Fundamentals of Molecular Biology. Part 1: Molecular Biology of the Cell - A.N. Ogurtsov 2011

Molecular Foundations of Protein Function
Protein Folding, Modification, and Degradation

The Mechanism of METABOLISM/35.html">Protein Biosynthesis will be discussed In the second part of this tutorial. Here, we will focus on the cellular systems responsible for the proper folding and maturation of protein chains.

The Cell possesses quality control systems that detect errors in Protein Synthesis AND folding. A misfolded protein chain lacks the specific activity characteristic of that protein and can cause disease. Errors in the folding process of a protein chain into a native globular protein—known as misfolding—are prevented by two distinct mechanisms.

First, the cell has systems that protect the protein chain from improper folding.

Second, all misfolded protein globules and any cytosolic Proteins no longer needed by the cell undergo degradation (are cleaved into Amino Acids) within the cell.

6.5.1. Protein Folding. A protein chain spontaneously folds into its functional native conformation, which is the most energetically favorable state, with all parameters determined exclusively by the Introduction/19.html">Primary Structure of the protein.

Experiments on the Denaturation and renaturation of protein chains induced by reversible changes in Temperature, environmental acidity, or the presence of specific chemicals that weaken Disulfide Bonds, for instance, have demonstrated that folding occurs spontaneously, requiring no additional protein or non-protein factors.

However, spontaneous folding is a very slow process, and it is inefficient for the cell to synthesize protein chains that take a long time to assume their active form. Furthermore, in vivo aggregation of protein chains can hinder proper folding.

The universal mechanism ensuring rapid and error-free folding involves chaperones—specialized proteins found in all Organelles across all organisms, from Bacteria to primates (Figure 121).

Generally speaking, chaperones fall into two categories.

1. Molecular chaperones, which bind to the protein chain, preventing its aggregation or degradation.

2. Chaperonins, which facilitate protein folding.

Molecular chaperones consist of Hsp70 proteins and their homologs:

- Hsp70 in the Cytosol and mitochondrial matrix,

- BiP in The Endoplasmic reticulum,

- DnaK in bacteria.

Hsp70 proteins are referred to as heat Shock proteins because they are actively synthesized by the cell upon heating. (Hsc70 is a constitutively expressed homolog of Hsp70).

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Figure 121 - Protein folding involving chaperones and chaperonins: a - folding scheme; b - chaperonin Conformations

ATP-bound Hsp70 attaches to a hydrophilic region of the protein chain. ATP Hydrolysis (ATP→ADP) permits the folding of the protein chain. The exchange of ADP for ATP leads to the dissociation of Hsp70 from the folded protein chain (Figure 121, top).

Chaperonins are large cylindrical macromolecular complexes consisting of two oligomeric rings (eight monomers each in the eukaryotic chaperonin TriC and seven monomers each in the chaperonins of bacteria, Mitochondria, and Chloroplasts, GroEL). They isolate the protein chain during the folding process (Figure 121, bottom). GroEL binding to ATP releases the folded protein. GroEL Functions with the assistance of the co-chaperonin GroES, which caps the chaperone cavity during folding.

6.5.2. Chemical modification of proteins. Nearly all proteins undergo chemical modification following their synthesis on the ribosome.

Such modifications, which alter a protein's activity, lifespan, or cellular localization, primarily involve the attachment of specific reactive groups either to the free -NH₂ or -COOH ends of the protein chain, or to the side chains of "internal" amino acid residues.

Although the cell utilizes only 20 amino acids during biosynthesis, protein analysis reveals up to 100 different amino acids within them. These "extra" Amino acids are generated in the protein molecule precisely As a result of chemical modification.

Examples of chemical modification. Acetylation—The addition of an acetyl group (CH₃CO) to the amino group at the N-terminus—occurs in up to 80% of all proteins (Figure 122(a)).

Acetylation protects proteins from rapid degradation. Acetyl and other chemical groups can attach to specific amino acid residues within proteins (Figure 122).

Phosphorylation and dephosphorylation of Serine, Threonine, Tyrosine, and Histidine represent a crucial reversible chemical modification that regulates The activity of numerous proteins (Figure 122(d)).

Glycosylation involves the attachment of linear or branched polysaccharide chains to asparagine, serine, and threonine.

Hydroxylation of Proline and Lysine in Collagen (Figure 8.8(d)).

Methylation of histidine in Membrane Receptors (Figure 122(b)).

γ-Carboxylation of glutamate in prothrombin, an essential Blood-clotting factor (Figure 122(e)).

Proteolysis. Another type of irreversible post-translational Modification of protein molecules does not involve changes in amino acid structure. It is frequently referred to as Processing. This typically involves the enzymatic "Cleavage" of a portion of the polypeptide chain at the C- or N-terminus by protease Enzymes.

Figure 122 - Typical chemical modifications of proteins: a - N-terminal acetylation; b - 3-methylhistidine; c - acetyllysine; d - 3-hydroxyproline; e - phosphoserine; f - γ-carboxyglutamic acid

Proteolytic cleavage (proteolysis) serves as a common activation mechanism for enzymes involved in Blood Coagulation, Digestion, and programmed cell death.

Proteolysis is also responsible for generating active protein Hormones, such as EGF and Insulin, from larger precursor Polypeptides.

Protein self-splicing is a relatively rare type of protein processing in which, unlike proteolysis and without the aid of specialized enzymes, a spontaneous (autocatalytic) excision of an internal segment of the protein chain occurs, followed by the ligation of the remaining parts into a new strand.

A variant of self-splicing is the self-Cleavage of the protein chain terminus ("tail-dropping").

A variant of self-splicing is self-cleavage of the protein chain terminus ("tail clipping").

6.5.3. Protein Degradation. Enzyme activity depends on their concentration, which is therefore determined by the balance between protein Synthesis and degradation rates within the cell.

The lifespan of proteins ranges from a few minutes (for instance, proteins driving mitosis, such as mitotic cyclins) to the entire lifetime of the Organism (such as the proteins in the eye lens).

Eukaryotic Cells employ several intracellular proteolytic Mechanisms for the degradation (turnover) of primarily the following Three types of proteins:

1) misfolded or denatured (unfolded) proteins,

2) "normal" proteins whose concentration needs to be reduced,

3) extracellular proteins taken up by the cell via processes such as endocytosis or phagocytosis.

The primary intracellular degradation mechanism is enzymatic proteolysis within Lysosomes, whose acidic environment is packed with hydrolytic enzymes (Figure 123).

Lysosomal degradation is primarily designed for the proteolysis of exogenous proteins entering the cell through

1) endocytosis,

2) phagocytosis,

3) the proteolysis of non-functional cell organelles that are sequestered and "digested" by the cell (autophagy).

Ubiquitination. Distinct from the lysosomal pathway, another cytosolic mechanism of protein degradation is carried out by modifying lysine residues within the protein through the attachment of ubiquitin, a 76-amino-acid polypeptide. Ubiquitinated proteins are subsequently degraded in proteasomes (Figure 124).

Figure 123 - Proteolysis in lysosomes

Ubiquitination occurs in three stages involving three distinct enzymes.

1. ATP-dependent activation of the ubiquitin-activating enzyme E1 by attaching a ubiquitin molecule to it.

2. Transfer of the ubiquitin molecule to the Cysteine residue of the ubiquitin-conjugating enzyme E2.

3. Formation of a peptide bond between the ubiquitin molecule on the E2 enzyme and a lysine residue on the target protein destined for degradation, a reaction catalyzed by ubiquitin ligase E3.

These three steps are repeated multiple times, with new ubiquitin molecules attaching to previously added ones, resulting in a polyubiquitin chain that is recognized by the proteasome.

Figure 124 - Ubiquitin-mediated proteolysis: a - stages of proteolysis; b - STRUCTURE OF THE proteasome

The proteasome features a hollow cylindrical core capped at both ends by regulatory particles. Inside the proteasome, ubiquitinated proteins are cleaved into short Peptides (7-8 residues long) by multiple proteases lining the inner walls, while ubiquitin molecules are detached from the proteins (step 5 in Figure 124).

Ubiquitin-dependent protein degradation serves two main functions.

1. Removal of cytosolic proteins whose intracellular concentration needs to be reduced.

For example, cyclins must only be present during specific Phases of the Cell Cycle. Phosphorylation of a cyclin alters its conformation, exposing an internal sequence—Arg-X-X-Leu-Gly-X-Ile-Gly-Asp/Asn (where X represents any amino acid)—that was initially buried within the protein globule, making it accessible to ubiquitination enzymes.

2. Proteolysis of misfolded protein molecules in the endoplasmic reticulum.

Misfolding, much like in the previous case, exposes hydrophobic Regions of the polypeptide chain that are normally tucked inside the globule during proper folding. These proteins are translocated into the cytosol, where they are recognized by ubiquitination enzymes.

In both scenarios, the proteins contain specific Amino acid sequences recognized by the ubiquitination machinery.