Medicine 1 - Proteins 2: Protein Folding & Function
Protein Folding & Function
Kuru and Prion Diseases
Kuru: A past affliction of the Fore people of the Eastern Highlands, primarily affecting women and children.
Symptoms: Truncal ataxia, joint pains, eventual memory loss, pneumonia, etc.
Incubation time: Highly varied, up to several decades.
The long incubation period makes it difficult to trace the source of infection and implement effective control measures.
Funeral practices involved mortuary cannibalism.
These practices facilitated the transmission of prions, the infectious agents, leading to the spread of the disease.
Distribution amongst the population reflected societal structure and gender roles.
Women and children, who were more involved in the preparation and consumption of the deceased, were disproportionately affected.
Prion Diseases: Transmissible and can lead to large epidemics.
Prion diseases are caused by misfolded prion proteins that can induce other normal proteins to misfold, leading to a cascade of protein aggregation and neuronal damage.
This transmissibility poses a significant threat to public health, requiring stringent measures to prevent the spread of infection.
Reference: Gajdusek, D. (1976) Nobel Lecture.
Learning Objectives
Understand the processes driving protein folding and how these relate to primary, secondary, and tertiary structure.
Explore the roles of chaperones, hydrophobic interactions, and disulfide bonds in guiding protein folding to its native state.
Explain how protein complexes form and describe their effects on protein function.
Investigate the mechanisms of protein-protein interactions, including electrostatic forces, hydrogen bonding, and van der Waals forces.
Understand how these interactions influence protein stability, activity, and regulation.
Recognize the role of mutations leading to altered proteins in some diseases, using hemoglobin as a model.
Analyze the impact of specific mutations on protein structure and function, and how these alterations contribute to disease pathogenesis.
Recognize common types of post-translational modification and their purposes.
Investigate the diverse array of post-translational modifications, such as phosphorylation, glycosylation, acetylation, and ubiquitination.
Understand how these modifications modulate protein activity, stability, localization, and interactions.
Protein Folding Reminder
Proteins are macromolecules.
Proteins are essential for virtually every biological process, including catalysis, transport, signaling, and structural support.
They are amino acid polymers.
Amino acids are the building blocks of proteins, linked together by peptide bonds to form polypeptide chains.
They form from one or more polypeptide chains.
Some proteins consist of a single polypeptide chain, while others are composed of multiple subunits that assemble into a functional complex.
Amino acids vary in their physiochemical properties.
The unique properties of each amino acid, such as size, charge, hydrophobicity, and hydrogen-bonding capacity, dictate how a protein folds and interacts with other molecules.
Proteins fold in 3D; their function depends on this 3D structure.
The specific three-dimensional conformation of a protein determines its ability to perform its biological function.
Misfolded proteins can lose their activity or even become toxic.
Conformation depends on the amino acid sequence, which is determined genetically.
The sequence of amino acids in a protein, encoded by its gene, ultimately determines its three-dimensional structure and function.
Folding: Primary & Secondary Structure
Primary (1°): The amino acid sequence (including S-S bonds).
The sequence of amino acids, linked together by peptide bonds, forms the primary structure of a protein.
Disulfide bonds, formed between cysteine residues, can stabilize the primary structure and influence folding.
The primary sequence determines which secondary structures form.
Specific amino acid sequences favor the formation of particular secondary structures, such as alpha helices and beta sheets.
Secondary (2°): Local packing & regular arrangements.
Secondary structures are local, regular arrangements of the polypeptide backbone, stabilized by hydrogen bonds between amino acid residues.
Common secondary structures include alpha helices, beta sheets, and turns.
The secondary structures then fold together…
The arrangement and interactions of secondary structure elements contribute to the overall three-dimensional structure of the protein.
Folding: Tertiary Structure
Tertiary (3°): The 3D packing of secondary structure elements.
Tertiary structure refers to the overall three-dimensional arrangement of all the atoms in a protein, including the side chains of amino acids.
Folding into the tertiary structure is driven by:
Interactions between 2° structure elements.
Interactions between alpha helices, beta sheets, and other secondary structure elements, such as hydrophobic interactions, hydrogen bonds, and electrostatic forces, drive the formation of tertiary structure.
Entropy (burying hydrophobic residues).
The tendency of hydrophobic amino acid residues to cluster together in the interior of a protein, away from water, contributes to the stability of the tertiary structure and increases entropy.
Structure Stabilization (Folding)
The protein fold forms during and after biosynthesis.
Protein folding can begin as the polypeptide chain is being synthesized on the ribosome, and continues after synthesis is complete.
The final conformation depends on:
The amino acid sequence.
The sequence of amino acids in a protein dictates its folding pathway and final three-dimensional structure.
Folding constraints from the position & extent of secondary structure.
The arrangement and interactions of secondary structure elements constrain the possible folding pathways and influence the final conformation of the protein.
The major stabilizing forces in proteins are:
Hydrophobic interactions.
The tendency of hydrophobic amino acid residues to cluster together in the interior of a protein, away from water, provides a major driving force for protein folding and stabilizes the tertiary structure.
Electrostatic attractions.
Electrostatic interactions, such as hydrogen bonds, ionic interactions, and van der Waals forces, contribute to the stability of the protein structure by promoting favorable interactions between amino acid residues.
Covalent linkages.
Covalent bonds, such as disulfide bonds between cysteine residues, can provide significant stability to the protein structure by cross-linking different parts of the polypeptide chain.
Structure Stabilization Continued
Hydrophobic Interactions:
The major driver for protein folding.
Hydrophobic interactions are the primary driving force for protein folding, as they promote the burial of hydrophobic amino acid residues in the interior of the protein, away from water.
Nonpolar residues are buried on the inside of a polypeptide’s interior, maximizing entropy.
By burying nonpolar residues in the protein's interior, the surrounding water molecules become more disordered, leading to an increase in entropy and contributing to the overall stability of the folded protein.
Electrostatic Interactions (collectively strong):
Van der Waal forces.
Van der Waals forces are weak, short-range attractive forces that arise from temporary fluctuations in electron distribution.
Although individually weak, these forces can collectively contribute to protein stability by promoting close packing of atoms.
Hydrogen bonds (drive 2° structure).
Hydrogen bonds are interactions between a hydrogen atom and an electronegative atom, such as oxygen or nitrogen.
They play a crucial role in stabilizing secondary structures, such as alpha helices and beta sheets, as well as promoting specific interactions between amino acid residues.
Ionic interactions (“salt-bridges”).
Ionic interactions, also known as salt bridges, are electrostatic interactions between oppositely charged amino acid residues.
These interactions can contribute to protein stability and influence protein-protein interactions and ligand binding.
Protein Folding & Domains
Longer polypeptides tend to fold into a number of smaller independent domains.
Modular units within a protein that fold and function independently.
These are often functionally-distinct units.
Each domain may have a specific function, such as binding a particular ligand or catalyzing a specific reaction.
The folding of individual domains follows the same principles.
The folding of individual domains is driven by the same forces, such as hydrophobic interactions, hydrogen bonds, and electrostatic forces, as the folding of the entire protein.
Example: Tissue plasminogen activator (tPA) is a clot-degrading protein.
Tissue plasminogen activator (tPA) is a serine protease involved in the breakdown of blood clots.
It contains five independent domains that perform specific functions.
Fibronectin type I domain, epidermal growth factor domain, kringle 1 domain, kringle 2 domain, and the serine protease domain.
Tertiary Structure Variability
A specific (primary) sequence will tend to fold into a specific 3D structure; however:
While a protein's primary sequence dictates its overall fold, there can be some variability in the tertiary structure.
Proteins can adopt multiple conformations (shapes), often related to their function.
The ability of a protein to adopt multiple conformations allows it to interact with different binding partners, respond to different stimuli, and perform its biological function.
Even if they have a single main conformation, proteins are flexible, with the extent of movement related to temperature.
The extent of protein flexibility depends on various factors, including temperature, pH, and the presence of ligands or binding partners.
Structural Flexibility
The atoms in proteins are not static – they “breathe.”
Atoms within a protein molecule are in constant motion, vibrating, rotating, and fluctuating in position.
Side chains and even domains can move by (2Å).
Side chains and domains can undergo significant movements, which are important for protein function and regulation.
Much larger movements are possible when ligands bind.
Ligand binding can induce conformational changes that propagate throughout the protein structure, leading to alterations in activity and interactions.
Quaternary Structure
Quaternary (4°): Number and arrangement of multiple polypeptides.
The quaternary structure of a protein describes the number and arrangement of multiple polypeptide chains, called subunits, within the protein complex.
Formation of protein-protein interfaces:
Quaternary structure is stabilized by non-covalent interactions between subunits that form Protein-protein interfaces
Interaction is usually non-covalent.
Non-covalent interactions drive quaternary structure formation.
Interfaces must be complimentary; protein-protein interaction is a pattern-recognition process.
For stable quaternary structure, interfaces of subunits must fit together closely and have complimentary properties.
This is achieved through a mix of H-bonds, van der Waal, and salt-bridges.
These interactions create patterns that allow subunits to bind correctly.
On average, interfaces are less hydrophilic than solvent-facing protein surfaces but more hydrophilic than protein interiors.
Because interfaces are less hydrophilic, they tend to be buried in the complex.
Why Form Complexes?
Efficiency/Ease of Construction:
Forming complexes allows proteins to improve function, and also control and repair complexes more easily.
Can expand function.
Subunits come together to add functionality to a protein, such as the case of multiple binding sites.
Easier repair and control.
It's easier for quality control systems to replace a faulty subunit than an entire protein.
Can assemble where required, not just in the nucleus.
In eukaryotes, proteins are translated in the cytoplasm. Having subunits produced separately allows the complexes to be built in specific parts of the cell where their function is best utilized.
Smaller genome size (can reuse subunits in different complexes).
Given just one gene for a subunit, a number of different proteins can be made.
Oligomers Can Make Better Enzymes:
Size increase can stabilize individual subunits
By increasing surface area, the complex is more stable overall.
Multiple binding/active sites.
Oligomers can have multiple active sites, allowing them to perform complex enzymatic reactions more efficiently.
Subunits permit regulation and cooperativity.
Multiple subunits allow for regulatory mechanisms, such as allosteric control, where the binding of one molecule to one subunit affects the activity of other subunits.
Complexes: Hemoglobin
Hemoglobin: The oxygen carrier of blood.
Hemoglobin (Hb) is a protein found in red blood cells that binds to oxygen in the lungs and transports it to tissues throughout the body.
4 “globin” subunits (i.e., tetrameric).
Hemoglobin is a tetrameric protein, containing four globin subunits, each of which can bind one molecule of oxygen.
HbA contains two alpha (α) and two beta (β) subunits;
Adult hemoglobin (HbA) consists of two alpha (α) subunits and two beta (β) subunits, which are arranged in a specific three-dimensional structure.
A heme group containing iron(II) within each subunit binds oxygen.
Each globin subunit contains a heme group, which consists of a porphyrin ring coordinated to an iron(II) ion.
The iron(II) ion is responsible for binding oxygen molecules.
Cooperativity & Affinity
DeoxyHb dimers are held together by salt bridges and H-bonds.
In deoxyhemoglobin (Hb without oxygen bound), the globin subunits are held together by a network of salt bridges and hydrogen bonds, which stabilize the T state.
These weaken when oxygen binds.
When oxygen binds to one subunit, it triggers a conformational change that weakens the salt bridges and hydrogen bonds, promoting the transition to the R state
The two forms are called T (taut) and R (relaxed) respectively.
The T state (taut) is the low-oxygen affinity state, while the R state (relaxed) is the high-oxygen affinity state.
Cooperativity & Affinity (2)
Hemoglobin:
Hemoglobin must efficiently bind oxygen to function properly
Must bind oxygen efficiently in the lungs (high ) and release it to tissues (low ).
To efficiently carry oxygen between the lungs and tissues, hemoglobin must be able to bind oxygen tightly in the lungs, where the partial pressure of oxygen is high, and release it readily in the tissues, where the partial pressure of oxygen is low.
A protein with constant high or low oxygen affinity would not work well.
A protein with constant high oxygen affinity would not release oxygen to the tissues, while a protein with constant low oxygen affinity would not bind oxygen efficiently in the lungs.
The Solution:
Cooperativity
Hb binds oxygen cooperatively; binding of one oxygen increases the affinity at the other sites; release of an oxygen does the reverse.
Cooperativity allows hemoglobin to efficiently bind oxygen in the lungs and release it in the tissues.
A sigmoidal curve is the signature of cooperativity.
The sigmoidal shape of the oxygen-binding curve for hemoglobin is a characteristic feature of cooperativity, reflecting the increasing affinity of hemoglobin for oxygen as more oxygen molecules bind.
Three Main Protein Classes
Fibrous:
Fibrous proteins only display a single secondary structure
Normally display single 2° structure.
Fibrous proteins are characterized by their elongated shape and structural function.
e.g., α-keratin from wool & hair (α-helical coiled coil).
Alpha-keratin is the main structural component of hair, wool, and nails.
It consists of two alpha-helices that are coiled together.
Globular:
Globular proteins are interspersed with irregular loops.
Exhibit 2° structure elements interspaced with irregular loops.
Globular proteins are characterized by their compact, spherical shape and diverse functions.
e.g., most cytoplasmic proteins.
Many enzymes, antibodies, and hormones are globular proteins.
Membrane:
Membrane proteins have hydrophobic domains
Integral membrane proteins contain one or more hydrophobic domains that span the lipid bilayer.
Membrane proteins are embedded in the lipid bilayer of cell membranes and play a crucial role in transporting molecules, transmitting signals, and maintaining cell integrity.
e.g., ion channels & most receptors.
Ion channels are membrane proteins that form pores through which ions can flow across the cell membrane.
Receptors are membrane proteins that bind to specific ligands, such as hormones or neurotransmitters, and trigger a cellular response.
Genetic Mutations & Disease
Evolution has optimized proteins for their roles.
Natural selection has shaped proteins to perform their specific functions with high efficiency and precision.
Mutations causing amino sequence changes are often deleterious.
Changes to the amino acid sequence of a protein can disrupt its structure and function, leading to a variety of diseases.
If a protein is absent or dysfunctional, the other allele may produce sufficient protein for the effect to be a recessive trait (if heterozygous).
In heterozygous individuals, where one allele is mutated and the other is normal, the normal allele may produce enough functional protein to compensate for the mutated allele, resulting in a recessive trait.
Mutations can have a range of effects depending on the function of the affected protein.
The impact of a mutation depends on the protein's role in the cell and the extent to which the mutation disrupts its function.
Genetic Mutations: Potential Effects
Effect | Details |
|---|---|
Disruption to a metabolic pathway | Loss of product(s) and accumulation of precursor. e.g., phenylketonuria (inability to metabolize dietary phenylalanine). |
Impairment or loss of defense against infection | Mutations within the innate or adaptive immune system can reduce their effectiveness or lead to inappropriate activation. |
Protein aggregation | Can lead to diseases such as Alzheimer’s and Huntingdon’s. |
Dysfunction of a regulatory protein or receptor | Can disrupt pathway to cause physiological or developmental defects. |
Example: Sickle-Cell Anemia
A genetic disease affecting millions worldwide, more common among people with African or Mediterranean ancestry.
Sickle-cell anemia is a genetic disorder that primarily affects individuals of African or Mediterranean descent.
Caused by a single mutation in the beta subunit of hemoglobin.
Sickle-cell anemia is caused by a single point mutation in the gene encoding the beta subunit of hemoglobin.
Glutamate (Glu/E) at position 6 becomes valine (Val/V).
The mutation results in the substitution of glutamate (Glu/E) with valine (Val/V) at position 6 of the beta-globin chain.
Individuals with a single βS copy are generally asymptomatic.
Individuals who inherit one copy of the sickle-cell gene (βS) and one copy of the normal gene (βA) are generally asymptomatic carriers.
With two copies, the hemoglobin aggregates.
Individuals who inherit two copies of the sickle-cell gene (βS/βS) produce abnormal hemoglobin that tends to aggregate, leading to the characteristic symptoms of sickle-cell anemia.
Sickle-Cell Anemia: Molecular Basis
HbS molecules carrying E6V mutation aggregate to form fibers under low oxygen conditions.
The abnormal hemoglobin molecules (HbS) in sickle-cell anemia aggregate to form long, fibrous polymers under low oxygen conditions.
These distort erythrocytes into sickle-shapes, which causes them to block capillary blood-flow.
The polymerization of HbS molecules causes red blood cells to distort into a sickle shape, which reduces their flexibility and makes them prone to blocking small blood vessels.
Folding & Disease
A protein’s function depends on it having a specific 3D structure.
The biological activity of a protein is determined by its precise three-dimensional structure, which enables it to interact with other molecules and perform its specific function.
Proteins can misfold during synthesis or simply over time (i.e., aging).
Proteins can misfold due to errors during synthesis, genetic mutations, environmental factors, or the accumulation of damage over time.
Cells have developed extensive mechanisms to prevent or remove misfolded proteins:
Chaperone and proteolytic systems
Chaperones to mediate folding.
Chaperone proteins assist in the proper folding of newly synthesized proteins and help to prevent misfolding and aggregation.
Proteasome (via ubiquitin) & lysosome (in autophagy) to mediate breakdown.
The proteasome and lysosome are cellular organelles responsible for degrading misfolded or damaged proteins.
Misfolding often exposes hydrophobic residues, making the protein more prone to aggregation.
Misfolded proteins often expose hydrophobic amino acid residues that are normally buried in the protein's interior, leading to aggregation and the formation of insoluble protein deposits.
Protein Misfolding Disorders (PMDs)
Where these controls fail, proteins can fold into non-native “conformers.”
When the cellular mechanisms for preventing and removing misfolded proteins are overwhelmed, proteins can fold into abnormal conformations that are non-native.
Above a certain threshold, these can become toxic.
Accumulation of misfolded proteins can lead to cellular dysfunction, inflammation, and cell death, contributing to the pathogenesis of various diseases.
Leads to protein-misfolding disorders (around 50 examples, including Alzheimer’s, Parkinson’s & Huntington’s diseases).
Protein-misfolding disorders (PMDs) are a diverse group of diseases characterized by the accumulation of misfolded proteins in cells and tissues.
Occur not just in the CNS (e.g., amyloidosis in the liver).
Protein misfolding can occur in various organs and tissues throughout the body, not just the central nervous system.
Many of these disorders are age-related, but there can be a genetic component.
The risk of developing protein-misfolding disorders increases with age.
Genetic factors can also play a role in the susceptibility to these disorders.
Example: Prion Diseases
Amongst the best-characterized protein-misfolding disorders.
Prion diseases are a group of fatal neurodegenerative disorders caused by the misfolding and aggregation of the prion protein (PrP).
Result from misfolding and aggregation of cellular prion protein (PrPc becoming PrPSc).
Prion diseases arise when the normal cellular prion protein (PrPc) misfolds into an infectious form called PrPSc.
Termed “transmissible spongiform encephalopathies” and can be genetic or acquired (e.g., CJD).
Prion diseases are also known as transmissible spongiform encephalopathies (TSEs) because they can be transmitted between individuals and cause a characteristic spongelike appearance in the brain.
The prion protein forms insoluble fibrillary aggregates (PrPSc) and small soluble oligomers.
Misfolded PrPSc molecules aggregate to form insoluble fibrillary structures, which are thought to contribute to neuronal damage.
These cause immune-mediated inflammation and ultimately cell death.
The accumulation of PrPSc aggregates triggers an immune response, leading to inflammation and neuronal death.
Protein Modifications
Proteins in eukaryotes are often covalently modified:
Covalent modifications alter the structure of proteins
During ribosomal synthesis (co-translational modification).
Co-translational modifications occur during protein synthesis on the ribosome and can influence protein folding, stability, and function.
Following synthesis (post-translational modification (PTM)).
Post-translational modifications (PTMs) occur after protein synthesis and can regulate a wide range of cellular processes.
Modifications include glycosylation, phosphorylation, and hydroxylation.
Glycosylation involves the attachment of carbohydrate moieties to proteins.
Phosphorylation involves the addition of phosphate groups to proteins.
Hydroxylation involves the addition of hydroxyl groups to proteins.
Changes can be reversible (e.g., phosphorylation) or irreversible (e.g., glycosylation, peptide-bond cleavage).
Reversible modifications, such as phosphorylation, can be dynamically regulated to quickly alter protein activity in response to cellular signals.
Irreversible modifications, such as glycosylation and peptide-bond cleavage, are more permanent and often involved in developmental processes or protein maturation.
Effects of Protein Modifications
Post-translational modification can regulate protein activity:
Protein activity is regulated through PTMs
Reversible changes can include phosphorylation, methylation & acylation.
Phosphorylation, methylation, and acylation are reversible modifications that can alter protein activity, stability, and interactions.
Irreversible changes can include cleavage.
Cleavage (proteolysis) is an irreversible modification that can activate or inactivate a protein by removing a specific domain or peptide.
It can also regulate localization:
Localization is determined by PTMs
e.g., lipidation & glycosylation.
Lipidation and glycosylation are PTMs that can target proteins to specific cellular compartments or membranes.
Effect depends on context (e.g., ubiquitination).
Ubiquitination can have different effects depending on the context.
It can target proteins for degradation, alter their activity, or modulate their interactions with other proteins.
Modifications: Glycosylation
Glycosylation is the attachment of carbohydrates.
Glycosylation is the enzymatic process of covalently attaching carbohydrate moieties (glycans) to proteins or lipids.
Common in secreted & cell-surface proteins (protects against digestion).
Glycosylation is particularly common in secreted proteins and cell-surface proteins, where it can protect against degradation by proteases and other enzymes.
Sugars are either N-linked (to asparagine) or O-linked (to serine or threonine).
N-linked glycosylation occurs at asparagine residues within the consensus sequence Asn-X-Ser/Thr, where X can be any amino acid except proline.
O-linked glycosylation occurs at serine or threonine residues.
Modifications: Glycosylation (2)
Glycosylation has a number of roles:
Glycosylation influences protein targeting and folding
It aids protein folding and can influence targeting of newly synthesized proteins to their destination.
Glycans can act as chaperones, assisting in the proper folding of newly synthesized proteins and preventing aggregation.
Glycosylation is involved in cell-to-cell recognition.
Glycans on the cell surface can interact with specific receptors on other cells, mediating cell-cell adhesion and signaling.
Example: Blood Groups
ABO blood group antigens are O-linked oligosaccharides on RBC proteins and lipids.
The ABO blood group antigens are carbohydrate structures that are attached to proteins and lipids on the surface of red blood cells.
All antigens share the O carbohydrate foundation.
The O antigen is the precursor to the A and B antigens, and it is found in individuals with blood type O.
A & B have an extra monosaccharide.
The A antigen has an additional N-acetylgalactosamine residue, while the B antigen has an additional galactose residue.
Modifications: Hydroxylation
Hydroxylation of collagen:
Hydroxylation adds stability to collagen
Collagen is a fibrous connective-tissue protein which contains multiple G-x-y repeats.
Collagen is a major structural protein in the extracellular matrix, providing strength and support to tissues such as skin, bone, and cartilage.
The third amino acid is often a hydroxylated proline.
The hydroxylation of proline residues is essential for the proper folding and stability of collagen fibers.
Collagen cross-linking also requires hydroxylated lysine.
Hydroxylation of lysine residues is required for the formation of cross-links between collagen molecules, which further strengthens the collagen matrix.
Both modifications require vitamin C (ascorbic acid).
Vitamin C is a cofactor for the enzymes that hydroxylate proline and lysine residues in collagen.
Deficiency leads to scurvy (minor hemorrhages, fatigue, eventual heart failure).
Scurvy is a disease caused by vitamin C deficiency, which leads to impaired collagen synthesis and a variety of symptoms.
Humans (and other primates) have lost the last enzyme within the ascorbic acid synthesis pathway.
Humans and other primates cannot synthesize vitamin C and must obtain it from their diet.
Summary
Proteins fold based on their primary and secondary structures, with hydrophobic side chains tending to be buried.
The amino acid sequence (primary structure) and local arrangements of the polypeptide backbone (secondary structure) guide the folding of proteins into their three-dimensional structures, with hydrophobic side chains typically buried in the protein's interior.
Proteins in complexes interact through interfaces formed from complimentary surfaces.
Proteins in complexes interact through interfaces formed from complimentary surfaces that facilitate specific and stable interactions.
Complex formation has many advantages, including cooperative binding.
Forming protein complexes has numerous benefits, including cooperative binding, enhanced stability, and the ability to perform complex functions.
Genetic mutations leading to alterations in protein structure, as well as chemical damage incurred over a protein’s lifetime can directly cause disease.
Genetic mutations that alter protein structure and chemical damage accumulated over a protein's lifetime can disrupt protein function and lead to various diseases.
Post-translational modification of proteins can alter their activity and/or structure.
Post-translational modifications, such as phosphorylation, glycosylation, and ubiquitination, can modulate protein activity, localization, and interactions, thereby regulating a wide range of cellular processes.