Cell Signaling: Structural Properties, Regulation, and Posttranslational Modifications of Signaling Proteins

Introduction

Instructor: Prof. Anca Dinischiotu
Affiliation: Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Bucharest

Modular Structure of Signaling Proteins

Multifunctionality:
- Signaling proteins are constructed from multiple signaling modules or domains.
- These modules can act independently or collectively, serving distinct roles in signaling.
Proteome Representation:
- Several signaling modules are found in various copies within the human proteome, used repeatedly for regulating distinct processes in a cell-type specific manner.
Evolutionary Innovation:
- Shuffling of modules or domains is believed to be a major source of evolutionary innovation in signaling behavior.
Types of Signaling Modules:
- Interaction Domains: Engage with downstream and upstream partners.
- PTM Sites: Serve as points for posttranslational modifications.
- Catalytic Domains: Activate enzymes and direct them to their targets.

Catalytic Domains

Function:
- Catalytic domains transmit signaling information, often through phosphorylation.
Activation Mechanism:
- Typically, the activity is low in the absence of a signal due to autoinhibition, inhibitory modifications, or inhibitor binding.
- Activation occurs through relief of inhibitory constraints in a signal-directed manner.

Targeting and Interaction Domains

Signaling Molecule Targeting:
- Distinct domains facilitate interaction with substrates, other signaling proteins, and cell membranes.
Importance of Membrane Targeting:
- Recruitment of signaling proteins to the inner side of the cell membrane is crucial, as signaling events often occur in close association with the membrane.

Regulatory Domains

Multivalency:
- Modules can interact simultaneously, sequentially, or in various subcellular locations.
Integration of Inputs:
- Signaling proteins receive and respond to multiple signals, leading to varied outputs depending on the cellular environment.

Unstructured, Flexible Sections

Role in Signaling:
- Unstructured regions can facilitate transitions from unstructured to structured states, essential for allosteric activation.
Example:
- Tumor suppressor protein p53 exhibits multiple PTMs in unstructured regions, regulating its function as a transcription factor.

Multivalency in Signaling Proteins

Functional Versatility:
- Module composition allows for the simultaneous reception of numerous signals, integrating them into differential responses.
Module Variants:
- Subtypes or splice variants increase functional multiplicity, affecting enzymatic activities and regulatory properties.

Modular Signaling Complexes

Assembly of Complexes:
- Form in response to signal input, involving many proteins and changing based on their modification status and context.
Efficiency in Signal Transmission:
- Interaction among multiple signaling components enhances signal transmission without the need for diffusion.

Specificity of Signal Transduction

Rapid Signal Coupling:
- Tight assembly ensures specific signal coupling and prevents unwanted signal dissipation.

Recruitment via Posttranslational Modifications (PTMs)

Dynamic Nature of Modifications:
- PTMs determine the composition and dynamics of signaling complexes, influencing signal outputs.

Regulation of Signaling Enzymes

Enzyme Structure:
- Generally modular, comprising catalytic, targeting, and regulatory domains.
Dominant Regulatory Mechanisms:
- Effector binding and PTMs, both involve allosteric conformational changes.

Allostery in Signaling Enzymes

Definition:
- Allostery allows enzymes to exist in multiple conformations, affecting activity based on activating or inhibitory signals.
Influence of Incoming Signals:
- Signals can change module interactions and enzyme packing, facilitating allosteric transitions.

Common Effector Molecules

Types:
- Primarily low-molecular-weight organic compounds or metal ions interacting with signaling enzymes, influencing activity.
Examples of Regulation:
- Feedback mechanisms in metabolic and signaling pathways, e.g., amino acids, purines, and glycolysis.

Posttranslational Modifications Overview

Classification:
- Stable PTMs: Disulfide formation, glycosylation, lipidation preserved longer.
- Transient PTMs: Serve dynamic, regulatory purposes.
Dynamic Nature of Modifications:
- Changes by allosteric interactions, recognized by interaction domains, facilitating target protein binding.

Common Regulatory PTMs

List of Important Modifications:
- Ser/Thr phosphorylation, Tyr phosphorylation, Lysine acetylation, Methylation, hydroxylation, and ubiquitination.
Specific Enzymatic Control:
- Enzymes exist for both PTM introduction and removal, highlighting regulatory control.

Modification Enzymes (Writers, Erasers, Readers)

Writers (Modifying Enzymes):
- Attach modifications to target proteins.
Erasers (Demodifying Enzymes):
- Remove modifications, restoring the original protein state.
Readers (Interaction Modules):
- Recognize and bind to specific modifications for downstream signaling.

Cross-Modifications and Recognition

Interaction with PTMs:
- Many regulatory functions are linked to multisite modifications, introducing specificity and redundancy in signaling pathways.

Antagonistic Interactions between PTMs

Characterization:
- Modifications may inhibit each other's function, e.g., acetylation versus phosphorylation on lysine residues.

Protein Phosphorylation as a Regulatory Mechanism

Mechanism Overview:
- Catalyzed by protein kinases, affecting enzyme activity, conformation, and interaction with other proteins.
Enzyme Specificity:
- Human genome has ~500 protein kinases, indicative of phosphorylation's central role in biochemistry.

Phosphorylation Mechanism and Impact

Effects of Phosphorylation:
- Conformational changes, binding site creation for downstream interactions, and dynamic regulation of cellular functions.
- Significant in signal transduction, influencing cellular response.

Lysine Acetylation Overview

Characteristics:
- Important in numerous cellular functions, widely prevalent, affects transcription, metabolism, and signaling pathways.
Dynamic Modification:
- Reversible, tightly linked to enzyme regulation by acetyltransferases and deacetylases.

Protein Methylation

Functionality:
- Methylation occurs on lysine and arginine residues, regulated by methyltransferases and demethylases, impacting transcriptional regulation and chromatin remodeling.

Ubiquitin Modification in Cellular Control

Functions:
- Ubiquitination serves primarily for protein degradation but also enables nonproteolytic functions such as trafficking and DNA repair.
Pathways of Degradation:
- Proteasomal and lysosomal pathways, with ubiquitin playing a central role in tagging proteins for destruction.

Ubiquitin Conjugation Process

Mechanism:
- Involves three enzymes (E1, E2, E3) and forms polyubiquitin chains essential for signaling and degradation signals.
Ubiquitin Code:
- Different linkages (e.g., K48, K63) lead to varied cellular responses.

Nonproteolytic Functions of Ubiquitination

Roles Beyond Degradation:
- Ubiquitin signaling is critical in various cellular pathways, including transcription and DNA repair through signaling complexity.

Summary of Lipid Modifications

Lipidation Mechanism:
- Attaches hydrophobic residues to proteins for stable membrane association, crucial for signaling pathways.
Types of Lipid Modifications:
- Myristoylation, palmitoylation, prenylation alter proteins' membrane interactions and signaling functions.

Glycosyl-Phosphatidyl-Inositol (GPI) Anchors

Functionality:
- Involved in cellular interactions, nutrient uptake, T-cell signaling; examples include various enzymes and receptors.
Dynamic Release:
- GPI-anchored proteins can be released from membranes into soluble forms for diverse cellular functions.