins. Do not assume a single universal function.
• A table in the lecture summarizes main types of covalent modifications, examples of functional consequences, and figure references in the textbook (useful as a reference, not a universal rule).

Phosphorylation (S/T/Y)

• Residues that can be phosphorylated: serine (S), threonine (T), and tyrosine (Y).

• Enzymes:

  • Kinases add phosphate groups.
  • Phosphatases remove phosphate groups.

• Phosphate groups are highly electronegative, driving conformational changes in the target protein.

• Consequences can include:

  • Promotion of protein complex assembly (e.g., signaling complexes).
  • Activation or inhibition of enzyme activity (context-dependent).
  • Recruitment of other proteins (e.g., adapter proteins) in signaling pathways, such as receptor tyrosine kinases leading to downstream signaling.

• Phosphorylation acts as a molecular switch: reversible on/off control of activity.

• Src protein kinase as a canonical example of phosphorylation-based regulation:

  • Src has a C-terminal kinase domain plus regulatory SH2 and SH3 domains.
  • In the inactive state, Src is phosphorylated at a tyrosine in its N-terminal tail; the phospho-tyrosine is bound by the SH2 domain, keeping Src in a closed, inactive conformation.
  • Dephosphorylation by a phosphatase loosens the structure; the SH2 domain can then bind a phosphotyrosine from an activating ligand on another protein.
  • The SH3 domain binds to a proline-rich activating ligand, providing partial activation.
  • Src autophosphorylates a tyrosine within its own sequence, achieving full activation and substrate phosphorylation.
  • This illustrates how phosphorylation integrates multiple signals (and multiple binding events) to regulate activity.

• Key takeaway: phosphorylation is a versatile switch and can be integrated with other signals to fine-tune activity.

• Tyrosine kinase receptors (RTKs) example preview: when activated, they become phosphorylated on cytosolic tyrosines and recruit adapter proteins to propagate signals downstream.

Other covalent modifications (gene expression and beyond)

• Methylation and acetylation

  • Critical in regulating gene expression; especially prevalent on histones.
  • Methylation/acetylation of histones affects DNA accessibility to transcription factors and RNA polymerase.
  • Further details to be explored in future lectures; foundational for chromatin regulation and epigenetics.

• Palmitoylation

  • Addition of a fatty acid chain to a protein.
  • Used to tether proteins to membranes.
  • Not heavily discussed in this module; will be explored further later.

• N-acetylglucosaminylation (GlcNAcylation)

  • Addition of GlcNAc residues (sugar residues) to proteins.
  • Not very common in all contexts; involvement in regulation of glucose homeostasis in various ways.

• Ubiquitination (ubiquitin system)

  • A distinct and highly versatile covalent modification where whole small proteins (ubiquitin) are conjugated to substrates.
  • Ubiquitin is a small protein of 76 amino acids and ~8.6 kDa; it is ubiquitous and essential for signaling and protein degradation.
  • Ubiquitination is not a single modification but a small set of related processes with different meanings depending on context.
  • Catalyzed by a cascade of enzymes (see below); the specificity and outcome depend on the ubiquitin code generated.
  • Ubiquitin-like proteins (e.g., SUMO) can also be conjugated to proteins in similar ways.

• Important caveat about ubiquitination and other PTMs:

  • The bottom edge of the ubiquitination diagram shows several example outcomes; these are not universal rules. The meaning of each ubiquitin linkage is highly context-dependent and protein-specific.

Ubiquitination in depth

• Ubiquitin’s basic properties

  • Ubiquitin is a 76-aa protein (~8.6 kDa).
  • It can be attached to substrates in multiple ways, generating a “ubiquitin code” that is read by other cellular proteins to determine a fate or activity change.

• Basic forms of ubiquitination

  • Monoubiquitination (or ubiquitylation): a single ubiquitin attached to a single lysine on the substrate.
  • Multiubiquitination / multimono-ubiquitination: multiple single ubiquitins attached to multiple lysines on the substrate.
  • Polyubiquitination: chains of ubiquitin attached to a substrate through ubiquitin’s own lysine residues; mainly two linkages are emphasized:
  • K_{48}-linked chains are typically kinked and target the substrate to proteasomes for degradation.
  • K_{63}-linked chains are more linear and often modulate activity or promote complex formation, including roles in DNA repair and signaling.
  • Other polyubiquitin linkages exist (e.g., K{6}, K{11}, K_{27}), but these are not typically the focus of exams in this course.

• Ubiquitin chain types and outcomes

  • K_{48} chains generally tag substrates for degradation by the proteasome.
  • K_{63} chains influence protein activity and/or the assembly of protein complexes; important for DNA repair and signaling networks.

• Ubiquitin phosphorylation and variability

  • Ubiquitin itself can be phosphorylated on residues such as Ser65, adding another layer to the “ubiquitin code.”
  • There can be phospho-ubiquitin conjugates (phosphoproteo-ubiquitin).

• Complexity and regulation

  • Ubiquitination is executed by a cascade of three enzyme activities:
  • E1 ubiquitin-activating enzyme
  • E2 ubiquitin-conjugating enzyme
  • E3 ubiquitin ligase
  • The trio provides substrate specificity, chain elongation, and linkage type.
  • The concept of a ubiquitin code means the combination and type of ubiquitin modifications determine the outcome for the substrate.

• Ubiquitin-like modifiers

  • Other small proteins similar to ubiquitin, such as SUMO, can be conjugated to substrates and exert regulatory effects.

• Practical example: p53 as a multi-modified substrate

  • p53 is a key transcription factor governing the cell cycle and cell death in response to DNA damage.
  • p53 can be ubiquitinated, acetylated, phosphorylated, and sumoylated at different sites.
  • Different enzymes add these modifications in response to distinct signals; the combination allows signal integration (a form of information processing).
  • Cross-talk exists: one modification can enable or block others nearby due to steric or allosteric effects.
  • In some cases, multiple modifications are required for full activation (or repression); single modifications may have little or no effect—an example of Boolean logic in signaling, such as an AND operation.

• Takeaway: the ubiquitin code and multiple PTMs enable combinatorial regulation and signal integration in the cell.

Covalent modification as integrated signaling and molecular switches

• Concept of signal integration
  • A protein can be decorated with several different PTMs at multiple sites; these signals can be added in a specific order and may influence each other.
  • Some modifications are prerequisites for others; certain modifications can block others nearby.
  • A protein may require multiple modifications to become fully active, illustrating a Boolean-logic-like control (e.g., an AND operation).
• Example of a simple switch: phosphorylation as a molecular switch
  • A phosphate can turn a protein on or off by changing its conformation and/or interactions.
  • This reversible switch allows rapid and tunable control of activity in response to cellular cues.

GTP-binding proteins as molecular switches

• Overview
  • GTP-binding proteins switch between an active (GTP-bound) and inactive (GDP-bound) state.
  • These proteins have intrinsic GTPase activity that hydrolyzes bound GTP to GDP, turning the switch off.
  • GDP dissociation is relatively slow; binding of a new GTP (fast) reactivates the protein.
• Regulation by GEFs and GAPs
  • GEFs (Guanine nucleotide Exchange Factors) promote GDP dissociation, allowing GTP binding and activation; functionally similar to kinases activating signaling cascades.
  • GAPs (GTPase-Activating Proteins) stimulate GTP hydrolysis, turning the protein off by promoting GDP formation.
• Significance
  • GTP-binding proteins are used widely as molecular switches in processes like nuclear import and vesicular trafficking, making them central to many regulatory networks.

Summary and key takeaways

• The most important covalent modifications in eukaryotic cells include phosphorylation and ubiquitination (and related PTMs like acetylation, methylation, sumoylation, GlcNAcylation).
• PTMs can act as molecular switches, toggling proteins between active and inactive states; they can serve as part of larger signal integration networks that require combinations of signals to produce a response.
• The same modification can have different outcomes depending on the protein context; do not assume a universal function.
• Ubiquitination is a multi-faceted system with a spectrum of outcomes depending on chain type and context; the ubiquitin code is interpreted by cellular machinery to regulate degradation, activity, localization, and signaling.
• The ubiquitination cascade (E1, E2, E3) creates substrate specificity and linkage types; ubiquitin-like modifiers (e.g., SUMO) add additional layers of regulation.
• p53 serves as a prime example of how multiple PTMs can be integrated to modulate transcriptional activity and cell fate decisions in response to DNA damage.
• GTP-binding proteins provide an alternative family of molecular switches, controlled by GEFs and GAPs, and functioning across diverse cellular processes.
• The next lecture will delve into protein–protein and protein–substrate interactions as they relate to these regulatory mechanisms.

Key terms to remember

• Post-translational modification (PTM)
• Kinase / phosphatase
• Phosphorylation: on S, T, Y residues
• Methylation / Acetylation (histone regulation and gene expression)
• Palmitoylation
• GlcNAcylation (N-acetylglucosaminylation)
• Ubiquitination / Ubiquitin code
• Monoubiquitination / Multiubiquitination / Polyubiquitination
• K{48}-linked ubiquitin chains / K{63}-linked ubiquitin chains
• E1 / E2 / E3 enzymes
• Ubiquitin-like proteins (e.g., SUMO)
• p53 and multi-site PTMs
• Boolean logic in signaling (e.g., AND operation)
• GTP-binding proteins / GTPases
• GEFs / GAPs
• Src protein kinase (SH2/SH3 regulation; tyrosine phosphorylation)
• Reversibility and context-dependence of PTMs